                  Federal Food, Drug, and Cosmetic Act (FFDCA)
             Considerations for Bacillus subtilis strain IAB/BS03 

                                Docket ID Number: EPA-HQ-OPP-2013-0574
                                               Date: January 5, 2015

Section 408(c)(2)(A)(i) of FFDCA allows the EPA to establish an exemption from the requirement for a tolerance (the legal limit for a pesticide chemical residue in or on a food) only if the EPA determines that the exemption is "safe." Section 408(c)(2)(A)(ii) of FFDCA defines "safe" to mean that "there is a reasonable certainty that no harm will result from aggregate exposure to the pesticide chemical residue, including all anticipated dietary exposures and all other exposures for which there is reliable information." This includes exposure through drinking water and in residential settings but does not include occupational exposure. Pursuant to FFDCA section 408(c)(2)(B), in establishing or maintaining in effect an exemption from the requirement of a tolerance, the EPA must take into account the factors set forth in FFDCA section
408(b)(2)(C), which require the EPA to give special consideration to exposure of infants and children to the pesticide chemical residue in establishing a tolerance exemption, and to "ensure that there is a reasonable certainty that no harm will result to infants and children from aggregate exposure to the pesticide chemical residue...." Additionally, FFDCA section 408(b)(2)(D) requires that the EPA consider "available information concerning the cumulative effects of [a particular pesticide's] . . . residues and other substances that have a common mechanism of toxicity."

The EPA performs a number of analyses to determine the risks from aggregate exposure to pesticide residues. First, the EPA determines the toxicity of pesticides. Second, the EPA examines exposure to the pesticide through food, drinking water, and through other exposures that occur as a result of pesticide use in residential settings.

I. Summary of Petitioned-for Tolerance Exemption

In the Federal Register of September 12, 2013 (78 FR 56185) (FRL-9399-7), the EPA issued a notice pursuant to FFDCA section 408(d)(3), 21 U.S.C. 346a(d)(3), announcing the filing of a pesticide tolerance petition (PP 3F8177) by Technology Sciences Group (712 Fifth St., Suite A, Davis, CA 95616) on behalf of IAB, S.L. [Investigaciones y Aplicaciones Biotechnologicas S.L.,Avda, Paret del Patriarca 11-B, Ap. 30, 46113 Moncada (Valencia) Spain. The petition requested that 40 CFR part 180 be amended by establishing an exemption from the requirement of a tolerance for residues of Bacillus subtilis strain IAB/BS03 in and on all food commodities. The notice referenced a summary of the petition prepared by the petitioner, IAB, S.L, which is available in Docket ID Number EPA-HQ-OPP-2013-0574 via http://www.regulations.gov.  
 
Three comments were filed in the Bacillus subtilis strain IAB/BS03 docket but none of these appeared to concern Bacillus subtilis strain IAB/BS03 or the requested exemption from the requirement of a tolerance.  The comments are not relevant to this tolerance action as these comments do not concern the safety of this microbe or have any bearing on the Agency's assessment under section 408 of the FFDCA.   

II. Toxicological Profile

Consistent with section 408(b)(2)(D) of FFDCA, the EPA reviewed the available scientific data and other relevant information on Bacillus subtilis strain IAB/BS03, and considered its validity, completeness, and reliability, as well as the relationship of this information to human risk. The EPA also considered available information concerning the variability of the sensitivities of major identifiable subgroups of consumers, including infants and children.

A.  Overview of Bacillus subtilis strain IAB/BS03 

Bacillus subtilis strain IAB/BS03 is a naturally occurring bacterium originally isolated from the rhizosphere of cucurbits in Valencia, Spain. The Bacillus subtilis species is a naturally occurring bacterium found in agricultural soils worldwide. 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. 
The four end-use products EPs containing Bacillus subtilis strain IAB/BS03 are proposed for use to control bacterial or fungal pests in various grain, small fruit and vegetable crops, tree fruit and nut crops, and grass seed crops, row crops such as sugarbeet, tobacco, sugarcane, hops, oil crops, greenhouse/shadehouse and landscape ornamental tree and plant crops.  Additional proposed uses include applications to garden and ornamental landscapes in home-owner and residential areas.

