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BIOPESTICIDES REGISTRATION ACTION DOCUMENT
Bacillus amyloliquefaciens strain D747
Pesticide Chemical (PC) Code: 016482
U.S. Environmental Protection Agency
Office of Pesticide Programs
Biopesticides and Pollution Prevention Division
December 8, 2011 version
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Bacillus amyloliquefaciens strain D747 Page 2 of 30
Biopesticides Registration Action Document
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
John L. Kough, Ph.D.
Gail Tomimatsu, Ph.D.
Shannon Borges
Regulations
Product Analysis, Human Health
Product Analysis, Human Health
Environmental Effects
Environmental Effects
Sheryl K. Reilly, Ph.D.
Susanne Cerrelli
Chief, Microbial Pesticides Branch
Regulatory Action Leader
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I. EXECUTIVE SUMMARY
Certis USA, LLC, ("Certis" or "applicant) submitted data to support the registration of Bacillus
amyloliquefaciens strain D747 is the active ingredient in two end-use pesticide products, CX-
9030 (EPA File Symbol 70051-107) and CX-9032 (EPA File Symbol 70051-108). 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 registration of the two aforementioned
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 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 labels, and in accordance with
good agricultural practices. Additional mammalian and nontarget organism toxicity data are not
required for the registered 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,
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there is a reasonable certainty that no harm will result to the United States population, including
infants and children, from aggregate exposure to residues of B. amyloliquefaciens strain D747.
No dietary risks (including drinking water) are expected from use of B. amyloliquefaciens strain
D747 as an active ingredient in pesticide products. The two end-use pesticide products, CX-
9030 and CX-9032 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 pesticide products
containing B. amyloliquefaciens strain D747, a new active ingredient, were subject to a 30-day
comment period. . During this comment period, no comments were received on EPA's
preliminary decision to register the two end-use pesticide products containing B.
amyloliquefaciens strain D747, CX-9030 and CX-9032. Therefore, EPA maintained that, based
upon the risk assessment and information submitted in support of registration of such pesticide
products, it was in the best interest of the public and the environment to issue the end-use
pesticide product registrations for B. amyloliquefaciens strain D747, CX-9030 and CX-9032. 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.
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
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conducted in accordance with test guidelines or comes from other sources, such as peer-
reviewed, scientific literature. The active ingredient or particular product is classified into
Toxicity Category I, II, III, or IV, where Toxicity Category I indicates the highest toxicity, and
Toxicity Category IV indicates the lowest toxicity (see 40 CFR § 156.62).
A. Product Analysis Assessment (40 CFR § 158.2120)
All product analysis data requirements for B. amyloliquefaciens strain D747 have been fulfilled
for the current 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. amyloliqaefaciens strain D747 have been
fulfilled for the current 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 (MRlDNo. 481657-04):
B. amyloliquefaciens strain D747 was administered once orally to 14 rats of both sexes (5-weeks
old) at a single dosage of 10 colony-forming units (CFU) per animals. No deaths occurred, and
no abnormalities (clinical signs, body weight) were observed, during the study or at necropsy.
3 5*
The test microbe was detected at 10 - 10 CFU/g in feces 1 day after administration of the test
material, but was not detected on dayl4. The examination for internal persistence did not detect
the test microbe in any organs or tissues, such as the kidney, brain, liver, lung, spleen, stomach,
small intestine (duodenum), large intestine (cecum), mesenteric lymph nodes, or blood,
throughout the experimental period. Fecal clearance occurred by day 14, and no viable
organisms were recovered from blood or other organs or tissues. The results of this acceptable
study demonstrated that B. amyloliquefaciens strain D747 was not infective, pathogenic, or toxic
to rats when orally dosed with 1.0x10 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
2 3
ended. The presence of the test microbes decreased in the lungs by approximately 10-10 CFU/g
from day 0 to day 60. The test microbes were detected in the bronchial lymph nodes on day 3.
