Bacillus subtilis strain IAB/BS03 Page 2 of 36

Biopesticides Registration Action Document

TABLE OF CONTENTS

I. EXECUTIVE SUMMAR Y

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

II. ACTIVE INGREDIEN T OVERVIEW

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

III. REGULATORY BACK GROUND

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

A. Applicatio ns for Pesticide Product Registration

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

B. Food Tolerance Exemption

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

IV. RISK ASSESSMENT

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

A. Product Analysis Assessment (40 CFR § 158.2120)

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

B. Human Health Assessment (40 CFR § 158.2140)

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

C. Environmental Assessment (40 CFR § 158.2150)

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

V. ENVIRONMENTAL JU STICE

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

VI. RISK MANAGEMENT DECISION

................................ ................................ ................................ ................. 1 9

VII. ACTIONS REQUIRE D OF THE REGISTRANT

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

A. Final Printed Labeling

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

B. Terms of Registration

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

VIII. GLOSSARY OF A CRONYMS AND ABBREVIA TIONS

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

IX. BIBLIOG RAPHY

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

A. Studies Submitted to Support the Active Ingredient, Bacillus subtilis strain IAB/BS03 and Pesticide Product Registrations for the (TGAI) product, Bacill us subtilis strain IAB/BS03 Technical Powder, and four

new end

- use products (EPs): MildoreTM; Mildore MaxTM; Milagrum PlusTM and FungiseiTM

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

B. Environmental Protection Agency Risk Assessment Memoranda

................................ ................................ ... 24

C. Other References

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

APPENDIX A. MICROBI AL PESTICIDES DATA R EQUIREMENTS

................................ ........................... 26

APPENDIX B. PESTICI DE PRODUCTS

................................ ................................ ................................ .............. 35Bacillus subtilis strain IAB/BS03 Page 3 of 36

Biopesticides Registration Action Document

BIOPESTICIDES REGISTRATION ACTION DOCUMENT TEAM

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

Microbial Pesticides Branch

Science Reviews

Ibrahim S. Barsoum, Ph.D. Product Analysis, Human Health

John L. Kough, Ph.D. Product Analysis, Human Health

Gail Tomimatsu , Ph.D. Environmental Effects Shannon Borges Environmental Effects

Regulat ory

Kimberly Nesci Chief, Microbial Pesticides Branch

ShaRon Carlisle Acting Associate Chief, Microbial

Pesticides Branch

Susanne Cerrelli Regulatory Action LeaderBacillus subtilis strain IAB/BS03 Page 4 of 36

Biopesticides Registration Action Document

I. EXECUTIVE SUMMARY

Background

On May 7, 2013, Technology Sciences Group on behalf of Investigaciones y Aplicaciones Biotecnologicas S.L. (IAB, S.L., Spain) submitted application s for a ne w technical grade active ingredient (TGAI) product

, Bacillus subtilis strain IAB/BS03 Technical Powder (E PA File Symbol: 89615

- R), and for two new end

- use products ( EPs ) : MildoreTM (EPA File Symbol

89615

- E) a wettable

- powder formulation with 1% a.i.; and Mildore MaxTM (EPA File Symbol

89615

- G) a wettable

- powder formulation with 10% a.i.

, to the United States Environmental Protection Agency (EPA) under section 3 of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). On May 21, 2014, Technology Sciences Group on behalf of Investigaciones y Aplicaciones Biotecnologicas S.L. (IAB, S.L., Spain) submitted application s for two additional

new EPs : Milagrum PlusTM (EPA File Symbol 89615

- U), a liquid formulation containing 0.3%

a.i. ; and FungiseiTM (EPA File Symbol, 89615

- L), a liquid formulation containing 0.08% a.i.

, under section 3 of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). These products all contain the new active ingredient, Bacillus subtilis strain IAB/BS03

.

Bacillus su btilis strain IAB/BS03 is a naturally occurring bacterium originally isolated from the rhizosphere of cucurbits in Valencia, Spain. The Bacillus subtilis species is a naturally occurring

bacteri um found in agricultural soils worldwide

. Bacillus subtilis is a rod

- shaped, gram

- positive, aerobic, motile bacterium which is ubiquitous in nature and has been recovered from water, soil, air and decomposing plant residue.

Bacillus subtilis strain IAB/BS03 is antagonistic to fungal plant pathogens through nutrient competition, site exclusion and colonization

.

In March 2010

, EPA completed a F inal R egistration R eview D ecision for Bacillus subtilis

, Registration Review Case 60 12 ( http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

OPP

- 2007

- 1026

- 0029 ). In its decision, EPA concluded that the standards of R egistration R eview

were met for Bacil lus subtilis and identified no general risk concerns . This strain will now be considered as part of the Bacillus subtilis Registration Review Case 6012.

Th e EP s containing Bacillus subtilis strain IAB/BS03 are proposed for use to control bacterial or fungal pests in various grain, small fruit and vegetable crops, tree fruit and nut crops, and grass seed crops, row crops such as sugarbeet, tobacco, sugarcane, hops, oil crops

, greenhouse/shadehouse and landscape ornamental tree and plant crops. Addi tional proposed uses include applications to garden and ornamental landscapes in home

- owner and residential

areas

.

EPA s cientists reviewed product analysis, toxicology, and nontarget organism data and information ( 40 CFR §§ 158.2120, 158.2140, and 158.21 50, respectively) submitted to su pport the registration of the proposed pesticide product s

. They found that, o verall, such data and information are adequate for risk assessment purposes, fulfill the current microbial pesticide data requirements, and support registration of the product under FIFRA section 3(c)(5)

.Bacillus subtilis strain IAB/BS03 Page 5 of 36

Biopesticides Registration Action Document

Product Analysis

For the purposes of FIFRA section 3(c)(5) registration, the product analysis data requirements

for the TGAI (also the manufacturing use product) and EP s were fulfilled by a cceptable guideline studies. These data requirements include product chemistry and composition, analysis of samples, and physical and chemical characteristics

.

Toxicology

The applicant submitted a dequate mammalian toxicology data and information to support the pesticide products. A n acute injection toxicity/pathogenicity study showed that Bacillus subtilis strain IAB/BS03 when injected intravenously at the dose administered is not toxic or p athogenic

via th is route of exposure

. The acute oral and dermal toxicity studies were acceptable and were designated EPA Toxicity Category III based on the maximum dose administered. The a cute
inhalation toxicity and acute eye irritation stud ies are accept able and rated as EPA Toxicity Category IV. In l ight of the results of the acute toxicity/pathogenicity data , EPA did not require testing at higher tiers (i.e., Tiers II and III).

Tolerance Exemption

In its application to the EPA in 201 3

, Technology Sciences Group on behalf of IAB, S.L. provided a petition, data and other information to support an exemption from the requirement of

a tolerance for residues of Bacillus subtilis strain IAB/BS03 in or on all food commodities.

Occupational Exposure

Despite the low toxicological profile of the active ingredient, Bacillus subtilis strain IAB/BS03

, the EPA is requiring baseline personal protective equipment (PPE) for handlers that, due to their occupation, may be subject to prolonged or repeated exposure to the active ingredient

. The label s

on the end

- use product s direct h andlers working with

, Bacillus subtilis strain IAB/BS03

, in agricultural settings to wear a long

- sleeved shirt, long pants, socks, shoe s, waterproof glove s
, 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

.

Nontarget Organisms

Bacillus subtilis has been found in water, soil, air and decomposing plant residue. The information provided is sufficient to satisfy most of the Tier I nontarget organism data requirements and for nontarget organism risk assessment for this new active ingredient and its proposed uses. The honeybee and nontarget insect test data requirements are incomplete. Since some risks might be expected to certain populations of nontarget insects and honeybees as a consequence of frequent applications of products containing Bacillus subtilis strain IAB/ BS03

, precautionary label language is re quired to protect these organisms from exposures to products containing Bacillus subtilis strain IAB/BS03 . It should be noted that adverse population effects cannot be characterized because of the uncertainty of the environmental persistence of Bacillus

subtilis strain IAB/BS03

.

T he nontarget organism data requirements have been adequately addressed f o r the purposes of FIFRA section 3(c)(5) registration, based on the submitted data, rationales, limited anticipatedBacillus subtilis strain IAB/BS03 Page 6 of 36

Biopesticides Registration Action Document

exposure to nontarget aquatic organisms , and required labeling

. The specific findings for each

nontarget organism are addressed in Section IV below.

Public Participation

On October 1, 2009, the EPA announced a new policy to provide a more meaningful oppo rtunity for the public to participate i n major registration decisions before they occur. According to this policy, the EPA intends to provide a public comment period prior to making a registration decision for, at minimum, the following types of appli catio ns: new active ingredients; first food uses; first outdoor uses; first residential use s; or any other registration actions for which the Agency believes there may be significant public interest

.

Consistent with the policy of making registration actions more transparent, the pesticide product s containing Bacillus subtilis strain IAB/BS03 are subject to a 15

- day comment period prior to registration of the manufacturing and end

- use products

. In addition to containing a new active ingredient, the registration of the pesticide product w ill result in the first outdoor and food and

residential uses for Bacillus subtilis strain IAB/BS03 . The docket identification number associated with these registration actions is EPA

- HQ

- OPP

- 2013

- 0575

, and may be acce ssed through either http://www.regulations.gov or http://www.epa.gov/pesticides/regulating/registration

- status.html

. The following documents are being made available for comment in EPA

- HQ

- OPP

- 2013

- 057 5 : (1) draft Bacillus subtilis strain

IAB/BS03 Biopesticides Registration Action Document (BRAD)

, (2) draft label for the manufacturing

- use product, (TGAI) product, Bacillus subtilis strain IAB/BS03 Technical Powder (EPA File Symbol: 89615

- R), (3) draft label for MildoreTM (EPA File Symbol 89615

- E)

, (4) draft label for Mildore MaxTM (EPA File Symbol 89615

- G )

, (5) draft label for Milagrum PlusTM (EPA File Symbol 89615

- U ), and (6) draft la bel for FungiseiTM (EPA File Symbol,

89615

- L)

.

Registration Decision

While a final decision on registration is contingent upon review and consideration of public comments, EPA presently believes that the risk assessment and information submitted support registration of the Bacillus subtilis strain IAB/BS03 Technical Powder, and the four end use Products: MildoreTM, Mildore MaxTM, Milagrum PlusTM, and Fungisei TM

, and that it is appropriate to issue these registrations

. The basis for this decision can be found in the risk

assessment for Bacillus subtilis strain IAB/BS03 which is characterized throughout this BRAD

.

II . ACTIVE INGREDIENT OVERVIEW

Biological Name: Bacillus subtilis strain IAB/BS03

Cultu re Deposit: Colecciόn Española de Cultivos Tipo CECT No. 7254

OPP Chemical Code: 006544

Type of Pesticide: Mi crobial Pesticide

– fungicide

See Appendix B for specific information (e.g., use sites, application rate s , methods of application, formulation type s , and target pests) regarding the pesticide products containing this active ingredient.Bacillus subtilis strain IAB/BS03 Page 7 of 36

Biopesticides Registration Action Document

III. REGULATORY BACKGROUND

A. Applications for Pesticide Product Registration

Technology Sciences Group ( 712 Fifth St., Suite A, D avis, CA 95616 ) on behalf of IAB, S.L.
[ Investigaciones y Apl icaciones Biotechnologicas S.L. ,Avda, Paret del Patriarca 11

- B, Ap. 30, 46113 Moncada (Valencia) Spain ] submitted application s for a new TGAI, Bacillus subtilis

strain IAB/BS03 Technical Powder ( EPA File Symbol: 89615

- R) and for two new EPs

, MildoreTM (EPA File Symbol 89615

- E) l for Mildore MaxTM (EPA File Symbol 89615

- G on May 7, 2013

, and two additional new EPs

, Milagrum PlusTM (EP A File Symbol 89615

- U) and FungiseiTM (EPA File Symbol, 89615

- L)

, on May 21, 2014

, to the EPA under section 3 of

FIFRA.

