memorandum subject: human health risk assessment for …

OFFICE OF CHEMICAL SAFETY

AND POLLUTION PREVENTION

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C. 20460

MEMORANDUM

SUBJECT: Human Health Risk Assessment for the New Active Ingredients Bacillus

subtilis strain RTI477 and Bacillus velezensis strain RTI301 in the Proposed

Manufacturing-use Products 279-OAUT, 279-OAUI and End-use Products 279-

OAUO, 279-OALN and 279-OALR for FIFRA Section 3 Registration with

Tolerance Exemption Petitions

Decision Numbers: 549769, 549770, 549772, 549771, 549767, 549773, 549774

DP Numbers: 451944, 451948, 451958, 452003, 452028, 457332, 457333

Submission Numbers: 1032814, 1032816, 1032837, 1032818, 1032828, 1032815,

1032817

EPA File Symbols: 279-OAUT, 279-OAUI, 279-OALR, 279-OALN, 279-OAUO

Active Ingredient Names: Bacillus subtilis strain RTI477 and Bacillus velezensis strain

RTI301

PC Codes: 006552, 006553

Tolerance Exemption

Petitions: 9F8749 (B. subtilis strain RTI477),

9F8750 (B. velezensis strain RTI301)

Associated MRIDs: 50354402, 50354403, 50354404, 50354405, 50354406, 50354415,

50354501, 50354503, 50354504, 50354505, 50354506, 50354515,

50354601, 50354602, 50354603, 50354701, 50354702, 50354703,

50354801, 50354802, 50354803, 50354804, 50354805, 50354414,

50925702, 51255601, 50354411, 50354412, 50354413, 50354418,

50354514, 50925701, 51255501, 50354511, 50354512, 50354513,

50354518, 50354705, 51391802, 50354707, 50354706, 50354704,

50354708, 50354605, 51391801, 50354607, 50354606, 50354604,

50354608, 50354808, 51391803, 50354809, 50354810, 50354811,

50354807, 50644801, 50923401, 50644901, 50923601, 50354812

FROM: Mark Perry, Risk Assessment Process Leader

Risk Assessment Branch

Biopesticides and Pollution Prevention Division

THRU: Shannon Borges, Branch Chief

John Kough, Ph.D., Senior Scientist

Risk Assessment Branch


Active Ingredients: B. subtilis strain RTI477 / B. velezensis strain RTI301 PC Codes: 006552, 006553

Action: Human Health Risk Assessment

2

TO: Monica Thapa, Risk Manager

Microbial Pesticides Branch


I. Action Requested

Under Section 3 of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), FMC

Corporation has requested registration of two new food-use active ingredients, Bacillus subtilis

strain RTI477 (hereafter “Bacillus subtilis RTI477”) and Bacillus velezensis strain RTI301

(hereafter “Bacillus velezensis RTI301”). This application includes two proposed manufacturing-

use products, Bacillus subtilis RTI477 Technical (EPA File Symbol 279-OAUT) and Bacillus

velezensis RTI301 Technical (EPA File Symbol 279-OAUI), as well as three end-use products.

The proposed end-use products are F4034-5 (EPA File Symbol 279-OALN) containing Bacillus

subtilis RTI477 and Bacillus velezensis RTI301, F4007-9 (EPA File Symbol 279-OAUO)

containing Bacillus velezensis RTI301, and F4092-3 (EPA File Symbol 279-OALR) containing

Bacillus subtilis RTI477, Bacillus velezensis RTI301 and bifenthrin as the active ingredients.

The three end-use products (F4034-5, F4007-9 and F4092-3) are proposed for use on a wide

variety of crops to protect seedlings against fungal diseases primarily caused by Rhizoctonia,

Fusarium, and Phytophthora. Since F4092-3 contains bifenthrin as an active ingredient, it also

has insecticidal uses. In support of registration, the applicant has submitted product analysis data,

mammalian toxicology data, Confidential Statements of Formula (CSFs) and data matrices for

the proposed manufacturing-use and end-use products. Since FMC Corporation has requested the

establishment of tolerance exemptions for Bacillus subtilis RTI477 and Bacillus velezensis

RTI301 in or on all food commodities, tolerance exemption petitions under 9F8749 (Bacillus

subtilis RTI477) and 9F8750 (Bacillus velezensis RTI301) have also been submitted.

II. Executive Summary

FMC Corporation has applied for registration of the new food-use active ingredients Bacillus

subtilis strain RTI477 and Bacillus velezensis strain RTI301. The application includes two

proposed manufacturing-use products; Bacillus subtilis strain RTI477 Technical (EPA File

Symbol 279-OAUT) and Bacillus velezensis strain RTI301 Technical (EPA File Symbol 279-

OAUI), as well as three end-use products; F4034-5 (EPA File Symbol 279-OALN), F4007-9

(EPA File Symbol 279-OAUO) and F4092-3 (EPA File Symbol 279-OALR). Bacillus subtilis

and Bacillus velezensis are environmentally ubiquitous bacteria that produce endospores under

certain environmental conditions and are naturally resident in soil and on plant undergrowth.

Both Bacillus subtilis RTI477 and Bacillus velezensis RTI301 were isolated from environmental

sources within the United States.

Bacillus subtilis RTI477 colonizes plant seed and root surfaces and obstructs development of

pathogenic organisms through competition for space and nutrients. There are multiple modes of

action for how this is achieved, including the ability of Bacillus subtilis RTI477 to produce



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volatile compounds and metabolites that enhance plant growth, as well as secondary metabolites

that are antagonistic against certain fungal pathogens. Bacillus subtilis RTI477 is also capable of

stimulating the host plant’s immune system, which results in Induced Systemic Resistance (ISR)

and Systemic Acquired Resistance (SAR) responses (MRID 50925702). The pesticidal modes of

action for Bacillus velezensis RTI301 are very similar to those for Bacillus subtilis RTI477, as

this microbe is also known to produce volatile compounds and metabolites that enhance plant

growth, in addition to secondary metabolites that are antagonistic against fungal pathogens.

Bacillus velezensis RTI301 is also capable of increasing ISR and SAR responses in the host plant

(MRID 50925701).

The end-use product, F4034-5 is proposed for use as a bio-fungicide to protect seedlings against

diseases caused by Rhizoctonia, Fusarium, and Phytophthora. F4034-5 contains 2.5% B. subtilis

RTI477 and 7.5% B. velezensis RTI301. The minimum viability of Bacillus subtilis RTI477 and

Bacillus velezensis RTI301 in this product is 1.15 x 109 CFU/mL and 1.5 x 1010 CFU/mL,

respectively. This product is intended for application to a variety of crops/seed including corn,

dried bean/pea, soybean, fruiting vegetables, cucurbit vegetable, cereal grains, oil seed,

root/tuber vegetables, cotton and brassica vegetables. The application methods include in-furrow,

at-transplant drench water treatment and seed treatment. Seed treatment application are

performed by mechanical, slurry, or mist-type seed treating equipment. The application rates

given for in-furrow applications and transplant drench water applications for all crops on the

proposed label are 4 to 10 ounces of product per acre diluted in a minimum of five gallons of

water. The application rates for seed treatment applications are given on a per seed weight basis

or on a per seed unit basis depending on the crop being treated, with a maximum rate of 0.27 fl.

oz./100,000 seeds.

F4007-9 is an end-use product intended only for use as a seed treatment to protect seedlings

against diseases caused by Rhizoctonia, Fusarium, and Phytophthora. This product contains

5.5% Bacillus velezensis RTI301 (minimum viability of 1.1 x 1010 CFU/mL) as the sole active

ingredient. F4007-9 is intended for application to seed for corn, dried bean/pea, soybean, fruiting

vegetables, cucurbit vegetable, cereal grains, oil seed, root/tuber vegetables and cotton.

Application rates for this product are given on a per seed weight basis or on a per seed unit basis

depending on the crop being treated, with a maximum rate of 0.34 fl. oz./100,000 seeds. The

application instructions indicate that the product may be applied to the seeds with mechanical,

slurry, or mist-type seed treating equipment.

