		

                                                       
                                       
                                       

                                       
                  BIOPESTICIDES REGISTRATION ACTION DOCUMENT
                                       
                                       
                                     DRAFT
                                       
                                       
                                       
                                       

                                       
                                       
                    Bacillus amyloliquefaciens strain D747
                                       
                                       
                     Pesticide Chemical (PC) Code: 016482
                                       
                                       
                                       
                                       
                                       

   
	U.S. Environmental Protection Agency
	Office of Pesticide Programs
                Biopesticides and Pollution Prevention Division
                                       
                                       
                           November 10, 2011 version
                                       
                                       
                               TABLE OF CONTENTS
I.  EXECUTIVE SUMMARY	4
II.  ACTIVE INGREDIENT OVERVIEW	5
III.	 REGULATORY BACKGROUND	6
A.  Applications for Pesticide Product Registration	6
B.  Food Tolerance Exemption	6
IV.	 RISK ASSESSMENT	6
A.  Product Analysis Assessment (40 CFR § 158.2120)	7
B.  Human Health Assessment (40 CFR § 158.2140)	7
C.  Environmental Assessment (40 CFR § 158.2150)	12
V.  ENVIRONMENTAL JUSTICE	14
VI.  RISK MANAGEMENT DECISION	15
VII.  ACTIONS REQUIRED BY THE REGISTRANT	16
VIII.  GLOSSARY OF ACRONYMS AND ABBREVIATIONS	16
IX.  BIBLIOGRAPHY	18
A.  Studies Submitted to Support Pesticide Product Registrations	18
B.  Environmental Protection Agency Risk Assessment Memoranda and Other Documents	20
C.  Other References	20
APPENDIX A.  MICROBIAL PESTICIDES DATA REQUIREMENTS	22
APPENDIX B.  PESTICIDE PRODUCTS	30
                                       
                                       
                                       

                BIOPESTICIDES REGISTRATION ACTION DOCUMENT TEAM
                                       
                                       
                      Office of Pesticide Programs (OPP)
                Biopesticides and Pollution Prevention Division
                          Microbial Pesticides Branch
                                       
                                       
                                Science Reviews
                                       
Joel V. Gagliardi, Ph.D.				   Product Analysis, Human Health
John L. Kough, Ph.D.					   Product Analysis, Human Health
Gail Tomimatsu, Ph.D.				  Environmental Effects
Shannon Borges					   Environmental Effects

                                  Regulations
                                       
Sheryl K. Reilly, Ph.D.				   Chief, Microbial Pesticides Branch
Susanne Cerrelli	Regulatory Action Leader

I.  EXECUTIVE SUMMARY

Certis USA, LLC, ("Certis" or "applicant) has proposed to register Bacillus amyloliquefaciens strain D747 as the active ingredient in two end-use pesticide products, CX-9030 (EPA File Symbol 70051-RNI) and CX-9032 (EPA File Symbol 70051-RNT). The products are intended for use to control fungi and bacteria in outdoor agricultural crops, greenhouses, nurseries, shadehouses, ornamentals, and turfgrass.  B. amyloliquefaciens strain D747 was initially identified by Certis as"Bacillus subtilis variant amyloliquefaciens strain D747," since B. subtilis and B. amyloliquefaciens were originally classified as subtypes or variants of the same species. B. amyloliquefaciens is now considered a separate species, and the correct taxonomic designation is used in this Biopesticides Registration Action Document ("BRAD") (Priest et. al., 1987; Logan and de Vos, 2009; and Murray et. al., 2007). 

EPA scientists reviewed product analysis, mammalian and nontarget organism toxicity data, and other information submitted by Certis to support the proposed product registrations. The product analysis data requirements for B. amyloliquefaciens strain D747, including product chemistry and composition, analysis of samples, and physical and chemical characteristics, were fulfilled by acceptable studies conducted in accordance with Agency guidelines. Mammalian toxicity data (acute oral, injection, and pulmonary toxicity/pathogenicity) and information from peer-reviewed scientific literature demonstrated that B. amyloliquefaciens strain D747 is not toxic, infective or pathogenic in laboratory rats. Acceptable nontarget organism data also demonstrated that B. amyloliquefaciens strain D747 is not toxic to estuarine and marine fish and invertebrates, nontarget insects (including honey bees), and nontarget plants. 

We have assessed human health and environmental risks from the proposed uses of B. amyloliquefaciens strain D747, and determined that the pesticide would not cause unreasonable adverse effects to nontarget organisms when used in accordance with the directions on the proposed labels, and in accordance with good agricultural practices. Additional mammalian and nontarget organism toxicity data are not required for the proposed uses and application methods.

Bacillus species, including B. amyloliquefaciens, are commonly found in soils, including agricultural settings, and are naturally present on fresh produce. The Manual of Clinical Microbiology (9[th] Edition) states that dried foods, such as spices, milk powder and grains, often contain large amounts of Bacillus spores. B. amyloliquefaciens is not known to produce any mammalian toxins, and no food-borne disease outbreaks associated with B. amyloliquefaciens have been reported. Given that the microorganism occurs naturally in soils, exposure to B. amyloliquefaciens from surface and groundwater may occur. No adverse effects have been reported, and none are expected from exposure to B. amyloliquefaciens through drinking water.

Despite the low toxicological profile of B. amyloliquefaciens strain D747, personal protective equipment (PPE) is required for pesticide handlers that are frequently exposed to the active ingredient for prolonged periods. Handlers will be directed to wear a long-sleeved shirt, long pants, socks, shoes, waterproof gloves, and a dust/mist filtering respirator meeting National Institute for Occupational Safety and Health (NIOSH) standards of at least N-95, R-95, or P-95. EPA may require additional PPE, other than the standard described above, if additional pesticide uses resulting in increased exposures are proposed in the future.  
 
EPA has concluded that, based upon the results of the toxicity tests and lack of adverse incidents, there is a reasonable certainty that no harm will result to the United States population, including infants and children, from dietary (including drinking water) and aggregate exposure to residues of B. amyloliquefaciens strain D747 from the proposed pesticide uses. The proposed products meet the standards for registration under Section 3(c)(5) of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA).

On October 1, 2009, 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, 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 EPA believes there may be significant public interest.
  
Consistent with the policy of making registration actions more transparent, the proposed pesticide products containing B. amyloliquefaciens strain D747, a new active ingredient, are subject to a 30-day comment period. In addition to containing a new active ingredient, the registration of these products would result in the first outdoor use, first residential use and first food use for B. amyloliquefaciens strain D747. 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-2011-0853. The following documents are available for comment in EPA-HQ-OPP-2011-0853: (1) draft B. amyloliquefaciens strain D747 Biopesticides Registration Action Document (BRAD); (2) environmental risk assessment for B. amyloliquefaciens strain D747; (3) draft product label for the end-use product, CX-9030 (EPA File Symbol 70051-RNI) ; and (4) draft product label for the end-use product, CX-9032 (EPA File Symbol 70051-RNT ). While a final decision on registration is contingent upon review and consideration of public comments, EPA presently believes that, based upon the risk assessment and information submitted in support of the CX-9030 and CX-9032 EPs, it is in the best interest of the public and the environment to issue these registrations. The basis for this preliminary decision can be found in the risk assessment for B. amyloliquefaciens strain D747, which is presented in this document.   


II. ACTIVE INGREDIENT OVERVIEW

   Biological Name:                  	Bacillus amyloliquefaciens strain D747 
 
   Culture Deposit:		Agricultural Research Service Culture Collection (also 
   			known as the Northern Regional Research Laboratory 
                                                  		(NRRL) Collection) in Peoria, Illinois, under Accession 
                                                  		Number NRRL B-50405.
 
 	OPP Chemical Code: 		016482	
 
	Type of Pesticide:				Microbial Pesticide  -  Insecticide

					See Appendix B for specific information (e.g.,
                        use sites, application rates, methods of application, 
                        formulation types, and target pests) regarding
                        the registered pesticide products containing this 
                        active ingredient.

