# i

EPA REGISTRATION DIVISION COMPANY NOTICE OF FILING FOR

PESTICIDE PETITIONS PUBLISHED IN THE FEDERAL REGISTER

EPA Registration Division contact: William Cutchin 703

- 305

- 7990

INSTRUCTIONS: Please utilize this outline in prepa ring the pesticide petition. In cases where the outline element does not apply, please insert “NA

- Remove” and maintain the outline. Please do not change the margins, font, or format in your pesticide petition. Simply replace the instructions that appear i n green , i.e., “ [ insert

company name ] ,” with the information specific to your action.

TEMPLATE:

BASF Corporation

IN10891

EPA has received a pesticide petition ( IN

- 10891 ) from BASF Corporation

, 26 Davis Drive, Research Triangle Park, NC 27709 request in g, pursuant to section 408(d) of the Federal Food, Drug, and Cosmetic Act (FFDCA), 21 U.S.C. 346a(d), to amend 40 CFR part 180 to establish an exemption from the requirement of a tolerance for Bacillus simplex strain BU288 in or on the raw agricultural com modity all food commodities

under 40 CFR 180.910 as an emulsifier

.

EPA has determined that the petition contains data or information regarding the elements set forth in section 408 (d)(2) of FDDCA; however, EPA has not fully evaluated the sufficiency o f the submitted data at this time or whether the data supports granting of the petition. Additional data may be needed before EPA rules on the petition.

A. Residue Chemistry

1. Plant metabolism. NA

- Remove

2. Analytical method. An analytical method for residues is not applicable. It is expected that, when used as proposed, Bacillus simplex strain BU288 would not result in residues in or on food that are of toxicological concern. Application of Bacillus simplex strain BU288 to seeds through seed treatm ent, in

- furrow, soil drench, or foliar applications is not expected to significantly increase the levels of the microbe in the

environment above natural concentrations. Bacillus simplex has been shown to occur naturally in soils around the world

.

3. Mag nitude of residues. [ NA

- Remove ]

B. Toxicological Profile2

1. Acute toxicity. Acute Oral Toxicity (OPPTS 870.1100) (MRID 49780704): The test substance, Bacillus simplex strain BU288, was evaluated for its acute oral toxicity potential in female albino r ats when administered via gavage at a dose level of 5000 mg/kg. There was no mortality. Animals gained weight throughout the study and there were no clinical signs of toxicity observed. Gross necropsy conducted at termination of the study revealed no obse rvable abnormalities. The acute oral LD 50 was estimated to be greater than 5000 mg/kg (Category IV).

1.A

. Acute Dermal Toxicity (OPPTS 870.1200) (MRID 49780705): The test substance, Bacillus simplex strain BU288, was evaluated for its dermal toxicity pote ntial following a single moistened dose of 5050 mg/kg applied to the intact skin of albino rats. No mortality occurred during the study. There were no clinical signs of toxicity. Very slight erythema was observed in 1/10 animals on day 1 only. Animals exhi bited weekly weight gain, with the exception of one female that failed to gain weight between Days 7 and14. Gross necropsy conducted at termination of the study revealed no observable abnormalities. The LD50 was determined to be greater than 5050 mg/kg (Ca tegory IV).

1.B. Acute Inhalation Toxicity (OPPTS 870.1300) (MRID 49780706): The test substance, Bacillus simplex strain BU288, was evaluated for its acute inhalation toxicity potential in albino rats. Five males and five females were exposed for four ho urs to an aerosol generated from the undiluted test substance (fine powder) at a level of 2.14 mg/L. There was no mortality. Clinical signs included activity decrease and/or piloerection in all animals, resolving by Day 4. Animals exhibited weekly weight g ain during the study except in two animals that lost weight between Days 0 and 7. Gross necropsy revealed no observable abnormalities. The acute inhalation LC50 is greater than 2.14 mg/L (Category IV).

1.C

. Acute IV Injection Toxicity/Pathogenicity Study in Rats (OPPTS 885.3200) (MRID 49780707): This study was conducted to evaluate the acute intravenous toxicity and infectivity of Bacillus simplex strain BU288 at a single high dose exposure and an adequate post

- exposure observation period. The active test substance or

heat

- inactivated test substance was injected into the tail vein of one of two groups of rats

at 1 x 10 8 CFU (colony forming units) per animal. A control group (untreated) was included concurrently. The animals were observed frequently on the day of dosing for mortality and clinical signs of toxicity and once daily thereafter for 42 days. Tissue and blood samples taken at interim sacrifices from the group receiving the active test substance were cultured to provide quantitative measurements of the test microbe’s clearance pattern. There was no mortality or clinical signs of toxicity in any group during the study. Gross necropsy conducted at termination of the study revealed no observable abnormalities. Relative spleen weight was statistically s ignificantly increased in females of the active test substance group, but this change was not observed in males nor observed in animals receiving the inactivated test substance. The active test organism had cleared completely from the blood, kidneys, mese nteric lymph nodes, lungs, brain, liver, spleen and cecum contents by Day 14. The test substance Bacillus simplex strain

