Aureobasidium pullulans strains DSM 14940 and 14941 Page 1 of 41 Biopesticides Registration Action Document

BIOPESTICIDES REGISTRATION ACTION DOCUMENT



## Aureobasidium pullulans strain DSM 14940

and Aureobasidium pullulans strain DSM 14941

Pesticide Chemical (PC) Code s : 0 46010 and 036010

U.S. Environmen tal Protection Agency Office of Pesticide Programs Biopesticides and Pollution Prevention Division

January, 2012Aureobasidium pullulans strains DSM 14940 and 14941 Page 2 of 41 Biopesticides Registration Action Document

TABLE OF CONTENTS

I. EXECUTIVE SUMMAR Y

................................ ................................ ................................ ...... 4

II. ACTIVE INGREDIE NT OVERVIEW

................................ ................................ ................ 6

III. REGULATORY BACKGROUN D

................................ ................................ ................. 6

A. Applications for Pesticide Registration

................................ ................................ ................................ ................ 6

B. Food Tolerance Exemption

................................ ................................ ................................ ................................ ... 6

IV. RISK ASSESSMENT

................................ ................................ ................................ ...... 6

A. Product Analy sis Assessment (40 CFR § 158.2120)

................................ ................................ ....................... 7

B. Human Health Assessment (40 CFR § 158.2140)

................................ ................................ ................................ 8

C. Environmental Assessment (40 CFR § 158.21 50)

................................ ................................ .............................. 14

V. ENVIRONMENTAL JU STICE

................................ ................................ .......................... 17

VI. RISK MANAGEMENT DECISION

................................ ................................ ................. 17

V II. ACTIONS REQUIRE D BY THE REGISTRANT

................................ ......................... 18

VIII. GLOSSARY OF A CRONYMS AND ABBREVIA TIONS

................................ ........... 19

A. Studies Submitted to Support the Aur eobasidium pullulans strains DSM 14940 and 14941 Pesticide Product Registrations

................................ ................................ ................................ ................................ ................ 20

B. Environmental Protection Agency Risk Assessment Memoranda

................................ ................................ ... 25

C. Other References

................................ ................................ ................................ ................................ ................. 25

APPENDIX A. MICROBI AL PESTICIDES DATA R EQUIREMENTS

............................ 28

APPENDIX B. PESTICI DE PRODUCTS

................................ ................................ ............... 41Aureobasidium pullulans strains DSM 14940 and 14941 Page 3 of 41 Biopesticides Registration Action Document

BIOPESTICIDES REGISTRATION ACTION DOCUMENT TEAM

Office of Pesticide Programs (OPP) Biopesticides and Pollution Prevention Division

Microbial Pesticides Branch

Science Reviews

Ibrahim Barsoum , Ph.D. Product Ana lysis, Human Health

John L. Kough, Ph.D. Product Analysis, Human Health Shannon Borges Environmental Effects

Gail Tomimatsu , Ph.D. Environmental Effects

Regulations

Sheryl K. Reilly, Ph.D. Chief, Microbial Pesticides Branch Alan Reynolds Team Leader, Microbial Pesticides Branch

Susanne Cerrelli Regulatory Action LeaderAureobasidium pullulans strains DSM 14940 and 14941 Page 4 of 41 Biopesticides Registration Action Document

I. EXECUTIVE SUMMARY

Aureobasidium pullulans strains DSM 14940 and DSM 14941 are naturally occur r ing fungi

, which were isolated from apple leaves and classified as Au reobasidium pullulans ( A. pullulans ) by the German Strain Collection for Microorganisms (DSMZ). T hese closely related fungi are neither mutant nor genetically modified strains

.

B ioferm GmbH submitted applications to register two manufacturing

- use pesticid e product s
, Aureobasidium pullulans strain DSM 14941 Technical (EPA File Symbol 86174

- E) and Aureobasidium pullulans strain DSM 14940 Technical (EPA File Symbol 86174

- R )

. The active ingredient is 80% w/w (minimum of 2 x 10 9 cfu/g) in each of these t echnic al products. B ioferm

GmbH also submitted applications to register two end

- use pesticide product s :

**•** Botector (EPA File Symbol 86174

- G)

, containing 40% w/w Aureobasidium pullulans

strain DSM 14940 (minimum of 5.3 x 10 8 cfu/g) and 40% w/w Aureobasidium pullula ns strain DSM 14941 (minimum of 5.3 x 10 8 cfu/g)

, to protect pome fruits from storage diseases; grapes and strawberries from Botrytis ; and stone fruit from Monilia ; and

**•** Blossom Protect (EPA File Symbol 86174

- U)

, to protect pome fruits against fire blig ht, containing the active ingredients of 25.8% w/w Aureobasidium pullulans strain DSM

14940 (minimum of 4.4 x 10 8 cfu/g) and 25.8% w/w Aureobasidium pullulans strain

DSM 14941 (minimum of 4.4 x 10 8 cfu/g).

A

. pullulans is a “yeast

- like” saprophytic fungu s found in the phyllosphere of many plants, and it

also has been isolated from soil and aquatic environments (Chi et al. 2009, Cooke 1959, Slepecky and Starmer 2009). It is commonly isolated from healthy grape (Fleet 2002, 2003)

, apple (Granado et al. 200 8), and other plants (Gao et al. 2005, Osono 2008, Slavikova et al. 2007, Suryanarayanan et al. 2005, Webb and Mundt 1978). T his fungus is generally recognized

as a n organism that derives its nourishment from non

- living or decaying organic matter ( saprobe )

and as a weak pathogen or parasite of certain plants (Kockova

- Kratochvilova et al. 1980, Cooke 1959). A. pullulans is an antagonist of several plant disease

- causing organisms, and it is an important producer of enzymes for biotechnological and industrial applications (Chi et al. 2009).

The applicant, bioferm GmbH, submitted product analysis, toxicology, and non

- target organism

data and other information sufficient to satisfy the data requirements for an unconditional registration of Aureobasidium p ullulans strains DSM 14940 and 14941 for the proposed pesticide use s

. The data, cited literature, and other information submitted by the applicant were reviewed by Agency scientists from the Microbial Pesticides Branch (MPB) in the Biopesticides

and Pollut ion Prevention Division (BPPD).

Th e submitted acute oral, pulmonary and subcutaneous injection toxicity/pathogenicity studies in laboratory rats showed that, at a single high dose, A. pullulans strain DSM 14941 is not toxic, infective, and/or pathogeni c. Since the p roduct analysis data demonstrate d that strain DSM 14941 is closely related to strain DSM 14940

, the results of these studies were applicable to both

strains. A second acute subcutaneous toxicity/pathogenicity study was submitted that was

con ducted with both A. pullulans strain s ( DSM 14940 and DSM 14941 ), and neither where

shown to be toxic, infective, or pathogenic in laboratory rats

.Aureobasidium pullulans strains DSM 14940 and 14941 Page 5 of 41 Biopesticides Registration Action Document

Although A

. pullulans infections in im mune

- compromised patients have been reported in published literature

, the Agency’s scientists found that the organisms responsible for these

infections were not closely related to the A

. pullulans strains (DSM 14941 and DSM 14940 ) proposed for registration as pesticides. Since neither of the strains can grow at temperatures above 34oC, their potential pathogenicity in humans , with an average body temperature of 37 oC, is virtually impossible. No dietary or non

- dietary exposure issues were identified to indicate that

the intended use of these active ingredients as pesticides would be harm ful to infants, children, and the general U.S. population.

The Agency’s scientists determined that adverse effects to non

- target organisms ( wild mammals, birds, non

- target plants, non

- target insects, honey bees, freshwater fish and inverteb rates) are not anticipated as a result of the proposed pesticide uses of A

. pullulans strain s DSM 14940 and

DSM 14941. A “No Effect” determination ha s been made for direct and indirect effects to listed species and their designated critical habitats result ing from the proposed uses of Aureobasidium pullulans strain s DSM 14940 and DSM 14941, as labeled.

I f post

- harvest use sites are proposed for these products in the future , a dditional product analysis data would be required

.

On October 1, 2009, EPA annou nced a new policy to provide a more meaningful opportunity for the public to participate in major registration decisions before they occur. According to this policy, EPA intends to provide a public comment period prior to making a registration decision

for , at minimum, the following types of applications: new active ingredients; first food uses; first outdoor uses; first residential uses; or any other registration actions for which EPA believes there may be significant public interest. Consistent with th is policy

, the proposed pe sticide products containing two new microbial pesticide active ingredients, A

. pullulans strains DSM

14941 and DSM 14940, were subject to a 30

- day comment period. During this comment period,

one comment was received in dicating suppor t of the proposed uses because strains of this species are commonly present in apple trees in the United States

, and the biopesticide shows “significant promise for control of fire blight,” which “may help replace the use of antibiotic ” for controlling thi s disease

.

The Agency’s risk assessments and conclusions that the standards for registration under FIFRA section 3(c)(5) have been met for the proposed active ingredients, Aureobasidium pullulans

strain DSM 14940 and Aureobasidium pullulans strain DSM 14 941

, are provided in this

B iopesticide Registration Action Document ( BRAD)

.Aureobasidium pullulans strains DSM 14940 and 14941 Page 6 of 41 Biopesticides Registration Action Document

II. ACTIVE INGREDIENT OVERVIEW

Biological Name: Aureobasidium pullulans strains DSM 14940 and 14941

Culture Deposit: German Strain Collection for Microorganisms (DSMZ) und er the

reference number DSM 14940 & DSM 14941

OPP Chemical Code: 046010 and 036010

Type of Pesticide: Microbial Pesticide

– Fungicide

See Appendix B for specific information (e.g., use sites, application rate s , metho ds of application, formulation type s , and target pests) regarding the registered pesticid e products containing this active ingredient.

III. REGULATORY BACKGROUND

A. Applications for Pesticide Registration

On September 18, 2009, bioferm GmbH, (addre ss: Konrad Lorenz Str. 20; A

- 3430; Tulin Austria) submitted applications to register two manufacturing use products, Aureobasidium pullulans strains DSM 14940 Tech n ical (EPA File Symbol 86174

- E) and Aureobasidium pullulans strains DSM 14941 Technical (EPA File Symbol 86174

- R); and two end

- use products, Blossom Protect (EPA File Symbol 86174

- U) and Botector (EPA File Symbol 86174

- G ) under

FIFRA section 3

. On M arch 10, 2010

, EPA announced receipt of these application s to register pesticide product s containin g new active ingredient s ( 75 Federal Register (FR) 11175 ) and opened a 3 0

- day public comment period pursuant to the provisions of FIFRA section 3(c)(4). No

comments were received fo llowing this publication

.

B. Food Tolerance Exemption

Concurrent with its registration application s
, and in accordance with the Federal Food, Drug, and Cosmetic Act (FFDCA) section 408(d), bioferm GmbH submitted a petition to establish an exemption fro m the requirement of a tolerance for Aureobasidium pullulans strains DSM 14940

and DSM 14941 (Pesticide Petition (PP) 9F7623 ). On March 10, 2010 EPA announced ( 75 FR

11171 ) that bio ferm GmbH proposed to establish an exemption from the requirement of a

tolerance for residues of the fungicides, Aureobasidium pullulans strains DSM 14940 and DSM 14941, in or on all food commodities

, and opened a 30

- day comment period. No comments were

re ceived following this publication.

IV. RISK ASSESSMENT

EPA issued a Final Rule ( 72 FR 61002 ) amending the data requirements to support registration of microbial pesticides and updated the definition of “ microbial pesticides ,” which became

effective on December 26, 2007. The data and scientific risk assessments presented in thisAureobasidium pullulans strains DSM 14940 and 14941 Page 7 of 41 Biopesticides Registration Action Document

BRAD were considered in light of these requirements.

Classification of Data Submissions

The classifi cations for each study or data submission are assigned by EPA science reviewers

, and

i ndicat e the usefulness of the information for risk assessment. A n “ acceptable ” rating indicates a study conducted in accordance with an established Agency guideline is sc ientifically sound and

useful for a risk assessment. A stud y may have certain aspects determined not to be scientifically acceptable

, but with additional information, it may be upgraded to “acceptable.” This study is

rated “ supplemental : upgradeable ,” and EPA provides information to the registration applicant concerning what must be provided to upgrade the study

. A “ supplemental ” rating the data provide some information useful for risk assessment, or it may mean that the data were not required. Both “ accept able” and “supplemental ” studies may be used in the risk assessment

process as appropriate. An “unacceptable ” rating indicates that new data need to be submitted.