F4092-3 is an end-use product intended for use as combination bio-fungicide and insecticide to

protect seedlings against nematodes as well as diseases caused by Rhizoctonia solani, Fusarium

spp., Pythium spp. and Phytophthora spp. F4092-3 contains Bacillus subtilis RTI477 (2.5%),

Bacillus velezensis RTI301 (2.5%) and bifenthrin (15.7%) as the active ingredients. The

minimum viability of Bacillus subtilis RTI477 and Bacillus velezensis RTI301 in this product is

5.0 x 109 CFU/mL for each microbe. The product is proposed for use on a variety of crops

including artichoke, brassica vegetables, berries, citrus, cucurbit, beans/peas, eggplant, grapes

and many tuber/root vegetables. Application methods include in-furrow at plant, pre-plant

incorporated, pre-emergent, lay-by in-furrow (with seed), as a T-band (over the open furrow), as



4

a broadcast (to soil) and in transplant drench water during setting. The field application rates

range from 2.9 to 42.5 fl. oz./acre. The product is applied in a minimum of five gallons of water

per acre for all field applications except for citrus which must be applied in a minimum of 40

gallons of water per acre. The directions for use on citrus crops instruct the applicator to apply

F4092-3 by ground equipment to bare soil beneath citrus trees from the trunk to the drip line of

the trees.

The generic and product-specific mammalian toxicology data performed with Bacillus subtilis

RTI477 and/or Bacillus velezensis RTI301 as the only active ingredients did not exhibit any

toxicity, infectivity or pathogenicity. While moderate toxicity/irritation was observed in the acute

oral, primary eye irritation and primary dermal irritation testing for the end-use product F4092-3

(EPA File Symbol 279-OALR), these results are very likely due to the presence of bifenthrin as

an active ingredient in this product.

Food and drinking water exposures to Bacillus subtilis RTI477 and Bacillus velezensis RTI301

are expected to be negligible as significant residues are not likely due to the low application rates

and the use of primarily soil-directed applications early in the growing season. There is potential

for occupational exposure, however, no toxicological endpoints have been identified in guideline

mammalian toxicology studies except for one end-use product containing bifenthrin as an active

ingredient (F4092-3; EPA File Symbol 279-OALR). Potential occupational risks resulting from

the use of F4092-3 should be adequately addressed through product label restrictions/warnings

and the use of required personal protective equipment (PPE). Residential exposure resulting from

the pesticidal use of these active ingredients is expected to be minimal. While residential use of

treated turf seed is allowed on two of the end-use product labels, the likelihood of significant

exposure from this use is very low. Considering the results of the mammalian toxicology data

review and the available exposure data/information, the Agency has determined that no further

studies are needed at this time on Bacillus subtilis RTI477 and Bacillus velezensis RTI301.

Based on the available toxicology and exposure information, no unreasonable adverse effects to

humans are expected from the use of Bacillus subtilis strain RTI477 and Bacillus velezensis

strain RTI301 as pesticides when used in accordance with the product label instructions and

requirements. Further, 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 RTI477 and Bacillus velezensis strain RTI301 resulting from the proposed

pesticidal uses.

III. Background

Bacillus subtilis RTI477 and Bacillus velezensis RTI301 are naturally occurring microorganisms

that have been discovered to have fungicidal and plant health promoting characteristics. Bacillus

subtilis and Bacillus velezensis are found in many environmental habitats including in/on organic

material/plant undergrowth, plant roots and in soil samples. These active ingredients are

proposed for use primarily in agricultural settings as food use bio-fungicides. Exposure to

Bacillus subtilis and Bacillus velezensis occurs naturally and is likely a common component of



5

the human diet via its presence on surfaces of plant-based foods. When used as pesticidal active

ingredients Bacillus subtilis RTI477 and Bacillus velezensis RTI301 are not expected to result in

significant residues in/on food or in water due to the specific use patterns and application

methods employed. According to the results of toxicity/infectivity and acute toxicity studies,

Bacillus subtilis RTI477 and Bacillus velezensis RTI301 have a low toxicity profile and no

toxicological endpoints were identified in studies containing these microorganisms as the only

active ingredients.

IV. Product Identity and Analysis Review

The applicant submitted data to comply with product identity and analysis data requirements

published in 40 CFR § 158.2120 in support of the registration of Bacillus subtilis RTI477 and

Bacillus velezensis RTI301, as well as the products containing these active ingredients. In

addition, certain product identity and analysis data requirements published in 40 CFR § 158.310

were provided to support EPA File Symbol 279-OALR which contains bifenthrin. The product

identity, manufacturing process, discussion of formation of unintentional ingredients, analysis of

samples, certification of limits, storage stability/corrosion characteristics, and physical/chemical

characteristics data submitted for these products are acceptable. See Appendix I for additional

details on the data supporting these requirements.

V. Summary of Toxicology Data

Tables 1 and 2 below provide the status of the generic or technical-grade active ingredient

(TGAI) data requirements for human health risk assessment as published in 40 CFR § 158.2140

for Bacillus subtilis RTI477 and Bacillus velezensis RTI301, respectively. Table 3 provides the

status of the product-specific data requirements for human health risk assessment as published in

40 CFR § 158.2140. Specifically, Table 3 summarizes the manufacturing-use (EPA File Symbols

279-OAUT and 279-OAUI) and end-use (EPA File Symbols 279-OALN, 279-OAUO and 279-

OALR) products containing Bacillus subtilis RTI477 and/or Bacillus velezensis RTI301 as active

ingredients. Note that scientific rationales were submitted to satisfy the generic acute oral

toxicity/pathogenicity and acute injection toxicity/pathogenicity data requirements for Bacillus

subtilis RTI477, and the acute injection toxicity/pathogenicity data requirement for Bacillus

velezensis RTI301. Additionally, scientific rationales were submitted to satisfy several of the

manufacturing-use product (MP) and end-use product (EP) data requirements.

The information provided satisfies the Tier I toxicology data requirements for the human health

risk assessment for Bacillus subtilis RTI477 and Bacillus velezensis RTI301, as well as the

associated pesticide products. Further testing at higher tiers is not required for the current uses

identified in the proposed pesticide labels. While moderate toxicity/irritation was observed in the

acute oral (870.1100), primary eye irritation (870.2400) and primary dermal irritation (870.2500)

testing for the end-use product F4092-3 (EPA File Symbol 279-OALR), these findings are very

likely due to the presence of bifenthrin as an active ingredient in this product. This conclusion is

supported by the fact that the testing for the MPs and EPs containing only Bacillus subtilis



6

RTI477 and/or Bacillus velezensis RTI301 as active ingredients did not demonstrate significant

acute oral toxicity or eye/dermal irritation.

Table 1. Summary of generic data submitted to comply with toxicology data requirements

published in 40 CFR § 158.2140 for support of the registration of products containing Bacillus

subtilis strain RTI477.

Data Requirement

OCSPP

Guideline No.

Summary, Results and Classification

MRID

Generic Bacillus subtilis RTI477 Toxicology Data

Acute Oral

Toxicity/Pathogenicity

885.3050 Testing waived based on the applicant’s scientific

rationale which cited the lack of toxicity and/or

pathogenicity in MRID Nos. 50354403 (acute oral

toxicity [870.1100] study conducted with the B. subtilis

MP) and 50354402 (acute pulmonary

toxicity/pathogenicity study conducted with the TGAI).

In addition, the applicant referenced supporting

literature which indicates that B. subtilis is very

unlikely to cause toxicity/pathogenicity in humans.

Classification: Acceptable

50354415

Acute Pulmonary


885.3150

A study was provided that demonstrated a pattern of

clearance for B. subtilis RTI477 from the organs of

treated animals. Not toxic, infective, and/or pathogenic

to rats when administered by intra-tracheal instillation

in a single dose of 3.08 x 108 CFU/animal.


50354402

Acute Injection


885.3200

Testing waived based on the applicant’s scientific

rationale which cited the lack of toxicity and/or

pathogenicity in MRID Nos. 50354403 (acute oral

toxicity [870.1100] study conducted with the B. subtilis

MP) and 50354402 (acute pulmonary

toxicity/pathogenicity study conducted with the TGAI).

