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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 preparing 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 in 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 requesting, 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 commodity 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 of 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 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. Bacillus simplex has been shown to occur 

naturally in soils around the world.

3. Magnitude of residues. [NA-Remove]

B. Toxicological Profile
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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 rats 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 observable abnormalities. The acute oral LD50 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 potential 

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 exhibited 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 (Category 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 hours 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 gain 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 108 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 significantly 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, mesenteric 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 109 CFU/g or 1.0 x 108 CFU/rat. 
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1.D. Acute 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 conjunctival 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 BU288. 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 irritation 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 incidents 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 infectivity”. 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 identified 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 context.

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-Remove
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3. Reproductive and developmental toxicity. NA-Remove

4. Subchronic toxicity. NA-Remove

5. Chronic toxicity. NA-Remove

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 endocrine 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 

endocrine-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 animals 

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 health 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 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.

i. Food. NA-Remove

ii. Drinking water. Similarly, exposure to humans 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 through 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 drinking-water facility including 

chlorination, pH adjustments, high temperatures and/or anaerobic conditions. 

The results of toxicity testing indicate there is no identifiable hazard to human 
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health or the environment from Bacillus simplex strain BU288 via 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 absence 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 as 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. Application 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 exposure 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 tolerance 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 ocular 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 mammalian 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 of 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 irritation. 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 effects 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 Bacillus 
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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. The results of toxicity testing indicate there 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 population 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.

International -

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