Acute toxicity studies are assigned Toxicity C ategories

, based on the hazard(s) identified f rom studies and/or other information pro vided or cited by the registration applicant

. The active ingredient and/ or product is classified as Toxicity Category I, II, III, or IV, where Toxicity Category I is assigned for the highest level of toxicity

, and T oxicity Category IV indicates the

lowest.

A. Product Analysis Assessment ( 40 CFR § 158.2120 )

A. p ullulans strains DSM 14940 and DSM 14941 were isolated from apple leaves and classified

as Aureobasidium pullulans by the German Strain Collection for Microorganisms (DSMZ) by means of morphological characterization of the colonies on yeast medium (YM) aga r plates. The two strains show similar colony characteristics on agar plates; they are identical on their 18S

r ibosomal RNA gene region; and they are also identical on their Internal Transcribed Spacer (ITS) regions ITS1 and ITS4, which are commonly used f or inter

- and intra

- species differentiation. Definitive differentiation can only be achieved by molecular biological methods focusing on the whole genome, either by conducting such analyses as RAPD PCR (Random Amplification of Polymorphic DNA Polymerase Ch ain Reaction) or by using recently developed

strain

- specific primer characteristics. xv 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 iso lates of A. pullulans and from other yeast

- like fungi. The subject strains are neither mutant strains nor genetically modified strains. [ Pesticide Petition (PP) 9F7623 ]

A

. pullulans can live in an environment offer ing very low levels of nutrients

. A

. pullulans can be found in environments with fluctuating water activities, such as leaf surfaces , bathrooms, food,

and feeds ; and highly osmotically stressed environments, such as hypersaline w aters in salterns and rocks and monuments. Due to the production of large quantities of yeast

- like propagules, this fungus disperses globally. [ Pesticide Petition (PP) 9F7623 ]

A ll product analysis data requirements for A

. pullulans strains DSM 14940 and 1 4941 have been

fulfilled to support current use sites . Refer to Tables 1 , 2, 3, 4 , 5, 6 and 7 in Appendix A for a brief summary of the data requirements, including both generic and product

- specific information.Aureobasidium pullulans strains DSM 14940 and 14941 Page 8 of 41 Biopesticides Registration Action Document

B. Human Heal th Assessment ( 40 CFR § 158.2140 )

1. Toxicity

All toxicology data requirements for A

. pullulans strains DSM 14940 and 14941 have been

fulfilled

. Acceptable Tier I mammalian toxicology data and information support registration of

the A

. pullulans strains DSM 14940 and 14941 pesticide products

. Furthermore, Tier II and Tier III studies were not require d for A

. pullulans strains DSM 14940 and 14941 b ecause of the lack of acute toxicity/pathogenicity in the Tier I studies.

For a compr ehensive summary of the generic toxicology data requirements described in sections IV(B)(1)(a) and IV(B)(1)(b), as well as additional product

- specific data submitted to support the individual registrations, refer to Table 8 in Appendix A

.

a. Acute Toxicity/Pathogenicity

– Tier I

Twenty

- four rats were given a single dose of A. pullulans strain DSM 14941. Three animals of

each sex were sacrificed on days 3, 7, and 14; the remainin g animals were sacrificed at the end of the study (day 21). Body tissues were examined for A. pullulans . All samples of feces, blood, brain, lung, liver, kidney, spleen, stomach, and intestine were negative. The lymph nodes of one animal sacrificed on day 7 tested positive. Based on the results of this study

, yeast isolate A

. pullulans DSM 14941 does not appear to be toxic, infective, and/or pathogenic in rats, when

dosed at 4 x 10 8 cfu/animal

. Acceptable

. Acute Oral Toxicity/Pathogenicity ( Office of Chemical Safety and Pol lution Prevention (OCSPP) Guideline 885.3050; Master Record Identification Number (MRID No. ) 47899302 ):

Acute Pulmonary Toxicity/Pathogenicity (OCSPP Gui de line 885.3150; MRID No. 47899303 ) : M ale and female rats were given an intratracheal dose of A. pullulans strain DSM 14941 Technical (4 x 10 8 cfu/g). Interim sacrifices were made 3 hours post

- dose and on study days 3, 7, and 14; the remaining animals were s acrificed at the end of the study (day 21). There was no mortality, and, except on the day of dosing when the entire test material group showed reduced motor activity and dyspnea, all animals appeared normal throughout the study. Tissue samples

were evalua ted for A. pullulans after sacrifice. The following tissues were negative for A. pullulans at all time points: feces, brain, kidney, and intestine. Blood samples were negative except for one male on day 0; lung samples were negative except for two males an d two females on day 0; liver samples were negative except for three males on day 0; spleen samples were negative except for two males on day 0; lymph node samples were negative except for one animal on day 7, and stomach samples were negative except for t wo males on day 0. Based on these results, A

. pullulans DSMZ 14941 does not appear to be toxic, infective, and/or pathogenic in rat, when dosed at 0.8 x 10 8 cfu/animal

. Acceptable

.

Acute Injection Toxicity/ Pathogenicity (OCSPP Guidel ine 885.3200; MRID No. 47871807 and 47871808) : M ale and female rats were injected with A . pullulans strain DSM 14940 (2.36 x 10 10 cfu/g) and DSM 14941 (1.5 x 10 10 cfu/g). Interim sacrifices were made on days 3, 7, and 14; the remaining

animals were sacrificed at the end of the study (day 21). There were no deaths, and all animals appeared normal throughout the study except for edema or slight erythema at the injection site. Samples of the following tissues were negative for A. pullulans at all time points: feces, blood, brain, lung, liver, kidney, spleen, stomach, and intestine. Lymph node samples were negativeAureobasidium pullulans strains DSM 14940 and 14941 Page 9 of 41 Biopesticides Registration Action Document

except for one animal sacrificed on day 7. A

. pullulans strains DSMZ 14940 and DSMZ 14941 did not appear to be toxic

. Acceptable

.

In a second acute subcutaneous toxicity /pathogenicity study (MRID 47871808), male and female

rats were injected with A . pullulans strain DSM 14941 (1.1 x 10 9 cfu/g). Interim sacrifices were made on days 1 and 7; the remaining animals were sacrificed at the end of the study (day 21).

. There were no deaths, and all animals appeared normal throughout the study except for severe edema or slight erythema at the injection site. Samples of the following tissues were negative for A. pullulans at all time points: brain, lung, spleen, kidney, lymph nodes, blood and urine. The skin was positive for A. pullulans in three males and three females from the test material group on day 1 and in two females of the same group on day 7. The liver of one male from the test material group was positive on day 1. The cec um contents were positive in one male and one female from the test material group on day 1, and in two females from the same group on day 7.

A

. pullulans DSM 14941 does not appear to be toxic, infective, and/or pathogenic in rats, when

dosed at 1.6 x 10 7 c fu/animal. Acceptable.

Hypersensitivity Incidents (OCSPP Guideline 885.3400; MRID No. 47 945023 ) No hypersensitivity incidents, including immediate

- type or delayed

- type reactions of humans

and domestic animals during research, development, or testing o f A

. pullulans strains DSM

14940 and 14941 were reported by the applicant . Any future hypersensitivity incidents must be reported to EPA (refer to OCSPP Guideline 885.3400)

. :

Cell Culture (OCSPP Guideline 885.3500) This study was not required because Aur eobasidium pullulans strains DSM 14940 and 14941 are not virus es (refer to test note #4 of 40 CFR § 158.2140(d)). :

Acute Oral Toxicity

Six rats received a single 2000 mg/kg bw dose of Blossom Protect [22% (2 x 10 10 cfu/g) A

. pullulans strain DSM 14940, 22% (2 x 10 10 cfu/g) A . pullulans strain DSM 14941, 25% sucrose, 6% water]. There was no mortality, and all animals appeared normal throughout the study. The

LD 50 was >2000 mg/kg. (OCSPP Guideline 870.1100 ; MRID No. 47869614) :

Acceptable; T oxicity C ategory I V.

Ten rats were dosed with 2000 mg/kg Blossom Protect (7 x 10 9 cfu/g each of DSM 14940 and DSM 14941). One male was humanely sacrificed on day 5 due to a ruptured abscess at the injection site; all other ani mals survived until study end on day 14. Necropsy revealed abscesses at all injection sites. The LD 50 was >2000 mg/kg. Acceptable. Acute Subcutaneous Toxicity (MRID 47869616) :

In an acute dermal toxicity study, 10 rats were dosed with 2000 mg/kg Blossom Protect (7 x 10 9

cfu/g each of strains DSM 14940 and DSM 14941). One male and one female exhibited chromodacryorrhea, but this was not considered to be toxicologically significant. All animals appeared healthy at necropsy. The LD 50 was >2000 mg/kg. Acute Dermal Toxicity (OCSPP Guideline 870.1200 ; MRID 47869615) :

Acceptable; T oxicity C ategory IV.Aureobasidium pullulans strains DSM 14940 and 14941 Page 10 of 41 Biopesticides Registration Action Document

In an acute inhalation toxicity study, 10 rats were exposed ( nose

- only ) to a 10% suspension of

Blossom Protect (7 x 10 9 cfu/g A. pullula ns strain DSM 14940 and 7 x 10 9 cfu/g A. pullulans strain DSM 14941) at 5.17 mg/L. The MMAD was 4.2 μm. There were no deaths and all animals appeared healthy throughout the study. Necropsy was unremarkable. The LC 50 was >5.17 mg/L. Acute Inhalation Toxicity (OCSPP Guideline 870.1300 ; MRID 47869617) :

Acceptable; T oxicity C a tegory IV.

Three rabbits were dosed with 0.1 mL (60

- 62 g) of Blossom Protect (7 x 10 9 cfu/g A. pullulans

strain DSM 14940 and 7 x 10 9 cfu/g A. pullulans strain DSM 14941). One animal had a score of 1 for conjunctival redness one hour after application. There were no other clinical signs, and all animals appeared normal at 24 hours. Blossom protect was considered to be virtually non

irritating. Acute Eye Irritation (OCSPP Guideline 870.2400; MRID 47869618) :

Acceptable; T oxicity C ategory IV.

T hree rabbits were dosed with 0.5 g Blossom Protect (7 x 10 9 cfu/g A. pullulans strain DSM

14940 and 7 x 10 9 cfu/g A. pullulans strain DSM 14941) moistened with 1.0 mL deionized

water. All animals a ppeared normal throughout the study. Blossom Protect was considered virtually non

- irritating. Acceptable; T oxicity C ategory IV. Primary Derm al Irritation (OCSPP Guideline 870.2500 ; MRID 47869619) :

In vivo Micronucleus (MRID 47899304)

Mice received single oral gavage doses of A. pullulans strain DSM 14941 (2000 mg/kg)

di ssolved in water. There were no clinical signs. The animals were sacrificed 24 or 48 hours

post

- dosing

, and the femoral bone marrow was immediately harvested. The ratio of polychromatic erythrocytes to total erythrocytes (mature and immature) was determine d, and 2000 immature erythrocytes/animal were scored for the incidence of micro nucleated immature erythrocytes. A. pullulans strain DSM 14941 did not produce a statistically significant increase in micro nucleated immature erythrocytes compared to the neg ative control animals. The response of the positive control animals (cyclophosphamide, IP injection, 10 mg/kg) was appropriate. Acceptable. :

The effects of temperature on the reproduction of A. pull ulans strains DSM 14940, DSM 14941, and CBS 626.85 in liquid culture or on agar plates were examined. All three strains grew well at 30°C. At 33°C, the number of duplications of DSM 14940 and DSM 1 4941 was less than one, while the number of duplications fo r CBS 626.85 was approximately five. Neither DSM 14940

nor DSM 14941 grew at 35 or 37°C. CBS 625.85 produced approximately two duplications at 35°C and one at 37°C. Based on the lack of growth at temperatures of 35°C and above, strains

DSM 14940 and DSM 14 941 are non

- pathogenic in humans. Acceptable. Influence of Temperature on Reproduction (MRID 47871833) :

Toxicity/Pathogenicity of Aureobasidium pullulans in Immunocompromised Patients (MRID 4787181631) : P ublished literature describing various infections caused by Aureobasidium pullulans in

immun e

- compromised p atients were reviewed. None of the infections were caused by the two

strains of A. pullulans (DSM 14941, 14940) submitted for registration.Aureobasidium pullulans strains DSM 14940 and 14941 Page 11 of 41 Biopesticides Registration Action Document

b. Acute Toxicology and Subchronic Toxicity/Pathogenicity

– Tier II; Reproductive Fertility Effects, Carci nogenicity, Immunotoxicity, and Infectivity/Pathogenicity Analysis

– Tier III

Tier II and Tier III studies were not required for A. pullulans strains DSM 14940 and 14941 based on the lack of acute toxicity/pathogenicity in the Tier I studies.

c . Endocrine Disruptors

As required under FFDCA section 408(p), EPA developed the Endocrine Disruptor Screening Program (EDSP) to determine whether certain substances (including pesticide active and other ingred ients) may have an effect in humans or wildlife similar to an effect produced by a “naturally occurring estrogen, or other such endocrine effects as the Administrator may designate.” The EDSP employs a two

- tiered approach to making the statutorily required determinations. Tier 1 consists of a battery of 11 screening assays to identify the potential of a chemical substance to interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal systems. Chemicals that go through Tier 1 screening and are fou nd to have the potential to interact with E, A, or T hormonal systems will proceed to the next stage of the EDSP wh ere EPA will determine which, if any, of the Tier 2 tests are necessary based on the available data. Tier 2 testing is designed to identify a ny adverse endocrine

- related effects caused by the substance, and establish a quantitative relationship between the dose and the E, A, or T effect.

