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TABLE OF CONTENTS

I. EXECUTIVE SUMMARY.....................................................................................................................................4

II. ACTIVE INGREDIENT OVERVIEW ................................................................................................................6

III. REGULATORY BACKGROUND......................................................................................................................7

A. Applications 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 JUSTICE .........................................................................................................................18

VI. RISK MANAGEMENT DECISION .................................................................................................................19

VII. ACTIONS REQUIRED OF THE REGISTRANT .........................................................................................20

A. Final Printed Labeling.........................................................................................................................................20

B. Terms of Registration ..........................................................................................................................................20

VIII. GLOSSARY OF ACRONYMS AND ABBREVIATIONS..........................................................................21

IX. BIBLIOGRAPHY ..............................................................................................................................................22

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

Product Registrations for the (TGAI) product, Bacillus 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. MICROBIAL PESTICIDES DATA REQUIREMENTS ...........................................................26

APPENDIX B. PESTICIDE PRODUCTS..............................................................................................................35
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BIOPESTICIDES REGISTRATION ACTION DOCUMENT TEAM

Office of Pesticide Programs (OPP)

Biopesticides and Pollution Prevention Division

Microbial Pesticides Branch

Science Reviews

Ibrahim 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

Regulatory

Kimberly Nesci Chief, Microbial Pesticides Branch

ShaRon Carlisle Acting Associate Chief, Microbial 

Pesticides Branch

Susanne Cerrelli Regulatory Action Leader
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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 applications for a new technical grade active 

ingredient (TGAI) product, Bacillus subtilis strain IAB/BS03 Technical Powder (EPA 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 applications 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 subtilis 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 

bacterium 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 Final Registration Review Decision for Bacillus subtilis, 

Registration Review Case 6012 (http://www.regulations.gov/#!documentDetail;D=EPA-HQOPP-2007-1026-0029). In its decision, EPA concluded that the standards of Registration Review 

were met for Bacillus subtilis and identified no general risk concerns. This strain will now be 

considered as part of the Bacillus subtilis Registration Review Case 6012. 

The EPs 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. Additional proposed 

uses include applications to garden and ornamental landscapes in home-owner and residential 

areas.

EPA scientists reviewed product analysis, toxicology, and nontarget organism data and 

information (40 CFR §§ 158.2120, 158.2140, and 158.2150, respectively) submitted to support 

the registration of the proposed pesticide products. They found that, overall, 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).
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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 EPs were fulfilled by acceptable 

guideline studies. These data requirements include product chemistry and composition, analysis 

of samples, and physical and chemical characteristics.

Toxicology

The applicant submitted adequate mammalian toxicology data and information to support the 

pesticide products. An acute injection toxicity/pathogenicity study showed that Bacillus subtilis

strain IAB/BS03 when injected intravenously at the dose administered is not toxic or pathogenic 

via this 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 acute 

inhalation toxicity and acute eye irritation studies are acceptable and rated as EPA Toxicity 

Category IV. In light 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 2013, 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 labels

on the end-use products direct handlers working with, Bacillus subtilis strain IAB/BS03, in 

agricultural settings to wear a long-sleeved shirt, long pants, socks, shoes, waterproof gloves, and

a dust/mist filtering respirator meeting National Institute for Occupational Safety and Health 

(NIOSH) standards of at least N-95, R-95, or P-95. 

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 required 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 nontarget organism data requirements have been adequately addressed for the purposes of 

FIFRA section 3(c)(5) registration, based on the submitted data, rationales, limited anticipated 
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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 opportunity 

for the public to participate in 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 applications: new active ingredients; first food 

uses; first outdoor uses; first residential uses; 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 products

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 will 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 accessed 

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-2013-0575: (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 label 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 FungiseiTM, 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

Culture Deposit: Colecciόn Española de Cultivos Tipo CECT No. 7254

OPP Chemical Code: 006544

Type of Pesticide: Microbial Pesticide –fungicide

See Appendix B for specific information (e.g., use sites, application rates, methods of 

application, formulation types, and target pests) regarding the pesticide products containing this

active ingredient.
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III. REGULATORY BACKGROUND

A. Applications for Pesticide Product Registration

Technology Sciences Group (712 Fifth St., Suite A, Davis, CA 95616) on behalf of IAB, S.L. 

