Tolclofos-methyl Human Health Risk Assessment DP No. 387242

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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND

 POLLUTION PREVENTION

MEMORANDUM

Date: March 22, 2012

SUBJECT: Tolclofos-methyl: Human Health Risk Assessment for Proposed New Seed 

Treatment Uses

PC Code: 128905 DP Barcode: D387242

Decision No.: 443921 Registration No.:

Petition No.: Regulatory Action: Section 3 Registration 

Risk Assessment Type: Single Chemical/No 

Aggregate

Case No.: None

TXR No.: None CAS No.: 57018-04-9

MRID No.: None 40 CFR:

FROM: Charles Smith, Senior Environmental Scientist/Risk Assessor

Elissa Reaves, Ph.D., Senior Toxicologist

Sheila Piper, Senior Chemist

Shalu Shelat, Industrial Hygienist

Risk Assessment Branch VI

Health Effects Division (7509P)

THROUGH: Felecia Fort, Chief

Risk Assessment Branch VI

Health Effects Division (7509P)

and

P.V. Shah, Ph.D.

Margarita Collantes

Risk Assessment Review Committee

Health Effects Division

TO: Lisa Jones/Mary Waller, Risk Management Team 21

Herbicide Branch

Registration Division (7505P)

Risk Management Division
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The Health Effects Division (HED) of the Office of Pesticide Programs (OPP) is charged with 

estimating the risk to human health from exposure to pesticides. Valent U.S.A. Corporation has 

requested registration of the new active ingredient (ai) tolclofos-methyl for seed treatment use on 

various crops, turf grass, ornamental flower, and conifers. The Registration Division (RD) of OPP 

has requested that HED evaluate hazard and exposure data and conduct dietary, occupational, and 

aggregate exposure assessments, as needed, to estimate the risk to human health that will result from 

the proposed seed treatment uses of tolclofos-methyl. 

HED has evaluated the toxicity and exposure databases for the new active ingredient, tolclofosmethyl, and has conducted a human health risk assessment in support of the proposed uses. Based 

on this assessment, HED has determined that there are no potential risk estimates of concern for the 

proposed uses of tolclofos-methyl. HED recommends that the requested uses be granted.

A summary of the findings and an assessment of human health risk resulting from the proposed uses

for tolclofos-methyl are provided in this document. The HED team members contributing to this 

risk assessment include Shalu Shelat (occupational/residential assessments), Sheila Piper (residue 

chemistry assessment), Elissa Reaves (hazard evaluation), and Charles Smith (risk assessment).
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1.0 Executive Summary................................................................................................................... 4

2.0 HED Recommendations ............................................................................................................ 7

2.1 Data Deficiencies ................................................................................................................... 7

2.2 Tolerance Considerations....................................................................................................... 8

2.2.1 Enforcement Analytical Method..................................................................................... 8

2.2.2 International Harmonization ........................................................................................... 8

2.2.3 Recommended Tolerances.............................................................................................. 8

2.3 Label Recommendations........................................................................................................ 8

2.3.1 Recommendations from Residue Reviews ..................................................................... 8

2.3.2 Recommendations from Occupational Assessment........................................................ 8

3.0 Ingredient Profile ....................................................................................................................... 9

3.1 Structure and Nomenclature................................................................................................... 9

3.2 Physical/Chemical Characteristics......................................................................................... 9

3.3 Summary of Existing/Proposed Uses................................................................................... 10

3.4 Anticipated Exposure Pathways........................................................................................... 11

3.5 Consideration of Environmental Justice .............................................................................. 11

4.0 Hazard Characterization/Assessment ...................................................................................... 11

4.1 Toxicology Studies Available for Analysis ......................................................................... 11

4.2 Absorption, Distribution, Metabolism, and Elimination (ADME) ...................................... 12

4.2.1 Dermal Absorption........................................................................................................ 13

4.3 Toxicological Effects ........................................................................................................... 13

4.4 Consideration of Toxicity to Children ................................................................................. 14

4.4.1 Completeness of the Toxicology Database................................................................... 14

4.4.2 Evidence of Neurotoxicity ............................................................................................ 15

4.4.3 Evidence of Sensitivity/Susceptibility in the Developing or Young Animal ............... 15

4.4.4 Residual Uncertainty in the Exposure Database........................................................... 16

4.4 Toxicity Endpoints and Points of Departure Selections....................................................... 16

4.5.1 Dose Response Assessment .......................................................................................... 16

4.5.2 Recommendation for Combining Routes of Exposure for Risk Assessment ............... 17

4.5.3 Cancer Classification and Risk Assessment Recommendations .................................. 17

4.5.4 Summary of Points of Departure and Toxicity Endpoints Used in Human Risk 

Assessment.................................................................................................................................. 17

5.0 Dietary Exposure and Risk Assessment .................................................................................. 18

5.1 Food Residue Profile............................................................................................................ 18

5.2 Water Residue Profile .......................................................................................................... 19

5.3 Dietary Risk Assessment...................................................................................................... 19

6.0 Residential (Non-Occupational) Exposure/Risk Characterization .......................................... 19

7.0 Aggregate Exposure/Risk Characterization............................................................................. 19

8.0 Cumulative Exposure/Risk Characterization........................................................................... 19

9.0 Occupational Exposure/Risk Characterization ........................................................................ 20

9.1 Short- and Intermediate-Term Handler Risk........................................................................ 20

9.2 Short- and Intermediate-Term Post-Application Risk ......................................................... 25

Appendix A: Toxicology Profile and Updated Executive Summaries........................................... 26

Appendix B: Review of Human Research...................................................................................... 49
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1.0 Executive Summary

Valent U.S.A. Corporation has requested registration of the active ingredient (ai) tolclofos-methyl 

for seed treatment use on various crops, turf grass, ornamental flower, and conifers. The 

Registration Division (RD) of OPP has requested that HED evaluate hazard and exposure data and 

conduct dietary, occupational, and aggregate exposure assessments, as needed, to estimate the risk 

to human health that will result from the proposed seed treatment uses of tolclofos-methyl. This 

document contains the human health exposure and risk assessment for the requested uses. 

Use Profile: Tolclofos-methyl (O-(2,6-dichloro-4-methylphenyl) O,O-dimethyl phosphorothioate) 

is a organophosphate fungicide used to protect plants from soil borne and seed borne fungal 

pathogens. Tolclofos-methyl is being proposed for seed treatment use on various crops, turf grass, 

ornamental flower, and conifers. The proposed formulated product is a liquid containing 42.0% 

tolclofos-methyl active ingredient (4.17 lbs tolclofos-methyl per gallon). The proposed label is for 

both commercial and on-farm seed treatment.

Hazard Assessment: Tolclofos-methyl is an organophosphate with several older toxicity studies 

(dating back to 1970s) submitted for consideration in risk assessment. Upon re-evaluation of these 

old toxicity studies conducted in the 1970 to 1980 period, several studies are now considered 

unacceptable: 90-day oral rat (MRID No. 00156135) (1982); developmental rat (MRID 

No.00156142-(1979); developmental rat MRID No.41422904 is acceptable)); developmental rabbit 

(MRID No. 00156143) (1982); 3-generation reproduction (MRID No.00156145) (1985); 

carcinogenicity mouse (MRID No.00156139) (1983), and chronic/carcinogenicity rat (MRID 

No.00156140) (1982). Based on the re-evaluation of these old toxicity studies, the toxicity database 

for tolclofos-methyl is not complete but adequate for the current non-food seed treatment use. If the 

registrant pursues food uses in the future additional studies relevant to the food use will be required.

Systemic toxicity from repeat exposure to tolclofos-methyl is primarily characterized by decreases in 

body weight, body weight gain, and reduced food consumption and/or efficiency in rats and dogs. 

Organ weight changes (kidney, liver, thyroid) observed at high doses typically did not correlate with 

histopathological changes. Neurotoxicity data from the recently submitted acute and subchronic 

neurotoxicity studies indicate tolclofos-methyl is not a potent neurotoxicant. The acute 

neurotoxicity study resulted in decreased locomotor activity (total and ambulatory counts) near the 

limit dose while the subchronic neurotoxicity study resulted only in decreased body weight/gain and 

food utilization. There was no evidence of increased susceptibility in the pre-natal study with rats. 

The mouse immunotoxicity study did not indicate immunotoxicity and tolclofos-methyl has not been 

associated with a genetic toxicity or mutagenicity concern.

In risk assessments for non-food uses that do not result in residential exposures, PoDs are selected 

for the dermal and inhalation routes of occupational exposure only. In the case of tolclofos-methyl, 

there is no dermal or systemic toxicity at the limit dose and there is low concern for the minimal 

developmental toxicity (i.e., a variation not a malformation) seen at the limit dose in the offspring. 

Therefore, quantification of dermal risk is not required. Quantification of inhalation risks was 

performed using two oral toxicity studies since inhalation data are currently unavailable. The 90-day

rat and 6- month dog studies provided the most appropriate PoDs for considerations and were used 

as co-critical studies since they yielded similar NOAELs and LOAELs for use in risk assessment. 
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These NOAELs are considered are protective of the endpoints of concern since the recently 

submitted acute and subchronic neurotoxicity studies resulted in no ChE inhibition or toxicity near 

the limit dose (1,000 mg/kg/day). Therefore, the NOAEL of 23 mg/kg/day from the 6-month dog 

study, which is similar to the RBC BMDL10 of 34 mg/kg/day from the 90-day rat study, is the most 

appropriate POD and protective of the short-term and intermediate-term durations of exposure. An 

uncertainty factor of 100x (10x to account for interspecies extrapolation and 10x for intraspecies 

variation) was applied to the POD. 

Food Quality Protection Act (FQPA) considerations do not apply to tolclofos-methyl as the proposed 

seed treatment uses have been deemed non-food uses. However, there are three outstanding studies 

according to revised 40 CFR Part 158 Toxicology Data Requirements. These studies include a 

development toxicity study in rabbits, a reproduction study in rats, and an adequate in vivo

mammalian cell assay. The Agency is not requesting any of these studies at this time and does not 

believe a UFDB is required for the lack of these studies. Based on the proposed uses, quantification 

of risk is limited to short- and intermediate-term inhalation exposure for workers. A waiver request 

for a 28-day inhalation study was denied to protect for any future proposed uses as tolclofos-methyl 

has potential to volatilize off of treated fields based on its vapor pressure (D390269, TXR0055976). 

However, the Agency is not requiring an inhalation study at this time since the proposed uses and 

the risk estimates for occupational handlers are sufficiently protective (MOES = 16,000 to 

26,000,000; see Section 9.0). This decision will be reevaluated based on future use requests.

There are no currently registered or proposed food uses for tolclofos-methyl. Carcinogenicity 

studies are not required for non-food use chemicals. Carcinogenicity studies in mice and rats are 

available for tolclofos-methyl and both of these studies were deemed unacceptable for use in the risk 

assessment. These cancer studies would be required under the Revised Part 158 Guidelines should a 

food use for tolclofos-methyl be requested in the future.

The acute toxicity profile of tolclofos-methyl consists of low toxicity via the oral, dermal, and 

inhalation routes of exposure (Category III and IV). Tolclofos-methyl is not an eye or skin irritant 

(Category IV) and it is not a dermal sensitizer. 

Food Residue Profile: The submitted total radioactive residues (TRR) data for soybean, corn, 

lettuce, cucumber, radish and cotton seeds are adequate to satisfy data requirements and support the 

seed treatment uses being classified as non-food uses. Valent met with EPA on October 2, 2008 to 

discuss tolclofos-methyl radiotracer studies which were conducted to support the non-food 

classification and the required toxicology data requirements which were based on Valent’s review of 

existing data and their interpretation of the 40 CFR Part 158 requirements for nonfood uses. 

Subsequently, HED’s Chemistry Science Advisory Council (ChemSAC) met on December 3, 2008 

to discuss Valent’s proposal for non-food use classification of the seed treatment uses of tolclofosmethyl on various crops and crop groups. As a result, the proposed seed treatment use of tolclofosmethyl on corn, soybean and cotton can be classified as non-food. Additionally, the registrant 

submitted radiotracer studies on lettuce and cucumber at 10 g.ai/100kg, and radish at 50 g.ai/100kg 

for a non-food use classification if the TRR are determined to be less than 5 ppb. The results 

showed that the lettuce, cucumber and radish plants grown for seeds treated with [phenyl14C]tolclofos-methyl did not take up the radiolabeled test substance residue.
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Dietary Exposure and Risk: A dietary exposure assessment is not required, since it is a non-food 

use chemical and the Agency does not expect the proposed seed treatment uses of tolclofos-methyl

to adversely impact ground water or surface water. 

Residential Exposure: There are currently no registered residential uses associated with tolclofosmethyl; therefore, a residential/non-occupational exposure assessment is not required at this time. 

Aggregate Risk: There are currently no registered food or residential uses for tolclofos-methyl and 

the Agency does not expect the proposed seed treatment uses of tolclofos-methyl to adversely impact 

ground water or surface water; therefore, an aggregate risk assessment is not required at this time.

Occupational Exposure/Risk:

Handler Exposure: Based on the proposed seed treatment uses of tolclofos-methyl, two types of 

occupational handler exposure are expected: 1) primary handler exposure is expected to result for 

handlers involved in commercial seed treatment and on-farm seed treatment and 2) secondary

handler exposure is expected to result from planting tolclofos-methyl treated seeds. The short- and 

intermediate-term inhalation risk estimates for all primary and secondary handler scenarios do not 

exceed HED’s level of concern (MOEs > 100) at some level of personal protective equipment (PPE).

Post-application Exposure: Occupational post-application exposure is not expected for the proposed 

seed treatment uses of tolclofos-methyl. As a result, no quantitative post-application assessment is 

required for exposure to treated seeds that have already been planted. The proposed tolclofosmethyl label has the appropriate REI of 12 hours based on the acute toxicity of tolclofos-methyl.

Review of Human Research: This risk assessment relies in part on data from studies in which 

adult human subjects were intentionally exposed to a pesticide or other chemical. These studies 

(listed in Appendix B) have been determined to require a review of their ethical conduct and all of 

the studies utilized in this assessment have received the appropriate review.
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2.0 HED Recommendations

HED has evaluated the toxicity and exposure databases for the active ingredient, tolclofos-methyl, 

and has conducted a human health risk assessment in support of the proposed uses. Based on this 

assessment, HED has determined that there are no potential risk estimates of concern for the 

proposed uses of tolclofos-methyl. HED recommends that the requested uses be granted.

2.1 Data Deficiencies

TOXICOLOGY

The existing toxicology database is not complete but is adequate for characterizing tolclofos-methyl 

toxicity and quantification of risk for a non-food seed treatment use. In accordance with the revised 

40 CFR Part 158 Toxicity Data Requirements, the tolclofos-methyl database lacks a development 

toxicity study in rabbits, a reproduction study in rats, and an adequate in vivo mammalian cell assay. 

The Agency is not requesting the developmental toxicity study in rabbits at this time for the 

following reasons: 1) it is unlikely that the use of this product at the low application rate (range 

0.000489 to 0.00000077 lb ai/lb seed) will result in significant human exposure over a portion of the 

human lifespan in terms of frequency, magnitude or duration of exposure (sub-bullet #26 from 40 

CFR Part 158 Toxicity Data Requirements Table); 2) the available developmental toxicity study in 

rabbits, in spite of its deficiencies, indicates that the rabbits are not the most sensitive species; and 3) 

the risk assessment is based on an endpoint observed in dogs, which is shown to be the most 

sensitive species for tolclofos-methyl induced toxicity. 

