Document ID: EPA-HQ-OPP-2010-0102-0009
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2011-04-22T04:00Z

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
                            WASHINGTON, D.C.  20460
                                       

                                  OFFICE OF CHEMICAL SAFETY AND
                                       POLLUTION PREVENTION

Date: April 13, 2011

MEMORANDUM

SUBJECT:		Triflusulfuron-methyl: Revised Human Health Risk Assessment for Use in Garden Beet.

PC Code:  129002
DP Barcode:  D388621
Decision No.: 425459
Registration No.:  352-569
Petition No.:  9E7669
Regulatory Action:  Section 3 Registration
Assessment Type:  Single Chemical Aggregate
Case No.:  NA
TXR No.:  NA
CAS No.:  126535-15-7
MRID No.:  47943201
40 CFR:  §180.492

FROM:	Elizabeth Holman, Chemist/Risk Assessor 
      Margarita Collantes, Biologist
      Linnea Hansen, Toxicologist
      Risk Assessment Branch 2
      Health Effects Division (7509P)

THROUGH:	Richard A. Loranger, Ph.D., Senior Scientist
      Christina Swartz, Branch Chief
      Risk Assessment Branch 2
		Health Effects Division (7509P)
					
TO:		Laura Nollen, Risk Manager Reviewer
		Barbara Madden, Review Manager #5
		John Hebert, Acting Chief
      Risk Integration, Minor Use, and Emergency Response Branch
		Registration Division (7505P)

The Registration Division (RD) requested that the Health Effects Division (HED) conduct a risk assessment for the active ingredient triflusulfuron-methyl to estimate the risk to human health that will result from the proposed use to control broadleaf weeds in garden beet.   The risk assessment completed 2/17/11 (E. Holman, D373493) was revised to provide additional characterization of the doses and severity at which hemolytic anemia was observed in studies conducted in the rat (E. Holman, 4/7/11, D388460).  This revised risk assessment has been further revised to address issues related to plant-back intervals (PBI) for rotational crops and restricted entry intervals (REI).  

                               Table of Contents
1.0	Executive Summary	4
2.0	Ingredient Profile	7
2.1	Summary of Proposed Uses	7
3.0	Hazard Characterization/Assessment	9
3.1	Hazard and Dose-Response Characterization	9
3.1.1	Database Summary	10
3.1.1.1	Studies available and considered (animal, human, general literature)	10
3.1.1.2	Mode of action, metabolism, toxicokinetic data	10
3.1.1.3	Sufficiency of studies/data	10
3.1.2	Toxicological Effects	11
3.1.3	Dose-response	12
3.2	Absorption, Distribution, Metabolism, Excretion (ADME)	12
3.3	FQPA Considerations	13
3.3.1	Adequacy of the Toxicity Database	13
3.3.2	Evidence of Neurotoxicity	13
3.3.3	Developmental Toxicity Studies	14
3.3.4	Reproductive Toxicity Study	14
3.3.5	Additional Information from Literature Sources	14
3.3.6	Pre-and/or Postnatal Toxicity	14
3.3.6.1	Determination of Susceptibility	14
3.3.6.2	Degree of Concern Analysis and Residual Uncertainties for Pre- and/or Postnatal Susceptibility	15
3.3.7	Recommendation for a Developmental Neurotoxicity Study	15
3.4	Safety Factor for Infants and Children	15
3.5	Hazard Identification and Toxicity Endpoint Selection	16
3.5	Hazard Identification and Toxicity Endpoint Selection	16
3.5.1	Acute Reference Dose (aRfD) - Females age 13-49 and the General Population	16
3.5.2	Chronic Reference Dose (cRfD)	16
3.5.5	Dermal Absorption	17
3.5.8	Residential and Occupational Dermal Exposure, Long-Term (>30 days)	17
3.5.9	Residential and Occupational Inhalation Exposure, Short-Term (1 to 30 days)	18
3.5.11	Residential and Occupational Inhalation Exposure (Long-Term)	18
3.5.12	Level of Concern for Margin of Exposure	18
3.5.2	Summary of Toxicological Doses and Endpoints for Use in Human Risk Assessments	19
3.5.12	Recommendation for Aggregate Exposure Risk Assessments	20
3.5.13	Classification of Carcinogenic Potential	20
3.6	Endocrine disruption	21
4.0	Public Health and Pesticide Epidemiology Data	21
5.0	Dietary Exposure/Risk Characterization	22
5.1	Pesticide Metabolism and Environmental Degradation	24
5.1.9	Drinking Water Residue Profile	26
5.2	Dietary Exposure and Risk	27
6.0	Residential (Non-Occupational) Exposure/Risk Characterization	28
7.0	Combined Risk Assessments and Risk Characterization	29
8.0	Cumulative Risk Characterization/Assessment	29
9.0	Occupational Exposure/Risk Pathway	29
10.0	Data Needs and Label Recommendations	31
10.1	Toxicology	31
10.2	Residue Chemistry	31
10.3	Occupational and Residential Exposure	32
References:	32
Appendix A:	Toxicology Assessment	33
A.1  Toxicology Data Requirements	33
A.2  Acute Toxicity Profile for Isoxaben	34
A.3  Executive Summaries	38
A.4  Rationale for Toxicology Data Requirements	50
 1.0	Executive Summary
	
Triflusulfuron-methyl is a sulfonylurea herbicide that inhibits the biosynthesis of branched-chain amino acids in plants (via inhibition of the plant enzyme acetolactate synthase).  Marketed as UpBeet(R), a dry flowable (DF) formulation containing 50% active ingredient (ai), triflusulfuron-methyl is currently approved for use on sugar beet commodities and chicory using broadcast (ground- or aerial-based) or band application methods at seasonal rates of up to 0.078 pounds ai per acre (lb ai/A).  UpBeet(R) is not to be applied via any type of irrigation system. The existing label for UpBeet(R) lists a pre-harvest interval (PHI) of 60 days for both sugar beet and chicory.  The registrant, E. I. du Pont de Nemours and Company, has submitted a supplemental label for a proposed use on garden beets.  HED has sufficient information to complete a human health risk assessment.

The maximum proposed single application rate on this supplemental label is 0.0156 lb ai/A for agricultural use on garden beets for control of broadleaf weeds, with a maximum seasonal application rate of 0.047 lb ai/A.  Consistent with the existing uses, the requested new use on garden beet involves aerial or groundboom application methods. The proposed PHI on garden beet is 30 days, a value that is well supported by the garden beet field trial data submitted for this new use petition.  The proposed supplemental label needs to be revised to reflect the 30 day PHI and specify that use of aerial application methods is allowed.  

The toxicology database is considered adequate for conducting a risk assessment in accordance with the Food Quality Protection Act (FQPA).  The toxicology database is largely complete, missing only an immunotoxicity study.  A subchronic inhalation study is not available but not required at this time due to low use rates, low vapor pressure and high MOEs associated with handler exposure; the need for this study may be reassessed once the Agency finalizes guidance on pesticide volatilization and postapplication exposure.  Toxicological effects observed in mammals were decreased body weight and body weight gain, alterations in hematology (decreases in red blood cell parameters, particularly in males), and increased interstitial cell hyperplasia in testes.  No toxicological effects were attributable to a single, oral (i.e., acute) exposure.  In dermal exposure studies, no effects were observed at the highest dose tested.  For assessment of inhalation exposures, route-to-route extrapolation from oral exposures has been used, assuming 100% absorption for inhalation exposures.  Triflusulfuron-methyl has been classified as a Category C "possible human carcinogen" and the chronic risk assessment based on the chronic Population Adjusted Dose (cPAD) is sufficiently protective of human health.  HED recommends that the 10x FQPA safety factor for infants and children be reduced to 1x for triflusulfuron-methyl because there is no evidence of increased susceptibility in rat or rabbit fetuses from the developmental toxicity studies, and no evidence of susceptibility in the rat reproductive toxicity study.  There were no neuropathological or neurobehavioral effects observed in the database, which included acute and subchronic neurotoxicity studies, except for an increased incidence of degeneration of the sciatic nerve in female rats in the chronic toxicity/carcinogenicity study at study termination.  However, HED considers this finding of low concern because it is commonly observed at high incidence in older rats and was not increased at the interim (1 year) sacrifice or in shorter-term studies.  A developmental neurotoxicity (DNT) study is therefore not required.   There was no evidence of direct immunotoxicity observed in the submitted studies.  Although mild hemolytic anemia was observed in rats and dogs, the observed hemolytic effects were generally sporadic and marginal except at higher doses, with more pronounced changes observed at doses >=100-fold above the selected point of departure (POD) in the rat, and >=15-fold above the selected POD in the dog.  The selected endpoints are protective of hematological effects and an additional 10x database uncertainty factor was therefore not retained to account for lack of the immunotoxicity study.

The residue chemistry database is complete and there are no residue chemistry issues that would preclude granting the requested new use and establishing permanent tolerances for residues in garden beet roots and tops.  For the purposes of this petition on garden beet, the previously reviewed sugar beet metabolism study is sufficient to support the requested use of triflusulfuron-methyl on garden beets.  The proposed tolerance expression in/on garden beets should include only the parent compound, triflusulfuron-methyl.  HED is recommending for the establishment of permanent tolerances for the requested crops.  Suitable analytical methods are available to enforce tolerances for residues of triflusulfuron-methyl.  

HED has assessed dietary and occupational exposures.  Risks from these exposures, including aggregate exposures, are below the level of concern.  The existing registered label (for sugar beet and chicory uses) and the proposed supplemental label for garden beet include a 4-hr Restricted Entry Interval (REI).  HED has no objections to these REI.  

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, which comprise the Pesticide Handlers Exposure Database (PHED), have been determined to require a review of their ethical conduct, have received that review, and have been determined to be ethically conducted.

Environmental Justice
Potential areas of environmental justice concerns, to the extent possible, were considered for this human health risk assessment, in accordance with US 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 USDA under the CSFII, and are used in pesticide risk assessments for all registered food uses of a pesticide.  These data are analyzed and categorized by subgroups based on age, season of the year, ethnic group, and region of the country.  Whenever appropriate, non-dietary exposures based on home use of pesticide products, associated risks for adult applicators, and for toddlers, youths, and adults entering or playing on treated areas post-application 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.

Proposed Tolerances

At this time, tolerances are not required for residues in livestock commodities.  For plant commodities, existing tolerances are in place for the previously assessed sugar beets and chicory.   New tolerances in/on garden beets are being requested in the petition under review (Petition 9E7669).  The registrant has proposed that tolerances for the residues of the herbicide triflusulfuron-methyl, or methyl 2-[[[[[4-(dimethylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-yl]amino]carbonyl]amino]sulfonyl]-3-methylbenzoate, be established on garden beet roots at 0.01 ppm (parent only) and sugar beet tops at 0.02 ppm (parent only).  For the purposes of this tolerance petition, the compound to be regulated is parent only.  

Note to RD:  According to HED's Interim Guidance on Tolerance Expressions (S. Knizner, 27 May 2009), the tolerance expression for triflusulfuron-methyl cited in 40CFR §180.492[a] should be revised to state:  

      Tolerances are established for residues of triflusulfuron-methyl, including its metabolites and degradates, in or on the commodities listed in the table below.  Compliance with the tolerance levels specified below is to be determined by measuring only triflusulfuron-methyl (methyl 2-[[[[[4-(dimethylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-yl]amino]carbonyl]amino]sulfonyl]-3-methylbenzoate) in or on the commodity.  

A summary of the petitioned-for tolerances and HED's recommendations is provided in Table 1, below.  The requested new use on garden beet is being assessed as part of a work share agreement with Canada's PMRA.  The recommended tolerance for garden beet roots is identical to that of PMRA's.  However, the recommended tolerance of 0.02 ppm for garden beet tops is not harmonized with PMRA's value of 0.01 ppm.  The rationale for this recommendation is discussed further in Section 5.1.11.       

Table 1.1  Tolerance Summary for Triflusulfuron-methyl
Commodity
                           Proposed Tolerance (ppm)
                          Recommended Tolerance (ppm)
Comments (correct commodity definition)
Beet, garden, roots
                                     0.01
                                     0.01
--
Beet, garden, tops
                                     0.02
                                     0.02
--

Provided the issues associated with the UpBeet label detailed in Sections 10.2 and 10.3 are addressed, HED can recommend for establishment of the tolerances in Table 1.1.  HED recommends that the registration of the use on garden beets be conditional on future submission of the immunotoxicity study (see Section 10.1).

2.0	Ingredient Profile	

2.1	Summary of Proposed Uses	
Summary of Existing Uses on Garden Beet and Chicory

For sugar beet, UpBeet(R) may be broadcast applied via ground or air equipment at up to 0.032 lb ai/A/application.  Multiple applications can be made at 5-10 day re-treatment intervals (RTIs) with the total application not to exceed 0.078 lb ai/A/season.  For chicory, two applications may be made, at 0.024 lb ai/A and at 5- to 10-day RTIs, for a maximum seasonal application rate of 0.048 lb ai/A.  This formulation may also be band-applied at rates equivalent to the broadcast rates (actual band rates are dependent upon crop row spacing).  UpBeet(R) is not to be applied via any type of irrigation system.  The existing label for UpBeet(R) lists a PHI of 60 days for both sugar beet and chicory, representing a potential mid-season use.  Plant-back intervals (PBIs) are 0 days for sugar beets, 14 days for all other crops except corn, and 21 days for corn.  A plant-back interval has not been proposed for chicory or garden beet.  HED is satisfied with the 14-day interval that these crops receive from being in the "other crop" category as listed on the current label.
 
Proposed Use on Garden Beet
Table 2.1 provides a summary of the proposed uses for DuPont(TM) UpBeet[(R)] Herbicide (DF, water-dispersible granule, WDG, containing 50% triflusulfuron-methyl by weight).  The maximum single application rate is 0.0156 lb ai/A for agricultural use on garden beets for control of broadleaf weeds.  Triflusulfuron-methyl (as UpBeet(R)) may be applied aerially or with groundboom equipment.  

TABLE 2.1 Summary of Proposed Directions for Use of Triflusulfuron-Methyl on Garden Beets.
Trade Name
                              Application Timing
                               Application Rate 
                                   (lb ai/A)
                          Max. No. Applic. per Season
                                    RTI[1]
                                    (days)
                          Max. Seasonal Applic. Rate
                                   (lb ai/A)
                                      PHI
                                    (days)
                     Use Directions and other Limitations
   (i.e. grazing, type of application/equipment, plant-back interval, other)
                                 Garden Beets
UpBeet(R) Herbicide
Apply as a post-emergent spray when garden beets are at the following stages: 2-4 leaf stage; 4-6 leaf stage; and 6-8 leaf stage.
                                    0.0156 
                      +                       NIS or COC
                                (at 0.25% v/v)
                                       3
                            See appli-cation timing
                                    0.047 
                                      30
- Apply UpBeet(R) Herbicide with NIS or COC.2
- Apply UpBeet(R) Herbicide via groundboom or aerial application equipment.  
- Do not apply UpBeet(R) Herbicide through any type of irrigation equipment.

[1] RTI = retreatment interval
[2] NIS = non-ionic surfactant; COC = crop oil concentrate

0.2 Structure and Nomenclature
TABLE 2.2	Test Compound Nomenclature.
Compound
Chemical Structure

Common name
Triflusulfuron-methyl
Company experimental name
DPX 66037
IUPAC name
methyl 2-[4-dimethylamino-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylcarbamoylsulfamoyl]-m-toluic acid
CAS name
methyl 2-[[[[[4-(dimethylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-yl]amino] carbonyl]amino]sulfonyl]-3-methylbenzoate
CAS #
126535-15-7
End-use product/(EP)
UpBeet Herbicide*
*The EP used in the submitted garden beet field trials under review was Safari 50DF, an EP that is equivalent to that of UpBeet(R).  
 

