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

December 15, 2011
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TABLE OF CONTENTS

I. EXECUTIVE SUMMARY ................................................................................................. 5

II. ACTIVE INGREDIENT OVERVIEW.............................................................................. 7

III. REGULATORY BACKGROUND.................................................................................... 7

A. Application for Pesticide Registration................................................................................................... 7

B. Food Tolerance Exemption..................................................................................................................... 7

IV. RISK ASSESSMENT ......................................................................................................... 7

A. Product Analysis Assessment (40 CFR § 158.2120)............................................................................ 8

B. Human Health Assessment (40 CFR § 158.2140)................................................................................ 8

C. Environmental Assessment (40 CFR § 158.2150) .............................................................................13

V. ENVIRONMENTAL JUSTICE....................................................................................... 15

VI. RISK MANAGEMENT DECISION................................................................................ 15

VII. ACTIONS REQUIRED OF THE REGISTRANT ......................................................... 16

A. Final Printed Labeling ..........................................................................................................................16

B. Terms of Registration............................................................................................................................16

C. Reporting of Adverse Effects and Hypersensitivity Incidents...........................................................16

VIII. GLOSSARY OF ACRONYMS AND ABBREVIATIONS............................................ 18

IX. BIBLIOGRAPHY.............................................................................................................. 19

A. Submitted and Cited Studies................................................................................................................19

B. EPA Risk Assessment Memoranda and Other Documents................................................................20

APPENDIX A. MICROBIAL PESTICIDES DATA REQUIREMENTS............................. 22

APPENDIX B. PESTICIDE PRODUCT.................................................................................. 26
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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
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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 CX9060 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, nonoccupational 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 
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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, the proposed

registration decision for the pesticide product containing Bacillus subtilis strain CX-9060, a new 

active ingredient, was subject to a 30-day comment period. In addition to containing a new active 

ingredient, the registration of this product results 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 were 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, none were received. 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. 
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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 CX9090 (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 
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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 1010 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. 
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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 
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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 nonoccupational exposures, including drinking water from ground water or surface water and 

exposure through pesticide use in gardens, lawns, or buildings (residential and other indoor 

uses).

Food Exposure and Risk Characterization: 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.
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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 foodrelated 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 otherwisecompromised 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. 
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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, 
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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 restrictedentry 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 
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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 108 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 CX9060 is estimated to be < 1.25 x 1010 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 CX9060. 

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 
Bacillus subtilis strain CX-9060 Page 15 of 28

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listed species and their designated critical habitats resulting from the proposed uses of the enduse 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 CX9060, 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 
Bacillus subtilis strain CX-9060 Page 16 of 28

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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.

3. A copy of the Organic Materials Review Institute (OMRI) certificate must be 

submitted to the Agency within one year of the date of this Notice of Pesticide 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
Bacillus subtilis strain CX-9060 Page 17 of 28

Biopesticides Registration Action Document

(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 18 of 28

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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 
Bacillus subtilis strain CX-9060 Page 19 of 28

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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-Jun1991

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-Jun1991

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-Jun1991

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-Mar1993

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-Jul2008

478203-01

Eyal, J. (2009) Bacillus subtilis Strain MBI 600 End-Use Product CX9090: 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-Aug2009

478203-02

Eyal, J. (2009) Bacillus subtilis Strain MBI 600 End-Use Product CX9090: 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-Aug2009

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-Aug2009

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-Aug2009
Bacillus subtilis strain CX-9060 Page 20 of 28

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. Unpublished study prepared 

by Stillmeadow, Inc. 17 p.

04-Aug2009

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-Aug2009

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-Aug2009

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-Aug2009

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-Aug2009

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-Aug2009

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-Aug2009

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-Jan2010

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 Systematic Bacteriology, Volume 3; 2nd 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.
Bacillus subtilis strain CX-9060 Page 21 of 28

Biopesticides Registration Action Document

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.
Bacillus subtilis strain CX-9060 Page 22 of 28

Biopesticides Registration Action Document

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
Bacillus subtilis strain CX-9060 Page 23 of 28

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Table 2. Summary of Human Health Toxicity Data for Certis’ Bacillus subtilis CX-9090

DATA REQUIREMENT

(OCSPP GUIDELINE)

MRID NO. TOXICITY AND CLASSIFICATION

TOXICITY

CATEGORYa

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 108 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 108 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 107 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
Bacillus subtilis strain CX-9060 Page 24 of 28

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 

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 108viable 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 

(NCIB112376) was not toxic, infective or pathogenic to rats at a dose of 2 x 108 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 108viable 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 108 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 107viable 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 107 CFU of the test material. (U.S. EPA 1993) 

CLASSIFICATION: Acceptable

 

1NCIB stands for “National Collections of Industrial Bacteria;” http://www.ncimb.com/culture.html. 
Bacillus subtilis strain CX-9060 Page 25 of 28

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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
Bacillus subtilis strain CX-9060 Page 26 of 28

Biopesticides Registration Action Document

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
Bacillus subtilis strain CX-9060 Page 27 of 28

Biopesticides Registration Action Document

Data Requirement OCSPP Guideline No. Results Summary and Classification MRID No.

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 CX9090, 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
Bacillus subtilis strain CX-9060 Page 28 of 28

Biopesticides Registration Action Document

Data Requirement OCSPP Guideline No. Results Summary and Classification MRID No.

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 108for 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 CX9090

25.0% End Use,

Water 

Dispersible 

Granular

Many 

food 

crop and 

ornament

al 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 Foliaror soil-applied 

spray

1 1/4 to 5 lbs./acre 

(foliar); 1/8 to 1 

lb./acre (soil)

Aerial 

(fixed or rotary 

winged)

Apply 1 1/4 to 5

lbs./acre in a 

minimum of 3 gal. 

of water/acre

Drip or sprinkler 

chemigation

1 to 3 lbs./100 gal. 

water