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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

January 3 1 ,2013



### SUBJECT: Pyroxasulfone Human Health Risk Assessment for Use of New Active Ingredient Pyroxasu lfon e on Soybeans.

PC Code: 090099 DP Barcode: 400783
Decision N o.: 462689 Registration No. : 63588-91, -92, -93 Petition No.: 2F8 0 05 Regulatory A ction: Section 3 Regi s trati on Risk Assessment Type: Si n g le C h e mic al Aggregate Case No.: NA

TXR N o.: NA CAS N o.: 447399-55-5
MRID No.: NA 40 CFR: 180.659

FROM: Thurston G. Morton, Chemist

# ,_.**~ ~** **r/"~** Jam es S. Miller

, E n v ironm ental Scientist**~** '"'/..--.**~"'--** Abdallah Khasawinah, PhD

., Toxicologi

, / (;:/

### (_(}~ Risk Assessment Branch 4 (RAB IV ) ·/.h .' ;/.

_ Ana Rivera-Lupianez, Chemist, RAB V**~** H ea l th Effects Division (7509 P )



### THROUGH: Elissa Reaves, Ph.D., Branch C hi ef

# &~2,...**~** Susan V. Hummel, Branch Senior

## ScientistC**tJ ,** **~** Risk Assessment Branch 4

, HED (7509P)



### TO:

Erik Kraft

Herbicide Branch Registration D iv ision (7505P)

This document provide s the revised Health Effects Division's (HED's) ri sk assessment of the

Section 3 use of pyroxasulfone on soybean.

Page 1 of80Page 2 of 80 1.0 EXECUTIVE SUMMARY ................................................................................................... 5

2.0 HED RECOMMENDATIONS ............................................................................................. 7

2.1 Data Deficiencies/Conditions of Registration................................................................... 7

2.2 Tolerance Considerations .................................................................................................. 7

2.2.1 Enforcement Analytical Method ................................................................................ 7

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

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

2.2.4 Revisions to Petitioned-For Tolerances ..................................................................... 9

2.3 Label Recommendations .................................................................................................... 9

3.0 INGREDIENT PROFILE ................................................................................................... 10

3.1 Chemical Identity ............................................................................................................. 10

3.2 Physical/Chemical Characteristics .................................................................................. 11

3.3 Pesticide Use Pattern ........................................................................................................ 11

3.3.1 Registered Products ................................................................................................... 11

3.3.2 Proposed New Uses .................................................................................................... 11

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

3.5 Considerations of Environmental Justice ...................................................................... 12

4.0 HAZARD CHARACTERIZATION/ASSESSMENT ...................................................... 12

4.1 Toxicology Studies Available for Analysis ..................................................................... 12

4.2 Absorption, Distribution, Metabolism and Excretion ................................................... 12

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

4.4 Safety Factor for Infants and Children (FQPA Safety Factor).................................... 15

4.4.1 Completeness of the Toxicology Database ............................................................... 15

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

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

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

4.5 Toxicity Endpoint and Point of Departure ..................................................................... 16

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

4.5.2 Recommendations for Combining Exposure Routes .............................................. 18

4.5.3 Classification of Carcinogenic Potential .................................................................. 18

4.5.4 Summary of Points of Departure Used in Risk Assessment .................................. 18Page 3 of 80 5.0 DIETARY AND DRINKING WATER EXPOSURE AND RISK ASSESSMENT ....... 20

5.1 Metabolite/Degradate Residue Profile ............................................................................ 20

5.1.1 Summary of Plant and Livest ock Metabolism Studies ........................................... 20

5.1.2 Comparison of Metabolic Pathways ......................................................................... 20

5.1.3 Environmental Fate and Transport ......................................................................... 21

5.1.4 Residues of Concern Summary and Rationale ........................................................ 21

5.2 Food Residue Profile ........................................................................................................ 2 2

5.2.1 Residues in Crops ....................................................................................................... 22

5.3 Water Residue Profile ...................................................................................................... 23

5.3.1 Estimated Drinking Water Concentrations............................................................. 23

5.4.1 Acute Dietary and Drinking Water Analysis .......................................................... 24

5.4.2 Chronic Dietary and Drinking Water Analysis ...................................................... 24

6.0 RESIDENTIAL EXPOSURE AND RISK ASSESSMENT ............................................. 25

6.1 Residential Bystander Post-application Inhalation Exposure ...................................... 25

6.2 Spray Drift.................................................................................................................... ..... 25

7.0 AGGREGATE EXPOSURE A ND RISK ASSESSMENT ............................................... 26

7.1 Acute & Chronic Aggregate Risk .................................................................................... 26

7.2 Short- and Intermediate-Term Aggregate Risk ............................................................ 26

7.3 Intermediate-Term Aggregate Risk ................................................................................ 26

8.0 CUMULATIVE RISK ......................................................................................................... 26

9.0 OCCUPATIONAL EXPOSURE/R ISK CHARACTERIZATION ................................. 27

9.1 Exposure Scenarios.......................................................................................................... 2 7

9.2 Handler Exposure ............................................................................................................. 27

9.2.1 Handler Exposure Scenarios ..................................................................................... 27

9.2.2 Handler Exposure Data ............................................................................................. 28

9.2.3 Handler Exposure Assumptions ............................................................................... 28

9.2.4 Handler Exposure and Risk Estimates .................................................................... 28

9.3 Post Application Exposure .............................................................................................. 30

9.3.1 Post Application Exposure Scenarios....................................................................... 30

9.3.2 Post Application Exposure Assumptions ................................................................. 31

9.3.3 Post-Application Exposure and Risk Estimates ...................................................... 31

9.3.4 Restricted Entry Interval .......................................................................................... 31

9.3.5 Inhalation Post-application Risk ..................................................................................... 32Page 4 of 80 10. REFERENCES .................................................................................................................... . 32

A TOXICOLOGY DATA SUMMARY ................................................................................. 33

A.1 Guideline Data Requirements ................................................................................... 33

A.3 Toxicological Endpoints ................................................................................................ 49

APPENDIX B. Chemical Names And Structures Of Metabolites ............................................ 77

APPENDIX C. Physical/Chemical Properties ............................................................................. 80

APPENDIX D. Studies Reviewed for Ethical Conduct ............................................................. 80Page 5 of 80 1.0 EXECUTIVE SUMMARY

This assessment provides information to support a Section 3 registration fo r the use of the active ingredient, pyroxasulfone on soybeans. This document addresses the exposures and estimated risk associated with the proposed new uses of pyroxasulfone. It provides an assessment of proposed tolerances (pesticide residu e limits in food) to ensure that they meet the safety standard established by the Food Quality Protection Act (FQPA) of 1996.

Use Profile

Pyroxasulfone (3-[5-(difluoromethoxy)-1 -methyl-3-(trifluoromethyl)pyrazol-4-yl methylsulfonyl]-4,5-dihydro-5,5-dimet hyl-1,2-oxazole) is a new herbicide belonging to a class of selective herbicides identified as pyrazole herbicid es. It is used to control a broad spectrum of broadleaf weeds and grasses, including glyphosate-re sistant varieties. Py roxasulfone is thought to act as an inhibitor of very-l ong-chain fatty acid biosynthesis. Applications may be made using ground or aerial equipment. There are no pyroxa sulfone products registered for homeowner use and there are no products registered for application to residential areas. Proposed tolerances are for combined residues of pyroxasulfone a nd its major metabolites in/on soybean.

K-I Chemical, U.S.A. Inc. is requesting a Sec tion 3 registration of tw o pyroxasulfone products; Pyroxasulfone 85 WG Herbicide, an 85% water-d ispersible granule (WG) formulation (EPA Reg. No. 63588-92), and V-10233 Herbicide, a 42.5% WG formulation (EPA Reg. No. 63588- 93) for use on soybeans. Applications to soybe ans are to be made preplant, preemergence, or early postemergence at an application rate of 0. 08-0.21 lb ai/A (depending on soil texture) with a
maximum seasonal rate of 0.267 lb ai/A. Pre-ha rvest intervals (PHIs) and/or pre-grazing intervals (PGIs) of 7-37 da ys have been proposed.

Hazard Identification

The toxicology database for pyroxasu lfone is adequate for evaluati ng and characterizing toxicity and selecting endpoints for purposes of this risk assessment. Py roxasulfone acute toxicity to mammals is low by all routes of exposure. Subchr onic and chronic oral studi es in mice, rats and dogs produced a variety of effect s including cardiac toxicity (inc reased cardiomyopathy), liver toxicity (centrilobular hepatoce llular hypertrophy, histopathological and/or clinical pathological indicators), kidney toxicity (n ephropathy), neurotoxicity (imp aired hind limb function, ataxia, tremors, sciatic nerve lesions, axonal/myelin dege neration in the sciatic nerve and spinal cord sections), skeletal muscle myopathy, urinary bladder mucosal hyperplasia, and urinary bladder transitional cell papillomas. Minimal to m ild cardiac myofiber degeneration and localPage 6 of 80 inflammation were also seen in a rat dermal t oxicity study. Neurotoxicit y was also seen in a developmental neurotoxicity study in rats (decre ased brain weight, decreased thickness of the hippocampus, corpus callosum and cerebellum in offspring). Dogs appear to be the most sensitive species to the neurotox ic effects of pyroxasulfone. I mmunotoxicity studies in rats and
mice show no evidence of immunotoxic effects fro m pyroxasulfone. There is evidence of fetal and offspring susceptibility in the developmental neurotoxicity study in rats as effects occurred
in the absence of matern al toxicity. There is no concern fo r reproductive toxicity as offspring effects occurred only at maternally toxic doses in the reproduction study. The FQPA safety factor was reduced to 1X because all the points of departure (POD) selected for risk assessment are protective of offspring effects seen in the developmental neurotoxicity study, the fetal/offspring effects occurred at high doses of administration, and a clear dose-response has
been well defined with NOAELs and LOAELs fo r all effects of concern. Thus, there are no residual uncertainties with regard to pre-and/or postnatal toxicity. Pyroxasulfone is classified as

“ Not Likely to be Carcinogenic to Humans ” at doses that do not cause crystals with subsequent calculi formation resulting in cellu lar damage of the urinary tract . The Agency has determined that the quantification of risk using a non-linear approach (i.e., RfD) will adequately account for all chronic toxicity, incl uding carcinogenicity, that could result from exposure to pyroxasulfone.

Dose Response Assessment

Toxicological points of departure (PODs) were selected for dietary/drinking water and occupational exposure scenarios for this assessmen t. Acute and chronic reference doses (RfDs)
were selected for assessment of food and dr inking water exposures. The population adjusted dose (PAD) is equivalent to the reference dose (RfD) divided by the additional FQPA Safety Factor, if applied. An acute RfD/PAD for a ll populations was selected from a developmental neurotoxicity study in rats base d on neurotoxicity characterized by decreased brain weights and morphometric brain changes in offspring in the DNT study. A chr onic RfD/PAD for all populations was selected from a chronic feedi ng study in dogs based on neurotoxic effects. A
POD for short- and intermediate -term inhalation exposure was selected from subchronic and chronic oral toxicity studies in dogs based on neurotoxic effects. The POD for short-and intermediate-term dermal exposure was chosen fro m a 28 day dermal toxicity study in rats based on mild cardiac myofiber degenera tion and local inflammation.
The POD for short- and intermediate-term inha lation exposure was chosen from a 90-day oral dog study based on axonal/myelin degeneration in the sciatic nerve and minimal to mild subacute
inflammation of skeletal muscle in males a nd females and impaired limb function. An uncertainty factor of 100x was applied to endpoi nts selected for all e xposure routes (10x for interspecies extrapolation, 10x fo r intraspecies variation).

Exposure/Risk Assessment a nd Risk Characterization

A dietary (food and water) exposure and risk assessment was conducted for soybean. HED assessed occupational exposure and risk from pr oposed uses of pyroxasulfone on corn, wheat and soybean commodities in anticipation of future registration of wheat and soybean if and when the registrant resubmits its a pplication. Worker exposures we re assessed for handler and post-Page 7 of 80 application activities. Screening level acute and chronic dietary a nd drinking water risk assessments for pyroxasulfone show that dietar y and drinking water exposure estimates for the
assessed corn use are below HED’s level of c oncern for the general population and all population subgroups. Occupational exposure and risk estimates indicate that worker handler and postapplication exposures are not of concern at th e maximum allowable app lication rates for the majority of scenarios. Exposure and risk estim ates indicate that for most exposure scenarios
non-cancer risks are not of concer n for occupational handler activ ities for the proposed new use (MOEs > 100). However, risks of concern exis t (MOE<100) for “Inhalation” exposure during mixing and loading activities for aer ial applications. Risks of c oncern exist for the “mixing and loading” of a water dispersible granular for aeri al applications (MOE = 71) with label specific PPE (long-sleeved shirt and long pants, chemi cal resistant gloves and shoes and socks). However, with a PF5 respirator, risks are not of concern (MOE = 350).

Use of Human Studies

This risk assessment relies in part on data from studies in which adult human subjects were intentionally exposed to a pesticide or other chemical. These studies, listed in Appendix D, have been determined to require a re view of their ethical conduct. Some of these studies are also subject to review by the Human St udies Review Board. All of the studies used have received the appropriate review.

2.0 HED RECOMMENDATIONS

2.1 Data Deficiencies/Condi tions of Registration

HED recommends for the establishment of to lerances to support the proposed use of pyroxasulfone on soybean commodities provided requirements in Section 2.1 are met. Deficiencies are stated below. The specific to lerance recommendations are discussed in Section 2.2, and label modifications ar e discussed in Section 2.3.

Note to PM:

Revised Section B & F required.

2.2 Tolerance Considerations

2.2.1 Enforcement Analytical Method

The petitioner has proposed high performance liq uid chromatography/triple quadrupole mass spectrometry (LC/MS/MS) methods for the enfor cement of tolerances fo r pyroxasulfone residues in/on crop commodities. The methods are adequate to determine residues of pyroxasulfone and metabolites M-1, M-3, M-25, and M-28 in soybean commodities. The validated limit of quantitation (LOQ) is 0.005 ppm fo r parent, M-3 and M-25 in most soybean commodities. ThePage 8 of 80 LOQ is 0.01 for M-1 and M-28 in most soybean commodities. Enforcement methods for livestock commodities are not needed to support the proposed uses.

2.2.2 International Harmonization

There are no Codex MRLs established for residues of pyroxasulfone in any commodities.
International harmonization is not an issue at this time.

2.2.3 Recommended Tolerances

HED has examined the residue chemistry databa se for pyroxasulfone. Pending submission of a revised Section B (see requirements under Dire ctions for Use), a revised Section F (see requirements under Proposed Tolerances) and anal ytical reference standards (see requirements under Submittal of Analytical Reference Standard s), there are no residue chemistry issues that would preclude granting condi tional Section 3 registration for the requested uses of pyroxasulfone, or establishment of toleran ces for residues of pyroxasulfone as follow:

Tolerances are established for residues of the herbicide pyroxa sulfone, including its metabolites and degradates, in or on the comm odities in the table below. Compliance with the tolerance levels specified below is to be determined by measuring only the sum of pyroxasulfone [3-[[[5-(difluoromethoxy)-1 -methyl-3-(trifluorom ethyl)-1H-pyrazol-4- yl]methyl]sulfonyl]-4,5-dihydro-5,5-dimet hylisoxazole] and its metabolites [5- (difluoromethoxy)-1-methyl-3-(trifluoromethyl )-1H-pyrazol-4-yl]methanesulfonic acid (M-1), 5-(difluoromethoxy)-1-methyl-3-(tr ifluoromethyl)-1H-pyrazo l-4-carboxylic acid (M-3), and [5-(difluoromethoxy)-3-(trifluor omethyl)-1H-pyrazol-4-yl]methanesulfonic acid (M-25), calculated as the stoichiometric equivalent of pyroxasulfone, in or on the commodity.

Soybean, forage............................................................. 1.0 ppm Soybean, hay ................................................................. 2.0 ppm

Tolerances are established for residues of the herbicide pyroxa sulfone, including its metabolites and degradates, in or on the comm odities in the table below. Compliance with the tolerance levels specified below is to be determined by measuring only the sum of pyroxasulfone [3-[[[5-(difluoromethoxy)-1 -methyl-3-(trifluorom ethyl)-1H-pyrazol-4- yl]methyl]sulfonyl]-4,5-dihydro-5,5-dimet hylisoxazole] and its metabolites [5- (difluoromethoxy)-1-methyl-3-(trifluoromethyl )-1H-pyrazol-4-carboxylic acid (M-3), [5-(difluoromethoxy)-3-(trifluor omethyl)-1H-pyrazol-4-yl]met hanesulfonic ac id (M-25), and M-28 (3-[1-carboxy-2-(5,5-di methyl-4,5-dihydroisoxazol- 3-ylthio)ethylamino]-3- oxopropanoic acid) calculated as th e stoichiometric equivalent of pyroxasulfone, in or on the commodity.

Soybean, seed .............................................................. 0.06 ppmPage 9 of 80 2.2.4 Revisions to Petitioned-For Tolerances

K-I Chemical proposed that tolerances for pyroxasulfone residues of concern in crop commodities be expressed in terms of combined residues of pyroxasulfone and its major metabolites M-1, M-3, M-25, and M-28, calculated as pyroxasulfone. K-I Chemical proposed tolerances for pyroxasulfone on soybean commod ities. HED’s recommended revisions to the tolerances submitted by K-I Chemical fo r soybean is listed in Table 1.

Table 1. Tolerance Summary fo r Pyroxasulfone for Soybean.
Commodity Proposed Tolerance (ppm) HED
Recommended Tolerance (ppm)* Comments; Correct Commodity Definition

Soybean Forage 1.5 1.0 Soybean, forage
Soybean Hay 2.0 2.0 Soybean, hay
Soybean Seed 0.07 0.06 Soybean, seed *Changes are due to the use of the Organization for Economic Cooperation and Development tolerance calculation procedures and use of all residue field trials, unless otherwise stated.

2.3 Label Recommendations

The following label revisions must be provi ded prior to a soybean tolerance being established for registration.

 Because applications to soybean are ba sed on defined growth stages, and crop commodities were harvested at appropriate crop growth stages in the field trials submitted with this petition, no PHIs or PGIs need to be specified for any soybean commodity. Therefore, the product label for the 85% WG formulation should be amended to remove the following: the 15-da y PHI for soybeans; and the 7-day PGI for soybean and soybean forage, hay, and straw.

 The petitioner must amend the product label for the 85% WG formulation to specify the following maximum seasonal application rates: (1) 0.186 lb ai/A for soybean grown in fine-, medium-fine-, and medium-textured so ils; (2) 0.112 lb ai/A for soybean grown in
coarse-textured soils

 For the 42.5% WG formulation, the petitioner must remove Weedmaster® from the list of tank mix partners for app lication to soybean, as this product (EPA Reg. No. 7969-133) is not registered for use on soybeans.Page 10 of 80  Risks of concern exist for the “mixing and lo ading” of a water disp ersible granular for aerial applications, therefore, the PPE for th is scenario must require a PF5 respirator.

3.0 INGREDIENT PROFILE

3.1 Chemical Identity

Structure and nomenclature are reported in Table 2.

Table 2. Pyroxasulfone Nomenclature.

Chemical structure

Common name Pyroxasulfone

Company experimental name KIH-485; AE 2196191

IUPAC name 3-[5-(difluoromethoxy)-1-methyl-3-(trifluoromethyl)pyrazol-4-ylmethylsulfonyl]- 4,5-dihydro-5,5-dimethyl-1,2-oxazole
CAS name 3-[[[5-(difluoromethoxy)-1-methyl-3-(trifluoromethyl)-1H-pyrazol-4- yl]methyl]sulfonyl]-4,5-dihydro-5,5-dimethylisoxazole
CAS registry number 447399-55-5

End-use product (EP) Pyroxasulfone 85 WG Herbicide (85% WG; EPA File Symbol 63588-OE) V-10233 Herbicide (42.5% WG; EPA File Symbol 63588-OG)

Chemical structure of M-1
metabolite

[5-(difluoromethoxy)-l-methyl-3-(trifluoromethyl)-lH-pyrazol-4- yl)]methanesulfonic acid

Chemical structure of M-3
metabolite

5-(difluoromethoxy)-l-methyl-3-(trifluo romethyl)-lH-pyrazole-4-carboxylic acidPage 11 of 80 Table 2. Pyroxasulfone Nomenclature.

Chemical structure of M-25
metabolite

[5-(difluoromethoxy)-3-(trifluoromethyl )-1H-pyrazol-4-yl)]methanesulfonic acid

3.2 Physical/Chemical Characteristics

A detailed description of the physicochemical properties of pyroxasulfone is provided in Appendix C. Pyroxasulfone exhibits relatively lo w solubility in water and higher solubility in organic solvents. It has a very low vapor pressu re. Pyroxasulfone is mobile and persistent in terrestrial and aquatic environments. Pyroxasulfone does not presen t significant concerns for bioaccumulation based on the lipophilicity of the compound.

3.3 Pesticide Use Pattern

3.3.1 Registered Products

Pyroxasulfone is an herbicide belonging to a new class of selective herbicides identified as pyrazole herbicides. Currently, Pyroxasulfone 85 WG Herbicide, an 85% water-dispersible granule (WG) formulation, and V-10233 Herbicide, a 42.5% WG formulation are registered for use on field corn, sweet corn, and pop corn Appl ications to soybeans are to be made preplant, preemergence, or early postemergence at an appl ication rate of 0.08-0. 21 lb ai/A (depending on soil texture) with a maximum seasonal rate of 0. 267 lb ai/A; pre-harvest in tervals (PHIs) and/or pre-grazing intervals (PGIs) of 7-37 days ha ve been proposed. Product registrations and proposed use patterns and formula tions are provided in Table 3.

3.3.2 Proposed New Uses

Table 3. Proposed Use Patterns And Formulations Pyroxasulfone
Formulation Use Sites Method of Application Max Single
A R Max
Seasonal AR Timing of Application

85% a.i. WG Reg # 63588-92 Soybeans broadcast ground 0.21 0.267 preplant, preemergence, or early postemergence

42.5% WG Reg # 63588-93 Soybeans broadcast aerial,
broadcast/band ground 0.11 0.267 preplant, preemergence

3.4 Anticipated Exposure Pathways

Dietary (food and water) and occupational exposu res via dermal and inhalation pathways are expected based on proposed use of pyroxasu lfone on soybean. A dietary (food and water)Page 12 of 80 exposure and risk assessment was conducted fo r the requested soybean use. HED assessed occupational exposure and risk from proposed us es of pyroxasulfone on soybean commodities. Worker exposures were assessed for handler a nd post-application activ ities. There are no residential uses for pyroxasulfone so residential exposure to childre n via incidental oral activities is not anticipated.

3.5 Considerations of Environmental Justice

Potential areas of environmental ju stice concerns, to the extent possi ble, were considered in this human health risk assessment, in accordance w ith U.S. Executive Order 12898, “Federal Actions
to Address Environmental Just ice in Minority Populations an d 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-esta blished procedures. In line with OPP policy, HED estimates risks to population subgroups fr om pesticide exposures that ar e based on patterns of that subgroup’s food and water consumption, and activ ities in and around the home that involve pesticide use in a residential se tting. Extensive data on food consumption patterns are compiled by the USDA under the Continuing Survey of Food Intake by Individuals (CSFII) and are used in pesticide risk assessments for all registered fo od 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. Additionally, OPP is able to assess di etary exposure to smaller, specialized subgroups and exposure assessments are performed when c onditions or circumstances warrant. Whenever appropriate, nondietary exposures based on home use of pesticide products and associated risks for adult applicators and for t oddlers, youths, and adults enteri ng or playing on treated areas post-application are evaluated. Further considera tions are currently in development as OPP has committed resources and expertise to the developm ent of specialized software and models that consider exposure to bystanders and farm worker s as well as lifestyle and traditional dietary patterns among specific subgroups.

4.0 HAZARD CHARACTERIZATION/ASSESSMENT

4.1 Toxicology Studies Available for Analysis

The studies available for consid eration of pyroxasulfone toxicity provide a comprehensive and complete database. All toxicity studies requ ired in accordance with the new 40 CFR Part 158 data requirements have been submitted.