The Agency is not requesting the reproduction study in rats at this time because it is unlikely that the 

use of this product at the low application rate (range 0.000489 to 0.00000077 lb ai/lb seed) will 

result in significant human exposure over a portion of the human lifespan in terms of frequency, 

magnitude or duration of exposure (sub-bullet #26 from 40 CFR Part 158 Toxicity Data 

Requirements Table).

The Agency is not requesting the in vivo mammalian cell assay as the Agency does not expect that 

the lack of this assay would impact the quantification of risk based on the other available tolclofosmethyl mutagenicity studies and the fact that organophosphates are not known to be mutagenic.

A waiver request for a 28-day inhalation study was denied to protect for any future proposed uses as 

tolclofos-methyl has potential to volatilize off of treated fields based on its vapor pressure 

(D390269, TXR0055976). However, the Agency is not requiring an inhalation study at this time 

since the proposed uses and the risk estimates for occupational handlers are sufficiently protective

(MOES = 16,000 to 26,000,000; see Section 9.0). This decision will be reevaluated based on future 

use requests.

It is noted that additional toxicity studies would be required to fulfill the Revised Part 158 guidelines 

for any other use than the seed treatment use, especially for a food use, and the registrant should 

consult with the Agency about any of the outstanding studies before requesting any additional uses 

(see Appendix A.1). 
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2.2 Tolerance Considerations

2.2.1 Enforcement Analytical Method

An adequate data collection method is available for tolclofos-methyl. Batches of radish, lettuce and 

cucumber seeds were treated with appropriate amounts of the test substance, [phenyl14C]tolclofosmethyl. To determine the total radioactive residue (TRR), aliquots of samples were combusted in a 

biological oxidizer followed by liquid scintillation counting (LSC) of the aliquot vials. HPLC was 

used to determine the radiochemical purity of the test substance before and after seed treatment. The 

limit of quantification (LOQ) is 0.003 ppm. The method was adequate for data collection based on 

acceptable concurrent method recoveries.

2.2.2 International Harmonization

There are currently no established or proposed tolerances for tolclofos-methyl in the United States as 

the proposed seed treatment uses has been deemed non-food uses. There are currently no established 

MRLs in Canada, however, there are currently Codex MRLs for lettuce, potato, and radish. Since 

there are no U.S. tolerances for tolclofos-methyl, there are no issues of compatibility with respect to 

the Codex MRLs.

2.2.3 Recommended Tolerances

Tolerances are not required for the proposed seed treatment uses of tolclofos-methyl. There are no 

residue chemistry issues that would preclude granting the tolcolfos-methyl seed treatment as nonfood uses (40 CFR Part 158).

2.3 Label Recommendations

2.3.1 Recommendations from Residue Reviews

No label revisions are needed with respect to residues and no recommendations are being made by

HED.

2.3.2 Recommendations from Occupational Assessment

No label revisions are needed for occupational purposes and no recommendations are being made by 

HED.
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3.0 Ingredient Profile

Tolclofos-methyl is a new organophosphate fungicide active ingredient used to protect plants from 

soil borne and seed borne fungal pathogens.

3.1 Structure and Nomenclature

Table 3.1: Tolclofos-methyl Nomenclature

Chemical Structure

Common Name tolclofos-methyl

IUPAC name O-2,6-dichloro-p-tolyl O,O-dimethyl phosphorothioate

CAS Name O-(2,6-dichloro-4-methylphenyl) O,O-dimethyl phosphorothioate

CAS Registry Number 57018-04-9

Chemical Class Organophosphate

3.2 Physical/Chemical Characteristics

Table 3.2: Toclofos-methyl Physical and Chemical Properties

Parameter Value

Molecular Weight 301.12 g/mol.

Melting point/range 78-80°C

Water solubility (25°C) 1.10 ± 0.08 mg/litre

Solvent solubility % w/w (25°C)

Acetone 50.2, 

Methyl isobutyl ketone 36.5

Cyclohexanone 36.0

Methanol 5.9

Ethyl cellosolve 11.8

Ethylene glycol 0.3

Ethyl acetate 38.9

Hexane 3.8

Xylene 36.0

Petroleum ether 2.3

Acetonitrile 33.1

Chloroform 49.0

Dimethyl formamide 48.7

Vapor pressure (25°C) 1.38 x 10-5 mm Hg

Octanol/water partition coefficient (25°C) 4.56

OP(OCH3)2

Cl

Cl

CH3

S
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3.3 Summary of Existing/Proposed Uses

Tolclofos-methyl is a new active ingredient being proposed for seed treatment use on various crops, 

turf grass, ornamental flower, and conifers. The proposed formulated product is a liquid containing 

42.0% tolclofos-methyl active ingredient (4.17 lbs tolclofos-methyl per gallon). The proposed label 

is for both commercial and on-farm seed treatment. The proposed label restricts the use of tolclofosmethyl in hopper-box or planter-box seed treatment systems at planting time. The proposed label 

also states that the product is only to be applied to “true seeds” and is not to be applied on other 

propagation parts. The application rates range from 0.000489 to 0.00000977 lb ai/lb seed.

Table 3.3: Use Profile for Proposed Tolclofos-methyl Uses

Rizolex Flowable Fungicide (Reg. No. XXX-XXX)

suspension concentrate (SC), 4.17 lb ai/gallon

Crops Application 

Equipment

Maximum

Application 

Rate

Notes

- Root and Tuber Vegetables (Crop 

Group 1A)

Commercial Seed 

Treatment Facility 

and On-Farm

Standard Slurry or 

Mist-Type

0.000489 lb 

ai/lb seed

except Burdock, Chervil, Turnip-Rooted; 

Ginseng; Horseradish; Salsify; Skirret;

- Cotton

- Bulb Vegetables (Crop Group 3-7)

Commercial Seed 

Treatment Facility 

and On-Farm

Standard Slurry or 

Mist-Type

0.00000977 lb 

ai/lb seed

except Daylily, Bulb; Elegans Hosta; 

Fritillaria, Bulb; Fritillaria, Leaves; Garlic, 

Bulb; Garlic, Great-Headed, Bulb; Garlic, 

Serpent, Bulb; Lily, Bulb; Onion, Chinese, 

Bulb; Onion, Macrostem; Onion, Tree, 

Tops; Onion, Welsh, Tops; Shallot, Bulb ; 

Shallot, Fresh Leaves

- Leafy Vegetables (Crop Group 4) except Brassica Vegetables

- Brassica (Cole) Leafy Vegetables 

(Crop Group 5)

- Legume Vegetables (Succulent or 

Dried) (Crop Group 6)

- Fruiting Vegetables (Crop Group 8-10)

- Cucurbit Vegetables (Crop Group 9)

- Cereal Grains (Crop Group 15) except Rice and Wildrice

- Grass Forage, Fodder, and Hay (Crop 

Group 17)

- Non-grass Animal Feeds (Forage, 

Fodder, Straw, and Hay) (Crop Group 

18)

- Herbs and Spices (Crop Group 19)

- Oilseeds (Crop Group 20) except cotton

- Ornamental Flowers

- Conifers
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3.4 Anticipated Exposure Pathways

The Registration Division has requested an assessment of human health risk to support the proposed 

new uses of tolclofos-methyl as a seed treatment use on various crops, turf grass, ornamental flower, 

and conifers. Humans are not expected to be exposed to tolclofos-methyl in their diet as the 

proposed uses of tolclofos-methyl for seed treatment have been deemed to be non-food uses based 

on the submitted residue chemistry data. Drinking water exposure is also not expected based on the 

proposed seed treatment uses of tolclofos-methyl. There are no residential uses of tolclofos-methyl, 

so exposure in residential and non-occupational settings is not likely. In an occupational setting, 

applicators may be exposed while handling the pesticide prior to application to the seed, as well as 

during the application to the seed. Additionally, there is a potential for exposure for workers who 

handle previously treated seed during planting activities. 

These are the first registrations proposed for tolclofos-methyl in the United States; therefore, this is 

the first risk assessment prepared by the Agency to address potential risks associated with the use of 

this new active ingredient.

3.5 Consideration of Environmental Justice

Potential areas of environmental justice concerns, to the extent possible, were considered in this 

human health risk assessment, in accordance with U.S. Executive Order 12898, “Federal Actions to 

Address Environmental Justice in Minority Populations and Low-Income Populations,” 

(http://www.eh.doe.gov/oepa/guidance/justice/eo12898.pdf. As a part of every pesticide risk 

assessment, OPP considers a large variety of consumer subgroups according to well-established 

procedures. In line with OPP policy, HED estimates risks to population subgroups from pesticide 

exposures that are based on patterns of that subgroup’s food and water consumption, and activities in 

and around the home that involve pesticide use in a residential setting. Extensive data on food 

consumption patterns are compiled by the USDA under the Continuing Survey of Food Intake by

Individuals (CSFII) and are used in pesticide risk assessments for all registered food uses of a 

pesticide. These data are analyzed and categorized by soccer20

subgroups based on age, season of the year, ethnic group, and region of the country. Additionally, 

OPP is able to assess dietary exposure to smaller, specialized subgroups and exposure assessments 

are performed when conditions or circumstances warrant. Whenever appropriate, non-dietary

exposures based on home use of pesticide products and associated risks for adult applicators and for 

toddlers, youths, and adults entering or playing on treated areas postapplication are evaluated. 

Further considerations are currently in development as OPP has committed resources and expertise 

to the development of specialized software and models that consider exposure to bystanders and 

farm workers as well as lifestyle and traditional dietary patterns among specific subgroups.

4.0 Hazard Characterization/Assessment

4.1 Toxicology Studies Available for Analysis

Tolclofos-methyl is a new active ingredient currently being considered by the Agency to be 

registered as a non-food seed treatment use for various crops, turf grass, ornamental flowers and 

conifers. Tolclofos-methyl is an organophosphate with several older toxicity studies (dating back to 
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1970s) submitted for consideration in risk assessment. Upon re-evaluation of these old toxicity

studies (1970s to 1980s), several studies are now considered unacceptable: 90-day oral rat (MRID 

No. 00156135) (1982); developmental rat (MRID No. 00156142) (1979); developmental rabbit 

(MRID No. 00156143) (1982); 3-generation reproduction (MRID No. 00156145) (1985); 

carcinogenicity mouse (MRID No.00156139) (1983), and chronic/carcinogenicity rat (MRID No. 

00156140) (1982). 

In accordance with the revised 40 CFR Part 158 Toxicity Data Requirements, the tolclofos-methyl 

database lacks a development toxicity study in rabbits, a reproduction study in rats, and an adequate 

in vivo mammalian cell assay. The Agency is not requesting any of these studies at this time and 

does not believe a UFDB is required for the lack of these studies (see Section 4.4.1 for more details). 

Based on the re-evaluation of these old toxicity studies, the toxicity database for tolclofos-methyl is 

not complete but adequate for the proposed non-food seed treatment use. The available

toxicological studies for tolclofos-methyl are listed in Appendix A. Data from the following studies 

were used to evaluate the hazard potential of tolclofos-methyl: 

- Acute lethality studies (oral, dermal, inhalation, primary eye and dermal irritation, dermal 

sensitization)

- Subchronic dietary (rat, dog)

- 9-month dietary (mouse)

- 104-week special Cholinesterase (rat)

- Developmental (rat, 41422904)

- Neurotoxicity (acute and subchronic) (rat)

- Metabolism (rat, mouse)

- Immunotoxicity (rat)

- Mutagenicty battery

Tolclofos-methyl is a new active ingredient although it has been registered in other countries 

(European Union countries) for several years. A search in PubMed (August 2011) with tolclofosmethyl as a search term resulted in no applicable toxicity studies for risk assessment and no toxicity

studies with humans were submitted.

4.2 Absorption, Distribution, Metabolism, and Elimination (ADME)

The absorption, distribution, metabolism and excretion (ADME) of tolclofos-methyl in rats and mice 

was characterized in a general metabolism study that was administered via stomach intubation in a 

single dose of 5 mg/kg. In rats, tolclofos-methyl was eliminated mainly in urine (65-70%) and feces 

(17-22%) by 7 days post-dosing. In mice, approximately 82-83% was eliminated in urine and 5-7% 

in the feces at 7 days post-dosing. Absorption and elimination were rapid with most of the 

radioactivity eliminated at 23 hours post-dosing. Tissue residues were small, in the ppb range. Total 

recovery of radioactivity was 87% to 91% of the dose. The main metabolite in rat urine was 3,5-

dichloro-4-hydroxybenzoic acid, accounting for 26% of the dose. In mice, 2, 6-dichloro-4-

hydroxybenzyl-glycine, O,O-dimethyl-1-O-(2,6-dichloro-4-carboxyphenyl) phosphate, and Omethyl-O-(2,6-dichloro-4-carboxyphenyl) phosphate each accounted for 8-13% of the dose in urine. 

Unchanged parent compound accounted for 5% and 1.3% of the dose in the feces of rats and mice, 

respectively. Tolclofos-methyl is metabolized via oxidative desulfuration of the thiophosphoryl 
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group to give the oxyphosphoryl analog (oxon), oxidation at the 4-methylphenyl group, and 

hydrolytic cleavage of the p-O-aryl and p-O-methyl linkages. This study was not administered at a 

high dose or repeat dosing for 14 days as required by guideline. 

4.2.1 Dermal Absorption

A dermal absorption study is not available for tolclofos-methyl. However, a non-guideline 21-day

dermal (rabbit) toxicity study is available and, therefore, dermal absorption information is not 

necessary for this assessment.

4.3 Toxicological Effects

Tolclofos-methyl is not acutely toxic and is a poor inhibitor of cholinesterase (ChE) compared to 

other organophosphate compounds. There was no inhibition observed in a time to peak rat study 

after a single oral dose up to the limit dose (2,000 mg/kg). ChE activity was therefore not measured 

in the main acute neurotoxicity (ACN) study. In the subchronic neurotoxicty (SCN) study, RBC 

ChE was not inhibited up to approximately 800 mg/kg/day in either males or females while the 

female brain BMDL10 was 659 mg/kg/day. In a special 104-week ChE rat study, tolclofos-methyl 

did not inhibit ChE activity in any compartment (plasma, RBC, or brain) in either sex up to the limit 

dose. For the older studies that did measure ChE activity, ChE inhibition was very sporadic with 

poor dose-response. For example, the female RBC ChE data from the 90-day rat study were the 

only data to fit BMD modeling (male RBC and male or female brain ChE did not produce a 

significant dose-response curve) with a BMDL10 of 34 mg/kg/day. The 9-month mouse toxicity

study resulted in a female RBC BMDL10 of 10 mg/kg/day with the male RBC and male or female 

brain data again not fitting a dose-response curve.

Neurotoxicity data from the newly submitted acute and subchronic neurotoxicity studies indicate 

tolclofos-methyl is not a potent neurotoxicant. The acute neurotoxicity study resulted in decreased 

locomotor activity (total and ambulatory counts) near the limit dose while the subchronic 

neurotoxicity study resulted only in decreased bodyweight/gain and food utilization.

Systemic toxicity from repeat exposure to tolclofos-methyl is primarily characterized by decreases in 

body weight, body gain, and reduced food consumption and/or efficiency in rats and dogs. Organ 

weight changes (kidney, liver, thyroid) observed at high doses typically did not correlate with 

histopathological changes. The acceptable developmental rat study resulted in marginal decreases in 

maternal bodyweight at the limit dose with unossified sternebra of fetuses occurring only at the limit 

dose. Changes in liver weight and liver enzymes were not consistent across the database but were 

sporadic. The rabbit developmental study is downgraded to unacceptable/non-guideline due to 

numerous critical study design and data reporting issues. The 3-generation reproduction study is 

also downgraded to unacceptable due to critical deficiencies resulting in no conclusion regarding the 

reproductive toxicity of tolclofos-methyl. The carcinogenicity studies in mouse and rat are 

unacceptable since the doses tested were determined to be not adequate to assess the carcinogenic 

potential of tolclofos-methyl.