0.3 Physical and Chemical Properties
      
TABLE 2.3	Physicochemical Properties of the Triflusulfuron-Methyl.
Parameter
Value
Reference
Melting point/range
                                  155-158[o]C
                       Petition Administrative Materials
pH
                                 Not indicated
                                       
Density (g/mL)
                                 1.45 absolute
                                  0.56 (Bulk)
                                       
Water solubility ( 25°C)
pH                            ppm
3                                 1.0
5                                 2.7
7                               110
9                          11 000
                                       

Low solubility at pH3 and pH 5; soluble at pH 7; very soluble at pH 9. Solubility increases with increasing pH.
                                       
Solvent solubility (mg/L)
solvent                        mg/L
hexane                      <0.0016
octanol                          0.026
toluene                              2.0
methanol                           7.0
ethyl acetate                       27
acetonitrile                         80
acetone                            120
chloroform                       160
methylene chloride          580
                                       
Vapor pressure at 25°C
                             <1 x 10[-7] mm Hg
                                       

A relatively non-volatile compound; low potential for losses due to volatilization.
                                       
Dissociation constant (pKa)
                                      4.4
                                       

This compound will dissociate and exist in its anionic form at most environmentally relevant pHs.
                                       
Octanol/water partition coefficient (KOW)
pH                       Kow at 25°C
5                                220
7                                 9.2
9                                 0.86
                                       

Low potential for bioaccumulation in biota.
                                       
UV/visible absorption spectrum
λmax = 395 nm
absorption at λ>400 nm is not anticipated
                                       

Potential to absorb sunlight in the UV/visible range of 290-400 nm.
                                       
 
3.0	Hazard Characterization/Assessment	

This hazard characterization updates the previous assessment for triflusulfuron-methyl, prepared February 12, 2002 for the Section 3 Registration on sugar beets (D260076).

3.1	Hazard and Dose-Response Characterization	

3.1.1	Database Summary	

3.1.1.1   Studies available and considered (animal, human, general literature)	

Studies considered for this assessment included acute lethality (oral, dermal and inhalation routes); primary eye and dermal irritation, dermal sensitization, subchronic oral toxicity (rat, mouse and dog), rabbit 21-day dermal toxicity, dog chronic toxicity, rat chronic toxicity and combined chronic toxicity/carcinogenicity, mouse carcinogenicity, developmental toxicity (rat and rabbit), rat two-generation reproductive toxicity, genotoxicity, rat metabolism and acute and subchronic neurotoxicity studies in the rat.  An in vitro mode of action study on hepatic aromatase activity and testosterone/estradiol levels in Leydig (interstitial) cells was also submitted.  Summaries of these studies are provided in Appendix A.3.

3.1.1.2	 Mode of action, metabolism, toxicokinetic data	

Triflusulfuron-methyl is a post-emergence sulfonylurea herbicide used to control broadleaf weeds.  In plants, it inhibits growth by preventing branched-chain amino acid formation in plants (via inhibition of acetolactate synthase), but does not affect germination.  A mode of action for mammalian toxicity has not been fully characterized.  Data were submitted to determine whether the Leydig cell tumors observed in male rats were due to treatment-related hormonal activity.  In an in vitro study in isolated rat Leydig cells, triflusulfuron-methyl induced hepatic aromatase activity and testosterone/estradiol levels, but the data were insufficient to demonstrate conclusively a hormonal mode of action. 

In metabolism studies conducted in the rat, triflusulfuron was well-absorbed and extensively metabolized; however, at high doses, the extent of absorption was somewhat lower and the parent compound was the major radiolabeled component.  The majority of the administered radioactivity was excreted in the urine at low doses, but at high doses, the major route of excretion was the feces.  The major urinary metabolite was N-desmethyl triflusulfuron-methyl.  

3.1.1.3	 Sufficiency of studies/data	

The available toxicity data are considered adequate for conducting this risk assessment for triflusulfuron-methyl.  However, in accordance with the recently revised 40 CFR Part 158 data requirements for conventional pesticides, an immunotoxicity study is required.  The rationale for the immunotoxicity data requirement is provided in Appendix A.4.  A 90-day inhalation toxicity study in the rat is not available but is not required to support registration at this time.  In the absence of inhalation data, an inhalation point of departure was selected for this assessment using route-to-route extrapolation of oral data and assuming 100% inhalation absorption.  Although inhalation exposure may occur (handling and postapplication exposures), use of an oral study to assess inhalation toxicity is not expected to be under protective for either scenario.  Triflusulfuron-methyl has low vapor pressure, shows low acute inhalation toxicity (category IV) and is used at a relatively low application rate.  The MOEs calculated for occupational handlers were at or above 56,000.  Therefore, even if an inhalation study were to provide a lower point of departure than route-to-route extrapolation from the oral study, it is not expected to have a significant impact on estimated risk.  A developmental neurotoxicity study is not required, based on lack of evidence of frank neurotoxicity in the available studies (see 3.1.2, below).  

3.1.2	Toxicological Effects	

The primary target organs identified for triflusulfuron-methyl are the liver, testes and red blood cells.  Hematological and histopathological changes consistent with mild hemolytic anemia were observed in the rat and the dog.  Increased incidence of testicular interstitial hyperplasia was observed following subchronic or chronic-term dietary administration to the rat; the incidence of testicular interstitial adenomas was also increased with chronic exposure.  In the dog, testicular atrophy and reduced size were observed following subchronic or chronic exposure.  Liver effects were observed in the dog (increased weight, altered serum enzyme profile and histopathology) and mouse (increased weight and histopathology), but not in the rat.  There was no indication of frank neurotoxicity in the available studies.  Although a statistically significant increase in sciatic nerve myelin/axon degeneration in high dose female rats was observed in the rat combined chronic toxicity/carcinogenicity study, the incidence was high in all dose groups, no increases were seen in the interim sacrifice groups or in shorter-term studies and the lesion is commonly observed in older rats.  Therefore, HED did not consider this finding to be evidence of frank neurotoxicity.  The only potential evidence of immunotoxicity observed for triflusulfuron-methyl was hemolytic anemia, an effect that is associated with exposure to some sulfonylurea compounds as an immune-mediated cytotoxicity.  However, there are currently no data demonstrating that hemolytic anemia from triflusulfuron-methyl is immune-mediated.  No effects of treatment were reported in a 21-day repeated application dermal toxicity study in the rabbit.  Dermal absorption studies were not submitted.

Triflusulfuron-methyl is of low acute toxicity when administered orally (Toxicity Category IV), dermally (Category III), or via inhalation (Category IV).  It is not a dermal sensitizer or irritant (Category IV) and causes only minor transient irritation to the eye (Category III).

There was no quantitative or qualitative evidence of increased pre- and/or postnatal susceptibility in rat or rabbit developmental toxicity studies or in the rat reproductive studies.  No developmental effects were seen in the rat developmental study, whereas maternal toxicity (decreased body weight gain, food consumption and feed efficiency) was observed at the highest two doses.  Abortions were observed in the rabbit developmental toxicity study at a dose that also caused significant maternal toxicity, including mortality, clinical signs, sharply reduced food consumption and decreased weight gain.  In the rat reproductive toxicity study, decreased parental body weight/weight gain and F1 pup weight during lactation were observed at the mid and high doses.  No reproductive effects were observed.

Triflusulfuron-methyl was classified as a "Group C" (possible human) carcinogen under the 1986 Agency Guidelines for Carcinogen Assessment.  The classification was based on an increased incidence of testicular interstitial cell adenomas in male rats at two doses and evidence of clastogenicity in some in vitro genotoxicity studies.  A special mechanistic study that evaluated hormonal changes in male rats provided insufficient information to establish a nonlinear mode of action for the formation of these tumors.  Although the incidence of hepatocellular adenomas was increased in mice, it was within historical control range and not considered as part of the weight-of-evidence for determination of cancer classification.  Cancer risk was not quantified using a cancer potency factor because the tumors were benign and seen only in one species; the use of the reference dose (RfD) in risk assessment was therefore considered to be protective of potential carcinogenicity.

3.1.3	Dose-response	

Several effects showed a dose-response in one or more species with subchronic or chronic exposures.  Body weight decreases in the dog studies were only seen at the highest doses tested, but the rat studies showed a dose-response, usually at the highest two dose levels.  Mild decreases in erythrocyte parameters in the clinical hematology evaluations were observed in the dog.  In the subchronic dog study, significant hematological effects were only observed at the high dose of 8000 ppm but mild effects (decreases of 10% or less below controls) were seen at 3500 ppm in the chronic study.  Mild hematological effects in the rat were observed at 750 and 1500 ppm in the chronic study  and at higher doses following  subchronic exposure (2000 ppm and above).  More pronounced hematological regenerative changes in the rat were seen at 10,000 and 15,000 ppm following subchronic exposure.

Testicular interstitial cell microscopic pathology was observed in the rat and the dog following subchronic and chronic exposure.  In the rat, testicular effects were observed only at the high dose of 15,000 ppm following subchronic exposure, but were seen at 750 and 1500 ppm in the chronic study.  Interstitial cell tumors observed in the chronic toxicity/carcinogenicity study in the rat were significantly increased at the two highest doses tested, although the incidence at these dose levels was the same.  Liver and testicular effects in the dog were observed at the highest two doses (4000 and 8000 ppm) following subchronic exposure, but were observed only at the highest dose in the chronic study (3500 ppm).  Liver effects in the mouse carcinogenicity showed a dose-response at the highest two doses tested (2500 and 7000 ppm).  An increased incidence of sciatic nerve myelin/axon degeneration was only observed in female rats in the chronic toxicity/carcinogenicity study at the highest dose (1500 ppm).

Abortions and maternal toxicity were observed in the rabbit developmental toxicity study at the LOAEL of 270 mg/kg/day, with a NOAEL of 90 mg/kg/day.  Abortions and maternal toxicity both showed a dose-response, with increased incidence and severity of maternal effects (including clinical signs and mortality) at the highest dose of 800 mg/kg/day.  In the reproductive toxicity study, parental and pup (lactation period) body weights were decreased at the highest two dose levels.

3.2	Absorption, Distribution, Metabolism, Excretion (ADME)	

The metabolism of triflusulfuron-methyl following oral dosing was evaluated in the rat at a single low dose of 25 mg/kg, a single high dose of 250 mg/kg, and after repeated low doses of 25 mg/kg/day for 14 days.  The rate of absorption was unaffected by dose or dosing regimen, but the extent of absorption was decreased at 250 mg/kg.  Urine was the major route of excretion (61 to 77% of the administered dose, or AD) at 25 mg/kg, but feces were the primary route of excretion at 250 mg/kg (43-63% of dose).  Excretion of radiolabel via expired air was negligible.  By 120 hr post-dosing, significant amounts of radioactivity remained in the liver.  Comparison of triflusulfuron-methyl triazine-label with the ester carbonyl-label indicated greater levels of the triazine-label radioactivity in tissues.

Several metabolites of triflusulfuron-methyl were identified.  At 25 mg/kg, the major urinary metabolite was N-desmethyl triflusulfuron-methyl (25-44% of AD), with females excreting higher levels than males of this metabolite.  The N-hydroxymethyl metabolite was also found in urine (10-16% of the dose).  Triazine metabolites (triazine amine, N-desmethyl triazine amine and N,N-bis-desmethyl triazine amine) were identified in liver as well as urine; in feces none of these metabolites represented more than 2% of AD.  In rats dosed with the ester carbonyl-labeled triflusulfuron-methyl, methyl saccharin was a major metabolite in the urine and liver.

3.3	FQPA Considerations	

3.3.1	Adequacy of the Toxicity Database	

The database is considered adequate for purposes of assessment of susceptibility of infants and children to the effects of triflusulfuron-methyl.  Acceptable developmental toxicity studies in two species (rat, rabbit) and a multigeneration reproductive toxicity study (rat) are available.  There was no frank evidence of neurotoxicity in the available studies (see 3.3.2, below).  Other than  hemolytic anemia as previously discussed  (see 3.1.2, above), there was no evidence of direct immunotoxicity observed in the available studies.  However, this effect is of low concern because effects were generally sporadic and marginal (except at high doses) and a large margin of safety was provided by the PODs selected for risk assessment.  In the rat, more pronounced hematological alterations and regenerative changes were observed at doses  >=100-fold higher than the selected PODs (>=15-fold higher in the dog).  Therefore, although an immunotoxicity study has not been submitted, it is not considered likely to identify a more sensitive endpoint (and lower points of departure) than those already selected, and an additional 10X safety factor to account for data gaps is not required.  An oral study was selected for inhalation exposure assessment using route-to-route extrapolation, with an assumption of 100% inhalation absorption and is considered adequate for that purpose at this time (see Section 3.1.1.3).

3.3.2	Evidence of Neurotoxicity	

Triflusulfuron-methyl did not show evidence of frank neurotoxicity in the available studies, which included rat acute and subchronic neurotoxicity studies.  In the rat chronic toxicity/carcinogenicity study, the incidence of myelin/axon degeneration in females at study termination was increased at the high dose only.  HED considers this finding to be of low concern because increases were only observed in females at the terminal sacrifice and not in animals examined at one year (or in shorter-term studies, including the acute and subchronic neurotoxicity studies).  Furthermore, it is typically observed at a high incidence in aged rats. Although an  increase was seen in high dose females, it was also observed at a high incidence in all dose groups.

3.3.3	Developmental Toxicity Studies	

Acceptable developmental toxicity studies in the rat and rabbit are available.  No developmental toxicity was observed in rats dosed by gavage at 0, 30, 120, 350 or 1000 mg/kg/day.  Maternal toxicity was observed at the LOAEL of 350 mg/kg/day, based on decreased body weight gain and food efficiency; the maternal NOAEL was 120 mg/kg/day.  In rabbits dosed by gavage at 0, 90, 270 or 800 mg/kg/day, developmental effects (decreased pup weight and abortions) were observed at the LOAEL of 270 mg/kg/day, a dose that also caused significant maternal toxicity including mortality, decreased body weight gain and clinical signs.  The frequency and severity of maternal toxicity was increased at 800 mg/kg/day, along with an increased number of abortions.  The maternal and developmental NOAELs were 90 mg/kg/day. 

3.3.4	Reproductive Toxicity Study	

An acceptable two-generation reproductive toxicity study in the rat is available.  Male and female rats were administered triflusulfuron-methyl continuously in the diet at 0, 10, 100, 750 or 1500 ppm (0, 0.588, 5.81, 44.0 or 89.5 mg/kg/day in males and 0, 0.764, 7.75, 58.0 or 115 mg/kg/day in females).  Decreased body weight/weight gain, food efficiency and food consumption were observed at the parental LOAEL of 44/58 mg/kg/day in males/females, respectively.  Decreased F1 pup body weights at lactation day 14 were observed at the offspring LOAEL of 44/58 mg/kg/day.  The NOAELs for parental and offspring toxicity were 5.81/7.75 mg/kg/day in males/females.  Reproductive toxicity was not observed at any dose tested.

3.3.5	Additional Information from Literature Sources	

A screening-level literature search was conducted for information on the toxicity of triflusulfuron.  No relevant studies providing additional information were identified.  However, immune-mediated hemolytic anemia in patients receiving sulfonylurea drugs as therapeutic agents for treatment of diabetes has been described in the literature. 

3.3.6	Pre-and/or Postnatal Toxicity	

3.3.6.1	Determination of Susceptibility	

There was no evidence of increased quantitative or qualitative pre- and/or postnatal susceptibility in developmental toxicity studies in two species or the rat multigeneration reproductive toxicity study.  Abortions in the rabbit developmental toxicity study occurred at a dose that caused significant maternal toxicity, including mortality, a sharp reduction in food consumption and decreased body weight gain.  All abortions occurred either post-dosing or during the later part of the dosing period.

3.3.6.2	Degree of Concern Analysis and Residual Uncertainties for Pre- and/or Postnatal Susceptibility	 

There is low concern and no residual uncertainty for postnatal susceptibility for the reasons discussed above (Section 3.3.6.1).