On October 30 , 201 3 ( 7 8 FR 64937 ) , EPA announced receipt of the application to register a pesticide product containing a new active ingredient for use as a f ungicide in g reenhouse s , field use, and h ome and garden use on various fruits and vegetables, cotton, hops, tobacco, fruit and nut trees, turf, and ornamentals. EPA opened a 3 0

- day public comment period pursuant to the provisions of FIFRA section 3(c)(4). No comme nts were received following thi s publication.

B. Food Tolerance Exemption

Concurrent with its registration applications and under Federal Food, Drug, and Cosmetic Act (FFDCA) section 408(d), Technology Sciences Group on behalf of IAB, S.L

. submitted a petition to establish an exemption from the requirement of a tolerance for residues of the pesticide active ingredient, Bacillus subtilis strain IAB/BS03 [Pesticide Petition (PP) 3 F81 77 ] in

or on all food commodities

. EPA published a Notice co ncerning the filing of this petition in the Federal Register of September 12, 2013 (78 FR 56185) and opened a 30

- day comment period.

Two comments were received in the docket for the petition for Bacillus subtilis strain IAB/BS03

t olerance exemption ( EPA

- HQ

- OPP

- 2013

- 0574) that do not appear to be specific to th is petition. One expressed general opposition to the establishment of a tolerance for pesticides, the other appeared to have been erroneously filed because it relates to a different pesticide

, d iflubenzuron

which is assigned a different docket. For Bacillus subtilis strain IAB/BS03 an exemption from the requirement of a tolerance was requested, not a tolerance and there is no diflubenzuron

contained in the Bacillus subtilis strain IAB/BS03 produ cts. Neither of these comments were specific to Bacillus subtilis strain IAB/BS03

. Both comment s were not found to be relevant to the proposed exemption from the requirement of a tolerance for Bacillus subtilis strain

IAB/BS03

.

IV . RISK ASSESSMENT

In the Federal Register of October 26, 2007 ( 72 FR 61002 )

, the EPA issued a Final Rule on the data requirements to support registration of microbial pesticides and updated the defi nition for microbial pesticides

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

The EPA classifies each data submission with an indication of the usefulness of the information contained in the documents for risk assessment. A rating of “ acceptable ” indicates the study isBacillus subtilis strain IAB/BS03 Page 8 of 36

Biopesticides Registration Action Document

scientifically sound and is useful for risk assessment. A “ supplemental ” rating indicates the data provide some information that can be usefu l 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 indication of what is lacking or

what can b e provided to change the rating to “ acceptable .” If there is simply a “ supplemental ” rating, the reviewer will often state that t he study is not required by 40 CFR P art 158. Both

“ acceptable” and “supplemental ” studies may be used in the risk assessment pr ocess as appropriate. An “unacceptable ” rating indicates that new data must 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 the E PA in support of a pesticide registration. The EPA classifies t he active ingredient or particular product into Toxicity Category I, II, III, or IV, where Toxicity Category I indicates the highest toxicity and Toxicity Category IV

indicates the lowest toxic ity (see 40 CFR § 156.62 )

.

A. Product Analysis Assessment ( 40 CFR § 158.2120 )

For purposes of registration under FIFRA section 3(c)(5), the EPA determined th at all product analysis data requirements for Bacillus subtilis strain IAB/BS03 have been fulfilled. Refer to

Tables 1

- 10

, in Appendix A for a summary of the data requirements, including both generic and product

- specific inform ation.

B. Human Health Assessment ( 40 CFR § 158.2140 )

1. Toxicity

All a ppli ca ble m a mm a li a n to x i c ology d a ta re qui r e m e nts suppo r ting the r e q u e st f or a n e x e mption

fr o m the re qui re m e nt of a tol er a n c e f or r e sidu e s of Bacillus subtilis strain IAB/BS03 i n or on a ll

f ood c ommoditi e s h a ve b e e n f ul f ill e d with d a ta submitt e d b y the applicant or with scientific

rationale. F urthermore, Tier II and Tier III studies were not required for Bacillus subtilis strain

IAB/BS03 because of the lack of acute toxicity/pathogenicity in the Tier I studies. The acute toxicological data submitted for the for Bacillus subtilis strain IAB/BS03 TGAI (EPA File Symbol 89615

- R ) were determined to be adequate to address the data requirements for the four

end

- use products: MildoreTM (EPA File Symbol 89615

- E)

, Mildore MaxTM (EPA File Symbol

89615

- G)

, Milagrum Pl usTM (EPA File Symbol 89615

- U) and FungiseiTM (EPA File Symbol,

89615

- L)

.

The to x i c i t y and pathogenicity t e sts ( acute oral, inhalation and d er m a l to x i c i t y ) a nd i r r it a tion

t e sts (ac ute e ye a nd p r im a r y d er m a l i rr it a tion) th a t a d d re ss pot e nti a l r out e s of e x posu r e to the

ac tive i n g r e di e nt a r e a ll c l a ssi f i e d in To x i c i t y C a t e g o r y I I I or I V ( s e e below or U.S. EPA 2014)

a nd re v ea l e d little to no to x i c i t y a tt r ib u t e d to Bacillus subtilis strain IAB/BS03

. F in a l l y
, the applicant re po r t e d th a t no h y p e r s e nsitivi t y in c id e n t s o cc u rr e d du r i n g the r e s earc h,

d e v e lopm e nt, a nd t e sting of this active ingredient.

The ov e r a ll c on c lusions f r om a ll to x i c olo g i ca l in f

o r m a tion submitt e d b y the applicant ar e b r i ef l y d e s cr ib e d b e low, in sections IV(B)(1)(a) and IV(B)(1)(b) (see pages 8

– 1 1 ), and summarized in

Table 11 in Appendix A

.Bacillus subtilis strain IAB/BS03 Page 9 of 36

Biopesticides Registration Action Document

a. Acute Toxicity/Pathogenicity

– Tier I

Acute Injection Toxicity/Pathogenicity

– Rat ( OCSPP Guideline Number 885.3200 ) (MRID 48969303)

The microbial pest control agent ( MPCA ) test substance or inactivated MPCA test substance was injected into the tail vein of one of two groups of rats at a high dose ( 1.4 x 10 8 CFU/mL or 1.4 x

10 7 CFU/rat ) . A control group was conducted concurrently. The animals were observed frequently on da y of dosing for mortality and signs of pharmacologic and/or toxicological effects and once daily thereafter for 21 days. Tissue and blood samples taken at interim sacrifices from the group receiving the active test substance were cultured to provide quant itative measurements of the test microbe’s clearance pattern. There was no mortality in any group during the study. During observations for clinical signs, all animals appeared normal for the duration of the study. The gross necropsy conducted at termin ation of the study revealed no observable abnormalities, with the exception of one white dot on the liver of a Group III

- MPCA animal. The test organism had cleared completely from the Group III

- MPCA blood by Day 3. Once clearance was established by t wo consecutive interim sacrifices that showed less than 100 CFU/gram tissue of the test organism in the organs plated, no further tissue samples were taken or cultured. The test organism had completely cleared from the kidneys by Day 14 and from the brain by Day 21. There was no test substance seen in the mesenteric lymph nodes or lungs at the Day 21 counts. Test organism was still present in the liver and spleen at Day 21 but was showing a pattern of clearance. Test organism was present in the cecum co ntents by Day 21. Based on the mortality of the animals, the test substance Technical Product Bacillus subtilis strain IAB/BS03 was

determined to be non

- toxic to rats when injected intravenously at a dose of 1.4 x 10 8 CFU/mL or

1.4 x 10 7 CFU/rat.

EPA determined this study to be acceptable

.

Acute Oral Toxicity/Pathogenicity

– Rat ( OCSPP Guideline Number 885.3050 ) and Acute Pulmonary Toxicity/Pathogenicity

– Rat (OCSPP Guideline Number 885.3150) (MRID 48969317) The registrant has submitted the follow ing rationale that existing data for the Acute IV Toxicity / Pathogenicity study on the TGAI be considered to satisfy the requirements for Acute Oral & Pulmonary Tox/Pathogenicity studies: Technical Product Bacillus subtilis strain IAB/BS03, was dosed at a single high dose exposure and followed for an adequate post

- exposure observation period. The test substance was injected into the tail vein of one of two groups of rats at a high dose. A control group (untreated) was include d. The animals were observed frequently on day of dosing for mortality and signs of pharmacologic and/or toxicological effects and once daily thereafter for 21 days. Tissue and blood samples taken at interim sacrifices from the group receiving the active t est substance were cultured to provide quantitative measurements of the test microbe’s clearance pattern. The EPA determined that existing data for the Acute IV Toxicity / Pathogenicity study was adequate and the rationale submitted is rated as acceptable.

Acute Oral Toxicity Study in Rats ( OCSPP Guideline Number 870.1100 ) (MRID 48969304)

The test item Bacillus subtilis IAB/BS03 Technical Powder was administered to a group of 6 female Sprague Dawley rats at the single dose of 2000 mg/kg body weight (mean : 7.8 x 10 9
CFU/animal). No mortality occurred during the study. No clinical signs related to the administration of the test item were observed. The body weight evolution of the animals remained normal throughout the study. The macroscopical examination o f the animals at the endBacillus subtilis strain IAB/BS03 Page 10 of 36

Biopesticides Registration Action Document

of the study did not reveal treatment related change. In conclusion, the LD 50 of the test item

Bacillus subtilis IAB/BS03 Technical Powder is higher than 2000 mg/kg body weight by oral

route in the rat. EPA classified the acute ora l toxicity of Bacillus subtilis IAB/BS03 as EPA Toxicity Category III. EPA determined this study to be acceptable.

Acute Dermal Toxicity Study in Rats (OCSPP Guideline Number 870. 1200 ) (MRID 48969305)

The test item Bacillus subtilis IAB/BS03 Technical Powder was applied onto the intact skin of 10 Sprague Dawley rats (5 males and 5 females) at the single dose of 2000 mg/kg body weight (mean : 8.8 10 9 CFU/animal). No mortality occurred during the study. Neither cutaneous reactions nor systemic clinical signs related to the administration of the test item were observed. A slight brown coloration was noted on the treatment site of all animals on days 1 and 2. The body weight evolution of the animals remained normal throughout the study. The macros copical examination of the animals at the end of the study did not reveal treatment

- related changes. In conclusion, the LD50 of the test item Bacillus subtilis IAB/BS03 Technical Powder is higher than 2000 mg/kg body weight by dermal route in the rat. EPA classified the acute dermal toxicity

of Bacillus subtilis IAB/BS03 as Toxicity Category III and rated this study as acceptable

.