In addition, the applicant referenced supporting

literature which indicates that B. subtilis is very

unlikely to cause toxicity/pathogenicity in humans.


50354415

Hypersensitivity

Incidents

885.3400

The applicant reported that no hypersensitivity incidents

or other adverse effects occurred during research,

development, manufacturing or handling of the TGAI.

Any future hypersensitivity incidents must be reported

to the EPA (refer to test note #3 of 40 CFR §

158.2140(d)).


50354415

Cell Culture 885.3500

Not required because B. subtilis RTI477 is not a virus

(refer to test note #4 of 40 CFR § 158.2140(d)).


50354415

Table 2. Summary of generic data submitted to comply with toxicology data requirements

published in 40 CFR § 158.2140 for support of the registration of products containing Bacillus

velezensis strain RTI301.



7

Data Requirement

OCSPP

Guideline No.

Summary, Results and Classification

MRID

Generic Bacillus velezensis RTI301 Toxicology Data

Acute Oral


885.3050 A study was provided that showed a pattern of

clearance for B. velezensis RTI301 from the organs of


to rats when administered orally in a single dose of 2.1

x 108 CFU/animal.


50354506

Acute Pulmonary


885.3150

A study was provided that showed a pattern of

clearance for B. velezensis RTI301 from the organs of


to rats when administered by intra-tracheal instillation

in a single dose of 5.27 x 108 CFU/animal.


50354501

Acute Injection


885.3200


rationale which cited the lack of toxicity/pathogenicity

in MRID Nos. 50354506 (acute oral toxicity/

pathogenicity study conducted with the TGAI),

50354501 (acute pulmonary toxicity/pathogenicity

study conducted with the TGAI) and 50354503 (acute

dermal toxicity on the B. velezensis MP). In addition,

the applicant referenced supporting literature which

indicates that B. velezensis (which is closely related to

B. subtilis) is very unlikely to cause

toxicity/pathogenicity in humans.


50354515

Hypersensitivity

Incidents

885.3400

The applicant reported that no hypersensitivity incidents





158.2140(d)).


50354515

Cell Culture 885.3500

Not required because B. velezensis RTI301 is not a

virus (refer to test note #4 of 40 CFR § 158.2140(d)).


50354515

Table 3. Summary of product-specific data submitted to comply with toxicology data

requirements (40 CFR § 158.2140) for support of the registration of manufacturing-use products

and end-use products containing B. subtilis strain RTI477 and/or B. velezensis strain RTI301.

Data Requirement

OCSPP

Guideline No.

Results Summary, Classification and Toxicity

Category

MRID

Product-specific (MP) Toxicology Data - Bacillus subtilis strain RTI477 Technical (EPA File Symbol

279-OAUT)

Acute Oral Toxicity 870.1100

Oral LD50 (female rats) > 5,000 mg/kg


Toxicity Category: IV

50354403



8

Data Requirement

OCSPP

Guideline No.


Category

MRID

Acute Dermal

Toxicity

870.1200

Dermal LD50 (male and female rats) > 5,000 mg/kg



50354404

Acute Inhalation

Toxicity

870.1300 Testing waived based on oral study with TGAI and

because this formulation contains inert ingredients that

are not expected to be of toxicological concern (refer to

test note #5 of 40 CFR § 158.2140(d)).



50354415

Primary Eye Irritation 870.2400 The product was mildly irritating to the eyes of rabbits.

No corneal opacity, iritis, conjunctivitis, or other signs

of ocular irritation were noted at 24 hours post

installation.



50354405

Primary Dermal

Irritation

870.2500 The product was slightly irritating to the skin of rabbits.

All animals were free of slight erythema or edema at 72

hours.



50354406

Hypersensitivity

Incidents

885.3400 The applicant reported that no hypersensitivity incidents





158.2140(d)).


50354415

Product-specific (MP) Toxicology Data – Bacillus velezensis strain RTI301 Technical (EPA File Symbol

279-OAUI)




in MRID Nos. 50354506 (acute oral toxicity/

pathogenicity study conducted with the TGAI),

50354501 (acute pulmonary toxicity/pathogenicity

study conducted with the TGAI) and 50354503 (acute

dermal toxicity on the B. velezensis MP). In addition,

the applicant referenced supporting literature which

indicates that B. velezensis is very unlikely to cause

toxicity/pathogenicity in humans, and inert ingredients

are not expected to be of toxicological significance.



50354515

Acute Dermal

Toxicity

870.1200

Dermal LD50 (male and female rats) > 5,000 mg/kg



50354503



9

Data Requirement

OCSPP

Guideline No.


Category

MRID

Acute Inhalation

Toxicity



in MRID Nos. 50354501 (acute pulmonary

toxicity/pathogenicity study conducted with the TGAI)

and 50354503 (acute dermal toxicity on the B.

velezensis MP). In addition, the applicant referenced

supporting literature which indicates that B. velezensis

is very unlikely to cause toxicity/pathogenicity in

humans, and inert ingredients are not expected to be of

toxicological significance.



50354515

Primary Eye Irritation 870.2400 The product was mildly irritating to the eyes of rabbits.

No positive effects (corneal opacity, iritis,

conjunctivitis, or other signs of ocular irritation) were

noted at any time point in the study.



50354504

Primary Dermal

Irritation

870.2500 The product was slightly irritating to the skin of rabbits.

All animals were free of significant erythema or edema

by 72 hours.



50354505

Hypersensitivity

Incidents






158.2140(d)).


50354515

Product-specific (EP) Toxicology Data - F4034-5 (EPA File Symbol 279-OALN)



rationale which cited studies performed with Bacillus

subtilis RTI477 (MRIDs 50354403, 50354404,

50354402) and Bacillus velezensis RTI301 (MRIDs

50354503, 50354506, 50354501), and because this

formulation contains inert ingredients that are not

expected to be of toxicological concern (refer to test

note #5 of 40 CFR § 158.2140(d)).



50354703



10

Data Requirement

OCSPP

Guideline No.


Category

MRID

Acute Dermal

Toxicity

870.1200




50354402, 50354406) and Bacillus velezensis RTI301

(MRIDs 50354503, 50354506, 50354501, 50354505),

and because this formulation contains inert ingredients

that are not expected to be of toxicological concern

(refer to test note #5 of 40 CFR § 158.2140(d)).



50354703

Acute Inhalation

Toxicity




50354402) and Bacillus velezensis RTI301 (MRIDs

50354503, 50354506, 50354501), and because this

formulation contains inert ingredients that are not

expected to be of toxicological concern (refer to test

note #5 of 40 CFR § 158.2140(d)).



50354703

Primary Eye Irritation 870.2400 Treated eyes were free of all irritation by 48 hours. The

product was minimally irritating to the rabbit eye.



50354701

Primary Dermal

Irritation

870.2500 The product elicited well-defined irritation on rabbit

skin at 72 hours.



50354702

Hypersensitivity

Incidents



development, manufacturing or handling of these

TGAIs. Any future hypersensitivity incidents must be

reported to the EPA (refer to test note #3 of 40 CFR §

158.2140(d)).


50354703

Product-specific (EP) Toxicology Data - F4007-9 (EPA File Symbol 279-OAUO)




velezensis RTI301 (MRIDs 50354503, 50354506,

50354501), and because this formulation contains inert

ingredients that are not expected to be of toxicological

concern (refer to test note #5 of 40 CFR § 158.2140(d)).



50354603



11

Data Requirement

OCSPP

Guideline No.


Category

MRID

Acute Dermal

Toxicity

870.1200




50354501, 50354505), and because this formulation

contains inert ingredients that are not expected to be of

toxicological concern (refer to test note #5 of 40 CFR §

158.2140(d)).



50354603

Acute Inhalation

Toxicity




50354501), and because this formulation contains inert

ingredients that are not expected to be of toxicological

concern (refer to test note #5 of 40 CFR § 158.2140(d)).