3.3.7	Recommendation for a Developmental Neurotoxicity Study	

A developmental neurotoxicity study is not recommended for triflusulfuron-methyl.  No developmental abnormalities were observed in the developmental toxicity studies at doses up to the limit dose, and increased pre- and/or postnatal susceptibility was not observed.  Other than the sciatic nerve degeneration in aged rats (described above; see 3.3.2), no evidence of neurotoxicity was observed in any studies in the available toxicology database, including acute and subchronic rat neurotoxicity studies.  

0.4 FQPA Safety Factor for Infants and Children	

HED recommends that the 10x FQPA safety factor for infants and children be reduced to 1x for triflusulfuron-methyl, for the following reasons:

      * The toxicology database is largely complete, missing only an immunotoxicity study.  There was no evidence of direct immunotoxicity in the available studies.  Although sulfonylurea compounds may cause anemia, a potential immune-mediated cytotoxic effect, the endpoints selected for this risk assessment are protective of hematological changes.  The observed hemolytic effects are considered to be of low concern because they were generally sporadic and marginal except at higher doses:  the more pronounced hematological and regenerative changes were observed at doses exceeding a 10X margin of safety (in the rat, >=100-fold above and in the dog, >=15-fold above the selected PODs).  An additional 10x database factor was therefore not included for lack of the immunotoxicity study.  An oral study of appropriate duration has been selected for assessment of risk from inhalation exposure and is considered adequately protective (see Section 3.1.1.3); additionally, there are no residential uses of triflusulfuron-methyl that would result in postapplication exposure to infants or young children.

      * Based on the lack of frank neurotoxicity observed in the available data, which include rat acute and subchronic neurotoxicity studies, a developmental neurotoxicity study is not needed.

      * There is no evidence of increased quantitative or qualitative pre- and/or postnatal susceptibility observed in developmental toxicity studies in the rat and rabbit or in a two-generation reproduction study in the rat.

      * The exposure assessment for food and drinking water will not underestimate potential exposure to triflusulfuron-methyl, and a conservative approach was used for aggregate risk assessment.  

	
0.5 Hazard Identification and Toxicity Endpoint Selection
	
3.5.1	Acute Reference Dose (aRfD)  -  All Populations (Including Infants and Children and Females age 13-49	)

      An acute reference dose was not selected because an appropriate endpoint resulting from a single exposure was not identified.  Abortions observed in the rabbit developmental toxicity study were not considered a potential single-dose effect.  They were not observed until post-dosing (Gestation Days 21-26) at the maternal/developmental toxicity LOAEL of 270 mg/kg/day (and during late dosing at the high dose of 800 mg/kg/day), and appeared to be secondary to significant maternal toxicity.  Two does died (GD 24 and 27) at this dose.  The animals that died and/or aborted showed evidence of significant gastrointestinal toxicity, based on sharp reductions in food consumption after GD 13, clinical signs (no stool or small stool) and necropsy findings (gastric ulceration, gas distension of intestines).  

3.5.2   Chronic Reference Dose (cRfD)	

      Study Selected:  Chronic Toxicity/Carcinogenicity in the Rat (Dietary)
      MRID No:  42991413
      Dose and Endpoint for Risk Assessment: NOAEL = 2.44 mg/kg/day, based on decreased body weight and weight gain in males and females, hematological changes (primarily in males) and testicular interstitial hyperplasia in males observed at the LOAEL of  30.6 mg/kg/day.
      Uncertainty Factor: 100x (10x interspecies extrapolation, 10x intraspecies variability)

                       Chronic RfD =  = 0.0244 mg/kg/day

      Comments about Study/Endpoint/Uncertainty Factors:   The study is of the appropriate duration and exposure route (i.e., long-term oral exposure) and represents the most conservative available endpoint and dose available in the database for assessment of chronic dietary exposure.  The FQPA safety factor was reduced from 10x to 1x, based on the lack of evidence of increased offspring susceptibility, and availability of clearly defined NOAELs for developmental and offspring toxicity.  An additional database uncertainty factor for lack of an immunotoxicity study was not applied.  Although sulfonamide compounds may cause anemia via an immune-mediated cytotoxic mechanism, there is currently no data demonstrating that the hematological effects of triflusulfluron-methyl are immune-mediated.  The hematological effects are of low concern because they were generally sporadic and marginal (except at high doses) and a large margin of safety was provided by the selected POD.     In the rat, more pronounced hematological alterations and regenerative changes were observed at doses >=100-fold greater (and in the dog, >=15-fold greater) than the selected POD.  There was no other evidence of immune effects in the available studies.  

3.0.3 Incidental Oral Exposure, All Durations
      
      An endpoint for incidental oral exposure was not selected because there are no residential uses for triflusulfuron-methyl.  
      
3.5.4	Dermal Absorption	

      There is no dermal absorption study available.  A dermal absorption factor was not estimated because a dermal study was selected to evaluate short- and intermediate-term dermal exposure.  In addition, based on that study, no dermal short- or immediate-term toxicity is expected (see 3.5.5 below).

5.0.5 Occupational Dermal Exposure, Short-Term (1 to 30 days) and Intermediate-Term (1 to 6 months)
      
      Study Selected:  21-Day Dermal Toxicity in the Rabbit
      MRID No:  42991409
      Dose and Endpoint for Risk Assessment:  An appropriate dose/endpoint was not selected due to lack of effects in the dermal study.
      Comments about Study/Endpoint/Uncertainty Factors:   No toxicity was observed up to the limit dose of 1000 mg/kg/day in the rabbit 21-day dermal toxicity study.  The endpoints of concern identified in the subchronic oral studies (decreased body weight gain and hematological changes) were not observed in rabbits following exposure via the dermal route.  
      
6.0.6 Occupational Dermal Exposure, Long-Term (>6 months) 	

      A long-term occupational risk assessment is not required because long-term dermal exposure is not anticipated from the proposed or existing uses.

3.5.7	Occupational Inhalation Exposure, Short-Term (1 to 30 days) and Intermediate-Term (1 to 6 months)	
      
      Study Selected:  90-Day Subchronic Toxicity in the Rat (Dietary)
      MRID No:  42991408
      Dose and Endpoint for Risk Assessment:  6.56 mg/kg/day, based on decreased body weight gain in males and hematological effects in females at 133 mg/kg/day.
      Comments about Study/Endpoint/Uncertainty Factors:   Since an inhalation toxicity study was not available, an oral toxicity study was used to select a dose and endpoint. The study selected showed decreases in body weight gain after the first week of dosing (and continuing to Week 13) and is therefore of an appropriate duration.  The dose selected is also protective of potential hematologic effects, which were observed at later times but were not evaluated at one month of treatment.  A second rat 90-day oral toxicity study (MRID 42991406) had a similar NOAEL (6.20 and 7.54 mg/kg/day in males and females, respectively) based on decreased body weight gain in both sexes by Week 1 (and other effects by Week 13).  The 90-day oral study in the dog (MRID 42991440) showed body weight decreases in females by Week 4 at a similar LOAEL (146.9 mg/kg/day), but had a lower NOAEL (3.7 mg/kg/day) that was due to dose spacing.  The selected endpoint is protective for offspring toxicity (decreased F1 pup weight during lactation) based on the offspring NOAEL of 5.81 and 7.52 mg/kg/day in males and females, respectively, in the rat two-generation reproduction toxicity study (MRIDs 42496843; 42991412).  The NOAEL for abortions observed in the rabbit developmental toxicity study was 90 mg/kg/day, which is much higher than other NOAELs being considered for short- and intermediate-term inhalation doses.  In the absence of inhalation data, inhalation absorption of 100% was assumed for route-to-route extrapolation from oral to inhalation exposure.  An additional uncertainty factor for lack of inhalation toxicity data is not needed; based on low acute inhalation toxicity, low vapor pressure and lack of persistence of triflusulfuron-methyl in the environment, exposure via volatilization is expected to be low.  A database uncertainty factor for lack of an immunotoxicity study is also not needed (see Comments in Section 3.5.2).
      
3.5.8	Occupational Inhalation Exposure, Long-Term (> 6 months)	
      
      A long-term occupational risk assessment is not required because long-term inhalation exposure is not anticipated from the proposed or existing uses.

3.5.9	Level of Concern for Margin of Exposure (MOE)	

Table 3.5.1  Summary of Levels of Concern for Risk Assessment.
Route
                                  Short-Term
                                 (1 - 30 Days)
                               Intermediate-Term
                                (1 - 6 Months)
                                   Long-Term
                                (> 6 Months)
                        Occupational (Worker) Exposure
Dermal
                                      N/A
                                      N/A
                                      N/A
Inhalation
                                      100
                                      100
                                      N/A

3.5.10	Summary of Toxicological Doses and Endpoints for Use in Human Risk Assessments	

Table 3.5.2a. Summary of Toxicological Doses and Endpoints for Triflusulfuron-Methyl for Use in Dietary and Non-Occupational Human Health Risk Assessments
Exposure/
Scenario
                              Point of Departure
                        Uncertainty/FQPA Safety Factors
                RfD, PAD, Level of Concern for Risk Assessment
                        Study and Toxicological Effects
Acute Dietary, General Population (including Infants and Children and Females age 13 to 49)
Not required.  An appropriate endpoint for this risk assessment was not identified.
Chronic Dietary (All Populations)
NOAEL= 2.44  mg/kg/day
UFA= 10x
UFH= 10x
FQPA SF= 1x   
Chronic RfD = 0.0244
mg/kg/day

cPAD = 0.0244 mg/kg/day
Chronic oral toxicity/carcinogenicity in the rat 
LOAEL = 30.6 mg/kg/day, based on decreased body weight/weight gain, hematological changes (primarily males) and increased interstitial cell hyperplasia (males).
Cancer (oral, dermal, inhalation)
Classification:  Group C (1986 Cancer Guidelines), based on increased incidence of testicular interstitial cell adenomas in rats.   The RfD is considered adequately protective of these effects and a quantitative assessment of cancer risk using a cancer potency factor is not required.
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).  UFL = use of a LOAEL to extrapolate a NOAEL.  UFS = use of a short-term study for long-term risk assessment.  UFDB = to account for the absence of key data (i.e., lack of a critical study).  FQPA SF = FQPA Safety Factor.  PAD = population adjusted dose (a = acute, c = chronic).  RfD = reference dose.  MOE = margin of exposure.  LOC = level of concern.  N/A = not applicable.

Table 3.5.2b Summary of Toxicological Doses and Endpoints for Triflusulfuron-Methyl for Use in Occupational Human Health Risk Assessments
Exposure/
Scenario
Point of Departure
Uncertainty Factors
Level of Concern for Risk Assessment
Study and Toxicological Effects
Dermal Short- Term (1 to 30 days) and Intermediate-Term (1 to 6 months)
Not required.  No toxicity was observed in the rabbit 21-day dermal toxicity study up to the limit dose of 1000 mg/kg/day.
Dermal Long-Term (> 6 months)
Not required.  Long-term dermal exposure is not anticipated from current and proposed uses.
Inhalation Short-Term (1  to 30 days) and Intermediate-Term (1 to 6 months)
NOAEL = 6.56 mg/kg/day

(inhalation absorption rate = 100%)
UFA=10x
UFH=10x

Occupational LOC for MOE = 100
90-day oral toxicity in the rat
LOAEL = 133 mg/kg/day (males)/153 mg/kg/day (females), based on decreased body weight gain (observed after the first week of dosing) and body weight, and in females, extramedullary hematopoiesis.
Inhalation Long-Term (> 6 months)
Not required.  Long-term inhalation exposure is not anticipated from current and proposed uses.
Cancer (oral, dermal, inhalation)
Classification:   Group C (1986 Cancer Guidelines), based on increased incidence of testicular interstitial cell adenomas in rats.   The RfD is considered adequately protective of these effects and a quantitative assessment of cancer risk using a cancer potency factor is not required.
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).  UFL = use of a LOAEL to extrapolate a NOAEL.  UFS = use of a short-term study for long-term risk assessment.  UFDB = to account for the absence of key data (i.e., lack of a critical study).  MOE = margin of exposure.  LOC = level of concern.  N/A = not applicable.

3.5.12	Recommendation for Aggregate Exposure Risk Assessments	

For chronic dietary assessments, exposure from food and drinking water should be combined.  There are no residential uses at this time.  Therefore, it is not necessary to address the suitability of combining exposures from different routes (e.g., oral and inhalation).    

3.5.13	Classification of Carcinogenic Potential	

In 1996, triflusulfuron-methyl was reviewed by the HED Cancer Peer Review Committee (TXR # 0011939).  Triflusulfuron-methyl was classified under the 1986 Agency Guidelines for Assessment of Carcinogenicity as a "Group C," possible human carcinogen, based on a statistically significant increase in the incidence of testicular interstitial cell adenomas in male rats in the rat two-year dietary study.  Although there was suggestive evidence of a hormonal basis for induction of these tumors, the data were considered insufficient to demonstrate conclusively a hormonal mode of action.  Genotoxicity data were mixed, with in vitro chromosomal aberration studies showing positive results but other studies, including a mouse micronucleus assay, being negative.  For risk assessment purposes, the use of the chronic RfD/PAD is considered to be protective of potential carcinogenicity since the tumors were benign.  The increased incidence of tumors was observed at doses that were >=12.5-fold greater than the study NOAEL.  No interstitial cell tumors were observed in the one-year interim sacrifice animals.  As a chemical class, the sulfonylurea compounds in general do not induce tumors, but treatment-related tumor increases have been reported for prosulfuron, primisulfuron-methyl (both Group D, not classifiable as to human carcinogenicity) and tribenuron-methyl (Group C, possible human carcinogen based on increased mammary gland tumors in female rats).

3.6	Endocrine disruption		

As required under FFDCA section 408(p), EPA has developed the Endocrine Disruptor Screening Program (EDSP) to determine whether certain substances (including all pesticide active and other ingredients) "may have an effect in humans or wildlife similar to an effect produced by a naturally occurring estrogen, or other such endocrine effects as the Administrator may designate."  The EDSP employs a two-tiered approach to making the statutorily required determinations.  Tier 1 consists of a battery of 11 screening assays to identify the potential of a chemical substance to interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal systems.  Chemicals that go through Tier 1 screening and are found to have the potential to interact with E, A, or T hormonal systems will proceed to the next stage of the EDSP where EPA will determine which, if any, of the Tier 2 tests are necessary based on the available data.  Tier 2 testing is designated to identify any adverse endocrine related effects caused by the substance, and establish a dose-response relationship between the dose and the E, A, or T effect.

Between October, 2009 and early 2010, EPA issued test orders/data call-ins for the first group of 67 chemicals, which contains 58 pesticide active ingredients and 9 inert ingredients.  This list of chemicals was selected based on the potential for human exposure through pathways such as food and water, residential activity, and certain post-application agricultural scenarios.  This list should not be construed as a list of known or likely endocrine disruptors.  Triflusulfuron-methyl is not among the group of 58 pesticide active ingredients on the initial list to be screened under the EDSP.  Under FFDCA sec. 408(p), the Agency must screen all pesticide chemicals.  Accordingly, EPA anticipates issuing future EDSP test orders/data call-ins for all pesticide active ingredients.  

For further information on the status of the EDSP, the policies and procedures, the list of 67 chemicals, the test guidelines and the Tier 1 screening battery, please visit our website:  http://www.epa.gov/endo/.

4.0	Public Health and Pesticide Epidemiology Data and Incident Reports	

There are no public health, pesticide epidemiology or incident data to report at this time.