4.2 Absorption, Distribution, Metabolism and Excretion

Pyroxasulfone was shown to be quickly absorbed and excreted after oral administration to rats with the half-life ranging from 15 – 173 hours; the longer half lif e corresponded to a high orally administered dose (700 mg/kg). At lower admini stered oral doses (10 mg/kg) absorption was fast (t
max of 1.1-7 hours with 77-88 % ab sorption) while at a higher dose (700 mg/kg) absorptionPage 13 of 80 was slower (t
max 7.0-17.3 hours, with 22-26% absorption). The main excretory routes were the urine and feces. Biliary excretion was demonstr ated. Following absorption pyroxasulfone was widely distributed, with high con centrations observed in the bla dder, prostate, adrenals, kidney, liver, abdominal fat, uterus, and blood.

There was no significant sex difference in the me tabolism of pyroxasulfone. There appeared to
be saturation or induction of metabolism at the hi gh dose, which resulted in an altered metabolic profile. The absorbed pyroxasulfone was extens ively metabolized to a number of compounds. The major metabolite in the urine of rats dosed with [Py- 14 C]- pyroxasulfone was M-13, accounting for up to 26% of the dos e at the low dose and up to 4% at the high dose. M-7 and M3 were the next most abundant metabolites. Most of the metabolites in urine were produced by the cleavage between the pyrazole and isoxazolin e ring structure. There were three major metabolites in urine in rats dosed with [Ix- 14 C]- pyroxasulfone; M-13 accounting for up to 20% of the dose, M-16 accounting for up to 19% of the dose and M-26 accounting for up to 6.4% of the dose. A major portion of th e radioactivity was highly pola r and multi-component, containing 5-methyl-5-isoxazoline carboxylic acid and M-30, and accounted for up to 34% of the dose.

A dermal absorption study with pyroxasulfone is not available. A dermal absorption factor can be derived by comparing the toxi city endpoints from a dermal toxic ity study and an oral toxicity study of comparative durations. In one 28-day oral toxicity study in rats (MRID 47889320), the LOAEL was 1000 ppm (75.4/84.4 mg/kg/day for M/F) based on myocardial degeneration/necrosis and the NOAEL is 100 ppm (7.3/8.3 mg/kg/day for M/ F). In the 28-day dermal toxicity study, the LOAEL was 1000 mg /kg/day based on minimal to mild cardiac myofiber degeneration with inflammation in males and females and the NOAEL was 100 mg/kg/day. By comparing the LOA ELs from both studies a dermal absorption factor of 7.5% is
derived.

4.3 Toxicological Effects

The toxicology database for pyr oxasulfone is adequate for ev aluating and characterizing pyroxasulfone toxicity and selec ting endpoints for purposes of this risk assessment. Subchronic and chronic toxicity testing of pyroxasulfone technical in mice, ra ts and dogs produced a variety
of adverse effects in seve ral target organs, but the most sensitive effect is neurotoxicity in dogs.
Effects seen in animal studies ranged from cardiac toxicity (increased cardiomyopathy in mice and rats), liver toxicity (c entrilobular hepatocellular hypert rophy, histopathol ogical and/or clinical pathological indicators) , neurotoxicity characterized by axonal/myelin degeneration in the sciatic nerve (dog, mouse and rat) and spinal cord sections (dog), skeletal muscle myopathy, kidney toxicity (increased incidence of chroni c progressive nephropathy in dogs and retrograde nephropathy in mice), urinary bladder mucosal hyperplasia, inflammati on, and urinary bladder transitional cell papillomas (rats). Decreased body weight, enzyme changes were noted in some studies. Toxic adverse effects (impaired hi nd limb function, ataxia, hind limb twitching and tremors; increased creatine kina se, aspartate aminotransferase; axonal/myelin degeneration of the sciatic nerve and spinal cord sections) in dogs occurred at ≥ 10 mg/kg/day doses while in the mouse toxic adverse effects (deg eneration of sciatic and trig eminal nerve axons and their associated myelin sheaths and chronic progr essive nephropathy, rena l tubular adenomas)Page 14 of 80 occurred at higher doses (131 mg/kg/day and above).

Comparing effects by route of administration, pyroxasulfone was moderately toxic to rats following a 4-week dermal exposure producing lo cal inflammation and systemic effects of minimal to mild cardiac myofiber degenera tion at the limit dose of 1000 mg/kg/day with a NOAEL of 100 mg/kg/day. No adve rse effects were noted in an inhalation study following exposure for 28 days at 200 mg/m 3 /day (equivalent to 52.2 mg/kg/ day oral dose), the highest
dose tested of an aerosol dust.

In cancer studies in mice and rats, renal tubula r adenomas were observed in male mice at a dietary dose of 0.6 and 255 mg/kg/da y (but not at an intermediate dose of 18 mg/kg/day) and urinary bladder transitional cell papillomas were observed in male rats at 42 and 84 mg/kg/day. The CARC met in May, 2011 to consider studies submitted by the registrant to support a mode
of action for bladder tumors and a re-evaluati on of the kidney tumors conducted by an external Pathology Working Group (PWG). Based on the evidence provided by the PWG, the CARC concluded that that the kidney adenomas in male mice were not treatment-related. The CARC
considered the transitional cell bladder tumors in male rats to be treatment-related based on statistically significant trends for urinary bladder transitional cell papillomas and combined papilomas and carcinomas, the occurrence of pren eoplastic lesions at 42 and 84 mg/kg/day and
the rare occurrence of bladder transitional cell tu mors. The CARC concluded that the mode of action for bladder tumors has been adequately established based on subm itted data that support both a dose-response and temporal concordance of the key events and bladder tumors. The Agency has determined that the quantification of risk using a non-linear approach (i.e., RfD) will adequately account for all chronic toxicity, including carcinogenicity, th at could result from exposure to pyroxasulfone.

Pyroxasulfone did not exhibit developmental toxi city in the rat guidel ine study at the limit dose of 1000 mg/kg/day and it exhibited slight developmental toxicity in rabbits (reduced fetal weight and resorptions) at the limit dos e of 1000 mg/kg/day. However, developmental effects were noted in PND 21 offspring at 300 mg/kg/day in the rat developmental neurotoxicity (DNT) study
characterized as decreased brain weight and morphometric changes. In a reproductive toxicity in rats reduced pup weight and body we ight gains during lactation) o ccurred at similar or higher doses causing pronounced maternal toxicity (r educed body weight, body we ight gain and food consumption and increased kidney weight, ca rdiomyopathy and urinary bladder mucosal hyperplasia with inflammation).

Pyroxasulfone did not produce immunotoxic eff ects in mice following dietary feeding for 28 days up to 4000 ppm (633/791 mg/kg/day, M/F) or in rats at dietary c oncentrations of 7500 ppm (529/570 mg/kg/day in M/F).

Acute toxicity studies show that pyroxasulfone ex hibits low acute toxicity by all exposure routes (EPA Toxicity Category III or IV) an d is not a dermal sensitizer.

The complete toxicity profile for pyr oxasulfone is provided in Appendix A.Page 15 of 80 4.4 Safety Factor for Infants and Children (FQPA Safety Factor)

4.4.1 Completeness of the Toxicology Database

The toxicity database for pyroxasulfone is sufficient for a full hazard evaluation and is considered adequate to evaluate risks to infant s and children. Acceptable developmental toxicity
studies in the rat and rabbit a nd acceptable multi- and single generation reproduction studies in the rat have been submitted. Acceptable acute, subchronic and developmental neurotoxicity
studies in the rat are availabl e. Acceptable immunotoxicity studies in mice and rats are
available.

4.4.2 Evidence of Neurotoxicity

Pyroxasulfone produced neurotoxic effects in dogs, mice and rats. Dogs appear to be the most sensitive species as minimal to mild axonal/myelin degeneration in the scia tic nerve occurred at

≥ 10 mg/kg/day for 90 days. The nerve effects in dogs were also accompanied by impaired limb function, body and limb hypotonia at a subchronic dose of 15 mg/kg/day. Chronic administration of pyroxasulfone at 10 mg/kg/day to dogs resulted in similar fi ndings of impaired hind limb function, ataxia, hind limb twitching and tremors and axonal/myelin degeneration of the sciatic nerve and spinal cord sections.

In rats, sciatic nerve lesions (minimal to mild axon/myelin degeneration w ithin the sciatic nerve sections) were observed in a reproduction study in parental females fe d pyroxasulfone at 5000 ppm (383 mg/kg/day) but not at lower doses. These lesions were also seen in female rats administered doses of 1000 ppm ( 54 mg/kg/day) and above after ch ronic feeding for two years, but not after one year of feeding. However, spec ific acute and subchronic neurotoxicity tests in
rats did not reveal neurotoxic effects following an acute oral dose of 2000 mg/kg or subchronic dietary administration of 2500 pp m (equivalent to 161/200 mg/kg/day (M/F), the HDT. In the rat DNT studies, pyroxasulfone did not produce beha vioral or maternal to xicity at the highest dose tested of 900 mg/kg/day. Ho wever, it was associated with offspring toxicity at 300 mg/kg/day resulting in decreased brain weight in male and female offspring accompanied with a
decrease in the thickness of the hippocampus, corpus callosum and cerebellum in PND 21 female offspring with a NOAEL of 100 mg/kg/day. Although the offspring are more sensitive, the doseresponse is well characterized for neur otoxicity and a NOAEL is identified.

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

Evidence of increased susceptibil ity of fetuses and offspring was seen in a DNT study in rats and a developmental study in rabbits following in utero or post natal exposure to pyroxasulfone. However, no susceptibility was seen in the rat de velopmental or reproducti on studies. In rabbits, developmental toxicity was only seen at the limit dose of 1000 mg/kg/day as reduced fetal weight and increased fetal resorptions with a NOAEL of 500 mg/kg/day for these effects, compared to no maternal toxicity at these doses. In a DNT study in rats, offspring toxicity was seen at 300 mg/kg/day compared to no maternal toxicity at 9 00 mg/kg/day. This increasedPage 16 of 80 susceptibility is occurring at high doses. NOAE Ls and LOAELs have been identified for all effects of concern and thus, a clear dose res ponse has been well defined. Therefore, residual uncertainties or concerns for pr e- and/or post-natal toxicity are minimal, and HED recommends the FQPA safety factor be reduced to 1x.

4.4.4 Residual Uncertainty in the Exposure Database

Since the dietary and non-dietary exposure es timates were based on several conservative assumptions, HED does not believe that the expo sure estimates are underestimated. The acute and chronic dietary assessments conducted with DEEM-FCID were screening level analyses.
The assessments utilized toleran ce values, default processing fact ors, and 100% crop treated was assumed for all commodities. The DEEM analysis also assumed that all drinking water will contain pyroxasulfone at the highest EDWC levels modele d by EFED for ground water or
surface water. For these reasons it can be conc luded that the DEEM-FCID analysis does not underestimate risk from acute or chronic expos ure to pyroxasulfone. Similarly, HED does not believe that the non-dietary occupational exposur es are underestimated b ecause they are also based on conservative assumptions, including ma ximum application rates, labeled PPE, and standard values for unit exposures , acreage treated/amount handled.

4.4.5 FQPA Factor for Infants and Children

HED recommends the FQPA SF be reduced to 1x based on the following:

- The toxicological data base is complete for FQPA assessment.

- There are no residual uncertain ties or concerns for pre- and/or post-natal toxicity. - Although an apparent increas ed quantitative su sceptibility was observed in fetuses and offspring based on toxicity at high dos es of administration, a clear dose-response
has been well defined with NOAELs and LOAE LS for all effects of concern and all points of departure (PODs) are protectiv e of the fetal/offspring effects.

4.5 Toxicity Endpoint and Point of Departure

4.5.1 Dose-Response Assessment

Toxicity endpoints and PODs for dietary (food and water) and occupati onal exposure scenarios are summarized below. A detailed description of the studies used as a basis for the selected endpoints are presented in Appendi x A. PODs for incidental or al, dermal and inhalation for nonoccupational (i.e., residential) exposure are not required to assess risk for the proposed uses because there are no pyroxasulfone products re gistered for homeowner use and there are no products registered for applica tion to residential areas.

An acute POD of 100 mg/kg/day (NOAEL) was se lected from a developmental neurotoxicityPage 17 of 80 study in rats based on decreased brain weight in both sexes and decr eased thickness of the hippocampus, corpus callosum and cerebellum in PND 21 female offspring occurring at 300 mg/kg/day. An uncertainty factor (UF) of 100x (10x to account for interspecies extrapolation and 10x for intraspecies variation) was applied to the NOAEL to obtain an acute reference dose (aRfD) of 1.0 mg/kg/day. Sin ce the FQPA factor ha s been reduced to 1X , the acute population adjusted dose (aPAD) is equivalent to the aRfD. The HED DNT committee considers morphometric changes in the hippocampus, corp us callosum and cerebellum during the first 21 days to result from a single acute exposure. This endpoint is considered to be protective of all populations including women of child-b earing age and nursing infants.

A chronic POD of 2.0 mg/kg/day ( NOAEL) was selected from the one year dog study based on impaired hind limb function, ataxia, hind limb twitching and tremors; clinical pathology: increased creatine kinase, aspart ate aminotransferase; axonal/myelin degeneration of the sciatic nerve and spinal cord sections at 10 mg/kg/day. An UF 100x (10x to account for interspecies extrapolation and 10x for intraspecies variation) was applied to the dose to obtain a chronic reference dose (cRfD) of 0.02 mg/kg/day. Since the FQPA factor has been reduced to 1X, the chronic population adjusted dose (cPAD) is equivalent to the cRfD. The dog was the most sensitive species for the neurotox ic effects of pyroxasulfone. Also the dose ranges used in the dog testing were the narrowest allowing for more accurate determination of a NOAEL. The 2 mg/kg/day NOAEL from this dog st udy is also protective from othe r adverse effects seen at higher doses in rats and mice such as cardiac a nd skeletal myopathy, live r, kidney and urinary bladder toxicity.

A POD of 100 mg/kg/da y (NOAEL) for short- and intermediate-term dermal exposure was selected from the 28-day dermal toxicity in th e rat based on cardiac myofiber degeneration with inflammation at 1000 mg/kg/day. The target margin of exposure (MOE) for dermal toxicity is 100. The 28-day dermal toxicity study is route sp ecific and is considered appropriate for this exposure scenario. This endpoint is considered protective of developmental effects seen in the DNT study at 300 mg/kg/day and in the rabbit de velopmental study at 1000 mg/kg/day. While
the effects aren’t seen until the limit dose of 1000 mg/kg/day, there is a 10-fold difference
between the dermal NOAEL and LOAEL. Th erefore, the true NOAEL is probably higher (between 100 and 1000 mg/kg/day). This POD is al so protective of the neurotoxic effects seen in the dog following chronic oral exposure to 10 mg/kg/day. Dermal absorption is estimated to be 7.5%, so the relative dermally absorbed dose for the dermal NOAEL of 100 mg/kg/day is approximately 7.5 mg/kg/day which is less than the oral LOAEL of 10 mg/kg/day identified in the dog study. In addition, rats in general are a more sensitive species for the dermal exposure route because of higher skin permeability.

A POD of 2.0 mg/kg/ day for short- and intermediate-term i nhalation exposure was selected from the oral 90 day dog study (MRIDs 47701696 -97) based on a LOAEL of 10 mg/kg/day for axonal/myelin degeneration in the sciatic nerve, minimal to mild subacute inflammation of skeletal muscle in males and females and impaired limb function, body and limb hypotonia, increase in serum creatine kinase activity. The ta rget MOE for inhalation toxicity is 100. This endpoint is more protective than the findings in the 28 day rat inhalation study (MRID 47701699) where no effects were seen at the high est dose tested for an aerosol dust of 200Page 18 of 80 mg/m 3 (equivalent to 52.2 mg/kg/da y) (NOAEL). Since mixer/loader exposures would be to both dust and aerosols (with aerosol exposures typically being more sensitive than dust exposures), HED believes the inha lation study was not protective of the neurotoxicity findings in the dog study.

4.5.2 Recommendations for Combining Exposure Routes

Under FQPA, when there are potenti al residential exposures to a pesticide, HED must aggregate exposures from three major sources: oral, derm al and inhalation exposures. These combined residential exposures must then be aggregated with exposure from food and water to determine aggregate risk. Since there are no residential incidental oral exposur es to pyroxasulfone, aggregate exposure from food and non-food oral exposures is not required. Although the dermal
and inhalation scenarios are based on muscle effect s, the cardiac muscle is histologically very different from skeletal muscle pa rticularly as it relates to its electrophysiology. Therefore, shortterm dermal and inhalation exposures to workers should be not aggregated with dietary (food and water) exposures because endpoints are not based on common toxicity effects.

4.5.3 Classification of Carcinogenic Potential

In accordance with EPA’s Final Guidelines for Carcinogen Risk Assessment (March 2005) , the CARC classified Pyroxasulfone as “Not Likely to be Carcinogenic to Humans” at doses that do not cause crystals with subsequent calculi forma tion resulting in cellular damage of the urinary tract (HED TXR 0055883). This cl assification was based on urinary bladder tumors seen in male rats. Crystal formation in the absence of calculi is not associat ed with hyperplasia or urinary bladder tumors; therefore, the formation of urinary bladder calculi is the prerequisite for subsequent hyperplasia and neoplasia. The require ment for calculi formation also supports a high-dose threshold phenomenon for the development of urinary bladder tumors, i.e., tumors do not develop at doses too low to produce calculi. There is no concer n for mutagenicity. As noted previously, the CARC concluded that the kidney adenomas in male mice were not treatmentrelated. The Agency has determined that the qua ntification of risk usi ng a non-linear approach (i.e., RfD) will adequately account for all chroni c toxicity, including carcinogenicity, that could result from exposure to pyroxasulfone. There is a clear thre shold of 1000 ppm (42.55 mg/kg/day) for tumorigenesis. A point of de parture (POD) of 50 ppm (2.0 mg/kg/day) is not expected to result in urinary bladder calculi fo rmation which is a prerequisite for subsequent hyperplasia and neoplasia.

4.5.4 Summary of Points of Departure Used in Risk Assessment

Toxicological doses/endpoints sele cted for the pyroxasulfone ri sk assessment are provided in
Tables 4 and 5.Page 19 of 80 Table 4. Toxicological Doses and Endpoints for Pyroxasulfone for Use in Dietary Human Health Risk Assessments

Exposure/
Scenario Point of Departure Uncertainty/ FQPA Safety
Factors RfD & PAD Study and Toxicological Effects

Acute Dietary (General Population, NOAEL= 100 mg/kg UF A = 10 x
UF H =10 x FQPA SF=1x Acute RfD = 1.0 mg/kg

aPAD = 1.0 mg/kg Developmental neurotoxicity study (DNT) in rats, MRID 47701724. The LOAEL of 300 mg/kg/day is based on decreased brain weight in both sexes, reduced thickness of the hippocampus, corpus callosum and cerebellum in PND 21 female offspring. Chronic Dietary (All Populations) NOAEL= 2 mg/kg/day UF A = 10 x
UF H =10 x FQPA SF=1x Chronic RfD
= 0.02 mg/kg/day

cPAD = 0.02 mg/kg/day One- year chronic dog study, MRID 47701707 The LOAEL of 10 mg/kg/day is based on impaired hind limb function, ataxia, hind limb twitching and tremors; clinical pathology: increased creatine kinase, aspartate aminotransferase; axonal/myelin degeneration of the sciatic nerve and spinal cord sections. Cancer (all routes) “Not Likely to be Carcinogenic to Humans” at doses that do not cause crystals with subsequent calculi formation resulting in cellular damage of the urinary tract. Risk is quantified using a non-linear (i.e., RfD) approach. Point of Departure (POD) = A data point or an estimated point that is derived from observed dos e-response data and used to mark the beginning of extrapolation to determine risk associat ed with lower environmentally relevant human exposures. NOAEL
= no observed adverse effect level. L OAEL = lowest observed adverse effect le vel. UF = uncertainty factor. UF A = extrapolation from animal to human (interspecies). UF H = potential variation in sensitivity among members of the human population (intraspecies). FQPA SF = FQPA Safety Factor. PAD = population adjusted dose (a = acute, c = chronic). RfD = reference dose. MOE = margin of exposure. LO C = level of concern. N/A = not applicable.

Table 5

. Toxicological Doses and Endpoints for Pyroxasulfone for Use in Occupational Human Health Risk
Assessments

Exposure/
Scenario Point of Departure Uncertainty/ FQPA Safety
Factors Level of
Concern Study and Toxicological Effects

Incidental Oral Not required. Incidental exposure not anticipated. There are no registered residential uses Dermal Short- (1- 30 days) &
Intermediate-Term (1-6 months) NOAEL = 100 mg/kg/day UF A = 10 x
UF H =10 x Occupational
MOE = 100 28-day dermal toxicity study in rats MRID 47701698 The LOAEL of 1000 mg/kg/da y was based on minimal to mild cardiac myofiber degeneration with in
inflammation in males and females

Inhalation Short- (1-30 days) &
Intermediate-Term (1-30 days) NOAEL = 2 mg/kg/day
100% absorption UF A = 10 x
UF H =10 x Occupational
MOE = 100 90-day toxicity study in dogs MRIDs 47701696 -97 LOAEL of 10 mg/kg/day: axonal/myelin degeneration in the sciatic nerve. Minimal to mild
subacute inflammation of skeletal muscle in males and females. Impaired limb function, body and limb hypotonia, increase in serum creatine kinase activity. Cancer (all routes) “Not Likely to be Carcinogenic to Humans” at doses that do not cause crystals with subsequent calculi formation resulting in cellular damage of the urinary tract. Risk is quantified using a non-linear (i.e., RfD) approach. Point of Departure (POD) = A data point or an estimated point that is derived from observed dos e-response data and used to mark the beginning of extrapolation to determine risk associat ed with lower environmentally relevant human exposures. NOAEL
= no observed adverse effect level. L OAEL = lowest observed adverse effect le vel. UF = uncertainty factor. UF A = extrapolation from animal to human (interspecies). UF H = potential variation in sensitivity among members of the human population (intraspecies). FQPA SF = FQPA Safety Factor. PAD = population adjusted dose (a = acute, c = chronic). RfD = reference dose. MOE = margin of exposure. LO C = level of concern. N/A = not applicable.Page 20 of 80 5.0 DIETARY AND DRINKING WATER EXPO SURE AND RISK ASSESSMENT

5.1 Metabolite/Degradate Residue Profile

5.1.1 Summary of Plant and Livestock Metabolism Studies

The metabolism studies submitted for pyroxasulfone are adequate to satisfy data requirements on the nature of the residue in plants (soybean) . The nature of the residu e in plants is adequately
understood based on the submitted studies. The metabolic pathway of pyroxasulfone involves cleavage of the methyl sulfone bridge of the parent compound, oxi dation and/or demethylation of the pyrazole cleavage product to yield meta bolites M-1 and M-25, and reaction of the isoxazoline cleavage product with cysteine a nd/or glutathione; fu rther demethylation, deamination, and/or conjugation of these metabol ites occurs, yield additional metabolites including M3 (chemical structures are provided in Appendix B).

The submitted livestock metabolism studies with goa ts and hens are adequate to satisfy data requirements for the nature of the residue in livesto ck. The nature of the residue in livestock is adequately understood based on submitted metabolis m studies. Metabolism of pyroxasulfone in livestock occurs primarily via cleavage between the rings, to form metabolites M-1, M-8, M-10, and/or M-15, followed by oxidati on, to yield metabolite M-3, a nd/or demethylation, to yield metabolites M-12 and M-9. The parent co mpound may also undergo demethylation, hydroxylation followed by oxidation, or oxi dation followed by ring cleavage.

5.1.2 Comparison of Metabolic Pathways

The proposed metabolic pathway for pyroxasulfone in plants is provided in Appendix B. The major pyroxasulfone metabolites in plants are M-1, M-3, M-25, M-28. All of the plant metabolites exhibit low acute toxicity (Toxicity Category IV). The M-1 and M-3 metabolites, which are also found in rat, were not toxic in rats afte r 14 days of oral dosing at 1000 mg/kg/day. Structure-activity relationship (SAR) analysis (D EREK) did not reveal any alerts for any of the major metabolites. All of the te sted metabolites were negative in the Ames test. M1, M25, and M-28 are potentially less toxic than the parent as they are lower molecular weight acids (i.e., more easily excreted). However, there are no da ta to demonstrate that they do not retain the toxic properties of the parent compound. Based on the limited testing data available, pyroxasulfone metabolites appear to be equal or less toxic than the parent material.

