Aureobasidium pullulans strains DSM 14940 and 14941 Page 1 of 43 Biopesticides Registration Action Document

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BIOPESTICIDES REGISTRATION ACTION DOCUMENT



## Aureobasidium pullulans strain s DSM 14940 and 14941

Pesticide Chemical (PC) Code: 0 36010

DRAFT DOCUMENT

U.S. Environmental Protection Agency

Office of P esticide Programs Biopesticides and Pollution Prevention Division

December 20 , 2011Aureobasidium pullulans strains DSM 14940 and 14941 Page 2 of 43 Biopesticides Registration Action Document

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TABLE OF CONTENTS

I. EXECUTIVE SUMMAR Y

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

II. ACT IVE INGREDIENT OVERV IEW

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

III. REGULATORY BACKGROUN D

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

A. Applications for Pesticide Registration

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

B. Food Tolerance Exemption

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

IV. RISK ASSESSMENT

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

A. Product Analysis Assessment (40 CFR § 158.2 120)

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

B. Human Health Assessment (40 CFR § 158.2140)

................................ ................................ ................................ 9

C. Environmental Assessment (40 CFR § 158.2150)

................................ ................................ .............................. 15

V. ENVIRONMENTAL JU STICE

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

VI. RISK MANAGEMENT DECISION

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

VII. ACTIONS REQUIR ED BY THE R EGISTRANT

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

VIII. GLOSSARY OF A CRONYMS AND ABBREVIA TIONS

................................ ........... 21

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

................................ ................................ ................................ ................................ ................ 22

B. Environmental Protection Agency Risk Assessment Memoranda

................................ ................................ ... 27

C. Other References

................................ ................................ ................................ ................................ ................. 27

APPENDIX A. MICROBI AL PESTICIDES DATA R EQUIREMENTS

............................ 30

APPENDIX B. PESTICI DE PRODUCTS

................................ ................................ ............... 43Aureobasidium pullulans strains DSM 14940 and 14941 Page 3 of 43 Biopesticides Registration Action Document

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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 Analysis, Human Health

John L. Ko ugh, 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 Pest icides Branch

Susanne Cerrelli Regulatory Action LeaderAureobasidium pullulans strains DSM 14940 and 14941 Page 4 of 43 Biopesticides Registration Action Document

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I. EXECUTIVE SUMMARY

Aureobasidium pullulans strains DSM 14940 and DSM 14941 were isolated from apple leaves

and classified as Aureobasidium pullulans by the German Strain Collection for Microorga nisms (DSMZ). A. pullulans strains DSM 14941 and DSM 14940 are neither mutant strains nor genetically modified strains, and they are not closely related to toxigenic human pathogens.

A

. pullulans strain DSM 14940 is very closely related to strain DSM 14941

. N o difference s were 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.

B ioferm GmbH has proposed to register two manufacturing

- use pesticide pro duct s
, Aureobasidium pullulans strain DSM 14941 Technical and Aureobasidium pullulans strain DSM

14940 Technical

. The active ingredient is 80% w/w Aureobasidium pullulans (minimum of x 10 9 cfu/g) in each of these t echnical products.

B ioferm GmbH has p roposed to also register two end use product s to prevent fire blight and storage rot on terrestrial food and non

- food plants:

**•** Botector, to protect pome fruits and citrus from storage diseases; grapes and strawberries

from Botrytis ; and stone fruit from M onilia

, containing the active ingredients: 40% w/w Aureobasidium pullulans strain DSM 14940 (minimum of 5 x 10 9 cfu/g) and 40% w/w Aureobasidium pullulans strain DSM 14941 (minimum of 5 x 10 9 cfu/g) ; and

**•** Blossom Protect

, to protect pome fruits against f ire blight, containing the active ingredients

of 25.8% w/w Aureobasidium pullulans strain DSM 14940 (minimum of 5 x 10 9 cfu/g)

and 25.8% w/w Aureobasidium pullulans strain DSM 14941 (minimum of 5 x 10 9 cfu/g).

A

. pullulans is a “yeast

- like” saprophytic f ungus 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) and apple (Granado et al. 2008) plants, as well as other plants (Gao et al. 2005, Osono 2008, Slavikova et al. 2007, Suryanarayanan et al. 2005, Webb and Mundt 1978). While it has been associated with disease in some plants, this fungus generally is recognized as a saprobe and weak pathogen or parasite of certain plants (Kockova

- Kratochvilova et al. 1980, Cooke 1959). It is known to be an antagonist of several plant disease causing organisms, and it also is an important producer of enzymes for biotechnological and industrial ap plications (Chi et al. 2009).

Environmental Protection Agency (EPA) scientists have reviewed product analysis, toxicology, and nontarget organism data and information (40 Code of Federal Regulations (CFR) §§ 158.2120, 158.2140, and 158.2150, respect ively) submitted to support the registration of th e four proposed Aureobasidium pullulans strain DSM 14941 and Aureobasidium pullulans strain DSM 14940 products

. Overall, such data and information are adequate for risk assessment purposes

, fulfill current data requirements

, and allow for registration under Section 3(c)(5) of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA)

.

A cceptable guideline studies to support the proposed pre

- harvest use of these products fulfilled

p roduct analysis data requirements for A

. pullulans strain s DSM 14941 and DSM 14940

, including product chemistry and composition, analysis of samples, and physical and chemical

characteri stics

. I f post

- harvest use sites are proposed for these products in the future , a dditiona lAureobasidium pullulans strains DSM 14940 and 14941 Page 5 of 43 Biopesticides Registration Action Document

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product analysis data would be required

.

