

EPA REGISTRATION DIVISION COMPANY NOTICE OF FILING FOR PESTICIDE PETITIONS PUBLISHED IN THE FEDERAL REGISTER  

EPA Registration Division contact: [insert name and telephone number with area code]

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:

AgroSpheres, Inc.

[IN-11585]

	EPA has received a pesticide petition ([IN-11585]) from AgroSpheres, Inc., 1180 Seminole Trail, Charlottesville, VA, USA, 22901 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 180.910.

(Options (pick one)
   
      1. by establishing a tolerance for residues of NA-Remove

	2. to establish an exemption from the requirement of a tolerance for Escherichia coli K-12 derived micelles, a biologically derived inert ingredient under 40 CFR §180.910 for pre- or post-harvest use without restriction when used as an inert ingredient for all agriculture uses, including to protect and retard the release of pesticidal active ingredients, pesticide formulations, and non-pesticide agriculture products, for use on all food, feed, and nonfood crops and agriculture commodities. 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.

in or on the raw agricultural commodity at parts per million (ppm).  NA-Remove

A. Residue Chemistry

	1. Plant metabolism.
AgroSpheres is a new inert ingredient product comprised of bacterial bilayer membrane spheres (micelles) derived from a non-viable, non-toxigenic and nonpathogenic, enfeebled strain of E. coli K-12. This inert is considered to be minimal risk and microbially-derived.  Due to the physical size and chemical composition of AgroSpheres, plants are not capable of absorption or uptake of this inert ingredient and therefore plant metabolism studies for AgroSpheres are not necessary to establish crop residue data. The company is requesting an exemption from the requirement of a tolerance, and therefore plant metabolism studies are not required.

	2. Analytical method. Data and literature provided in support of the inert submission and this Petition, supports that the AgroSpheres inert qualifies for an exemption from the requirement of a tolerance without restriction.  Establishing methods for removal and detection of residues are therefore not necessary for this product.  

 	3. Magnitude of residues. There is no basis to evaluate the residue remaining on the food or on requiring an analytical method for the detection of the AgroSpheres inert ingredient.    

B. Toxicological Profile

	1. Acute toxicity. Toxicological analyses for AgroSpheres included literature on the safety of E. coli K-12 and constituents in current use, safety reviews by EPA and FDA of living E. coli K-12 and constituents, and empirical data using AgroSpheres in oral and inhalation toxicity studies, as well as margin-of-exposure calculations.  

As confirmatory data for the finished AgroSpheres product, an acute oral toxicity test was conducted with rats to determine the potential for the AgroSpheres inert to produce toxicity from a single dose via the oral route of exposure.  An initial limit dose of 5,000 mg/kg was administered.  All animals survived the test substance administration, gained body weight, and appeared active and healthy during the study.  Under the conditions of this study, the acute oral LD50 of the test substance is greater than 5,000 mg/kg of body weight in female rats (EPA Category IV). 

An additional acute inhalation toxicity test was conducted with rats to determine the potential for AgroSpheres to produce toxicity from a single exposure via the inhalation (nose-only exposure) route.  All animals survived exposure to the test atmosphere and gained body weight during the study. Under the conditions of this study, the acute inhalation LC50 of the test substance is >0.55 mg/L in male and female rats (EPA Category III).

A safety assessment was also conducted for lipopolysaccharide activity in the AgroSpheres K-12-derived inert, that considered the routes and extent of exposure from the intended use, the results of oral and inhalation toxicity studies for AgroSpheres, and the established literature regarding endotoxin safety including its ubiquitous presence from gram-negative microorganisms in the human gut, agricultural soil and air.  This assessment concluded that there are no adverse toxigenic effects associated with the proper use of the AgroSpheres inert, even when exposed at very high doses.

Additional toxicity and metabolism studies were deemed to be unnecessary based on the biological and nontoxic nature of the inert material, extensive history of safe use and prior regulatory review of the parental E. coli K-12 organism, the acute toxicity studies using the AgroSpheres inert material, and based on relevant exposures for the intended use as an inert ingredient in pesticidal and nonpesticidal agriculture formulations.  Taken together, this information provides sufficient evidence that the use of this inert ingredient does not pose any unreasonable risk to humans.

