	

                                                       
                                       
                                       

                                   Proposed
                  BIOPESTICIDES REGISTRATION ACTION DOCUMENT
                                       
                                       
                                       
                                       
                                       
                                       
                                       

                                       
                                       
                       Bacillus subtilis strain CX-9060 
                                       
                     Pesticide Chemical (PC) Code: 016480
                                       
                                       
                                       
                                       

   
	U.S. Environmental Protection Agency
	Office of Pesticide Programs
                Biopesticides and Pollution Prevention Division
                                       
                                       
                                       
                                November, 2011
                                       
                                       
                                       
                               TABLE OF CONTENTS
I. 	EXECUTIVE SUMMARY	4
II. 	ACTIVE INGREDIENT OVERVIEW	6
III.	 REGULATORY BACKGROUND	6
      A. Application for Pesticide 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	13
VI. 	RISK MANAGEMENT DECISION	14
VII. 	ACTIONS REQUIRED of THE REGISTRANT	15
      A. Final Printed Labeling	15
      B. Terms of Registration	15
      C. Reporting of Adverse Effects and Hypersensitivity Incidents	15
VIII. GLOSSARY OF ACRONYMS AND ABBREVIATIONS	16
IX. 	BIBLIOGRAPHY	17
      A. Submitted and Cited Studies	17
      B. EPA Risk Assessment Memoranda and Other Documents	18
APPENDIX A. MICROBIAL PESTICIDES DATA REQUIREMENTS	20
APPENDIX B. PESTICIDE PRODUCT	24
                                       
                                       
                                       
                                       
                                       
                BIOPESTICIDES REGISTRATION ACTION DOCUMENT TEAM
                                       
                                       
                      Office of Pesticide Programs (OPP)
                Biopesticides and Pollution Prevention Division
                          Microbial Pesticides Branch
                                       
                                       
                                Science Reviews
                                       
John Kough, Ph.D., Senior Scientist	Product Analysis, Human Health
Kathleen Martin, M.S.E.S.	Product Analysis, Human Health
Gail Tomimatsu, Ph.D.	Environmental Effects 



                                  Regulations
                                       
Sheryl K. Reilly, Ph.D.				   Chief, Microbial Pesticides Branch
Denise Greenway	Regulatory Action Leader
I.  EXECUTIVE SUMMARY

On July 30, 2009, Certis U.S.A., L.L.C. (Certis) submitted an application for an end-use pesticide product, Bacillus subtilis CX-9090 (EPA File Symbol 70051-RNL), to the United States Environmental Protection Agency (EPA or the Agency). Bacillus subtilis CX-9090 contains a new active ingredient, Bacillus subtilis strain CX-9060, and is proposed for fungicide and bactericide uses on food crop and ornamental plants. 

Bacillus subtilis is a rod-shaped, gram-positive, aerobic, motile (peritrichous flagella) bacterium, which is ubiquitous in nature and has been recovered from water, soil, air, and decomposing plant residues. The bacterium commonly produces proteases and other enzymes; as a result, strains of the bacterium are often used for industrial production of enzymes and other chemicals. The bacterium also produces an endospore that allows it to endure extreme conditions of heat and desiccation in the environment. Bacillus subtilis is not considered toxic or pathogenic to humans, animals, or plants (U.S. EPA 1997). Several strains of Bacillus subtilis are used predominantly as fungicidal active ingredients within various pesticide formulations registered with the Agency.   

A new strain, Bacillus subtilis strain CX-9060, proposed for pesticide use by Certis, is the subject of this Biopesticides Registration Action Document (BRAD). Bacillus subtilis strain CX-9060 was isolated from a peat medium containing a naturally occurring strain of the Bacillus subtilis bacterium. The progenitor strain is a currently registered pesticide. Data and information, submitted by Certis and reviewed by the Agency, demonstrated the similarity of the two strains. The established level of equivalency is such that citation of existing data on the progenitor strain is a valid approach to support the Bacillus subtilis strain CX-9060 registration application. 

EPA scientists have reviewed product analysis, toxicology, and nontarget organism data and information submitted or cited to support registration of the pesticide product containing Bacillus subtilis strain CX-9060. Product analysis data requirements for Bacillus subtilis strain CX-9060, including product chemistry and composition, analysis of samples, and physical and chemical characteristics, were adequately fulfilled by acceptable guideline studies. In support of mammalian and nontarget organism toxicology, pathogenicity, infectivity and irritation testing, the Bacillus subtilis strain CX-9060 application included both newly conducted Bacillus subtilis strain CX-9060 studies and the citation of existing data (conducted with the progenitor strain). EPA found such scientific information submitted or cited by the applicant to fulfill the relevant toxicology and nontarget organism data requirements. 

Bacillus subtilis strain CX-9060 is not expected to pose any singular or cumulative risks to humans, including infants and children, or to other mammals through exposure in the food supply, from drinking or surface waters, or through other non-dietary exposures. Previously reviewed studies showed no infectivity or pathogenicity of the progenitor strain and were assigned the EPA Toxicity Category IV rating for all tested routes of administration. There have been no reports of hypersensitivity in over 15 years of registered uses of the progenitor strain, and Bacillus subtilis, in general, is ubiquitous in soils and common in agricultural environments. In addition, non-occupational exposure is considered unlikely for Bacillus subtilis strain CX-9060 as all currently proposed uses occur in distinctly agricultural or commercial settings; there are no proposed uses for residential areas. Overall, EPA concludes that there is a reasonable certainty that no harm will result to the United States (U.S.) population, including infants and children, from aggregate exposure to residues of Bacillus subtilis strain CX-9060.

Despite the low toxicological profile of Bacillus subtilis strain CX-9060, baseline personal protective equipment (PPE) is required for handlers that may be exposed to the active ingredient and a restricted-entry interval is required after pesticide application. Handlers working with Bacillus subtilis strain CX-9060 in occupational and agricultural settings are directed to wear a long-sleeved shirt, long pants, socks, shoes, and 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. In accordance with the criteria set forth in Pesticide Registration Notice 95-3 (http://www.epa.gov/PR_Notices/pr95-3.html), a reduced restricted-entry interval of four hours for all uses is appropriate based on the submitted or cited toxicology/pathology data.
 