5.0	Dietary Exposure/Risk Characterization	
(Triflusulfuron-methyl.  Petition for the Establishment of Permanent Tolerances and Registration
for Use on Garden Beets (PP 9E7669).  Summary of Analytical Chemistry and Residue Data; D376350; E. Holman;
February 2011)

5.1	Pesticide Metabolism and Environmental Degradation

5.1.1	Metabolism in Primary Crops

For the purposes of this petition (9E7669) on garden beet, the previously reviewed sugar beet metabolism study is sufficient to support the new requested uses of triflusulfuron-methyl on garden beets.  The proposed tolerance expression should include only the parent compound, triflusulfuron-methyl, as determined by HED [Per memo of J. Stokes (February 1996), meeting of 16 January 1996].

5.1.2	Metabolism in Livestock

HED previously reviewed the submitted goat metabolism study and cited deficiencies to be addressed if additional Section 3 registrations were pursued by the petitioner.  However, HED has determined that measurable triflusulfuron-methyl residues above the limit of detection (LOD; 0.005 ppm) will not be detectable in milk, meat, fat, muscle, or meat byproducts from the existing use on sugar beets and the new proposed use on garden beet.  Therefore, the deficiencies cited in the HED memorandum of 7 June 1993 (J. Herndon) do not need to be addressed for the purpose of this new food use on garden beet.  However, if future uses on large-acreage crops such as wheat, corn, or soybean are proposed, and consequently involve relevant livestock feedstuffs, then an adequate ruminant metabolism study should be provided.

5.1.3	Metabolism in Rotational Crops

Additional rotational crop studies or rotational crop tolerances are not needed at this time.  Such studies may be needed if uses are proposed on major crops (e.g., wheat, cotton, corn, soybeans) due to the observed uptake of triazine-derived soil residues into wheat straw in the confined study.  [See memo of J. Stokes (31 January 1996) for HED meeting of 16 January 1996].

5.1.4	Environmental Degradation

Triflusulfuron-methyl is not a very persistent sulfonylurea herbicide.  The principal pathway of dissipation in the environment is through aerobic and anaerobic metabolism and to a lesser degree hydrolysis.  Direct photolysis on soil and in water is not a very important pathway, but indirect photolysis may play a minor role in its degradation on soils.  Biotically, triflusulfuron-methyl degradation follows a bi-phasic pattern with an approximate soil half-life of 6 days initially, followed by a secondary half-life of approximately 170 days in aerobically incubated soil.  Under anaerobic soil conditions, triflusulfuron-methyl degraded with a half-life of 20 days.  Triflusulfuron-methyl is mobile in all soils tested; Kd's are less than 1.3.  However, the parent was not detected below 14 inches in the two field dissipation studies and decreased in concentration over the period of the study indicating biotic degradation.  Sorption was not well correlated to organic matter, but was better correlated to the soil pH.
5.1.5	Comparative Metabolic Profile

Triflusulfuron-methyl degrades through cleavage of the sulfonylurea bridge to form degradates containing either the triazine or phenyl ring.  The principle degradates seen in the laboratory studies are methyl saccharin and various triazine amines.  Methyl saccharin is more mobile than the parent with Kd's all less than 1.0 (KOC's <25).  In several of the soils, binding was so low that analysis could not be performed to determine the extent of binding.  The half-life of methyl saccharin is 50-days under aerobic conditions and further degrades via mineralization.  The other major degradates, triazine-amines, are less mobile than the parent.  Among the triazine-amines, mobility increases with a higher degree of demethylation.  The bis-NDM-triazine amine is the most mobile with Kd's up to 3.72 (KOC's <220) and the least mobile, triazine-amine, had Kd's up to 10 (KOC's <2175).  For NDM-triazine-amine, Kd's were up to 4.88  (KOC's <300). The half-life of triazine amine is approximately 40 days and further degrades by demethylation.  Both degradates were detected up to the 36-inch segment in one or both of the field dissipation studies.  Concentrations of methyl saccharin and triazine amine were very similar during the studies and dissipated with very similar patterns.  

In a rat metabolism study, the liver, (as well as the ovaries and skin at the high dose), had a significant proportion of residual radioactivity at 120 hours post-dose at all dose levels.  Urine was the major route of excretion at the low dose, with feces being the major route at the high dose.  The major urinary metabolite (N-desmethyl triflusulfuron-methyl) composed between 25-44% of the dose at the low dose.  Female rats showed higher urinary levels than males of this metabolite.  The N-hydroxymethyl metabolite was also found in urine, but did not vary significantly with dose.  The triazine metabolites of triflusulfuron-methyl (Triazine amine, N-desmethyl triazine amine, N,N-bis-desmethyl triazine amine) were found in urine and liver.   The parent chemical was found as a major component only in feces and liver from high dose treatments. 

In plants, triflusulfuron-methyl undergoes cleavage of the sulfonylurea bridge to form triazine urea and ester sulfonamide.  This cleavage occurs readily in soil and on the leaf surface, leaving no measurable residues of parent in sugar beets (whole plant) after 7 days post treatment.  Thus, there are no metabolites observed in plant metabolism studies that were not observed in the rat metabolism studies.  Due to low total radioactivity in mature sugar beets and no quantifiable residues of the parent or three triazine metabolites observed in field trials with exaggerated use rates (2-8X), HED previously determined that the parent compound, triflusulfuron-methyl, is the only residue of concern for risk assessment (See memo of J. Stokes (31 January 1996) for HED meeting of 16 January 1996).
 

5.1.6	Pesticide Metabolism and Degradates of Concern 	
 
 Based on the available metabolism studies, HED concluded that for these uses, the nature of the residue has been adequately delineated for tolerance enforcement and risk assessment (Table 5.1.6).  
 
 
Table 5.1.6:  Residue Definition for Triflusulfuron-methyl
Matrix
Risk Assessment
Compliance Monitoring
Plants
Primary Crop
Triflusulfuron-methyl
Triflusulfuron-methyl

Rotational Crop
Not applicable at this time (no rotational crops associated with this use)
Not applicable at this time 
Livestock
Ruminant
Not applicable at this time (no livestock commodities associated with this use)
Not applicable this time (under 40 CFR 180.6 (a)(3))

Poultry
Not applicable at this time (no poultry feed items associated with this use)
Not applicable at this time (under 40 CFR 180.6 (a)(3))
Drinking Water
Triflusulfuron-methyl
Not Applicable

5.1.8	Analytical Methodology

The review of Method 4480 was originally completed by PMRA, but has been reviewed by HED and is consistent with OCSPP policy.  An adapted version of Method 4480 was utilized in the garden beet field trials submitted for this new use petition.  

The independent laboratory validation (ILV) of Method 4480 successfully demonstrated the reliability of the HPLC/MS/MS method for the analysis of residues of triflusulfuron-methyl in sugar beet foliage and sugar beet roots on the first attempt.  Method 4480 also included a scientifically acceptable rationale to waive the requirement of a radiovalidation study for this method, based on the similarities in extraction steps between this method and those employed in a corresponding sweet potato metabolism study (received and reviewed by PMRA).  Therefore, method 4480 is considered acceptable as a data-gathering and enforcement method for the determination of triflusulfuron-methyl in plant matrices (including garden beets).  

The enforcement method on record for triflusulfuron-methyl is method AMR 1930-91 (MRID 42496873).  Method AMR 2021-91 (MRID 42496872) is very similar to AMR 1930-91 and was used in the previously submitted sugar beet and chicory field trials.  Method 4480 is also similar to both these methods, with the primary difference being the addition of a tandem mass spectrometric detection protocol in Method 4480.  In addition, the ILV of Method 4480 successfully demonstrated the reliability of the method on the first attempt.  The registrant should clarify whether they want to replace the existing enforcement method with Method 4480 when applying for future new uses, as this newer method appears to be an acceptable enforcement method.        

9.3.9 Food Residue Profile 

Crop Field Trials and Processing Studies:	The crop field trials for chicory and sugar beets were previously classified acceptable and satisfied the guideline requirement for crop field trials (M. Doherty, et al., D260076, 12 February 2002).  

The newly submitted crop field trials are adequate to support the requested new use of triflusulfuron-methyl on garden beet.  The review of these field trials was originally conducted by PMRA under a work share agreement, but their conclusions have been reviewed by HED and are consistent with OCSPP policy.  The freezer storage stability of triflusulfuron-methyl residues in/on garden beets was demonstrated concurrently, with samples spiked at 0.10 ppm stored frozen for 436 days. Analysis of these samples showed no significant degradation of residues occurred throughout this time interval. The maximum frozen storage time for garden beet samples was 381 days, a duration that is shorter than the demonstrated residue stability period.  Therefore, there are no concerns with the stability of residues over time in this study.

The data indicate that residues of triflusulfuron-methyl in/on garden beet roots and tops were all <= 0.01 ppm when treated at total rates of 0.047-0.049 lb ai/A (using a NIS/COC or not) and harvested at PHIs of 22-56 days.  It should be noted that the petition under review requests a 30-day PHI for garden beet, whereas a 60-day PHI is currently approved for sugar beets.  The requested 30-day PHI for garden beet is supported by the submitted field trial data.

Based on the available data, the field trial and residue decline data demonstrate that triflusulfuron-methyl is rapidly degraded after application to sugar beet plants.  Even at exaggerated rates (8X), no triflusulfuron-methyl was found in the sugar beet roots (RAC) or processed commodities; therefore concentration of residues in processed commodities is unlikely at the currently approved use rates on sugar beets.  Additional residue decline data were not submitted for the garden beet proposed use currently under review.  There are no known processed commodities for garden beets in which residues are expected to concentrate; thus, processed food and feed should not be an issue for this new food use petition.  

Meat, Milk, Poultry and Eggs:	Using information in the goat metabolism studies, residues of triflusulfuron-methyl (as defined by the livestock residue definition) are expected to be below the LOQ of a reasonable livestock analytical method.  Hence a 40 CFR 180.6(a)(3) situation (no expectation of finite residues) exists.  Neither feeding studies nor tolerances in livestock commodities are required at this time.  Feeding studies and analytical methods may be required to support future uses of triflusulfuron-methyl that involve significant livestock feed items. 

Rotational Crops:	Additional rotational crop studies or rotational crop tolerances are not needed at this time.  Such studies may be needed if uses are proposed on major crops (e.g., wheat, cotton, corn, soybeans) due to the observed uptake of triazine-derived soil residues into wheat straw in the confined study.  [See memo of J. Stokes (31 January 1996) for HED meeting of 16 January 1996].

5.1.10	Drinking Water Residue Profile	
(Estimated Drinking Waters Concentrations (EDWCs) of Triflusulfuron-methyl for the Use in the Human Health
Risk Assessment: New Use (IR-4) of the Chemical on Garden Beets; J. Meléndez, D373494; July 22, 2010)

The registrant is proposing the use of the chemical on garden beets.  Based on an inspection of the new use of triflusulfuron-methyl, it was found that sugar beet still represents the scenario with the highest exposure with respect to potential residues in drinking water.  The previously modeled use on sugar beets covers the proposed new use on garden beets.  Therefore, the drinking water assessment results do not change from the previous ones (DP Barcode D260078).  Triflusulfuron-methyl may be found in surface water used as a source of drinking water at peak concentrations up to 0.42 ug/L and on a long-term basis (mean) at 0.005 ug/L from use on sugar beets in Minnesota.  Ground water concentrations, independent of the use area, may be found at up to 0.5 ug/L for both peak and mean concentrations.  Triflusulfuron-methyl will not persist in the environment (accumulate between repeated yearly applications) due to its short initial half-life (bi-phasic) and hydrolytic half-life at environmental pH's.  The low adsorption coefficients may facilitate triflusulfuron's leaching to ground water under favorable conditions (shallow depth to groundwater, coarse soils, or soils with significant preferential flow). Due to triflusulfuron-methyl's structure and physical properties, it is not likely to be significantly affected by common treatment methods employed at most community water systems.  

Triflusulfuron-methyl is currently registered for use on sugar beets.  The drinking water concentrations used to estimate exposure via drinking water are included in Table 5.1.10.  

Table 5.1.10  Estimated Drinking Water Concentrations (EDWCs) for human health risk assessment for aerial application of triflusulfuron-methyl on sugar beets
                                       
                                   Chemical
                                       
                     Acute Surface Water PRZM/EXAMS (ppb)
                                       
                    Chronic Surface Water  PRZM/EXAMS (ppb)
                                       
                Acute and Chronic Ground Water  SCI-GROW (ppb)
                             Triflusulfuron-methyl
                                     0.42
                                     0.005
                                     0.50

5.1.11	International Residue Limits

The requested new use on garden beet is being assessed as part of a work share agreement with Canada's PMRA.  Therefore, implemented tolerances stemming from this new use will be harmonized with the PMRA review.  The recommended tolerance for garden beet roots of 0.01 ppm is identical to that of PMRA's, with all garden beet roots having concentrations less than the LOQ of 0.01 ppm in observed field trials.  However, the recommended tolerance of 0.02 ppm for garden beet tops is not harmonized with PMRA's value of 0.01 ppm.  Based on the submitted garden beet field trial data, garden beet tops had concentrations equal to 0.01 ppm at a single field trial in Texas (Trial 08043.05-TX*19).  Given the level of uncertainty for quantification around the LOQ of 0.01 ppm and the limited number of field trials, HED does not consider setting the garden beet top tolerance at 0.01 ppm to be appropriate.  Therefore, HED is concurring with the registrant-proposed tolerance of 0.02 ppm for garden beet tops.     

5.2	Dietary Exposure and Risk	
(Triflusulfuron-methyl: Chronic Aggregate Dietary Exposure (Food and Drinking Water) and Risk Assessment for
the Section 3 Registration on Garden Beet; E. Holman; D376268; February 2011)
 
A chronic aggregate dietary risk assessment (food and drinking water) was conducted using the Dietary Exposure Evaluation Model (DEEM-FCID(TM), Version 2.03), which uses food consumption data from the U.S. Department of Agriculture (USDA's) Continuing Surveys of Food Intakes by Individuals (CSFII) from 1994-1996 and 1998.  The analysis was performed to support a Section 3 registration for use of triflusulfuron-methyl [methyl 2-[[[[[4-(dimethylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-yl]amino]carbonyl]amino]sulfonyl]-3-methylbenzoate] on garden beets.  Triflusulfuron-methyl is currently registered for use on sugar beets and chicory.  This chronic analysis is based on tolerance-level residues and 100% crop treated assumptions.  For chronic assessments, HED is concerned when dietary risk exceeds 100% of the PAD.  The chronic dietary exposure analysis results are reported in Table 5.2 and show all population exposures are well below HED's level of concern.

No appropriate endpoint attributable to a single dose was identified.  As no acute dietary endpoint was identified, an acute exposure analysis was not performed.

The chronic dietary risk estimates are less than 1% of the cPAD for the U.S. population and all population subgroups, including those of infants and children.

  Table 5.2  Summary of Dietary Exposure and Risk for Triflusulfuron-Methyl.

                             Population Subgroup*

                                 Acute Dietary

                                Chronic Dietary

                                    Cancer
                                       

                         Dietary Exposure (mg/kg/day)

                                    % aPAD

                               Dietary Exposure
                                  (mg/kg/day)

                                    % cPAD

                               Dietary Exposure
                                  (mg/kg/day)

                                     Risk
General U.S. Population
                                      N/A
                   No acute dietary endpoint was identified
                                      N/A
                                   0.000025
                                    <1%
                                       
                                      N/A
                                       
                                      N/A
All Infants (< 1 year old)
                                       
                                       
                                  0.000050   
                                    <1%
                                       
                                       
Children 1-2 years old
                                       
                                       
                                   0.000052
                                    <1%
                                       
                                       
Children 3-5 years old
                                       
                                       
                                   0.000056
                                    <1%
                                       
                                       
Children 6-12 years old
                                       
                                       
                                   0.000041
                                    <1%
                                       
                                       
Youth 13-19 years old
                                       
                                       
                                   0.000023
                                    <1%
                                       
                                       
Adults 20-49 years old
                                       
                                       
                                   0.000020
                                    <1%
                                       
                                       
Adults 50+ years old
                                       
                                       
                                   0.000019
                                    <1%
                                       
                                       
Females 13-49 years old
                                       
                                       
                                   0.000021
                                    <1%
                                       
                                       

5.2.1	Cancer Dietary Risk

Triflusulfuron-methyl is a Group C (1986 Cancer Guidelines - possible human carcinogen) chemical, based on increased incidence of testicular cell adenomas in rats.  The chronic RfD is considered adequately protective of these effects and indicates no concern for cancer risk.  Therefore, a quantitative assessment of cancer risk using a cancer potency factor is not required.  