III.	 REGULATORY BACKGROUND

A.  Applications for Pesticide Product Registration

Certis USA, LLC (address:  9145 Guilford Road, Suite 175, Columbia, MD, 21046), submitted applications to register two end-use pesticide product, CX-9032 and CX-9030, under FIFRA section 3(c)(5) on July 26, 2010. EPA announced receipt of these applications to register pesticide products containing a new active ingredient on February 2, 2011 (76 Federal Register (FR) 5805), and 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 the Federal Food, Drug, and Cosmetic Act (FFDCA) section 408(d), Certis USA, LLC, submitted a petition to establish an exemption from the requirement of a tolerance for Bacillus subtilis variant amyloliquefaciens strain D747 (Pesticide Petition (PP) 0F7760). In the Federal Register of February 4, 2011 (76 FR 6465), EPA announced that Certis USA, LLC, proposed to establish an exemption from the requirement of a tolerance for residues of the insecticide, Bacillus subtilis variant amyloliquefaciens strain D747, in or on all food commodities, and opened a 30-day comment period. No comments were received following this publication. The correct taxonomic designation of the microorganism is Bacillus amyloliquefaciens strain D747; therefore, the tolerance exemption, when established, will reflect the correct name of the active ingredient.


IV.	 RISK ASSESSMENT

In the Federal Register of October 26, 2007, EPA issued a Final Rule establishing changes to the data requirements that support the registration of microbial pesticides. The rule also updated the definition for microbial pesticides (72 FR 61002), and became effective on December 26, 2007. The data and information evaluated for this BRAD were considered in accordance with these requirements.

The classifications or ratings that are given for each data requirement in this BRAD were assigned by the EPA scientists who reviewed the data, and convey the usefulness of the information for the human health and nontarget risk assessment purposes. "Acceptable" indicates that a study is scientifically sound and is useful for risk assessment. A "supplemental" classification indicates the studies provided some information that can be useful for risk assessment. "Supplemental" ratings are given to studies that are not required for registration, as well as those that are required. In the latter case, the study lacks information that should have been obtained if it was conducted according to the guideline associated with the data requirement. Sometimes the missing information would not add anything necessary to inform a risk assessment, and the study classification remains "supplemental." A classification of "supplemental:upgradable" indicates the  study lacks necessary information, but if it is made available by the applicant, the study may be upgraded to "acceptable." An "unacceptable" rating indicates that new data must be submitted.

Toxicity categories are assigned to acute toxicity studies, based upon any signs of toxicity (hazards) observed in the test animals during the course of the study, whether the study was conducted in accordance with test guidelines or comes from other sources, such as peer-reviewed, scientific literature. The active ingredient or particular product is classified 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)

All product analysis data requirements for B. amyloliquefaciens strain D747 have been fulfilled for the proposed product registrations. Refer to Tables 1, 2, 3, and 4 in Appendix A for a brief summary of the data requirements, including both generic and product-specific information. 

B.  Human Health Assessment (40 CFR § 158.2140)

1.  Toxicity

All Tier I mammalian toxicity data requirements for B. amyloliquefaciens strain D747 have been fulfilled for the proposed product registrations. Refer to Tables 5, 6 and 7. Based on the lack of acute toxicity/pathogenicity, Tier II and Tier III studies were not required. 

For a comprehensive summary of the generic and product-specific toxicity data requirements described below, refer to Tables 5, 6 and 7 in Appendix A.

      a. Acute Toxicity/Pathogenicity  -  Tier I

Acute Oral Toxicity/Pathogenicity (Office of Chemical Safety and Pollution Prevention (OCSPP) Guideline 885.3050; Master Record Identification Number (MRID No. 481657-04):  
B. amyloliquefaciens strain D747 was administered once orally to 14 rats of both sexes (5-weeks old) at a single dosage of 10[8] colony-forming units (CFU) per animals. No deaths occurred, and no abnormalities (clinical signs, body weight) were observed, during the study or at necropsy. The test microbe was detected at 10[3]  -  10[5] CFU/g in feces 1 day after administration of the test material, but was not detected on day14. The examination for internal persistence did not detect the test microbe in any organs or tissues, such as the kidney, brain, liver, lung, spleen, stomach, small intestine (duodenum), large intestine (cecum), mesenteric lymph nodes, or blood, throughout the experimental period.   Fecal clearance occurred by day 14, and no viable organisms were recovered from blood or other organs or tissues. The results of this acceptable study demonstrated that B. amyloliquefaciens strain D747 was not infective, pathogenic, or toxic  to rats when orally dosed with 1.0x10[8] CFU / animal.  
 
Acute Pulmonary Toxicity/Pathogenicity (OCSPP Guideline 885.3150; MRID No.481657-06): 
B. amyloliquefaciens strain D747 was administered once intratracheally to 20 male and female Sprague-Dawley rats (5-week old) at a dosage of 10[7] CFU per animal. No deaths occurred, and no abnormalities (clinical signs, body weight) were observed during the study or at necropsy. The examination for internal persistence showed that the test microbe was detected shortly after administration in the lung, trachea, and nasal cavity.  The test microbes were not detected in the nasal cavity on day 7, and almost completely cleared from the trachea by day 60, when the study ended. The presence of the test microbes decreased in the lungs by approximately 10[2]-10[3] CFU/g from day 0 to day 60. The test microbes were detected in the bronchial lymph nodes on day 3. Since many test microbes remained in the trachea on day 3, their presence in the bronchial lymph nodes on day 3 was attributed to transfer by macrophages from the respiratory tract to the lymph nodes. This conclusion was supported by the observation that a particle larger than 1 m in diameter administered into the trachea and bronchi was phagocytized by a macrophage and carried to a lymph node. The presence of the microbes in the lungs, though decreasing throughout the observation period, was attributed to the continuous transfer of viable test organisms to the bronchial lymph nodes. This result is not unusual for spore-forming bacteria, since bacterial spores are extremely tolerant of adverse conditions, and take a longer time for the immune system to clear than bacteria that do not form spores. Since a pattern of clearance was demonstrated, the remaining viable cells were considered to be spores, which take longer for a healthy immune system to clear. This acceptable study demonstrated that B. amyloliquefaciens strain D747 was not toxic or pathogenic to rats when dosed intratracheally at 1.0 x10[7] CFU/ animal.

Acute Injection Toxicity/Pathogenicity (Intravenous)  -  Rat (OCSPP Guideline 885.3200; MRID No. 481657-05): In an acute intravenous injection toxicity and pathogenicity study, groups of 17 male and female Sprague-Dawley rats (5-weeks old) were injected with B. amyloliquefaciens strain D747 at a dosage of 10[7] CFU per animal, and the influence on the animals was investigated. No deaths occurred, and there were no observed abnormalities (clinical signs, body weight) during the study or at necropsy. The examination for internal persistence of the test microbe showed that the test microbes were mainly detected in the kidney, liver, spleen, and blood shortly after administration. The test microbe decreased after that and was not detected in the blood from day14; clearance from the kidney occurred by day 60.  McClintock et al. previously reported that when B. thuringiensis and B. subtilis, which are both spore-forming bacteria, were administered intravenously to rats, clearance from the liver and spleen is difficult. In this study, the test microbe did not completely disappear from these organs by day 60. No test microbes were detected in the brain, and only a few viable microbes at the limit level were sporadically detected in the small and large intestines, and lymph nodes, but were cleared by day 60. This acceptable study demonstrated that B. amyloliquefaciens strain D747 was not toxic, infective, or pathogenic to rats when injected intravenously with 1.0 x10[7] CFU/ animal.

Hypersensitivity Incidents (OCSPP Guideline 885.3400; MRID No. 481655-05): No hypersensitivity incidents in humans have been reported, and none occurred during research, development, or testing of B. amyloliquefaciens strain D747. Should hypersensitivity or other adverse incidents in humans occur in the future, Certis must report them to EPA, in accordance with FIFRA section 6(a)(2).

Cell Culture (OCSPP Guideline 885.3500): This study was not required because B. amyloliquefaciens strain D747 is not a virus (refer to test note #4 of 40 CFR § 158.2140(d)).

	b. Acute Toxicity and Subchronic Toxicity/Pathogenicity  -  Tier II;
	    Reproductive Fertility Effects, Carcinogenicity, Immunotoxicity, and 
	   Infectivity/Pathogenicity Analysis  -  Tier III

Tier II and Tier III studies were not required for B. amyloliquefaciens strain D747 based on the lack of acute toxicity/pathogenicity in the Tier I studies. 


	c. Endocrine Disruptors
	
As required under FFDCA section 408(p), EPA has developed the Endocrine Disruptor Screening Program (EDSP) to determine whether certain substances (including pesticide active and other ingredients) may have an effect in humans or wildlife similar to an effect produced by a "naturally occurring estrogen, or other such endocrine effects as the Administrator may designate." The EDSP employs a two-tiered approach to making the statutorily required determinations. Tier 1 consists of a battery of 11 screening assays to identify the potential of a chemical substance to interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal systems. Chemicals that go through Tier 1 screening and are found to have the potential to interact with E, A, or T hormonal systems will proceed to the next stage of the EDSP where EPA will determine which, if any, of the Tier 2 tests are necessary based on the available data. Tier 2 testing is designed to identify any adverse endocrine-related effects caused by the substance, and establish a quantitative relationship between the dose and the E, A, or T effect.

