BIOPESTICIDES REGISTRATION ACTION DOCUMENT

DRAFT

Bacillus amyloliquefaciens strain D747

Pesticide Chemical (PC) Code: 016482

U.S. Environmental Protection Agency

Office of Pesticide Programs

Biopesticides and Pollution Prevention Division

November 10, 2011 version
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TABLE OF CONTENTS

I. EXECUTIVE SUMMARY...................................................................................................... 4

II. ACTIVE INGREDIENT OVERVIEW ................................................................................ 5

III. REGULATORY BACKGROUND................................................................................. 6

A. Applications for Pesticide Product Registration.................................................................................................6

B. Food Tolerance Exemption ...................................................................................................................................6

IV. RISK ASSESSMENT ...................................................................................................... 6

A. Product Analysis Assessment (40 CFR § 158.2120)............................................................................................7

B. Human Health Assessment (40 CFR § 158.2140)................................................................................................7

C. Environmental Assessment (40 CFR § 158.2150)..............................................................................................12

V. ENVIRONMENTAL JUSTICE .......................................................................................... 14

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

VII. ACTIONS REQUIRED BY THE REGISTRANT ......................................................... 16

VIII. GLOSSARY OF ACRONYMS AND ABBREVIATIONS........................................... 16

IX. BIBLIOGRAPHY................................................................................................................ 18

A. Studies Submitted to Support Pesticide Product Registrations.......................................................................18

B. Environmental Protection Agency Risk Assessment Memoranda and Other Documents............................20

C. Other References .................................................................................................................................................20

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

APPENDIX B. PESTICIDE PRODUCTS............................................................................... 30
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BIOPESTICIDES REGISTRATION ACTION DOCUMENT TEAM

Office of Pesticide Programs (OPP)

Biopesticides and Pollution Prevention Division

Microbial Pesticides Branch

Science Reviews

Joel V. Gagliardi, Ph.D. Product Analysis, Human Health

John L. Kough, Ph.D. Product Analysis, Human Health

Gail Tomimatsu, Ph.D. Environmental Effects

Shannon Borges Environmental Effects

Regulations

Sheryl K. Reilly, Ph.D. Chief, Microbial Pesticides Branch

Susanne Cerrelli Regulatory Action Leader
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I. EXECUTIVE SUMMARY

Certis USA, LLC, (“Certis” or “applicant) has proposed to register Bacillus amyloliquefaciens

strain D747 as the active ingredient in two end-use pesticide products, CX-9030 (EPA File 

Symbol 70051-RNI) and CX-9032 (EPA File Symbol 70051-RNT). The products are intended 

for use to control fungi and bacteria in outdoor agricultural crops, greenhouses, nurseries, 

shadehouses, ornamentals, and turfgrass. B. amyloliquefaciens strain D747 was initially

identified by Certis as“Bacillus subtilis variant amyloliquefaciens strain D747,” since B. subtilis

and B. amyloliquefaciens were originally classified as subtypes or variants of the same species.

B. amyloliquefaciens is now considered a separate species, and the correct taxonomic designation 

is used in this Biopesticides Registration Action Document (“BRAD”) (Priest et. al., 1987; 

Logan and de Vos, 2009; and Murray et. al., 2007). 

EPA scientists reviewed product analysis, mammalian and nontarget organism toxicity data, and 

other information submitted by Certis to support the proposed product registrations. The product 

analysis data requirements for B. amyloliquefaciens strain D747, including product chemistry

and composition, analysis of samples, and physical and chemical characteristics, were fulfilled

by acceptable studies conducted in accordance with Agency guidelines. Mammalian toxicity data

(acute oral, injection, and pulmonary toxicity/pathogenicity) and information from peer-reviewed 

scientific literature demonstrated that B. amyloliquefaciens strain D747 is not toxic, infective or 

pathogenic in laboratory rats. Acceptable nontarget organism data also demonstrated that B.

amyloliquefaciens strain D747 is not toxic to estuarine and marine fish and invertebrates, 

nontarget insects (including honey bees), and nontarget plants. 

We have assessed human health and environmental risks from the proposed uses of B.

amyloliquefaciens strain D747, and determined that the pesticide would not cause unreasonable 

adverse effects to nontarget organisms when used in accordance with the directions on the 

proposed labels, and in accordance with good agricultural practices. Additional mammalian and 

nontarget organism toxicity data are not required for the proposed uses and application methods.

Bacillus species, including B. amyloliquefaciens, are commonly found in soils, including 

agricultural settings, and are naturally present on fresh produce. The Manual of Clinical 

Microbiology (9th Edition) states that dried foods, such as spices, milk powder and grains, often 

contain large amounts of Bacillus spores. B. amyloliquefaciens is not known to produce any 

mammalian toxins, and no food-borne disease outbreaks associated with B. amyloliquefaciens

have been reported. Given that the microorganism occurs naturally in soils, exposure to B.

amyloliquefaciens from surface and groundwater may occur. No adverse effects have been 

reported, and none are expected from exposure to B. amyloliquefaciens through drinking water.

Despite the low toxicological profile of B. amyloliquefaciens strain D747, personal protective 

equipment (PPE) is required for pesticide handlers that are frequently exposed to the active 

ingredient for prolonged periods. Handlers will be directed to wear a long-sleeved shirt, long 

pants, socks, shoes, waterproof gloves, and a dust/mist filtering respirator meeting National 

Institute for Occupational Safety and Health (NIOSH) standards of at least N-95, R-95, or P-95.

EPA may require additional PPE, other than the standard described above, if additional pesticide 

uses resulting in increased exposures are proposed in the future. 

EPA has concluded that, based upon the results of the toxicity tests and lack of adverse incidents,

there is a reasonable certainty that no harm will result to the United States population, including 
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infants and children, from dietary (including drinking water) and aggregate exposure to residues 

of B. amyloliquefaciens strain D747 from the proposed pesticide uses. The proposed products

meet the standards for registration under Section 3(c)(5) of the Federal Insecticide, Fungicide, 

and Rodenticide Act (FIFRA).

On October 1, 2009, EPA announced a new policy to provide a more meaningful opportunity for 

the public to participate in major registration decisions before they occur. According to this 

policy, EPA intends to provide a public comment period prior to making a registration decision 

for, at minimum, the following types of applications: new active ingredients; first food uses; first 

outdoor uses; first residential uses; or any other registration actions for which EPA believes there 

may be significant public interest.

 

Consistent with the policy of making registration actions more transparent, the proposed 

pesticide products containing B. amyloliquefaciens strain D747, a new active ingredient, are

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

registration of these products would result in the first outdoor use, first residential use and first 

food use for B. amyloliquefaciens strain D747. The docket identification number, associated with 

these registration actions and accessed through either http://www.regulations.gov/ or 

http://www.epa.gov/pesticides/regulating/registration-status.html, is EPA-HQ-OPP-2011-0853. 

The following documents are available for comment in EPA-HQ-OPP-2011-0853: (1) draft B.

amyloliquefaciens strain D747 Biopesticides Registration Action Document (BRAD); (2) 

environmental risk assessment for B. amyloliquefaciens strain D747; (3) draft product label for 

the end-use product, CX-9030 (EPA File Symbol 70051-RNI) ; and (4) draft product label for 

the end-use product, CX-9032 (EPA File Symbol 70051-RNT ). While a final decision on 

registration is contingent upon review and consideration of public comments, EPA presently

believes that, based upon the risk assessment and information submitted in support of the CX9030 and CX-9032 EPs, it is in the best interest of the public and the environment to issue these 

registrations. The basis for this preliminary decision can be found in the risk assessment for B.

amyloliquefaciens strain D747, which is presented in this document. 

II. ACTIVE INGREDIENT OVERVIEW

Biological Name: Bacillus amyloliquefaciens strain D747

Culture Deposit: Agricultural Research Service Culture Collection (also 

known as the Northern Regional Research Laboratory

 (NRRL) Collection) in Peoria, Illinois, under Accession 

 Number NRRL B-50405.

OPP Chemical Code: 016482

Type of Pesticide: Microbial Pesticide – Insecticide

See Appendix B for specific information (e.g.,

use sites, application rates, methods of application, 

formulation types, and target pests) regarding

the registered pesticide products containing this 

active ingredient.
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III. REGULATORY BACKGROUND

A. Applications for Pesticide Product Registration

Certis USA, LLC (address: 9145 Guilford Road, Suite 175, Columbia, MD, 21046), submitted 

applications to register two end-use pesticide product, CX-9032 and CX-9030, under FIFRA 

section 3(c)(5) on July 26, 2010. EPA announced receipt of these applications to register 

pesticide products containing a new active ingredient on February 2, 2011 (76 Federal Register 

(FR) 5805), and opened a 30-day public comment period, pursuant to the provisions of FIFRA 

section 3(c)(4). No comments were received following this publication. 