[Investigaciones y Aplicaciones Biotechnologicas S.L.,Avda, Paret del Patriarca 11-B, Ap. 30, 

46113 Moncada (Valencia) Spain] submitted applications 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 (EPA 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, 2013 (78 FR 64937), EPA announced receipt of the application to register a 

pesticide product containing a new active ingredient for use as a fungicide in greenhouses, field 

use, and home and garden use on various fruits and vegetables, cotton, hops, tobacco, fruit and 

nut trees, turf, and ornamentals. EPA opened a 30-day public comment period pursuant to the 

provisions of FIFRA section 3(c)(4). No comments were received following this publication. 

B. Food Tolerance Exemption

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) 3F8177] in 

or on all food commodities. EPA published a Notice concerning 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

tolerance exemption (EPA-HQ-OPP-2013-0574) that do not appear to be specific to this 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, diflubenzuron

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 products. Neither of these comments were

specific to Bacillus subtilis strain IAB/BS03. Both comments 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 definition 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 is 
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scientifically sound and is useful for risk assessment. A “supplemental” rating indicates the data 

provide some information that can be useful for risk assessment. The studies may have certain 

aspects determined not to be scientifically acceptable (“supplemental: upgradeable”). If a study 

is rated as “supplemental: upgradeable,” EPA always provides an indication of what is lacking or 

what can be provided to change the rating to “acceptable.” If there is simply a “supplemental” 

rating, the reviewer will often state that the study is not required by 40 CFR Part 158. Both 

“acceptable” and “supplemental” studies may be used in the risk assessment process as 

appropriate. An “unacceptable” rating indicates that new data must be submitted.

For the acute toxicity data requirements, Toxicity Categories are assigned based on the hazard(s) 

identified from studies and/or other information submitted to the EPA in support of a pesticide 

registration. The EPA classifies the 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 toxicity (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 that 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 information.

B. Human Health Assessment (40 CFR § 158.2140)

1. Toxicity

All applicable mammalian toxicology data requirements supporting the request for an exemption 

from the requirement of a tolerance for residues of Bacillus subtilis strain IAB/BS03 in or on all 

food commodities have been fulfilled with data submitted by the applicant or with scientific 

rationale. Furthermore, 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 PlusTM (EPA File Symbol 89615-U) and FungiseiTM (EPA File Symbol, 

89615-L).

The toxicity and pathogenicity tests (acute oral, inhalation and dermal toxicity) and irritation 

tests (acute eye and primary dermal irritation) that address potential routes of exposure to the

active ingredient are all classified in Toxicity Category III or IV (see below or U.S. EPA 2014)

and revealed little to no toxicity attributed to Bacillus subtilis strain IAB/BS03. Finally, the

applicant reported that no hypersensitivity incidents occurred during the research, 

development, and testing of this active ingredient.

The overall conclusions from all toxicological information submitted by the applicant are briefly

described below, in sections IV(B)(1)(a) and IV(B)(1)(b) (see pages 8–11), and summarized in

Table 11 in Appendix A. 
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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 108 CFU/mL or 1.4 x 

107 CFU/rat). A control group was conducted concurrently. 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 test substance were cultured to provide quantitative 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 termination 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 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 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 contents 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 108 CFU/mL or 

1.4 x 107 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 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 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 included. 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 test 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 109

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 of the animals at the end 
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of the study did not reveal treatment related change. In conclusion, the LD50 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 oral 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 : 8.8 109 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 macroscopical 

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 (LC50) 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 LC50 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 weight 

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 exposure 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 the 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 LC50 for the product Bacillus subtilis IAB/BS03 in rats is higher than 4.7 mg/L in air during 

4 hours. EPA classified 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 109 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 chemosis 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.
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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.

