**BIOPESTICIDES REGISTRATION ACTION DOCUMENT** 

### DRAFT



## Bacillus amyloliquefaciens strain D747 Pesticide Chemical (PC) Code: 016 482

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

**November 10**  **, 2011 version** Bacillus amyloliquefaciens strain D747 Page 2 of 30

Biopesticides Registration Action Document **TABLE OF CONTENTS I. EXECUTIVE SUMMAR**  **Y ................................**  **................................ ................................**  **...... 4**  **II. ACTIVE INGREDIE NT OVERVIEW**  **................................ ................................**  **................ 5**  **III. REGULATORY BACKGROUN** **D ................................**  **................................ .................**  **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. Hum an Health Assessment (40 CFR § 158.2140)**  **................................ ................................**  **................................ 7**  **C. Environmental Assessment (40 CFR § 158.2150)**  **................................ ................................**  **.............................. 12**  **V. ENVIRONMENTAL JU STICE**  **................................ ................................**  **.......................... 14**  **VI. RISK MANAGEMENT DECISION**  **................................ ................................**  **................. 15**  **VII. ACTIONS REQUIR ED BY THE REGISTRANT**  **................................ .........................**  **16 VIII. GLOSSARY OF A**  **CRONYMS AND ABBREVIA TIONS**  **................................ ...........**  **16 IX. BIBLIOGRAPHY**  **................................ ................................**  **................................ ................**  **18 A. Studies Submitted to Support Pesticide Product Registrations**  **................................ ................................**  **....... 18**  **B. Environm ental Protection Agency Risk Assessment Mem**  **oranda and Other Documents ............................**  **20 C. Other**  **References ................................**  **................................ ................................**  **................................ .................**  **20 APPENDIX A. MICROBI** **AL PESTICIDES DATA R EQUIREMENTS**  **............................ 22**  **APPENDIX B. PESTICI DE PRODUCTS**  **................................ ................................**  **............... 30** Bacillus amyloliquefaciens strain D747 Page 3 of 30

Biopesticides Registration Action Document

**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 Anal ys is, Human Health John L. Kough, Ph.D. Product Anal ys is, Human Health Gail Tomimat su , Ph.D. Environmental Effects Shannon Borges Environmental Effects **Regulations** Sheryl K. Reill y, Ph.D. Chief, Microbial Pesticides Branch Susanne Cerrelli Regulatory Action LeaderBacillus amyloliquefaciens strain D747 Page 4 of 30

Biopesticides Registration Action Document **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 Sy mbol 70051
- RNI) and CX
- 9032 (EPA File S ymbol 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 ,” sin ce B. subtilis and B. amyloliquefaciens were originally classified as subty

pes 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; Lo gan and de Vos , 2009; and Murray et. al
. , 2007)
. EPA scientists reviewed product anal ys is, mammalian and nontarget organism toxicity data, and other information submitted by Certis to support the proposed product registration s . The p roduct anal ys is data requirements for B. amyloliquefaciens strain D747 , including product chemistry and composition, analysis of samples, and phy sical and chemical characteri stics , were fulfilled by acceptable studies conducted in accordance with Agency guidelines. M amm alian toxic ity 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 pathogeni c 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 an d environmental risk s 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 label s, 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 naturall y present on fresh produce. The Manual of Clinical Microbiology

(9 th E dition) states that dried food s, such as spices, mi lk 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 frequentl y 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, includingBacillus amyloliquefaciens strain D747 Page 5 of 30

Biopesticides Registration Action Document 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 product s 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 i n 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 appli cations: new active ingredients; first food uses; first outdoor uses; first residential use s; 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 c ontaining 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 Sy mbol 70051
- RNI) ; and (4) draft product label for the end
- use product, CX
- 9032 (EPA File Sy mbol 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 CX
9030 and CX
- 9032 EP s , 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 NRRLB
- 50405
. **OPP Chemical Code:** 016 482 **Type of Pesticide:** Microbial Pesticide