B. Food Tolerance Exemption

Concurrent with its registration applications, and under the Federal Food, Drug, and Cosmetic 

Act (FFDCA) section 408(d), Certis USA, LLC, submitted a petition to establish an exemption 

from the requirement of a tolerance for Bacillus subtilis variant amyloliquefaciens strain D747

(Pesticide Petition (PP) 0F7760). In the Federal Register of February 4, 2011 (76 FR 6465), EPA 

announced that Certis USA, LLC, proposed to establish an exemption from the requirement of a 

tolerance for residues of the insecticide, Bacillus subtilis variant amyloliquefaciens strain D747, 

in or on all food commodities, and opened a 30-day comment period. No comments were 

received following this publication. The correct taxonomic designation of the microorganism is

Bacillus amyloliquefaciens strain D747; therefore, the tolerance exemption, when established, 

will reflect the correct name of the active ingredient.

IV. RISK ASSESSMENT

In the Federal Register of October 26, 2007, EPA issued a Final Rule establishing changes to the 

data requirements that support the registration of microbial pesticides. The rule also updated the 

definition for microbial pesticides (72 FR 61002), and became effective on December 26, 2007. 

The data and information evaluated for this BRAD were considered in accordance with these 

requirements.

The classifications or ratings that are given for each data requirement in this BRAD were

assigned by the EPA scientists who reviewed the data, and convey the usefulness of the 

information for the human health and nontarget risk assessment purposes. “Acceptable” indicates 

that a study is scientifically sound and is useful for risk assessment. A “supplemental” 

classification indicates the studies provided some information that can be useful for risk 

assessment. “Supplemental” ratings are given to studies that are not required for registration, as 

well as those that are required. In the latter case, the study lacks information that should have 

been obtained if it was conducted according to the guideline associated with the data 

requirement. Sometimes the missing information would not add anything necessary to inform a 

risk assessment, and the study classification remains “supplemental.” A classification of

“supplemental:upgradable” indicates the study lacks necessary information, but if it is made 

available by the applicant, the study may be upgraded to “acceptable.” An “unacceptable” rating 

indicates that new data must be submitted.
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Toxicity categories are assigned to acute toxicity studies, based upon any signs of toxicity

(hazards) observed in the test animals during the course of the study, whether the study was 

conducted in accordance with test guidelines or comes from other sources, such as peerreviewed, scientific literature. The active ingredient or particular product is classified into 

Toxicity Category I, II, III, or IV, where Toxicity Category I indicates the highest toxicity, and 

Toxicity Category IV indicates the lowest toxicity (see 40 CFR § 156.62). 

A. Product Analysis Assessment (40 CFR § 158.2120)

All product analysis data requirements for B. amyloliquefaciens strain D747 have been fulfilled

for the proposed product registrations. Refer to Tables 1, 2, 3, and 4 in Appendix A for a brief 

summary of the data requirements, including both generic and product-specific information. 

B. Human Health Assessment (40 CFR § 158.2140)

1. Toxicity

All Tier I mammalian toxicity data requirements for B. amyloliquefaciens strain D747 have been 

fulfilled for the proposed product registrations. Refer to Tables 5, 6 and 7. Based on the lack of 

acute toxicity/pathogenicity, Tier II and Tier III studies were not required. 

For a comprehensive summary of the generic and product-specific toxicity data requirements 

described below, refer to Tables 5, 6 and 7 in Appendix A.

a. Acute Toxicity/Pathogenicity – Tier I

Acute Oral Toxicity/Pathogenicity (Office of Chemical Safety and Pollution Prevention (OCSPP) 

Guideline 885.3050; Master Record Identification Number (MRID No. 481657-04): 

B. amyloliquefaciens strain D747 was administered once orally to 14 rats of both sexes (5-weeks

old) at a single dosage of 108colony-forming units (CFU) per animals. No deaths occurred, and 

no abnormalities (clinical signs, body weight) were observed, during the study or at necropsy. 

The test microbe was detected at 103– 105 CFU/g in feces 1 day after administration of the test 

material, but was not detected on day14. The examination for internal persistence did not detect 

the test microbe in any organs or tissues, such as the kidney, brain, liver, lung, spleen, stomach, 

small intestine (duodenum), large intestine (cecum), mesenteric lymph nodes, or blood,

throughout the experimental period. Fecal clearance occurred by day 14, and no viable 

organisms were recovered from blood or other organs or tissues. The results of this acceptable 

study demonstrated that B. amyloliquefaciens strain D747 was not infective, pathogenic, or toxic 

to rats when orally dosed with 1.0x108 CFU / animal. 

Acute Pulmonary Toxicity/Pathogenicity (OCSPP Guideline 885.3150; MRID No.481657-06): 

B. amyloliquefaciens strain D747 was administered once intratracheally to 20 male and female

Sprague-Dawley rats (5-week old) at a dosage of 107 CFU per animal. No deaths occurred, and 

no abnormalities (clinical signs, body weight) were observed during the study or at necropsy. 

The examination for internal persistence showed that the test microbe was detected shortly after 

administration in the lung, trachea, and nasal cavity. The test microbes were not detected in the 

nasal cavity on day 7, and almost completely cleared from the trachea by day 60, when the study

ended. The presence of the test microbes decreased in the lungs by approximately 102-103 CFU/g 

from day 0 to day 60. The test microbes were detected in the bronchial lymph nodes on day 3.
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Since many test microbes remained in the trachea on day 3, their presence in the bronchial lymph 

nodes on day 3 was attributed to transfer by macrophages from the respiratory tract to the lymph 

nodes. This conclusion was supported by the observation that a particle larger than 1 m in 

diameter administered into the trachea and bronchi was phagocytized by a macrophage and 

carried to a lymph node. The presence of the microbes in the lungs, though decreasing 

throughout the observation period, was attributed to the continuous transfer of viable test 

organisms to the bronchial lymph nodes. This result is not unusual for spore-forming bacteria, 

since bacterial spores are extremely tolerant of adverse conditions, and take a longer time for the 

immune system to clear than bacteria that do not form spores. Since a pattern of clearance was 

demonstrated, the remaining viable cells were considered to be spores, which take longer for a 

healthy immune system to clear. This acceptable study demonstrated that B. amyloliquefaciens

strain D747 was not toxic or pathogenic to rats when dosed intratracheally at 1.0 x107 CFU/ 

animal.

Acute Injection Toxicity/Pathogenicity (Intravenous) – Rat (OCSPP Guideline 885.3200; MRID 

No. 481657-05): In an acute intravenous injection toxicity and pathogenicity study, groups of 17 

male and female Sprague-Dawley rats (5-weeks old) were injected with B. amyloliquefaciens 

strain D747 at a dosage of 107 CFU per animal, and the influence on the animals was 

investigated. No deaths occurred, and there were no observed abnormalities (clinical signs, body 

weight) during the study or at necropsy. The examination for internal persistence of the test 

microbe showed that the test microbes were mainly detected in the kidney, liver, spleen, and 

blood shortly after administration. The test microbe decreased after that and was not detected in 

the blood from day14; clearance from the kidney occurred by day 60. McClintock et al. 

previously reported that when B. thuringiensis and B. subtilis, which are both spore-forming 

bacteria, were administered intravenously to rats, clearance from the liver and spleen is difficult. 

In this study, the test microbe did not completely disappear from these organs by day 60. No test 

microbes were detected in the brain, and only a few viable microbes at the limit level were 

sporadically detected in the small and large intestines, and lymph nodes, but were cleared by day

60. This acceptable study demonstrated that B. amyloliquefaciens strain D747 was not toxic, 

infective, or pathogenic to rats when injected intravenously with 1.0 x107 CFU/ animal.

Hypersensitivity Incidents (OCSPP Guideline 885.3400; MRID No. 481655-05): No 

hypersensitivity incidents in humans have been reported, and none occurred during research, 

development, or testing of B. amyloliquefaciens strain D747. Should hypersensitivity or other 

adverse incidents in humans occur in the future, Certis must report them to EPA, in accordance 

with FIFRA section 6(a)(2).

Cell Culture (OCSPP Guideline 885.3500): This study was not required because B.

amyloliquefaciens strain D747 is not a virus (refer to test note #4 of 40 CFR § 158.2140(d)).

b. Acute Toxicity and Subchronic Toxicity/Pathogenicity – Tier II;

 Reproductive Fertility Effects, Carcinogenicity, Immunotoxicity, and 

 Infectivity/Pathogenicity Analysis – Tier III

Tier II and Tier III studies were not required for B. amyloliquefaciens strain D747 based on the 

lack of acute toxicity/pathogenicity in the Tier I studies.
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c. Endocrine Disruptors

As required under FFDCA section 408(p), EPA has developed the Endocrine Disruptor 

Screening Program (EDSP) to determine whether certain substances (including pesticide active 

and other ingredients) may have an effect in humans or wildlife similar to an effect produced by 

a “naturally occurring estrogen, or other such endocrine effects as the Administrator may

designate.” The EDSP employs a two-tiered approach to making the statutorily required 

determinations. Tier 1 consists of a battery of 11 screening assays to identify the potential of a 

chemical substance to interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal 

systems. Chemicals that go through Tier 1 screening and are found to have the potential to 

interact with E, A, or T hormonal systems will proceed to the next stage of the EDSP where EPA 

will determine which, if any, of the Tier 2 tests are necessary based on the available data. Tier 2 

testing is designed to identify any adverse endocrine-related effects caused by the substance, and

establish a quantitative relationship between the dose and the E, A, or T effect.