The mouse immunotoxicity study did not indicate immunotoxicity and tolclofos-methyl has not been 

associated with a genetic toxicity or mutagenicity concern.
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4.4 Consideration of Toxicity to Children

Food Quality Protection Act (FQPA) considerations do not apply to tolclofos-methyl as the proposed 

seed treatment uses have been deemed non-food uses. However, there are three outstanding studies 

according to revised 40 CFR Part 158 Toxicology Data Requirements. These studies include a 

development toxicity study in rabbits, a reproduction study in rats, and an adequate in vivo

mammalian cell assay. The Agency is not requesting any of these studies at this time and does not 

believe a UFDB is required for the lack of these studies. Based on the proposed uses, quantification 

of risk is limited to short- and intermediate-term inhalation exposure for workers. The PoD used for 

assessing repeated inhalation exposure is based on the most sensitive endpoint in the most sensitive 

species (dogs) and supported by the observation of similar effects at a comparable dose in the rat. 

Consequently, the risk assessment does not underestimate potential risks to occupational workers 

and a total uncertainty factor of 100 is adequate.

4.4.1 Completeness of the Toxicology Database

The existing toxicology database is not complete but is adequate for characterizing tolclofos-methyl 

toxicity and quantification of risk for a non-food seed treatment use. In accordance with the revised 

40 CFR Part 158 Toxicity Data Requirements, the tolclofos-methyl database lacks a development 

toxicity study in rabbits, a reproduction study in rats, and an adequate in vivo mammalian cell assay. 

The Agency is not requesting the developmental toxicity study in rabbits at this time for the 

following reasons: 1) it is unlikely that the use of this product at the low application rate (range 

0.000489 to 0.00000077 lb ai/lb seed) will result in significant human exposure over a portion of the 

human lifespan in terms of frequency, magnitude or duration of exposure (sub-bullet #26 from 40 

CFR Part 158 Toxicity Data Requirements Table); 2) the available developmental toxicity study in 

rabbits, in spite of its deficiencies, indicates that the rabbits are not the most sensitive species; and 3) 

the risk assessment is based on an endpoint observed in dogs, which is shown to be the most 

sensitive species for tolclofos-methyl induced toxicity. 

The Agency is not requesting the reproduction study in rats at this time because it is unlikely that the 

use of this product at the low application rate (range 0.000489 to 0.00000077 lb ai/lb seed) will 

result in significant human exposure over a portion of the human lifespan in terms of frequency, 

magnitude or duration of exposure (sub-bullet #26 from 40 CFR Part 158 Toxicity Data 

Requirements Table).

The Agency is not requesting the in vivo mammalian cell assay as the Agency does not expect that 

the lack of this assay would impact the quantification of risk based on the other available tolclofosmethyl mutagenicity studies and the fact that organophosphates are not known to be mutagenic.

A waiver request for a 28-day inhalation study was denied to protect for any future proposed uses as 

tolclofos-methyl has potential to volatilize off of treated fields based on its vapor pressure 

(D390269, TXR0055976). However, the Agency is not requesting an inhalation study at this time 

since the proposed uses and the risk estimates for occupational handlers are sufficiently protective

(MOES = 16,000 to 26,000,000; see Section 9.0. This decision will be reevaluated based on future 

use requests.
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4.4.2 Evidence of Neurotoxicity

The rat acute neurotoxicity study resulted in decreased locomotor activity in both sexes at 700 and 

2000 mg/kg. ChE inhibition was not inhibited after a single dose up to 2000 mg/kg. In the 

subchronic neurotoxicity study, decreased bodyweight, bodyweight gain, and food utilization were 

observed at 735 mg/kg/day with the female brain BMDL10 of 659 mg/kg/day. There was no effect 

on brain weight and there were no clinical signs in the subchronic neurotoxicity study following 

dietary administration. Neurotoxicity was not a clinical sign or pathological feature of the chronic 

rat, mouse or dog studies. Since cholinesterase inhibition is not a consistent and sensitive endpoint 

for tolclofos-methyl, a comparative cholinesterase study that evaluates cholinesterase inhibition 

compared to juvenile rodents is not needed.

The tolclofos-methyl risk assessment team considered the submission of a data waiver for the 

developmental neurotoxicity (DNT) study and determined that a DNT study is not needed at this 

time for the following reasons: 1) there is no evidence of treatment-related neurological effects in 

adult animals (i.e., clinical signs of neurotoxicity, neuropathology or functional or behavioral 

effects) in the subchronic neurotoxicity study at doses up to 762 m/k/g; 2) there is no evidence of 

neurotoxicity in the other studies in the data base; and 3) there is no evidence of treatment-related 

neurological effects in developing animals following pre natal exposure (i.e., nervous system 

malformations or neuropathology) in the developmental rat study. 

4.4.3 Evidence of Sensitivity/Susceptibility in the Developing or Young Animal

Developmental toxicity. There was no clear sensitivity observed in the rat developmental study. 

The maternal NOAEL was established at the limit dose (≥1000 m/kg/day) even though a marginal 

decrease in bodyweight gains (28%) was observed from GD6-11 at the limit dose. Although there 

was no fetal mortality or external malformations, the developmental LOAEL (1000 mg/kg/day) was 

established at the limit dose based on a non-dose responsive increased incidence of unossified 6th

sternebra only.

The developmental rabbit study is considered unacceptable due to critical study and data reporting 

flaws. However, the maternal LOAEL of 1000 mg/kg/day was based on decreased food 

consumption and food efficiency at the mid (1000 mg/kg/day) and the highest dose (3000 

mg/kg/day) tested as well as decreased spleen and kidney weights. Although several key

measurements were not provided for the developmental toxicity, based on maternal toxicity seen at 

doses including and higher than the limit dose it is evident that the rabbits are less sensitive than 

rats which lessens the concern for potential developmental toxicity in this species.

Reproductive toxicity. The rat reproduction study is now considered unacceptable since several 

factors preclude the characterization of the reproductive potential of tolclofos-methyl. The low 

pregnancy rate in the controls precludes a comparison with treatment groups. Although this study is 

required for a non-food use, the maternal toxicity or developmental/reproductive toxicity will likely 

not be lower than the POD (23 mg/kg/day) established for the current occupational assessment. 

Also, it is unlikely that the use of this product at the low application rate (range 0.000489 to 

0.00000077 lb ai/lb seed) will result in significant human exposure over a portion of the human 
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lifespan in terms of frequency, magnitude or duration of exposure (sub-bullet #26 from 40 CFR Part 

158 Toxicity Data Requirements Table).

4.4.4 Residual Uncertainty in the Exposure Database

There are no residual uncertainties in the exposure data base based on the following: 1) there are no 

currently registered or proposed residential uses of tolcofos-methyl in the U.S.; 2) the proposed uses 

are considered non-food uses; and 3) adequate exposure data are available to assess the occupational 

exposures resulting from the proposed uses.

4.4 Toxicity Endpoints and Points of Departure Selections

4.5.1 Dose Response Assessment

The details for selecting toxicity endpoints and points of departure (PODs) are presented in 

Appendix A2. In risk assessments for non-food uses that do not result in residential exposures, 

PODs are selected for the dermal and inhalation routes of exposure only. In this case, there is no 

dermal toxicity and therefore a dermal POD has not been selected and a quantitative dermal 

assessment not performed. A quantitative assessment was only performed for the inhalation route. 

PODs for the acute and chronic dietary and incidental oral exposures are not required to assess risk 

for non-food uses that do not result in residential exposures, and therefore have not been selected.

Acute and Chronic Dietary

The current proposed use is a non-food seed treatment. Acute and chronic PODs have not been 

established for this risk assessment.

Short/Intermediate Dermal

The 21-day dermal rabbit study (non-guideline) was re-evaluated to ensure consistency with current 

HED policy and practices. Based upon this re-evaluation, systemic toxicity and dermal irritation 

was not observed at concentrations up to the limit dose (1,000 mg/kg/day) after 3 weeks of exposure 

(5 days/week for 3 weeks). Therefore a quantitative short- and intermediate-term dermal assessment 

has not been conducted for tolclofos-methyl.

Short/Intermediate Inhalation

The short- and intermediate-term inhalation POD was based on oral toxicity studies since route 

specific inhalation data are currently unavailable. The 90-day rat and 6- month dog studies provide 

the most appropriate PODs to consider for risk assessment. The 90-day rat and 6-month dog studies 

were selected as co-critical since they yield similar NOAELs and LOAELs for use in risk 

assessment. The NOAEL of 23 mg/kg/day from the 6-month dog study, which is similar to the RBC 

BMDL10 of 34 mg/kg/day from the 90-day rat study, is the most appropriate POD and protective of 

the short-term and intermediate-term durations of exposure. An uncertainty factor of 100x (10x to 

account for interspecies extrapolation and 10x for intraspecies variation) was applied to the POD. 

This POD is considered health protective since the recently submitted ACN and SCN studies 

resulted in no ChE inhibition or toxicity near the limit dose of 1,000 mg/kg/day.
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4.5.2 Recommendation for Combining Routes of Exposure for Risk Assessment

As part of conducting a human health risk assessment process, the Agency considers risks from 

individual routes of exposure (oral, dermal, and inhalation). Additionally, the Agency must assess 

aggregate risks from dietary and residential exposures. In order to conduct an aggregate risk 

assessment a determination is made if it is appropriate to combine across routes of exposure and 

aggregate dietary risks with residential (non-occupational) risks. At this time, there are no 

residential uses for tolclofos-methyl and the proposed uses have been deemed non-food uses, so a 

discussion of combining routes of exposure is not pertinent to this assessment. 

4.5.3 Cancer Classification and Risk Assessment Recommendations

There are no currently registered or proposed food uses for tolclofos-methyl. Carcinogenicity 

studies are not required for non-food use chemicals. Carcinogenicity studies in mice and rats are 

available for tolclofos-methyl and both of these studies were deemed unacceptable for use in the risk 

assessment. These cancer studies would be required under the Revised Part 158 Guidelines should a 

food use for tolclofos-methyl be requested in the future.

4.5.4 Summary of Points of Departure and Toxicity Endpoints Used in Human Risk 

Assessment

Table 4.5.4. Summary of Toxicological Doses and Endpoints for Tolclofos-Methyl for Non-Food Use 

Occupational Risk Assessments

Exposure/

Scenario

Point of 

Departure 

(POD)

Uncertainty 

Factors

Level of 

Concern Study and Toxicological Effects

Acute Dietary (General 

Population, including 

Infants and Children)

The proposed seed treatment use is non-food.

Chronic Dietary (All 

Populations) The proposed seed treatment use is non-food.

Dermal 

(Short/Intermediate) Systemic toxicity was not observed up to the limit dose of 1,000 mg/kg/day.

Inhalation

(Short/Intermediate)

NOAEL= 

23 mg/kg

UFA = 10X

UFH = 10X MOE = 100

Co-Critical:

1. 6-month dog (00156138); 

LOAEL = 70 mg/kg/day, based on decreased 

bodyweight and gain, hematocrit, hemoglobin, 

and erythrocyte count in both sexes. In 

addition, ↓relative thyroid weight, ↑ALK and 

↑absolute and relative liver wt at high dose.

A consistent dose-response for ChE was not 

observed.

2. 90-day rat (41422901)

RBC BMDL10=34 MKD

RBC BMD10=72 MKD

Cancer (oral, dermal, 

inhalation) Not required for non-food use pesticides.

Point of Departure (POD) = A data point or an estimated point that is derived from observed dose-response data and used to mark the beginning of 

extrapolation to determine risk associated with lower environmentally relevant human exposures. NOAEL = no observed adverse effect level. 

LOAEL = lowest observed adverse effect level. UF = uncertainty factor. UFA = extrapolation from animal to human (interspecies). UFH = potential 

variation in sensitivity among members of the human population (intraspecies).
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5.0 Dietary Exposure and Risk Assessment

5.1 Food Residue Profile

MRIDs 48341956 and 48341957; DP 395999

Tolerances are not required for the proposed seed treatment uses of tolclofos-methyl. HED’s 

Chemistry Science Advisory Council (ChemSAC) met on December 3, 2008 to discuss Valent’s 

proposal for non-food use classification of the seed treatment uses of tolclofos-methyl on various 

crops and crop groups. ChemSAC concluded that if all TRR results are less than 5 ppb (based on 

memorandum of Chemistry SAC-HED -7509C titled “Classification of Seed Treatments as Food or 

Nonfood Uses” dated Oct 28, 1999), then the proposed seed treatment use of tolclofos-methyl on 

various crops can be classified as non-food (D362358, S.Piper, 2/27/09). 

Batches of soybean, corn, lettuce, cucumber, radish and cotton seeds were treated with formulated 

[phenyl14C]tolclofos-methyl. The target treatment rates were 50 g/100 kg of seeds for cotton and 

radish, and 10 g/100 kg of seeds for corn, soybean, lettuce and cucumber. The results showed that 

the seeds treated with [phenyl14C]tolclofos-methyl did not take up the radiolabeled test substance 

residue (<5 ppb). Only the radish top samples grown from seeds treated at the rate of 51.6 g/100 kg 

showed TRR level showed residues above 0.005 ppm. The radish tops grown from seeds contained 

residues at 0.02 – 0.03 ppm and additional characterization of the residues was provided. The 

treated radish top samples (TRR = 0.029 ppm and TRR = 0.020 ppm) were spiked with radiolabeled 

tolclofos-methyl and analyzed by HPLC to determine the efficiency of the extraction method. Both 

acetonitrile and aqueous extracts showed some polar radioactive residues, but did not contain any

detectable amount of tolclofos-methyl. There are no residue chemistry issues that would preclude 

granting the tolclofos-methyl seed treatment as non-food uses (40 CFR Part 158). Table 5.0 shows 

the data that was translated for the proposed seed treatment use of tolclofos-methyl.

Table 5.0 Translation of Tolclofos-methyl Seed Treatment as a Nonfood Uses

Crop Crop Group Number Crop Group Name

Corn* 15 Cereal grains

16 Forage, fodder and straw

Soybean* 6 Legume vegetable

7 Foliage 

20 Oilseeds

Cucumber* 8 Fruiting vegetable (except cucurbits)

9 Cucurbit

Lettuce* 4 Leafy vegetable (except Brassica 

vegetables)

5 Brassica cole leafy vegetables

Radish** 1 Root and tuber vegetables (except 

potatoes)

2 Leaves of root and tuber vegetables 

(human food and animal feed)

3 Bulb vegetables

Cotton** --

*application rate: 10 g ai/100 kg seed

**application rate: 50 g ai/100 kg seed
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5.2 Water Residue Profile

A qualitative drinking water assessment of tolclofos-methyl was performed for this risk assessment. 

Tolclofos-methyl may transport to nearby surface water through runoff and erosion, especially if 

tolclofos-methyl treated seeds are planted coinciding with or closely followed by a rain event. After 

discussing the very low proposed seed treatment application rates, environmental fate data, and 

modeling estimates with the Ecological Fate and Effects Division (EFED); the Agency does not 

expect the proposed seed treatment uses of tolclofos-methyl to adversely impact ground water or 

surface water.

5.3 Dietary Risk Assessment

Dietary exposure (food + drinking water) is not expected for tolclofos-methyl, since it is a non-food 

use chemical and the Agency does not expect the proposed seed treatment uses of tolclofos-methyl

to adversely impact ground water or surface water.

6.0 Residential (Non-Occupational) Exposure/Risk Characterization

Residential exposures and risk are not assessed in this document because the proposed uses of 

tolclofos-methyl do not involve applications by homeowners or commercial applicators in residential 

settings at this time and there are no existing residential uses.

