**Proposed BIOPESTICIDES REGISTRATION ACTION DOCUMENT** 

Bacillus subtilis s train CX - 906 0 Pesticide Chemical (PC) Code: 016480

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

November , 2011Bacillus subtilis strain CX - 9060 Page 2 of 27

Biopesticides Registration Action Document **TABLE OF CONTENTS I**  **. EXECUTIVE SUMMARY**  **................................ ................................**  **................................ .**  **4 II.**  **ACTIVE INGREDIENT OV ERVIEW**  **................................ ................................**  **.............. 6**  **II I.**  **REGULATORY BACKGROUN D**  **................................ ................................**  **.................... 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 .**  **Hum an Health Assessment (40 CFR § 158.2140)**  **................................ ................................**  **................ 7**  **C .**  **Environmental Assessment (40 CFR § 158.2150)**  **................................ ................................**  **............. 12**  **V .**  **ENVIRONMENTAL JUSTIC E**  **................................ ................................**  **....................... 13**  **VI .**  **RISK MANAGEMENT DECI SION**  **................................ ................................**  **................ 14**  **VII. ACTIONS REQUIRED**  **OF THE REGISTRANT**  **................................ .........................**  **15 A**  **. Final Printed Labeling**  **................................ ................................**  **................................ ..........................**  **15 B. Terms of Registration**  **................................ ................................**  **................................ ............................**  **15 C**  **. Repor**  **ting of Adverse Effects and Hypersensitivity Incidents ................................**  **........................... 15**  **VIII. GLOSSARY OF AC RONYMS AND ABBREVIAT**  **IONS ................................**  **............ 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** Bacillus subtilis strain CX - 9060 Page 3 of 27

Biopesticides Registration Action Document

**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 Anal ys is, Human Health Kathleen Martin, M.S.E.S. Product Anal ys is, Human Health Gail Tomimatsu , Ph.D. Environmental E ffects **Regulations** Sheryl K. Reill y, Ph.D. Chief, Microbial Pesticides Branch Denise Greenway Regulatory Action LeaderBacillus subtilis strain CX - 9060 Page 4 of 27

Biopesticides Registration Action Document **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 re cove red from water, soil, air, and decomposing plant residues. The bacterium commonly produces proteases and other enzy mes; as a result, strains of the bacterium are often used for industrial production of enz ym es and other chemicals. The bacterium also produc es 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 pre dominantly

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 naturall y occurring strain of the Bacillus subtilis bacterium . The progenitor strain is a currently registered pesticide. Data and information , submitted by Certis and reviewed b y the Agency , demonstrated the similarity of the two strains. Th e 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 regi stration application. EPA scientists have reviewed product anal ys is, toxicology , and nontarget organism data and information submitted or cited to support registration of the pesticide product containing Bacillus subtilis strain CX - 9060 . P roduct anal ys is data requirements for Bacillus subtilis strain CX - 9060 , including product chemistry and composition, analy sis of samples, and physical and chemical characteri stics, were adequately fulfilled by acceptable guideline studies. In support of mammalian and nontarget organism toxicology , pathogenicity, inf ectivity and irritation testing, the Bacillus subtilis strain CX - 9060 application included both newl y conducted Bacillus subtilis strain CX - 9060 studies and the citation of existing data ( condu cted with the progenitor strain) . EPA found such scientific information submitted or cited by the applicant to fulfill the relevant toxicology

and nontarget organism data requirement s . 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. P reviously

reviewed studies s how ed no infectivity or pathogenicity of the progenitor strain and were assigned the EPA T oxicity C ategory IV rating for all tested routes of administration. There have been no reports of hy persensitivity

in over 15 ye ars of registered uses of the progenitor strain , and Bacillus subtilis , in general, is ubiquitous in soils and common in agricultural environments. Inaddition , non occupational exposure is considered unlikel y for Bacillus subtilis strain CX - 9060 as all currentl y proposed uses occur in distinctly agricultural or commercial settings; there are no proposed uses for residential areas. Overall, E PA concludes that there is a reasonable certainty that no harm will result to the United States (U.S.) population, including infants and children, f rom aggregate exposure to residues of Bacillus subtilis strain CX - 9060.Bacillus subtilis strain CX - 9060 Page 5 of 27