B.  Microbial Pesticide Toxicology Data Requirements

All applicable mammalian toxicology data requirements supporting the request for an exemption from the requirement of a tolerance for residues of Bacillus subtilis strain IAB/BS03 in or on all food commodities have been fulfilled with data submitted by the petitioner or scientific rationale. An acute injection toxicity/pathogenicity study showed that Bacillus subtilis strain IAB/BS03 when injected intravenously at the dose administered is not toxic or pathogenic via this route of exposure. The acute oral and dermal toxicity were designated EPA Toxicity Category III based on the maximum dose administered. The acute inhalation toxicity and acute eye irritation studies were classified in EPA Toxicity Category IV (see below or Ref. 1, and 2) and revealed little to no toxicity attributed to Bacillus subtilis strain IAB/BS03.  Finally, the petitioner reported that no hypersensitivity incidents occurred during the research, development, and testing of this active ingredient.

The overall conclusions from all toxicological information submitted by the petitioner are briefly described below, and more in-depth synopses of study results can be found in the associated draft Biopesticides Registration Action Document (BRAD) for Bacillus subtilis strain IAB/BS03, provided as a reference in Section VII of this document (Ref. 1).

   1. Acute Injection Toxicity/Pathogenicity  -  Rat  (OCSPP Guideline Number 885.3200) (MRID 48969303) 
The microbial pest control agent (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 (1.4 x 10[8] CFU/mL or 1.4 x 10[7] CFU/rat).  A control group was conducted concurrently.  The animals were observed frequently on day of dosing for mortality and signs of pharmacologic and/or toxicological 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.  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 white dot on the liver of a Group III - MPCA animal.  The test organism had cleared completely from the Group III - MPCA blood by Day 3.  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 had completely cleared from the kidneys by Day 14 and from the brain by Day 21.  There was no test substance seen in the mesenteric lymph nodes or lungs at the Day 21 counts.  Test organism was still present in the liver and spleen at Day 21 but was showing a pattern of clearance.  Test organism was present in the cecum contents by Day 21. Based on the mortality of the animals, the test substance Technical Product Bacillus subtilis strain IAB/BS03 was determined to be non-toxic to rats when injected intravenously at a dose of 1.4 x 10[8] CFU/mL or 1.4 x 10[7] CFU/rat. EPA determined this study to be acceptable. 

   2. Acute Oral Toxicity/Pathogenicity  -  Rat (OCSPP Guideline Number 885.3050) and Acute Pulmonary Toxicity/Pathogenicity  -  Rat (OCSPP Guideline Number 885.3150)  (MRID 48969317)
The registrant has submitted the following rationale that existing data for the Acute Injection Toxicity/ Pathogenicity study on the TGAI be considered to satisfy the requirements for Acute Oral & Pulmonary Tox/Pathogenicity studies: Technical Product Bacillus subtilis strain IAB/BS03 was dosed at a single high dose exposure and followed for an adequate post-exposure observation period. The test substance was injected into the tail vein of one of two groups of rats at a high dose. A control group (untreated) was included. The animals were observed frequently on day of dosing for mortality and signs of pharmacologic and/or toxicological 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 acute injection toxicity/ pathogenicity study on the TGAI was adequate to show the microorganism was not infective or toxic at high doses, and the data from the acute oral and acute inhalation studies demonstrated low toxicity (EPA toxicity category III and IV respectively). The EPA determined that existing data for the Acute Injection Toxicity /Pathogenicity study was adequate and the rationale submitted is rated as acceptable.  