7.0 Aggregate Exposure/Risk Characterization

Aggregate risk assessments evaluate the combined risk from dietary exposure (food and water) and 

any non-occupational (residential) uses. However, there are currently no registered food or 

residential uses for tolclofos-methyl and the Agency does not expect the proposed seed treatment

uses of tolclofos-methyl to adversely impact ground water or surface water; therefore, an aggregate 

risk assessment is not required at this time.

8.0 Cumulative Exposure/Risk Characterization

Section 408(b)(2)(D)(v) of the Federal Food, Drug, and Cosmetic Act (FFDCA), as amended by

the Food Quality Protection Act (1996) stipulates that when determining the safety of a pesticide

chemical, EPA shall base its assessment of the risk posed by the chemical on, among other

things, available information concerning the cumulative effects to human health that may result

from dietary, residential, or other non-occupational exposure to other substances that have a

common mechanism of toxicity. Tolclofos-methyl is a member of the organophosphate (OP) class 

of pesticides. Other members of this class of pesticides are numerous and include azinphos methyl, 

diazinon, chlorpyrifos, dichlorvos, dicrotophos, dimethoate, disulfoton, methamidophos, 

methidathion, monocrotophos, naled, oxydemeton-methyl, phorate, phosmet, pirimiphos-methyl, and 

trichlorfon to name a few. EPA considers organophosphates to express toxicity through a common 

biochemical interaction with cholinesterase which may lead to a myriad of cholinergic effects and, 
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consequently the organophosphate pesticides should be considered as a group when performing 

cumulative risk assessments.

The Agency has completed a cumulative risk assessment for OPs, (US EPA, 2001), a revised

cumulative risk assessment for OPs, (US EPA, 2002), and an updated OP cumulative risk

assessment (US EPA, August 2006) which can be found on the Agency's web site

http://www.epa.gov/pesticides/cumulative/rra-op/. As the proposed uses of tolclofos-methyl are the 

first United States registrations of the chemical, tolclofos-methyl was not considered or included in 

the previous OP cumulative assessment. OPP is in the process of evaluating the most current 

methods and data for suitability of use in the next version of the OP cumulative risk assessment. 

Tolclofos-methyl will be considered for inclusion in any revised OP cumulative risk assessment.

9.0 Occupational Exposure/Risk Characterization

Based on the proposed seed treatment uses of tolclofos-methyl, two types of occupational handler 

exposure is expected: 1) primary handler exposure is expected to result for handlers involved in 

commercial seed treatment and on-farm seed treatment and 2) secondary handler exposure is 

expected to result from planting tolclofos-methyl treated seeds. Occupational post-application 

exposure is not expected for the proposed seed treatment uses of tolclofos-methyl.

9.1 Short- and Intermediate-Term Handler Risk

Tolclofos-methyl is being proposed for both commercial and on-farm seed treatment use on various 

crops, turf grass, ornamental flower, and conifers. The proposed formulated product is a liquid 

containing 42.0% tolclofos-methyl active ingredient (4.17 lbs tolclofos-methyl per gallon). The 

proposed maximum application rates and use sites are summarized above in Table 3.3. Based on the 

proposed use pattern, there is potential for exposure to toclofos-methyl to primary handlers (i.e., 

activities related to treating the seed) and secondary handlers (i.e., planting treated seed).

Primary Handler (Treating Seed)

Potential occupational exposure scenarios from the use of tolclofos-methyl as a commercial seed 

treatment include:

 Mixing, loading, applying liquid formulations;

 Bagging treated seed;

 Sewing bags; and

 Multiple activities.

Typically, for large-scale commercial seed treatments, workers perform only those specific 

individual tasks listed above; however, it is assumed that workers also may perform multiple 

activities throughout the day. As a result a “multiple activities” scenario (i.e. where one worker 

performs all seed treatment tasks such as loading/applying, sewing, bagging, cleaning, calibration, 

repair, forklift driver, etc.) was addressed.

On-farm seed treatment utilizes similar machinery as commercial seed treatment with the only

difference being the size of the equipment. The proposed label restricts use of tolclofos-methyl in 
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hopper-boxes and planter-boxes thus the commercial seed treatment risk estimates should be 

considered protective of any on-farm seed treatment scenarios.

Secondary Handler (Planting Treated Seed)

Potential occupational exposure scenarios from the use of tolclofos-methyl as a commercial seed 

treatment include planting treated seed (secondary handler).

It is the policy of HED to use the best available data to assess handler exposure. For assessing 

commercial seed treatment and seed planting activities, unit exposure data were taken from HED 

ExpoSAC Policy 14: SOPs for Seed Treatment. The amount of active ingredient handled depends 

on the application rate (lb ai/lb seed), the pounds of seed treated in a day, and the pounds of seed that 

can be planted in a day, all of which vary depending upon the seed type. Values for the amount of 

seed treated and planted per day were obtained from a Biological and Economic Analysis Division 

memo entitled, “Acres Planted per Day and Seeding Rates of Crops Grown in the United States” (J. 

Becker and S. Ratnayake; 3/24/11).

Primary and secondary handler exposure is expected to be short- or intermediate-term in duration 

based on information provided on the proposed label. The short- and intermediate-term 

toxicological endpoints are the same for the respective routes of exposure; therefore, the estimates of 

risk for short-term duration exposures are protective of those for intermediate-term durations. Longterm exposures are not expected; therefore, a long-term assessment was not conducted. 

Summaries of the risk estimates for primary and secondary handlers are included in Tables 9.1.1 and 

9.1.2, respectively. The short- and intermediate-term inhalation risk estimates for all primary and 

secondary handler scenarios do not exceed HED’s level of concern (MOEs > 100) at some level of 

personal protective equipment (PPE).
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Table 9.1.1: Summary of Short- and Intermediate-Term Primary Handler Exposure and Risk Estimates for Tolclofos-methyl

Seed Type

Inhal UEa Max App Rateb Amt Treatedc

Inhal Dosed

Inhal MOEe

mg/lb lb ai/lb seed lb seed/day mg/kg/day LOC = 100

LOADER/APPLICATOR

Root and Tuber Vegetables (Crop Group 1) 0.00034 0.000489 5,500 1.14E-05 2,000,000

- Bulb Vegetables (Crop Group 3-01)

- Leafy Vegetables (except brassica vegetables) (Crop Group 4)

- Brassica (Cole) Leafy Vegetables (Crop Group 5)

- Legume Vegetables (Succulent or Dried) (Crop Group 6)

- Fruiting Vegetables (Crop Group 8-10)

- Cucurbit Vegetables (Crop Group 9)

- Herbs and Spices (Crop Group 19)

0.00034 0.00009773 5,500 2.28E-06 10,000,000

- Cereal Grains (Crop Group 15, except rice and wildrice)

- Grass Forage, Fodder, and Hay (Crop Group 17)

- Non-grass Animal Feeds (Forage, Fodder, Straw, and Hay) (Crop Group 18)

- Oilseeds (Crop Group 20, except Cotton)

0.00034 0.00009773 718,000 2.98E-04 77,000

Cotton 0.00034 0.000489 160,000 3.33E-04 69,000

Conifers 0.00034 0.00009773 2,000 8.31E-07 28,000,000

Ornamental Flowers 0.00034 0.00009773 5,500 6.65E-05 350,000

SEWER

Root and Tuber Vegetables (Crop Group 1) 0.00023 0.000489 5,500 7.73E-06 3,000,000

- Bulb Vegetables (Crop Group 3-01)

- Leafy Vegetables (except brassica vegetables) (Crop Group 4)

- Brassica (Cole) Leafy Vegetables (Crop Group 5)

- Legume Vegetables (Succulent or Dried) (Crop Group 6)

- Fruiting Vegetables (Crop Group 8-10)

- Cucurbit Vegetables (Crop Group 9)

- Herbs and Spices (Crop Group 19)

0.00023 0.00009773 5,500 1.55E-06 15,000,000

- Cereal Grains (Crop Group 15, except rice and wildrice)

- Grass Forage, Fodder, and Hay (Crop Group 17)

- Non-grass Animal Feeds (Forage, Fodder, Straw, and Hay) (Crop Group 18)

- Oilseeds (Crop Group 20, except Cotton)

0.00023 0.00009773 718,000 2.02E-04 110,000

Cotton 0.00023 0.000489 160,000 2.25E-04 100,000

Conifers 0.00023 0.00009773 2,000 5.62E-07 41,000,000

Ornamental Flowers 0.00023 0.00009773 5,500 4.50E-05 510,000
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Table 9.1.1: Summary of Short- and Intermediate-Term Primary Handler Exposure and Risk Estimates for Tolclofos-methyl

Seed Type

Inhal UEa Max App Rateb Amt Treatedc

Inhal Dosed

Inhal MOEe

mg/lb lb ai/lb seed lb seed/day mg/kg/day LOC = 100

BAGGER

Root and Tuber Vegetables (Crop Group 1) 0.00016 0.000489 5,500 5.38E-06 4,300,000

- Bulb Vegetables (Crop Group 3-01)

- Leafy Vegetables (except brassica vegetables) (Crop Group 4)

- Brassica (Cole) Leafy Vegetables (Crop Group 5)

- Legume Vegetables (Succulent or Dried) (Crop Group 6)

- Fruiting Vegetables (Crop Group 8-10)

- Cucurbit Vegetables (Crop Group 9)

- Herbs and Spices (Crop Group 19)

0.00016 0.00009773 5,500 1.08E-06 21,000,000

- Cereal Grains (Crop Group 15, except rice and wildrice)

- Grass Forage, Fodder, and Hay (Crop Group 17)

- Non-grass Animal Feeds (Forage, Fodder, Straw, and Hay) (Crop Group 18)

- Oilseeds (Crop Group 20, except Cotton)

0.00016 0.00009773 718,000 1.40E-04 160,000

Cotton 0.00016 0.000489 160,000 1.56E-04 150,000

Conifers 0.00016 0.00009773 2,000 3.91E-07 59,000,000

Ornamental Flowers 0.00016 0.00009773 5,500 3.13E-05 740,000

MULTIPLE ACTIVITIES

Root and Tuber Vegetables (Crop Group 1) 0.0016 0.000489 5,500 5.38E-05 430,000

- Bulb Vegetables (Crop Group 3-01)

- Leafy Vegetables (except brassica vegetables) (Crop Group 4)

- Brassica (Cole) Leafy Vegetables (Crop Group 5)

- Legume Vegetables (Succulent or Dried) (Crop Group 6)

- Fruiting Vegetables (Crop Group 8-10)

- Cucurbit Vegetables (Crop Group 9)

- Herbs and Spices (Crop Group 19)

0.0016 0.00009773 5,500 1.08E-05 2,100,000

- Cereal Grains (Crop Group 15, except rice and wildrice)

- Grass Forage, Fodder, and Hay (Crop Group 17)

- Non-grass Animal Feeds (Forage, Fodder, Straw, and Hay) (Crop Group 18)

- Oilseeds (Crop Group 20, except Cotton)

0.0016 0.00009773 718,000 1.40E-03 16,000

Cotton 0.0016 0.000489 160,000 1.56E-03 15,000

Conifers 0.0016 0.00009773 2,000 3.91E-06 5,900,000

Ornamental Flowers 0.0016 0.00009773 5,500 3.13E-04 74,000

a Unit Exposures from HED Exposure Science Advisory Council Policy 14 (baseline inhalation = no respirator).

b Application Rates based on proposed label uses for tolclofos-methyl.

c HED default for lb seed treated per day from HED Exposure Science Advisory Council Policy 15.

d Daily Inhalation Dose (mg/kg/day) = daily inhalation unit exposure (mg/lb ai) x application rate (lb ai/lb seed) x amount treated (lb seed/day) x absorption factor (100%) / body 

weight (80 kg adult).

e Inhalation MOE = NOAEL (23.0 mg/kg/day for short- and intermediate-term exposure) / Inhalation Dose (mg/kg/day). Level of concern = 100.
Tolclofos-methyl Human Health Risk Assessment DP No. 387242

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Table 9.1.2: Summary of Secondary Handler (Planter) Exposures and Risk Estimates for Tolclofos-methyl

Seed Type Represented by

Max lbs of 

seed/acre Inhal UEa Max App Rateb Amt of Seed 

Planted/Dayc Inhalation Dosed

Inhalation MOEe

lb seed/acre mg/lb lb ai/lb seed lb seed/day mg/kg/day LOC = 100

Root and Tuber Vegetables (Crop Group 1) radish 33 3.4 0.000489 2,614 5.43E-05 420,000

Bulb Vegetables (Crop Group 3-01) onion, dry blub 13 3.4 0.00009773 1,004 4.17E-06 5,500,000

Leafy Vegetables (except brassica vegetables) 

(Crop Group 4) spinach 9 3.4 0.00009773 697 2.89E-06 8,000,000

Brassica (Cole) Leafy Vegetables (Crop Group 

5) broccoli 3 3.4 0.00009773 211 8.76E-07 26,000,00

Legume Vegetables (Succulent or Dried) (Crop 

Group 6) pea, garden 412 3.4 0.00009773 32,920 1.37E-04 170,000

Fruiting Vegetables (Crop Group 8-10) pepper 3 3.4 0.00009773 209 8.68E-07 26,000,000

Cucurbit Vegetables (Crop Group 9) watermelon 9 3.4 0.00009773 726 3.02E-06 7,600,000

Cereal Grains (Crop Group 15, except rice and 

wildrice) wheat 188 3.4 0.00009773 37,500 1.56E-04 150,000

Grass Forage, Fodder, and Hay (Crop Group 17) bluegrass, 

kentucky 35 3.4 0.00009773 2,800 1.16E-05 2,000,000

Non-grass Animal Feeds (Forage, Fodder, Straw, 

and Hay) (Crop Group 18) lupin 160 3.4 0.00009773 12,800 5.32E-05 430,000

Herbs and Spices (Crop Group 19) parsely 10 3.4 0.00009773 836 3.47E-06 6,600,000

Oilseeds (Crop Group 20, except Cotton) safflower 35 3.4 0.00009773 2,800 1.16E-05 2,000,000

Cotton cotton 13 3.4 0.000489 2,667 5.54E-05 420,000

Ornamental Flowers flowers- varied 11 3.4 0.00009773 440 1.83E-06 12,600,000

Conifers dust 10 3.4 0.00009773 2,000 8.31E-06 2,770,000

a Information from “Acres Planted per Day and Seeding Rates of Crops Grown in the United States” (J. Becker and S. Ratnayake; 3/24/11).: Number of seeds per acre (Table 10) 

÷ Number of seeds per lb (Table B-1) = Max lbs of seed per acre

b Application Rates based on proposed label uses for tolclofos-methyl.

c Incorporation of acres treated per day from HED Exposure Science Advisory Council Policy 15 and “Acres Planted per Day and Seeding Rates of Crops Grown in the United 

States” (J. Becker and S. Ratnayake; 3/24/11)

d Daily Inhalation Dose (mg/kg/day) = daily inhalation unit exposure (mg/lb ai) x application rate (lb ai/lb seed) x amount planted (lb seed/day) x absorption factor (100%) / body 

weight (80 kg adult).

e Inhalation MOE = NOAEL (23 mg/kg/day for short- and intermediate-term exposure) / Inhalation Dose (mg/kg/day). Level of concern = 100.
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9.2 Short- and Intermediate-Term Post-Application Risk

Occupational post-application exposure is not expected for the proposed seed treatment uses of 

tolclofos-methyl. Sustained levels of contact with treated seed after it has been placed in the soil or 

other planting media would not be expected because no routine cultural practice required for the 

production of agricultural commodities involves such an activity as defined in the no/low contact 

criteria in the Worker Protection Standard (WPS). As a result, no quantitative post-application 

assessment is required for exposure to treated seeds that have already been planted.

