**BIOPESTICIDES REGISTRATION ACTION DOCUMENT**



## Bacillus subtilis strain QST 713 variant Soil Pesticide Chemical (PC) Code: 006 5 79

**U.S. Environmental Protection Agency Office of Pesticide Programs**  **Biopesticides and Pollution Prevention Division**

DRAFT

– October 3 , 2012Bacillus subtilis strain QST 713 variant Soil Page 2 of 28

Biopesticides Registration Action Document **TABLE OF CONTENTS**
**I. EXECUTIVE SUMMARY**  **................................ ................................**  **................................ ...........................**  **4 II.**  **ACTIVE INGREDIENT OV ERVIEW**  **................................ ................................**  **................................ .......**  **7 III.**  **REGULATORY BACKGROUN D**  **................................ ................................**  **................................ ..............**  **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.**  **Hum an Health Assessment (40 CFR § 158.2140)**  **................................ ................................**  **........... 8**  **C. Environmental Assessment (40 CFR §**  **158.2150) ................................**  **................................ .........**  **14 V.**  **ENVIRONMENTAL JUSTIC E**  **................................ ................................**  **................................ .................**  **16 VI.**  **RISK MANAGEMENT DECI SION**  **................................ ................................**  **................................ .........**  **16 VII.**  **ACTIONS REQUIRED OF THE REGISTRANT**  **................................ ................................**  **................... 17**  **VIII. GLOSSARY OF ACRONYMS**  **AND ABBREVIATIONS ................................**  **................................ ......**  **18 IX.**  **BIBLIOGRAPHY ................................**  **................................ ................................**  **................................ .......**  **19**

**A. Studies Submitted**  **to Support the Active Ingredient,**  **Bacillus subtilis strain QST 713 variant**  **Soi l, and**  **Pesticide Product , Serenade Soil DPZ,**  **Registration ................................**  **................................ ...............**  **19**

**B. Environm ental Protection Agency Risk Assessme**  **nt Mem oranda**  **................................ .............**  **22 C. Other References**  **................................ ................................**  **................................ ..........................**  **22 3**  **APPENDIX A. MICROBI AL PESTICIDES DATA R** **EQUIREMENTS ................................**  **........................... 24**  **APPENDIX B. PESTICI DE PRODUCT**  **................................ ................................**  **................................ ................**  **28**Bacillus subtilis strain QST 713 variant Soil Page 3 of 28

Biopesticides Registration Action Document

**BIOPESTICIDES REGISTRATION ACTION DOCUMENT TEAM**

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

**Microbial Pesticides Branch**
**Science Reviews**  Ib rahim S. Barsoum, Ph.D. Product Anal ys is, Human Health John L. Kough, Ph.D. Product Anal ys is, Human Health Shannon Borges Environmental Effects **Regulations** Kimberly Nesci Acting Chief, Microbial Pesticides Branch Mic hael Glikes Regulatory Action LeaderBacillus subtilis strain QST 713 variant Soil Page 4 of 28

Biopesticides Registration Action Document **I. EXECUTIVE SUMMARY**  Background In July 2011, Agraquest, Inc., submitted an application for a new end

- use pesticide product, Serenade Soil DPZ (EPA File Sy mbol 69592

- EI ), containing a new active ingredient, Bacillus subtilis strain QST 713 variant S oil, to the United States Environmental Protection Agency (EPA) under section 3 of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). Concurrentl y with this application, Agraquest, Inc., filed a petition for an amendment to an existing exemption from the requirement of a tolerance for Bacillus subtilis strain QST to include residues of products containing the active ingredient Bacillus subtilis strain QST variant S oil. Bacillus subtilis strain QST 713 variant S oil is a naturally occurring variant of Bacillus subtilis strain QST 713 . In 2000, EPA first registered the latt er as a pesticide active ingredient for use on several minor crops to treat a variety of plant diseases and fungal pathogens including gray mold, powdery mildew, earl y and late blight, fire blight, scab, sour rot, bacterial spot, and walnut blight. Bacillus subtilis is commonly found in various ecological niches including soil, water and air , and it does not have a history of pathogenici ty

from contact in the environment. Trade names for the registered end

- use products include: Serenade ® , Rhapsody® , Rhapsody® ASO, Serenade ® ASO, Serenade ® MAX TM , Serenade Biofungicide Wettable Powder, Serenade ® AS, Serenade ® Garden Disease Control Concentrate, and Serenade ® Garden Disease Control Ready To Use
. In addition, there are other strains of B acillus subtilis registered as microbial pesticides. The bacterium, Bacillus subtilis , is prevalent in soils and has been found in a variety of habitats worldwide. The QST 713 strain of Bacillus subtilis is known to be antagonistic toward many fungal plant pathogens. This antagonism may be achieved in several ways including nutrient competition, site exclusion, colonization, and attachment of the bacteria to the fungal pathogen. In addition, Agraquest reports that the QST 713 strain of Bacillus subtilis has been shown to induce plants' natural systemic resistance or systemic acquired resistance (SAR) of certain plants against bacterial pathogens. These b acteria can stop plant pathogen spores from germinating, disrupt germ tube growth, and inhibit attachment of the plant pathogen to the leaf. The applicant submitted data to the EPA demonstrating that Bacillus subtilis strain QST 713 variant Soil is stabl e and does not revert back to the parent strain , Bacillus subtilis strain QST 713 , over multiple generations. When compared to the parent strain , the variant can be identified by its distinct rough (sandpaper), adherent phenoty pic colony

morphology
. This p henoty pe is associated with enhanced biofilm formation, which enhances growth promotion and disease protection of the parental strain. Th is gene has been identified as an essential gene for Bacillus to move over solid substrates
. A fter researchers transferred the test substance in flasks 15 times, n o Bacillus subtilis strain QST 713 wild ty pe colonies were found when they plat ed Bacillus subtilis strain QST 713 variant Soil
. These results demonstrate that the particular genetic sequence mutant is st able in Bacillus subtilis strain QST 713 variant Soil and , hence, the strain breeds true

to - form . The EPA determined that the data are acceptable. The proposed end

-use product (EP) containing th is active ingredient , Bacillus subtilis strain QST 713 variant S oil , is Serenade Soil DPZ, a biofungicide to be primarily applied as a soil drench or in

- furrow spray . Th is EP is intended for use in field applications, greenhouses, glasshouses, nurseries, shad e houses, landscapes, interiors capes , seedling production sites, and forest seedling production sites, as well as turf and home and garden sites.Bacillus subtilis strain QST 713 variant Soil Page 5 of 28

Biopesticides Registration Action Document EPA s cientists reviewed product anal ys is, toxicology , and nontarget organism data and information ( 40 CFR §§ 158.2120, 158.2140, and 158.2150, respectivel y) submitted to su pport the registration of the proposed Bacillus subtilis strain QST 713 variant S oil pesticide product. They found that, o verall, such data and information are adequate for risk assessment purposes, fulfill the current microbial pesticide data requirements, and support registration of the proposed product under FIFRA section 3(c)(5)
. Product Anal ys is For the purposes of FIFRA section 3(c)(5) registration, the product anal ys is data requirements for Bacillus subtilis strain QST 713 variant Soil, including product chemistry and composition, anal ys is of samples, phy sical and chemical characteristics, and additional data concerning genetic stability , were fulfilled by acceptable guideline studies. Toxicology The applicant submitted adequate mammalian toxicology data and information to support the pesticide products. A cute oral, pulmonary , and injection tox icity /pathogenicity

studies showed that, at a single high dose, Bacillus subtilis strain QST 713 variant Soil , is not toxic and/or p athogenic via these route s of exposure
. Moreover, the applicant reported that no hy persensitivity

incidents occurred during rese arch, development, or testing of Bacillus subtilis strain QST 713 variant Soil
. Inl ight of the results of the acute toxicity /pathogenicity

data and the absence of hypersensitivity incidents, testing at higher tiers (i.e., Tiers II and III) was not required. EPA concluded that Bacillus subtilis strain QST 713 and Bacillus subtilis strain QST 713 variant Soil are substantially similar for the purposes of assessing toxicity , pathogenicity , and infectivity

( Review of Product Chemistry and Acute Toxcity Studies, Memorandum from Barsoum to Glikes, July