Adequate mamm alian toxicology data and information were submitted to support A

. pullulans

strains DSM 14941& DSM 14940 pesticide products.

V arious infections caused by Aureobasidium pullulans in immunocompromise d patients were reported in published literature . T hese infections , however, were not caused by the two strains of A. pullulans (DSM 14941, 14940) submitted for registration

, and t he acut e oral, pulmonary an d

sub

- cutaneous injection toxicity/pathogenicit y stud ies showed t hat, at a single high dose, A

. pullulans strain DSM 14941 is not toxic , infective, and/or pathogenic

. Product analysis data

demonstrate that A

. pullulans strain DSM 14941 is closely related to strain DSM 14940 . T he Agency determined that the findings of these DSM 14941 acute toxicity/pathogenicity studies support both strains. In another acute subcutaneous toxicity/pathogenicity study, rats were injected with both A. pullulans strain DSM 14940 and DSM 14941 , and the results indicate that

both stra ins are not toxic, infective, and/or pathogenic. A study on the i nfluence of temperature on reproduction also supports the finding that DSM 14940 and DSM 14941 strains are not pathogenic to humans based on the lack of growth of these strains at temperature s of 35°C and

above. N o hypersensitivity incidents, during research, development, or testing of A. pullulans

strain DSM 14940 and DSM 14941 were reported by the applicant . In light of the se results of acute toxicity testing and the absence of hypersensitiv ity incidents, testing at higher tiers (i.e.,

Tiers II and III) was not required for either strain

.

EPA has made a preliminary conclusion that there is a reasonable certainty that no harm will result to the United States population, including infants and children, from aggregate exposure to

residues of A

. pullulans strains DSM 14941 and DSM 14940

. No dietary risks are expected from

use of A

. pullulans strains DSM 14941 and DSM 14940 as an active ingredient in pesticide products.

Despite the low toxicologic al profile of A

. pullulans strains DSM 14941 and DSM 14940

, baseline personal protective equipment (PPE) is required for handlers who may be exposed to the active ingredient, for prolonged periods or numerous times . Handlers working with A

. pullulans

strai ns DSM 149 41 and DSM 14940 in agricultural settings are directed to wear a long

- sleeved shirt, long pants, socks, shoes, waterproof gloves, and a dust/mist filtering respirator meeting National Institute for Occupational Safety and Health (NIOSH) standards of at least N

- 95, R

- 95,

or P

- 95. EPA may require additional PPE, other than the standard described above, on a product

specific basis.

Scientific rationale s w ere submitted to satisfy data requirements for freshwater fish toxicity/pathogenicity testing , nontarget insect testing, and nontarget plant testing. Toxicity/pathogenicity studies with terrestrial plants ( Triticum spp., Glycine max

, Latuca sativa ), Japanese quail ( Coturnix coturnix japonica ), rainbow trout ( Oncorhynchus mykiss ), Daphnia

magna , a nd honey bee ( Apis mellifera ) were also submitted to satisfy data requirements.

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 magnitude lower. A spray buf fer of 25 f ee t is required around aquatic areas to limit concentrations of the active ingredients in aquatic environments to within the range ofAureobasidium pullulans strains DSM 14940 and 14941 Page 6 of 43 Biopesticides Registration Action Document

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concentrations observed in nature (e.g., see Slavikova and Vadkertiova 1997). Therefore, the proposed applicati ons of A

. pullulans strain DSM 14940 and strain DSM 14941 are not expected to result in concentrations in terrestrial or aquatic environments above those found in the

environment.

S everal guideline studies and scientific rationale s show that toxicity or pathogenicity is not expected with wild mammals, birds, nontarget plants, nontarget insects, honey bees, freshwater fish (toxicity only), and freshwater invertebrates. The information provided is sufficient to satisfy the Tier I nontarget organism data re quirements and nontarget organism risk assessment for the active ingredient s . Further testing of nontarget organisms at higher tier levels is not required. Based on the data, literature citations, and data waiver rationale provided by the applicant, EPA

h as determined that adverse effects to nontarget organisms are not anticipated as a result of the proposed uses of A

. pullulans strain DSM 14940 and strain DSM 14941. A “No Effect”

determination ha s also been made for direct and indirect effects to listed s pecies and their designated critical habitats resulting from the proposed uses of A

. pullulans strain DSM 14940 and strain DSM 14941, as labeled.

On October 1, 2009, EPA announced a new policy to provide a more meaningful opportunity for the public to pa rticipate 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; firs t 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 the policy of making registration actions more transparent, the proposed

pe sticid e products containing A

. pullulans strains DSM 14941 and DSM 14940, two new active ingredient s
, are subject to a 30

- day comment period. In addition to containing a new active ingredient, the registration of each of these products would result in the first outdoor use , first

residential use and first food use for A

. pullulans strains DSM 14941 & DSM 14940. The docket

identification number s associated with these registration actions and accessed through either http:// www.regulations.gov/ or http://www.epa.gov/pesticides/regulating/registration

- status.html

,

are EPA

– HQ

– OPP

– 2010

– 0106 for Aureobasidium pullulans strain DSM 14941 Technical and

EPA

– HQ

– OPP

– 2010

– 0100 for Aureobasidium pullulans strain DSM 14940 Technical. The following documents are available for comment in EPA