Restricted-Entry Interval (REI)

The proposed tolclofos-methyl label has the appropriate REI of 12 hours based on the acute toxicity

of tolclofos-methyl. It also includes the appropriate WPS language related to early worker entry if 

there will be no worker contact with the soil subsurface or treated seed.
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Appendix A: Toxicology Profile and Updated Executive Summaries

A.1 Toxicology Data Requirements

The requirements (40 CFR 158.340) for non-food use of tolclofos-methyl are shown in the table 

below. Use of the new guideline numbers does not imply that the new (1998) guideline protocols 

were used.

Test Technical

Required Satisfied

870.1100 Acute Oral Toxicity.......................................................

870.1200 Acute Dermal Toxicity ..................................................

870.1300 Acute Inhalation Toxicity..............................................

870.2400 Primary Eye Irritation....................................................

870.2500 Primary Dermal Irritation ..............................................

870.2600 Dermal Sensitization......................................................

yes

yes

yes

yes

yes

yes

Yes

yes

yes

yes

yes

yes

870.3100 Oral Subchronic (rodent)...............................................

870.3150 Oral Subchronic (nonrodent).........................................

870.3200 21/28-Day Dermal .........................................................

870.3250 90-Day Dermal ..............................................................

870.3465 90-Day Inhalation..........................................................

CR

CR

no

yes

CR

Yes

yes

yes

yes

--

870.3700a Developmental Toxicity (rodent)...................................

870.3700b Developmental Toxicity (nonrodent).............................

870.3800 Reproduction .................................................................

yes

yes

yes

yes

NO 1

NO 1

870.4100a Chronic Toxicity (rodent)..............................................

870.4100b Chronic Toxicity (nonrodent)........................................

870.4200a Oncogenicity (rat)..........................................................

870.4200b Oncogenicity (mouse)....................................................

870.4300 Chronic/Oncogenicity....................................................

CR

no

CR

CR

CR

NO 1

--

--

--

--

870.5100 Mutagenicity—Gene Mutation - bacterial.....................

870.5300 Mutagenicity—Gene Mutation - mammalian................

870.5375 Mutagenicity—Structural Chromosomal Aberrations...

870.5550 Mutagenicity—Other Genotoxic Effects.......................

yes

yes

yes

yes

NO 1

NO 1

yes

NO 1

870.6100a Acute Delayed Neurotox. (hen).....................................

870.6100b 90-Day Neurotoxicity (hen)...........................................

870.6200a Acute Neurotox. Screening Battery (rat) .......................

870.6200b 90-Day Neuro. Screening Battery (rat)..........................

870.6300 Develop. Neuro..............................................................

CR

CR

yes

yes

CR

Yes

--

Yes

Yes

--

870.7485 General Metabolism ......................................................

870.7600 Dermal Penetration........................................................

870.7800 Immunotoxicity...............................................

CR

CR

yes

No

--

Yes

Special Studies for Ocular Effects

Acute Oral (rat)...........................................................

Subchronic Oral (rat) ..................................................

Six-month Oral (dog)..................................................

no

no

no

--

--

--

--Not applicable

1

 The Agency is not requesting any of these studies at this time and does not believe a UFDB is required for the lack of 

these studies (see Section 4.4.1). 
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Appendix A.2. Toxicity Profiles

A.2. Toxicity Profiles

Table A.2.1. Acute Toxicity Profile for Tolclofos-methyl*

Guideline 

No. Study Type MRID Results Toxicity Category

870.1100

Acute oral- rat

Acute oral-mouse

Acute oral-dog

00156124 (1985)

00156128 (1978)

00156125 (1978)

Combined LD50 > 5 g/kg, rats#

3500 mg/kg (2760-4800), mice

>1000 mg/kg/day

IV

III

III

870.1200 Acute dermalrabbit

00156126 (1985)

Water vehicle

1/5 died by day 5

No clinical signs after dosing of 

surviving rabbits.

LD50 > 2 g/kg

III

870.1300 Acute inhalation 

- rat

41410501 (1986)@

Powder,nonacceptable/nonguideline

41445702 (1983) @

25% flowable, 

unacceptable/nonguideline

LC50≥ 3.32 mg/L

Particles 0.3-5.5μm

%MMAD 1μm or less not met

LC50≥ 0.146 mg/L

MMAD not determined

III

NA

870.2400

Primary eye 

irritation- rabbit 00156129 (1985)

Transient slight conjunctival 

reaction (24 hrs or less). Not an eye 

irritant

IV

870.2500

Primary Dermal 

irritation- rabbit 00156131 (1985) PIS=0, not an irritant IV

870.2600

Skin 

sensitizationguinea pig00156131 (1985) Not a contact sensitizer N/A

*Acute summaries found in TXR006933

#Clinical signs (piloerection, hunched posture) in all animals were observed shortly after dosing with complete recovery 

by 5 days post-dosing.

@

Studies found in TXR008856
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Table A.2.2. Tolclofos-methyl: Subchronic, Chronic and Other Toxicity Profile

Guideline 

No. 

Study Type MRID No. (year)/ 

Classification /Doses

Results

870.3200 21-Day dermal

(rabbit)

41506302 (1986)

Acceptable/non-guideline

0 (acetone), 30, 300, 1000 

mg/kg

5/sex/dose

(previous TXR008890)

Systemic NOAEL ≥ 1000 mg/kg/day

Systemic LOAEL not established.

*A dose-response of the ChE data resulted in 

No reliable fit for BMD analysis.*

Irritation NOAEL ≥ 1000 mg/kg/day

Irritation LOAEL not established.

870.3100 90-Day oral 

toxicity (rat)

4 weeks

Sprague-Dawley

TXR006933

00156135 (1982)

Purity not reported

Unacceptable/nonguideline

0, 200, 1000, 5000, 

20,000 ppm

M: 0, 16, 79, 414, 1636 

mg/kg/day

F: 0, 18, 88, 452, 1829 

mg/kg/day

No purity information.

HDT: Increased kidney and liver wts (males) 

and females (mid and HDT) as well as 

increased spleen and thyroid wts, enlarged 

hepatocytes HDT male and females. Decreased 

brain ChE at all dose levels.

870.3100 90-Day oral 

toxicity (rat)

Sprague-Dawley

TXR009047

41796501 (1990)

Acceptable/guideline

0, 100, 1,000, 10,000 ppm

0, 6.8, 69, 675 mg/kg/day, 

M&F

RBC ChE

Male RBC ChE data did not provide a reliable 

fit for modeling.

Female RBC BMD10= 72.1 mg/kg/day

Female RBC BMDL10 = 34.01 mg/kg/day

Male and Female brain ChE data did not 

provide a reliable fit.

Systemic NOAEL= 69 mg/kg

Systemic LOAEL = 675 m/kg, based on 

decreased bw gain and increased cholesterol 

and phosphor lipids.

870.3150 6-month (rat)

TXR006933

00156136 (1985)

Unacceptable/nonguideline

0, 300, 1000, 3000, 

10,000 ppm

M:0,16, 51, 164, 540 

MKD

F:0, 18, 65, 184, 623 

MKD

Homogeneity, concentration, and stability 

not provided.

No histopathology provided for mammary 

gland.

NOEL=300 ppm (16/18 mg/kg/day)

LOEL=1,000 ppm (51/65 mg/kg/day), based on 

increased kidney and liver weight, and fatty 

bone marrow infiltrates in females at 1,000 

ppm and above.
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Table A.2.2. Tolclofos-methyl: Subchronic, Chronic and Other Toxicity Profile

Guideline 

No. 

Study Type MRID No. (year)/ 

Classification /Doses

Results

870.3150 6 month (26 weeks)

diet (dog)

Beagle

TXR006933

00156138 (1979)

Acceptable/guideline

0, 200, 600, 2000 ppm

M:0, 7, 23, 70 mg/kg/day

F: 0, 6, 20, 63 mg/kg/day

RBC & plasma ChE 

measured at 4, 8, 13 & 26 

weeks.

The LOAEL is 2,000 ppm (equivalent to 

70/63 mg/kg/day M/F), based upon decreased 

body weight gains in males, increased thyroid 

weights in males and females, and decreased

cholesterol levels in males and females.

The NOAEL is 600 ppm (23/20 mg/kg/day 

M/F).

No consistent dose-response was observed for 

any of the ChE parameters.

870.3250 90-Day Dermal Fulfilled by 21-day rabbit

870.3465 90-Day Inhalation 

toxicity (rat)

NA Conditionally Required

Data Waiver Denied- A waiver request for a 

28-day inhalation study was denied to protect 

for any future proposed uses as tolclofosmethyl has potential to volatilize off of treated 

fields based on its vapor pressure (D390269, 

TXR0055976). This inhalation study is not 

required to assess the proposed uses and the 

risk estimates for occupational handlers are 

sufficiently protective. 

870.3700a Prenatal 

developmental in 

(rat)

Fischer 344

TXR006933

00156142 (1979)

Unacceptable/nonguideline

0, 5, 15, 50 mg/kg/day

Maternal NOAEL = HDT (50 mg/kg).

LOAEL not established > 50 mg/kg

Developmental NOAEL could not be 

established since external examination data 

were not provided.

870.3700a Prenatal 

developmental in 

(rat)

Crl:CD(SD)BR

TXR009333

41422901 (1987)

Acceptable/guideline

0, 100, 300, 1000 

mg/kg/day

(TXR009333)

Maternal NOAEL = >1000 mg/kg/day

Maternal LOAEL = not established. 

Developmental NOAEL = 300 mg/kg/day

Developmental LOAEL = 1,000 mg/kg/day 

based on increased incidence of unossified 6th

sternebra (p≤0.01).

870.3700b Prenatal 

developmental in 

(rabbit)

New Zealand 

White

TXR006933

00156143 (1982)

Unacceptable/nonguideline

0, 300, 1000, 3000 

mg/kg/day

Maternal NOAEL = 300 mg/kg/day

Maternal LOAEL = 1000 mg/kg/day based on 

decreased food consumption and food 

efficiency at mid and HDT, decreased spleen 

and kidney wts at HDT (histology not 

conducted).

Developmental NOAEL could not be 

determined due to critical study design and data 

reporting issues.
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Table A.2.2. Tolclofos-methyl: Subchronic, Chronic and Other Toxicity Profile

Guideline 

No. 

Study Type MRID No. (year)/ 

Classification /Doses

Results

870.3800 3- Gen 

Reproduction 

(rat) SpragueDawley

TXR006933

00156145 (1985)

Unacceptable/nonguideline

0, 100, 300, 1000 ppm

Study is UNACCEPTABLE.

1. Low pregnancy rates in the controls.

2. Number of viable litters in these control 

groups was low (16/generation).

3. The combined effect of low numbers of 

pregnancies and viable litters precludes an 

evaluation of test results because of the 

potential of the inadequate control data

masking compound effects.

4. Unusually high incidence of kidney lesions, 

which are generally seen in older animals, 

occurred in weanlings from all groups; the 

significance of these findings is unclear.

Similarly, no conclusion can be reached 

regarding the increased incidence of 

cytoplasmic vacuolation of adrenals in the F2 

dams of all dosage groups. Although the 

incidence particularly in the high dose group is 

suggestive of a compound effect, the frequency 

of this finding was not dosage related.

870.3150 9- month oral 

toxicity (mouse)

ddY

TXR006933

00156137 (1978)

Acceptable/non-guideline

0, 10, 30, 100, 3000 ppm

0, 1.2, 4, 12, 513(M) 

0, 1.4, 4, 14, 564 (F) 

mg/kg/day

Concentration, Homogeneity, and Stability 

data were not submitted.

Systemic NOAEL = 100 ppm (12M/14F 

mg/kg/day)

Systemic LOAEL = 3000 ppm (513/564 

mg/kg/day), based on decreased body weight 

and lower body weight gains in both males and 

females, as well as decreased heart and liver 

wts (males and females), decreased kidney 

weight (females), and decreased testes weights 

all at 3,000 ppm.

ChE BMD analysis: ChE RBC and brain data 

at 2, 4, and 13 weeks did not provide a reliable 

fit. No dose-response. However, female RBC 

ChE at 40 weeks data did provide a reliable fit.

Female RBC BMD10= 13.5 mg/kg/day (week 

40 only)

Female RBC BMDL10 = 10.0 mg/kg/day

(week 40 only)
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Table A.2.2. Tolclofos-methyl: Subchronic, Chronic and Other Toxicity Profile

Guideline 

No. 

Study Type MRID No. (year)/ 

Classification /Doses

Results

870.4200 Carcinogenicity

(mouse)

TXR006933

00156139 (1983)

UNACCEPTABLE

MTD not achieved

0, 10, 50, 250, 1000 ppm

M: 0, 1.3, 6.5, 32.2, 134 

F: 0, 1.3, 6.9, 34.1, 137 

mg/kg/day

-Stability and homogeneity analyses of the test 

diet were not reported. 

-Data from the pilot study were not reported. 

-The study authors reported that the first two 

analyses of the test diet concentration revealed 

that the control diet was contaminated with 1.8 

to 3.0 μg/g of S3349; the study authors 

indicated that these results may not be accurate. 

-A large amount of the individual data was 

handwritten and illegible; therefore, validation 

of many parameters (body weights, 

hematology, clinical chemistry, urinalyses, and 

organ weights) was not possible. 

-Individual data were not reported for clinical

observations or palpable mass observation.

870.4300 Chronic/ 

Carcinogenicity

(rat)

Sprague-Dawley

TXR006933

00156140 (1982)

UNACCEPTABLE

Acceptable/guideline 

0, 100, 300, 1000 ppm

mg/kg/day:

M: 0, 4.6, 13.8, 47.0

F: 0, 5.4, 16.3, 54.5

MTD not established. 

Special 104-week ChE 

study (rat)

Fischer 344

TXR006933

00156141 (1985)

Acceptable/non-guideline

0, 100, 300, 1000 ppm

4.5/5.2, 13.5/16.7, 

45.6/52.7 mg/kg/day

NOAEL > 1000 ppm

No decrease in plasma, RBC, or brain ChE at 

any dose in either sex.

870.5100 DNA Repair

TXR006933

00156147 (1985)

Unacceptable

DNA repair: Unacceptable

870.5100 Salmonella 

typhimurium. & E. 

coli

Reverse gene 

mutation

TXR006933

00156147 (1985)

Acceptable

Salmonella & E.coli: Acceptable

870.5375 In vitro Chinese 

Hamster Lung 

Forward Mutation

TXR006933

00156148 (1981)

Acceptable/ guideline

Negative in V79 cells with or without S9 

activation at doses ranging from 5x10-9to 5x10-

6 M.

870.5375 In vivo 

chromosome 

aberration

TXR006933

00156149 (1981)

Unacceptable/guideline

Unacceptable.
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Table A.2.2. Tolclofos-methyl: Subchronic, Chronic and Other Toxicity Profile

Guideline 

No. 

Study Type MRID No. (year)/ 

Classification /Doses

Results

870.5375 Dominant Lethal 

Rats

TXR006933

00156515 (1981)

Unacceptable

MTD not achieved.

870.5375 UDS

TXR006933

41760801 (1990)

Acceptable/guideline

Negative to primary rat hepatocytes up to 40 

μg/ml.

870.5375 Chromosome 

Aberration (CHOK1)

TXR006933

41506301 (1990)

Acceptable/guideline

Negative with or without metabolic activation.

870.5375 Chromosome 

Aberration 

TXR006933

00156149 (1981)

Unacceptable 

Data not presented, corn oil not recommended, 

both sexes not used.

870.5550 Unscheduled DNA 

Synthesis in 

Mammalian 

Cultured Cells

TXR006933

00156148 (1981)

Unacceptable

Inadequate dose, HeLa cells not recommended.