11, 2012 ). Tolerance Exemption The EPA is amending the existing exemption for the requirement of a tolerance for Bacillus subtilis strain QST at 40 CFR 180.1209 to include residues of the active ingredient Bacillus subtilis strain QST variant S oil in or on all agricultural commodities. The EPA conclude d that there is reasonable certainty that no harm will result to the U.S. population, including infants and children, from aggregate exposure to residues of Bacillus subtilis strain QST 713 variant Soil
. Data and information submitted by the petitioner support the conclusion that n o dietary risks are expected to occur as a result of the use of Bacillus subtilis strain QST 713 variant Soil as an active ingredient in pesticide products. The EPA concludes that there will not be s ignificant exposure to Bacillus subtilis strain QST 713 variant Soil through food and drinking water . This conclusion is based on : (1) the propos ed application methods of the end

- use pesticide products (soil directed, soil incorporated , and/or seed directed; no aquatic applications ) ; (2) the filtering effect of many particulate soil ty pes; and (3) the conditions (e.g., filtration and pH adjustments) water is subjected to in wastewater treatment s ys tems and drinking water facilities. Based on their review of the acute oral toxicity and pathogenicity

d ata, the EPA determined that, even if Bacillus subtilis strain QST 713 variant Soil were present on food or in drinking water, human exposure to this bacterium would not result in unreasonable adverse effects.Bacillus subtilis strain QST 713 variant Soil Page 6 of 28

Biopesticides Registration Action Document Occupational Exposure Despite the low toxicological profile of Bacillus subtilis strain QST 713 variant Soil , 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 product label directs h andlers working with Bacillus subtilis strain QST 713 variant Soil in agricultural settings to wear a long

- sleeved shirt, long pants, socks, and shoes. For future products, EPA may require additional PPE (e.g., protective eyewear and/or 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 ) on a product

- specific basis. Nontarget Organisms The applicant submitted rational e bridging studies previously submitted for the registration of Bacillus subtilis strain QST 713 to support the Agency ’s waiving nontarget organism testing for the new active ingredient. Because the two variants of the bacterium are closely related, the EPA accepted th e bridging rational e and determined that the data are sufficient to fulfill the relevant microbial pesticide data requirements and for risk assessment purposes. Further testing of nontarget organisms at higher tier levels (i.e., Tiers II, III, and IV) is not required. The EPA performed an environment al risk assessment , based on data and other information provided by the applicant, and determined that adverse effects to nontarget organisms are not anticipated from the proposed pesticidal uses of Bacillus subtilis strain QST 713 variant Soil. Moreover, the EPA made a “No Effect” determination for direct and indirect effects to listed species and their designated critical habitats resulting from these same proposed pesticidal uses. Public Participation On October 1, 2009, th e EPA announced a new policy to provide a more meaningful opportunity for the public to participate i n major registration decisions before they occur. According to this policy , the EPA intends to provide a public comment period prior to making a registrati on decision for, at minimum, the following types of appli cations: new active ingredients; first food uses; first outdoor uses; first residential use s; or any other registration actions for which the Agency believes there may be significant public interest
. Consistent with the policy of making registration actions more transparent, the proposed pesticide product containing Bacillus subtilis strain QST 713 variant Soil , a new active ingredient, is subject to a 15
- day comment period. In addition to containi ng a new active ingredient, the registration of the pesticide product would result in the first outdoor and food uses for Bacillus subtilis strain QST 713 variant Soil . The docket identification number associated with these registration actions , and access ed through either http://www.regulations.gov or http://www.epa.gov/pesticides/regulating/registration

-status.html , is EPA

- HQ
- OPP
- 201 1
- 0 774
. The following documents are available for comment in EPA

- HQ
- OPP
- 201 1
- 0 774 : (1) draft Bacillus subtilis strain QST 713 variant Soil Biopesticide Registration Action Document (BRAD); (2) environmental risk assessment for Bacillus subtilis strain QST 713 variant Soil ; and (3) draft label for the end

- use pesticide product, Serenade Soil DPZ (EPA File Sy mbol 69592

- EI ). While a final decision on registration is contingent upon review and consideration of public comments, EPA believes that, based upon the dat a and information submitted in support of Serenade Soil DPZ, it is appropriate to issue the registration. The basis for this preliminary decision can be found in the risk assessment for Bacillus subtilis strain QST 713 variant Soil ,Bacillus subtilis strain QST 713 variant Soil Page 7 of 28

Biopesticides Registration Action Document which is characterized throughout this BRAD. **II. ACTIVE**  **INGREDIENT OVERVIEW Biological Name:**  Bacillus subtilis strain QST 713 variant soil **Culture Deposit:** USDA Agricultural Research Service Patent Culture Collection: NRLL B

- 50421 **OPP Chemical Code:**  006 5 79 **Type of Pesticide:** Microbial Pesticide

– Biofungicide See Appendix B for specific information (e.g., use sites, application rate s , methods of application, formulation ty

pe s , and target pests) regarding the proposed pesticide products containing this active ingredient. **III. REGULATORY BACKGROUND A. Applications for Pesticide Product Registration**  In July 2011 , Agraquest , Inc. (address: 1540 Drew Avenue; Davis, California 95618) submitted an application to register an end

- use pesticide product, Serenade Soil DPZ (EPA File Sy mbol 69592
- EI ), under FIFRA section 3. On October 12, 2011 ( 76 FR 63298 ), EPA announced receipt of the application to register a pesticide product containing a new active ingredient for use in field applications, greenhouses, glasshouses, nurseries, shade houses, landscapes, interiors capes, seedling production sites, and forest seedling production sites , as well as turf and home and garden sites. EPA opened a 3 0
- day public comment period pursuant to the provisions of FIFRA section 3(c)(4). No comments were received following this publication. **B. Food Tolerance Exemption** Concurrent with its registration application s and under Federal Food, Drug, and Cosmetic Act (FFDCA) section 408(d), Agraquest, Inc., submitted a petition to amend the exemption from the requirement of a tolerance for Bacillus subtilis strain QST 713 to include residues of B acillus subtilis strain QST 713 variant Soil (Pesticide Petition (PP) IF7896)
. In the Federal Register of September 7, 2011 ( 76 FR 55329 ), the EPA announced that Agraquest, Inc., proposed to amend the exemption from the requirement of a tolerance for resid ues of the microbial pesticide, Bacillus subtilis strain QST 713, in or on all raw agricultural crops , to include residues of Bacillus subtilis strain QST 713 variant Soil and the Agency opened a 30

- day comment period. No comment s w ere received following this publication
. **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.Bacillus subtilis strain QST 713 variant Soil Page 8 of 28

Biopesticides Registration Action Document 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 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 scientificall y acceptable (“ supplemental : upgradeable ”). If a study is rated as “supplemental: upgradeable,” EPA alway s 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 no t required b y 40 CFR P art 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 C ategories are assigned based on the hazard(s) identified from studies and/or other infor mation 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, o r IV, where Toxicity Category Iindicates 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 anal ys is data requirements for Bacillus subtilis strain QST 713 have been fulfilled. Refer to Tables 1, 2, 3, and 4 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 toxicology

data requirements for Bacillus subtilis strain QST 713 variant Soil have been fulfilled. Acceptable Tier Imammalian toxicology data and information support registration of the pesticide product, Serenade Soil DPZ
. EPA conclu ded that Bacillus subtilis strain QST 713 and Bacillus subtilis strain QST 713 variant Soil are substantially similar for the purposes of assessing toxicity , pathogenicity , and infectivity ( Review of Product Chemistry and Acute Toxcity Studies, Memorandum from Barsoum to Glikes, July 11, 2012 )
. Furthermore, Tier II and Tier III studies were not required for Bacillus subtilis strain QST 713 variant Soil because of the lack of acute toxicity

/pathogenicity

in the Tier I studies. For a summary

of the toxicology

data described in sections IV(B)(1)(a) and IV(B)(1)(b) (see pages 8

– 10 ), refer to Table 5 in Appendix A
. For additional information on the generic toxicology

data on Bacillus subtilis strain QST 7 13, see the March 24, 2010, Final Registration Review Decision for Bacillus subtilis available in EPA’s docket at www.regulations.gov , docket number EPA