	2. Genotoxicty. [NA-Remove]

	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. [NA-Remove]


C. Aggregate Exposure

	1. Dietary exposure. 

AgroSpheres is intended as an inert ingredient in agriculture formulations and potential dietary exposures pose no identifiable hazards.  Evaluation of the risks associated with exposure to AgroSpheres includes the extensive history of safe use of the parental organism for over 50 years; two preexisting comprehensive regulatory reviews of E. coli K-12; literature confirming that that oral exposure to either living E. coli K-12 or to lipopolysaccharides/endotoxins from E. coli K-12 is nonhazardous and nonpathogenic; information that nonpathogenic strains of E. coli are a normal part of the gastrointestinal tract of humans; knowledge that measurable residues of large quantities of deactivated E. coli K-12 components applied in agriculture fields disappear within weeks; and the results of acute toxicity studies on the AgroSpheres inert establishing large margins of exposures by either oral or inhalation routes exposure.  
 
	i. Food. Oral exposure, use rates, residues. 
AgroSpheres is intended as an inert ingredient in agriculture formulations. Potential dietary exposures based on the highest anticipated use rate of 5kg AgroSpheres ingredient per acre were analyzed and found to pose no unreasonable risks. The determination of residues was not deemed necessary due to the low hazard of the material and the anticipated short lifespan of bacterial membrane derived material in the agriculture environment.  An extensive body of literature and the EPA review of E. coli K-12 have identified that the live organism and constituents thereof pose no identified oral hazard to humans.  An acute oral study was conducted using AgroSpheres in rats to confirm the prior safety assessments of E. coli K-12 apply to the AgroSpheres inert.  The acute oral LD50 of AgroSpheres is greater than 5000 mg/kg in female rats (EPA Category IV).  Therefore, the presence of AgroSpheres in or on food at any reasonable level within good agriculture practices poses no identifiable hazard.  
 
	ii. Drinking water. NA-Remove 

	2. Non-dietary exposure. 
Nonfood exposures could occur in handling treated agriculture products, by inhalation during mixing/loading operations, and by end-product application within the anticipated use range between ~0.25 and 5kg AgroSpheres ingredient per acre. Contact exposure is expected to be limited by formulators and handlers when used as part of a pesticide or other agriculture formulations which likely include personal protective equipment (PPE) and specific handling requirements for those products.  Considering a potential inhalation exposure, an acute inhalation toxicity test was conducted with rats to determine the potential for AgroSpheres to produce toxicity from a single exposure via the inhalation route.  All animals survived exposure to the test atmosphere and gained body weight during the study and appeared active and healthy for the remainder of the 14-day observation period.  Under the conditions of this study, the acute inhalation LC50 of the test substance is greater than 0.55 mg/L in male and female rats.

D. Cumulative Effects.  AgroSpheres is a labile biological material and will be quickly broken down into basic nutrients by sunlight and microbial action in the field or after harvest. AgroSpheres would not accumulate or bioaccumulate in plants, animals, or the environment due to its molecular characteristics and its organic chemical nature as food for microorganisms.  

E. Safety Determination

	1. U.S. population. This inert submission request for an exemption from the requirement of a tolerance establishes and confirms the safety of AgroSpheres for the intended use as an inert ingredient in agriculture products.  The key elements of the safety determination for the AgroSpheres inert ingredient and its intended use, is based on the significant precedence based on E. coli K-12 and product specific information.  As a result of these considerations, the microbial-derived inert ingredient is considered to be of minimal risk and appropriate for the exemption from the need for food tolerances.  This petition establishes that there is reasonable certainty of safety, to health and the environment, from the use of AgroSpheres inert ingredient under conditions of its intended use as a microbial-derived inert ingredient in pesticide formulations.  

Large margins of exposure were calculated for potential inhalation by workers exposed to the AgroSpheres inert ingredient.  Margin of exposures (MOE) for the inhalation route of exposure were based on the No Effect Level observed in the acute inhalation study in rats and potential exposures in a work environment (mixers/loaders and applicators) using the maximum anticipated use rate.  The MOE for workers who mixed/loaded product for an application on 100 acres were in excess of 600X and substantially higher when respiratory PPE was used.  For applicators, the MOEs were substantially higher.  Considering oral consumption, the MOEs are sufficiently large for all population subgroups.  For the 99[th] percentile values, the range was from 1,985 (Children, 1-2 years) to 9,451 (Male, 20+ years).  The MOE value for the Total U.S. Population is 4,662 using very conservative high exposure assumptions.  Thus, the actual MOEs for the AgroSpheres inert ingredient would be substantially higher than these worst-case calculated values.  

It is concluded that the information provided in this petition and accompanying toxicology and ecotoxicology data and analysis supports the use of the AgroSpheres inert product be granted an exemption from the requirement of a tolerance for the intended uses without restriction. 

	2. Infants and children.  Large safety Margins of Exposure (1,985X minimum) were calculated for dietary exposures for infants and children using highly conservative estimates of exposure. 

F. International Tolerances.  NA-Remove


