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**EPA BIOPESTICIDES AND POLLUTION PREVENTION DIVISION COMPANY**  **NOTICE OF FILING FOR PESTICIDE PETITIONS PUBLISHED IN THE**  **FEDERAL REGISTER EPA**  **Biopesticides and Pollution Prevention Division contact:**  **[ Kimberly Nesci;**  **(703) 308 -**  **80 59**  **] 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 chang e the margins, font, or format**  **in your pesticid**  **e petition . Simply replace the instructions that appear in**  **green , i.e., “**  **[ insert**  **company name ]**  **,” with the information specific to your action. SUBMISSION:**  **Email the completed template to:**  **hollis .**  **linda @epa.gov.**  **TEMPLATE:** [ IAB, S.L . (Investigaciones y Apl icaciones Biotecnologicas S.L.) ] [Insert petition number] EPA has received a pesticide petition ( [insert petition number] ) from [ IAB, S.L
. (Investigaciones y Aplicaciones Biotecnologicas S.L .) ] , [ Avda. Paret del Patriarca 11
- B, Ap. 30 , 46113 Moncada (Val encia), SPAIN ] 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 180to establish an exemption from the requirement of a tolerance for the microbial pesticide [Bacillus subtilis strain IAB/BS03 ]
. Pursuant to section 408(d)(2)(A)(i) of FFDCA, as amended, [ IAB, S.L . (Investigaciones y Aplicaciones Biotecnologicas S.L .) ] has submitted the following summary of information, data, and arguments in support of their pesticide petition. This summary was prepared b y [ IAB, S.L. (Investigaciones y Aplicaciones Biotecnologicas S.L.) ] and EPA has not fully

evaluated the merits of the pesticide petition. The summary may have been edited by EPA if the terminology

used was unclear, the summ ary contained extraneous material, or the summary unintentionally made the reader conclude that the findings reflected EPA’s position and not the position of the petitioner.
I. [ IAB, S.L. (Investigaciones y Aplicaciones Biotecnologicas S.L.) ] Petition Summary [Insert petition number] A. Product Name and Proposed Use Practices2
**[** Bacillus subtilis strain IAB/BS03 Technical Powder, a Technical Grade Active In gredient (TGAI), is proposed for manufacturing use only , for further formulation into registered end
-use products. MildoreTM (1% ai) and MildoreTM Max (10% ai) are formulated end
-use products for use to control fungal pathogens on growing crops in greenhouse and agricultural use sites, and in home and garden use sites. **]** B. Product Identity/Chemistry 1. Identity of th e pesticide and corresponding residues

. [Bacillus subtilis strain IAB/BS03 (CAS No. Not applicable). Information regarding the name, identity and composition has been submitted to EPA and can be found in MRID No. 489693
- 01. Bacillus subtilis is a natural ly o ccurring bacteria found in agricultural soils worldwide
. Per EPA, Bacillus subtilis is a rod
- shaped, gram -positive, aerobic, motile bacterium which is ubiquitous in nature and has been recovered from water, soil, air and decomposing plant residue (EPA Registration Review Final Work Plan

– Bacillus subtilis Case 6012). Bacillus subtilis strain IAB/BS03 is antagonistic to fungal plant pathogens through nutrient competition, site exclusion and colonization (MRID No. 489693
- 01). ] 2. Magnitude of residues at the time of harvest and method used to determine the residue

. [NA
- Remove ] 3. A statement of why an analytical method of detecting and measuring the levels of the pesticide residue are not needed. [ An analy tical method for residues is not applicabl e. It is expected that, when used as proposed, Bacillus subtilis strain IAB/BS03, would not result in residues that are of toxicological concern. ] C. Mammalian Toxicological Profile [ Studies to evaluate the safety to mammals were conducted on the technical grade active ingredient (TGAI) and are summarized as follows: 1
. Acute Oral Toxicity ( OCSPP 870.1100) : Bacillus subtilis strain IAB/BS03 Technical Powder was not toxic in an acute oral study in female rats that received a dose of 5,000 mg/kg test article by g avage (MRID No 489693
- 04). There were no clinical signs of toxicity and no mortality

in any o f the subjects during the study
. The acute oral LD50 for Bacillus subtilis strain IAB/BS03 Technical Powder was identified as > 5,000 mg/kg, which is E PA Toxicity Category IV. 2. Acute Dermal Toxicity ( OCSPP 870.1200) : Bacillus subtilis strain IAB/BS03 Technical Powder was not toxic in an acute dermal study in albino rats that received a single dose of 2,000 mg/kg test article moistened with 1.0 ml water applied to the skin (MRID No. 489693
- 05). The treated area on each rat was covered with gauze and wrapped with a veterinary b andage for 24 hours. After3 24
- hours the wrappings were removed and the sites were gentl y washed. There were no clinical signs o f toxicity or dermal irritation during the observation period and no mortality during the study
. The acute dermal LD50 was determined to be >2,000 mg/kg bw, which is EPA Toxicity Category I II. 3. Acute IV Toxicity /Pathogenicity