Acute Inhalation Toxicity Study in Rats (OCSPP Guideline Number 870. 1300) (MRID 48969407 ) This study was performed to assess the toxic potential of a substance by single inhalation dose, and estimate the lethal concentration (LC 50 ) in rats at a level of concentration of 4.7 mg/L in air during 4 hours of exposure to Bacillus subtilis IAB/BS03 . The test substance was administered by a system of resuspension of dust which provided the material in a concentration of 4.7 mg/L in air. 10 rats (5 for each sex) of the Sprague

- Dawley strain were exposed to the product within an exposure chamber for a period of 4 hours. The results showed a mortality of 0% and a L C 50 of level upper of 4.7 mg/L in air during 4 hours. The clinical observations were conducted daily, looking for signs of toxicity or mortality in the population over a 14 day period . Body wei ght was recorded weekly during the study. At the end, the surviving animals were submitted to necropsy. The animals exposed by inhalation route to 4.7 mg/L of the product Bacillus subtilis

IAB/BS03 in air during 4 hours showed no symptoms associated with e xposure of the product during the 14 day period of observation. The macroscopic pathology examination of the animals exposed to the test substance showed moderate pulmonary congestion, mild petechia in lung and moderate to severe hepatic congestion. All th e animals showed a weight increase during the period of the study. This increase was 29.5% in male and 8.5% in female. This study estimated

that L C 50 for the product Bacillus subtilis IAB/BS03 in rats is higher than 4.7 mg/L in air during

4 hours. EPA clas sified the acute inhalation toxicity of Bacillus subtilis IAB/BS03 as Toxicity Category IV and rated this study as acceptable

Primary Eye Irritation

– Rabbit ( OCSPP Guideline Number 870.2400 ) (MRID 48969306)

The test item Bacillus subtilis IAB/BS03 Technical Powder was instilled, as supplied, into the eye of 3 New Zealand rabbits at the dose of 0.1 g (2 x 10 9 CFU)/animal. The ocular conjunctivae reactions observed during the study have been slight to moderate and totally reversible . A slight

to moderate redness was noted 1 hour after the test item instillation and was totally reversible between days 2 and 4 . This redness was associated with a slight chemosis noted 1 hour after the

test item instillation in two animals and this slight c hemosis was totally reversible on day 1. EPA classified the primary eye irritation of Bacillus subtilis IAB/BS03 as Toxicity Category IV and rated this study as acceptable

.Bacillus subtilis strain IAB/BS03 Page 11 of 36

Biopesticides Registration Action Document

Primary Dermal Irritation

– Rabbit ( OCSPP Guideline Number 870.2500 ) (MRID 48969307)

The test item Bacillus subtilis IAB/BS03 Technical Powder was applied, as supplied, at the dose of 0.5 g (1010 CFU /animal), under semi

- occlusive dressing during 4 hours on an undamaged skin area of 3 rabbits. A slight erythema was noted on the treated area of one animal, 1 and 24 hours after the patch removal. This reaction was totally reversible on day 2. EPA classified the primary dermal irritation of Bacillus subtilis IAB/BS03 as Toxicity Category IV and rated this study as acceptable.

H y p e rs e nsiti v ity in c id

e nts ( OCSPP Guideline Number 885.3400 ) To date, no incidents of hypersensitivity or any other adverse effects have occurred through the research, development, testing or commercialization of the active ingredient Bacillus subtilis

strain IAB/BS03 Technical Powder and its related end

- use products. Should any incidents occur, they must be reported per FIFRA Section 6(a)(2).

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 for Bacillus subtilis strain IAB/BS03 based on the lack of acute toxicity/pathogenicity in the Tier I data requirements.

c

. Endocrine Disruptors

As required by the Administrator under the Federal Food, Drug, and Cosmetic Act ( FFDCA )

, the EPA has developed the Endocrine Disruptor Screening Program (EDSP) and has begun to implement the screening program that is to be used to test all pesticides 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 endocrine effects as the Administrator may designate.” FFDCA section 408(p)(4) authorizes the Administrator, by order, to exempt from the requirements of the Endocrine Disruptor Screening Program a biologic su bstance or other substance if a determination is made that the substance is anticipated to not produce any effect in humans similar to an effect produced by a naturally occurring estrogenic substance. Between October 2009 and February 2010, EPA issued tes t
orders/data call

- ins for the first group of 67 chemicals, which contains 58 pesticide active ingredients and 9 inert ingredients. Bacillus subtilis strain IAB/BS03 is not among the group of 58 pesticide active ingredients on the initial list to be screen ed under the EDSP.

The EPA believes that for Bacillus subtilis strain IAB/BS03 is a substance that would not produce any effect in humans similar to an effect produced by a naturally occurring estrogenic substance. As such, and pursuant to Section 408(p)(4), the EPA will determine in the future whether it can exempt for Bacillus subt ilis strain IAB/ BS03 from t he requirements of the Section 408(p)(4) EDSP. In the event the EPA does determine to exempt this substance from the EDSP,

an order will be issued. For further information on the status of the EDSP, the policies and procedures, t he list of 67 chemicals, the test guidelines and the Tier 1 screening battery, please

visit our website: http://www.epa.gov/endo/

.Bacillus subtilis strain IAB/BS03 Page 12 of 36

Biopesticides Registration Action Document

2. Federal Food, Drug, and Cosmetic Act (FFDCA) Considerations

Section 408(c)(2)(A)(i) of FFDCA allows the EPA to establish an exemption from the requirement for a tolerance (the legal limit for a pesticide chemical residue in or on a food) only

if the EPA determines that the exemption is “safe.” Section 408(c)(2)(A)( ii) of FFDCA defines “safe” to mean that “there is a reasonable certainty that no harm will result from aggregate exposure to the pesticide chemical residue, including all anticipated dietary exposures and all other exposures for which there is reliable in formation.” This includes exposure through drinking water and in residential settings

, but it 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, the EPA must take into account the factors set forth in section 408(b)(2)(C) of FFDCA, which require the EPA to give special consideration to exposure of infants and children to the pesticide chemical residue in establishing a tolerance exempti on
, 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 the EPA consider “available in formation concerning the cumulative effects of [a particular pesticide's] . . . residues and other substances that have a common mechanism of toxicity.”

The EPA performs a number of analyses to determine the risks from aggregate exposure to pesticide resi dues. First, the Agency determines the toxicity of a pesticide . Second, the 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 section 408(b)(2)(D) of FFDCA, the EPA has reviewed the available scientific data and other relevant information, and considered its validity, completeness, and reliability and the relationship of this information

to human risk. The Agency 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 previously and

pres ented in Table 11 in Appendix A , t he data required for a FFDCA risk assessment for

Bacillus subtilis strain IAB/BS03 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 wate r and exposure through pesticide use in gardens, lawns, or buildings (residential and other indoor uses).

Food Exposure and Risk Characterization : The EPA found that increased d ietary exposure to

Bacillus subtilis strain IAB/BS03

, a naturally occurring bacterium

, is anticipated to be negligible

. Bacillus subtilis strain IAB/BS03 and other closely related Bacillus subtilis bacteria already exist

in the soil environment

. The EPA concluded that t he risk posed to adults, infants, and children is likely to be minimal because of the low acute toxicity/ p athogenicity potential of Bacillus subtilis

strain IAB/BS03

.

Drinking Water Exposure and Risk Characterization : Bacillus subtilis is naturally present in soils ( Logan et. al., 2009) ; therefore, Bacillus subtilis may occur in surface water and possibly groundwater. Bacillus species are often detected in drinking water even after going throughBacillus subtilis strain IAB/BS03 Page 13 of 36

Biopesticides Registration Action Document

acceptable water treatment processes, largely because the spores are resistant to these

disinfection processes ( Wor ld Health Organization, 2011) Should this microbial pesticide be present, no adverse effects are expected from exposure to Bacillus subtilis strain IAB/BS03

, through drinking water, based on the results of toxicity studies described in in sections IV(B)(1) (a), and summarized in Table 11 in Appendix A

.

Non

- occupational, Residential Risk Characterization : Because Bacillus subtilis strain IAB/BS03

use sites do include residential settings and the bacterium is naturally

- occurring

, some non

occupational exposure to the bacterium is likely

. Repeated exposure s to the Bacillus subtilis

strain IAB/BS03 microorganism from pesticidal applications do not exceed EPA’s level of concern, particularly in light of available data that demonstra te Bacillus subtilis strain IAB/BS03

is not toxic (acute injection toxicity /pathogenicity and acute oral, dermal, and inhalation toxicity ), is non

- irritating (primary dermal irritation , primary eye irritation ), and is not pathogenic when used as labeled in accordance with good agricultural practices ( acute injection toxicity/pathogenicity ) (see section IV(B)(1)(a) on pages 8

– 1 1 and Table 11 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 exemption, the EPA consider “available information concerning the cumulative effects of [a particular pesticide’s] . . . residues and other substances that have a common mechanism of toxicity.” The EPA has not found Bacillus subtilis strain IAB/BS03 to share a common mechanism of toxicity with other substances. Bacillus subtilis strain IAB/BS03 does not appear to be toxic to humans via dietary, dermal and pulmonary exposure. For the purposes of the tolerance action, therefore, the EPA has assumed that Bacillus subtilis strain

IAB/BS03 does not have a common mechanism of toxicity with other substances. Thus, section 408(b)(2)(D)(v) o f the FFDCA does not apply. For information regarding the EPA’s efforts to determine which chemicals have a common mechanism of toxicity and to evaluate the cumulative effects of such chemicals, see the EPA’s website at http://www.epa.gov/pesticides/cumulative

.

c. Determination of Safety for the United States Population, Infants and Children

In considering the establishment of a tolerance or tolerance exemption for a pesticide chemical residue, FFDCA section 408 (b) (2) (C) provides that the 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 i nfants and children of the residues and other substances with a common mechanism of toxicity. In addition,

FFDCA section 408 (b) (2) (C) provides that the EPA shall apply an additional tenfold (10X) margin of exposure ( safety ) for infants and children in t he case of threshold effects to account for prenatal and postnatal toxicity and the completeness of the database on toxicity and exposure

unless the EPA determines that a different margin of exposure ( safety ) will be safe for infants

and children. This add itional margin of exposure ( safety ) is commonly referred to as the Food Quality Protection Act Safety Factor. In applying this provision, the EPA either retains the default value of 10X or uses a different additional safety factor when reliable data availa ble to

the EPA support the choice of a different factor.

Based on the acute toxicity an d pathogenicity data/information discussed in section IV(B)(1)(a) (see pages 8

– 1 1 ) and Table 11 in Appendix A

, t he EPA concludes that there are no thresholdBacillus subtilis strain IAB/BS03 Page 14 of 36

Biopesticides Registration Action Document

effects of concern to infants, children, or adults when Bacillus subtilis strain IAB/BS03 is used as labeled in accordance with good agricultural practices. As a result, the EPA concludes that no additional margin of exposure (safety) is necessary to protect infants and children and that not adding any additional margin of exposure (safety) will be safe for infants and children.

Moreover, based on the same data /information and EPA analysis as presented directly above, the Agency is able to conclude that there is a reasonable certainty that no harm will result to the

United States population, including infants and children, from aggregate exposure to the residues

of Bacillus subtilis strain IAB/BS03 when it is used

— as labeled and in accordance with good agricultural practices

— as a n herbicide . Such exposure includes all anticipated dietary exposures and all other exposures for which there is reliable information. The EPA has a rrived at this conclusion because, considered collectively, the data and information available on Bacillus

subtilis strain IAB/BS03 do not demons trate toxic or pathogenic potential to mammals, including

infants and children.

3. Occupational Exposure and Risk Characterization

The EPA does not expect h andler exposure to Bacillus subtilis strain IAB/BS03 to pose any
undue risk. Based on the acute toxicity category III noted in the acute oral and acute dermal toxicity studies, and because r epeated exposures to high concentrations of microbial proteins can cause allergic reactions appropriate personal protective equipment (PPE) and precautionary statements are required on pesticide product labels to mitigate any potential risks to pesticide

h andlers due to prolonged or numerous exposures. Handlers applying end use products containing Bacillus subtilis strain IAB/BS03 in agricultural settings must wear a dust/mist filtering respirator, waterproof gloves, long

- sleeved shirt, long pants, socks and shoes.