870.6100 Acute delayed 

neurotoxicity 

(hen)

TXR006933

00156134 (1982)

Acceptable/guideline

10/group

0, 8000 x 2 mg/kg

Decreased motor activity observed immediately 

after dosing but resolved by day 5 or 6. 

Decreased food consumption. Plasma ChE

inhibited 42% by 24 hrs but returned to normal 

by day 8 on study. Negative for axonal 

degeneration or demyelination. (Brain and RBC 

ChE not measured).

870.6200a Acute 

neurotoxicity: time 

to peak (rat)

Crl:CD(SD)

TXR0055856

48341941 (2009)

Acceptable/non-guideline

Adults only

2,000 mg/kg

1, 2, 4, 8 hrs

A single oral dose of 2000 mg/kg to rats did not 

produce clinical deficits as well as plasma, 

RBC and whole brain cholinesterase activity 

significantly different from the control group; a 

time to peak effect (peak cholinesterase 

inhibition) of tolclofos-methyl was not 

established.

870.6200a Acute 

neurotoxicity: 

range-finding (rat)

Crl:CD(SD)

TXR0055856

48341940 (2010) 

Acceptable/guideline

5/sex/dose

0, 1000, 2000 mg/kg

Adults only

Clinical observations (ease of removal, 

handling, piloerection, grooming, etc) taken 

every hr from 1-8 hrs. No clinical signs at any 

dose (thru 8 hrs post-dose).

ChE activity not measured.

Doses of 200, 700, and 2000 mg/kg selected for 

main ACN.
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Table A.2.2. Tolclofos-methyl: Subchronic, Chronic and Other Toxicity Profile

Guideline 

No. 

Study Type MRID No. (year)/ 

Classification /Doses

Results

870.6200a Acute neurotoxicity 

(rat)

Crl:CD(SD)

TXR0055856

48341951 (2010) 

Acceptable/guideline

Adults only 12/sex/dose

0, 200, 700, 2000 

mg/kg/day

Time to peak: 4 hrs

ChE not measured.

Clinical observations taken at 4 hrs post-dose

NOAEL = 200 mg/kg/day 

LOAEL = 700 mg/kg/day, based on decreased 

locomotor activity (total and ambulatory 

counts) in both sexes as well as in the HDT.

ChE not measured since no inhibition in the 

time to peak inhibition study (48341941).

870.6200b Subchronic 

neurotoxicity (rat)

(Alpk:APfSD)

Diet

TXR0055856

48341952 (2007)

Acceptable/Nonguideline

Wistar-derived Adults 

12/sex/dose

0, 300, 1800, 10000ppm

M:0, 21, 122, 736 mg/kg

F:0, 23, 136, 763 mg/kg

Satelitte 5/dose for ChE at 

2, 5, 9, 14 weeks

M:0, 23, 130, 720 mg/kg

F:0, 24, 144, 817 mg/kg

The NOAEL is 122.3 M/135.8 F mg/kg/day. 

The LOAEL is 735.7M /762.7F mg/kg/day, 

based on decreased bodyweight and 

bodyweight gain, and food utilization. 

Brain ChE

Adult Female Brain ChE BMD10 = 956.4 

mg/kg/day

Adult Female Brain ChE BML10 = 659.2 

mg/kg/day

The adult male brain ChE data did not produce 

a dose-response and therefore did not provide a 

reliable fit for BMD analysis.

RBC ChE

The RBC ChE data in both males and 

females failed to provide a dose response and 

therefore did not provide a reliable fit for 

BMD analysis. 

870.6300 Developmental 

Neurotoxicity NA 

Conditionally Required

Waiver Granted at this time.

870.7485 Metabolism (rats & 

mice)

TXR006933

00156150 (1980)

Acceptable/non-guideline

Rats and mice were given a single oral dose of 

5 mg/kg. Absorption and elimination was 

almost complete by 24 hrs post-dosing. 13 

metabolites found in mice and rats. Unchanged 

parent accounted for only 5 and 1.3% of dose in 

feces of rats and mice, respectively.

*Repeat or high doses were not administered.

870.7800 Immunotoxicity

28-day Diet (mice)

TXR0055856

48341954 (2010)

Acceptable/guideline 

0, 91, 273, 811 mg/kg/day

Systemic NOAEL = 273 mg/kg/day

Systemic LOAEL = 811 mg/kg/day, based on 

decreased body weights/gains, and food 

consumption.

Immunotoxicity NOAEL = HDT (811 

mg/kg/day), LOAEL not established.
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A.3 HAZARD IDENTIFICATION AND ENDPOINT SELECTION

A.3.1 Acute Reference Dose (aRfD) – Females age 13-49 and

A.3.2 Acute Reference Dose (aRfD) general population, including infants and children

A Study and PoD is not required for this risk assessment.

A.3.3. Chronic Reference Dose (cRfD)

A Study and PoD is not required for this risk assessment.

A.3.4 Incidental Oral Exposure (short and intermediate term)

A study and POD is not required for this risk assessment.

A.3.5 Dermal Exposure (short and intermediate term)

Study Selected: None Selected

MRID: NA

Summary: No systemic or dermal irritation was observed at concentrations up to the limit dose 

(1,000 mg/kg/day) after 3 weeks of exposure (5 days/week for 3 weeks; MRID41506302). 

Therefore, a dermal assessment for the short- and intermediate-term durations is not required for 

tolclofos-methyl.

A.3.6 Inhalation Exposure (short and intermediate term)

Study Selected: Since an inhalation study is currently unavailable, an oral study was selected for 

the short- and intermediate-term inhalation assessment.

MRID: 6-month dog (00156138)

Summary: The LOAEL of 2,000 ppm (equivalent to 70/63 mg/kg/day M/F) was based upon 

decreased body weight gains in males, increased thyroid weights in males and females, and 

decreased cholesterol levels in dogs of both sexes. The NOAEL is 600 ppm (23/20 mg/kg/day

M/F). 

Dose and Endpoint for Risk Assessment: 70 mg/kg/day.

Uncertainty Factor(s): 100x [10x interspecies; 10x intraspecies]

Comments about Study/Endpoint/Uncertainty Factor: The duration of exposure of this 

subchronic oral study is appropriate for both the short (1-30 days) and intermediate (1-6 months) 

durations of exposure. Furthermore, the endpoints identified in the dog are protective of effects 

observed in the rat, which is the typical species for organosphate compounds.
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A.3.7 Dermal and Inhalation Exposures (Long-Term)

The current non-food seed treatment use is short and intermediate-term, not long-term. Therefore 

long-term dermal and inhalation risk assessments were not performed.

A.4 UPDATED EXECUTIVE SUMMARIES

The executive summaries presented here are either from the critical studies used in the assessment 

and required an updated executive summary or newly submitted studies which required a data 

evaluation record (DER). 

DERMAL:

EXECUTIVE SUMMARY: In a 21-day dermal toxicity study (MRID 41506302), tolclofosmethyl (97.7% a.i.; Lot No. 40810) was applied to the clipped skin of 5/sex/dose New Zealand 

White albino rabbits at dose levels of 0, 30, 300, or 1000 mg/kg bw/day for 6 hours/day and 5 

days/week for 3 weeks (total 15 days of exposure). The rabbits were restrained with plexiglass neck 

collars during exposure. The collars were removed and the test sites wiped clean with gauze after 

the exposure period. Doses were prepared by combining the appropriate finely powdered test 

material with the desired volume of acetone. The low- and mid-dose levels constituted a solution and 

the high-dose level a suspension. Animals were inspected twice daily for signs of toxicity and 

mortality. Ophthalmologic and urinalysis examinations were not performed.

The stability and concentration of the material in the solutions were not analyzed but are reported to 

be on file with the sponsor. 

No deaths were reported as well as clinical signs of toxicity. There were no treatment related effects 

on bodyweight or food consumption. Re-evaluation of the hematology data suggest that there was 

not a biologically significant increase in eosinophil counts in HDT males (Table 1), unlike the 

previous review. These eosinophil counts are within control ranges and are therefore not considered 

to be of biological significance. In addition a positive dose-related trend in eosinophil levels was not 

observed, unlike the previous review. 

Re-evaluation of the clinical chemistry resulted in all parameters within the normal range published 

for this species and age of rabbits. Absolute organ weights were not affected by treatment. Relative 

kidney weights, however, were increased (20%) in HDT females (Table 2). There were no 

compound-related histopathological changes observed in the kidneys, however, and therefore not of 

biological significance.

The ChE data were submitted for BMD analysis and the data were not fit for analysis since a doseresponse was lacking for both males and females. Therefore the ChE data are not appropriate for 

establishing a systemic LOAEL. 

Based on the lack of biologically significant effects, the systemic LOAEL was not identified. The 

systemic NOAEL is ≥ 1,000 mg/kg/day.

The dermal irritation scores were located in Table 4 of the original study report and were reevaluated for establishment of the irritation NOAEL and LOAEL. Very slight erythema was 

observed in some animals of every treatment group (Table 3). However, the incidence of the 
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erythema decreased with increasing dose. Likewise, the compound was observed to build up on the 

skin in every treatment group with the first signs in the HDT (6 days) followed by the mid-dose (8 

days) and low-dose (14 days). This slight erythema, however, does not constitute an adverse 

irritation effect. 

The irritation LOAEL was not identified. The irritation NOAEL is ≥1000 mg/kg/day.

This study is classified as acceptable/guideline and satisfies the guideline requirements (OPPTS 

870.3200; OECD 410) for a 28-day dermal toxicity study in Rabbits.

ORAL: Subchronic Rat

EXECUTIVE SUMMARY: In a 90-day study (MRID 41796501) Rizolex (tolclofos-methyl, S3349) (96.6% w/w, lot # 90437 - M) was administered continuously in the diet for 90-days to

12/sex/dose Crj:CD (SD) rats, at dose levels of 0, 100, 1,000, and 10,000 ppm (equivalent to 0, 6.8, 

68.6, 674.5 mg/kg/day, respectively). Food consumption was determined for 48 hours each week 

throughout the treatment period and mean daily diet consumption was calculated. Animals were 

inspected once daily for signs of toxicity and mortality. Animals were weighed on the day 1, 3, and 

once weekly thereafter and before necropsy. Blood was collected on the day of sacrifice for 

hematology, clinical analysis and cholinesterase determinations from all animals. Brain 

cholinesterase levels were determined in the left half of a mid-line section of the whole brain taken 

immediately after death from all rats in each group. All animals that died and sacrificed on schedule 

were subject to gross pathological examination, but only tissues from the controls and 10,000 ppm 

groups were subjected to histological examination (only lung, liver, and kidneys). The ovaries, 

adrenals, thyroid (parathyroid) and pituitary were weighed after 23 hour fixation in 10% neutral 

buffered formalin. 

No mortality or clinical signs of toxicity were observed. Body weight gains of males (25-65%) and 

females (27-61%) were significantly (P≤0.01) reduced, in the 10,000 ppm group, during the entire 

treatment period when compared to controls. At termination, the mean cumulative body weight gain 

of males and females was decreased by approximately 25% and 27% (P ≤ 0.01) respectively, when 

compared to the controls (Table 2). The food consumption also decreased significantly (P≤0.01) in 

the males (17-46%) and females (13-19%), throughout the treatment period, at 10,000 ppm, when 

compared to the controls (Table 3). 

In males, the activated partial thromboplastin time (APTT) was increased (8.5% or 5 minutes) and 

the MCHC was decreased (1.5%) significantly, when compared to the controls. At the same dose in 

females, the hemoglobin (HGB), mean corpuscular HGB, and prothrombin time were decreased 

3.6%, 2.8%, and 2% respectively, when compared to controls. Although these decreases were 

significant statistically, they do not represent biologically meaningful changes to consider as adverse 

effects. 

Males and females at the 10,000 ppm exhibited statistically significant increases in total cholesterol 

(33% M and 50% F) and phospholipids (27% M and 39% F), which may be due to disturbed fat 

metabolism (Table 4). Also total protein, GGT, SGOT and ALP significantly increased/decreased in 

males and females. 
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Males and females in the 10,000 ppm group exhibited significant decreases in RBC, plasma and 

brain cholinesterase levels. A BMD analysis resulted only in female RBC data fitting a doseresponse model. The male ChE data not provide a reliable fit for modeling as well as the female 

brain ChE data. The ChE data are provided in Table5 as well as the BMD model outputs for the 

female RBC ChE analysis.

The histopathology and weight of organs (spleen, brain, heart, and kidney) did not change 

significantly due exposure to tolclofos-methyl. Treatment related effects on liver were evidenced by

dark, discolored livers grossly and hypertrophy of hepatocytes microscopically. 

The above information supports a LOAEL of 10, 000 ppm (674.5 mg/kg/day), based on 

significantly reduced bodyweight gains and increased total cholesterol and phospholipids. The 

NOAEL is 1,000 ppm (68.6 mg/kg/day).

Cholinesterase:

Female RBC BMD10= 72.1 mg/kg/day

Female RBC BMDL10 = 34.01 mg/kg/day

Male and Female brain ChE data did not provide a reliable fit.

The study is classified as Acceptable/Guideline and satisfies the guideline requirement for a 

subchronic neurotoxicity study in rats (870.6200b). 

Subchronic Dog

EXECUTIVE SUMMARY: In a subchronic oral toxicity study (MRID 00156138), tolclofosmethyl (98.7%, white powder, Lot#4, LH No 17,631B) was administered to 6 beagle dogs/sex/dose 

in the diet at doses of 0, 200, 600, or 2000 ppm (equivalent to 0/0, 7/6, 23/20, and 70/63 mg/kg/day 

in males/females) for 6 months (26 weeks). No interim sacrifices were performed in this study. 

Ophthalmological examinations were performed on all dogs prior to study start and at study 

termination.

All animals survived the study and no treatment-related clinical signs were reported. 

Ophthalmological examinations were normal in all treatment groups. There were no significant 

differences in body weights or food consumption with the exception of reduced body weight gain in 

high dose males (↓54%). 

Absolute and relative liver (37%, 44%) and thyroid (23%, 31%) weights were increased in HDT 

males compared to controls, respectively (Table 1). In females, absolute (30%) and relative (39%) 

liver weights were also increased as well as relative thyroid weight (18%). However, 

histopathological findings did not reveal changes in the liver or thyroid.

Unlike the previous review of the hematology, the hematocrit, hemoglobin, and erythrocyte values 

for the treated groups were similar to controls in males and females. Therefore, there are no 

biologically significant changes in hematology parameters (Table 2). 
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The clinical chemistry parameters were also re-evaluated. Alkaline phosphatase (ALK) levels were 

consistently increased from week 12 thru week 24 in HDT males (58-70%) and females (64-72%). 

Cholesterol was reduced in HDT males from week12 through week 20 (9-21%) but similar to 

controls at week 24. In females, cholesterol was decreased throughout the entire study and ranged 

from 4-33%.

The LOAEL is 2,000 ppm (equivalent to 70/63 mg/kg/day M/F), based upon decreased body 

weight gains in males, increased thyroid weights in males and females, and decreased cholesterol 

levels in males and females. The NOAEL is 600 ppm (23/20 mg/kg/day M/F). 

It is noted that the increased liver weights and increased ALK values are considered adaptive since 

no histopathological changes were observed in the liver.

This study is classified as acceptable/guideline and satisfies the guideline requirement for a 90-day

oral toxicity study (OPPTS 870.3150; OECD 409) in dogs.