Biopesticides Registration Action Document 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 . Inaccordance 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 fulfill ed the curr ent data requirements and were adequate f

or risk assessment purposes. EPA has concluded that the proposed use of Bacillus subtilis strain CX - 9060 meets the standard for registration under s ection 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, th is proposed registration decision for the pe sticide product containing Bacillus subtilis strain CX - 9060 , a new active ingredient, i s 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 doc ket 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 - 20 1 0 - 0 10 3 . The f ollowing 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 Sy mbol 7 0051 - 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 .Bacillus subtilis strain CX - 9060 Page 6 of 27

Biopesticides Registration Action Document **II .**  **ACTIVE INGREDIENT OVERVIEW**  **Biological Name:** Bacillus subti li s s train CX - 906 0 **Culture Deposit:** Agricultural Research Service Culture Collection (also known as the Northern Regional Research Laboratory Collection) in Peoria, Illinois under Accession Number NRRLB - 50287 **OPP Chemical Code:** 016480 **Type of Pesticide:** Microbial Pesticide

– Fung icide and Bactericide See Appendix B for specific information ( e.g., use sites, application rates, methods of application, formulation ty pes, 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 C X 9090 (EPA File Sy

mbol 70051 - RNL) , under FIFRA section 3. On M arch 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 3 0 - day public comment period pursuant to the provisions of FIFRA section 3(c)(4). No comments were received following this publication . **B .**  **Food Tolerance Exemption**  O n 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 s train CX - 9060 (Pesticide Petition (PP) 9 F 7 643 ). In the Federal Register of March 10 , 20 1 0 ( 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 s train 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 definiti on 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 requirem ents. The classifications that are found for each data submission are assigned b y EPA scienceBacillus subtilis strain CX - 9060 Page 7 of 27

Biopesticides Registration Action Document 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 (“ su

pplemental : upgradeable ”). If a study is rated as “ supplemental : upgradeable ,” EPA alway s provides an indication of what is lacking or what can be provided to change the rating to “ acceptable .” If there is simply a “ supplemental ” rating, the reviewer will often state that the study is not required b y 40 C FR P art 158. Both “ acceptable ” and “ supplemental ” studies may be used in the risk assessment process as appropriate. An “unacceptable ” rating indicates that new data must be submitted. For the acute toxici ty

data requirements, Toxicity C ategories 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 Iindicates the highest toxicity and Toxicity

Category IV indicates the lowest toxicity ( 40 CFR § 156.62 ) . **A .**  **Product Analysis Assessment (** **40 CFR § 158.2120**  **)** A ll product anal ys is data requirements for Bacillus subtilis strain CX - 9060 have been fulfilled ( Master Record Identification Numbers ( MRID Nos . ) 47 8203 - 01, 47 8203 - 02 and 47 8203 - 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 b y the Age ncy . Acceptable Tier I mammalian toxicology information found therein support s 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 No s . 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 f or a brief summary

of the data requirements.Bacillus subtilis strain CX - 9060 Page 8 of 27

Biopesticides Registration Action Document **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) determin ation s 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 employ s 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 sy stems. Chemicals that go through Tier 1 screening and are found to have the potential to interact with E, A, or T hormonal sy stems 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 pathway s 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 i s 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. Accordingl y, EPA anticipates issuing future EDSP orders/data call - ins for all p esticide 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 .**  **F ederal 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 F FDCA 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 informatio n.” 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 aBacillus subtilis strain CX - 9060 Page 9 of 27

Biopesticides Registration Action Document 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 th at 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 anal ys es to determine the risks from aggregate exposure to pesticide residues. First, EPA determines th e 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 - 90 60 of Bacillus subtilis is derived from a naturall y occurring isolate of the genus Bacillus , which was originally isolated from faba bean plant s grown at the Nottingham University

School of Agriculture in the United Kingdom). As a result, dietary exposure to background levels of the microbe likel y 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 pe sticidal uses , when compared to existing exposure to background levels of Bacillus subtilis . As discussed above, dietary exposure to Bacillus subtilis strain CX - 9060 l ikely is already occurring and likely

will continue to occur. Notably , similar Bacillus s ubtilis 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 commerc ial bakeries and so the Agency considers this particular food exposure scenario to beBacillus subtilis strain CX - 9060 Page 10 of 27