   3. Acute Oral Toxicity Study in Rats (OCSPP Guideline Number 870.1100) (MRID 48969304)
The test item Bacillus subtilis IAB/BS03 Technical Powder was administered to a group of 6 female Sprague Dawley rats at the single dose of 2000 mg/kg body weight (mean : 7.8 x 10[9] CFU/animal). No mortality occurred during the study. No clinical signs related to the administration of the test item were observed. The body weight evolution of the animals remained normal throughout the study. The macroscopical examination of the animals at the end of the study did not reveal treatment related change. In conclusion, the LD50 of the test item Bacillus subtilis IAB/BS03 Technical Powder is higher than 2000 mg/kg body weight by oral route in the rat. EPA classified the acute oral toxicity of Bacillus subtilis IAB/BS03 as EPA Toxicity Category III. EPA determined this study to be acceptable.

   4. Acute Dermal Toxicity Study in Rats (OCSPP Guideline Number 870.1200)
 (MRID 48969305) 
The test item Bacillus subtilis IAB/BS03 Technical Powder was applied onto the intact skin of 10 Sprague Dawley rats (5 males and 5 females) at the single dose of 2000 mg/kg body weight (mean : 8.8 10[9] CFU/animal). No mortality occurred during the study. Neither cutaneous reactions nor systemic clinical signs related to the administration of the test item were observed. A slight brown coloration was noted on the treatment site of all animals on days 1 and 2. The body weight evolution of the animals remained normal throughout the study. The macroscopical examination of the animals at the end of the study did not reveal treatment-related changes. In conclusion, the LD50 of the test item Bacillus subtilis IAB/BS03 Technical Powder is higher than 2000 mg/kg body weight by dermal route in the rat. EPA classified the acute dermal toxicity of Bacillus subtilis IAB/BS03 as Toxicity Category III and rated this study as acceptable.

   5. Acute Inhalation Toxicity Study in Rats (OCSPP Guideline Number 870.1300)
    (MRID 48969407)
      This study was performed to assess the toxic potential of a substance by single inhalation dose, and estimate the lethal concentration (LC50) in rats at a level of concentration of 4.7 mg/L in air during 4 hours of exposure to Bacillus subtilis IAB/BS03. The test substance was administered by a system of resuspension of dust which provided the material in a concentration of 4.7 mg/L in air. 10 rats (5 for each sex) of the Sprague-Dawley strain were exposed to the product within an exposure chamber for a period of 4 hours. The results showed a mortality of 0% and a LC50 of level upper of 4.7 mg/L in air during 4 hours. The clinical observations were conducted daily, looking for signs of toxicity or mortality in the population over a 14 day period. Body weight was recorded weekly during the study. At the end, the surviving animals were submitted to necropsy. The animals exposed by inhalation route to 4.7 mg/L of the product Bacillus subtilis IAB/BS03 in air during 4 hours showed no symptoms associated with exposure of the product during the 14 day period of observation. The macroscopic pathology examination of the animals exposed to the test substance showed moderate pulmonary congestion, mild petechia in lung and moderate to severe hepatic congestion. All the animals showed a weight increase during the period of the study. This increase was 29.5% in male and 8.5% in female. This study estimated that LC50 for the product Bacillus subtilis IAB/BS03 in rats is higher than 4.7 mg/L in air during 4 hours. EPA classified the acute inhalation toxicity of Bacillus subtilis IAB/BS03 as Toxicity Category IV and rated this study as acceptable
   6. Primary Eye Irritation  -  Rabbit   (OCSPP Guideline Number 870.2400) (MRID 48969306)
The test item Bacillus subtilis IAB/BS03 Technical Powder was instilled, as supplied, into the eye of 3 New Zealand rabbits at the dose of 0.1 g (2 x 10[9] CFU)/animal. The ocular conjunctivae reactions observed during the study have been slight to moderate and totally reversible. A slight to moderate redness was noted 1 hour after the test item instillation and was totally reversible between days 2 and 4. This redness was associated with a slight chemosis noted 1 hour after the test item instillation in two animals and this slight chemosis was totally reversible on day 1. EPA classified the primary eye irritation of Bacillus subtilis IAB/BS03 as Toxicity Category IV and rated this study as acceptable.