50354603

Primary Eye Irritation 870.2400 The product elicited very mild irritation in rabbit eyes at

24-hours after treatment. No corneal opacity, iritis,

conjunctivitis, or other signs of ocular irritation was

noted at 48-hours after treatment.



50354601

Primary Dermal

Irritation

870.2500 The product was a slight irritant to the skin of rabbits.

All animals were free of slight erythema at 7 days after

treatment.



50354602

Hypersensitivity

Incidents



development, manufacturing or handling of the TGAI

(B. velezensis RTI301). Any future hypersensitivity

incidents must be reported to the EPA (refer to test note

#3 of 40 CFR § 158.2140(d)).


50354603

Product-specific (EP) Toxicology Data - F4092-3 (EPA File Symbol 279-OALR)


Oral LD50 (female rats) 748.8 mg/kg


Toxicity Category: III

50354801

Acute Dermal

Toxicity

870.1200

This study requirement is waived; the acute toxicity

category is based on the acute oral toxicity study

results. Classification: Acceptable


50354806

Acute Inhalation

Toxicity

870.1300 Inhalation LC50 (male and female rats) > 2.04 mg/L



50354802



12

Data Requirement

OCSPP

Guideline No.


Category

MRID

Primary Eye Irritation 870.2400 The product elicited redness and chemosis in all rabbit

eyes at 24 hours. No corneal opacity, iritis,

conjunctivitis, or other signs of ocular irritation were

noted by 72 hours after treatment.



50354804

Primary Dermal

Irritation

870.2500 The product elicited moderate to severe erythema and

moderate edema at 72 hours. All animals were free of

abnormalities by day 14.


Toxicity Category: II

50354803

Dermal Sensitization

870.2600 The study results indicate that the product is a dermal

sensitizer under the test parameters of the Local Lymph

Node Assay (LLNA).


Toxicity Category: Sensitizer (Positive)

50354805

A. Toxicology Study Summaries

1. Generic (TGAI) Toxicology Data for Bacillus subtilis RTI477

Study Title: 885.3150; Acute Pulmonary Toxicity/Pathogenicity (B. subtilis RTI477)

MRID No.: 50354402


Study Summary: In this study, rats received a dose of 3.08 x 108 viable spores in 0.1 ml

of dosing solution by intra-tracheal administration, while a control group remained untreated.

Lung, brain, liver, spleen, kidney, lymph nodes, cecum contents and blood were collected 4,

8, 15, 22, 36, 57, and 85/86 days after dosing, and lung samples were collected soon after

dosing on Day 1. There was no mortality in the study animals and no significant effect on

body weight or body weight gain associated with strain RTI477. A pattern of microbial clearance

from the organs of treated animals was demonstrated during the study. Although this study

demonstrated low level persistence in the lungs on days 85/86, this finding is not uncommon for

spore-forming microbes. Further, with the absence of adverse clinical signs and the

establishment of a pattern of clearance, this persistence is not considered significant.

2. Generic (TGAI) Toxicology Data for Bacillus velezensis RTI301

Study Title: 885.3050; Acute Oral Toxicity/Pathogenicity (B. velezensis RTI301)

MRID No.: 50354506


Study Summary: In this study, male and female rats received an oral dose of 2.1 x 108

CFU/animal. Various tissue samples were collected 2, 3, 7, 14, and 21 days after dosing and

cultured for detecting Bacillus velezensis strain RTI301. There were no mortalities or test

substance-related clinical signs observed during the study. No macroscopic findings at scheduled



13

sacrifice. Bacillus velezensis strain RTI301 was completely cleared from all cultured tissue

samples evaluated during the study.

Study Title: 885.3150; Acute Pulmonary Toxicity/Pathogenicity (B. velezensis RTI301)

MRID No.: 50354501


Study Summary: In this study, male and female rats received a dose of 5.27 x 108 spores

inactivated (Group 2) or viable (Group 3) in 0.1 ml of dosing solution by intra-tracheal

administration, while a third group (Group 1) remained untreated. No mortality was observed in

the study animals and no test substance-related effects on body weight or body weight gain for

either male or female rats during the study. A pattern of microbial clearance from the organs of

treated animals was demonstrated for B. velezensis RTI301during the study. Although this study

demonstrated low level persistence in the lungs on days 85/86, this finding is not uncommon for

spore-forming microbes. Further, with the absence of adverse clinical signs and the

establishment of a pattern of clearance, this persistence is not considered significant.

3. Product-specific (MP) Toxicology Data – Bacillus subtilis strain RTI477

Technical (EPA File Symbol 279-OAUT)

Study Title: 870.1100; Acute Oral Toxicity

MRID No.: 50354403



Study Summary: Three fasted Sprague-Dawley-derived female rats were treated by oral gavage

with Bacillus subtilis strain RTI477 as a 20% w/w mixture in distilled water at a dose level of

5000 mg/Kg bw (24.20 mL/Kg volume) divided into two approximately equal portions and

administered two hours apart. There was no perceptible change in viable test substance during

the observation period. There were no deaths, abnormal clinical signs, or abnormal gross

necropsy findings. All the animals gained weight during both weeks of the study. The acute oral

lethal dose of the test substance is greater than 5000 mg/Kg bw.

Study Title: 870.1200; Acute Dermal Toxicity

MRID No.: 50354404



Study Summary: Five male and five female fasted young adult Sprague-Dawley derived albino

rats were dermally exposed for 24 hours to Bacillus subtilis strain RTI477 as a 55% w/w mixture

with distilled water applied to a 6-square-inch area of previously shaved, intact skin (~9.6% to

12.8% of the body surface area) at a dose of 5000 mg/Kg bw. Following exposure on Day 0, the

animals were observed for a period of 14 days. There were no deaths, abnormal clinical signs, or

abnormal gross necropsy findings. All the animals gained weight during both weeks of the study.

The acute dermal lethal dose of Bacillus subtilis strain RTI477 is greater than 5000 mg/Kg bw.

Study Title: 870.2400; Primary Eye Irritation



14

MRID No.: 50354405



Study Summary: A 0.1 mL volume (0.056 g) of Bacillus subtilis strain RTI477 was instilled

into the conjunctival sac of the unanesthetized right eye of three 17-week-old female New

Zealand White rabbits, and the upper and lower lids were held shut for approximately one

second, while the untreated left eye of each animal served as control. Eyes were scored for ocular

irritation according to the Draize method at 1, 24, 48, and 72 hours after instillation, with

fluorescein staining done at 24 hours. There were no observations of corneal opacity, iritis, or

“positive” conjunctival findings at any time during the study. Based on the MMTS of 6.0 (and

non-zero scores at 48 hours), Bacillus subtilis strain RTI477 is mildly irritating to the eye (Kay

and Calandra, 1962). Bacillus subtilis strain RTI477 caused minimal effects that cleared within

24 hours.

Study Title: 870.2500; Primary Dermal Irritation

MRID No.: 50354406



Study Summary: Three young adult female New Zealand albino rabbits were dermally exposed

for 4 hours to 500 mg of Bacillus subtilis strain RTI477 in distilled water (60% w/w; 0.83 g of

mixture) applied via a 4-ply gauze pad to intact, previously clipped application sites measuring 6

cm2 and then covered by a semi-occlusive dressing. The application sites were scored for

irritation according to the Draize method at 30-60 minutes and at 1, 24, 48, and 72 hours after

patch removal. At 30-60 minutes following patch removal, all three sites showed very slight,

barely perceptible erythema (score=1), and two sites also had very slight, barely perceptible

edema (score=1). At 24 hours, the three sites had very slight erythema without edema. At 48 and

72 hours, all sites were free of erythema, edema, and other signs of dermal irritation. The

Primary Dermal Irritation Index was 0.67. Based on the PDII, the test material is a slight irritant.

4. Product-specific (MP) Toxicology Data – Bacillus velezensis strain RTI301

Technical (EPA File Symbol 279-OAUI)

Study Title: 870.1200; Acute Dermal Toxicity

MRID No.: 50354503



Study Summary: Five male and five female fasted young adult Sprague-Dawley derived albino

rats were dermally exposed for 24 hours to Bacillus velezensis Strain RTI301 manufacturing-use

product as an 80% w/w mixture with distilled water applied to a 6-squareinch area of previously

shaved, intact skin (~8.4% to 12.9% of the body surface area) at a dose of 5000 mg/Kg bw.

