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                Bacillus subtilis strain QST 713 variant Soil  
                                       
                     Pesticide Chemical (PC) Code: 006579
                                       
                                       
                                       
                                       

   
	U.S. Environmental Protection Agency
	Office of Pesticide Programs
                Biopesticides and Pollution Prevention Division
                                       
                                       
                           DRAFT  -  October 3, 2012
                                       
                                       
                                       
                               TABLE OF CONTENTS
I.  	EXECUTIVE SUMMARY	4
II.  	ACTIVE INGREDIENT OVERVIEW	7
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	16
VI.  	RISK MANAGEMENT DECISION	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 Soil, and Pesticide Product, Serenade Soil DPZ, Registration	19
				B.  Environmental Protection Agency Risk Assessment Memoranda	22
				C.  Other References	23
APPENDIX A.  MICROBIAL PESTICIDES DATA REQUIREMENTS	24
APPENDIX B.  PESTICIDE PRODUCT	28
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                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
Shannon Borges					   Environmental Effects

                                  Regulations

Kimberly Nesci					   Acting Chief, Microbial Pesticides Branch
Michael Glikes	Regulatory Action Leader

I.  EXECUTIVE SUMMARY

Background

In July 2011, Agraquest, Inc., submitted an application for a new end-use pesticide product, Serenade Soil DPZ (EPA File Symbol 69592-EI), containing a new active ingredient, Bacillus subtilis strain QST 713 variant Soil, to the United States Environmental Protection Agency (EPA) under section 3 of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). Concurrently 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 Soil.

Bacillus subtilis strain QST 713 variant Soil is a naturally occurring variant of Bacillus subtilis strain QST 713. In 2000, EPA first registered the latter 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, early 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 pathogenicity from contact in the environment. Trade names for the registered end-use products include: Serenade[(R)], Rhapsody[(R)], Rhapsody(R) ASO, Serenade(R) ASO, Serenade(R) MAXTM , Serenade Biofungicide Wettable Powder, Serenade(R) AS, Serenade(R) Garden Disease Control Concentrate, and Serenade(R) Garden Disease Control Ready To Use. In addition, there are other strains of Bacillus 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 bacteria 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 stable 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 phenotypic colony morphology. This phenotype is associated with enhanced biofilm formation, which enhances growth promotion and disease protection of the parental strain. This gene has been identified as an essential gene for Bacillus to move over solid substrates. After researchers transferred the test substance in flasks 15 times, no Bacillus subtilis strain QST 713 wild type colonies were found when they plated Bacillus subtilis strain QST 713 variant Soil. These results demonstrate that the particular genetic sequence mutant is stable 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 this active ingredient, Bacillus subtilis strain QST 713 variant Soil, is Serenade Soil DPZ, a biofungicide to be primarily applied as a soil drench or in-furrow spray. This EP is intended for use in field applications, greenhouses, glasshouses, nurseries, shade houses, landscapes, interiorscapes, seedling production sites, and forest seedling production sites, as well as turf and home and garden sites. 

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 Bacillus subtilis strain QST 713 variant Soil pesticide product. 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 proposed product under FIFRA section 3(c)(5).

Product Analysis

For the purposes of FIFRA section 3(c)(5) registration, the product analysis data requirements for Bacillus subtilis strain QST 713 variant Soil, including product chemistry and composition, analysis of samples, physical 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. Acute oral, pulmonary, and injection toxicity/pathogenicity studies showed that, at a single high dose, Bacillus subtilis strain QST 713 variant Soil, is not toxic and/or pathogenic via these routes of exposure. Moreover, the applicant reported that no hypersensitivity incidents occurred during research, development, or testing of Bacillus subtilis strain QST 713 variant Soil. In light 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 Soil in or on all agricultural commodities. The EPA concluded 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 no 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 significant exposure to Bacillus subtilis strain QST 713 variant Soil through food and drinking water. This conclusion is based on: (1) the proposed 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 types; and (3) the conditions (e.g., filtration and pH adjustments) water is subjected to in wastewater treatment systems and drinking water facilities. Based on their review of the acute oral toxicity and pathogenicity data, 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.



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 handlers 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 rationale 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 the bridging rationale 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 environmental 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, 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 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 containing 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 accessed through either http://www.regulations.gov or http://www.epa.gov/pesticides/regulating/registration-status.html, is EPA-HQ-OPP-2011-0774. The following documents are available for comment in EPA-HQ-OPP-2011-0774: (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 Symbol 69592-EI). While a final decision on registration is contingent upon review and consideration of public comments, EPA believes that, based upon the data 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, 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: 		006579	
 
	Type of Pesticide:				Microbial Pesticide  -  Biofungicide

					See Appendix B for specific information (e.g.,
                        use sites, application rates, methods of application, 
                        formulation types, 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 Symbol 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, interiorscapes, seedling production sites, and forest seedling production sites, as well as turf and home and garden sites. 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), 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 Bacillus 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 residues 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 comments were 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.