4. Human Health Risk Characterization

The EPA considered human exposure to Bacillus subtilis strain IAB/BS03 in light of the standard for registration in FIFRA and the relevant safety factors in FFDCA. The Agency

determin ed that no unrea sonable adverse effects to the United States population in general, and to infants and children in particular, will result when products containing Bacillus subtilis strain

IAB/BS03 are used in accordance with EPA

- approved labeling.

C. Environmental Asse ssment ( 40 CFR § 158.2150 )

Ten original guideline studies conducted with the TGAI and three sets of scientific rationale or scientific literature were submitted to meet data requirements for nontarget organisms per 40

CFR Part 158.2150 for Bacillus subtilis strain IAB/BS03

. The information provided is sufficient to satisfy most of the Tier I nontarget organism data requirements and for nontarget organism

risk assessment for this new active in gredient and its proposed uses. The honeybee and nontarget insect test data requirements are incomplete. Since some risks might be expected to certain populations of nontarget insects and honeybees as a consequence of frequent applications of products containing Bacillus subtilis strain IAB/BS03 , precautionary label language is

re quired to protect these organisms from exposures to products containing Bacillus subtilis strain

IAB/BS03 . It should be noted that adverse population effects cannot be characterized because of the uncertainty of the environmental persistence of Bacillus subtilis strain IAB/BS03 . The precautionary labeling noted above, however, will mitigate potential adverse effects; therefore higher

- tier studies that would enable a more complete characterization of potential ris ks are notBacillus subtilis strain IAB/BS03 Page 15 of 36

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required.

For a summary of the generic data requirements described in sections IV(C)(1) (see pages 14

– 1 8 ), refer to Table 12 in Appendix A

.

1. Ecological Exposure and Risk Characterization

Based on the proposed use p attern, which includes uses on many food and non

- food crops

, the proposed applications are expected to result in direct contact of the a.i. with foliar surfaces and soil, and because spray drift is expected to occur, exposure to terrestrial and aquatic nontarget organisms is possible. Based on current labeled information concerning the amount of active ingredient 1 in Mildore MaxTM (10% a.i.)

, t he EP with the highest concentration of a.i.

, the Agency estimates terrestrial maximum environmental concentratio ns (EC) of 181.2 g a.i./A. An aquatic maximum EC was estimated at 0.3 mg a.i./L of water (or, ≥ 3 x 10 7 cfu/L for Mildore MaxTM). Because applications are proposed for every 7 to 1 0 days for many of the labeled crops, constant exposures of the biological c ontrol agent to nontarget organisms are anticipated throughout the season. Presently there are no data or information regarding the environmental stability and persistence of Bacillus subtilis strain IAB/BS03

.

2. Terrestrial Animals and Plants

Birds and Mammals (OCSPP Guideline Numbers 885.4050, 885.4100

, 885.4150

) ( MRIDS 48969308, 48969317 and 48969303)

Birds and mammals may be exposed to Bacillus subtilis strain IAB/BS03 primarily through ingestion or contact with treated surfaces. Information present ed to BPPD indicates low toxicity to birds and minimal toxicity to wild mammals as a result of the proposed applications. Tests required by 40 CFR § 158.2140 are adequate and appropriate for assessment of hazards to wild mammals. The TGAI, Bacillus subtili s strain IAB/BS03 was determined to be non

- toxic to rats when injected intravenously at a dose of 1.4 x 10 8 CFU/mL (or 1.4 x 10 7 CFU/rat)

.

The avian oral study was of insufficient duration and did not have appropriate controls to test for infectivity/pathogenicity hazards. Current microbial pesticide guideline s for testing hazard potential to avian species recommend study durations of at least a 25

- day observation period after dosing for 5 days, and testing of a sterile filtrate (cell

- free) control IAB/BS03 . Although the Agency is unaware of reports of infectivity or pathogenesis by B acillus subtil i s to avian species, the lack of information regarding maximum temperatures for growth and/or survival of Bacillus

subtilis strain IAB/BS03 and th e lack of testing does not preclude potential adverse effects as a

result of untested infectivity

. However , available data indicate the a.i. did not produce treatment

related effects in birds at the tested dose level (14 day LD 50 > 5000 mg/a.i./kg)

. In add ition,

b ecause available avian batch data are related to the test substance used in the fish and aquatic invertebrate studies, it is likely

, that the test substance used in the avian study was also sufficiently stable and viable . Existing data were sufficient to conclude that m inimal adverse effects are anticipated for most nontarget avian or wild mammal species exposed to Bacillus

subtilis strain IAB/BS03 as a result of the proposed labeled applications

.

1 Terrestrial maximum EC calculation : EEC based on Aerial applications : 4 lbs product/A * 454 g/lb * 10% a.i. per A ≈ 181.2g a.i/A per application of EP Mildore MaxTM

. ; aquatic maximum EC: 4 lbs/A * 454,000 m g/lb *
1A/40,468,564 cm 2 = 0.045 mg/cm 2  15 cm = 0.003 mg/mL * 1000 ml/L =3.0 mg/L *10.0 % = 0.3 mg a.i./L.Bacillus subtilis strain IAB/BS03 Page 16 of 36

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Nontarget Insects (OCSPP Guideline Number 88 5.4340 ) (MRIDS 48969312, 48969313

and 48969314 )

Nontarget insect testing conducted on the parasitic wasp and the predatory mite used a

formulated end

- use product. Notably, these studies may have been of insufficient duration, or the experimental design was of insufficient range to observe a significant median LC 50

. Because the testing laboratories did not confirm the test substance in the exposure solutions, it is not possible to determine whether these exposures were conducted at maximum hazard

concent rations. The testing conducted on the ladybird beetle was inconclusive, since nearly equivalent mortalities were reported in the untreated control and treatment groups. The nontarget insect test data requirement is incomplete and there is uncertainty in t he toxicity of Bacillus

subtilis strain IAB/BS03 to nontarget insects. There were no observations or tests for potential infectivity to these test insects. As a result of the continuous and frequent exposures (i.e., application intervals of every 7 to 10 d ays) of wild populations of nontarget insects, adverse effects to certain species could be reasonably expected. There are reports of Bacillus subtil i s strains that are apparently pathogenic to mosquitos (U.S. EPA, 1997).

Nontarget insect populations will be exposed to frequent applications of Bacillus subtilis strain

IAB/BS03 ; however, the Agency cannot reasonably determine if the risk(s) to populations of insects are low with any certainty. Submitted studies suggested a potential for delayed toxicity or pathogenicity; however

, additional research is necessary to obtain statistically valid biological endpoints (e.g., median LC 50 values). Precautionary language is required on end

- use products regarding risk to non

- target insects (as specified in section VII B.) because of the expected continual exposures to insects as a result of frequent applications

.

Honeybees (OCSPP Guideline Number 885.4380 ) (MRID 48969315 and 48969316)

The honey bee (

Apis mellifera ) study classified Supplemental (MRID 48969315) provided LD 50 values from contact and oral exposures. T he test material did not produce statistically significant mortality compared to the untreated control through the oral route of exposure. However, bee mortality significantly increased in the 10 μg a. i./bee group on day 4 and in both the 1 μg a.i./bee and 10 μg a.i./bee groups on day 5. Because a biologically significant dose was not tested to characterize the potential hazard(s) of products containing Bacillus subtilis strain IAB/BS03

, there is uncert ainty if the highest dose tested (i.e., estimated average of 10 μg a.i./bee) in the contact exposure part of the test is representative of maximum hazard doses or representative of some multiple of expected environmental concentrations of Bacillus subtilis strain IAB/BS03

.

Because the end

- use products are presently labeled for applications frequently throughout the season (i.e., every 7 to 10 days for some uses), there are likely to be exposures to bees. Honeybee populations will be exposed to frequent app lications of Bacillus subtilis strain IAB/BS03

, however, the Agency cannot reasonably determine if the risk(s) to honeybees are low with any certainty. Submitted studies suggested a potential for delayed toxicity; however additional research is necessary t o obtain statistically valid biological endpoints (e.g., median LC 50 values). Because there is uncertainty in the toxicity and environmental persistence of Bacillus subtilis

strain IAB/BS03 , potential adverse effects could be reasonably expected as a resul t of frequent applications of one week intervals.Bacillus subtilis strain IAB/BS03 Page 17 of 36

Biopesticides Registration Action Document

Precautionary language is required on end

- use products to protect bees and non

- target insects because of the expected continual exposures as a result of frequent applications (as specified in section VII B. below)

.

Nontarget Plants (OCSPP Guideline Number 885.4300 ) (MRID 48969317)

Adverse effects are not anticipated to populations of nontarget plants as a result of commercial uses of products containing Bacillus subtilis s train IAB/BS03. Existing data from the open literature was considered sufficient to satisfy the conditional requirement for Nontarget Plant Studies, Tier I. Bacillus subtilis effects on crop plants have been reported as beneficial and can act as biological control agents for some plant

- pathogenic fungi. Furthermore, there are no reports of phytopathogenicity by Bacillus subtil i s in the open literature, and the registrant claims there were no symptoms of phytotoxicity during research and development of Bacil lus subtilis s train

IAB/BS03.

3. Aquatic Animals and Plants

Freshwater Fish and Invertebrates (OCSPP Guideline Numbers 885.4200 and

885.4240 ) ( MRIDs 48969309, 48969311 and 48969310)

Adverse effects from short term exposures to Bacillus subtilis strain IAB/BS03 through spray drift or soil mobility are not anticipated to populations of freshwater fish or aquatic invertebrates.

Bacillus subtilis IAB/BS03 is determined practically non

- toxic to freshwater fish and invertebrates, for short term exposures . Populations of the MPCA could be reasonably expected to increase slightly soon after application to terrestrial crops, and subsequently reduce to natural levels of total bacilli because of nutrient cycling and decomposition. The aquatic EEC was

estimated as ~ 0.3 mg a.i./L, or ~ 3 x 10 7 cfu/L for Mildore MaxTM

. Since there are no labeled sites with direct applications to aquatic systems, the Agency does not anticipate adverse effects

to n ontarget freshwater fish or aquatic invertebrates over the long

- term.

Marine/Estuarine Fish and Invertebrates (OCSPP Guideline Number 885.4280) (MRID 48969317)

A scientific rationale and published literature were submitted in lieu of hazard testing on

marine/estuarine fish and invertebrates. Adverse effects from short term exposures to Bacillus

subtilis strain IAB/BS03 through spray drift or soil mobility are not anticipated to populations of marine/estuarine fish and invertebrates. Populations of the MPCA could be reasonably expected

to incre ase slightly soon after application to terrestrial crops, and subsequently reduce to natural levels of total bacilli because of nutrient cycling and decomposition. Since t here are no labeled sites with direct applications to marine/estuarine systems, the A gency does not anticipate adverse effects to nontarget fish or aquatic invertebrates over the long

- term.