The metabolic pathway of pyroxasulfone in plan ts mainly involves cl eavage of the methyl sulfone bridge of the parent compound followe d by oxidation and/or demethylation of the pyrazole cleavage product to yield metabolites M-1 and M-25, and reac tion of the isoxazoline cleavage product with cysteine a nd/or glutathione; further deme thylation, deamination, and/or conjugation of these metabolites occurs, yi eld additional metabolites including M-3.Page 21 of 80 The metabolism of pyroxasulfone in rats appears to proceed by two main routes. One route, similar to the metabolic pathway seen in plan ts, involves cleavage of the sulfonyl group and subsequent sequential oxidations of this group on the pyrazole moiety or glutathione conjugation and subsequent hydrolysis of the isoxazoline moiety. The second rout e involves sequential oxidations of the parent compound.

5.1.3 Environmental Fate and Transport

Pyroxasulfone is mobile (average K
oc = 94.75) and persistent (t 1/2 = 142 to 533 days) in terrestrial and aquatic environments There are two ma jor degradation products (>10% of applied radioactivity) and 6 minor degrad ation products (<10 % of applie d radioactivity) detected in laboratory metabolism studies. Additionally, there is a high percentage of applied radioactivity (8-25% of applied radioactivity) identified as “bound” soil/sediment re sidues. Because the extraction of pyroxasulfone residues was conducte d using only acetonitrile/ water without the use of sequentially harsher soil extractants, there is un certainty regarding the identity and availability
of the “bound” soil residues.

The Environmental Fate and Effects Division ( EFED) conducted a drinking water assessment for the proposed uses of pyroxasulfone on corn, soyb eans, wheat, fallow land, bare ground, and noncropped areas. EFED provided modeling-based drinking wa ter exposure estimates using the Tier II PRZM GW drinking water assessment from su rface water sources for the proposed uses of pyroxasulfone. Residues of concern for drinking water for risk assessment purposes were parent, M1, and M3. Estimated drinking water residue s for peak concentration of 0.210 mg/L (ppm)
were used in the acute assessment. Es timated 30 yr annual average drinking water concentrations of 0.174 mg/L (ppm) were used the chronic assessment.

5.1.4 Residues of Concern Summary and Rationale

Previously, in order to harmonize with Canada an d Australia, HED has c oncluded the residues of concern for Corn, field, grain a nd Corn, sweet, kernel plus cob with husks removed consist of pyroxasulfone and its metabolite M-3. Residue s of concern for drinking water for risk assessment purposes were parent, M-1, and M-3. The modeling was conducted for pyroxasulfone and total pyroxasulfone residues (parent+M-1+M-3, and total pyroxasulfone residues (parent+M-1+M-3+M-5+M-6+M-8+ M-9+M-10+M-11+unidentified bound soil residues).

Another acidic metabolite, M3, was proposed as a residue of concer n by the petitioner. Although not a major component of the total radioactive residue (TRR), ROCKS recommends
that M3 be included in the residu es of concern since it is analy zed concurrently with the parent compound in the proposed analytical enforcement method.Page 22 of 80 Table 6. Compounds to be Included in the Risk Assessment and Tolerance Expression.

Matrix Residues included in Risk Assessment Residues included in Tolerance Expression for Compliance Monitoring

Plants Cereal Grains Pyroxasulfone, M1, M3, M25 Pyroxasulfone, M1, M3, M25***

Soybeans Pyroxasulfone, M1, M3, M25, M28** Pyroxasulfone, M1, M3, M25, M28*
Drinking Water Pyroxasulfone, M1, M3 Not Applicable
Pyroxasulfone = 3-[[[5-(difluoromethoxy)-1-methyl-3-(trifluoromethyl)-1 H -pyrazol-4-yl]methyl]sulfonyl]-4,5- dihydro-5,5-dimethylisoxazole. M1 = [5-(difluoromethoxy)-l-methyl-3-(trifluoromethyl)-l H -pyrazol-4-yl)]methanesulfonic acid. M3 = 5-(difluoromethoxy)-l-methyl-3-(trifluoromethyl)-l H -pyrazole-4-carboxylic acid. M25 = [5-(difluoromethoxy)-3-(trifluoromethyl)-1 H -pyrazol-4-yl)]methanesulfonic acid. M28 = 3-[1-carboxy-2-(5,5-dimethyl-4,5-dihydroisoxazol-3-ylthio)ethylamino]-3-oxopropanoic acid. Metabolite C = 3-[1-carboxy-2-(5,5-dimethyl-4,5-dihydr oisoxazol-3-ylthio)ethenylamino]-3-oxopropanoic acid. * reanalysis of the seed samples showed residues of M2 8 and therefore M28 needs to be included in both the tolerance expression and risk assessment as a residue of concern in soybean seed. M-1 is included for soybean forage and hay. ** residues of concern for soybean seed is pyroxasu lfone and metabolite M-3, M-25, & M-28 for both risk
assessment and toleran ce enforcement purposes. ***residues of concern for corn, field, grain and corn, sweet, kernel plus cob with husks removed is pyroxasulfone and metabolite M-3.

5.2 Food Residue Profile

5.2.1 Residues in Crops

K-I Chemical submitted new field trial data from soybean field trials conducted in the U.S. The purpose of this study was to obtain Raw Agricu ltural Commodity (RAC) residue data for pyroxasulfone (KIH-485) and its metabolites M-1, M-3, M-25, and M-28 (in seed only) on soybeans following a single post-emergence appl ication of pyroxasulfone 85 WG herbicide at the third trifoliate leaf stage (V3 growth stage) with and without adjuvant and fertilizer to determine if adding fertilizer a nd/or adjuvant to the spray solu tion affected the residues in the harvested crop. The results from these field trials along with the previously submitted field trial
data are discussed below and summarized in Table 7.

For all studies, HED converted residues of metabo lites to parent equivalents for calculation of total residues; molecular weight conversi on factors of 1.262 for M-1, 1.504 for M-3, 1.321 for M-25, and 1.286 for M-28 were used. The full LOQ value was used for residues <LOQ for calculating total residues.Page 23 of 80 Table 7. Summary of Residue Data from Crop Field Trials with Pyroxasulfone.
Crop matrix Target Applic.

Rate (lb ai/A) [g ai/ha] PHI (days) Total Residues of Pyroxasulfone and Metabolites M-1, M-3, M25, and M-28 (ppm) 1

n Min. Max. HAFT 2 Median Mean Std.
Dev. 3

SOYBEAN (proposed use = 0.267 lb ai/A total application rate, 15-day PHI for seed, 7-day PGI for forage, hay, or straw)
Soybean forage 0.112-0.186 [125-209] 5 15-44 44 <0.027 0.781 0.724 0.115 0.181 0.192
Soybean hay 21-63 44 <0.030 1.901 1.85 0.138 0.249 0.378
Soybean seed 88-137 44 <0.045 <0.065 <0.063 0.045 0.048 0.006

1 For the purposes of calculating total residues, HAFT, median, mean, and standard deviation, the LOQ was used for residues reported as <LOQ. Metabo lite residues were converted to parent equivalents. M-28 was only included in soybean seed calculations.

2 HAFT = Highest average field trial result.

3 NA = Not applicable.

4 Applications were made at early po stemergence of the crop (~V4 growth st age). Actual application rates ranged 97.6-102.9% of the target rate.

5 Applications were made at early post emergence of the crop (third trifoliate leaf stage). Actual application rates ranged 96.4-103.9% of the target rate.

6 Actual application rates were within 1.6% of target rate.

5.3 Water Residue Profile

5.3.1 Estimated Drinking Water Concentrations

Pyroxasulfone is mobile (average K
oc = 94.75) and persistent (t 1/2 = 142 to 533 days) in terrestrial and aquatic environments There are two ma jor degradation products (>10% of applied radioactivity) and 6 minor degrad ation products (<10 % of applie d radioactivity) detected in laboratory metabolism studies. Additionally, there is a high percentage of applied radioactivity (8-25% of applied radioactivity) identified as “bound” soil/sediment re sidues. Because the extraction of pyroxasulfone residues was conducte d using only acetonitrile/ water without the use of sequentially harsher soil extractants, there is un certainty regarding the identity and availability
of the “bound” soil residues.

The Environmental Fate and Effects Division ( EFED) conducted a drinking water assessment for the proposed uses of pyroxasulfone on corn, soyb eans, wheat, fallow land, bare ground, and noncropped areas. EFED provided modeling-based drinking wa ter exposure estimates using the Tier II PRZM GW drinking water assessment from su rface water sources for the proposed uses of pyroxasulfone. Residues of concern for drinking water for risk assessment purposes were parent, M1, and M3. Estimated drinking water residue s for peak concentration of 0.210 mg/L (ppm)
were used in the acute assessment. Estimated average drinking water concentrations of 0.174 mg/L (ppm) were used the chronic assessment.Page 24 of 80 5.4 Dietary and Drinking Water Exposure and Risk

HED conducted screening-level acu te and chronic dietary and dr inking water exposure and risk assessments using the Dietary Exposure Evalua tion Model with the Food Commodity Intake Database (DEEM-FCIDTM). Dietary risk assessment incorporat es both exposure and toxicity of a given pesticide. For acute and chronic diet ary assessments, the risk is expressed as a percentage of a maximum acceptable dose (i.e., the dose which HED has concluded will result in
no unreasonable adverse health eff ects). This dose is referred to as the populati on adjusted dose (PAD). The PAD is equivalent to the refere nce dose (RfD) divided by the additional FQPA Safety Factor, if applied. For acute and non-can cer chronic exposures, HED is concerned when estimated dietary risk ex ceeds 100% of the PAD.

Proposed use on soybean was assessed for di etary and drinking water exposure. DEEM ® residue inputs for soybean commodities used maximum resi dues from specific use patterns for the acute and chronic dietary exposure analyses. Pe rcent crop treated was set at 100 %.

5.4.1 Acute Dietary and Drinking Water Analysis

A new dietary assessment was conducted for the propos ed use. The proposed use results dietary risk estimates below HED’s level of concern; see Table 8. The highest is All Infants <1 year resulting in
3.6% of the aPAD.

Table 8. Results of Acute Dietary Exposure Analysis for Food and Drinking Water

Population Subgroup aPAD (mg/kg/day) 95th Percentile Exposure (mg/kg/day) % aPAD General U.S. Population

1.0 0.011572 1.2 All Infants (< 1 year old) 0.036099 3.6

Children 1-2 years old 0.017929 1.8 Children 3-5 years old 0.014714 1.5 Children 6-12 years old 0.011088 1.1 Youth 13-19 years old 0.009640 1.0 Adults 20-49 years old 0.011374 1.1 Adults 50-99 years old 0.010165 1.0 Females 13-49 years old 0.011527 1.2

5.4.2 Chronic Dietary and Drinking Water Analysis

A new dietary assessment was conducted for the pr oposed use of pyroxasulfone and results in dietary
risk estimates below HED’s leve l of concern; see Table 9.Page 25 of 80 Table 9. Results of Chronic Dietary Expo sure Analysis for Food and Drinking Water

Population Subgroup cPAD (mg/kg/day) Exposure (mg/kg/day) % cPAD General U.S. Population

0.02 0.003728 19 All Infants (< 1 year old) 0.009569 48 Children 1-2 years old 0.005463 27 Children 3-5 years old 0.004527 23 Children 6-12 years old 0.003333 17 Youth 13-19 years old 0.002747 14 Adults 20-49 years old 0.003706 19 Adults 50+ years old 0.003644 18 Females 13-49 years old 0.003690 18

6.0 RESIDENTIAL EXPOSURE AND RISK ASSESSMENT

A residential assessment is not required because there are no pyroxasulfon e products registered
for homeowner use and there are no products registered for applic ation to residential areas.

6.1 Residential Bystander Post-application Inhalation Exposure

Based on the Agency's current practices, a quant itative residential bystander post-application inhalation exposure assessment wa s not performed for [chemical] at this time. However, volatilization of pesticid es may be a potential source of pos t-application inhalation exposure to individuals nearby to pesticide applications. The Agency s ought expert advice and input on issues related to volatilization of pesticides from its Federal Ins ecticide, Fungicide, and
Rodenticide Act Scientific Advi sory 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 approp riate, develop policies and procedures, to identify the need for and, subsequently, the way to incorporate 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 fo r a quantitative postapplic ation inhalation exposure assessment for pyroxasulfone.

6.2 Spray Drift

Spray drift is always a potential source of expos ure to residents nearby to spraying operations. This is particularly the case with aerial appl ication, but, to a lesser ex tent, could also be a potential source of exposure from the ground ap plication method employed for [chemical]. The Agency has been working with the Spray Drift Task Force, EPA Regional Offices and State Lead Agencies for pesticide regulation and ot her parties to develop the best spray drift management practices (see the Agency’s Spra y Drift website for more information at http://www.epa.gov/opp00001/factsheets/ spraydrift.htm). On a chemical by chemical basis, the Agency is now requiring interim mitigation measures for aerial applications that must be placed on product labels/labeling. The Agency has comp leted its evaluation of the new database submitted by the Spray Drift Task Force, a membership of U.S. pesticide registrants, and isPage 26 of 80 developing a policy on how to appr opriately apply the data and th e AgDRIFT computer model to its risk assessments for pesticides applied by ai r, 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 drif t with specific products with significant risks
associated with drift.

7.0 AGGREGATE EXPOSURE AND RISK ASSESSMENT

In accordance with the FQPA, when there are potential residentia l exposures to a pesticide, aggregate risk assessment must consider exposur es from three major routes: oral, dermal, and
inhalation. There are three s ources for these types of expos ures: food, drinking water, and residential uses. In an aggregate assessment, e xposures from relevant s ources are added together and compared to quantitative estimates of hazard (e.g., a NOAEL or PAD), or the risks themselves can be aggregated. When aggrega ting exposures and risks from various sources,
HED considers both the route and duration of e xposure. The proposed/registered pyroxasulfone uses are not expected to result in residential exposure . Therefore, the acute and chronic exposure estimates provided in the Dietary Exposure Section represent aggregate exposure.

7.1 Acute & Chronic Aggregate Risk

Acute and chronic aggregate exposures include food plus drinking water exposures. As demonstrated under Section 5.4, acute and chro nic aggregate risks are not of concern.

7.2 Short- and Intermediate-Term Aggregate Risk

7.3 Intermediate-Term Aggregate Risk

8.0 CUMULATIVE RISK

Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to establish, modify, or revoke a tolerance, the Agency consider “a vailable information” concerning the cumulative effects of a particular pesticid e's residues and “other substances that have a common mechanism of toxicity.”

EPA does not have, at this time, available data to determine whether pyroxasulfone has a common mechanism of toxicity w ith other substances. Unlike other pesticides for which EPA has followed a cumulative risk approach base d on a common mechanism of toxicity, EPA has
not made a common mechanism of to xicity finding as to pyroxasul fone and any other substances and, pyroxasulfone does not appear to produce a toxic metabolite produced by other substances
which have tolerances in the U. S. For th e purposes of this tole rance reassessment action, therefore, EPA has not assumed that pyroxasulf one has a common mechanis m of toxicity with other substances. For information regarding EPA’ s efforts to determine which chemicals have aPage 27 of 80 common mechanism of toxicity and to evaluate th e cumulative effects of such chemicals, see the policy statements released by EPA’s OPP con cerning common mechanism determinations and procedures for cumulating effects from subs tances found to have a common mechanism on
EPA’s website at http://www.epa.gov/fedrgstr /EPA_PEST/2002/January/Day_16/

.

9.0 OCCUPATIONAL EXPOSURE /RISK CHARACTERIZATION

9.1 Exposure Scenarios

Occupational handler and post-ap plication exposure scenarios we re assessed for the proposed use on soybeans. Based on the product labels a nd information provided by the registrant, shortand intermediate-term exposure is assessed fo r occupational handlers and post-application activities. Dermal and inhalation exposures to workers are not aggreg ated for pyroxasulfone because the toxicity endpoints for these exposur e routes are not based on common toxicological effects. Labels for both pyroxasulfone formulati ons require that applicators and other handlers wear long-sleeved shirt and long pants, chemi cal resistant gloves and shoes and socks.

9.2 Handler Exposure

The term “handler” applies to individuals who mix, load, and apply the pesticide product. There is a potential for exposure to pyroxasu lfone during mixing, load ing, and application activities through the dermal and inhalation routes . Pyroxasulfone is a pplied in the spring at preplant and preemergence growth stages and in the fall to minimum or no tillage fields to be planted the following spring. Pyroxasulfone produc ts are applied using aer ial, right of way and or handgun sprayers, as well as groundboom equipment. Th e following handler exposure
scenarios evaluated for this assessment are based on information provided in the proposed labels.

9.2.1 Handler Exposure Scenarios

The term “handler” applies to individuals who mix, load, and apply the pesticide product. There is a potential for exposure to pyroxasu lfone during mixing, load ing, and application activities through the dermal and inhalation routes . Pyroxasulfone is a pplied in the spring at preplant and preemergence growth stages and in the fall to minimum or no tillage fields to be planted the following spring. Pyroxasulfone produc ts are applied using aer ial, right of way and or handgun sprayers, as well as groundboom equipment. Th e following handler exposure
scenarios evaluated for this assessment are based on information provided in the proposed labels.

9.2.1.1 Mixing and Loading Scenarios

• Mixing and Loading a water-di spersible granule formulation for groundboom application. • Mixing and Loading a water-dispersi ble granule formulation for aerial application.Page 28 of 80 9.2.1.2 Application Scenarios

• Applying a water-dispersible granule formulation with groundboom equipment. • Applying a water-dispersibl e granule formulation with aerial equipment.

• Flagging for aerial application scenarios.

9.2.2 Handler Exposure Data

No chemical-specific handler exposure data were submitted in support of this registration. It is HED’s policy to use data from the USEPA OPP Occupational Pesticide Handler Unit Exposure Surrogate Reference Table (5/4/11). http://www.epa.gov/pesticides /science/handler-exposuredata.html ).

9.2.3 Handler Exposure Assumptions

• Average body weight of an adult handler is 80 kg. • Exposure duration short-term (1- 30 da ys), intermediate-term (1-6 months) • Maximum label application rates:

- 0.21 lb ai/A for soybean
• Area treated:

- 1200 acres per day for aerial application

- 200 acres per day for groundboom application.

- 350 acres for flagging during aeri al application scenarios.

- 9.2.4 Handler Exposure and Risk Estimates

A target LOC or MOE of 100 is considered ad equate for dermal and inhalation exposure. All worker exposures are assessed as short-term based on label prescribed uses and expected exposure durations. Exposure and risk estimates indicate that for most exposure scenarios noncancer risks are not of concern for occupational handler act ivities for the proposed new use (MOEs > 100). However, risks of concern exis t (MOE<100) for “Inhalation” exposure during mixing and loading activities for aerial applica tions A summary of occupational handler exposure and risk calculati ons, assumptions, and results is provided in Table 10.Page 29 of 80 Table 10. Short and Intermediate-Term Occupation al Handler Exposure and Risk for Pyroxasulfone

Exposure
Scenario 1 Crop or Target App
Rate (lb ai/A) 2 Acres
Treated Daily 3 Unit Exposure 3 Dose (mg/kg/Day) 4,5 Risk Estimation

Dermal (mg/lb ai) Inhalation (μg/lb ai) Dermal Inhal MOEs 6,7

Dermal Inhal

BL Resp.
PF5
Mixer/Loader ( water-dispersible granule)

Groundboom

Baseline

Soybean 0.21 200 0.227 8.96 0.119 0.0047 840 430

Groundboom
Gloves 0.052 8.96 0.0271 0.0047 3,700 430

Aerial
Baseline 1200 0.227 8.96 0.715 0.0283 140 71 350

Aerial
Gloves 0.052 8.96 0.163 0.0283 610 71 350

Applicator ( water-dispersible granule)

Groundboom

Baseline Soybean 0. 21 200 0.0786 0.34 0.0413 0.0002 2,400 110,000

Groundboom
Gloves 0.0161 0.34 0.00845 0.0002 120,000 110,000

Aerial Protected by Groundboom Application

Flagging

Aerial Soybean 0.21 350 0.011 0.35 0.0101 0.0003 9,900 6,200

1. Baseline and PPE unit exposure values ar e reported USEPA OPP Occupa tional Pesticide Handler Un it Exposure Surrogate
Reference Table

2 Application rates based on proposed label rate.

3. Amount treated based on information provided ExpoSAC Policy 9

4. Dermal dose (mg/kg/event) = [unit dermal exposure (mg/lb ai) * application rate (lb ai/A) * Amount treated / body weight (80 kg)].

5 Inhalation dose (mg/kg/event) = [unit exposure (kg/lb ai) * (1mg/1000 kg) conversion * appl. rate (lb ai/lbs seed) * Amount treated / body weight (80 kg)].

6. Dermal MOE = short-term endpoint for dermal (NOAEL 100 mg/kg/day)/Dermal Dose

7. Inhalation MOE = short-term endpoint for de rmal (NOAEL 2 mg/kg/da y)/Inhalation Dose

Note; Risks of concern exist for the “mixing and loading” of a water dispersible granular for aerial applications (MOE = 71) with label specific PPE (long- sleeved shirt and long pants, chemical resistant gloves and shoes and socks). Ho wever, with a PF5 respir ator, risks are not of concern (MOE = 350).Page 30 of 80 9.3 Post Application Exposure

HED uses the term “post-application” to desc ribe those individuals who can be exposed to pesticides after entering areas previously treated with pesticides and performing certain tasks or activities (also often referred to as reentry exposure). Post-applic ation exposures are expected to occur primarily via the dermal route. Based on current Agency's practices, a quantitative postapplication inhalation exposure assessment was no t performed. However, the Agency is in the process of evaluating available data and informa tion on post-application i nhalation exposure. If new policies or procedures are put into place based on that evaluation, the Agency may revisit the need for a quantitative occupational post- application inhalation e xposure assessment for pyroxasulfone.

Minimal exposure during harvesting or other late season activities is expected for pre-emergent applications of pyroxasulfone for the following re asons. Preemergence herbicides are applied to the surface soil directly or are incorporated into the soil we ll before the crops mature. Pyroxasulfone is proposed for use on the high ac reage field crop soybean. This crop is mechanically planted early in the season which minimizes the potential for dermal contact. Based on these considerations, HED does not re quire a post-application occupational exposure assessment for pre-emergent herbicides used on crops that are mechanically planted (HED Exposure Science Advisory Council Policy No. 008). However, there is potential exposure to persons entering treated sites after applicati on of pyroxasulfone as an early post-emergence herbicide to soybean (application up to third trifo liate leaf stage). Cons equently, post-application exposure from early post-emergence uses is assumed to occur during minimum foliage development for soybean (ExpoSAC Policy No. 003.1).

9.3.1 Post Application Exposure Scenarios

The following post-application exposure s cenarios were assessed for proposed postemergence uses of pyroxasulfone.

• Hand weeding soybean at low crop he ight and minimum foliage development

Note: Based on label specific directions, the application method and timing for soybeans is preplant and pre-emergence only. Therefor e the TC of 70 for “hand weeding” was appropriate because the crop height is lo w with minimal foliage. However, since pyroxasulfone is used to contro l a broad spectrum of broadleaf weeds and grasses, therfore scouting will be the activity associated with this TC.

In accordance with the Worker Protection Standard (WPS), a 12-hr restricted entry interval (REI) is required fo r chemicals classified under T oxicity Category III/IV.Page 31 of 80 9.3.2 Post Application Exposure Assumptions

No chemical-specific handler exposure data were submitted in support of this registration. Therefore, agricultural transfer coefficients used for this assessment are taken from HED’s Science Advisory Council for Exposure Po licy on Agricultural Transfer Coefficients (SOP # 003.1, 8/7/00).