Between October 2009 and February 2010, EPA issued test orders/data call-ins for the first 

group of 67 chemicals, which contains 58 pesticide active ingredients and 9 inert ingredients. 

This list of chemicals was selected based on the potential for human exposure through pathways 

such as food and water, residential activity, and certain post-application agricultural scenarios. 

This list should not be construed as a list of known or likely endocrine disruptors.

B. amyloliquefaciens strain D747 is not among the group of 58 pesticide active ingredients on the 

initial list to be screened under the EDSP. Under FFDCA section 408(p), EPA must screen all 

pesticide chemicals. Accordingly, EPA anticipates issuing future EDSP orders/data call-ins for 

all pesticide active ingredients. 

For further information on the status of the EDSP, the policies and procedures, the list of 67 

chemicals, the test guidelines and the Tier 1 screening battery, please visit our website: 

http://www.epa.gov/endo/.

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

Section 408(c)(2)(A)(i) of FFDCA allows EPA to establish an exemption from the requirement 

for a tolerance (the legal limit for a pesticide chemical residue in or on a food) only if EPA 

determines that the exemption is “safe.” Section 408(c)(2)(A)(ii) of FFDCA defines “safe” to 

mean that “there is a reasonable certainty that no harm will result from aggregate exposure to the 

pesticide chemical residue, including all anticipated dietary exposures and all other exposures for 

which there is reliable information.” This includes exposure through drinking water and in 

residential settings but does not include occupational exposure. Pursuant to section 408(c)(2)(B) 

of FFDCA, in establishing or maintaining in effect an exemption from the requirement of a 

tolerance, EPA must take into account the factors set forth in section 408(b)(2)(C) of FFDCA, 

which require EPA to give special consideration to exposure of infants and children to the 

pesticide chemical residue in establishing a tolerance and to “ensure that there is a reasonable 

certainty that no harm will result to infants and children from aggregate exposure to the pesticide 

chemical residue....” Additionally, section 408(b)(2)(D) of FFDCA requires that EPA consider 

“available information concerning the cumulative effects of [a particular pesticide's] . . . residues 

and other substances that have a common mechanism of toxicity.”
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EPA performs a number of analyses to determine the risks from aggregate exposure to pesticide 

residues. First, EPA determines the toxicity of pesticides. Second, EPA examines exposure to the 

pesticide through food, drinking water, and through other exposures that occur as a result of 

pesticide use in residential settings.

Consistent with section 408(b)(2)(D) of FFDCA, EPA has reviewed the available scientific data 

and other relevant information, and considered its validity, completeness, and reliability and the 

relationship of this information to human risk. EPA has also considered available information 

concerning the variability of the sensitivities of major identifiable subgroups of consumers, 

including infants and children. All of the data requirements have been fulfilled, as described 

previously, and are presented in Table 5 in Appendix A. The following summarizes the results of 

EPA’s dietary risk assessment for B. amyloliquefaciens strain D747.

a. Aggregate Exposure

In examining aggregate exposure, section 408 of FFDCA directs EPA to consider 

available information concerning exposures from the pesticide residue in food and all other nonoccupational exposures, including drinking water from ground or surface water, and through 

pesticide use in residential and other indoor uses.

Food Exposure and Risk Characterization: Bacillus species, including B. amyloliquefaciens, are 

commonly found in the soil in agricultural settings, and are present on fresh produce of all kinds 

with no known adverse effects. The Manual of Clinical Microbiology (9th edition) mentions that 

dried food such as spices, milk powder and grains often contain large amounts of B. spores. B.

amyloliquefaciens is not known to produce any mammalian toxins, and no foodborne disease 

outbreaks associated with B. amyloliquefaciens have been reported. 

Based on the data and other information submitted to satisfy the data requirements for 

registration of the manufacturing-use and end-use pesticide products containing the active 

ingredient, B. amyloliquefaciens strain D747, no toxicity, infectivity, pathogenicity or other 

adverse effects from dietary exposure to are expected (see section IV(B)(1)(a), above, and Table 

5 in Appendix A).

Drinking Water Exposure and Risk Characterization: B. amyloliquefaciens is naturally present in 

soils (Logan and de Vos, 2009); therefore, B. amyloliquefaciens may occur in surface and 

possibly groundwater. According to the World Health Organization, Bacillus species are often 

detected in drinking water even after going through acceptable water treatment processes, largely 

because the spores are resistant to these disinfection processes (World Health Organization, 

2011). Should this microbial pesticide be present, no adverse effects are expected from exposure 

to B. amyloliquefaciens through drinking water, based on the data and other information 

submitted to satisfy the data requirements for registration of the manufacturing-use and end-use 

pesticide products containing the active ingredient, B. amyloliquefaciens strain D747 (see section 

IV(B)(1)(a), above, and Table 5 in Appendix A).

Non-occupational, Residential Risk Characterization: The use sites for these products include 

residential garden sites and agricultural sites. As stated previously, B. amyloliquefaciens is 

naturally present in soil, and based on the data and other information submitted to satisfy the data 

requirements for registration of the manufacturing-use and end-use pesticide products containing

the active ingredient, B. amyloliquefaciens strain D747, no toxicity, infectivity, pathogenicity or 
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other adverse effects from non-occupational exposure are expected (see section IV(B)(1)(a), 

above, and Table 5 in Appendix A).

b. Cumulative Effects from Substances with a Common Mechanism of Toxicity

Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to establish, modify, 

or revoke a tolerance exemption, EPA consider “... available information concerning the 

cumulative effects of [a particular pesticide’s] ... residues and other substances that have a 

common mechanism of toxicity.”

EPA has found that B. amyloliquefaciens strain D747 does not share a common mechanism of 

toxicity with any other microorganism, pesticidal or toxic substance. EPA concludes that there 

are no cumulative effects associated with B. amyloliquefaciens strain D747 that need to be 

considered. For information regarding how EPA determines common mechanisms of toxicity

and evaluates cumulative effects, see EPA’s website at: 

http://www.epa.gov/pesticides/cumulative.

c. Determination of Safety for the United States (U.S.) Population, Infants and 

 Children

FFDCA section 408(b)(2)(C) provides that EPA shall assess the available information about 

consumption patterns among infants and children, special susceptibility of infants and children to 

pesticide chemical residues, and the cumulative effects on infants and children of the residues 

and other substances with a common mechanism of toxicity. In addition, FFDCA section 

408(b)(2)(C) provides that EPA shall apply an additional tenfold (10X) margin of safety for 

infants and children in the case of threshold effects to account for prenatal and postnatal toxicity

and the completeness of the database on toxicity and exposure, unless EPA determines that a 

different margin of safety will be safe for infants and children. This additional margin of safety is 

commonly referred to as the Food Quality Protection Act Safety Factor. In applying this 

provision, EPA either retains the default value of 10X or uses a different additional safety factor 

when reliable data available to EPA support the choice of a different factor.

EPA concludes that, based upon the results of the toxicity data and other information considered 

and described in this document, there is a reasonable certainty that no harm will result to the U.S.

population, including infants and children, from aggregate exposure to the residues of B.

amyloliquefaciens strain D747. Such exposure includes all anticipated dietary and other 

exposures for which there is reliable information. With no threshold effects of concern, an

additional margin of safety is not required for infants and children. 

3. Occupational Exposure and Risk Characterization

Occupational exposure to B. amyloliquefaciens strain D747 is not expected to undue risks to 

pesticide handlers (mixer/loader/applicators), but EPA requires appropriate personal protective 

equipment and precautionary statements to mitigate any potential risks (e.g., respiratory

allergenicity) to pesticide handlers from prolonged or repeated exposures. Handlers applying B.

amyloliquefaciens strain D747 end-use products in agricultural settings must wear a long-sleeved 

shirt, long pants, socks, shoes, waterproof gloves, and a dust/mist filtering respirator meeting 

NIOSH standards of at least N-95, R-95, or P-95. 
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4. Human Health Risk Characterization

EPA considered human exposure to B. amyloliquefaciens strain D747 in light of the registration

standards of FIFRA and the relevant FFDCA safety factors for allowable pesticide residues in 

food and animal feed commodities. EPA has determined that no unreasonable adverse effects to 

the U.S. population in general, and to infants and children in particular, will result when the 

proposed pesticide products containing B. amyloliquefaciens strain D747 as the active ingredient 

are used in accordance with the proposed labeling.

C. Environmental Assessment (40 CFR § 158.2150)

The primary habitat of Bacillus species is soil, although they have also been isolated from a wide 

variety of other habitats. B. amyloliquefaciens has a wide-spread distribution, owed in part to its 

ability to form endospores that are resistant to greater variation in environmental conditions than 

the vegetative cells, should transfer to other climates occur. B. amyloliquefaciens has been 

isolated from internal tissues of healthy plants, and is known to promote plant growth. It is not 

recognized as a pathogen among Bacillus species (Logan and de Vos, 2009). 

