Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 3 of 107 7.0 A GGREGATE E XPOSURE /R ISK C HARACTERIZATION ................................................................. 27
8.0 C UMULATIVE E XPOSURE /R ISK C HARACTERIZATION ............................................................... 27
9.0 O CCUPATIONAL E XPOSURE /R ISK C HARACTERIZATION ........................................................... 28
9.1 Short- and Intermediate -Term Handler Risk .................................................................... 28
9.2 Short- and Intermediate-Ter m Post-Application Risk ...................................................... 30
10.0 R EFERENCES ...................................................................................................................... 31
A PPENDIX A. T OXICOLOGY P ROFILE AND E XECUTIVE S UMMARIES ............................................ 32
A.1 Toxicology Data Requirements ........................................................................................ 32
A.2 Toxicity Profiles ............................................................................................................. ... 33
A.3 Hazard Identification and Endpoint Selection .................................................................. 44
A.4 Executive Summaries........................................................................................................ 47
A PPENDIX B. M ETABOLISM S UMMARY T ABLE ............................................................................. 95
A PPENDIX C. P HYSICAL /C HEMICAL P ROPERTIES .......................................................................... 98
A PPENDIX D. S UMMARY OF F LUENSULFONE R ESIDUE D ATA FOR C UCURBIT AND F RUITING V EGETABLES ............................................................................................................................ 99
A PPENDIX E. R EVIEW OF H UMAN R ESEARCH ............................................................................. 101
A PPENDIX F. S UMMARY OF S UBMITTED S TUDIES ....................................................................... 102Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 4 of 107 1.0 Executive Summary

Makhteshim-Agan of North America (MANA) ha s petitioned the Agency to register the nematicide fluensulfone for use on cucurbit vege tables (Crop Group 9) and fruiting vegetables (Crop Group 8-10) and to establish permanent tole rances for residues resulting from those uses. This document presents the HED aggregate human health risk assessment for fluensulfone.

Fluensulfone {5-Chloro-2-[(3,4,4-tr ifluoro-3-buten-1-yl)sulfonyl]thi azole} is the first nematicide in the fluoroalkenyl class and it is currently undergoing global join t review [review partners are
the United States Environmental Protection Agency (U.S. EPA), the Pest Management Regulatory Agency (PMRA) of Canada and the Au stralian Pesticides a nd Veterinary Medicines Authority (APVMA)]. The submitted studies make up a complete database for purposes of conducting an aggregate human health risk assessment.

The proposed use is a single pre-plant applic ation of an emulsifiable concentrate (EC) formulation (4 lb a.i./gal, 480 g/L) by drip irrigation, banded spray, or broadcast spray. Treatment occurs at least 7 days prior to transp lanting, at a maximum rate of 3.5 lb a.i./A (3.84 kg a.i./ha). Based on the pre-plan t-incorporated nature of the us e pattern, occupational exposures are expected for persons invol ved in handling and applicatio n activities but not for postapplication activities. Based on the use sites, there is the potential for dietary exposure to
fluensulfone and/or its breakdow n products via consumption of tr eated crops and from drinking water. There are no proposed or existing uses that would result in resi dential exposure including exposure from volatization.

In mammals, exposure to fluensulfone results in effects on the hematopoi etic system (decreased platelets, increased white blood cells, hematocr it, and reticulocytes), kidneys, and lungs. Changes in clinical chemistry as well as in body weight were also observed across multiple studies and species. An acute neurotoxicity screening assay showed evidence of potential neurotoxicity; however, neurotox ic effects were not observed in any other study, including a subchronic neurotoxicity study. Evidence of qualitative, but not qua ntitative, increased susceptibility of infants and children to the e ffects of fluensulfone was observed in the twogeneration reproduction study in rats , wherein pup loss was observed at a dose that resulted in body weight effects in the dams. No evidence of either qualitative or qua ntitative su sceptibility was observed in developmental toxic ity studies in rats or rabbits. Dietary and inhalation studies in rats showed evidence of portal-of-entry e ffects in the forestomach, pharynx, epiglottis, and nasal cavity. Overall, the most sensitive endpo ints for assessing human health risk are the increased pup-loss effects for acute dietary e xposure; body weight, hematological and clinical chemistry changes for chronic dietary as well as dermal exposures (short/intermediate term); and clotting time, decreased thymus weight, and por tal-of-entry effects (histopathology of the epiglottis and nasal cavity) for inhalation exposur es (short/intermediate term). For assessing occupational oral and dermal expos ures, the standard inter- and in tra-species uncertainty factors of 10X are appropriate and result in a level of concern (LOC) of 100. For assessing occupational inhalation exposure, the use of a reference conc entration for humans (R fC) supports reducing the interspecies factor to 3X; however, an additiona l 10X factor is required because a no-observedadverse-affect level (NOAEL) was not found for the portal-of-entry effects, resulting in an LOC of 300. Given the different endpoints at the regu latory points of departure for the dermal andFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 5 of 107 inhalation routes of exposure, ri sk estimates from these two routes should not be combined. Based on the completeness of the to xicity data, the use of the mo st sensitive endpoints for risk assessment and the resulting low degree of concern regarding the observed qualitative susceptibility, and the conservatisms in the e xposure assessments, HE D is reducing the Food Quality Protection Act Safety Factor (FQPA SF) from 10X to 1X.

Crop metabolism studies and environmental fate studi es demonstrate that fluensulfone is rapidly and, in the case of crops, quantit atively broken down into smalle r molecules. In crops, the predominant residues are two sulfonic acid metabolites, referred to as BSA and TSA 1 . As a result, dietary exposures from tr eated crops will be primarily to metabolites of the parent compound and not the parent compound itself. In water, the predominant residues are expected to be a mixture of the parent compound, dech lorinated parent compound, a methyl-sulfone metabolite (MeS 1 ), and/or the sulfonic acid metabolit es, with the relative amounts of each dependent on the elapsed time after application and on the source of the water (ground water vs. surface water). HED has determined that the su lfonic acid metabolites ar e not of toxicological
concern based on the available data from acute toxicity, 28-day oral t oxicity, and ecotoxicity
studies based on no occurances of adverse effects. Therefore, human health exposure and risk estimates are based on residues of the parent compound, the dechlorinated parent compound, and on the methyl-sulfone metabolite, with the latter two being significant only in drinking water. Regulatory enforcement for tolerances in crops, however, is based on BS A only due to lack of the parent compound in harvested crops and th e need for separate analyses for BSA and
fluensulfone. Enforcement of tolerances can be accomplished by a validated analytical method with a limit of quantitation (LOQ) of 0.01 ppm.

Following application of fluensulfone according to the directions for use on the proposed label, residues of BSA in cucurbit vegetables a nd fruiting vegetables range from < 0.01 to approximately 0.4 ppm and are relatively consistent across all five representative commodities (cucumbers, melons, squash, pepper, and tomato). The consistency of the residues supports crop group tolerances at 0.80 ppm for cucurbit vegeta bles (Crop Group 9) an d 0.70 ppm for fruiting vegetables (Crop Group 8-10).

Dietary (food and water) risk estimates for flue nsulfone are below HED’s level of concern. Based on screening-level inputs, risk estimates across all population groups ranged from 2.0% to 7.4% of the acute population-adjusted dose (aP AD) and from 2.7% to 9.5% of the chronic population-adjusted dose (cPAD). For both acute and chronic durations, the maximum exposure estimates were for the “all infants” group. As ther e are no residential uses for fluensulfone, there are no residential exposure contribu tions to the aggregat e risk estimates. Th erefore, aggregate risk estimates are equivalent to the dietary ri sk estimates and are also below HED’s level of

concern.

For occupational scenarios, long-term exposures (> 6 months) are not expected to occur; however, short-term (1-30 days) and intermedia te-term (1-6 months) exposures may occur.
Provided the fluensulfone label is revised to specify additiona l personal protective equipment

1 BSA refers to the butene sulfonic acid metabolite (3,4,4-trifluoro-but-3-ene-1-sulfonic acid; Company Code M3627); TSA refers to the thiazole sulfonic acid metabo lite (5-chloro-thiazole-2-sulfonic acid; Company Code M3625); MeS refers to the methyl sulfone metabolite (5-c hloro-2-methyl sulfonyl thi azole; Company Code M-3626).Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 6 of 107 (PPE; e.g., PF5 respirator, engineering controls (enclosed-systems)), there are no risks of concern associated with mixing, loading, or ap plying fluensulfone via groundboom equipment. A potential dermal risk of concern was identi fied [margin of exposure (MOE) = 60, LOC = 100] for persons mixing/loading liquids for applicat ion via chemigation, even with maximum PPE [i.e., engineering controls (enclo sed systems)]. The maximum singl e application rate to achieve an MOE of 100 for mixing/loading liquids for chemigation is 2.0 lb a.i./acre usi ng engineering controls as the level of mitiga tion. Post-application exposures are expected to be negligible based on the pre-plant-incorporated nature of th e use pattern and the mechanical equipment and precautions associated with transplanting operations.

Aside from the potential occupational risk issu e noted above, there are no human health risk concerns that would preclude granting the requested regist rations for fluensulfone and establishing tolerances for residue s at the recommended levels.

Potential areas of environmental justice concerns were considered in th is human health risk assessment to the extent possible. For more information, see Section 3.5.

This risk assessment relies in part on data from studies in which adult human subjects were intentionally exposed to a pesticide or other chemical. Those st udies are subject to ethics review, have received that review, and are complia nt with applicable ethics requirements.

2.0 HED Recommendations

2.1 Data Deficiencies/Conditions of Registration

None.

2.2 Tolerance Considerations

2.2.1 Enforcement Analytical Method

A suitable method for tolerance enforcement has been developed and independently validated (Method ID PTRL-West 1997W). For all matrices, th e LOQ, defined as the lowest spiking level where acceptable precision and accuracy data were obtained, was determined to be 0.01 mg/kg.
The limit of detection was defined to be 30% of the LOQ (i.e., 0.003 mg/kg). The method uses solvent extraction, solid-phase extraction and clean-up, and anal ysis by reverse-phase HPLCMS/MS. Sufficient ion transitions ar e available for residue confirmation.

The FDA multi-residue methods are not suitable for detection and enforcement of fluensulfone residues (as the BSA metabolite) in non-fatty matrices.

2.2.2 International Harmonization

Fluensulfone is the subject of a global joint review by the U.S. EPA, PMRA, and APVMA, and
scheduled for review at the 2014 Join t Meeting on Pesticide Residues.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 7 of 107 At this time, the residue definition for enfo rcement is harmonized between the U.S. and Australia. For Canadian MRLs, PMRA is te ntatively recommending th e parent compound plus BSA as the residue definition for enforcement. The recommended regulatory levels for the
commodities in this assessment are harmonized am ong the joint review partners regardless of whether or not the parent compound is included in the residue definition.

2.2.3 Recommended Tolerances

HED has reviewed the available residue data and has determined the appropriate tolerance levels for residues of fluensulfone (Table 2.2.3). The recommended tolerance expression is as follows:
Tolerances are established for residues of the nematicide fluensulfone, 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 3,4,4- trifluoro-but-3-ene-1-sulfonic acid, calculated as the stoichiometric equivalent of
fluensulfone.

Table 2.2.3. Tolerance Summary for Fluensulfone.
Commodity Proposed Tolerance,

ppm HED-Recommended Tolerance, ppm Comments ( correct commodity definition )

Cucurbits 1.0 0.80 Vegetables, cucurbit, group 9
Fruiting vegetables 0.6 0.70 Vegetables, fruiting, group 8-10

2.2.4 Revisions to Petitioned-For Tolerances

The proposed tolerance levels differ from those being recommended by HED. Although both the petitioner and HED have used the OECD calculati on procedures to obtain tolerance levels, the residue definitions being used are different. The petitioner’s proposed levels are based on residues of BSA and TSA, combined and expres sed as parent fluensul fone whereas the HED
recommendations are based on residues of onl y the BSA metabolite, expressed as parent fluensulfone. Furthermore, the petitioner comb ined residue data from the representative commodities to obtain their proposed tolerances. In accordance with current policy, HED calculated separate tolerance levels for each re presentative commodity a nd then selected the maximum tolerance estimate within each crop group as the recommended level.

2.3 Label Recommendations

At the maximum single application rate, there are handler dermal and inhalation risks of concern at the label-specified PPE (i.e., long-sleeved sh irt, long pants, socks, shoes, and chemicalresistant gloves). Additional PPE (i.e., PF5 respir ator) and levels of mitigation (i.e., engineering controls (enclosed-systems)) are required on the proposed label to be protective of occupational handler exposure or the applicati on rate should be reduced (see Sect ion 9.1). Note that even at the engineering-controls level of mitigation, ther e is still a potential risk from dermal exposure for workers involved in mixing and lo ading activities for chemigation.Fluensulfone Human Health Risk Assessment DP No. D403767

Page 8 of 107 3.0 Introduction

3.1 Chemical Identity

Table 3.1. Fluensulfone Nomenclature. Compound Chemical Structure

Common name Fluensulfone Company experimental name MCW-2 IUPAC name 5-chloro-1,3-thiazol-2-yl 3,4,4-trifluorobut-3-en-1-yl sulfone CAS name 5-chloro-2-[(3,4,4-trifluoro-3-buten-1-yl)sulfonyl]thiazole

CAS # 318290-98-1 End-use product/EP 66222-EUG -- Emulsifiable Concentrate (40% a.i.) Chemical Class Fluoroalkenyl Known Impurities of Concern None

3.2 Physical/Chemical Characteristics

Technical-grade fluensulfone is a solid at r oom temperature but may be a liquid at warmer, environmentally relevant temperatures. The co mpound is moderately volatile; thus, there is some likelihood for exposure to fluensulfone in the vapor phase. The technical-grade material has a log K OW of 2.2 indicating a somewhat-fat-solubl e compound, but not to the degree that bioaccumulation is of particular concern. A listing of the physical/c hemical properties of fluensulfone are provided in Appendix C.

The exposure profile from dietary sources is primarily to the two main metabolites BSA and TSA rather than to the parent compound (whi ch was generally <LOQ in crop metabolism and field trials). Physicochemical properties were not provided for either BSA or TSA. As sulfonic acids, these two metabolites are expected to be much more hydrophilic than the parent compound and also less volatile.

3.3 Pesticide Use Pattern

The proposed use pattern specified in the end-us e product label is summarized in Table 3.3. Fluensulfone 480 EC may be applied as a pre-pl ant band/broadcast spray (incorporated by water or mechanical means) using ground or chemiga tion (i.e., drip irrigation), equipment. The proposed Restricted Entr y Interval (REI) is 12 hours for all crops.

The PPE statement on the proposed label requires applicators and other ha ndlers to wear longsleeved shirt and long pants, socks, shoes, and ch emical-resistant gloves, such as barrier laminate
or Viton ≥ 14 mils.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 9 of 107 Table 3.3. Summary of Directions for Use of Fluensulfone.
Applic. Timing, Type, and Equip. Formulation [EPA Reg. No.] Max. Single Applic. Rate Max. No. Applic. per

Season A Max.
Seasonal Applic. Rate,
lb a.i./A PHI, days Use Directions and
Limitations B

Cucurbit Vegetables - crop group 9 includes cucumbers, melons, (cantaloupes, watermelon, honeydew) squash;
and Fruiting Vegetables - crop group 8-10 includes bell pepp er, eggplant, groundcherry, hot pepper, pepino, pepper (except black), pimento, sweet pepper, tomatillo, tomatoes for processing

Pre-Plant: Drip irrigation, Band applicator, Broadcast spray Fluensulfone
480 EC (40% a.i.) (4.0 lb a.i./gal)

[66222-EUG] 1.75-3.50 lb a.i./A (56-112 oz/A) [1.92-3.84 kg a.i./ha] 1 appl. /year 3.5 lb a.i./A (112 oz/A)

[3.84 kg a.i./ha] N/A Apply with a
minimum of seven days before transplanting.

Do not plant any crops not on this
label into treated land for 365 days after application of the product.

A) Max. Single Application Rate = (4.0 lb a.i./gal )x(1 gal/ 128 fl oz)x(112 fl oz/A) = 3.5 lb a.i./A.

B) Minimum spray volume is 40 gal/A. (3.5 lb a.i./A)/ (40 gal/A) = 0.0875 lb a.i./gal spray concentration.

3.4 Anticipated Exposure Pathways

Fluensulfone has been proposed for use on cucurb it vegetables (e.g., cucumbers, melons, squash) and fruiting vegetables (e.g., tomatoes, peppers, eggpl ant). The product is meant to be applied to soil prior to transplant ing crops into the field. Humans may, therefore, be exposed to
fluensulfone-related residues from crops grown in treated soil and from migration of residues to surface and/or ground water source s of drinking water. From crops, the exposure will be to metabolites of fluensulfone (primarily BSA a nd TSA). In drinking water, the residues may
include BSA and TSA as well as parent fluensulfone, dechlorina ted fluensulfone, and the MeS
metabolite. The distribution of residues be tween these compounds will be dependent on the source of the drinking water (s urface or ground water) as well as the amount of elapsed time since application of the product.

At this time, there are no existing or proposed re sidential uses of fluensulfone and exposure via residential pathways is not expected.

In an occupational setting, app licators may be exposed while ha ndling the pesticide prior to application, as well as during application. The use of fluensulfone is appl ication to the soil with immediate incorporation to a depth of 6-8 inches seven days prior to transplanting. Given that use pattern and that large-scale transplant ope rations are with mechanical equipment which minimizes contact with soil, HED does not expect there to be significan t dermal or inhalation post-application exposures.