• Average body weight of an adult handler is 80 kg. • Exposure duration is short-term (1- 30 days) • Maximum label application rates:

- 0.21 lb ai/A for soybean
• Transfer Coefficients:

- 70cm 2 /hour for scouting immature soybean crop
• Initial fraction of ai re tained on foliage is 25%

• Exposure is assumed to occur on the day of application (day 0)

9.3.3 Post-Application Exposure and Risk Estimates

A target LOC or MOE of 100 is considered adeq uate for dermal exposure. Exposure and risk estimates indicate post-applicati on risks are not of concern (MOE s > 100) as shown on Table 11.

Table 11. Estimated Pyroxasulfone Exposure & MOEs for Occupational Post-Application Exposure

Exposure Scenario App Rate (lb ai/A) 1 DFR (μg/cm 2 ) 2 TC (cm 2 /hr) 3 Exposure Duration (hrs/day) Dermal dose (mg/kg/day) 4 MOE 5

Soybean - Scouting immature plants 0.21 0.589 70 8 0.00412 24,000

1 Application rates are based on maximu m values based on proposed label.

2 DFR (mg/cm 2 ) = Dislodgeable Foliar Residues corresponding to day 0. A pplication Rate (lb ai/A) x CF (4.54E+5 mg/lb) x CF (2.47E-8 A/ cm 2 ) x 25% (initial fraction of ai retained on foliage)

3 TC cm 2 /hr = Transfer coefficients and associat ed activities (ExpoSAC Policy Memo #003.1)

4 Dermal Dose (mg/kg/day) = DFR (mg/cm 2 ) x TC (cm 2 /hr) x 8 (hrs/day) / Body weight (80 kg).

5 Dermal MOE = short-term endpoint for dermal (NOAEL 100 mg/kg/day)/Dermal Dose

9.3.4 Restricted Entry Interval

Pyroxasulfone has been classified in Toxicity Category III for acute dermal and Category IV for eye irritation and primary skin irritation. Per the Worker Protection Standard (WPS), a 12-hr restricted entry interval (REI) is required for chemicals classi fied under Toxicity Category III/IV.Page 32 of 80 9.3.5 Inhalation Post-application Risk

Based on the Agency's current practices, a quant itative occupational pos tapplication inhalation exposure assessment was not performed for pyroxasu lfone at this time. However, there aremultiple potential sources of post-application inhalation exposure to individuals performing postapplication activities in previously treated fiel ds. These potential sour ces include volatilization of pesticides and resuspension of dusts and/or pa rticulates that contain pesticides. The Agency sought expert advice and input on is sues related to volatilization of pesticides from its Federal Insecticide, Fungicide, and Rode nticide 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/SA P/meetings/2009/120109meeting.html ). The Agency is in the process of evaluating the SAP report as well as available post-applicat ion inhalation exposure data generated by the Agricultural Reentry Task Force and may, as appropriate, develop policies and procedures, to identify the need for and, s ubsequently, the way to incorporate occupational post-application inhalation exposur e into the Agency's risk asse ssments. If new policies or procedures are put into place, the Agency may revisit the need for a quantitative occupational post-application inhalation expos ure assessment for pyroxasulfone.

10. REFERENCES

Pyroxasulfone. Application for Section 3 Registration for Use on Soybean – T. Morton, D408764, 1/31/13

Pyroxasulfone. Acute and Chronic Aggregate Dietary Exposure and Risk Assessments for the Section 3 Registration Request for Use of Pyroxasulfone on Soybean. – T. Morton, D406657, 1/30/13

Pyroxasulfone, Herbicide End-Use Product: V-10233 Herbic ide, Pyroxasulfone 85 WG Herbicide, KIH-485 W85, and V10233 Herbicide Water Dispersible Granule (Commercial).Refined Ground Water Drinking Water Modeling of Pyroxasulfone Proposed Use on Corn, Soybeans, Wheat, Fallow Land, Bare Ground, and Non-Cropped Areas – J. Hetrick, D390221, 6/1/2011

Pyroxasulfone: Occupational and Residential Exposure Assessment for the Use of Pyroxasulfone on Soybean – J. S. Miller, D408787, 1/31/13Page 33 of 80 APPENDICES

A TOXICOLOGY DATA SUMMARY

A.1 Guideline Data Requirements The requirements (40 CFR 158.340) for food us e for pyroxasulfone are in Table 1.

Guideline
No. Study Type Technical MRID No. Required Submitted

870.3100

870.3150

870.3200
870.3250
870.3465 Subchronic (Oral) To xicity - Rodent ............................

Subchronic (Oral) Toxi city - Non-Rodent....................

21/28-Day Dermal Toxicity ......................................... 90-Day Dermal Toxicity 90-Day Inhalati on Toxicity .......................................... Y

Y

N
N
N Y

Y

Y
N
Y 47701692
47701693
47701694
47701695
47701696
47701697
47701698

--- 47701699

870.3700a

870.3700b

870.3800 Prenatal Developmental Toxicity - Rodent ..................

Prenatal Developmental To xicity - Non-Rodent ..........

Reproduction and Fe rtility E ffects ............................... Y

Y

Y Y

Y

Y 47701701
47701702
47701703
47701704
47746840
47701705
47701706

870.4100a
870.4100b
870.4200a
870.4200b
870.4300 Chronic (Oral) Toxi city - R odent ................................. Chronic (Oral) Toxicity - Non-Rodent (Dog) .............. Carcinogenicity - Rat Carcinogenicity - Mouse
Combined Chronic Toxic ity /Carcinogenicity ............. Y
N
N
Y
Y Y
Y
N
Y
Y 47701708
47701707

--- 47701710
47701709

870.6100a
870.6100b
870.6200a
870.6200b
870.6300

870.7800 Neurotoxicity - Acute Delayed Neurotox.- Hen ........... Neurotoxicity - Su bchronic - Hen ............................... Neurotoxicity Screening Battery - Acute - Rat ............ Neurotoxicity Screening Battery -Subchronic - Rat ..... Developmental Neurotoxicity..................

Immunotoxicity............................. ...................... N
N
Y
Y
N

Y N
N
Y
Y
Y

Y ---

--- 47701721
47701722
47701723
47701724
47701731
47701732Page 34 of 80 A.2 TOXICITY PROFILES

A.2.1 Acute Toxicity

Table A.2.1.1 Acute Toxicity Profile – Pyroxasulfone
Guideline
No. Study Type MRID(s) Results Toxicity Category

870.1100 Acute oral – rat (females) 47701677 LD 50 = >2000 mg/kg III
870.1200 Acute dermal – rat 47701684 LD 50 >2000 mg/kg
M & F III

870.1300 Acute inhalation – rat 47701685 LC 50 >6.56 mg/L M & F IV 870.2400 Acute eye irritation – rabbit 47701686 Minimally irritating IV 870.2500 Acute dermal irritation – rabbit 47701687 Non-irritating IV 870.2600 Skin sensitization – local lymph
node - mice 47701688 No sensitization -

TableA.2.1.2 Acute Toxicity Prof ile – Pyroxasulfone Metabolites
Guideline
No. Study Type MRID(s) Results Toxicity Category

870.1100 Acute oral – rat (females) M-1 47701678 LD 50 = >2000 mg/kg III 870.1100 Acute oral – rat (females) M-3 47701679 LD 50 = >2000 mg/kg III 870.1100 Acute oral – rat (females) M-25 47701680 LD 50 = >2000 mg/kg III 870.1100 Acute oral – rat (females) M-28 47701755 LD 50 = >2000 mg/kg III 870.1100 Acute oral – rat (females) I-3 47701681 LD 50 = >2000 mg/kg III 870.1100 Acute oral – rat (females) I-4 47701682 LD 50 = >2000 mg/kg III 870.1100 Acute oral – rat (females) I-5 47701683 LD 50 = >2000 mg/kg IIIPage 35 of 80 A.2.2 Subchronic, Chronic, Other Toxicity

Table A.2.2 Subchronic, Chronic and Oth er Toxicity Profile of Pyroxasulfone

Guideline No. Study Type MRID No. (year)/
Classification /Doses Results

870.3050 28-Day Dietary toxicity – Han Wistar

Rat

5 rat/sex/dose 47701689 (2003), Covance Labs 0, 500, 5000, 20,000 ppm

M: 64.3, 649.6, 2677.5 mg/kg/day

F: 62.2, 673.9, 2584.7 mg/kg/day

Acceptable/guideline NOAEL = 500 ppm (64.3/62.2 mg/kg/day in males/females)
LOAEL = 5000 ppm (650/674 mg/kg/day in males/females) based on hepatocyte and myocardial vacuolation; myocardial degeneration.

Non-Guideline Special Study 28-Day Dietary toxicity – Rat
Myocardial toxicity and enzyme markers

5 rat/sex/dose 47889320 (2004)

Covance Labs

0, 100, 1000, 5000, 20000

ppm M: 7.3, 75.4, 367.2, and 1364.0 mg/kg/day

F: 8.3, 84.4, 388.0, and 1676.1 mg/kg/day

Acceptable/Non-Guideline Aspartate aminotransferase was increased by 138% in the 20,000 ppm females on Day 22. On Day 29, increases in aspartate aminotransferase we re noted in the 5000 (incr 150%) and 20,000 (incr 93%) ppm females. On Day 29, increases in lactate dehydrogenase were observed in the 5000 ppm group (incr 119-167%) and 20,000 ppm group (incr 57-87%). Decreases (p ≤ 0.05) in alanine aminotransferase and creatinine phosphokinase were observed, but were not considered toxicologically
relevant. hepatic lesions were noted at 1000 ppm and above and 20,000 ppm females myocardial degeneration/necrosis in the 1000 ppm and above males and females, vacuolation in 5000 and 20,000 ppm males and fibrosis in 20,000 ppm males and
females. CPK enzymes were not predictive markers of myocardial toxicity.

870.3100 90-Day oral toxicity -
Han Wistar Rat

10 rats/sex/dose,

5 rats/sex/dose for

recovery 47701692(2005); Covance
Labs 0, 100, 500, 2500, 5000 ppm; M: 0, 8.9, 43.9, 221.4, 451.2 mg/kg/day; F: 0, 10.2, 48.9, 255.8, 514.2 mg/kg/day Acceptable/guideline NOAEL 500 ppm (44/49 mg/kg/day in M/F) LOAEL = 2500 ppm (221/256 mg/kg/day in M/F) based on either altered organ weights, histopathological indicators or clinical pathological indicat ors in liver, skeletal and cardiac muscle and the pancreas (liver centrilobular hepatocyte hypertrophy and eosinophilia in males, a dose related increased incidence of myocardial degeneration or
necrosis in the heart of males and skeletal muscle myopathy in females). Full or partial recovery was evident in all the above effects apart from kidney weights
in females.Page 36 of 80 Table A.2.2 Subchronic, Chronic and Oth er Toxicity Profile of Pyroxasulfone

Guideline No. Study Type MRID No. (year)/
Classification /Doses Results

870.3100 90-Day oral toxicity - Crl:CD®(SD)IGS BR

rats

10 rats/sex/dose 47701693 (2005) Haskell
Labs

0, 25, 250, 2500 ppm

M: 0, 1.7, 16.4, 171.2 mg/kg/day

F: 0, 2.20, 20.6, 205.4 mg/kg/day

Acceptable/guideline NOAEL = 250 ppm (16/21 mg/kg/day in males/females)
LOAEL = 2500 PPM (171/205 mg/kg/day in males and females, based on increased liver weight, centrilobular hepatocellular hypertrpophy, focal/multifocal myofiber degeneration and inflammation, urinary bladder diffuse mucosal hyperplasia.

870.3100 90-Day oral toxicity
B6C3F1 mouse

12 mice/sex/dose 47701694 (2005); Covance
Labs 0, 500, 1000, 5000, 10000/7500 ppm; M: 0, 103, 206, 1424, 2494 mg/kg/day; F: 0, 96, 202, 1228, 1941mg/kg/day Acceptable/guideline NOAEL = 1000 ppm (equivalent to 206/202 mg/kg/day, M/F) LOAEL = 5000 ppm (1424/1228 mg/kg/day in males/females) based decreased body weight, changes in clinical chemistry parameters, changes in organ weights (including increased kidney and liver weights), and microscopic effects in the kidney (cortical tubular nephropathy) and heart (myocardial degeneration/fibrosis). Target organs at 5000 ppm and above are heart, liver, kidney.

870.3100 90-Day oral toxicity
B6C3F1 mouse

10 mice/sex/dose 47701695 (2005); Haskell
Labs

0, 25, 250, 2500 ppm

M: 4.0, 39.8, 394 mkd

F: 5.4, 51.2, 531.3 mkd

Acceptable/guideline NOAEL = 250/2500 ppm in females /males (51.2/394 mg/kg/day in females /males). LOAEL = 2500 ppm in females (equivalent to 531 mg/kg/day) based on an increased incidence of chronic progressive nephropathy (minimal to mild), multifocal tubules lined by basophilic epithelium indicative of tubular regeneration in females. LOAEL in males is >2500 ppm (394 mg/kg/day).

870.3150 90- day oral toxicity – Dog
4 dogs/sex/dose 47701696 (2004); Biosafety Research Center, Japan
0, 0.2, 2.0, 10.0 mg/kg/day in gelatin caps

Acceptable/guideline NOAEL = 2 mg/kg/day LOAEL =10 mg/kg/day. Slight muscle fiber degeneration of the diaphragm and hyperplasia of satellite cells; nerve fiber degeneration, in addition to the similar adverse effects occurring at 15 mg/kg/day dose in a subsequent 90-day dog study (MRID 47701697).

870.3150 90- day oral toxicity – Dog
4 dogs/sex/dose 47701697 (2006); Haskell
0, 15 mg/kg/day in gelatin

caps Acceptable/guideline LOAEL = 15 mg/kg/day: Decreased BW gain ( ↓ 40%) in males; Minimal to mild axonal/myelin degeneration in the
sciatic nerve of all males and 3⁄4 females. Minimal to mild
subacute inflammation of skeletal muscle in males and females. Impaired limb function, body and limb hypotonia, increase in seru m creatine kinase activity.

870.4100 1-yr oral toxicity – Dog
4/dog/sex/dose 47701707 (2008) MPI Research (Mattawan Michigan)
0, 0.2, 2.0, 10.0 mg/kg/day in gelatin caps

Acceptable/guideline NOAEL = 2 mg/kg/day LOAEL = 10 mg/kg/day based on impaired hind limb function, ataxia, hind limb twitching and tremors; clinical pathology: increased creatine kinase, aspartate aminotransferase; axonal/myelin degeneration of the sciatic nerve and spinal cord sectionsPage 37 of 80 Table A.2.2 Subchronic, Chronic and Oth er Toxicity Profile of Pyroxasulfone

Guideline No. Study Type MRID No. (year)/
Classification /Doses Results

870.3465 4- week inhalation –

Rat

10/sex/dose 47701699 (2008) – Dupont
0, 10 40, 200 mg/m 3 /day aerosol dust/nose only

Acceptable/guideline NOAEL = 200 mg/ m 3 /day based on lack of adverse toxic
effects at this HDT.

870.3200 28-Day dermal – Rat
10/sex/dose 47701698 (2005)
Dupont 0, 20, 100, 1000 mg/kg/day Acceptable/guideline NOAEL = 100 mg/kg/day LOAEL = 1000 mg/kg/day based on minimal to mild cardiac myofiber degeneration with inflammation in
males and females

870.3700a Prenatal developmental in (rat) Crj:CD(SD)IGS[SPF] Range finding

7/dose 47701701 (2004) Biosafety Res. Ctr. Japan 0, 125, 250, 1000 mg/kg GD
5-19 Acceptable/non-guideline NOAEL for maternal and developmental toxicity = 1000 mg/kg/day (HDT) unossified hyoids at 1000 mg/kg/day and this finding was not considered to be an unequivocally treatment-related
effect

870.3700a Prenatal developmental in Crl:CD(SD) (rat)

22 rats/dose 47701702 (2007) Haskell 0, 100, 50, 1000 mg/kg/day
on GD 6-20 Acceptable/guideline NOAEL for maternal and developmental toxicity = 1000 mg/kg/day (HDT)

870.3700b Prenatal developmental in (rabbit) ) Range finding

7/dose 47701703 (2004) Covance 0, 250, 500, 1000 mg/kg/day
on GD 7-28 Acceptable/non-guideline NOAEL for maternal and developmental toxicity = 1000 mg/kg/day (HDT)

870.3700b Prenatal developmental in (rabbit) 22/dose 47701704 (2007) Haskell 0, 250, 500, 1000 mg/kg/day
on GD 7-28 Acceptable/guideline Maternal toxicity NOAEL = 1000 mg/kg/day (HDT) Developmental toxicity NOAEL = 500 mg/kg/day LOAEL = 1000 mg/kg/day reduced fetal weight and increased fetal resorptionsPage 38 of 80 Table A.2.2 Subchronic, Chronic and Oth er Toxicity Profile of Pyroxasulfone

Guideline No. Study Type MRID No. (year)/
Classification /Doses Results

870.3800 Reproduction and fertility effects (rat) Crl:CD®(SD)IGS BR One-Gen Study

12/sex/dose Range-finding 47701705 (2006) Dupont Haskell
0, 25, 250, 5000 ppm

P M premating 1.69,16, 339 mg/kg/day
P F premating 1.98, 19.6, 383 mg/kg/day
P F gestation 1.78, 18, 332 mg/kg/day
P F lactation 3.82, 37.9, 728 mg/kg/day
F1 M postweanling 2.73, 27.9, 564 mg/kg/day
F1 F postweanling 2.99, 30.4, 643 mg/kg/day Acceptable/nonguideline Parental toxicity NOAEL = 250 ppm (16/20 mg/kg/day M/F) LOAEL = 5000 ppm, (339/383 mg/kg/day) reduced body weight and gain and food consumption, cardiac myofiber and sciatic nerve lesions, micr oscopic effects in the heart, liver, muscle, and sciatic nerve of males and females Reproductive toxicity NOAEL = 5000 ppm (339 mg/kg/day) (HDT) Offspring toxicity NOAEL = 250 ppm (16/20 mg/kg/day) LOAEL = 5000 ppm (339 mg/kg/day) reduced pup weight during lactation (27%-30% lower than control mean) and delayed sexual maturation in both sexes.

870.3800 Reproduction and fertility effects (rat) Crl:CD®(SD)IGS BR 2-gen study

30/sex/dose 47701706 (2008) Dupont Haskell
0, 5, 100, 2000 ppm

Mean substance intake 0.4/0.4, 7.2/8.4, 144/165 mg/kg/day for
Males/Females Acceptable/guideline Parental toxicity NOAEL = 100 ppm (7.2/8.4mg/kg/day) LOAEL = 2000 ppm (144/165mg/kg/day) reduced body weight, body weight gain and food consumption and increased kidney weight, cardiomyopathy and urinary bladder mucosal hyperplasia with inflammation. Reproductive toxicity NOAEL = 2000 ppm (135 mg/kg/day) (HDT) Offspring toxicity NOAEL = 100 ppm (11 mg/kg/day) LOAEL = 2000 ppm (144/165 mg/kg/day) reduced pup weights and body weight gains during lactationPage 39 of 80 Table A.2.2 Subchronic, Chronic and Oth er Toxicity Profile of Pyroxasulfone

Guideline No. Study Type MRID No. (year)/
Classification /Doses Results

870.4200b Carcinogenicity (mouse) Crl:CD1(ICR) mice

50+10/sex/dose 47701710 (2009) Dupont Haskell
0, 5, 150, 2000 ppm
After 14 months 2000 ppm reduced to 1000 ppm for M and 500 ppm for F.

M: 0, 0.63/0.61, 18.33/18.56, 255.13/131.34 mg/kg/day

F: 0, 0.88/0.71, 26.33/22.44, 371.64/76.49 mg/kg/day

Overall mean

0, 0.62/0.84, 18.4/25.5, 228/306, M/F

Acceptable/guideline NOAEL = 150 ppm (18.4/25.5mg/kg/day) (M/F) LOAEL = 228/306 mg/kg/day M/F, based on degeneration of sciatic and trigeminal nerve axons and their associated myelin sheaths and an increase in the incidence of chronic progressive nephropathy (females). Renal tubular adenomas at this dose in 3 of 50 main study males, in the presence of a 17% reduction of body weight gain compared to control males. The test substance was considered initially oncogenic
in male mice. Revaluat ion of the renal tubular adenomas of the male mice was conducted by an independent kidney expert pathologist (MRID 48354403) and reviewed by a Pathology Working Group (MRID 48354405). The renal tubular adenomas observed in male mice were considered by
the PWG to be incidental and unrelated to the administration of pyroxas ulfone. A dose-related increase in retrograde nephro pathy in males was also
identified.

870.4100 One year chronic (Rat)

20 rats/sex/dose 47701708 (2008) Dupont Haskell
0, 5, 50, 1000, 2000 ppm
M: 0, 0.22, 2.22, 46.2, 91.91 mg/kg/day
F: 0, 0.3, 3.12, 60.80, 121.33 mg/kg/day Acceptable/guideline NOAEL = 50 ppm mg/kg/day. LOAEL = 1000 ppm or 46.2/60.8 mg/kg/day (M/F) based on reduced body weight parameters in males ( ↓ 5-10%) and microscopic findings in the liver (centrilobular hepatocellular hypertrophy (m ales)), heart (increased cardiomyopathy (females)) and urinary bladder (mucosal hyperplasia (males and females)). At 2000 ppm, these effects were accompanied by increased clinical observations of red discharge from the penis and associated red-stained cage boards as well as increased liver and kidney weights.

870.4200a Carcinogenicity (rat)

70/sex/dose 47701709 (2009) Dupont Haskell 0, 5, 50, 1000, 2000 ppm
M: 0, 0.21, 2.05, 42.55, 84.55 mg/kg/day F: 0, 0.28, 2.69, 54.28, 106.74 mg/kg/day Acceptable/guideline NOAEL = 50 ppm or 2.05/2.69 (M/F) mg/kg/day LOAEL = 1000 ppm or 42.55/54.28 (M/F) mg/kg/day based on reduced body weight parameters (males and females), reduced food consumption (females), and increased red-stained urine on cageboards (males) at 2000 ppm and reduced body weight and food consumption parameters (females) and increased red-stained urine on cageboards (males) at 1000 ppm. Increased cardiomyopathy (males and females); urinary bladder mucosal hyperplasia, inflammation, and transitional cell papillomas (males); and sciatic nerve degeneration (females) at 1000 ppm and above.
Based on a low incidence in urinary bladder transitional cell papillomas in the 1000 ppm and 2000 ppm males , the test substance was considered oncogenic in male rats at exposures of 1000 ppm and above.Page 40 of 80 Table A.2.2 Subchronic, Chronic and Oth er Toxicity Profile of Pyroxasulfone

Guideline No. Study Type MRID No. (year)/
Classification /Doses Results

870.5100 Bacterial reverse
mutation test 47701712 (2003), Covance TA98, TA1535 and TA1537- S typhimurium & WR2 uvrA
of

E coli Acceptable/guideline Test article has no reverse mutagenic potential under these experimental conditions either in the presence or absence
of metabolic activities.
Precipitation of the test article was observed on all E coli test plates treated at 1 581 μg/plate, and on all S typhimurium test plates treated at 632.4 μg/plate and
above.

870.5380 In vivo studies in germ cells -
Mammalian spermatogonial
chromosomal
aberration test NA Because all other genotoxicity studies are negative, no studies for this data point were performed with regulatory agency concurrence per 28 February 2009 OECD meeting.

870.5550 In vivo genotoxicity testing – Unscheduled DNA synthesis NA Because all other genotoxicity studies are negative, no studies for this data point were performed with regulatory agency concurrence per 28 February 2009 OECD meeting.

870.5300 In vitro mammalian cell gene mutation

test: Mouse Lymphoma L5178Y
Cells 47889311 (2009) Huntingdon Acceptable/guideline Pyroxasulfone TGAI was tested up to the limit of solubility and up to cytotoxic effects and was not mutagenic in the presence or ab sence of S9-activation.

870.5375 In vitro mammalian
cell chromosome aberration test, CHO
cells 47701719 (2003) , Covance Acceptable/guideline Test article did not induce chromosome aberrations in cultured (CHO) cells when tested up to its limit of solubility in both the absence and presence of metabolic activation (S-9).