The data, literature citations, and data waiver rationale submitted by the applicant to support the 

pesticide products containing B. amyloliquefaciens strain D747 fulfilled the Tier I nontarget

organism data requirements, and were sufficient for risk assessment purposes. Further testing of 

nontarget organisms at higher tier levels (i.e., Tiers II, III, and IV) is not required for the 

proposed uses. EPA performed an environmental risk assessment, and has determined that the 

proposed use of B. amyloliquefaciens strain D747 are not expected to cause unreasonable 

adverse effects to nontarget organisms. 

For a comprehensive summary of the generic data requirements described in sections IV(C)(1) of 

this BRAD, refer to Table 8 in Appendix A.

1. Ecological Exposure and Risk Characterization 

a. Terrestrial Animals and Plants

The end-use products include a water-dispersible granular formulation and an aqueous 

suspension, proposed for application to agricultural crops, nurseries, ornamental plants, turfgrass, 

greenhouses, and shadehouses. Applications can be made to both foliar surfaces and soil, so

exposure to nontarget organisms is possible. The proposed maximum application rates were used 

in the assessment of nontarget risk. 

Data on the naturally occurring levels of B. amyloliquefaciens are not available. Many factors 

influence the environmental fate of microbial pesticides, and resulting population levels in the 

environment cannot be predicted. EPA expects that B. amyloliquefaciens strain D747 may

survive after application if conditions are favorable, but the strain would not significantly add to 

the overall levels of B. amyloliquefaciens already present in the environment. 

Birds (OCSPP Guideline 885.4050) and Mammals (OCSPP Guideline 885.4150): 

A supplemental study showed that the acute oral LD50 for B. amyloliquefaciens strain D747 is > 

4.5 x 1011 spores/kg BW or > 8 x 109spores/bird in Northern bobwhite (Colinus virginianus; 

MRID 48165712). Additionally, B. amyloliquefaciens is intentionally included in some 
Bacillus amyloliquefaciens strain D747 Page 13 of 30

Biopesticides Registration Action Document

domestic avian food diets as a nutritional additive (e.g., European Food Safety Authority, 2010;

Wizna et al., 2009), and it is not known to be pathogenic to animals (Logan and de Vos, 2009). 

An extensive literature search in several databases returned no reports of toxicity or 

pathogenicity of B. amyloliquefaciens in birds. Based on these lines of evidence, B. 

amyloliquefaciens strain D747 is not expected to pose risk of adverse effects in birds. 

A study with laboratory rats (MRID 48165704) also showed that B. amyloliquefaciens strain 

D747 is not toxic, infective, or pathogenic at the maximum hazard dose of 1.0 x 108 CFU/animal. 

Therefore, adverse effects to wild mammals are also not expected as a result of the proposed 

applications of B. amyloliquefaciens strain D747. 

Nontarget Insects (OCSPP Guideline 885.4340) and Honey Bees (OCSPP Guideline 885.4380): 

Studies with Orius stricollis, Crysoperla carnea, and Phytoseiulus persimilis were submitted for 

the nontarget insect data requirement (MRID 48165716). While no effects of B. 

amyloliquefaciens strain D747 were observed in these studies, they were not acceptable for use 

in the ecological risk assessment. Two honey bee studies were also submitted that showed no 

adverse effects of B. amyloliquefaciens D747 after 48 hours (MRID 48165717) and 17 days (no 

MRID currently assigned). These studies were rated Supplemental because they were not of 

sufficient duration (30 days); however, except in rare cases, bacteria that are pathogenic to 

insects typically produce toxins that kill the insect within a few days (Tanada and Kaya, 1993). 

Adverse effects resulting from exposure to B. amyloliquefaciens strain D747, therefore, would 

likely have been evident in the bee studies, especially after 17 days. Scientific rationale was 

submitted to show that adverse effects are not expected to nontarget insects (MRIDs 48621502 

and 48621503). Entomopathogenic B. species (e.g., B. thuringiensis, B. sphaericus) have been 

extensively studied, and their pathobiology is well-known. B. amyloliquefaciens is not among the 

B. species recognized as frank pathogens to insects or other animals (Logan and de Vos, 2009). 

There are some accounts in the literature of effects of B. subtilis on insects; however, none of 

these were associated with B. amyloliquefaciens or B. subtilis var. amyloliquefaciens. Therefore, 

based on the studies and other information provided, B. amyloliquefaciens strain D747 is not 

expected to pose risk to honey bees and other nontarget insects as a result of its proposed 

applications.

Nontarget Plants (OCSPP Guideline 885.4300):

Studies with plants exposed to B. amyloliquefaciens strain D747 were unacceptable, based on 

several deficiencies (MRID 48165715). B. amyloliquefaciens is not taxonomically related to any 

known plant pathogens. As discussed previously, however, the microorganism has been isolated 

from tissues of healthy plants and is known as a plant growth-promoting rhizobacterium. It is,

therefore, not expected to pose risk to nontarget plants as a result of the proposed applications. 

b. Aquatic Animals and Plants 

B. amyloliquefaciens strain D747 is not intended to be applied directly to water, but some of the 

applied product may reach aquatic habitats through runoff or spray drift. Spray drift at 

application is the primary mechanism by which the pesticide is expected to reach water. A spray

drift analysis was included in the aquatic risk assessment to determine exposure, and further 

detail is provided in the environmental risk assessment for B. amyloliquefaciens strain D747. 

Freshwater Fish (OCSPP 885.4200) Guideline and Invertebrates (OCSPP Guideline 885.4240): 

In a 30-day study with rainbow trout (Oncorhynchus mykiss), the LC50 for B. amyloliquefaciens
Bacillus amyloliquefaciens strain D747 Page 14 of 30

Biopesticides Registration Action Document

strain D747 was 8.1 x 1010 CFU/L, and the NOEC based on sub-lethal effects was 1.44 x 1010

CFU/L. If the proposed maximum broadcast application rate (4 fluid ounces/1000 ft2or 2.55 x 

106 CFU/cm2) is applied directly to a 1-ha body of water 15 cm deep (the EPA Standard 

Wetland), the resulting concentration would be 1.7 x 108 CFU/L. This would be the maximum 

possible aquatic concentration at this application rate, but is not a proposed use. The NOEC and 

LC50 are approximately 85X and 476X higher than this concentration, respectively. Exposure in 

freshwater environments will be well below the concentrations that would produce adverse 

effects, and the proposed applications of B. amyloliquefaciens strain D747 are not expected to 

pose risks to freshwater fish. 

A study with Daphnia magna provided an EC50 based on mortality/immobility of 3.7 x 1010

CFU/L, and a NOEC for sub-lethal effects of 2.84 x 108 CFU/L. Based on the spray drift 

analysis, the NOEC would be approximately 20X the expected environmental concentration 

(EEC), and the EC50 would be 218X the EEC; therefore, the proposed applications of B. 

amyloliquefaciens strain D747 are not expected to result in adverse effects to freshwater 

invertebrates. 

Marine/Estuarine Fish (OCSPP 885. 4280) and Invertebrates (OCSPP 885.4240): 

Concentrations reaching marine or estuarine areas are expected to be less than those calculated 

above for freshwater animals, due to further dilution in deeper water. B. amyloliquefaciens strain 

D747 is not expected to reach marine or estuarine environments in significant concentrations, 

and risk to animals in these environments is not anticipated.

Aquatic Plants (OCSPP Guideline 885.4300): 

B. amyloliquefaciens strain D747 is not related to known plant pathogens; therefore, adverse 

effects to aquatic plants are not anticipated.

2. Environmental Fate Data 

The information provided for the proposed uses and application methods was sufficient to satisfy

the Tier I nontarget organism data requirements and for nontarget organism risk assessment for 

B. amyloliquefaciens strain D747; further testing at higher tier levels (i.e., Tiers II, III, and IV) is 

not required. 

3. Threatened and Endangered Species Assessment

Since EPA has determined that no effects are anticipated for any nontarget species exposed to B. 

amyloliquefaciens strain D747 as a result of the proposed applications, effects to federally listed 

threatened and endangered species and their designated critical habitats are also not expected. 

Therefore, a “No Effect” determination is made for direct and indirect effects to listed species 

and their designated critical habitats resulting from the proposed uses of B. amyloliquefaciens

strain D747. 

V. ENVIRONMENTAL JUSTICE

EPA seeks to achieve environmental justice—the fair treatment and meaningful involvement of 

all people regardless of race, color, national origin, or income—with respect to the development, 

implementation, and enforcement of environmental laws, regulations, and policies. Fair 

treatment means that no group of people, including racial, ethnic, or socioeconomic groups, 
Bacillus amyloliquefaciens strain D747 Page 15 of 30

Biopesticides Registration Action Document

should bear a disproportionate share of the negative environmental consequences resulting from 

industrial, municipal, and commercial operations or the execution of federal, state, local, and 

tribal environmental programs and policies. Meaningful involvement means that (1) potentially

affected community residents have an appropriate opportunity to participate in decisions about a 

proposed activity that will affect their environment and/or health; (2) the public’s contribution 

can influence the regulatory agency’s decision; (3) the concerns of all participants involved will 

be considered in the decision-making process; and (4) the decision-makers seek out and facilitate 

the involvement of those potentially affected. EPA has this goal for all communities and persons 

across the United States. 