- HQ
- OPP
- 2007
- 1026.
**a. Acute Toxicity/Pathogenicity** 

**– Tier I**  Acute Oral Toxicity/Pathogenicity

– Rat (Harmonized Guideline 885.3050; Master Record Identification Number (MRID No. 446519

- 06 ) : QST 713 Technical was administered orall y to male and female rats at a dose of approximately 1.13 x 10 8 colony

- forming units ( cfu)/animal inBacillus subtilis strain QST 713 variant Soil Page 9 of 28

Biopesticides Registration Action Document a l millileter (mL) volume. No adverse clinical signs were observed, and gross necrops y did not reveal any abnormalities. The organism cleared from all tissues by Day 14. This study

was rated “ACCEPTABLE for risk assessment purposes (U.S. EP A 2006; Watson et al. 1999)
. Acute Pulmonary Toxicity/Pathogenicity

– Rat (H armonized Guideline 885.3150; MRID No. 446519

- 09

) : Male and female rats were dosed intratracheally with QST 713 technical powder as a suspension in purified water at a concentration of 1.2 x 10 8 cfu/animal. There was no mortality , and other than a mottle lung parenchym a on Day 0, no other adverse effects were observed via gross necropsy . The test micro be (pre

- and post

- heat treatment) was detectable in lungs of rats through Day 35, although at significantly less levels when compared to Day 0. The organism (post

- heat treatment) was cleared from the spleen, liver, and kidneys of all animals by Day 21. Thi s study

was rated “ACCEPTABLE” for risk assessment purposes (U.S. EPA 2006; Watson et al. 1999). Acute Injection Toxicity/Pathogenicity (Intra venous )

– Rat (Harmonized Guideline 885.3200; MRID No 485309

- 09 ) : Researchers exposed thirty 49
- to 84
- day

- old Sprague
- Dawley , albino rat s (15 per sex) by

intravenous route to Bacillus subtilis strain QST 713 variant Soil at doses of 6.6 x 10 8 cfu/rat. They

then observed the rats for up to 21 day s. No animals were killed from dosing, and all animals appeared normal for the duration of the study . The researchers observed no abnormalities, except for 2 enlarged and/or discoloured spleens, at the end of the study . The test organism had cleared completely from treatment group blood and brains by day 21 and showed a pattern of clearance from other organs. They determined the test substance to be non

toxic, non

- infective and non

- pathogenic to rats when injected intravenously at a dose of 6.6 x 10 8 cfu/rat
. The EPA classified this intravenous toxicity /pathogenicity study as acceptable. Hypersensitivity Incidents (Harmonized Guideline 885.3400; MRID 485309

- 02 ) : The applicant reported that n o hy

persensitivity

incidents, including immediate

- ty pe or delayed

- ty

pe reactions of human s and domestic animals , occurred during research, development, or testing of Bacillus subtilis strain QST713 variant Soil . Any future hypersensitivity

incidents must be reported to EPA (refer to test note #3 of 40 CFR § 158.2140(d)). Cell Culture (Harmoni

zed Guideline 885.3500) : This study is not required because Bacillus subtilis strain QST713 variant Soil is not a virus (refer to test note #4 of 40 CFR § 158.2140(d)). Acute Oral Toxicity

– Rat (Harmonized Guideline 870.1100; Master Record Identification Number (MRID No.) 485309

- 10 ) : Researchers fed three 56
- to 84
- day

- old female Sprague
Dawley , albino rat s a single dose of Serenade Soil DPZ ( 1 .72 %, undiluted test article , Bacillus subtilis strain QST 713 variant Soil ) at approximately 2.87 x 10 9 cfu/g at doses of 4.89 mL/kg , or 5000 mg/kg b ody w eight (bw)
. After 14 day s, they observed no deaths or abnormalities. Based on the results of this study , Bacillus subtilis strain QST 713 variant Soil does not appear to be tox ic and/or pathogenic in rats when dosed at approximately 5000 mg/kg bw. The EPA classified the acute oral toxicity

/pathogenicity

as Toxicity

Category IV, the lowest toxicity

category , and the Agency

rated the study

as acceptable
. Acute Inhalation Toxicity

– Rat (Harmonized Guideline 870

- 1300; MRID No. 485309

- 12 ) : Researchers exposed groups of 56

- to 84
- day

- old Sprague
- Dawley , albino rat s (5 per sex) nose only via the inhalation route to Serenade Soil DPZ at a dose of 2.87 x 10 9 cfu/g ( 1.72 % a.i. ) i n undiluted test article s for 4 hours at concentrations of 5.15 mg/L. They observed a few s ymptoms that did not continue after the fourth day . N one of the rats died after 14 days. The EPA classifiedBacillus subtilis strain QST 713 variant Soil Page 10 of 28

Biopesticides Registration Action Document the acute inhalation toxicity as Toxicity Category IV , th e lowest toxicity

category , and the Agency rated the study

as a cceptable Acute Dermal Toxicity

- Rat (Harmonized Guideline 870.1200; MRID No 485309

- 11) : Researchers treated ten 56
- to 84
- day

- old Sprague
- Dawley , albino rat s (5 per sex) to Serenade Soil DPZ, Bacillus subtilis strain QST 713 variant Soil at doses of 4.94 mL/kg , or 5 , 050 mg/kg body weight (bw) , for 24 hours
. They observed the animals one hour after exposure and at periodic intervals for 14 day s. The researchers did not find signs of dermal irritation during any of these observations, and they did not observe any

abnormalities after the rats were sacrificed and dissected. The EPA classified the dermal toxicity as Toxicity

Category IV, and the Agency rated the study as ac ceptable. Acute Eye Irritation Study

- Rabbit (Harmonized Guideline 870.2400; MRID No 485309

- 13) : Researchers instilled 0.1ml of Serenade Soil DPZ ( Bacillus subtilis strain QST 713 variant Soil, 2.87 x 10 9 cfu/g or 1.72 % a.i. into the conjunctival sac of the right eye of 3 female, 84

- day

- old, albino rabbits for 24 hours. They observed the animals for 3 day s and rated the effects with a maximum average irritation score of 2.0, meaning that the formulation is practicall y non irritating to the eye . S ince there were no positive effects and minimal effects cleared in less than 24 hours , the EPA classified the eye irritation as Toxicity Category IV, and the Agency

rated the study as acceptable. Acute Dermal Irritation Study

- Rabbit (Harmonized Guideline 870.2500; MRID No 485309

- 14) : Researchers applied 0.5 mL of Serenade Soil DPZ ( Bacillus subtilis strain QST713 variant Soil, 2.87 x 10 9 cfu/g, 1.72 % a.i. ) to the trunks of 2 male and 1 female albino rabbits and then covered the exposed skin for 4 hours. After observ ing the rabbits for 3 day s (72 hours) , the researchers determined that there was no dermal irritati on to the skin , based on a maximum irritation score of 0 at 24, 48 and 72 h our obs ervations
. The EPA classified the primary dermal irritation as Toxicity

Category IV and rated the study as acceptable. **b. Acute Toxicology and Subchronic Toxicity/Pathogenicity** 

**– Tier II;**  **ReproductiveFertility Effects, Carcinogenicity, Immunotoxicity, and**  **Infectivity/Pathogenicity Analysis** 

**– Tier III**  Tier II and Tier III studies were not required for Bacillus subtilis strain QST 713 variant Soil , based on the lack of acute toxicity /pathogenicity i n the Tier I studies for both Bacillus subtilis strain QST 713 and Bacillus subtilis strain QST 713 variant Soil
. **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 hav e 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 testBacillus subtilis strain QST 713 variant Soil Page 11 of 28