Study in Rats (OCSPP 885.3 050) : It has been demonstrated that Bacillus subtilis strain IAB/BS03 Technical Powder is not toxic or infective to mammals in a guideline IV toxicity /pathogenicity

study (MRID No. 489693
- 03). This study w as conducted to evaluate the acute intravenous toxicity

and infectivity

o f a microbial pest control agent (MPCA); Technical Product Bacillus subtilis strain IAB/BS03, at a single high dose exposure and an adequate post - exposure observation period. The MPCA test substance or inactivated MPCA test substance was injected into the tail vein of one of two groups of rats at a high dose. Ac ontrol group (untreated) was conducted concurrently
. The animals were observed frequentl y on day o f dosing for mortality

and signs of pharmacologic and/or toxicologic eff ects and once daily thereafter for 21 days. T issue 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. The dose verification analysis of the test substance solution resulted in a count of 1.4 x 10 8 CFU/mL or 1.4 x 10 7 CFU dosed per rat. The Day 0analysis of the inactivated test substance verified 0 CFU/mL of the MPCA. There was no mortality in any group during the study
. During observations for clinical signs, all animals appeared normal for the duration of the study
. The gross necropsy conducted at termination of the study revealed no observable abnormalities, with the exception of one MPCA group animal found with one white dot on liver. Once clearance was established by two consecutive interim sacrifices that showed less than 100 CFU/gram tissue of the test organism in the organs plated, no further tissue samples were taken or cultured. The test organism was considered completely

c leared from the Group III
- MPCA blood by D ay 3
. The test organism was considered completely c leared from the Group III
- MPCA kidneys by D ay 21. The test organism was considered completely c leared from the Group III
- MPCA brain after Day 2 1. The test organism showed a pattern of clearance from the Group III lungs, liver, spleen and mesenteric ly mph nodes, but the test organism had not cleared from the Group III cecum contents by D ay 2 1. The test substance, Bacillus subtilis strain IAB/BS03 Technical Powder was determined to be non
-toxic to rats when injected intravenousl y ata dose of 1.4 x 10 7 CFU/rat
. A reason for the lack of clearance of the organism in cecum contents was investigated. B ased on an extensive literature search it was determined that Bacillus subtilis is a transient resident of the gut and the organism can act as a probiotic like common Lactobacillus

– ty pe probiotics (MRID No. 489693
- 17).4

5. Primary

E ye Irritation (OCSPP 870.2400) : Aprimary e ye irritation study

u sing Bacillus subtilis strain IAB/BS03 Technical Powder was conducted on rabbits by instilling 0.1g (2 X 10 9 CFU/animal) of test article in the conjunctival sac of one eye of three rabbits (MRID No. 489693
- 06). The other eye of each served as the untreated control. The degree of ocular irritation was characterized and recorded at 1, 24, 48 and 72 hours after treatment. The ocular conjunctivae reactions observed during the study w ere slight to moderate and totally reversible. The test substance wa s identified as minimally irritating to the eyes and assigned to Toxicity Category IV. 6. Primary

D ermal Irritation (OCSPP 870.2500) : Aprimary d ermal irritation study using Bacillus subtilis strain IAB/BS03 Technical Powder was conducted on rabbits b y application of 0.5 g (10 10 CFU)/animal of test article moistened with 0.5 ml of deionized water to clipped sites on the dorsal skin three rabbits (MRID No. 489693
- 07). The contralateral dorsal skin of each rabbit served as the untreated control. The treated area on each rabbit was covered with gauze and loosely wrapped with a semipermeable membrane for 4 hours. After 4
- hours the wrappings were removed and the sites were gently w ashed to remove the test substance. The test sites were observed for erythema and edema or other signs of dermal irritation at 1, 24, 48 and 72 hours after removal of the wrappings. V ery slight erythema was observed through the first 24 hours, but no edema was observed during the study and the irritation was reversible by day 2
. No other signs of irritation were reported during the study
. The test substance was identified as slightl y irritating and assigned to Toxicity Category IV. 7. Hy

persensitivity

Incidents (OCSPP 885.3400) : The registrant has noted that no incidents of hy

persensitivity

or any other adverse effects have occurred through 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) (MRID No. 48969 3
- 17). Literature searches have demonstrated that there are no reports of ecological or human health hazards caused Bacillus subtilis strain. Itdoes not produce recognized toxins, enzy mes, or virulence factors normally a ssociated with mammalian invasiveness or toxicity
. The results of toxicity

t esting show there is no risk to human health from the active ingredient. Bacillus subtilis strain IAB/BS03 is not toxic, pathogenic, infective or irritating to mammals. ] D