870.5395 Mammalian erythrocyte
micronucleus test 47701720 (2007), Dupont 500, 1000, 2000 mg/kg orally to male and female
mice Acceptable/guideline Under the conditions of this study, KIH-485TGAI did not induce biologically relevant increases in micronucleated polychromatic erythrocytes in animal bone marrow. The test substance was concluded to be negative in this in vivo study.

870.6200a Acute neurotoxicity screening battery
Crl:CD(SD) rat

10/sex/dose 47701721 (2007), Dupont 0, 20, 200, 2000 mg/kg Acceptable/guideline NOAEL = 2000 mg/kg/day in males and females (HDT).

870.6200b Subchronic neurotoxicity screening battery
Crl:CD(SD) rat

12/sex/dose 47701722 (2007), Dupont 0, 25, 250, 2500 ppm M: 0, 1.56, 15.85, and 161.48 mg/kg/day F: 0, 1.92, 19.60, and 199.59 mg/kg/day Acceptable/guideline Systemic NOAEL = 2500 ppm or 161/200 mg/kg/day (M/F), the HDT Neurotoxicity NOAEL = 2500 ppm or 161/200 mg/kg/day (M/F), the HDTPage 41 of 80 Table A.2.2 Subchronic, Chronic and Oth er Toxicity Profile of Pyroxasulfone

Guideline No. Study Type MRID No. (year)/
Classification /Doses Results

870.6300 DNT Study
Crl:CD(SD) rat
Preliminary Range Finding

8 mated females/dose 47701723 (2007), Huntingdon 0, 100, 300, 600, 1000 mg/kg/day GD6-LD7; Offspring Day 7-20 of age Acceptable/nonguideline There was no effect of mate rnal treatment, or direct treatment of the offspring with KIH-485, on the clinical condition, survival, or macropathology findings of the dams or the offspring.
Treatment of adult females at 1000 mg/kg/day was associated with up to 16 % lower mean numbers of implantations and litter sizes at birth and on Day 4. The use of a dose above 1000 mg/kg/day may be associated with a more marked reduction in the implantation rate and litter size sufficient to preclude a meaningful evaluation of developmental neurotoxicity in the offspring. Treatment at 600 mg/kg/day was associated with up to 8% lower mean numbers of implantations and litter size at birth.

870.6300 DNT Study
Crl:CD(SD) rat

24 mated
females/dose 47701724 (2008), Huntingdon 0, 100, 300, 900 mg/kg/day GD6-LD7; Offspring Day 7- 20 of age (EPA reviewed protocol) Acceptable/guideline Maternal NOAEL = 900 mg/kg bw/day (HDT), the
LOAEL was >900 mg/kg/day.

Functional development NOAEL = 900 mg/kg/day.
Offspring NOAEL = 100 mg/kg/day based on decrease in brain weight in male and female offspring accompanied with a decrease in the thickness of the hippocampus, corpus callosum and cerebellum in PND 21 female offspring at 300 mg/kg/day

870.7800 Immunotoxicity Feeding Study in
mice

10/mice/sex/dose 47701732 (2008) Dupont 0, 40, 400, 4000 ppm for 28 days M: 0, 6, 61, 633 mg/kg/d F: 0, 8, 77, 791 mg/kg/d Acceptable/guideline Systemic toxicity
NOAEL = 61/77 (M/F) mg/kg/day
LOAEL = 633/791 (M/F) mg/kg/day based on reduction in body weight (-5 to -9%) and body weight gain (-32%)
Immunotoxicity:
NOAEL = 633/791 (M/F) mg/kg/day (HDT) based on
absence of immunotoxic effects.

870.7800 Immunotoxicity Feeding Study in rats

10/rats/sex/dose 47701731 (2006) Dupont 0, 25, 250, or 7500 ppm for 28 days (0/0, 2/2, 18/19, and 529/570 mg/kg/day in males/females). Acceptable/guideline Systemic toxicity
NOAEL = 250 ppm (18/19 mg/kg/day M/F)
LOAEL = 7500 ppm (529/570 (M/F) mg/kg/day based on reduction in body weight gain (-30% to -56%) and food consumption (-19% to – 52%)
Immunotoxicity:
NOAEL = 529/570 (M/F) mg/kg/day (HDT) based on
absence of immunotoxic effects.

870.7600 Dermal penetration NA Not required for US or Australia and waived for Canada per 28 February 2009 OECD meeting.Page 42 of 80 Table A.2.2 Subchronic, Chronic and Oth er Toxicity Profile of Pyroxasulfone

Guideline No. Study Type MRID No. (year)/
Classification /Doses Results

870.7485 Metabolism and pharmacokinetics
(beagle dog) 47701726 (2005),
Covance One female dog: single oraldose of 10 mg of pyrazole14 C /kg bw, urine and feces
were collected over 120 h. Sequential blood samples were and the dog was killed at 120 h after dosing. The
level of radioactive residues was determined in the blood, plasma, and selected tissues (heart, kidney and liver) at necropsy. The excreta were analyzed to determine the number and nature of any metabolites present using
HPLC and HPLC/MS. Acceptable/nonguideline Overall recovery of radioactivity was 98%. Oral absorption of radioactivity was at least 51%. The majority of the radioactivity (76.9%) was eliminated in the first 24 h. The radioactivity was equally excreted in urine and
feces. C

max was of 3.954 and 2.116 μg equivalents/g at 8 h in blood and plasma respectively and were still quantifiable at 120 h. The terminal half-life was of 89.58 and 40.80 h in blood and plasma respectively. The levels of radioactivity in the tissues were highest in the liver and blood (0.334 and 0.327 μg equivalents/g, respectively). The kidney, plasma and heart contained the next highest concentrations. The feces contained only unchanged KIH-485. Urine contained no parent compound. HPLC chromatography showed that there were several metabolites, some of which were changed following treatment with ß-D-glucuronidase. Metabolites M-3, M-7 and M-8 were identified in the dog, though M-3 was only a minor metabolite. Structures were proposed for three
other metabolites.

870.7485 Metabolism and pharmacokinetics
(Mouse) 47701730 (2007) Huntingdon single oral dose of 10 mg pyrazole- 14 C pyroxasulfone/kg bw, Autoradiography: 4 ♀
Excretion/balance:3 ♀
Tissue distribution: 12 ♀ Acceptable/nonguideline KIH-485 was rapidly absorbed, metabolized and
eliminated. ~ 78% excreted in the urine after 120 hours (about 73% was excreted within the first 24 hours). The remainder in the feces. Only about 0.2% of the dose was
retained in the tissues. Fifteen metabolites were resolved by the chromatographic and three products resulting from oxidative phase I processes (KIH-485-MI, KIH-485-M3 and KIH-485-13) were identified by co-chromatography. One minor metabolite was identified as a glucuronide or sulfate conjugate. KI H-485 itself was not present in urine. Concentrations of total radioactivity in the liver and heart were highest at the earliest sacrifice time of 2 hours: 3.186μg KIH-485 equivalents/g in the liver and 0.603 μg KIH485 equivalents/g in the heart. After 48 hours these levels had declined to 0.430 and 0.031 μg KIH485 equivalents/g, respectively.
Whole-body autoradiography showed. At 2 hours after dosing, radioactivity had been absorbed from the GI tract and was distributed into the vast majority of tissues. High
concentrations in the liver a nd gallbladder were consistent with an appreciable first-pass elimination of 14 C-KIH-485 and/or its metabolites by the liver, followed by biliary excretion. Low radioactivity concentrations were
associated with the bone surfa ces, brain, spinal cord and the lens of the eye. By 24 hours after dosing, radioactivity concentrations in the majority of tissues (including the central nervous system) were close to, or indistinguishable from, background levels.Page 43 of 80 Table A.2.2 Subchronic, Chronic and Oth er Toxicity Profile of Pyroxasulfone

Guideline No. Study Type MRID No. (year)/
Classification /Doses Results

870.7485 Metabolism and pharmacokinetics
(rat) 4/sex/group 47701728 (2008)
Covance Single oral dose 10, 700 mg/kg of either the pyrazole- 14 C or the
isoxzzoline- 14 C radiolabel Acceptable/guideline 91-104% recovery. Absorption at the low dose based on bile and urine >76% AD. Cmax 2 hrs at low dose, 11 hrs at the high dose. T 1/2 longer for the IX label. Saturation of absorption with increasing dose; a 70-fold dose level increase produced a 10 -fold increase in C

max and a 20 to
50-fold increase in AUC (0-t) with [Py- 14 C]-KIH-485, and a
20-fold increase in C

max and a 10-fold increase in AUC (0-t) with [Ix- 14 C]-KIH-485. Excretion rapid and complete
within 48-72 hrs. Low dose an imals’ excretion mostly in urine. High dose animals mostly in feces. Residual in
carcass <7% in low dose and <0.5% in high dose. No significant sex differences in elimination or metabolism. No significant 14 CO2 elimination. Liver and kidney highest residues. Extensive metabolism ~30 chromatographic regions in urine, 19 in feces, 31 in bile. Major metabolite of [Py- 14 C] urine was M-13 (up to 26% of the low dose level and up to 4% at the high dose level) followed by M-7 and M-3. Cleavage between the pyrazole and isoxazoline ring structure. Major metabolites of [Ix- 14 C]-compound in the urine were M-13 (20% of the dose), M-16 (19% of the dose) and M-26 (6.4% of the dose). The major fecal radioactivity was unabsorbed KIH-485 (10% of the dose at the low dose level and up to 78% at the high dose level). The next most abundant metabolite was M-13 (up to 3% of the dose).
In bile of the [Py- 14 C] rats a sulphate conjugate of hydroxy KIH-485 and M-13 glucuronide accounted for up to 3.1% of the dose each plus free M-13 metabolite (~ 1.6% of the AD).

[Ix- 14 C]-KIH-485 bile, M-26 (10% of the AD) tentatively identified and was produced by the cleavage of the pyrazole ring and subsequent loss of the fluorine moieties. A sulphate conjugate of hydroxy KIH-485 (7%) and the other M-13 glucuronide (3%). A glutamyl cysteine conjugate of the isoxazoline ring (4%) was tentatively
identified.Page 44 of 80 Table A.2.2 Subchronic, Chronic and Oth er Toxicity Profile of Pyroxasulfone

Guideline No. Study Type MRID No. (year)/
Classification /Doses Results

870.7485 Metabolism and pharmacokinetics (rat) 47701729 (2008)
Covance Steady State: 10mg/kg/day to 4 females of the pyrazole- 14 C for 14 days
Pharmacokinetics: 10 mg/kg/day to 4 females for 7days followed by 10 mg/kg of either radiolabel
Excretion Balance: 10 mg/kg/day to 4 females for 7days followed by 10 mg/kg of either radiolabel
Tissue Distribution: 10 mg/kg/day to 10 females for 7days followed by 10 mg/kg of either radiolabel Acceptable/guideline Steady state reached after 7 doses. Recoveries ranged from between 92.4 to 99.6% of administered radioactivity. Absorption, based on urinary excretion, was >69% of the
dose. Absorbed radioactiv ity was rapidly excreted, primarily in the urine. KIH-485 or its radiolabeled
metabolites were associated with the cellular fraction of blood. For the Py- 14 C, Cmax 1 hr. There were no apparent differences in the t

max in plasma and blood but
the t 1⁄2 was ca 7-fold greater in blood; the t 1⁄2 was 16.8 hours in plasma and 112 hours in blood. For the Ix- 14 C,
Cmax ca 1.5 hours in plasma and at ca 7.5 hours in blood.
The t

max in blood was approximately double the levels in plasma and the t 1⁄2 was ca 5-fold greater in blood; the t 1⁄2 was 60.9 hours in plasma and 302 hours in blood.
Absorbed radioactivity was rapidly distributed throughout tissues and rapidly cleared without accumulation of radioactivity in the tissues. KIH-485 underwent extensive metabolism with the major metabolite being M-13. Excretion was rapid with 85-95% of radioactivity being
excreted within 24 hours. Th ere was minimal difference in
the routes and rates of excretion between the two
radiolabeled forms. Eliminat ion half life in plasma was extended following [Ix- 14 C]-KIH-485 administration.

Compared with single dose animals, there was saturation
or induction of metabolism which resulted in an altered metabolic profile, particularly for the [Py- 14 C] label, and this may also have resulted in small shift (6%) from urinary to fecal excretion in repeat dose animals. Otherwise, repeat dosing did not discernibly affect the overall pharmacokinetics of KIH-485. There was no
discernible difference in the tissue distribution between single dose and repeat dosing.

------------- Metabolism and pharmacokinetics Transfer to milk (rat) 47701727 (2006)
Covance single oral dose of 250 mg [ 14 C]-KIH-485/kg to 18 female lactating rats
Milk collected from 3 rats each interval at 0.5, 2, 8, 24, 48, 96 hours, then sacrificed
for blood collection acceptable/non-guideline The concentrations of radioactivity in the milk and plasma reached maximal concentrations of 23.48 and 47.89 μg equivalents/g respectively at 2 h post-dose before decreasing to 0.667 and 0.184 μg equivalents/g respectively at 96 h post-dose.

The elimination half-lives of the test substance from the milk and plasma were similar at 21.4 h and 22.5 h, respectively. The milk: plasma ratio of levels of radioactivity increased throughout the study, from 0.189 at 30 min after dosing, reaching near parity at 24 h, to 3.625 at 96 h after dosing.Page 45 of 80 Table A.2.2 Subchronic, Chronic and Oth er Toxicity Profile of Pyroxasulfone

Guideline No. Study Type MRID No. (year)/
Classification /Doses Results

870.7485 Metabolism and pharmacokinetics Transfer to offspring (rat) 4770125(2006) Huntingdon
3 female rats dose at 10 mg/kg/day of pyrazole-5-

14 C]-KIH-485 from GD 6 until Day 9 after parturition.
Ten mated undosed rats provided naïve pups. Three naïve pups at each interval of 3, 5, 7, 9, 10, 11, 13 days post parturition replaced the pups of the dosed mothers
and allowed to suckle for 1 hour, then sacrificed and stomach contents analyzed. acceptable/non-guideline During the dosing period at Days 3 - 9 after parturition, concentrations in maternal plasma (1.31 – 1.76 μg equivalents/g ) were ~ double those in pup stomach contents (0.672 – 1.013 μg equivalents/g).
After cessation of dosing (1 - 4 days after last dose) concentrations dropped rapidly for both matrices (13 days after parturition, pup stomach contents: <0.028 μg equivalents/g; maternal plasma: <0.060 μg equivalents/g).
Changes in concentrations of radioactivity in both
matrices mimicked each other. The ratio of the mean concentrations of pup stomach contents to maternal plasma remained relatively constant with an overall mean value, both during and after dosing, of 0.49.

Toxicity of Metabolites

870.3050 14-Day oral gavage toxicity – Han Wistar

Rat

3 females/dose 47701690 (2005) Safepharm Labs 0, 1000 mg/kg/day of
KIH-485 M-1 acceptable/non-guideline NOAEL = 1000 mg/kg/day no observed adverse effects.

870.3050 14-Day oral gavage toxicity – Han Wistar

Rat

3 females/dose 47701691 (2008) Safepharm Labs 0, 100, 1000 mg/kg/day of
KIH-485 M-3 acceptable/non-guideline NOAEL = 1000 mg/kg/day no observed adverse effects.

870.5100 Bacterial reverse
mutation test 47701713 (2005), Safepharm Labs TA98, TA1535, TA100
and TA1537- S typhimurium & WP2
uvrA of E coli 50-5000 μg/plate
KIH-485 M-1 acceptable/guideline Test article has no reverse mutagenic potential under the experimental conditions either in the presence or absence
of metabolic activities.

870.5100 Bacterial reverse
mutation test 47701714 (2004), Safepharm Labs TA98, TA1535, TA100
and TA1537- S typhimurium & WP2
uvrA of E coli 50-5000 μg/plate
KIH-485 M-3 acceptable/guideline Test article has no reverse mutagenic potential under the experimental conditions either in the presence or absence
of metabolic activities.Page 46 of 80 Table A.2.2 Subchronic, Chronic and Oth er Toxicity Profile of Pyroxasulfone

Guideline No. Study Type MRID No. (year)/
Classification /Doses Results

870.5100 Bacterial reverse
mutation test 47701715 (2008), RCC Ltd., Germany TA98, TA1535, TA100
and TA1537- S typhimurium & WP2
uvrA of E coli 33-5000 μg/plate
KIH-485 M-25 acceptable/guideline Test article has no reverse mutagenic potential under the experimental conditions either in the presence or absence
of metabolic activities.

870.5100 Bacterial reverse
mutation test 47701756 (2009), Huntingdon TA98, TA1535, TA100
and TA1537- S typhimurium & WP2
uvrA of E coli 50-5000 μg/plate
KIH-485 M-28 acceptable/guideline Test article has no reverse mutagenic potential under the experimental conditions either in the presence or absence
of metabolic activities.

870.5100 Bacterial reverse
mutation test 47701716 (2007), Safepharm Labs TA98, TA1535, TA100
and TA1537- S typhimurium & WP2
uvrA of E coli 15-5000 μg/plate
KIH-485 I-3 acceptable/guideline Test article has no reverse mutagenic potential under the experimental conditions either in the presence or absence
of metabolic activities.

870.5100 Bacterial reverse
mutation test 47701717 (2007), Safepharm Labs TA98, TA1535, TA100
and TA1537- S typhimurium & WP2
uvrA of E coli 50-5000 μg/plate
KIH-485 I-4 acceptable/guideline Test article has no reverse mutagenic potential under the experimental conditions either in the presence or absence
of metabolic activities.

870.5100 Bacterial reverse
mutation test 47701718 (2007), Safepharm Labs TA98, TA1535, TA100
and TA1537- S typhimurium & WP2
uvrA of E coli 50-5000 μg/plate
KIH-485 I-5 acceptable/guideline Test article has no reverse mutagenic potential under the experimental conditions either in the presence or absence
of metabolic activities.Page 47 of 80 Table A.2.2 Subchronic, Chronic and Oth er Toxicity Profile of Pyroxasulfone

Guideline No. Study Type MRID No. (year)/
Classification /Doses Results

NA DEREK Evaluation 47701757 (2009)
LSR Associates Metabolites: M-1, M-3, M-9, M-25, M-29 and parent Pyroxasulfone All available toxicity endpoints were searched (carcinogenicity, chromoso me damage, genotoxicity, hepatotoxicity, HERG channel inhibition, irritation, mutagenicity, reproductive toxicity, respiratory sensitization, thyroid toxicity and other miscellaneous endpoints). Toxicity to humans, mammals and bacteria was assessed. Neither pyroxasulfone nor the five metabolites evaluated fired any structural alerts for toxicity.

NA (Special Study: A Toxicological Mechanism Study) Effects on motor systems in mice after 14- day dietary exposure using a glutathione depletion animal model. 48046901 (2009)
Female CD1 mice (3/group) were administered 0, 4500 ppm pyroxasulfone + 20 mM BSO, 20000 ppm pyroxasulfone + 20 mM
BSO and 20 mM BSO alone for 14 days. acceptable/non- guideline Gross pathological observation showed wasted body muscle volumes in the 20000 ppm pyroxasulfone + BSO group animals. Histopatho logical examination showed degeneration/necrosis, calcification, cellular infiltration and the features of regenerating muscular tissue, namely proliferation of satellite cells in skeletal muscle in all BSO treated animals. Pyroxasulfone appeared to augment the depletion of the intracellular GSH in muscular tissues.

NA (Special Study: A Toxicological Mechanism Study) Heart histopathology for a toxicological mechanism study ofPyroxasulfone (KIH485): effects on motor systems in mice after 14- days dietary exposure using a glutathione depletion animal model 48046902 (2010) mice (3 females/group)
were treated with pyroxasulfone at 0, 4500 + 20 mM BSO, and 20000
+ 20 mM BSO or 20 mM BSO alone mg/kg/day for 14 days. acceptable/non- guideline Hearts (2 slices/animal) of all animals in control and treated groups were routinely fixed, processed, embedded in paraffin wax, sectioned at 4 μ m, stained and examined microscopically. Pyroxasu lfone induced very mild myocardial toxicity in mice at the dietary level of 20000 ppm under the GSH depleted conditions of the study

NA (Special Study: A Toxicological Mechanism Study) Investigation for Cell Proliferation Activity
and Oxidative Stress in Rat Urinary Bladder After Dietary Exposure for 14 Days 48046903 (2009) Male rats at 0, 2000, and 20000 ppm Pyroxasulfone (160 and 1632 mg/kg/day) acceptable/non- guideline Both the 2000 and 20000 ppm groups showed an increase in Ki-67 labeling index associated with hyperplasia in the urinary bladder epithelium.

NA (Special Study: A Toxicological Mechanism Study) Investigation for Cell Proliferation Activity
and Oxidative Stress in Mouse Kidneys After Dietary Exposure for 14 Days 48046904 (2009) Male Crlj:CD1 (ICR) mice (8 /group) at 2000 or 15000 ppm pyroxasulfone (292 and 2199 mg/kg/day) acceptable/non- guideline The Ki-67 labeling indices, an indicator of cell proliferation activity, in all regions of both treatment groups were almost equal to those in the control groups.Malondialdehyde contents in the kidneys of both KIH485-treated groups from confirmation of the oxidative stress were comparable to that in the control group. It is unlikely that KIH-485 treatment is involved in cell proliferation activity and oxidative stress in the mouse kidney under the conditions of the present study.Page 48 of 80 Table A.2.2 Subchronic, Chronic and Oth er Toxicity Profile of Pyroxasulfone

Guideline No. Study Type MRID No. (year)/
Classification /Doses Results

NA (Special Study: A Toxicological Mechanism Study) Electron Microscopic
Examination of Rat Urinary Bladder Treated with Pyroxasulfone (KIH-485) for 14 Days 48354402 (2011) Male Crl:CD(SD) rats (5/ group) at 0, 500, 2000 or 20000 ppm pyroxasulfone (44, 177 and 1765 mg/kg/day) Doses of 2,000 and 20,000 ppm of KIH-485 (pyroxasulfone) induced morphological changes (suggesting hyperplastic and toxic effects) in the surface of the bladder epithelium.

NA (Special Study) Rat In Vivo Comet Test. 48046906 (2010) Male rats at 0, 500, 1000 and 2000 mg/kg pyroxasulfone : one dose at 0 time, 2 nd dose after 21
hrs. Positive control group received ethyl methanesulphonate at 200 mg/kg. acceptable/non-guideline A statistically significant increase in the tail intensity was
observed in the liver of rats treated with Pyroxasulfone at 2000 mg/kg/day ( p <0.001), compared to vehicle control values, Statistically significant increases in the tail intensity were observed in the bladder of rats treated with Pyroxasulfone at 1000 and 2000 mg/kg/day ( p <0.001),

NA (Special Study) Mouse In Vivo Comet
Test. 48046907 (2010) Male mice at 0, 500, 1000 and 2000 mg/kg pyroxasulfone : one dose at 0 time, 2 nd dose after 21
hrs. Positive control group received ethyl methanesulphonate at 200 mg/kg. acceptable/non-guideline A statistically significant increase in the tail intensity was
observed in the liver of CD 1 mice treated at 500, 1000 and 2000 mg/kg/day ( p <0.01), compared to vehicle control values. Group mean va lues are within historical control range, considered no t biologically significant. Statistically significant increases in the tail intensity were observed in the kidney at 1000 ( p <0.05) and 2000 mg/kg/day ( p <0.001).

NA (Special Study) One week oral cytotoxicity study in
male rats 48354404 (2010) Male rats 918/group) at 0, 50, 1000 or 2000 ppm pyroxasulfone. 6 rats/group sacrificed at 1, 3 or 7 days, SEM of urinary bladders acceptable/non-guideline This study provides evidence to the cytotoxicity of pyroxasulfone the urinary bladder epithelium of male rats.Page 49 of 80 A.3 Toxicological Endpoints

A.3.1 Acute Reference Dose (aRfD) – General Population

Study Selected: Developmental Neur otoxicity Study- Rats
MRID No: 47701724 Dose and Endpoint for Risk Assessment : 100 mg/kg/day based on decr eased brain weight in
both sexes and decreased thic kness of the hippocampus, corpus callosum and cerebellum in PND 21 female offspring occu rring at 300 mg/kg/day. Uncertainty Factor: 100x (10x interspecies extrapolation, 10x intras pecies variability. 1X FQPA safety factor)

ൌ ࡰࢌࡾࢇ 1 00 mg/kg/day ሺNOAELሻ

૚૙૙ ሺ ࡲࢁ ሻ ൌ 1 .0 mg/kg

ൌ ࡰ࡭ࡼࢇ 1 mg/kg/day ሺaRfDሻ

૚ ሺ ࡭ࡼࡽࡲ ࡲࡿ ሻ ൌ 1 .0 mg/kg

Comments: The HED DNT committee considers morpho metric changes in the hippocampus, corpus callosum and cerebellum during the first 21 days to result from a single acute exposure.