Following exposure on Day 0, the animals were observed for a period of 14 days. There were no

deaths, abnormal clinical signs, or abnormal gross necropsy findings. All animals gained weight

during both weeks of the study. The acute dermal lethal dose of Bacillus velezensis Strain

RTI301 is greater than 5000 mg/Kg bw.



15


MRID No.: 50354504



Study Summary: A 0.1 mL volume (0.044 g) of Bacillus velezensis Strain RTI301 was instilled

into the conjunctival sac of the unanesthetized right eye of three 11-week-old female New

Zealand White rabbits, and the upper and lower lids were held shut for approximately one

second, while the untreated left eye of each animal served as control. Eyes were scored for ocular



“positive” conjunctivitis at any time during the study. “Non-positive” conjunctivitis was noted in

the three treated eyes: one-hour post instillation, conjunctival redness and discharge were seen in

all three treated eyes, and one treated eye also had chemosis (all scores=1); at 24 hours, chemosis

was noted in all three treated eyes, with redness and discharge each noted in 2/3 eyes (all

scores=1). All eyes were clear of abnormal findings at 48 and 72 hours. Bacillus velezensis

Strain RTI301 is minimally irritating to the eye.


MRID No.: 50354505




for 4 hours to 0.5 g of Bacillus velezensis Strain RTI301 as an 80% w/w mixture with distilled

water applied to intact, previously clipped application sites measuring 6 cm2 then covered by a

semi-occlusive dressing. Following patch removal, any residual test substance was removed via

gentle cleansing with a 3% soap solution followed by tap water and a clean paper towel. The

application sites were scored for irritation according to the Draize method at 30-60 minutes, at 1,

24, 48, and 72 hours, and at 7 and 10 days after patch removal. At 30-60 minutes following patch

removal, all three sites had very slight erythema, and one site also had very slight edema. At 24,

48, and 72 hours, two sites exhibited both very slight erythema and very slight edema, and the

third site was normal. On Day 7, one site had very slight erythema without edema, and two sites

were normal. All sites were free of erythema, edema, and/or other signs of skin irritation on Day

10. The Primary Dermal Irritation Index was 1.33.

5. Product-specific (EP) Toxicology Data - F4034-5 (EPA File Symbol 279-OALN)


MRID No.: 50354701



Study Summary: A 0.1 mL volume of undiluted F4034-5 was instilled into the conjunctival sac

of the unanesthetized right eye of three 17-week-old female New Zealand White rabbits, and the

upper and lower lids were held shut for approximately one second, while the untreated left eye of



16

each animal served as control. Eyes were scored for ocular irritation according to the Draize

method at 1, 24, 48, and 72 hours after instillation, with fluorescein staining done at 24 hours.

There were no observations of corneal opacity or iritis at any time during the study. Fluorescein

staining at 24 hours verified the absence of corneal opacity. The Maximum Mean Total Score

(MMTS) of F4034-5 was 6.7, noted one-hour post treatment.


MRID No.: 50354702




for 4 hours to 0.5 mL of undiluted F4034-5 applied to intact, previously clipped application sites

measuring 6 cm2 and covered by a semi-occlusive dressing. Following patch removal, any

residual test substance was removed via gentle cleansing with a 3% soap solution followed by

tap water and a clean paper towel. The application sites were scored for irritation according to

the Draize method at 30-60 minutes, at 1, 24, 48, and 72 hours, and at 7 days after patch

removal. At 30-60 minutes following patch removal, all three sites showed very slight or well-

defined erythema with very slight or slight edema. The erythema persisted, unchanged, on the

three application sites through 72 hours and resolved by Day 7. At 24 and 48 hours, one site was

free of edema, and the other two sites had very slight edema or slight edema. At 72 hours, very

slight edema was present on two sites, and all sites were free of edema by Day 7. The Primary

Dermal Irritation Index was 2.7.

6. Product-specific (EP) Toxicology Data – F4007-9 (EPA File Symbol 279-OAUO)


MRID No.: 50354601



Study Summary: A 0.1 mL volume of undiluted F4007-9 was instilled into the conjunctival sac

of the right eye of three young adult female New Zealand White rabbits, and the upper and lower

lids were held shut for approximately one second, while left eye of each animal was not treated

with the test material and served as a control. Topical anesthetic was not used for the first animal

treated. Because the initial treated animal exhibited signs of distress following dosing, an

unspecified topical anesthetic was used for the remaining two animals; it is unknown whether the

contralateral eyes of these two animals were also anesthetized. Eyes were scored for ocular



“positive” conjunctivitis at any time during the study. Fluorescein staining at 24 hours verified

the absence of opacity. The Maximum Mean Total Score (MMTS) of F4007-9 was 4.0, noted

one-hour post treatment.


MRID No.: 50354602



17



Study Summary: Three young adult female New Zealand albino rabbits dermally exposed for 4

hours to 0.5 mL of undiluted F4007-9 applied to intact, previously clipped application sites

measuring 6 cm2 and covered by a semi-occlusive dressing. Following patch removal, any

residual test substance was removed via gentle cleansing with a 3% soap solution followed by

tap water and a clean paper towel. The application sites were scored for irritation according to

the Draize method at 30-60 minutes, at 1, 24, 48, and 72 hours, and at 7 days after patch

removal. At 30-60 minutes following patch removal, all three sites showed very slight or well-

defined erythema with very slight or slight edema. At 24 hours, one site had well-defined

erythema with very slight edema, one site had very slight erythema without edema, and one site

was free of abnormal findings. At 48 hours, very slight erythema was present without edema on

two sites, and the third site remained free of abnormal findings. At 72 hours, one site had very

slight erythema without edema, one site had desquamation without other abnormalities, and one

site was normal. All sites were free of erythema, edema, and other signs of skin irritation by Day

7. The Primary Dermal Irritation Index was 1.3.

7. Product-specific (EP) Toxicology Data - F4092-3 (EPA File Symbol 279-OALR)

Study Title: 870.1100; Acute Oral Toxicity

MRID No.: 50354801



Study Summary: Eight fasted 8-10-week-old female Sprague-Dawley-derived rats were given

single oral gavage doses of undiluted F4092-3 at dose levels of 110 mg/Kg bw (1 animal), 350

mg/Kg bw (2 animals), 1100 mg/Kg bw (3 animals), or 2000 mg/Kg bw (2 animals) with dose

selection, progression, and stopping criteria in accordance with AOT 425 (limit dose: 2000

mg/Kg bw; assumed LD50: 1100 mg/Kg bw; sigma: 0.5). Dosing was on Day 0, and the animals

were observed for up to 14 days. The animal treated with 110 mg/Kg bw remained active and

healthy throughout the study duration. One 350-mg/Kg animal was found dead on Day 1 after

exhibiting irregular respiration and tremors on Day 0. The surviving 350-mg/Kg animal

exhibited hypoactivity and irregular respiration following dosing on Day 0, had irregular

respiration, slight tremors, and reduced fecal volume on Day 1, and appeared active and healthy

on Day 2 and thereafter. One 1100-mg/Kg animal was found dead on Day 1 after exhibiting

irregular respiration and tremors on Day 0. The two surviving 1100-mg/Kg animals exhibited

hypoactivity and irregular respiration on Days 0 and/or 1, with reduced fecal volume and

piloerection or hypersensitivity on Day 1; both recovered by Day 3 and appeared active and

healthy thereafter. Both 2000-mg/Kg animals died: one was found dead on Day 1 after exhibiting

irregular respiration and hypoactivity on Day 0; and the other was found dead on Day 2 without

any preceding abnormal clinical signs. The surviving animals gained weight during both weeks

of the study.