3.5 Consideration 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 ActionsFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 10 of 107 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 c onsumer subgroups according to well-established procedures. In line with O PP policy, HED estimates risks to population subgroups from pesticide exposures that are ba sed on patterns of that subgrou p’s food and water consumption,
and activities in and around th e home that involve pesticide use in a residential setting. Extensive data on food consumption pattern s are compiled by the USDA under the National Health and Nutrition Survey/What We Eat in America (NHANES/WWEIA) and are used in pesticide risk assessments for all registered food us es of a pesticide. Thes e data are analyzed and categorized by subgroups based on age and ethnic group. Additionally, OPP is able to assess dietary exposure to smaller, specialized s ubgroups and exposure assessments are performed
when conditions or circumstances warrant. When ever appropriate, non-dietary exposures based on home use of pesticide products and associated risks for adult applicators and for toddlers, youths, and adults entering or pl aying on treated areas post-application are evaluated. Further considerations are currently in development as OPP has committed resources and expertise to the development of specialized software and models that consider exposure to bystanders and farm workers as well as lifestyle and traditi onal dietary patterns among specific subgroups.

4.0 Hazard Characterization and Dose-Response Assessment

4.1 Toxicology Studies Available for Analysis

The toxicology database for flue nsulfone is complete and acce ptable for selecting toxicity endpoints and points of departure for risk assessment. The ava ilable toxicology studies include the following: (1) subchronic oral toxicity stud ies in rats, mice and dogs; (2) a 28-day dermal toxicity study in rats; (3) a 90-da y inhalation study in ra ts; (4) developmental toxicity studies in rats and rabbits; (5) a 2-generati on reproduction study in rats; (6) chr onic toxicity studies in rats and dogs; (7) carcinogenicity studie s in mice and rats; (8 ) a battery of genotoxi city studies; (9) four metabolism studies in rats; (10) a de rmal penetration study in rats and an in vitro comparative dermal absorption study with rat a nd human skin (testing an EC formulation of fluensulfone); (11) acute and subchronic ne urotoxicity studies in rats; and (12) an immunotoxicity study in mice.

For the BSA and TSA metabolites, toxicology studie s include acute oral st udies in rats, 28-day oral studies in rats, and bacterial a nd mammalian cell mutage nicity studies.

4.2 Absorption, Distribution, Metab olism, & Elimination (ADME)

In rats, fluensulfone is rapidly absorbed follo wing oral dosing. The time to maximum plasma levels (t max ) was 2-4 hours in males and females at the low dose (5 mg/kg) and 1-8 hours at the high dose (500 mg/kg/day). The time to maximum whole blood levels (t max ) was longer at 2-8 hours at the low dose and 48 hours at the high do se. The difference in whole blood and plasma

t max is related to fluensulfone reacting with th e free thiol moiety of globin in the blood. The majority of the dose was absorbed, as indicated by recovery of about 85% of the applied dose in the urine (including cage wash) at both doses te sted (5 and 500 mg/kg). Urine was the major route of excretion accounting for 75-88% of the administered dose (AD) with feces accountingFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 11 of 107 for 5-13% AD. Most of the AD (~80%) was excr eted during the first 24 hours at the 5 mg/kg dose level. Fluensulfone-related compounds did not accumulate in tissues; the total residual radioactivity in tissues /organs was ~3.1% AD at 51 hours post-dos ing. The highest tissue levels were observed in the liver, kidney, and gastrointestinal tr act. Identification of compounds in the urine (Table 4.2) showed no fluensulfone and indi cates that fluensulfone cleaves at the sulfonyl bridge. The metabolic pathway begins with flue nsulfone reacting with glutathione to displace
butene sulfinic acid which is then oxidized to BS A. The glutathione addu ct of the thiazole ring can then be cleaved to the cysteine conjugate, which is acetylated to the mercapturate. The glutathione ring is also cleaved to thiazole thiol (not detected in metabolism studies), which can either be oxidized to TSA or conjugated with gl ucuronic acid to give the two glucuronides. No major sex-related differences were observed in absorption, distribution, metabolism, or
excretion.

Table 4.2. Summary of Compounds in Rat Urine Following Dosing with 14 C-Fluensulfone. Chemical Identity % of Applied Dose
Butene-Radiolabel Thiazole-Radiolabel 5 mg/kg 500 mg/kg 5 mg/kg 500 mg/kg

Fluensulfone ≤ 0.1 ≤ 0.1 ≤ 0.1 ≤ 0.1
Butene sulfinic acid 32-35 53-57 -- -- Butene sulfonic acid (BSA) 4-5 4 -- -- Thiazole mercapturate -- -- 38-40 49-54 Thiazole glucuronide I and II -- -- 24-27 20-22 Thiazole sulfonic acid (TSA) -- -- 3-5 1-3

-- = this metabolite was not detectable b ecause it did not contain the radiolabel.

4.2.1 Dermal Absorption

An in vivo dermal absorption study in the rat and an in vitro dermal absorption study using rat
and human skin were submitted. Both studies used fluensulfone in an emulsifiable-concentrate (EC) formulation as the test material. The da ta demonstrated an inverse relationship between dosing concentration and pe rcent absorption. Based on in vivo dermal absorption observed in the
rat and in vitro comparative rat:human absorption data, an estimated human dermal absorption factor (DAF) was obtained as follows (all ab sorption values adjust ed for recovery):

(1) In the rat in vivo dermal absorption study, a dermal absorption of 58.0% (directly absorbed plus absorbable) was found at 24 hrs post-exposure using an application of 0.0953 mg/cm 2 ; (2) In vitro exposure of split-thickness rat skin under the same exposure and assessment conditions gave a dermal absorption of 93.5% (directly absorbed plus absorbable); (3) The ratio of the rat in vitro to the rat in vivo absorption is 1.6 (93.5/58.0) which is close to 1, indicating that the in vitro data is predictive of in vivo absorption; (4) Based on this ratio, a DAF for humans may be calculated using in vitro human dermal absorption (65.4%, directly absorb ed plus absorbable) observed in vitro under the same exposure conditions. The DAF fo r humans is therefore 65.4%/1.6 = 40.9%.

4.3 Toxicological EffectsFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 12 of 107 The hematopoietic system was the major target of fluensulfone activity in rats, dogs, and mice. Effects on the hematopoietic system included increased white blood cells (rats and mice), increased hematocrit (mice), decreased platelets (mice), and increased reticulocytes (dogs) in subchronic dietary studies at the lowest observable effect levels (LOAEL) with additional effects on the hematopoietic system observed at higher dose s. Effects on the hematopoietic system were also observed in chronic dietary studies for all species and, in the case of the chronic rat and dog studies, these effects were obser ved at the LOAELs. Effects on the hematopoietic system are
consistent with results from rat metabolism studi es which demonstrated that fluensulfone can
react with the free thio l moiety of globin, forming a covale nt linkage to the thiazole group displacing butene sulfinic acid. This led to slower elimination of the thiazole portion of fluensulfone from whole blood (time from dose to 50% of peak concentration, DT 50 = 6-9 days) compared to plasma (DT 50 = 16-17 hours).

Other effects commonly observed in mice, rats, a nd dogs included changes in clinical chemistry parameters and decreased body weights, both of which were often seen at the LOAELs in
subchronic and chronic studies. Altered clinic al chemistry parameters included decreased plasma alanine amino transferase (ALAT, rats an d dogs), increased choles terol (rats), increased triglycerides (rats), increased phospholipids (rats), increase d (mice) or decreased (dogs) bilirubin, decreased protein ( dogs), decreased albumin and al bumin/globulin ratio (dogs), decreased glucose (dogs), and increased TS H (dogs) in subchronic studies. For the hematological, clinical chemistry, and body weight effects observed in all three species, the dog appeared to be slightly more sensitive than mice and rats.

Fluensulfone also caused effect s on the kidney in male rats; howev er, the effects are consistent
with accumulation of α -2-microglobulin in the kidneys which is a male rat specific process that
is not relevant for human health risk assessment (USEPA 1991).

Effects on the lung were observed following chro nic dietary exposure in mice and rats. These
effects consisted of an increased incidence and se verity of lung interstiti al inflammation in the 2- year rat study and an increased incidence of br onchiolization (change fr om flattened epithelium to cubodial epithelium) in the 18-month mouse study.

Fluensulfone also appeared to cause portal-of -entry effects in rats . This included the forestomach (diffuse basal cell hyperplasia) a nd pharynx (mononuclear infiltrates) in dietary subchronic studies; the epiglottis (squamous metaplasia, epithelial hyperplasia, and focal mononuclear cell infiltrates) and nasal cavity (squamous epithelial hyperplasia) in the 90-day inhalation study; and an increase d incidence/severity of acanthos is/hyperkeratosis in the 28-day dermal study. The dermal effects were consider ed adaptive due to the moderate degree of acanthosis/hyperkeratosis observed.

There was no evidence of immunotoxicity in the ra t. Following a single oral administration, rats
exhibited decreased locomotor activity, decreased spontaneous activity, decreased rearing, and impaired righting response only on Day 1 in the acute neurotoxicity study. No evidence of neurotoxicity was observed in any other st udy, including a subchronic neurotoxicity study. Following dermal exposure for 28-days, systemic effects including increased reticulocytes, andFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 13 of 107 increased cholesterol were observed, but only at a very high dose ( 2000 mg/kg/day, which is twice the highest dose required by th e guidelines for a dermal study).

No evidence of increased quantitative or qualita tive susceptibility was seen in developmental toxicity studies in rats and ra bbits. In the rat developmental toxicity study, decreased fetal weight was observed in the presen ce of decreased body weight in maternal animals. In the rabbit developmental toxicity study, decreased fetal weight and incomplete ossification of digit 5 of the medial phalanx in both forelimbs were observed in the presence of decreased body weight in maternal animals. In the two generation reproduction study, ther e was no evidence of quantitative susceptibility; evid ence for qualitative susceptibility was manifested as decreased pup weight, decreased spleen weight, and an in crease in post-natal pup lo ss [post-natal days (PND) 1-4] which were seen at the same dose that caused decreases in body weight in the parental animals.

Chronic dietary exposure to fluensulfone in fema le mice resulted in an increased incidence of lung adenomas and carcinomas. The Cancer Assessment Review Committee (CARC) classified fluensulfone as having “Suggestive Evidence of Carcinogenicity” based on an increased
incidence of alveolar/b ronchiolar adenomas and carcinomas in the lung of mid- and high-dose female mice in the carcinogenicity study. A ll genotoxicity studies that were conducted on fluensulfone and three of its metabolites (thiaz ole sulfonic acid; TSA, but ene sulfonic acid; BSA, and methyl sulfone; MeS) were negative, except for MeS with Salmonella typhimurium strain
TA 100 in the absence of metabolic activation du ring a bacterial reverse mutation assay. There is little concern for the positive results as it was abolished by the presence of S9 and was not
observed in the other tester stra ins specific for base-pair changes. Based on the results of the genetic toxicology studies, it was concluded that there is no mutagenic concern for fluensulfone
or the fluensulfone metabolites tested.

Testing in acute lethality studies with fluensulfone technical-gra de material resulted in low toxicity classifications via the oral (Toxicity Category III), dermal (Toxicity Category III), and inhalation (Toxicity Category IV) ro utes of exposure. Fluensulfone technical-grade material was not an irritant to eyes (Toxici ty Category IV) or skin (Toxici ty Category IV) but was a skin
sensitizer. Similar results were obtained in acute lethality studies with an emulsifiable concentrate formulation (480 EC, Table A.2.2 in the Appendix).

Oral acute lethality studies were also conducted w ith the metabolites MeS, BSA, or TSA, each at 2000 mg/kg bw. By the oral route of exposure, MeS was of moderate toxicity (Toxicity Category II) and both BSA and TSA were of low t oxicity (Toxicity Category III). HED notes
that all animals treated with BS A or TSA survived the entire st udy period and that the toxicity category for those metabolites is based on the dosing level rath er than on actual LD 50 estimates. Furthermore in the BSA and TSA acute toxicity st udies, body weights of all animals were within the normal range, no macroscopic findings were reco rded at necropsy, and no clinical signs were observed except for three animals which exhi bited slightly ruffled fur two hours after administration of TSA, which persisted up to the three-hour observation in two animals. In 28- day oral toxicity studies with BSA and TSA, no treatment-relate d effects were observed at any dose (limit-dose = 1000 mg/kg/day). Based on the av ailable data addressing toxicity of the BSA and TSA metabolites, the Agency has determined that they are not of toxicological concern.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 14 of 107 4.4 Safety Factor for Infants and Children (FQPA Safety Factor)

The toxicology and the exposure databases for fl uensulfone support a redu ction of the required 10X FQPA safety factor to 1X based on the fo llowing considerations: the completeness of the toxicity database including adequa te studies to assess the potential suscepti bility in the young; there is no indication of quantitat ive susceptibility in the developm ental and reproductive toxicity studies, and there are no residua l uncertainties concerning pre- or post-natal toxicity; the endpoints and doses chosen for risk assessment are protective of the qua litative susceptibility observed in the 2-generation re production study; acute and subc hronic neurotoxicity studies (ACN and SCN) are available. In addition, all endpoints used in the risk assessment are protective of the evidence of neurotoxicity observed in the ACN, and a developmental neurotoxicity study is not requ ired. The dietary assessment is based on reliable data and modeled drinking water residues, and will not un derestimate exposure. Finally, there are no registered or requested uses for fluensulfone that would result in resi dential exposure.

4.4.1 Completeness of the Toxicology Database

The toxicology database is consid ered complete and is adequate for the purpose of assessing preand postnatal susceptibility. Acceptable/guidelin e developmental toxicity studies in rats and rabbits and a reproduction study in rats, as well as acute and subchronic ne urotoxicity studies in rats are available for FQPA assessment. A deve lopmental neurotoxicity study is not required.

4.4.2 Evidence of Neurotoxicity

Fluensulfone primarily targets the hematopoietic system, likely by inter acting with the thiol groups in globin as observed in the rat meta bolism study. Decreased locomotor activity in females, and decreased spontaneous activity, d ecreased rearing, and impaired righting response
in both sexes were observed in the acute neurotox icity study at the lowest dose tested. However, no other evidence for neurotoxicity was observed in the other studi es in the toxicity database, including a subchronic neurotoxic ity study. The doses and endpoint s chosen for risk assessment are all protective of the effects seen in the ac ute neurotoxicity study. For example, the endpoint used for acute dietary exposure (NOAEL = 16 mg /kg/day) from the 2-generation reproduction study is 6-times lower than the dose at which eff ects were observed in the acute neurotoxicity study (100 mg/kg). A developmen tal neurotoxicity study is not required at this time.

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

No evidence of quantitative or qualitative sus ceptibility was seen in developmental toxicity
studies in rats and rabbits. Offs pring effects in those studies occu rred in the presence of maternal toxicity and were not considered more severe th an the parental effects. However, there was evidence of increased qualitative, but not quantitative, susceptibil ity of pups in the 2-generation reproduction study in rats. Maternal effects obser ved in that study were decreased body weight and body weight gain; at the same dose, effect s in offspring were decreased pup weights, decreased spleen weight, and increased pup loss (PND 1-4).Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 15 of 107 Although there is eviden ce of increased qualitative sus ceptibility in the 2-generation reproduction study in rats, there are no residual uncerta inties with regard to pre- and/or post-natal toxicity following in utero exposure to rats or rabbits and pre- and/or post-natal exposures to rats. Considering the overall toxicity profile, the clear NOAEL for the pup effects observed in the 2- generation reproduction study, and that the doses a nd endpoints selected for risk assessment are equal to or less than the NOAEL from that study, the degree of concern for the susceptibility is
low.

4.4.4 Residual Uncertainty in the Exposure Database

There is no residual uncertainty in the exposure database. The current dietary assessment is based on high-end assumptions such as toleranc e-equivalent residue le vels of the parent compound in foods, 100% crop treated, and modeled estim ates of residues in drinking water. All of the exposure estimates are based on conser vative assumptions and are not likely to underestimate risk. Furthermore, ther e are no proposed residential uses.

4.5 Toxicity Endpoint and Point of Departure Selections

4.5.1 Dose-Response Assessment

The toxicity endpoints and point s of departure are presente d in Table 4.5.4.1 and Table 4.5.4.2, and the details for selecting toxicity endpoints and points of departure for various exposure scenarios are presented in Appendix A.

For fluensulfone, HED has select ed endpoints from the available data to establish points of departure (PODs) for the risk assessment that are protective of the obs erved adverse effects
across the entire database. Two studies with e ffects after one or two doses are available at similar LOAELs: pup mortality in the 2-gene ration reproduction toxicity study in rats (122 mg/kg/day) and decreased motor activity in th e ACN in rats (100 mg/kg/day). The POD for acute dietary exposure was chosen from the 2-generation reproductive toxicity study in rats. In this study, an increase in pup loss between PND 1 and 4 in the F1 and F2 offspring was observed at 122.0/169.1 mg/kg/day (M/F) w ith a NOAEL of 16.2/23.0 mg/kg/day (M/F). The majority of the pup deaths occurred on post-na tal day 2, although there were also a number that occurred on post-natal day 1. Therefore, the deaths may be a ttributable to a single dose. Effects observed in the ACN in rats also support the POD for acute di etary risk assessment. In this study, decreased motor activity in females and decreased spontan eous activity, decreased rearing, and impaired righting response in both sexes were observed at the lowest dose tested (100 mg/kg) on the day of dosing only. The decrease in motor activity had a steep dose-response curv e, with a decrease of 26% at 100 mg/kg/day, 42% at 400 mg/kg/day, and 58% at 1200 mg/kg/day. The POD was chosen from the 2-generation reproduction st udy because it had a clear NOAEL for the pup death and is protective of the effects observed at 100 mg/kg/day in the ACN.

The dog studies were chosen over the chronic dietary studies from others species because the dog was the most sensitive species for effects of fluensulfone having the lowest NOAELs and LOAELs amongst the chronic dietary studies. The POD for chronic dietary exposure was chosen from the chronic study in dogs, base d on a NOAEL of 3.1 mg/kg/day with decreasedFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 16 of 107 body weight, increased mean hemoglobin concentra tion distribution width, increased relative and absolute reticulocyte counts in both sexes, de creased prothrombin time in males and increased platelets in females at the LOAEL of 16 mg/ kg/day. The 90-day dog study was considered cocritical due to the findings of similar toxic effects occurring at the same LOAEL (17 mg/kg/day) with no progression of toxicity over time. In the 90-day dog study, increased relative and absolute reticulocyte counts, decreased bilir ubin, albumin, and A/G rati o, and pigmented Kupffer cells in both sexes, decreased body weight in females, and decr eased protein in males were observed at the LOAEL. While the 90-day dog study had a slightly lower NOAEL than the 1- year dog study (1.6 mg/kg/day), this is due to do se spacing differences betw een the two studies.