A.3.2 Chronic Reference Dose (cRfD )

Study Selected: 1-year oral t oxicity study - Dog
MRID No: 47701707 Dose and Endpoint for Risk Assessment : 2 mg/kg/day based on impaired hind limb function, ataxia, hind limb twitching and trem ors; clinical pathology: increased creatine kinase, aspartate aminotransferase; axonal/myelin degeneration of the sciatic nerve and spinal cord sections at 10 mg/kg/day. Uncertainty Factor: 100x (10x interspecies extrapolation, 10x intras pecies variability, 1X FQPA safety factor)

ࢉ ࡰࢌࡾ ൌ 2 mg/kg/day ሺNOAELሻ

૚૙૙ ሺ ࡲࢁ ሻ ൌ 0 .0

2 mg/kg

࡭ࡼࢉ ࡰ ൌ 0.02 mg/kg/day ሺ cRfD ሻ

૚ ሺ ࡲࢁ ሻ ൌ 0 .0

2 mg/kg

Comments about Study/Endpoint/Uncertainty Factors: In the various species tested, the dog
exhibited adverse toxic effects at lower doses of exposure to pyroxasu lfone. Also the dose ranges used in the dog testing were the narrowest allowing for more accurate determination of aPage 50 of 80 NOAEL. The 2 mg/kg/day NOAEL from this dog st udy is also protective from other adverse effects seen at higher doses in rats and mice such as cardiac and skeletal myopathy, liver, kidney and urinary bladder toxicity and developmental effects in the DNT and rabbit developmental studies. This endpoint is considered to be pr otective of all populations including women of child-bearing age and nursing infants.

A.3.3 Incidental Oral Exposure

Pyroxasulfone has not been proposed for resi dential uses; therefor e, selection of a toxicity endpoint is not necessary for this exposure scenario at this time.

A.3.4 Dermal Absorption

A dermal absorption study with pyrox asulfone is not available.

A.3.5 Dermal Exposure (Short- & Intermediate-Term)

Critical Study Selected: 28-day Dermal -- Rat
MRID No: 47701698 Dose and Endpoint for Risk Assessment: Dermal NOAEL= 100 mg/kg/day based on cardiac myofiber degeneration/inflammation cutaneous myofiber degenerati on/inflammation at 1000 mg/kg/day.

Uncertainty Factor: 100x (10x interspecies extrapolat ion, 10x intraspecies variability)

Comments about Study/Endpoint/Uncertainty Factors: A 28-day dermal toxicity study was selected as for the dose and endpoint for short and intermediate term dermal exposure. This study is route specific and is appropriate for this exposure scenario. The LOAEL of 1000 mg/kg/ day is based on minimal to mild cardiac myofiber degeneration with inflam mation in males and females. This endpoint is also considered protective of developmental e ffects in DNT at 300 mg/kg/day and rabbit developmental study at 1000 mg/kg/day. This POD is also protective of the neurotoxic effects seen in the dog following chronic oral expos ure to 10 mg/kg/day. Dermal absorption is estimated to be 7.5%, so the relative derma lly absorbed dose for the dermal NOAEL of 100 mg/kg/day is approximately 7.5 mg/kg/day which is less than the oral LOAEL of 10 mg/kg/day identified in the dog study. In addition, rats in general are a more sensitive species for the dermal exposure route because of higher skin permeability.

A.3.6 Inhalation Exposure (Short- & Intermediate-Term)

Critical Study Selected: 90 day oral toxicity study in dogs.
MRID No: 47701696 and 47701697 Dose and Endpoint for Risk Assessment: 2 mg/kg/day based on a LOAEL of 10 mg/kg/day for axonal/myelin degeneration in the sciatic nerve, minimal to mild subacute inflammation of skeletal muscle in males and females and impaired limb function, body and limb hypotonia,Page 51 of 80 increase in serum creatine kinase activity.

. The assumed inhalation ab sorption factor is 100%.

Uncertainty Factor: 100x (10x interspecies extrapolat ion, 10x intraspecies variability)

Comments about Study/Endpoint/Uncertainty Factors: A 28 day inhalation toxicity study in rats (M RID 47701699) was considered for selecting an endpoint of toxicity for assessi ng this route specific exposure. However the endpoint based on the 90-day dog study is more prot ective than the findings in th e 28 day inhalation toxicity study in rats where the NOAEL was 200 mg/m3 (equivalent to 52.2 mg/kg/day or al dose), the highest dose tested using an aerosol dust of pyroxasulfone, not an aerosol. Since no effects were seen at the highest dose tested in the inhalation study an d since mixer/loader exposures would be to both dust and aerosols (with aerosol exposures typica lly being more sensitive than dust exposures), HED believes that a dust exposure study was not as protective. Since no e ffects were seen at the highest dose tested in the inhalation study, use of the oral dog would compensate for this uncertainty, and is protective of neurot oxicity. The inhalation NOAEL of 200 mg/m 3 /day is equivalent to 52.2 mg/kg/day oral dose using the following formula:

mg/kg/day = mg/L x A x CF x D x AF

where

A is absorption factor us ing 1 as a default value
CF is a conversion factor in L/hr/kg to account for respirat ory volume and body weight for a given species and strain. For the rat stra in used in this study it is 43.5
D is duration of daily exposure in hours. A 6 hour daily exposure was used.
AF is activity factor. The anim al default factor of 1 was used.

MOE: 100Page 52 of 80 A.4 EXECUTIVE SUMMARIES

A.4.1 Subchronic Toxicity

870.3100 90-Day Oral Toxicity – Rat

In a subchornic oral toxi city study (MRID 47701692), Pyroxa sulfone (KIH-485; 99.1% a.i.; Lot/Batch No. RCT301) was administered in the diet to Crl:WI(Glx/BRL/Han)BR rats (10/sex/dose) for up to 13 weeks at doses of 0, 100, 500, 2500, or 5000 ppm (equivalent to 0/0, 8.9/10.2, 43.9/48.9, 221.4/255.9, and 451.2/514.2 mg/kg bw/day in males/females). In the control and 5000 ppm group, an additional 5 rats/sex /group were fed the test diets for 13 weeks followed by a 4-week recovery period.

Assessment of toxicity was based on mortality, clinical observati ons, and clinical and anatomic pathology evaluations. Ther e were no effects on food consumption, ophthalmoscopy, or functional observational battery parameters.

The target organs were liver, skeletal muscle , cardiac muscle, and the pancreas based on altered organ weights, histopathological indicators, and/or c linical pathological indicators. Reduced body weight gain was observed in the 5000 ppm group.

At 2500 and 5000 ppm, slight decreases in overall body weight gain were noted in both sexes (decr 12-15%). Additionally, the following adverse findings were observed that each may

indicate an adverse effect on muscle tissue: (i) decreased am bulation and total movement in males (decr 24-38%); (ii) decreased creatinine in males (decr 11-12%); (iii) increased minimal to slight myocardial degeneration in both sexes (8- 10/10 treated vs 0-1/10 controls); (iv) increased minimal quadriceps myopathy in males (6/10 each treated vs 3/10 controls); and (vi) increased minimal to slight myopathy in skeletal muscle s other than quadriceps in both sexes (4-9/10 treated vs 1/10 controls). In th e females, increased aspartate am inotransferase (AST; incr 122%, each dose) and increased liver weights (decr 10-23%) were also noted.

Additionally at 5000 ppm, the following adverse findi ngs were observed: (i) pale teeth in both sexes; (ii) decreased body weight and body weight gain in both sexes; (iii) increased AST in males; (iv) increased urine volume in both sexes; (v) increased liver weights in males; (vi) gross changes in the liver (large, mottled, and/or pale) in both sexes; (vii) dark pancreas in females; (viii) microscopic changes in the liver (agonal congestion/hemorrhage, centrilobular hypertrophy/eosinophilia, and/or hepa tocyte vacuolation) in both sexe s; and (ix) degranulation in the pancreas of females.

After the recovery period, increas ed incidences in agonal conges tion/hemorrhage in the liver was observed at 5000 ppm in males (3/5 treated vs 0/5 c ontrols), and pale teeth was noted in 1/5 ratsPage 53 of 80 in each sex (vs 0 controls). Decreased body wei ghts were noted in the 5000 ppm males (decr 7- 19%) with body weights remainin g decreased during the rec overy period (decr 10-12%), although a slight increase in body weight gain wa s noted during the recovery period. However, all other microscopic findings in the liver, heart, mu scle and pancreas in the 5000 ppm animals were comparable to control rats.

The LOAEL is 2500 ppm (equivalent to 221/256 mg /kg/day in males/females), based on the findings detailed above. The NOAEL is 500 ppm (equivalent to 44/49 mg/kg/day in males/females).

This study is classified as acceptable/guideline and satisfies the guideline requirements (OPPTS 870.3100; OECD 408) for a subchronic or al toxicity study in the rat.

COMPLIANCE: Signed and dated GLP Compliance, Qual ity Assurance, Data Confidentiality, and Flagging statements were provided. The GL P Compliance statement indicated that the study was conducted in accordance with OECD rather than FIFRA, 40 CFR 160.

870.3150 90-Day Oral Toxicity – Dog

The purpose of this study was to evaluate the potential toxi city of KIH-485 TGAI when administered via oral capsule dose administ ration. Four dogs per sex per dose level were administered the test substance via gelatin capsules at concentrations of 0.2, 2.0, and 10.0 mg/kg/day for the 90 day (13 week) in-life test period. An additional group of four dogs per sex served as the control and received the empty ge latin capsules. The dogs were observed twice daily for signs of toxicity. B ody weights and food consumption were measured weekly and a battery of urinalysis, hematological and clin ical pathology examina tions was conducted at initiation and at six and thirteen weeks. Ophtha lmological examinations were done at initiation and at 13 weeks. The post-life phase of the st udy included organ weight measurements, gross and pathologic assessments.

No deaths occurred during the study period. There were no tr eatment related changes in body weight, food consumption, hematology, blood ch emistry, coagulatio n, ophthalmological or urinalysis parameters. Pathological examin ation (organ weight measurement and gross observation), found no effects attr ibutable to the administratio n of the test substance.

Slight muscle fiber degeneration of the musculat ure portion of the diaphr agm, slight hyperplasia of satellite cell of the muscle, and slight nerve fiber degeneration of the sciatic nerve were observed in one 10 mg/kg/day male dog. In a subsequent 90-day dog toxicity study (MRID 47701697), dogs administered pyroxasulfone at 15 mg/kg/day developed minimal to mild axonal/myelin degeneration in the sciatic nerve of all males (4/4) and 3⁄4 females in addition to

minimal to mild subacute inflammation of skeletal muscle in males and females. ConsideringPage 54 of 80 these two studies together, the LOAEL for thes e adverse effects in dogs is 10 mg/kg/day and the NOAEL is 2 mg/kg/day.

When reviewed in conjunction with MRID 47 701697 (concurrently review ed), this study is

classified acceptable/guideline and satisfies the guideline requirements (OPPTS 870.3150; OECD 409) for a subchronic oral toxicity study in dogs.

COMPLIANCE : Signed and dated Data Confidentiality, GLP Co mpliance, Flagging, and Quality Assurance
statements were provided.

870.3200 21/28-Day Dermal Toxicity – Rat

In a 29-day dermal toxicity study (MRID 47701698) , Pyroxasulfone (97.2% a.i.; Lot No. L001) in deionized water was applied to the shaved, intact skin of male and female Crl: CD ® (SD) IGS BR rats (10 sex/dose) at dose levels of 0, 20, 100, or 1000 mg/kg bw/day for 6 hours/day for 29 consecutive days.

No adverse, treatment-related effects was obs erved on mortality, clin ical signs, body weight, body weight gain, food consumption, food effici ency, hematology, clinical chemistry, organ weights, or gross pathology.

An increased incidence of minimal to mild fo cal/multifocal cardiac myofiber degeneration with

inflammation was observed in the hearts of the 1000 mg/kg/day males (90% treated vs 0% controls) and females (40% treated vs 10% c ontrols). An increased incidence of minimal focal/multifocal cutaneous myofiber degeneration with inflammation was observed in the treated skin of 1000 mg/kg/day male rats (60% treated vs 0% controls).

The LOAEL is 1000 mg/kg/day (the limit dose), based on cardiac myofiber degeneration/inflammation in bo th sexes and cutaneous myofiber degeneration/inflammation in ma les. The NOAEL is 100 mg/kg/day.

This 29-day dermal toxicity study is classified as acceptable/guideline and satisfies the guideline requirement for a 28-day dermal toxi city study (OPPTS 870.3200; OECD 410) in rats.

COMPLIANCE : Signed and dated GLP, Quality Assurance, and Data Confidentiality statements were provided.

870.3465 90-Day Inhalation – Rat

- NA -Page 55 of 80 A.4.2 Prenatal Developmental Toxicity

870.3700a Prenatal Developmental Toxicity Study – Rat

The purpose of this study (MRID 47701702) was to determine the potential maternal and developmental toxicity of KIH-485 (Pyroxa sulfone; 99.1%; Lot #05TG005) in rats, when administered orally, by gavage. Time-mated female Crl:CD(SD) rats were assigned to four groups (22 animals/group). Each group receiv ed dose preparations containing the control article or 100, 500 or 1000 mg of test article/kg of body weigh t/day (mg/kg/day) at a dose volume of 10 ml/kg of body weight from Days 6 to 20 of gestation. All dams were observed for general conditions. Body weights and food cons umption were recorded during the gestation period. On GD 21, caesarean section and necropsy were conducted on the dams, and the fetuses were examined for external, visceral, a nd skeletal malformations and variations.

There were no maternal deaths and no dose relate d clinical signs or necropsy findings. There was no adverse effect of treatment on gr oup mean body weights or food intake.

The maternal LOAEL was not observed. Th e maternal NOAEL is 1000 mg/kg/day (limit dose).

There were no abortions, premature deliveries, dead fetuses, late resorptions, or complete litter resorptions and no effects of treatment on the number s of litters, live fetuses, or early resorptions. Additionally, sex ratio and mean litt er weight in the treated groups were comparable to controls. There were no external, visceral, or skelet al defects attributable to treatment.

The developmental LOAEL was not observ ed. The developmental NOAEL is 1000 mg/kg/day (limit dose).

This study is classified acceptable/guideline and satisfies the guideline requirement (OPPTS 870.3700a; OECD 414) for a developmen tal toxicity study in rats.

COMPLIANCE : Signed and dated Data Confiden tiality, GLP Compliance, Flagging, and Quality Assurance statements were provided.

Rat Developmental Study (GLN

In a rat developmental to xicity study (MRID 47701702), 22 female rats/dose group were administered 0, 100, 500 or 1000 mg of pyroxasulf one from gestation day (GD) 6-20. There

were no maternal deaths and no dose related clinical signs or necropsy findings. There was no adverse effect of treatment on group mean body we ights or food intake. The maternal LOAEL

was not observed. The maternal NOAEL is 10 00 mg/kg/day (limit dose). There were no abortions, premature deliveries, d ead fetuses, late resorptions, or complete litter resorptions andPage 56 of 80 no effects of treatment on the numbers of litters, li ve fetuses, or early resorptions. Sex ratio and mean litter weight in the treated groups were co mparable to controls. There were no external, visceral, or skeletal defects attributable to treatment. The developmental LOAEL was not observed. The developmental NOA EL is 1000 mg/kg/day (limit dose).

870.3700b Prenatal Developmenta l Toxicity Study – Rabbit

In a developmental study (MRID 47701704), time mated female Hra:(NZW)SP rabbits (22 animals/group) were administered by oral gavage 0, 250, 500 or 100 0 mg of KIH-485 TGAI (Pyroxasulfone; Lot/Batch #05TG005; 99.1%)/kg of body weight/day at a dose volume of 5 ml/kg of body weight from Gestation Day (GD) 7 to 28. All dams were observed for general conditions. Body weights and food consumption were recorded dur ing the gestation period. On GD 29, caesarean section and necropsy were conducted. Fetuses were examined for external, visceral, and skeletal abnormalities.

Seven females died or were euthanized prior to scheduled necropsy, 3 due to premature abortion and 4 because of intubation injury. One abortio n occurred at 250 mg/kg/day and 2 occurred at 1000 mg/kg/day.

There were no test substance-rela ted deaths, clinical signs of toxicity, or gross post-mortem findings in dams at any dosage. There were no test-substance related re ductions in body weight or food consumption parameters at any level tested. The LOAEL for maternal toxicity was not

observed. The NOAEL for maternal toxi city is 1000 mg/kg/day (limit dose).

There were test substance-related effects on feta l weights and the number of resorptions (total and early) at 1000 mg/kg/day; these effects were re latively slight, but st atistically significant.

The mean number of corpora lutea, implantation sites, live fetuses, and sex ratio were comparable across all groups. Totals of 164, 164, 170 and 125 fetuses were examined for abnormalities and variations at 0, 250, 500 and 1000 mg/kg/day, respectively. There were no test substance-related fetal external, vi sceral, or skeletal malformations or variations observed at any dosage.

The LOAEL for developmental toxicity is 1 000 mg/kg/day (limit dose) based on reduced fetal weight and increased fetal resorptions. Th e NOAEL for developmental toxicity is 500 mg/kg/day.

This study is classified acceptable/guideline and satisfies the guideline requirement (OPPTS 870.3700a; OECD 414) for a developmen tal toxicity study in rats.

COMPLIANCE : Signed and dated Data Confiden tiality, GLP Compliance, Flagging, and Quality Assurance statements were providedPage 57 of 80 A.4.3 Reproductive Toxicity

870.3800 Reproduction and Fertility Effects – Rat

The purpose of this study (MRID #47701705) was to determine the reproductive toxicity of the test article, KIH-485TGAI (Pyr oxasulfone; 97.2%; Lot/Batch #L 001), following oral (dietary) administration to the rat. This study was c onducted as a pilot study for a multigenerational reproduction study. Crl:CD®(SD) IGS BR rats (12/se x/concentration) were fed diets containing 0, 25, 250, or 5000 ppm KIH-485TGAI for at least 28 days prior to mating to produce the F1

litters. Dams were allowed to deliver and rear their offspring until wean ing (postpartum day 21). At weaning, 12 F1 rats/sex/group were randomly selected to continue on study until post-natal day (PND) 60 and were fed the same dietar y concentration level as their parents.

There was a significant (p<0.05) tr end for stained perineum in th e P dams during lactation, with 3/12 animals affected at 5000 pp m compared to 0/11 controls.

In P males test substance-related reductions in we ight gain were observed at 250 and 5000 ppm. At 5000 ppm, weekly mean body weight gains were decreased throughout most of the study,most notably on Days 0-7 and 21-28 and resulted in reductions in overall weight gain for the premating period and the entire exposure period. At 250 ppm, weekly mean body weight gains were also decreased throughout the st udy, and, except for Days 0-7, the reductions were of similar magnitude as those in the 5000 ppm group, de spite the 20-fold difference in dietary concentration. Overall weight ga ins for the pre-mating period a nd the entire exposure period were decreased in this group. In P females , there were test substanc e-related reductions in body weights at 5000 ppm and weight gain at 250 and 5000 ppm. At 5000 ppm, weekly body weight gains were decreased during pre- mating and during the first and la st week of gestation. As a result of these decreases in weekly weight ga in, overall weight gain for the pre-mating and gestation periods were 36-38% lower than the control group mean. In addition, weekly mean body weights were decreased at the end of th e pre-mating period and throughout gestation and lactation. At 250 ppm, body weight gain was decr eased during the last w eek of gestation, which

resulted in a decrease in overall weight gain during gestation.

In F 1 adult males and females , test substance-related reducti ons in body weights and weight gain were observed at 5000 ppm. Overall mean body weight gain for the post-weaning period for this group was 20% lower than the controls in males and 18% lower in females as a result of decreased weekly body weight gains throughout th e post-weaning period. These reductions in weekly weight gains were reflec ted in weekly mean body weights th at were lower than controls throughout the post-weaning period. In the 5000 ppm F 1 adult males and females , weekly decreases in food consumption th roughout most of the post-weaning period resulted in decreases of 16-18% in overall food consumption for the post-weaning period (Days 0-39).Page 58 of 80 In P males , food efficiency was decreased during pr e-mating at 5000 ppm and reflected lower food efficiency throughout the pre-mating pe riod, most notably on Days 0-7 and 21-28. In P

females , there were test-substance related effects on food efficiency at 250 and 5000 ppm. At 5000 ppm, weekly food efficiency was decreased throughout pre-mating a nd gestation. At 250 ppm, weekly food efficiency was de creased during the last week of ge station; as a result of this decrease, overall food efficiency during gestation in this group was 14% lower than control.

Dietary exposure to 5000 ppm of the test substa nce resulted in increased mean relative liver weights (%body wt.) in both P and F 1 adult males and females. The increased liver weights correlated with microscopic hepa tocellular hypertrophy (in P adults) and were interpreted to be

the result of non-adverse inducti on of hepatocellular enzymes by a xenobiotic. All other organ weight differences were interpreted to be th e result of general body weight loss and not indicative of organ-specific effects.

In the 5000 ppm P adult rats, there were test substance -related microscopic findings in the liver, quadriceps muscle, heart, and sciatic nerve. Minimal centrilobular he patocellular hypertrophy was observed in the livers of five (5/12) male and three (3/12) female rats exposed to 5000 ppm in the P generation. Locally extensive, minimal to mild atrophy of the quadriceps muscle was observed in all of the 5000 ppm females in the P animals and in none of the other rats examined. The atrophy consisted of a minimal (10/12 rats) to mild (2/12 rats) decrease in the cross-sectional area of the muscle fibers, as compared to the co ntrol tissue. Minimal to mild focal and multifocal degeneration of heart myofibers (i.e., myocardium) was pr esent in five (5/12) 5000 ppm P females and was considered a heart-specific adverse effect. Minimal to mild axon/myelin degeneration within the sciati c nerve sections was observed in all (12/12) of the 5000 ppm

female rats. The occurrence of m yocardial and peripheral nerve fi ber degeneration exclusively in females treated at 5000 ppm may be related to s ubstantially increased food consumption, and test substance dose, during lactation.

The LOAEL for parental toxicity is 5000 ppm (equivalent to 3 39/383 mg/kg/day in males/females, respectively) based and microscop ic effects in the heart, liver, muscle, and sciatic nerve of males and females, decreased bo dy weight gains in the P males and females and decreased food efficiency in the P dams . The NOAEL for parental toxicity is 250 ppm (equivalent to 16/20 mg/kg/day in males/female s, respectively). The decreased body weigh gains at the 250 ppm dose were transient and mo stly not statistically significant, and the bodyweights were not affected. The eff ects at 250 ppm are therefore equivocal.

There were no treatment-related effects on litter size, sex ratio, or pup survival. At 5000 ppm, mean pup weights were decreased by 27-30% comp ared to controls throughout the post-natal period. There were test substance-related effects on develo pmental landmarks at 5000 ppm. The mean day of achievement of preputial separation was increased by 3 days in this group comparedPage 59 of 80 to the control group, and the mean age of achievement of vagina l patency was increased by 1.8 and 2.7 days in the 250 a nd 5000 ppm groups, respectively; change s that were cons idered due to reductions in body weight and we ight gain in these groups.

The LOAEL for offspring toxicity is 5000 ppm (equivalent to 339/383 mg/kg/day in males/females, respectively) based on decrease d pup weight and delaye d sexual maturation in both sexes. The NOAEL for offspring toxicity is 250 ppm (equivalent to 16.0/19.6 mg/kg/day in males/fema les, respectively).

There were no test substance-re lated effects on mating, fertility, gestation duration, number of implantation sites, or implantation efficiency in P adult females at any concentration.