6.0	Residential (Non-Occupational) Exposure/Risk Characterization	

There are no current or pending residential uses associated with triflusulfuron; therefore, a residential exposure assessment was not performed nor required.

6.2	Spray Drift

Spray drift is a potential source of exposure for residents living in close proximity to spraying operations.  This situation is particularly the case with aerial application.  However, to a lesser extent, spray drift resulting from the ground application of triflusulfuron-methyl could also be a potential source of exposure.  The Agency has been working with the Spray Drift Task Force (a membership of US pesticide registrants), EPA Regional Offices, State Lead Agencies for pesticide regulation, and other parties to develop the best spray drift management practices.  The Agency is now requiring interim mitigation measures for aerial applications that must be placed on product labels/labeling.  The Agency has completed its evaluation of the new database submitted by the Spray Drift Task Force, and is developing a policy on how to appropriately apply the data and the AgDRIFT computer model to its risk assessments for pesticides applied by air, orchard airblast, and ground hydraulic methods.  After the policy is in place, the Agency may impose further refinements in spray drift management practices to reduce off-target drift, and risks associated with pesticide application.  

7.0	Aggregate Risk Assessments and Risk Characterization	

There is no acute dietary exposure endpoint; therefore, an acute aggregate risk assessment was conducted and there is no concern with acute risks   

Additionally, because there are no residential uses and only dietary (food + water) pathways of exposure are being considered, short- and intermediate-term aggregate risk assessments are not required.  Other than dietary exposure, there are no other sources of aggregate exposure that constitute a long-term exposure scenario; therefore, long-term aggregate exposure and risk estimates are equivalent to the chronic dietary exposure (food+water) and risk estimates summarized in Table 5.2 and are below HED's level of concern.

8.0	Cumulative Risk Characterization/Assessment	

FQPA (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.  The reason for consideration of other substances is due to the possibility that low-level exposures to multiple chemical substances that cause a common toxic effect by a common mechanism could lead to the same adverse health effect as would a higher level of exposure to any of the other substances individually.  A person exposed to a pesticide at a level that is considered safe may, in fact, experience harm if that person is also exposed to other substances that cause a common toxic effect by a mechanism common with that of the subject pesticide, even if the individual exposure levels to the other substances are also considered safe.

EPA does not have, at this time, available data to determine whether triflusulfuron-methyl has a common mechanism of toxicity with other substances or how to include this pesticide in a cumulative risk assessment.  For the purposes of this tolerance action, EPA has not assumed that triflusulfuron-methyl has a common mechanism of toxicity with other substances.  At such time as the Agency has a scientific basis for evaluating cumulative risk, the petitioner must submit, upon EPA's request and according to a schedule determined by the Agency, such information as the Agency directs to be submitted in order to evaluate cumulative risk issues for triflusulfuron-methyl.  At that time, the Agency will determine whether any tolerances for triflusulfuron-methyl need to be modified or revoked.

9.0	Occupational Exposure/Risk Pathway	
(Triflusulfuron-methyl: Occupational and Residential Exposure Assessment for Use on Garden Beets; 
M. Collantes: January 2011; D376267)

The registrant, E.I. du Pont de Nemours and Company, has requested a Section 3 registration for the use of the herbicide triflusulfuron methyl on garden beets.  The proposed product, UpBeet[(R)], is a herbicide recommended for selective postemergence control of broadleaf and grass weeds.  It is a dry flowable formulation currently registered to control weeds in sugar beets.  The supplemental label proposed an application rate of 0.0156 lbs. of active ingredient (a.i.) per acre to be applied by ground and/or aerial equipment.  A previous occupational exposure assessment was performed for the use of triflusulfuron methyl on sugar beets at a higher application rate of 0.032 lbs a.i. per acre using ground and aerial applications (S. Wang, D260076, 2/12/02).  Occupational exposure and risk resulting in MOEs greater than or equal to 100 will not be of concern to HED.   No systemic toxicity was seen at the limit dose (1000 mg/kg/day) of a 28-day dermal study in the rabbit.  Therefore, neither a quantitative dermal handler nor postapplication exposure assessment was performed.  The inhalation handler exposure assessment resulted in MOEs greater than 100, which are not of concern.  

Since the new proposed use of triflusulfuron methyl on garden beets will result in the same agricultural practice and exposure scenarios as with sugar beets, only at a lower application rate (0.0156 vs. 0.032 lb a.i./acre), the previous occupational exposure assessment for sugar beets will be protective of exposures resulting from use of triflusulfuron on garden beets.  Therefore, a new quantitative assessment for occupational handler exposure is not required.

Based on the Agency's current practices, a quantitative post-application inhalation exposure assessment was not performed for triflusulfuron-methyl at this time primarily because of the low acute inhalation toxicity (Toxicity Category IV), low vapor pressure (less than 1.0 x 10[-][7] mm Hg), and the low proposed use rate (0.0156 lb ai/A).  However, there are multiple potential sources of post-application inhalation exposure to individuals performing post-application activities in previously treated fields.  These potential sources include volatilization of pesticides and resuspension of dusts and/or particulates that contain pesticides.  The Agency sought expert advice and input on issues related to volatilization of pesticides from its Federal Insecticide, Fungicide, and Rodenticide Act Scientific Advisory Panel (SAP) in December 2009, and received the SAP's final report on March 2, 2010 (http://www.epa.gov/scipoly/SAP/meetings/2009/120109meeting.html).  The Agency is in the process of evaluating the SAP report as well as available post-application inhalation exposure data generated by the Agricultural Reentry Task Force and may, as appropriate, develop policies and procedures, to identify the need for and, subsequently, the way to incorporate occupational post-application inhalation exposure into the Agency's risk assessments.  If new policies or procedures are put into place, the Agency may revisit the need for a quantitative occupational post-application inhalation exposure assessment for triflusulfuron-methyl.

Restricted Entry Interval
Since postapplication risks were not a concern on day 0 (12 hours following application), the restricted entry interval (REI) is based on the acute toxicity of triflusulfuron-methyl technical material.  Triflusulfuron-methyl is classified as Toxicity Category III by the dermal route, Toxicity Category IV for eye irritation potential, and Toxicity Category IV for skin irritation potential.  Under the Worker Protection Standard for Agricultural Pesticides, active ingredients classified as acute toxicity categories III or IV for these routes are usually assigned a 12-hour REI.  However, triflusulfuron-methyl meets the criteria in PR Notice 95-3 for low risk pesticides that are permitted to have a 4-hour REI.  Therefore, HED has no objections to the 4-hour REIs on the existing registered label (for sugar beet and chicory uses) and the proposed supplemental label for garden beet. 

10.0	Data Needs and Label Recommendations	

3.1      Toxicology	

An immunotoxicity study (OCSPP 870.7800) is required for triflusulfuron-methyl under the revised 40 CFR Part 158 Guidelines for toxicology data requirements.  HED recommends that future submission of this study be a condition of registration for the use on garden beets.

Based on the available toxicity database and the Agency's current practices, the inhalation risk for triflusulfuron-methyl was assessed using route-to-route extrapolation from an oral toxicity study.  The Agency sought expert advice and input on issues related to this route-to-route extrapolation approach (i.e. the use of oral toxicity studies for inhalation risk assessment) from its Federal Insecticide, Fungicide, and Rodenticide Act Scientific Advisory Panel (SAP) in December 2009.  The Agency received the SAP's final report on March 2, 2010 (http://www.epa.gov/scipoly/SAP/meetings/2009/120109meeting.html).  The Agency is in the process of evaluating the SAP report and may, as appropriate, re-examine and develop new policies and procedures for conducting inhalation risk assessments, including route-to-route extrapolation of toxicity data.  At this time a subchronic inhalation study is not required for triflusulfuron-methyl (see Section 3.1.1.3).  If any new policies or procedures are developed, the Agency may revisit the need for an inhalation toxicity study for triflusulfuron-methyl and/or a re-examination of the inhalation toxicity risk assessment.

10.2	Residue Chemistry	

The residue chemistry data are complete with the exception of:   

1. The following label amendments are needed: a) The requested 30-day PHI for garden beet needs to be added to the supplemental label.  b) The use of the aerial application method needs to be clarified in the supplemental label.  The submitted supplemental label for garden beet does not include the petition-requested 30-day PHI, nor does it delineate the use of the aerial application method.   Aerial application method instructions are included in the existing label for sugar beets. 

2. Analytical Method  -  An adapted version of DuPont Method 4480 (MRID 48207101) was used in the submitted garden beet field trials under review in this petition.  The enforcement method on record for triflusulfuron-methyl is method AMR 1930-91 (MRID 42496873), an HPLC-UV method.  Method AMR 2021-91 (MRID 42496872) is very similar to AMR 1930-91, with the addition of an automatic switching column protocol, and was used in the previously submitted sugar beet and chicory field trials.  Method 4480 is also similar to both these methods, with the primary difference being the addition of a tandem mass spectrometric detection protocol in Method 4480.  In addition, the independent laboratory validation (ILV) of Method 4480 successfully demonstrated the reliability of the method on the first attempt.  The registrant should clarify whether they want to replace the existing enforcement method with Method 4480 when applying for future new uses, as this newer method does appear to be an acceptable enforcement method.  

Should the registrant decide to request Method 4480 be used for enforcement purposes, they will need to ensure that the standards are made available to the USEPA's Analytical Chemistry Branch (ACB) in the event that they differ from those used in method AMR 1930-91.  

10.3	Occupational and Residential Exposure	

1.  The petition under review (9E7669, pg. 17) for the new use on garden beets lists both ground and aerial application methods.  The existing label for sugar beets covers both of these application methods.  The proposed supplemental label for garden beets covers only the ground application method.  The sponsor has confirmed that aerial application is requested for this new use.  Therefore, the aerial application method needs to be added to the supplemental label.  

11.0	References

   1. Triflusulfuron-methyl: Occupational and Residential Exposure Assessment for Use on Garden Beets; M. Collantes: D376267; January 2011.
   2. Triflusulfuron-methyl: Chronic Aggregate Dietary Exposure (Food and Drinking Water) and Risk Assessment for the Section 3 Registration on Garden Beet; E. Holman; D376268; February 2011.
   3. Estimated Drinking Waters Concentrations (EDWCs) of Triflusulfuron-methyl for the Use in the Human Health Risk Assessment: New Use (IR-4) of the Chemical on Garden Beets; J. Meléndez; D373494; July 2010.
   4. Triflusulfuron-methyl.  Petition for the Establishment of Permanent Tolerances and Registration for Use on Garden Beets (PP 9E7669).  Summary of Analytical Chemistry and Residue Data; E. Holman; D376350; February 2011.
   5. Triflusulfuron-methyl: Human Health Risk Assessment for Use in Garden Beet.  E.Holman; D373493; February 2011.  

Appendix A:	Toxicology Assessment	

A.1	Toxicology Data Requirements 

The requirements (40 CFR 158.340) for food use for are shown below in the following table. Use of the new guideline numbers does not imply that the new (1998) guideline protocols were used.

                                     Test 

                       Triflusulfuron-methyl, 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-Day Dermal	
870.3250    90-Day Dermal	
870.3465    90-Day Inhalation	
                                      yes
                                      yes
                                      yes
                                      no
                                      no
                                      yes
                                      yes
                                      yes
                                       -
                                       -
870.3700a  Developmental Toxicity (rodent)	
870.3700b  Developmental Toxicity (nonrodent)	
870.3800    Reproduction	
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
870.4100a  Chronic Toxicity (rodent)	
870.4100b  Chronic Toxicity (nonrodent)	
870.4200a  Oncogenicity (rat)	
870.4200b  Oncogenicity (mouse)	
870.4300    Chronic/Oncogenicity	
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
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
                                      yes
                                      yes
                                      yes
                                      yes
870.6100a  Acute Delayed Neurotoxicity (hen)	
870.6100b  90-Day Neurotoxicity (hen)	
870.6200a  Acute Neurotoxicity Screening Battery (rat)	
870.6200b  90-Day Neurotoxicity Screening Battery (rat)	
870.6300    Develop. Neurotoxicity	
                                      no
                                      no
                                      yes
                                      yes
                                      no
                                      --
                                      --
                                      yes
                                      yes
                                      --
870.7485    General Metabolism	
870.7600    Dermal Penetration	
                                      yes
                                      no
                                      yes
                                      no
870.7800     Immunotoxicity series.........................................................
                                      yes
                                      no
Special Studies for Ocular Effects
         Acute Oral (rat)	
         Subchronic Oral (rat)	
         Six-month Oral (dog)	
                                       
                                       no
                                       no
                                       no
                                       
                                      --
                                      --
                                      --

A.2.1 Acute Toxicity Profile for Triflusulfuron-methyl (technical) 

 A.2.1	Acute Toxicity Profile  -  Triflusulfuron technical
Guideline No.
Study Type
MRID(s)
                                    Results
                               Toxicity Category
870.1100
Acute oral - rat
                                   42496819
LD50 > 5000 mg/kg (both sexes)
                                      IV
870.1200
Acute dermal - rabbit
                                   42496826
LD50 > 2000 mg/kg (both sexes)
                                      III
870.1200
Acute dermal - rat
                                   42496825
LD50 > 2000 mg/kg (both sexes)
                                      III
870.1300
Acute inhalation - rat
                                   42496828
LC50 > 5.1 mg/L (both sexes)
                                      IV
870.2400
Primary eye irritation - rabbit
                                   42496830
Slight irritation of cornea and conjunctive clearing within 72 hrs.
                                      III
870.2500
Primary dermal irritation - rabbit
                                   42496834
Slight irritation clearing within 48 hrs.
                                      IV
870.2600
Skin sensitization - guinea pig
                                   42496837
Not a sensitizer (Buehler method)
                                      N/A
870.6200a
Acute neurotoxicity - rat
                                   43398601
NOAEL>=2000 mg/kg (HDT)
                                      N/A

Table A.2.2	Subchronic, Chronic and Other Toxicity Profile for Triflusulfuron-Methyl
                                Guideline No. 
                                  Study Type
                    MRID No. (year)/ Classification /Doses
                                    Results
870.3100

90-Day oral toxicity (rat)
MRID  42991408 (1992)
Acceptable/guideline
0, 100, 2000, 10,000 or 15,000 ppm
(0, 6.56, 133, 658 or 1036 mg/kg/day in males;
0, 7.71, 153, 783 or 1124 mg/kg/day in females)
NOAEL = 6.56/7.71 mg/kg/day in m/f  
LOAEL = 133/153 mg/kg/day in m/f, based on decreased body weight gain and food efficiency in males; increased incidence of extramedullary hematopoiesis in females.  Mild regenerative anemia was observed at 10,000 ppm.  
870.3100
90-Day oral toxicity (rat)
MRID 42991406 and -07 (1990)
Acceptable/guideline
0,  100, 2000, 10,000 or 15,000 ppm 
(males 0, 6.20, 127, 646 or 965 mg/kg/day;
Females 0, 7.54, 150, 774 or 1070 mg/kg/day)
NOAEL =  6.2/7.54 mg/kg/day in m/f
LOAEL = 127/150 mg/kg/day in m/f, based on decreased body weight and weight gain and food efficiency, mild regenerative anemia in males and hemosiderin in the renal proximal tubular epithelium of females.  At 15,000 ppm, testicular tubular atrophy in males and renal tubular cell atrophy in females were observed.
870.3150

90-Day oral toxicity (dog)
MRID 42991440 (1991)
Acceptable/guideline
0, 100, 4000 or 8000 ppm in the diet
(males 0, 3.9, 146.9 or 267.6 mg/kg/day;
females 0, 3.7, 159.9 or 250.7 mg/kg/day) 
NOAEL = 3.9/3.7mg/kg/day in m/f 
LOAEL = 146.9/159.9 mg/kg/day in m/f, based on  increased SGOT, SGPT and alkaline phosphatase, increased liver weight, decreased testes weight, and microscopic changes in the testes and liver.  Decreased body weights and effects on hematologic parameters were seen at 8000 ppm only.
870.3200

21-Day dermal toxicity (New Zealand white rabbit)
MRID 42991409 (1993)
Acceptable/guideline
0, 50, 350 or 1000 mg/kg/day applied for 6 hr/day, 21 days
NOAEL = 1000 mg/kg/day (HDT). 
LOAEL was not observed (>1000 mg/kg/day).  