Between October 2009 a nd February 2010, EPA issued test orders/data call

- ins for the first group of 67 chem icals, which contains 58 pesticide active ingredients and 9 inert ingredients. This list of chemicals was selected based on the potential for human exposure through pathways such as food and water, residential activity, and certain post

- application agricul tural scenarios. This list should not be construed as a list of known or likely endocrine disruptors.

A. pullulans strains DSM 14940 and 14941 are not among the group of 58 pesticide active ingredients on the initial list to be screened under the EDSP. Un der FFDCA section 408(p), EPA must screen all pesti cide chemicals. Accordingly, EPA anticipates issuing future EDSP

orders/data call

- ins for all pesticide active ingredients.

For further information on the status of the EDSP, the policies and procedures, the list of 67 chemicals, the test guidelines and the Tier 1 screening battery, please visit our website: http://www.epa.gov/endo/

.

2

. Food Quality Protection Act (FQPA) Considerations

Section 408(c)(2)(A)(i) of F FDCA allows EPA to establish an exemption from the requirement for a tolerance (the legal limit for a pesticide chemical resi due in or on a food) only if 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 t hrough drinking water and in residential settings but does not include occupational exposure. Pursuant to section 408(c)(2)(B) of FFDCA, in establishing or maintaining in effect an exemption from the requirement of a tolerance, EPA must take into account t he factors set forth in section 408(b)(2 )(C) of FFDCA, which require EPA to give special consideration to exposure of infants and children to theAureobasidium pullulans strains DSM 14940 and 14941 Page 12 of 41 Biopesticides Registration Action Document

pesticide chemical residue in establishing a tolerance and to “ensure that there is a reasonable certainty tha t no harm will result to infants and children from aggregate exposure to the pesticide chemical residue....” Additionally, section 408(b)(2)(D) of FFDCA requires that EPA consider “available information concerning the cumulative effects of [a particular pe sticide's] residues and other substances that have a common mechanism of toxicity.”

EPA performs a number of analyses to determine the risks from aggregate exposure t o pesticide residues. First, EPA determines the tox icity of pesticides. Second, EPA examin es exposure to the pesticide through food, drinking water, and through other exposures that occur as a result of pesticide use in residential settings.

Consistent with s ection 408(b)(2)(D) of FFDCA, EPA has reviewed the available scientific data

and other relevant information, and considered its validity, completeness, and reliability and the relationship of thi s information to human risk. EPA has also considered available information concerning the variability of the sensitivities of major identifiable sub groups of consumers, including infants and children.

Based on the acute toxicity/pathogenicity data and information discussed previously and presented in Table 8 in Appendix A , t he data required for a FQPA risk assessment for A. pullulans strains DSM 14940 and 14941 have been fulfilled

.

a. Aggregate Exposure

In examining aggregate exposure, section 408 of FFDCA directs 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).

Dietary Exposure and Risk Characterization : A. pullulans is lik ely to be commonly present on fresh produce. According to Webb and Mundt (1978) A. pullulans is “a major resident on most green plants.” In a study with several species of crop plants, they determined A. pullulans to be among the most abundant (71%

- 85%) of all fungi present on plant surfaces. A. pullulans made up an average of 77.1% of the mold species isolated on green beans, and A. pullulans and other fungi occurred at high levels (up to 2.7 x 10 4 cfu/cm 2 ) on cucumbers and squash. Dietary

exposure to A. pullulans strains DSM 14940 and 14941 is possible from ingestion of

s trawberries, gr apes, p ome and stone fruits harvested from plants treated with pesticide containing these active ingredients

. N one of the studies indicated that toxic or pathogenic effec ts
will result if dietary exposure occurs. In addition

, neither strain of A. pullulans can grow at human body temperature ( 37°C ), even if dietary exposure occurred, there is a reasonable certainty th at no harm will result from exposure to such residues bec ause of the lack of toxicity, infectivity, and or pathogenicity in the previously described toxicological studies (see section IV(B)(1)(a) and Table 8 in Appendix A )

.

Drinking Water Risk Characterization : Naturally occurring A. pullulans is widely distributed in

many environmental habitats (e.g., in soil, water, leaves and other above ground plant parts, wood and many other plant materials, rocks monuments and limestone; Chi et al. 2009). Human

exposure to residues of pesticide s containing A. pullulans strains DSM 14940 and 14941 in consumed drinking water is highly unlikely , however, since t he proposed use s do not include direct application to aquatic environments , and neither strain is

. A

. pullulans strains DSMAureobasidium pullulans strains DSM 14940 and 14941 Page 13 of 41 Biopesticides Registration Action Document

14940 and 149 41 known to inhabit aquatic environments

. Should A. pullulans strains DSM

14940 and 14941 be transferred to surface or ground water intended for human consumption, however, the fungi would not survive the conditions water is subjected to in a drinking w ater treatment facility, including high temperatures, chlorination, pH adjustments, and/or filtration. Even if exposure should occur through consumed drinking water, there is a reasonable certainty

th at no harm will result from exposure to such residues

, because of the lack of toxicity, infectivity, and / or pathogenicity in the previously described toxicological studies (see section IV(B)(1)(a) and Table 8 in Appendix A )

.

Non

- occupational, Residential Risk Characterization : The u se sites for th ese product s include agricultural and residential garden s

. B ased on the data and other information submitted to satisfy data requirements for registration of the manufacturing

- use and end

- use pesticide product s containing the active ingredie nt s A. pullulans strains DSM 14940 and 14941

, no toxicity, infectivity, pathogenicity or other adverse effects from non

- occupational exposure are expected (see section IV(B)(1)(a) , above, and Table 8 in Appendix A ).

b. Cumula tive 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 exemption

, EPA consider “available information concerning the

cumulative effects of [a particular pesticide’s] residues and other substances that have a common mechanism of toxicity.”

A. pullulans strains DSM 14940 and 14941 do not appear to produce toxic metabolite s against

target pest s and do not share a common mechanism of t oxicity with other substances. For the purposes of this tolerance action, therefore, EPA has assumed that A. pullulans strains DSM

14940 and 14941 do not have a common mechanism of toxicity with other substances. For

information re garding EPA ’s efforts to determine which chemicals have a common mechanism of toxicity and to evaluate the cumulative ef fects o f such chemicals, see EPA ’s website at http://www.epa.gov/pesticides/cumulative

.

c. Determinatio n of Safety for the U.S. Population, Infants and Children

FFDCA sectio n 408(b)(2)(C) provides that EPA shall assess the available information about consumption patterns among infants and children, special susceptibility of infants and children to pestici de 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 sectio n 408(b)(2)(C) provides that EPA shall apply an additional tenfold (10X) margin of s afety for infants and children in the case of threshold effects to account for prenatal and postnatal toxicity and the complet eness of the database on toxicity and exposure unless EPA determines that a different margin of safety will be safe for infants an d children. This additional margin of safety is commonly referred to as the FQPA Safety Factor. In applying this provision, EPA either retains the default value of 10X or uses a different additional safety factor when reliable data available to EPA support the choice of a different factor.

Based on the acute toxicity and pathogenicity data summarized in section IV(B)(1)(a) and Table

5 in Appendix A

, 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 the residues of A. pullulans strains DSM 14940 and 14941 . This includes all anticipated dietary exposures and allAureobasidium pullulans strains DSM 14940 and 14941 Page 14 of 41 Biopesticides Registration Action Document

other exposures for which there is reliable information. EPA has arrived at this conclusion

because the data and information available on A. pullulans strains DSM 14940 and 14941 do not demonstrate toxic, pathogenic, and/ or infective potential to mammals. T here are no threshold effects of concern and, as a result, the p rovision requiring an additional margin of safety does not apply.

3. Occupational Exposure and Risk Characterization

Handler exposure to A. pullulans strains DSM 14940 and 14941 is not expected to pose any undue risk. However, p ublished literature was submitted concerning various infections caused by Aureobasidium pullulans in immun e

- compromised patients (MRID 47871816

- 31). These infections were not caused by the two strains of A. pullulans (DSM 14941, 14940) submitted for registration. As a precaution ary measure
, the Agency is requiring that the label include specified personal protective equipment ( PPE) to protect workers from repeated exposure to Aureobasidium pullulans strains DSM 14941 & DSM 14940 . A ppropriate personal protective equipment and pr ecautionary statements are required on pesticide product labels to mitigate any potential risks to pesticide handlers due to prolonged or numerous exposures. Handlers applying

A. pullulans strains DSM 14940 and 14941 end

- use products in agricultural settin gs must wear a long

- sleeved shirt, long pants, socks, shoes, waterproof gloves, and a dust/mist filtering respirator meeting NIOSH standards of at least N

- 95, R

- 95, or P

- 95. Additional PPE, other than the standard described above, may be required on a prod uct

- specific basis.

4. Human Health Risk Characterization

EPA considered human exposure to A. pullulans strains DSM 14940 and 14941 in light of the

standard s for registration in FIFRA and the relevant safety factors in FFDCA N o unreasonab le

adverse effect s to the U.S. population in general, and to infants and children in particu lar, will

result when A. pullulans strains DSM 14940 and 14941 pesticide products are used in

accordance with EPA

- approved labeling.

C. Environmental Assessment ( 40 CFR § 158.2150 )

The Agency has performed an environmental risk assessment for the proposed uses of A. pullulans strain DSM 14940 and 14941.

The data and data waiver rationale, submitted by the applicant to support the pesticide products containing A. pullulans strains DSM 14940 and 14941 fulfill the Tier I non

- target organism data requirements and are sufficient for risk assessment purposes

. Further testing of non

- target organisms at higher tier levels (i.e., Tiers II, III, and IV) is not required for the current uses and application methods. EPA performed an environmental risk assessment based on the data and

dat a waiver rationale provided by the applicant and determined that the proposed use of A. pullulans strains DSM 14940 and 14941 does not pose significant risk to non

- target organisms when used according to label directions.

