1 Federal Food, Drug, and Cosmetic Act (FFDCA) Considerations for Bacillus pumilus strain BU F
- 33
Docket ID Number: EPA
- HQ
- OPP
- 201 2
- 0264 Date: May 22, 2013 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 “th ere 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 d rinking 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 fact ors 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 informat ion 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. F irst, 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 28, 2012 ( 7 7 FR 59578 ) , 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 2F7978 ) by Becker Underwood , Inc., 801 Dayton Ave ., P.O. Box 667, Ames, IA 50010 . The petition requested that 40 CFR part 180 be amended by establishing an exemption from the requirement of a tolerance for resid ues of Bacillus pumilus strain BU F

- 33. The notice referenced a summary of the petition prepared by the petitioner, Becker Underwood , Inc., which is available in D ocket ID Number EPA

- HQ

- OPP

- 2012

- 0264 via http://www.regulations.gov

. 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 pumilus strain BU F

- 33 , 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 co nsumers, including infants and children. A. Overview of Bacillus pumilus strain BU F

- 33 Bacillus pumilus are Gram

- positive, endospore

- forming bacteria that are ubiquitous in nature and have been identified and isolated from samples of a variety of media, including soil, water, aquatic invertebrates, and growing plants (Refs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, an d 11). Since 2001, three isolates of Bacillus pumilus (i.e., Bacillus pumilus strain GB34, Bacillus pumilus strain QST 2808, and Bacillus pumilus strain GHA 180) have been used in registered pesticide products to suppress plant pathogens such as Pythium ul timum ,2 Fusarium oxysporum , Rhizoctonia solani , and Alternaria species (Ref. 2). In conjunction with some of these registered pesticide products, the EPA established the following exemptions from the requirement of a tolerance: (1) Bacillus pumilus strain GB34 ( 40 C

. F

. R . § 180.1224 )

– see the Federal Registers of April 2, 2003 ( 68 FR 15958 ) and December 22, 2004 ( 69 FR 76619 )

. (2) Bacillus pumil us strain QST 280 8 ( 40 C

. F

. R

. § 180.1255 )

– see the Federal Register of November 3, 2004 ( 69 FR 63 950 )

. (3) Bacillus pumilus strain GHA 180 ( 40 C

. F

. R . § 180.1313 )

– see the Federal Register of March 30, 2012 ( 77 FR 19109 )

. Bacillus pumilus strain BU F

- 33 was isolated from inside the stem of a healthy cucumber plant at an Auburn University facility outside Tallassee, Alabama (Refs. 1 and 11). Data demonstrate that Bacillus pumilus strain BU F

- 33 elicits systemic changes in plants, thereby enhancing their defense against a spectrum of pathogens, including viruses, fungi, and bacteria (Refs. 1 and 11). This increased protection results in more vigorous root and shoot systems in treated plants and leads to more uniform stands and greater yields (Refs. 1 and 11). In its January 11, 2012 submission to the EPA, Becker Underwood, Inc., proposed to r egister an end

- use pesticide product that contains Bacillus pumilus strain BU F

- 33 as an active ingredient and that would be commercially applied to the seed of several plants to elicit induced systemic resistance. 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 pumilus strain BU F

- 33 in or on all food commodities have been fulfilled with data sub mitted by the petitioner or data waiver requests that have been granted by the EPA. The toxicity tests (acute oral, dermal, and inhalation toxicity) and irritation tests (acute eye and primary dermal irritation) that address potential routes of exposure to the active ingredient are all classified in Toxicity Category IV (see Section II of Ref. 12) and revealed little to no toxicity or irritation attributed to Bacillus pumilus strain BU F

- 33. Moreover, when the skin was bypassed as a protective barrier dur ing an acute injection toxicity/pathogenicity test, Bacillus pumilus strain BU F

- 33 was not found to be toxic, infective, and/or pathogenic via the intravenous route of exposure. Finally, the petitioner reported that no hypersensitivity incidents occurred during the research, development, and testing of this bacterium. 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 provided as a reference in Section IV of this document (Ref. 11).
1. Acute oral toxicity

– rat (Harmonized Guideline 870.1100; Master Record Identification Number (MRID No.) 487164