The submitted or cited data and other information fulfilled the current data requirements and were adequate for risk assessment purposes. EPA has concluded that the proposed use of Bacillus subtilis strain CX-9060 meets the standard 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, this proposed registration decision for the pesticide product containing Bacillus subtilis strain CX-9060, a new active ingredient, is subject to a 30-day comment period. In addition to containing a new active ingredient, the registration of this product would result in both the first outdoor use and first food use for Bacillus subtilis strain CX-9060. 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-2010-0103. The following documents are available for comment in EPA-HQ-OPP-2010-0103: (1) proposed Bacillus subtilis strain CX-9060 BRAD; (2) environmental risk assessment for Bacillus subtilis strain CX-9060; and (3) proposed product label for the end-use pesticide product, Bacillus subtilis CX-9090 (EPA File Symbol 70051-RNL). While a final decision on registration is contingent upon review and consideration of public comments, EPA believes that, based upon the risk assessment and information submitted in support of Bacillus subtilis CX-9090 (the end-use pesticide product), it is in the best interest of the public and the environment to issue this registration. The basis for this decision can be found in the risk assessment for Bacillus subtilis strain CX-9060 (the new active ingredient), which is characterized throughout this BRAD.  












II.  ACTIVE INGREDIENT OVERVIEW

    Biological Name:  	Bacillus subtilis strain CX-9060
    
    Culture Deposit:		Agricultural Research Service Culture Collection (also 
    		known as the Northern Regional Research Laboratory 
    		Collection) in Peoria, Illinois under Accession 
    		Number NRRL B-50287
                                        
 	OPP Chemical Code: 		016480	
 
	Type of Pesticide:				Microbial Pesticide  -  Fungicide and Bactericide

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

III.	 REGULATORY BACKGROUND

A. Application for Pesticide Registration

On July 30, 2009, Certis U.S.A., L.L.C. (address: 9145 Guilford Road, Suite 175, Columbia, MD 21046) submitted an application to register an end-use pesticide product, Bacillus subtilis CX-9090 (EPA File Symbol 70051-RNL), under FIFRA section 3. On March 10, 2010, EPA announced receipt of this application to register a pesticide product containing a new active ingredient (75 Federal Register (FR) 11175) 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

On November 13, 2009, and under Federal Food, Drug, and Cosmetic Act (FFDCA) section 408(d), Certis U.S.A., L.L.C. submitted a petition to establish an exemption from the requirement of a tolerance for Bacillus subtilis strain CX-9060 (Pesticide Petition (PP) 9F7643). In the Federal Register of March 10, 2010 (75 FR 11171), EPA announced that Certis U.S.A., L.L.C. proposed to establish an exemption from the requirement of a tolerance for residues of the microbial pesticide, Bacillus subtilis strain CX-9060, in or on all food commodities, including residues resulting from post-harvest uses, and opened a 30-day comment period. No comments were received following this publication. 


IV.	 RISK ASSESSMENT

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

The classifications that are found for each data submission are assigned by EPA science reviewers and are 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 EPA in support of a pesticide registration. 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 (40 CFR § 156.62). 

A. Product Analysis Assessment (40 CFR § 158.2120)

All product analysis data requirements for Bacillus subtilis strain CX-9060 have been fulfilled (Master Record Identification Numbers (MRID Nos.) 478203-01, 478203-02 and 478203-03). Refer to Table 1 in Appendix A for a brief summary of the data requirements. 

The active ingredient is 25.0 % Bacillus subtilis strain CX-9060 (minimum of 
5 x 10[10] colony-forming units (CFU) per gram).

 B. Human Health Assessment (40 CFR § 158.2140)

1. Toxicity

All toxicology data requirements for Bacillus subtilis strain CX-9060 have been fulfilled either by the submission of valid Bacillus subtilis strain CX-9060 studies or by the citation of acceptable studies, conducted using an equivalent Bacillus subtilis strain, previously submitted to and reviewed by the Agency. Acceptable Tier I mammalian toxicology information found therein supports registration of the Bacillus subtilis CX-9090 end-use pesticide product. Furthermore, Tier II and Tier III studies were not required for Bacillus subtilis strain CX-9060 based on the data cited for Tier I toxicity/pathogenicity studies. Refer to Table 2, as well as its associated text, in Appendix A for a brief summary of the data requirements. 

      a. Acute Toxicity/Pathogenicity  -  Tier I

The applicant cited data adequate to fulfill requirements for toxicology, pathogenicity, and infectivity testing (MRID Nos. 419074-02, 419074-04, and 419074-05) for Bacillus subtilis strain CX-9060, and submitted acceptable toxicology and irritation data/information (MRID Nos. 478203-04, 478203-05, and 478203-06) for Bacillus subtilis CX-9090. Refer to Table 2, as well as its associated text, in Appendix A for a brief summary of the data requirements. 




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

Tier II and Tier III studies were not required for Bacillus subtilis strain CX-9060 based on the lack of acute toxicity/pathogenicity demonstrated by cited data that fulfilled the Tier I data requirements, and the Toxicity Category III (ocular) and IV (all other exposure routes) determinations made for the Bacillus subtilis CX-9090 end-use pesticide product.

	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.

Bacillus subtilis strain CX-9060 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 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 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.
Based on the acute toxicity/pathogenicity information discussed previously and presented in Table 2 (as well as its associated text) in Appendix A, the data required for a FFDCA risk assessment for Bacillus subtilis strain CX-9060 have been fulfilled.