Between October 2009 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. This list of chemicals was selected based on the potential for human exposure through pathways such as food and water, residential activity, and certain post-application agricultural scenarios. This list should not be construed as a list of known or likely endocrine disruptors.

B. amyloliquefaciens strain D747 is not among the group of 58 pesticide active ingredients on the initial list to be screened under the EDSP. Under FFDCA section 408(p), EPA must screen all pesticide chemicals. Accordingly, EPA anticipates issuing future EDSP orders/data call-ins for all pesticide active ingredients. 

For further information on the status of the EDSP, the policies and procedures, the list of 67 chemicals, the test guidelines and the Tier 1 screening battery, please visit our website: http://www.epa.gov/endo/.

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

Section 408(c)(2)(A)(i) of FFDCA allows EPA to establish an exemption from the requirement for a tolerance (the legal limit for a pesticide chemical residue in or on a food) only if 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, EPA must take into account the factors set forth in section 408(b)(2)(C) of FFDCA, which require EPA to give special consideration to exposure of infants and children to the pesticide chemical residue in establishing a tolerance and to "ensure that there is a reasonable certainty that no harm will result to infants and children from aggregate exposure to the pesticide chemical residue...." Additionally, section 408(b)(2)(D) of FFDCA requires that EPA consider "available information concerning the cumulative effects of [a particular pesticide's] . . . residues and other substances that have a common mechanism of toxicity."
EPA performs a number of analyses to determine the risks from aggregate exposure to pesticide residues. First, EPA determines the toxicity of pesticides. Second, EPA examines exposure to the pesticide through food, drinking water, and through other exposures that occur as a result of pesticide use in residential settings.
Consistent with section 408(b)(2)(D) of FFDCA, EPA has reviewed the available scientific data and other relevant information, and considered its validity, completeness, and reliability and the relationship of this information to human risk. EPA has also considered available information concerning the variability of the sensitivities of major identifiable subgroups of consumers, including infants and children. All of the data requirements have been fulfilled, as described previously, and are presented in Table 5 in Appendix A. The following summarizes the results of EPA's dietary risk assessment for B. amyloliquefaciens strain D747.
	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 non-occupational exposures, including drinking water from ground or surface water, and through pesticide use in residential and other indoor uses.
 
Food Exposure and Risk Characterization: Bacillus species, including B. amyloliquefaciens, are commonly found in the soil in agricultural settings, and are present on fresh produce of all kinds with no known adverse effects. The Manual of Clinical Microbiology (9[th] edition) mentions that dried food such as spices, milk powder and grains often contain large amounts of B. spores. B. amyloliquefaciens is not known to produce any mammalian toxins, and no foodborne disease outbreaks associated with B. amyloliquefaciens have been reported. 

Based on  the data and other information submitted to satisfy the data requirements for registration of the manufacturing-use and end-use pesticide products containing the active ingredient, B. amyloliquefaciens strain D747, no toxicity, infectivity, pathogenicity or other adverse effects from dietary exposure to are expected (see section IV(B)(1)(a), above, and Table 5 in Appendix A). 

Drinking Water Exposure and Risk Characterization: B. amyloliquefaciens is naturally present in soils (Logan and de Vos, 2009); therefore, B. amyloliquefaciens may occur in surface and possibly groundwater.  According to the World Health Organization, Bacillus species are often detected in drinking water even after going through acceptable water treatment processes, largely because the spores are resistant to these disinfection processes (World Health Organization, 2011).  Should this microbial pesticide be present, no adverse effects are expected from exposure to B. amyloliquefaciens through drinking water, based on the data and other information submitted to satisfy the data requirements for registration of the manufacturing-use and end-use pesticide products containing the active ingredient, B. amyloliquefaciens strain D747 (see section IV(B)(1)(a), above, and Table 5 in Appendix A). 

Non-occupational, Residential Risk Characterization: The use sites for these products include residential garden sites and agricultural sites. As stated previously, B. amyloliquefaciens is naturally present in soil, and based on the data and other information submitted to satisfy the data requirements for registration of the manufacturing-use and end-use pesticide products containing the active ingredient, B. amyloliquefaciens strain D747, no toxicity, infectivity, pathogenicity or other adverse effects from non-occupational exposure are expected (see section IV(B)(1)(a), above, 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, EPA consider "... available information concerning the cumulative effects of [a particular pesticide's] ... residues and other substances that have a common mechanism of toxicity."
EPA has found that B. amyloliquefaciens strain D747 does not share a common mechanism of toxicity with any other microorganism, pesticidal or toxic substance. EPA concludes that there are no cumulative effects associated with B. amyloliquefaciens strain D747 that need to be considered. For information regarding how EPA determines common mechanisms of toxicity and evaluates cumulative effects, see EPA's website at:  http://www.epa.gov/pesticides/cumulative.

	c. Determination of Safety for the United States (U.S.) Population, Infants and 
                Children
	
FFDCA section 408(b)(2)(C) provides that EPA shall assess the available information about consumption patterns among infants and children, special susceptibility of infants and children to pesticide chemical residues, and the cumulative effects on infants and children of the residues and other substances with a common mechanism of toxicity. In addition, FFDCA section 408(b)(2)(C) provides that EPA shall apply an additional tenfold (10X) margin of safety for infants and children in the case of threshold effects to account for prenatal and postnatal toxicity and the completeness of the database on toxicity and exposure, unless EPA determines that a different margin of safety will be safe for infants and children. This additional margin of safety is commonly referred to as the Food Quality Protection Act Safety Factor. In applying this provision, EPA either retains the default value of 10X or uses a different additional safety factor when reliable data available to EPA support the choice of a different factor.
EPA concludes that, based upon the results of the toxicity data and other information considered and described in this document, there is a reasonable certainty that no harm will result to the U.S. population, including infants and children, from aggregate exposure to the residues of B. amyloliquefaciens strain D747. Such exposure includes all anticipated dietary and other exposures for which there is reliable information. With no threshold effects of concern, an additional margin of safety is not required for infants and children. 

3.  Occupational Exposure and Risk Characterization

Occupational exposure to B. amyloliquefaciens strain D747 is not expected to undue risks to pesticide handlers (mixer/loader/applicators), but EPA requires appropriate personal protective equipment and precautionary statements to mitigate any potential risks (e.g., respiratory allergenicity) to pesticide handlers from prolonged or repeated exposures. Handlers applying B. amyloliquefaciens strain D747 end-use products in agricultural settings must wear a long-sleeved shirt, long pants, socks, shoes, waterproof gloves, and a dust/mist filtering respirator meeting NIOSH standards of at least N-95, R-95, or P-95. 

4.  Human Health Risk Characterization
EPA considered human exposure to B. amyloliquefaciens strain D747 in light of the registration standards of FIFRA and the relevant FFDCA safety factors for allowable pesticide residues in food and animal feed commodities. EPA has determined that no unreasonable adverse effects to the U.S. population in general, and to infants and children in particular, will result when the proposed pesticide products containing B. amyloliquefaciens strain D747 as the active ingredient are used in accordance with the proposed labeling.

C.  Environmental Assessment (40 CFR § 158.2150)

The primary habitat of Bacillus species is soil, although they have also been isolated from a wide variety of other habitats. B. amyloliquefaciens has a wide-spread distribution, owed in part to its ability to form endospores that are resistant to greater variation in environmental conditions than the vegetative cells, should transfer to other climates occur. B. amyloliquefaciens has been isolated from internal tissues of healthy plants, and is known to promote plant growth.  It is not recognized as a pathogen among Bacillus species (Logan and de Vos, 2009). 

The data, literature citations, and data waiver rationale submitted by the applicant to support the pesticide products containing B. amyloliquefaciens strain D747 fulfilled the Tier I nontarget organism data requirements, and were sufficient for risk assessment purposes. Further testing of nontarget organisms at higher tier levels (i.e., Tiers II, III, and IV) is not required for the proposed uses. EPA performed an environmental risk assessment, and has determined that the proposed use of B. amyloliquefaciens strain D747 are not expected to cause unreasonable adverse effects to nontarget organisms.  

