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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: Sheryl Reilly, Branch 

Chief, 703-308-8269

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: E-mail the completed template to: hollis.linda@epa.gov.

TEMPLATE:

AgraQuest, Inc.

[1F7896]

EPA has received a pesticide petition (insert petition number) from AgraQuest, 

Inc., 1540 Drew Avenue, Davis, CA 95618 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

(Options (pick one)

3. to establish an amendment/expansion of an existing tolerance exemption for the

(Options (pick one)

1. microbial pesticide Bacillus subtilis strain QST 713 variant Soil

Pursuant to section 408(d)(2)(A)(i) of FFDCA, as amended, AgraQuest, Inc. has 

submitted the following summary of information, data, and arguments in support of their 
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pesticide petition. This summary was prepared by AgraQuest, Inc. 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. AgraQuest, Inc. Petition Summary

[1F7896]

A. Product Name and Proposed Use Practices

Bacillus subtilis strain QST 713 is the active ingredient in the formulated end-use 

product Serenade® ASO (EPA Reg. No. 69592-12), with uses as a foliar spray on many 

agricultural field and greenhouse crops, soil treatment for selected agricultural field 

corps, foliar and soil drench treatments for ornamentals including turf, in fields, 

greenhouses and nurseries. Serenade ASO is also sold under an alternate brand name 

into the consumer home and garden market for use on a variety of crops for foliar 

applications. 

Bacillus subtilis strain QST 713 variant Soil is the active ingredient in the proposed 

formulated end-use product Serenade®Soil DPZTM. The formulated product containing 

Bacillus subtilis strain QST 713 variant Soil is intended for use as a fungicide for the 

control or suppression of various soil diseases including Rhizoctonia spp., Pythium spp., 

Fusarium spp., Verticillium spp., and Phytophthora spp. Treatment with Serenade Soil 

DPZ enhances plant growth and plant health. The end-use product, Serenade Soil DPZ, 

consists of a 1.57% active ingredient aqueous suspension formulation that is mixed with 

water and applied as a soil drench or in-furrow spray with conventional application 

equipment. It may also be shaked-in or injected into the soil prior to-, at-, or postplanting/transplanting of crops alone or with most types of liquid nutrients. Serenade 

Soil DPZ can be mixed with chemical fungicides registered for soil applications. It may

be applied as a water-based slurry alone or with other registered seed treatment 

insecticides and fungicides through standard slurry or mist commercial seed treatment 

equipment. The product would be applied at a maximum application rate of 6 quarts per 

acre for field applications. 

B. Product Identity/Chemistry

1. Identity of the pesticide and corresponding residues. Bacillus subtilis strain 

QST 713 variant Soil is a microbial pesticide active ingredient. Serenade Soil 

DPZ’s fermentation and formulation processes and inert ingredients are the 

same as for Serenade ASO (EPA Reg. No. 69592-12); therefore no new 

impurities are produced as the strains differ in a single point mutation which 

results in enhanced root colonization by Bacillus subtilis strain QST 713 variant 

Soil compared to the parent strain Bacillus subtilis strain QST 713. Therefore, 

the active ingredient Bacillus subtilis strain QST 713 and Bacillus subtilis strain 
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QST 713 variant Soil are substantially similar and data supporting the existing 

registration of Serenade ASO is supportive of the proposed product Serenade 

Soil DPZ. 

The parent strain Bacillus subtilis strain QST 713 is a EPA- registered microbial 

pesticide active ingredient and has an exemption from tolerance (40 CFR 

§180.1209). Bacillus subtilis strain QST 713 variant Soil has the same active 

component and has been shown to have the same activity spectrum as Bacillus 

subtilis strain QST 713 with enhanced plant growth and promotion of plant health 

when applied in the soil prior to-, at-, or post-planting/transplanting of crops or as 

a seed treatment.