To help address potential environmental justice issues, EPA seeks information on any groups or 

segments of the population who, as a result their location, cultural practices, or other factors, 

may have atypical, unusually high exposure to B. amyloliquefaciens strain D747, compared to 

the general population. Please comment if you are aware of any subpopulations that may have 

atypical, unusually high exposure compared to the general population.

For additional information regarding environmental justice issues, please visit EPA’s web site at 

http://www.epa.gov/compliance/environmentaljustice/index.html.

VI. RISK MANAGEMENT DECISION

Section 3(c)(5) of FIFRA permits for the registration of a pesticide provided that all the 

following determinations are made:

(1) Its composition is such as to warrant the proposed claims for it;

(2) Its labeling and other material required to be submitted comply with the

 requirements of FIFRA;

(3) It will perform its intended function without unreasonable adverse effects on 

 the environment; AND

(4) When used in accordance with widespread and commonly recognized 

 practice, it will not generally cause unreasonable adverse effects on the 

 environment.

To satisfy criterion 1, the B. amyloliquefaciens strain D747 pesticide products have well-known 

properties. EPA has no knowledge that would contradict the claims made for these products, the 

CX-9030 and CX-9032 EP labels, and we have concluded that such products are not expected to 

cause unreasonable adverse effects on the environment when used according to the label 

instructions. Criterion 2 is satisfied by the current product labels, as well as the data and 

information presented in this document. It is believed that the B. amyloliquefaciens strain D747

pesticide products will not cause any unreasonable adverse effects on the environment, and CX9030 and CX-9032 (end-use pesticide product), in particular, are likely to provide protection 

against fungal and bacterial pests as claimed, satisfying criterion 3. Criterion 4 is satisfied in that

the B. amyloliquefaciens strain D747 pesticide products are not expected to cause unreasonable 

adverse effects when used according to label instructions. Therefore the end-use products, CX9030 and CX-9032, containing B. amyloliquefaciens strain D747 as a new active ingredient, are

eligible for registration under FIFRA section 3(c)(5) for the labeled uses. Should uses that are 

more extensive be proposed in the future (e.g., aquatic uses), EPA will likely require that 

additional data be submitted.
Bacillus amyloliquefaciens strain D747 Page 16 of 30

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VII. ACTIONS REQUIRED BY THE REGISTRANT

A. Final Printed Labeling

Before releasing pesticide products containing B. amyloliquefaciens strain D747 for shipment, 

the registrant is required to provide appropriate final printed labeling to EPA prior to shipment of 

product.

B. Terms of Registration

As a term of the registration CX-9032 EP, the registrant must submit the following data within 

one year of this product’s registration:

(1) Storage Stability (OCSPP Guideline 830.6317) and Corrosion Characteristics (OCSPP 

Guideline 830.6320): The results of a one-year storage stability and corrosion 

characteristics study. 

C. Reporting of Adverse Effects and Hypersensitivity Incidents

Notwithstanding the information stated previously, it should be clearly understood that certain 

specific data are required to be reported to EPA as a requirement for maintaining the federal 

registration for a pesticide product. A brief summary of these types of data are described below.

Reports of all incidents of adverse effects to the environment must be submitted to EPA under 

the provisions stated in FIFRA section 6(a)(2). Additionally, all incidents of hypersensitivity

(including both suspected and confirmed incidents) must be reported to EPA under the 

provisions of 40 CFR § 158.2140(d).

VIII. GLOSSARY OF ACRONYMS AND ABBREVIATIONS

ASAE American Society of Agricultural Engineers

BPPD Biopesticides and Pollution Prevention Division

BRAD Biopesticides Registration Action Document

CFR Code of Federal Regulations

CFU colony-forming unit(s)

cfu/kg colony-forming units per kilogram

cfu/mL colony-forming units per milliliter

cP centipoise

EDSP Endocrine Disruptor Screening Program

EP end-use product

EPA Environmental Protection Agency (the “Agency”)

FFDCA Federal Food, Drug, and Cosmetic Act

FIFRA Federal Insecticide, Fungicide, and Rodenticide Act

FQPA Food Quality Protection Act

FR Federal Register

g/mL gram per milliliter

LC50 median lethal concentration. A statistically derived concentration of a 

substance that can be expected to cause death in 50% of test animals. It is 
Bacillus amyloliquefaciens strain D747 Page 17 of 30

Biopesticides Registration Action Document

usually expressed as the weight of substance per weight or volume of water, 

air, or feed (e.g., mg/L, mg/kg, or ppm).

LD50 median lethal dose. A statistically derived single dose that can be expected 

to cause death in 50% of the test animals when administered by the route 

indicated (oral, dermal, or inhalation). It is expressed as a weight of 

substance per unit weight of animal (e.g., mg/kg).

mg/kg milligrams per kilogram

mg/L milligrams per liter

MP manufacturing-use product

mPa·s milliPascal-second, term used as the unit of dynamic viscosity.

MRID No. Master Record Identification Number

NIOSH National Institute for Occupational Safety and Health

NRRL Northern Regional Research Laboratory

OCSPP Office of Chemical Safety and Pollution Prevention

OPP Office of Pesticide Programs

PC Code Pesticide Chemical Code

PP Pesticide Petition

PPE personal protective equipment

ppm parts per million

TGAI technical grade of the active ingredient

U.S. United States
Bacillus amyloliquefaciens strain D747 Page 18 of 30

Biopesticides Registration Action Document

IX. BIBLIOGRAPHY

A. Studies Submitted to Support Pesticide Product Registrations

48165500 Certis U.S.A., LLC (2010) Submission of Product Chemistry and Toxicity Data in 

Support of the Application for Registration of CX-9032. Transmittal of 6 Studies.

48165501 Chen, C. (2010) Bacillus subtilis Var. amyloliquefaciens Strain D747 (CX-9032): 

Product Identity, Manufacturing Process, Discussion of Formation of Unintentional 

Ingredients: Analysis of Samples: Certification of Limits. Unpublished study

prepared by Certis USA, LLC. 120 p.

48165502 DeVane, W. (2010) Bacillus subtilis var. amyloliquefaciencs Strain D-747 End-Use 

Product: Color: Physical State: Odor: Stability to Normal and Elevated Temperatures, 

Metals, and Metal Ions: Storage Stability of the Liquid Concentrate: Miscibility: 

Corrosion Characteristics: pH: Viscosity: Bulk Density. Unpublished study prepared 

by Certis USA, LLC. 8 p.

48165503 Simpson, A. (2010) Bacillus subtilis var. amyloliquefaciens Strain D-747 End-Use 

Product: 830.6317 Storage Stability - Interim Study Report. Unpublished study

prepared by Certis USA, LLC. 6 p.

48165504 Dively, C. (2010) Bacillus subtilis var. amyloliquefaciens Strain D747: CX-9032: 

Acute Oral Toxicity: Acute Dermal Toxicity: Acute Inhalation Toxicity: 

Hypersentivity (Sic). Unpublished study prepared by Certis USA, LLC. 6 p.

48165505 Kuhn, J. (2010) Bacillus subtilis CX-9032: Acute Eye Irritation Study in Rabbits: 

Final Report. Project Number: 14046/10. Unpublished study prepared by

Stillmeadow, Inc. 17 p.

48165506 Kuhn, J. (2010) Bacillus subtilis CX-9032: Acute Dermal Irritation Study in Rabbits: 

Final Report. Project Number: 14047/10. Unpublished study prepared by

Stillmeadow, Inc. 11 p.

48165700 Certis U.S.A., L.L.C. (2010) Submission of Product Chemistry and Toxicity Data in 

Support of the Petition for Tolerance of Bacillus subtilis var. amyloliquefaciens and 

the Application for Registration of CX-9030. Transmittal of 17 Studies.

48165701 Chen, C. (2010) Bacillus subtilis var. amyloliquefaciens Strain D747: (CX-9030): 

Product Identity: Manufacturing Process: Discussion of Formation of Unintentional 

Ingredients: Analysis of Samples: Certification of Limits. Unpublished study

prepared by Certis USA, LLC. 145 p.

48165702 DeVane, W. (2010) Bacillus subtilis var. amyloliquefaciens Strain D-747 End-Use 

Product (ID Code CX-9030): Product Properties: Color: Physical State: Odor: 

Stability to Normal and Elavated Temperatures, Metals, and Metal Ions: Storage 

Stability of the TGAI: Miscibility: Corrosion Characteristics: pH: Viscosity: Bulk 

Density. Unpublished study prepared by Certis USA, LLC. 10 p.

48165703 Simpson, A. (2010) Bacillus subtilis var. amyloliquefaciens Strain D-747 End-Use 

Product (ID Code CX-9030): Microbial Pesticide Data Requirements: 830.6317 

Storage Stability. Unpublished study prepared by Certis USA, LLC. 6 p.