For a compr ehensive summary of t he generic data requirements described in sections IV( C)(1)

, refer to Table 9 in Appendix A

.Aureobasidium pullulans strains DSM 14940 and 14941 Page 15 of 41 Biopesticides Registration Action Document

1. Ecological Exposure and Risk Characterization

a. Terrestrial Animals and Plants

Birds (OCSPP Guideline 885.4050) and Mammals (OCSPP Guideline 885.4150) ; MRID

47871811 The applicant submitted guideline studies and scientific rationale to satisfy data requirements for

non

- target organism risk assessment with the active ingredient. Submitted studies show that

adverse effects to birds and mammals do not occur at maximum hazard doses ( 1.0 x 10 9

cfu/ animal for birds and 4 x 10 8 cfu/animal for mammals) after 30 and 21 days of exposure, respectively. Additionally, information was submitted to show that A. pullulans strain s DSM

14940 and DSM 14941 do not grow at avian or mammalian body temperatures. :

A guideline study with honey bees showed no toxicity or pathogenicity after 22 days of exposure

at 1.0 x 10 6 cfu/bee. Scientific rationale discussed additional evidence that A. pullulans is not toxic, pathogenic, or infective to other non

- target insects. A. pullulans has been reported as part

of the normal flora of insect s ( Pagnocca et al. 2008, Sacchi and Vaughan

- Martini 2002, 2003) and stingless bees (Rosa et al. 2003) . I t is commonly found in the environment, and it is not known as an entomopathogen. Therefore, A. pullulans strain s DSM 14940 and DSM 14941 are not expected to cause ad verse effects in non

- target insects and honey bees. Non

- target Insects (OCSPP Guideline 885.4340) and Honey Bees (OCSPP Guideline 885.4380) ; MRID 48531003:

Non

- target Plants (OCSPP Guideline 885.4300 ): Based on published literature, normal populations of A. pullulans on plants occur at up to 10 5 cfu/cm 2 (e.g., see Osono 2008). The end use products (EPs) are applied at rates that are within the range of naturally occurring levels on plants. Spray drift analysis indicates that the amounts deposited at a point immediately adjacent to the treated area in terrestrial environments are several orders of magnit ude lower. A spray buffer of 25 ft is required around aquatic areas, to limit concentrations of the active ingredients in aquatic environments to within the range of concentrations observed in nature (e.g., see

Slavikova and Vadkertiova 1997 ). Therefore, t he proposed applications of Aureobasidium pullulans strain s DSM 14940 and DSM 14941 are not expected to result in concentrations in terrestrial or aquatic environments above those found in the environment. Data waiver rationale

was submitted to fulfill th e non

- target plant data requirement.

A ureobasidium pullulans is regarded as a weak plant pathogen (Deshpande et al. 1992, Kockova

Kratochvilova et al. 1980). Instances where disease is known to occur have required vectors (e.g., stigmatomycosis in pista chio) or where A. pullulans was intentionally applied at very high concentration (10 16 cfu/mL) for weed biocontrol. A. pullulans also is reported as a naturally occurring epi

- or endophyte from a wide range of plant species but does not caus e any signs of disease (Deshpande et al. 1992, see also Gao et al. 2005, Granado et al 2008, Osono 2008, Slavikova et al. 2007, Smith et al. 1989, Suryanarayanan et al. 2005, Webb and Mundt 1978). A. pullulans also is very common on the phylloplane of plants (Chi et al. 2009). BPPD performed a literature search in several databases and did not find any reports of Aureobasidium pullulans as a pathogen in plants. Based on the available data, BPPD concluded that A. pullulans strains

DSM 14940 and DSM 14941 have low potent ial to cause toxicity or pathogenicity in plants.Aureobasidium pullulans strains DSM 14940 and 14941 Page 16 of 41 Biopesticides Registration Action Document

b. Aquatic Animals and Plants

Freshwater Fish (OCSPP Guideline 885.4200) and Freshwater Invertebrates (OCSPP Guideline

885.4240 ) ; MRID 47871812 : A freshwater fish study showed that A. pullulans stra ins DSM 14940 and 14941 do not produce toxic effects at 100 mg/L ( 4 .1 x 10 3 cfu/mL measured )

. Additional rationale and a spray drift analysis were sufficient to show that significant concentrations (i.e., above naturally occurring levels) will not reach aq uatic environments when applied according to the proposed label. A 21

- day Daphnia study showed that the active ingredients are not toxic or pathogenic to aquatic invertebrates at concentrations (approx. 2

– 24 cfu/mL) that may occur in water as a result of the proposed applications (EC 50 > 1.3 x 10 6

cfu/mL, NOEC = 1.3 x 10 6 cfu/mL). Based on this information, adverse effects are not expected

to freshwater fish and invertebrates. Testing on m arine/estuarine fish and invertebrate and aquatic plant s was not re quired because significant concentrations are not expected to reach these organisms

.

Estuarine/Marine Fish and Invertebrates (OCSPP Guideline 885.4280) : As determined above, the proposed applications are not expected to reach aquatic areas in significan t amounts with the 25 foot spray buffer requirement in place. Therefore, adverse effects to marine or estuarine fish and invertebrates are not expected to result from the proposed applications of A. pullulans strain s 14940 and 14941.

c. Open Literatu re Information

EPA performed l iterature searches to determine if other effects have been reported that were not contained within the information provided by the registrant. A search was performed using the

Environmental Information Search service availab le to the Agency. This search tool simultaneously queries the Agricola

, Biosis Previews

, CAB Abstracts

, Energy, Science, and Technology, General Science Abstracts, and National Technical Information Service databases within the years of 1926

– present. T he Web of Knowledge database also was searched for all years available. Searches were performed with the term ‘ Aureobasidium pullulans ’ combined with each of the terms, ‘bird,’ ‘mammal,’ insect,’ ‘bee,’ ‘arthropod,’ ‘aquatic,’ ‘invertebrate,’ ‘fish,’ ‘rep tile,’ ‘amphibian,’ ‘plant,’ ‘algae,’ ‘freshwater,’ ‘marine,’ ‘estuarine,’ ‘soil,’ and ‘pathogen. None of the records returned in these searches indicated potential adverse effects on

non

- target organisms.

2. Environmental Fate Data

As t he information provided , for the current uses and application methods, is sufficient to both satisfy the Tier I non

- target organism data requirements and for non

- target organism risk

asse ssment for A. pullulans strain s DSM 14940 and DSM 14941 , f urther testing at higher tier

levels (i.e., Tiers II, III, and IV) is not required.

3

. Threatened and Endangered Species Assessment

The proposed applications of A. pullulans strain s DSM 14940 and DSM 14941 are not expected

to increase the amount of A. pullulans above levels t hat are found naturally in the environment. Additionally, several guideline studies and scientific rationale show that toxicity or pathogenicity is not expected with wild mammals, birds, non

- target plants, non

- target insects, honey bees, freshwater fish ( toxicity only), and freshwater invertebrates. The information provided isAureobasidium pullulans strains DSM 14940 and 14941 Page 17 of 41 Biopesticides Registration Action Document

sufficient to satisfy the Tier I non

- target organism data requirements and for non

- target organism risk assessment for the active ingredient. Further testing of non

- target organisms at higher tier levels is not required. B ased on the data, literature citations, and data waiver rationale provided by the applicant

, BPPD has determined that adverse effects to non

- target organisms are not anticipated as a result of the proposed uses of A ureobasidium pullulans strain s DSM 14940 and

DSM 14941

. A “No Effect” determination is also made for direct and indirect effects to listed species and their designated critical habitats resulting from the proposed uses of Aureobasidium pullulans strain s DS M 14940 and DSM 14941, as labeled.

V. ENVIRONMENTAL JUSTICE

EPA seeks to achieve environmental justice

— the fair treatment and meaningful involvement of all people regardless of race, color, national origin, or income

— with respect to the development,

i mplementation, and enforcement of environmental laws, regulations, and policies. Fair treatment means that no group of people, including racial, ethnic, or socioeconomic groups, should bear a disproportionate share of the negative environmental consequence s resulting from industrial, municipal, and commercial operations or the execution of federal, state, local, and tribal environmental programs and policies. Meaningful involvement means that (1) potentially affected community residents have an appropriate opportunity to participate in decisions about a proposed activity that will affect their environment and/or health; (2) the public’s contribution can influence the regulatory agency’s decision; (3) the concerns of all participants involved will

be consider ed in the decision

- making process; and (4) the decision

- makers seek out and facilitate the involvement of those potentially affected. EPA has this goal for all communities and persons

across the United States.

EPA sought information on any groups or segm ents of the population who, as a result their location, cultural practices, or other factors, may have atypical, unusually high exposure to A. pullulans strain s DSM 14940 and DSM 14941 , compared to the general population. No public

comments were received o n this particular matter.

For additional information regarding environmental justice issues, please visit EPA’s web site at http://www.epa.gov/compliance/environmentaljustice/index .html

.

V I

. RISK MANAGEMENT DECISION

Section 3(c)(5) of FIFRA permits for the registration of a pesticide provided that all the following determinations are made:

(1) Its composition is such as to warrant the proposed claims for it; (2) Its labeling and other material required to be submitted comply with the requirements of FIFRA; (3) It will perform its intended function without unreasonable adverse effects on the environment; AND (4) When used in accordance with widespread and commonl y recognized practice, it will not generally cause unreasonable adverse effects on the

environment.

To satisfy criterion 1, the Aureobasidium pullulans strain DSM 14940 and AureobasidiumAureobasidium pullulans strains DSM 14940 and 14941 Page 18 of 41 Biopesticides Registration Action Document

pullulans strain DSM 14941 pesticide products have wel l

- known properties. EPA has no knowledge that would contradict the claims made on the Aureobasidium pullulans strain DSM

14940 Technical, Aureobasidium pullulans strain DSM 14941 Technical, Botector, and Blossom

Protect EP label s . These products are not ex pected to cause unreasonable adverse effects on the

environment when used according to label instructions. Criterion 2 is satisfied by the current product label s, as well as the data and information presented in this document. It is believed that

the Aureo basidium pullulans strain DSM 14940 and Aureobasidium pullulans strain DSM 14941 will not cause any unreasonable adverse effects on the environment, and Botector, and Blossom

Protect EP s (end

- use product s ) , in particular, are likely to protect against fung al pests as claimed, thereby satisfying criterion 3. Criterion 4 is satisfied in that the Aureobasidium pullulans strain

DSM 14940 and Aureobasidium pullulans strain DSM 14941 pesticide products are not expected

to cause unreasonable adverse effects when u sed according to label instructions. Therefore, Aureobasidium pullulans strain DSM 14940 Technical, Aureobasidium pullulans strain DSM

14941 Technical, Botector, and Blossom Protect (all containing Aureobasidium pullulans strain

DSM 14940 and /or Aureobasid ium pullulans strain DSM 14941 as two new active ingredient s )

are eligible for registration under FIFRA section 3(c)(5) for labeled uses. If the applicant

proposes uses that are more extensive in the future (e.g., p ost

- harvest applications )

, EPA will

likel y require that additional data be submitted in conjunction with a registration amendment

request

.

V I I

. ACTIONS REQUIRED BY THE REGISTRANT

A. Final Printed Labeling

Before releasing pesticide products containing Aureobasidium pullulans strain DSM 14940 and Aureobasidium pullulans strain DSM 14941 for shipment, the registrant is required to provide appropriate final printed labeling to EPA

.

B . Reporting of Adverse Effects and Hypersensitivity Incidents

Notwithstanding the information stated in the pre vious sections, it should be clearly understood that certain, specific data are required to be reported to EPA as a requirement for maintaining the federal registration for a pesticide product. A brief summary of these types of data are described

below.

R eports of all incidents of adverse effects to the environment must be submitted to EPA under the provisions stated in FIFRA section 6(a)(2). Additionally, all incidents of hypersensitivity (including both suspected and confirmed incidents) must be reported to EPA under the provisions of 40 CFR § 158.2140(d).Aureobasidium pullulans strains DSM 14940 and 14941 Page 19 of 41 Biopesticides Registration Action Document

VIII. GLOSSARY OF ACRONYMS AND ABBREVIATIONS

ASAE American Society of Agricultural Engineers

BPPD Biopesticides and Pollution Prevention Division

BRAD Biopesticides Registration Action Documen t
CFR Code of Federal Regulations

c fu colony

- forming unit (s) cfu/kg colony

- forming units per kilogram

cfu/mL colony

- forming unit s per milliliter

cP centipoise

DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen (the German Strain

Collection for Mi croorganisms)

EDSP Endocrine Disruptor Screening Program

EP end

- use product

EPA Environmental Protection Agency (the “Agency”)

FFDCA Federal Food, Drug, and Cosmetic Act

FIFRA Federal Insecticide, Fungicide, and Rodenticide Act FQPA Food Quality Protectio n Act

FR Federal Register g/ mL gram per milliliter

LC 50 median lethal concentration. A statistically derived concentration of a substance that can be expected to cause death in 50% of test animals. It is usually expressed as the weight of substance per weight or volume of water, air, or feed (e.g., mg/L, mg/kg, or ppm).