For a comprehensive summary of the generic data requirements described in sections IV(C)(1) of this BRAD, refer to Table 8 in Appendix A.

1.  Ecological Exposure and Risk Characterization  

		a. Terrestrial Animals and Plants

The end-use products include a water-dispersible granular formulation and an aqueous suspension, proposed for application to agricultural crops, nurseries, ornamental plants, turfgrass, greenhouses, and shadehouses. Applications can be made to both foliar surfaces and soil, so exposure to nontarget organisms is possible. The proposed maximum application rates were used in the assessment of nontarget risk. 

Data on the naturally occurring levels of B. amyloliquefaciens are not available. Many factors influence the environmental fate of microbial pesticides, and resulting population levels in the environment cannot be predicted. EPA expects that B. amyloliquefaciens strain D747 may survive after application if conditions are favorable, but the strain would not significantly add to the overall levels of B. amyloliquefaciens already present in the environment.   

Birds (OCSPP Guideline 885.4050) and Mammals (OCSPP Guideline 885.4150): 
A supplemental study showed that the acute oral LD50 for B. amyloliquefaciens strain D747 is > 4.5 x 10[11] spores/kg BW or > 8 x 10[9] spores/bird in Northern bobwhite (Colinus virginianus; MRID 48165712).  Additionally, B. amyloliquefaciens is intentionally included in some domestic avian food diets as a nutritional additive (e.g., European Food Safety Authority, 2010; Wizna et al., 2009), and it is not known to be pathogenic to animals (Logan and de Vos, 2009). An extensive literature search in several databases returned no reports of toxicity or pathogenicity of B. amyloliquefaciens in birds. Based on these lines of evidence, B. amyloliquefaciens strain D747 is not expected to pose risk of adverse effects in birds.  

A study with laboratory rats (MRID 48165704) also showed that B. amyloliquefaciens strain D747 is not toxic, infective, or pathogenic at the maximum hazard dose of 1.0 x 10[8] CFU/animal. Therefore, adverse effects to wild mammals are also not expected as a result of the proposed applications of B. amyloliquefaciens strain D747.    

Nontarget Insects (OCSPP Guideline 885.4340) and Honey Bees (OCSPP Guideline 885.4380): 
Studies with Orius stricollis, Crysoperla carnea, and Phytoseiulus persimilis were submitted for the nontarget insect data requirement (MRID 48165716). While no effects of B. amyloliquefaciens strain D747 were observed in these studies, they were not acceptable for use in the ecological risk assessment. Two honey bee studies were also submitted that showed no adverse effects of B. amyloliquefaciens D747 after 48 hours (MRID 48165717) and 17 days (no MRID currently assigned). These studies were rated Supplemental because they were not of sufficient duration (30 days); however, except in rare cases, bacteria that are pathogenic to insects typically produce toxins that kill the insect within a few days (Tanada and Kaya, 1993). Adverse effects resulting from exposure to B. amyloliquefaciens strain D747, therefore, would likely have been evident in the bee studies, especially after 17 days. Scientific rationale was submitted to show that adverse effects are not expected to nontarget insects (MRIDs 48621502 and 48621503).  Entomopathogenic B. species (e.g., B. thuringiensis, B. sphaericus) have been extensively studied, and their pathobiology is well-known. B. amyloliquefaciens is not among the B. species recognized as frank pathogens to insects or other animals (Logan and de Vos, 2009). There are some accounts in the literature of effects of B. subtilis on insects; however, none of these were associated with B. amyloliquefaciens or B. subtilis var. amyloliquefaciens. Therefore, based on the studies and other information provided, B. amyloliquefaciens strain D747 is not expected to pose risk to honey bees and other nontarget insects as a result of its proposed applications.

Nontarget Plants (OCSPP Guideline 885.4300):
Studies with plants exposed to B. amyloliquefaciens strain D747 were unacceptable, based on several deficiencies (MRID 48165715). B. amyloliquefaciens is not taxonomically related to any known plant pathogens.  As discussed previously, however, the microorganism has been isolated from tissues of healthy plants and is known as a plant growth-promoting rhizobacterium. It is, therefore, not expected to pose risk to nontarget plants as a result of the proposed applications. 

      b. Aquatic Animals and Plants 

B. amyloliquefaciens strain D747 is not intended to be applied directly to water, but some of the applied product may reach aquatic habitats through runoff or spray drift. Spray drift at application is the primary mechanism by which the pesticide is expected to reach water. A spray drift analysis was included in the aquatic risk assessment to determine exposure, and further detail is provided in the environmental risk assessment for B. amyloliquefaciens strain D747.  

Freshwater Fish (OCSPP 885.4200) Guideline and Invertebrates (OCSPP Guideline 885.4240):  
In a 30-day study with rainbow trout (Oncorhynchus mykiss), the LC50 for B. amyloliquefaciens strain D747 was 8.1 x 10[10] CFU/L, and the NOEC based on sub-lethal effects was 1.44 x 10[10] CFU/L. If the proposed maximum broadcast application rate (4 fluid ounces/1000 ft[2] or 2.55 x 10[6] CFU/cm[2]) is applied directly to a 1-ha body of water 15 cm deep (the EPA Standard Wetland), the resulting concentration would be 1.7 x 10[8] CFU/L. This would be the maximum possible aquatic concentration at this application rate, but is not a proposed use. The NOEC and LC50 are approximately 85X and 476X higher than this concentration, respectively. Exposure in freshwater environments will be well below the concentrations that would produce adverse effects, and the proposed applications of B. amyloliquefaciens strain D747 are not expected to pose risks to freshwater fish.  

A study with Daphnia magna provided an EC50 based on mortality/immobility of 3.7 x 10[10] CFU/L, and a NOEC for sub-lethal effects of 2.84 x 10[8] CFU/L. Based on the spray drift analysis, the NOEC would be approximately 20X the expected environmental concentration (EEC), and the EC50 would be 218X the EEC;  therefore, the proposed applications of B. amyloliquefaciens strain D747 are not expected to result in adverse effects to freshwater invertebrates.  

Marine/Estuarine Fish (OCSPP 885. 4280) and Invertebrates (OCSPP 885.4240):      
Concentrations reaching marine or estuarine areas are expected to be less than those calculated above for freshwater animals, due to further dilution  in deeper water. B. amyloliquefaciens strain D747 is not expected to reach marine or estuarine environments in significant concentrations, and risk to animals in these environments is not anticipated.

Aquatic Plants (OCSPP Guideline 885.4300):  
B. amyloliquefaciens strain D747 is not related to known plant pathogens; therefore, adverse effects to aquatic plants are not anticipated. 

2.  Environmental Fate Data 

The information provided for the proposed uses and application methods was sufficient to satisfy the Tier I nontarget organism data requirements and for nontarget organism risk assessment for B. amyloliquefaciens strain D747; further testing at higher tier levels (i.e., Tiers II, III, and IV) is not required.  

3.  Threatened and Endangered Species Assessment

Since EPA has determined that no effects are anticipated for any nontarget species exposed to B. amyloliquefaciens strain D747 as a result of the proposed applications, effects to federally listed threatened and endangered species and their designated critical habitats are also 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 uses of B. amyloliquefaciens strain D747. 

V.  ENVIRONMENTAL JUSTICE

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. EPA has this goal for all communities and persons across the United States. 

To help address potential environmental justice issues, EPA seeks information on any groups or segments of the population who, as a result their location, cultural practices, or other factors, may have atypical, unusually high exposure to B. amyloliquefaciens strain D747, compared to the general population. Please comment if you are aware of any subpopulations that may have atypical, unusually high exposure compared to the general population.

For additional information regarding environmental justice issues, please visit EPA's web site at http://www.epa.gov/compliance/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 B. amyloliquefaciens strain D747 pesticide products have well-known properties. EPA has no knowledge that would contradict the claims made for these products, the CX-9030 and CX-9032 EP labels, and we have concluded that such products are not expected to cause unreasonable adverse effects on the environment when used according to the label instructions. Criterion 2 is satisfied by the current product labels, as well as the data and information presented in this document. It is believed that the B. amyloliquefaciens strain D747 pesticide products will not cause any unreasonable adverse effects on the environment, and CX-9030 and CX-9032  (end-use pesticide product), in particular, are likely to provide protection against fungal and bacterial pests as claimed, satisfying criterion 3. Criterion 4 is satisfied in that the B. amyloliquefaciens strain D747 pesticide products are not expected to cause unreasonable adverse effects when used according to label instructions. Therefore the end-use products, CX-9030 and CX-9032, containing B. amyloliquefaciens strain D747 as a new active ingredient, are eligible for registration under FIFRA section 3(c)(5) for the labeled uses. Should uses that are more extensive be proposed in the future (e.g., aquatic uses), EPA will likely require that additional data be submitted.
VII. ACTIONS REQUIRED BY THE REGISTRANT

A.  Final Printed Labeling

Before releasing pesticide products containing B. amyloliquefaciens strain D747 for shipment, the registrant is required to provide appropriate final printed labeling to EPA prior to shipment of product. 