For short- and intermediate-term occupational exposure scenarios involving dermal exposure, HED did not chose the 28-day dermal toxicity study in rats conducte d with the technical material. Although that study is of the appropria te route and duration, the results do not appear to reflect the high dermal absorption (53-92 %) of the emulsifiable concentrate (480 EC)
observed in the in vivo and in vitro dermal penetrations studies. Therefore, HED chose the cocritical oral dog studies discussed above, with a 40.9% dermal absorption factor (see Section 4.2.1), to derive the point-of-departure. The two dog studies are appropriate for short- and intermediate-term exposure because the effects (hematology and clinical chemistry parameters) were seen starting at Week 6 in both studies and the lack of progr ession in the toxicity database (subchronic and chronic studi es of all species had sim ilar NOAELs and LOAELs).

For short- and intermediate-term occupational exposure scenarios involvi ng inhalation exposure, the POD was chosen from the 90-day inhalation toxi city study in rats. In this study, decreased body weight, decreased thymus weight, and histop athological changes in the nasal cavity of males; prolonged prothrombin time in females; a nd histopathological changes in the epiglottis in
both sexes were observed at the lowest dose te sted (0.04 mg/L). Since a NOAEL could not be identified in this study, an a dditional 10X uncertainty factor for the use of a LOAEL to extrapolate to a NOAEL was applied to short- and intermediate-term inhalation occupational exposure scenarios.

The methods and dosimetry equations describe d in EPA’s reference concentration (RfC) guidance and OPP’s guidance on the applica tion of the RfC methodology are suited for calculating human-equivalent concentrations (HECs) based on the i nhalation toxicity NOAEL for use in margin of exposure (MOE) calculatio ns. The regional-deposited-dose ratio (RDDR), which accounts for the particulate diameter (mass median aerodynamic diameter [MMAD] and geometric standard deviation [  g ] of aerosols), can be used to estimate the different dose fractions deposited along the resp iratory tract. The RDDR is also based on interspecies differences in ventilation and respiratory-tract surface areas. Thus, the RDDR can be used to adjust an observed inhalation particulate expos ure of an animal to the predicted inhalation exposure for a human. For the 90-day inhalati on study with fluensulfone, an RDDR for the extrathoracic region (based on e ffects observed in the nasal cav ity) was estimated at 0.143 based on the reported MMAD of 1.02 μm, standard deviation (  g ) of 1.59, and the respiratory-tract
surface area of the rat rela tive to the human. Since the rat in halation study was carried out for 6 hr/day for 5 days/week, the only exposure adjust ment was in the assumption of 8 hrs/day for human occupational exposures. The resulti ng HEC is 0.00429 mg/L. The level of concern (LOC) for this scenario is 300, which includes interspecies (3X), intr aspecies (10X), andFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 17 of 107 LOAEL-to-NOAEL (10X) extrapolations. The inters pecies factor was reduced from 10X to 3X due to the HEC calculation accounting for pharmac okinetic (not pharmacodynamic) interspecies
differences.

Endpoint for incidental oral expos ure were not selected because th ere are no residential uses of fluensulfone. Long-term dermal and inhalation exposures are not expect ed; therefore, endpoints
and doses were not selected.

4.5.2 Recommendation for Combining Routes of Exposures for Risk Assessment

Since the toxic effects selected as the basis fo r the dermal and inhalatio n risk assessments are different, these routes of exposure should not be combined in the risk assessment.

4.5.3 Cancer Classification and Ri sk Assessment Recommendation Report of the Cancer Assessment Review Committee, TXR No. 0056419, J. Rowland; K. Middleton, 7 May 2014

The Cancer Assessment Review Committee (CARC) classified fluensulfone as “Suggestive Evidence of Carcinogenicity” based on an increase d incidence of alveolar/bronchiolar adenomas and carcinomas in the lung of mid- and high- dose female mice in the oncogenicity study.
Tumors were not observed in the rat chronic toxicity/carcinogenici ty study in either sex or in male mice. Mutagenicity is not of concern. Although the mouse study showed that fluensulfone
was associated with alveolar/bronchiolar adenom as and carcinomas in the female, the EPA has determined that quantification of risk using a non-linear approach (i.e., RfD) for fluensulfone will adequately account for all chronic toxicity, including carcinogenicity, that could result from exposure to fluensulfone. That conclusion is based on the following c onsiderations: (1) the tumors occurred in only one sex in one species ; (2) fluensulfone and its metabolites are not mutagenic; (3) no carcinogenic resp onse was seen in either sex in the rat; and (4) the tumors in the mouse study were observed at a dose (39 mg/ kg/day) that is 12.6-time s higher than the dose chosen for risk assessment (3.1 mg/kg/day).

4.5.4 Summary of Points of Departure and T oxicity Endpoints Used in Human Risk
Assessment

Table 4.5.4.1. Summary of Toxicological Doses and Endpoints for use in Fluensulfone Dietary and NonOccupational Human Health Risk Assessments. Exposure/
Scenario Point of Departure Uncertainty/ FQPA Safety
Factors RfD, PAD,
Level of
Concern for
Risk
Assessment Study and Toxicological Effects

Acute Dietary (All Populations, including
Infants and
Children and
Females 13-49 years of age) Offspring
NOAEL= 16.2mg/kg/da

y UF A = 10x
UF H =10x FQPA SF= 1x Acute RfD = 0.16 mg/kg/day

aPAD = 0.16 mg/kg/day 2-generation reproduction – rat Offspring LOAEL = 122.0mg/kg/day based on an increase in pup loss between PND 1 and 4 in the F1 and F2 offspring with the majority of deaths occurring on day 2.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 18 of 107Table 4.5.4.1. Summary of Toxicological Doses and Endpoints for use in Fluensulfone Dietary and NonOccupational Human Health Risk Assessments. Exposure/
Scenario Point of Departure Uncertainty/ FQPA Safety
Factors RfD, PAD,
Level of
Concern for
Risk
Assessment Study and Toxicological Effects

Chronic Dietary (All Populations) NOAEL = 3.1 mg/kg/day UF A = 10x
UF H =10x FQPA SF= 1x Chronic RfD =
0.03 mg/kg/day

cPAD = 0.03 mg/kg/day Co-critical 90-day dog and chronic dog

Chronic: LOAEL = 16 mg/kg/day based on decreased body weight, increased mean hemoglobin concentration distribution width, and increased relative and absolute reticulocyte counts in both sexes, decreased prothrombin time in males and increased platelets in females.

Subchronic: NOAEL = 1.6 mg/kg/day LOAEL = 17.1 mg/kg/day based on decreased body weight in females and
increased relative and absolute reticulocyte counts, decreased bilirubin, decreased albumin, decreased A/G ratio, increased TSH, and pigmented Kupffer cells in both sexes.
Incidental Oral
All Durations A dose and endpoint for incidental oral exposure were not selected becaus e there are presently no
residential uses for fluensulfone.
Dermal All
Durations There are presently no residential uses for fluensulfone.

Inhalation All
Durations There are presently no residential uses for fluensulfone.

Cancer Classification: Suggestiv e evidence of carcinogenicity in humans based on lung tumors in female rats. The RfD is protective of potential cancer effects. Point of Departure (POD) = A data point or an estimated poin t that is derived from observed dose-response data and used to mark the beginning of extrapolation to determine risk associated with lower en vironmentally relevant human exposures. NOAEL = no observed adverse effect level. LOAEL = lowest observed adverse effect level. UF = uncertainty factor. UF A = extrapolation from animal to human (interspecies). UF H = potential variation in sensitivity among members of the human population (intraspecies). UF L = use of a LOAEL to extrapolate a NOAEL. FQPA SF = FQPA Safety F actor. PAD = population adjusted dos e (a = acute, c = chronic). RfD = reference dose. MOE = margin of exposure. LOC = level of concern.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 19 of 107 Table 4.5.4.2. Summary of Toxicological Doses and Endpoints for use in Fluensulfone Occupational Human
Health Risk Assessments. Exposure/
Scenario Point of Departure Uncertainty
Factors Level of
Concern for
Risk
Assessment Study and Toxicological Effects

Dermal Shortand
IntermediateTerm (1-30 days and 1-6 months) NOAEL= 3.1 mg/kg/day

DAF = 40.9% UF A =10x
UF H =10x Occupational
LOC for MOE
= 100 Co-critical 90-day dog and chronic dog

Chronic: LOAEL = 16 mg/kg/day based on decreased body weight, increased mean hemoglobin concentration distribution width, and
increased relative and absolute reticulocyte counts in both sexes, decreased prothrombin
time in males and increased platelets in females.

Subchronic: NOAEL = 1.6 mg/kg/day LOAEL = 17.1 mg/kg/day based on decreased body weight in females and
increased relative and absolute reticulocyte counts, decreased bilirubin, decreased albumin, decreased A/G ratio, increased TSH, and pigmented Kupffer
cells in both sexes.
Inhalation
Short- and
IntermediateTerm (1-30 days and 1-6 months) NOAEL= not established (<0.04 mg/L)

b HEC = 0.00429 mg/L

c HED = 0.244 mg/kg/day (low breathing rate), = 0.491 mg/kg/day (medium breathing rate), = 0.853 mg/kg/day (high breathing rate) UF A =3x
UF H =10x
UF L =10x Occupational
LOC for MOE
= 300 90-day inhalation toxicity – rat LOAEL = 0.04 mg/L (lowest dose tested) based on weight loss in males, prolonged prothrombin time in females, decreased thymus weight in males, histopathological changes in the epiglottis in both sexes, and histopathological changes in the nasal cavity of
males. Cancer Classification: Suggestiv e evidence of carcinogenicity in humans based on lung tumors in female mice. The RfD is protec tive of potential cancer effects.

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). UF L = use of a LOAEL to extrapolate a NOAEL. MOE = margin of exposure. LOC = level of concern. DAF = dermal absorption factor

b HEC = rat NOAEL x (daily duration adjustment) x weekly daily duration adjustment x RDDR =0.04 mg/L x (6 hr/8 hr) x (1) x 0.143 = 0.00429 mg/L.

c HED = HEC x human specific conversion factor x activity factor for the exposure scenario x daily duration = 0.00429 mg/L x 11.8 L/hr-kg x (0.6, 1.2, and 2.1 for low, medium, and high, respectively) x 8 hr.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 20 of 107 5.0 Dietary Exposure and Risk Assessment

5.0.1 Special Note on Fluoride

HED noted some concern about fluensulfone and its potential to result in increased fluoride exposure. In two subchronic toxici ty studies in rats, tooth discol oration was noted. In a 90-day oral study, several male rats at the high dose showed evidence of teeth discoloration. In a 90-day inhalation study, male rats had disc oloration or whitening of their teeth at the mid- and high-dose
and females had similar effects at all doses. Other studies in th e fluensulfone database did not identify similar effects but did show incr eased fluoride levels in teeth and bone.

It is important to note that the tooth discoloration found in the 90-day oral rat study was not reproduced in two recent subchroni c oral studies, a 90-day neurot oxicity study in rats and a 2- generation reproduction study in rats. These newe r and more detailed studies had similar dose
levels to the earlier subchronic rat study but showed no effects on teeth or any histopathological effects of fluorosis. Further, none of the chro nic studies show any disc oloration effects on teeth or histopathological effects on teet h or bones as would be expected if there was exposure to free
fluoride. Increased fluoride leve ls in teeth and bone that is f ound in the subchronic and chronic studies does not necessarily indicate exposure to free fluoride because th e analytical method for measuring fluoride levels includes fluorine bound in fluensulfone and its metabolites in its total
of fluoride residues. The tooth discolorati on seen in the 90-day inhalation study remains unexplained. The petitioner has hypothesized that this discoloration is due to decreased iron
stores as was observed with anot her pesticide, fluxapyroxad. While this hypothesis is plausible, unlike with fluxapyroxad, the petitioner here has not submitted data on iron levels in teeth to support the hypothesis. Nonetheles s, the overall weight of the t oxicity data as an indirect indicator of free fluoride exposure is not substantial. The inhala tion study raises some possibility of free fluoride exposure, but this study carries the l east weight for analyz ing potential residues from exposure to fluensulfone in or on food due to the significantly different metabolic effects
that can result from inhalation co mpared to oral exposures.

Given that the tooth discolora tion or whitening might be fluoros is caused by exposure to free
fluoride and that fluensulfone contains several fluorine atom s, EPA investigated whether
residues of fluensulfone in food might reasonably be expected to result in expos ure to finite
residues of free fluoride. Metabo lism studies in plants and animal s did not show that metabolism
of fluensulfone resulted in th e release of free fluoride as a me tabolite. However, these studies
are not definitive in that not all radio-labeled material in the studies was identified. More
conclusive is a statistical anal ysis of the fluoride levels in treated crops, soil, and water performed by the petitioner. That analysis show ed no statistically different levels of fluoride between treated and untreated samples. The i ndirect evidence suggest ing the possibility of fluensulfone resulting in free fl uoride exposure seen in the toxici ty studies does not outweigh this
direct examination of fluoride residues in food. Considering all of the direct and indirect information bearing on the potenti al for free fluoride exposure, a nd relying particularly on the statistical analysis of fluoride residues in crops, soil, and wate r, HED concludes that there is no reasonable expectation of exposure to finite resi dues of free fluoride resulting from exposure to
fluensulfone residues.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 21 of 107 5.1 Metabolite/Degradate Residue Profile

5.1.1 Summary of Plant and Animal Metabolism Studies

Studies depicting the metabolism of fluensulfone in lettuce, potato, and tomato as primary crops are adequate to delineate the residues of concer n for the currently proposed uses. The studies were conducted at approximately a 1X application rate but with a shorter pr e-plant interval (0 days) than specified on the label (7 days). Matu re and immature lettuce and potatoes and mature tomatoes were harvested and analyzed for residue s. The only major residues in the harvested
matrices were the BSA and TSA metabolites an d, with the exception of trace levels of
fluensulfone in immature lettuce and mature potato, no parent compound was detected. The short pre-plant interval is not expected to have significantly altered th e residue profile in the harvested

crops.

Rotational crop studies were conducte d with radish, lettuce, and whea t at plant-back intervals of 30, 120, and 360 or 390 days. As with primary crops, the major residues were the BSA and TSA metabolites. The similarity in the results is not unexpected given that for both types of studies, application of the test compound was to the soil early in the pl anting season. In the rotational crop studies, a low leve l of the parent compound was observed in lettuce, radish root, radish foliage, and wheat forage, hay, and straw (but not grain). Fluensulfone, when found, was typically 1 to 2 orders of magnitude less than the BSA or TSA residue levels. In all cases,
residues of fluensulfone and BS A were not quantifiable after th e 120-day plant-back interval whereas residues of TSA persis ted at quantifiable levels for at least one year.

Studies with laying hens and la ctating goats showed uptake a nd subsequent elimination of radiolabeled fluensulfone. In hens, radioactiv e residues in excreta accounted for approximately 80% of the dosed material for both label positions. Total radioactivity in eggs did not plateau within the eight dosing days of the hen study. Ra dioactive residues in eggs were not identified; most residues were aqueous phases in the ex traction schemes. In liver, 20-30% of the radioactivity was associated w ith proteins and/or amino aci ds and approximately 3% was
identified as TSA. Fluensulfone was a ma jor residue (21-55% TRR) in poultry fat.

In goats, the amount of radioactive residue in feces was similar between the two labels (approximate 14% of dose); however, the amount in urine appeared to be dependent on the label location (37% of dose for the thiazole-label, 70% of dose for the butene-label). In other matrices, the highest levels of radioactivity were associated with liver, kidney, milk fat, and blood. In excreta, the major iden tified residues were trifluorobut ene sulfinic acid and the MeS metabolite. No BSA, TSA, or fluensulfone was detected in any goat matrix. Radioactivity in milk and tissues was associated with gl ucose, lactose, proteins/amino acids, and triglycerides/fatty acids and the radioactivity appears to be due to incorporation of the
radiolabeled carbon.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 22 of 107 5.1.2 Summary of Environmental Degradation

In environmental fate studies, fluensulfone s howed different degradation outcomes depending on the environmental system. Fluensulfone is st able to hydrolysis under environmental conditions (pH 4-9) but is pr one to photolysis (t 1/2 of 0.6-0.7 days in water and t 1/2 of 39 days in soil). In soils, the rate of degradation is dependent on th e aerobic state of the sy stem and some parent compound persisted in both the aerobic and anaer obic soil metabolism studies. In an aerobic soil metabolism study, major degradation products were BS A, TSA, and MeS. In an anaerobic soil, fluensulfone degraded slowly (t 1/2 of 397 days) and the major degradates were BSA and TSA. In aquatic systems, the time to 50% dissipation (DT 50 ) was less dependent on the aerobic state, with estimates of 39-42 days under aerobic conditi ons and 26-28 days under anaerobic conditions. Under anaerobic conditions, the major degradates were deschloro-fluensul fone, MeS, BSA, and
trifluorobutene sulfinic acid. DT 50 estimates for the TSA and MeS metabolites under aerobic
soil conditions are 421 and 33 days, respectively. In field dissipation studies, DT 50 estimates for parent fluensulfone were in close agreement across all study sites: 10.1 days (California), 8.7 days (Florida), 10.8 days (Texas), and 7.4 days (Ontario). Maximum metabolite detections occurred in the top 15 cm of soil at 14 days afte r application for BSA and TSA and 28 days after application for MeS.

Fluensulfone is classified as “moderately mob ile” in soil. The BSA and TSA metabolites are both classified as “highly mobile” and the MeS metabolite as “mobile.”

5.1.3 Comparison of Metabolic Pathways

The available data indicate that fluensulfone has differe nt metabolic outcomes following application to environmental systems in comparis on to that following ingestion by animals. In target crops, rotational crops, and soil systems, the predominant residues are BSA and TSA, with
lesser amounts of MeS in soils. In aquatic systems, those three degradates were major residues along with fluensulfone, deschl oro-fluensulfone, deschloro-th iazole sulfonic acid, and
trifluorobutene sulfinic acid.