Hypersensitivity incidents (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 substance 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 test 

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 screened 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 subtilis strain IAB/BS03 from the 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, the list of 67 chemicals, the test guidelines and the Tier 1 screening battery, please 

visit our website: http://www.epa.gov/endo/.
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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 information.” 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 exemption, 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 information 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 residues. 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 

presented in Table 11 in Appendix A, the 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 water 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 dietary 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 the risk posed to adults, infants, and children is 

likely to be minimal because of the low acute toxicity/pathogenicity 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 through 
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acceptable water treatment processes, largely because the spores are resistant to these 

disinfection processes (World 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 nonoccupational exposure to the bacterium is likely. Repeated exposures 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 demonstrate 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 8–11 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) of 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 infants 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 the 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 additional 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 available to 

the EPA support the choice of a different factor.

Based on the acute toxicity and pathogenicity data/information discussed in section IV(B)(1)(a) 

(see pages 8–11) and Table 11 in Appendix A, the EPA concludes that there are no threshold 
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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 an herbicide. Such exposure includes all anticipated dietary exposures 

and all other exposures for which there is reliable information. The EPA has arrived at this 

conclusion because, considered collectively, the data and information available on Bacillus 

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

infants and children. 

3. Occupational Exposure and Risk Characterization

The EPA does not expect handler 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 repeated 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 

handlers 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 

determined that no unreasonable 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 Assessment (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 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 

required 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 risks are not 
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required.

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

18), refer to Table 12 in Appendix A.

1. Ecological Exposure and Risk Characterization 

Based on the proposed use pattern, 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 

ingredient1in Mildore MaxTM (10% a.i.), the EP with the highest concentration of a.i., the

Agency estimates terrestrial maximum environmental concentrations (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 107cfu/L for Mildore 

MaxTM). Because applications are proposed for every 7 to 10 days for many of the labeled crops, 

constant exposures of the biological control 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 presented 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 subtilis strain IAB/BS03 was determined to be non-toxic to rats 

when injected intravenously at a dose of 1.4 x 108 CFU/mL (or 1.4 x 107 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 guidelines 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 Bacillus subtilis to avian species, 

the lack of information regarding maximum temperatures for growth and/or survival of Bacillus 

subtilis strain IAB/BS03 and the 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 LD50 > 5000 mg/a.i./kg). In addition, 

because 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 minimal 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 mg/lb * 

1A/40,468,564 cm2 = 0.045 mg/cm2  15 cm = 0.003 mg/mL * 1000 ml/L =3.0 mg/L *10.0 % = 0.3 mg a.i./L. 
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Nontarget Insects (OCSPP Guideline Number 885.4340) (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 LC50. 

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 

concentrations. 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 the 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 days) of wild populations of nontarget insects, adverse 

effects to certain species could be reasonably expected. There are reports of Bacillus subtilis

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 LC50 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 LD50

values from contact and oral exposures. The 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 uncertainty 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 applications 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 to obtain statistically valid biological endpoints (e.g., median LC50 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 result of frequent 

applications of one week intervals.
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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 strain 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 subtilis in the open literature, and the registrant claims there were 

no symptoms of phytotoxicity during research and development of Bacillus subtilis strain 

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 107cfu/L for Mildore MaxTM. Since there are no labeled 

sites with direct applications to aquatic systems, the Agency does not anticipate adverse effects 

to nontarget 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 increase slightly soon after application to terrestrial crops, and subsequently reduce to natural 

levels of total bacilli because of nutrient cycling and decomposition. Since there are no labeled 

sites with direct applications to marine/estuarine systems, the Agency 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 strain 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. 
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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 proteases 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 Bacillus 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 

reasonably 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 population levels of 106to 107per 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 by 

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. Threatened 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, terrestrial 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, and 

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 effects 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 

determination 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, implementation, and enforcement of environmental laws, regulations, and policies. 
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Fair treatment means that no group of people, including racial, ethnic, or socioeconomic groups, 

should bear a disproportionate share of the negative environmental consequences resulting from 

industrial, municipal, and commercial operations or the execution of federal, state, local, and 

tribal environmental programs and policies. Meaningful involvement means that (1) potentially 

affected community residents have an appropriate opportunity to participate in decisions about a 

proposed activity that will affect their environment and/or health; (2) the public’s contribution 

can influence the regulatory agency’s decision; (3) the concerns of all participants involved will 

be considered in the decision-making process; and (4) the decision-makers seek out and facilitate 

the involvement of 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 exposure 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.