Study Title: 870.1300; Acute Inhalation Toxicity

MRID No.: 50354802



18



Study Summary: Five male and five female 9-10-week-old Sprague-Dawley-derived rats were

exposed by the nose-only inhalation route to aerosolized undiluted F4092-3 for four hours at a

mean gravimetric concentration of 2.04 mg/L (mean MMAD 2.98 μm and GSD of 2.23).

Exposure was on Day 0, and the animals were observed for 14 days. There were no deaths or

abnormal gross necropsy findings. Upon removal from the chamber and/or beginning on Day 0,

all the animals had moist rales, irregular respiration, and hypoactivity, with abnormal gait and

tremors noted from each of two animals (four animals in total). All the animals subsequently

exhibited anogenital staining, with or without red nasal discharge, beginning on Day 1. Tremors

were noted in four additional animals with onset on Day 3, and one female exhibited

hypersensitivity to sound on Days 3-4. Most of the animals recovered by Days 5-8, but one

female exhibited anogenital staining through Day 13. All the animals lost weight during Days 0-

1, and one female lost additional weight during Days 1-3. All the animals had net body weight

gains for Days 0-14.


MRID No.: 50354804



Study Summary: A 0.1 mL volume of F4092-3 was instilled into the conjunctival sac of the

unanesthetized right eye of three 17-week-old female New Zealand White rabbits, and the upper

and lower lids were held shut for approximately one second, while the untreated left eye of each

animal served as a control. Eyes were scored for ocular irritation according to the Draize method

at 1, 24, 48, and 72 hours after instillation, with fluorescein staining done at 24 hours. There

were no observations of corneal opacity or iritis at any time during the study. One hour after

instillation, the three treated eyes exhibited conjunctival redness (scores of 1-2) and chemosis

(scores of 1-2). At 24 hours, 3/3 eyes had chemosis (scores of 1-2), with redness noted in 2/3

(scores of 1) and discharge noted in 2/3 (scores of 1). At 48 hours, abnormalities were limited to

chemosis (score=1) in 3/3 eyes. All eyes were free of abnormal findings (all scores of zero) at 72

hours.


MRID No.: 50354803


Toxicity Category: II


for 4 hours to 0.5 mL of undiluted F4092-3 applied to intact, previously clipped application sites

measuring 6 cm2 and covered by a semi-occlusive dressing. The application sites were scored

for irritation according to the Draize method at 30-60 minutes, at 1, 24, 48, and 72 hours, and at

7 and 10 days after patch removal. At 30-60 minutes and at 24 hours following patch removal,

all three sites showed well-defined or moderate-to-severe erythema with slight edema. One site

had well-defined erythema with very slight edema at 48 and 72 hours; on this site, the edema

resolved by Day 7, and erythema resolved by Day 10. The other two sites had moderate-to-



19

severe erythema at 48 and 72 hours, with slight edema on both sites at 48 hours and slight or

moderate edema at 72 hours. For the latter two sites, edema resolved by Day 10, and erythema

resolved by Day 10 or Day 14. The three sites were free of abnormalities on Day 14. The

Primary Dermal Irritation Index was 4.58.

Study Title: 870.2600; Dermal Sensitization

MRID No.: 50354805


Toxicity Category: Positive Sensitizer

Study Summary: Groups of five female CBA/J mice were tested using the Local Lymph Node

Assay (individual animal approach). Concentrations of 0% (vehicle only), 25%, 50%, or 100%

were evaluated. An additional positive control group of five animals was treated with Alpha-

Hexylcinnamaldehyde (≥95% a.i.) administered at a 25% concentration in 1% Pluronic® L92.

The stimulation indices (SIs) of the groups treated with 25%, 50%, and 100% concentrations of

the test material were 5.04, 8.38, and 7.63, respectively, while the SI of the positive control

group was 8.63. The mean radioactive DPM (disintegrations per minute) values of the groups

treated with the 50% and 100% concentrations of the test material were significantly greater than

control (p<0.05), and the mean DPM of the positive controls also was significantly greater

(p<0.01).

VI. Human Exposure and Risk Characterization Assessment

A. Description of Uses

1. F4034-5; EPA File Symbol 279-OALN - The application methods for F4034-5 include

in-furrow, at-transplant drench water treatment and seed treatment. Seed treatment

application are performed by mechanical, slurry, or mist-type seed treating equipment in

commercial facilities or on-farm treatments. The application rates given for in-furrow

applications and transplant drench water applications for all crops on the proposed label

are 4 to 10 ounces of product per acre diluted in a minimum of five gallons of water. The

application rates for seed treatment applications are given on a per seed weight basis or

on a per seed unit basis depending on the crop being treated, with a maximum rate of

0.27 fl. oz./100,000 seeds.

2. F4007-9; EPA File Symbol 279-OAUO - F4007-9 is intended for seed treatment only.

Application rates for this product are given on a per seed weight basis or on a per seed

unit basis depending on the crop being treated, with a maximum rate of 0.34 fl.

oz./100,000 seeds. The application instructions indicate that the product may be applied

to the seeds with mechanical, slurry, or mist-type seed treating equipment in commercial

facilities or on-farm treatments.

3. F4092-3; EPA File Symbol 279-OALR - The application methods for F4092-3 include

in-furrow at plant, pre-plant incorporated, pre-emergent, and lay-by in-furrow (with the

seed), as a T-band (over the open furrow), as a broadcast application, as a band over the



20

row or in transplant drench water during setting. The product application rates range from

2.9 to 42.5 fl. oz./acre. The directions for citrus crops instruct the applicator to apply

F4092-3 by ground equipment to bare soil beneath citrus trees from the trunk to the drip

line of the trees.

B. Federal Food, Drug, and Cosmetic Act (FFDCA) Considerations

Section 408(c)(2)(A)(i) of FFDCA allows EPA to establish an exemption from the requirement

of a tolerance (the legal limit for a pesticide chemical residue in or on a food) only if 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, EPA must take into account the factors set forth in FFDCA section 408(b)(2)(C) and

(D), which require EPA to give special consideration to exposure of infants and children to the

pesticide chemical residue in establishing a tolerance or 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 EPA consider “available information concerning the cumulative effects of [a

particular pesticide’s]… residues and other substances that have a common mechanism of

toxicity.”

As a result, EPA performs analyses to determine the risks from aggregate exposure to pesticide

residues. First, for microbial pesticides, EPA determines the pathogenicity and toxicity of the

pesticide. Second, EPA examines exposure to the pesticide through food, drinking water, and

other exposures that occur as a result of pesticide use in residential settings, as well as other non-

occupational exposure to the substance or substances.

1. Aggregate Exposure and Risk Characterization

In examining aggregate exposure, FFDCA section 408 directs 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). However, since no adverse effects of concern were observed in toxicological tests

performed with Bacillus subtilis RTI477 and Bacillus velezensis RTI301, the EPA did not

conduct a quantitative exposure assessment.

a. Food Exposure and Risk Characterization

Although Bacillus subtilis RTI477 and Bacillus velezensis RTI301 are intended for direct food

use, significant dietary exposure to these organisms resulting from these uses is not expected.