Since many test microbes remained in the trachea on day 3, their presence in the bronchial lymph
nodes on day 3 was attributed to transfer by macrophages from the respiratory tract to the lymph
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nodes. This conclusion was supported by the observation that a particle larger than 1 |j,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
n
strain D747 was not toxic or pathogenic to rats when dosed intratracheally at 1.0 xlO 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
n
strain D747 at a dosage of 10 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 dayl4; 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,
n
infective, or pathogenic to rats when injected intravenously with 1.0 xlO CFU/ animal.
Hypersensitivity Incidents (OCSPP Guideline 885.3400; MRID No. 481655-05): No
hypersensitivity incidents in humans have been reported, and none occurred during research,
development, or testing of B. amyloliquefaciens strain D747. Should hypersensitivity or other
adverse incidents in humans occur in the future, Certis must report them to EPA, in accordance
with FIFRA section 6(a)(2).
Cell Culture (OCSPP Guideline 885.3500): This study was not required because B.
amyloliquefaciens strain D747 is not a virus (refer to test note #4 of 40 CFR § 158.2140(d)).
b. Acute Toxicity and Subchronic Toxicity/Pathogenicity — Tier II;
Reproductive Fertility Effects, Carcinogenicity, Immunotoxicity, and
Infectivity/Pathogenicity Analysis — Tier III
Tier II and Tier III studies were not required for B. amyloliquefaciens strain D747 based on the
lack of acute toxicity/pathogenicity in the Tier I studies.
c. Endocrine Disruptors
As required under FFDCA section 408(p), EPA has developed the Endocrine Disruptor
Screening Program (EDSP) to determine whether certain substances (including pesticide active
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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:
htty://www. eya. gov/endo/.
2. Federal Food, Drug, and Cosmetic Act (FFDCA) Considerations
Section 408(c)(2)(A)(i) of FFDCA allows EPA to establish an exemption from the requirement
for a tolerance (the legal limit for a pesticide chemical residue in or on a food) only if EPA
determines that the exemption is "safe." Section 408(c)(2)(A)(ii) of FFDCA defines "safe" to
mean that "there is a reasonable certainty that no harm will result from aggregate exposure to the
pesticide chemical residue, including all anticipated dietary exposures and all other exposures for
which there is reliable information." This includes exposure through drinking water and in
residential settings but does not include occupational exposure. Pursuant to section 408(c)(2)(B)
of FFDCA, in establishing or maintaining in effect an exemption from the requirement of a
tolerance, EPA must take into account the factors set forth in section 408(b)(2)(C) of FFDCA,
which require EPA to give special consideration to exposure of infants and children to the
pesticide chemical residue in establishing a tolerance and to "ensure that there is a reasonable
certainty that no harm will result to infants and children from aggregate exposure to the pesticide
chemical residue..." Additionally, section 408(b)(2)(D) of FFDCA requires that EPA consider
"available information concerning the cumulative effects of [a particular pesticide's] . . . residues
and other substances that have a common mechanism of toxicity."
EPA performs a number of analyses to determine the risks from aggregate exposure to pesticide
residues. First, EPA determines the toxicity of pesticides. Second, EPA examines exposure to the
pesticide through food, drinking water, and through other exposures that occur as a result of
pesticide use in residential settings.
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Biopesticides Registration Action Document
Consistent with section 408(b)(2)(D) of FFDCA, EPA has reviewed the available scientific data
and other relevant information, and considered its validity, completeness, and reliability and the
relationship of this information to human risk. EPA has also considered available information
concerning the variability of the sensitivities of major identifiable subgroups of consumers,
including infants and children. All of the data requirements have been fulfilled, as described
previously, and are presented in Table 5 in Appendix A. The following summarizes the results of
EPA's dietary risk assessment for B. amyloliquefaciens strain D747.
a. Aggregate Exposure
In examining aggregate exposure, section 408 of FFDCA directs EPA to consider
available information concerning exposures from the pesticide residue in food and all other non-
occupational exposures, including drinking water from ground or surface water, and through
pesticide use in residential and other indoor uses.