VI. 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 

of FIFRA;

(3) It will perform its intended function without unreasonable adverse effects on the 

environment; AND

(4) When used in accordance with widespread and commonly recognized practice, it will 

not generally cause unreasonable adverse effects on the environment.

To satisfy criterion 1, the Bacillus subtilis strain IAB/BS03 products, the TGAI and EPs, have

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

support the claims made on the labels. 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 unreasonable 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 instructions.

Criterion 4 is satisfied as the Bacillus subtilis strain IAB/BS03 products 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 FungiseiTM which contain Bacillus subtilis strain IAB/BS03 as a new 

active ingredient, are eligible for registration under FIFRA section 3(c)(5) for the labeled uses. 
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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 products: MildoreTM, Mildore MaxTM, Milagrum PlusTM, and FungiseiTM, 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, the 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 Hazard section of each label: “This product may harm beneficial 

insects and honey 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-MaxTM, Milagrum 

PlusTM, and FungiseiTM End-use Products (EPA File Symbols 89615-G, -U, and -L):

Storage Stability and Corrosion Characteristics (Harmonized Guidelines 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 studies on Mildore-MaxTM, Milagrum 

PlusTM, and FungiseiTM.

Analysis of Samples (Harmonized Guideline 885.1400) 

Additional analysis of sample data for Mildore-MaxTM (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).
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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

LD50 median lethal dose. A statistically derived single dose that can be expected 

to cause death in 50% of the test animals when administered by the route 

indicated (oral, dermal, or inhalation). It is expressed as a weight of 

substance per unit weight of animal (e.g., mg/kg).

mg/kg 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 States
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IX. BIBLIOGRAPHY

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

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

IAB/BS03 Technical Powder, and four new end-use products (EPs): MildoreTM; Mildore 

MaxTM; Milagrum PlusTM and FungiseiTM 

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 Eye 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). Project 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. Project 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 

Number: 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/OCR. Unpublished study prepared by Jai Research Foundation. 39p.
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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 Report. 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 Determine 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 Research 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 Mileson, 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: 

BT078/11. 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 

Acute 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 prepared 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 Response 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.html
Bacillus 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 TGAI 

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 York. 

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 

Guideline 

Number

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

885.1500

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. 48969401

885.1200 Manufacturing 

Process Submitted data fulfill the requirement for manufacturing process. 48969401

885.1300

Discussion of 

Formation of 

Unintentional 

Ingredients

Submitted data fulfill the requirement for preventing and detecting any 

microbial contaminants. 48969401 

885.1400 Analysis of Samples Submitted data fulfill the requirement for analysis of samples. 48969401

885.1500 Certification of 

Limits Submitted data fulfill the requirement for certification of limits. CSF dated May 7, 2013
Bacillus 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. 48969401

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, 

2013
Bacillus 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. 49130601

49130602

885.1200 Manufacturing 

Process Submitted data fulfill the requirement for manufacturing process. 49130601

885.1300

Discussion of 

Formation of 

Unintentional 

Ingredients

Submitted data fulfill the requirement for preventing and detecting any 

microbial contaminants.

49130601

49275701

885.1400 Analysis of Samples Submitted data fulfill the requirement for analysis of samples. 49130601

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

830.6304 Odor Slight fermentation odor 

830.6313

Stability to Normal and 

Elevated

Temperatures, Metals,

and Metal Ions

Stable at normal temperatures for up to two years. Stable at 

elevated temperatures as endospores of Bacillus subtilis are 

heat tolerant. Expected to be stable in contact with metal and 

metal ions

48969301

830.6317 Storage Stability

Data on the TGAI/MUP is not available, as it is formulated 

immediately into end-use products during the manufacturing 

process (continuous process). Formulated products are stable 

for up to 3 months at RT and 6 months at 8o C.