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 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 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 I mammalian toxicology data and information support registration of the pesticide product, Serenade Soil DPZ. 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). 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 713, 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 orally to male and female rats at a dose of approximately 1.13 x 10[8] colony-forming units (cfu)/animal in a l millileter (mL) volume. No adverse clinical signs were observed, and gross necropsy 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. EPA 2006; Watson et al. 1999).  

Acute Pulmonary Toxicity/Pathogenicity  -  Rat (Harmonized 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 parenchyma on Day 0, no other adverse effects were observed via gross necropsy. The test microbe (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. This study was rated "ACCEPTABLE" for risk assessment purposes (U.S. EPA 2006; Watson et al. 1999).   

Acute Injection Toxicity/Pathogenicity (Intravenous)  -  Rat (Harmonized Guideline 885.3200; MRID No 485309-09 ): Researchers exposed thirty 49- to 84-day-old Sprague-Dawley, albino rats (15 per sex) by intravenous route to Bacillus subtilis strain QST 713 variant Soil at doses of 6.6 x 108 cfu/rat. They then observed the rats for up to 21 days. 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 108 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 no hypersensitivity incidents, including immediate-type or delayed-type reactions of humans 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 (Harmonized 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 rats 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 body weight (bw).  After 14 days, 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 toxic 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 rats (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.) in undiluted test articles for 4 hours at concentrations of 5.15 mg/L. They observed a few symptoms that did not continue after the fourth day. None of the rats died after 14 days. The EPA classified the acute inhalation toxicity as Toxicity Category IV, the lowest toxicity category, and the Agency rated the study as acceptable

Acute Dermal Toxicity - Rat (Harmonized Guideline 870.1200; MRID No 485309-11): Researchers treated ten 56- to 84-day-old Sprague-Dawley, albino rats (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 days. 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 acceptable.

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 days and rated the effects with a maximum average irritation score of 2.0, meaning that the formulation is practically non- irritating to the eye. Since 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 observing the rabbits for 3 days (72 hours), the researchers determined that there was no dermal irritation to the skin, based on a maximum irritation score of 0 at 24, 48 and 72 hour observations. 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 in 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 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 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 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 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) 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 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 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 residues of Bacillus subtilis strain QST variant soil.

	
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 dietary exposure to Bacillus subtilis strain QST 713 variant Soil, a naturally 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 is likely to be minimal because of the low acute oral toxicity/pathogenicity potential of Bacillus subtilis strain QST 713." The EPA concluded, based on the similarity in product composition and production, measured physical/chemical, and pathogenicity/infectivity 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 Water Exposure and Risk Characterization: Exposure to residues of Bacillus subtilis strain QST 713 variant Soil in consumed drinking water is unlikely. The proposed use patterns for Serenade Soil DPZ are soil directed and/or soil incorporated, thereby limiting contact with surface water by 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 likely be filtered out by the particulate nature of many soil types. 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 systems or drinking water facilities, it likely would not survive the conditions water is subjected to in such systems or facilities, including chlorination, pH adjustments, filtration, and/or occasionally high temperatures (Centers for Disease Control and Prevention 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 systems or facilities), its target pest specificity and available data indicate no toxicity and/or pathogenicity is likely to occur with any drinking water exposure to Bacillus subtilis strain QST 713 variant Soil that results from pesticide applications made in accordance with good agricultural practices (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 likely 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).
	
	
	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 pests. 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 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 - 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 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 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 collectively, the data and information available on Bacillus subtilis strain QST 713 and Bacillus subtilis strain QST 713 variant Soil do not demonstrate toxic and/ 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 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 respirators) 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 determined 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 organisms at higher tier levels (i.e., Tiers II, III, and IV) is not required. The EPA performed an environmental risk assessment based on data and other information provided by 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. 4853909-15): 

While applications of the proposed end use product containing Bacillus subtilis strain QST 713 variant Soil are soil directed, birds and wild mammals could be exposed. Such exposure 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 Bacillus subtilis strain QST 713 variant Soil. The percentage of the a.i. in the proposed end use product is similar to that of Serenade ASO, a currently-registered end use product containing Bacillus 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 previously submitted studies are sufficient for determining risk. Birds and wild mammals were not adversely affected 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 Bacillus subtilis strain QST 713 variant Soil, or if this a.i. is present on food items. Studies submitted for the registration of Bacillus 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 ladybird beetle (Hippodamia convergens), but the dead insects were not caused by treatment with the a.i. Several 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 from 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. 4853909-15):

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). These applications could result in some 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 exposed to high levels of the a.i.; however, the reviewers determined that these deaths resulted from water fouling by high concentrations of the test material and not from toxicity or pathogenicity. The reviewers observed similar problems in a study on Daphnia magna, and they determined that mortalities at high exposure levels were also due to fouling of the water (MRID 48530905). Therefore, 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, based on its analysis of freshwater animals, that high concentrations of Bacillus 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 species as a result of the proposed applications of Bacillus subtilis strain QST 713 variant Soil is minimal.