Aquatic Plants (OCSPP Guideline Number 885.4300 ) (MRID 48969317)

Adverse effects are not anticipated to populations of aquatic plants as a result of commercial uses of products containing Bacillus subtilis s train IAB/BS03. As discussed above, Bacillus subtilis

strain IAB/BS03 is not related to known plant pathogens; and there are no labeled sites with direct applications to aquatic systems.Bacillus subtilis strain IAB/BS03 Page 18 of 36

Biopesticides Registration Action Document

4. Environmental Persistence and Fate

Bacillus subtilis is a common saprophytic inhabitant of soils and is thought to contribute to nutrient cycling due to the variety of protease s and other enzymes members of the species are capable of producing. Under adverse environmental conditions, Bacillus subtilis produces endospores that are resistant to heat and desiccation (U.S. EPA, 1997). Specific data comparing the survivability of industrial and wildtype strains of Bacillus subtilis were not available in the existing literature. However, the ability of Bacil lus subtilis to produce highly resistant spores and grow under a wide range of conditions indicates that released strains are likely to survive outside

of containment. Since the natural habitat for Bacillus subtilis is soil, long

- term survival in soil is

r easonably expected to occur.

There were no data or information regarding quantitative estimates of viable Bacillus subtilis

strain IAB/BS03 populations after application in the submission. It is widely accepted however

that Bacillus spp. occur at populati on levels of 10 6 to 10 7 per gram of soil (Alexander, 1977), residing in the endospore stage. It is thought that 60 to 100% of soil bacilli populations exist in the inactive spore state (Alexander, 1977). A wide variety of antibiotic compounds is produced b y Bacillus subtilis strains in culture media, however the importance of antibiotic production in the environment is unknown (Alexander, 1977).

Frequent applications of products containing Bacillus subtilis strain IAB/BS03 may increase total bacilli soon after application; total bacilli populations will likely subsequently decrease. It should be noted that the level of persistence of pesticide products containing Bacillus subtilis is presently

uncertain at this time.

5. Thr eatened and Endangered Species Assessment

EPA has determined that no adverse effects are anticipated for most nontarget species exposed to

Bacillus subtilis strain IAB/BS03 as a result of the proposed labeled applications, including mammals, birds, terres trial and aquatic plants, and freshwater and marine/estuarine animals. Therefore, a “No Effect” determination is made for direct and indirect effects to federally listed endangered and threatened (“listed”) mammals, birds, terrestrial and aquatic plants, a nd freshwater and marine/estuarine animals and for their designated critical habitat.

Although sustained population

- level effects to honeybees and nontarget insects are not anticipated, results of the supplemental honeybee and nontarget insect testing suggest uncertainties in the toxicity and environmental persistence of the MPCA. Direct ef fects to individuals of listed insect species cannot be precluded, nor can indirect effects to listed species that depend specifically on a listed insect.

Additional analysis of the potential effects to these nontarget organisms is necessary to make a

de termination for these species.

V. ENVIRONMENTAL JUSTICE

The EPA seeks to achieve environmental justice

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

— with respect to the development, i mplementation, and enforcement of environmental laws, regulations, and policies.Bacillus subtilis strain IAB/BS03 Page 19 of 36

Biopesticides Registration Action Document

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

be consider ed in the decision

- making process; and (4) the decision

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

To help address potential environmental justice issues, during the 15

- day public participation comment period, the EPA is seeking 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 expos ure to Bacillus subtilis strain IAB/BS03

, 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 the EPA’s website

at http://www.epa.gov/compliance/environmentaljustice/index.html

.

V I

. RISK MANAGEMENT DECISION

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

(1) Its composition is such as to warrant the claims for it; (2) Its labeling and other material required to be submitted comply with the requirements

o f FIFRA; (3) It will perform its intended function without unreasonable adverse effects on the environment; AND (4) When used in accordance with widespread and commonly recognized practice, it will not generally cause unreasonable adv erse effects on the environment.

To satisfy criterion 1, the Bacill us subtilis strain IAB/BS03 product s, the TGAI and EP s
, ha ve well

- known properties. The data and other information submitted to and reviewed by EPA

support the claims made on the label s

. Criterion 2 is satisfied in that the proposed labels, data, and other materials comply with FIFRA requirements. Criterion 3 is satisfied based on the data

submitted and presented in this document which indicate that products will perform as intended

without unreasona ble adverse effects

. With the addition of precautionary label statements to
protect bees and other nontarget and pollinating insects

, products are not expected to cause unreasonable adverse effects on the environment when used according to label instructio ns. Criterion 4 is satisfied as the Bacillus subtilis strain IAB/BS03 product s are not expected to cause unreasonable adverse effects when used according to label instructions and commonly recognized practices for applying these types of products. Therefore, the MP

, Bacillus subtilis

strain IAB/BS03 Technical Powder), and the four end use products: MildoreTM, Mildore MaxTM, Milagrum PlusTM, and Fungise i TM which contain Bacillus subtilis strain IAB/BS03 as a new active ingredient, are eligible for regi stration under FIFRA section 3(c)(5) for the labeled uses.Bacillus subtilis strain IAB/BS03 Page 20 of 36

Biopesticides Registration Action Document

VII . ACTIONS REQUIRED OF THE REGISTRANT

A. Final Printed Labeling

Before the manufacturing

- use product, Bacillus subtilis strain IAB/BS03 Technical Powder, and

the four end use p roducts: MildoreTM , Mildore MaxTM, Milagrum PlusTM, and Fungisei TM , may be released for shipment, the applicant is required to provide appropriate final printed labeling to

the EPA

.

B. Terms of Registration

In addition to the final printed labeling mentioned above, t he terms of registration for Bacillus

subtilis strain IAB/BS03 products include product specific data, and required label statements as listed below

.

Required EP Label Statements : Because of the frequency of application and the possibility of delayed toxicity to honeybees, all

end

- use products containing Bacillus subtilis strain IAB/BS03 must include the following

statement in the Environmental H azard section of each label: “This product may harm beneficial

insects and h oney bees. Do not apply this product when bees or other pollinating insects are actively foraging.” In addition, the Directions for Use section that provides application instructions must prominently include the statement : “ Do not apply this product when bees or other pollinating insects are actively foraging .”

EP Data Requirements

The following data requirements will be terms of registration for the Mildore

- M ax TM , M ilagr u m
P lus TM , and Fungisei TM End

- use Products ( EPA File Symbols 89615

- G,

- U, and

- L ):

Storage Stability and Corrosion Characteristics (Harmonized Guideline s 830.6317 and

830.6320 ) : Within one year of registration, IAB, S.L., must provide the EPA with the results of a

one

- year storage stability and corrosion characteristics stud ies on Mildore

- Max TM , Milagr u m Plus TM , and Fungisei TM

.

Analysis of Samples (Harmonized Guideline 885.1400)

A ddition al analysis of sample data for Mildore

- Max TM ( EPA File Symbol 89615

- G) are required

to be submitted when this product is manufactured, within one year of registration.

C. Reporting of Adverse Effects and Hypersensitivity Incidents

Notwithstanding the information stated in the previous sections, it should be clearly understood

that certain specific data must be reported to the EPA as a requirement for maintaining the federal registration for a pesticide product. Reports of all incidents of adverse effects to the

environment must be submitted to the EPA under the provisions stated in FIFRA section 6(a)(2). Additionally, all incidents of hypersensitivity (including both suspected and confirmed incidents) must be reported to the EPA under the provisions of 40 CFR § 158.2140(d).Bacillus subtilis strain IAB/BS03 Page 21 of 36

Biopesticides Registration Action Document

VIII. GLOSSARY OF ACRONYMS AND ABBREVIATIONS

BRAD Biopesticides Registration Action Document

CFR Code of Federal Regulations

CFU colony forming units

cSt centistokes

EDSP Endocrine Disruptor Screening Program

EP end

- use pesticide product

EPA United States Environmental Protection Agency (the “Agency”)

FFDCA Federal Food, Drug, and Cosmetic Act

FIFRA Federal Insecticide, Fungicide, and Rodenticide Act

FR Federal Register g/mL grams per milliliter lb/gal pounds per gallon

LD 50 median lethal dose. A statistically derived single dose that can be expected

to cause death in 50% of the test animals when administer ed by the route indicated (oral, dermal, or inhalation). It is expressed as a weight of substance per unit weight of animal (e.g., mg/kg). mg/kg milligrams per kilogram

MP manufacturing

- use pesticide product

MPCA microbial pest control agent

MRID No. Master Record Identification Number

NIOSH National Institute for Occupational Safety and Health

OPP Office of Pesticide Programs

PC Code Pesticide Chemical Code

PP Pesticide Petition

PPE personal protective equipment

TGAI technical grade of the active ingredient

U.S. United StatesBacillus subtilis strain IAB/BS03 Page 22 of 36

Biopesticides Registration Action Document

I X. BIBLIOGRAPHY

A. Studies Submitted to Support the Active Ingredient, Bacillus subtilis strain IAB/BS03

and Pesticide Product Registration s for the (TGAI) product, Bacillus subtilis strain

IAB/BS03 Technical Powder, and four new end

- use products (EPs): MildoreTM; Mildore MaxTM; Milagrum PlusTM and Fungisei TM

MRID No. Study Information

48969301 Roberts, A. (2013) Product Chemistry for Bacillus subtilis Strain IAB/BS03

Technical Powder. Unpublished study prepared by Technology Sciences Group, Inc. 65p.

48969302 Coranelli, S. (2012) Determination of Contaminants and Pathogens on Two Batches

of Bacillus subtilis Strain IAB/BS03 Technical Product, One Batch of Bacillus

subtilis IAB/BS03 EP Formulation and One Batch of Inert Ingredient. Project Number: BT187/12/OCR. Unpublished study prepared by BioTechnologie Bt Srl. 60p.

48969303 Doig, A. (2013) Technical Product Bacillus subtilis Strain IAB/BS03 Acute

Intravenous Toxicity / Pathogenicity Study in Rats. Project Number: 16801/12/OCR. Unpublished study prepared by Stillmeadow, Inc. 32p.

48969304 Richeux, F. (2011) Bacillus subtilis IAB/BS03 Technical Powder Evaluation of

Acute Oral Toxicity in Rats. Project Number: TAO423/PH/11/0245/OCR. Unpublished study prepared by Phycher Bio Developpement. 23p.

48969305 Richeux, F. (2011) Bacillus subtilis IAB/BS03 Technical Powder Evaluation of

Acute Dermal Toxicity in Rats. Project Number: TAD/PH/11/0245/OCR. Unpublished study prepared by Phycher Bio Developpement. 23p.

48969306 Richeux, F. (2011) Bacillus subtilis IAB/BS03 Technical Powder Assessment of

Acute Ey e Irritation (in Rabbits). Project Number: IO/OCDE/PH/11/0245/OCR. Unpublished study prepared by Phycher Bio Developpement. 21p.

48969307 Richeux, F. (2011) Bacillus subtilis IAB/BS03 Technical Powder Assessment of

Acute Dermal Irritation (in Rabbits). Pr oject Number: IC/OCDE/PH/11/0245/OCR. Unpublished study prepared by Phycher Bio Developpement. 18p.

48969308 Halappa, R. (2011) Acute Oral Toxicity (LD50) Study of Bacillus subtilis, Strain

IAB/BS03 Technical Product in Japanese Quail: Final Report. Proje ct Number: 516/3/08/3132/OCR. Unpublished study prepared by Jai Research Foundation. 37p.

48969309 Halappa, R. (2012) Acute Toxicity Study of Bacillus subtilis, Strain IAB/BS03

Technical Product in Common Carp, Cyprinus carpio: Final Report. Project

Numbe r: 504/3/07/4152/OCR. Unpublished study prepared by Jai Research Foundation. 37p.