Food Exposure and Risk Characterization. Bacillus species, including B. amyloliquefaciens, are
commonly found in the soil in agricultural settings, and are present on fresh produce of all kinds
with no known adverse effects. The Manual of Clinical Microbiology (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)(l)(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)(l)(a), above, and Table 5 in Appendix A).
Non-occuyational Residential Risk Characterization: The use sites for these products include
residential garden sites and agricultural sites. As stated previously, B. amyloliquefaciens is
naturally present in soil, and based on the data and other information submitted to satisfy the data
requirements for registration of the manufacturing-use and end-use pesticide products containing
the active ingredient, B. amyloliquefaciens strain D747, no toxicity, infectivity, pathogenicity or
other adverse effects from non-occupational exposure are expected (see section IV(B)(l)(a),
above, and Table 5 in Appendix A).
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b. Cumulative Effects from Substances with a Common Mechanism of Toxicity
Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to establish, modify,
or revoke a tolerance exemption, EPA consider "... available information concerning the
cumulative effects of [a particular pesticide's] ... residues and other substances that have a
common mechanism of toxicity."
EPA has found that B. amyloliquefaciens strain D747 does not share a common mechanism of
toxicity with any other microorganism, pesticidal or toxic substance. EPA concludes that there
are no cumulative effects associated with B. amyloliquefaciens strain D747 that need to be
considered. For information regarding how EPA determines common mechanisms of toxicity
and evaluates cumulative effects, see EPA's website at:
http://www. epa.gov pesticides cumulative.
c. Determination of Safety for the United States (U.S.) Population, Infants and
Children
FFDCA section 408(b)(2)(C) provides that EPA shall assess the available information about
consumption patterns among infants and children, special susceptibility of infants and children to
pesticide chemical residues, and the cumulative effects on infants and children of the residues
and other substances with a common mechanism of toxicity. In addition, FFDCA section
408(b)(2)(C) provides that EPA shall apply an additional tenfold (10X) margin of safety for
infants and children in the case of threshold effects to account for prenatal and postnatal toxicity
and the completeness of the database on toxicity and exposure, unless EPA determines that a
different margin of safety will be safe for infants and children. This additional margin of safety is
commonly referred to as the Food Quality Protection Act Safety Factor. In applying this
provision, EPA either retains the default value of 10X or uses a different additional safety factor
when reliable data available to EPA support the choice of a different factor.
EPA concludes that, based upon the results of the toxicity data and other information considered
and described in this document, there is a reasonable certainty that no harm will result to the U.S.
population, including infants and children, from aggregate exposure to the residues of B.
amyloliquefaciens strain D747. Such exposure includes all anticipated dietary and other
exposures for which there is reliable information. With no threshold effects of concern, an
additional margin of safety is not required for infants and children.
3. Occupational Exposure and Risk Characterization
Occupational exposure to B. amyloliquefaciens strain D747 is not expected to undue risks to
pesticide handlers (mixer/loader/applicators), but EPA requires appropriate personal protective
equipment and precautionary statements to mitigate any potential risks (e.g., respiratory
allergenicity) to pesticide handlers from prolonged or repeated exposures. Handlers applying B.
amyloliquefaciens strain D747 end-use products in agricultural settings must wear a long-sleeved
shirt, long pants, socks, shoes, waterproof gloves, and a dust/mist filtering respirator meeting
NIOSH standards of at least N-95, R-95, or P-95.