2.  Environmental Fate Data 

As the data and information provided are sufficient to fulfill the Tier I nontarget organism data requirements and allow for nontarget organism risk assessment for Bacillus subtilis strain QST 713 variant Soil, further 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 critical habitats 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 variant 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, implementation, and enforcement of environmental laws, regulations, and policies. Fair treatment means that no group of people, including racial, ethnic, or socioeconomic groups, should bear a disproportionate share of the negative environmental consequences resulting from industrial, municipal, and commercial operations or the execution of federal, state, local, and tribal environmental programs and policies. Meaningful involvement means that (1) potentially affected community residents have an appropriate opportunity to participate in decisions about a proposed activity that will affect their environment and/or 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, 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 subtilis 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.

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

To satisfy criterion 1, the Bacillus subtilis strain QST 713 variant Soil product has well-known properties. The EPA has no knowledge that would contradict the claims made on the Serenade Soil DPZ label, and this pesticide 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 document. The Agency believes that the Bacillus subtilis strain QST 713 variant Soil pesticide product will not cause any unreasonable adverse effects on the environment. In fact, Serenade Soil DPZ is likely 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. 

VII.  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 terms 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 incidents of hypersensitivity (including both suspected and confirmed incidents) must be reported to EPA under the provisions of 40 CFR § 158.2140(d).


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
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
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






















IX.  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 
				prepared by Chemical Consultants International, Inc. 4 p.

44659104		Bellet, E. (1998) Manufacturing and Analytical 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) Sensitivity 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/Pathogenicity Testing of QST 713 Following 																																Acute 				Acute Oral Challenge in Rats: Lab Project Number: L08726SN4. Unpublished 	
				study prepared by IIT Research Institute. 126 p.

44651907 		Findlay, J. (1998) Acute Dermal Toxicity/Pathogenicity Study of QST 713 in 		
				Rabbits: Lab Project Number: L08726SN7. Unpublished study prepared by IIT 	
				Research Institute. 27 p.

44651909		Harrington, K. (1998) Toxicity/Pathogenicity Testing of QST 713 Following 		
				Acute Intratracheal 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 Number: 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
				(Oncorhynchus mykiss): 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.

MRID No.	                                             Study Information

45455203	Drottar, K.; Flaggs, R.; Krueger, H. (2001) QST 713 Technical: A 21-Day Life-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.

45455205	Bryan, R.; Porch, J.; Krueger, H. (2001) Bacillus Subtilis Strain QST 713: A Dietary Pathogenicity and Toxicity Study with the Parasitic Hymenopteran (Nasonia vitripennis): Final Report: Lab Project Number: 489-107B. Unpublished study prepared by Wildlife International, Inc. 25 p.

44651915	Hoxter, K.; Krueger, H.; Palmer, S. (1998) A Dietary Pathogenicity Toxicity Study with the Ladybird Beetle (Hippodamia convergens): (QST 713): Lab Project Number: 489-103B. Unpublished study prepared by Wildlife International Ltd. 24 p.

44651916	Hoxter, K.; Krueger, H.; Palmer, S. (1998) A Dietary Pathogenicity Toxicity Study with the Green Lacewing Larvae (Chrysoperia 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 Honeybee 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 Honeybee Studies Conducted with QST 713 Technical and Serenade Products. Unpublished study prepared by Agraquest Inc and California State Polytechnic College and Montana State University. 237 p.

45385301	Van Koppenhagen, J.; Hilbig, L. (2001) Product Chemistry of Serenade AS: Lab 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, LLC. 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, Inc. 8 p.


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.

48530900	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.

48530901	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.

48530902	Heins, S.; Thurston, J.; Guilhabert-Goya, M.; et al. (2011) Product Chemistry of Bacillus subtilis Strain QST 713 Variant Soil (TGAI). Project Number: 110509/2. Unpublished study prepared by AgraQuest, Inc. 107 p.

48530903	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.

48530904 	Monds, K. (2011) QST 713 Strain of Bacillus subtilis Variant Soil: Identification and Enumeration of a Microbial Pest Control Agent (MPCA): Final Report. Project Number: 15246/11. Unpublished study prepared by Stillmeadow, Inc. 10 p.

48530905 	Heins, S.; Thurston, J.; Guilhabert-Goya, M.; et al. (2011) Product Chemistry of Serenade Soil DPZ (EP). Project Number: 110509/1. Unpublished study prepared by AgraQuest, Inc. 70 p.