B. Terms of Registration

As a term of the registration CX-9032 EP, the registrant must submit the following data within one year of this product's registration:

   (1) Storage Stability (OCSPP Guideline 830.6317) and Corrosion Characteristics (OCSPP Guideline 830.6320): The results of a one-year storage stability and corrosion characteristics study. 

C.  Reporting of Adverse Effects and Hypersensitivity Incidents

Notwithstanding the information stated previously, it should be clearly understood that certain specific data are required to be reported to EPA as a requirement for maintaining the federal registration for a pesticide product. A brief summary of these types of data are described below.

Reports of all incidents of adverse effects to the environment must be submitted to EPA under the provisions stated in FIFRA section 6(a)(2). Additionally, all incidents of hypersensitivity (including both suspected and confirmed incidents) must be reported to EPA under the provisions of 40 CFR § 158.2140(d).


VIII. GLOSSARY OF ACRONYMS AND ABBREVIATIONS
                                       
ASAE	American Society of Agricultural Engineers
BPPD 	Biopesticides and Pollution Prevention Division
BRAD	Biopesticides Registration Action Document
CFR	Code of Federal Regulations
CFU	colony-forming unit(s)
cfu/kg	colony-forming units per kilogram
cfu/mL	colony-forming units per milliliter
cP	centipoise
EDSP	Endocrine Disruptor Screening Program
EP	end-use product
EPA	Environmental Protection Agency (the "Agency")
FFDCA	Federal Food, Drug, and Cosmetic Act
FIFRA	Federal Insecticide, Fungicide, and Rodenticide Act
FQPA 	Food Quality Protection Act
FR	Federal Register
g/mL	gram per milliliter
LC50	median lethal concentration. A statistically derived concentration of a 
	substance that can be expected to cause death in 50% of test animals. It is 
	usually expressed as the weight of substance per weight or volume of water, 
	air, or feed (e.g., mg/L, mg/kg, or ppm).
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
mg/L	milligrams per liter
MP	manufacturing-use product
mPa·s 	milliPascal-second, term used as the unit of dynamic viscosity.
MRID No.	Master Record Identification Number
NIOSH 	National Institute for Occupational Safety and Health
NRRL	Northern Regional Research Laboratory
OCSPP	Office of Chemical Safety and Pollution Prevention
OPP	Office of Pesticide Programs
PC Code	Pesticide Chemical Code
PP	Pesticide Petition
PPE	personal protective equipment
ppm	parts per million	
TGAI 	technical grade of the active ingredient
U.S. 		   United States







IX. BIBLIOGRAPHY

A.  Studies Submitted to Support Pesticide Product Registrations
48165500
Certis U.S.A., LLC (2010) Submission of Product Chemistry and Toxicity Data in Support of the Application for Registration of CX-9032. Transmittal of 6 Studies.
48165501
Chen, C. (2010) Bacillus subtilis Var. amyloliquefaciens Strain D747 (CX-9032): Product Identity, Manufacturing Process, Discussion of Formation of Unintentional Ingredients: Analysis of Samples: Certification of Limits. Unpublished study prepared by Certis USA, LLC. 120 p.
48165502
DeVane, W. (2010) Bacillus subtilis var. amyloliquefaciencs Strain D-747 End-Use Product: Color: Physical State: Odor: Stability to Normal and Elevated Temperatures, Metals, and Metal Ions: Storage Stability of the Liquid Concentrate: Miscibility: Corrosion Characteristics: pH: Viscosity: Bulk Density. Unpublished study prepared by Certis USA, LLC. 8 p.
48165503
Simpson, A. (2010) Bacillus subtilis var. amyloliquefaciens Strain D-747 End-Use Product: 830.6317 Storage Stability - Interim Study Report. Unpublished study prepared by Certis USA, LLC. 6 p.
48165504
Dively, C. (2010) Bacillus subtilis var. amyloliquefaciens Strain D747: CX-9032: Acute Oral Toxicity: Acute Dermal Toxicity: Acute Inhalation Toxicity: Hypersentivity (Sic). Unpublished study prepared by Certis USA, LLC. 6 p.
48165505
Kuhn, J. (2010) Bacillus subtilis CX-9032: Acute Eye Irritation Study in Rabbits: Final Report. Project Number: 14046/10. Unpublished study prepared by Stillmeadow, Inc. 17 p.
48165506
Kuhn, J. (2010) Bacillus subtilis CX-9032: Acute Dermal Irritation Study in Rabbits: Final Report. Project Number: 14047/10. Unpublished study prepared by Stillmeadow, Inc. 11 p.
48165700
Certis U.S.A., L.L.C. (2010) Submission of Product Chemistry and Toxicity Data in Support of the Petition for Tolerance of Bacillus subtilis var. amyloliquefaciens and the Application for Registration of CX-9030. Transmittal of 17 Studies.
48165701
Chen, C. (2010) Bacillus subtilis var. amyloliquefaciens Strain D747: (CX-9030): Product Identity: Manufacturing Process: Discussion of Formation of Unintentional Ingredients: Analysis of Samples: Certification of Limits. Unpublished study prepared by Certis USA, LLC. 145 p.
48165702
DeVane, W. (2010) Bacillus subtilis var. amyloliquefaciens Strain D-747 End-Use Product (ID Code CX-9030): Product Properties: Color: Physical State: Odor: Stability to Normal and Elavated Temperatures, Metals, and Metal Ions: Storage Stability of the TGAI: Miscibility: Corrosion Characteristics: pH: Viscosity: Bulk Density. Unpublished study prepared by Certis USA, LLC. 10 p.
48165703
Simpson, A. (2010) Bacillus subtilis var. amyloliquefaciens Strain D-747 End-Use Product (ID Code CX-9030): Microbial Pesticide Data Requirements: 830.6317 Storage Stability. Unpublished study prepared by Certis USA, LLC. 6 p.
48165704
Onishi, Y. (2003) Bacillus subtilis var. amyloliquefaciens Strain D747: D747 Single Oral Administration Toxicity Study in Rats: Acute Oral Toxicity/Pathogenicity: Final Report. Project Number: B030504. Unpublished study prepared by Mitsubishi Chemical Safety Institute, Ltd. 25 p.
48165705
Onishi, Y. (2003) Bacillus subtilis var. amyloliquefaciens Strain D747: D747 Single Intravenous Administration Toxicity in Rats: Acute Injection Toxicity/Pathogenicity: Final Report. Project Number: B030506. Unpublished study prepared by Mitsubishi Chemical Safety Institute, Ltd. 29 p.
48165706
Onishi, Y. (2003) D747 Single Endotracheal Administration Toxicity Study in Rats: Acute Pulmonary Toxicity/Pathogenicity: Final Report. Project Number: B030505. Unpublished study prepared by Mitsubishi Chemical Safety Institute, Ltd. 29 p.
48165707
Kuhn, J. (2010) Acute Oral Toxicity Study (UDP) in Rats: Bacillus subtilis CX-9030: Final Report. Project Number: 13847/10. Unpublished study prepared by Stillmeadow, Inc. 11 p.
48165708
Kuhn, J. (2010) Acute Dermal Toxicity Study in Rats: Bacillus subtilis CX-9030: Final Report. Project Number: 13848/10. Unpublished study prepared by Stillmeadow, Inc. 12 p.
48165709
Doig, A. (2010) Acute Inhalation Toxicity Study in Rats: Bacillus subtilis Cx-9030: Final Report. Project Number: 13820/10. Unpublished study prepared by Stillmeadow, Inc. 18 p.
48165710
Kuhn, J. (2010) Acute Eye Irritation Study in Rabbits: Bacillus subtilis CX-9030: Final Report. Project Number: 13821/10. Unpublished study prepared by Stillmeadow, Inc. 16 p.
48165711
Kuhn, J. (2010) Acute Dermal Irritation Study in Rabbits: Bacillus subtilis CX-9030: Final Report. Project Number: 13822/10. Unpublished study prepared by Stillmeadow, Inc. 11 p.
48165712
Hubbard, P.; Beavers, J. (2010) Bacillus subtilis CX-9025: An Acute Oral Toxicity Study with the Northern Bobwhite. Project Number: 671/102. Unpublished study prepared by Wildlife International, Ltd. 33 p.
48165713
Cockroft, R. (2004) Freshwater Fish Toxicity/Pathogenicity (Prolonged Toxicity to Microbial Pesticides to Fish) (Oncorhynchus mykiss): Bacillus subtilis var. amyloliquefaciens Strain D747: Final Report. Project Number: KCI/275/042551. Unpublished study prepared by Huntingdon Life Sciences, Ltd. 37 p.
48165714
Cockroft, R. (2010) (Prolonged Toxicity of Microbial Pesticides to Daphnia magna): Freshwater Invertebrate Toxicity/Pathogenicity: Bacillus subtilis var. amyloliquefaciens Strain D747: Final Report. Project Number: KCI/276/042552. Unpublished study prepared by Huntingdon Life Sciences, Ltd. 51 p.
48165715
Takagaki, M. (2004) Bacillus subtilis var. amyloliquefaciens Strain D747: Non-Target Plant Test: Final Report. Unpublished study prepared by Kumiai Chemical Industry Co. 13 p.
48165716
Dively, C. (2002) Bacillus subtilis var. amyloliquefaciens Strain D747: Non-Target Insect Testing: Final Report. Unpublished study prepared by Japan Plant Protection Assoc. Research Institute. 17 p.
48165717
Dively, C. (2003) Bacillus subtilis var. amyloliquefaciens Strain D747: An Acute Dietary Pathogenicity and Toxicity Study with the Honey Bee (Apis mellifera): Final Report. Unpublished study prepared by Life Science Research Institute. 5 p.
48621501
Dively, C. (2011) Bacillus subtilis var. Amyloliquefaciens Strain D747: Avian Oral Toxicity/Pathogenicity (in Northern Bobwhites): Addendum to MRID 48165712. Unpublished study prepared by Certis USA, LLC. 10p.
48621502
Dimock, M. (2011) Bacillus subtilis var. amyloliquefaciens Strain D747: Non-Target Insect Testing: Addendum to MRID 48165716. Unpublished study prepared by Certis USA, LLC. 20p.
48621503
Dively, C. (2011) Bacillus subtilis var. amyloliquefaciens Strain D747: Acute Dietary Pathogenicity and Toxicity Study with the Honey Bee (Apis mellifera): Addendum to MRID 48165717. Unpublished study prepared by Certis USA, LLC. 9p.