LD 50 median lethal dose. A statistically derived single dose that can be expected to cause death in 50% of the test animals when administered by the route indicated (oral, dermal, or inhalation). It is expressed as a weight of substance per unit weight of animal (e.g., mg/kg). mg/kg milligram s per kilogram mg/L milligram s per liter

MP manufacturing

- use product

MRID No. Master Record Identification Number

NIOSH National Institute for Occupational Safety and Health

NRRL Northern Regional Research Laboratory

OCSPP Office of Chemical Safety and Pollution Prevention

OPP Office of Pesticide Programs

PC Code Pesticide Chemical Code

PP Pesticide Petition

PPE personal protective equipment

ppm parts per million

TGAI technical grade of the active ingredient

ULV ultra low volumeAureobasidium pullulans strains DSM 14940 and 14941 Page 20 of 41 Biopesticides Registration Action Document

I X. BIBLIOGRAPHY

A. Studies Submitted to Support the Aureobasidium pullulans strains DSM 14940 and 14941 Pesticide Product Registrations

MRID Citation Reference

47869500 Bio

- Fern GmbH (2009) Submission of Product Chemistry and Toxicity Data in Support of the Application for Registration of Botector. Transmittal of 3

Studies.

47869501 Smith, F. (2009) Botector: Product Identity and Composition, Des cription of Beginning Materials, Description of Formulation Process, Discussion of the Formation of Impurities, and Certified Limits. Unpublished study prepared by SciReg, Inc. 39 p.

47869502 Smith, F. (2009) Botector: Physical

- Chemical Properties. Unpubl ished study prepared by SciReg, Inc. 7 p.

47869503 Smith, F. (2009) Botector: Acute Toxicity Summary. Unpublished study prepared by SciReg, Inc. 11 p.

47869600 Bio

- Ferm GmbH (2009) Submission of Product Chemistry and Toxicity Data in Support of the Appli cation for Registration of Blossom Protect. Transmittal of

19 Studies.

47869601 Smith, F. (2009) Blossom Protect: Product Identity and Composition, Description of Beginning Materials, Description of Formulation Process, Discussion of the Formation of Impu rities, and Certified Limits. Unpublished study prepared by SciReg, Inc. 33 p.

47869602 Smith, F. (2009) Blossom Protect: Physical

- Chemical Properties. Unpublished study prepared by SciReg, Inc. 9 p.

47869603 Kiss, G. (2006) Determination of Flammability of Component B of Blossom Protect FB: Final Report. Project Number: 06/262/356AN. Unpublished study prepared by LAB International Research Centre Hungary, Ltd. 12 p.

47869604 Kiss, G. (2009) Determination of the pH

- Values of Blossom Protect (Component B): Final Report. Project Number: 09/097/338AN. Unpublished study prepared by LAB International Research Centre Hungary, Ltd. 12 p.

47869605 Kiss, G. (2006) Determination of the Relative Self

- Ignition Temperature of Component B of Blossom Protect FB: Fina l Report. Project Number: 06/262/355AN. Unpublished study prepared by LAB International Research Centre Hungary, Ltd. 12 p.

47869606 Moller, M. (2007) Component B of Blossom Protect FB: Wettability: Final Report. Project Number: 20060875/03. Unpublished s tudy prepared by Siemens Axiva GmbH & Co. KG. 10 p.

47869607 Moller, M. (2007) Component B of Blossom Protect FB: Persistent Foaming: Final Report. Project Number: 20060875/01. Unpublished study prepared by Siemens Axiva GmbH & Co. KG. 9 p.Aureobasidium pullulans strains DSM 14940 and 14941 Page 21 of 41 Biopesticides Registration Action Document

47869608 Moll er, M. (2007) Component B of Blossom Protect FB: Suspensibility: Final Report. Project Number: 20060875/02. Unpublished study prepared by Siemens Axiva GmbH & Co. KG. 10 p.

47869609 Moller, M. (2007) Component B of Blossom Protect FB: Dispersibility: Fina l Report. Project Number: 20060875/04. Unpublished study prepared by Siemens Axiva GmbH & Co. KG. 10 p.

47869610 Kiss, G. (2007) Wet Sieve Test of Component B of Blossom Protect FB: Final Report. Project Number: 06/262/342AN. Unpublished study prepared by LAB International Research Centre Hungary, Ltd. 12 p.

47869611 Kiss, G. (2006) Determination of the Particle Size Distribution of Component B of Blossom Protect FB: Final Report. Project Number: 06/262/363AN. Unpublished study prepared by LAB Internation al Research Centre Hungary, Ltd. 14 p.

47869612 Moller, M. (2007) Component B of Blossom Protect FB: Dustiness of Granules: Final Report. Project Number: 20060875/05. Unpublished study prepared by Siemens Axiva GmbH & Co. KG. 10 p.

47869613 Smith, F. (20 09) Blossom Protect: Acute Toxicity Summary. Unpublished study prepared by SciReg, Inc. 5 p.

47869614 Wolf, T. (2003) "BP2042": Acute Oral Toxicity Study with Rats (Acute Toxic Class Method). Project Number: ARC/UL/0608. Unpublished study prepared by

Aust rian Research Center (ARC). 18 p.

47869615 Bernat, E. (2003) "BP2042": Acute Dermal Toxicity Study with Rats. Project Number: ARC/UL/0614. Unpublished study prepared by Austrian Research Center (ARC). 20 p.

47869616 Wolf, T. (2003) "BP2042": Acute Subcut aneous Toxicity Study with Rats. Project Number: ARC/UL/0609. Unpublished study prepared by Austrian Research Center (ARC). 22 p.

47869617 Weniger, P. (2003) "BP2042": Acute Inhalation Toxicity in Rats. Project Number: OEFZS/UL/0638. Unpublished study pre pared by Austrian Research Center (ARC). 30 p.

47869618 Wolf, T. (2003) "BP2042": Acute Eye Irriation/Corrosion Study with Rabbits. Project Number: ARC/UL/0611. Unpublished study prepared by Austrian Research Center (ARC). 16 p.

47869619 Wolf, T. (2003) "BP2042": Acute Dermal Irritation/Corrosion Study with Rabbits. Project Number: ARC/UL/0610. Unpublished study prepared by Austrian Research Center (ARC). 16 p.

47871700 Bio

- Ferm GmbH (2009) Submission of Product Chemistry Data in Support of the Applicati on for Registration of Aureobasidium pullulans Strains DSM 14940

Technical. Transmittal of 3 Studies.

47871701 Smith, F. (2009) Aureobasidium pullulans Strain DSM 14940: Product Identity and Composition, Description of Beginning Materials, Description of Production Process, Discussion of the Formation of Impurities, and CertifiedAureobasidium pullulans strains DSM 14940 and 14941 Page 22 of 41 Biopesticides Registration Action Document

Limits. Unpublished study prepared by SciReg, Inc. 95 p.

47871702 Smith, F. (2009) Aureobasidium pullulans Strain DSM 14940: Analysis of Samples. Unpublished study prepared by Sc iReg, Inc. 10 p.

47871703 Smith, F. (2009) Aureobasidium pullulans Strain DSM 14940: Summary of Physical

- Chemical Properties. Unpublished study prepared by SciReg, Inc. 11 p.

47871800 Bio

- Fern GmbH (2009) Submission of Product Chemistry, Toxicity, Effica cy, and Exposure and Risk Data in Support of the Application for Registration of Aureobasidium pullulans Strain DSM 14941 Technical, and the Petition for Tolerance of Aureobasidium pullulans for Food Use. Transmittal of 26 Studies.

47871802 Smith, F. (20 09) Aureobasidium pullulans Strain DSM 14941: Analysis of Samples. Unpublished study prepared by SciReg, Inc. 10 p.

47871803 Smith, F. (2009) Aureobasidium pullulans Strain DSM 14941: Summary of Physical

- Chemical Properties. Unpublished study prepared by SciReg, Inc. 11 p.

47871804 Smith, F. (2009) Storage Stability of Aureobasidium pullulans Strains DSM 14940 and DSM 14941. Unpublished study prepared by SciReg, Inc. 8 p.

47871805 Smith, F. (2009) Summary of Tier 1 Data Requirements: (Aureobasidium pullu lans Strain DSM 14941 Technical). Unpublished study prepared by SciReg, Inc. 13 p.

47871807 Muller, W. (2005) Determination of the Toxic/Infectious/Pathogen Behaviour of Two Strains of Aureobasidium pullulans (DSM 14940 and 14941) in the Rat

after Subcuta neous Administration: Final Report. Project Number: KBL/2004/1474/RSC. Unpublished study prepared by Kesla Forschung & Service GmbH + Co. KG. 60 p.

47871808 Muller, W. (2006) Determination of the Toxic/Infectious/Pathogen Behaviour of Aureobasidium pullul ans DSMZ 14941 in the Rat after Subcutaneous Administration: Final Report. Project Number: KBL/2006/1540/RSC. Unpublished study prepared by Kesla Forschung & Service GmbH + Co. KG. 60 p.

47871811 Gaty, S. (2009) Avian Oral Pathogenicity and Toxicity Study of Aureobasidium pullulans DSM 14941 (CF40) on Japanese Quail (Coturnix coturnix japonica): Final Report. Project Number: 09/159/115FU. Unpublished study prepared by LAB Research, Ltd. 40 p.

47871812 Hernadi, D. (2007) Fish Acute Toxicity Study with Comp onent B of Blossom Protect fb on Rainbow Trout (Oncorhynchus mykiss): Final Report. Project Number: 06/282/009H. Unpublished study prepared by LAB International Research Centre Hungary, Ltd. 23 p.

47871816 Redondo

- Bellon, P.; Idoate, M. ; Rubio, M. ; et a l. (1997) Chromoblastomycosis Produced by Aureobasidium pullulans in an Immunosuppressed Patient. Archives of Dermatology 133:663

- 664.

47871817 Tan, H. ; Wahlstrom, H. ; Zamora, J. ; et al. (1997) Aureobasidium pneumonia in a Post Liver Transplant Recipie nt: A Case Report. Hepato

- Gastroenterology

44:1215

- 1218.Aureobasidium pullulans strains DSM 14940 and 14941 Page 23 of 41 Biopesticides Registration Action Document

47871818 Clark, E.; Silver, S.; Hollick, G.; et al. (1995) Continuous Ambulatory Peritoneal Complicated by Aureobasidium pullulans Peritonitis. American Journal of Nephrology 15:353

- 355.

47871819 C aporale, N.; Calegari, L.; Perez, D.; et al. (1996) Peritoneal Catheter Colonization and Peritonitis with Aureobasidium pullulans. Peritoneal Dialysis International 16(1):97

- 98.

47871820 Matsumoto, T.; Padhye, A.; Ajello, L. (1987) Medical Significance of the So

Called Black Yeasts. European Journal of Epidemiology 3(2):87

- 95.

47871821 Hoog, G. (1996) Risk Assessment of Fungi Reported from Humans and Animals. Mycoses 39:407

- 417.

47871822 Bolignano, G.; Criseo, G. (2003) Disseminated Nosocomial Fungal Inf ection by Aureobasidium pullulans Var. melanigenum: A Case Report. Journal of Clinical Microbiology 41(9):4483

- 4485.

47871823 Fostel, J.; Lartey, P. (2000) Emerging Novel Antifungal Agents. Drug Discovery Today 5(1):25

- 32.

47871824 Schrattenholz, A.; Fle sch, P. (1993) Isolation, Structural and Toxicological Characterization of Three New Mycotoxins Produced by the Fungus Aureobasidium pullulans. Mycotoxin Research 9:13

- 21.

47871825 Sanchez, D.; Calla, M.; Martin

- diaz, M.; et al. (2006) Subcutaneous Mycosi s Produced by Aureobasidium pullulans in a Renal Transplant Recipient. Journal of European Academy of Dermatology and Venereology 20:229

- 230.

47871826 Koppang, H.; Olsen, I.; Stuge, U.; et al. (1990) Aureobasidium Infection of the

Jaw. Journal of Oral Pat hology and Medicine 20:191

- 195.

47871827 Hawkes, M.; Rennie, R.; Sand, C.; et al. (2005) Aureobasidium pullulans Infection: Fungemia in an Infant and a Review of Human Cases. Diagnostic Microbiology and Infectious Disease 51:209

- 213.

47871828 Taylor, P.; Esch, R.; Flagan, R.; et al. (2005) Identification and Possible Disease

Mechanisms of an Under

- Recognized Fungus, Aureobasidium pullulans. International Archives of Allergy and Immunology 139:45

- 52.