48165704 Onishi, Y. (2003) Bacillus subtilis var. amyloliquefaciens Strain D747: D747 Single 

Oral Administration Toxicity Study in Rats: Acute Oral Toxicity/Pathogenicity: Final 
Bacillus amyloliquefaciens strain D747 Page 19 of 30

Biopesticides Registration Action Document

Report. Project Number: B030504. Unpublished study prepared by Mitsubishi 

Chemical Safety Institute, Ltd. 25 p.

48165705 Onishi, Y. (2003) Bacillus subtilis var. amyloliquefaciens Strain D747: D747 Single 

Intravenous Administration Toxicity in Rats: Acute Injection Toxicity/Pathogenicity: 

Final Report. Project Number: B030506. Unpublished study prepared by Mitsubishi 

Chemical Safety Institute, Ltd. 29 p.

48165706 Onishi, Y. (2003) D747 Single Endotracheal Administration Toxicity Study in Rats: 

Acute Pulmonary Toxicity/Pathogenicity: Final Report. Project Number: B030505. 

Unpublished study prepared by Mitsubishi Chemical Safety Institute, Ltd. 29 p.

48165707 Kuhn, J. (2010) Acute Oral Toxicity Study (UDP) in Rats: Bacillus subtilis CX-9030: 

Final Report. Project Number: 13847/10. Unpublished study prepared by

Stillmeadow, Inc. 11 p.

48165708 Kuhn, J. (2010) Acute Dermal Toxicity Study in Rats: Bacillus subtilis CX-9030: 

Final Report. Project Number: 13848/10. Unpublished study prepared by

Stillmeadow, Inc. 12 p.

48165709 Doig, A. (2010) Acute Inhalation Toxicity Study in Rats: Bacillus subtilis Cx-9030: 

Final Report. Project Number: 13820/10. Unpublished study prepared by

Stillmeadow, Inc. 18 p.

48165710 Kuhn, J. (2010) Acute Eye Irritation Study in Rabbits: Bacillus subtilis CX-9030: 

Final Report. Project Number: 13821/10. Unpublished study prepared by

Stillmeadow, Inc. 16 p.

48165711 Kuhn, J. (2010) Acute Dermal Irritation Study in Rabbits: Bacillus subtilis CX-9030: 

Final Report. Project Number: 13822/10. Unpublished study prepared by

Stillmeadow, Inc. 11 p.

48165712 Hubbard, P.; Beavers, J. (2010) Bacillus subtilis CX-9025: An Acute Oral Toxicity

Study with the Northern Bobwhite. Project Number: 671/102. Unpublished study

prepared by Wildlife International, Ltd. 33 p.

48165713 Cockroft, R. (2004) Freshwater Fish Toxicity/Pathogenicity (Prolonged Toxicity to 

Microbial Pesticides to Fish) (Oncorhynchus mykiss): Bacillus subtilis var. 

amyloliquefaciens Strain D747: Final Report. Project Number: KCI/275/042551. 

Unpublished study prepared by Huntingdon Life Sciences, Ltd. 37 p.

48165714 Cockroft, R. (2010) (Prolonged Toxicity of Microbial Pesticides to Daphnia magna): 

Freshwater Invertebrate Toxicity/Pathogenicity: Bacillus subtilis var. 

amyloliquefaciens Strain D747: Final Report. Project Number: KCI/276/042552. 

Unpublished study prepared by Huntingdon Life Sciences, Ltd. 51 p.

48165715 Takagaki, M. (2004) Bacillus subtilis var. amyloliquefaciens Strain D747: NonTarget Plant Test: Final Report. Unpublished study prepared by Kumiai Chemical 

Industry Co. 13 p.

48165716 Dively, C. (2002) Bacillus subtilis var. amyloliquefaciens Strain D747: Non-Target 

Insect Testing: Final Report. Unpublished study prepared by Japan Plant Protection 

Assoc. Research Institute. 17 p.
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48165717 Dively, C. (2003) Bacillus subtilis var. amyloliquefaciens Strain D747: An Acute 

Dietary Pathogenicity and Toxicity Study with the Honey Bee (Apis mellifera): Final 

Report. Unpublished study prepared by Life Science Research Institute. 5 p.

48621501 Dively, C. (2011) Bacillus subtilis var. Amyloliquefaciens Strain D747: Avian Oral 

Toxicity/Pathogenicity (in Northern Bobwhites): Addendum to MRID 48165712. 

Unpublished study prepared by Certis USA, LLC. 10p.

48621502 Dimock, M. (2011) Bacillus subtilis var. amyloliquefaciens Strain D747: Non-Target 

Insect Testing: Addendum to MRID 48165716. Unpublished study prepared by Certis 

USA, LLC. 20p.

48621503 Dively, C. (2011) Bacillus subtilis var. amyloliquefaciens Strain D747: Acute Dietary

Pathogenicity and Toxicity Study with the Honey Bee (Apis mellifera): Addendum to 

MRID 48165717. Unpublished study prepared by Certis USA, LLC. 9p.

B. Environmental Protection Agency Risk Assessment Memoranda and Other Documents

U.S. EPA. 2004. Primer for Municipal Wastewater Treatment Systems. EPA 832-R-04-001. 

Available from http://www.epa.gov/npdes/pubs/primer.pdf.

U.S. EPA. 1996. Microbial Pesticide Test Guidelines – Background for Residue Analysis of 

Microbial Pest Control Agents (OPPTS 885.2000). Available from 

http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/series885.htm

U.S. EPA. 2010. Bacillus subtilis Case 6012. Final Registration Review Decision; Docket 

Number EPA-HQ-OPP-2007-1026

 

C. Other References

Benitez, LB, et. al. Antimicrobial factor from Bacillus amyloliquefaciens inhibits PaeniB. 

agent of American foulbrood. Arch. Microbiology August 20, 2010

Centers for Disease Control and Prevention. 2009. Drinking Water - Water Treatment. Available 

from http://www.cdc.gov/healthywater/drinking/public/water_treatment.html.

Dedj. S., et.al. Effectiveness of honeybees in delivering the biocontrol agent Bacillus subtilis to 

blueberry flowers to suppress mummy berry disease. Biol. Control 31:422-427.

European Food SafetyAuthority. 2010. Scientific opinion on the compatibility of Ecobiol 

(Bacillus amyloliquefaciens CECT 5940) for chickens for fattening with coccidiostats. EFSA 

Journal 8: 1918.

Logan, N.A., and P. de Vos. 2009. Genus I. Bacillus, pp. 21-128 In: P. de Vos, G.M. Garrity, D. 

Jones, N.R. Krieg, W. Ludwig, F.A. Rainey, K.H. Schleifer, and W. Whitman (Eds.) Bergey’s 

Manual of Systematic Bacteriology, Volume 3, 2nd Ed. Springer, New York.

Maccagnami, B., et. al. Apis mellifera and Osmia cornuta as carriers for the secondary spread of 

Bacillus subtilis on apple flowers. Biocontrol 54: 123-133.
Bacillus amyloliquefaciens strain D747 Page 21 of 30

Biopesticides Registration Action Document

McClintock, JT., Schaffer, CR., Sjoblad, RD., A comparative review of the mammalian toxicity

of Bacillus thuringensis-based pesticides. Pestic Sci 1995; 45: pp 95 – 105.

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

edition, 2007.

Priest, F.G., Goodfellow, M., Shute, L.A, and Berkeley, R.C.W. 1987. Bacillus 

amyloliquefaciens sp. nov., nom. rev. International Journal of Systematic Bacteriology, 37: 69-

71.

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

Wizna, R. Yose, H., Abbas, A., Dharma, I.P. Kompiang. 2009. Influence of dietary fermented 

tapioca by-products on the performance of broilers and ducklings. International Journal of 

Poultry Science 8: 902-904.

World Health Organization, Guidelines for Drinking-water Quality (2011) Fourth Edition.
Bacillus amyloliquefaciens strain D747 Page 22 of 30

Biopesticides Registration Action Document

APPENDIX A. MICROBIAL PESTICIDES DATA REQUIREMENTS 

(40 CFR PART 158 – SUBPART V)

TABLE 1. Product Analysis Data Requirements for the End-Use Product (EP), CX-9030

(40 CFR § 158.2120)

OCSPP Guideline 

Number

Data Requirement Results MRID No.

885.1100 Product Identity Submitted data fulfill the requirement for product 

identity. CX-9030 contains 25.0% by weight 

Bacillus amyloliquefaciens strain strain D747

(minimum of 5x 1010 CFU/g)

481655-01

CSF dated

10/18/2011

885.1200 Manufacturing Process Submitted data fulfill the requirement for 

manufacturing process.

481655-01

Not applicable Deposition of a Sample in a 

Nationally Recognized 

Culture Collection

Submitted data fulfill the requirement for

deposition. Culture on deposit under Accession 

Number NRRL B-50405.

481655-01

885.1300 Discussion of Formation of 

Unintentional Ingredients

Submitted data fulfill the requirement for 

discussion of formation of unintentional 

ingredients.

481655-01

885.1400 Analysis of Samples Submitted date fulfill the requirement for analysis 

of samples. 