	a. Aggregate Exposure

In examining aggregate exposure, section 408 of FFDCA directs EPA to consider available information concerning exposures from the pesticide residue in food and all other non-occupational 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: Bacillus subtilis is ubiquitous in the environment (U.S. EPA. 2008), especially in soils (Bergey. 2009) and agricultural environments (indeed, strain CX-9060 of Bacillus subtilis is derived from a naturally occurring isolate of the genus Bacillus, which was originally isolated from faba bean plants grown at the Nottingham University School of Agriculture in the United Kingdom). As a result, dietary exposure to background levels of the microbe likely is already occurring and likely will continue to occur. Because of the ubiquitous presence of Bacillus subtilis in the environment, the Agency expects there to be no increase in exposure to Bacillus subtilis strain CX-9060, resulting from the proposed pesticidal uses, when compared to existing exposure to background levels of Bacillus subtilis.
As discussed above, dietary exposure to Bacillus subtilis strain CX-9060 likely is already occurring and likely will continue to occur. Notably, similar Bacillus subtilis strains are used internationally in the production of food grade products and in fermented foods in Japan and Thailand. Reports in the literature, implicating Bacillus subtilis (as distinguished from the specific strain, Bacillus subtilis strain CX-9060, at issue in this BRAD) in food-borne illness, do not describe any pathogen or toxin production, but rather simple spoilage from Bacillus subtilis growth in dough. Such low-quality dough would not be suitable for bread production by commercial bakeries and so the Agency considers this particular food exposure scenario to be unlikely and the risk to be negligible. The risk posed to adults, infants and children from food-related exposures to Bacillus subtilis strain CX-9060 is minimal due to the expected lack of acute oral toxicity/pathogenicity associated with the microbial pesticide. Based on the evaluation of the submitted and cited data, there are no dietary risks that exceed the Agency's Level of Concern (LOC).
Drinking Water Exposure and Risk Characterization: Because Bacillus subtilis is ubiquitous in the environment, exposure to the microbe through drinking water may already be occurring and likely will continue to occur. While the proposed use sites do not include direct application to aquatic environments, the intended use of Bacillus subtilis strain CX-9060 is treatment of growing crops (including roots and cuttings) for the control of plant disease. If such uses were to result in pesticide spray drift or runoff that were to reach surface or ground waters, there is the potential for human exposure to Bacillus subtilis strain CX-9060 residues, albeit likely greatly diluted, in drinking water. Municipal drinking water treatment processes and deep water wells, however, should both further reduce any such residues. More importantly, even if oral exposure to this ubiquitous microbe should occur through drinking water, due to its expected lack of acute oral toxicity/pathogenicity, the Agency concludes that there is a reasonable certainty that no harm will result from such exposure (see section IV(B)(1)(a) and Table 2, as well its associated text, in Appendix A).  
Non-occupational, Residential Exposure and Risk Characterization: The pesticide uses of Bacillus subtilis strain CX-9060 are limited to commercial agricultural and horticultural settings. There are no residential uses. Nonetheless, because Bacillus subtilis is naturally occurring and ubiquitous in the environment, the potential for non-dietary, non-occupational exposure to its residues for the general population, including infants and children, is likely since populations have probably been previously exposed (and likely will continue to be exposed) to background levels of the microbe. Neither such common human exposures to similar Bacillus subtilis strains naturally present in soils, waters and plants, nor exposures associated with those Bacillus subtilis strains used internationally in producing food-grade products and fermented foods, however, have resulted in reports of disease or other effects. Finally, while the literature includes accounts of Bacillus subtilis infections in humans (which consistently are reported only in otherwise-compromised individuals), those reports are most notable for their rare and exceptional nature.  

EPA's evaluation of the required high-dose Tier I acute toxicity and pathogenicity tests that were cited resulted in the assignment of Toxicity Category IV (least toxic), as well as determinations of not infective and not pathogenic, for all exposure routes for the active ingredient. No toxicological end points of concern were identified. There are no dietary endpoints that exceed the Agency's LOC. Therefore, the Agency has determined that any additional exposure to the microbe resulting from residues attributable to Bacillus subtilis strain CX-9060 pesticide use will not result in additional aggregate non-occupational risk from dermal and inhalation exposures. This conclusion, based solely on non-occupational exposures, is consistent with EPA's determination that no occupational risks exceed the Agency's LOC, meaning that even regular occupational exposures associated with this active ingredient pose negligible risk.
	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."
No mechanism of toxicity in mammals has been identified for Bacillus subtilis strain CX-9060. For the purposes of this tolerance action, therefore, EPA has assumed that Bacillus subtilis strain CX-9060 does not have a common mechanism of toxicity with other substances. Therefore, section 408(b)(2)(D)(v) does not apply. For information regarding EPA's efforts to determine which chemicals have a common mechanism of toxicity and to evaluate the cumulative effects of such chemicals, see EPA's website at http://www.epa.gov/pesticides/cumulative/.
      c. Determination of Safety for the 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.

Based on the acute toxicity and pathogenicity submission and citations summarized in section IV(B)(1)(a) and Table 2 (as well as its associated text) in Appendix A, EPA concludes that 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 Bacillus subtilis strain CX-9060. Such exposure includes all anticipated dietary exposures and all other exposures for which there is reliable information. EPA has arrived at this conclusion because the information available on or cited in support of Bacillus subtilis strain CX-9060 does not demonstrate toxic, pathogenic, and/or infective potential to mammals. Thus, there are no threshold effects of concern and, as a result, the provision requiring an additional margin of safety is not necessary. 

3. Occupational Exposure and Risk Characterization

Handler exposure to Bacillus subtilis strain CX-9060 is not expected to pose any undue risk, but appropriate personal protective equipment and precautionary statements are required on the pesticide product label to mitigate any potential risks to pesticide handlers due to prolonged or numerous exposures. Handlers applying the Bacillus subtilis strain CX-9060 end-use pesticide product in commercial and 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. Furthermore, in accordance with the criteria set forth in Pesticide Registration Notice 95-3 (http://www.epa.gov/PR_Notices/pr95-3.html), a reduced restricted-entry interval of four hours for all uses is appropriate based on the submitted or cited toxicology/pathology data.
 
4. Human Health Risk Characterization
EPA considered human exposure to Bacillus subtilis strain CX-9060 in light of the standard for registration in FIFRA and the relevant safety factors in FFDCA. A determination has been made that no unreasonable adverse effects to the U.S. population in general, and to infants and children in particular, will result when the Bacillus subtilis strain CX-9060 pesticide product is used in accordance with EPA-accepted labeling.

C. Environmental Assessment (40 CFR § 158.2150)

The data and information submitted or cited by the applicant to support the pesticide product containing Bacillus subtilis strain CX-9060 are sufficient to fulfill the Tier I nontarget organism 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 for the proposed uses and application methods. EPA has performed an environmental risk assessment based on the data and information provided by the applicant and has determined that the proposed use of Bacillus subtilis strain CX-9060 does not pose significant risk to nontarget organisms when used according to label directions. 

For a comprehensive summary of the generic data requirements described in section IV(C)(1), refer to Table 3 in Appendix A.

1. Ecological Exposure and Risk Characterization  

	a. Terrestrial Animals and Plants 

Bacillus subtilis strain CX-9060 was identified as a member of naturally occurring and widely prevalent microorganisms that are not known pathogens to animals or plants. Several end-use pesticide products containing different strains of Bacillus subtilis and Bacillus subtilis var. amyloliquefaciens were evaluated in the Registration Review of March 2010 (U.S. EPA 2010) and are used commercially on a variety of crops, including apples, pears, beans, broccoli, carrots, cherry, cucurbits, grapes, hops, leafy vegetables, mint, onions, garlic, peanuts, peppers, potatoes, strawberries, tomatoes, walnuts, and wasabi. No known adverse effects of these strains on nontarget plants have been reported. In the applicant's greenhouse and field trials, which included evaluation of phytotoxicity and plant/crop status, no adverse effects to nontarget plants were observed. Although a literature search of Agricola, TOXLINE, Biological Abstracts, CHEMTOX, and TOXNET did not reveal any reports of adverse effects to mammals (either via acute toxicity or endocrine effects) or plants, there was a report of cyclic lipopeptides secreted by two Bacillus subtilis strains that had a limited effect on mosquito (Culex quinquefasciatus) larvae. No other effects on insects were found. Nontarget insect studies are not required unless an active ingredient controls the target insect pest by a mechanism of infectivity (40 CFR § 158.2150, Test Note 8). Because there are no insecticidal pest claims for Bacillus subtilis CX-9090, nontarget insect testing (OCSPP 885.4340) was not required.