Biopesticides Registration Action Document 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 expec ted 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 L evel of C oncern (LOC) . Drinking W

ater 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 greatl y diluted, in drinking water. Municipal drinking water treatment processes and deep water wells, however, should both further reduce any such residues. More importantl y, even if oral exposure to this ubiquitous microbe should occur through drinking water, due to its expected lack of acute oral tox icity

/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, b ecause 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 probabl y been previously exposed (and likely

will continue to be exposed) to background levels of the microbe. N either 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 - 90 6 0 pesticide use will not result in additional aggregate non - occupational risk from dermal and inhalati on 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 po se 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 .”Bacillus subtilis strain CX - 9060 Page 11 of 27

Biopesticides Registration Action Document 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 EP A’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 subst ances 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 to xicity 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 F ood 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 do es 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 appl ying 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 ci ted 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 m ade that no unreasonable adverse effects to the U.S. population in general, and to infants and childrenBacillus subtilis strain CX - 9060 Page 12 of 27

Biopesticides Registration Action Document in particular, will result when the Bacillus subtilis strain CX - 9060 pesticide product i s used in accordance with EPA - accept ed labeling. **C .**  **Environmental Assessment (** **40 CFR § 158.2150**  **)** The data and information s ubmitted 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 b y 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 naturall y occurring and widel y prevalent microorganisms that are not known pathogens to animals or plants. Several end - use pesticide products containing different strains of B acillus subtilis and B acillus subtili s 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 applic ant’s greenhouse and field trials, which included evaluation of phyt otoxicity 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 B acillus 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 B acillus subtili s CX - 9090, nontarget insect testing (OCSPP 885.4340) was not requir ed. A different median lethal dose ( LD 50 ) determination will be used f or the hazard assessment of hon ey bees to B acillus subtilis strain CX - 90 6 0, given that the submitted study (MRID No. 478203 - 11 ) was unacceptable for regulatory risk assessment purposes and the LD 50 was inconclusive . The LD 50 for a similar strain, B acillus subtilis MBI600 , was determined to be > 1 x 10 8 CFU B acillus subtili s/milliliter (mL) in an acceptable 20 - day study conducted at a single limit dose (MRID 474724 - 04) . The environmental concentration of B acillus subtilis strain CX 90 6 0 is estimated to be < 1.25 x 10 10 CFU B acillus subtilis/gram ( g ) unless “sufficient water” is clarified in the following section found on page 10 of the November 9, 2009 B acillus subtilis CX - 9090 label : “Agricultural crops, For control of diseases on foliage, flowers or fruit . ”Bacillus subtilis strain CX - 9060 Page 13 of 27

Biopesticides Registration Action Document **b. Aquatic Animals and Plants**  Baci llus subtilis strain CX - 9060 is a member of bacterial genera th at 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 - 90 90, 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 OCSP P guidelines for microbial pesticides, there are no known toxicity or pathogenicity

endpoints for freshwater organisms by Bacillus subtilis . The 96 - hour median lethal concentration ( LC 50 ) 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 ( EC 50 ) for Daphnia magna neonates was calculated as 326.64 mg/L (95% confiden ce 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 asse ssment for Bacillus subtilis strain CX - 9060 , f urther 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 - 90 6 0 as a result of the proposed labeled applications, effects to federally listed , threatened and endangered species and their designated critical habitats a re 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 - 909 0 , as l abeled. **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) potentiallyBacillus subtilis strain CX - 9060 Page 14 of 27

Biopesticides Registration Action Document 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 se ek 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 th e 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 th at may have aty pical, 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 . **V I**  **. RISK**  **MANAGEMENT DECISION** Pursuant to s ection 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 generall y cause unreasonable adverse effects on the environment. With respect to criterion 1, the proposed pesticide product contain s Bacillus subtilis strain CX 9060 , which is simil ar to currentl y registered Bacillus subtilis s trains 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 ef fects on the environment when used according to the label instructions. Regarding c riterion 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 pestici de 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 disease s 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.Bacillus subtilis strain CX - 9060 Page 15 of 27

Biopesticides Registration Action Document **V I**  **I .**  **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 term s 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 s tudies 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. Accordin gl y , the currentl y existing storage stability information only supports a nine - month storage time frame . 2. Analy sis 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 hy persensitivity (including both suspected and confirmed incidents) must be reported to EPA under the provisions of 40 CFR § 158.2140( d).Bacillus subtilis strain CX - 9060 Page 16 of 27