48969310 Desai, Y. (2012) Acute Immobilisation Study of Bacillus subtilis, Strain IAB/BS03

Technical Product in Daphnia magna: Final Report. Project Number:

502/3/07/3130/OC R. Unpublished study prepared by Jai Research Foundation. 39p.Bacillus subtilis strain IAB/BS03 Page 23 of 36

Biopesticides Registration Action Document

48969311 Desai, Y. (2012) Reproductive Output of Daphnia magna Exposed to Different

Concentrations of Bacillus subtilis, Strain IAB/BS03 Technical Product over a Period of 21 Days: Final Repor t. Project Number: 509/3/07/3133/OCR. Unpublished study prepared by Jai Research Foundation. 75p.

48969312 Aucejo, S. (2012) Extended Laboratory Study to Determine the Side Effects of the

Test Item Bacillus subtilis Strain IAB/BS03 WP on the Non

- target Arthropod Typhlodrmus pyri (Acari: Phytoseiidae) in Spain 2012. Project Number: 106SRES11C7/OCR. Unpublished study prepared by Recerca Agricola/ SynTech Research Spain. 32p.

48969313 Aucejo, S. (2013) Extended Laboratory Study to Det ermine the Side Effects of the

Test Item Bacillus subtilis Cepa IAB/BS03 WP on the Non

- target Arthropod Aphidius rhopalosiphi (Hymenoptera: Braconidae) in Spain 2012. Project Number: 106SRES11C6/OCR. Unpublished study prepared by Recerca Agricola/ SynTech Research Spain. 32p.

48969314 Younger, C. (2012) WP Formulation Bacillus subtilis Strain IAB/BS03 Ladybird

Beetle Non

- target Insect Microbial Testing. Project Number: 16458/12/OCR. Unpublished study prepared by Stillmeadow, Inc. 12p.

48969315 Aucejo, S. (2012) Study to Test the Side Effects of the Test Item Bacillus subtilis

Cepa IAB/BS03 WP on Aphis mellifera L.: Acute Oral and Contact Toxicity, in Spain 2012. Project Number: 106SRES11C5/OCR. Unpublished study prepared by Recerca Agricola/ SynTech Resear ch Spain. 40p.

48969316 Younger, C. (2013) Technical Product Bacillus subtilis Strain IAB/BS03 Honey

Bee, Apis mellifera, Non

- target Insect Microbial Testing. Project Number: 16460/12/OCR. Unpublished study prepared by Stillmeadow, Inc. 15p.

48969317 Mil eson, B. (2013) Response to Tier 1 Microbial Pesticide Data Requirements for

Bacillus subtilis Strain IAB/BS03 Technical Powder. Unpublished study prepared by Technology Sciences Group, Inc. 69p.

48969401 Roberts, A. (2013) Product Chemistry for Mildore. Unpublished study prepared by

Technology Science Group, Inc. 32p.

48969402 Aversa, S. (2011) Physical

- Chemical Properties of Product Bacillus subtilis

IAB/BS03 WP Formulation Before and After Accelerated Storage at 40+

- 2 (Degrees Celsius) for 8 Weeks. Project Number: BT079/11. Unpublished study prepare by BioTechnologie Bt Srl. 42p.

48969403 Aversa, S. (2012) Interim Report of Study BT080/11

- 12 Months Check Point.

Project Number: BT080/11. Unpublished study prepared by BioTechnologie Bt Srl. 23p.

48969404 Aversa, S. (2012) Physical

- Chemical Properties: Bulk Density and Persistent

Foaming of Product Bacillus subtilis IAB/BS03 WP Formulation. Project Number:

B T078/11. Unpublished study prepared by BioTechnologie Bt Srl. 32p.

48969405 Richeux, F. (2011) Bacillus subtilis IAB/BS03 Formulation (WP) Evaluation of

Acute Oral Toxicity in Rats. Project Number: TAO423/PH/11/0246. Unpublished study prepared by Phycher Bio Developpement. 23p.Bacillus subtilis strain IAB/BS03 Page 24 of 36

Biopesticides Registration Action Document

48969406 Richeux, F. (2011) Bacillus subtilis IAB/BS03 Formulation (WP) Evaluation of

Acute Dermal Toxicity in Rats. Project Number: TAD/PH/11/0246. Unpublished study prepared by Phycher Bio Developpement. 23p.

48969407 Lemus, I. (2012) (Bacillus subtilis IABBs03) Acute Inhalation Toxicity

- Code

-

1358

- TI (in Rats). Project Number: 03564/12. Unpublished study prepared by Salimax, Ltda. 28p.

48969408 Richeux, F. (2011) Bacillus subtilis IAB/BS03 Formulation (WP) Assessment of

Acut e Eye Irritation (in Rabbits). Project Number: IO/OCDE/PH/11/0246. Unpublished study prepared by Phycher Bio Developpement. 21p.

48969409 Richeux, F. (2011) Bacillus subtilis IAB/BS03 Formulation (WP) Assessment of

Acute Dermal Irritation (in Rabbits). Project Number: ICO/OCDE/PH/11/0246. Unpublished study prepared by Phycher Bio Developpement. 18p.

49122601 Plato Roberts, A. (2013) Product Chemistry for Mildore Max. Unpublished study

prepared by Technology Sciences Group, Inc. 25p.

49122602 Mileson, B. (2013) Response to Tier 1 Microbial Pesticide Data Requirements for

Mildore Max. Unpublished study prepared by Technology Sciences Group, Inc. 23p.

49130501 Roberts, A. (2013) Product Chemistry for Milagrum Plus. Unpublished study

prepared by Technology Science Group Inc. 22p.

49130502 Patton, L. (2013) Response to Tier 1 Microbial Pesticide Data Requirements for

Milagrum Plus. Unpublished study prepared by Technology Sciences Group, Inc. 110p.

49130601 Roberts, A. (2013) Product Chemistry for Fungisei. Unpublished study prepared by

Technology Science Group, Inc. 21p.

49130602 Patton, L. (2013) Response to Tier 1 Microbila Pesticide Data Requirements for

Fungisei. Unpublished study p repared by Technology Sciences Group, Inc. 76p.

49275601 del Val, R. (2013) Determination of Microbial Contamination in Milagrum Plus.

Project Number: IAB001/13. Unpublished study prepared by IAB, S.L(Investigaciones y Aplicaciones Biotecnologicas S.L.). 19p.

49275701 del Val, R. (2013) Determination of Microbial Contamination in Fungisei. Project

Number: IAB002/13. Unpublished study prepared by IAB, S.L(Investigaciones y Aplicaciones Biotecnologicas S.L.). 19p.

49275801 Roberts, A. (2013) Supplemental R esponse to Tier 1 Microbial Pesticide Data

Requirements for Bacillus subtilis strain IAB/BS03 Technical Powder. Unpublished study prepared by IAB,S.L. 22p.

B. Environmental Protection Agency Risk Assessment Memoranda

US Environmental Protection Agency ( U.S. EPA ) , 1997. Bacillus subtilis Final Risk Assessment, Biotechnology Program under the Toxic Substances Control Act (TSCA) Dated February, 1997

. http://www.epa.gov/biotech_rule/pubs/fra/fra009.htm lBacillus subtilis strain IAB/BS03 Page 25 of 36

Biopesticides Registration Action Document

U.S. EPA

. 2010. Bacillus subtilis Final Registration Review Decision Case 6012. Dated March 24, 2010. Available from http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPP

- 2007

- 1026

- 0030

U.S. EPA . 2014. Memorandum from I. Barsoum (OPP/BPPD) to S. Cerrelli, Re: Review of Product Chemistry, and Acute Toxicity Studies for Section 3 registration of a new TG AI

Bacillus subtilis strain IAB/BS03 ( EPA. Reg. No. 89615

- R) Dated July 29, 2014

C. Other References

Alexander, M. 1977. Introduction to Soil Microbiology. John Wiley and Sons, Inc., New York.

Logan, N.A., and P. de Vos. 2009. Genus I. Bacillus, Pp. 21

- 128 In: P. de Vos, G.M. Garrity, D. Jones, N.R. Krieg, W. Ludwig, F.A. Rainey, K.H. Schleifer, and W. Whitman (Eds.) Bergey's Manual of Systematic Bacteriology, Volume 3, 2nd Ed. Springer, New Yor k.

U.S. EPA. 2004. Primer for Municipal Wastewater Treatment Systems. EPA 832

- R

- 04

- 001.

Available from http://www.epa.gov/npdes/pubs/primer.pdf

World Health Organization, Guidelines for Drinking

- water Quality. (2011) 4th Ed. http://www.who.int/water_sanitation_health/publications/2011/dwq_guidelines/en/Bacillus subtilis strain IAB/BS03 Page 26 of 36

Biopesticides Registration Action Document

APPENDIX A. MICROBIAL PESTICIDES DATA REQUIREMENTS

(40 CFR PART 158

– SUBPART V)

TABLE 1. Product Analysis Data Requirements for the Active Ingredient (TGAI),

Bacillus subtilis strain IAB/BS03 Technical Powder (EPA File Symbol 89615

- R) (40 CFR § 158.2120)

Harmonized

G uideline

N umber Data Requirement Results MRID No.

885.1100 Product Identity Submitted data fulfill the requirement for product identity and composition. 48969301

885.1200 Manufacturing

Process Submitted data fulfill the requirement for manufacturing process. 48969301

885.1250 Deposition of a

Sample in a Nationally Recognized Culture

Collection Bacillus subtilis strain IAB/BS03 is on deposit at Colecciόn Española de Cultivos Tipo as CECT No 7254

48969301

885.1300 Discussion of

Formation of

Unintentional Ingredients Submitted data fulfill the requirement for preventing and detecting any

microbial contaminants. 48969301

and

48969302

885.1400 Analysis of Samples Submitted data fulfill the requirement for analysis of samples. 48969301

8 85 .1 50 0 Certification of

Limits Submitted data fulfill the requirement for certification of limits. 48969301

TABLE 2 . Product Analysis Data Requirements for MildoreTM (EPA File Symbol 89615

- E) (40 CFR § 158.2120)

Harmonized

Guideline

Number Data Requirement Results MRID No.

885.1100 Product Identity Submitted data fulfill the requirement for product identity and composition. 48969 4 01

885.1200 Manufacturing

Process Submitted data fulfill the requirement for manufacturing process. 48969 4 01

885.1300 Discussion of

Formation of

Unintentional Ingredients Submitted data fulfill the requirement for preventing and detecting any

microbial contaminants. 48969 4 01

885.1400 Analysis of Samples Submitted data fulfill the requirement for analysis of samples. 48969 4 01

885.1500 Certification of

Limits Submitted data fulfill the requirement for certification of limits. CSF dated May 7, 2013Bacillus subtilis strain IAB/BS03 Page 27 of 36

Biopesticides Registration Action Document

TABLE 3 . Product Analysis Data Requirements for Mildore MaxTM (EPA File Symbol 89615

- G) (40 CFR § 158.2120)

Harmonized

Guideline

Number Data Requirement Results MRID No.

885.1100 Product Identity Submitted data fulfill the requirement for product identity and composition. 49122601

885.1200 Manufacturing

Process Submitted data fulfill the requirement for manufacturing process. 49122601

885.1300 Discussion of

Formation of

Unintentional Ingredients Submitted data fulfill the requirement for preventing and detecting any

microbial contaminants. 49122601

885.1400 Analysis of Samples Submitted data fulfill the requirement for analysis of samples. 48969 4 01

885.1500 Certification of

Limits Submitted data fulfill the requirement for certification of limits. CSF dated

December
16, 2013

TABLE 4 . Product Analysis Data Requirements for Milagrum PlusTM (EPA File Symbol 89615

- U) (40 CFR § 158.2120)

Harmonized

Guideline

Number Data Requirement Results MRID No.