A different median lethal dose (LD50) determination will be used for the hazard assessment of honeybees to Bacillus subtilis strain CX-9060, given that the submitted study (MRID No. 478203-11) was unacceptable for regulatory risk assessment purposes and the LD50 was inconclusive. The LD50 for a similar strain, Bacillus subtilis MBI 600, was determined to be > 1 x 10[8] CFU Bacillus subtilis/milliliter (mL) in an acceptable 20-day study conducted at a single limit dose (MRID 474724-04). The environmental concentration of Bacillus subtilis strain CX-9060 is estimated to be < 1.25 x 10[10] CFU Bacillus subtilis/gram (g) unless "sufficient water" is clarified in the following section found on page 10 of the November 9, 2009 Bacillus subtilis CX-9090 label: "Agricultural crops, For control of diseases on foliage, flowers or fruit."  
 
	b. Aquatic Animals and Plants 
      
Bacillus subtilis strain CX-9060 is a member of bacterial genera that commonly colonize soil and leaf litter. According to the submitted waiver rationale and request to waive additional testing to aquatic organisms, the end-use product, Bacillus subtilis CX-9090, is not for direct application to water and there are no aquatic food crops (except for perhaps watercress) on the label of November 9, 2009. Although hazard tests for freshwater fish and freshwater invertebrates were not conducted in accordance to OCSPP guidelines for microbial pesticides, there are no known toxicity or pathogenicity endpoints for freshwater organisms by Bacillus subtilis.

The 96-hour median lethal concentration (LC50) for rainbow trout juveniles was calculated as 127.23 milligrams per liter (mg/L) (95% confidence limits of 91.58-212.15 mg/L), and the No Observed Effect Concentration (NOEC) for survival was 62.5 mg/L. The 48-hour median effective concentration (EC50) for Daphnia magna neonates was calculated as 326.64 mg/L (95% confidence limits of 148.43-733.75 mg/L), and the NOEC for mobility was determined as 200 mg/L. Therefore, directions for aerial applications to open water must be avoided, and precautionary label language is required to avoid direct applications to open waters such as farm ponds, irrigation canals, lakes and streams and to prohibit applications to aquatic crops.    

2. Environmental Fate Data 

As the information provided for the proposed uses and application methods is sufficient to fulfill the Tier I nontarget organism data requirements and for the purposes of nontarget organism risk assessment for Bacillus subtilis strain CX-9060, further testing at higher tier levels (i.e., Tiers II, III, and IV) is not required. 

3. Threatened and Endangered Species Assessment

EPA has determined that adverse effects to any nontarget organisms are not anticipated as a result of the proposed labeled applications of the active ingredient, Bacillus subtilis strain CX-9060. 

Since EPA has determined that no effects are anticipated for any nontarget species exposed to Bacillus subtilis strain CX-9060 as a result of the proposed labeled 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 the end-use pesticide product, Bacillus subtilis CX-9090, as labeled.


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 Bacillus subtilis strain CX-9060, 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

Pursuant to section 3(c)(5) of FIFRA, EPA may register 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.

With respect to criterion 1, the proposed pesticide product contains Bacillus subtilis strain CX-9060, which is similar to currently registered Bacillus subtilis strains having well-known properties. EPA has no knowledge that would contradict the claims made on the Bacillus subtilis CX-9090 label, and the pesticide product is not expected to cause unreasonable adverse effects on the environment when used according to the label instructions. Regarding criterion 2, the current product label, as well as the data and information presented in this document, comply with the requirements of FIFRA. It is believed that the pesticide product containing Bacillus subtilis strain CX-9060 will not cause any unreasonable adverse effects on the environment, and Bacillus subtilis strain CX-9060 is likely to provide protection against fungal and bacterial plant diseases as claimed, thus satisfying criterion 3. Criterion 4 is satisfied in that the pesticide product containing Bacillus subtilis strain CX-9060 is not expected to cause unreasonable adverse effects when used according to label instructions. Bacillus subtilis CX-9090, containing Bacillus subtilis strain CX-9060 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 releasing Bacillus subtilis CX-9090 for shipment, the registrant is required to provide appropriate final printed labeling to EPA.

B. Terms of Registration

EPA has determined the following terms must be met for the registration of Bacillus subtilis strain CX-9060 as a new microbial active ingredient.   

 1.  Storage Stability (OCSPP 830.6317) and Corrosion Characteristics (OCSPP 830.6320) data from on-going studies of one-year duration, and conducted with the end-use pesticide product containing Bacillus subtilis strain CX-9060, must be submitted within 90 days of the date of registration to support the labeled viability claim of the product. Although the submitted storage stability study was deemed acceptable, only nine months of observations were reported in such study. Accordingly, the currently existing storage stability information only supports a nine-month storage time frame. 

             2. Analysis of Samples (OCSPP 885.1400) testing, conducted with five batches of the end-use pesticide product containing Bacillus subtilis strain CX-9060, must be submitted within one year of the date of registration.

C. Reporting of Adverse Effects and Hypersensitivity Incidents

Notwithstanding the information stated in the previous sections, it should be clearly understood that certain, specific data 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
                                       
BRAD
Biopesticides Registration Action Document
CFR
Code of Federal Regulations
CFU
Colony-forming units
CSF
Confidential statement of formula
EC50
Effective Concentration 50% of Population
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
FR
Federal Register
g
Gram
g/mL
Gram per milliliter
gal.
Gallon(s)
lb
Pound
LC50
Median Lethal Concentration 50% of Population
LD50
Median Lethal Dose  50% of Population
LOC
Level of Concern
mg/L
Milligram per liter
mL
Milliliter
MP
Manufacturing-use product
MRID No.
Master Record Identification Number
NCIB
National Collections of Industrial Bacteria
NIOSH
National Institute for Occupational Safety and Health
NOEC
No Observed Effect Concentration
OCSPP
Office of Chemical Safety and Pollution Prevention
OPP
Office of Pesticide Programs
PC Code
Pesticide Chemical Code
TGAI
Technical Grade of the Active Ingredient
U.S. 
United States
USDA - ARS - NCAUR
United States Department of Agriculture - Agricultural Research Service - National Center for Agricultural Utilization Research  