- HQ

- OPP 2010

– 0106 and EPA

– HQ

– OPP

– 2010

– 0100: (1) draft Aureobasidium pullulans strains DSM 14940 and 14941 Biopesticides Re gistration Action Document (BRAD) ; (2) environmental risk assessment for Aureobasidium pullulans strains DSM 14940 and 14941 ; (3) draft product label s for the manufacturing

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

– R) and Aureobasidium pullulans strain DSM 14940 Technical (EPA File Symbol: 86174

– E ); and ( 4 ) draft product label s for the end

- use product s
, Botector. (EPA File Symbol 86174

- G ) and Blossom Protect (EPA File Symbol 86174

– U) . While a final decision on registration is contingent upon review and consideration of public comments, EPA presently

believes t hat, based upon the risk assessment and informat ion submitted in support of Aureobasidium pullulans strains DSM 14940 and 14941 T echnicals and Blossom Pro tect and

Botector EP, it is in the best interest of the public and the environment to issue these registrations. The basis for this preliminary decision can be found in the risk assessment for A

. pullulans strains DSM 14940 and 14941 , which is characterize d throughout this BRAD.Aureobasidium pullulans strains DSM 14940 and 14941 Page 7 of 43 Biopesticides Registration Action Document

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II. ACTIVE INGREDIENT OVERVIEW

Biological Name: Aureobasidium pullulans strains DSM 14940 and 14941

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

reference number DSM 14940 & DSM 14941

OP P Chemical Code: 036010

Type of Pesticide: Microbial Pesticide

– Fungicide

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

III. REGULATORY BACKGROUND

A. Applications for Pesticide Registration

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

- 3430; Tulin Austria) submitted ap plications 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 Protec t (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 containing a new active ingredient ( 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 following this publication

.

B. Food Tolerance Exemption

Conc urrent with its registration application s and under Federal Food, Drug, and Cosmetic Act (FFDCA) section 408(d), bioferm GmbH submitted a petition to establish an exemption from the requirement of a tolerance for Aureobasidium pullulans strains DSM 14940 and DSM 14941 (Pesticide Petition (PP) 9F7623 ). In the Federal Register of March 10, 2010 ( 75 FR 11171 ), EPA

announced that bioferm 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 received following this publication.

IV. RISK ASSES SMENT

In the Federal Register of October 26, 2007, EPA issued a Final Rule on the data requirements to support registration of microbial pesticides and updated the definition for microbial pesticidesAureobasidium pullulans strains DSM 14940 and 14941 Page 8 of 43 Biopesticides Registration Action Document

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( 72 FR 61002 )

. The rule became effective on December 26, 2007. The data and information evaluated for this BRAD were considered in light of these requirements.

The classifications for each data submission are assigned by EPA science rev iewers and i ndicat e the usefulness of the information contained in the documents for risk assessment. A rating of

“ acceptable ” indicates the study is scientifically sound and is useful for risk assessment. A

“ supplemental ” rating indicates the data provide some information that can be useful for risk assessment. The studies may have certain aspects determined not to be scientifically acceptable (“ supplemental : upgradeable ”). If a study is rated as “supplemental: upgradeable ,” EPA always provides an indicati on of what is lacking or what can be provided to change the rating to

“ acceptable .” If there is simply a “ supplemental ” rating, the reviewer often will state that t he study is not required by 40 C FR P art 158. Both “ acceptable” and “supplemental ” studies ma y be used in the risk assessment process as appropriate. An “unacceptable ” rating indicates that new

data need to be submitted.

For the acu te toxicity data requirements, Toxicity C ategories are assigned based on the hazard(s) identified from studies and/ or other infor mation submitted to EPA in support of a pesticide registration. The active ingredient or particular product is classified as Toxicity Category I, II, III, or IV, where Toxicity Category I indicates the highest toxicity and Toxicity Category I V indicates the lowest toxicity.

A. Product Analysis Assessment ( 40 CFR § 158.2120 )

Both strains, DSM 14940 and DSM 14941, were isolated from apple leaves and classified as Aureobasidium pullulans by the German Strain Collection for Microorganisms (DSMZ). For this organism, no sexual reproduction is known. Aureobasidium is classified into the phylum

As comycota.

As discussed in Pesticide Petition (PP) 9F7623

, Aureobasidium pullulans strain DSM 14940 is very closely related to strain DSM 14941. 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 regions ITS1 and ITS4, which are commonly used

for inter

- and intra

- species differentiation. Definitive differentiation from each other can only be achieved by molecular biological m ethods focusing on the whole genome, by conducting such analyses as RAPD PCR (Random Amplification of Polymorphic DNA Polymerase Chain Reaction) or by using recently developed strain

- specific primer characteristics. A

. pullulans

strains DSM 14940 and DSM 1 4941 were identified at DSMZ by means of morphological characterization of the colonies on yeast medium (YM) agar plates. Further confirmation of these strains can be achieved via PCR. PCR reveals a specific band that distinguishes A. pullulans

strains DSM 14940 and DSM 14941 from other isolates of A. pullulans and from other yeast

- like fungi. The subject strains are neither mutant strains nor genetically modified strains.

A

. pullulans can live in an environment offer i ng 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 stre ssed environments, such as hypersaline waters in salterns and rocks and monuments. Due to the production of large quantities of yeast

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

A ll product analysis data requirements for A

. pullulans strains DSM 14940 and 14941 have beenAureobasidium pullulans strains DSM 14940 and 14941 Page 9 of 43 Biopesticides Registration Action Document

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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.