481655-01

885.1500 Certification of Limits Limits listed on the confidential statement of 

formula are adequate/acceptable

CSF dated

10/18/2011

Additional Studies

830.1800 Enforcement Analytical 

Method

Submitted data fulfill the requirement for an 

enforcement analytical method

481655-01

TABLE 2. Product Analysis Data Requirements for the End-Use Product (EP), CX-9032

(40 CFR § 158.2120)

OCSPP Guideline 

Number

Data Requirement Results MRID No.

885.1100 Product Identity Submitted data fulfill the requirement for product 

identity. CX-9030 contains 98.95% by weight 

Bacillus amyloliquefaciens strain strain D747

(minimum of 1x 1010 CFU/g)

481655-01

CSF dated

10/18/2011

885.1200 Manufacturing Process Submitted data fulfill the requirement for 

manufacturing process.

481657-01

481655-01

Not applicable Deposition of a Sample in a 

Nationally Recognized 

Culture Collection

Submitted data fulfill the requirement for

deposition. Culture on deposit under Accession 

Number NRRL B-50405.

481657-01

481655-01

885.1300 Discussion of Formation of 

Unintentional Ingredients

Submitted data fulfill the requirement for 

discussion of formation of unintentional 

ingredients.

481657-01

481655-01

885.1400 Analysis of Samples Submitted date fulfill the requirement for analysis 

of samples. 

481655-01

481657-01

885.1500 Certification of Limits Limits listed on the confidential statement of 

formula are adequate/acceptable

CSF dated 

10/18/2011

Additional Studies

830.1800 Enforcement Analytical 

Method

Submitted data fulfill the requirement for an 

enforcement analytical method

481655-01
Bacillus amyloliquefaciens strain D747 Page 23 of 30

Biopesticides Registration Action Document

TABLE 3. Physical and Chemical Characteristics for the Technical Grade of the Active Ingredient (TGAI) Bacillus.

amyloliquefaciens strain strain D747 / CX-9030 (EP). (40 CFR § 158.2120)

OCSPP 

Guideline 

Number 

Data Requirement

Results MRID No.

TGAI CX-9030 (EP)

830.6302 Color Beige Not Applicable 481657-02 

830.6303 Physical State Fine powder Not applicable 481657-02

830.6304 Odor Yeast odor Not applicable 481657-02

830.6313 Stability to Normal and Elevated

Temperatures, Metals,

and Metal Ions

Spores inactivated at 

54oC 

Not applicable 481657-02

830.6314 Oxidation/Reduction:

Chemical Incompatibility

Not applicable, the product does not contain oxidizing 

or reducing agents

481657-02

830.6315 Flammability Not applicable, the product does not contain flammable 

ingredients

481657-02

830.6316 Explodability Not applicable, the product does not contain explosive 

ingredients

481657-02

830.6317 Storage Stability Stable up to one year at 25oC without loss of viability

.

481657-02

481657-03

830.6319 Miscibility Not applicable, the product is not an emulsifiable liquid.

(refer to test note #2 of 40 CFR § 158.2120(d)).

481657-02

830.6320 Corrosion Characteristics Not applicable, the product is a powder. 481657-02

830.6321e Dielectric Breakdown Voltage Not applicable, the product is not for use around 

electrical equipment

481657-02

830.7000 pH 6.5 – 7.0 (1% w/w) 7.5- 8.0 481657-02

830.7100 Viscosity Not applicable, the 

product is a powder.

Not Applicable.CX-9030 is 

not a liquid. 

481657-02

830.7300 Density/Relative Density/Bulk 

Density (Specific Gravity)

0.307- 0.375 g/ml 0.60-.0.78 g/ cm3481657-02
Bacillus amyloliquefaciens strain D747 Page 24 of 30

Biopesticides Registration Action Document

TABLE 4. Physical and Chemical Characteristics for CX-9032 (EP). (40 CFR § 158.2120)

OCSPP 

Guideline 

Number 

Data Requirement

Results MRID No.

TGAI CX-9030 (EP)

830.6302 Color Beige Light brown 481655-02 

830.6303 Physical State Fine powder Liquid 481655-02

830.6304 Odor Yeast odor Yeast odor 481655-02

830.6313 Stability to Normal

& Elevated

Temperatures, Metals,

& Metal Ions

Spores inactivated at 54oC.. 481655-02

830.6317 Storage Stability Stable up to one year at 

25oC without loss of 

viability

Stable up to 78 days at 25oC 

without loss of viability. As a 

term of the registration, EPA 

will require the results of a 

one-year storage stability and 

corrosion characteristics 

study be submitted within one 

year.

481655-02

830.6319 Miscibility Not applicable; product is not an emulsifiable liquid.

(refer to test note #2 of 40 CFR § 158.2120(d)).

481655-02

830.6320 Corrosion Characteristics Not applicable; product is a 

powder.

None evident to polyethylene 

packaging after 78 days. As a 

term of the CX-9032 (EP)

registration, EPA will require 

submission of the results of a 

one-year storage stability & 

corrosion characteristics 

within one year.

481655-02

830.7000 pH 6.5 – 7.0 (1% w/w) 4.2 - 4.3 (1% w/w). 481655-02

830.7100 Viscosity Not applicable, the product 

is a powder.

4.6 - 16.0 milliPascal-second

(mPa s) at 25oC 

 

481655-02

830.7300 Density/Relative 

Density/Bulk Density 

(Specific Gravity)

0.307- 0.375 g/ml 1.02 – 1.03 g/mL 481655-02
Bacillus amyloliquefaciens strain D747 Page 25 of 30

Biopesticides Registration Action Document

TABLE 5. Toxicology Data Requirements for the Technical Grade of the Active Ingredient (TGAI) (40 CFR § 158.2140)

OCSPP Guideline 

Number

Data Requirement Results MRID No.

TGAI

Tier I

885.3050 Acute Oral 

Toxicity/Pathogenicity

No evidence of infectivity, pathogenicity or toxicity was found 

from oral administration of 1.0x108 CFU Bacillus 

amyloliquefaciens strain D747 to rats. Clearance from fecal 

material occurred by day 14 and no viable organisms were 

recovered from blood or any other organ or tissue.

Classification: Acceptable

481657-04

885.3150 Acute Pulmonary 

Toxicity/Pathogenicity

No evidence of infectivity, pathogenicity or toxicity was found 

from intratracheal administration of 1.0x107spores B.

amyloliquefaciens strain D747 to rats.

Classification: Acceptable

481657-06

885.3200 Acute Injection 

Toxicity/Pathogenicity 

(Intravenous)

Not toxic, infective, and/or pathogenic to rats when dosed

intravenously at 1.0x107spores per animal.

Classification: Acceptable

481657-05

885.3400 Hypersensitivity 

Incidents

No hypersensitivity incidents, including immediate-type or 

delayed-type reactions of humans and domestic animals that 

occurred during research, development, or testing of the 

TGAI/MP, were reported. Future hypersensitivity incidents must 

be reported (For reporting format: OCSPP Guideline 885.3400). 

479450-23

885.3500 Cell Culture Not required. B. amyloliquefaciens strain D747 is not a virus

(Test note #4 of 40 CFR § 158.2140(d)).

Tiers II and III

Not required for Bacillus amyloliquefaciens strain D747 based on the lack of acute toxicity/pathogenicity in the Tier I studies.
Bacillus amyloliquefaciens strain D747 Page 26 of 30

Biopesticides Registration Action Document

TABLE 6. Toxicology Data Requirements for the End-Use Product (EP), CX-9030

(40 CFR § 158.2140)

OCSPP Guideline Number Data Requirement Results MRID No.

885.3400 Hypersensitivity Incidents No hypersensitivity incidents, including 

immediate-type or delayed-type 

reactions of humans and domestic 

animals that occurred during research, 

development, or testing of the EP, were 

reported by the applicant. Any future 

hypersensitivity incidents must be 

reported per OCSPP Guideline 

885.3400.

870.1100 Acute Oral Toxicity Oral LD50 > 5000 mg/Kg 

Classification: Acceptable

Toxicity Category IV

481657-07

870.1200 Acute Dermal Toxicity Dermal LD50 > 5050 mg/Kg 

Classification: Acceptable

TOXICITY CATEGORY IV

481657-08

870.1300 Acute Inhalation Toxicity Inhalation LC50 > 2.18 mg/L

Classification: Acceptable

TOXICITY CATEGORY IV

481657-09

870.2400 Acute Eye Irritation The maximum average irritation score of 

18.3 obtained 1 hour after treatment 

declined to 17.33 after 24 hours, 2 after 

48 hours and 0 after 72 hours following 

ocular administration of 0.1 mL B.

amyloliquefaciens strain D747 to New

Zealand White rabbits in a 72 hour 

observation period.

Classification: Acceptable

TOXICITY CATEGORY III

481657-10

870.2500 Primary Dermal Irritation No evidence of irritation was found from 

dermal administration of 500 mg B.

amyloliquefaciens strain D747 CX-9030 

to rabbits during the 4 hour exposure and 

72 observation periods. The dermal 

irritation score for B. amyloliquefaciens 

strain D747 CX-9030 was 0.00.