2. Magnitude of residues at the time of harvest and method used to determine the 

residue. Since Bacillus subtilis is a ubiquitous organism, it is commonly recovered from 

soil, water and decomposing plant residue. It is found at population levels of 106to 107

per gram of soil (EPA Risk Assessment of Bacillus subtilis, February, 1997); therefore,

any residues of Bacillus subtilis strain QST 713 variant Soil on crops at the time of 

harvest are not of concern due to the low toxicity and non-pathogenic nature of these 

strains, and the long history of human exposure to Bacillus subtilis spores including use 

of the strain as a probiotic. In addition, common food handling practices would further 

reduce human exposure. It would be difficult to routinely determine if any Bacillus 

subtilis strain QST 713 variant Soil residue on crops were from the natural environment 

or from applications of the microbial pest control agent. Studies indicate that the MPCA 

concentration is elevated directly after foliar application to crops but then declines 

rapidly1, though Bacillus spores may persist at low levels on the plant surfaces and in the 

soil community. Environmental factors can reduce populations on plant surfaces due to 

UV radiation, excess moisture, and competition for nutrients from other microbes. 

Bacillus subtilis strain QST 713 variant Soil applied to the soil would be expected to 

become part of the soil community and have no negative effect on the indigenous soil 

community.

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 detecting Bacillus subtilis strain QST 713 variant Soil is 

not applicable. It is expected that, when used as proposed, Bacillus subtilis strain 

QST 713 variant Soil would not result in residues that are of toxicological 

concern.

C. Mammalian Toxicological Profile

Bacillus subtilis strain QST 713 and Bacillus subtilis strain QST 713 variant Soil 

are produced under the same conditions; therefore, the resulting products are substantially 

 

1 Knudsen and Dandurand. 1995. Model For Dispersal And Epiphytic Survival Of Bacteria Applied To Crop Foliage. Department Of 

Plant, Soil And Entomological Sciences University Of Idaho, Moscow, ID. The 7th Symposium On Environmental Releases Of 

Biotechnology Products. June 6-8, 1995
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similar. It is not unreasonable to conclude, based on the similarity in product 

composition and production, measured physical/chemical, and pathogenicity / infectivity

toxicity data that data generated for Bacillus subtilis strain QST 713 can be used to bridge 

any data gaps for which there are no corresponding Bacillus subtilis strain QST 713 

variant Soil toxicity data. (This concept was proposed to and accepted by BPPD at a 

December 14, 2010 pre-registration meeting with AgraQuest.) At the request of the 

Agency a study to evaluate the safety to mammals and demonstrate equivalence of the 

strains has been conducted with the Bacillus subtilis strain QST 713 variant Soil and

compared to the same study conducted Bacillus subtilis strain QST 713, and is 

summarized as follows: 

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 two enlarged and/or discoloured spleens from the test group. The test 

organism had cleared completely from treatment group blood and brains by Day 21 and 

showed a pattern of clearance for the lungs, liver, spleen, kidneys mesenteric lymph 

nodes and cecum contents. 

The test substance was determined to be non-toxic, non-infective and non-pathogenic to 

rats when injected intravenously at a dose of 6.6 x 108cfu/rat.

Due to the similarity of the results for both Intravenous Toxicity / Pathogenicity Study in 

Rats conducted with Bacillus subtilis strain QST 713 and Bacillus subtilis strain QST 713 

variant Soil, it is not unreasonable to conclude, that data generated for Bacillus subtilis

strain QST 713 can be used to bridge any data gaps for which there are no corresponding 

Bacillus subtilis strain QST 713 variant Soil data. Therefore, the battery of OPPTS series 

885 studies that were conducted on Bacillus subtilis strain QST 713 technical powder to 

determine the potential detrimental effects to humans by the strain are representative of 

the toxicological / pathogenicity profile of the strain Bacillus subtilis strain QST 713 

variant Soil. Results from studies on file with Agency are summarized below.

OPPTS 

Guideline 

Number 

(OPP 

Number)

Study Title Test 

Substance Study Result Tox.Cat.