   7. Primary Dermal Irritation  -  Rabbit  (OCSPP Guideline Number 870.2500) 
(MRID 48969307)
The test item Bacillus subtilis IAB/BS03 Technical Powder was applied, as supplied, at the dose of 0.5 g (1010 CFU /animal), under semi-occlusive dressing during 4 hours on an undamaged skin area of 3 rabbits. A slight erythema was noted on the treated area of one animal, 1 and 24 hours after the patch removal. This reaction was totally reversible on day 2. EPA classified the primary dermal irritation of Bacillus subtilis IAB/BS03 as Toxicity Category IV and rated this study as acceptable.

   8. Hypersensitivity incidents (OCSPP Guideline Number 885.3400)
To date, no incidents of hypersensitivity or any other adverse effects have occurred through the research, development, testing or commercialization of the active ingredient Bacillus subtilis strain IAB/BS03 Technical Powder and its related end-use products. Should any incidents occur, they must be reported per FIFRA Section 6(a)(2).

III. Aggregate Exposure

In examining aggregate exposure, section 408 of FFDCA directs EPA to consider 
available information concerning exposures from the pesticide residue in food and all other nonoccupational exposures, including drinking water from ground water or surface water and exposure through pesticide use in gardens, lawns, or buildings (residential and other indoor uses).

Food Exposure and Risk Characterization: Bacillus subtilis strain IAB/BS03 and other closely related Bacillus subtilis bacteria already exist in the soil environment. The EPA found that increased dietary exposure to Bacillus subtilis strain IAB/BS03, a naturally occurring bacterium, from pesticide use is anticipated to be negligible because  it is anticipated that the concentrations of Bacillus subtilis strain IAB/BS03 on treated plants may be elevated immediately after application but will rapidly decline to environmental background levels. The EPA concluded that the risk posed to adults, infants, and children is likely to be minimal because of the low acute toxicity/pathogenicity potential of Bacillus subtilis strain IAB/BS03 based on the results of toxicity studies described in in sections II of this document.. 

Drinking Water Exposure and Risk Characterization: Bacillus subtilis is naturally present in soils (Ref. 3); therefore, Bacillus subtilis may occur in surface water and possibly groundwater. There are no use sites for the Bacillus subtilis strain IAB/BS03 containing pesticides with direct applications to water, but there is a possibility of spray drift from aerial applications or runoff of prepared fields and rangelands into surface waters.  Bacillus species are often detected in drinking water even after going through acceptable water treatment processes, largely because the spores are resistant to these disinfection processes (Ref. 4).and it is possible that spores of Bacillus subtilis strain IAB/BS03 could be found in drinking water along with naturally occurring Bacilli spores found in soils. Should this microbial pesticide be present, no adverse effects are expected from exposure to Bacillus subtilis strain IAB/BS03, through drinking water, based on the results of toxicity studies described in in sections II of this document.

Non-occupational, Residential Risk Characterization:  Because Bacillus subtilis strain IAB/BS03 use sites do include residential settings and the bacterium is naturally-occurring, some non-occupational exposure to the bacterium is likely. Repeated exposures to the Bacillus subtilis strain IAB/BS03 microorganism from pesticidal applications do not exceed EPA's level of concern, particularly in light of available data that demonstrate Bacillus subtilis strain IAB/BS03 is not toxic (acute injection toxicity/pathogenicity and acute oral, dermal, and inhalation toxicity), is non-irritating (primary dermal irritation, primary eye irritation), and is not pathogenic when used as labeled in accordance with good agricultural practices (acute injection toxicity/pathogenicity) (see additional discussion in Section II of this document).  