Biopesticides Registration Action Document orders/data call

- ins for the first group of 67 chemicals, which contains 58 pesticide active ingredients and 9 inert ingredients. Bacillus subtilis is not among the group of 58 pesticide active ingredients on the initial list to be screened under the EDSP. The EPA believes that Bacillus subtilis is a substance that would not produce any effect in humans similar to an effect produced b y 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 Bacillus subtilis 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/
. **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) onl y if the EPA determines that the exemption is “safe.” Section 408(c)(2)(A)(ii) of FFDCA defines “safe” to mean that “th ere 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 d rinking water and in residential settings , but does not include occupational exposure. Pursuant to section 408(c)(2)(B) of FFDCA, in establishing or maintaining in effect an exemption from the requirement of a tolerance, 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 c ertainty 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 analy ses 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 th e 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 5 in Appendix A , the data required for a FFDCA risk assessment for Bacillus subtilis strain QST 713 variant Soil have been fulfilled. Hence, the EPA amended the existing exemption for the requirement of a tolerance for Bacillus subtilis strain QST to include residu es of Bacillus subtilis strain QST variant soil.Bacillus subtilis strain QST 713 variant Soil Page 12 of 28

Biopesticides Registration Action Document
**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 diet ary exposure to Bacillus subtilis strain QST 713 variant Soil , a naturall y occurring soil bacterium, is anticipated to be negligible
. Due to the ubiquitous nature of the organism, and the concentrations of Bacillus subtilis and other closely related Bacillus species that already exist in the environment, the likelihood of increased risk to humans or animals from the use of Serenade Soil DPZ is low. The EPA in its registration review decision concluded “the risk posed to adults, infants, and children i s likely to be minimal because of the low acute oral toxicity / p athogenicity potential of Bacillus subtilis s train QST 713.” The EPA conclude d , based on the similarity in product composition and production, measured phy sical/chemical, and pathogenicity /infe ctivity

toxicity data, that the risk from any potential exposure to Bacillus subtilis strain QST 713 variant Soil in Serenade Soil DPZ also would be minimal. Drinking W

ater Exposure and Risk Characterization : Exposure to residues of Bacillus subtilis strai n QST 713 variant Soil in consumed drinking water is unlikel y. The proposed use patterns for Serenade Soil DPZ are soil directed and/or soil incorporated , thereby limiting contact with surface water b y drift and runoff. Furthermore, ground water is not expected to have significant exposure to Bacillus subtilis strain QST 713 variant Soil since, like other microorganisms, this microbial pesticide would like ly be filtered out by the particulate nature of many soil ty pes. If it were to be transferred to surface or ground waters that are intended for eventual human consumption (e.g., through spray drift or runoff) and directed to wastewater treatment sy stems or drinking water facilities, it likely would not survive the conditions water is subjected to in such sy stems or facilities, including chlorination, pH adjustments, filtration , and/or occasionall y high temperatures (Centers for Disease Control and Preventio n 2009; U.S. EPA 2004). In the remote likelihood that Bacillus subtilis strain QST 713 variant Soil is present in drinking water (e.g., water not subject to treatment sy stems or facilities), its target pest specificity and available data indicate no toxici ty and/or pathogenicity is likely to occur with any drinking water exposure to Bacillus subtilis strain QST 713 variant Soil that results from pesticide application s made in accordance with good agricultural practice s (see section IV(B)(1)(a) on pages 8

–9 and Table 5 in Appendix A). Non

- occupational, Residential Risk Characterization : Given Bacillus subtilis strain QST 713 variant Soil ’s natural occurrence in soil, EPA determined that non - occupational exposure to the bacterium likel y is already occurring . Additional exposure to the microorganism , due to pesticidal applications, is not expected because such exposures do not exceed EPA’s level of concern, particularly

in light of available data that demonstrate Bacillus subtilis strain QST 713 and Bacillus subtilis strain QST 713 variant Soil are not toxic (acute dermal toxicity and acute pulmonary

toxicity /pathogenicity), non - irritating (primary dermal irritation), and not pathogenic (acute pulmonary toxicity /pathogenicity) (see section IV(B)(1)(a) on pages 8

–9 and Table 5 in Appendix A).Bacillus subtilis strain QST 713 variant Soil Page 13 of 28

Biopesticides Registration Action Document **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 QST 713 variant Soil to share a common mechanism of toxicity

with other substances, and Bacillus subtilis strain QST 713 variant Soil does not appear to produce a toxic metabolite against target pest s . For the purposes of the tolerance action, therefore, the EPA has assumed that Bacillus subtilis strain QST 713 variant Soil does not have a common mechanism of toxicity with other substances. Thus, section 4 08(b)(2)(D)(v) of the FFDCA does not appl y. 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 chemic al 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 chil dren 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 commonl y referred to as the Food Quality Protection Act Safety Factor. In appl yi ng 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 an d pathogenicity data/information discussed in section IV(B)(1)(a) (see pages 8

–9) and Table 5 in Appendix A , the EPA concludes that there are no threshold effects of concern to infants, children, or adults when Bacillus subtilis strain QST 713 variant Soil is used as labeled in accordance with good agricultural practices. As a result, the EPA concludes that no additional margin of expo sure (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 anal ys is as presented directl y 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 QST 713 variant Soil when it is used

—as labeled and in accordance with good agricultural practices

—as a biofungicide . 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 collectivel y, the data and information available on Bacillus subtilis strain QST 713 and Bacillus subtilis strain QST 713 variant Soil do notBacillus subtilis strain QST 713 variant Soil Page 14 of 28

Biopesticides Registration Action Document demons trate toxic and/ pathogenic potential to mammals , including infants and children. **3. Occupational Exposure and Risk Characterization** The EPA does not expect h andler exposure to Bacillus subtilis strain QST 713 variant Soil to pose any undue risk. Regardless, 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 Serenade Soil DPZ in agricultural settings must wear a long

- sleeved shirt, long pants, socks, and shoes. For future products containing this microorganism as an active ingredient , the EPA may require additional PPE (e.g., protective eyewear and/or dust/mist filtering respirator s) on a product

specific basis. **4. Human Health Risk Characterization**  The EPA considered human exposure to Bacillus subtilis strain QST 713 variant Soil in light of the standard for registration in FIFRA and the relevant safety factors in FFDCA. The Agency determin ed that no unreasonable adverse effects to the United States population in general, and to infants and children in particular, will result when Bacillus subtilis strain QST 713 variant Soil is used in accordance with EPA

- approved labeling. **C. Environmental Assessment**  **( 40 CFR §**  **158.2150 )**  Data and other information (e.g., scientific literature) submitted by the applicant to support requests to waive nontarget organism testing for Bacillus subtilis strain QST 713 variant Soil are sufficient to fulfill the relevant microbial pesticide data requirements and for risk assessment purposes. Further testing of nontarget organis ms at higher tier levels (i.e., Tiers I I, III, and IV) is not required. The EPA performed an environmental risk assessment based on data and other information provided b y the applicant, and the Agency determined that adverse effects to nontarget organisms are not anticipated from the proposed pesticidal uses of Bacillus subtilis strain QST 713 variant Soil . Moreover, the EPA has made a “No Effect” determination for direct and indirect effects to listed species and their designated critical habitats resulting from these same proposed pesticidal uses. For a summary

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

– 17) , refer to Table 6 in Appendix A
. **1. Ecological Exposure and Risk Characterization** 
**a. Terrestrial Animals and Plants**  Birds (Harmonized Guideline 885.4050), Nontarget Insects (Harmonized Guideline 885.4340), and Honey Bees (Harmonized Guideline 885.4380) (MRID No. 48 53909

- 1 5 ) : While applications of the proposed end use product containing B acillus subtilis strain QST 713 variant Soil are soil directed, birds and wild mammals could be exposed. Such e xposure is expected to occur through ingestion of food items that contact the active ingredient, or it may occur through incidental ingestion of soil or drinking water (e.g., puddles) that contain B acillus subtilis strain QST 713 variant Soil. The percentage of the a.i. in the proposed end use product isBacillus subtilis strain QST 713 variant Soil Page 15 of 28

Biopesticides Registration Action Document similar to that of Serenade ASO, a currentl y- registered end use product containing B acillus subtilis strain QST 713
. Since the potency and maximum application rates are the same for both products , the proposed uses of the end