– Insecticide See Appendix B for specific information (e.g., use sites, application rate s , methods of application, formulation ty

pe s , and target pests) regarding the registered pesticide products containing this active ingredient.Bacillus amyloliquefaciens strain D747 Page 6 of 30

Biopesticides Registration Action Document **III. REGULATORY BACKGROUND**  **A. Applications for Pesticide Product**  **Registration** Certis USA , LLC (address: 9145 Guilford Road, Suite 175, Columbia, MD , 21046) , submitted application s 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 application s to register pesticide product s containing a new active ingredient on February 2, 2011 ( 76 Federal Register (FR) 5805 ) , and opened a 3 0
- day public comment period , pursuant to the provisions of FIFRA section 3(c)(4). No comments were received following this publication. **B. Food Tolerance Exemption** Concurrent with its registration application s , 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 perio d. No c omments 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 thes e requirements. The classifications or ratings that are given for each data requirement in this BRAD were assigned b y the EPA scientists who reviewed the data, and convey the usefulness of the information for the human health and nontarget risk assessment purposes . “A cceptable ” indicates that a study

is scientifically

sound and is useful for risk assessment. A “ supplemental ” classification indicates the studies provide d some information that can be useful for risk assessment . “ Supplemental ” ratings are giv en to studies that are not required for registration, as well as those that are required. In the latter case, the study lack s information that should have been obtained if it was conducted according to the guideline associated with the data requirement
. So metimes the missing information would not add any thing necessary

to inform a risk assessment, and the study classification remains “supplemental.” A classification of “ s upplemental: u

pgradable” indicates the study lacks necessary information, but if it is made available b y the applicant , the study may be upgraded to “ acceptable
. ” An “unacceptable ” rating indicates that new data must be submitted.Bacillus amyloliquefaciens strain D747 Page 7 of 30

Biopesticides Registration Action Document Toxicity c ateg ories 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 peer
reviewed, scientific literature. The active ingredient or particular product is classified int o Toxicity Category I , II, III, or IV, where Toxicity Category Iindicates the highest toxicity, and Toxicity Category IV indicates the lowest toxicity (see 40 CFR § 156.62 )
. **A. Product Analysis Assessment**  **( 40 CFR § 158.2120**  **)** A ll product anal ys is 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 Assessm**  **ent (** **40 CFR § 158.2140 )**  **1. Toxicity**  All Tier I mammalian toxic ity 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 Tie r III studies were not required. For a comprehensive summary of the generic and product
- specific toxic ity data requirements described below , refer to Tables 5, 6 and 7 in Appendix A
. **a. Acute Toxicity/Pathogenicity**

**– Tier I**  Acute Oral Toxicity/Pathogenicity (O ffice of Chemical Safety and Pollution Prevention (O CSPP ) Guideline 885.3050; Master Record Identification Number ( MRID No. 481657

- 04

) : B. amyloliquefaciens strain D747 was administered once orall y to 14 rats of both sexes (5
- week s old) at a single dosage of 10 8 colony
- forming units ( CFU) per animals
. No deaths occurred , and no abnormalities (clinical signs, body weight) were observed , during the study or at necrops y. The test microbe was detected at 10 3

– 10 5 CFU/g in feces 1 day after administration of the test material , but was no t detected on day 14. 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 l ymph nodes, or blood , throughout the experiment al period. Fecal c learance occurred by day 14 , and no viable organisms were recovered from blood or other organ s or tissue s . The results of this acceptable study demonstrated that B. amyloliquefaciens strain D747 was not infective , pathogenic , or toxic to rats when oral ly

dosed with 1. 0 x10 8 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 10 7 CFU per animal
. N o deaths occurred , and no abnormalities (clinical signs, body weight) were observed during the study or at necrops y. The examination for internal persistence showed that the test microbe was detected shortl y after administration in the lung, trachea, and nasal cavity
. The test microbes w ere 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 b y approximately 10 2