In rat, poultry, and ruminant st udies, the majority of the dose was absorbed and then excreted, (primarily in urine for mammals). Contrary to the environmental studies (crop/soil/aquatic),
BSA and TSA were minor residues in animals. Th e major residues in at least one animal matrix were fluensulfone (poultry fat), MeS (ruminant urine), trifluorobutene su lfinic acid (rat and ruminant urine), thiomercapturate (rat urine), and thi azole glucaronides I a nd II (rat urine).

The commonality of the residues in the target crop, rotational crop, and so il metabolism studies may reflect metabolism of the parent compound in the soil and uptake of the BSA and TSA metabolites by the growing crops; however, the da ta are insufficient to make a conclusive
determination.

5.1.4 Residues of Concern Summary and Rationale Holman, E. 27 August 2013, D412114

The Residues of Concern Knowledgebase S ubcommittee (ROCKS) has recommended that
enforcement for residues of fluensulfone be de termined by analysis of the BSA metabolite onlyFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 23 of 107 and that risk assessment be based on pare nt compound as well as MeS and deschlorofluensulfone, with the latter two for drinking water only (T able 5.1.4; D412114, E. Holman, 27 August 2013).

Table 5.1.4. Summary of Metabolites and Degradates to be included in the Risk Assessment and Tolerance Expression of Fluensulfone.
Matrix Residues Included in Risk Assessmen t Residues Included in Tolerance Expression Plants Primary Crop Fluensulfone BSA * , as parent equivalent Rotational Crop Fluensulfone BSA * , as parent equivalent
Livestock Ruminant Not determined Not determined Poultry Not determined Not determined Drinking Water Fluensulfone + MeS * + deschlorofluensulfone, expressed as parent equivalent Not Applicable

* BSA refers to the butene sulfonic acid metabolite (3,4 ,4-trifluoro-but-3-e ne-1-sulfonic acid; Company Code M3627); MeS refers to the methyl sulfone metabolite (5 -chloro-2-methyl sulfonyl thiazole; Company Code M3626).

Plants (Primary Crops): For dietary risk assessment of primary crops, parent compound should be the only residue included as a residue of concern. The metabolites TSA and BSA are the primary residues observed in metabolism and field trials. No quantifiable residues of parent
fluensulfone were observed in the crop field trials and only trace residues of parent fluensulfone were observed in potato tubers and immature lettuce from meta bolism studies. At levels expected in foods, the TSA and BSA metabolites ar e considered to be rela tively non-toxic.

Enforcement of tolerances should be via meas urement of residues of BSA only. The parent compound alone is a poor indicator of misuse due to the lack of quantif iable residues. The metabolites TSA and BSA both appear at quantifiabl e levels in the crop field trials and are good candidates for marker compounds. The TSA metabol ite may persist from one year to the next; therefore, the potential for TSA to accumulate makes BSA a better indicator of misuse.

Rotational Crops: Rotational crop data indicate that metabolites TSA and BSA are the primary anticipated residues, with minimal residues of fluensulfone observed. Although TSA was a primary residue in rotational crop studies, it may not be an adequate indicat or of misuse given its persistence. For tolerance enfo rcement, residues of BSA, expre ssed as parent equivalents, are considered an adequate indicator of misuse, as discussed above for primary crops.

Livestock: There are no livestock feed items associated with the current petition. Therefore, no decision is being made at this time.

Water: The ROCKS recommends including parent fluensulfone as well as the two transformation products MeS and de schloro-fluensulfone in the ri sk assessment. Although there are several major metabolites, acute toxicity da ta for the sulfonic acid metabolites (TSA and BSA) and MeS indicate that the acids, specifica lly TSA and BSA, are significantly less toxic than the parent fluensulfone and that MeS ha s similar toxicity to the parent compound. Furthermore, concern for toxicity of the acid metabolites (TSA, BSA, butene sulfinic acid, and deschloro thiazole sulfonic acid) is diminished because in contrast to the parent chemical, the sulfonic acid moiety in these metabolites si gnificantly increases th e likelihood of rapid bodyFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 24 of 107 excretion. First, the sulfonic ac id moiety significantly decreases lipophilicity relative to the parent chemical and thus the likelihood for bioaccumulation (p redicted Log P = -1.06 versus 1.96 for the parent chemical). Second, the sulfonic acid functional group is predic ted to be charged in the body at physiological pH (predicted pKa = -7. 11) and thus readily excretable by renal anion transporters. In addition, the rat metabolism study shows that parent fluensulfone is metabolized to TSA and BSA, consistent with these metabolit es being detoxification products. In contrast,
MeS and deschloro-fluensul fone are expected to be more simila r to parent fluensulfone in terms of excretion by the body. Theref ore, only fluensulfone, deschlor o-fluensulfone, and MeS were
included for risk assessment.

5.2 Food Residue Profile Doherty, M. 4 June 2014, D403813

The submitted residue chemistry studies were generally well conducted and are adequate for supporting regulatory conclusions, es tablishing appropriate tolerance levels for enforcement, and for purposes of risk assessment. The number of trials is sufficient and the geographic distribution of the trials captures the major fr uiting-vegetable and cucurbit-vegetable growing regions in the U.S., per OCSPP Series 860 Guidelines . Data from the trials are representative of worst-case residues expected to re sult from application of fluens ulfone according to the proposed use patterns, and the residue data are supporte d by adequate data from analytical method validation and storage stability studi es. For regulatory enforcement, analysis of residues can be accomplished through standard analytical technique s, and residues do not show any trends for dissipation during frozen storage.

Quantifiable residues of parent fluensulfone are not expected from applic ation patterns addressed by the field trials, including the pre-plant applicat ion listed on the proposed label, as well as preplant + post-transplant applicati ons. In biological environments , fluensulfone undergoes rather rapid cleavage at the sulfonyl moiety via glut athione conjugation to yield the BSA and TSA breakdown products. This, combined with its ea rly season use profile, re sults in the nearly complete absence of parent compound in any resi due studies to date. Quantifiable residues of the metabolites BSA and TSA are likely to occur a nd appear at similar levels across all of the examined crops (Appendix D); altho ugh there is a rather high degr ee of variability within each crop (relative standard deviations are on the order of 100%). As previously noted, the occurrence of BSA and TSA in crops may reflect uptake of those metabolites from soil. The data indicate that application by broadcast spray results in high er residues than application by drip irrigation; however, there are too few data points from th e drip irrigation trials for a meaningful statistical comparison. In melon, residues of BS A and TSA were lower in the pulp than in the peel. On a whole- fruit basis, the concentrations in pulp are expected to be, on average, 0.62x (BSA) and 0.50x (TSA) of those in the whole melon.

Under the currently proposed use pattern, tolerances are not needed for rotational crop commodities; however, HED notes that if future us es have higher application rates, limited field rotational crop data may be required. Also, if there are future us es on crops that are significant livestock feedstuffs, then livestock feeding studies may be necessary.

In the submitted tomato processing study, all anal ytes (fluensulfone, BSA, TSA, MeS) were <0.01 ppm in all commodities. As a result, the re sidue data cannot be used to derive processingFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 25 of 107 factors for tomato commodities. In a separa te study provided by the Australian extension of Makhteshim-Agan (Farmoz), quantifiable residu es of BSA occurred in the raw commodity making it possible to calculate the following pr ocessing factors for tomato. Typically, HED makes recommendations regarding tolerances for resi dues in tomato juice, paste, and puree. For those commodities, the processing factors are ≤ 1, indicating that tolerances for residues of
fluensulfone in tomato are sufficient to address residues in th e processed products.

Due to the dose-dependent accumulation of fluoride in teeth and bones observed in multiple toxicity studies, the petitioner provided the Agency with analys es of total fluoride residues in treated and untreated crop samples from the field trials as well as water and soil samples from the trial sites. Statistical analys es of the results were conducted to examine differences in total fluoride levels between samples from control and treated plots, controlling for combinations of location, time post application, and depth effects, as appropriate. Significant effects were found for location for the crop and soil matrices (locati on was not assessed for the water samples). For all matrices, the analyses found that fluensulfone treatment had no significant effect on total
fluoride levels.

5.3 Water Residue Profile Lin, J. 1 August 2013, D411774 Lin, J. 30 January 2014, D417634

The Environmental Fate and Effects Division (E FED) provided HED with modeled estimates of
fluensulfone residues in dr inking water (D411774, J. Lin, 1 August 2013 and D417634, J. Lin, 30 January 2014). The initial estimates (D 411774) are for total residues of concern (fluensulfone + deschloro-fluensulfone + MeS) in surface water (via Tier II PRZM-EXAMS) and ground water (via Tier I PRZM-GW and SCI-G ROW). Of those estimates, the highest estimated drinking water concentrations (EDWCs) were from PRZM-GW and resulted in dietary risk estimates of concern. Refined gr ound water EDWCs (D417634) we re provided and are
based on concentrations of fluensulfone and MeS modeled separately and then added together.
Deschloro-fluensulfone was not observed in the soil metabolism studies and was, therefore, omitted from the refined ground water analysis . Aside from those two items, no other modifications were made to the analysis a nd, even with the refinement, the ground water EDWCs represent high-end estimates. The EDWCs are summarized in Table 5.3.

Table 5.3. Fluensulfone Estimated Drinking Water Concentrations for Dietary Risk Assessment (D411774+D417634) Residue Source (Tier: Model) Use Rate, lb a.i./A/yr Acute EDWC, μg/L Chronic EDWC, μg/L Surface Water (Tier II: PRZM-EXA MS; D411774) 3.57 11.8 0.173 Ground water (Tier I: PRZM-GW; D417634) 3.57 77.6 52.5Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 26 of 107 5.4 Dietary Risk Assessment Doherty, M. 5 June 2014, D418204

5.4.1 Description of Residue Data Used in Dietary Assessment

For both the acute and chronic assessments for fl uensulfone, LOQ-level residues of fluensulfone (0.01 ppm) are assumed for all crops. For processed tomato commodities, empirical data for juice, puree, paste, and dried tomato show no concentration of parent residues. For all assessments, HED assumed that 100% of the crops are treated with fluensulfone.

5.4.2 Dietary Exposure Risk Estimates

Dietary exposure (food + drinki ng water) and risk estimates we re developed for the following population subgroups: the general U.S. population, all infants (< 1 year old), children 1-2, children 3-5, children 6-12, yout h 13-19, adults 20-49, females 13-49, and adults 50+ years old. Generally, HED is concerned when dietary risk estimates exceed 100% of the populationadjusted dose (PAD) for either acu te or chronic exposure scenarios. For fluensulfone, all dietary
risk estimates were well below 100% of either PAD (Table 5.4.3). The maximum exposure and
risk estimates occurred for th e “all infants” populati on subgroup and occupied 7.4% of the aPAD
and 9.5% of the cPAD.

Fluensulfone is classified as having “suggestive evidence of carcinogenic poten tial.” Risk
assessments based on the chronic RfD are cons idered adequate to address cancer risks.

5.4.3 Dietary Risk Assessment Summary Table

Table 5.4.3. Summary of Acute and Chronic Dietary Exposure and Risk Estimates for Fluensulfone.

Population Subgroup Acute (95 th Percentile) Chronic Cancer Exposure, mg/kg/day Risk, %
aPAD Exposure, mg/kg/day Risk, %
cPAD Total US Population 0.004252 2.4 0.001111 3.7 See Chronic
All Infants 0.013276 7.4 0.002845 9.5
Children 1-2 0.006543 3.6 0.001611 5.4
Children 3-5 0.005340 3.0 0.001360 4.5
Children 6-12 0.004084 2.3 0.000979 3.3
Youth 13-19 0.003548 2.0 0.000811 2.7
Adults 20-49 0.004176 2.3 0.001107 3.7
Adults 50-99 0.003754 2.1 0.001095 3.6
Female 13-49 0.004245 2.4 0.001103 3.7

Bold entries are maximum exposure and risk estimates.

6.0 Residential (Non-Occupational) Exposure/Risk Characterization

There are no existing or proposed uses of fluens ulfone that are define d as residential uses; therefore, a quantitative residentia l exposure assessment was not conducted.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 27 of 107 6.1 Residential Bystander Post-Application Inhalation Exposure

Volatilization of pes ticides may be a source of post-ap plication inhala tion exposure to individuals nearby pesticide app lications. The agency sought expert advice and input on issues
related to volatilization of pesticides from its Federal Insec ticide, Fungicide, and Rodenticide Act Scientific Advisory Panel (S AP) in December 2009, and received the SAP’s final report on March 2, 2010 ( http://www.epa.gov/scipoly/SA P/meetings/2009/120109meeting.html ). The agency has evaluated the SAP report and has de veloped a Volatilization Screening Tool and a subsequent Volatilization Screening Analysis ( http://www.regulations.gov/#!doc ketDetail;D=EPA-HQ-OPP-2014-0219 ). During Registration Review, the agency will utilize this analysis to determine if data (i.e., fl ux studies, route-specific inhalation toxicological studies ) or further analysis is required for fluensulfone.

6.2 Spray Drift

Spray drift is a potential source of exposure to those nearby pest icide applications. This is particularly the case with aerial application, but, to a lesser exte nt, spray drift can also be a potential source of exposure from the gr ound application methods (e.g., groundboom) employed for fluensulfone. The agency has been working with the Spray Drift Task Force (a task force composed of various registrants which was deve loped as a result of a Data Call-In issued by EPA), EPA Regional Offices and St ate Lead Agencies for pesticid e regulation and other parties to develop the best spray drift management prac tices (see the agency’s Spray Drift website for more information). The agency has also devel oped a policy on how to appropriately consider spray drift as a potential source of exposure in risk assessments for pesticides. The potential for spray drift will be quantitatively evaluated for each pesticide during the Registration Review process which ensures that all uses for that pe sticide will be considered concurrently. The approach is outlined in the revised (2012) Standa rd Operating Procedures for Residential Risk Assessment (SOPs) - Residential Exposure Assessm ent Standard Operating Procedures Addenda 1: Consideration of Spray Drift. This docum ent outlines the quantific ation of indirect nonoccupational exposure to drift.

7.0 Aggregate Exposure/Risk Characterization

In accordance with the FQPA, HED must consider and aggregate (add) pesticide exposures and risks from three major sources: food, drinking water, and residential exposures. In an aggregate assessment, exposures from relevant sources are added together and compared to quantitative estimates of hazard (e.g., a NOAEL or PAD), or th e risks themselves can be aggregated. When aggregating exposures and risks from various sources, HED consider s both the route and duration of exposure.

As previously noted, there are no re sidential uses for fluensulfone at this time; therefore, dietary (food and water) exposure is the only pathway a ppropriate for assessment. Aggregate risk estimates are equivalent to the dietary risk estim ates discussed in Section 5 and are below HED’s
level of concern.

8.0 Cumulative Exposure/Ri sk CharacterizationFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 28 of 107 Unlike other pesticides for which EPA has fo llowed a cumulative risk approach based on a common mechanism of toxicity, EPA has not made a common mechanism of toxicity finding as to fluensulfone and any other substances and fluensulfone does not a ppear to produce a toxic metabolite produced by other substances. For the purposes of this tolerance action, therefore,
EPA has not assumed that fluensulfone has a common mechanism of toxicity with other substances. For information regarding EPA’s efforts to determine which chemicals have a common mechanism of toxicity and to evaluate th e cumulative effects of such chemicals, see the policy statements released by EPA’s Office of Pesticide Programs concerning common mechanism determinations and proc edures for cumulating effects from substances found to have
a common mechanism on EPA’s website at http://www.epa.gov/pestic ides/cumulative/.

9.0 Occupational Exposure/ Risk Characterization Figueroa, Z. 18 July 2014, D403814

There is a potential for occupational exposure as sociated with handler activities (i.e., mixing, loading, and applying). Based on the use patter n and application timing (i.e., soil-incorporated application seven days before transplanting) , no post-application exposure is expected for workers re-entering treated areas. Based on the use patterns (i.e., 1 application per season) and resistance management practices, the duration of exposure for the proposed food uses is expected to be short-term (1-30 days) and intermediate-ter m (1-6 months). Long-term exposures (greater than 6 months) are not anticipated.

9.1 Short- and Intermediate-Term Handler Risk

It is HED policy to use the best available data to assess handler exposure. Sources of generic handler data, used as surrogate data in the absence of chemical-specific data, include the Pesticide Handlers Exposure Database Version 1.1 (PHED 1.1) and the Agricultural Handler Exposure Task Force (AHETF) database, or ot her registrant-submitted occupational exposure studies. Some of these data are proprietary (e.g., AHETF data), and subject to the data protection provisions of FIFRA. Default assu mptions established by HED’s Science Advisory Council for Exposure were used for parameters such as body weight, acres treated and amount
handled.

The maximum single application rates on the pro posed labels range from 1.75-3.50 lb a.i./A, and
were used to assess the handler exposure scenarios. Refer to Ta ble 3.3 for a complete use pattern description. The inputs for area treated were based on information in ExpoSAC Policy 9.1.

The short- and intermediate-term PODs are th e same for dermal and inhalation exposure. Therefore, the quantitative short-term handler assessment represents both exposure durations and is protective of intermediate-term exposure. A ll inhalation occupational handler exposure risk estimates were not of concern (MOEs > 300) usi ng some level of personal protective equipment (PPE; i.e., no respirator or PF5 re spirator). Furthermore, dermal risk estimates were also not of concern when using some level of PPE [e.g., ch emical-resistant gloves, engineering-controls (enclosed-systems)] with one exception. The mi xing/loading liquids for chemigation scenario resulted in a potential dermal ri sk of concern (MOE=60) using engi neering-controls (Table 9.1). The maximum single application rate to achieve an MOE of 100 for mixi ng/loading liquids forFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 29 of 107 chemigation is 2.0 lb a.i./acre using engineering cont rols as level of mitiga tion. At the request of the registration division, Table 9.1 includes an applicati on rate of 2.5 lb ai/A in addition to the
minimum and maximum labeled use rates.