870.3700a

Prenatal developmental in Crl:CD(BR) rat
MRID  42496841 (1991)
Acceptable/guideline
0, 30, 120, 350 or 1000 mg/kg/day by gavage
Maternal NOAEL = 120 mg/kg/day
Maternal LOAEL = 350 mg/kg bw/day, based on reduced body weight gain and lower food efficiency.

Developmental NOAEL = 1000 mg/kg /day
Developmental LOAEL was not observed >1000 mg/kg/day).
870.3700b

Prenatal developmental in New Zealand white rabbit
MRID  42496842 (1991)
Acceptable/guideline
0, 15, 90, 270 or 800 mg/kg/day by gavage
Maternal NOAEL = 90 mg/kg/day 
Maternal LOAEL = 270 mg/kg/day, based on maternal death and abortions, clinical signs and decreased weight gain.  

Developmental NOAEL = 90 mg/kg/day 
Developmental LOAEL = 270 mg/kg/day, based on increased abortions and decreased mean fetal body weight.  
870.3800

Reproduction and fertility effects
(rat) 
MRID 42496843; 42991412 (1993)
Acceptable/guideline
0, 10, 100, 750 or 1500 ppm in the diet (males 0, 0.588, 5.81, 44.0 or 89.5 mg/kg/day; 
females 0, 0.764, 7.75, 58.0 or 115 mg/kg/day)

Parental NOAEL = 5.81/7.75 mg/kg/day in m/f
Parental LOAEL = 44/58 mg/kg/day in m/f, based on decreased body weight/body weight gain. 

Offspring NOAEL = 5.81/7.75 mg/kg/day in m/f
Offspring LOAEL = 44/58 mg/kg/day in m/f, based on decreased F1 pup body weight during lactation.

Reproductive NOAEL = 89.5/115 mg/kg/day in m/f
Reproductive LOAEL = not determined (>89.5/115 mg/kg/day in m/f)
870.4100a

Chronic toxicity
(rat)  -  
See 870.4300, MRID 42991413
870.4100b

Chronic toxicity (dog)
MRID 42991410 (1993)
Acceptable/guideline
0, 35, 875 or 3500 ppm in the diet
(males 0, 1.0, 26.9 or 111.6 mg/kg/day;
 females 0, 1.2, 27.7 or 95.5 mg/kg/day)
NOAEL = 26.9/27.7 mg/kg/day in  m/f
LOAEL = 111.6/95.5 mg/kg/day in m/f, based on increased alkaline phosphatase (males), liver weight and incidence of minimal centrilobular hepatocellular hypertrophy.  Marginal reductions in RBC parameters were also observed.

870.4200a

Carcinogenicity
(rat)

See 870.4300, below
870.4200b

Carcinogenicity
(CD-1mouse)
 MRID 42991411 (1993)
Acceptable/guideline
0, 150, 2500 or 7000 ppm in the diet 
(males 0, 1.37, 20.9, 349 or 1024 mg/kg/day ;
females 0, 1.86, 27.7, 488 or 1360 mg/kg/day)
NOAEL = 20.9/27.7 mg/kg/day in m/f
LOAEL = 349/488 mg/kg/day in m/f, based on increased liver weights and hepatocellular microscopic findings (necrosis and intracellular pigment accumulation in Kupffer cells/macrophages in males; intrahepatocellular erythrocytes in males and females).  At 7000 ppm, body weight gain was decreased only during the first 90 days for males and from days 0-364 and 0-546 for females.

No evidence of carcinogenicity.  A statistically significant increase in hepatocellular adenomas observed in males at 2500 and 7500 ppm was within historical control range.
870.4300
Combined carcinogenicity/chronic toxicity (rat)
MRID  42991413 (1993)
Acceptable/guideline
0, 10, 100, 750 or 1500 ppm in the diet 
(males 0, 0.406, 2.44, 30.6 or 64.5 mg/kg/day; 
females 0, 0.546, 3.28, 41.5 or 87.7 mg/kg/day)
NOAEL = 2.44/3.28 mg/kg/day in m/f
LOAEL = 30.6/41.5 mg/kg/day in  m/f, based on decreased body weight and weight gain, decreases in hematology parameters (primarily in males) and increased incidence of testicular interstitial cell hyperplasia.  
Evidence of carcinogenicity was observed as an increased incidence of testicular interstitial cell adenomas at 750 and 1500 ppm.
870.5100
Bacterial Gene Reverse Mutation 
S. typhimurium Ames Assay, strains TA 98, 100, 1535, 1537, 1538
MRID  42496846  (1992) Acceptable/guideline
62.5, 125, 250, 500 or 1000 μg/plate (with and without S9 activation)
Negative.  There was no evidence of induced reverse mutations with or without metabolic activation in 5 strains of Salmonella.

870.5100
Bacterial Gene 
Reverse Mutation
S. typhimurium TA98, 100, 1535, 1537 and 1538
42496849 (1991)
Acceptable/guideline
50, 100, 250, 500, 1000 or 3000 ug/plate (with and without S9 activation)
Negative.  There was no evidence of induced reverse mutation with or without metabolic activation in 5 strains of Salmonella.

870.5300
In Vitro Mammalian Cell Forward Gene Mutation
Chinese Hamster Ovary (CHO) cells
42496850 (1991) Acceptable/guideline
Tested up to 2000 μg/mL (with and without S9 activation)
Negative.  There was no evidence of induced forward mutations with or without activation.

870.5375
In Vitro Mammalian Chromosome Aberration 
Human lymphocytes cells
42496852 (1991)
Acceptable/guideline
100 to 2000 ug/mL (with and without S9 activation)
Positive for induction of a treatment-related increase in the frequency of chromosomal aberrations at concentrations  >=1700 ug/mL in the presence of metabolic activation; inconclusive in the absence of metabolic activation.
870.5375
In Vitro Mammalian Chromosome Aberration 
Human lymphocytes cells
42496845 (1992)
Acceptable/guideline

100 to 2000 ug/mL (with and without S9 activation)
Positive for induction of a treatment-related increase in the frequency of chromosomal aberrations at 2000 ug/mL in the presence of metabolic activation; inconclusive in the absence of metabolic activation.  Follow-up assay to MRID 42496852.
870.5395
In vivo Mammalian Micronucleus Assay 
Mouse (male and female)
42496853 (1991)
Acceptable/guideline
Tested up to 5000 mg/kg
Negative.  There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow of mice up to the limit dose at 24, 48 or 72 hr postdosing.
870.6200b
Subchronic neurotoxicity (rat)
43398602 (1994)
Acceptable/guideline
0, 100, 750, 1500 or 3000 ppm in the diet
(males 0, 6.1, 46.1, 92.7 or 186.2 mg/kg/day;
females 0, 7.1, 51.6, 104.1 or 205.2 mg/kg/day)
NOAEL = 92.7 mg/kg/day (males) and 
                 7.1 mg/kg/day (females)
LOAEL = 186.2 mg/kg/day (males) and 
                 51.6 mg/kg/day (females), based on decreased body weight/body weight gain.
No evidence of neurotoxicity.
870.7485

Metabolism and pharmacokinetics
(rat)
42991416 (1992)
Acceptable/guideline

[14]C-triazine labeled triflusulfuron-methyl was administered as :
Low dose 25 mg/kg single oral dose; repeated low dose 25 mg/kg/day for 14 days; high dose 250 mg/kg, single oral dose.  Ester [14]C-carbonyl labeled triflusulfuron-methyl was also administered at 250 mg/kg, single oral dose.

Evaluated for absorption, distribution, metabolism and excretion
Rate of absorption was unaffected by dose or dosing regimen but the extent of absorption was decreased at 250 mg/kg.  Urine was the major route of excretion at 25 mg/kg but the primary route of excretion at 250 mg/kg was the feces.  Excretion of radioactivity via expired air was negligible.  By 120 hr post-dosing, significant amounts of radioactivity remained in the liver.  At 25 mg/kg the major urinary metabolite was N-desmethyl triflusulfuron-methyl (25-44% of AD), with females excreting higher levels than males of this metabolite.  The N-hydroxymethyl metabolite was also found in urine (10-16% of AD).  Triazine metabolites (triazine amine, N-desmethyl triazine amine and N,N-bis-desmethyl triazine amine) were identified in liver as well as urine; in feces none of these metabolites represented more than 2% of AD.  In rats dosed with the ester carbonyl-labeled triflusulfuron-methyl, methyl saccharin was a major metabolite in the urine and liver.  
Nonguideline
Evaluation of Mode of Action of Leydig Tumor Cell Induction
42991414 (1993)
Acceptable/nonguideline (as supplemental information)
(1)0, 1000, 1500 or 2000 mg/kg/day for 15 days to male rats; examined for  ;
(2)In vitro studies-induction of male rat hepatic microsomal aromatase activity;
(3)in vitro evaluation of testosterone, progesterone and estradiol in rat Leydig cells +-hCG stimulation

(1) male rats showed decreased body weight, decreased absolute/relative accessory sex gland weights, decreased estradiol levels.  No effects on beta-oxidation or aromatase activity, or cytochrome p-450 content;
(2) hepatic microsomal activity showed dose-dependent treatment-related decreases;
(3)in vitro incubation with triflusulfuron-methyl +-hCG caused decreased synthesis of estradiol and  - hCG increased synthesis of testosterone .

A.3  Executive Summaries

A.3.1	Subchronic Toxicity

        5.3100 a   90-Day Oral Toxicity - Rat

(1) In a subchronic oral rat toxicity study (MRID 42991406), 10 Crl:CD[(R)]BR rats/sex/group were administered DPX-66037 in the diet for 90 days at dosages of 0, 100, 2000, 10,000 or 15,000 ppm (equivalent to average daily intakes of 0, 6.2, 127, 646 or 965 mg/kg/day, males and 0, 7.54, 150, 774 or 1070 mg/kg/day, females, respectively).  

Overall mean body weight and mean body weight gain were significantly decreased in males and females at >=2000 ppm.  The range of decreases in body weight in the 2000 to 15,000 ppm group males was 9% to 40%; the range in females was 16% to 35%.  The decreases in body weight gain were more pronounced and ranged from 16% to 72% in males and from 41% to 82% in females.  Overall mean food consumption was significantly decreased in males in the 10,000 and 15,000 ppm groups and in females in the 2000, 10,000 and 15,000 ppm groups.  In general, the magnitude of the decreases in food consumption was less than the decreases in body weight and weight gain.  Significant decreases in overall mean food efficiency were seen in the three highest dosage groups of both sexes.  Evidence of a mild regenerative anemia was seen in males and females in the 10,000 and 15,000 ppm groups and in males in the 2000 ppm group in the form of significantly decreased RBC counts, HGB and HCT, and increased reticulocyte counts.  Changes in clinical chemistry parameters (decreased glucose, phosphate, total protein and globulin) in males and females in the two highest dosage groups were thought to be caused by decreased food intake.  The only finding on gross necropsy was an increase in the number of males in the 15,000 ppm group with small testes.  The absolute weight of most of the organs evaluated decreased, most likely due to decreased body weights.  The relative weights of several organs were also increased in these groups; the toxicological significance of these findings is unknown.  The only histopathology lesions which appeared to be treatment-related were bilateral atrophy/degeneration of the seminiferous tubules of the testes and oligospermia of the epididymides in the 15,000 ppm group males, and hemosiderin deposition in the proximal tubular epithelium of the kidneys in males and females in the 10,000 and 15,000 ppm groups, and females in the 2000 ppm group.  Females in the 15,000 ppm group also had an increased incidence of atrophy of the tubular epithelium.  The testicular effects were possibly secondary to poor nutrition.  The LOAEL is 2000 ppm (127 mg/kg/day in males and 150 mg/kg/day in females), based on decreased mean body weight and mean body weight gain, decreased mean food consumption (females), mean food efficiency, altered hematology parameters (males) and hemosiderin deposition in the kidneys (females).  The NOAEL is 100 ppm (6.2 mg/kg/day in males and 7.54 mg/kg/day in females).

This study is classified as acceptable/guideline and satisfies the guideline requirement for a subchronic toxicity study in the rat (OCSPP 870.3100).

(2) In a subchronic oral toxicity study (MRID 42991408), 10 Crl:CD[(R)]BR rats/sex/group were administered DPX-66037-59 (triflusulfuron-methyl technical, 98.7% a.i., lot no. DPX-66037-59) at dietary concentrations of 0, 100, 2000, 10,000 or 15,000 ppm (equivalent to average daily intakes of 0, 6.56, 133, 658 or 1036 mg/kg/day in males and 0, 7.71, 153, 783 or 1124 mg/kg/day in females, respectively).

Mean body weights were significantly decreased throughout the study in male and female rats treated at concentrations of 10,000 and 15,000 ppm.  The decreases at Day 91 in the males at 10,000 and 15,000 ppm were 12% and 19%, respectively.  Mean body weights of females at Day 91 were decreased by 14% and 17% for those in the 10,000 and 15,000 ppm groups.  Overall mean body weight gains of male rats were decreased 11%, 18% and 28% for the 2000, 10,000 and 15,000 ppm groups, respectively.  Weight gains of females in the 10,000 and 15,000 ppm groups were decreased by 26% and 33%, respectively.  The overall mean food consumption of males in the 15,000 ppm group was decreased by 10% in comparison to the controls.  Decreases in the 10,000 and 15,000 ppm group females were 13% and 17%, respectively.  Decreases in food efficiency were observed in males treated at 2000, 10,000 and 15,000 ppm and in females treated at 10,000 and 15,000 ppm.  Significant alterations in hematology parameters (decreases in RBC, HGB and HCT and increases in reticulocyte counts) seen in the 10,000 and 15,000 ppm group males and females at the 45- and 90-day evaluations indicated that the chemical produced a regenerative anemia.  Terminal mean body weights were significantly decreased for males and females in the 10,000 and 15,000 ppm groups.  The absolute weights of all organs from the treated animals were comparable to the control group.  The mean relative weights of the majority of the organs were increased in the 10,000 and 15,000 ppm group males and females.  The increase in the relative weight of the liver was considered to be compound-related.  There were increased incidences of microscopic changes in the kidney (pigment accumulation) and spleen (extramedullary hematopoiesis) which were considered secondary to compound-related hemolysis.  Pigment accumulation in the proximal tubules of the kidney was present in males and females in the 10,000 and 15,000 ppm groups.  Increased incidences of extramedullary hematopoiesis were present in males treated at 10,000 ppm and above, and in females at 2000 ppm and above.  This study was done to compare the toxicity of the chemical produced by the cyanate method (DPX-66037-59) to that produced by the carbamate method (DPX-66037-8).  A prior subchronic rat study (MRID 42991406) was conducted with DPX-66037-8 at the same dosage levels.  Actual mg/kg/day intake in the studies was very similar.  The results of the two studies demonstrate that DPX-66037-8 affected some of the quantitative measures (body weight, body weight gain, food consumption and food efficiency) more severely than DPX-66037-59.  The LOAEL is 2000 ppm (133 mg/kg/day) in males, based on decreased body weight gain and food efficiency and 153 mg/kg/day in females, based on increased incidence of histopathological changes.  The NOAEL is 100 ppm (6.56 mg/kg/day in males and 7.71 mg/kg/day in females).