B.  Environmental Protection Agency Risk Assessment Memoranda and Other Documents

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.

U.S. EPA. 1996. Microbial Pesticide Test Guidelines  -  Background for Residue Analysis of 
		Microbial Pest Control Agents (OPPTS 885.2000). Available from 
		http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/series885.htm

U.S. EPA. 2010.  Bacillus subtilis Case 6012. Final Registration Review Decision; Docket Number  EPA-HQ-OPP-2007-1026

   
C.  Other References

Benitez, LB, et. al. Antimicrobial factor from Bacillus amyloliquefaciens inhibits PaeniB.      agent of American foulbrood.  Arch. Microbiology August 20, 2010

Centers for Disease Control and Prevention. 2009. Drinking Water - Water Treatment. Available from http://www.cdc.gov/healthywater/drinking/public/water_treatment.html.

Dedj. S., et.al. Effectiveness of honeybees in delivering the biocontrol agent Bacillus subtilis to blueberry flowers to suppress mummy berry disease. Biol. Control 31:422-427.

European Food SafetyAuthority.  2010.  Scientific opinion on the compatibility of Ecobiol (Bacillus amyloliquefaciens CECT 5940) for chickens for fattening with coccidiostats.  EFSA Journal 8: 1918.

Logan, N.A., and P. de Vos.  2009.  Genus I. Bacillus, pp. 21-128 In: P. de Vos, G.M. Garrity, D. Jones, N.R. Krieg, W. Ludwig, F.A. Rainey, K.H. Schleifer, and W. Whitman (Eds.) Bergey's Manual of Systematic Bacteriology, Volume 3, 2[nd] Ed. Springer, New York.

Maccagnami, B., et. al. Apis mellifera and Osmia cornuta as carriers for the secondary spread of Bacillus subtilis on apple flowers. Biocontrol 54: 123-133.
McClintock, JT., Schaffer, CR., Sjoblad, RD., A comparative review of the mammalian toxicity of Bacillus thuringensis-based pesticides. Pestic Sci 1995; 45:  pp 95  -  105.

Murray, P.R, et al., Manual of Clinical Microbiology. Washington, D. C.: ASM Press; 9th edition, 2007.

Priest, F.G., Goodfellow, M., Shute, L.A, and Berkeley, R.C.W. 1987.  Bacillus amyloliquefaciens sp. nov., nom. rev.  International Journal of Systematic Bacteriology, 37: 69-71.

Tanada, Y., and. Kaya, H.K.  1993.  Insect Pathology.  Academic Press, New York. 666 pages.

Wizna, R. Yose, H., Abbas, A., Dharma, I.P. Kompiang.  2009.  Influence of dietary fermented tapioca by-products on the performance of broilers and ducklings.  International Journal of Poultry Science 8: 902-904.

World Health Organization, Guidelines for Drinking-water Quality (2011) Fourth Edition.





             APPENDIX A.  MICROBIAL PESTICIDES DATA REQUIREMENTS 
                        (40 CFR PART 158  -  SUBPART V)
                                       
TABLE 1.  Product Analysis Data Requirements for the End-Use Product (EP), CX-9030 
                             (40 CFR § 158.2120)
                            OCSPP Guideline Number
                               Data Requirement
                                    Results
                                   MRID No.
                                   885.1100
                               Product Identity
Submitted data fulfill the requirement for product identity. CX-9030 contains  25.0% by weight Bacillus amyloliquefaciens strain strain D747 (minimum of  5x 10[1]0 CFU/g)
                                   481655-01
                                       
                                   CSF dated
                                  10/18/2011
                                   885.1200
                             Manufacturing Process
Submitted data fulfill the requirement for manufacturing process.
                                   481655-01
                                Not applicable
     Deposition of a Sample in a Nationally Recognized Culture Collection
Submitted data fulfill the requirement for deposition.  Culture on deposit under Accession  Number NRRL B-50405.
                                   481655-01
                                   885.1300
             Discussion of Formation of Unintentional Ingredients
Submitted data fulfill the requirement for discussion of formation of unintentional ingredients.
                                   481655-01
                                   885.1400
                              Analysis of Samples
Submitted date fulfill the requirement for analysis of samples.  
                                   481655-01
                                   885.1500
                            Certification of Limits
Limits listed on the confidential statement of formula are adequate/acceptable
                                   CSF dated
                                   10/18/2011
                              Additional Studies
                                   830.1800
                         Enforcement Analytical Method
Submitted data fulfill the requirement for an enforcement analytical method
                                   481655-01

TABLE 2.  Product Analysis Data Requirements for the End-Use Product (EP), CX-9032 
                             (40 CFR § 158.2120)
                            OCSPP Guideline Number
                               Data Requirement
                                    Results
                                   MRID No.
                                   885.1100
                               Product Identity
Submitted data fulfill the requirement for product identity. CX-9030 contains  98.95% by weight Bacillus amyloliquefaciens strain strain D747 (minimum of  1x 10[1][0] CFU/g)
                                   481655-01
                                   CSF dated
                                  10/18/2011
                                   885.1200
                             Manufacturing Process
Submitted data fulfill the requirement for manufacturing process.
                                   481657-01
                                   481655-01
                                Not applicable
     Deposition of a Sample in a Nationally Recognized Culture Collection
Submitted data fulfill the requirement for deposition.  Culture on deposit under Accession Number NRRL B-50405.
                                   481657-01
                                   481655-01
                                   885.1300
             Discussion of Formation of Unintentional Ingredients
Submitted data fulfill the requirement for discussion of formation of unintentional ingredients.
                                   481657-01
                                   481655-01
                                   885.1400
                              Analysis of Samples
Submitted date fulfill the requirement for analysis of samples.  
                                   481655-01
                                   481657-01
                                   885.1500
                            Certification of Limits
Limits listed on the confidential statement of formula are adequate/acceptable
                             CSF dated  10/18/2011
                              Additional Studies
                                   830.1800
                         Enforcement Analytical Method
Submitted data fulfill the requirement for an enforcement analytical method
                                   481655-01
TABLE 3.  Physical and Chemical Characteristics for the Technical Grade of the Active Ingredient (TGAI) Bacillus. amyloliquefaciens strain strain D747 / CX-9030 (EP). (40 CFR § 158.2120)
                            OCSPP Guideline Number 
                               Data Requirement
                                    Results
                                       