IX.  BIBLIOGRAPHY 

A. Submitted and Cited Studies 
                                   MRID No.
                                   Citation
                                 Receipt Date
419074-02
Hossack, D.; Allan, S.; Baker, M. (1989) Acute Oral Toxicity and Infectivity/Pathogenicity to Rats of MBI 600: Lab Project Number : 89396/AGC 1/0/AC. Unpublished study prepared by Huntingdon Research Centre Ltd. 29 p.
04-Jun-1991
41907404
Hossack, D.; Allan, S.; Baker, M. (1989) Acute Pulmonary Toxicity and Infectivity/Pathogenicity to Rats of MBI 600: Lab Project Number: 89397D/AGC: 1/2/AC. Unpublished study prepared by Huntingdon Research Centre Ltd. 37 p.
04-Jun-1991
419074-05
Hossack, D.; Allan, S.; Baker, M. (1989) Acute Intravenous Toxicity and Infectivity/Pathogenicity to Rats of MBI 600: Lab Project Number: 8939D/AGC: 1/3/AC. Unpublished study prepared by Hunti- ngdon Research Centre Ltd. 29 p.
04-Jun-1991
426829-01
Campbell, S.; Grimes, J.; Jaber, M. (1993) Bacillus subtilis Strain MBI 600: An Avian Oral Pathogenicity and Toxicity Study in the Bobwhite: Lab Project Number: 93-001: 301-102. Unpublished study prepared by Wildlife International Ltd. 23 p.
02-Mar-1993
474724-04
Mori, K. (1997) Effect of IK-1080 WP on Honeybee. Unpublished study prepared by Japan Plant Protection Assoc. Research Institute. 10 p.
09-Jul-2008
478203-01
Eyal, J. (2009) Bacillus subtilis Strain MBI 600 End-Use Product CX-9090: Product Identity: Manufacturing Process: Discussion of Formation of Unintentional Ingredients: Analysis of Samples: Certification of Limits. Project Number: QC/89001. Unpublished study prepared by Certis U.S.A., L.L.C. 108 p.
04-Aug-2009
478203-02
Eyal, J. (2009) Bacillus subtilis Strain MBI 600 End-Use Product CX-9090: Color: Physical State: Odor: Stability to Normal and Elevated Temperatures, Metals, and Metal Ions: Miscibility: Corrosion Characteristics: pH: Viscosity: Bulk Density. Unpublished study prepared by Certis U.S.A., L.L.C. 9 p.
04-Aug-2009
478203-03
Simpson, A. (2009) Bacillus subtilis Strain MBI 600 End-Use Product CX-9090: Storage Stability. Unpublished study prepared by Certis U.S.A., L.L.C. 6 p.
04-Aug-2009
478203-04
Kuhn, J. (2008) CX-9090: Acute Dermal Irritation Study in Rabbits: Final Report. Project Number: 12177/08. Unpublished study prepared by Stillmeadow, Inc. 12 p.
04-Aug-2009
478203-05
Kuhn, J. (2008) CX-9090: Acute Eye Irritation Study in Rabbits: Final Report. Project Number: 12176/08. Unpublished study prepared by Stillmeadow, Inc. 17 p.
04-Aug-2009
478203-06
Dively, C. (2009) Bacillus subtilis Strain MBI 600 End-Use Product CX-9090: Acute Toxicity Data Waivers. Unpublished study prepared by Certis U.S.A., L.L.C. 9 p.
04-Aug-2009
478203-07
Hartwell, T. (2008) CX-9060 (Bacillus subtilis Technical Powder): Daphnia magna 48-Hour Acute Toxicity Test: Final Report. Project Number: 12308/08. Unpublished study prepared by Stillmeadow, Inc. 14 p.
04-Aug-2009
478203-08
Hartwell, T. (2008) CX-9060 (Bacillus subtilis Technical Powder): Rainbow Trout (Oncorhynchus mykiss) 96-Hour Acute Toxicity Test: Final Report. Project Number: 12297/08. Unpublished study prepared by Stillmeadow, Inc. 14 p.
04-Aug-2009
478203-09
Dively, C. (2009) Bacillus subtilis Strain MBI 600 End-Use Product CX-9090: Aquatic Organism Non-Target Data Waivers. Unpublished study prepared by Certis U.S.A., L.L.C. 5 p.
04-Aug-2009
478203-10
Dively, C. (2009) Bacillus subtilis Strain MBI 600 End-Use Product CX-9090: NonTarget Wild Mammal, Insect, and Plant Studies. Unpublished study prepared by Certis U.S.A., L.L.C. 9 p.
04-Aug-2009
478203-11
Porch, J.; Sindermann, A.; Krueger, H. (2009) Bacillus subtilis: A Dietary Pathogenicity and Toxicity Study with the Honey Bee (Apis mellifera): Final Report. Project Number: 671/101A. Unpublished study prepared by Wildlife International, Ltd. 34 p.
04-Aug-2009
479463-01
Eyal, J.; Dively, C. (2009) Bacillus subtilis Strain CX-9060: Discussion of Formation of Unintentional Ingredients. Unpublished study prepared by Certis U.S.A., L.L.C. 57 p.
06-Jan-2010
484161-01
Chen, C. (2011) Bacillus subtilis Strain CX-9060 End Use Product CX-9090 Microbial Pesticide Data Requirements: Product Identity: Addendum to MRID 478203-01. Unpublished study prepared by Certis U.S.A., L.L.C. 23 p.
11-Mar-2011
B. EPA Risk Assessment Memoranda and Other Documents

Bergey. 2009. Bergey's Manual of Systematic Bacteriology, Volume 3; 2[nd] Ed. Springer. New York.  
Hossack, D. et al.  1989a. Acute Oral Toxicity and Infectivity/Pathogenicity to Rats of MBI 600:  Lab Project Number: 89396/AGC 1/0/AC. Unpublished study prepared by Huntingdon Research Centre Ltd. 29 p. January 30, 1989. EPA MRID 419074-02.
Hossack, D. et al. 1989b. Acute Pulmonary Toxicity and Infectivity/Pathogenicity to Rats of MBI 600: Lab Project Number: 89397D/AGC: 1/2/AC. Unpublished study prepared by Huntingdon Research Centre Ltd. 37 p. February 2, 1989. EPA MRID 419074-04.  
Hossack, D. et al. 1989c. Acute Intravenous Toxicity and Infectivity/Pathogenicity to Rats of MBI 600: Lab Project Number: 8939D/AGC: 1/3/AC. Unpublished study prepared by Huntingdon Research Centre Ltd. 29 p. January 31, 1989. EPA MRID 419074-05.  