There are handler dermal and inhalation risks ofconcern at the label-specified PPE (i.e., longsleeved shirt, long pants, socks, shoes, and chem ical-resistant gloves). Additional PPE (i.e., PF5 respirator) and levels of mitigation (i.e., engine ering controls (enclosed-systems)) should to be required on the proposed label to be protective of handler exposure or the application rate should
be reduced.

Table 9.1. Short- and Interm ediate-Term Occupational Handler Non-Cancer E xposure and Risk Estimates for Fluensulfone. Exposure
Scenario Mitigation
Level a Unit Exposure b (μg/lb ai) Application
Rate c Area
Treated d Dermal Inhalation

Dermal Inhalation Dose e (mg/kg/day) MOE f Dose g

(mg/kg/day) MOE h

Mixer/Loader Mixing/ Loading Liquids for Chemigation (AHETF/ PHED) Baseline 220 0.219 1.75 lb ai/A 350 A 0.69 5 0.00168 290 Engineering
Controls 8.6 0.0438 0.0269 120 0.000635 770

Baseline 220 0.219 2.50 lb ai/A 0.9870 3.1 0.002400 200 Engineering
Controls 8.6 0.0438 0.0385 81 0.000908 540

Baseline 220 0.219 3.50 lb ai/A 1.38 2 0.003350 150 Engineering
Controls 8.6 0.0438 0.0537 60 0.001280 380

Mixing/ Loading Liquids for
Groundboom (AHETF/ PHED) Baseline 220 0.219 1.75 lb ai/A 80 A 0.157 20 0.000384 1,300 Single Layer, Gloves, No Respirator 37.6 0.219 0.0269 120 0.000384 1,300

Baseline 220 0.219 2.50 lb ai/A 0.2250 14 0.000548 900 Single Layer, Gloves, No Respirator 37.6 0.219 0.0384 81 0.000548 900

Baseline 220 0.219 3.50 lb ai/A 0.315 10 0.000766 640 Engineering
Controls 8.6 0.083 0.0123 250 0.000290 1,700

Applicator Applying Liquids for
Groundboom Application (AHETF/ PHED) Baseline 78.6 0.34 1.75 lb ai/A 80 A 0.0562 60 0.000595 410 Single Layer, Gloves, No Respirator 16.1 0.34 0.0115 270 0.000595 410

Baseline 78.6 0.34 2.50 lb ai/A 0.0803 40 0.00085 290 Single Layer, Gloves, No Respirator 16.1 0.34 0.0165 190 0.00085 290

Baseline 78.6 0.34 3.50 lb ai/A 0.1120 30 0.00119 210 Single Layer, Gloves, PF5 Respirator 16.1 0.068 0.0231 130 0.000238 1,000

a. Mitigation level or PPE. Baseline = long-sleeved shirt, long pants, shoes, socks, no gloves, no respirator. b. Unit Exposures based on AHETF/PHED. c. Application Rates based on proposed use on label (3.5 lb ai/A for all crops). d. Exposure Science Advisory Council Policy No. 9.1. e. Dermal Dose (mg/kg/day) = Daily Unit Exposure (μg/lb ai) x De rmal Absorption Factor (40.9%) x Application Rate (lb ai/gal) x Area Treated / Body Weight (80 kg). f. Dermal MOE = NOAEL (3.1 mg/kg/day) / De rmal Daily Dose (mg/kg/day). LOC = 100. g. Inhalation Dose (mg/kg/day) = Daily Unit Exposure (μg/lb ai) x Application Rate (lb ai/acre) x Acres Treated / Body Weight ( 80 kg). h. Inhalation MOE = HED (0.491 mg/kg/day M/L or 0.244 mg/kg/day A ) / Inhalation Daily Dose (mg/kg/day). LOC = 300.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 30 of 107 9.2 Short- and Intermediate-T erm Post-Application Risk

Inhalation

There are multiple potential sources of post-app lication inhalation exposure to individuals performing post-applicati on activities in previously treated fields. These potential sources
include volatilization of pesticides and resuspension of dusts and/or particulat es that contain pesticides. The agency sought expert advice an d input on issues relate d to volatilization of pesticides from its Federal Insecticide, Fungicide , and Rodenticide Act Scie ntific Advisory Panel (SAP) in December 2009, and received th e SAP’s final report on March 2, 2010 ( http://www.epa.gov/scipoly/SA P/meetings/2009/120109meeting.html ). The agency has evaluated the SAP report and has developed a Vo latilization Screening Tool and a subsequent Volatilization Screening Analysis ( http://www.regulations.gov/ #!docketDetail;D=EPA-HQOPP-2014-0219 ). During Registration Review, the agency will utilize this analysis to determine if data (i.e., flux studies, route-specific inhalation toxicological studies) or further analysis is required for fluensulfone

In addition, the Agency is continuing to eval uate the available post- application inhalation exposure data generated by the Ag ricultural Reentry Task Force. Given these two efforts, the Agency will continue to identify the need for and, subsequently, the way to incorporate occupational post-application inhalation expos ure into the agency's risk assessments.

Although a quantitative occupational post-applic ation inhalation exposure assessment was not performed, an inhalation exposure assessment was performed for occupational/commercial handlers. Handler exposure resultin g from application of pesticides outdoors is likely to result in higher exposure than post -application exposure. Therefore, it is expect ed that these handler inhalation exposure estimates would be protec tive of most occupatio nal post-application inhalation exposure scenarios.

Dermal

Occupational quantitative dermal post-application risk estimates for agricultural workers following treatments to agricultural crops were no t assessed because fluensulfone is applied directly to the soil as a pre-plant/pre-emergent br oadcast spray or via drip-line chemigation seven days before transplanting (i.e., no crops are present when chemical is applied and the potential for workers to contact foliage b earing residues is minimal).

Furthermore, there is a potential for workers to contact treated soil during mechanically assisted cucurbit and fruiting vegetables transplanting activiti es. However, this event is likely to result in negligible exposures because tr ansplanting activities involve moving starter plants onto a rotating wheel while riding upon an implement being moved across a field. In such cases, there would be minimal contact with machinery, whic h may carry small amounts of treated soil, because of the possibility of mechanical injury. Also, commercial cultivation is typically in tarped, raised beds with small planting holes pun ched in the tops of the raised beds. This agricultural machinery also minimize the contact potential with treated soil. Even thoughFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 31 of 107 fluensulfone is stable under re levant laboratory conditions, the parent compound has half-life of approximately 10 days in field dissipation studi es, which further decreases the potential for occupational exposures with treated soil. Thes e conclusions are appropriate for the proposed use-pattern of fluensulfone. Therefore, dermal post-applica tion exposures are considered negligible and a quantitative dermal post-applic ation exposure assessm ent was not conducted.

Restricted Entry Interval

Fluensulfone is classified as T oxicity Category III via the dermal route and Toxicity Category IV for skin and eye irritation potenti al. It is a skin sensitizer. Since a quantitative dermal postapplication exposure assessment was not conducte d, the REI specified on the proposed label is based on the acute toxicity of fl uensulfone. Under 40 CFR 156.208 (c) (2) (iii), ai’s classified as
Acute III or IV for acute dermal , eye irritation and primary skin irritation are assigned a 12-hour REI. Therefore, the [156 subpart K] Worker Pr otection Statement interim REI of 12 hours listed in the proposed label is adequate to protect agricultural workers from post-application exposures
to fluensulfone.

10.0 References

Doherty, M. 4 June 2014, D403813. Fluensulfone . Petition for the Establishment of Permanent Tolerances and Registration fo r Use of the New Active Ingredient on Fruiting Vegetables (Crop Group 8-10) a nd Cucurbit Vegetables (Crop Group 9). Summary of Analytical Ch emistry and Residue Data. Doherty, M. 5 June 2014, D418204. Fluensuflone Acute and Chronic Aggregate Dietary (Food and Drinking Water) Exposure and Risk Asse ssments for the First Food Use Section 3 Registration Action on Fruiti ng and Cucurbit Vegetables. Figueroa, Z. 18 July 2014, D403814. Fluensulfone . Occupational and Residential Exposure and Risk Assessment to Support the Regist ration of the New Active Ingredient on Cucurbits and Fruiting Vegetables. Holman, E. 27 August 2013, D412114. Fluensulfone . Report of the Residues of Concern Knowledgebase Subcommittee (ROCKS). Lin, J. 1 August 2013, D411774. Fluensulfone: Drinking Water Exposure Assessment for Proposed New Registration for uses on cucurbits and fruiting vegetables. Lin, J. 30 January 2014, D417634. Fluensulf one: Refined Drinking Water Exposure Assessment for Proposed New Registrati on for Uses on Cucurbits and Fruiting Vegetables Based on Ground Water Sources. Kitchens, B. 2014, Fluensulfone OECD Monogr aph Volume 3, Annex B, Parts 1 and 2. Rowland, J. and Middleton, K. 7 May 2014, TXR NO. 0056419. Report of the Cancer
Assessment Review Committee.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 32 of 107 Appendix A. Toxicology Profil e and Executive Summaries

A.1 Toxicology Data Requirements The toxicology data requirements (40 CFR 158.340) for fluensulfone are presented in Table 1. Use of the new guideline numbers does not imply that the new (1998) guideline protocols were used.

Test Technical Fluensulfone Required Satisfied 870.1100 Acute Oral Toxicity 870.1200 Acute Dermal Toxicity 870.1300 Acute Inhalation Toxicity 870.2400 Primary Eye Irritation 870.2500 Primary Dermal Irritation
870.2600 Dermal Sensitization yes
yes
yes
yes
yes
yes yes
yes
yes
yes
yes
yes 870.3100 Oral Subchronic (rodent) 870.3150 Oral Subchronic (nonrodent) 870.3200 21/28-Day Dermal 870.3250 90-Day Dermal 870.3465 90-Day Inhalation yes
yes
yes

no
yes yes
yes
yes

- yes 870.3700a Developmental Toxicity (rodent) 870.3700b Developmental Toxicity (nonrodent) 870.3800 Reproduction yes
yes
yes yes
yes
yes 870.4100a Chronic Toxicity (rodent) 870.4100b Chronic Toxicity (nonrodent) 870.4200a Oncogenicity (rat) 870.4200b Oncogenicity (mouse) 870.4300 Chronic/Oncogenicity yes
yes
yes
yes
yes yes
yes
yes
yes
yes 870.5100 Mutagenicity—Gene Mutation - bacterial 870.5300 Mutagenicity—Gene Mutation - mammalian 870.5375 Mutagenicity—Structural Chromosomal Aberrations 870.5550 Mutagenicity—Other Genotoxic Effects yes
yes
yes
yes yes
yes
yes
yes 870.6100a Acute Delayed Neurotoxicity (hen) 870.6100b 90-Day Neurotoxicity (hen) 870.6200a Acute Neurotoxicity Screening Battery (rat) 870.6200b 90-Day Neurotoxicity Screening Battery (rat) 870.6300 Developmental Neurotoxicity no

no
yes
yes

no --

-- yes
yes

-- 870.7485 General Metabolism
870.7600 Dermal Penetration 870.7800 Immunotoxicity yes
yes
yes yes
yes
yesFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 33 of 107 A.2 Toxicity Profiles

Acute Toxicity Profile - Fluensulfone technical Guideline No. Study Type MRID(s) Results Toxicity Category

870.1100 Acute oral - rat 48574745 LD 50 = 671 III

870.1100 Acute oral - rat 48574746 LD 50 > 300 mg/kg but < 2000 mg/kg III

870.1200 Acute dermal - rat 48575035 LD 50 > 2000 mg/kg III
870.1300 Acute inhalation - rat 48574748 LC 50 > 5.1 mg/L IV 870.2400 Acute eye irritation - rabbit 48574750 Non-irritant IV
870.2500 Acute dermal irritation - rabbit 48574749 Non-irritant IV 870.2600 Skin sensitization - guinea pig 48574751 Sensitizer N/A

Acute Toxicity Profile - Fl uensulfone EC480 Formulation Guideline No. Study Type MRID(s) Results Toxicity Category

870.1100 Acute oral - rat 48574924 LD 50 > 2000 mg/kg III

870.1100 Acute oral - rat 48574918 LD 50 > 300 mg/kg but < 2000 mg/kg III

870.1200 Acute dermal - rat 48574962 LD 50 > 2000 mg/kg III
870.1300 Acute inhalation - rat 48574920 LC 50 > 6.0 mg/L IV 870.2400 Acute eye irritation - rabbit 48574926 Slightly irritating III 870.2400 Acute eye irritation - rabbit 48574922 Slightly irritating III
870.2500 Acute dermal irritation - rabbit 48574925 Non-irritant IV
870.2500 Acute dermal irritation - rabbit 48574921 Non-irritant IV 870.2600 Skin sensitization - guinea pig 48574923 Sensitizer N/A

Acute Oral Toxicity Profil e - Fluensulfone Metabolites Metabolite Study Type MRID(s) Results Toxicity Category

TSA Acute oral - rat 48574783 LD 50 > 2000 mg/kg III
MeS Acute oral - rat 48574787 LD 50 > 300 mg/kg but < 2000 mg/kg II

BSA Acute oral - rat 48574792 LC 50 > 2000 mg/kg III

Toxicity Profile - Fluensulfone Guideline Study Type Doses Tested/Study
Classification MRID Results

870.3100 2003-90-day dietary-rat mg/kg/day = M: 0, 4.3, 8.3, 34.9, 139.0 F: 0, 4.8, 11.7, 53.1, 148.7

Acceptable/Guideline 48575035 NOAEL (mg/kg/day): M=8.3, F=11.7 LOAEL (mg/kg/day): M=34.9, F=53.1 based on kidney effects, observations in the pharynx and forestomach, increased cholesterol in females, and decreased body weight and body weight gain in males.

870.3100 2002-28-day dietary-rat mg/kg/day= M: 0, 10.4, 42.5, 152.0 F: 0, 12.2, 37.2, 165.9

Acceptable/Guideline 48575023 NOAEL (mg/kg/day): M=10.4, F=12.2 LOAEL (mg/kg/day): M=42.5, F=37.2 based on decreased body weight gain in both sexes, increased WBC in females, increased cholesterol in males and renal pathology in males.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 34 of 107 Toxicity Profile - Fluensulfone Guideline Study Type Doses Tested/Study
Classification MRID Results

870.3100 2003-90-day dietary-mouse mg/kg/day = M: 0, 11, 51, 228 F: 0, 18, 68, 252

Acceptable/Guideline 48574753 NOAEL (mg/kg/day): M=11, F=18 LOAEL (mg/kg/day): M=51, F=68 based on increased bilirubin, hematology effects in males, and reduced food consumption in females. 870.3100 2003-28-day dietary-mouse mg/kg/day = M: 0, 30, 101, 375 F: 0, 41, 155, 353

Acceptable/Guideline 48575024 NOAEL (mg/kg/day): M=30, F=41 LOAEL (mg/kg/day): M=101, F=155 based on decreased food consumption, decreased platelets in males, decreased absolute heart weight in males, and increased relative liver weight in
females. 870.3150 2002-90-day dietary-dog mg/kg/day = M: 0, 0.2, 1.6, 17.1 F: 0, 0.2, 1.8, 18.0

Acceptable/Guideline 48574755 NOAEL (mg/kg/day): M=1.6, F= 1.8 LOAEL (mg/kg/day): M=17.1, F=18.0 based on decreased plasma and liver ALAT, liver effects, hematological findings, altered clinical chemistry, increased TSH, decreased body weight gains and decreased body weight in
females. 870.3150 2002-28-day dietary-dog mg/kg/day = M: 0, 1.9, 7.3, 31 F: 0, 2.0, 7.9, 30

Acceptable/Guideline 48574752 NOAEL (mg/kg/day): M=7.3, F= 7.9 LOAEL (mg/kg/day): M=31, F=30 based on slight decreases in body weight and slight increases in liver and thyroid weight. 870.3200 2011-28-day
dermal-rat mg/kg/day = 0, 80, 400, 2000

Acceptable/Guideline 48574758 Systemic: NOAEL (mg/kg/day):
400 LOAEL (mg/kg/day): 2000 based reduced body weight gain, increased prothrombin time and increased relative spleen weight in males; increased reticulocytes and
increased cholesterol in females.

870.3465 2012-90-day
inhalation-rat mg/L/day = 0, 0.04, 0.2, 1.0

Acceptable/Guideline 48575045 NOAEL (mg/L/day):
Not determined LOAEL (mg/kg/day): 0.04 based on weight loss in males, prolonged prothrombin time in females, decreased thymus weight in males, histopathological changes in the epiglottis in both sexes, and histopathological changes in the nasal cavity of males.

870.3700 2003- developmental

tox-rat mg/kg/day = 0, 8, 50, 300

Acceptable/Guideline 48574771 Maternal: NOAEL (mg/kg/day): 50 LOAEL (mg/kg/day): 300 based on decreased body weight and food consumption.

Developmental: NOAEL (mg/kg/day): 50Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 35 of 107 Toxicity Profile - Fluensulfone Guideline Study Type Doses Tested/Study
Classification MRID Results

LOAEL (mg/kg/day): 300 based on decreased body weight.

870.3700 2009- developmental
tox-rabbit mg/kg/day = 0, 2.5, 10, 40

Acceptable/Guideline 48574770 Maternal: NOAEL (mg/kg/day): 10 LOAEL (mg/kg/day): 40 based on transient weight loss and decreased food consumption, GD6-9.

Supported by significant toxicity at 50 mg/kg/day (marked body weight loss, cold ears, decreased food intake, and discolored urine) observed in the range finding study.

Developmental: NOAEL (mg/kg/day): 10 LOAEL (mg/kg/day): 40 based on reduced body weight and incomplete ossification of digit 5 of the medial phalanx in both
forelimbs.

870.3800 2011-2- generationreproductionrat mg/kg/day = P, male: 0,1.9, 15.4, 114.2, P, female: 0, 2.7, 22.8,
164.8 F1, male: 0, 2.0, 17.0,
129.7 F1, female: 0, 2.9, 23.3,
169.1

Acceptable/Guideline 48574769 Parental: NOAEL (mg/kg/day): M=16.2, F=23.0 LOAEL (mg/kg/day): M=122.0, F=169.1 based on decreased body weight

Reproductive: NOAEL (mg/kg/day): M=16.2, F=23.0 LOAEL (mg/kg/day): M=122.0, F=169.1 based on an increase in post-natal pup loss in both P and F1 generations.