B. Human Health Assessment ( 40 CFR § 158.2140 )

1. Toxicity

All toxicolog y 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 required 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 administered a single dose of A. pullulans strain DSM 14941. Three

animals of each sex were sac rificed on days 3, 7, and 14; the remaining animals were sacrificed at test end on 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 o f 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

. This study was rated acceptabl e. Acute Oral Toxicity/Pathogenicity ( Office of Chemical Safety and Pollution Prevention (OCSPP) Guideline 885.3050; Master Record Identification Number (MRID No. ) 47899302 ):

Acute Pulmonary Toxicity/Pathogenicity (OCSPP Gui deline 885.3150; MRID No. 47899303 ) : M ale and female animals were exposed intratracheal ly with 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 sacrificed at study end on 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 th roughout the study. Tissue samples were

evaluated 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 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. 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

. This study was rated acceptable

.

Acute I njection Toxicity/ Pathogenicity (OCSPP Guidel ine 885.3200; MRID No. 47871807 and 47871808) : In an acute subcutaneous toxicity/pathogenicity study (MRID 47871807), male and female rats

were injected with A . pullulans strain DSM 14940 (2.36 x 10 10 cfu/g) an d DSM 14941 (1.5 x

10 10 cfu/g). Interim sacrifices were made on days 3, 7, and 14; the remaining animals wereAureobasidium pullulans strains DSM 14940 and 14941 Page 10 of 43 Biopesticides Registration Action Document

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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 a t 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 negative except for one animal sacrificed on day 7. A

. pullulans strains DSMZ 14940 and DSMZ 14941 did not appear to be toxic, infective, and/or pathogenic in rats, when dosed at 1.95 x 10 7 or 1.12 x 10 7 cfu/animal, respectively. This study was rated acceptable

.

In a second acute subcutaneous toxicity/path ogenicity 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 test end on day 21. There were no deaths, and a ll 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 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 cfu/animal. This study was rated 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 tes ting of A

. pullulans strains DSM

14940 and 14941 were reported by the applicant . Any future hypersensitivity incidents must be reported per OCSPP Guideline 885.3400. :

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

Acute Oral Toxicity In an acute oral toxicity study, six animals 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. This study was rated acceptable

, and

the EP is classified as toxicity category III

. (OCSPP Guideline 870.1100 ; MRID No. 47869614) :

In an acute subcutaneous toxicity study, 10 animals were dosed with 2000 mg/kg Blossom Protect (7 x 10 9 cfu/g each of DSM 14940 an d DSM 14941). One male was humanely sacrificed on day 5 due to a ruptured abscess at the injection site; all other animals survived until study end on day 14. Necropsy revealed abscesses at all injection sites. The LD 50 was >2000 mg/kg. This
study was rat ed acceptable. Acute Subcutaneous Toxicity (MRID 47869616) :

In an acute dermal toxicity study, 10 animals 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 exhib ited chromodacryorrhea, but this was not considered to be toxicologically significant. All animals Acute Dermal Toxicity (OCSPP Guideline 870.1200 ; MRID 47869615) :Aureobasidium pullulans strains DSM 14940 and 14941 Page 11 of 43 Biopesticides Registration Action Document

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appeared healthy at necropsy. The LD 50 was >2000 mg/kg. This study was rated acceptable

, and

the EP is classified as toxicity category IV.

In an acute inhalation toxicity study, 10 animals were exposed nose

- only to a 10% suspension 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 149 41) 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 Inhalatio n Toxicity (OCSPP Guideline 870.1300 ; MRID 47869617) :

This study was rated acceptable

, and the EP is classified as toxicity category IV.

In an eye irritation study, 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

a nimal 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. This study was rated acceptable

, an d the EP is classified as toxicity

category IV. Ac ute Eye Irritation (OCSPP Guideline 870.2400; MRID 47869618) :

In a dermal irritation study, three 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 appeared normal throughout the study. Blossom Protect was considered virtually non

- irritating. This study was rated acceptable

, and the EP is classified as toxicity category IV. Primary Dermal Irritation (OCSPP Guideline 870.2500 ; MRID 47869619) :

In vivo Micronucleus (MRID 47899304) Groups of mice received single oral gavages of 2000 mg/kg A. pullulans strain DSM 14941

dissolved in water. There were no in

- life clinical signs. The animals were sacrificed 24 or 48 hours post

- dosing

, an d 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 scored for the incidence of micro nucleated immature erythro cytes. 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. This study was rated acceptable. :

The effects of temperature on the reproduction of A. pullulans strains DSM 14940, DSM 14941, and CBS 626.85 in liquid culture or on agar plate s 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 for CBS 626.85 was approximately five. Neither DSM 14940

nor DSM 14941 w ere able to gro w at 35 or 37°C. CBS 625.85 produced about two duplications at 35°C and about one duplication at 37°C. Based on the lack of growth at temperatures of 35°C and above, strains DSM 14940 and DSM 14941 are considered non

- pathogenic in humans. This study was ra ted acceptable. Influence of Temperature on Reproduction (MRID 47871833) :

Toxicity/Pathogenicity of Aureobasidium pullulans in Immunocompromised Patients (MRID 4787181631) : The applicant ( bioferm GmbH ) has submitted reviews of published literature concerning variousAureobasidium pullulans strains DSM 14940 and 14941 Page 12 of 43 Biopesticides Registration Action Document

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infections caused by Aureobasidium pullulans in immunocompromised patients. These infections 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. Based on the findings in these published literature

, 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 as a preventive measure

.

