i

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) 308-8059]

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.

SUBMISSION: Email the completed template to: hollis.linda@epa.gov.

TEMPLATE:

[IAB, S.L. (Investigaciones y Aplicaciones 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 (Valencia), 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 by [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 summary 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 Practices
2

[Bacillus subtilis strain IAB/BS03 Technical Powder, a Technical Grade Active 

Ingredient (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 the 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 naturally occurring 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 analytical method for residues is not 

applicable. 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 gavage (MRID No 489693-04). There were 

no clinical signs of toxicity and no mortality in any of 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 EPA 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 bandage for 24 hours. After 
3

24-hours the wrappings were removed and the sites were gently washed. There 

were no clinical signs of 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 III.

3. Acute IV Toxicity/Pathogenicity Study in Rats (OCSPP 885.3050): 

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 was conducted to evaluate the acute 

intravenous toxicity and infectivity of 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. A control group (untreated) was 

conducted concurrently. The animals were observed frequently on day of dosing 

for mortality and signs of pharmacologic and/or toxicologic effects and once daily 

thereafter for 21 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. 

The dose verification analysis of the test substance solution resulted in a count of 

1.4 x 108 CFU/mL or 1.4 x 107 CFU dosed per rat. The Day 0 analysis 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 cleared from the Group III - MPCA blood by Day 3. The test 

organism was considered completely cleared from the Group III - MPCA kidneys 

by Day 21. The test organism was considered completely cleared from the Group 

III - MPCA brain after Day 21. The test organism showed a pattern of clearance 

from the Group III lungs, liver, spleen and mesenteric lymph nodes, but the test 

organism had not cleared from the Group III cecum contents by Day 21. The test 

substance, Bacillus subtilis strain IAB/BS03 Technical Powder was determined to 

be non-toxic to rats when injected intravenously at a dose of 1.4 x 107 CFU/rat.

A reason for the lack of clearance of the organism in cecum contents was 

investigated. Based 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 – type probiotics (MRID No. 489693-17). 
4

5. Primary Eye Irritation (OCSPP 870.2400): A primary eye irritation study using 

Bacillus subtilis strain IAB/BS03 Technical Powder was conducted on rabbits by 

instilling 0.1g (2 X 109 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 were slight to moderate and totally reversible. The test 

substance was identified as minimally irritating to the eyes and assigned to 

Toxicity Category IV. 

6. Primary Dermal Irritation (OCSPP 870.2500): A primary dermal irritation study 

using Bacillus subtilis strain IAB/BS03 Technical Powder was conducted on 

rabbits by application of 0.5 g (1010CFU)/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 washed 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. Very

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 slightly irritating and assigned to Toxicity Category IV. 

7. Hypersensitivity Incidents (OCSPP 885.3400): The registrant has noted that no 

incidents of hypersensitivity or any other adverse effects have occurred through 

the research, development or testing of the active ingredient and its related enduse product. Should any incidents occur, they will be reported per FIFRA Section 

6(a)(2) (MRID No. 489693-17).

Literature searches have demonstrated that there are no reports of ecological or human 

health hazards caused Bacillus subtilis strain. It does not produce recognized toxins, 

enzymes, or virulence factors normally associated with mammalian invasiveness or 

toxicity. 

The results of toxicity testing 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 exposure.

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 of 
5

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, enzymes, or virulence factors normally associated 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. Potential 

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 strain 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. Additionally 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. 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 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. 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 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.]
6

E. Cumulative Effects

[It is 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, enzymes, or virulence factors normally associated 

with mammalian invasiveness or toxicity. The absence of acute toxicity or pathogenicity 

in laboratory animals demonstrates the benign nature of this strain.]

F. Safety 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 caused by this strain of Bacillus subtilis. 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 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 of no harm for infants and children 

from exposure to Bacillus subtilis strain IAB/BS03 from the proposed uses.]

G. Effects on the Immune and Endocrine 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 tolerance 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.]