47871829 Salkin, I.; Martinez, J.; Kemna, M. (1986) Opp ortunistic Infection of the Spleen Caused by Aureobasidium pullulans. Journal of Clinical Microbiology 23(5):828

- 831.

47871830 Kaczmarski, E.; Liu, J.; Tooth, J.; et al. (1986) Systemic Infection with Aureobasidium pullulans in a Leukaemic Patient. Journa l of Infection 13:289

- 291.

47871831 Buzina, W.; Braun, H.; Freudenschuss, K.; et al. (2003) Fungal Biodiversity

- as Found in Nasal Mucus. Medical Mycology 41:149

- 161.

47871832 Perez, R.; Chacon, J.; Martin, J.; et al. (2009) Peritonitis by Aureobasidium pullulans in Conditions Ambulatory Peritoneal Dialysis. Archives of Dermatoloy 133:1544

- 1545.Aureobasidium pullulans strains DSM 14940 and 14941 Page 24 of 41 Biopesticides Registration Action Document

47871833 Kunz, S. (2009) Influence of Temperature on the Reproduction on Different Strains of the Species Aureobasidium pullulans. Unpublished study prepared by

Bio

- Protect GmbH. 11 p.

47899300 Bio

- Ferm GmbH (2009) Submission of Product Chemistry Data in Support of the Application for Registration of Aureobasidium pullulans Strains DSM 14941 Technical and the Petition for Tolerance of Aureobasidium pullulans (St rains DSM 14941 and DSM 14941). Transmittal of 7 Studies.

47899301 Smith, F. (2009) Aureobasidium pullulans strain DSM 14941: Product Identity and Composition, Description of Beginning Materials, Description of Production Process, Discussion of the Format ion of Impurities, and Certified Limits. Unpublished study prepared by SciReg, Inc. 144 p.

47899302 Zeljenkova, D. (2006) Acute Oral Toxicity/Pathogenicity in Rats According to EPA Microbial Pesticide Test Guidelines: (Aureobasidium pullulans): Final Repo rt. Project Number: E/318/2006. Unpublished study prepared by Slovak Medical University. 33 p.

47899303 Zeljenkova, D.; Kazar, J. (2007) Acute Pulmonary Toxicity/Pathogenicity in Rats According to EPA Microbial Pesticide Test Guidelines: (Aureobasidium

pu llans): Final Report. Project Number: E/400/2006. Unpublished study prepared by Slovak Medical University. 54 p.

47899304 Szabova, E. (2006) Mutagenicity

- in Vivo Mammalian Erythrocyte Micronucleus Test: (Aureobasidium pullulans): Final Report. Project N umber: 319/06. Unpublished study prepared by Slovak Medical University. 24 p.

47899305 Fritz, J.; Pruckner, M. (2009) Extended Report: Toxicity Test with Water Fleas: (Blossom Protect, Produktkomponente B). Project Number: BIOFERM/DAPHNIA. Unpublished stu dy prepared by University of Natural Resources and Applied Life Sciences, Vienna. 8 p.

47899306 Fritz, J.; Pruckner, M. (2009) Extended Report: Toxicity Test with Plants: (Blossom Protect). Project Number: BIOFERM/PLANTS. Unpublished study prepared by Uni v. of Natural Resources and Applied Life Sciences, Vienna. 21

p.

47899307 Donat, C. (2009) Statement on Toxicity of the Active Substance of Blossom Protect on Bees. Unpublished study prepared by Bio

- ferm GmbH. 10 p.

48531000 Bio

- Ferm GmbH (2011) Submissi on of Toxicity and Product Chemistry Data in Support of the Applications for Registration of Aureobasidium pullulans Strain DSM 14940 Technical, Aureobasidium pullulans Strain DSM 14941, Botector

and Blossom Protect. Transmittal of 3 Studies.

48531002 Gat y, S. (2009) Reproduction Test with Blossom Protect (Component B) on Daphnia magna. Project Number: 09/097/124/DA. Unpublished study prepared by LAB International Research Centre Hungary, Ltd. 46 p.

48531003 Gaty, S. (2009) Oral Toxicity Test with Blossom Protect (Component B) and with Tank Solution on Honey Bees (Apis mellifera). Project Number: 09/127/116MT. Unpublished study prepared by LAB International Research Centre Hungary, Ltd. 26 p.Aureobasidium pullulans strains DSM 14940 and 14941 Page 25 of 41 Biopesticides Registration Action Document

48531004 Smith, F. (2011) Blossom Protect: Certified Limits. Un published study prepared by SciReg, Inc. 7 p.

48547201 Donat, C. (2011) Storage Stability: Blossom Protect Component B at 20 Degrees Celsius. Unpublished study prepared by Bio

- Ferm GmbH. 15p.

B. Environmental Protection Agency Risk Assessment Memoran da

U.S. EPA

. 2011 a

. Environmental risk assessment for the FIFRA Section 3 registration of Aureobasidium pullulans strain DSM 14940 and strain DSM 14941 for control of diseases in pome fruits, stone fruits, grapes, and strawberries . Memorandum from S. Borges, to S. Cerrelli dated November 2, 2011

.

U.S. EPA

. 2011 b

. Review of Product Chemistry, Manufacturing Process, and Acute Toxicity Studies for section 3 registration of two end use products (EP) Botector (EPA Reg. No. 86174

- G) and Blossom Protect (EP A Reg. No. 86174

- U) containing two new TGAI: Aureobasidium pullulans strain DSM 14941 and Aureobasidium pullulans strain DSM 14940. Memorandum

from I

. S. Barsoum, Ph.D . and J

. L. Kough, Ph.D., to S. Cerrelli dated May 1, 2011.

U.S. EPA

. 2011 c

. Review o f Product Chemistry, Manufacturing Process, and Acute Toxicity Studies for section 3 registration of two TGAI Aureobasidium pullulans strains DSM 14941 (EPA Reg. No. 86174

- R) and strain DSM 14940 (EPA Reg. No. 86174

- E) Memorandum from: I.S. Barsoum, , Ph.D . and J. L. Kough, Ph.D., to S. Cerrelli dated May 1, 2011.

U.S. EPA

. 2011 d

. Review of the Registrant’s response to the deficiencies found by the Agency in its review of product chemistry, manufacturing process, and acute toxicity studies for the registr ation of two new EPs: Botector and Blossom Protect containing two new TGAIs

Aureobasidium pullulans strains DSM 14940 & DSM 14941. Memorandum from: I. S. Barsoum

, Ph.D and J. L. Kough, Ph.D., to S. Cerrelli dated September 29, 2011.

C. Other References

Andrews, J.H., R.N. Spear, E.V. Nordheim. 2002. Population biology of Aureobasidium pullulans on apple leaf surfaces. Can. J. Microbiol. 48: 500

- 513.

Chi, Z., F. Wang, Z. Chi, L. Yue, G. Liu, T. Zhang. 2009. Bioproducts from Aureobasidium pullulans , a b iotechnologically important yeast. Appl. Microbiol. Biotechnol., 82: 793

- 804.

Cooke, W.B. 1959. An ecological life history of Aureobasidium pullulans (De Bary) Arnaud. Mycopathologia, 12: 1

- 45.

Deshpande, M.S., V.B. Rale, and J.M. Lynch. 1992. Aureobasi dium pullulans in applied microbiology: a status report. Enzyme Microbiol. Technol. 14: 514

- 527.

Eskalen, A., M. Kusek, L. Dani

- ti, and S. Karada. 1999. Fungal diseases in pistachio trees in

East

- Mediterranean and Southeast Anatolian regions. 11. GREMPA S eminar on Pistachios and Almonds, Sanliurfa (Turkey), 1

- 4 Sept. 1999.Aureobasidium pullulans strains DSM 14940 and 14941 Page 26 of 41 Biopesticides Registration Action Document

Fleet, G.H. 2002. The yeast ecology of wine grapes. Pp. 1

- 17 in Biodiversity and Biotechnol. Of Wine Yeasts (Ciani, M., ed .). Research Signpost, Kerala, India.

Fleet, G.H. 2003. Yeast interactions and wine flavor. Int. J. Food Microbiol., 86: 11

- 22.

Gao, X.

- X., H. Zhou, D.

- Y. Xu, C.

- H. Yu, Y.

- Q. Chen, L.

- H. Qu. 2005. High diversity of endophytic fungi from the pharmaceutical plant, Heterosmilax japonica Kunth revealed by

cultivation

- i ndependent approach. FEMS Microbiol. Letters, 249: 255

- 266.

Granado, J., B. Thurig, E. Kieffer, L. Petrini, A. Fliessbach, L. Tamm, F.P. Weibel, G.S. Wyss. 2008. Culturable fungi of stored ‘Golden Delicious’ apple fruits: a one

- season comparison study of organic and integrated production systems in Switzerland. Microb. Ecol., 56: 720

- 732.

Kockova

- Kratochvilova, A., M. Cernakova, and E. Slavikova. 1980. Morphological changes during the life cycle of Aureobasidium pullulans (de Bary) Arnaud. Folia Microbio logica,

25:

Leibinger, W., B. Breuker, M. Hahn, and K. Mendgen. 1997. Control of postharvest pathogens and colonization of the apple surface by antagonistic microorganisms in the field. Phytopathology, 97: 1103

- 1110.

Osono, T. 2008. Endophytic and epiph ytic phyllosphere fungi of Camellia japonica : seasonal and leaf age

- dependent variations. Mycologia, 100: 387

- 391.

Pagnocca, F.C., A. Rodrigues, N.S. Nagamoto, M. Bacci, Jr. 2008. Yeasts and filamentous fungi carried by the gynes of leaf

- cutting ants. Ant onie van Leeuwenhoek , 94: 517

- 526.

Prashanthi, S.K. and S. Kulkarni. 2005. Aureobasidium pullulans , a potential mycoherbicide for biocontrol of eupatorium [ Chromolaena odorata (L.) King and Robinson] weed. Current Science, 88: 18

- 21.

Rosa, C.A., M.

- A. La chance, J.O.C. Silva, A.C.P. Teixeira, M.M. Marini, Y. Antonini, R.P. Martins. Yeast communities associated with stingless bees. FEMS Yeast Res. 4: 271

- 275.

Slavikova, E., R. Vadkertiova, and D. Vranova. 2007. Yeasts colonizing the leaf surfaces. J.

Basi c Microbiol. 47: 344

- 350.

Slepecky, R.A. and W.T. Starmer. 2009. Phenotypic plasticity in fungi: a review with

observations on Aureobasidium pullulans . Mycologia 101: 823

- 832.

Smith, C.S., T. Chand, R.F. Harris, J.H. Andrews. 1989. Colonization of a subm ersed aquatic plant, Eurasian water milfoil ( Myriophyllum spicatum ), by fungi under controlled conditions. Appl. and Environm. Microbiol. 55: 2326

- 2332.

Suryanarayanan, T.S., S.K. Wittlinger, and S.H. Faeth. 2005. Endophytic fungi associated with

cacti in Arizona. Mycol. Res. 109: 635

- 639.Aureobasidium pullulans strains DSM 14940 and 14941 Page 27 of 41 Biopesticides Registration Action Document

Webb, T.A. and J.O. Mundt. 1978. Molds on vegetables at the time of harvest. Appl. Environ. Microbiol., 35: 655

- 658.

Woody, S.T., R.N. Spear, E.V. Nordheim, A.R. Ives, and J.H. Andrews. 2003. Single

- leaf

resolution of the temporal population dynamics of Aureobasidium pullulans on apple leaves. Appl. Environm. Microbiol., 69: 4892

- 4900.