b. Acute Toxicology and Subchronic Toxicity/Pathogenicity

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

– Tier III

Tier II and Tier III studies were not required fo r 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 has developed the Endo crine Disruptor Screening Program (EDSP) to determine whether certain substances (including pesticide active and other ingredients) may have an effect in humans or wildlife similar to an effect produced by a “naturally occurring estrogen, or other such end ocrine 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 found 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 det ermine which, if any, of the Tier 2 tests are necessary based on the available data. Tier 2 testing is designed to identify any 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 chemicals, which contains 58 pesticide active ingredients and 9 inert ingredients. This list of chemicals was selected based on the pot ential for human exposure through pathways such as food and water, residential activity, and certain post

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

A. pullulans strains DSM 149 40 and 14941 are not among the group of 58 pesticide active ingredients on the initial list to be screened under the EDSP. Under FFDCA section 408(p), EPA must screen all pesti cide chemicals. Accordingly, EPA anticipates issuing future EDSP

orders/data ca ll

- 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 FFDCA allows EPA to establish an exemption from the requirement for a tolerance (the legal limit for a pesticide chemical resi due i n or on a food) only if EPAAureobasidium pullulans strains DSM 14940 and 14941 Page 13 of 43 Biopesticides Registration Action Document

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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, includin g all anticipated dietary exposures and all other exposures for which there is reliable information.” This includes exposure through 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 the 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 the pesticide chemical residue in establishing a tolerance and to “ensure that there is a reasonable certainty that 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 pesticide'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 examines 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 subgroups of consumers, including infants and children.

Based on the acute toxicity/pathogenicity data and information discussed prev iously 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 nonoccupational exposures, including drinking water from ground water or surface water and exposure through pesticide use in garde ns, lawns, or buildings (residential and other indoor uses).

Dietary Exposure and Risk Characterization : A. pullulans is likely to be commonly present on fresh produce. According to Webb and Mundt (1978) A. pullulans is “a major resident on most

green p lants.” 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 the 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 certain fruits (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 these fungicidal active ingredients

. N one of the studies have indicated that i f strains DSM 14940 and

14941 of A. pullulans are ingested, any pathogenic effects will result in mammals. In addition

,

no growt h for A. pullulans strains DSM 14940 and 14941 has been noted at the normal human body temperature of 37°C. Even if oral exposure should occur by ingesting harvested fruits

, EPA concludes that there is a reasonable certainty th at no harm will result from e xposure to suchAureobasidium pullulans strains DSM 14940 and 14941 Page 14 of 43 Biopesticides Registration Action Document

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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 )

.

Drinking Water Risk Characterizat ion : Naturally occurring, A. pullulans is known to be widely distributed in the environment in many 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. 2 009). Exposure of humans to residues of pesticides containing A. pullulans strains DSM

14940 and 14941 in consumed drinking water is unlikely. The proposed use patterns, use sites, and application methods for A. pullulans strains DSM 14940 and 14941 do no t include direct application to aquatic environments, thereby limiting contact with surface water

. Aureobasidium pullulans strains DSM 14940 and 14941 are not likely to be present in aquatic environments

since they are not known to inhabit aquatic environ ments

. In the event that Aureobasidium pullulans strains DSM 14940 and 14941 is transferred to surface or ground water intended for human consumption, the fungi would not survive the conditions water is subjected to in a drinking water treatment facility, including high temperatures, chlorination, pH adjustments,

and/or filtration. Even if oral 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

o f 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 use sites for th ese pr oduct s include residential garden sites and agricultural sites. As stated previously, A. pullulans is naturally

present in many habitats , and b ased on the data and other information submitted to satisfy data requirements for registration of the manufacturi ng

- use and end

- use pesticide product s containing

the active ingredient 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. Cumulative 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 exem ption

, EPA consider “available information concerning the cumulative effects of [a particular pesticide’s] residues and other substances that have a common mechanism of toxicity.”

EPA has not found A. pullulans strains DSM 14940 and 14941 to share a common mechanism of toxicity with any other substances, and A. pullulans strains DSM 14940 and 14941 do not appear to produce a toxic metabolite as its mode of action against target pest s . For the purposes of this tolerance action, therefore, EPA has assumed tha t 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. Determination 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 pesticide chemical residues, and the cumulative effects on infants and children of the residuesAureobasidium pullulans strains DSM 14940 and 14941 Page 15 of 43 Biopesticides Registration Action Document

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and other subst ances 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 safety for infants and children in the case of threshold effects to account for prenatal and postnatal to xicity 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 and children. This additional margin of safety is commonly referred to as the FQPA Safety Factor. In apply ing 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 sect ion 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. pullulan s strains DSM 14940 and 14941 . This includes all anticipated dietary exposures and all 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. Thus, there are no threshold effects of concern and, as a result, the provision 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 immunocompromised pa tients (MRID 47871816

- 31). These infections were not caused by the two strains of A. pullulans (DSM 14941, 14940) submitted for registration. As a precautionary measure

, the Agency is requiring that the label include specified personal protective equipmen t ( PPE) to protect workers from repeated exposure to Aureobasidium pullulans strains DSM 14941 & DSM 14940 . A ppropriate personal protective equipment and precautionary 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 settings must wear a long

- sleeved shirt, long pants, socks, shoes, waterproof gloves, and a dust/mist fi ltering 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 product

- specific basis.