U.S. EPA 2011. Memorandum from Kathleen Martin to Denise Greenway.  Review of Product Analysis and Tier 1 Toxicology Data to Support the Section 3 Registration of End-use Product CX-9090. November 3, 2011.

U.S. EPA 2011. Memorandum from Gail Tomimatsu to Denise Greenway.  November 2, 2011.  Environmental Risk Assessment of the Active Ingredient, Bacillus subtilis Strain CX-9060: Nontarget Organism Data and Waiver Rationale for End-Product CX-9090. November 2, 2011.

U.S. EPA. 2010. Bacillus subtilis Registration Review Documents. Available from http://www.epa.gov/oppsrrd1/registration_review/bacillus_subtilis/index.htm

U.S. EPA. 2008. Memorandum from Joel V. Gagliardi to Denise Greenway. December 23, 2008. Bacillus subtilis MBI 600.

U.S. EPA 1997. Bacillus subtilis Final Risk Assessment. Available from http://www.epa.gov/oppt/biotech/pubs/fra/fra009.htm.

U.S. EPA. 1993. Memorandum from Cindy Schaffer to Clarence Lewis/Julie Fairfax. SAB Review of Data Submitted by Gustafson, Incorporated to Support the Registration of GUS 376 Concentrate Biological Fungicide, Bacillus subtilis (I.D. No.: 007501-RUT; Submission No.: S446779; DP Barcode No.: D194490). October 20, 1993.



              APPENDIX A. MICROBIAL PESTICIDES DATA REQUIREMENTS 
                        (40 CFR PART 158  -  SUBPART V)


Table 1.  Product Analysis Data Requirements 
                                  Requirement
                               (OCSPP Guideline)
                                   MRID No.
                                     Value
                                Classification
                                       
                                       
                                       
Product identity (885.1100)
                                   478203-01
                                   484161-01
                 Bacillus subtilis strain CX-9060 is closely 
                     related to Bacillus subtilis MBI 600.
                   All the inert ingredients are cleared for
                      pre- and post-harvest use on food.
                                  Acceptable
Manufacturing process (885.1200)
                                   478203-01
                         The manufacturing process is
                             adequately described.
                                  Acceptable
Deposition of a sample in a nationally recognized culture collection
                                   478203-01
                      The culture is deposited at:  USDA
                      ARS-NCAUR; the accession number is
                                 NRRL-B-50287.
                                  Acceptable
                                       
Discussion of formation of unintentional ingredients (885.1300)
                                   478203-01
                                   479463-01
                    Formation of unintentional ingredients 
                        is sufficiently characterized.
                                  Acceptable
Analysis of samples (885.1400)
                                   478203-01
                                       
                         TGAI:  A five-batch analysis 
                          for the TGAI was provided.
                                  Acceptable

                                     none
                       EP:  Outstanding data requirement
                             Needs to be submitted
Certification of limits (885.1500)
                                   478203-01
                     The certified limits, as provided on 
                      the CSF, comport with the standard 
                             at 40 CFR § 158.350.
                                  Acceptable
Color (830.6302)
                                   478203-02
                                     beige
                                  Acceptable
Physical state (830.6303)
                                   478203-02
                                    powder
                                  Acceptable
Odor (830.6304)
                                   478203-02
                                     yeast
                                  Acceptable
Stability to normal and elevated temperatures, metals and metal ions (830.6313)
                                   478203-02
                    Stable at normal temperatures; at 54°C
CX-9060 (the TGAI) deactivates. The product is not expected to come in contact with metals during its storage and use.
                                  Acceptable
Storage stability (830.6317)
                                   478203-02
                    TGAI:  stable over a 12-month interval
                                  Acceptable

                                   478203-03
                      EP:  The submitted data support a 
nine-month storage interval. This study is being upgraded to include the stability through 12 months 
                                  Acceptable
Miscibility (830.6319)
478203-02
                   Immiscible with hydrocarbon oil at 25°C.
                                  Acceptable
Corrosion Characteristics (830.6320) 
                                     none
                         Outstanding data requirement
                             Needs to be submitted
pH (830.7000)
478203-02
                                      6.7
                                  Acceptable
Density/relative density/ bulk density (specific gravity) (830.7300 )
478203-02
                                  0.512 g/mL
                                  Acceptable







Table 2.  Summary of Human Health Toxicity Data for Certis' Bacillus subtilis CX-9090
                               Data Requirement
                               (OCSPP Guideline)
                                   MRID No.
                          Toxicity and Classification
                                   Toxicity
                                  Category[a]


Acute Oral Toxicity/ Pathogenicity (885.3050)
                                   419074-02
            Bacillus subtilis MBI 600 was not toxic, infective or 
           pathogenic to rats at a dose of 2 x 10[8] CFU per animal.
Classification:  Acceptable
                                      IV
Acute Pulmonary 
Toxicity/ Pathogenicity
( 885.3150)
                                   419074-04
Bacillus subtilis MBI 600 was not toxic, infective or pathogenic to rats when dosed intratracheally with 3.4 x 10[8] CFU of the test material.
Classification:  Acceptable
                                      IV
Acute Injection (Intravenous)Toxicity/ Pathogenicity (885.3200)
                                   419074-05
Bacillus subtilis  MBI 600 was not toxic, infective or pathogenic to rats when dosed intravenously with approximately 4 x 10[7] CFU of the test material.  
Classification:  Acceptable
                                     none
Hypersensitivity 
Incidents (885.3400)
                                   478203-06
          No such incidents have been reported for this product. The 
         applicant acknowledges that repeated exposure to the end-use 
product could cause a certain degree of hypersensitivity. If such incidents are seen, Certis will report them to the Agency.
Classification:  Acceptable
                                     none
Acute Oral Toxicity
(870.1100)
                                   478203-06
         Based on results from the acute oral toxicity/pathogenicity 
            study (MRID No. 419074-02), EPA expects that the acute 
               oral toxicity would be in Toxicity Category IV.  
Classification:  Acceptable
                                      IV
Acute Dermal Toxicity
(870.1200)
                                   478203-06
Based on the low toxicity of the inert ingredients and slight dermal irritation, EPA expects that acute dermal toxicity would be in Toxicity Category IV.
Classification:  Acceptable
                                      IV
Acute Inhalation Toxicity 
(870.1300)
                                   478203-06
       Based on results from the acute pulmonary toxicity/pathogenicity 
            study (MRID No. 419074-04), EPA expects that the acute 
             inhalation toxicity would be in Toxicity Category IV.
Classification:  Acceptable
                                      IV
Acute Eye Irritation (870.2400)
                                   478203-05
                               Mildly irritating
Classification:  Acceptable
                                      III
Primary Dermal 
Irritation (870.2500)
478203-04
                Slight erythema was observed in one animal at 
                      24 hours; it resolved by 48 hours.
Classification:  Acceptable
                                      IV
[a] See 40 CFR § 156.62 for a description of the Toxicity Categories