                                   MRID No.
                                       
                                       
                                     TGAI
                                 CX-9030 (EP)
                                       
                                   830.6302
                                       
                                     Color
                                     Beige
                                Not Applicable
                                  481657-02 
                                   830.6303
                                Physical State
                                  Fine powder
                                Not applicable
                                       
                                   481657-02
                                   830.6304
                                     Odor
                                  Yeast odor 
                                Not applicable
                                   481657-02
                                   830.6313
                       Stability to Normal and Elevated
                             Temperatures, Metals,
                                and Metal Ions
                        Spores inactivated at 54[o]C  
                                Not applicable
                                   481657-02
                                   830.6314
                             Oxidation/Reduction:
Chemical  Incompatibility
            Not applicable, the product does not contain oxidizing 
or reducing agents 
481657-02
                                   830.6315
                                 Flammability
Not applicable, the product does not contain flammable ingredients
481657-02
                                   830.6316 
                                 Explodability
Not applicable, the product does not contain explosive ingredients
481657-02
                                   830.6317
                               Storage Stability
           Stable up to one year at 25[o]C without loss of viability
                                       .
                                   481657-02
                                   481657-03
                                   830.6319
                                  Miscibility
          Not applicable, the product is not an emulsifiable liquid.
 (refer to test note #2 of 40 CFR § 158.2120(d)).
                                   481657-02
                                   830.6320
                          Corrosion  Characteristics
Not applicable, the product is a powder.
                                   481657-02
                                   830.6321e
                         Dielectric Breakdown Voltage
    Not applicable, the product is not for use around electrical equipment
                                   481657-02
                                   830.7000
                                      pH
                             6.5  -  7.0 (1% w/w)
                                   7.5- 8.0
                                   481657-02
                                   830.7100
                                   Viscosity
                   Not applicable, the product is a powder.
                   Not Applicable.CX-9030 is not a liquid.  
                                   481657-02
                                   830.7300
           Density/Relative Density/Bulk Density (Specific Gravity)
                               0.307- 0.375 g/ml
                              0.60-.0.78 g/ cm[3]
                                   481657-02

TABLE 4.  Physical and Chemical Characteristics for CX-9032 (EP). (40 CFR § 158.2120)
                            OCSPP Guideline Number 
                               Data Requirement
                                    Results
                                       
                                   MRID No.
                                       
                                       
                                     TGAI
                                 CX-9030 (EP)
                                       
                                   830.6302
                                       
                                     Color
                                     Beige
                                  Light brown
                                  481655-02 
                                   830.6303
                                Physical State
                                  Fine powder
                                    Liquid
                                   481655-02
                                   830.6304
                                     Odor
                                  Yeast odor 
                                  Yeast odor
                                   481655-02
                                   830.6313
                              Stability to Normal
                                & Elevated
                             Temperatures, Metals,
                               & Metal Ions
                       Spores inactivated at 54[o]C..  
                                   481655-02
                                   830.6317
                               Storage Stability
           Stable up to one year at 25[o]C without loss of viability
Stable up to 78 days at 25[o]C without loss of viability.  As a term of the registration, EPA will require the results of a one-year storage stability and corrosion characteristics study be submitted within one year.
481655-02
                                   830.6319
                                  Miscibility
Not applicable; product is not an emulsifiable liquid.
 (refer to test note #2 of 40 CFR § 158.2120(d)).
                                   481655-02
                                   830.6320
                          Corrosion  Characteristics
                     Not applicable; product is a powder.
None evident to polyethylene packaging after 78 days.  As a term of the CX-9032 (EP) registration, EPA will require submission of the results of a one-year storage stability & corrosion characteristics within one year.
                                   481655-02
                                   830.7000
                                      pH
                             6.5  -  7.0 (1% w/w)
                              4.2 - 4.3 (1% w/w).
                                   481655-02
                                   830.7100
                                   Viscosity
                   Not applicable, the product is a powder.
               4.6 - 16.0 milliPascal-second (mPa s) at 25[o]C 
                                        
                                   481655-02
                                   830.7300
           Density/Relative Density/Bulk Density (Specific Gravity)
                               0.307- 0.375 g/ml
                              1.02  -  1.03 g/mL 
                                   481655-02

TABLE 5.  Toxicology Data Requirements for the Technical Grade of the Active Ingredient (TGAI) (40 CFR § 158.2140)
                            OCSPP Guideline Number
                               Data Requirement
                                    Results
MRID No.
                                       
                                       
                                     TGAI
                                       
                                    Tier I
                                   885.3050
                       Acute Oral Toxicity/Pathogenicity
No evidence of infectivity, pathogenicity or toxicity was found from oral administration of 1.0x10[8] CFU Bacillus amyloliquefaciens strain D747 to rats. Clearance from fecal material occurred by day 14 and no viable organisms were recovered from blood or any other organ or tissue.
Classification: Acceptable
                                   481657-04
                                       
                                       
                                   885.3150
                    Acute Pulmonary Toxicity/Pathogenicity
 No evidence of infectivity, pathogenicity or toxicity was found from intratracheal administration of 1.0x10[7] spores B. amyloliquefaciens strain D747 to rats. 
Classification: Acceptable
                                   481657-06
                                       
                                   885.3200
             Acute Injection Toxicity/Pathogenicity (Intravenous)
Not toxic, infective, and/or pathogenic to rats when dosed intravenously at 1.0x10[7] spores per animal.
Classification: Acceptable 
                                   481657-05
                                       
                                   885.3400
                               Hypersensitivity 
                                   Incidents
No hypersensitivity incidents, including immediate-type or delayed-type reactions of humans and domestic animals that occurred during research, development, or testing of the TGAI/MP, were reported. Future hypersensitivity incidents must be reported (For reporting format: OCSPP Guideline 885.3400). 
                                   479450-23
                                   885.3500
                                 Cell Culture
Not required. B. amyloliquefaciens strain D747 is not a virus
 (Test note #4 of 40 CFR § 158.2140(d)).
                                       
                               Tiers II and III
Not required for Bacillus amyloliquefaciens strain D747 based on the lack of acute toxicity/pathogenicity in the Tier I studies.









 TABLE 6.  Toxicology Data Requirements for the End-Use Product (EP), CX-9030
                              (40 CFR § 158.2140)
                            OCSPP Guideline Number
                               Data Requirement
                                    Results
                                   MRID No.
                                   885.3400
                          Hypersensitivity Incidents
No hypersensitivity incidents, including immediate-type or delayed-type reactions of humans and domestic animals that occurred during research, development, or testing of the EP, were reported by the applicant. Any future hypersensitivity incidents must be reported per OCSPP Guideline 885.3400.
                                       
                                   870.1100
                              Acute Oral Toxicity
                          Oral LD50  > 5000 mg/Kg 
Classification: Acceptable
Toxicity Category IV 
                                   481657-07
                                       
                                   870.1200
                             Acute Dermal Toxicity
Dermal LD50 > 5050 mg/Kg Classification: Acceptable
TOXICITY CATEGORY IV 
                                   481657-08
                                       
                                   870.1300
                           Acute Inhalation Toxicity
Inhalation LC50 > 2.18 mg/L
Classification: Acceptable
TOXICITY CATEGORY IV
                                       
                                   481657-09
                                   870.2400
                             Acute Eye Irritation
The maximum average irritation score of 18.3 obtained 1 hour after treatment declined to 17.33 after 24 hours, 2 after 48 hours and 0 after 72 hours following ocular administration of 0.1 mL B. amyloliquefaciens strain D747 to New Zealand White rabbits in a 72 hour observation period.
Classification: Acceptable
TOXICITY CATEGORY III
                                       
                                   481657-10
                                   870.2500
                           Primary Dermal Irritation
No evidence of irritation was found from dermal administration of 500 mg B. amyloliquefaciens strain D747 CX-9030 to rabbits during the 4 hour exposure and 72 observation periods. The dermal irritation score for B. amyloliquefaciens strain D747 CX-9030 was 0.00.
Classification: Acceptable
TOXICITY CATEGORY IV
                                   481657-11