- 03) . An acceptable acute oral toxicity study demonstrated that Bacillus pumilus strain BU F

33 was not toxic to female rats when dosed at 5,000 milligrams per kilogram (mg/kg) of body weight. The oral m edian lethal dose (LD 50 ), which is a statistically derived single dose that can be expected to cause death in 50% of test animals, was greater than 5,000 mg/kg for female rats (Toxicity Category IV) (Refs. 1 and 11).3 2. Acute oral toxicity/pathogenicity ( Harmonized Guideline 885.3050; MRID No. 487164

- 10) . Upon consideration of results of other definitive toxicological data submitted by the petitioner, the EPA waived acute oral toxicity/pathogenicity testing for Bacillus pumilus strain BU F

- 33. An acute ora l toxicity study conducted on female rats (MRID No. 487164

- 03) demonstrated that Bacillus pumilus strain BU F

- 33 was not toxic (LD 50 greater than 5,000 mg/kg; Toxicity Category IV), while an acute injection toxicity/pathogenicity study conducted on rats (M RID No. 487164

- 09) showed that Bacillus pumilus strain BU F

- 33 was not toxic, infective, and/or pathogenic when the skin was bypassed as a barrier. The EPA believes these data, when taken together, indicate that this bacterium would not be toxic, infective , and/or pathogenic through the oral route of exposure and that further testing is not necessary (Refs. 1 and 11).
3. Acute inhalation toxicity

– rat (Harmonized Guideline 870.1300; MRID No. 487164

- 05) . An acceptable acute inhalation toxicity study demons trated that Bacillus pumilus strain BU F

- 33 was not toxic to male and female rats when exposed to 2.22 milligrams per liter (mg/L). The inhalation median lethal concentration (LC 50 ), which is a statistically derived concentration that can be expected to ca use death in 50% of test animals, was greater than 2.22 mg/L for female rats, male rats, and both female and male rats combined (Toxicity Category IV) (Refs. 1 and 11).
4. Acute pulmonary toxicity/pathogenicity (Harmonized Guideline 885.3150; MRID No. 487 164

- 10) . Upon consideration of results of other definitive toxicological data submitted by the petitioner, the EPA waived acute pulmonary toxicity/pathogenicity testing for Bacillus pumilus strain BU F

- 33. An acute inhalation toxicity study conducted on ra ts (MRID No. 487164

- 05) demonstrated that Bacillus pumilus strain BU F

- 33 was not toxic (LC 50 greater than 2.22 mg/L; Toxicity Category IV), while an acute injection toxicity/pathogenicity study conducted on rats (MRID No. 487164

- 09) showed that Bacillus p umilus strain BU F

- 33 was not toxic, infective, and/or pathogenic when the skin was bypassed as a barrier. The EPA believes these data, when taken together, indicate that this bacterium would not be toxic, infective, and/or pathogenic through the inhalatio n route of exposure and that further testing is not necessary (Refs. 1 and 11).
5. Acute injection toxicity/pathogenicity (intravenous)

– rat (Harmonized Guideline 885.3200; MRID Nos. 487164

- 02 and 487164

- 09) . The petitioner submitted data from two separa te acute injection toxicity/pathogenicity tests. The results of the first acceptable study demonstrated that Bacillus pumilus strain BU F

- 33 was not infective or pathogenic to rats when administered intravenously in a single dose of 6.1 x 10 10 colony

- formi ng units (CFU) per animal; however, because of decreased body weight gain of active

- and inactive

- treated male rats and the increased spleen and mesenteric lymph node weights in active

- and inactive

- treated animals of both sexes with correlated gross chang es to the spleen, toxicity could not definitely be ruled out. It is known that intravenous injection with a high dose of bacteria (e.g., 6.1 x 10 10 CFU) can cause toxicity in animals. (In fact, the guideline calls only for a dose level of at least 10 7 unit s of microbial pest control agent per test animal (Ref. 13), and this particular dose level is three orders of magnitude lower than what was tested in the first study.) The second acceptable study, which used a lower dose (5.2 x 10 7 CFU) more comparable to what is specified in the guideline and was conducted for a longer time period than the first study (42 vs. 21 days), demonstrated that Bacillus pumilus strain BU F