Classification: Acceptable

TOXICITY CATEGORY IV

481657-11
Bacillus amyloliquefaciens strain D747 Page 27 of 30

Biopesticides Registration Action Document

TABLE 7. Toxicology Data Requirements for the End-Use Product (EP), CX-9032

(40 CFR § 158.2140)

OCSPP Guideline Number Data Requirement Results MRID No.

885.3400 Hypersensitivity Incidents No hypersensitivity incidents including 

immediate-type or delayed-type reactions 

of humans and domestic animals that 

occurred during research, development, 

or testing of the EP were observed or 

reported. All hypersensitivity incidents 

must be reported per OCSPP Guideline 

885.3400.

870.1100 Acute Oral Toxicity Waiver request submitted. Requirement 

satisfied by submitted MP data. Oral 

exposure to the MP showed no adverse 

effects including infectivity, 

pathogenicity and toxicity up to the limit 

dose. Inerts are exempt from tolerance.

No additional oral toxicity is expected 

from inerts.

Classification: Acceptable

481655-04

870.1200 Acute Dermal Toxicity Waiver request submitted. Requirement 

adequately addressed by CX-9030 EP 

data as well as, dermal irritation testing 

on CX-9032-EP. Dermal toxicity and 

irritation testing for the CX-9030 EP and 

dermal irritation data on CX-9032 EP

showed no adverse effects up to the limit 

doses. No additional dermal toxicity is 

expected from inerts.

Classification: Acceptable

481655-04

481655-06 

481655-11

481657-08

870.1300 Acute Inhalation Toxicity Waiver request submitted. Pulmonary 

exposure to the MP showed no adverse 

effects, including infectivity, 

pathogenicity or toxicity though slight 

toxicity lasting 2 days from a 4-hour 

aerosol inhalation administration of 2.18 

mg/L, where inert ingredients were also 

present, was noted in a CX-9030 EP 

study. No additional toxicity is expected 

from this EP’s inert ingredients. 

Classification: Acceptable

481655-04

481657-06

481657-09

870.2400 Acute Eye Irritation The maximum average irritation score of 

0.667 at 1 hour after treatment declined to 

0 after 24 hours following ocular 

administration of 0.1 mL 

B. amyloliquefaciens strain D747 CX9032 to New Zealand White rabbits (72-

hour observation period).

Classification: Acceptable

TOXICITY CATEGORY IV

481655-05
Bacillus amyloliquefaciens strain D747 Page 28 of 30

Biopesticides Registration Action Document

TABLE 7. Toxicology Data Requirements for the End-Use Product (EP), CX-9032

(40 CFR § 158.2140)

OCSPP Guideline Number Data Requirement Results MRID No.

870.2500 Primary Dermal Irritation No irritation occurred from dermal 

administration of 0.5 mL undiluted B.

amyloliquefaciens strain D747 CX-9032 

to shaved skin of rabbits during the 4 

hour exposure and 72 observation 

periods. The dermal irritation score for B.

amyloliquefaciens strain D747 CX-9032 

was 0.00.

Classification: Acceptable

TOXICITY CATEGORY IV

481655-06

TABLE 8. Nontarget Organism Toxicity and Environmental Fate Data Requirements for the Technical Grade of the 

Active Ingredient (TGAI), Bacillus amyloliquefaciens strain D747 (40 CFR § 158.2150)

OCSPP Guideline 

Number

Data Requirement Results MRID No.

Tier I

885.4050 Avian Oral Toxicity A study showed that B. amyloliquefaciens D747 

is not toxic to birds at 8.9 x 109spores/bird.

Classification: Supplemental

Scientific rationale is sufficient to conclude that 

B. amyloliquefaciens D747 is not expected to 

pose a hazard to birds.

Classification: Acceptable

48165712

48621501

885.4100 Avian Inhalation 

Toxicity/Pathogenicity

Not required. B. amyloliquefaciens D747 is not 

considered to be pathogenic to birds

885.4150 Wild Mammal 

Toxicity/Pathogenicity

Tests required by 40 CFR § 158.2140 are 

adequate/ appropriate for assessment of hazards 

to wild mammals. B. amyloliquefaciens D747 

was not infective, toxic or pathogenic to 

laboratory rats 1.0 x 108 CFU/animal 

48165704

885.4200 Freshwater Fish 

Toxicity/Pathogenicity

A 30-day study shows that the LC50 to rainbow

trout is 8 x 1010 CFU/L. The NOEC for sublethal 

effects is 1.44 x 1010 CFU/L.

Classification: Acceptable

48165713

885.4240 Freshwater Invertebrate 

Toxicity/Pathogenicity

A 21-day study shows that the EC50 to Daphnia 

magna based on mortality is 3.7 x 1010 CFU/L. 

The NOEC for sublethal effects is 2.84 x 108

CFU/L.

Classification: Acceptable

48165714

885.4280 Estuarine/Marine Fish and 

Invertebrate Testing

Not required. B. amyloliquefaciens D747 is not 

to be applied directly to water and is not expected 

to reach estuarine or marine environments in 

significant quantities.

885.4300 Nontarget Plant Testing A study submitted was determined to be 

unacceptable; however, testing is not required 

because. B. amyloliquefaciens is not related to 

known plant pathogens

48165715
Bacillus amyloliquefaciens strain D747 Page 29 of 30

Biopesticides Registration Action Document

TABLE 8. Nontarget Organism Toxicity and Environmental Fate Data Requirements for the Technical Grade of the 

Active Ingredient (TGAI), Bacillus amyloliquefaciens strain D747 (40 CFR § 158.2150)

OCSPP Guideline 

Number

Data Requirement Results MRID No.

885.4340 Nontarget Insect Testing Studies with three species of arthropods were 

determined to be unacceptable. 

Additional scientific rationale was submitted to 

show that B. amyloliquefaciens is not expected to 

be toxic or pathogenic to nontarget insects.

Classification: Acceptable

48165716

48621502

885.4380 Honey Bee Testing Two studies showed that B. amyloliquefaciens

D747 is not toxic to honey bees. 

Classification: Supplemental

Additional rationale was sufficient to show that 

pathogenicity to honey bees is not expected as a 

result of exposure to B. amyloliquefaciens D747. 

Classification: Acceptable

48165717

48621503

Tiers II, III, and IV

Not required for Bacillus amyloliquefaciens strain D747 based on the current uses and application methods.
Bacillus amyloliquefaciens strain D747 Page 30 of 30

Biopesticides Registration Action Document

APPENDIX B. PESTICIDE PRODUCTS

TABLE 9. Table Title?

EPA 

Registration 

Number

Registration 

Name

Percentage 

Active 

Ingredient

Formulation 

Type

Use Site(s) Method(s) 

of 

Application

Application 

Rate

Target Pest

70051-107 CX-9032 98.35% End Use –

Aqueous 

Suspension

Various 

agricultural 

and 

greenhouse 

crops (e.g., 

vegetables, 

tree fruits, 

berries, 

grapes and 

tropical fruit, 

tree nuts, 

herbs and 

spices, 

coffee, 

tobacco, 

hops, 

forestry 

seedlings 

ornamentals, 

and turf) 

Tractor 

mounted 

boom, 

airblast, 

hose-end, 

backpack 

and other 

pressurized 

sprayers; 

foggers or 

mist 

blowers; 

water wheel 

and other 

drench 

applicators; 

soil 

injection; 

aerial; and 

chemigation 

with drip or 

sprinkler 

irrigation 

and cutting 

or root dip

Rate listed on 

label varies 

depending on 

application

method from 

O.5 pints 

/acre to 6 

quarts/acre 

Various fungal and 

bacterial pests listed 

on the label 

including:

Alternaria,

Botrytis cinerea 

Didymella bryoniae

Phoma 

cucurbitacearum

Erisphe, 

Fusarium, 

Macrophomina 

phaseoli 

Monosporascus 

cannonballus

Peronospora , 

Phytophthora 

Pseudomonas 

syringae pv. tomato 

Pseudoperonospora

spp.

Puccinia spp.

Pythium,

Rhizoctonia, 

Sphaerotheca spp.

Verticillium, spp.

Xanthomonas spp.

70051-108

CX-9030 25.0% Water 

Dispersible 

Granule 

Various 

agricultural

and 

greenhouse 

crops (e.g., 

vegetables, 

ornamentals, 

and turf)

Tractor 

mounted 

boom, 

airblast, 

hose-end, 

backpack 

and other 

pressurized 

sprayers; 

foggers or 

mist 

blowers; 

water wheel 

and other 

drench 

applicators; 

shank or 

other soil 

injection 

equipment; 

aerial; and 

chemigation 

with drip or 

sprinkler 

irrigation 

and cutting 

or root dip 

0.25 –3 

pounds per 

acre

Various fungal and 

bacterial pests listed 

on the label 

including:

Alternaria,

Botrytis cinerea 

Didymella bryoniae

Phoma 

cucurbitacearum

Erisphe 

Fusarium, 

Macrophomina 

phaseoli 

Monosporascus 

cannonballus

Peronospora, 

Phytophthora 

Pseudomonas 

syringae pv. tomato 

Pseudoperonospora

spp.

Puccinia spp.

Pythium,

Rhizoctonia, 

Sphaerotheca spp.

Verticillium, spp.,

Xanthomonas spp.