Zacchi, L. and A. Vaughan

- Martini. 2002. Yeasts associated with insects in agricultural areas of Perugia, Italy. Ann. Microbiol. 52: 237

- 244.Aureobasidium pullulans strains DSM 14940 and 14941 Page 28 of 41 Biopesticides Registration Action Document

A PPENDIX A. MICROBIAL PESTICIDES DATA REQUIREMENTS (40 CFR PART 158

– SUBPART V )

TABLE 1. Product Analysis Data Requirements for the Technical Grade of the Active Ingredient (TGAI) /Manufacturing

- Use Product ( MP )

, Aureobasidium pullul ans strain DSM 14940 Technical (40 CFR § 158.2 120 )

OCSPP

G uideline

N umber Data Requirement Results MRID No. TGAI MP

885.1100 Product Identity Not applicable Submitt ed data fulfill the requirement for product identity. 47871701

885.1200 Manufact uring Process Submitted data fulfill the requiremen t for manufacturing

process

. 47871701

Not a pplicable Deposition of a Sample in a Nationally Recognized Culture

Collection Aureobasidium pullulans

strain DSM 14940 is on deposit with the Deutsche Sammlung von Mikroorganismen und

Zellkulturen ( DSMZ

, the

German Strain Collection for Microorganisms ) under the

reference number DSM

14940 Not applicable 47871701

885.1300 Discussion of Formation

of Unintentional Ingredients Submitted data fulfill the require men t for discussion of formation of unintentional ingredients for the current use

sites

. 47871701

885.1400 Analysis of S amples Submitted data fulfill the requirement for analysis of samples. 4787170 2

8 85 .1 50 0 Certification of L imits Not applicable Li mits listed on the

confidential statement of

formula are adequate/acceptable. CSF dated July 7, 2011

Additional Studies

830.1800 Enforcement Analytical

M ethod Not applicable Submitted data fulfill the requiremen t for an enforcement analytical

method

. 4 7869601Aureobasidium pullulans strains DSM 14940 and 14941 Page 29 of 41 Biopesticides Registration Action Document

TABLE 2

. Product Analysis Data Requirements for the Technical Grade of the Active Ingredient (TGAI)/Manufacturing

- Use Product (MP), and Aureobasidium pullulans strain DSM 14941 Technical (40 CFR § 158.2120)

OCSPP

G uideline

N umber Data Req uirement Results MRID No. TGAI MP

885.1100 Product Identity Not applicable Submitt ed data fulfill the requirement for product identity. 47899301

885.1200 Manufacturing Process Submitted data fulfill the requiremen t for manufacturing

process

. 4789 9301

Not applicable Deposition of a Sample in a Nationally Recognized Culture

Collection Aureobasidium pullulans

strain DSM 1494 1 is on deposit with the DSMZ ( the

German Strain Collection for Microorganisms) under the

reference number DSM

14941 Not appli cable 47871701

885.1300 Discussion of Formation

of Unintentional Ingredients Submitted data fulfill the requiremen t for discussion of formation of unintentional ingredients for the current use

sites

. . 47899301

885.1400 Analysis of S amples Submitted da ta fulfill the requirement for analysis of samples. 47871802

8 85 .1 50 0 Certification of L imits Not applicable Limits listed o n the

confidential statement of

formula are adequate/a cceptable. CSF dated July 7, 2011

Additional Studies

830.1800 Enforcement Analytical

M ethod Not applicable Submitted data fulfill the requiremen t for an enforcement analytical

method

. 47869601Aureobasidium pullulans strains DSM 14940 and 14941 Page 30 of 41 Biopesticides Registration Action Document

TABLE 3

. Product Analysis Data Requirements for the End

- Use Product ( E P), Blossom Protect (40 CFR § 158.2120)

OCSPP Guideline Nu mber Data Requirement Results MRID No.

885.1100 Product Identity Submitt ed data fulfill the requirement for product identity

. 47869601

885.1200 Manufacturing Process Submitted data fulfill the requirement for manufacturing process. 47869601

Not applicab le Deposition of a Sample in a Nationally Recognized

Culture Collection Not applicable Not applicable

885.1300 Discussion of Formation of Unintentional Ingredients Submitted data fulfill the requirement for

discussion of formation of unintentional ingredi ents for the current use sites

. . 47869601

885.1400 Analysis of Samples Results of a five

- batch preliminary analysis for the active ingredient in

Blossom Protect were not submitted. Each of the active ingredients is being registered concurrently. 47869 601

885.1500 Certification of Limits Limits listed on the confidential statement of formula are adequate/acceptable. CSF dated January 26, 2012

Additional Studies

830.1800 Enforcement Analytical

Method Submitted data fulfill the requirement for

an enfo rcement analytical method. 47869601

TABLE 4 . Product Analysis Data Requirements for the End

- Use Product (EP), Botector (40 CFR § 158.2120)

OCSPP Guideline Number Data Requirement Results MRID No.

885.1100 Product Identity Submitted data fulfill th e requirement for product identity. 47869501

885.1200 Manufacturing Process Submitted data fulfill the requirement for manufacturing process. 47869501

Not applicable Deposition of a Sample in a Nationally Recognized

Culture Collection Not applicable No t applicable

885.1300 Discussion of Formation of Unintentional Ingredients Submitted data fulfill the requirement for

discussion of formation of unintentional ingredients for the current use sites

. 47869501

885.1400 Analysis of Samples Results of a fi ve

- batch preliminary analysis for the active ingredient in

Botector were not submitted. Each of the active ingredients is being registered concurrently. 47869501

885.1500 Certification of Limits Limits listed on the confidential statement

of formula are adequate/acceptable. CSF dated

J anuary 26, 2012

Additional Studies

830.1800 Enforcement Analytical

Method Submitted data fulfill the requirement for an enforcement analytical method. 47869 5 01Aureobasidium pullulans strains DSM 14940 and 14941 Page 31 of 41 Biopesticides Registration Action Document

TABLE 5 . Physical and Chemical Characteristics for the T echnical Grade of the Active Ingredient (TGAI)/Manufacturing

- Use Product (MP), Aureobasidium pullulans strain DSM 14940 Technical (40 CFR § 158.2120)

OCSPP

G uideline

N umber Data Requirement Results MRID No.
TGAI MP

830.6302 Color Pale pink to pa le brown Not applicable 47871703

830.6303 Physical State Powder Not applicable 47871703

830.6304 Odor Bread

- like Not applicable 47871703

830.6313 Stability to Normal and

Elevated Temperatures, Metals,

and Metal Ions Expected to be stable during applicat ion or

when in contact with

metals or metal ions Not applicable 47871703

830.6317 Storage Stability The registrant is relying on the storage stability for Blossom Protect (EP) which was stable at 20°C for 12 months. 48547201

830.6319 Miscibility Not ap plicable Not required because A. pullulans

strain DSM 14940 is not an

e mulsifiable form of microbial pesticide (refer to test note #2 of 40 CFR § 158.2120(d)). Not applicable

830.6320 Corrosion

Characteristics Not applicable Blossom protect data cited are adequate to support this data requirement. 48547201

830.7000 pH 5.66

- 6.25 (1% solution in deionized water) Not applicable 47871703

830.7100 Viscosity Not applicable Not applicable, the product is a

solid. 47871703

830.7300 Density/Relative Density/B ulk Density

(Specific Gravity) 0.69 g/mL Not applicable 47871703

TABLE 6 . Physical and Chemical Characteristics for the End

- Use Product (EP), Blossom Protect (40 CFR § 158.2120)

OCSPP Guideline Number Data Requirement Results MRID No.

83 0.6317 Storage Stability the product is considered to be stable for

12 months at 20°C 48547201

830.6319 Miscibility Not applicable, the product is not an emulsifiable liquid. 47869602

830.6320 Corrosion Characteristics No damage of the packaging materia l

was observed during 15 month storage stability study. 48547201

830.7100 Viscosity Not applicable, the product is a solid 47869602

830.7300* Density/Relative Density/Bulk Density (Specific Gravity) 0.60

- 0.69 g/mL b 47869602 and

CSF dated July 7, 2011Aureobasidium pullulans strains DSM 14940 and 14941 Page 32 of 41 Biopesticides Registration Action Document

TABLE 7 . Physical and Chemical Characteristics for the End

- Use Product (EP), Botector (40 CFR § 158.2120)

OCSPP Guideline Number Data Requirement Results MRID No.

830.6317 Storage Stability T he registrant is relying on the storage stability for Bl ossom Protect (EP) which was

stable at 20°C for 1 2 months. 48547201

830.6319 Miscibility Not applicable, the product is not an emulsifiable liquid. 47869502

830.6320 Corrosion Characteristics C ited Data on Blossom Protect are adequate to support this E P. No damage of the packaging material was observed during this 15 month storage stability study. 48547201

830.7100 Viscosity Not applicable, the product is a solid 47869502

830.7300* Density/Relative Density/Bulk Density (Specific Gravity) 0.64 kg/L

0.6 0

- 0.69 g/mL b 47869502 and

CSFAureobasidium pullulans strains DSM 14940 and 14941 Page 33 of 41 Biopesticides Registration Action Document

TABLE 8. Toxicology Data Requirements for the Technical Grade of the Active Ingredient (TGAI)/Manufacturing

- Use Product (MP), Aureobasidium pullulans

strain DSM 14940 and strain DSM 14941 Technicals and for t he End

- Use Products (EP), Botector and Blossom Protect (40 CFR § 158.2140)

OCSPP

Guideline

Number Data Requirement Results

DSM 14941 TGAI DSM 1494 0

TGAI DSM 14941

&

DSM 1494 0

Technicals

(MPs) Botector (EP) Blossom Protect (EP) MRID No.

Tier I

88 5.3050 Acute Oral Toxicity/ Pathogenicity 24 rats were administered a single dose of A. pullulans strain DSM 14941. Three animals /sex were sacrificed on days 3, 7, and 14; the remaining animals were sacrificed at test end on day 21. Body

tissues were exam ined for A. pullulans . All samples of feces, blood, brain, lung, liver, kidney, spleen, stomach, and intestine were negative. The lymph nodes of one animal sacrificed on day 7 tested positive. Classification: Acceptable A. pullulans

strain DSM

14941 data

Cited 1 Not applicable Not applicable Not applicable 47899302

885.3150 Acute Pulmonary Toxicity/ Pathogenicity Male and female animals were dosed with A.

pullulans strain DSM 14941 Technical (4 x 10 8

cfu/g). There was no mortality, and, except on the day o f dosing when the entire test material group showed reduced motor activity and dyspnea, all animals appeared normal throughout the study. Tissue samples were evaluated for A. pullulans after sacrifice. The following tissues were negative for A. pullulans a t all time points: feces, brain, kidney, and intestine. Blood samples were negative except for one male on day 0; lung samples were negative except for two males and two females on day 0; liver samples were negative except for three males on day 0; spleen samples were negative except for two males on day 0; lymph node samples were negative except for one animal on day 7, and stomach samples were negative except for two males on day 0. A. pullulans

strain DSM

14941 data

Cited 1 Not applicable Not applicable Not applicable 47899303Aureobasidium pullulans strains DSM 14940 and 14941 Page 34 of 41 Biopesticides Registration Action Document

TABLE 8. Toxicology Data Requirements for the Technical Grade of the Active Ingredient (TGAI)/Manufacturing

- Use Product (MP), Aureobasidium pullulans

strain DSM 14940 and strain DSM 14941 Technicals and for t he End

- Use Products (EP), Botector and Blossom Protect (40 CFR § 158.2140)

OCSPP

Guideline

Number Data Requirement Results

DSM 14941 TGAI DSM 1494 0

TGAI DSM 14941

&

DSM 1494 0

Technicals

(MPs) Botector (EP) Blossom Protect (EP) MRID No.

Classification: Acceptable

885.3200 Acute Injection Toxicity/ Pathogenicity M ale and female rats were dosed subcutaneously with A. pullulans

strain DSM 14940 (2.36 x 10 10 cfu/g) and DSM 14941 (1.5 x 10 10

cfu/g). Interim sacrifices w ere made on days 3, 7, and 14; the remaining animals were sacrificed at test end on day 21. There were no deaths, and all animals appeared normal throughout the study except for edema or slight erythema at the injection site. Samples of the following tissu es were negative for A. pullulans at all time points: feces, blood, brain, lung, liver, kidney, spleen, stomach, and intestine. Lymph node samples were negative except for one animal sacrificed on day 7. Classification: Acceptable Not applicable Not applic able Not applicable 47871807

M ale and female rats were dosed subcutaneously

with A. pullulans strain DSM 14941 (1.1 x 10 9

cfu/g). Interim sacrifices were made on days 1 and 7; the remaining animals were sacrificed at test end on day 21. There were no de aths, and all animals appeared normal throughout the study except for severe edema or slight erythema at the injection site. Samples of the following tissues were negative

for A. pullulans at all time points: brain, lung, spleen, kidney, lymph nodes, blood and urine. The skin was positive for A. pullulans in three males and three females from the test material group on day 1 and in two females of the same group on day 7. The liver of one male from the test material group was positive on day 1. The cecum con tents were positive

in one male and one female from the test material group on day 1, and in two females from the same group on day 7. Classification: Acceptable A. pullulans

strain DSM

14941 Cited 1 47871808

885.3400 Hypersensitivity No incid ents of hypersensitivity associated with A. pullulans strains DSM 14940 or DSM 14941 have been reported by theAureobasidium pullulans strains DSM 14940 and 14941 Page 35 of 41 Biopesticides Registration Action Document

TABLE 8. Toxicology Data Requirements for the Technical Grade of the Active Ingredient (TGAI)/Manufacturing

- Use Product (MP), Aureobasidium pullulans

strain DSM 14940 and strain DSM 14941 Technicals and for t he End

- Use Products (EP), Botector and Blossom Protect (40 CFR § 158.2140)

OCSPP

Guideline

Number Data Requirement Results

DSM 14941 TGAI DSM 1494 0

TGAI DSM 14941

&

DSM 1494 0

Technicals

(MPs) Botector (EP) Blossom Protect (EP) MRID No.

