Document ID: EPA-HQ-OPP-2013-0574-0004
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2014-10-28T04:00Z

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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) 308-8059]

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

	[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.

	Mildore™ (1% ai) and Mildore™ 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 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).  

 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 end-use 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 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.]

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.]

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