21

Both Bacillus subtilis and Bacillus velezensis are ubiquitous environmental microbes that are

common in soils and on plant surfaces. As pesticides, Bacillus subtilis RTI477 and Bacillus

velezensis RTI301 will be applied by seed treatment and soil-directed applications early in the

growing season. Due to the relatively low application rates proposed for the end-use products,

the pesticidal use of these microbes would not be expected to significantly increase the levels of

B. subtilis and B. velezensis naturally present in the treated environment. Further, since food

crops undergo a post-harvest washing process to remove soil and surface residues, it is unlikely

that significant amounts of these organisms would remain on treated crops. Should these

organisms happen to remain on some food commodities at substantial levels, the results of the

guideline mammalian toxicology testing performed with these microorganisms demonstrated a

lack of significant adverse effects (toxicity, pathogenicity or infectivity). Therefore, as a result of

the minimal likelihood of significant food exposure and low hazard findings, quantitative food

exposure assessments were not performed for these microbial active ingredients.

b. Drinking Water Exposure and Risk Characterization

Although Bacillus subtilis RTI477 and Bacillus velezensis RTI301 are intended for agricultural

use, significant drinking water exposure to these organisms resulting from these uses is not

expected to occur. Both B. subtilis and B. velezensis are ubiquitous environmental microbes that

are common in soils and on plant surfaces. As pesticides, Bacillus subtilis RTI477 and Bacillus

velezensis RTI301 will be applied by seed treatment and direct soil applications which reduce the

likelihood of off-site airborne movement of these active ingredients. If these active ingredients

were to enter surface or ground water to some degree, considering the relatively low application

rates they would likely be present at levels below that of the naturally occurring B. subtilis and

B. velezensis organisms. Further, if these active ingredients were to ultimately enter drinking

water systems, they would be even further reduced in numbers by way of standard drinking

water treatment processes. In addition, the results of the guideline mammalian toxicology testing

performed with these organisms demonstrated a lack of significant adverse effects (toxicity,

pathogenicity or infectivity). Therefore, as a result of these low exposure and hazard findings,

quantitative drinking water exposure assessments were not performed for these microbial active

ingredients.

c. Residential, Non-occupational Exposure and Risk Characterization

As previously noted, Bacillus subtilis RTI477 and Bacillus velezensis RTI301 are intended for

agricultural use. However, there is some potential for indirect residential exposure as F4034-5

(EPA File Symbol 279-OALN) and F4007-9 (EPA File Symbol 279-OAUO) allow for treatment

of turf seed that could be used in residential locations. Residential exposures resulting from this

use would likely be infrequent and would pose minimal concern due to the small amount of

active ingredient involved. As a result, this use scenario is not expected to result in significant

residential exposure. Further, since Bacillus subtilis RTI477 and Bacillus velezensis RTI301 will

be applied agriculturally by seed treatment and direct soil applications, other non-occupational

exposures through drift or other means are also considered very unlikely. Should significant

residential or non-occupational exposures occur, the results of the guideline mammalian

toxicology testing performed with these active ingredients demonstrated a lack of significant



22

adverse effects (toxicity, pathogenicity or infectivity). Therefore, quantitative non-

occupational/residential exposure assessments were not performed for these microbial active

ingredients.

2. Cumulative Effects

Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to establish, modify,

or revoke a tolerance, EPA consider “available information concerning the cumulative effects of

[a particular pesticide’s] . . . residues and other substances that have a common mechanism of

toxicity.”

Bacillus subtilis RTI477 and Bacillus velezensis RTI301are not toxic and do not have a common

mechanism of toxicity with other substances. Consequently, FFDCA section 408(b)(2)(D)(v)

does not apply.

3. Determination of Safety for U.S. Population, Infants and Children

a. U.S. Population

For all the reasons discussed previously, EPA concludes that there is reasonable certainty that no

harm will result to the U.S. population, including infants and children, from aggregate exposure

to residues of Bacillus subtilis RTI477 and Bacillus velezensis RTI301. This includes all

anticipated dietary exposures and all other exposures for which there is reliable information.

b. Infants and Children

FFDCA section 408(b)(2)(C) provides that 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 EPA

determines based on reliable data 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, EPA either retains the default value of 10X or uses

a different additional safety factor when reliable data available to EPA support the choice of a

different factor. As discussed previously, EPA has concluded that Bacillus subtilis RTI477 and

Bacillus velezensis RTI301 are not toxic, pathogenic, or infective to mammals, including infants

and children. Because there are no threshold levels of concern to infants, children, and adults

when Bacillus subtilis RTI477 and Bacillus velezensis RTI301 are used in accordance with label

directions and good agricultural practices, EPA concludes that no additional margin of safety is

necessary to protect infants and children.

c. Occupational Exposure and Risk Characterization

Based on the agricultural use pattern proposed for Bacillus subtilis RTI477 and Bacillus

velezensis RTI301, there is potential for occupational exposure to applicators and other handlers



23

through the dermal, ocular, and inhalation routes. However, the supporting mammalian

toxicological testing has shown a lack of irritation, toxicity, infectivity, or pathogenicity

associated with these active ingredients. Although one of the proposed end-use products, F4092-

3 (EPA File Symbol 279-OALR), did demonstrate significant eye and skin irritation, the irritant

effects were likely due to the presence of bifenthrin in the product formulation. As a result, the

product labeling for F4092-3 will require the appropriate precautions and protective equipment

deemed necessary to address these hazards. In addition, all three end-use products proposed for

registration with this application include labeling that specifies use of a National Institute for

Occupational Safety and Health-approved respirator, long-sleeved shirt, long pants, gloves,

socks, and shoes while handling these products. Therefore, since occupational exposure to

Bacillus subtilis RTI477 and Bacillus velezensis RTI301 are not expected to exceed any toxicity

thresholds when pesticide handlers follow the precautions and requirements identified on the

product label, quantitative occupational assessments were not performed.

4. Human Health Conclusions

EPA does not expect significant dietary (food and drinking water) or other non-occupational

risks from use of Bacillus subtilis RTI477 and Bacillus velezensis RTI301 as active ingredients

in the proposed pesticide products. The required toxicology data demonstrated that Bacillus

subtilis RTI477 and Bacillus velezensis RTI301 are not toxic, pathogenic or infective. Any risks

to handlers resulting from exposure to Bacillus subtilis RTI477, Bacillus velezensis RTI301 or

the proposed end-use product containing bifenthrin are expected to be minimized by use of the

required personal protective equipment. Based on the available toxicology and exposure

information, EPA concludes that no unreasonable adverse effects to humans are expected from

the use of Bacillus subtilis strain RTI477 and/or Bacillus velezensis strain RTI301 as pesticides,

when EPA-approved product label instructions are followed. Further, 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 RTI477 and/or Bacillus velezensis strain

RTI301 resulting from the proposed pesticidal uses. In addition, residues of Bacillus subtilis

strain RTI477 and Bacillus velezensis strain RTI301 will be covered by exemptions from the

requirement of a tolerance in or on food commodities.



24

Appendix I

Table 4. Summary of data submitted to comply with product analysis data requirements

published in 40 CFR § 158.2120 for support of the registration of products containing [Microbial

active ingredient].

Data Requirement

OCSPP

Guideline No.

Results and Classification (As Applicable)

MRID No.

Product Analysis Data for Bacillus subtilis RTI477 Technical (EPA File Symbol 279-OAUT)

Product Identity 885.1100 Submitted data fulfill the requirement for product

identity.


50354414,

50925702,

51255601

Manufacturing

Process

885.1200 Submitted data fulfill the requirement for

manufacturing process.


51255601*

Deposition of a

Sample in a

Nationally

Recognized Culture

Collection

885.1250

Bacillus subtilis RTI477 is on deposit with the ATCC;

PTA-121167 (Deposit at American Type Culture

Collection) and DSM 32474 (Deposit at Deutche

Sammlung von Mikroorganismen und Zellkulturen)


50354414,

50925702,

51255601

Discussion of

Formation of

Unintentional

Ingredients

885.1300

Submitted data fulfill the requirement for discussion of

formation of unintentional ingredients.


50354414,

50925702,

51255601

Analysis of Samples 885.1400

Submitted data fulfill the requirement for analysis of

samples.


50354411,

50354412,

51255601

Certification of Limits 885.1500 Submitted data fulfill the requirement for certification

of limits.


50354412,

51255601

Color 830.6302

Brownish beige 50354413,

51255601

Physical State 830.6303 Solid powder 50354413,

51255601

Odor 830.6304 Slightly sweet, musty oder 50354413,

51255601

Stability to Normal

and Elevated

Temperatures, Metals,

and Metal Ions

830.6313 The TGAI product was stable for at least 12 months at

25°C. It is expected to be stable at elevated

temperatures since the optimal growth temperature

range for Bacillus subtilis strain RTI477 is 28°C - 30°C.

51255601

Storage Stability 830.6317 The two-year storage stability study is ongoing.

The interim results showed that the TGAI

product was stable when stored in foil pockets at

25°C for at least 12 months.

50354418,

51255601

Miscibility 830.6319 Not applicable NA



25

Data Requirement

OCSPP

Guideline No.