4. Human Health Risk Characterization
EPA considered human exposure to B. amyloliquefaciens strain D747 in light of the registration
standards of FIFRA and the relevant FFDCA safety factors for allowable pesticide residues in
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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
pesticide products containing B. amyloliquefaciens strain D747 as the active ingredient are used
in accordance with EPA-approved 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 EPA-
approved use sites. EPA performed an environmental risk assessment, and has determined that
the use of B. amyloliquefaciens strain D747 is 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, 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 maximum application rates from the labels of
registered products 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 fOCSPP Guideline 885.4050) and Mammals fOCSPP 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 109 spores/bird in Northern bobwhite (Colinus virginianus\
MRID 48165712). Additionally, B. amyloliquefaciens is intentionally included in some
domestic avian food diets as a nutritional additive (e.g., European Food Safety Authority, 2010;
Wizna et al., 2009), and it is not known to be pathogenic to animals (Logan and de Vos, 2009).
An extensive literature search in several databases returned no reports of toxicity or
pathogenicity of B. amyloliquefaciens in birds. Based on these lines of evidence, B.
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Bacillus amyloliquefaciens strain D747
Biopesticides Registration Action Document
Page 13 of 30
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
o
D747 is not toxic, infective, or pathogenic at the maximum hazard dose of 1.0 x 10 CFU/animal.
Therefore, adverse effects to wild mammals are also not expected as a result of the applications
of B. amyloliquefaciens strain D747 in accordance with label instructions.
Nontarset Insects (OCSPP Guideline 885.4340) and Honey Bees (OCSPP Guideline 885.4380) :
Studies with Orius stricollis, Crysoperla carnea, and Phytoseiuluspersimilis 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 when applied in
accordance with label instructions..
Nontar set 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 t applications made in
accordance with label instructions.
b. Aquatic Animals and Plants
B. amyloliquefaciens strain D747 is not intended to be applied directly to water, but some of the
applied product may reach aquatic habitats through runoff or spray drift. Spray drift at
application is the primary mechanism by which the pesticide is expected to reach water. A spray
drift analysis was included in the aquatic risk assessment to determine exposure, and further
detail is provided in the environmental risk assessment for B. amyloliquefaciens strain D747.
Freshwater Fish (OCSPP 885.4200) Guideline and Invertebrates (OCSPP Guideline 885.4240):
In a 30-day study with rainbow trout (Oncorhynchus mykiss), the LC50 for B. amyloliquefaciens
strain D747 was 8.1 x 1010 CFU/L, and the NOEC based on sub-lethal effects was 1.44 x 1010
CFU/L. If the maximum broadcast application rate (4 fluid ounces/1000 ft2 or 2.55 x 106
CFU/cm2) is applied directly to a 1-ha body of water 15 cm deep (the EPA Standard Wetland),
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Bacillus amyloliquefaciens strain D747
Biopesticides Registration Action Document
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o
the resulting concentration would be 1.7 x 10 CFU/L. This would be the maximum possible
aquatic concentration at this application rate, but this direct application to water is not on the EP
labels. 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 applications of B. amyloliquefaciens strain D747 as
presented on EPA- approved labels 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 applications of B. amyloliquefaciens
strain D747 D747 as presented on EPA- approved labels 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 to support uses and application methods that are presented on EPA-
approved labels 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 applications made in accordance with EPA-
approved labels, 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 approved uses of B. amyloliquefaciens strain D747.
V. ENVIRONMENTAL JUSTICE
EPA seeks to achieve environmental justice—the fair treatment and meaningful involvement of
all people regardless of race, color, national origin, or income—with respect to the development,
implementation, and enforcement of environmental laws, regulations, and policies. Fair
treatment means that no group of people, including racial, ethnic, or socioeconomic groups,
should bear a disproportionate share of the negative environmental consequences resulting from
industrial, municipal, and commercial operations or the execution of federal, state, local, and
tribal environmental programs and policies. Meaningful involvement means that (1) potentially
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Biopesticides Registration Action Document
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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.
EPA sought 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. No public comments were
received on this particular matter.
For additional information regarding environmental justice issues, please visit EPA's web site at
http: www.epa.isov compliance environmental justice index.html.