Offspring: NOAEL (mg/kg/day): M=16.2, F=23.0 LOAEL (mg/kg/day): M=122.0, F=169.1 based on decreased pup and spleen weights. 870.4100 2011-1-year dietary-dog mg/kg/day = M: 0, 0.1, 1.5, 3.1, 16.0 F: 0, 0.1, 1.5, 3.3, 16.2

Acceptable/Guideline 48574756 NOAEL (mg/kg/day): M=3.1, F= 3.3 LOAEL (mg/kg/day): M=16.0, F=16.2 M: based on decreased body weight and changes in hematological and clinical chemistry parameters in both sexes.
870.4200 2011-18-month carcinogenicity

-mouse mg/kg/day = M: 0, 4.2, 27.6, 152.3 F: 0, 6.4, 39.0, 188.4

Acceptable/Guideline 48574766 NOAEL (mg/kg/day): M=4.2, F=6.4 LOAEL (mg/kg/day): M=27.6, F=39.0 based on an increased incidence of lung bronchiolization and decreased body weight and body weight gain in males. Increased incidence of lung adenomas
above historical controls was observed inFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 36 of 107 Toxicity Profile - Fluensulfone Guideline Study Type Doses Tested/Study
Classification MRID Results

females; combined adenoma/carcinomas was also increased (carcinoma only increased trend). 870.4300 2011-2-year toxicity/carcino genicity-rat mg/kg/day =

Oncogenicity group M: 0, 1.4, 9.6, 57.7 F: 0, 1.7, 11.6, 69.3

Chronic toxicity group M: 0, 1.6, 11.0, 66.3 F: 0, 1.9, 13.1, 75.2

Acceptable/Guideline 48574765 NOAEL (mg/kg/day): M=9.6, F=11.6 LOAEL (mg/kg/day): M=57.7, F=69.3 based on decreased body weight in males, and hematology and clinical chemistry findings and histopathological changes in the lungs and esophagus in both sexes.

870.5100 2002-Bacterial

reverse mutation assay (MCW-2) Test 1: 0, 16, 50, 158, 500, 1581, or 5000  g/plate ( Salmonella typhimurium ) in the presence and
absence of metabolic activation (+ S9). Test 2: 0, 1000, 2000, 3000, 4000, 5000, or 6000

 g/plate ( Salmonella typhimurium ) in the presence and absence of metabolic activation (+ S9). Test 3: 0, 375, 750, 1300, 2600, or 5200  g/plate ( Salmonella typhimurium )
in the absence of metabolic activation (-S9).

Acceptable/Guideline 48574759/
48575015 Negative with strains TA1535, TA1537, TA98, and TA102.

Weakly positive with strain TA 100.

870.5100 2008-Bacterial

reverse mutation assay (MCW-2) Test 1: 0, 3, 10, 33, 100, 333, 1000, 2500, or 5000

 g/plate ( Salmonella typhimurium-Escherichia
coli ) in the presence and
absence of metabolic activation (+ S9). Test 2: 0, 33, 100, 333, 1000, 2500, or 5000

 g/plate ( Salmonella typhimurium-Escherichia
coli ) in the presence and
absence of metabolic activation (+ S9).

Acceptable/Guideline 48574760 NegativeFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 37 of 107 Toxicity Profile - Fluensulfone Guideline Study Type Doses Tested/Study
Classification MRID Results

870.5100 2011-Bacterial

reverse mutation assay (MCW-2) Test 1: 0, 10, 32, 100, 316, 1000, 2500, or 5000

 g/plate ( Salmonella typhimurium-Escherichia
coli ) in the presence and
absence of metabolic activation (+ S9). Test 2: 0, 3, 10, 32, 100, 316, 1000, 2500, or 5000

 g/plate ( Salmonella typhimurium-Escherichia
coli ) in the presence and
absence of metabolic activation (+ S9).

Acceptable/Guideline 48574761 Negative

870.5100 2010-Bacterial

reverse mutation assay (MCW-2
metabolite #3625) Test 1: 0, 3, 10, 33, 100, 333, 1000, 2500, or 5000

 g/plate ( Salmonella typhimurium-Escherichia
coli ) in the presence and
absence of metabolic activation (+ S9). Test 2: 0, 33, 100, 333, 1000, 2500, or 5000

 g/plate ( Salmonella typhimurium-Escherichia
coli ) in the presence and
absence of metabolic activation (+ S9).

Acceptable/Guideline 48574784 Negative

870.5100 2010-Bacterial

reverse mutation assay (MCW-2
metabolite #3626) Test 1: 0, 3, 10, 33, 100, 333, 1000, 2500, or 5000

 g/plate ( Salmonella typhimurium-Escherichia
coli ) in the presence and
absence of metabolic activation (+ S9). Test 2: 0, 33, 100, 333, 1000, 2500, or 5000

 g/plate ( Salmonella typhimurium-Escherichia
coli ) in the presence and
absence of metabolic activation (+ S9). Test 3: 0, 100, 333, 1000, 2500, 3750, or 5000

 g/plate ( Salmonella typhimurium-Escherichia
coli ) in the presence and
absence of metabolic 48574788 Negative, except positive with Salmonella typhimurium strain TA100 in the absence
of metabolic activationFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 38 of 107 Toxicity Profile - Fluensulfone Guideline Study Type Doses Tested/Study
Classification MRID Results

activation (+ S9).

Acceptable/Guideline
870.5100 2010-Bacterial

reverse mutation assay (MCW-2
metabolite #3627) Test 1: 0, 3, 10, 33, 100, 333, 1000, 2500, or 5000

 g/plate ( Salmonella typhimurium-Escherichia
coli ) in the presence and
absence of metabolic activation (+ S9). Test 2: 0, 33, 100, 333, 1000, 2500, or 5000

 g/plate ( Salmonella typhimurium-Escherichia
coli ) in the presence and
absence of metabolic activation (+ S9).

Acceptable/Guideline 48574793 Negative

870.5300 2003- In vitro mammalian cell gene mutation assay (MCW-2) Without activation: 0, 24, 32, 40, 48, 56, 64, 72

 g/ml With activation: 0, 20, 30, 40, 50, 60, 70, 80  g/ml

Acceptable/Guideline 48574763 Negative

870.5300 2011- In vitro mammalian cell gene mutation assay (MCW-2
metabolite #3626) Without activation: test1 = 0, 3.8, 7.5, 15, 30, 40, 50,

60  g/ml, test 2 = 0, 5.5, 10.9, 21.9, 43.8, 87.5, 131.3, 175  g/ml

With activation: test 1 = 0, 37.5, 75, 150, 300, 600, 800, 1000  g/ml, test 2 = 0, 87.5, 175, 350, 700, 800, 900, 1000  g/ml

Acceptable/Guideline 48574789 Negative

870.5375 2010- In vitro mammalian cell
chromosome
aberration test (MCW-2) 0, 62.5, 125, 250, 500, 1000 μg/mL with and
without metabolic
activation

Acceptable/Guideline 48574762 Negative

870.5375 2010- In vitro
mammalian cell
chromosome
aberration test (MCW-2
metabolite Test 1: 0, 9.3, 18.5, 37, 74.1, 148.1, 296.3, 592.5, 1185, 2370 μg/mL with
and without metabolic
activation 48574785 NegativeFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 39 of 107 Toxicity Profile - Fluensulfone Guideline Study Type Doses Tested/Study
Classification MRID Results

#3625) Test 2: 0, 9.3, 18.5, 37, 74.1, 148.1, 296.3, 592.5, 1185, 2370 μg/mL without
metabolic activation and 148.1, 296.3, 592.5, 1185, 2370 μg/mL with
metabolic activation

Acceptable/Guideline

870.5375 2010- In vitro
mammalian cell
chromosome
aberration test (MCW-2
metabolite #3627) Test 1: 0, 8.3, 16.6, 33.3, 66.6, 133.2, 266.4, 532.8, 1065.5, 2131 μg/mL with
and without metabolic
activation

Test 2: 0, 8.3, 16.6, 33.3, 66.6, 133.2, 266.4, 532.8, 1065.5, 2131 μg/mL
without metabolic activation and 133.2, 266.4, 532.8, 1065.5, 2131 μg/mL with metabolic
activation

Acceptable/Guideline 48574794 Negative

870.5395 2003-
mammalian erythrocyte
micronucleus test (MCW-2) mg/kg/day = 0, 75, 150,
300

Acceptable/Guideline 48574764 Negative

870.5395 2011-
mammalian erythrocyte
micronucleus test (MCW-2
metabolite #3625) mg/kg/day = 0, 500, 1000, 2000

Acceptable/Guideline 48574786 Negative

870.5395 2011-
mammalian erythrocyte
micronucleus test (MCW-2
metabolite #3626) mg/kg/day = 0, 125, 250,
500

Acceptable/Guideline 48574791 Negative

870.5395 2010-
mammalian erythrocyte
micronucleus test (MCW-2
metabolite #3627) mg/kg/day = 0, 500, 1000, 2000

Acceptable/Guideline 48574795 Negative

870.5550 2011- mg/kg/day = 0, 250, 500 48574790 NegativeFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 40 of 107 Toxicity Profile - Fluensulfone Guideline Study Type Doses Tested/Study
Classification MRID Results

unscheduled DNA synthesis (MCW-2
metabolite #3626) Acceptable/Guideline

870.6200 2010-Acuteneurotoxicityrat mg/kg/day = 0, 100, 400,
1200

Acceptable/Guideline 48574773 NOAEL (mg/kg/day): not identified LOAEL (mg/kg/day): 100 based on decreased spontaneous activity, decreased rearing, impaired righting response, and reduced locomotor activity in females. 870.6200 2011-90-dayneurotoxicityrat mg/kg/day = M: 0, 6, 31, 153 F: 0, 7, 34, 162

Acceptable/Guideline 48574774 NOAEL (mg/kg/day): M=31, F=34 LOAEL (mg/kg/day): M=153, F=162 based on decreased food consumption, decreased body weight gain, and decreased body weight in males.

870.7485 2011-
Metabolism and excretion, single oral-rat([thiazole14 C]MCW-2and [butene14 C]MCW-2) mg/kg/day =
5 and 500

Acceptable/Guideline 48574742 The recovery of radioactivity was 91.3-
97.3% of the administered dose for all groups of rats. The disposition and
excretion of radiolabel for males and females were generally similar. Urine was the major route of excretion for both radiolabel positions; 75-88% of the
administered dose was excreted in urine depending on the dosing. Unmetabolized
fluensulfone was not detected as a significant residue in either urine or feces. The study investigators indicated that initially the parent compound probably reacts with glutathione to displace butene sulfinic acid, the major urinary metabolite.

870.7485 2011-
Metabolism and excretion, repeat oral-rat ([butene14 C]MCW-2) mg/kg/day =
5

Acceptable/Guideline 48574744 The recovery of excreted radiolabel was
94-99% for individual rats. Radiolabel was found mainly in urin e (71-83%), feces (9- 11%) and the cage wash (6-16%). One day after dosing with radiolabeled fluensulfone, an average of 0.6-0.8% of the administered radiolabel was in liver and 0.1% in kidney
with 1.7-2.1% in th e carcass (selected tissues removed) and 4.5-4.7% remained in the gastro-intestinal tract. By 7 days after treatment with radiolabeled fluensulfone, an average of 0.1-0.2% of the administered
radiolabel remained in liver and 0.03% in kidney.There were no significant sex
differences in either elimination or tissue residues. Analysis of urine showed four major metabolites identified as thiazole sulfonic acid (TSA), glucuronide I, glucuronide II and th iazole mercapturate.

870.7485 2010- pharmacokinetics, single oralmg/kg/day =
5 and 500 48574741 The absorption of fluensulfone-derived 14 C was rapid at both doses. The kinetic parameters for males and females wereFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 41 of 107 Toxicity Profile - Fluensulfone Guideline Study Type Doses Tested/Study
Classification MRID Results

rat ([thiazole14 C]MCW-2and [butene14 C]MCW-2) Acceptable/Guideline generally similar for the butene radiolabel, while for the thiazole label, male rats demonstrated a slightly higher absorption
based on AUC values in whole blood.
Fluensulfone reacts with the free thiol moiety of globin (protein of hemoglobin) to form a covalent linkage to the thiazole group and displaces butene sulfinic acid.

870.7485 2011-
toxicokinetic study-rat([thiazole14 C]MCW-2and [butene14 C]MCW-2) mg/kg/day =
5 and 500

Acceptable/Guideline 48574743 Residues were generally similar between
males and females. For both labels and both dose levels, the gastrointestinal tract, liver and kidney were among the tissues containing the highest radioactivity levels
at C
max for both sexes. As predicted by the pharmacokinetic study, radioactivity from the thiazole label was slowly eliminated
from red blood cells consistent with the derivatization of the hemoglobin cysteine
thiol. The ratio of residues at the C
max compared to the DT 90 was near the expected 10% for plasma, but for the thiazole label (low and high doses) was >25% for numerous tissues/organs. For the butene label (low and high doses), the ratio
of residues at C
max compared to DT 90 was also >25% for numerous tissues/organs except those involved with the digestive system. The slow elimination of radioactivity with the thiazole and butene labels may reflect metabolism of [ 14 C] fluensulfone to 1- and 2-carbon fragments with reincorporation in natural products (e.g. protein, fatty acids) with long

turnover rates.
870.7600 2011 Dermal penetration in
vivo – male rat Mg/cm 2 = 0.1 (label
dilution of commercial formulation) and 4.8 (undiluted commercial formulation) 480 EC formulation. Equivalent to 10 g a.i./L and 480 g a.i./L, respectively. All animals exposed for 10 hours, then monitored at 24, 72 or 120 hrs. 48574949 MCW-2 as the MCW-2 480 EC formulation penetrated rat skin at both high and low dose levels at a relatively high level (63.71 and 19.16% AD at 10 hours, respectively), which was mainly excreted in the urine. The maximum potentially
absorbable % AD for th e low and high dose groups is 64% at 10 hrs and 21% at 24 h, respectively. After five days, 2.2% of low dose and 0.66% of high dose radioactivity
remained in the animals. The calculated penetration rates did not increase in a proportional fashion with dose. 870.7800 2011-28-day immunotoxicity
-mice mg/kg/day = 0, 17, 86, 204

Acceptable/Guideline 48574779 Systemic: NOAEL (mg/kg/day):
86 LOAEL (mg/kg/day): 204 based on mortality, clinical signs and decreased body weight and food consumption.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 42 of 107 Toxicity Profile - Fluensulfone Guideline Study Type Doses Tested/Study
Classification MRID Results

Immunotoxicity: NOAEL (mg/kg/day):
204 LOAEL (mg/kg/day):
not determined Special study 2011 – 3 or 7- day dietary study evaluating
BrdU incorporation in lungs – female
mice 0 or 1200 ppm

Acceptable/nonguideline 48574767 At 1200 ppm increased cell proliferation was observed in the lungs as determined by BrdU incorporation. The effect was transient, observed at 3 days but not after 7 days.

Special study 2011 – Comparative
biotransformati
on in human vs. mouse lung
microsomes 2 or 20 μM, in vitro incubation

Acceptable/nonguideline 48574768 Fluensulfone was extensively metabolized by mouse lung microsomes but not by human lung microsomes. Mouse Cyp2f2 appeared to be involved in the metabolism.

Special study 2009 –
Inhibition of
Alanineaminotransferas e (ALAT) in a 28-day dog dietary study 0, 50, 200 or 900 ppm

Acceptable/nonguideline 48574775 Liver, serum and liver cytosolic fractions of untreated dogs, spiked with serum orcytosolic fractions from fluensulfonetreated dogs, did not show inhibition of ALAT activity in the untreated dogs. The decrease in ALAT activity was therefore not likely to be due to direct inhibition of the enzyme by fluensulfone/ metabolites, or inhibition of the ALAT gene or protein expression in the liver. Special study 2011 – 2-week dietary study in dog –
evaluation of
MOA for
decreased ALAT activity 0 or 500 ppm

Acceptable/nonguideline 48574776 Reduced ALAT activity was not due to decreased mRNA or protein expression, or decreased cofactor pyridoxal 5’-phosphate.

Special study 2010 – in vitro
inhibition of ALAT activity by fluensulfone
from liver
microsomes of untreated dogs Up to approximately 19.1 μM, in vitro incubation

Acceptable/nonguideline 48574777 Fluensulfone did not cause a direct inhibition of ALAT activity when incubated with dog liver homogenates.

Special study 2009 –
Determination of hepatic
ALAT in untreated dogs Untreated animals 48574778 An historical database for adult male and female beagle dogs was established based
on data collected between 6/09 and 9/09

Special Study 2011- comparative in
vitro dermal absorption mg/cm 2 = 0.1 and 4.8 480 EC
formulation. 48574950 The total absorption was calculated to be 92.11% and 81.26% (low and high dose), and 62.46% and 4.65% (low and high dose) for rat and human skin membranes,Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 43 of 107 Toxicity Profile - Fluensulfone Guideline Study Type Doses Tested/Study
Classification MRID Results

(MCW-2 [Ring-U- 14 C]) Acceptable/nonguideline respectively. Based on percentages absorbed, the ratio between human and rat
skin membrane was 1:1.5 for the low dose level and 1:18 for the high dose level.

Special Study 2014-28-Day Dietary – rat [BSA] mg/kg/day = M: 0, 6.4, 30, 82, 732 F: 0, 8.6, 39, 120, 1024

Acceptable/nonguideline 49387602 NOAEL (mg/kg/day): None LOAEL (mg/kg/day): None

Aside from marginal (<25%) decreases in mean serum aspartate aminotransferase
levels in both sexes and alanine aminotransferase levels in males at the high dose of 10,000 ppm, no treatment-related effects were observed in this study up to the highest dose tested. Special Study 2014-28-Day Dietary – rat [TSA] mg/kg/day = M: 0, 10, 41, 113, 1194 F: 0, 12, 43, 123, 1779

Acceptable/nonguideline 49387601 NOAEL (mg/kg/day): None LOAEL (mg/kg/day): None

No treatment-related effects were observed in this study up to the highest dose tested.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 44 of 107 A.3 Hazard Identification and Endpoint Selection

A.3.1 Acute Reference Dose (aRfD) – General Population Including Females age 13-49

Study Selected: 2-generation repro ductive toxicity study.