Biopesticides Registration Action Document **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 EC 50 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 LC 50 Median Lethal Concentration 50% of Population LD 50 Median Lethal Dose 50% of Population LOC Level of Concern mg/L Milligram per liter mL Milliliter MP M anufacturing - use product MRID No. Master Record Identification Number NCIB National Collections of Industrial Bacteria NI OSH 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 T echnical Grade of the A ctive Ingredient U.S. United States USDA - ARS - NCAUR United States Department of Agriculture - Agricultural Research Service - National Center for Agricultural Utilization ResearchBacillus subtilis strain CX - 9060 Page 17 of 27

Biopesticides Registration Action Document **IX .**  **BIBLIOGRAPHY A**  **. Submitted**  **and Cited Studies**  **MRID No.**  **Citation Receipt**  **Date** 419074 - 02 H o ssack, D.; Allan, S.; Baker, M. (1989) Acute Oral Toxicity and In fectivity/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 b y Hunti - ngdon Research Centre Ltd. 29 p. 04 - Jun 1991

426829 - 01 Campbell, S.; Grimes, J.; Jaber, M. (19 93) 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 b y Wildlife International Ltd. 23 p. 02 - Mar1993

474724 - 04 Mori, K. (1997) Effect of IK- 1080 WP on Honey bee. 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: Discussi on of Formation of Unintentional Ingredients: Anal ys is 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 b y Certis U . S . A . , L . L . C. 9 p. 04 - Aug 2009

478203 - 03 Simpson, A. (2009) Bacillus subtilis Strain MBI600 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 2009Bacillus subtilis strain CX - 9060 Page 18 of 27

Biopesticides Registration Action Document **MRID No.**  **Citation Receipt**  **Date** 478203 - 05 Kuhn, J. (2008) CX - 9090: Acute Eye Irritation Study in Rabbits: Final Report. Project Number: 12176/08. Unpubl ished 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 S train 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 Pathogenic ity

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:

Discus sion of Formation of Unintenti on al 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 - Mar2011 **B .**  **EPA Risk Assessment Memoranda**  **and Other Documents** Bergey . 2009. Bergey ’s Manual of Sy stematic 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 b y 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 byBacillus subtilis strain CX - 9060 Page 19 of 27

Biopesticides Registration Action Document Hunting don 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 Re search Centre Ltd. 29 p. January 31, 1989. EPA MRID 419074 - 05. U.S. EPA 2011 . Memorandum from Kathleen Martin to D enise Greenway . Review of Product Analy sis 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 D enise 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 MBI600. U . S . EPA 1997. Bacillus subtilis Final Risk Assessment. Availa ble 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.Bacillus subtilis strain CX - 9060 Page 20 of 27

Biopesticides Registration Action Document

**A PPENDIX**  **A .**  **MICROBIAL PESTICIDES DATA REQUIREMENTS**

**(40 CFR PART 158**  **– SUBPART**  **V )**  **Table 1** . Product Analy sis Data Requirements

REQUIREMENT ( OCSPP GUIDELINE ) MRID N O . VALUE C LASSIFICATION **Product i**  **dentity** (885.1100) 478203 - 01 484161 - 01 B acillus 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 (8 85.1400 ) 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. 63 13 ) 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 (8 30.6319 ) 478203 - 02 Immiscible with hydrocarbon oil at 25°C . Acceptable Corrosion Characteristics (8 30.6320 ) 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 - 9090Bacillus subtilis strain CX - 9060 Page 21 of 27

Biopesticides Registration Action Document D ATA R EQUIREMENT ( OCSPP G UIDELINE ) MRID N O . T OXICITY AND C LASSIFICATION T OXICITY C ATEGORY 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. **C LASSIFICATION**  **:** Acceptable IV Acute Pulmonary To xicity/ Pathogenicity ( 885.3150) 419074 - 04 B acillus 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. **C LASSIFICATION**  **:** Acceptable IV Acute Injection ( I ntravenous)Toxicity/ Pathogenicity (885.3200) 419074 - 05 B acillus 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. **C LASSIFICATION**  **:** Acceptable none Hypersensitivity I ncidents (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. **C LASSIFIC**  **ATION** : 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. **C LASSIFICATION**  **:** 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. **C LASSIFICATION**  **:** 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 To xicity Category IV. **C LASSIFICATION**  **:** Acceptable IV Acute Eye Irritation (870.2400) 478203 - 0 5 Mildly irritating **C LASSIFICATION**  **:** Acceptable III Primary Dermal Irritation (870.2500) 478203 - 04 Slight erythema was observed in one animal at 24 hours; it resolved by 48 hours. **C LASSIFICATION**  **:** Acceptable IV

a See 40 CFR § 156.62 for a description of the Toxicity CategoriesBacillus subtilis strain CX - 9060 Page 22 of 27