MRID #

885.3100

Acute Dermal 

Toxicity / 

Pathology

Rabbits

QST 713 

Technical 

Powder

LD50 >2000 

mg/kg;

Doses: 2.3E +11 –

1.73E+11 cfu 

/animal

no deaths 

occurred;

no signs of 

systemic toxicity

III 44651907
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885.3200

Acute 

Intravenous 

(Injection) 

Toxicity / 

Pathogenicity 

Rats 

QST 713 

Technical 

Powder

LD50 >9.4E+06 

cfu/animal 

not toxic or 

pathogenic; 

no mortality and 

no adverse clinical 

signs

N/A 44651908

885.3150

Acute 

(Intratracheal) 

Pulmonary

Toxicity / 

Pathogenicity

Rats

QST 713 

Technical 

Powder

LD50 >1.2E+08 

cfu/animal

in 0.1 ml

not toxic or 

pathogenic;

no mortality and 

no adverse effects 

N/A 44651909 

885.3400 Hypersensitivity

Incidents

QST 713 

Technical 

Powder

none reported

N/A

885.3050

(152-30)

Acute Oral 

Toxicity / 

Pathogenicity

Rats

QST 713 

Technical 

Powder

LD50 >1.13E+08 

cfu/animal

not toxic or 

pathogenic

IV 44651906 

870.2400 Acute Eye 

Irritation Rabbits

QST 713 

Technical 

Powder

mild or slight 

irritant III 44664602 

870.2500 Acute Dermal 

Irritation Rabbits

QST 713 

Technical

Powder

slight irritant

IV 44664601 

Toxicity Data on End-Use Product

Both Serenade ASO (EPA Reg. No. 69592-12), Serenade AS (EPA Reg. No. 695925-8) 

and Serenade Soil DPZ (EPA Reg. No. 69592-EI) contain the same amount of active 

ingredient, and based on the similarity in product composition and production, measured 

physical/chemical, and pathogenicity / infectivity toxicity data, it is not unreasonable to 

conclude that the end-use product toxicity data for Serenade AS, non-organic formulation 

and Serenade ASO, eligible for organic production under NOP regulations, support 

Serenade Soil DPZ acute toxicity Tier 1 870 series data requirements. Results for the 

currently registered products to support Serenade Soil DPZ are as follows:

OPPTS 

Guideline 

Number Study Title

Test 

Substance Study Result

Toxicity

Category MRID #

OPPTS 

870.1100

Acute Oral 

Toxicity Serenade AS LD50 >5000 mg/kg IV 45385304

OPPTS 

870.1200

Acute Dermal 

Toxicity Serenade AS LD50 >5000 mg/kg IV 45385305

OPPTS 

870.1300

Acute 

Inhalation 

Toxicity Serenade AS LC50 >1.45 mg/L III 45385306

OPPTS Acute Eye Serenade AS nonirritant IV 45385307
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870.2400 Irritation

OPPTS 

870.2500

Acute Dermal 

Irritation Serenade AS

mild or slight 

irritant IV 45385308

OPPTS 

870.2600

Dermal 

Sensitization Serenade AS not a sensitizer N/A 45385309

Serenade ASO contains the same certified limits of active ingredient as Serenade AS. All 

of the inert ingredients in Serenade ASO are from EPA’s Inert List, 4(a) or 4(b). Under 

the USDA’s National Organic Program the active ingredient also qualifies as an organic 

material, thus Serenade ASO is labeled in the USA for use in organic agriculture. 

Serenade ASO should be considered even more toxicologically benign and 

environmentally sound than Serenade AS. A comparison of the additives for the two 

products is located in MRID 46012201. By extension the data generated on Serenade AS 

is supportive of Serenade Soil DPZ.

The results of toxicity testing with Bacillus subtilis strain QST 713 and Bacillus subtilis

strain QST 713 variant Soil and information found in published literature, and EPA 

Bacillus subtilis Case 6012, Final Registration Review Decision (March 2010) on this 

strain and other strains of Bacillus subtilis indicate that no foreseeable risks to human 

health are to be expected by Bacillus subtilis strain QST 713 variant Soil.