4. Human Health Risk Characterization

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

standard for registration in FIFRA and the relevant safety factors in FFDCA. A determination

has been made that no unreasonab le adverse effects to the U.S. population in general, and to

infants and childr en 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. A. pullulans is a “yeast

- like” sap rophytic fungus that is common in the phyllosphere of many plants, and it also has been isolated from many environments, including soil and aquatic environments (Chi et al. 2009, Cooke 1959, SlepeckyAureobasidium pullulans strains DSM 14940 and 14941 Page 16 of 43 Biopesticides Registration Action Document

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and Starmer 2009). It is commonly isolated from healthy grape (Fleet 2002, 2003) and apple (Granado et al. 2008) plants, as well as other plants (Gao et al. 2005, Osono 2008, Slavikova et al. 2007, Suryanarayanan et al. 2005, Webb and Mundt 1978). While it has been associated with disease in some plants, it i s generally recognized as a saprobe and weak pathogen or parasite of certain plants ( Kockova

- Kratochvilova et al. 1980, Cooke 1959). It is known to be an antagonist of several plant disease causing organisms, and it also is an important producer of enzyme s for biotechnological and industrial applications (Chi et al. 2009).

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 nontarget organi sm data requirements and are sufficient for risk assessment purposes

. Further testing of nontarget 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 ri sk assessment based on the data and data 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 nontarget organisms when used according to label directio ns. Based on study results, there are concerns of toxicity to terrestrial arthropods, aquatic invertebrates, and honey bees.

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

, refer to Table 9 in Appendix A

.

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 scient ific rationale to satisfy data requirements for nontarget 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. pull ulans is not toxic, pathogenic, or infective to other nontarget 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 commo nly 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 adverse effects in nontarget insects and honey bees. Nontarget Insects (OCSPP Guid eline 885.4340) and Honey Bees (OCSPP Guideline 885.4380) ; MRID 48531003:

Nontarget 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 magnitude lower. A sprayAureobasidium pullulans strains DSM 14940 and 14941 Page 17 of 43 Biopesticides Registration Action Document

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buffer of 25 ft is required around aquatic areas, to limit concentrations of the ac tive ingredients in aquatic environments to within the range of concentrations observed in nature (e.g., see

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

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

Kratochvilova et al. 1980). Instances where disease is known to occur have required vectors (e.g., stigmatomycosis in pistachio) 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 Aureob asidium pullulans as a pathogen in plants. Based on the available data, BPPD concluded that A. pullulans strains

DSM 14940 and DSM 14941 have low potential to cause toxicity or pathogenicity in plants.

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 strains DSM 14940 and 14941 do not produce toxic effects at 100 mg/L ( 4 .1 x 10 3 cfu/mL measured )

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

- day Daphnia study showed that th e 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 informat ion, adverse effects are not expected

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

.

Estuarine/Mar ine Fish and Invertebrates (OCSPP Guideline 885.4280) : As determined above, the proposed applications are not expected to reach aquatic areas in significant 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 Literature Information

EPA performed l iterature searches to determine if other effects have been reported t hat were not contained within the information provided by the registrant. A search was performed using the Environmental Information Search service available to the Agency. This search tool simultaneously queries the Agricola

, Biosis Previews

, CAB Abstra cts

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

– present. The Web of Knowledge database also was searched for allAureobasidium pullulans strains DSM 14940 and 14941 Page 18 of 43 Biopesticides Registration Action Document

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years available. Searches were performed wit h the term ‘ Aureobasidium pullulans ’ combined with each of the terms, ‘bird,’ ‘mammal,’ insect,’ ‘bee,’ ‘arthropod,’ ‘aquatic,’ ‘invertebrate,’ ‘fish,’ ‘reptile,’ ‘amphibian,’ ‘plant,’ ‘algae,’ ‘freshwater,’ ‘marine,’ ‘estuarine,’ ‘soil,’ and ‘pathogen. No ne of the records returned in these searches indicated potential adverse effects on nontarget 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 nontarget 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 Ass essment

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 that are found naturally in the environment. Additionally, several guideline studies and scientific rat ionale show that toxicity or pathogenicity is not expected with wild mammals, birds, nontarget plants, nontarget insects, honey bees, freshwater fish (toxicity only), and freshwater invertebrates. The information provided is sufficient to satisfy the Tier I nontarget organism data requirements and for nontarget organism risk assessment for the active ingredient. Further testing of nontarget organisms at higher tier levels is not required. B ased on the data, literature citations, and data waiver rationale p rovided by the applicant

, BPPD has determined that adverse effects to nontarget organisms are not anticipated as a result of the proposed uses of Aureobasidium pullulans strain s DSM 14940 and

DSM 14941

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

V. ENVIRONMENTAL JUSTICE

EPA seeks to achieve environmental justice

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

— with respect to the development, implementation, 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 consequences 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 h ealth; (2) the public’s contribution can influence the regulatory agency’s decision; (3) the concerns of all participants involved will

be considered in the decision

- making process; and (4) the decision

- makers seek out and facilitate

the involvement of tho se potentially affected. EPA has this goal for all communities and persons

across the United States.

To help address potential environmental justice issues, EPA seeks information on any groups or segments 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

,Aureobasidium pullulans strains DSM 14940 and 14941 Page 19 of 43 Biopesticides Registration Action Document

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compared to the general population. Please comment if you are aware of any subpopulations that may have atypical, unusually high exposure compared to the general population.

For additional information regarding environmental justice issues, please visit EPA’s web site at http://www.epa.gov/compliance/en vironmentaljustice/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 fo r 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 wit h widespread and commonly recognized practice, it will not generally cause unreasonable adverse effects on the

environment.