BU288 was determined to be non

- toxic to rats when injected intravenously at a dose of

1.0 x 10 9 CFU/g or 1.0 x 10 8 CFU/rat.3

1.D

. Acu te Eye Irritation (OPPTS 870.2400) (MRID 49780708): An acute ocular irritation study was conducted with three albino rabbits using test substance Bacillus simplex strain BU288. The test substance, 0.1 mL by volume (94.8 mg), was placed into the conjunctiv al sac of the right eye of each animal selected for testing. All treated eyes were washed with room temperature deionized water for one minute immediately after recording the 24

- hour observation. Minimal ocular irritation was observed in each animal during the 24

- hr treatment period, with clearance by 48 hrs (Category III).

1.E

. Acute Dermal Irritation (OPPTS 870.2500) (MRID 49780709): A primary dermal irritation study was conducted on three albino rabbits using test substance Bacillus simplex strain BU2 88. Each dermal test site was treated with 500 mg of test

substance moistened with 0.5 mL of deionized water and covered with a semi

- permeable dressing. The test substance was maintained in contact with the skin for 4 hours.

Observations for dermal irritat ion and skin defects were made at 1, 24, 48 and 72 hours after removal of the dressings. Very slight erythema was observed in each animal 1 hour following removal of the dressing, with clearance by 24 hours (Category IV).

1.F

. Hypersensitivity Incidents (OPPTS 885.3400): BASF Corporation has noted that no incidents of hypersensitivity or any other adverse effects have occurred in humans during the research, development or testing of the active ingredient and its

related end

- use product. Should any inci dents occur, they will be reported per FIFRA Section 6(a)(2).

A PubMed literature search was performed for Bacillus simplex on November 17, 2015 using the search terms “ Bacillus simplex toxicity”, “ Bacillus simplex pathogenicity”, and

“ Bacillus simplex in fectivity”. Results of this search are attached in Appendix 3. The search yielded no hits for pathogenicity or infectivity. There was no indication of toxicity specific to the Bacillus simplex strain BU288. Two published papers ( Ref 11 ) ( Ref 12 ) were i dentified that indicate the potential for toxin production or in vitro cytotoxicity in cell culture models testing to supernatants enriched in Bacillus simplex of unidentified strain. However, given the results from in vivo toxicity studies conducted

with our specific strain of Bacillus simplex (BU288; also attached and summarized above) that indicate a very low order of acute toxicity (including irritation) with this strain, the results of the two in vitro studies are not generalizable to the in vivo cont ext.

In summary, acute, in vivo toxicity testing has not identified a potential hazard for Bacillus simplex strain BU288 that would be relevant to human health risk assessment, other than slight ocular or dermal irritation. Bacillus simplex strain BU288 was non

- toxic via the oral, dermal, and inhalation routes when tested up to limit doses, and was cleared from the blood and all organs within 14 days following a large intravenous bolus dose in

rats. Bacillus simplex strain BU288 would not be expected to be toxic, pathogenic, or infective in humans or other mammals under the proposed uses of this petition. ]

2. Genotoxicty. NA

- Remove4

3. Reproductive and developmental toxicity. NA

- Remove

4. Subchronic toxicity. NA

- Remove

5. Chronic toxicity. NA

- Remov e

6. Animal metabolism. NA

- Remove

7. Metabolite toxicology

. NA

- Remove

8. Endocrine disruption

. To date there is no evidence to suggest that Bacillus simplex strain BU288 functions in a manner similar to any known hormone, or that it acts

as an endocr ine disrupter. Results of acute toxicity studies conducted with this strain, as well as the results of a PubMed literature search conducted on November 23, 2015 using

the search terms “ Bacillus simplex and endocrine”, do not indicate the potential for

end ocrine

- mediated effects with the species Bacillus simplex

.

C. Aggregate Exposure

1. Dietary exposure. Bacillus simplex and other closely related endospore

- forming Bacillus species are ubiquitous in the environment. Dietary exposure levels from the use

of Bacillus simplex strain BU288, as proposed, are not expected to be greater than existing exposures to background levels. If dietary exposure were to fall above natural background levels, the absence of acute toxicity or pathogenicity in laboratory anim als demonstrates the overall benign nature of this strain and would support the establishment of an exemption from the requirement of a tolerance for Bacillus simplex strain BU288.