48969301

830.6319 Miscibility Not applicable (chemical is not an emulsifiable liquid and is 

not diluted with petroleum solvents.).

830.6320 Corrosion 

Characteristics

Data on the TGAI/MUP is not available, as it is formulated 

immediately into end-use products during the manufacturing 

process (continuous process). No degradation seen in 

packaging integrity during a two-year storage stability study at 

ambient temperatures.

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)

0.74 g/ml 48969301
Bacillus 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

830.6303 Physical State Fine powder 48969401

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

830.6303 Physical State Fine powder 49122601

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 49122601
Bacillus 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

830.6303 Physical State Viscous liquid 49130501

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

830.6303 Physical State Viscous liquid 49130601

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 49130601
Bacillus 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 Product Bacillus subtilis strain IAB/BS03, was dosed at 

a single high dose exposure and followed for an adequate postexposure 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 included. 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 test 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 from 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 present 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 108 CFU/mL or 

1.4 x 107 CFU/rat.

Classification: Acceptable

48969303

 

2Acute 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 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 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 LD50 .female rats.> 2000 mg/kg body weight. 

Classification: Acceptable

TOXICITY CATEGORY III

48969304

870.1200 Acute Dermal Toxicity

Dermal LD50 Males >2000 mg/kg bw; 

Females > 2000 mg/kg bw; 

Combined > 2000 mg/kg bw

Classification: Acceptable

TOXICITY CATEGORY III

48969305

870.1300 Acute Inhalation 

Toxicity

Inhalation LC50> 4.7 mg/L in air during 4 hours.

Classification: Acceptable

TOXICITY CATEGORY IV

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 

Irritation

A slight erythema was noted on the treated area of one animal, 1 

and 24 hours after removal. 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 LD50 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 study (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 108 CFU/mL (or 1.4 x 107 CFU/rat).

Classification: Acceptable

48969303

885.4200 Freshwater Fish 

Toxicity/Pathogenicity

The 96-hr LD50 > 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/BS03to 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 EC50 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-toxic 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 EC50 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 Daphnia 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 subtilis. 

Classification: Acceptable.

48969317

885.4300 Nontarget Plant Testing

Submitted literature indicates that some strains of Bacillus subtilis offer 

biocontrol of certain fungal plant diseases. There are no reports of 

phytopathogenicity by Bacillus subtilis 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

48969317
Bacillus 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 LC50 ≈ 1012 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 LR50 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 dose 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 LD50 > 88.235 μg a.i./adult bee (or 2.9 x 109cfu/mL), 

and the 5-day contact LD50 was 177.38 μg a.i./adult bee (or 8.9 x 108

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 laboratory. Classification: Supplemental.

48969315

The toxicity of Bacillus subtilis strain IAB/BS03 to honeybees is 

uncertain because of uncertainties in the test material and problems with 

the experimental design. The results are inconclusive since honeybee 

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 PRODUCTS

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% wettablepowder

Artichoke

Asparagus

Berries

Bulb Vegetables

various Cereal 

grains,

Citrus,

Cole Crops

Corn

Cotton

Curcurbits

Fruiting 

Vegetables

Grapes

Grass seed

Herbs/Spices

Hops

Leafy Vegetables 

Legumes

Mint

Oil Seed Crops

Olives

Ornamental tree 

and Ornamental 

plant crop, 

Peanuts

Pome Fruits

Rice,

Root tuber and 

Corm Vegetables

Soybean 

Strawberry

Ttree fruit and nut 

crops, and Sugar

Beets, , sugarcane, 

Tobacco , Tropical 

Fruits 

greenhouse/shade

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% wettablepowder

89615-U

Milagrum 

PlusTM 0.3% liquid

89615-L FungiseiTM 0.08% liquid 
Bacillus 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 Leaf Spot