- 33 was not toxic, infective, and/or pathogenic to rats when administered intravenously in a single dose of 5.2 x 10 7 CFU per animal. In summary, it is known that intravenous injection with a high dose of bacteria causes toxicity in animals (first study). But, when animals were injected with a lower dose of Bacillus pumilus strain BU F

- 33 that me t the specifications of the associated guideline, no toxicity was observed (second study) (Refs. 1 and 11).
6. Hypersensitivity incidents (Harmonized Guideline 885.3400; MRID No. 487164

- 10) . The petitioner reported that no hypersensitivity incidents, incl uding immediate

- type or delayed

- type reactions of humans4 and domestic animals, occurred during research, development, or testing of Bacillus pumilus strain BU F

33 (Refs. 1 and 11).
7. Acute dermal toxicity

– rat (Harmonized Guideline 870.1200; MRID No. 487164

- 04) . An acceptable acute dermal toxicity study demonstrated that Bacillus pumilus strain BU F

- 33 was not toxic to male and female rats when dosed at 5,050 mg/kg of body weight for 24 hours. The dermal LD 50 was greater than 5,050 mg/kg for female rat s, male rats, and both female and male rats combined (Toxicity Category IV) (Refs. 1 and 11).
8. Primary dermal irritation

– rabbit (Harmonized Guideline 870.2500; MRID No. 487164

- 07 ). An acceptable primary dermal irritation study demonstrated that Bacill us pumilus strain BU F

- 33 was a slight irritant to the skin of rabbits (all animals free of slight erythema at 72 hours; Toxicity Category IV) (Refs. 1 and 11).
9. Acute eye irritation

– rabbit (Harmonized Guideline 870.2400; MRID No. 487164

- 06) . An acce ptable eye irritation study demonstrated that Bacillus pumilus strain BU F

- 33 was non

- irritating to the eyes of rabbits (no corneal opacity, iritis, conjunctivitis, or other signs of ocular irritation were noted at any time point in the study; Toxicity Cat egory IV) (Refs. 1 and 11).
III. Aggregate Exposure In examining aggregate exposure, FFDCA section 408 directs the EPA to consider available information concerning exposures from the pesticide residue in food and all other non

- occupational exposures, i ncluding 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 : Exposure to residues of Bacillus pumilus strain BU F

- 33, a naturally occurring bacterium (Refs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11), in consumed food is anticipated to be possible but negligible. Bacillus pumilus strain BU F

- 33 is to be applied to various plant seeds within commercial seed tr eatment facilities. Subsequently, treated seeds would be bagged, sold to growers, and likely deposited into soil in agricultural settings. Since Bacillus pumilus strain BU F

- 33 colonizes and grows with developing plant roots, it is possible that food commo dities that develop underground (e.g., root and tuber vegetables) or in close proximity to the soil/ground (e.g., cucumber and pumpkin) could be directly exposed to Bacillus pumilus strain BU F

- 33. Further, treated seed could be diverted for food or feed p urposes or processed into oil; therefore, exposure to Bacillus pumilus strain BU F

- 33 in this situation is also plausible. Although residues from the use of pesticides containing Bacillus pumilus strain BU F

- 33 may be present on certain food commodities, p rocessing procedures (e.g., peeling and washing) may reduce such residues (Ref. 14). Regardless of the scenario, should Bacillus pumilus strain BU F

- 33 be present on food, supporting toxicological data indicate that no toxicity, pathogenicity, and/or infec tivity is likely to occur with any food exposure resulting from the use of Bacillus pumilus strain BU F

- 33 as a pesticide when applied in accordance with label directions and good agricultural practices (see additional discussion in Section II of this docu ment ). Drinking Water Exposure : Like consumed food, exposure to residues of Bacillus pumilus strain BU F

- 33, a naturally occurring bacterium (Refs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11), in consumed drinking water is anticipated to be possible but negligible. The proposed use pattern for Bacillus pumilus strain BU F

- 33 involves commercial seed t reatment that takes place indoors. Commercial seed treaters are required to avoid contamination of water when disposing of equipment washwaters or rinsate and, for applications involving container sizes greater than or equal to 5 gallons or 50 pounds, to o btain a National Pollutant5 Discharge Elimination System permit and to notify the permitting authority in writing prior to discharge into lakes, streams, ponds, estuaries, oceans, and other waters (see sections II, III, and VII of Ref. 15). Thus, for larger container sizes, it is possible that commercial seed treatment facilities could discharge effluent containing Bacillus pumilus strain BU F