	

      A.  Acute Oral Toxicity/Pathogenicity (OCSPP 885.3050; MRID No. 419074-02). For this data requirement, Certis cites MRID No. 419074-02 -- Acute Oral Toxicity and Infectivity/Pathogenicity to Rats of Bacillus subtilis MBI 600 (Hossack et al. 1989a). In this study, male and female CD rats were gavaged with a single dose of the test substance, which contained at least 10[8] viable spores per animal. The animals were observed for three weeks, with sacrifice on Day 22.  
Clinical signs were minimal. All rats exhibited piloerection within ten minutes of dosing; by the end of Day 1, it resolved and no further clinical signs were observed over the duration of the study. Slightly low bodyweight gains were recorded for a few of the animals; the others achieved anticipated bodyweight gains throughout the study. Although the microbe was found in the stomach, small intestines, caecum, feces, and urine of the treated rats, the test material was cleared from all organs by Day 21 of the study. None of the animals died and at terminal autopsy, findings were normal. Based on the submitted data, Bacillus subtilis MBI 600 (NCIB12376) was not toxic, infective or pathogenic to rats at a dose of 2 x 10[8] CFU per animal. (U.S. EPA 1993)
      Classification:  Acceptable; Toxicity Category IV

	B.  Acute Pulmonary Toxicity/Pathogenicity (OCSPP 885.3150; MRID 419074-04). For this data requirement, Certis cites MRID No. 419074-04 -- Acute Pulmonary Toxicity and Infectivity/Pathogenicity to Rats of Bacillus subtilis MBI 600 (Hossack et al. 1989b). Male and female CD rats were given a single dose of Bacillus subtilis MBI 600 at a concentration of at least 10[8] viable spores per animal via the inhalation route (using an endotracheal tube). The animals were observed for three weeks, with sacrifice on Day 22.  
Although a high mortality was observed (24% in males, 15% in females), death was not attributed to the microorganism tested; autopsy of rats that died revealed no macroscopic abnormalities. By three weeks after dosing, a distinct clearance pattern was noted throughout the study in all organs. Based on the submitted data, Bacillus subtilis MBI 600 (NCIB 12376) was not toxic, infective or pathogenic to rats when dosed intratracheally with 3.4 x 10[8] CFU of the test material. (U.S. EPA 1993) 
      Classification:  Acceptable; Toxicity Category IV

	C.  Acute Injection (Intravenous) Toxicity/Pathogenicity (OCSPP 885.3200; MRID No. 419074-05). For this data requirement, Certis cites MRID No. 419074-05 -- Acute Intravenous Toxicity and Infectivity/Pathogenicity to Rats of Bacillus subtilis MBI 600 (Hossack et al. 1989c). Male and female CD rats were given a single intravenous dose of Bacillus subtilis MBI 600 at a concentration of at least 10[7] viable spores per animal. The animals were observed for three weeks, with sacrifice on Day 22.  

Although the microbe was found in all the organs of the treated rats, the test material displayed a pattern of clearance from all organs during the study. Based on the submitted data, Bacillus subtilis MBI 600 (NCIB 12376) was not toxic, infective or pathogenic to rats when dosed intravenously with approximately 4 x 10[7] CFU of the test material. (U.S. EPA 1993) 
      Classification:  Acceptable
      
      D.  Hypersensitivity Incidents (OCSPP 885.3400; MRID No. 478203-06). 40 CFR § 158.2140 requires that "[h]ypersensitivity incidents, including immediate type and delayed-type reactions of humans or domestic animals, [that] occur during the testing or production of the TGAI, MP, or EP, or are otherwise known to the applicant must be reported if they occur."  
Certis acknowledges that repeated exposure to the end-use product could cause a certain degree of hypersensitivity; they will report all hypersensitivity incidents to the Agency if they occur.
      Classification:  Acceptable

	E., F. and G.  Acute Oral, Dermal, and Inhalation Toxicity (OCSPP 870.1100, 870.1200, and 870.1300; MRID No. 478203-06).  Certis has requested that acute oral, dermal, and inhalation toxicity testing be waived for Bacillus subtilis CX-9090. As justification, they argue that Bacillus subtilis is a naturally occurring soil and leaf litter colonizer; this microbe is ubiquitous in the environment; the active ingredient is nontoxic to human health; and there are no reports of adverse effects via these routes in the literature. With respect to the inert ingredients, Certis asserts that they have all been "cleared by EPA for use in/on food, are all List 4A or 4B, and have been previously evaluated by EPA in other products with similar acute toxicity profiles."  
Bacillus subtilis strain CX-9060 is derived from Bacillus subtilis MBI 600. For the acute oral toxicity/ pathogenicity study, Certis cites a study (MRID No. 419074-02) submitted to support the registration of a peat-based pesticide product containing Bacillus subtilis MBI 600 as an active ingredient. This study showed that Bacillus subtilis MBI 600 is not acutely toxic when administered via the oral route. Regarding acute dermal toxicity, test note 5 to the table at 40 CFR § 158.2140 states "[t]he 870 series studies . . . [may be waived] for any or all of these studies when the applicant can demonstrate that the combination of inert ingredients is not likely to pose any significant human health risks." The inert ingredients in the end-use product, Bacillus subtilis CX-9090, are not expected to be acutely toxic -- all are exempt from the requirement of tolerance when used pre- or post-harvest, with no restrictions. Further, only slight erythema, which resolved within 24 hours, was observed in the submitted dermal irritation study (MRID No. 478203-04); no other adverse effects were seen. Therefore, EPA would expect that Bacillus subtilis CX-9090 would not be acutely toxic via the dermal route. Regarding acute inhalation toxicity, the acute pulmonary toxicity/pathogenicity study (MRID No. 419074-04) showed that the test material (Bacillus subtilis MBI 600) was not toxic, infective or pathogenic to rats.  
	Classification for Acute Oral Toxicity:  Acceptable; Toxicity Category IV
	Classification for Acute Dermal Toxicity:  Acceptable; Toxicity Category IV
      Classification for Acute Inhalation Toxicity:  Acceptable; Toxicity Category IV