In a nine-month oral toxicity study (MRID00156137), tolclofos-methyl (S 3349), (97% a.i, Batch 

# 524) was administered in diet to 5 week old, (ddY) mice 15/sex/group at the dietary levels of 0, 

10, 30, 100 and 3,000 ppm (calculated to be 1.2/1.42, 3.78/4.14, 12.2/13.8, and 513/564 mg/kg/day, 

respectively for male and female mice) for 9 months. The highest dose level of 3,000 ppm was the 

maximum tolerable dose determined by a preliminary study. There were 3 satellite groups (5 

animals/sex/group) to determine time-course changes of plasma, RBC and brain ChE levels. Body 

weight, food and water consumption were recorded weekly. Clinical signs and mortality were 

observed daily. Organs and tissues were fixed with 10% formol-saline, embedded in paraffin wax, 

sliced at 5μ in thickness, stained with hematoxylin and eosin and examined under light-microscope.

Neither abnormal clinical signs nor dead animals were found throughout the feeding period. Body 

weights of both males (12-21%) and females (17-28%) treated with the highest dose of tolclofosmethyl (3,000 ppm) were lower than controls (week 10 – week 40). Highest dose females (30%) 

and males (33%) had significantly less weight gain (p<0.01). Although weight gain appeared lower 

in the other dose groups, the control males were small at the beginning of the study. For females, 

body weight gain did not follow a dose-response up to 100 ppm. 

Ophthalmological examinations were performed on all surviving animals at termination of exposure 

(week 41) to the test compound and did not find significant changes in irises, lenses, and retinas. 

Some of the mice including controls had cornea opacity (1/15, 1/15, 2/15 and 0/15 HDT compared to 

1/15 controls).

Hematological analysis found statistically significant decreases in leucocytes for all treated male 

mice, however in a reverse dose-response with ↓57% at the lowest dose to ↓25% at the highest dose. 

For treated female mice, an increase in leucocytes (lymphocytes) was found but with no doseresponse pattern. 

Cholinesterase levels were measured on 5 mice/sex/group of the satellite animals at each time point 

(2, 4, 13 weeks), and 5 mice/sex/group of the main group at study termination (week 40) (Table 6 B 

in the report). BMD analyses revealed no reliable fit of the data in males or females at any time 
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point (weeks 2, 4, 13, or 40) expect for female RBC ChE at 40 weeks. Female plasma ChE was 

inhibited significantly (p<0.05) at 100 ppm in week 2 and 30 ppm and above in week 40. 

Gross pathology revealed hydronephrosis, defect of pelvis in kidney, pneumonia, hyperplasia of 

mesenteric lymph node and spleen, and rough-surfaced liver in some of the mice. Heart (10%, 13%) 

and liver (12%, 16%) weight were significantly lower in high dose males and females, respectively. 

Kidney weight was significantly lower in high dose females (20%) while testes weight (11%) was 

significantly lower in high dose males (p≤0.05). Ovarian weight was increased in 100 ppm females

(19%), but not at any other dosage level. Pituitary weight was increased in 10 ppm (47%) and 30 

ppm (23%) males, but not in any other dosage groups. Pituitary weight was increased in 10 (31%), 

30 (31%), 100 (22%) and 3000 (37%) ppm females.

Histological evaluation revealed adenomatous hyperplasia and adenocarcinoma of lungs in one male 

and one female at 3,000 ppm, respectively. Lymphocytic infiltration (one control male) and fatty 

change in liver (five control males) were found only in controls and in two of the highest dosed 

males (one with lymphocytic infiltration, and one with both fatty change and lymphocytic 

infiltration) and 7 females (one female with fatty change, two females with focal necrosis, and four 

female with lymphocytic infiltrates (note: one female had both fatty change and lymphocytic 

infiltrate)]. Kidney lesions were found in 4 high dose males (1 hydronephrosis; 1 hyperplasia; and 2 

pelvic defects) and none in the other treatment groups while lesions were found in 1 control (atrophy 

of kidney) and two high dose females (1 cystic change of tubuli and 1 glomerular hyalinosis). Fatty

change of adrenal medulla was found in 10 control females and 12 females at 3000 ppm group; 

while none were observed in males. Interstitial nephritis, vacuolation in cytoplasm of epithelial cells 

were found in a few mice; cystic changes of tubuli and glomerular hyalinosis were sporadically 

found. Urinary bladder erosion and edema were found in 3,000 ppm females. Atrophy and 

hyperplasia of interstitial cells were found in a few animals of both control and the treatment group. 

Other organs including brain, spinal cord, and sciatic nerve had no remarkable changes.

ChE Inhibition

Tolclofos-methyl in the diet for 9 months did not consistently produce ChE inhibition over the time 

course of the study in mice. A BMD analysis produced a reliable fit for the 40 week RBC ChE data 

in females only. The female RBC (week 40 only) BMD10 is 13.5 mg/kg/day and the BMDL10 is 

10.0 mg/kg/day. 

Systemic

Systemic LOAEL = HDT (3,000 ppm), based on decreased body weight and lower body weight 

gains in both males and females, as well as decreased heart and liver wts (males and females), 

decreased kidney weight (females), and decreased testes weights all at 3,000 ppm. 

Systemic NOAEL = 100 ppm (12.2M/13.8 F mg/kg/day)

This updated Executive Summary updates the NOAEL and LOAEL conclusions for the 

9 month study for tolclofos-methyl and supersedes the previous conclusions in TXR 006933. 

Deficiencies:
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The analysis for concentration, homogeneity, and stability was not provided in the study report. 

However, since effects were observed in mice at relatively lower concentrations than other studies 

for tolclofos-methyl, then the mice were exposed to sufficient tolclofos-methyl to elicit these 

responses. The study, however, is considered non-guideline.

This study is classified as Acceptable/Non-guideline. This study does not satisfy a guideline 

requirement for tolclofos-methyl. 

Chronic/Cancer Rat

EXECUTIVE SUMMARY: In a chronic toxicity study (MRID 00156140), S-3349 (three batches 

were used: batch no. 0523, purity 94.9%; batch no.4, purity: 98.7%; batch no.523, purity: 94.9%) 

was administered as a lifetime dietary administration at 100, 300 and 1000 ppm (equivalent to 0, 

4.6/5.4, 13.8/16.3, 47/54.4 mg/kg/day for M/F respectively) to Fischer 344 rats (until termination,

for males at week 122 and 123; females at week 130). The test material was incorporated on a 

weight-per-weight basis into the basal diet by adding the appropriate amount to 250 g of basal diet. 

Diet was prepared weekly. Storage conditions were not specified. Samples of diets were collected 

weekly beginning at week 68 and samples were assayed at 4-week intervals. Animals were 

assigned randomly to the 0, 100, 300, and 1000 ppm test groups. There were 55animals/sex/group in 

main study group and 10 animals/sex/group in interim sacrifice group (termination at 52 weeks). 

Animals were inspected twice daily for signs of toxicity and mortality. Observations were recorded 

weekly for the first 26 weeks, biweekly for the next 26 weeks, and every week for the remainder of 

the study. Individual food consumption was recorded weekly for the first 26 weeks, biweekly for 

the next 26 weeks, and every 4 weeks for the remainder of the study. Individual body weight gains 

were analyzed at weeks 0, 4, 14, and 50 in male rats and at weeks 0, 4, 16, and 52 in female rats. 

Growth rates, total food consumption, and clinical pathology data generated through week 52 and 

organ weight data at week 52 were compared between dosed and control groups of the same sex by

Bartlett’s test for homogeneity of variance and the one-way classification analysis of variance 

(ANOVA). Scheffe’s multiple pairwise comparison procedure was used when ANOVA showed 

significance. Mean body weights at weeks 0 and 52 were also analyzed using a multiple pairwise 

comparison procedure. Ophthalmology examinations were performed on all rats initially and at 

weeks 26, 52, 78 and 104; on all male rats at week 122, and on all female rats at week 129. Blood 

was collected at 1, 4, 13, 26, 52, 78, and 104 weeks for hematology and clinical chemical analysis 

from 10 rats/sex/group, and from 10 males/group at week 122 and 10 females/group at week 129. 

Blood was collected by segmental amputation of the tail for hematology determinations and by

orbital sinus puncture under ether anesthesia for clinical chemistry determinations. Urine samples 

were collected by housing rats overnight in metabolism cages and pooling the samples by sex within 

each group. Interim sacrifice was done at 52 weeks, and terminal sacrifice was done at week 122 

and week 123 for main study males and at week 130 for main study females. Gross pathology and 

histology examinations were done on all animals that died and that were sacrificed on schedule.

Incidental signs such as hunched and/or thin appearance, labored respiration, enlarged testes in 

males, and/or ocular changes occurred with comparable frequency in control and treated rats. There 

was no compound related effect on mortality. At 120 weeks, survival ranged from 44 to 59% in 

treated male rats, compared to 51% in control male rats. At 128 weeks, survival ranged from 41 to 

56% in treated female rats, compared to 48% in control female rats. No dose-related trends in 

survival or clinical signs were observed. 
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Stability and homogeneity analyses conducted on diet samples prepared after study termination 

showed that stability ranged from 89.3 to 106.0% of target levels and homogeneity ranged from 87.6 

to 110.3% of target levels. Mean food consumption in male rats fed low and mid dose diets, and in 

female rats fed mid and high dose diets were comparable to control values. An analysis of mean 

efficiency of food utilization for the first year of the study did not reveal any significant compound 

effects. Weekly group water consumption was recorded during weeks 3, 12, 25, 51, 77 and 103. 

Terminal water consumption values were recorded the first 4 days of week 122 for males and all of 

week 124 for females. Individual water consumption values were calculated by dividing group 

consumption by the number of rats/group. Water consumption was consistently higher in high-dose 

males at all intervals although it was not statistically significant after week 51.

There were no compound-related changes in the ocular findings.

No compound related changes in hematology values were noted. There were some statistically 

significant differences between controls and compound-treated rats at sporadic intervals; however, 

the changes were not consistent with time or dose. 

Mean alkaline phosphatase values were lower in treated group than control males; significant 

decreases were measured at all three dose levels. No consistent significant decrease in alkaline 

phosphatase was measured in treated female rats. 

Cholinesterase (ChE) levels were significantly lower in 100, 300, and 1,000 ppm males and females 

at week 52. Also significantly lower ChE levels were found in 300 ppm males (at terminal/week 

122); and 100, and 300 ppm females (at terminal/week 129). These changes were, however, not 

dose related. It is noted that, standard deviations levels were higher than means in females at all 

three dose levels at week 52; and females at 100 and 300 ppm at terminal/week 129. No consistent 

significant changes in plasma or RBC ChE values were detected. At the terminal sacrifice, values 

for plasma and RBC ChE were similar in dosed and control groups but standard deviations were 

high. Examination of the individual data in all the groups indicated that 30-60% of the blood 

samples were hemolyzed. 

Mean alkaline phosphatase values were significantly lower in all three dose levels males and 

females.

No significant compound-related effects were determined by urinalysis.

No significant compound-related organ or tissue changes were observed. There were no differences 

in organ weights between compound-treated rats and control group at the interim sacrifice and the 

terminal sacrifice. Decreased spleen weights, increased thyroid weights and decreased adrenal 

weights were noted for selected dose levels, however, no significant dose-relate pattern was evident. 

Histopathology examination at week 52 found areas of interstitial cell hyperplasia of the testes, 

hyperplasia in the pars anterior of the pituitary, chronic myocarditis and myocardial degeneration, 

peribronchial lymphoid infiltration, and focal areas of hemorrhage and intimal mineralization of the 

pulmonary vessels in the respiratory system to be among the more frequently occurring lesions. 

However, their prevalence was comparable between compound-treated groups and control groups. 
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In the urinary system both groups showed chronic interstitial nephritis, tubular dilatation, and tubular 

eosinophilic proteinaceous casts with equal frequency. In the digestive system, hemosiderosis of the 

spleen, lymphoid hyperplasia and lymphoid necrosis of the Peyer’s patches occurred with 

comparable frequency in control and compound-treated rats. A summary tabulation of histologic 

findings at the 12-month sacrifice was not presented; however, tabulation of findings for individual 

animals supported the authors’ descriptive text on findings. No dose-related trends for nonneoplastic lesions were apparent. Neoplastic lesions were observed with comparable frequency in 

control and compound treated rats with a few sporadic exceptions. There were no apparent doserelated trends nor was there any difference in degree of histologic differentiation with increasing 

dietary compound levels. Neoplastic lesions are summarized in Table ... 

Deficiencies: 

Examination of individual data in all the groups indicated that 30-60% of the blood samples were 

hemolyzed. 

Dose levels used were not adequate to produce biologically relevant changes in life parameters, 

pathology findings, or cholinesterase activity. 

Brain ChE values in all groups including controls had an excess of outlying values (approximately

0.1 of mean value). For example, in females receiving 0, 100, 300, 1000 ppm there were two, seven, 

seven and seven outliers at 12 months and two, six, six, and three outliers at termination 

(10rats/group). 

In addition, mean values in controls were about 5 to 8 times lower than normally expected. The 

sponsor’s summary indicted that there was a technical error in the cholinesterase determinations. 

This study is classified as Unacceptable/Non-guideline. This study does not satisfy a guideline 

requirement for tolclofos-methyl. 

Cancer Mouse

In a Chronic Toxicity/Oncogenicity Feeding Study in Mice (MRID 00156139), S-3349, tolclofosmethyl (94.3% a.i., Lot # of Batch # not specified) at dose levels 10, 50, 250 and 10,000 ppm were 

fed in the diet to male and female B6C3F1 mice for 24 months. There was one week acclimation 

period and animals were assigned to the above test dose groups by stratified randomization. Mice 

were individually housed in an environmentally controlled room with a 12-hr light/dark cycle. The 

test compound was mixed monthly with the basal diet at the appropriate test concentrations based on 

the results of the pilot study. However, data from the pilot study were not reported. The test diet 

was stored in a cold room of unspecified temperature. Concentration analyses of the test diet were 

performed monthly for 18 months at the Institute for Biological Sciences of Sumitomo Chemical 

Company. Animals received food and water ad libitum. There were 50 animals/sex/group in main 

study groups and 10 animals/sex/group in satellite groups. Animals were inspected twice daily for 

signs of morbidity and mortality. All mice were individually examined for palpable masses at 

weekly intervals. Main study mice were weighed weekly at weeks 1 to 13 and monthly thereafter 

until study termination. Mice of the satellite group were weighed monthly. Mean daily diet 

consumption was calculated at weekly intervals. Efficiencies of food utilization were determined 

monthly at months 1 to 6 and biannually thereafter to study termination. Efficiency and compound 

intake were calculated from the consumption and body weight gain data. Water consumption every

48 hours was determined weekly. Ophthalmological examinations were performed on controls and 

high-dose males and females at 12 and 24 months. Blood was collected from the caudal vein or 
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right ventricle for hematological analysis at 12 and 24 months and for clinical chemistry analyses at 

6, 12 and 24 months from 10animals/sex/group. Urine was collected from 10animals/sex/group at 6 

and 12 months from mice of the satellite group, and 10animals/sex/group at 18 months from mice of 

the main group, and all survivors at 24 months. All animals that died and that were sacrificed on 

schedule were subject to gross pathological examination and the tissues were collected for 

histological examination. Student’s T test was used to analyze the numerical data: body weight, food 

consumption, clinical pathology, and organ weight data. Chi-square test was used to analyze 

mortality and gross microscopic pathology. 

Body weights of males receiving 250 and 1000 ppm were found to be slightly but significantly

(p<0.01, p<0.001) decreased from weeks 1 to 12 and from weeks 1 to 32, respectively; thereafter, 

body weights were found to be similar to those of concurrent controls. Body weights of females 

receiving 50 ppm were found to be slightly but significantly (p<0.05, p<0.01, p<0.001) decreased 

from weeks 1-52, while body weights of females receiving 250 and 1000 ppm were slightly but 

significantly (p<0.05, p<0.01, p<0.001) decreased at most weighing intervals throughout the study 

when compared to concurrent controls. Dosed male and female mice of the satellite groups 

exhibited slight fluctuations in body weights. 