- 10 3 CFU/g from day 0 to day 60. The test microbes were detected in the bronchial l ymph node s on day 3.Bacillus amyloliquefaciens strain D747 Page 8 of 30

Biopesticides Registration Action Document Since many

test microbes remained in the trachea on day 3, their presence in the bronchial ly mph nodes on day

3 was attributed to transfer by macrophages from the respiratory tract to the l ymph nodes. This conclusion was supported by the observation that a particle larger than 1 m in diameter administered into the trachea and bronchi was phagocy tized by a macrophage and carried to a ly

mph node. The presence of the microbes in the lungs, though decreasing throughout the observation period , was attributed to the c ontinuous transfer of viable test organisms to the bronchial ly mph no de s . 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 s ys tem to clear. This a cceptable study demonstrated that B. amyloliquefaciens strain D747 was not toxic or pathogenic to rats when dosed intratracheally at 1.0 x10 7 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
- week s old) were injected with B. amyloliquefaciens strain D747 at a dosage of 10 7 CFU per animal , and the influence on the animals was investigated. No deaths occurred , and there were no observed abnormal iti es (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 day 14 ; 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 intravenousl y to rats, clearance from the liver and spleen is difficult
. In this study, the test microbe did not completel y disappear from these organs by day 60. No test microbes w ere detected in the brain, and onl y a few viable microbes at the limit level were sporadicall y detecte d in the small and large intestine s, and l ymph 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 x10 7 CFU/ animal. Hypersensitivity Incidents (OCSPP Guideline 885.3400; MRID No. 481655

- 05 ) : No hy persensitivity

incidents in humans have been reported, and none occurred during research, development, or testing of B. amyloliquefaciens strain D747 . Should hy persensitivity 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 D74 7 is not a virus (refer to test note #4 of 40 CFR § 158.2140(d)). **b. Acute Toxic** **ity and Subchronic Toxicity/Pathogenicity** 

**– Tier II;**  **Reproductive Fert**

**ility 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.Bacillus amyloliquefaciens strain D747 Page 9 of 30

Biopesticides Registration Action Document
**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 eff ect produced b y a “naturally

occurring estrogen, or other such endocrine effects as the Administrator may designate.” The EDSP employ s a two - tiered approach to making the statutorily required determinations. Tier 1 consists of a battery of 11 screening ass ays to identify the potential of a chemical substance to interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal sy stems. Chemicals that go through Tier 1 screening and are found to have the potential to interact with E, A, or T hormonal sy s tems will proceed to the next stage of the EDSP wh ere 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 k nown 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 pesti cide chemicals. Accordingl y, 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 resi due 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 resi dential 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 re quire 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 comm on mechanism of toxicity .”Bacillus amyloliquefaciens strain D747 Page 10 of 30

Biopesticides Registration Action Document EPA performs a number of anal ys es to determine the risks from aggregate exposure to pesticide residues. First, EPA determines the tox icity 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 s ection 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 thi s 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 re sidential 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 (9 th edition) mentions that dried food such as spices, milk powder and grains often contain large amounts of B. spores. B. amyloliquefaciens i s 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 product s 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 W

ater 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, largel y 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, b ased on the data and other information submitted to satisfy

the data requirements for registration of the manufacturing
- use and end
- use pesticide product s containing the active ingredient, B. amyloliq uefaciens strain D747 (see section IV (B)(1)(a) , above, and Table 5 in Appendix A). Non

- occupational, Residential Risk Characterization : The use sites for th ese product s include residential garden sites and agricultural sites. As stated previousl y, B. amyloliquefaciens is naturally present in soil , and b ased on the data and other information submitted to satisfy the data requirements for registration of the manufacturing
- use and end
- use pesticide product s containing the active ingredient, B. amyloliquefaciens strain D74 7 , no toxicity, infectivity , pathogenicity orBacillus amyloliquefaciens strain D747 Page 11 of 30