The LOAEL for reproductive toxicity was no t observed. The NOAEL for reproductive toxicity is 5000 ppm (equivalen t to 339/383 mg/kg/day in males/ females, respectively), the highest concentration tested.

This study is classified acceptable/non-guideline provides data for use in dose selection for the definitive 2-generation reproducti ve toxicity study in rats.

COMPLIANCE : Signed and dated Data Confiden tiality, GLP Compliance, Flagging, and Quality Assurance statements were provided.

A.4.4 Chronic Toxicity

870.4100a (870.4300) Chronic Toxicity – Rat

In a chronic feeding toxicity study (M RID 47701708), KIH485 TGAI (pyroxasulfone), was administered to male and female rats Crl:CD(SD) from Charles River Lab. Twenty rats per sex per level were administered test material by dail y feeding at concentrations of 0, 5, 50, 1000 and 2,000 ppm for the one year. Data for body weights and food consumption clinical observations and ophthalmological examinations were collected. A full battery of hema tological and clinical pathology examinations was conducted at three, si x and twelve months. The post-life phase of the study included organ weight measuremen ts and gross and microscopic pathologic

assessments.

The mean daily intake of KIH-485 TGAI in males was 0, 0.22, 2.22, 46.20 and 91.91 mg/kg/day at 0, 5, 50, 1000 and 2000 ppm, respectively. In fema les, the mean daily intake of the test substance was 0, 0.30, 3.12, 60.80 and 121.33 mg/kg/day at the same respective dietary levels.

Test substance-related effect s occurred at 1000 ppm and a bove that included reduced body weight parameters and microscopic findings. These findings were observed in the liver (centrilobular hepatocellular hypertrophy (males)), heart (increased cardiomyopathy (females))Page 60 of 80 and urinary bladder (mucosal hype rplasia (males and females)). At 2000 ppm, these effects were accompanied by increased clinical observations of red discharge from the penis and associated red-stained cageboards as well as increased liver and kidney weights. A sli ght increase in liver and kidney weights was observed in the 2000 pp m male and female di etary exposure groups.

The lowest-observed-adverse-effect level (L OAEL) of KlH-485 TGAI, given as a dietary mixture, was 1000 ppm (46.20 and 60.80 mg/kg/day for males and females, respectively). The

no-observed-adverse-effect leve l (NOAEL) was 50 ppm (2.22 a nd 3.12 mg/kg/day for males and females, respectively).

This study is classified as acceptable/guideline and satisfies the guideline requirements (OPPTS 870.4100a; OECD 452) for a chronic study in rats.

COMPLIANCE: Signed and dated GLP Compliance, Qual ity Assurance, Data Confidentiality, and Flagging statements were provided.

870.4100b Chronic Toxicity – Dog

In a 1-year dog study (MRID: 47701707), KIH-485, ( 99.1%) was administered daily to 4 beagle dogs/sex/dose in capsules at dose levels of 0.0, 0.2, 2.0, and 10.0 mg/kg bw/day) for 364 consecutive days.

No effects of treatment were observed on mo rtality, body weights, body weight gains, food consumption, food efficiency, ophthalmoscopic exam inations, electrocardi ograpic examinations, urinalysis, organ weight s, or gross pathology.

Toxicity was observed at 10 mg/kg/day. Impaired hind limb function and tremors was noted in two males, and impaired hind limb function was no ted in one female. One of the males was also observed with hind limb twitching and ataxia. Dogs in this group (3 males and one female) also had abnormal, test article-relate d neurobehavioral observations in cluding mild, moderate, and/or severe limb weakness, primarily in the hind limb s. Limb tone was sometimes noted as hypotonic. Abnormal gait, mobility, and limb position were noted. Clonic (quivering) or tonic (rigid limbs) movements were noted infrequentl y. Increases were observed in as partate aminotransferase (not statistically significant) and cr eatine kinase (p < 0.05) starting during Week 13 and continuing throughout the treatment period. Females approached control values by Week 52. All males (4 of 4) and females (4 of 4) had minimal to mild axonal/myelin degeneration of the sciatic nerve. Minimal axonal/myelin degeneration was observed in the spinal cord as follows: (i) in the cervical cord of two (2 of 4) males and two (2 of 4) females; (ii) in the lumbar cord of all (4 of 4) males and one (1 of 4) female; and (iii) in the thoracic co rd of two (2 of 4) males and three (3 of 4) females.Page 61 of 80 Additionally at 10 mg/kg/day, minimal myofiber degeneration/necrosis was observed in the following skeletal muscles: (i) in the biceps femoris of two (2 of 4) males; (ii) in the diaphragm of one (1 of 4) male; and (iii) in the quadriceps of one (1 of 4) males. Skeletal muscles of the 10 mg/kg/day females were unremarkable. Although el evations of aspartate aminotransferase and creatine kinase in both sexes indicated a possi ble effect on skeletal muscle, the microscopic findings were minimal in severity, and the clinical chemistry findings appeared to be returning to normal levels by Week 52. These observations suggest ed that any test artic le-related effects on

muscle were transient and/or reversible.

Beginning at Week 13 in females only, there was a mild, dose-dependent trend towards decreased erythrocyte mass as illustrated by sequential decreases in erythrocytes ( ↓ 8-16%) , hemoglobin ( ↓ 8-14%), and hematocrit ( ↓ 7-12%) relative to controls at 2.0 and 10.0 mg/kg/day. However, group means were within expected rang es, and statistical signi ficance was rarely and sporadically attained. Therefore, thes e findings were considered equivocal.

The LOAEL is 10.0 mg/kg/day, based on clinical and neuromuscular observations of the hind limbs as described above, increased seru m aspartate aminotransferase and serum creatine kinase, minimal to mild axonal/myelin degeneration of the sciatic nerve, and minimal axonal/myelin degenerati on in all three levels of th e spinal cord in males and females. The NOAEL is 2.0 mg/kg/day.

This study is classified acceptable/guideline and satisfies the guideline requirements (OPPTS 870.4100; OECD 452) for a chronic or al toxicity study in dogs.

COMPLIANCE : Signed and dated Data Confidentiali ty, GLP Compliance, Flagging, and Quality Assurance statements were provided.

A.4.5 Carcinogenicity

870.4200a Carcinogenicity/Chronic Study – RatPage 62 of 80 In this oncogenicity study (MRID # 47701709), Crl:CD®(SD)IGS BR rats (70/sex/group) were

administered diets that c ontained 0, 5, 50, 1000 or 2000 ppm of pyroxasulfone (M: 0, 0.21, 2.05, 42.55, 84.55 mg/kg/day; F: 0, 0.28, 2.69, 54.28, 106.74 mg/kg/day) for two years. Males were exposed for at least 99 weeks and females were exposed for at least 97 and surviving males and females were euthanized and examined for gross and microscopic pathologic alterations. The decision to terminate these groups prior to the intended termination at 104 weeks was necessitated by the expected surv ival of the rats. Unscheduled deaths were approaching the minimum deemed adequate for ensuring robust data sets and regulat ory compliance. All surviving rats were sacrificed and subjected to gross and microscopic pathological examination. Assessment of toxicity was based on mortality, cl inical observations, and clinical and anatomic pathology evaluations.

Test substance-related effects occurred at 1000 and 2000 ppm. At 2000 ppm, effects included reduced body weight parameters (males and fema les), reduced food consum ption (females), and increased red-stained urine on cageboards (mal es). At 1000 ppm, effects included reduced body weight and food consumption parameters (fem ales) and increased red-stained urine on cageboards (males). Dietary exposure of male and female rats to 1000 ppm or more of the test substance, for approximately two years, resu lted in increased cardiomyopathy (males and females); urinary bladder mucosal hyperplasia, inflammation, and transi tional cell papillomas (males); and sciatic nerve dege neration (females); and liver pe ribiliary fibrosis/inflammation (females).

An increase in urinary bladder transitional ce ll papillomas was observed in males at 1000 ppm

and above in association with a marked increase in mucosal hyperp lasia. A slight increase in mucosal inflammation also appeared to be associ ated with the mucosal hyperplasia. There were no test substance-related urinary bladder effects in females. Therefore, the test substance was interpreted to be oncogenic in male but not female rats.

An increase in the background inci dence and severity of cardiom yopathy occurred in both sexes at 1000 ppm and above. The lesion included myocar dial degeneration, infl ammation, and fibrosis and resembled naturally occurring cardiomyopathy of control rats of th is age and strain.

An increase in the background inci dence of sciatic nerve degeneration was observed in female rats at 1000 ppm and above. The lesion was grad ed minimal in most cases. Test substancerelated sciatic nerve degeneration was not observed in males.

An increase in liver pe ribiliary fibrosis/inflammation was ob served in females at 1000 ppm and above. An increase in bile duct hyperplasia was observed in females at 2000 ppm.

There were no test substance related effects on causes of death, organ weights or gross observations made at necropsy. There were no treatment-related change s in white blood cellPage 63 of 80 relative differential counts at a ny dietary concentration tested.

Under the conditions of the current study, test su bstance-related effects that were considered adverse were observed at 1000 and 2000 ppm. There were no test substance-related or adverse effects at either 5 or 50 ppm. Therefore, the no-observed-adverse-eff ect level (NOAEL) was 50 ppm. Based on an increase in urinary bladder transitional cell papillomas in the 1000 ppm and 2000 ppm males, the test substance was consid ered oncogenic in males at exposures of 1000 ppm and above.

This study is classified as acceptable/guideline and satisfies the guideline requirements (OPPTS 870.4200a; OECD 451) for a carcino genicity study in rats.

COMPLIANCE : Signed and dated GLP Compliance, Qual ity Assurance, Data Confidentiality, and Flagging statements were provided.

870.4200b Carcinogenicity (feeding) – Mouse

The purpose of this study (MRID: 47701710) wa s to determine oncogeni c potential of KIH-485 in mice, following oral (dietary) administration to the mouse for 18 months. Four groups of young adult male and female Crl:CD1(ICR) mice ( 60/sex/group) were admi nistered diets that contained 0, 5, 150, or 2000 ppm or 4000 pp m (males)/4000 ppm (females) substance (equivalent to 0, 0.62/0.84, 18.4/25.5, 228/306, and 465/668 mg/kg/day in males/females) of the test substance for up to 18 months. Tr eatment at 4500/4000 ppm exceeded the maximum tolerated dose, and this group was terminated prior to the scheduled sacrifice. After approximately 14 months on study, dietary concen trations for the 2000 ppm groups were reduced to 1000 and 500 ppm for males and females, resp ectively, in order to insure a satisfactory

survival rate at the scheduled sa crifice. To provide interim data at one year, 10 mice per sex per group were sacrificed and given gross and mi croscopic pathological examinations. After approximately 18 months of dietary exposure, al l surviving mice were sacrificed and given a gross and microscopic pathological examination. Assessment of toxicity was based on mortality, clinical observations, and clinical and anatom ic pathology evaluations.

In the 4500/4000 ppm group, marked body weight lo sses occurred during the first 2 months of the study, which warranted the reduction of the dietary concentration to 3000 ppm. This reduction appeared to stabilize the body weight s. However, after a bout 10 (females) or 12 (males) months on the study, clinical observation s (principally, affects on the hindlimbs) began to be noted with a frequency and severity that was dose-related. These effects included abnormal hindlimb gait, low carriage, curled toes, extend ed hindlimbs, and splayed limbs. The progression of effect on the hindlimbs began to interfere with th e mice access to food a nd water. Therefore, this group was terminated before the scheduled sacrifice.Page 64 of 80 Test substance-related adverse effects were observed in males and females at 2000/1000 and 2000/500 ppm, respectively. No adverse effects were observed in males or females at 150 ppm

or lower. In males and females at 2000/1000 and 2000/500 ppm, respectively, test substancerelated effects during the in-life period incl uded reductions in body weight parameters and increases in adverse clinical signs. The clin ical signs consisted of hindlimb gait/stance abnormalities and included abnormal hindlimb ga it, low carriage, curled toes, extended hindlimbs, and splayed limbs. These clinical ob servations were not ove rtly obvious until about 10 months on study and correlate with the hist opathologic lesions of the nervous system

described below.

At 2000 ppm, the one-year interim s acrifice data indicate d that daily dietary exposure of male and female mice had adverse effects on the kidneys and nerve fibers in both sexes. Minimal to mild tubular nephrosis in the pars recta, and an associated decrease in ki dney weight parameters,

was observed in both sexes. Minimal to mild de generation of nerve fiber axons and myelin was

observed in the sciatic nerve of some mice. Mi nimal trigeminal nerve degeneration was observed

in one male and one female mouse. There we re no test substance-related effects on gross pathology.

At 2000 ppm, the 18 month terminal s acrifice data indicated test su bstance-related changes in the kidneys, sciatic nerve, and trigeminal nerve (det ailed below). Test substa nce-related causes of death were limited to five 2000 ppm female mice that were euthanized due to clinical signs secondary to test substance-rela ted sciatic nerve degeneration.

At one year, minimal to mild tubular nephrosis in the pars recta, and an associated decrease in kidney weight parameters, was observed in both sexes at 2000 ppm. Minimal to mild degeneration of nerve fiber axons and myelin was observed in th e sciatic nerve of some mice, observed in one male and one female mouse, also at 2000 ppm.

Minimal to mild hepatocellula r hypertrophy in the 2000 ppm females was associated with a small increase in mean liver weight parameters. Both liver effects were consistent with pharmacological enzyme induction and were interpreted to be not adverse.

Organ weight effects at 2000 pp m included lower mean kidney (both sexes), as compared to controls. Lower mean kidney weights in both sexes correlated with adverse microscopic changes. At necropsy, there was a small increase in gros s kidney deformity in males. Microscopic kidney effects in males included the pres ence of an intratubular precipitat e as well as increased tubular epithelial nephrosis (deg eneration/necrosis), hyperplasia, a nd adenoma. An increase in the incidence (females) and severity (males) of chronic progressive nephropathy (CPN) was also

observed.

At 2000 ppm, microscopic nervous tissue effects in both sexes in cluded degeneration of sciaticPage 65 of 80 and trigeminal nerve axons and their associated myelin sheaths. Degeneration of axons in the dorsal funiculi of the spinal cord and the trigemin al tract in the pons of the brain (females only) were interpreted to be an extension of the scia tic and trigeminal nerve lesions, respectively.

The LOAEL is 2000 ppm (equivalent to 228/306 mg/kg/day in males/females, based on decreased body weight and body weight gain, n ervous system toxicity, and renal toxicity in both sexes. The NOAEL was 150 ppm (equivalent to 18/25 mg/kg/day in males/females).

Three (3/50) benign tubular adenomas in th e 2000/1000 ppm male kidneys were observed and may have been related to the test substance -related degenerative a nd hyperplastic changes observed in the 2000 ppm males. Based on this finding, the test subs tance was initially considered oncogenic in male mice. Revaluation of the renal tubular aden omas of the male mice was conducted by an independent kidney expert pathologist (MRID 48354403) and reviewed by a Pathology Working Group (MRID 48354405). The re nal tubular adenomas observed in male mice were considered by the PWG to be incide ntal and unrelated to the administration of pyroxasulfone. HED CARC considered the evid ence presented by the PWG and concluded that
the renal tubular adenomas observed in this mouse study were not treatment related.

Dosing was considered adequate based on decreas ed body weight and body weight gain, nervous system toxicity, and renal toxicity in both sexes.

This study is classified as acceptable/guideline and satisfies the guideline requirements (OPPTS 870.4200b; OECD 451) for a carcino genicity study in mice.Page 66 of 80 A.4.6 Mutagenicity

Gene Mutation
Guideline # 870.5100 Bacterial assay

Guideline #870.5300, In vitro mammalian cell gene mutation test
MRID 47889311 Acceptable Guideline Not mutagenic

Not mutagenic in the presence or abse nce of S9-activation up to the limit of solubility and cytotoxic effects.

Cytogenetics

Guideline # 870.5375, Clastogenicity in mammalian cells
MRID 47701719 Acceptable Guideline

Guideline #870.5395
Micronucleus test in bone marrow
MRID 47701720 Acceptable Guideline No evidience of clastogenicity up to its limit of solubility

No evidence of genotoxicity

A.4.7 Neurotoxicity

870.6100 Delayed Neurotoxicity Study – Hen - NA

870.6200 Acute Neurotoxicity Screening Battery - Rat

In an acute neurotoxicity study (MRID # 4770 1721), 10 Sprague-Dawley rats/sex/dose were administered a single oral (g avage, 10 mL/kg) dose of KIH- 485 TGAI (99.1% purity) suspended in 0.5% methylcellulose and Tween 80 at doses of 0, 20, 200, or 2000 mg/kg (limit dose) and

were observed for 2 weeks. A functional observa tional battery (FOB) and motor activity testing were performed during the pretest period (basel ine), and on Days 1 (app roximately 2 hours postdosing, time of peak effect), 8, and 15. At termination, 6 rats/sex/dose underwent whole-body in

situ perfusion, and tissues from the control and 2000 mg/kg groups were processed for neuropathology. References to 5 positive control studies were cited; however, no data were provided. No compound-related effects on mort ality, clinical signs, body weight, body weight gain, food consumption, and gross or neuropathol ogy were observed at any dose in either sex. No evidence of neurotoxicity was observed in the FOB or motor activity assessments.

The LOAEL was not observed. The NOAEL is 2000 mg/kg (limit dose) in rats.

This study is classified as acceptable/guideline and satisfies the guideline requirement (870.6200; OECD 424) for an acute neurotoxicity study in rats.Page 67 of 80 COMPLIANCE : Signed and dated Data Confiden tiality, GLP Compliance, Flagging, and Quality Assurance statements were provided.

870.6200 Subchronic Neurotoxicity Screening Battery

In a subchronic neurotoxic ity study (MRID# 47701722), 12 Sprague -Dawley rats/sex/dose were administered KIH-485 TGAI (99.1% purity) in th e diet at concentrations of 0, 25, 250, or 2500 ppm (equivalent to 0/0, 1.56/1.92, 15.85/19.60, a nd 161.48/199.59 mg/kg/day [M/F]) for 13 weeks. A functional observational battery (FOB ) and motor activity testing were performed during the pretest period (bas eline), and during Weeks 4, 8, and 13. At termination, 6

rats/sex/dose unde rwent whole-body in situ perfusion, and tissues from the control and 2500 ppm groups were processed for neuropathology. References to 5 positive control studies were cited; however, no data were provided.

No compound-related effects on mortality, clini cal signs, body weight, body weight gain, food consumption, food efficiency, and gross or neuropa thology were observed at any dose in either sex. No evidence of neurotoxicity was observe d in the FOB or motor activity assessments.

The LOAEL is 2500 ppm (equivalent to 161.4 8/199.59 mg/kg/day) based on muscular degeneration observed in females. The NOAEL is 250 ppm (equivalent to 15.85/19.60 mg/kg/day).

The study is classified as acceptable/guideline and satisfies the Guideline requirements (870.6200b) for a subchronic neurot oxicity study in the rat.

Compliance: Signed and dated Data Confidentialit y, GLP Compliance, Flagging, and Quality Assurance statements were provided.

870.6300 Developmental Neurotoxicity Study - Rat

In a developmental neurotoxicity study (MRID# 47701724), KIH-485 (Pyroxasulfone, 99.1% a.i., Batch# 05TG005) in 0.5% (w/v) sodium car boxymethylcellulose was administered daily by gavage (5 mL/kg) to groups of 24 mated female Sprague-Dawley rats/dos e at dose levels of 0, 100, 300, or 900 mg/kg/day from gestation day (GD) 6 through lactation day (LD) 7, inclusive. Subsequently, the pups were direc tly dosed via gavage at the sa me doses as their dams from post-natal day (PND) 7 to 20 or 21, inclusive.

Dams were allowed to deliver naturally and we re killed on LD 21, following weaning of their respective litters. On PND 4, at least twenty litters of appropriate size ( ≥ 8 pups/litter) were

available. These litters were standardized to 8 pups/litter; the remaining offspring and dams were

sacrificed and discarded without further examinations. Subsequently, 10-12 pups/sex/group werePage 68 of 80 allocated to Subsets 1-6 for ne urobehavioral testing (Subsets 2, 3, 4, and 5), neuropathological examination (Subsets 1 and 2). References for 7 positive control studies that provided data that validated the procedures and observers of the performing lab to assess motor activity, neurotoxicity and behavioral eff ects were provided; however, data from these studies were not provided.

Treatment of pregnant and lactating female rats with KIH-485 from GD 6 to LD 7 at doses up to 900 mg/kg/day was well tolerated. There were no trea tment-related effects on mortality, clinical signs of toxicity, body weight, body weight gain , food consumption, FOB parameters, gestation length, macropathology findings, or brain weights. Additionally, there was no effect of treatment on the mean number of implantations or litter size.

The maternal LOAEL was not observed. The maternal NOAEL is 900 mg/kg bw/day.

No effects on offspring survival or bodywei ght performance of the offspring to PND 7 (commencement of direct treatment of the offspr ing) were observed at any dose. Additionally, no effects on clinical condition or mean bodyweight gain were observed in the offspring during

direct administration of KI H-485 from PND 7 to 20/21.

Sexual maturation, assessed by the time of vagi nal opening or balano-p reputial separation was unaffected by treatment at all dos e levels. There was no clear e ffect of treatment on the FOB performance including all activity measures, au ditory startle response habituation, pre-pulse inhibition, or on the learning and memory of the offspring at any dose level.

Motor activity assessments on PND 13 and 17 of age indicated several differences in the activity of treated offspring of both sexes relative to concurrent controls. However, reported motor activity measurements were very highly vari able making conclusions difficult to make.
Coefficients of variation exceeded 50% and reached above 200%.

There were no treatment-related macroscopic find ings observed at scheduled termination and no histopathological changes in th e tissues presented for neuropa thological examination on PND 21

or 66.

The mean absolute brain weights of male o ffspring in the 900 mg/ kg/day group and female offspring in the 900 and 300 mg/kg/day groups on PND 21 were significantly lower (p<0.05)

than concurrent controls and the laboratory historical control, and this decr ease was dose-related.

There was no effect of treatment on the mean le ngth and width of the brain of males or females

on either PND 21 or 66.

Although the mean brain morphometric changes were stated to be within the limits of accuracy of the measurement, no valid posit ive control data were available to demonstrate that the testingPage 69 of 80 laboratory was capable of detecting changes in brain morphometry with known positive control substances (e.g., methimazole). The measurements in the treated groups were less than HC, as were the concurrent control values; however, a consistent pattern in reduced thicknesses was

observed. This is better seen when brain mo rphometric values in PND 21 males and females were compared to the conducting laboratory hist orical control data fr om four studies. The hippocampus thickness was decreased in males (-11%) and females (-8%) in the 900 mg/kg/day dose groups and in females at the 300 mg/kg/day dose (-6%). In female PND21 offspring this was accompanied by reduction in the thickness of the cerebellum (-19 to -20%) in the 300 and 900 mg/kg/day groups and a reductio n in the thickness of the cor pus callosum (-38%) in the 300 and 900 mg/kg/day dose groups. The decrease in the cerebellum thickness at the 100 mg/kg/day

dose in females was not considered to be tr eatment related since it was not accompanied by a decrease in the hippocampus thickness and the lack of effect on PND 66 and the absence of

associated neurobehavioral effects.

It is concluded that the LOA EL for offspring toxicity deri ved from this DNT study is 300 mg/kg/day based on decrease in brain weight in male and female offspring accompanied with a

decrease in the thickness of the hippocampus, corpus callosum and cerebellum in PND 21 female offspring. The NOAEL is 100 mg/kg/day.

With the deficiencies reported above, this study is classified acceptable/guideline and satisfies the guideline requir ement (OPPTS 870.6300; OECD 426) for a developmental neurotoxicity study in rats.

A.4.8 Metabolism

870.7485 Metabolism – Rat – Single Dose

In a rat metabolism study (MRID 47701728), four Sprague Dawley [Crl:CD(SD)] rats/sex/group were gavaged with a single dose of one of tw o radiolabeled forms of KIH-485 in 0.5% aqueous carboxymethylcellulose: [pyrazole- 14 C]-KIH-485 or [isoxazoline- 14 C]-KIH-485 at doses of either 10 mg/kg (low dose) or 700 mg/kg (hi gh dose) in a nominal dose volume of 10 mL/kg. Urine and feces were collected up to 96 hours afte r dosing, and expired air was collected in one group for up to 48 hours after dosing. For tissue distribution studies, 9 rats/sex/group were treated, and 3 rats/sex/gr oup were sacrificed at 3 (10 mg/kg) or 9 (700 mg/kg), 54 (both doses), and 96 (both doses) hours after treatment. Upon sacr ifice, selected tissue/ organ samples, as well as the residual carcass, were collected. All thes e samples were radioassayed, and selected fecal and urine samples were pooled and analy zed to determine a metabolic profile.