MRID No: 48574769

Executive Summary: See Appendix A.4, Guideline 870.3800

Dose and Endpoint for Risk Assessment : Offspring NOAEL = 16.2/23.0 mg/kg/day (M/F) based on increased post-natal pup loss by Day 2 in the P and F1 generations at the LOAEL of 122.0/169.1 mg/kg/day (M/F).

Uncertainty Factor: 100x (10x interspecies ex trapolation, 10x intraspeci es variability, 1x FQPA Safety Factor)

Acute RfD = ) ( 100 / / 2

. 16

UF day kg mg = 0.16 mg/kg/day

Comments about Study/Endpoint/Uncertainty Factor: The endpoint of concern in this study is protect ive of all populations and is appropriate for the exposure duration of concern. This study has a clearly defined NOAEL and the effect could be
considered to be the result of a single dose. The study is also protective of the effects observed in the ACN in rats at 100 mg/kg based on consid eration of the dose response curve at the doses tested, and absence of findings at 100 mg /kg in the range-finding study for the ACN.

A.3.2 Chronic Reference Dose (cRfD)

Study Selected: Co-critical chronic toxicity (dog) and 90-day toxicity (dog).

MRID No:
48574756 and 48574755

Executive Summary: See Appendix A4, Guideline 870.3150 and 870.4100b

Dose and Endpoint for Risk Assessment : NOAEL = 3.1 mg/kg/day based on hematological and clinical chemistry changes, and decrease d body weights at the LOAEL of 16 mg/kg/day
observed in both the chronic and subchronic stud ies. The higher NOAEL from the chronic dog study was used as the point of departure since th e differences in doses are an artifact of dose
selection.

Uncertainty Factor: 100x (10x interspecies extrapolat ion, 10x intraspecies variability)Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 45 of 107 Chronic RfD = ) ( 100 / / 1

. 3

UF day kg mg = 0.03 mg/kg/day

Comments about Study/Endpoint/Uncertainty Factor: The endpoint of concern in the chronic and subchronic studies is prot ective of all popul ations and appropriate for the exposure duration of concern. The 90-day and chroni c dog toxicity studies exhibit sim ilar toxic effect s occurring at the same LOAEL (16-17 mg/kg/day) with no prog ression in toxicity over time. The NOAEL of 3.1 mg/kg is also protective of the effects obser ved in the rat and mouse carcinogenicity studies, as well as potential developmental effects.

A.3.3 Incidental Oral Exposure (Short- and Intermediate-Term)

A dose and endpoint for incidental oral expos ure were not identified because there are no
residential uses for fluensulfone at this time.

A.3.4 Dermal Absorption

An in vivo dermal absorption study in the rat and an in vitro dermal absorption study using rat
and human skin were submitted. Both studies used fluensulfone in an emulsifiable-concentrate (EC) formulation as the test material. The da ta demonstrated an inverse relationship between dosing concentration and pe rcent absorption. Based on in vivo dermal absorption observed in the
rat and in vitro comparative rat:human absorption data, an estimated human dermal absorption factor (DAF) was obtained as follows (all ab sorption values adjust ed for recovery):

(1) In the rat in vivo dermal absorption study, a dermal absorption of 58.0% (directly absorbed plus absorbable) was found at 24 hrs post-exposure using an application of 0.0953 mg/cm 2 ; (2) In vitro exposure of split-thickness rat skin under the same exposure and assessment conditions gave a dermal absorption of 92.1% (directly absorbed plus absorbable); (3) The ratio of the rat in vitro to the rat in vivo absorption is 1.6 (92.1/58.0) which is close to 1, indicating that the in vitro data is predictive of in vivo absorption; (4) Based on this ratio, a DAF for humans may be calculated using in vitro human dermal absorption (62.5%, directly absorb ed plus absorbable) observed in vitro under the same exposure
conditions. The DAF for humans is therefore 62.5/1.6 = 39.1%.

A.3.5 Dermal Exposure (Short-, Intermediate- and Long-Term)

Study Selected: Co-critical chronic toxicity (dog) and 90-day toxicity (dog).

MRID No:
48574756 and 48574755

Executive Summary: See Appendix A4, Guideline 870.3150 and 870.4100b

Dose and Endpoint for Risk Assessment : NOAEL = 3.1 mg/kg/day based on hematological and clinical chemistry changes, and decrease d body weights at the LOAEL of 16 mg/kg/dayFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 46 of 107 observed in both the chronic and sub-chronic st udies. The higher NOAEL from the chronic dog study was used as the point of departure since th e differences in doses are an artifact of dose
selection.

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

Comments about Study/Endpoint/Uncertainty Factors: The endpoint of concern in the chronic and sub-chronic studies is protective of all populations and appropriate for the exposure
duration of concern. These two studies are a ppropriate for short- and intermediate-term exposures because of the timing of the effect s (effects on hematology and clinical chemistry parameters were seen starting at week 6 in both studies) and the lack of progression in the toxicity database (subchronic and chronic studies of all species had similar NOAELs and LOAELs).

For short- and intermediate-term occupational exposure scenarios involving dermal exposure, HED did not chose the 28-day dermal toxicity study in rats conducte d with the technical material. Although that study is of the appropria te route and duration, the results do not appear to reflect the high dermal absorption (53-92 %) of the emulsifiable concentrate (480 EC)
observed in the in vivo and in vitro dermal penetrations studies. The dermal toxicity study shows minimal toxicity following exposure of 2000 mg/kg/ day to technical-grade fluensulfone, and has a NOAEL of 400 mg/kg/day. At that NOAEL, the oral-equivalent dose assuming 60% absorption (approximate rat in vivo value at 10 hours) is 240 mg/ kg/day, which is approximately 6-fold higher than the LOAELs from oral subchronic toxicity studies with rats. Given the lack of toxic response in the dermal t oxicity study, HED has concluded that it is not representative of potential systemic exposure to fluensulfone from the 480 EC formulation. Therefore, HED chose the co-critical oral dog st udies discussed above, with a 40.9% dermal absorption factor (see Section 4.2.1), to derive the point-of-departure. The two dog studies are appropriate for short- and intermediate-term exposure because the effects (hematology and clinical chemistry parameters) were seen starting at Week 6 in both studies and the lack of progression in the toxicity database (subchronic and chronic studies of all species had similar NOAELs and LOAELs).

A.3.6 Inhalation Exposure (Short-, Intermediate- and Long-Term)

Study Selected: 90-day inhalation toxici ty study in rats.

MRID No: 48575045

Executive Summary: See Appendix A.4, Guideline 870.3465

Dose and Endpoint for Risk Assessment : NOAEL = not identified based on decreased weight gain in males, increased prothrombin time in fe males, decreased thymus weight in males and histopathology in the epiglottis in both sexes seen at LOAEL= 0.04 mg/L (lowest dose tested).Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 47 of 107 HEC: rat NOAEL x (daily duration adjustment) x weekly daily duration adjustment x RDDR = 0.04 mg/L x (6 hr/8 hr) x (1) x 0.143 = 0.00429 mg/L.

HED: HEC x CF x AF x A x D; HEC = human equi valent concentration in mg/L (0.00429); CF = conversion factor for breathing ra te in L/hr-kg bw (11.8); AF = ac tivity factor specific to the exposure scenario (0.6, 1.2, and 2.1 for low, me dium, and high, respectively); A = absorption ratio (1); D = human daily exposure duration in hours (8). See table 4.5.4.3 for HED values for various exposure scenarios.

Uncertainty Factor: 300x (3x interspecies extrapolation, 10x intras pecies variability, 10x extrapolation from LOAEL to NOAEL)

Comments about Study/Endpoint/Uncertainty Factors: The 90-day inhalation toxicity study in
rats was chosen because it is th e only study available of the appr opriate route and duration. An additional uncertainty factor of 10x for the extrap olation from a LOAEL to a NOAEL was applied. The interspecies uncertainty factor was reduced to 3x because of adjustment for species differences in pharmacokinetics.

A.4 Executive Summaries

A.4.1 Acute Toxicity

870.1100 Acute Oral Toxicity –TGAI (2009)

In an acute oral toxicity study performed according to the Acute Toxic Class Method, three groups, each of 3 fasted female HanRcc:WIST (S PF) rats, were treated with fluensulfone (MCW-2 tech.; 96.78% a.i.) by oral gavage at a dose of 2000 mg/kg bw or 300 mg/kg bw. PEG
300 was used as the vehicle a nd dosing volume was 10 mL/kg bw.

Two animals treated at 2000 mg/kg bw were found dead on test day 2; all other animals survived until the end of the study period. Slightly ruffled fur, slight sedation, slightly poor coordination, hunched posture and almost to completely closed eyes were the clinical signs associated with administration of the test item, present in the surviving animal of th e 2000 mg/kg bw dose group up to day 5, and in animals dosed with 300 mg/kg bw only on the day of treatment.

The only surviving female among those treated at 2000 mg/kg bw lost 4% of body weight during the first week after treatment but recovered by the end of the study and its body weight at study termination was considered within the range commonl y recorded for rats of this strain and age. Distended stomach with liquid contents was noted at necropsy of decedent animals; otherwise, no macroscopic findings were recorded in the surviving animals at necropsy.

The median lethal oral dose of MCW-2 t ech. was calculated to be 300 mg/kg bw < LD 50 (female rat) < 2000 mg/kg bw (LD 50 cut-off value: 1000 mg/kg bw). According to the acute toxicity classification criteria for PMRA, MCW-2 tech. is of slight toxicity via the or al route. This hazard classification warrants the signal word and hazard statement “Caution – Poison” on the Canadian
label. Based on the EPA classification criteria , MCW-2 tech. falls under Category III for acute oral toxicity. Under OCS criteria fo r acute toxicity endpoints, the test material is considered to be of low acute oral toxicity.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 48 of 107 870.1100 Acute Oral Toxicity –TGAI (2010)

In an acute oral toxicity st udy performed according to the Acut e Toxic Class Method, groups of 3 fasted female Wistar rats, were treated with fluensulfone (MCW-2 tech., 96.5% a.i.) by oral gavage at doses of 2000, 1500 or 300 mg/kg bw . 0.8% aqueous hydroxypropylmethylcellulose was used as the vehicle and dosing volume was 10 mL/kg bw. In total, 3 animals were treated with 2000 mg/kg bw of fluensulfone, and 6 anim als each were treated with 300 or 1500 mg/kg
bw.

Death occurred in 2 animals treated at 2000 mg/ kg bw and in all 6 anim als treated at 1500 mg/kg bw. One animal treated at 2000 mg/kg bw as we ll as all 6 animals treated at 300 mg/kg bw
survived until the end of the study period. Ataxia, reduced mobility and reduced muscle tone were the most commonly recorded clinical signs . All surviving animals gained the expected weight throughout the study period. No abnormality wa s recorded at necropsy of either decedent or surviving rats.

Using the Probit analysis according to Finney, the oral LD 50 of fluensulfone wa s calculated to be 671 mg/kg bw.

According to the acute toxicity classification cr iteria for the PMRA, MCW-2 tech. is moderately acutely toxic via the oral route based on the LD 50 value of 671 mg/kg bw in female rats. This hazard classification warrants the signal word and hazard statement “Warning – Poison” on the
Canadian label. Based on the EPA classificati on criteria, MCW-2 tech. falls under Category III for acute oral toxicity (signal word “Caution”). Under OCS criteria for ac ute toxicity endpoints, the test material is considered to be of low acute oral toxicity.

870.1100 Acute Oral Toxicity –480 EC (2009)

In an acute oral toxic cl ass study, 11 week old fasted RccHan:WIST (SPF) rats (6 ♀ /dose) were given a single oral dose of MCW-2 480 EC (497 g a. i. /L) in purified water by gavage at doses of 300 or 2000 mg/kg bw and observed for 14 days.

Oral LD 50 Females: 300 mg/kg bw < LD 50 < 2000 mg/kg bw (LD 50 cut off = 1000 mg/kg bw)

According to the acute toxicity classificati on criteria for the PMRA , MCW-2 480 EC is of Slight Toxicity via the oral route of exposure based on the LD 50 cut off of 1000 mg/kg bw. This hazard classification warrants the signal word and hazard statement “Caution – Poison” on the Canadian label. According to the US EPA criteria, MCW- 2 EC would be assigned to category III, signal
word “Caution.” Under OCS criteria for acute toxici ty endpoints, the test ma terial is considered to be of low acute oral toxicity.

Three out of six animals treated with 2000 mg/kg bw of the test material were found dead twenty-four hours post dose, and one animal ha d to be killed in extremis 28 hours following dosing. All other animals in this dose group surv ived to study termination. Clinical signs of toxicity observed in some or all of the anim als in the high dose group not surviving to study termination include: poor coordination, ventra l recumbancy, colourless urine, cloudy eyes, hypothermia, yellowish discoloured ears, cyanosis, and no reflexes. Clinical signs of toxicityFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 49 of 107 observed in all animals in the high dose group, from 0.5 to 5 hours post dose and persisting until Day 5 or 6, include: slight to moderate sedation, ruffled fur, hunched posture and eyes almost to completely closed. Clinical signs of toxicity were observed in animals in the 300 mg/kg bw dose group on the day of dosing (Day 1) and Day 2, in cluding: ruffled fur, sedation, hunched posture, and almost to completely closed eyes. No clinical signs of toxicity were noted in any animals in this dose group from Day 3 to study termination.

There were no treatment related effects not ed on body weight and no treatment related macroscopic observations.

This acute oral study is classifi ed acceptable (fully reliable). This study satisfies the guideline requirement for an acute oral study (OPPTS 870.1100; OECD 423) in the rat.

870.1100 Acute Oral Toxicity –480 EC (2010)

In an acute oral toxicity study performed acco rding to the Acute Toxic Class method, one group of three, followed by another group of three fema le RccHan: WIST(SPF) rats, were treated with MCW-2 480 EC (473.6 g/L) by oral gavage at a dose of 2000 mg/ kg bw. The test substance was formulated in purified water at a concentration of 0.2 g/mL and administered at a dosing volume of 10 mL/kg bw. Treatment was followed by an observation period of 14 days.

No deaths occurred during the study.

All animals showed slight to se vere clinical signs on test day 1 including swaying gait, dragging of front and/or rear limbs, decreased activity, ru ffled fur and a hunched posture. All signs reverted by test day 2 and were no longer present up to test day 15, the end of the observation period.

Body weight gain was within the range commonly reco rded for rats of the st rain and age used in this study.

No macroscopic findings we re noted at necropsy.

The LD 50 of MCW-2 480 EC after single oral administration to female rats was greater than 2000 mg/kg bw.

MCW-2 480 EC is not classified under GHS CLP.

According to US EPA criteria, MCW-2 480 EC is assigned to category III, signal word ‘Caution’.

870.1100 Acute Oral Toxicity – thiazole sulfonic acid

In an acute oral toxicity study performed according to the Acute Toxic Class Method, two groups, each of 3 fasted female HanRcc:WIST (S PF) rats, were treated with fluensulfone metabolite thiazole sulfonic acid (MCW-2 meta bolite #3625) by oral gavage at a dosage of 2000 mg/kg bw. Purified water was used as th e vehicle and dosing vol ume was 10 mL/kg bw.

All animals survived unt il the end of the study period. No clin ical signs were observed during the course of the study with the excep tion of 3 animals, in which sli ghtly ruffled fur was observed 2 hours after administration, persisting up to the 3-hour observation in 2 animals.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 50 of 107 The body weight of the animals was within the range commonly recorded for rats of this strain and age (all animals gained weight over the study period) and no macr oscopic findings were recorded at necropsy.

The oral LD 50 of thiazole sulfonic acid was greate r than 2000 mg/kg bw. Based on PMRA’s acute toxicity classification system , thiazole sulfonic acid (metabol ite #3625) is considered to be of low acute toxicity via the oral route. Based on the EPA classifi cation criteria, thiazole sulfonic acid falls under Category III for acute oral t oxicity. Under OCS criteria for acute toxicity endpoints, thiazole sulfonic acid is consider ed to be of low acute oral toxicity.

The acute oral toxicity of fluensulfone meta bolite #3625 is lower than that of fluensulfone.

870.1100 Acute Oral Toxicity – methyl sulfone

In an acute oral toxicity study performed according to the Acute Toxic Class Method, three groups, each of 3 fasted female HanRcc:WIST (S PF) rats, were treated with fluensulfone metabolite methyl sulfone (MCW-2 metabolite # 3626) by oral gavage at a dosage of 2000 mg/kg bw or 300 mg/kg bw. Corn oil was used as the vehicle and dosing volume was 10 mL/kg bw.

The 3 animals dosed at 2000 mg/kg died or were sacrificed because of ove rt toxicity within 2 hours of dosing. All 6 animals dosed at 300 mg/ kg survived until the end of the observation period.

Clinical signs were recorded during the course of the study and include d swaying gait, sedation, hunched posture, labored breathing, tightly shut ey es and/or ruffled fur. All clinical signs completely reverted by test day 3 for all surviving animals.

The body weight of the animals was within the range commonly recorded for rats of this strain and age. All surviving animals ga ined weight over the study period.

Congested lungs were recorded at necropsy for the 3 animals dosed at 2000 mg/kg bw, while no macroscopic findings were noted for th e 6 animals dosed at 300 mg/kg bw.

The acute median lethal dose of me thyl sulfone was 300 mg/kg bw < LD 50 < 2000 mg/kg bw (LD 50 cut-off value: 500 mg/kg bw). Based on PMRA ’s acute toxicity classification system, methyl sulfone (metabolite #3626) is considered to be of moderate toxicity via the oral route.
Based on the EPA classification cr iteria, methyl sulfone falls unde r Category II for acute oral toxicity. Under OCS criteria for ac ute toxicity endpoints, methyl su lfone is considered to be of low to moderate acute oral toxicity.