Biopesticides Registration Action Document 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 re garding how EPA determine s common mechanism s of toxicity and evaluate s cumulative ef fects , 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 sectio n 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 sectio n 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 complet eness 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 risk s 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 exposure s . Handlers appl ying 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 NI OSH standards of at least N
- 95, R
- 95, or P
- 95.Bacillus amyloliquefaciens strain D747 Page 12 of 30

Biopesticides Registration Action Document **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 unreasonab le 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 patho gen 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 fulfill ed 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- dispers ible granul ar formulation and an aqueous suspension, proposed for application to agricultural crops, nurseries, ornamental plants, turf grass , greenhouses , and shadehouses. Applications can be made to both foliar surfaces and soil , so exposure to nontarget organisms is possible. The proposed m aximum application rates were used in the assessment of nontarget risk
. Data on the natural ly

occurring levels of B. amyloliquefaciens are not available. Many factors influence the environmental fate of microbial pesticides, and resulting population leve ls 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 significantl y 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 LD 50 for B. amyloliquefaciens strain D747 is > 4.5 x 10 11 spores/kg BW or > 8 x 10 9 spores/bird in Northern bobwhite ( Colinus virginianus ; MRID 48165712). Additionally , B. amyloliquefaciens is intentionally included in someBacillus 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 i s 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 10 8 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 Ori us 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 day s (no MRID currently

assigned). These studies were rated S upplemental because th ey were not of sufficient duration (30 day s); however, except in rare cases, bacteria that are pathogenic to insects ty pically produce toxins that kill the insect within a few days (Tanada and Kaya , 1993). A dverse 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)
. Entomop athogenic 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 previousl y , however, the microorganism has been isolated from tissues of healthy plants and is k nown 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 directl y 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 b y which the pesticide is expected to reach water . A spray drift anal ys is 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 LC 50 for B. amyloliquefaciensBacillus amyloliquefaciens strain D747 Page 14 of 30

Biopesticides Registration Action Document strain D747 was 8.1 x 10 10 CFU/L , and the NOEC based on sub
- lethal effects was 1.44 x 10 10 CFU/L . I f the proposed maximum broadcast application rate (4 fluid ounces/1000 ft2 or 2.55 x 10 6 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 10 8 CFU/L . This would be the maximum possible aquatic concentration at this application rate , but is not a proposed use. The NOEC and LC 50 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 risk s to freshwater fish. A study with Daphnia magna provided an EC 50 based on mortality/immobility of 3.7 x 10 10 CFU/L , and a NOEC for sub
- lethal effects of 2.84 x 10 8 CFU/L . Based on the spray drift anal ys is, the NOEC would be approximately 20X the expected environmental concentration (EEC) , and the EC 50 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 asse ssment for B. amyloliquefaciens strain D747 ; f urther testing at higher tier levels (i.e., Tiers II, III, and IV) is not required. **3 .**  **Threatened and Endangere**  **d 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, a nd 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 th at 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 p opulation 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 aty pical, unusually

high exposure compared to the general population. For additional information regarding environmental justice issues, please visit EPA’s web site at http://www.epa.gov/compliance/environmentaljustice/index.html
. **V I**  **. RISK MANAGEMENT DECISION**  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 generall y cause unreasonable adverse effects on the environment. To satisfy criterion 1, the B. amyloliquefaciens strain D747 pesticide products have we ll
- known properties. EPA has no knowledge that would contradict the claims made for these products , the CX
- 9030 and CX
- 9032 EP label s, 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 label s, as well as the data and information presented in this document. It is believed that the B. amyloliquefaciens strain D747 pesticide products will not cau se any unreasonable adverse effects on the environment, and CX
9030 and CX
- 9032 (end
- use pesticide product) , in particular, are likel y 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.Bacillus amyloliquefaciens strain D747 Page 16 of 30