- 33 into surface waters. Additionally, Bacillus pumilus strain BU F

- 33 on treated seed that is planted by growers wil l colonize and grow with the roots of developing plants; therefore, its association with plant roots in the soil may result in exposure to ground and surface waters. If Bacillus pumilus strain BU F

- 33 were to be transferred to ground or surface waters as
a result of effluent discharged from commercial seed treatment facilities or treated seed planted by growers, it is possible that this bacterium could survive the conditions water is subjected to in wastewater treatment systems and drinking facilities (e.g. , pH adjustments and chlorination). According to the World Health Organization, Bacillus species, such as pumilus , 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. 16). In the remote likelihood that Bacillus pumilus strain BU F

- 33 is present in drinking water, supporting toxicological data indicate that no toxicity, pathogenicity, and/or infectivity is likely to occur with any dr inking water exposure resulting from the use of Bacillus pumilus strain BU F

- 33 as a pesticide when applied in accordance with label directions and good agricultural practices (see additional discussion in Section II of this document ).
Other Non

- occupation al Exposure : Given Bacillus pumilus ’ natural presence in the environment (Refs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11), dermal and inhalation non

- occupational exposures to Bacillus pumilus strain BU F

- 33 almost certainly are already occurring. Additional e xposure to Bacillus pumilus strain BU F

- 33 due to pesticidal applications is not expected because the proposed end

- use pesticide product is labeled only for use inside of commercial seed treatment facilities. Subsequently, treated seeds would be bagged, so ld to growers, and likely deposited into soil in agricultural settings; diverted for food or feed purposes; or processed into oil. Even if additional non

- occupational exposures were to occur (e.g., through eventual expansion of use sites or use patterns), no adverse effects attributed to Bacillus pumilus strain BU F

- 33 are anticipated, particularly in light of testing that indicate s that this bacterium is not toxic (acute inhalation toxicity and acute dermal toxicity tests), is only slightly irritating (pri mary dermal irritation test), and is not pathogenic or infective (acute injection toxicity/pathogenicity test) at high doses (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, 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 pumilus strain BU F

- 33 to share a common mechanism of toxicity with any other substances, and Bacillus pumilus strain BU F

- 33 does not appear to produce a toxic metaboli te produced by other substances. For the purposes of this tolerance action, therefore, the EPA has assumed that Bacillus pumilus strain BU F

- 33 does not have a common mechanism of toxicity with other substances. Following from this, therefore, the EPA conc ludes that there are no cumulative effects associated with Bacillus pumilus strain BU F

- 33 that need to be considered. For information regarding the EPA’s efforts to determine chemicals that have a common mechanism of toxicity and to evaluate the cumulativ e 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 FFDCA section 408(b)(2)(C) provides that, in considering the establishment of a tolerance or tolerance6 exemption for a pesticide chemical residue, 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 E PA shall apply an additional tenfold (10X) margin of 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 th at a different margin of safety will be safe for infants and children. This additional margin of 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, o r uses a different additional or no safety factor when reliable data are available to support a different additional or no safety factor. Based on the acute toxicity and pathogenicity data/information discussed in Section II of this document , the EPA con cludes that there are no threshold effects of concern to infants, children, or adults when Bacillus pumilus strain BU F

- 33 is used as labeled in accordance with good agricultural practices. As a result, the EPA concludes that no additional margin of exposu re (safety) is necessary. Moreover, based on the same data and the EPA analysis as presented directly above, the Agency conclude s that there is a reasonable certainty that no harm will result to the U.S. population, including infants and children, from ag gregate exposure to the residues of Bacillus pumilus strain BU F

- 33 when it is used as labeled and in accordance with good agricultural practices as an elicitor of induced systemic resistance in plants. Such exposure includes all anticipated dietary exposu res 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 pumilus strain BU F

- 33 do not demonstrate toxic, pathogenic, and/or infective 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 o f Bacillus pumilus strain BU F

- 33. Therefore, an exemption from the requirement of a tolerance is established for residues of Bacillus pumilus strain BU F