Absorption was at least 77-88% of the administ ered dose (AD) at 10 mg/kg, based on residues isolated in the urine, bile, and carcass. Abso rption was at least 22-26 % AD at 700 mg/kg, based on residues isolated in the urine, carcass, and ti ssues. Although bile was not collected in the 700Page 70 of 80 mg/kg animals, the parent was isolated in feces at 61-77% AD suggesting that limited absorption was occurring. (Absorbed parent compound is ex tensively metabolized as detailed below.)

Absorption was relatively quick at 10 mg/kg, with a t
max of 1.1-7.0 h in the blood and plasma of both sexes; at 700 mg/kg, the t
max was 7.0-17.3 hours. A 70-fold increase in dose resulted in an increase in radioactivity concentrat ions in the whole blood and plasma (C
max ) of 9-12 fold in both sexes treated with [pyrazole- 14 C]-KIH-485 and the [isoxazoline- 14 C]-KIH-485 treated females and 17-18 fold in the [isoxazoline- 14 C]-KIH-485 treated males. One possible explanation for this observation is limited absorption at the high dose, an explanati on that was corroborated by the large amount of parent compound is olated in the feces. An appare nt sex-related difference was noted in the [isoxazoline- 14 C]-KIH-485 animals. Half-lives were highly variable and ranged from 15-173 hours. Half-lives were always greater in the blood than in the plasma, suggesting the possibility of protein binding.

Excluding the gastrointestinal trac t (GIT), the highest tissue con centrations (μg equiv/g) were initially found in the bladder (males: 245-227; fe males: 12-19 at low dose and 178 at high dose) at both doses and labels. Initially, very high tissu e concentrations were also isolated in the prostate (16-47) at both doses and labels. High co ncentrations were also noted in the adrenals, kidney, liver, abdominal fat, uterus, blood, and pl asma at both doses, sexes (except uterus), and labels. After 96 hours, the concentration (μ g equiv/g) in blood was 0.08-2.49 (4.58 in the [isoxazoline- 14 C]-KIH-485 treated females) and in kidney was 0.10-1.30 in each sex, dose, and

label.

Following administration of [Ix- 14 C]-KIH-485, it was noticeable that concentrations in the stomach and stomach contents were much greater than in the [pyrazole- 14 C]-KIH-485 group. This suggested the possibility of active secre tion of radioactivity in to the stomach following administration of [Ix- 14 C]-KIH-485.

Total recovery was 91-105% AD. Most of the dose was excreted in th e urine within 24 hours regardless of sex, dose, or radiolabel; although, excretion was slowest in the 700 mg/kg males. Excretion was essentially complete by 48 hours (approximately 90% AD recovered in all the groups).

In the 10 and 700 mg/kg [pyrazole- 14 C]-KIH-485 group; slightly less residues were excreted in the urine and more residues in the feces in males compared to females; however, no sex

difference was noted when the other radiolabel was used. In the excretion balance groups, the excretion profiles were generally similar regardless of radiolab el. At 10 mg/kg, residues were found primarily in the urine (63-76% AD) a nd feces (13-29% AD). Less than 5% AD was isolated in the carcass and tissues, and <7% AD a nd was found in the cage wash and debris. Only negligible amounts of residues (<0.2% AD) were found in the expired air.Page 71 of 80 The excretion profile was diffe rent at 700 mg/kg compared to 10 mg/kg. At 700 mg/kg, residues were found primarily in the feces (70-80% AD) and urine (22-26% AD). Less than 0.3% AD was isolated in the carcass and tissues, and <5% AD and was found in the cage wash and debris.

In the 10 mg/kg bile-duct cannulated animals, there was a differing metabolic profile depending on the radiolabel. In the [pyrazole- 14 C]-KIH-485 group, residues we re found in the urine (62- 63% AD), bile (13-16% AD), f eces (6-10% AD), cage wash (6-7% AD), and cage debris and carcass (<1.2% AD). In the [isoxazoline- 14 C]-KIH-485 group, residues we re found primarily in the urine (44-50% AD), bile (33-37% AD), feces (1-6% AD), cage wash (7-9% AD), and cage debris and carcass (6-8% AD).

Techniques used in the isolation and identifi cation of the metabolites included the use of metabolite standards with HPLC, TLC, LC-MS, and GC-MS; enzyme deconjugation was used with some samples. KIH-485 underwent extensive metabolism following oral administration with up to 30 chromatographic regions of interest seen in the urine, up to 19 in the fecal extracts and up to 31 in bile. There was no significan t sex difference in the metabolism of KIH-485. Compared with low dose animals, there appeared to be saturation or in duction of metabolism at the high dose, which resulted in an altered metabolic profile.

The major metabolite in the ur ine of rats dosed with [Py- 14 C]-KIH-485 was M-13, accounting for up to 26% of the dose at the low dose level and up to 4% at the high dose level. M-7 and M-3

were the next most abundant metabolites. Most of the metabolites in urin e were produced by the cleavage between the pyrazole and isoxazoline ring structure.

When the rats were dosed with [Ix- 14 C]-KIH-485, there were three ma jor metabolites in urine,M-13 accounting for up to 20% of the dose, M-16 accounting for up to 19% of the dose and M26 accounting for up to 6.4% of the dose. A major portion of the radioactivity was highly polar and multi-component, containing 5-methyl-5 -isoxazoline carboxylic acid and M-30, and accounted for up to 34% of the dose.

The major component of the feces following dosi ng with both radiolabels was unabsorbed KIH485 accounting for up to 10% of the dose at the low dose level and up to 78% at the high dose level. The next most abundant metabolite was M-13, accounting for up to 3% of the dose.

In bile, the major metabolites of rats dosed with [Py- 14 C]-KIH-485 were two conjugated compounds one of which co-eluted with a sulpha te conjugate of hydrox y KIH-485 and the other co-eluted with M-13 glucuronide. These two regi ons each accounted for up to 3.1% of the dose. Another metabolite was M-13, accounti ng for up to 1.6% of the dose.

When the rats were dosed with [Ix- 14 C]-KIH-485, the major metabolite in bile was tentatively identified as M-26 accounting for up to 10% of the administered dose and was produced by thePage 72 of 80 cleavage of the pyrazole ring and su bsequent loss of the fluorine moieties. There were also two conjugated compounds one of which co-eluted w ith a sulphate conjugate of hydroxy KIH-485 and the other co-eluted with M-13 glucuronide. These two regions accounted for up to 7% and
3% of the dose each. There was one further me tabolite with a molecular weight of 347 accounting for up to 4% of the dose, which was te ntatively identified as a glutamyl cysteine conjugate of the isoxazoline ring.

This metabolism study in the rat is classified acceptable/guideline and satisfies the guideline requirement for a metabolism study [OPPTS 870.7485, OECD 417] in rats when considered in conjunction with the concurrently submitted metabolism study in rats (MRID 47701729).

870.7485 Metabolism – Rat – Repeat Dose

In a rat metabolism study (MRID 47701729), non-radiol abeled KIH-485 was or ally administered to four female Sprague Dawley (Crl:CD(SD) stra in rats per group at a nominal dose level of 10 mg/kg/day for 7 days, followed by a dose radiolabeled with 14 C at either the pyrazole (Py) or isoxazole (Ix) ring in 0.5% aqueous carboxymet hylcellulose and Tween 80 (<0.02% v/v): in a volume of 10 mL/kg. Additionally, one group of 4 rats were administered 10 mg/kg [pyrazole14 C]-KIH-485 in an oral gavage dose for 14 cons ecutive days. Urine and f eces were collected up to 96 hours after dosing. For ti ssue distribution studies, 12 ra ts/group were treated, and 3 rats/sex/group were s acrificed at 1, 20, 48, and 72 hours after treatment with [pyrazole- 14 C]-KIH485 or 2, 65, 168, and 288 hours after treatment with [isoxazoline- 14 C]-KIH-485. Upon sacrifice, selected tissue/organ samples, as well as the residual carcass, were collected. All these samples were radioassayed, and selected fecal and urine samples were pooled and analyzed to determine a metabolic profile.

This regime was based on an initial experiment in which four females received 14 daily doses of [Py- 14 C]-KIH-485. Blood and plasma concentrations had essentially ac hieved steady state conditions after 7 repeated or al administrations of [Py- 14 C]-KIH-485.

Absorption was at least 69% of the administered dose (AD) in all animals, based on residues isolated in the urine. Absorption was relatively quick at 10 mg/kg, with a t
max of 0.9-1.5 h in the blood and plasma (or 7.0 h in the blood of the of [isoxazoline- 14 C]-KIH-485 treated animals). Half-lives were highly variable (17-302 h) and s eemed to be higher in bl ood and higher in the [isoxazoline- 14 C]-KIH-485 treated animals. As half-lives were always greater in the blood than in the plasma, protein binding may be occurring. The following parameters were higher in the [isoxazoline- 14 C]-KIH-485 treated animals than the [pyrazole- 14 C]-KIH-485 treated animals in blood and plasma: t
max , t 1/2 , and AUC; C
max was higher in the blood only.

Excluding the gastrointestinal trac t (GIT), the highest tissue con centrations (μg equiv/g) were initially found in the bladder (20.7) and remain ed higher than plasma at 96 hours after dosingPage 73 of 80 (0.04 in bladder vs 0.03 in plasma). After 96 hour s post-dose, the highest residues were found in the erythrocytes, regardless of radiolabels. As indicated by pharmacokinetic parameters, the concentration of radioactivity remaining in the ti ssues at 96 hours after treatment were higher in the [isoxazoline- 14 C]-KIH-485 treated animals than the [pyrazole- 14 C]-KIH-485 treated animals.

Following administration of [Ix- 14 C]-KIH-485, it was noticeable that concentrations in the stomach and stomach contents were much greater than in the [pyrazole- 14 C]-KIH-485 group. This suggested the possibility of active secre tion of radioactivity in to the stomach following administration of [Ix- 14 C]-KIH-485.

Total recovery was 92-100% AD. Most of th e dose was excreted within 24 hours (>80% AD), predominantly in the urine (>60% AD). Afte r 96 hours, 0.5-3.9% AD remained in the carcass and tissues (higher in the [isoxazoline- 14 C]-KIH-485 treated animals) . The excretion profiles were generally similar regardless of radiolabel in that urine was the predominant route of excretion, feces was a major but s econdary route, and re latively little residues were isolated in the other samples.

KIH-485 is extensively metabolized , and there were up to 16 re gions of interests found during chromatography of the urine. Up to 17 propos ed structures are presented in the proposed metabolic pathway of KIH-485. The major metabolit es (structures presented in this report) include M-3, M-7, and M-16 in urine and M13 in the urine and feces. Thus, metabolism in the repeated dose animals was similar to that of th e single dose animals. Differences were noted, but these differences typically involved minor me tabolites. Overall, th e metabolism of KIH-485 appears to proceed by two main routes. One ro ute involved the cleavage of the sulfonyl group and subsequent sequential oxidations of this group on the pyrazole moiety or glutathione conjugation and subsequent hydrol ysis of the isoxazoline moie ty. The second route involved sequential oxidations of the parent compound.

This metabolism study in the rat is classified acceptable/guideline and satisfies the guideline requirement for a metabolism study [OPPTS 870.7485, OECD 417] in rats when considered in conjunction with the concurrently submitted metabolism study in rats (MRID 47701728).

COMPLIANCE : Signed and dated GLP Complian ce, Quality Assurance, and Data Confidentiality statements were provided. This study was conducted in accordance with the GLP standards of the United Kingdom and OECD (ENV/MC/CHEM (98) 17) . It was stated that the study was not conducted in accordance with 40 CFR Part 160; however, it was also stated that the GLP standards under which the study was c onducted are acceptable in Japan (JMAFF) and the USA (40 CFR Part 160).Page 74 of 80 870.7600 Dermal Absorption – Rat - NA

A.4.9 Immunotoxicity

870.7800 Immunotoxicity – Rat

The purpose of this study was to evaluate the potential of KIH-485 TGAI to suppress the primary humoral immune response following exposure via th e diet for 28 days. Groups of 10 rats per sex

were administered the test s ubstance at dietary levels of 0, 25, 250, or 7500 ppm (equivalent to 0/0, 2/2, 18/19, and 529/570 mg/kg/day in males/fe males). Body weights, food consumption measurements, and clinical observations were recorded during the in- life period. Prior to sacrifice, the immune system was stimulated by injecting sheep red blood cells (SRBC) on test day 22 and blood samples were collected from each rat on test day 28. The serum samples were assayed for their concentration of SRBC-speci fic IgM antibodies to provide a quantitative assessment of humoral immune response. Seru m from animals similarly challenged with a positive control immunosuppressive agent was anal yzed concurrently to provide confirmation that the assay performance was acceptable for detection of immunosuppression. At sacrifice, each animal was examined grossly and selected organs were weighe d (brain, spleen, and thymus).

There were no test substance-related effects obser ved on clinical observati ons, organ weights, or gross pathology.

Systemic toxicity was observed at 7500 pp m. Body weights and food consumption were decreased in the males throughout the study, resulting in an overall (Days 0-28) decrease in body weight gains of 30% and an ove rall decrease in food consumpti on of 13%. In the females, body weights were decreased on Days 21 and 28, resulting in an overall decrease in body weight gains of 56%, and food consumption was slightly decr eased throughout the study, resulting in an overall decrease of 8%. Overall food efficiency was also decreased by 19% in males and by 52%

in females.

There was no evidence of test substance-related t oxicity in male or fema le rats at 250 ppm or

lower.

The LOAEL for systemic toxicity is 7500 ppm (equivalent to 529/570 mg/kg/day in males/females), based on reduced body weights, body weight gains, food consumption, and food efficiency in both sexes. The NOAEL for systemic toxicity is 250 ppm (equivalent to 18/19 mg/kg/day in males/females).

There was no test substance-related evidence of immunosuppression in either male or female rats at any dietary concentration tested. The IgM tite rs were generally comparable across all groups.Page 75 of 80 The serum samples for the positive contro l groups that receiv ed 20 mg/kg/day of cyclophosphamide for 6 consecutive days demo nstrated 60 and 68% decreases in the IgM antibody response to SRBC for male and female rats respectively.

The LOAEL for immunotoxicity was not obs erved. The NOAEL for immunotoxicity was 7500 ppm (equivalent to 529/570 mg/k g/day in males/females).

This study is classified acceptable/guideline and satisfies the guideline requirements (OPPTS 870.7800) for an immunotoxicity study in rats.

COMPLIANCE : Signed and dated Data Confidentia lity, GLP Compliance, and Quality Assurance statements were provided.

870.7800 Immunotoxicity – Mouse

The purpose of this study was to evaluate the potential of KIH-485 TGAI to suppress the primary humoral immune response following exposure via the diet for 28 days. Groups of 10 mice per

sex were administered the test substance at dietary levels of 0, 40, 400, or 4000 ppm (equivalent to 0/0, 6/8, 61/77, and 633/791 mg/kg/day in male s/females). Body weights, food consumption measurements, and clinical observations were recorded during the in- life period. Prior to sacrifice, the immune system was stimulated by injecting sheep red blood cells (sRBC) on test day 23 (males) and 24 (females). Blood samples collected from each mouse on test day 28 (males) and 29 (females) were assayed for their concentration of sRBC-s pecific IgM antibodies to provide a quantitative assessment of humora l immune response. Serum from the animals similarly challenged with a positive control immunosuppressive agent (cyclophosphamide) was analyzed concurrently to provide confirmati on that the assay performance was acceptable for detection of immunosuppression. At sacrifice, ea ch animal was examined grossly and selected organs were weighed (bra in, spleen, and thymus).

There were no test substance-related eff ects observed on clinical observations, food consumption, organ weights, or gross pathology.

At 4000 ppm, body weights were decreased in th e males throughout the study by 7-9%, resulting in an overall (Days 0-28) decrease in body weight gains of 31%. Decreased (p<0.05) food efficiency was noted in the males on Days 0-7 ( ↓ 42%) and overall (Days 0-28; ↓ 31%); this was considered a reflection of the effects on body wei ght. In the females, body weights were slightly decreased throughout the study, resu lting in an overall decrease in body weight gains of 32%.

There was no evidence of test substance-related toxicity in male or female mice at 400 ppm or

lower.Page 76 of 80 The LOAEL for systemic toxicity is 4000 ppm (equivalent to 633/791 mg/kg/day in males/females), based on reduced body weights and body weight gains in both sexes, and decreased food efficiency in males. The NOAEL for systemic toxicity is 400 ppm (equivalent to 61/77 mg/kg/day in males/females).

There was no test substance-related evidence of immunosuppression in either males or females at any dietary concentration tested. The IgM titers were generally comparable across all groups. In contrast, male and female mice injected for fi ve consecutive days with 50 mg/kg/day of the known immunosuppressive agent, cyclophosphamide , demonstrated 100% and 92% inhibition, respectively, of the IgM antibody response to SRBC.

The LOAEL for immunotoxicity was not obs erved. The NOAEL for immunotoxicity was 4000 ppm (equivalent to 633/791 mg/k g/day in males/females).

This study is classified acceptable/guideline and satisfies the guideline requirements (OPPTS 870.7800) for an immunotoxicity study in mice.

COMPLIANCE : Signed and dated Data Confidentia lity, GLP Compliance, and Quality Assurance statements were provided.Page 77 of 80 APPENDIX B. Chemical Names A nd Structures Of Metabolites

B.1 Chemical Names and Structures of Pyroxasulfone and Metabolites.

Code IUPAC Name Common Name Chemical Structure

KIH-485 3-[[[5-(difluoromethoxy)-1-methyl-3- (trifluoromethyl)-1 H -pyrazol-4- yl]methyl]sulfonyl]-4,5-dihydro-5,5- dimethylisoxazole Pyroxasulfone

M-1 [5-(Difluoromethoxy)-1-methyl-3- (trifluoromethyl)-1 H -pyrazol-4- yl]methanesulfonic acid N/A

M-3 5-(Difluoromethoxy)-1-methyl-3- (trifluoromethyl)-1 H -pyrazol-4-carboxylic acid N/A

M-5 3-(5-Difluoromethoxy-3-trifluoromethyl-1 H

pyrazol4-ylmethanesulfonyl)-4,5-dihydro-5,5- dimethylisoxazole N/A

M-6 3-(5-Difluoromethoxy-1-methyl-3- trifluoromethyl-1 H -pyrazol-4- ylmethanesulfonyl)-4,5-dihydro-5,5- dimethylisoxazol-4-ol N/A

M-8 (5-Difluoromethoxy-1-methyl-3-trifluoromethyl1 H -pyrazol-4-yl)methanol N/A

M-9 5-Difluoromethoxy-3-trifluoromethyl-1 H

pyrazole-4-carboxylic acid N/A F F O N N CF 3

CH 3 S

O O

N O CH 3

C H

3

F F O N N CF 3

S

O O O H

CH 3

F F O N N CF 3

CH 3 O H

OPage 78 of 80 B.1 Chemical Names and Structures of Pyroxasulfone and Metabolites.

Code IUPAC Name Common Name Chemical Structure

M-9 malonyl

glucoside (RT26) 1 None provided NA

N-glycoside
of M-9 (Unknown @

13 mins.) 1 None provided NA

M-10 5-Difluoromethoxy-1-methyl-3-trifluoromethyl1 H -pyrazole-4-carbaldehyde N/A

M-11 [3-(5-Difluoromethoxy-1-methyl-3- trifluoromethyl-1 H -pyrazol-4- ylmethanesulfonyl)-4,5-dihydro-5- methylisoxazol-5-yl] methanol N/A

M-12 (5-Difluoromethoxy-3-trifluoromethyl-1 H

pyrazol-4-yl) methanol N/A

M-13 3-(5-Difluoromethoxy-1-methyl-3- trifluoromethyl-1 H -pyrazol-4- ylmethanesulfonyl)-4,5-dihydro-5- methylisoxazole-5-carboxylic acid N/A

M-15 2-Amino-5-[1-(carboxymethylamino)-3-(5,5- dimethyl-4,5-dihydroisoxazol-3-ylthio)-1- oxopropan-2-ylamino]-5-oxopentanoic acid N/A

M-16 2-Acetylamino-3-(5,5-dimethyl-4,5- dihydroisoxazol-3-ylthio)propanoic acid N/A H H

H O O H

H O

O H
OH O O

H OH O

N H N CF 3

F 2 HCO

O

N N

O F

F F

F F

H C

184 O

O H

O OH

OH

OH

OHPage 79 of 80 B.1 Chemical Names and Structures of Pyroxasulfone and Metabolites.

Code IUPAC Name Common Name Chemical Structure

M-22 5,5-Dimethyl-4,5-dihydroisoxazole N/A

M-25 [5-(Difluoromethoxy)-3-(trifluoromethyl)-1 H

pyrazol-4-yl]methanesulfonic acid N/A

M-28 3-[1-Carboxy-2-(5,5-dimethyl-4,5- dihydroisoxazol-3-ylthio)ethylamino]-3- oxopropanoic acid N/A

M-29 3-(5,5-Dimethyl-4,5-dihydroisoxazol-3-ylthio)-2- hydroxypropanoic acid N/A

M-30 2 3-cyano-2-hydroxy-2-methylpropanoic acid N/A

Metabolite A 1 None provided (cysteine glucose conjugate of isoxazoline-ring metabolite) N/A

Metabolite B 1 None provided (dimethylpropyl cysteine sulfoxide derivative) N/A

Metabolite C 1 None provided (malonyl dehydrogenated cysteine derivative of isoxazoline group) N/A

Metabolite D 1 None provided (cysteine conjugate of dimethyl acrylic acid) N/A

P1 1 None provided (malonyl cysteine conjugate of isoxazoline-ring products following reaction of pyroxasulfone or metabolites with glutathione) N/A F F O N H N CF 3

S

O O O H

O

H C H 3 COOH

CN

S O

C H 3 CH 3

NH 2 O

O-Glucose

S

NH 2 O

OH

O C H

3 CH 3

S

NH 2 O

OH C H

3 O O H

S N O C H 3 N H

O H O OH

O O

OHPage 80 of 80 APPENDIX C. Physical/Chemical Properties

Appendix C. Physicochemical Pr operties of Pyroxasulfone.

Parameter Value Reference 1

Melting point 130.7 oC MRID 47701615
pH 5.03 at 22 oC MRID 47701613
Density 1.60 g/cM-3 MRID 47701616
Water solubility 3.49 x 10 -3 g/L at 20.0 ± 0.5 oC MRID 47701619
Solvent solubility g/L at 20 oC
Methanol: 11.4
Acetone: > 250 Ethyl acetate: 97.0
n-Hexane: 0.0721
Toluene: 11.3
Dichloromethane: 151 MRID 47701620

Vapor pressure 2.4 x 10 -6 Pa at 25 oC MRID 47701621
Dissociation constant, pK

a Not applicable (pyroxasulfone Ncontaining rings are not expected to be protonated in a practical pH range) MRID 47701617

Octanol/water partition coefficient, Log(P O W ) log P OW = 2.39 at 25 oC, pH 8.7 MRID 47701618

UV/visible absorption spectrum at pH 1.13:
λ

max = 225.5 nm; ε = 7291 Lmol -1 cm -1 at pH 7.23:
λ

max = 225.0 nm; ε = 7340 Lmol -1 cm -1 at pH 10.91:
λ

max = 225.5 nm; ε = 7334 Lmol -1 cm -1 MRID 47701614

APPENDIX D. Studies Reviewed for Ethical Conduct

The PHED Task Force, 1995. The Pe sticide Handlers Exposure Database, Version 1.1. Task Force members Health Canada, U.S. Environmental Protection Agency, and the National Agricultural Chemicals Association, released February, 1995.

Agricultural Re-entry Task Force (ARTF) data base (SOP #3.1)