MRID No.

Corrosion

Characteristics

830.6320 No signs of corrosion were noted on the commercial

packaging (foil

pockets) during the 12-month storage stability study at

25°C.

50354418,

51255601

pH 830.7000 8.09 (1% w/v aqueous suspension) at 20°C 50354413,

51255601

Viscosity 830.7100 Not applicable NA

Density/Relative

Density/Bulk Density

830.7300 0.60 g/mL (pour density), 0.63 g/mL (tap density) 50354413,

51255601

Product Analysis Data for Bacillus velezensis RTI301 Technical (EPA File Symbol 279-OAUI)


identity.


50354514,

50925701,

51255501

Manufacturing

Process

885.1200 Submitted data fulfill the requirement for



51255501*

Deposition of a

Sample in a

Nationally

Recognized Culture

Collection

885.1250

Bacillus velezensis RTI301 is on deposit with the

ATCC; PTA-121165 (Deposit at American Type

Culture Collection) and DSM 32473 (Deposit at

Deutche Sammlung von Mikroorganismen und

Zellkulturen) Classification: Acceptable

50354514,

50925701,

51255501

Discussion of

Formation of

Unintentional

Ingredients

885.1300




50354514,

50925701,

51255501

Analysis of Samples 885.1400

Submitted data fulfill the requirement for analysis of

samples.


50354511,

50354512,

51255501


of limits.


50354512,

51255501

Color 830.6302

Brownish beige 50354513,

51255501

Physical State 830.6303 Solid powder 50354513,

51255501

Odor 830.6304 Sweet, yeast-like odor 50354513,

51255501

Stability to Normal

and Elevated

Temperatures, Metals,

and Metal Ions

830.6313 The TGAI product was found to be stable for at

least 12 months at 25°C. It is expected to be

stable at elevated temperatures since the optimal

growth temperature range for Bacillus velezensis

strain RTI301 is 30°C - 40°C.

51255501

Storage Stability 830.6317 The TGAI product was found to be stable for at

least 12 months at 25°C. It is expected to be

stable at elevated temperatures since the optimal

growth temperature range for Bacillus velezensis

strain RTI301 is 30°C - 40°C.

50354518,

51255501




26

Data Requirement

OCSPP

Guideline No.


MRID No.

Corrosion

Characteristics

830.6320 No signs of corrosion were noted on the

commercial packaging (foil pockets) during the

12-month storage stability study at 25°C.

50354518,

51255501

pH 830.7000 8.01 (1% w/v aqueous suspension) at 20°C 50354513,

51255501

Viscosity 830.7100 Not applicable NA

Density/Relative


830.7300 0.34 g/mL (pour density), 0.44 g/mL (tap

density)

50354513,

51255501

Product Analysis Data for F4034-5 (EPA File Symbol 279-OALN)


identity.


50354705

51391802

Manufacturing

Process

885.1200

Submitted data fulfill the requirement for



50354705

51391802*

Discussion of

Formation of

Unintentional

Ingredients

885.1300




50354705

51391802

Analysis of Samples 885.1400 Submitted data fulfill the requirement for analysis of

samples.


50354707

50354706


of limits.


50354705

51391802

Color 830.6302 Brownish beige 50354704

Physical state 830.6303 Liquid suspension 50354704

Odor 830.6304 Slightly sweet musty odor 50354704

Storage Stability 830.6317 Product was stable when stored in HDPE screw top

bottles at 25°C for at least 12 months.

50354708


Corrosion

Characteristics

830.6320 No signs of corrosion were noted during the 12-month

storage stability study at 25°C.

50354708

pH 830.7000 6.81 (1% w/v aqueous suspension) at 20°C;

6.29 (neat sample) at 20°C

50354704

Viscosity 830.7100 20°C: 604.9 mPa·s (30 rpm), 446.3 mPa·s (50 rpm),

97.1 mPa·s (60 rpm); 40°C: 454.0 mPa·s (30 rpm),

350.4 mPa·s (50 rpm), 316.9 mPa·s (60 rpm)

50354704

Density/Relative


830.7300 1.16 g/mL at 20.5°C 50354704

Product Analysis Data for F4007-9 (EPA File Symbol 279-OAUO)


identity.


50354605

51391801

Manufacturing

Process

885.1200




50354605

51391801*



27

Data Requirement

OCSPP

Guideline No.


MRID No.

Discussion of

Formation of

Unintentional

Ingredients

885.1300




50354605

51391801


samples.


50354607

50354606


of limits.


50354605

51391801



Odor 830.6304 Sweet musty 50354604

Storage Stability 830.6317 Stable at least 12 months in commercial packaging at

25°C

50354608


Corrosion

Characteristics

830.6320 Stable via visual inspection at least 12 months in

commercial packaging at 25°C

50354608

pH 830.7000 6.76 (neat); 7.98 (1% in deionized water) 50354604

Viscosity 830.7100 609.6 mPa s 20°C 30 rpm; 451.3 mPa s 20oC 50 rpm;

408.6 mPa s 20°C 60 rpm; 625.7 mPa s 40oC 30 rpm;

465.0 mPa s 40°C 50 rpm; 414.8 mPa s 40oC 60 rpm

50354604

Density/Relative


830.7300 1.14 g/mL at 20°C 50354604

Product Analysis Data for F4092-3 (EPA File Symbol 279-OALR)

Product Identity 885.1100,

830.1550

Submitted data fulfill the requirement for product

identity.


50354808

51391803

Materials used to

Product the Product

830.1600 Submitted data fulfill the requirement for materials used

to produce the product.


50354808

51391803

Manufacturing

Process,

Production Process,

Formulation Process

885.1200,

830.1620,

830.1650


manufacturing/production process.


50354808

51391803*

Discussion of

Formation of

Unintentional

Ingredients/ Impurities

885.1300,

830.1670


formation of unintentional ingredients/impurities.


50354808

51391803


samples.


50354809

50354810

Certification of Limits 885.1500,

830.1750

Submitted data fulfill the requirement for certification

of limits.


50354808

51391803

Enforcement

Analytical Method

830.1800 Submitted data fulfill the requirement for enforcement

analytical method.


50354809

50354811



28

Data Requirement

OCSPP

Guideline No.


MRID No.



Odor 830.6304 Not applicable NA

Stability to normal

and elevated temps,

metals, etc.

830.6313 Product is expected to be stable under the storage and

use conditions

50644801,

50923401,

50644901,

50923601

Oxidation/Reduction 830.6314 Product does not contain strong oxidizing/reducing

agents

50354807

Flammability 830.6315 Product does not contain combustible materials 50354807

Explodability 830.6316 Product does not contain high-energy materials 50354807

Storage Stability 830.6317 Product was stable when stored in HDPE screw top

bottles at 25°C for at least 12 months.

50354811,

50354812

Miscibility 830.6319 Not applicable 50354807

Corrosion

Characteristics

830.6320 No signs of corrosion were noted during the 12-month

storage stability study at 25°C.

50354811,

50354812

Dielectric breakdown

voltage

830.6321 Not applicable 50354807

pH 830.7000 5.97 (1% w/v aqueous suspension) at 20°C; 5.97 (neat

sample) at 20°C

50354807

Viscosity 830.7100 Viscosity at 20°C: 668.1 mPa·s (30 rpm), 533.3 mPa·s

(50 rpm), 488.3 mPa·s (60 rpm); Viscosity at 40°C:

577.1 mPa·s (30 rpm), 420.9 mPa·s (50 rpm), 371.4

mPa·s (60 rpm)

50354807

Density/Relative


(Specific Gravity)

830.7300 1.16 g/mL at 20.5°C

50354807

* See reference 1

REFERENCES

1. Memorandum: Addendum to Data Evaluation Records for the Active Ingredients Bacillus

subtilis strain RTI477 and Bacillus velezensis strain RTI301 contained in the Proposed

Manufacturing-use Products (File Symbols 279-OAUT and 279-OAUI); M. Perry

(BPPD, RAB) to M. Thapa (BPPD, MPB), 2/9/2021.

memorandum subject: human health risk assessment for …

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