1

Federal Food, Drug, and Cosmetic Act (FFDCA) Considerations for Bacillus pumilus strain BU F-33

Docket ID Number: EPA-HQ-OPP-2012-0264

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 “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 28, 2012 (77 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 residues 

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 Docket 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 consumers, 

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, and 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 ultimum, 
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 pumilus strain QST 2808 (40 C.F.R. § 180.1255) – see the Federal Register of 

November 3, 2004 (69 FR 63950).

(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 register 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 submitted 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 during 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 F33 was not toxic to female rats when dosed at 5,000 milligrams per kilogram (mg/kg) of body weight. The 

oral median lethal dose (LD50), 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 oral toxicity study 

conducted on female rats (MRID No. 487164-03) demonstrated that Bacillus pumilus strain BU F-33 was 

not toxic (LD50 greater than 5,000 mg/kg; Toxicity Category IV), while an acute injection 

toxicity/pathogenicity study conducted on rats (MRID 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 demonstrated 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 

(LC50), which is a statistically derived concentration that can be expected to cause 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. 487164-10). 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 rats (MRID No. 487164-05) demonstrated that Bacillus pumilus strain BU F-33 

was not toxic (LC50 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 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 inhalation 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 separate 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 1010 colony-forming 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 changes 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 1010 CFU) can cause toxicity in animals. (In fact, the guideline calls only for a dose level of at least 

107 units 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 107 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 107 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 met 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, including immediate-type or delayed-type reactions of humans 
4

and domestic animals, occurred during research, development, or testing of Bacillus pumilus strain BU F33 (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 LD50 was greater than 

5,050 mg/kg for female rats, 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 Bacillus 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 acceptable 

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 Category 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, 

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: 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 treatment 

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 commodities 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 purposes 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, processing 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 infectivity 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 document). 

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 treatment 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 obtain a National Pollutant 
5

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 will 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 drinking 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-occupational 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 exposure 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, sold 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 indicates that this bacterium is not toxic (acute 

inhalation toxicity and acute dermal toxicity tests), is only slightly irritating (primary 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 metabolite 

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 concludes 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 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

FFDCA section 408(b)(2)(C) provides that, in considering the establishment of a tolerance or tolerance 
6

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 EPA 

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 that 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, or 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 concludes 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 exposure (safety) is necessary.

Moreover, based on the same data and the EPA analysis as presented directly above, the Agency 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 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 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 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 of 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 used 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, Sands 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 

exposed 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 45:302–316.

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 and 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. Monitoring 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 (available 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) (Dated February 1996). Available from 

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

14. U.S. EPA. 1996b. Microbial Pesticide Test Guidelines – Background for Residue Analysis of Microbial 

Pest Control Agents (OPPTS 885.2000) (Dated February 1996). Available from 

http://www.epa.gov/ocspp/pubs/frs/publications/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/oppfead1/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.