VI. RISK MANAGEMENT DECISION
Section 3(c)(5) of FIFRA permits for the registration of a pesticide provided that all the
following determinations are made:
(1) Its composition is such as to warrant the proposed claims for it;
(2) Its labeling and other material required to be submitted comply with the
requirements of FIFRA;
(3) It will perform its intended function without unreasonable adverse effects on
the environment; AND
(4) When used in accordance with widespread and commonly recognized
practice, it will not generally cause unreasonable adverse effects on the
environment.
To satisfy criterion 1, the B. amyloliquefaciens strain D747 pesticide products have well-known
properties. EPA has no knowledge that would contradict the claims made for these products, the
CX-9030 and CX-9032 EP labels, and we have concluded that such products are not expected to
cause unreasonable adverse effects on the environment when used according to the label
instructions. Criterion 2 is satisfied by the current product labels, as well as the data and
information presented in this document. It is believed that the B. amyloliquefaciens strain D747
pesticide products will not cause any unreasonable adverse effects on the environment, and CX-
9030 and CX-9032 (end-use pesticide product), in particular, are likely to provide protection
against fungal and bacterial pests as claimed, satisfying criterion 3. Criterion 4 is satisfied in that
the B. amyloliquefaciens strain D747 pesticide products are not expected to cause unreasonable
adverse effects when used according to label instructions. Therefore the end-use products, CX-
9030 and CX-9032, containing B. amyloliquefaciens strain D747 as a new active ingredient, are
eligible for registration under FIFRA section 3(c)(5) for the labeled uses. Should uses that are
more extensive be proposed in the future (e.g., aquatic uses), EPA will likely require that
additional data be submitted.
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Bacillus amyloliquefaciens strain D747 Page 16 of 30
Biopesticides Registration Action Document
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
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Biopesticides Registration Action Document
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LD
50
mg/kg
mg/L
MP
mPas
MRID No.
NIOSH
NRRL
OCSPP
OPP
PC Code
PP
PPE
ppm
TGAI
U.S.
usually expressed as the weight of substance per weight or volume of water,
air, or feed (e.g., mg/L, mg/kg, or ppm).
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).
milligrams per kilogram
milligrams per liter
manufacturing-use product
milliPascal-second, term used as the unit of dynamic viscosity.
Master Record Identification Number
National Institute for Occupational Safety and Health
Northern Regional Research Laboratory
Office of Chemical Safety and Pollution Prevention
Office of Pesticide Programs
Pesticide Chemical Code
Pesticide Petition
personal protective equipment
parts per million
technical grade of the active ingredient
United States
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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. lip.
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
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Bacillus amyloliquefaciens strain D747
Biopesticides Registration Action Document
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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. lip.
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. lip.
48165712 Hubbard, P.; Beavers, J. (2010) Bacillus subtilis CX-9025: An Acute Oral Toxicity
Study with the Northern Bobwhite. Project Number: 671/102. Unpublished study
prepared by Wildlife International, Ltd. 33 p.
48165713 Cockroft, R. (2004) Freshwater Fish Toxicity/Pathogenicity (Prolonged Toxicity to
Microbial Pesticides to Fish) (Oncorhynchus mykiss): Bacillus subtilis var.
amyloliquefaciens Strain D747: Final Report. Project Number: KCI/275/042551.
Unpublished study prepared by Huntingdon Life Sciences, Ltd. 37 p.
48165714 Cockroft, R. (2010) (Prolonged Toxicity of Microbial Pesticides to Daphnia magna):
Freshwater Invertebrate Toxicity/Pathogenicity: Bacillus subtilis var.
amyloliquefaciens Strain D747: Final Report. Project Number: KCI/276/042552.
Unpublished study prepared by Huntingdon Life Sciences, Ltd. 51 p.