D. Aggregate Exposure

1. Dietary exposure. Due to the ubiquitous nature of the organism, the 

concentrations of Bacillus subtilis and other closely related Bacillus species that already 

exist in the environment, the likelihood of increased risk to humans or animals from the 

use of Serenade Soil DPZ is low. EPA in its registration review decision concluded “the 

risk posed to adults, infants, and children is likely to be minimal because of the low acute 

oral toxicity/Pathogenicity potential of Bacillus subtilis strain QST 713.” Common food 

handling practices such as washing and peeling would also reduce any residues on crops. 

It is not unreasonable to conclude, based on the similarity in product composition and 

production, measured physical/chemical, and pathogenicity / infectivity toxicity data, that 

the risk from any potential exposure to Bacillus subtilis strain QST 713 variant Soil in 

Serenade Soil DPZ would be minimal.

i. Food. Due to the ubiquitous nature of the organism, the concentrations of 

Bacillus subtilis and other closely related Bacillus species that already exist in the 

environment, the likelihood of increased risk to humans or animals from the use of 

Serenade Soil DPZ is low. EPA in its registration review decision concluded “the risk 

posed to adults, infants, and children is likely to be minimal because of the low acute oral 

toxicity/Pathogenicity potential of Bacillus subtilis strain QST 713.” It is not 

unreasonable to conclude, based on the similarity in product composition and production, 

measured physical/chemical, and pathogenicity / infectivity toxicity data, that the risk 

from any potential exposure to Bacillus subtilis strain QST 713 variant Soil in Serenade 

Soil DPZ would be minimal.
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ii. Drinking water. As discussed in literature and EPA Bacillus subtilis Case 

6012, Final Registration Review Decision (March 2010), intake of low levels of 

ubiquitous Bacillus subtilis in drinking water may occur but exposure would represent a 

minimal risk due to the low toxicity of the strain. Similarly exposure to other strains of 

Bacillus subtilis would not represent a greater risk. It is not unreasonable to conclude, 

based on the similarity in product composition and production, measured 

physical/chemical, pathogenicity / infectivity toxicity data, that the risk from any

potential exposure to Bacillus subtilis strain QST 713 variant Soil in Serenade Soil DPZ 

would be minimal and equivalent to the risk from exposure to Bacillus subtilis strain QST 

713.

2. Non-dietary exposure. The use sites for Serenade Soil DPZ containing Bacillus 

subtilis strain QST 713 variant Soil include both agricultural and residential garden sites. 

Based on the toxicity information on file with the Agency, aggregate exposure to Bacillus 

subtilis strain QST 713 variant Soil would be below the levels in the safety testing 

conducted on Bacillus subtilis strain QST 713 and would not represent an undue risk due 

to the lack of residues of toxicological concern and the low toxicity of the product and 

active ingredient. Therefore, the non-dietary exposures to the general population, 

including infants and children would not be expected to pose any quantifiable risk due to 

a lack of residues of toxicological concern. Serenade Soil DPZ in the manufacturing 

plant (fermentation facility) will be minimal due to rigorous Good Manufacturing 

Practices and quality controls put in place to minimize contamination, cross 

contamination, and exposure to the workers, and also due to protective equipment worn 

by manufacturing plant workers. Therefore, inadvertent releases in the workplace would 

not be expected to increase the risk to workers, especially since high levels of the 

organism already exist in this environment. The EPA Risk Assessment of Bacillus 

subtilis (February, 1997) concludes that ``human health and environmental hazards of 

Bacillus subtilis are low'' and ``the number of microorganisms released from the 

fermentation facility is low''. Furthermore exposure to Bacillus subtilis strain QST 713 

variant Soil would be minimized by use of required personal protection equipment by

applicators, handlers and manufacturing workers. It is not unreasonable to conclude, 

based on the similarity in product composition and production, measured 

physical/chemical, and pathogenicity / infectivity toxicity data, that the risk from any 

potential exposure to Bacillus subtilis strain QST 713 variant Soil in Serenade Soil DPZ 

would be minimal and equivalent to the risk from exposure to Bacillus subtilis strain QST 

713 which has been fully assessed by the Agency. 