To satisfy criterion 1, the Aureobasidium pullulans strain DSM 14940 and Aureobasidium pullulans strain DSM 14941 pes ticide products have well

- 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 . T hese products are not expected 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 Aureobasidium 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 fungal 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 unreasonabl e adverse effects when used 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 Aureobasidium 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 appli cations )

, EPA will likely 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 pu llulans strain DSM 14940 and Aureobasidium pullulans strain DSM 14941 for shipment, the registrant is required to provide appropriate final printed labeling to EPA

.Aureobasidium pullulans strains DSM 14940 and 14941 Page 20 of 43 Biopesticides Registration Action Document

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B . Reporting of Adverse Effects and Hypersensitivity Incidents

Notwithstanding the infor mation stated in the previous 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.

Reports 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 inc idents) must be reported to EPA under the provisions of 40 CFR § 158.2140(d).Aureobasidium pullulans strains DSM 14940 and 14941 Page 21 of 43 Biopesticides Registration Action Document

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VIII. GLOSSARY OF ACRONYMS AND ABBREVIATIONS

ASAE American Society of Agricultural Engineers

BPPD Biopesticides and Pollution Prevention Division

BRAD Biopesticides Reg istration Action Document

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 Microorganisms)

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 Protection 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

i ndicated (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

NI OSH 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 person al protective equipment

ppm parts per million

TGAI technical grade of the active ingredient

ULV ultra low volumeAureobasidium pullulans strains DSM 14940 and 14941 Page 22 of 43 Biopesticides Registration Action Document

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I X. BIBLIOGRAPHY

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

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 Ident ity and Composition, Description 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

- Che mical Properties. Unpublished 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 Application 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 Impurities, 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) Deter mination 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 Blo ssom 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: Final 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: 20 060875/03. Unpublished study 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 23 of 43 Biopesticides Registration Action Document

- DRAFT

47869608 Moller, 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: Final 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. Unpub lished 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 pre pared by LAB International 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. (2009) 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. Unpublishe d study prepared by Austrian 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 Subcutaneous 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/063 8. Unpublished study prepared 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.

4 7869619 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 Application 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 Ma terials, Description of Production Process, Discussion of the Formation of Impurities, and CertifiedAureobasidium pullulans strains DSM 14940 and 14941 Page 24 of 43 Biopesticides Registration Action Document

- DRAFT

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

47871702 Smith, F. (2009) Aureobasidium pullulans Strain DSM 14940: Analysis of Samples. Unpublis hed study prepared by SciReg, 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 Che mistry, Toxicity, Efficacy, 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. (2009) 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. Unpubl ished 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 Requiremen ts: (Aureobasidium pullulans 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 Subcutaneous 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 pullulans 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 Pathogen icity 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 Component 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.; Idoat e, M. ; Rubio, M. ; et al. (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 Recipient: A Case Report. Hepato

- Gastroenterology

44:1215

- 1218.Aureobasidium pullulans strains DSM 14940 and 14941 Page 25 of 43 Biopesticides Registration Action Document

- DRAFT

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 Caporale, 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) Disseminat ed Nosocomial Fungal Infection 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.

4787182 4 Schrattenholz, A.; Flesch, 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. (2 006) Subcutaneous Mycosis 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 Pathology 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

- 21 3.

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) Opportunistic 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 L eukaemic Patient. Journal 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) Peri tonitis by Aureobasidium pullulans in Conditions Ambulatory Peritoneal Dialysis. Archives of Dermatoloy 133:1544

- 1545.Aureobasidium pullulans strains DSM 14940 and 14941 Page 26 of 43 Biopesticides Registration Action Document

- DRAFT

47871833 Kunz, S. (2009) Influence of Temperature on the Reproduction on Different Strains of the Species Aureobasidium pullulans. Unpub lished 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 Aur eobasidium pullulans (Strains 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 Formation 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: (Aureobasidiu m pullulans): Final Report. 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 Guide lines: (Aureobasidium pullans): 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 Number: 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 study 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. Unpublish ed study prepared by Univ. 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

- F erm GmbH (2011) Submission 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 Gaty, 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 To xicity 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 27 of 43 Biopesticides Registration Action Document

- DRAFT

48531004 Smith, F. (2011) Blossom Prote ct: Certified Limits. Unpublished 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 Memoranda

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 strawberr ies . 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 (EPA 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 of 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 fr om: 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 registration 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 Aure obasidium pullulans , a biotechnologically 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. Aureobasidium 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 Anatoli an regions. 11. GREMPA Seminar on Pistachios and Almonds, Sanliurfa (Turkey), 1

- 4 Sept. 1999.Aureobasidium pullulans strains DSM 14940 and 14941 Page 28 of 43 Biopesticides Registration Action Document

- DRAFT

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 r evealed by

cultivation

- independent 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

- sea son 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 Microbiologica,

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. 20 08. Endophytic and epiphytic 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 o f leaf

- cutting ants. Antonie 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

- 2 1.

Rosa, C.A., M.

- A. Lachance, 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 th e leaf surfaces. J.