      H.  Acute Eye Irritation (OCSPP 870.2400; MRID No. 478203-05).  The concentration of the test material was not measured by Stillmeadow Labs -- it was provided by Certis along with a Certificate of Analysis attesting to the concentration of the test material. Certis did not provide information on the stability of the test material. This is not a concern because Stillmeadow Labs started the study one month after Certis certified the concentration of the test material (the Certificate of Analysis is dated July 10, 2008 and Stillmeadow Labs ran the study in August, 2008). The test species (white albino rabbit), number of animals (at least three), and test dose (0.1 mL) are what the test guideline recommends. One hour after treatment, one animal showed signs of corneal opacity; by 24 hours, it cleared. No animals developed iritis. Chemosis was exhibited by one animal at 1 and 24 hours after treatment; at 48 hours, it cleared. The maximum average irritation score (14.0) corresponds to a Draize rating of "mildly irritating."  
	Classification:  Acceptable; Toxicity Category III

	I.  Primary Dermal Irritation (OCSPP 870.2500; MRID No. 478203-04).  The concentration of the test material was not measured by Stillmeadow Labs -- it was provided by Certis along with a Certificate of Analysis attesting to the concentration of the test material. Certis did not provide information on the stability of the test material. This is not a concern because Stillmeadow Labs started the study one month after Certis certified the concentration of the test material (the Certificate of Analysis is dated July 10, 2008 and Stillmeadow Labs initiated the study on August 5, 2008). The test species (rabbit) is appropriate, and they are older than 12-weeks-old at the start of the study (OCSPP 870.2500 recommends that rabbits be at least 12-weeks-old). The average weight of each female is within 20% of the average female weight. The Guideline recommends at least five animals per sex per dose be used; the study investigator provided no justification for using fewer animals.  
	Classification:  Acceptable; Toxicity Category IV


Table 3.  Summary of Reviewed Nontarget Organism Studies, Citations and Rationale to Waive Testing
Data Requirement
OCSPP Guideline No.
Results Summary and Classification
MRID No.
Avian Oral Toxicity
885.4050
Acceptable 
426829-01

Avian Inhalation Toxicity/Pathogenicity
885.4100
Not required
N/A
Wild Mammal Toxicity/Pathogenicity
885.4150
Submitted rationale and cited data are sufficient to support a waiver for Wild Mammal Testing, Tier I. An acceptable infectivity study (MRID 419074-05) shows no concern for adverse effects to mammalian health. 
Classification: Acceptable
478203-10
419074-05
Freshwater Fish Toxicity/Pathogenicity
885.4200
The information provided is sufficient to support a waiver for Freshwater Fish Testing for the end-use product, Bacillus subtilis CX-9090. 
Classification: Acceptable
478203-09

Freshwater Fish Toxicity
850.1075
In a non-renewal static laboratory toxicity study, the 96-hour LC50 was calculated as 127.23 mg/L (95% confidence limits of 91.58-212.15 mg/L). The NOEC for survival was 62.5 mg/L.

Supplemental for scientific evaluation of hazards to freshwater fish to a viable microbial pesticide. Acceptable for end-use pesticide product labeling that has no direct aquatic uses. 

478203-08
Freshwater Invertebrate Toxicity/Pathogenicity
885.4240
The information provided is sufficient to support a waiver request for Aquatic Invertebrate Testing for the end-use pesticide product, Bacillus subtilis CX-9090, if there are no direct aquatic uses on the label.  Classification: Acceptable
478203-09

Acute Toxicity Freshwater Invertebrates
850.1010
In a non-renewal static laboratory bioassay, the 48-hour EC50 was calculated as 326.64 mg/L, and the NOEC for mobility was 200 mg/L.

Supplemental for scientific evaluation of hazards to aquatic invertebrates to a viable microbial pesticide. The duration of the study is insufficient for evaluation of viable microbial pesticides to aquatic invertebrates. Acceptable for end-use pesticide product labeling that has no directed aquatic uses.
478203-07

Estuarine/Marine Fish and Invertebrate Testing
885.4280
Data are not required. Bacillus subtilis strain CX-9060 will not be applied directly to marine/estuarine waters and is not expected to enter marine/estuarine environments in amounts that are significantly higher than naturally occurring concentrations.
N/A
Nontarget Plant Testing 
 
885.4300
A waiver rationale was provided for Nontarget Plant Testing, Tier I for Certis' Bacillus subtilis CX-9090. Because the pesticide is not taxonomically related to a known plant pathogen, nontarget plant testing was not required.
478203-10
Nontarget Insect Testing
885.4340
A waiver rationale was provided for Non-Target Insect Testing, Tier I for Certis' Bacillus subtilis CX-9090. Because there are no intended insecticidal uses for Bacillus subtilis CX-9090, nontarget insect testing was not required. 

478203-10
Honey Bee Testing
885.4380
Submitted testing (MRID No. 478203-11) required by 40 CFR §158.2140 are not sufficient for assessment of hazards to honey bees. The inconclusive LD50 and the short duration of the study provide insufficient data and information requisite to evaluate potential risks of Bacillus subtilis technical powder 9065 to honeybees. 
Classification: Unacceptable for regulatory risk assessment.

An LD50 > 1 x 10[8] for a similar strain of Bacillus subtilis was determined from another acceptable study (MRID 474724-04).  
478203-11
474724-04

                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       

                         APPENDIX B. PESTICIDE PRODUCT
                               EPA File Symbol 
                               Registration Name
                         Percentage Active Ingredient
                               Formulation Type
                                  Use Site(s)
                           Method(s) of Application
                               Application Rate
                                  Target Pest
                                   70051-RNL
                           Bacillus subtilis CX-9090
                                     25.0%
                                   End Use,
                          Water Dispersible Granular
                     Many food crop and ornamental plants
                                  Greenhouse
                     Foliar, soil drench, cutting/root dip
1 to 3 lbs./100 gal. water (foliar and soil drench), (1/2) lb. per gal. of water (dips)
                      Fungal and bacterial plant diseases
                                       
                                       
                                       
                                       
                                       
                     Outdoor Foliar- or soil-applied spray
            1 1/4 to 6 lbs./acre (foliar); 1/8 to 1 lb./acre (soil)
                                       
                                       
                                       
                                       
                                       
                                       
                                    Aerial 
                           (fixed or rotary winged)
        Apply 1 1/4 to 6 lbs./acre in a minimum of 3 gal. of water/acre
                                       
                                       
                                       
                                       
                                       
                                       
                         Drip or sprinkler chemigation
                          1 to 3 lbs./100 gal. water
                                       


