                 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

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, 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.

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).


	
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

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.  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 tolclofos-methyl on various crops and crop groups.  As a result, the proposed seed treatment use of tolclofos-methyl 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 [phenyl-[14]C]tolclofos-methyl did not take up the radiolabeled test substance residue.

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 tolclofos-methyl; 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 tolclofos-methyl 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.

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 tolclofos-methyl 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). 

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, [phenyl-[14]C]tolclofos-methyl. 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 non-food 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.

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

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 tolclofos-methyl 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
                                       
                                       


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 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 tolclofos-methyl 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 O-methyl-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 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.
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 tolclofos-methyl 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.

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 6[th] 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 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.

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).
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 [phenyl-[14]C]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 [phenyl-[14]C]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
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, 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 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.  Long-term 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).
Table 9.1.1:  Summary of Short- and Intermediate-Term Primary Handler Exposure and Risk Estimates for Tolclofos-methyl
                                   Seed Type
                                  Inhal UE[a]
                                Max App Rate[b]
                                Amt Treated[c]
                                 Inhal Dose[d]
                                 Inhal MOE[e]
                                       
                                     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


Table 9.1.1:  Summary of Short- and Intermediate-Term Primary Handler Exposure and Risk Estimates for Tolclofos-methyl
                                   Seed Type
                                  Inhal UE[a]
                                Max App Rate[b]
                                Amt Treated[c]
                                 Inhal Dose[d]
                                 Inhal MOE[e]
                                       
                                     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.
      
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 UE[a]
                                Max App Rate[b]
                          Amt of Seed Planted/Day[c]
                              Inhalation Dose[d]
                               Inhalation MOE[e]
                                       
                                       
                                 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.

0.2 
0.3 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.


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).  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 dermal- rabbit
                                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,non-acceptable/non-guideline
                                       
                               41445702 (1983) @
                   25% flowable, unacceptable/non-guideline
                               LC50>= 3.32 mg/L
                             Particles 0.3-5.5um
                          %MMAD 1um 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 sensitization- guinea pig
                                00156131 (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
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/non-guideline
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/non-guideline
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.
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 tolclofos-methyl 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/non-guideline
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 6[th] sternebra (p<=0.01).
870.3700b

Prenatal developmental in (rabbit)
New Zealand White
TXR006933
00156143 (1982)
Unacceptable/non-guideline
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.
870.3800

3- Gen Reproduction 
(rat) Sprague-Dawley
TXR006933
 00156145 (1985)
Unacceptable/non-guideline
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)
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 ug/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[-9] to 5x10[-6] M.
870.5375 
In vivo chromosome aberration
TXR006933
00156149 (1981)
Unacceptable/guideline
Unacceptable.
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 ug/ml.
870.5375
Chromosome Aberration (CHO-K1)
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.
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/Non-guideline
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.


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.

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), tolclofos-methyl (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 dose-response 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 re-evaluated 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 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, S-3349) (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.  

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 dose-response 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), tolclofos-methyl (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). 
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 5u 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 tolclofos-methyl (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 dose-response 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 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:
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.  

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.  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 non-neoplastic 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 dose-related 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, tolclofos-methyl (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 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 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 3.0ug/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.  

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 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.

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 4x10[8] cells/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/106 viable 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 PFC response. The numbers of PFC/106 viable 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 anti-SRBC 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).
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.