IV. Cumulative Effects from Substances with a Common Mechanism of Toxicity

Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to establish, modify, or revoke a tolerance exemption, the EPA consider "available information concerning the cumulative effects of [a particular pesticide's] . . . residues and other substances that have a common mechanism of toxicity."  The EPA has not found Bacillus subtilis strain IAB/BS03 to share a common mechanism of toxicity with other substances. Bacillus subtilis strain IAB/BS03 does not appear to be toxic to humans via dietary, dermal and pulmonary exposure. For the purposes of the tolerance action, therefore, the EPA has assumed that Bacillus subtilis strain IAB/BS03 does not have a common mechanism of toxicity with other substances. Thus, section 408(b)(2)(D)(v) of the FFDCA does not apply. For information regarding the EPA's efforts to determine which chemicals have a common mechanism of toxicity and to evaluate the cumulative effects of such chemicals, see the EPA's website at http://www.epa.gov/pesticides/cumulative.

V.  Determination of Safety for the United States Population, Infants and Children
In considering the establishment of a tolerance or tolerance exemption for a pesticide chemical residue, FFDCA section 408 (b) (2) (C) provides that the EPA shall assess the available information about consumption patterns among infants and children, special susceptibility of infants and children to pesticide chemical residues, and the cumulative effects on infants and children of the residues and other substances with a common mechanism of toxicity. In addition, FFDCA section 408(b) (2) (C) provides that the EPA shall apply an additional tenfold (10X) margin of exposure (safety) for infants and children in the case of threshold effects to account for prenatal and postnatal toxicity and the completeness of the database on toxicity and exposure unless the EPA determines that a different margin of exposure (safety) will be safe for infants and children. This additional margin of exposure (safety) is commonly referred to as the Food Quality Protection Act Safety Factor. In applying this provision, the EPA either retains the default value of 10X or uses a different additional safety factor when reliable data available to the EPA support the choice of a different factor.

Based on the acute toxicity and pathogenicity data/information discussed in section II above (and Ref. 1 and 2), the EPA concludes that there are no threshold effects of concern to infants, children, or adults when Bacillus subtilis strain IAB/BS03 is used as labeled in accordance with good agricultural practices. As a result, the EPA concludes that no additional margin of exposure (safety) is necessary to protect infants and children and that not adding any additional margin of exposure (safety) will be safe for infants and children.

Moreover, based on the same data/information and EPA analysis as presented directly above, the Agency is able to conclude that there is a reasonable certainty that no harm will result to the United States population, including infants and children, from aggregate exposure to the residues of Bacillus subtilis strain IAB/BS03 when it is used -- as labeled and in accordance with good agricultural practices -- as an herbicide. Such exposure includes all anticipated dietary exposures and all other exposures for which there is reliable information. The EPA has arrived at this conclusion because, considered collectively, the data and information available on Bacillus subtilis strain IAB/BS03 do not demonstrate toxic or pathogenic potential to mammals, including infants and children. 

.  
VI. Conclusions

The EPA concludes that there is a reasonable certainty that no harm will result to the U.S. population, including infants and children, from aggregate exposure to residues of Bacillus subtilis strain IAB/BS03. Therefore, an exemption from the requirement of a tolerance is established for residues of Bacillus subtilis strain IAB/BS03 in or on all food commodities when applied or used in accordance with label directions and good agricultural practices.

VII. References


   1. Biopesticides Registration Action Document (BRAD) for Bacillus subtilis strain IAB/BS03

   2. U.S. EPA. 2014a.  Memorandum from I. Barsoum (OPP/BPPD) to S. Cerrelli, Re:  Review of Product Chemistry and Acute Toxicity Studies for Section 3 registration of new TGAI Bacillus subtilis strain IAB/BS03 ( EPA Reg. No. 89615-R). Dated July 29, 2014.

   3. Logan, N.A., and P. de Vos. 2009. Genus I. Bacillus, Pp. 21-128 In: P. de Vos, G.M. Garrity, D. Jones, N.R. Krieg, W. Ludwig, F.A. Rainey, K.H. Schleifer, and W. Whitman (Eds.) Bergey's Manual of Systematic Bacteriology, Volume 3, 2nd Ed. Springer, New York. 

   4. World Health Organization, Guidelines for Drinking-water Quality. (2011) 4th Ed. http://www.who.int/water_sanitation_health/publications/2011/dwq_guidelines/en/
