48165715 Takagaki, M. (2004) Bacillus subtilis var. amyloliquefaciens Strain D747: Non-
Target Plant Test: Final Report. Unpublished study prepared by Kumiai Chemical
Industry Co. 13 p.
48165716 Dively, C. (2002) Bacillus subtilis var. amyloliquefaciens Strain D747: Non-Target
Insect Testing: Final Report. Unpublished study prepared by Japan Plant Protection
Assoc. Research Institute. 17 p.
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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. lOp.
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.isov 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.isov ocspp pubs frs publications Test (juidelines seriesHH5.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.isov healthywater drinkims 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 Safety Authority. 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.
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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.
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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
MRU) 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
MRU) 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 lx 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
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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
MRU) No.
TGAI
CX-9030 (EP)
830.6302
Color
Beige
Not Applicable
481657-02
830.6303
Physical State
Fine powder
Not applicable
481657-02
830.6304
Odor
Yeast odor
Not applicable
481657-02
830.6313
Stability to Normal and Elevated
Temperatures, Metals,
and Metal Ions
Spores inactivated at
54°C
Not applicable
481657-02
830.6314
Oxidation/Reduction:
Chemical Incompatibility
Not applicable, the product does not contain oxidizing
or reducing agents
481657-02
830.6315
Flammability
Not applicable, the product does not contain flammable
ingredients
481657-02
830.6316
Explodability
Not applicable, the product does not contain explosive
ingredients
481657-02
830.6317
Storage Stability
Stable up to one year at 25°C without loss of viability
481657-02
481657-03
830.6319
Miscibility
Not applicable, the product is not an emulsifiable liquid,
(refer to test note #2 of 40 CFR § 158.2120(d)).
481657-02
830.6320
Corrosion Characteristics
Not applicable, the product is a powder.
481657-02
830.6321e
Dielectric Breakdown Voltage
Not applicable, the product is not for use around
electrical equipment
481657-02
830.7000
pH
6.5 - 7.0 (1% w/w)
7.5-8.0
481657-02
830.7100
Viscosity
Not applicable, the
product is a powder.
Not Applicable.CX-9030 is
not a liquid.
481657-02
830.7300
Density/Relative Density/Bulk
Density (Specific Gravity)
0.307- 0.375 g/ml
0.60-.0.78 g/ cm3
481657-02
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Bacillus amyloliquefaciens strain D747
Biopesticides Registration Action Document
Page 24 of 30
TABLE 4. Physical and Chemical Characteristics for CX-9032 (EP). (40 CFR § 158.2120)
OCSPP
Guideline
Number
Data Requirement
Results
MRU) No.
TGAI
CX-9030 (EP)
830.6302
Color
Beige
Light brown
481655-02
830.6303
Physical State
Fine powder
Liquid
481655-02
830.6304
Odor
Yeast odor
Yeast odor
481655-02
830.6313
Stability to Normal
& Elevated
Temperatures, Metals,
& Metal Ions
Spores inactivated at 54°C..
481655-02
830.6317
Storage Stability
Stable up to one year at
25°C without loss of
viability
Stable up to 78 days at 25°C
without loss of viability. As a
term of the registration, EPA
is requiring submission of 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 is requiring
submission of the results of a
one-year storage stability &
corrosion characteristics
within one year.
481655-02
830.7000
pH
6.5 - 7.0 (1% w/w)
4.2-4.3 (1% w/w).
481655-02
830.7100
Viscosity
Not applicable, the product
is a powder.
4.6 -16.0 milliPascal-second
(mPa s) at 25°C
481655-02
830.7300
Density/Relative
Density/Bulk Density
(Specific Gravity)
0.307- 0.375 g/ml
1.02- 1.03 g/mL
481655-02
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Bacillus amyloliquefaciens strain D747
Biopesticides Registration Action Document
Page 25 of 30
TABLE 5. Toxicology Data Requirements for the Technical Grade of the Active Ingredient (TGAI) (40 CFR § 158.2140)
OCSPP Guideline
Number
Data Requirement
Results
MRU) No.