- use product, Serenade Soil DPZ , are expected to result in similar field exposures and the maximum hazard doses used in previousl y submitted studies are sufficient for determining risk. B irds and wild mammals were not adversely a ffected when exposed at high dosage rates , as demonstrated in bird studies with B. subtilis strain QST 713 ( MRID 44651910), and adverse effects were not observed in rats (MRID 44651906). Therefore, adverse effects to birds and wild mammals are not expected if they are exposed to Bacillus subtilis strain QST 713 variant Soil as a result of the proposed labeled uses. Nontarget insects and honey bees may

be exposed if they

contact surfaces, including soil, treated with B acillus subtilis strain QST 713 variant Soil, or if this a.i. is present on food items. Studies submitted for the registration of B acillus subtilis strain QST 713 show that adverse effects to several species of nontarget insects and honey bees do not occur when the bees are exposed at high levels. Researchers observed mortalities in studies with green lacewing ( Chrysoperla carnea ) and lady

bird beetle (

Hippodamia convergens ), but the dead insects were not caused b y treatment with the a.i. S everal supplemental field studies demonstrated that B. subtilis strain QST 713 applied at field rates is not hazardous to honey bees. Based on the results of these studies and expected field exposure levels, EPA scientists do not expect adverse effects to nontarget insects and honey

bees f rom the proposed applications of B. subtilis strain QST 713 variant Soil. The EPA determined that, since Bacillus subtilis strain QST 713 variant Soil is not taxonomically

related to known plant pathogens, adverse effects to nontarget plants are not expected. **b. Aquatic Animals and Plants**  Freshwater Fish (Harmonized Guideline 885.4200, Freshwater Invertebrates (Harmonized Guideline 885.4240) , Estuarine/Marine Fish and Invertebrates (Harmonized Guideline 885.4280) (MRID No. 485390 9

- 15) : Freshwater fish and invertebrates could be exposed to the Bacillus subtilis strain QST 713 variant Soil from runoff and drift into aquatic areas. While most of the proposed applications are soil directed, some applications are allowed to occur via spray equipment (e.g., for turf uses)
. Th ese applications could result in s ome runoff and drift of the applied Bacillus subtilis strain QST 713 variant Soil into aquatic areas , but the concentrations resulting from drift and runoff are expected to be a small fraction of the amount applied. A study conducted with rainbow trout (MRID 45445202) demonstrated that fish died when expos ed to high levels of the a.i.; however, the reviewers determined that these deaths result ed from water fouling b y high concentrati ons of the test material and not from toxicity or pathogenicity
. The reviewers observed s imilar problems i n a study on Daphnia magna , and they determined that mortalities at high exposure levels were also due to fouling of the water ( MRID 48530905). Theref ore, adverse effects to freshwater fish and invertebrates are not expected from proposed applications of Bacillus subtilis strain QST 713 variant Soil as labeled. The EPA determined, b ased on its analy sis of freshwater animals, that high concentrations of B acillus subtilis strain QST 713 variant Soil are not expected to be deposited and persist in estuarine or marine areas. Therefore, estuarine and marine fish and invertebrates are not likely to be exposed , and the risk to these speci es as a result of the proposed applications of B acillusBacillus subtilis strain QST 713 variant Soil Page 16 of 28

Biopesticides Registration Action Document subtilis strain QST 713 variant Soil is minimal
. **2. Environmental Fate Data**  As the data and information provided are sufficient to fulfill the Tier Inontarget organism data requirements and allow for nontarget organism risk asse ssment for Bacillus subtilis strain QST 713 variant Soil , f

urther testing at higher tier levels (i.e., Tiers II, III, and IV) is not required. **3. Threatened and Endangered Species Assessment** Since the EPA has determined that no effects are anticipated for any nontarget species exposed to Bacillus subtilis strain QST 713 variant Soil as a result of the proposed applications, effects to federally listed threatened and endangered species and their designated criti cal ha bitats also are not expected. Therefore, a “No Effect” determination is made for direct and indirect effects to listed species and their designated critical habitats resulting from the proposed pesticidal uses of Bacillus subtilis strain QST 713 vari ant Soil , as labeled. **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, implementati on, and enforcement of environmental laws, regulations, and policies. Fair treatment means that no group of people, including racial, ethnic, or socioeconomic groups, should bear a disproportionate share of the negative environmental consequences resulting from industrial, municipal, and commercial operations or the execution of federal, state, local, and tribal environmental programs and policies. Meaningful involvement means that (1) potentially affected community residents have an appropriate opportunity to participate in decisions about a proposed activity that will affect their environment and/or 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 d ecision

- 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, the EPA seeks information on any groups or segments of the population who, as a result of their location, cultural practices, or other factors, may

have atypical, unusually

high exposure to Bacillus s ubtilis strain QST 713 variant Soil , 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 web site at http://www.epa.gov/compliance/environmentaljustice/index.html
. **V I**  **. RISK MANAGEMENT DECISION**  Section 3(c)(5) of FIFRA permits for the registration of a pesticide provided that all the following determinations are made: (1) Its composition is such as to warrant the proposed claims for it;Bacillus subtilis strain QST 713 variant Soil Page 17 of 28

Biopesticides Registration Action Document (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 generall y cause unr easonable adverse effects on the environment. To satisfy criterion 1, the Bacillus subtilis strain QST 713 variant Soil product ha s well

- known properties. The EPA has no knowledge that would contradict the claims made on the Serenade Soil DPZ label , and this p esticide product is not expected to cause unreasonable adverse effects on the environment when used according to label instructions. Criterion 2 is satisfied by the current product label , as well as the data and information presented in this do cument. The Agency believe s that the Bacillus subtilis strain QST 713 variant Soil p esticide product will not cause any unreasonable adverse effects on the environment . In fact , Serenade Soil DPZ is likel y to provide protection against many plant diseases , satisfying criterion 3. Criterion 4 is satisfied in that the Bacillus subtilis strain QST 713 variant Soil pesticide product is not expected to cause unreasonable adverse effects when used according to label instructions. Therefore, Serenade Soil DPZ , containing Bacillus subtilis strain QST 713 variant Soil as a new active ingredient, is eligible for registration under FIFRA section 3(c)(5) for the labeled uses. **V II**  **. ACTIONS REQUIRED OF THE REGISTRANT**  **A. Final Printed Labeling** Before Serenade Soil DPZ

— the pesticide product containing Bacillus subtilis strain QST 713 variant Soil

–may be released for shipment, the registrant will be required to provide appropriate final printed labeling to the EPA
. **B. Terms of Registration** As a term of a Serenade Soil DPZ registration, Agraquest , Inc. will be required to submit additional information (confirmatory) on the product within six months of registration. Additionally , as term s of a Serenade Soil DPZ registration, Agraquest , Inc. will be required to submit within one year of the date of registration a study on storage stability ( OCSPP 830.6317 ) and a study demonstrating that Bacillus subtilis strain QST 713 variant Soil does not revert to Bacillus subtilis strain QST 713. **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 incident s of hy persensitivity

(including both suspected and confirmed incidents) must be reported to EPA under the provisions of 40 CFR § 158.2140(d).Bacillus subtilis strain QST 713 variant Soil Page 18 of 28

Biopesticides Registration Action Document **VIII. GLOSSARY OF ACRONYMS AND ABBREVIATIONS**  BRAD Biopesticides Registration Action Document CFR Code of Federal Regulations cSt centistokes EDSP Endocrine Disruptor Screening Program EP end

- use pesticide product EPA United States Environmental Protection Agency (the “Agency”) FFDCA Federal Food, Drug, and Cosmetic Act FIFRA Federal Insecticide, Fungicide, and Rodenticide Act FR Federal Register g/mL grams per milliliter lb/gal pounds per gallon LD 50 median lethal dose. A statistically derived single dose that can be expected to cause death in 50% of the test animals when 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 MRID No. Master Record Identification Number NI OSH National Institute for Occupational Safety and Health OPP Office of Pesticide Programs PC Code Pesticide Chemical Code PP Pesticide Petition PPE personal protective equipment TGAI technical grade of the active ingredient U.S. United StatesBacillus subtilis strain QST 713 variant Soil Page 19 of 28

Biopesticides Registration Action Document **I X. BIBLIOGRAPHY**  **A. Studies Submitted to Support the**  **Active Ingredient, Bacillus subtilis**  **strain QST 713 variant Soil**  **, and Pesticide Product**  **Registration , Serenade Soil DPZ.**  MRID No. Study Information 44651901 Bellet, E. (1998) Product Chemistry for QST 713 Technical. Unpublished study p repared b y Chemical Consultants International, Inc. 4 p. 44659104 Bellet, E. (1998) Manufacturing and Anal ytical Data for Technical QST 713.