Mean test compound intake was reported to be 1.28, 6.45, 32.20, and 134.15 mg/kg/day for males 

receiving 10, 50, 250 and 1000 ppm concentrations, respectively. Mean test compound intake for 

females receiving 10, 50, 250, and 1000 ppm concentrations was reported to be 1.32, 6.86, 34.10, 

and 136.91 mg/kg/day, respectively. Food consumption of treated males was similar to concurrent 

controls. Food consumption of females receiving 1000 ppm was found to be decreased from week 

30 to study termination. The food consumption of other treated females was similar to that of 

concurrent controls. 

Ophthalmological findings were similar in dosed and control mice. 

Serum and RBC cholinesterase (ChE) activities were significantly decreased in males and females 

receiving 250 and 1000 ppm S-3349 throughout the study when compared to concurrent controls; 

this decreased was more pronounced in females receiving 1000 ppm. Brain ChE activity was 

significantly decreased in males receiving 250 and 1000 ppm at 52 and 104 weeks and females 

receiving 50, 250, and 1000 ppm at 28 weeks, 250 and 1000 ppm at 52 weeks, and 1000 ppm at 104 

weeks. Brain cholinesterase activity was most extensively reduced in females receiving 1000 ppm at 

28 weeks.

Other clinical chemistry parameters were similar for control and dosed males and females and there 

were no effects of dosing on any urinary parameter.

Organ weights of control and dosed males and females were similar at 28 weeks. At 12 months, 

slightly but significantly (p<0.05) decreased absolute and relative pituitary weights were found in 

males receiving 250 ppm. Females receiving 1000 ppm had significantly increase (p<0.05) absolute 

and relative pituitary weights. Absolute brain weights were significantly (p<0.01) increased in 

males receiving 1000 ppm at 24 months; absolute and relative brain weights were slightly but 

significantly (p<0.05) increased in females receiving 250 ppm at this time period. Absolute kidney 

weights were slightly but significantly (p<0.05, p< 0.01) increased at 12 months in males receiving 
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250 ppm; absolute and relative kidney weights were significantly (p<0.05, p<0.01) increased in 

males receiving 1000 ppm and in females receiving 250 and 1000 ppm. The incidence of kidney

discoloration was increased in males receiving 10 and 1000 ppm and in females receiving 250 ppm. 

Hematomas of the pituitary were not found in control females but were found in one, four, and five 

females receiving 10, 50 and 1000 ppm, respectively; this finding was not exhibited in treated males. 

At 24 months, the incidence of liver nodules was found to be increased in 50 and 1000 ppm males 

and females at 1000 ppm when compared to concurrent controls; liver masses were found in 8 

control males and 17, 14, 11 and 13 males receiving 10, 50, 250, and 1000 ppm, respectively. 

Histological examination found that, at 24 months, the incidences of lymphomas were higher in 

females, and incidence of adenomas and adenocarcinomas of the liver and lung were higher in 

males. However, these findings were similar in dosed and control mice with the exception of 

adenomas of the liver which were increased in males treated with 10 ppm (9/50), 50 ppm (11/50) 

and 1000 ppm (9/50) when compared to concurrent controls (5/50). Non-neoplastic lesions such as 

lymphocytic infiltration of the kidneys were increased in dosed males when compared to controls, 

and pyelonephritis of the kidneys was increased in females receiving 10000 ppm when compared to 

controls. Hemorrhage of the lymph nodes were found in 2 control males and 10, 4, 3, and 7 males 

receiving 10, 50, 250 and 1000 ppm, respectively; the incidence at 10 ppm was significantly

(p<0.05) increased. Fibrosis of the tongue was reported in 4 control females and 8, 8, 5, and 12 

females receiving 10, 50, 250 and 1000 ppm, respectively; the incidence at 1000 ppm was 

significantly (p<0.05) increased. However, since there was no descriptive narrative in histologic 

lesions, it was unable to differentiate between malignant and benign lesions (could not determined if 

adenocarcinomas of the liver were actually hepatocellular carcinomas and if lung tumors were 

alveolar bronchiolar pulmonary lesions). 

Based on decreased serum, erythrocyte, and brain cholinesterase activities, MTD of 1000 ppm 

(highest dose tested) for this study was obtained. The systemic LOEL is 250 ppm and the systemic 

NOEL is 50 ppm S-3349, based on decreased serum, erythrocyte, and brain cholinesterase activities. 

The LOEL and NOEL for histopathology cannot be definitively determined.

Deficiencies:

Stability and homogeneity analyses of the test diet were not reported. Data from the pilot study were 

not reported. The study authors reported that the first two analyses of the test diet concentration 

revealed that the control diet was contaminated with 1.8 to 3.0μg/g of S3349; the study authors 

indicated that these results may not be accurate. 

A large amount of the individual data was handwritten and illegible; therefore, validation of many 

parameters (body weights, hematology, clinical chemistry, urinalyses, and organ weights) was not 

possible. 

Individual data were not reported for, clinical observations or palpable mass observation. 

The report’s section on conclusions (page#48 of the study report) indicated that ChE activities 

increased in males and females receiving 1000 ppm, when these parameters, in fact decreased. 
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One animal death was incorrectly identified in Table 43-3 of the study report (animal no. 1324 was 

reported with adenocarcinoma of liver when animal no.1334, a male receiving 50 ppm, was reported 

to have died with this finding in Table 47-3-1 of the study report). 

The numbers of organs with pathology reported in this study were compared to the number of 

animals sacrificed rather than the number of organs examined (which is appropriate to do). Also 

there was no descriptive narrative in histologic lesions and was unable to differentiate between 

malignant and benign lesions; as such, histopathology results could not be conclusively determined. 

The LOEL and NOEL for histopathology cannot be definitively determined. 

Based on the number of deficiencies this study is classified as Unacceptable/Non-guideline. This 

study does not satisfy a guideline requirement for tolclofos-methyl. 

Neurotoxicity Rat

Time to Peak

In a time to peak effect study (MRID 48341941), tolclofos-methyl (technical grade 97.5% a.i., Lot 

no. #080712G) in corn oil (vehicle) was administered orally by gavage as a single dose (2000 

mg/kg) to one group of adult (approximately 7 weeks old) Sprague-Dawley (Crl:CD(SD)) rats. A 

concurrent control group received the vehicle on a comparable regimen. The dose volume was 5 

ml/kg for all groups. Each group consisted of 20 rats/sex. For analysis of plasma, red blood cell 

(RBC) and whole brain cholinesterase activity, 5 animals/sex/group were euthanized by carbon 

dioxide at approximately 1, 2, 4, and 8 hours following dose administration. Blood samples were 

collected from the inferior vena cava, the whole brains were collected and weighed and plasma, red 

blood cell, and brain homogenates were analyzed for cholinesterase activity. 

All animals survived to the scheduled euthanasia, there were no test substance-related clinical 

findings noted. Cholinesterase activity (RBC, plasma, whole brain) was unaffected for males and 

females in the 2000 mg/kg group at approximately 1, 2, 4 and 8 hours (no different from the control 

group across all time points observed). The absence of consistent inhibition of cholinesterase 

through 8 hours following dose administration precluded the determination of a time of peak effect 

of cholinesterase inhibition. 

This study is classified as Acceptable/Non-guideline. This study does not satisfy a guideline 

requirement for tolclofos-methyl. 

ACN

In an acute neurotoxicity study (MRID # 48341940), groups of non-fasted, six weeks-old, 

Crl:CD(SD) rats, 12/sex/dose were given a single oral dose of tolclofos-Methyl (97.5% a.i., lot 

#080712G) in corn oil at doses of 0, 200, 700, or 2000 mg/kg bw and observed for 14 days. 

Neurobehavioral assessment (functional observational battery and motor activity testing) was 

performed in 12 animals/sex/group prior to the initiation of dose administration (study day -8), at the 

time of peak effect on study day 0 (considered to be approximately 4 hours after dose 

administration), and on study days 7 and 14. Cholinesterase activity was not measured. At study

termination, 12 animals/sex/group were euthanized and perfused in situ for neuropathological 

examination. Of the perfused animals, 6 animals/sex from the control group and the 2000 mg/kg 
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group were selected randomly and subjected to histopathological evaluation of brain and peripheral 

nervous system tissues. 

There were no test substance-related clinical findings observed during the study. All animals survived to 

the scheduled euthanasia (study day 15). Mean body weights and body weight gains were unaffected 

by treatment. Mean overall locomotor activity (total and ambulatory counts) was decreased for males 

and females in the 700 and 2000 mg/kg groups at the time of peak effect on study day 0, but was 

similar to the control group on study days 7 and 14. Locomotor activity was

unaffected by test substance administration at a dose level of 200 mg/kg. There were no test 

substance-related macroscopic or microscopic changes. Brain weights and measurements were 

unaffected by administration of the test compound at any dose level.

Based on the effects seen in this study, the LOAEL is 700 mg/kg bw/day based on decreases in 

overall locomotor activity (total and ambulatory counts) in males and females in the 700 and 

2000 mg/kg groups at the time of peak effect on study day 0. The NOAEL is 200 mg/kg 

bw/day.

Plasma, erythrocyte, and brain cholinesterase levels were not measured in this study.

This neurotoxicity study is classified as acceptable, guideline, and satisfies the guideline requirement 

for an acute neurotoxicity study in rats (870.6200; OECD 424). 

SCN

In a subchronic neurotoxicity study (MRID 48341952) tolclofos-methyl (Technical grade) (96.8% 

w/w, batch # 041128G) was administered continuously in the diet for 90 days to male and female 

Alpk:APfSD (Wistar-derived) rats 12/sex/group at dose levels of 0, 300, 1800, or 10,000 ppm 

(equivalent to 0, 20.6, 122.3, 735.7 mg/kg bw/day for males; 0, 23.1, 135.8, and 762.7 mg/kg/day for 

females). Neurobehavioral assessment (functional observational battery and motor activity testing) 

was performed in all main study animals 12 rats/sex/group in week -1, and in weeks 2, 5, 9 and 14. 

Cholinesterase (ChE) activity was determined at the Central Toxicology Laboratory (Clinical 

Pathology) in 5 rats/sex/dose, in brain and erythrocytes (RBC) at weeks 2, 5 and 9 (satellite phase) 

and week 14 (main study). Plasma ChE was not evaluated. At study termination (14 weeks), 5 

animals/sex/group were euthanized and perfused with formol saline for neuropathological 

examination. Brain was weighed from all of the perfused animals as was a histopathological 

evaluation of brain and peripheral nervous system tissues. 

There were no treatment-related clinical abnormalities recorded during the functional observation 

battery and on motor activity. Body weight was statistically significantly lower than control for both 

sexes fed 10,000 ppm throughout the study (maximum difference of 17% in males and 11% in 

females, starting from week 2). Body weight gain was also statistically significantly lower than the 

controls throughout the study in both sexes fed 10,000 ppm [range 4-82%]. Food utilization for both 

males and females at 10,000 ppm was lower than control in weeks 1 to 4 (80-98% of control) and for 

males over all (weeks 1-13; maximum 84% of controls). There were no effects on brain weights in 

the main study animals. There were no treatment-related neuropathology and microscopic findings 

in the tissues examined.
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A BMD analysis of the ChE data revealed a lack of dose response such that a reliable fit was not 

obtained for adult male RBC and whole brain ChE data. For females, the whole brain ChE data 

were fit to the exponential model, with a BMD10 of 956.4 mg/kg/day and BMDL10 of 659.2 

mg/kg/day. However, female RBC did not provide a reliable fit. 

Brain ChE

Adult Female Brain ChE BMD10 = 956.4 mg/kg/day

Adult Female Brain ChE BML10 = 659.2 mg/kg/day

The adult male Brain ChE data failed to produce a dose-response and therefore did not provide a 

reliable fit for BMD analysis.

RBC ChE

The RBC ChE data in both males and females failed to provide a dose response and therefore 

did not provide a reliable fit for BMD analysis. 

The NOAEL is 122.3 M/135.8 F mg/kg/day. The LOAEL is 735.7M /762.7F mg/kg/day, based on 

decreased bodyweight and bodyweight gain, and food utilization. 

The study is classified as Acceptable/Non-Guideline and satisfies the guideline requirement for a 

subchronic neurotoxicity study in rats (870.6200b). 

Immunotoxicity

In an immunotoxicity study (MRID 48341954), tolclofos-methyl (97.5% a.i., lot number: 

080712G) was administered to female Crl:CD1(ICR) mice (10/dose) via diet at dose levels of 0, 500, 

1500, or 4500 ppm (0, 91, 273, or 811 mg/kg/day, respectively) for 28 days. The positive control 

consisted of 8 female mice which were administered 20 mg/kg/day cyclophosphamide via gavage on 

days 22-26. On Day 25, all animals were immunized with a 0.5 mL intravenous injection of sheep 

red blood cells (SRBCs) at a concentration of 4x108cells/mL. On Day 29, all animals were 

sacrificed by carbon dioxide asphyxiation. The necropsy included the examinations of all major 

organs, tissues and body cavities. Spleens from all animals were harvested for evaluation of spleen 

anti-SRBC antibody response with a plaque-forming cell (PFC) assay. Other parameters evaluated 

were: clinical observation, mortality, body weight, body weight gain, food consumption, spleen and 

thymus weights.

There were no unscheduled deaths or treatment-related clinical signs. The mean final body weight 

(Day 29) for animals receiving 4500 ppm was lower (-11.2%) than controls. Overall mean body 

weight gains (Days 1 to 29) for animals receiving 4500 ppm were lower (-40%) than the controls.

Food consumption for females receiving 4500 ppm was lower (-11%) than the control. There was no 

treatment-related effect on water consumption, organ weights and macropathology.

The systemic toxicity NOAEL is 1500 ppm (273 mg/kg/day); the LOAEL was 4500 ppm (811 

mg/kg/day) based on decreased body weights, body weight gains and food consumption.

For immunotoxicity, there were no statistically significant changes in the numbers of cells/spleen, 

PFC/106viable cells or PFC/spleen in any of the tolclofos-methyl treated groups when compared to 

the control. The positive control with Cyclophosphamide resulted in a significant reduction of the 
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PFC response. The numbers of PFC/106viable cells and PFC/spleen (both p < 0.001) were 

significantly reduced when compared to the control. A high inter-individual variability was noted in 

all the treatment groups as well as in the control group. Evaluation of individual animal data of this 

study did not show any trend or distribution that would demonstrate significant suppression of antiSRBC plaque-forming-cell response. The positive control group demonstrated a statistical decrease 

in PFC response as compared to the vehicle controls.

The Natural Killer (NK) cells activity was not evaluated in this study. The toxicology database for 

tolclofos-methyl does not show any evidence of treatment-related effects on the immune system. The 

overall weight of evidence suggests that this chemical does not directly target the immune system. 

Under the HED guidance, a NK cells activity assay is not required at this time.

Under conditions of this study, the immunotoxicity NOAEL for anti-SRBC PFC response is 

4500 ppm (811 mg/kg/day); the LOAEL was not established.

This immunotoxicity study is classified acceptable/guideline and satisfies the guideline requirement 

for an immunotoxicity study (OPPTS 870.7800).
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Appendix B: Review of Human Research

This risk assessment relies in part on data from studies in which adult human subjects were 

intentionally exposed to a pesticide to determine their dermal and inhalation exposure. These 

studies, which have been utilized in the HED Seed Treatment SOPs 14 and 15, have been 

determined to require a review of their ethical conduct, have received that review, and have been 

determined to be ethical.