TGAI
Tier I
885.3050
Acute Oral
T oxicity/Pathogenicity
No evidence of infectivity, pathogenicity or toxicity was found
from oral administration of 1.0x10s 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
T oxicity/Pathogenicity
No evidence of infectivity, pathogenicity or toxicity was found
from intratracheal administration of l.OxlO7 spores B.
amyloliquefaciens strain D747 to rats.
Classification: Acceptable
481657-06
885.3200
Acute Injection
Toxicity/Pathogenicity
(Intravenous)
Not toxic, infective, and/or pathogenic to rats when dosed
intravenously at l.OxlO7 spores per animal.
Classification: Acceptable
481657-05
885.3400
Hypersensitivity
Incidents
No hypersensitivity incidents, including immediate-type or
delayed-type reactions of humans and domestic animals that
occurred during research, development, or testing of the
TGAI/MP, were reported. Future hypersensitivity incidents must
be reported (For reporting format: OCSPP Guideline 885.3400).
479450-23
885.3500
Cell Culture
Not required. B. amyloliquefaciens strain D747 is not a virus
(Test note #4 of 40 CFR § 158.2140(d)).
Tiers II and III
Not required for Bacillus amyloliquefaciens strain D747 based on the lack of acute toxicity/pathogenicity in the Tier I studies.
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Bacillus amyloliquefaciens strain D747
Biopesticides Registration Action Document
Page 26 of 30
TABLE 6. Toxicology Data Requirements for the End-Use Product (EP), CX-9030
(40 CFR § 158.2140)
OCSPP Guideline Number
Data Requirement
Results
MRU) 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
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Bacillus amyloliquefaciens strain D747
Biopesticides Registration Action Document
Page 27 of 30
TABLE 7. Toxicology Data Requirements for the End-Use Product (EP), CX-9032
(40 CFR § 158.2140)
OCSPP Guideline Number
Data Requirement
Results
MRU) No.
885.3400
Hypersensitivity Incidents
No hypersensitivity incidents including
immediate-type or delayed-type reactions
of humans and domestic animals that
occurred during research, development,
or testing of the EP were observed or
reported. All hypersensitivity incidents
must be reported per OCSPP Guideline
885.3400.
870.1100
Acute Oral Toxicity
Waiver request submitted. Requirement
satisfied by submitted MP data. Oral
exposure to the MP showed no adverse
effects including infectivity,
pathogenicity and toxicity up to the limit
dose. Inerts are exempt from tolerance.
No additional oral toxicity is expected
from inerts.
Classification: Acceptable
481655-04
870.1200
Acute Dermal Toxicity
Waiver request submitted. Requirement
adequately addressed by CX-9030 EP
data as well as, dermal irritation testing
on CX-9032-EP. Dermal toxicity and
irritation testing for the CX-9030 EP and
dermal irritation data on CX-9032 EP
showed no adverse effects up to the limit
doses. No additional dermal toxicity is
expected from inerts.
Classification: Acceptable
481655-04
481655-06
481655-11
481657-08
870.1300
Acute Inhalation Toxicity
Waiver request submitted. Pulmonary
exposure to the MP showed no adverse
effects, including infectivity,
pathogenicity or toxicity though slight
toxicity lasting 2 days from a 4-hour
aerosol inhalation administration of 2.18
mg/L, where inert ingredients were also
present, was noted in a CX-9030 EP
study. No additional toxicity is expected
from this EP's inert ingredients.
Classification: Acceptable
481655-04
481657-06
481657-09
870.2400
Acute Eye Irritation
The maximum average irritation score of
0.667 at 1 hour after treatment declined to
0 after 24 hours following ocular
administration of 0.1 mL
B. amyloliquefaciens strain D747 CX-
9032 to New Zealand White rabbits (72-
hour observation period).