Unpublished study

prepared by

Chemical Consultants International, Inc. 48 p. 45206101 Hilbig, L. (2000) Manufacturing Process Amendment for QST 713 Technical

Powder. Unpublished study prepared by

AgraQuest, Inc. 23 p. 44651905 Gingras, B. (1998) S ensitivity of Detection of Bacillus subtilis Strain QST 713 for Toxicity /Pathogenicity Testing in Rats: Lab Project Number: L08726SN3. Unpublished study

prepared by

IIT Research Institute. 30 p 44651906 Harrington, K. (1998) Toxicity /Pathogenic ity

Testing of QST 713 Following Acute Oral Challenge in Rats: L ab Project Number: L 08726SN4. Unpublished study prepared by

IIT Research Institute. 126 p. 44651907 Findlay , J. (1998) Acute Dermal Toxicity /Pathogenicity

Study of QST 713 in Ra bbits: Lab Project Number: L 08726SN7. Unpublished study prepared by

I IT Research Institute. 27 p. 44651909 Harrington, K. (1998) Toxicity /Pathogenicity

Testing of QST 713 Following Acute Intratrache al Challenge in Rats: Lab Project Number: L08726SN6. Unpublished study

prepared by

IIT Research Institute. 137 p. 44651908 Harrington, K. (1998) Toxicity /Pathogenicity

Testing of QST 713 Following Acute Intravenous Challenge in Rats: Lab Project Number: L08726SN5. Unpublished study

prepared by

IIT Research Institute. 119 p. 44651910 Foster, J.; Grimes, J.; Beavers, J. (1998) An Avian Oral Pathogenicity and

Toxicity Study in the Northern Bobwhite: (QST 713): Lab Project Numbe r: 489
- 101. Unpublished study

prepared by

Wildlife International Ltd. 28 p. 45455202 Drottar, K.; Flaggs, R.; Krueger, H. (2001) QST 713 Technical: A Five

Concentration Toxicity

and Pathogenicity Test with the Rainbow Trout (Oncorhyn chus my

kiss): Final Report: Lab Project Number: 489A

- 108. Unpublished study

prepared by

Wildlife International, Ltd. 39 p. 44651912 Drottar, K.; Krueger, H. (1998) A 48

- Hour Static Acute Toxicity Test with the Cladoceran (Daphnia Magna): (QST 713): Final Report: Lab Project Number: 489A
- 103. Unpublished study prepared by

Wildlife International Ltd. 22 p.Bacillus subtilis strain QST 713 variant Soil Page 20 of 28

Biopesticides Registration Action Document MRID No. Study Information 45455203 Drottar , K.; Flaggs, R.; Krueger, H. (2001) QST 713 Technical: A 21

- Day L ife

Cycle Toxicity

and Pathogenicity

Test with the Cladoceran (Daphnia magna): Final Report: Lab Project Number: 489

- 107A. Unpublished study prepared by Wildlife International, Ltd. 39 p. 4 5455205 Bryan, R.; Porch, J.; Krueger, H. (2001) Bacillus Subtilis Strain QST 713: A

Dietary Pathogenicity and Toxicity Study with the Parasitic Hy

menopteran (Nasonia vitripennis): Final Report: Lab Project Number: 489

- 107B. Unpublished study prepared by

W ildlife International, Inc. 25 p
. 44651915 Hoxter, K.; Krueger, H.; Palmer, S. (1998) A Dietary Pathogenicity Toxicity Study with the Lady

bird Beetle (Hippodamia convergens): (QST 713): Lab Project Number: 489

- 103B. Unpublished study prepared b y Wildlife International Ltd. 24 p. 44651916 Hoxter, K.; Krueger, H.; Palmer, S. (1998) A Dietary Pathogenicity Toxicity Study with the Green Lacewing Larvae (Chry soperia carnea): (QST 713): Lab Project Number: 489

- 104. Unpublished study prepared by

Wildlife International Ltd. 17 p. 46172902 Jimenez, D.; Richards, K. (2004) Evaluation of the Dietary Effect(s) of QST 713 Technical Powder on Larval Honey bee Development (Apis mellifera L.). Project Number: CAR/158/03. Unpublished study prepared by

California Agricultural Research Inc. 70 p. 46172901 Jimenez, D. (2004) Discussion of the Results of Honey bee Studies Conducted with QST 713 Technical and Serenade Products. Unpublished study prepared by Agraquest Inc and California State Poly technic College and Montana State University . 237 p. 45385301 Van Koppenhagen, J.; Hilbig, L. (2001) Product Chemistry of Serenade AS: L ab Project Number: QC

- 012301. Unpublished study prepared by

AgraQuest, Inc. 95 p. 45385309 Freeman, D. (2000) Dermal Sensitization Study (Closed

- Patch Repeated Insult) in Guinea Pigs with Serenade AS: Lab Project Number: 012774

- 1. Unpublished study prepared by

Ricerca, LL C. 36 p. 46012201 Lin, J.; Martinez, T.; Czochor, L. (2003) Product Chemistry of Serenade ASO. Project Number: AC/F/007/1. Unpublished study prepared by

AgraQuest Inc. 79 p. 45662301 Czochor, L . (2002) Amendment to MRID No. 45385301, Manufacturing Process:

Submission of Replacement Page 17. Unpublished study prepared by

AgraQuest, In c. 8 p
.Bacillus subtilis strain QST 713 variant Soil Page 21 of 28

Biopesticides Registration Action Document MRID No. Study Information 47720601 Heins, S. (2009) Product Chemistry of QRD 146. Project Number: 090225/1. Unpublished study

prepared by

AgraQuest, Inc. 67 p. 48 5 30900 AgraQuest, Inc. (2011) Submission of Product Chemistry and Toxicity

Data in Support of the Application for Registration of Serenade Soil DPZ. Transmittal of 15 Studies
. 48 5 30901 Heins, S.; Joo, D. (2011) Supplemental Information on Bacillus Identification as

Discussed in Amended Final Report 14676

- 10 and Final Report 15246

- 11. Project Number: REG110624/1. Unpublished study prepared by

AgraQuest, Inc. 126 p. 48 5 30902 Heins, S.; Thurston, J.; Guilhabert

- Go ya, M.; et al. (2011) Product Chemistry of

Bacillus subtilis Strain QST 713 Variant Soil (TGAI). Pro ject Number: 110509/2. Unpublished study

prepared by

AgraQuest, Inc. 107 p. 48 5 30903 Kaminsky , M. (2011) QST 713 Strain of Bacillus subtilis Variant Soil (TGAI):

Product Chemistry : Amended Final Report. Project Number: 15001/11. Unpublished study

prepared by

Stillmeadow, Inc. 19 p. 48 5 3 0 904 Monds, K. (2011) QST 713 Strain of Bacillus subtilis Variant Soil: I dentification

and Enumeration of a Microbial Pest Control Agent (MPCA): Final Report. Project Number: 15246/11. Unpublished study prepared by

Stillme adow, Inc. 10 p. 48 5 30905 Heins, S.; Thurston, J.; Guilhabert

- Go ya, M.; et al. (2011) Product Chemistry of

Serenade Soil DPZ (EP). Project Number: 110509/1. Unpublished study prepared by AgraQuest, Inc. 70 p. 48 5 30906 Kaminsky , M. (2011) Serenade Soil DPZ (EP): Product Chemistry : Final Report.