Incidents applicant. Any future hypersensitivity incidents must be reported per OCSPP Guideline 885.3400.

885.3500 Cell Culture Not required because A. p ullulans strain DSM 14940 and strain

14941 are not viruses (refer to test note #4 of 40 CFR § 158.2140(d)).Not applicable Not applicable Not applicable Not applicable

870.1100 Acute Oral Toxicity Not applicable Not applicable Oral LD 50 was >2000 mg/kg.

Classification: Acceptable

TOXICITY

CATEGORY III 47869614

870.1200 Acute Dermal Toxicity Not applicable Not applicable The LD 50 was >2000 mg/kg.

Classification: Acceptable

TOXICITY

CATEGORY III 47869615

870.1300 Acute Inhalation Toxicity Not applicabl e Not applicable The LC 50 was >5.17 mg/L.

Classification: Acceptable

TOXICITY

CATEGORY IV 47869617

870.2400 Acute Eye

Irritation Not applicable Not applicable Three female rabbits

were treated with

0.1 mL test material.

No corneal opacity, iritis, or positive conjunctival

irritation was noted on any rabbit 47869618Aureobasidium pullulans strains DSM 14940 and 14941 Page 36 of 41 Biopesticides Registration Action Document

TABLE 8. Toxicology Data Requirements for the Technical Grade of the Active Ingredient (TGAI)/Manufacturing

- Use Product (MP), Aureobasidium pullulans

strain DSM 14940 and strain DSM 14941 Technicals and for t he End

- Use Products (EP), Botector and Blossom Protect (40 CFR § 158.2140)

OCSPP

Guideline

Number Data Requirement Results

DSM 14941 TGAI DSM 1494 0

TGAI DSM 14941

&

DSM 1494 0

Technicals

(MPs) Botector (EP) Blossom Protect (EP) MRID No.

throughout the study. Blossom

Protect was not irritating to the eye.

Classification: Acceptable; Toxicity

TOXICITY

CATEGORY IV

870.2500 Primary Dermal

Irritation Not app licable Not applicable Three female rabbits

were treated with 0.5 g test material

for 4 hours. No

dermal irritation was noted on any rabbit.

Classification: Acceptable; Toxicity

TOXICITY

CATEGORY IV 47869619

Additional Studies

Acute

Subcutaneous

Toxi city/ Pathogenicity 10 animals were

dosed with 2000 mg/kg Blossom

Protect. One male was humanely sacrificed on day 5 due to a ruptured

abscess at the injection site; all 47869616Aureobasidium pullulans strains DSM 14940 and 14941 Page 37 of 41 Biopesticides Registration Action Document

TABLE 8. Toxicology Data Requirements for the Technical Grade of the Active Ingredient (TGAI)/Manufacturing

- Use Product (MP), Aureobasidium pullulans

strain DSM 14940 and strain DSM 14941 Technicals and for t he End

- Use Products (EP), Botector and Blossom Protect (40 CFR § 158.2140)

OCSPP

Guideline

Number Data Requirement Results

DSM 14941 TGAI DSM 1494 0

TGAI DSM 14941

&

DSM 1494 0

Technicals

(MPs) Botector (EP) Blossom Protect (EP) MRID No.

other animals survived until study end on day 14. Necropsy revealed

abscesses at all injection sites.

The LD 50 was >2000 mg/kg.

Classification: Acceptable

In vivo

Micronucleus Groups of mice received a single oral gavages of 2000 mg/kg A. pullulans strain DSM 14941

dissolved in water. There were no in

- life clinical signs. Th e animals were sacrificed 24 or 48 hours

post

- dosing and the femoral bone marrow was immediately harvested. The ratio of polychromatic erythrocytes to total erythrocytes (mature and immature) was determined, and 2000 immature erythrocytes/animal were score d for the incidence of micro nucleated immature erythrocytes. A. pullulans strain DSM 14941 did not produce a statistically significant increase in micro nucleated immature erythrocytes compared to the negative control animals. The response of the positive control animals (cyclophosphamide, IP injection, 10 mg/kg) was appropriate. Classification: Acceptable 47899304

Influence of Temperature on Reproduction The effect of temperature on the reproduction of Aureobasidium pullulans strains DSM 14940, DSM 14941, and CBS 626.85 was examined. Strain CBS 626.85 was isolated from the peritoneal dialysis fluid of a human. In Liquid Culture 47871833

- Neither DSM 14940 nor DSM 14941 was able to grow at 35 or 37°C. CBS 625.85 produced about two duplications atAureobasidium pullulans strains DSM 14940 and 14941 Page 38 of 41 Biopesticides Registration Action Document

TABLE 8. Toxicology Data Requirements for the Technical Grade of the Active Ingredient (TGAI)/Manufacturing

- Use Product (MP), Aureobasidium pullulans

strain DSM 14940 and strain DSM 14941 Technicals and for t he End

- Use Products (EP), Botector and Blossom Protect (40 CFR § 158.2140)

OCSPP

Guideline

Number Data Requirement Results

DSM 14941 TGAI DSM 1494 0

TGAI DSM 14941

&

DSM 1494 0

Technicals

(MPs) Botector (EP) Blossom Protect (EP) MRID No.

35°C and ab out one duplication at 37°C. The pH of the media for the two DSM strains was unchanged, while the CBS 625.85 medium pH fell from 6.3 to 5.0. On Agar Plates

Classification: Accepta ble – At 27°C, colonies of all three strains were visible after 48 hours. At 37°C, only CBS 626.85 prod uced colonies. Neither DSM 14940 nor DSM 14941 produced any colonies after five days

at 37°C. Based on the lack of growth at temperatures of 35°C and above, strains DSM 14940 and DSM 14941 can be

considered non

- pathogenic in humans.

Toxicity/ Pathogenicity of

Aureobasidium pullulans in Immunocompro

mised Patients

Published

Literature The registrant has submitted reviews of various infections caused by Aureobasidium pullulans in immunocompromised patients. These infectio ns were not caused by the two strains of A. pullulans (DSM 14941, 14940) submitted for registration. Also, these infections were successfully treated in the immunocompromised patients by antifungal drugs. 47871816

– 47871831

1 Data listed can be cited b ecause microbial c haracteristics are very similar and no difference was noted between A. pullulans strain DSM 14940

and A. pullulans strain DSM 14941 in their toxicity, irritation, or pathogenic potential to a variety of species.Aureobasidium pullulans strains DSM 14940 and 14941 Page 39 of 41 Biopesticides Registration Action Document

TABLE 9

. Non

- target Organism Toxicity and Environmental Fate Data Requirements for the Technical Grade of the Active Ingredient (TGAI), Aureobasidium pullulans strain DSM 14940 and strain DSM 14941. (40 CFR § 158.2150)

OCSPP Guideline

Number Data Requirement Results MRID No.

Tier I

885.4050 Avian Oral Toxicity The L D 50 for A. pullulans DSM 14941 (CF40) in Japanese quail was >2000 mg/kg bw (1.0 x 10 9

cfu/animal ). BPPD determined that bridging to

DSM 14940 was acceptable

. Classification: Acceptable 478 718 11

885.410 0 Avian Inhalation Toxicity/Pathogenicity Not required; A. pullulans is not known or shown to be an avian pathogen N/A

885.4150 Wild Mammal Toxicity/Pathogenicity Tests required by 40 CFR § 158.2140 are adequate and appropriate for assess ing hazards of

A. pullulans strains DSM 14940 and 14941 to

wild mammals. An acute oral toxicity/pathogenicity study with laboratory rats

indicates no adverse effects at 4 x 10 8 cfu/animal.

Classification : Acceptable for wild mammal

risk assessment 47899302

885.4200 Fre shwater Fish Toxicity/Pathogenicity The 96

- hour LC 50 was >100mg/L (estimated to

be 3.2 x 10 5 cfu/mL nominal; m easured conc .’s ranged up to 4.1 x 10 3 cfu/mL ) . The test was insufficient to determine pathogenicity Classification: Supplemental

Additional rat ionale was given that A. pullulans

strains DSM 14940 and 14941 will not reach aquatic areas in significant concentration. Classification: Acceptable rationale 47871812

No MRID

885.4240 Freshwater Invertebrate Toxicity/Pathogenicity In a 48

- hour stati c toxicity test with Daphna

magna , control mortality was 28%, indicating possible study design problems. Classification: Unacceptable

A 21

- day static

- renewal study with Daphnia

magna showed the mortality

- based EC 50 is >1.3 x

10 6 cfu/mL and the reproduct ion

- based NOEC is

1.3 x 10 6 cfu/mL

. Classification: Acceptable 47899305

48531002

885.4280 Estuarine/Marine Fish and Invertebrate Testing Not required

– significant concentrations are not expected to reach marine/estuarine environments

as a result of the proposed uses. N/A

885.4300 Non

- target Plant Testing Wheat, soybean, and lettuce plants were exposed

to A. pullulans strains DSM 14940 and DSM 14941. The study is inadequate to determine if the test material may be pathogenic to non

- target plants.

C lassification: Unacceptable Additional rationale was provided that was sufficient for use in ecological risk assessment. Classification: Acceptable rationale 47899306

No MRID

885.4340 Non

- target Insect Testing Not required

– A. pullulans does not infect or create an epizootic condition in insects. N/AAureobasidium pullulans strains DSM 14940 and 14941 Page 40 of 41 Biopesticides Registration Action Document

885.4380 Honey Bee Testing Descriptions of several field trials were submitted, but the provided information was

insufficient to determine the hazard to honey

bees. Classification: Unacceptable

A 2 2

- day toxicity/pathogenicity laboratory study with honey bees determined the LD 50 to be 1.0 x

10 6 cfu/bee.

Classification: Supplemental

Additional pathogenicity testing data for this guideline were waived based on scientific rationale and published lite rature that reports A. pullulans a s part of the normal flora of insects and stingless bees and as commonly found in the

environment

. A. pullulans was not reported in publications as an entomopathogen. Classification: Acceptable rationale 47899307

48531003

(Pagnocca et al. 2008, Sacchi and Vaughan

Martini 2002, 2003) and (Rosa et al. 2003)

Tiers II, III, and IV Not required for A. pullulans strains DSM 14940 and DSM 14941 based on the current uses and application methods.Aureobasidium pullulans strains DSM 14940 and 14941 Page 41 of 41 Biopesticides Registration Action Document

APPENDIX B. PESTICIDE PRODUCTS

EPA File Symbol Registration

Name Percentage

Active Ingredient Formulation Type Use Site(s) Method(s)

of Application Application

Rate Target Pest

86174

- R Aureobasidium pullulans strain

DSM 14940

Technical 80. 00% Technical N/A N/A N/ A N/A

86174

- E Aureobasidium pullulans strain

DSM 14941

Technical 80.00% Technical N/A N/A N/A N/A

86174

- G Botector 40% strain DSM

14940

40% strain DSM

14941 End

- use Product Pome and

stone fruits

S trawberries

Grapes Preharvest

spray As directed

on label P enicillium Botrytis

Monilia

Nectria
Pezicula

86174

- U Blossom Protect 25.8% strain

DSM 14940

25.8% strain

DSM 14941 End

- use Product Pome Fruits Spray 1.25 lbs/acre

As directed

on label Erwinia amylovora (Fire Blight)