Biopesticides Registration Action Document **V I**  **I .**  **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 follo wing 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
- ye ar storage stability and corrosion characteristics study. **C . Reporting of Adverse Effects and Hypersensitivity Incidents**  Notwithstanding the information stated previousl y , it should be clearl y 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 typ es of data are described below. Reports of all incidents of adverse effects to the environment must be submitted to EPA under the provisions stated in FIFRA section 6(a)(2). Additionally, all incidents of hy persensitivity (including both suspected and confirmed incidents) must be reported to EPA under the provisions of 40 CFR § 158.2140(d).
**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 unit s 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 LC 50 median lethal concentration. A statistically derived concentration of a substance that can be expected to cause death in 50% of test animals. It isBacillus 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). LD 50 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 milligram s per kilogram mg/L milligram s per liter MP manufacturing
- use product mPa·s milliPascal
- second , term used as the unit of dynamic viscosity

. MRID No. Master Record Identification Number NI OSH 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 StatesBacillus amyloliquefaciens strain D747 Page 18 of 30

Biopesticides Registration Action Document **I X. 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 subtili s Var. amyloliquefaciens Strain D747 (CX
- 9032): Product Identity , Manufacturing Process, Discussion of Formation of Unintentional In gredients: Anal ys is of Samples: Certification of Limits. Unpublished study prepared b y 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: Visc osity : 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 b y 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 : Hy persentivity

(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 Irri tation 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 Baci llus 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 In gredients: Anal ys is of Samples: Certification of Limits. Unpublished study prepared b y Certis USA, LLC. 145 p. 48165702 DeVane, W. (2010) Bacillus subtilis var. amyloliquefaciens Strain D
- 747 End
- Use

Product (ID Code CX
- 903 0): 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 : FinalBacillus 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, In c. 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: 13821/1 0. 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 b y Wildlife International, Ltd. 33 p. 48165713 Cockroft, R. (2004) Freshwater Fish Toxi city /Pathogenicity

(Prolonged Toxicity

to Microbial Pesticides to Fish) (Oncorhyn chus my kiss): 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. Unpublish ed 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 b y J apan Plant Protection Assoc. Research Institute. 17 p.Bacillus amyloliquefaciens strain D747 Page 20 of 30

Biopesticides Registration Action Document 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

L ife 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
- T arget

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 m ellifera): Addendum to MRID 48165717. Unpublished study prepared b y Certis USA, LLC. 9p. B. Environmental Protection Agency Risk Assessment Memoranda and Other Documents U.S. EPA. 2004. Primer for Municipal Wastewater Treatment S ys tems. 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 Anal ys is of

Microbial Pest Control Agents (OPP TS 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 Paeni B. agent of American foulbrood. Arch. Microbiology August 20, 2010 Centers for Disease Control and Prevention. 2009. Drinking Water
- Water Treatment. Available from http://www.cd

c.gov/healthywater/drinking/public/water_treatment.html
. Dedj. S., et.al
. Effectiveness of honey bees 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. Lo gan, 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 Sy stematic Bacteriology , Volume 3, 2 nd Ed. Springer, New York. Maccagnami, B., et. al
. Apis mellifera and Osmia cornuta as carriers for the secondary sp read 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; 9 th 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 P athology . 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

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

**(40 CFR PART 158** 

**– SUBPART**  **V )**  **TABLE 1**  **. Product Analysis Data Requirem**  **ents for the End**  **- Use Product (**  **E P),**  **CX -**  **9030 (40 CFR § 158.2120)**  **OCSPP Guideline Number**  **Data Requirement Results**  **MRID No.**
885.1 1 00 Product Identity Submitted data fulfill the requirement for product identity . CX
- 9030 contain s 25.0% by weight Bacillus amyloliquefaciens strain strain D747 (minimum of 5 x 10 1 0 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 Requirem ents for the End**  **- Use Product (EP),**  **CX -**  **9032 (40 CFR § 158.2120)**  **OCSPP Guideline Number**  **Data Requirement Results**  **MRID No.** 885.1 1 00 Product Identity Submitted data fulfill the requirement for product identity . CX
- 9030 contain s 98.95% by weight Bacillus amyloliquefaciens strain strain D747 (minimum of 1 x 10 1 0 CFU/g) 481655
- 01 CSF dated 10/18/2011 885.1200 Manufacturing Process Submitted data fulfill the requirement for manufacturing process. 48165 7
- 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. 48165 7
- 01 481655
- 01 885.1300 Discussion of Formation of