. Aggregate Exposure 1. Dietary exp osure. i. Food. [ Dietary

exposure from use of Bacillus subtilis strain IAB/BS03, as proposed, is minimal. The intended use of Bacillus subtilis strain IAB/BS03 is as a biological fungicide to growing agricultural crops and home gardens for the purposes of5

disease control. The results of toxicity

testing indicate there is no risk to human health or the environment from Bacillus subtilis strain IAB/BS03. There are no reports of ecological or human health hazards caused by this strain of Bacillus su btilis
. Itdoes not produce recognized toxins, enzy

mes, or virulence factors normally a ssociated with mammalian invasiveness or toxicity
. The absence of acute toxicity or pathogenicity in laboratory animals demonstrates the benign nature of this strain. ] ii. Drinking water
. [ Similarly, exposure to humans from residues of Bacillus subtilis strain IAB/BS03 in consumed drinking water would be unlikely
. P otential exposure to surface water would be negligible and exposure to drinking water (well or ground water) would be impossible to measure. Bacillus subtilis occurs naturally in soils and has long been identified in association with plant roots, organic material and inorganic material in soils (MRID No. 489693
- 17). The intended use of Bacillus subtilis s train IAB/BS03 is to growing agricultural crops and home gardens for the purposes of disease control. The risk of the microorganism passing through the soil to ground water is minimal to unlikely
. A dditionally the fungus would not tolerate the 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 risk to human health or the environment from Bacillus subtilis strain IAB/BS03. There are no reports of ecological or human health hazards caused by this strain of Bacillus subtilis
. Itdoes not produce recognized toxins, enzy

mes, or virulence factors normally a ssociated with mammalian invasiveness or toxicity. The absence of acute toxicity or pathogenicity in laboratory animals demonstrates the benign nature of this strain. ] 2. Non

- dietary exposure
. [ The intended use of Bacillus subtilis strain IAB/BS03 is to growing agricultural crops and home gardens for the purposes of disease control. Personal Protective Equipment (PPE) mitigates the potential for exposure to applicators and handlers of the proposed products, when used in agricultural settings. Bacillus subtilis occurs naturally

in soils and has long been identified in association with plant roots, organic material and inorganic material in soils (MRID No. 489693
- 17). The results of toxicity

testing indicate there is no risk to human health or the environment from Bacillus subtilis strain IAB/BS03. There are no reports of ecological or human health hazards caused by this strain of Bacillus subtilis
. Itdoes not produce recognized toxins, enzy

mes, or virulence factors normally a ssociated with mammalian invasiveness or toxicity
. The absence of acute to xicity or pathogenicity in laboratory animals demonstrates the benign nature of this strain. N on - dietary exposures would not be expected to pose any quantifiable risk due to a lack of residues of toxicological concern .]6

E. Cumulative Effects [ Iti s not expected that, when used as proposed, Bacillus subtilis strain IAB/BS03 would result in residues that are of toxicological concern. The intended use of Bacillus subtilis strain IAB/BS03 is to growing agricultural crops and home gardens for the purposes of disease control. The results of toxicity testing indicate there is no risk to human health or the environment from Bacillus subtilis strain IAB/BS03. There are no reports of ecological or human health hazards caused by this strain of Bacillus subtilis
. It does not produce recognized toxins, enzy mes, or virulence factors normally a ssociated with mammalian invasiveness or toxicity
. The absence of acute toxicity or pathogenicity in laboratory

animals demonstrates the benign nature of this strain .] F . Safet y Determination 1. U.S. population. [ Acute toxicity studies have shown that Bacillus subtilis strain IAB/BS03 is not toxic, pathogenic, infective or irritating to mammals. The intended use of Bacillus subtilis strain IAB/BS03 is to growing agricultural crops and home gardens for the purposes of disease control. The results of toxicity testing indicate there is no risk to human health or the environment from Bacillus subtilis strain IAB/BS03. There are no reports of ecological or human health hazards cau sed by this strain of Bacillus subtilis
. It does not produce recognized toxins, enzy mes, or virulence factors normally a ssociated with mammalian invasiveness or toxicity
. The absence of acute toxicity or pathogenicity in laboratory

animals demonstrates th e benign nature of this strain. There is a reasonable certainty of no harm to the general US population from exposure to this active ingredient .] 2. Infants and children
. [ As mentioned above, it is not expected that, when used as proposed, Bacillus subtilis strain IAB/BS03 would result in residues that are of toxicological concern. There is a reasonable certainty o f no harm for infants and children from exposure to Bacillus subtilis strain IAB/BS03 from the proposed uses .] G. Effects on the Immune and E ndocrine Systems [ To date there is no evidence to suggest that Bacillus subtilis strain IAB/BS03 functions in a manner similar to any known hormone, or that it acts as an endocrine disrupter .] H. Existing Tolerances [ There is no US EPA tolerance or to lerance exemption for Bacillus subtilis strain IAB/BS03 .] I . International Tolerances [ A Codex Alimentarium Commission Maximum Residue Level (MRL) is not established for Bacillus subtilis strain IAB/BS03 .]