Biopesticides Registration Action Document **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 In fectivity/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; b y the end of Day

1 , it resolved and no further clinical signs were observed o ver the duration of the study . Slightly

low bodyweight gains were recorded for a few of the animals; the others achieved anticipated bodywei ght gains throughout the study. Although the microbe was found in the stomach, small intestines, caecum, feces, and urine of the tre ated rats, the test material was cleared from all organs by D ay 21 of the study. None of the animals died and at terminal autopsy , findings were normal. Based on the submitted data, B acillus subtilis MBI600 (NCIB 1 12376) was not toxic, infective or pathogenic to rats at a dose of 2 x 10 8 CFU per animal. (U.S. EPA 1993 ) C LASSIFICATION : 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 In fectivity/Pathogenicity

to Rats of B acillus subtilis MBI 600 ( Hossack et al. 1989 b ) . M ale and female CD rats were given a single dose of B acillus subtilis MBI600 at a concentration of at least 10 8 viable sp ores per animal vi a 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; autops y 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, B acillus subtilis MBI600 (NCIB 12376) was not toxic, infective or pathogenic to rats when dosed intratracheall y with 3.4 x 10 8 CFU of the test material. (U.S. EPA 1993 ) C LASSIFICATION : Acceptable; Toxicity Category IV **C. Acute Injection ( I**  **ntravenous) Toxicity/Pathogenicity**  ( OCSPP 885.3200; MRID No. 419074 - 05 ). Fo r this data requirement, Certis cites MRID No. 419074 - 05

— Acute Intravenous Toxicity

and Infectivity/Pathogenicity to Rats of B acillus subtilis MBI 600 ( Hossack et al. 1989 c ) . Male and female CD rats were given a single intravenous dose of B acillus 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, B acillus subtilis MBI600 (NCIB 12376) was not toxic, infective or patho genic to rats when dosed intravenously

with approximately

4 x 10 7 CFU of the test material. (U.S. EPA 1993 ) C LASSIFICATION : Acceptable

1 NCIB stands for “National Collections of Industrial Bacteria;” http://www.ncimb.com/culture.html .Bacillus subtilis strain CX - 9060 Page 23 of 27

Biopesticides Registration Action Document **D. Hypersensitivity Incidents** ( OCSPP 885.3400 ; MRID No. 478203 - 06 ). 40 CFR § 158.2140 requires that “[ h ] yp ersensitivi ty incidents, including immediate type and delayed - ty pe 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 hy persensitivity ; they

will report all hy

persensitivity

incidents to the Agency

if they occur. C LASSIFICATION : 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 B acillus subtilis CX - 9090 . As justification, they argue that B acillus subti lis 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 b y EPA in other products with similar acute toxicity profiles.” B acillus subtilis strain CX - 9060 is derived from B acillus subtilis MBI600. 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 B acillus subtilis MB I 600 as an activ e ingredient. This study showed that B acillus subtilis MBI600 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 s ignificant human health risks. ” The inert ingredients in the end - use product , Bacillus subtilis CX - 9090 , are not expecte d to be acutel y toxic

—all are exempt from the requirement of tolerance when used pre - or post - harvest, with no restrictions. Further, onl y slight erythema, which resolved within 24 hours, was observed in the submitted dermal irritation study (MRID No. 4782 03 - 04); no other adverse effects were seen. Therefore, EPA would expect that B acillus subtilis CX - 9090 would not be acute ly toxic via the dermal route. Regarding acute inhalation toxicity, the acute pulmonary toxicity /pathogenicity

study (MRID No. 419074 - 04) showed that the test material ( B acillus subtilis MBI 600) was not toxic, infective or pathogenic to rats. C LASSIFICATION FOR A CUTE O RAL T OXICITY : Acceptable; Toxicity Category IV C LASSIFICATION FOR A CUTE D ERMAL T OXICITY : Acceptable; Toxicity Category IV C LASSIFICATION FOR A CUTE INHALATION T OXICITY : Acceptable; Toxicity Category IV **H . Acute Eye Irritation**  ( OCSPP 870.2400 ; MRID No. 478203 - 05 ) . The concentration of the test material was not measured b y Stillmeadow Labs