885.1100 Product Identity Submitted data fulfill the requirement for product identity and composition. 49130501

49130502

885.1200 Manufacturing

Process Submitted data fulfill the requirement for manufacturing process. 49130501

885.1300 Discussion of

Formation of

Unintentional Ingredients Submitted data fulfill the requirement for preventing and detecting any

microbial contaminants. 49130501

49275601

885.1400 Analysis of Samples Submitted data fulfill the requirement for analysis of samples. 49130501

49275601

885.1500 Certification of

Limits Submitted data fulfill the requirement for certification of limits. CSF dated May 22

, 2013Bacillus subtilis strain IAB/BS03 Page 28 of 36

Biopesticides Registration Action Document

TABLE 5 . Product Analysis Data Requirements for FungiseiTM (EPA File Symbol, 89615

- L) (40 CFR § 158.2120)

Harmonized

Guideline

Number Data Requirement Results MRID No.

885.1100 Product Identity Submitted data fulfill the requirement for product identity and composition. 49130 6 01

49130602

885.1200 Manufacturing

Process Submitted data fulfill the requirement for manufacturing process. 49130 6 01

885.1300 Discussion of

Formation of

Unintentional Ingredients Submitted data fulfill the requirement for preventing and detecting any

microbial contaminants. 491 306 01

49275701

885.1400 Analysis of Samples Submitted data fulfill the requirement for analysis of samples. 49130 6 01

49275701

885.1500 Certification of

Limits Submitted data fulfill the requirement for certification of limits. CSF dated May 22

, 2013

TABLE 6

. Physical and Chemical Characteristics for the Technical Grade of the Active Ingredient (TGAI)

, Bacillus subtilis strain

IAB/BS03 Technical (40 CFR § 158.2120)

Harmonized

Guideline Number Data Requirement Results MRID No.

830.6302 Color White

48969301 830.6303 Physical State Solid powder

830.630 4 Odor Slight fermentation odor

830.6313 Stability to Normal and

Elevated Temperatures, Metals,

and Metal Ions St ab le a t n o rm a l t e m pe r a t u r e s f

o r u p to t w o y e a rs. St ab le at

e l e v a t e d t e m

p e r a t u r e s a s en do s p o r e s o f B a cill u s s u b ti l is a r e hea t t o l e r an t. E x p e ct e d to b e st ab le in c o n t a ct w ith m e t a l a nd

m e t a l i o n s 48969301

830.6317 Storage Stability D a ta o n t h e TGAI/MUP is no t a v a il a b l e
, a s i t is f o rm u l a t ed
imm ed i a t e l y i n to end

- u se p r o d u cts du ri n g t h e m anu f a ct u r i ng
p r o c e ss (c o n ti n uou s p r o c e ss). F o rm u l a t e d p r o du c t s a re st ab le
f o r u p to 3 months at RT and 6 months at 8 o C. 48969301

830.6319 Miscibility Not applicable (chemical is n o t a n e m u lsif i a b le l iq u id a n d is

n o t d il u t e d w ith pe tr o l eu m s o lv e n ts. ).

830.6320 Corrosion

Characteristics D a ta o n t h e TGAI/MUP is no t a v a il a b l e
, a s i t is f o rm u l a t ed

imm ed i a t e l y i n to end

- u se p r o d u cts du ri n g t h e m anu f a ct u r i ng

p r o c e ss (c o n ti n uou s p r o c e ss). No deg r a da t i o n s e e n in

pa ck a g i n g i n t e g ri t y d u r i n g a t w o

- y e a r st o r a g e st ab i l i t y st u d y at

a m b i en t t e m p e r a t u r e s. 48969301

830.7000 pH 5 .0

– 9 .0 48969301

830.7100 Viscosity Not applicable to a solid

830.7300 Density/Relative

Density/Bulk Density (Specific Gravity) 0

. 7 4 g /ml 48969301Bacillus subtilis strain IAB/BS03 Page 29 of 36

Biopesticides Registration Action Document

TABLE 7 . Physical and Chemical Characteristics for MildoreTM (EPA File Symbol 89615

- E) (40 CFR § 158.2120)

Harmonized

Guideline Number Data Requirement Results MRID No.

830.6302 Color White

48969401 830.6303 Physical State Fine powder

830.6304 Odor odorless

830.6313 Stability to Normal and

Elevated Temperatures, Metals,

and Metal Ions Not applicable per 40 CFR 158.2120(c)

– Test substance is not

a TGAI.

830.6317 Storage Stability Stable for up to two

- years at ambient temperatures 48969401

830.6319 Miscibility Not applicable (chemical is not an emulsifiable liquid and is not diluted with petroleum solvents. ).

830.6320 Corrosion

Characteristics Not corrosive and no degradation of packaging integrity during 12 months of storage stability testing 48969401

830.7000 pH 6.78 (1% aqueous solution) 48969401

830.7100 Viscosity Not applicable to a solid

830.7300 Density/Relative Density/Bulk Density (Specific Gravity) 0.395 g/mL 48969401

TABLE 8 . Physical and Chemical Characteristics for Mildore MaxTM (EPA File Symbol 89615

- G) (40 CFR § 158.2120)

Harmonized

Guideline Number Data Requirement Results MRID No.

830.6302 Color White

49122601 830.6303 Physical State Fine powder

830.6304 Odor odorless

830.6313 Stability to Normal and

Elevated Temperatures, Metals,

and Metal Ions Not applicable per 40 CFR 158.2120(c)

– Test substance is not

a TGAI.

830.6317 Storage Stability A one year study will be initiated and submitted to EPA

830.6319 Miscibility Not applicable (chemical is not an emulsifiable liquid and is not diluted with petroleum solvents. ).

830.6320 Corrosion

Characteristics Corrosion Characteristics will be initiated and submitted to

EPA

830.7000 pH 6.78 (1% aqueous solution) 49122601

830.7100 Viscosity Not applicable to a solid

830.7300 Density/Relative Density/Bulk Density (Specific Gravity) 0.395 g/mL 49122601Bacillus subtilis strain IAB/BS03 Page 30 of 36

Biopesticides Registration Action Document

TABLE 9 . Physical and Chemical Characteristics for Milagrum PlusTM (EPA File Symbol 89615

- U) (40 CFR § 158.2120)

Harmonized

Guideline Number Data Requirement Results MRID No.

830.6302 Color Brown

49130501 830.6303 Physical State Viscous liquid

830.6304 Odor Characteristic odor

830.6313 Stability to Normal and

Elevated Temperatures, Metals,

and Metal Ions Not applicable per 40 CFR 158.2120(c)

– Test substance is not

a TGAI.

830.6317 Storage Stability A one year study will be initiated and submitted to EPA

830.6319 Miscibility Not applicable (chemical is not an emulsifiable liquid and is not diluted with petroleum solvents. ).

830.6320 Corrosion

Characteristics Corrosion Characteristics will be initiated and submitted to

EPA

830.7000 pH 5.0

- 6.0 49130501

830.7100 Viscosity Not applicable to a solid

830.7300 Density/Relative Density/Bulk Density (Specific Gravity) 1.015 g/mL 49130501

TABLE 10 . Physical and Chemical Characteristics for FungiseiTM (EPA File Symbol, 89615

- L) ) (40 CFR § 158.2120)

Harmonized

Guideline Number Data Requirement Results MRID No.

830.6302 Color Brown

49130601 830.6303 Physical State Viscous liquid

830.6304 Odor Characteristic odor

830.6313 Stability to Normal and

Elevated Temperatures, Metals,

and Metal Ions Not applicable per 40 CFR 158.2120(c)

– Test substance is not

a TGAI.

830.6317 Storage Stability A one year study will be initiated and submitted to EPA

830.6319 Miscibility Not applicable (chemical is not an emulsifiable liquid and is not diluted with petroleum solvents. ).

830.6320 Corrosion

Characteristics Corrosion Characteristics will be initiated and submitted to

EPA

830.7000 pH 8.1 49130601

830.7100 Viscosity Not applicable to a solid

830.7300 Density/Relative Density/Bulk Density (Specific Gravity) 1.231 g/mL 49130601Bacillus subtilis strain IAB/BS03 Page 31 of 36

Biopesticides Registration Action Document

TABLE 11

. Toxicology Data Requirements for the Active Ingredient Bacillus subtilis strain IAB/BS03 Technical (EPA File Symbol 89615

- R ), (40 CFR § 158.2140) 2

Harmonized

Guideline

Number Data Requirement Results MRID No.

Tier I

885.3050 Acute Oral

Toxicity/Pathogenicity The registrant has submitted the following rationale that existing data for the Acute IV Toxicity / Pathogenicity study on the TGAI be considered to satisfy the requirements for Acute Oral & Pulmonary Tox/Pathogenicity studies:

Technical Produc t Bacillus subtilis strain IAB/BS03, was dosed at a single high dose exposure and followed for an adequate post

exposure observation period. The test substance was injected into the tail vein of one of two groups of rats at a high dose. A control group (untreated) was include d. The animals were observed frequently on day of dosing for mortality and signs of pharmacologic and/or toxicological effects and once daily thereafter for 21 days. Tissue and blood samples taken at interim sacrifices from the group receiving the active t est substance were cultured to provide quantitative measurements of the test microbe’s clearance pattern. Classification: Acceptable 48996317 885.3150 Acute Pulmonary

Toxicity/Pathogenicity

885.3200 Acute Injection

Toxicity/ Pathogenicity No abnormalities were seen during daily observations and only one

white dot on a Group III

– MPCA liver was seen at necropsy. There was no mortality in any group during the study.

The test organism had cleared completely from the Group III

– MPCA blood Day 3. Once clearance was established by two

consecutive interim sacrifices that showed less than 100
CFU/gram tissue of the test organism in the organs plated, no
further tissue samples were taken or cultured. The test organism
had completely cleared f

rom the kidneys by Day 14 and from the
brain by Day 21. There was no test substance seen in the MLN or
lungs at the Day 21 counts. Test organism was present in the liver
and spleen at Day 21 but was showing a pattern of clearance. Test
organism was pres ent in the cecum contents by Day 21.

Based on lack of mortality, the test substance Technical Product

Bacillus subtilis strain IAB/BS03 was determined to be non

- toxic to
rats when injected intravenously at a dose of 1.4 x 10 8 CFU/mL or

1.4 x 10 7 CFU/rat.
Classification: Acceptab le 48969303

2 Acute Toxicity data submitted for Technical also support the end

- use products: MildoreTM (EPA File Symbol

89615

- E), Mildore MaxTM (EPA File Symbol 89615

- G), Milagrum PlusTM (EPA File Symbol 89615

- U) and FungiseiTM (EPA File Symbol, 89615

- L.Bacillus subtilis strain IAB/BS03 Page 32 of 36

Biopesticides Registration Action Document

TABLE 11

. Toxicology Data Requirements for the Active Ingredient Bacillus subtilis strain IAB/BS03 Technical (EPA File Symbol 89615

- R ), (40 CFR § 158.2140) 2

Harmonized

Guideline

Number Data Requirement Results MRID No.

885.3400 Hypersensitivity

Incidents The applicant reported that t o date, no incidents of hypersensitivity or any other adverse effects have occurred through the research, development, testing or commercialization of the active ingredient Bacillus subtilis strain IAB/BS03

Technical Powder and its related end

- use products . Should any incidents occur, they would be reported per FIFRA Section 6(a) (2). Any future hypersensitivity incidents must be reported to EPA (refer to test note #3 of 40 CFR § 158.2140(d)).