Basic 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 submersed 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 29 of 43 Biopesticides Registration Action Document

- DRAFT

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. S ingle

- 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, Ita ly. Ann. Microbiol. 52: 237

- 244.Aureobasidium pullulans strains DSM 14940 and 14941 Page 30 of 43 Biopesticides Registration Action Document

- DRAFT

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 ( M P )

, Aureobasidium pullulans 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. 4787 1701

885.1200 Manufacturing 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 wi th 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 requiremen 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 Limits 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

enforcem ent analytical

method

. 47869601Aureobasidium pullulans strains DSM 14940 and 14941 Page 31 of 43 Biopesticides Registration Action Document

- DRAFT

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 u ideline

N umber Data Requirement 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 man ufacturing

process

. 47899301

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 num ber DSM

14941 Not applicable 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 data 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 Studi es
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 32 of 43 Biopesticides Registration Action Document

- DRAFT

TABLE 3

. Product Analysis Data Requirements for the End

- Use Product ( E P), Blossom Protect (40 CFR § 158.2 120)

OCSPP Guideline Number 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 applicable 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 ingredients 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 registe red concurrently. 47869601

885.1500 Certification of Limits Limits listed on the confidential statement of formula are adequate/acceptable. CSF dated July 7, 2011

Additional Studies

830.1800 Enforcement Analytical

Method Submitted data fulfill the r equirement for an enforcement 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 Sub mitted data fulfill the 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 Collec tion Not applicable Not 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 Sa mples Results of a five

- 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 sta tement of formula are adequate/acceptable. CSF dated July 7, 2011

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 33 of 43 Biopesticides Registration Action Document

- DRAFT

TABLE 5 . Physical and Chemical Chara cteristics for the Technical 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 C olor Pale pink to pale 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 st able during application or

when in contact with

metals or metal ions Not applicable 47871703

830.6317 Storage Stability Stable at 20°C for 10 months 47871804

830.6319 Miscibility Not applicable Not required because A. pullulans

strain DSM 14940 is not a n

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/Bulk 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.

830.6317 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 material

was observed during 15 month storage stability study. 48547201

83 0.7100 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 34 of 43 Biopesticides Registration Action Document

- DRAFT

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 the technical products. The technical products were stable at 20°C fo r 10

months. 47869502

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 EP. No damage of the packaging material was obser ved 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

0.60

- 0.69 g/mL b 47869502 and

CSFAureobasidium pullulans strains DSM 14940 and 14941 Page 35 of 43 Biopesticides Registration Action Document

- DRAFT

T ABLE 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 the 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

885.3050 Acute Oral Toxicity/ Pathogenicity 24 ra ts 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 examined for A. pullulans . All samples of feces, bl ood, 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 applic able 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 of dosing when the entire test material group

sh owed 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 at all time points: feces, brain, kidney, and

in testine. 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 d ay 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 478993 03Aureobasidium pullulans strains DSM 14940 and 14941 Page 36 of 43 Biopesticides Registration Action Document

- DRAFT

T ABLE 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 the 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 were made on days 3, 7, and 14; the remaining an imals 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 tissues were negative for A. pullulans at all time

p oints: 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 applicable 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 deaths, and all animals appeared normal throughou t 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. pullul ans 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 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. Classification: Acceptable A. pullulans

strain DSM

14941 Cited 1 47871808

885.3400 Hypersensitivity No incidents of hypersensitivity associated with A. pul lulans strains DSM 14940 or DSM 14941 have been reported by theAureobasidium pullulans strains DSM 14940 and 14941 Page 37 of 43 Biopesticides Registration Action Document

- DRAFT

T ABLE 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 the 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. pullulans 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 I II 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 applicable Not applicable The LC 50 was >5.17 mg/L.

Cla ssification: 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

irrit ation was noted on any rabbit 47869618Aureobasidium pullulans strains DSM 14940 and 14941 Page 38 of 43 Biopesticides Registration Action Document

- DRAFT

T ABLE 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 the 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 applicable Not applicable Three female rabbits

w ere 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 Toxicity/ Pathogenicity 10 animals were

dosed w ith 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 39 of 43 Biopesticides Registration Action Document

- DRAFT

T ABLE 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 the 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. The animals were sacrificed 24 or 48 hours

post

- d osing 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 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 negative control animals. The response of the positive control animals (cyclophosphamide, IP injectio n, 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 40 of 43 Biopesticides Registration Action Document

- DRAFT

T ABLE 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 the 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 about one duplication at 37°C. The pH of the medi a 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: Acceptable – At 27°C, colonies of all three strains were visible after 48 hours. At 37°C, only CBS 626.85 produced 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

Aureo basidium pullulans in Immunocompro

mised Patients

Published

Literature The registrant has submitted reviews of various infections caused by Aureobasidium pullulans in immunocompromised patients. These infections were not caused by the two strains of A. pul lulans (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 because microbial c haracteristics are very simil ar 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 41 of 43 Biopesticides Registration Action Document

- DRAFT

TABLE 9

. Nontarget 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 T he 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

885.4100 Avian Inhalation Toxicity/Pathogenicity Not re quired; 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 m ammals. 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 Freshwater 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 rationale was given that A. pullulans

strains DSM 1 4940 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 static toxicity test with Daphna

magna , control morta lity 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 reproduction

- based NOEC is

1.3 x 10 6 cfu/mL

. Classificati on: 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 Nontarget Plan t 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 nontarget plants. Classification: Unacceptable

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

No MRID

885.4340 Nontarget 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 42 of 43 Biopesticides Registration Action Document

- DRAFT

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 22

- 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 literature that reports A. pullulans a s part of the no rmal 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 Vaug han

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 43 of 43 Biopesticides Registration Action Document

- DRAFT

APPENDIX B. PESTICIDE PRODUCTS

EPA File Symbol Registration

N ame 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 1 4941

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 Penicillium Botrytis

Monilia

Nectria
Pezicula

8617 4

- 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)