—it was provided by Certis along with a Certificate of Analy sis 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 L abs started the study one month after Certis certified the concentration of the test material (the Certificate of Anal ys is 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 do se (0.1 mL) are what the test guideline recommends. One hour after treatment , one animal showed signs of corneal op acity ; by 24 hours , it cleared. No animals developed iritis. Chemosis was exhibited by

one animal at 1 and 24 hours after treatment; at 48 ho urs , it cleared. TheBacillus subtilis strain CX - 9060 Page 24 of 27

Biopesticides Registration Action Document maximum average irritation score (14.0) corresponds to a Draize rating of “mildly irritating.” C LASSIFICATION : Acceptable; Toxicity Category III **I . Primary Dermal Irritation**  ( OCSPP 870.2500; MRID No. 478203 - 04 ) . The concentrati on of the test material was not measured by Stillmeadow Labs

—it was provided b y 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 Anal ys is is dated July 10, 2008 and Stillmeadow Labs initiated the study

on August 5, 2008). The test sp ecies (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. C LASSIFICATION : Acceptable; Toxicity Category IV **Table 3**  **.** Summary of Reviewed Nontarget Organism Studi es , Citations and Rationale to Waive Testing **Data Requirement OC**  **S PP**  **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) show s 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, B acillus subtili s CX - 9090. **Classification: Acce ptable**  478203 - 09 Freshwater Fish Toxicity 850.1075 In a non - renewal static laboratory toxicity study, the 96 - hour LC 50 wa s calculated as 1 27.23 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 ha s no direct aquatic uses. 478203 - 08

Freshwater Invertebrate Toxicity/Pathogenicity 885.4240 The information pro vided is sufficient to support a waiver request for Aquatic Invertebrate Testing for the end -use pesticide product, B acillus subtili s CX 9090, if there are no direct aquatic uses on the label. **Classification: Acceptable** 478203 - 09Bacillus subtilis strain CX - 9060 Page 25 of 27

Biopesticides Registration Action Document **Data Requirement OC**  **S PP**  **Guideline No. Results Summary and Classification**  **MRID No.** Ac ute Toxicity Freshwater Invertebrates 850.1010 In a non - renewal static laboratory bioassay , the 48 - hour EC 50 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 pesticid e. The duration of the study is insufficient for evaluation of viable microbial pesticides to aquatic invertebrates. **Acceptable** for end - use pesticide product labeling that ha s no directed aquatic uses. 478203 - 07

Estuarine/Marine Fish and Invertebrate Testing 885.4280 Data are not required. B acillus subtili s strain CX - 9060 wi ll not be applied directly to marine/estuarine water s and is not expected to enter marine/estuarine environments in amounts that are significantly higher than naturally occurring concentrations. N/A

Nontarget P lant Testing 885.4300 A waiver rationale wa s provided for Nontarget Plant Testing, Tier I for Certis’ B acillus subtilis CX - 9090 . Because the pesticide is not taxonomically related to a known plant pathogen, no ntarget plant testing wa s not requir ed. 478203 - 10

Nontarget Insect Testing 885.4340 A waiver rationale wa s provided for Non Target Insect Testing, Tier I for Certis’ B acillus subtilis CX - 9090 . Because there are no intended insecticidal uses for B acillus subtili s CX - 9090, nontarget insect testing wa s not requir ed . 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 LD 50 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 LD 50 > 1 x 10 8 for a similar strain of B acillus subtili

s was determined from another acceptable study (MRID 474724 - 04 ) . 478203 - 11 474724 - 04Bacillus subtilis strain CX - 9060 Page 26 of 27

Biopesticides Registration Action DocumentBacillus subtilis strain CX - 9060 Page 27 of 27

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

**Name Percentage** 

**Active Ingredient**  **Formulation**

**Type Use Site(s)**  **Method(s) of Application**  **Application Rate 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 g al . water

(foliar and soil drench), 1⁄2 lb. per g al . 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 g al . of water/acre Drip or sprinkler chemigation 1 to 3 lbs./100 g al . water