Project Number: 15000/11. Unpublished study prepared by

Stillmeadow, Inc. 9 p. 48 5 30907 Sehn, A.; Heins , S. (2007) Serenade ASP Storage Stability at 25 Degrees Celsius. Project Number: AQ/040514/2, AQ/061121/1. Unpublished study prepared by AgraQuest, Inc. 42 p. 48 5 30908 Heins, S. (2011) Bacillus subtilis Strain QST 713 Variant Soil: Request for Acute

Mammalian Toxicity

/ Pathogenicity

Study Waivers. Project Number: REG110602/1. Unpublished study prepared by

AgraQuest, Inc. 11 p. 48350909 Monds, K. (2011) QST 713 Strain of Bacillus subtilis Variant Soil: Intravenous

Toxicity / Pathogenicity Study

in Rats : Amended Final Report. Project Number: 14676/10. Unpublished study prepared by

Stillmeadow, Inc. 42 p. 48530910 Monds, K. (2011) QST 713 Strain of Bacillus subtilis Variant Soil: Intravenous

Toxicity / Pathogenicity Study

in Rats: Amended Final Report. Project Number: 14676/10. Unpublished study prepared by

Stillmeadow, Inc. 42 p.Bacillus subtilis strain QST 713 variant Soil Page 22 of 28

Biopesticides Registration Action Document MRID No. Study Information 48530911 Kuhn, J. (2011) Serenade Soil DPZ: Acute Dermal Toxici ty Study in Rats: Final Report. Project Number: 14932/11. Unpublished study prepared by

Stillmeadow, In c. 13 p. 48530912 Doig, A. (2011) Serenade Soil DPZ: Acute Inhalation Toxicity Study in Rats: Final Report. Project Number: 14933/11. Unpublished study prepared by Stillmeadow, Inc. 18 p. 48530913 Kuhn, J. (2011) Serenade Soil DPZ: Acute Eye Irritation Study in Rabbits: Final Report. Project Number: 14934/11. Unpublished study prepared by

Stillmeadow, In c. 17 p. 48530914 Kuhn, J. (2011) Serenade Soil DPZ: Acute Dermal Irritation Study in Rabbits: Final Report. Project Number: 14935/11. Unpublished study prepared by Stillmeadow, Inc. 11 p. 48530915 Heins, S. (2011) Bacillus subtilis Strain QST 713 Variant Soil: Nontarget

Organism Toxicity

/Pathogenicity

Study Waiver Requests. Project Number: REG110603/2. Unpublished study prepared by

AgraQuest, Inc. 15 p. 48906300 AgraQuest, Inc. (2012) Submission of Product Chemistry Data in Support of the Application for Registration of Serenade Soil DPZ. Transmittal of 1 Study. 48906301 Curtis, D. (2012) Bacillus subtilis Strain QST 713 Variant Soil (TGAI): Product

Id entity : Further Information on the Characterization of the MPCA. Project Number: 120810/AQ/REG/01. Unpublished study prepared b y AgraQuest , Inc. 6p. **B. Environmental Protection Agency Risk Assessment Memoranda** U.S. EPA. 201 2 a. Review of Product Chemistry and Acute Toxicity

Studies for section 3 registration of a new TGAI Bacillus subtilis s train QST 713 v ariant Soil and a new EP Serenade Soil DPZ (EPA Reg. no. 69592

- EI ). Memorandum from Ibrahim S. Barsoum, Ph.D. and John L. Kough, Ph.D. to Michael Glikes , dated July 11 , 2012. U.S. EPA. 201 2 b. Environmental Risk Assessment f or a FIFRA Section 3 Registration of Bacillus subtilis s train QST 713 v ariant Soil (EPA File Sy mbol 69592
- EI ). Memorandum from Shannon Borges to Michael Glikes , dated August 2 , 2012. U.S. EPA. 2012c. Review of additional data concerning the genetic stability of Bacillus subtilis strain QST 713 variant Soil for section 3 registration of the TGAI and a new EP Serenade Soil DPZ (EPA Reg. no. 69592

- EI ). Memorandum from Ibrahim Barsoum, Ph.D. and John L . Kough, Ph.D. to Michael Glikes, dated August 23, 2012. U.S. EPA. 2010. Bacillus subtilis Case 6012 Final Registration Review Decision, March 2010.Bacillus subtilis strain QST 713 variant Soil Page 23 of 28

Biopesticides Registration Action Document **C. Other References**  Centers for Disease Control and Prevention. 2009. Drinking Water

- Water Treatment. Available from http://www.cdc.gov/healthywater/drinking/public/water_treatment.html
. DeFelice K, Wollenhaupt N, Buchholz D. 1993. Aquifers and Soil Filter Effect. Available from http://extension.missouri.edu/p/W Q24
. U.S. EPA. 2004. Primer for Municipal Wastewater Treatment S ys tems. EPA 832
- R
- 04
- 001. Available from http://www.epa.gov/npdes/pubs/primer.pdf
.Bacillus subtilis strain QST 713 variant Soil Page 24 of 28

Biopesticides Registration Action Document

**APPENDIX A. MICROBIAL PESTICIDES DATA REQUIREMENTS** 

**(40 CFR PART 158**

**– SUBPART V)**  **TABLE 1. Product Analysis Data Requirem ents for the Active Ingredient (TGAI),**  **Bacillus subtilis strain QST 713 variant Soil**  **(40 CFR § 158.2120) Harm**  **onized G**  **uideline N**  **umber Data**  **Requirement Results**  **MRID No.**
885.1100 Product Identity Submitt ed data fulfill the requirement for product identity. Bacillus subtilis strain QST 713 variant Soil is a naturally occurring variant of Bacillus subtilis strain QST 713
. 446519
- 01
885.1200 Manufacturing

Process Submitted data fulfill the requirement for manufacturing process
. 446519
- 01, 446419
- 04

N/A Deposition of a

Sam ple in a Nationally Recognized Culture Collection Bacillus subtilis strain QST 713 variant Soil is on deposit with the USDA

Agricultural Research Service Patent Culture Collection: NRLL B

- 50421
. 446519
- 04

885.1300 Discussion of

Formation of Unintentional Ingredients **As a term of registration, EPA is requiring additional inform**  **ation (confirm**  **atory) on this data requirement.**  446519
- 04

885.1400 Analysis of S amples Submitted data fulfill the requirement for analysis of samples. 446519
- 02
8 85 .1 50 0 Certification of

L imits Submitted data fulfill the requirement for manufacturing process. 450845
- 01

**TABLE 2 .**  **Product Analysis Data Requirem ents for the End**  **- Use Pesticide Product (EP),**  **Serenade Soil DPZ (40 CFR § 158.2120)**  **Harm onized**  **Guideline Number**  **Data Requirement Results**  **MRID No.**

885.1100 Product Identity Submitted data fulfill the requirement for product identity. Serenade Soil DPZ contains 1.57% by weight Bacillus subtilis strain QST 713 variant Soil (contains at least 1 x 10 9 cfu per gram). 485309
- 05

885.1200 Manufacturing Process Submitted data fulfill the requirement for manufacturing process. 485309
- 05
N/A Deposition of a Sample in a

Nationally Recognized Culture Collection N/A N/A 885.1300 Discussion of Formation of

Unintentional Ingredients **As a term of registration, EPA is requiring additional**  **inform ation (confirmatory) on this data requirement.**  485309
- 05 885.1400 Analysis of Samples Submitted data fulfill the requirement for analysis of samples for purposes of FIFRA section 3(c)(5) registration. 485309
- 05 885.1500 Certification of Limits Limits listed on the confidential statement of formula are adequate/acceptable. 485309
- 05Bacillus subtilis strain QST 713 variant Soil Page 25 of 28

Biopesticides Registration Action Document **TABLE 3**  **. Physical and Chemical Characteristics for the Technical Grade of the Active Ingredient (TGAI)**  **, Bacillus subtilis**  **strain QST 713 variant Soil**  **(40 CFR § 158.2120)**  **Harm onized**  **G uideline**  **N umber**  **Data Requirement Results**  **MRID No.** 830.6302 Color B rown or light brown 48 5309
- 03 830.6303 Phy sical State Liquid 830.630 4 Odor Like yeas t
830.6313 Stability to Normal and E levated Tem peratures, Metals, and Metal I ons Unstable to elevated temperatures and to metals and metal ions. 485309
- 03
830.6317 Storage Stability **As a term of registration, EPA is requiring additional**  **inform ation (confirmatory) on this data requirement.**  485309
- 03 830.6319 Miscibility Non emulsifiable liquid. N/A 830.6320 Corrosion

Characteristics N/A N/A 830.7000 pH 7.34 (1% solution in distilled water) 485309
- 03 830.7100 Viscosity N/A (not a homogenous solution) 485309
- 03 830.7300 Density/Relative