The results of in vivo toxicity testing identified no potential human hea lth hazard following oral exposure to Bacillus simplex strain BU288. There are no reports of ecological or human health hazards caused by Bacillus simplex strain BU288. It is unlikely that the strain produces recognized toxins, enzymes, or virulence factor s normally associated with mammalian invasiveness or toxicity. The absence of acute toxicity or pathogenicity in laboratory animals demonstrates the overall benign nature of

this strain.

i. Food. NA

- Remove

ii. Drinking water

. S imilarly, exposure to hu mans from residues of Bacillus simplex strain BU288 in consumed drinking water would be unlikely. The proposed use

sites of Bacillus simplex strain BU288 do not include direct application to aquatic environments. The risk of the microorganism passing thr ough the soil to ground water is minimal, since, like other microorganisms, it would likely be filtered out by the particulate nature of many soil types. Additionally, the bacteria would not tolerate the treatment conditions water is subjected to in a drin king

- water facility including chlorination, pH adjustments, high temperatures and/or anaerobic conditions.

The results of toxicity testing indicate there is no identifiable hazard to human5

health or the environment from Bacillus simplex strain BU288 v ia the oral route. There are no reports of ecological or human health hazards caused by Bacillus simplex strain BU288. It does not produce recognized toxins, enzymes, or virulence factors normally associated with mammalian invasiveness or toxicity. The ab sence of acute toxicity or pathogenicity in laboratory animals demonstrates the overall benign nature of this strain.

2. Non

- dietary exposure

. The potential for non

- dietary exposure to the general population, including infants and children, is unlikely a s the proposed use sites are agricultural. For occupational exposure scenarios, Personal Protective Equipment (PPE) will mitigate the potential for exposure to applicators and handlers of the proposed products, when used in agricultural settings. Applica tion of Bacillus simplex strain BU288 to seeds through seed treatment, in

- furrow, soil drench, or foliar applications is not expected to significantly increase the levels of the microbe in the environment above

natural concentrations. If non

- dietary expos ure were to fall above natural background levels, the absence of acute toxicity or pathogenicity in laboratory animals demonstrates the overall benign nature of this strain and would support the establishment of an exemption from the requirement of a toler ance for Bacillus simplex strain BU288.

The results of toxicity testing indicate there is no identifiable hazard to human

health or the environment from Bacillus simplex strain BU288 other than the potential for slight ocular irritation. Any potential o cular irritation would be mitigated by the use of PPE. There are no reports of ecological or human health hazards caused by Bacillus simplex strain BU288. It does not produce recognized toxins, enzymes, or virulence factors normally associated with mammali an invasiveness or toxicity. The absence of acute toxicity or pathogenicity in laboratory animals demonstrates the overall benign

nature of this strain. Non

- dietary exposures would not be expected to pose any quantifiable risk due to a lack of residues o f toxicological concern.

D. Cumulative Effects

The results of in vivo toxicity testing identified no potential human health or environmental hazard following multiple routes of exposure to Bacillus simplex strain BU288, other than possible ocular irrita tion. There are no reports of ecological or human health hazards caused by Bacillus simplex strain BU288. It is unlikely that the strain produces recognized toxins, enzymes, or virulence factors normally associated with mammalian invasiveness or toxicity. The absence of acute toxicity or pathogenicity in laboratory animals demonstrates the overall benign nature of this strain.

A common mechanism of toxicity in mammals has not been identified for Bacillus simplex strain BU288. Therefore, cumulative effe cts with other related organisms would not be anticipated.

E. Safety Determination

1. U.S. population. Acute toxicity studies have shown that Bacillus simplex strain BU288 is not toxic, pathogenic, or infective in mammals. Application of Bacillus6

simpl ex strain BU288 to seeds through seed treatment, in

- furrow, soil drench, or foliar applications is not expected to significantly increase the levels of the microbe in the environment above natural concentrations. The results of toxicity testing indicate t here is

no risk to human health or the environment from Bacillus simplex strain BU288. There are no reports of ecological or human health hazards caused by Bacillus simplex strain BU288. It does not produce recognized toxins, enzymes, or virulence factors normally associated with mammalian invasiveness or toxicity. The absence of acute toxicity or pathogenicity in laboratory animals demonstrates the overall benign nature of this strain. There is a reasonable certainty of no harm to the general US populati on from exposure to this inert ingredient.

2. Infants and children

. As mentioned above, it is not expected that, when used as proposed, Bacillus simplex strain BU288 would result in residues that are of toxicological concern to this subpopulation. There is a reasonable certainty of no harm for infants and children from exposure to Bacillus simplex strain BU288 from the proposed uses.

F. International Tolerances

US EPA Tolerance

- There is no US. EPA Tolerance for Bacillus simplex strain BU288.

Intern ational

- A Codex Alimentarium Commission Maximum Residue Level (MRL) is not required for Bacillus simplex strain BU288.

Canada listing of Bacillus simplex on the CDSL (confidential portion of the Domestic Substances List)

Identifier: 18847

- 1

Published: August 12, 2015