885.3500 Cell Culture N/A

870.1100 Acute Oral Toxicity No mortality occurred during the study. No clinical signs related

to the administration of the test item were observed.

Oral LD 50

. female rats.> 2000 mg/kg body weight

. Classification: Acceptable

TOXICITY CATEGORY III 48969304

870.1200 Acute Dermal Toxicity Dermal LD 50 Males >2000 mg/kg bw; Females > 2000 mg/kg bw; Combined > 2000 mg/kg bw Classification: Acceptable

TOXICITY CATEGORY I II 48969305

870.1300 Acute Inhalation

Toxicity Inhalation LC 50 > 4.7 mg/L in air during 4 hours.

Classification: Acceptable

TOXICITY CATEGORY I V 48969407

870.2400 Acute Eye Irritation The ocular conjunctivae reactions observed during the study have been slight to moderate and totally reversible: a slight to moderate

redness noted 1 hour after the test item instillation and totally reversible between days 2 and 4, associated with a slight chemosis

noted 1 hour after the test item instillation in two animals and totally reversible on day 1. Classification: Acceptable

TOXICITY CATEGORY IV 48969306

870.2500 Primary Dermal

Irritati o n A slight erythema was noted on the treated area of one anim al, 1

and 24 hours after r em oval . This reaction was totally reversible on day 2.

TOXICITY CATEGORY IV 48969307

Tiers II and III

Not required for Bacillus subtilis strain IAB/BS03 Technical based on the lack of acute toxicity/pathogenicity in the Tier I

studies.Bacillus subtilis strain IAB/BS03 Page 33 of 36

Biopesticides Registration Action Document

TABLE 12

. Nontarget Organism Toxicity and Environmental Fate Data Requirements for the Technical Grade of the Active

Ingredient TGAI

, Bacillus subtilis strain IAB/BS03 Technical

. ( 40 CFR § 158.2150)

Harmonized

Guideline

Number Data Requirement Results MRID No.

Tier I

885.4050 Avian Oral

Toxicity /pathogenicity

The 14 The 14

- day LD 50 value > 5000 mg a.i./kg body weight, which indicates a lack of toxicity for Japanese quail. The study is insufficient to determine potential infectivity or pathogenicity for a viable microbial pesticide (OPPTS Guideline 885.4050) Classification: Supplemental 48969308

885.4100 Avian Inhalation

Toxicity/Pathogenicity Scientific rationale was partially sufficient to satisfy the data requirement. Bacillus subtilis strain IAB/BS03 is not considered toxic to birds through the oral route of exposure. There is uncertainty in the pathogenicity potential of Bacillus subtilis strain IAB/BS03 to birds following inhalation because of the lack of data and results from the

submitted oral stu dy (48969308). Classification: Supplemental. 48969317

885.4150 Wild Mammal

Toxicity/Pathogenicity Tests required by 40 CFR § 158.2140 are adequate and appropriate for

assessment of hazards to wild mammals . The TGAI, Bacillus subtilis

strain IAB/BS03 was determined to be non

- toxic to rats when injected intravenously at a dose of 1.4 x 10 8 CFU/mL (or 1.4 x 10 7 CFU/rat). Classification: Acceptable 48969303

885.4200 Freshwater Fish

Toxicity/Pathogenicity The 96

- hr LD 50 > 100 mg Bacillus subtilis , Strain IAB/BS03 /L under

semi

- static conditions. Bacillus subtilis IAB/BS03 is determined is practically non

- toxic to carp, for short term exposures. However, the pathogenic potential of Bacillus subtilis IAB/BS03 to freshwater fish is uncertain, because the study was not sufficiently long (30 days) to determine potential infectivity/pathogenicity.

Classification: Supplemental. 48969309

885.4240 Freshwater Invertebrate

Toxicity/Pathogenicity The 21

- day LOEC for reproductive output was 1.6 mg a.i./L and the NOEC was 0.5 mg a.i./L. The 21

- day EC 50 for reproductive output was 7.85 mg a.i./L (95% C.I: 1.80

– 34.22 mg a.i./L), under semi

- static conditions. Under conditions of the study, Bacillus subtilis strain IAB/BS03 is practically non

- to xic to daphnia populations. The potential pathogenicity of Bacillus subtilis strain IAB/BS03 is uncertain, as a sterile filtrate control was not tested and viability of test material was not confirmed or recovered from test suspensions.

Classification: Supplemental. 48969311

The 24

- and 48

- hr EC 50 was > 100 mg Bacillus subtilis IAB/BS03 per L in this static bioassay. Under conditions of the study, Bacillus subtilis strain IAB/BS03 is practically non

- toxic to D aphnia populations.

Classification: Supplemental. 48969310

885.4280 Estuarine/Marine Fish

and Invertebrate Testing Submitted literature indicated that no adverse effects to estuarine or

marine fish and invertebrates have been reported from research investigations using other strains of Bacillus subtil i s

. Classification: Acceptable. 48969317

885.4300 Nontarget Plant Testing Submitted literature indicates that some strains of Bacillus subtil i s offer biocontrol of certain fungal plant diseases. There are no reports of phytopathogenicity by Bacillus subtili s in the open literature, and the registrant claims there were no symptoms of phytotoxicity during research and development of Bacillus subtilis Strain IAB/BS03. Classification: Acceptable 4896 9317Bacillus subtilis strain IAB/BS03 Page 34 of 36

Biopesticides Registration Action Document

TABLE 12

. Nontarget Organism Toxicity and Environmental Fate Data Requirements for the Technical Grade of the Active

Ingredient TGAI

, Bacillus subtilis strain IAB/BS03 Technical

. ( 40 CFR § 158.2150)

Harmonized

Guideline

Number Data Requirement Results MRID No.

885.4340 Nontarget Insect

Testing (predatory mite, Typhlodromus pyri ) There is uncertainty in the toxicity and environmental persistence of

Bacillus subtilis strain IAB/BS03

. Under conditions of this study, the 7

- day LC 50 ≈ 10 12 cfu/A. The results also suggested that additional mortality might be expected after seven days. There was no confirmation for microbial viability at test initiation, or verification of survival in the test solutions or from treated plants. Maximum hazard testing was not confirmed. Classification: Supplemental. 48969312

885.4340 Nontarget insect testing

(parasitic wasp

– Aphidius rhopalosiphi ) The reported 24

- and 48

- hr LR 50 values for a formulated product of

Bacillus subtilis strain IAB/BS03 were extrapolated values and were not tested in this study. There is uncertainty in the toxicity of Bacillus

subtilis strain IAB/BS03 WP to parasitic wasps. Maximum hazard testing was not confirmed. About 32.5% wasp mortality was observed at the highest d ose tested (12,500 g of formulated product) and there were no significant increases in percent mortality among the reported concentration range (2500 g

– 12,500 g formulated product). Classification: Supplemental. 48969313

885.4340 Nontarget insect testing

(ladybird beetle

, Hippodamia

convergens ) The results are inconclusive and there are uncertainties relative to the

toxicity potential of Bacillus subtilis IAB/BS03 to ladybird beetles. Maximum hazard dosing was not confirmed. The experimental design was flawed and there was no distinct or remarkable differences in lady bird beetles in the treatment groups nor the untreated control group. Classification: Unacceptable

. 48969314

885.4380 Honey Bee Testing The 5

- day oral LD 50 > 88.235 μg a.i./adult bee (or 2.9 x 10 9 cfu/mL),

and the 5

- day contact LD 50 was 177.38 μg a.i./adult bee (or 8.9 x 10 8 cfu/mL). The toxicity of Bacillus subtilis strain IAB/BS03 to honeybees is uncertain, since a biologically significant dose was not tested to characterize the potential hazard(s) of products containing Bacillus

subtilis strain IAB/BS03 . Maximum hazard testing was not verified by the testing labora tory. Classification: Supplemental. 48969315

The toxicity of Bacillus subtilis s train IAB/BS03 to honeybees is uncertain because of uncertainties in the test material and problems with the experimental design. The results are inconclusive since h oneybee mortalities were noted in all groups (including the untreated control). Classification: Unacceptable. 48969316

Tiers II, III, and IV Not required for based on the acceptability of the data and other information provided for Tier I

.Bacillus subtilis strain IAB/BS03 Page 35 of 36

Biopesticides Registration Action Document

APPENDIX B. PESTICIDE PRODUCT S

EPA

File Symbol Registration

Name Percentage

Active Ingredient Formulation

Type Use Site(s) Method(s) of Application Application

Rate (s) Target Pest

89615

- R Bacillus subtilis

strain IAB/BS03

Technical

Powder 100% Technical Manufacturing use only N/A N/A

89615

- E MildoreTM 1% wettable

powder Artichoke

Asparagus

Berries Bulb Vegetables

various Cereal grain s
, Citrus, Cole Crops

Corn

Cotton

Curcurbits Fruiting Vegetables Grapes

Grass seed Herbs/Spices Hops Leafy Vegetables Legumes

Mint Oil Seed Crops

Olives

O rnamental tree

and O rnamental plant crop

, Peanuts

Pome Fruits Rice,

Root tuber and Corm Vegetables Soybean Strawberry

T tree fruit and nut crops, and S ugar
B eet s , , sugarcane,
T obacco , Tropical

Fruits greenhouse/shad e Home and Garden

Uses Foliar Ground,

Aerial, Chemigation, Dip treatment at Planting Drench,

In

- furrow,

In

- furrow seed

treatment at planting

,

Plant dip,

Shanked

- in Soil Injected

Seed treatments As specified

on label Aerial Web blight

Alternaria, spp. Anthracnose

Anthracnose Boll Rot Apple Scab Asian Soybean Rust Ascochyta Blight,

Boll Rot Bacterial Blight and

Streak

Bacterial Blast

Bacterial Canker

Bacterial Pustule Bacterial Speck Bacterial Spot

Bitter Rot

Black Root Rot

Black Rot Black Spot

Blue mold

Bot Rot Botrytis Blight Brooks Spot Brown rot, Leaf

Brown Rot Blossom Blight Brown Spot Bull’s Eye Rot Cercospora Blight and Leaf Spot Cherry leaf rot

Common Scab

Club root Diplodia Boll rot Dry Rot Downy mildew Eastern Filbert Blight Eutpa Fire Blight Fly Speck Frog

- eyed leaf Spot

Fusarium spp. Gray Mold Greasy Spot

Green Mold

Hardlock Boll Rot

Jacket Rot Late Blight

Leaf Rust Leaf Spot

Melanose

Mucor rot MummyBerry

Olive knot Onion Purple Blotch 89615

- G Mildore MaxTM 10% wettable

powder

89615

- U Milagrum

PlusTM 0.3% liquid

89615

- L FungiseiTM 0.08% liquidBacillus subtilis strain IAB/BS03 Page 36 of 36

Biopesticides Registration Action Document

EPA

File Symbol Registration

Name Percentage

Active Ingredient Formulation

Type Use Site(s) Method(s) of Application Application

Rate (s) Target Pest

Phoma Blight Phomopsis Leaf Phomopsis Fruit Rot Phytophthora spp. Pink rot Pod and Stem Blight Postbloom Fruit Drop Powdery mildew Pythium spp. Ramularia Leaf Spot
Rhizoctonia spp. Ripe Rot

Rust

Scab Septonia Brown

Shot hole

Silver Scurf Spot Sooty Blotch

Sour rot Spot Pin Rot complex Spur Blight Stemphylium Leaf Blight Spots and Smuts.

Rice Blast Septonia Leaf Spot Sheath spot and Blight

Stem rot Target Spot

Verticillium spp. Walnut Blight

White rot

White mold

Xanthomonas L eaf Spot