 TABLE 7.  Toxicology Data Requirements for the End-Use Product (EP), CX-9032
                              (40 CFR § 158.2140)
                            OCSPP Guideline Number
                               Data Requirement
                                    Results
                                   MRID No.
                                   885.3400
                          Hypersensitivity Incidents
No hypersensitivity incidents including immediate-type or delayed-type reactions of humans and domestic animals that occurred during research, development, or testing of the EP were observed or reported. All hypersensitivity incidents must be reported per OCSPP Guideline 885.3400.
                                       
                                   870.1100
                              Acute Oral Toxicity
Waiver request submitted. Requirement satisfied by submitted MP data. Oral exposure to the MP showed no adverse effects including infectivity, pathogenicity and toxicity up to the limit dose. Inerts are exempt from tolerance. No additional oral toxicity is expected from inerts. 
Classification: Acceptable
                                   481655-04
                                       
                                   870.1200
                             Acute Dermal Toxicity
Waiver request submitted. Requirement adequately addressed by CX-9030 EP  data as well as, dermal irritation testing on CX-9032-EP. Dermal toxicity and irritation testing for the CX-9030 EP and dermal irritation data on CX-9032 EP showed no adverse effects up to the limit doses. No additional dermal toxicity is expected from inerts. 
Classification: Acceptable
                                   481655-04
                             481655-06  481655-11
                                  481657-08 
                                       
                                   870.1300
                           Acute Inhalation Toxicity
Waiver request submitted. Pulmonary exposure to the MP showed no adverse effects, including infectivity, pathogenicity or toxicity though slight toxicity lasting 2 days from a 4-hour aerosol inhalation administration of 2.18 mg/L, where inert ingredients were also present, was noted in a CX-9030 EP study. No additional toxicity is expected from this EP's inert ingredients. Classification: Acceptable
                                   481655-04
                                   481657-06
                                   481657-09
                                       
                                   870.2400
                             Acute Eye Irritation
The maximum average irritation score of 0.667 at 1 hour after treatment declined to 0 after 24 hours following ocular administration of 0.1 mL 
B. amyloliquefaciens strain D747 CX-9032 to New Zealand White rabbits (72- hour observation period).
Classification: Acceptable
TOXICITY CATEGORY IV
                                   481655-05
                                   870.2500
                           Primary Dermal Irritation
No irritation occurred from dermal administration of 0.5 mL undiluted B. amyloliquefaciens strain D747 CX-9032 to shaved skin of rabbits during the 4 hour exposure and 72 observation periods. The dermal irritation score for B. amyloliquefaciens strain D747 CX-9032 was 0.00.
Classification: Acceptable
TOXICITY CATEGORY IV
                                   481655-06
                                       
                                        





TABLE 8.  Nontarget Organism Toxicity and Environmental Fate Data Requirements for the Technical Grade of the Active Ingredient (TGAI), Bacillus amyloliquefaciens strain D747 (40 CFR § 158.2150)
                            OCSPP Guideline Number
                               Data Requirement
                                    Results
                                   MRID No.
                                    Tier I
                                   885.4050
                              Avian Oral Toxicity
A study showed that B. amyloliquefaciens D747 is not toxic to birds at 8.9 x 10[9] spores/bird.
Classification:  Supplemental
Scientific rationale is sufficient to conclude that B. amyloliquefaciens D747 is not expected to pose a hazard to birds.
Classification:  Acceptable 
                                   48165712
                                   48621501
                                       
                                       
                                       
                                   885.4100
                    Avian Inhalation Toxicity/Pathogenicity
Not required.  B. amyloliquefaciens D747 is not considered to be pathogenic to birds 
                                       
                                       
                                   885.4150
                      Wild Mammal Toxicity/Pathogenicity
Tests required by 40 CFR § 158.2140 are adequate/ appropriate for assessment of hazards to wild mammals.  B. amyloliquefaciens D747 was not infective, toxic or pathogenic to laboratory rats 1.0 x 10[8] CFU/animal 
                                   48165704
                                   885.4200
                    Freshwater Fish Toxicity/Pathogenicity
A 30-day study shows that the LC50 to rainbow trout is 8 x 10[10] CFU/L.  The NOEC for sublethal effects is 1.44 x 10[10] CFU/L.
Classification:  Acceptable 
                                   48165713
                                   885.4240
                Freshwater Invertebrate Toxicity/Pathogenicity
A 21-day study shows that the EC50 to Daphnia magna based on mortality is 3.7 x 10[10] CFU/L. The NOEC for sublethal effects is 2.84 x 10[8] CFU/L.
Classification:  Acceptable
                                   48165714
                                   885.4280
                Estuarine/Marine Fish and Invertebrate Testing
Not required.  B. amyloliquefaciens D747 is not to be applied directly to water and is not expected to reach estuarine or marine environments in significant quantities.
                                       
                                   885.4300
                            Nontarget Plant Testing
A study submitted was determined to be unacceptable; however, testing is not required because. B. amyloliquefaciens is not related to known plant pathogens
                                   48165715
                                   885.4340
                           Nontarget Insect Testing
Studies with three species of arthropods were determined to be unacceptable.  
Additional scientific rationale was submitted to show that B. amyloliquefaciens is not expected to be toxic or pathogenic to nontarget insects.
Classification:  Acceptable
                                   48165716
                                       
                                   48621502
                                       
                                   885.4380
                               Honey Bee Testing
Two studies showed that B. amyloliquefaciens D747 is not toxic to honey bees.   
Classification:  Supplemental
Additional rationale was sufficient to show that pathogenicity to honey bees is not expected as a result of exposure to B. amyloliquefaciens D747. 
Classification:  Acceptable
                                   48165717
                                       
                                   48621503
                             Tiers II, III, and IV
Not required for Bacillus amyloliquefaciens strain D747 based on the current uses and application methods.










                        APPENDIX B.  PESTICIDE PRODUCTS
TABLE 9. Table Title?
                           EPA Registration Number 
                               Registration Name
                         Percentage Active Ingredient
                               Formulation Type
                                  Use Site(s)
                           Method(s) of Application
                               Application Rate
                                  Target Pest
                                       
70051-107
                                       
                                    CX-9032
                                       
                                    98.35%

End Use  -  Aqueous Suspension
Various agricultural and greenhouse crops (e.g., vegetables, tree fruits, berries, grapes and tropical fruit, tree nuts, herbs and spices, coffee, tobacco, hops, forestry seedlings ornamentals, and turf) 
Tractor mounted boom, airblast, hose-end, backpack and other pressurized sprayers; foggers or mist blowers; water wheel and other drench applicators; soil injection; aerial; and chemigation with  drip or sprinkler irrigation  and cutting or root dip
Rate listed on label varies depending on application method  from  O.5 pints /acre to 6 quarts/acre 
       Various fungal and bacterial pests listed on the label including:
                                  Alternaria,
          Botrytis cinerea Didymella bryoniae Phoma cucurbitacearum 
                                   Erisphe, 
                                  Fusarium, 
 Macrophomina phaseoli Monosporascus cannonballus
                          Peronospora , Phytophthora 
            Pseudomonas syringae pv. tomato Pseudoperonospora spp.
                                 Puccinia spp.
                                   Pythium,
                        Rhizoctonia, Sphaerotheca spp.
                      Verticillium, spp. Xanthomonas spp.

                                       
                                   70051-108
                                   CX-9030 
                                     25.0%
                          Water Dispersible Granule 
                                       
Various agricultural and greenhouse crops (e.g., vegetables, ornamentals, and turf)
Tractor mounted boom, airblast, hose-end, backpack and other pressurized sprayers; foggers or mist blowers; water wheel and other drench applicators; shank or other soil injection equipment; aerial; and chemigation with  drip or sprinkler irrigation and cutting or root dip 
                           0.25  - 3 pounds per acre
       Various fungal and bacterial pests listed on the label including:
                                  Alternaria,
          Botrytis cinerea Didymella bryoniae Phoma cucurbitacearum 
                                   Erisphe  
                                  Fusarium, 
 Macrophomina phaseoli Monosporascus cannonballus
                          Peronospora, Phytophthora 
            Pseudomonas syringae pv. tomato Pseudoperonospora spp.
                                 Puccinia spp.
                                   Pythium,
                        Rhizoctonia, Sphaerotheca spp.
                     Verticillium, spp., Xanthomonas spp.
                                       