Classification: Acceptable
TOXICITY CATEGORY IV
481655-05
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Bacillus amyloliquefaciens strain D747
Biopesticides Registration Action Document
Page 28 of 30
TABLE 7. Toxicology Data Requirements for the End-Use Product (EP), CX-9032
(40 CFR § 158.2140)
OCSPP Guideline Number
Data Requirement
Results
MRU) 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
MRU) No.
Tier I
885.4050
Avian Oral Toxicity
A study showed that B. amyloliquefaciens D747
is not toxic to birds at 8.9 x 109 spores/bird.
Classification: Supplemental
Scientific rationale is sufficient to conclude that
B. amyloliquefaciens D747 is not expected to
pose a hazard to birds.
Classification: Acceptable
48165712
48621501
885.4100
Avian Inhalation
T oxicity/Pathogenicity
Not required. B. amyloliquefaciens D747 is not
considered to be pathogenic to birds
885.4150
Wild Mammal
T oxicity/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
T oxicity/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
T oxicity/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
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Bacillus amyloliquefaciens strain D747
Biopesticides Registration Action Document
Page 29 of 30
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
MRU) 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.
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Bacillus amyloliquefaciens strain D747 Page 30 of 30
Biopesticides Registration Action Document
APPENDIX B. PESTICIDE PRODUCTS
TABLE 9. Table Title?
EPA
Registration
Percentage
Formulation
Use Site(s)
Method(s)
Application
Target Pest
Registration
Name
Active
Type
of
Rate
Number
Ingredient
Application
Various
Tractor
Rate listed on
Various fungal and
70051-107
CX-9032
98.35%
End Use -
agricultural
mounted
label varies
bacterial pests listed
Aqueous
and
boom,
depending on
on the label
Suspension
greenhouse
airblast,
application
including:
crops (e.g.,
hose-end,
method from
Alternaria,
vegetables,
backpack
0.5 pints
Botrytis cinerea
tree fruits,
and other
/acre to 6
Didymella bryoniae
berries,
pressurized
quarts/acre
Phoma
grapes and
sprayers;
cucurbitacearum
tropical fruit,
foggers or
Erisphe,
tree nuts,
mist
Fusarium,
herbs and
blowers;
Macrophomina
spices,
water wheel
phaseoli
coffee,
and other
Monosporascus
tobacco,
drench
cannonballus
hops,
applicators;
Peronospora,
forestry
soil
Phytophthora
seedlings
injection;
Pseudomonas
ornamentals,
aerial; and
syringae pv. tomato
and turf)
chemigation
Pseudoperonospora
with drip or
spp.
sprinkler
Puccinia spp.
irrigation
Pythium,
and cutting
Rhizoctonia,
or root dip
Sphaerotheca spp.
Verticillium, spp.
Xanthomonas spp.
CX-9030
25.0%
Water
Various
Tractor
0.25 -3
Various fungal and
70051-108
Dispersible
agricultural
mounted
pounds per
bacterial pests listed
Granule
and
boom,
acre
on the label
greenhouse
airblast,
including:
crops (e.g.,
hose-end,
Alternaria,
vegetables,
backpack
Botrytis cinerea
ornamentals,
and other
Didymella bryoniae
and turf)
pressurized
Phoma
sprayers;
cucurbitacearum
foggers or
Erisphe
mist
Fusarium,
blowers;
Macrophomina
water wheel
phaseoli
and other
Monosporascus
drench
cannonballus
applicators;
Peronospora,
shank or
Phytophthora
other soil
Pseudomonas
injection
syringae pv. tomato
equipment;
Pseudoperonospora
aerial; and
spp.
chemigation
Puccinia spp.
with drip or
Pythium,
sprinkler
Rhizoctonia,
irrigation
Sphaerotheca spp.
and cutting
Verticillium, spp.,
or root dip
Xanthomonas spp.
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