Unintentional Ingredients Submitted data fulfill the requirement for discussion of formation of unintentional ingredients. 48165 7
- 01 481655
- 01 885.1400 Analysis of Samples Submitted date fulfill the requirement for analysis of samples
. 481655
- 01 48165 7
- 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
- 01Bacillus 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** **.**

**am yloliquefaciens strain**  **strain D747 / CX** **- 90**  **30 (EP)** 
. **(40 CFR § 158.2120) OCSPP**  **G uideline**  **N umber**  **Data Requirement Results**  **MRID No. TGAI**  **CX -**  **90 30 (**  **E P**  **)** 830.6302 Color Beige Not Applicable 481657
- 02 830.6303 Phy sical State Fine powder Not applicable 481657
- 02 830.6304 Odor Yeast odor Not applicable 481657
- 02 830.6313 Stability to Normal and E levated

Tem peratures, Metals, and Metal I ons Spores inactivated at 54 o 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 o 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
- 02 830.6321 e Dielectric Breakdown Voltag e 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
- 02Bacillus amyloliquefaciens strain D747 Page 24 of 30

Biopesticides Registration Action Document **TABLE 4**  **. Physical and Chemical Characteristics for**  **CX -**  **90 32 (EP)** 
. **(40 CFR § 158.2120) OCSPP**  **G uideline**  **N umber**  **Data Requirement Results**  **MRID No. TGAI**  **CX -**  **90 30 (**  **EP)** 830.6302 Color Beige Light brow n 48165 5
- 02 830.6303 Phy sical State Fine powder Liquid 48165 5
- 02 830.6304 Odor Yeast odor Yeast odor 481655
- 02 830.6313 Stability to Normal & E levated Tem peratures, Metals, & Metal I ons Spores inactivated at 54 o C

. . 48165 5
- 02

830.6317 Storage Stability Stable up to one year at 25 o C without loss of viability Stable up to 78 days at 25 o C

without loss of viability
. **As a term** **of the registration, EPA will require the results of a**  **one -**  **year storage stability and c**  **orrosion characteristics study**  **be submitted within one**

**year.** 48165 5
- 02

830.6319 Miscibility Not applicable; product is not an emulsifiable liquid. (refer to test note #2 of 40 CFR § 158.2120(d)). 48165 5
- 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 sub**  **mission of the results of**  **a one**  **- year storage stability**  **& c**  **orrosion characteristics within one year.**  48165 5
- 02

830.7000 pH 6.5

– 7.0 (1% w/w) 4.2
- 4.3 (1% w/w). 48165 5
- 02 830.7100 Viscosity Not applicable , the product is a powder. 4.6
- 16. 0 milliPascal
- second ( mP a s ) at 25 o C 481655
- 02
830.7300 Density/Relative

Density/Bulk Density (Specific Gravity) 0.307
- 0.375 g/ml 1.02

– 1.03 g/mL 48165 5
- 02Bacillus amyloliquefaciens strain D747 Page 25 of 30

Biopesticides Registration Action Document **T ABLE**  **5 .**  **Toxicology Data Requirements**  **for the Technical Grade of the Active Ingredient (TGAI) (40 CFR § 158.2140)**  **OCSPP Guideline N**  **umber Data Requirement**  **Results MRID**  **N o.**  **TGAI Tier I**  885.3050 Ac ute Oral

Toxicity/Pathogenicity No evidence of infectivity, pathogenicity or toxicity was found from oral administration of 1.0x10 8 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 . **Cla ssification: Acceptable**  481657
- 04