This study is classified as acceptable/guideline and satisfies the guideline requirements (OCSPP 870.3100) for a subchronic oral toxicity study in the rat.

        3100.3150 90-Day Oral Toxicity  -  Dog

In a subchronic oral toxicity study (MRID 42496840), DPX-66037-24 (triflusulfuron-methyl technical, 95.6% a.i., lot no. 02) was administered to male and female dogs at nominal dose levels of 0, 100, 4000 or 8000 ppm (equivalent to average daily intakes of 0, 3.9, 146.9 or 267.6 mg/kg/day in males and 0, 3.7, 159.9 or 250.7 mg/kg/day in females).

Toxic effects from administration of technical DPX-66037-24 to male and female dogs were observed at the 4000 ppm dose level and above.  At 4000 ppm, increased plasma SGOT and SGPT, increased liver and decreased testes weights were observed.  These findings were supported by microscopic effects in the liver (brown pigment in sinusoidal macrophages and in females, bile stasis) and testes/epididymes (tubular atrophy, aspermia/oligospermia, cellular debris).  At 8000 ppm, decreased mean body weight and body weight gain, decreased erythrocytes, hemoglobin and hematocrit, increased SGOT, SGPT and alkaline phosphatase, increased absolute and relative weight of the liver and testes, and microscopic abnormalities of the testes and liver were observed.  Bone marrow of the sternum and femur showed hypercellularity in both sexes. The LOAEL is 4000 ppm (146.9 mg/kg/day in males and 159.9 mg/kg/day in females), based on hematological and liver enzyme changes, increased liver and decreased testes weights, and microscopic changes in the liver and testes.  The NOAEL is 100 ppm (3.9 mg/kg/day in males and 3.7 mg/kg/day in females).

This study is classified acceptable/guideline and meets the guideline requirements for a subchronic oral toxicity study in the dog (OCSPP 870.3150).

	870.3200	21-Day Dermal Toxicity  -  Rabbit

In a 21-day dermal toxicity study (MRID 42991409), 5 New Zealand White rabbits/sex/group were treated topically with dosages of 0, 50, 300 or 1000 mg/kg/day of DPX-66037 (triflusulfuron-methyl technical, 95.6% a.i., batch no. DPX-66037-24) for 21 days, 6 hrs/day.  

There were no differences between the control and treated groups in any of the parameters measured.  Signs of dermal irritation and the finding of inflammation and acanthosis on histopathology were reported in both the control and treated groups and were attributed to the mechanical trauma of frequent washing and clipping.  The systemic toxicity NOAEL is >=1000 mg/kg/day (HDT).  A LOAEL for systemic toxicity was not determined (>1000 mg/kg/day).  The dermal toxicity NOAEL is >=1000 mg/kg/day.  A LOAEL for local dermal toxicity was not determined (>1000 mg/kg/day).

This study is classified as acceptable/guideline and satisfies the guideline requirement for a 21-day dermal toxicity study in the rabbit.

A.3.2	Prenatal Developmental Toxicity

	870.3700a Prenatal Developmental Toxicity Study - Rat

In an oral developmental toxicity study, DPX-66037-24 (triflusulfuron-methyl technical,95.6% a.i., lot no. 18649-02) was administered by gavage  in 0.5% aqueous methylcellulose vehicle (10 mL/kg) to 25 Crl:CD[(R)]BR albino rats at dose levels of 0, 30, 120, 350 or 1000 mg/kg/day from gestation day (GD) 7 through 16, inclusive.

Maternal toxicity:  At 350 mg/kg/day, decreased body weight gain (about 20% less than controls during dosing) and a slight reduction in food efficiency (2.6%) were observed.  At 1000 mg/kg/day, effects were more pronounced and decreased weight gain and food consumption were statistically significant.  The maternal toxicity LOAEL is 350 mg/kg/day, based on decreased body weight gain, food consumption and food efficiency.  The NOAEL is 120 mg/kg/day.

Developmental toxicity:  No developmental effects were observed in this study.  The developmental toxicity NOAEL is >=1000 mg/kg/day.  A LOAEL was not observed (>1000 mg/kg/day).

This study is classified as acceptable/guideline and satisfies the guideline requirement for a developmental toxicity study in the rat (OCSPP 870.3700a).

	870.3700b Prenatal Developmental Toxicity Study - Rabbit

In an oral developmental toxicity study, DPX-66037-24 (triflusulfuron-methyl technical, 95.6% a.i., lot no. 18649-02) was administered by gavage in 0.5% aqueous methylcellulose vehicle (8 mL/kg) to 20 HRA(NZW)SPF rabbits at dose levels of 0, 15, 90, 270 or 800 mg/kg/day from gestation day (GD) 7 through 19, inclusive.

Maternal toxicity:  At 270 mg/kg/day, clinical signs of toxicity included absent/reduced stool and stained fur, postdosing maternal death (10%), increased abortions (40%), mean body weight loss (-82.9 g vs. gain of 165 g in controls) and lower food efficiency (4.1% vs. 8.5%, controls).  Abortions and mortality did not occur until post-dosing.  At 800 mg/kg/day, effects were more severe, with mortality reaching 45% and abortions 60% (both findings first observed on GD 15).  Postmortem findings indicated gastrointestinal effects, including ulcerations of the gastric mucosa, lack of formed feces in the large intestine, and gaseous distension in various parts of the digestive tract; these findings were observed in all of the animals that died during the study.  The maternal toxicity LOAEL is 270 mg/kg/day, based on mortality, clinical signs of toxicity, abortions and body weight loss.  The NOAEL is 90 mg/kg/day.

Developmental toxicity:  At 270 mg/kg/day, decreased mean fetal weight (-11% less than controls) and increased abortions were observed.  At 800 mg/kg/day, the incidence of abortions was increased.  Abortions were considered secondary to significant maternal toxicity.  The developmental toxicity LOAEL is 270 mg/kg/day, based on decreased mean fetal body weight and abortions (secondary to maternal toxicity).  The LOAEL is 90 mg/kg/day.

This study is classified as acceptable/guideline and satisfies the guideline requirement for a developmental toxicity study in the rabbit (OCSPP 870.3700b).

A.3.3	Reproductive Toxicity

	870.3800 Reproduction and Fertility Effects  -  Rat

In a two-generation reproduction study (MRID 42991412), 30 CD rats/sex/dose received DPX-66037-24 continuously in the diet at concentrations of 0, 10, 100, 750 or 1500 ppm (equivalent to average daily intakes of 0, 0.588, 5.81, 44.0 or 89.5 mg/kg/day in males and 0, 0.764, 7.75, 58.0 or 115 mg/kg/day in females, respectively).  The protocol was for a standard reproduction study with litters culled to 4 animals per sex per dose on postpartum Day 4.

Parental toxicity:  At 750 and 1500 ppm, mean body weight and body weight gain were significantly decreased in males during the premating period of both the F0 and F1 generations. Body weights for the 750 ppm and 1500 ppm F0 males were 94% and 90% of the controls, respectively, at the end of the premating period.  Mean body weight gains for these groups during this time period were 87% and 81% of controls, respectively.  Mean body weight during the premating period was significantly decreased for the F1 generation females at 750 and 1500 ppm (93% and 90% of controls, respectively).  Mean body weight gain during the premating period was decreased for the 750 and 1500 ppm groups of the F0 generation and the 1500 ppm group of the F1 generation (86% and 82%, respectively, for the F0 generation and 89% for the F1 generation).  Mean daily food consumption for the F0 males in the 1500 ppm group was significantly reduced during the premating period (94% of controls).  Overall mean food efficiency during this period was significantly reduced for the F0 males in the 750 and 1500 ppm groups (91% and 83% of controls, respectively).  Food consumption in the F1 males during the premating period was comparable to the controls.  However, food efficiency was significantly lower in the 1500 ppm group (94% of controls).  Decreases in food efficiency were seen in the 750 and 1500 ppm group females of the F0 generation during this period (90% and 86% of controls, respectively).  Food consumption in the F1 female rats in the 750 and 1500 ppm groups was significantly decreased during the premating period (95% and 93% of controls, respectively).  There was a slight reduction in food efficiency that was not statistically significant.  The LOAEL for parental/systemic toxicity is 750 ppm (44 mg/kg/day in males and 58 mg/kg/day in females), based on decreased body weight and body weight gain and food efficiency.  The NOAEL is 100 ppm (5.81 mg/kg/day in males and 7.75 mg/kg/day in females).

Offspring toxicity:  At 750 and 1500 ppm (44.0 mg/kg/day in males and 58.0 mg/kg/day in females), the mean body weights of male F1 pups were significantly decreased on Day 14 post-partum (90.5-92.6% of controls); body weights at Day 21 were slightly lower but did not achieve statistical significance.   At 1500 ppm, the mean combined male and female pup body weights were significantly decreased (90%-93% of controls).  The LOAEL for offspring toxicity is 750 ppm (44 mg/kg/day in males and 58 mg/kg/day in females), based on decreased mean F1 pup body weight in males during lactation.  The NOAEL is 100 ppm (5.81 mg/kg/day in males and 7.75 mg/kg/day in females).

Reproductive toxicity:  No reproductive toxicity was observed in this study, which tested up to doses causing maternal and offspring toxicity.  The NOAEL for reproductive toxicity is 1500 ppm (89.5 mg/kg/day in males and 115 mg/kg/day in females).  A LOAEL was not determined (>1500 ppm).

A.3.4	Chronic Toxicity

	870.4100a (870.4300) Chronic Toxicity  -  Rat

 See 870.4300

	870.4100b Chronic Toxicity  -  Dog

In a chronic oral toxicity study (MRID 42991410), DPX-66037-24 (technical, 95.6% a.i., Lot No. 02) was administered daily in the diet to 5 beagle dogs/sex/dose at concentrations of 0, 35, 875 or 3500 ppm (equivalent to average daily intakes of 0, 1.0, 26.9 or 111.6 mg/kg/day in males and 0, 1.2, 27.7 or 95.5 mg/kg/day in females, respectively).

At 3500 ppm, one male and one female were sacrificed in moribund condition during the first five months of the study.  On necropsy, the female was diagnosed with a pulmonary bacterial infection; the male was found to have acute hemorrhage in the pleural cavity and gastrointestinal tract compounded with thymic necrosis.  These deaths were not considered treatment-related.  Mean body weight gain of the 3500 ppm group males was significantly lower than controls at Weeks 7, 11, 12 and 13 only (approximately 50% of the control value).  There was no treatment-related effect on food consumption or food efficiency.  Mean total erythrocyte counts and hematocrit values for the 3500 ppm group males and females were slightly lower than controls throughout the study; however, the differences were statistically significant only at the 3 month evaluation.  Mean alkaline phosphatase (AP) values were increased relative to controls at all evaluation times; differences were significant at Months 6, 9 and 12.  A significant increased at 3500 ppm in females at Month 3 resulted from an exceptionally high value (259 IU/L) in the female that was euthanized.  On necropsy, there was a treatment-related increase in mean absolute, relative liver:body and relative liver:brain weights in males and in relative weight in females.  Minimal centrilobular hepatocellular hypertrophy was observed in 3/4 males and 2/4 females.  The LOAEL was 3500 ppm (111.6 mg/kg/day in males and 95.5 mg/kg/day in females), based on increased alkaline phosphatase (males, liver weight and incidence of minimal centrilobular hepatocellular hypertrophy.  The NOAEL was 875 ppm (26.9 mg/kg/day, males and 27.7 mg/kg/day, females).

This study is classified as acceptable/guideline and satisfies the guideline requirement for a chronic toxicity study in the dog.

A.3.5	Carcinogenicity

      870.4200b Carcinogenicity (feeding)  -  Mouse
      
In an oral carcinogenicity study (MRID 42991411), 80 CD-1 mice/sex/dose were administered DPX-66037-24 in the diet at concentrations of 10, 50, 2500 or 7000 ppm (equivalent to average daily intakes of 0, 1.37, 20.9, 349 or 1024 mg/kg/day in males and 0, 1.86, 27.7, 488 or 1360 mg/kg/day in females).  An additional ten groups of 30 mice/sex (5 groups for each sex) were designated for a cell proliferation, cytochrome P-450 and beta-oxidation substudy.

Mean body weight gain for the first 90 days was statistically significantly decreased (13%) in the 7000 ppm group males.  There was a statistically significant increase of body weight gain in the 150 ppm group males and the 2500 ppm group females during this time period.  Males in the 10 ppm group had a significant body weight gain increase over the course of the study.  The 7000 ppm group females had significant body weight decreases during the periods 0-364 days and 0-546 days.  The 7000 ppm group males had slightly decreased food efficiency (15.4%) over the first 91 days of the study as compared to the control group.  There were increases in the hepatic cytochrome P-450 levels in the 7000 ppm group males and females at two weeks and three months; this finding was statistically significant at both time points in females and at three months for the males.  There was a treatment-related increase in the absolute and relative liver weights of males and females in the 2500 and 7000 ppm groups.  There was an increase in the incidence of hepatic microscopic alterations in both male and female mice in the 2500 and 7000 ppm groups.  There was a statistically significant increase in hepatocellular adenomas in male mice in the 2500 and 7000 ppm groups (16% and 19% incidence in the 2500 and 7000 ppm groups, respectively, vs. 12% in the controls) using the Cochran-Armitage trend test.  The incidence of the combined adenomas and carcinomas was statistically increased in the 7000 ppm group males (20% in the treated group vs. 15% in the controls) and 2500 ppm group females (6% in the treated group vs. 0% in the controls).  However, these findings were within the historical range of these tumors, alone and combined.  Dosing was considered adequate based on and testing up to the limit dose of 1000 mg/kg/day.  The LOAEL is 2500 ppm (349 mg/kg/day) based on increases in liver weights and increased incidence of microscopic changes in the liver.  The 150 ppm dose was adjusted to the lowest concentration level for the chemical in stability studies (70%).  The NOAEL is 105 ppm (14.6 mg/kg/day).  

This study is classified acceptable/guideline and satisfies the guideline requirement for a carcinogenicity study in the mouse (OCSPP 870.4200b).

	870.4300 Carcinogenicity Study - rat

In a combined chronic toxicity/carcinogenicity study (MRID 42991413), 62 male and 62 female CD rats per group were treated with triflusulfuron-methyl in the diet for 96 weeks at dosages of 0, 10, 100, 750, or 1500 ppm (0, 0.406, 4.06, 30.6 or 64.5 mg/kg/day in males and 0, 0.546, 5.47, 41.5, or 87.7 mg/kg/day in females respectively).  The study was terminated early so that there were enough animals for the terminal necropsy.  Survival in the treated animals was comparable to that of the controls.  Mean body weights in the 750 and 1500 ppm group males and females were statistically significantly decreased in comparison to the control group at a majority of the time points evaluated.  Body weight gains for the first three months were statistically significantly decreased in the 1500 and 750 ppm group males (≈21% and 11%, respectively) and females (21% and 8%, respectively); the decreases persisted at the 1 year evaluations.  There were no statistical differences in mean food consumption and mean food efficiency.  Statistically significant decreases in hematology parameters (RBC, HGB and HCT) were occasionally seen in the 750 and 1500 ppm group males at the 3-, 6- and 12-month sampling periods.  The only organ weight change for which there were also microscopic changes was an increase in the relative weight of the testes in the 1500 ppm group males at the interim and terminal sacrifices.  There was a statistically significant increase in the incidence of Leydig (interstitial) cell hyperplasia in the testes of males in the 750 and 1500 ppm groups at the terminal sacrifice only.  There was a statistically significant increase in myelin/axon degeneration of the sciatic nerve in females at 1500 ppm (control to high dose, 52, 61, 67, 69 and 86%, respectively), but the lesion is commonly observed in older rats and was not increased in the interim sacrifice animals (males showed an incidence of 93, 84, 85, 80 and 92%, control to high dose, respectively).  The LOAEL for chronic toxicity is 750 ppm (30.6 mg/kg/day) for males and females based on decreased body weight and body weight gain, alterations in the hematology parameters (males predominantly) and an increased incidence of interstitial cell hyperplasia in males.  The 100 ppm dose was adjusted to the lowest concentration level of the chemical at this dosage (60%); therefore, the NOAEL is 60 ppm (2.44 mg/kg/day). 