E. Cumulative Effects

According the EPA Bacillus subtilis Case 6012, Final Registration Review 

Decision (March 2010), no toxic mechanism has been associated with the mode of action 

of Bacillus subtilis strain QST 713; therefore, no cumulative toxic effect would be 

expected for this strain or with other related strains of Bacillus subtilis. Therefore, it is 

not unreasonable to conclude, based on the similarity in product composition and 

production, measured physical/chemical, and pathogenicity / infectivity toxicity data that 

no cumulative toxic effect would result from exposure to Bacillus subtilis strain QST 713 
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variant Soil in Serenade Soil DPZ. Therefore, the likely hood of adverse dietary effects 

is expected to be minimal for the general population and to infants and children.

F. Safety Determination

1. U.S. population. Bacillus subtilis strain QST 713 is not pathogenic or infective 

to mammals. There have been no reports of toxins associated with the organism and 

acute toxicity/pathogenicity data demonstrate that Bacillus subtilis strain QST 713 is nontoxic, non-pathogenic, and non-irritating or slightly irritating to the skin and eyes 

depending on the formulation type. Therefore, it is not unreasonable to conclude, based 

on the similarity in product composition and production, measured physical/chemical, 

and pathogenicity / infectivity toxicity data for Bacillus subtilis strain QST 713 and 

Bacillus subtilis strain QST 713 variant Soil that there is reasonable certainty of no harm 

to the general US population from exposure to residues of the substantially similar active 

ingredient Bacillus subtilis strain QST 713 variant Soil in Serenade Soil DPZ, when used 

as proposed.

2. Infants and children. As stated above, it is not expected that, when used as 

proposed, Bacillus subtilis strain QST 713 variant Soil would result in residues of 

toxicological concern. This conclusion is supported by the data on the substantially

similar strain Bacillus subtilis strain QST 713, public literature and EPA’s Bacillus 

subtilis Case 6012, Final Registration Review Decision (March 2010). There is 

reasonable certainly of no harm to infants and children from exposure to Bacillus subtilis

strain QST 713 variant Soil from the proposed uses. 

G. Effects on the Immune and Endocrine Systems

Bacillus subtilis strain QST 713 and Bacillus subtilis strain QST 713 variant 

Soil are naturally occurring, non-pathogenic organisms. There is no evidence to suggest 

that Bacillus subtilis strain QST 713 or the substantially similar Bacillus subtilis strain 

QST 713 variant Soil will compromise the immune or endocrine systems, or that it 

functions in a manner similar to any known hormone or endocrine disruptor.

H. Existing Tolerances

While there are no existing tolerances for Bacillus subtilis strain QST 

713 variant Soil, there is an exemption from tolerance for the substantially similar 

Bacillus subtilis strain QST 713. On July 5, 2000, EPA established an exemption 

from the requirement of a tolerance for residues of the Bacillus subtilis strain 

QST 713 in or on all raw agricultural commodities when applied/used according 

to label instructions in 40 CFR §180.1209 which reads as follows; 

An exemption from the requirement of a tolerance is established for residues of 

the microbial pesticide Bacillus subtilis strain QST 713 when used in or on all 

food commodities.
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FR Doc. 00-16803 Filed 7-3-00; 8:45 am

BILLING CODE 6560-50-F.

I. International Tolerances

There is no Codex alimentarius commission maximum residue level for 

Bacillus subtilis strain QST 713. 

EU - Maximum Residue Levels (Reg. (EC) No 396/2005) (MRLs) as amended by

Commission Regulation (EC) No 839/2008 of 31 July 2008 to include in Annex IV 

Bacillus subtilis strain QST 713 as an active substance for which no maximum 

residue levels (MRLs) in or on food and feed of plant and animal origin are 

required.