- 33 in or on all food commodities when applied to elicit induced systemic resistance in plants and use d in accordance with label directions and good agricultural practices.
VII . References 1. U.S. EPA. 2012a. Review of Product Chemistry and Acute Toxicity Studies for Section 3 Registration of a New TGAI Bacillus pumilus strain BU F

- 33 (EPA Reg. No. 71840

- RE). Memorandum from I.S. Barsoum, Ph.D. through J.L. Kough, Ph.D. to J. Kausch , dated November 6, 2012.
2. U.S. EPA. 2012b. Bacillus pumilus Preliminary Work Plan and Summary Document (signed December 18, 2012) (available as a “Supporting Document” within docket ID number EPA

- HQ

- OPP

- 2012

- 0857 at http://www.regulations.gov ). 3. Juhnke ME, Mathre DE, San ds DC. 1987. Identification and characterization of rhizosphere

- competent bacteria of wheat. Applied and Environmental Microbiology 53(12):2793

– 2799. 4. Halverson LJ, Jones TM, Firestone MK. 2000. Release of intracellular solutes by four soil bacteria exp osed to dilution stress. Soil Science Society of America Journal 64:1630

– 1637.7 5. Garbeva P, van Veen JA, van Elsas JD. 2003. Predominant Bacillus spp. in agricultural soil under different management regimes detected via PCR

- DGGE. Microbial Ecology 45:30 2

– 316. 6. Yasinok AE, Biran S, Kocabas A, Bakir U. 2010. Xylanase from a soil isolate, Bacillus pumilus : gene isolation, enzyme production, purification, characterization and one

- step separation by aqueous

- two

- phase system. World Journal of Microbiology a nd Biotechnology 26:1641

– 1652. 7. Puente ME, Bashan Y, Li CY, Lebsky VK. 2004. Microbial populations and activities in the rhizoplane of rock

- weathering desert plants. I. Root colonization and weathering of igneous rocks. Plant Biology 6(2004):629

– 642.
8 . Ivanova EP, Vysotskii MV, Svetashev VI, Nedashkovskaya OI, Gorshkova NM, Mikhailov VV, Yumoto N, Shigeri Y, Taguchi T, Yoshikawa S. 1999. Characterization of Bacillus strains of marine origin. International Microbiology 2:267

– 271. 9. Merina M, Lipton AP , Wesley SG. 2011. Isolation, characterization and growth response of biofilm forming bacteria Bacillus pumilus from the sea grass, Halodule pinifolia , off Kanyakumari coast. Indian Journal of Marine Sciences 40(3):443

– 448. 10. Oguntoyinbo FA. 2007. Monit oring of marine Bacillus diversity among the bacteria community of sea water. African Journal of Biotechnology 6(2):163

– 166.
11. U.S. EPA. 2013. Draft Bacillus pumilus strain BU F

- 33 Biopesticides Registration Action Document dated May 9, 2013 (availabl e as a “Supporting Document” within docket ID number EPA

- HQ

- OPP

- 2012

- 0265 at http://www.regulations.gov ). 12. U.S. EPA. 2012c. Chapter 7 of the Label Review Manual (Precautionary Statements) (Revised July 2012) . Available from http://www.epa.gov/oppfead1/labeling/lrm/chap

- 07.pdf

. 13. U.S. EPA. 1996a. Microbial Pesticide Test Guidelines

– Acute Injection Toxicity/Pathogenicity (OPPTS 885.3200) (D ated February 1996)

. Available from http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/series885.htm

. 14. U.S. EPA. 1996b. Microbial Pesticide Test Gui delines

– Background for Residue Analysis of Microbial Pest Control Agents (OPPTS 885.2000) (Dated February 1996) . Available from http://www.epa.gov/ocspp/pubs/frs/p ublications/Test_Guidelines/series885.htm

. 15. U.S. EPA. 2012d. Chapter 8 of the Label Review Manual (Environmental Hazards) (Revised September 2012) . Available from http://www.epa.gov/opp fead1/labeling/lrm/chap

- 08.pdf

. 16. World Health Organization. 2011. Guidelines for Drinking

- water Quality (Fourth Edition). Available from http://whqlibdoc .who.int/publications/2011/9789241548151_eng.pdf

.