885.3150 Ac ute Pulmonary

Toxicity/Pathogenicity No evidence of infectivity, pathogenicity or toxicity was found from intratracheal administration of 1.0x10 7 spores B
. amyloliquefaciens strain D747 to rats. **Classification: Acceptable** 481657
- 06

885.3200 Ac ute Injection

Toxicity/Pathogenicity (Intravenous) N ot toxic, infective, and/or pathogenic to rats when dosed

intravenously at 1.0x10 7 spores per animal
. **Classification: Acceptable** 481657
- 05
885.3400 Hy

persensitivity Incidents No hypersensitivity incidents, including immediate
- type or

delayed
- ty

pe reactions of humans and domestic animals that occurred during research, development, or testing of the TGAI/MP , were reported
. F uture 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**  **Num ber**  **Data Requirement Results**  **MRID No.** 885.3400 Hy persensitivity Incidents No hypersensitivity incidents, including immediate
- ty

pe 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 Ac ute Oral Toxicity Oral LD 50 > 5000 mg/Kg **Classification: Acceptable** Toxicity Category IV 481657
- 07
870.1200 Ac ute Dermal Toxicity Dermal LD 50 > 5050 mg/Kg **Classification: Acceptable TOXICITY CATEGORY**  IV 481657
- 08
870.1300 Ac ute Inhalation Toxicity Inhalation LC 50 > 2.18 mg/L **Classification: Acceptable TOXICITY**  **CATEGORY I V**  481657
- 09 870.2400 Ac ute 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 period s . The dermal irritation score for B
. amyloliquefaciens strain D747 CX
- 9030 was 0.00. **Classification: Acceptable TOXICITY CATEGORY IV**  481657
- 11Bacillus 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 Hy persensitivity Incidents No hypersensitivity incidents including immediate
- ty

pe or delayed
- type reactions of humans and domestic animals that occurred during research, de velopment, or testing of the EP we re observed or reported
. All hypersensitivity incidents must be reported per OCSPP Guideline 885.3400. 870.1100 Ac ute 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. Inert s are exempt from tolerance
. N o additional oral toxicity is expected from inert s . **Classification: Acceptable** 481655
- 04

870.1200 Ac ute 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 inert s. **Classification: Acceptable** 481655
- 04 481655
- 06 481655
- 11 481657
- 08

870.1300 Ac ute 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. N o additional toxicity is expec ted from this EP’s inert ingredients
. **Classification: Acceptable** 481655
- 04 481657
- 06 481657
- 09

870.2400 Ac ute 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 I V**  481655
- 05Bacillus 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 period s . The dermal irritation score for B. amyloliquefaciens strain D747 CX
- 9032 wa s 0.00. **Classification: Acceptable**  **TOXICITY CATEGORY I V**  481655
- 06

**TABLE 8**  **. Nontarget Organism Toxicity and Environmental Fate Data Requirem ents**  **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 10 9 spores/bird. **Classification: Supple mental**  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 wa s not infective, toxic or pathogenic to laboratory rats 1.0 x 10 8 CFU/animal 48165704

885.4200 Freshwater Fish

Toxicity/Pathogenicity A 30
- day study shows that the LC 50 to rainbow trout is 8 x 10 10 CFU/L. The NOEC for sublethal effects is 1.44 x 10 10 CFU/L. **Classification: Acceptable** 48165713

885.4240 Freshwater Invertebrate

Toxicity/Pathogenicity A 21
- day study shows that the EC 50 to Daphnia magna based on mortality is 3.7 x 10 10 CFU/L. The NOEC for sublethal effects is 2.84 x 10 8 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 48165715Bacillus amyloliquefaciens strain D747 Page 29 of 30

Biopesticides Registration Action Document **TABLE 8**  **. Nontarget Organism Toxicity and Environmental Fate Data Requirem ents**  **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: Supple mental**  Additional rationale was sufficient to show that pathogenicity to honey bees is not expected as a result of exposure to B. amyloliquefaciens D747. **Classification: Accepta ble**  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; fogg ers 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, ornamental s , 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 chemi gation 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.