A statistically significantly and treatment-related increase in the incidence of interstitial cell adenomas was observed at 750 and 1500 ppm.  From control to high dose, incidence was 0, 4.3, 2.1, 14.0 and 13.7%, respectively.  

This study is classified acceptable/guideline and satisfies the guideline requirement for a combined chronic toxicity/carcinogenicity study in the rat (OCSPP 870.4300).

A.3.6

	870.6200a Acute Neurotoxicity Screening Study  -  Rat

In an acute oral neurotoxicity screening study (MRID 43398601), DPX-66037-24 (triflusulfuron-methyl technical, 95.6% a.i., Lot No. H-18649-02) was administered to 10 Crl:CD[(R)]BR VAF/Plus[(R)]  rats in single gavage doses of 0, 500, 1000 or 2000 mg/kg in 0.5% aqueous (w/v methylcellulose vehicle (10 mL/kg).  Neurobehavioral screening, consisting of functional observational battery (FOB) and motor activity (MA) evaluations, was performed before treatment on Days 1 (2 hrs post-dosing), 2-8 and 15.  Six animals/sex/dose were anesthetized and perfusion-fixed in situ for neuropathological evaluation; the remaining animals were examined grossly.

Mean body weights, body weight gains and absolute and relative food consumption of treated animals were comparable to controls.  All animals survived to terminal sacrifice without the appearance of any treatment-related clinical signs.  Neurobehavioral evaluations did not reveal any treatment-related changes in the FOB or in motor activity.  Neuropathological evaluations of high dose animals were comparable to controls.  The NOAEL is >=2000 mg/kg/day (highest dose tested.  A LOAEL was not determined (>2000 mg/kg).

This study is classified as acceptable/guideline and satisfies the guideline requirements for an acute neurotoxicity screening study in the rat (OCSPP 870.6200a).

	870.6200b  Subchronic Neurotoxicity Screening Study - Rat

In a subchronic oral neurotoxicity screening study, 11 Crl:CD[(R)]BR VAF/Plus[(R)]  rats were administered DPX-66037-24 (triflusulfuron-methyl technical, 95.6% a.i., Lot No. H-18649-02) in the diet at concentrations of 0, 100, 750, 1500 or 3000 ppm (equivalent to average daily intakes of 6.1, 46.1, 92.7 or 186.2 mg/kg/day in males and 0, 7.1, 51.6, 104.1 or 205.2 mg/kg/day in females).  Neurobehavioral screening (functional observational battery or FOB, and motor activity or MA) was performed prior to treatment and during weeks 4, 8 and 13.  Six animals/sex/dose were anesthetized and perfusion fixed in situ for neuropathological evaluation.

With the exception of one control female which was sacrificed in moribund condition on Days 52, all other animals survived to terminal sacrifice without the appearance of any treatment-related clinical signs.  At 3000 ppm, biological (>10%) and/or statistically (p<=0.01) significant decreases in the mean body weights were observed in males at weeks 8 and 13 and in females at weeks 4, 8 and 13; biologically significant decreases were observed at 750 and 1500 ppm females at week 13.  Mean body weight gains were lower in 3000 ppm males between days 1-8, 43-50, 64-71 and 1-92, and in females at >=750 ppm for days 1-8 and 1-92.  Results of the FOB and MA evaluations did not reveal any neurobehavioral effects in any of the treated animals; neuropathological evaluations of high-dose animals were comparable to controls.  The LOAEL is 3000 ppm in males (186.2 mg/kg/day) and 750 ppm (51.6 mg/kg/day) in females, based on decreased body weight/weight gains.  The NOAEL is 1500 ppm in males (92.7 mg/kg/day) and 100 ppm in females (7.1 mg/kg/day).

This study is classified acceptable/guideline and satisfies the guideline requirements for a subchronic neurotoxicity screening battery in the rat (OCSPP 870.6200b).

A.3.7	Mutagenicity

	Gene Mutation
870.5100 Bacterial Gene Reverse Mutation in S. typhimurium TA 98, 100, 1535,1537, 1538
MRID 42496846
Acceptable/guideline
0, 62.5, 125, 250 or 1000 ug/plate with and without S9 activation.
Negative up to precipitating concentrations.
870.5100 Bacterial Gene Reverse Mutation, S. typhimurium TA98, 100, 1535, 1537 and 1538
MRID 42496848
Acceptable/guideline
0, 50, 100, 250, 500, 1000 or 3000 ug/plate with and without S9 activation.
Negative up to precipitating concentrations.
870.5300 in Vitro Forward Mutation, Chinese hamster ovary (CHO) cells
MRID 42496850
Acceptable/guideline
0 to 2000 ug/mL with and without activation.
Negative up to cytotoxic concentrations.

	Cytogenetics
870.5375 In Vitro Chromosomal Aberration in human lymphocyte cells
MRID 42496852
Acceptable/guideline
0 to 2000 ug/mL with and without activation.
Positive for induction of a treatment-related increase in the frequency of chromosomal aberrations at concentrations >=1700 ug/mL in the presence of S9 activation; inconclusive in the absence of metabolic activation.
870.5375 In Vitro Chromosomal Aberration in human lymphocyte cells
MRID 42496845
Acceptable/guideline
0 to 2000 ug/mL with and without activation.
Positive for induction of a treatment-related increase in the frequency of chromosomal aberrations up to 2000 ug/mL in the presence of S9 activation; inconclusive in the absence of metabolic activation.  Follow-up assay to MRID 42496852.
870.5395 In Vivo Micronucleus Assay in Male Mice
MRID 42496853
Acceptable/guideline 
0 to 5000 mg/kg
Negative for induction of an increase in frequency of micronucleated polychromatic erythrocytes at 24, 48 hrs or 72 h.

A.3.8	Metabolism

	870.7485	Metabolism  -  Rat

In a rat metabolism study (MRID 42991416), [triazine-(U-[14]C)] DPX-66037 (triflusulfuron-methyl technical, radiochemical purity 95-97%, Lot No. 2622-175) was administered orally in PEG 200 vehicle to 5-6 Sprague-Dawley rats/sex/dose at the following dose levels:  a single low oral dose of 25 mg/kg, a repeated low oral dose of 25 mg/kg/day for 14 consecutive days, or a single high dose of 250 mg/kg.  An additional group of 10 rats/sex received a single oral dose of [ester carbonyl-[14]C]-DPX-66037 at 250 mg/kg.  Urine and feces were collected at 0-6 hr, 6-24 hr and at 24 hr post-dose.  Metabolite analysis was performed in urine and feces of all dose groups, and in liver of high dose females.  Pilot experiments showed less than 0.01% of the dose excreted through expired air, which was not measured in the main study.

The rate of absorption (as suggested by the time of peak urinary excretion) was not affected by dosing regimen or dose of test chemical.  The extent of absorption was decreased at the high dose as suggested by the increase in percentage of radioactivity excreted in feces and by the increased percentage of parent chemical observed in feces at the high dose.   The majority of residual radioactivity at 120 hours post-dose was found in the liver in all dose groups, and was observed to be composed of parent DPX-66037 (41.5-62.3% of the sample radioactivity), and the metabolites N-desmethyl DPX-66037 (18.7% - 24.8% of the sample radioactivity), triazine amine (8.4%-15.9% of the sample radioactivity), N,N-bis-desmethyl triazine amine (0.7%-2.3% of the sample radioactivity), and methyl saccharin (18.4% of the sample radioactivity).  The skin and ovaries were also found with measurable amounts of residual radioactivity at the high dose of 250 mg/kg.  Whole blood:plasma ratios were approximately 1 for the single and repeated low dose groups, but at the high dose, the ratio increased to approximately 2 for the triazine labeled material.  For the ester carbonyl labeled material, the ratio at the high dose was less than 1.  
Urine was the major route of excretion at the low dose, with between 61%-77% excreted via this route at 25 mg/kg.  Repeated oral dosing had no effect on the percentage of radioactivity excreted through urine.  At the 250 mg/kg dose, fecal excretion increased to between 43%-64% of the administered dose.  The percentage excreted through urine was higher in female rats than male rats in all dosing regimens.  The major urinary metabolite of DPX-66037 in urine was N-desmethyl DPX-66037, found to compose 25%-44% of the administered dose at the low dose level (single and repeated).  At the high dose, the percentage of this metabolite was decreased in urine (14%-28%).  Female rats showed higher urinary levels than males of this metabolite.  The N-hydroxymethyl metabolite of DPX-66037 was also found in urine, but did not vary significantly with dose (10%-16% of the dose).  Female rats also showed higher percentages of this metabolite in urine vs. males.  The triazine metabolites of DPX-66037 (triazine amine or T9, N-desmethyl triazine amine or T5, N,N-bis-desmethyl triazine amine or T3) were found in urine and liver.  In urine, only T3 was in significant percentage (3.6%-6.8%); T5 and T9 were less than 1.0% of the administered dose.  In feces, none of these metabolites represented more than 2.0% of the administered dose.  Parent compound was found as a major component only in high dose feces and liver.  Methyl saccharin was found as a major metabolite in urine and liver of rats dosed with the ester carbonyl labeled test material.  Based on the identified metabolites, a scheme for metabolism of DPX-66037 was proposed.

This study is classified acceptable/guideline and satisfies the guideline requirement for a general metabolism study in the rodent (OCSPP 870.7485).

A.3.9  Additional Studies

	Nonguideline mechanistic study

In a 2-week in vivo study, CD male rats were administered DPX-66037-24 at dosages of either 0, 1,000, 1,500 or 2,000 mg/kg/day for fifteen days. An additional untreated control group was pair-fed to the 2,000 mg/kg/day group. Ten animals in the unpaired control group and the 2,000 mg/kg/day group were administered 100 IU of human chorionic gonadotropin one hour prior to sacrifice on test day 16. Various clinical parameters and serum hormonal levels were measured, along with determinations of beta-oxidation activity, cytochrome P-450 content and aromatase activity on liver homogenates at necropsy. The body weight, body weight gain, food consumption and food efficiency results demonstrated that decreases in these parameters for the treated groups were due to reduced intake. Final body weights for the treated groups and the pair-fed control were decreased in comparison to the unpaired control. Only decreases in the absolute and relative weights of the accessory sex glands, decrease in absolute weight of the liver and increase in relative weight of the liver in the 2,000 mg/kg/day group was significantly different from the pair-fed control. The only hormone level which was statistically significantly different from either the pair-fed or unpaired control group was the serum estradiol level. There were significant decreases in the serum estradiol levels in the 1,000, 1,500 and 2,000 mg/kg/day groups (31.9, 13.6 and 13.4% of the unpaired control level). No differences were found with the interstitial estradiol levels. In the 2,000 mg/kg/day group rats treated with hCG, serum testosterone levels were significantly elevated and serum estradiol levels were significantly decreased. There were no treatment-related effects on beta-oxidation activity, cytochrome P-450 content or aromatase activity.

In an in vitro experiment, the hepatic microsomal fraction from ammonium perfluorooctanoate-induced male rats was studied to determine if DPX-66037-24 had an effect on aromatase activity. There was a dose-dependent statistically significant decrease in aromatase activity in the microsomes treated with 0.01, 0.02, 0.05, 0.1, 0.2 or 0.5 uM DPX-66037-24.

Hormonal analyses were also conducted on the male rats treated with DPX-66037-24 in the combined chronic toxicity/carcinogenicity study. The following statistically significant trends in hormonal levels were observed in the study: increasing serum testosterone levels, decreasing serum estradiol levels and increasing serum FSH levels. LH levels were not affected.

In another study, isolated Leydig cells from 11-week old rats were incubated for two hours with increasing concentrations of DPX-66037-24. Some of the cultures were then treated with hCG for three hours prior to analysis for testosterone, estradiol and progesterone.  There was no treatment-related effect on hCG-induced testosterone biosynthesis. However, Leydig cells incubated with 1,000 uM DPX-66037-24 and no hCG stimulation produced testosterone at levels 198.8% of control. Cells incubated with 100 and 1,000 uM DPX-66037-24 had significant decreases in estradiol, both with and without hCG stimulation. There was no treatment-related effect on progesterone production.

The studies demonstrated that DPX-66037-24 produces a dose-dependent decrease in aromatase activity in vitro. However, the effects of the chemical on the enzyme in vivo are not conclusive since no inhibition of activity at extremely high dose levels after a two-week exposure period was observed. Further, the hypothesis that this effect is mediated by a chronic depression in estradiol altering the negative feedback mechanism for LH upon the Leydig cells of the testes has been suggested but not clearly demonstrated. A trend but not pair-wise statistical significance has been shown for either the 750 or 1500 ppm serum levels of testosterone or estradiol after one year of exposure. In addition, no elevation in serum levels of LH was noted at either dose level.

This study is classified acceptable/nonguideline.  It was not submitted to satisfy/does not satisfy guideline requirements for triflusulfuron-methyl, but provides supplemental information evaluating effects on cytochrome p450 enzyme activities and hormonal levels in male rats
A.4  Rationale for Toxicology Data Requirements
Table A.4.1       Guideline Number:  870.7800
Study Title:  Immunotoxicity 
                       Rationale for Requiring the Data
The immunotoxicity study is a new data requirement under 40 CFR Part 158 as a part of the data requirements for registration of a pesticide (food and non-food uses). 

The Immunotoxicity Test Guideline (OPPTS 870.7800) prescribes functional immunotoxicity testing and is designed to evaluate the potential of a repeated chemical exposure to produce adverse effects (i.e., suppression) on the immune system. Immunosuppression is a deficit in the ability of the immune system to respond to a challenge of bacterial or viral infections such as tuberculosis (TB), Severe Acquired Respiratory Syndrome (SARS), or neoplasia.  Because the immune system is highly complex, studies not specifically conducted to assess immunotoxic endpoints are inadequate to characterize a pesticide's potential immunotoxicity.  While data from hematology, lymphoid organ weights, and histopathology in routine chronic or subchronic toxicity studies may offer useful information on potential immunotoxic effects, these endpoints alone are insufficient to predict immunotoxicity.  
                         Practical Utility of the Data
                                       
How will the data be used?

Immunotoxicity studies provide critical scientific information needed to characterize potential hazard to the human population on the immune system from pesticide exposure. Since epidemiologic data on the effects of chemical exposures on immune parameters are limited and are inadequate to characterize a pesticide's potential immunotoxicity in humans, animal studies are used as the most sensitive endpoint for risk assessment.  These animal studies can be used to select endpoints and doses for use in risk assessment of all exposure scenarios and are considered a primary data source for reliable reference dose calculation. For example, animal studies have demonstrated that immunotoxicity in rodents is one of the more sensitive manifestations of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) among developmental, reproductive, and endocrinologic toxicities.  Additionally, the EPA has established an oral reference dose (RfD) for tributyltin oxide (TBTO) based on observed immunotoxicity in animal studies (IRIS, 1997).

How could the data impact the Agency's future decision-making? 

If the immunotoxicity study shows that the test material poses either a greater or a diminished risk than that given in the interim decision's conclusion, the risk assessments for the test material may need to be revised to reflect the magnitude of potential risk derived from the new data.
 
If the Agency does not have these data, a 10X database uncertainty factor may be applied for conducting a risk assessment from the available studies.