48530906 	Kaminsky, M. (2011) Serenade Soil DPZ (EP): Product Chemistry: Final Report. Project Number: 15000/11. Unpublished study prepared by Stillmeadow, Inc. 9 p.

48530907 	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.

48530908 	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.

MRID No.	                                             Study Information

48530911 	Kuhn, J. (2011) Serenade Soil DPZ: Acute Dermal Toxicity Study in Rats: Final Report. Project Number: 14932/11. Unpublished study prepared by Stillmeadow, Inc. 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, Inc. 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 Identity: Further Information on the Characterization of the MPCA. Project Number: 120810/AQ/REG/01. Unpublished study prepared by AgraQuest, Inc. 6p.

B.  Environmental Protection Agency Risk Assessment Memoranda

U.S. EPA. 2012a. Review of Product Chemistry and Acute Toxicity Studies for section 3 registration of a new TGAI  Bacillus subtilis strain QST 713 variant 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. 2012b. Environmental Risk Assessment for a FIFRA Section 3 Registration of Bacillus subtilis strain QST 713 variant Soil (EPA File Symbol 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.
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/WQ24.

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.



             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 QST 713 variant Soil
                              (40 CFR § 158.2120)
                          Harmonized Guideline Number
                               Data Requirement
                                    Results
                                   MRID No.
                                       
                                   885.1100
                                       
                               Product Identity
Submitted 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 Sample 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 information (confirmatory) on this data requirement.
                                   446519-04
                                   885.1400
                              Analysis of Samples
                                       
        Submitted data fulfill the requirement for analysis of samples.
                                       
                                       
                                   446519-02
                                   885.1500
                            Certification of Limits
       Submitted data fulfill the requirement for manufacturing process.
                                   450845-01


TABLE 2.  Product Analysis Data Requirements for the End-Use Pesticide Product (EP), Serenade Soil DPZ 
                             (40 CFR § 158.2120)
                          Harmonized 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 information (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.
                                   485309-05

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)
                          Harmonized Guideline Number
                                       
                               Data Requirement
                                    Results
                                       
                                   MRID No.
                                   830.6302
                                     Color
                             Brown or light brown
                                   485309-03
                                   830.6303
                                Physical State
                                    Liquid
                                       
                                   830.6304
                                     Odor
                                  Like yeast
                                       
                                   830.6313
                       Stability to Normal and Elevated
                             Temperatures, Metals,
                                and Metal Ions
        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 information (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)
                                     1.02
                                   485309-03

TABLE 4.  Physical and Chemical Characteristics for the End-Use Pesticide Product (EP), Serenade Soil DPZ
                             (40 CFR § 158.2120)
                          Harmonized Guideline Number
                               Data Requirement
                                    Results
                                   MRID No.
                                  830.6302[1]
                                     Color
                                 Medium brown
                                      N/A
                                   485309-06
                                  830.6303[1]
                                Physical 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 information (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 Soil DPZ, it is summarized appropriately in this table.


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)
                          Harmonized Guideline Number
                                       
                               Data Requirement
                                       
                                    Results
                                       
                                   MRID No.
                                    Tier I
                                   885.3050
                       Acute 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
                                   446519-06
                                   489309-08
                                   885.3150
                    Acute 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
                           Acute 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
             Acute Injection Toxicity/Pathogenicity (Intravenous)
No mortality and no adverse clinical signs in rats dosed with QST 713 Technical powder. The test microbe 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
                               Hypersensitivity
                                   Incidents
The applicant reported that no hypersensitivity incidents, including immediate-type 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
                              Acute Oral Toxicity
Oral LD50 female rats > 5,000 mg/kg body weight (bw)
Classification: Acceptable
TOXICITY CATEGORY IV
                                   485309-10
                                   870.1200
                             Acute Dermal Toxicity
Dermal LD50 combined (male and female rats) > 5,050 mg/kg bw
Classification: Acceptable
TOXICITY CATEGORY IV
                                   485309-11
                                   870.1300
                           Acute Inhalation Toxicity
LC50 combined (male and female rats) >5.15 mg/L
Classification: Acceptable
TOXICITY CATEGORY III
                                   483509-12
                                   870.2400
                             Acute 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 IV
                                   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.







TABLE 6.  Nontarget Organism Toxicity and Environmental Fate Data Requirements for the Technical Grade of the Active Ingredient (TGAI), Bacillus subtilis strain QST 713 variant Soil (40 CFR § 158.2150)
                          Harmonized 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
                                   485309-15
                                   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 mammal 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
                                   485309-15
                                   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
                                   485309-15
                                   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.









APPENDIX B.  PESTICIDE PRODUCT

                               EPA File Symbol 
                               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