Density/Bulk Density (Specific Gravity) 1.0 2 485309
- 03 **TABLE 4. Physical and Chemical Characteristics for the End -**  **Use Pesticide**  **Product (EP), Serenade Soil DPZ**  **(40 CFR § 158.2120) Harm**  **onized Guideline Number**  **Data Requirement Results**  **MRID No.** 830.6302 1 Color Medium brown N/A 485309
- 06 830.6303 1 Phy sical State Liquid 485309
- 06 830.6304 1 Odor Yeast like odor 481460
- 06 830.6313 1 Stability to Normal and

Elevated Temperatures, Metals, and Metal Ions Unstable to elevated temperatures and to metals and metal ions. 460122
- 01 830.6317 Storage Stability **As a term of registration, EPA is requiring additional**  **inform ation (confirmatory) on this data requirement.**  485309
- 08
830.6319 Miscibility Not required because the end

- use pesticide product, Serenade Soil DPZ, is not an emulsifiable liquid form of microbial pesticides (refer to test note #2 of 40 CFR § 158.2120(d)). N/A 830.6320 Corrosion Characteristics N/A 460122
- 01 830.7000 pH 5.2
- 5.4 485309
- 06 830.7100 Viscosity 10

– 100 cPs 485309
- 06 830.7300 Density/Relative

Density/Bulk Density (Specific Gravity) 1.000

– 1.075 g/cc 485309
- 03
1 According to 40 CFR § 158.2120, these data are only required for the technical grade of the active ingredient. Since Agraquest , Inc. included this information with its application for Serenade S oil DPZ , it is summarized appropriately in this table.Bacillus subtilis strain QST 713 variant Soil Page 26 of 28

Biopesticides Registration Action Document
**TABLE 5. Toxicology**  **Data Requirements for the**  **Active Ingredient Bacillus subtilis**  **strain QST 713 variant Soil**

**and the End Use Pesticide Product Serenade Soil DPZ (40 CFR § 158.2140)**  **Harm onized**  **Guideline Number**  **Data Requirement Results**  **MRID No.**  **Tier I** 885.3050 Ac ute Oral

Toxicity/Pathogenicity Waived based on the results of MRID No. 446519

- 06, conducted on

Bacillus subtilis strain QST 713 **Classification: Acceptable TOXICITY**  **CATEGORY IV** 4 46519
- 06 489309
- 08

885.3150 Ac ute Pulmonary

Toxicity/Pathogenicity Waived based on the results of MRID No. 446519

- 09, conducted on

Bacillus subtilis strain QST 713 No mortality and no adverse effects. The test microbe was detectable (both pre - and post

- heat treatments) in the lung through day 35, but at significantly reduced levels compared to day 0. **Classification: Acceptable** 446519
- 09 489309
- 08

885.3200 Ac ute Injection

Toxicity/ Pathogenicity No mortality and no adverse clinical signs in rats. The test substance

(Bacillus subtilis strain QST 713 variant Soil) cleared from all organs by day 21. **Classification: Acceptable** 483509
- 09

885.3200 Ac ute Injection

Toxicity/Pathogenicity (Intravenous) No mortality and no adverse clinical signs in rats dosed with QST 713

Technical powder. The test mi crobe cleared from most organs by day 35; however, low

levels were detected in the spleen and liver after 35 days. **Classification: Acceptable** 446519
- 08

885.3400 Hy

persensitivity Incidents The applicant reported that no hypersensitivity incidents, including

immediate

- ty

pe or delayed

- type reactions of humans and domestic animals, occurred during research, development, or testing of the TGAI. Any future hypersensitivity incidents must be reported to EPA (refer to test note #3 of 40 CFR § 158.2140(d))
. 48530902

885.3500 Cell Culture Not required because **Bacillus subtilis strain QST 713 variant Soil**  is not a virus (refer to test note #4 of 40 CFR § 158.2140(d)). N/A 870.1100 Ac ute Oral Toxicity Oral LD 50 female rats > 5,000 mg/kg body weight (bw) **Classification: Acceptable TOXICITY CATEGORY I**  **V** 485309
- 10
870.1200 Ac ute Dermal Toxicity Dermal LD 50 com bined (male and female rats) > 5,050 mg/kg bw **Classification: Acceptable TOXICITY CATEGORY**  **IV** 485309
- 11
870.1300 Ac ute Inhalation

Toxicity LC 50 com bined (male and female rats) >5.15 mg/L **Classification: Acceptable TOXICITY**  **CATEGORY III** 483509
- 12
870.2400 Ac ute Eye Irritation Serenade Soil DPZ was essentially non

- irritating to the eyes of rabbits. **Classification: Acceptable TOXICITY CATEGORY IV**  485309
- 13
870.2500 Primary Dermal

Irritation The EP test substances were essentially non

- irritating to the skin of rats.

**Classification: Acceptable TOXICITY CATEGORY I**  **V** 485309
- 14

**Tiers II and III** Not required for **Bacillus subtilis strain QST 713 variant Soil**  , based on the lack of acute toxicity/pathogenicity in the Tier I studies.Bacillus subtilis strain QST 713 variant Soil Page 27 of 28

Biopesticides Registration Action Document **TABLE 6**  **. Nontarget Organism Toxicity and Environmental Fate Data Requirem**  **ents for the Technical Grade of the Active** 

**Ingredient (TGAI), Bacillus subtilis**  **strain QST 713 variant Soil (40 CFR § 158.2150)**  **Harm onized**  **Guideline Number**  **Data Requirement**  **Results MRID No.**  **Tier I** 

885.4050 Avian Oral Toxicity Data and other information provide sufficient information to determine that toxicity/pathogenicity to avian wildlife is not expected as a result of the proposed pesticidal uses. **Classification: Acceptable** 48 5309
- 15

885.4100 Avian Inhalation Toxicity/Pathogenicity Not required as the nature of the microbial pesticide does not indicate potential pathogenicity to birds or relatedness to any known bird pathogens (refer to test note #3 of 40 CFR § 158.2150(e)). N/A

885.4150 Wild Mammal Toxicity/Pathogenicity Tests required by 40 CFR § 158.2140 are adequate and appropriate for assessment of hazards to wild mammals. Studies submitted with laboratory rats indicate no adverse effects due to oral exposure. **Classification: Acceptable for wild m ammal**  **risk assessment** N/A

885.4200 Freshwater Fish Toxicity/Pathogenicity Data and other information provide sufficient information to determine that toxicity/pathogenicity to freshwater fish and invertebrates is not expected as a result of the proposed pesticidal uses. **Classification: Acceptable** 48 5309
- 15 885.4240 Freshwater Invertebrate Toxicity/Pathogenicity

885.4280 Estuarine/Marine Fish and Invertebrate Testing Not required as the microbial pesticide will not be applied directly to water and is not expected to enter marine/estuarine environments in amounts that would result in significant concentrations (refer to test note #6 of 40 CFR § 158.2150(e)). N/A

885.4300 Nontarget Plant Testing Not required as the microbial pesticide is not taxonomically related to any known plant pathogens (refer to test note #7 of 40 CFR § 158.2150(e)). N/A
885.4340 Nontarget Insect Testing Data and other information provide sufficient information to determine that toxicity/pathogenicity to honey bees and nontarget insects is not expected as a result of the proposed pesticidal uses. **Classification: Acceptable** 48 5309
- 15 885.4380 Honey Bee Testing **Tiers II, III, and IV** Not required for Bacillus subtilis strain QST 713 variant Soil based on the acceptability of the data and other information provided for Tier I
.Bacillus subtilis strain QST 713 variant Soil Page 28 of 28

Biopesticides Registration Action Document **APPENDIX B. PESTICIDE PRODUCT**
**EPA File**  **Sym bol**  **Registration**

**Name Percentage** 

**Active Ingredient**  **Formulation**

**Type Use Site(s)**  **Method(s) of Application**  **Application Rate (s)**  **Target Pest**

69592
- EI Serenade

Soil DPZ 1.57 % End

-use product Agricultural

Seed treatment Residential Ground application

Aerial Chemicagation Tank mix Seed treatment Hose
- end sprayer 1.5

– 14.7 fluid ounces/ 1000 row
- ft. Soil drench: 1

- 6 qt./acre Seed treatment: 1

- 10 oz./100 lbs. of seed Home and Garden: 2
- 8 fl. oz. per 1,000 square feet Soil diseases