The acute oral toxicity of fluensulfone meta bolite #3626 is similar to that of fluensulfone.

870.1100 Acute Oral Toxicity – butene sulfonic acid

In an acute oral toxicity study performed according to the Acute Toxic Class Method, two groups, each of 3 fasted female HanRcc:WIST (S PF) rats, were treated with fluensulfone metabolite butene sulfonic acid (MCW-2 metabo lite #3627) by oral gavage at a dosage of 2000 mg/kg bw. Purified water was used as th e vehicle and dosing vol ume was 10 mL/kg bw.

All animals survived unt il the end of the study period. No clin ical signs were observed during the course of the study. The body weight of the anim als was within the range commonly recordedFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 51 of 107 for rats of this strain and ag e (all animals gained weight over the course of the study) and no macroscopic findings were recorded at necropsy.

The LD 50 of butene sulfonic acid was greater than 2000 mg/kg bw. Based on PMRA’s acute toxicity classification system, but ene sulfonic acid (metabolite #3627) is considered to be of low acute toxicity via the oral rout e. Based on the EPA classificati on criteria, butene sulfonic acid falls under Category III for acute or al toxicity. Under OCS criteria for acute toxicity endpoints, metabolite butane sulfonic acid is considered to be of low acute oral toxicity.

The acute oral toxicity of fluensulfone meta bolite #3627 is lower than that of fluensulfone.

870.1200 Acute Dermal Toxicity – TGAI

In an acute dermal toxicity st udy, a group of 5 male and 5 female HanRcc:WIST (SPF) rats were treated with fluensulfone (MCW-2 tech., 96.78% a.i.) at 2000 mg/kg bw by dermal application.
The test substance was formulated in PEG 300 at the concentration of 0.5 g/mL and applied to the shaved backs of the animals (approximately 10% of the total body surface) at the dosage volume of 4 mL/kg bw. The exposure period was 24 hours. Treatment was followed by an observation period of 14 days.

No deaths occurred during the st udy, and no clinical signs, either systemic or local, were noted. Between test days 8 and 15 one male rat gained 0.1 g and between test days 1 and 8 one female rat did not gain weight while another gained only 0.5 g. Otherw ise, the body weights of the animals were within the range commonly record ed for rats of this strain and age. No macroscopic findings were observed at necropsy.

The acute dermal median lethal dose of fl uensulfone was greater than 2000 mg/kg bw.

According to the acute toxicity classification cr iteria for PMRA, MCW-2 tech. (fluensulfone) is of low acute toxicity via the derm al route (based on the dermal LD 50 value for males and females). Based on the EPA classification criter ia, MCW-2 tech. falls under Category III for acute dermal toxicity (signal word “Caution”). Under OCS criteria for ac ute toxicity endpoints, the test material is considered to be of low acute dermal toxicity.

870.1200 Acute Dermal Toxicity – 480 EC

In an acute dermal limit test, five male and fi ve female RccHan:WIST (SPF) rats were treated with MCW-2 480 EC at 2000 mg/kg bw by derm al application. The test substance was formulated in purified water at the concentration of 0.5 g/mL and applied to the shaved backs of the animals (approximately 10% of the total bod y surface) at the dosage volume of 4 mL/kg bw. The exposure period was 24 hours. Treatment was followed by an observation period of 14 days.

There were no deaths, and no clinic al signs were observed. Very s light to well-defined erythema was observed at the application si te of all animals from removal of the dressing (test day 2) to test day 3, 4 or 5. Additionally, scaling was noted in all animals from test day 3 to 5 or to 11. Slight body weight loss (1.4 to 7.3 g, 0.67% to 3.57% bw) was noted in three of five females from days 1 to 8 but overall, body weight gain wa s within the range commonly recorded for rats of the strain and age used in the study. No abnormality was detected at necropsy.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 52 of 107 The dermal LD 50 of MCW-2 480 EC was greater than 2000 mg/kg bw.

MCW-2 480 EC is assigned category 5 and does not require any la beling under GHS CLP. According to US EPA criteria, MCW-2 480 EC is assigned to categor y III, signal word ‘Caution’. According to the acute toxicity classi fication criteria for PMRA , MCW-2 480 EC is of low acute toxicity via the dermal route (based on the dermal LD 50 value for males and females).
Under OCS criteria for acute toxici ty endpoints, the test material is considered to be of low acute dermal toxicity.

870.1300 Acute Inhalation Toxicity – TGAI

In an acute inhalation toxicity study, a group of 5 male and 5 female HanRcc:WIST(SPF) rats was exposed by inhalation by nose-only , flow-past exposure, for four hours to fluensulfone (MCW-2 Tech., 96.2% a.i.) at the chemically determined mean concentration of 5.1 mg/L air (MMAD
obtained at three times duri ng exposure were 2.34, 2.18 and 2.16 μm, with GSD values of 1.74, 1.67 and 1.67, respectively). Exposure was followed by an observation period of 14 days.

All animals survived the end of the observation period. There we re no clinical signs during exposure or the observation period. There were no effects on the body weight development that were considered to be related to exposure, and no macroscopic findings we re noted at necropsy.

The 4-hour LC 50 of fluensulfone was estimated to be greater than 5.1 mg /L air (chemically determined mean aerosol concentration).

According to the acute toxicity classification cr iteria for the PMRA, MCW-2 tech. (fluensulfone) is of low acute toxicity via th e inhalation route (based on the LC 50 value in male and female rats). Based on the EPA classification system, MCW-2 t ech. falls under Category IV for acute inhalation toxicity. Under OCS criteria for acute toxicity endpoints, the test mate rial is considered to be of low acute inhalation toxicity.

870.1300 Acute Inhalation Toxicity – 480 EC

In an acute inhalation toxicity study, groups of 5 male and 5 female HanRcc:WIST(SPF) rats were exposed by nose-only, flow-past exposure, for four hours to MCW-2 480 EC (497 g a.i./L) at the chemically determined mean c oncentration of 6.0 mg/L air (MMAD = 1.8 μ m, GSD = 2.12). The mean gravimetric concentration was 5.3 mg/L. Exposure was followed by an observation period of 14 days.

No mortality occurred during th e study. Salivation and ruffled fu r were observed in all animals during or after exposure. Decreased activity was recorded in all ma les and two females at the end of the exposure period and/or one hour after e xposure. Rales were reco rded in two males one hour after the end of exposure. There were no c linical signs in any an imals from test day 2.

Slight body weight loss was noted in four males and all females between days 1 and 4. No body weight gain was recorded in this period in the remaining male. Thereafter normal body weight gain was recorded in all animals, although one fe male failed to regain the initial body weight by study termination.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 53 of 107 No abnormality was detected at necropsy.

The acute inhalation LC 50 of MCW-2 480 EC was greater than 6.0 mg/L air (chemically determined mean aerosol concentration).

MCW-2 480 EC is assigned category 5 and does not require any la beling under GHS CLP.

According to US EPA criteria, MCW- 2 480 EC is assigned to category IV.

According to the acute toxicity classification criteria for the PMRA, MCW-2 480 EC is of low acute toxicity via the inha lation route based on the LC 50 value in male and female rats.

Under OCS criteria for acute to xicity endpoints, MCW-2 480 EC is considered to be of low acute inhalation toxicity.

870.2400 Primary Eye Irritation – TGAI

In a primary eye irritation study, 0.1 g of fluens ulfone (MCW-2 tech., 96. 78% a.i.) was instilled into the left eye of each of three young adult New Zealand White rabbits . Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours after instillation.

No mortality occurred during the study and there were no clinical signs of toxicity recorded, nor were there any notable changes to the animals’ body weights.

All irritation scores were zero. No abnormal findings were observed in the cornea, the iris, the conjunctivae or the sclera of any animal at a ny of the examinations. No corrosion was observed at any of the scoring intervals.

No staining of the treated eyes by the test item was observed and no clinical signs were observed.

The test item did not induce significant or irreversible damage to the rabbit eye.

Based on the results of this study, MCW-2 tech. (f luensulfone) is considered non-irritating to the rabbit eye according to the PM RA’s classification scheme, a nd falls under Category IV for primary eye irritation according to the EPA’s clas sification criteria. Unde r OCS criteria for acute toxicity endpoints, the test materi al is not considered to be an irritant of the eye of rabbits.

870.2400 Primary Eye Irritation – 480 EC (2009)

In a primary eye irritation study, 0.1 ml of MCW-2 480 EC (497 g a.i. / L) was instilled into the conjunctival sac of the left eye of 13 week old New Zealand White rabbits (1 male, 2 females). Eyes were not washed post treatment. Animal s were observed for 10 days. Eye reactions were assessed according to the Draize scale.

Slight to marked reddening and chemosis of the conjunctivae, a nd slight reddening of the sclera was observed in all animals up to 72 hours after treatment. One animal showed corneal opacity affecting over half the eye at 24 to 48 hours pos t dose. This animal also had delayed/reduced light reflex at the 24 hour obser vation point. One animal show ed transient corneal opacity affecting less than one quarter of the area at 48 and 72 hours post treatment. All scores were zeroFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 54 of 107 by 7 days post treatment, except for slight reddeni ng of the sclera persisting in one animal. The scleral reddening was absent by day 10 post dose.

The Maximum Average Score (MAS) for 24, 48 and 72 hours, used by the PMRA for acute toxicity classification, was calculated to be 17, and the Maximum Irritation Score (MIS) was calculated to be 22 (recorded at 24 hours). In this study, MCW-2 480 EC is moderately irritating to the eye based on the MAS score of 17 and the persistence of reddening of the sclera in one animal on Day 7. This level of hazard co rresponds to the following label signal words and hazard statement: “Warning – Eye Irrita nt” for the Canadian label.

According to the US EPA criteria, the test materi al would be assigned to category III, with signal
word “Caution.”

Under OCS criteria for acute toxi city endpoints, MCW-2 480 EC is considered to be a moderate irritant to the eye of rabbits.

This study is classified as acceptable (fully re liable). This study satisfies the guideline requirement for a primary eye irritation st udy (OPPTS 870.2400; OECD 405) in the rabbit.

870.2400 Primary Eye Irritation – 480 EC (2011)

In a primary eye irritation study, 0.1 mL of MCW- 2 480 EC was instilled into the left eye of each of three young adult New Zealand White ra bbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours, as well as 7, 10 and 14 days after ins tillation.

The instillation of MCW-2 480 EC into the eye re sulted in slight corn eal opacity, slight to moderate reddening and slight to marked chemos is of the conjunctivae, slight to moderate reddening of the sclera and slight to moderate ocular discharge. These effects were reversible and were no longer evident 10 da ys (2 animals) or 14 days (t he remaining one animal) after
treatment. The cornea of all animals showed positive fluorescein staining from 24 to 72 hours after treatment and reverted 7 days after treat ment. No abnormal findings were observed for the iris light reflex and no corrosion was observed at any of the exam inations. No staining of the treated eyes by the test item was obser ved. No clinical si gns were observed.

The Maximum Average Score (MAS) for 24, 48 and 72 hours was determined to be 24, and the Maximum Irritation Score (MIS) was calculated to be 31 (recorded at 1 hour and 24 hours).

MCW-2 480 EC is assigned category 2 (H319: ca uses serious eye irritation) under GHS CLP.

As corneal effects had resolved, and no positive gr ades for the conjunctivae were recorded in any animal by day 7, according to US EPA criteria MCW-2 480 EC is assigned to category III, signal
word ‘Caution’.

Based on the MAS and the persistent conjunctival redness in all animals and scleral reddening in one animal 7 days post treatment, MCW-2 480 EC is considered moderately irritating to the rabbit eye according to the acute toxicity classification criteria for the PMRA. This level of hazard corresponds to the following label signa l words and hazard statement: “Warning – Eye
Irritant” for the Canadian label.

Under OCS criteria for acute toxi city endpoints, MCW-2 480 EC is considered to be a moderateFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 55 of 107 irritant to the eye of rabbits.

870.2500 Primary Dermal Irritation – TGAI

In a primary skin irritation study, 0.5 g of fl uensulfone (MCW-2 tech., 96.78% a.i.) moistened with ~0.5 mL purified water was ap plied to the intact left flan k of each of three young adult New Zealand White rabbits (1 male and 2 females) for 4 hours. No mortality or clinical signs were noted during the study. One female had a slight body weight loss (-14 g or -0.6%) between treatment and the day of termination.

The scoring of skin reactions was performed 1, 24, 48 and 72 hours, as well as 7 days after removal of the semi-occlusive dressing and rema ining test substance. The mean score was calculated across 3 scoring times (24, 48 and 72 hour s after patch removal) for each animal for erythema/eschar grades and for oedema grades, separately. The mean erythema/eschar score of the three animals was 1.00, 0.33 and 0.33, respectiv ely and the mean oedema score was 0.00 for
all animals. The mean irritation score at 72 hours was 0.33.The application of fluensulfone to the skin resulted in very s light signs of irritation, such as eryt hema. This effect was reversible and was no longer evident 7 days afte r treatment, the end of the obser vation period for all animals. The test item caused no staining of the treated skin. No corrosive effe cts were noted on the treated skin of any animal at a ny of the measuring intervals and no clinical signs were observed.

The Maximum Average Score (MAS) of 24, 48 and 72 hours was calculated to be 0.56 and the Maximum Irritation Score (MIS) was calculated to be 1.0, at 1 and 24 hours. According to the acute toxicity classification criteria for PMRA, MCW-2 tech. (fluensulfone) is minimally irritating to the skin, and based on the EPA cl assification criteria, MC W-2 tech. falls under Category IV for primary skin irritation. Under OC S criteria for acute toxic ity endpoints, the test
material is considered to be a sli ght irritant to the skin of rabbits.

870.2500 Primary Dermal Irritation – 480 EC (2009)

In a primary dermal irritation study, young adult New Zealand White rabbits were dermally exposed to 0.5 ml of MCW-2 480 EC (497 g a.i. /L) as supplied by the Sponsor, for 4 hours, to approximately 6 cm 2 of the body surface area. Animals were then observed for 14 days. Irritation was assessed according to the Draize scale.

Slight to well-defined erythema and very slight to slight oedema were observed in all animals from 1 to 72 hours post exposure. Very slight erythema persisted in one female 7 days post exposure. Scaling was observed in one female 72 hours post dosing and in all animals from 7 to 14 days post exposure. No corrosi ve effects were noted on the trea ted skin and no clinical signs were observed. The Maximum Average Score (MAS) for 24, 48 and 72 hours, used by the PMRA for classification purposes, was determin ed to be 3.4. The Maximum Irritation Score (MIS) was calculated to be 3.7, which wa s observed at 24 and 48 hours post dose.

In this study, MCW-2 480 EC is moderately irritating to the skin based on the MAS of 3.4. This level of hazard corresponds to the follo wing label signal words and hazard statement: “Warning – Skin Irritant” on the Canadian label. According to the US EPA criteria, the testFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 56 of 107 material would be assigned to category IV. U nder OCS criteria for acute toxicity endpoints,
MCW-2 480 EC is considered to be a mode rate irritant to the skin of rabbits.

This study is classified as acceptable (fully re liable). This study satisfies the guideline requirement for a primary dermal irritation study (OPPTS 870.2500; OECD 404) in the rabbit.

870.2500 Primary Dermal Irritation – 480 EC (2011)

In a primary skin irritation st udy, 0.5 mL of undiluted MCW-2 480 EC was applied to the intact left flank of each of three young female New Zeal and White rabbits for 4 hours. The scoring of skin reactions was performed at 1, 24, 48 and 72 hours, as well as 7, 10 and 14 days after removal of the semi-occlusive dressing.

Well-defined erythema and very slight to slight oedema was observed in all animals 1 hour after treatment. Very slight to well-defined erythe ma persisted in all animals until 10 days after treatment. Very slight oedema persisted until 72 hours in two animals. Two animals showed slight desquamation 10 days after treatment. A ll skin reactions had reverted 14 days after treatment, the end of the obser vation period for all animals.

The test item did not cause any staining of the tr eated skin. No corrosive effects were noted on the treated skin of any animal at any of the measuring intervals and no clinical signs were
observed.

The Maximum Average Score (MAS) for 24, 48, and 72 hours, used by the PMRA for acute toxicity classification, was calculated to be 2.33, and the Maximum Irritation Score (MIS) at 1hour post dose was calculated to be 3.33.

MCW-2 480 EC is not classified under GHS CLP for skin irritation in rabbits.

According to US EPA criteria, MCW- 2 480 EC is assigned to category IV.

According to the acute toxicity classification criteria of the PMRA, MCW-2 480 EC is mildly irritating to the skin based on the MAS of 2.33. This level of hazard corresponds to the following label signal words and hazard stat ement: “Caution – Skin Irritant” on the Canadian label. Under OCS criteria for acute toxicity endpoints, MCW-2 480 EC is considered to be a moderate irritant
to the skin of rabbits.

870.2600 Dermal Sensitization – TGAI

In a skin sensitization study conducted accordi ng to the Maximization method, 10 male guinea pigs were induced by intradermal administration of fluensulfone (MCW-2 tech., 96.78% a.i.) at 3% in PEG 300 and in parallel in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline, followed 8 days later by an epidermal exposure to the test substance for 48 hours under occlusion at 75% in PEG 300, follo wing pretreatment of the test areas with 10% Sodium-Lauryl-Sulfate (SLS) approximately 24 hours prior to application. The 5 male animals of the control group were treated in tradermally with PEG 300 and FCA/physiological saline and epidermally with PEG 300 under occlus ion. Two weeks after ep idermal induction, the test animals were challenged by epidermal app lication of fluensulfone at 75% in PEG 300 and PEG 300 alone under occlusive dressing. Control animals received the same treatment.
Cutaneous reactions were evaluated at 24 a nd 48 hours after removal of the dressing.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 57 of 107 Ten and 6 out of 10 test animals (100% and 60%, respectively) showed di screte/patchy erythema at 24 and 48 hours, respectively af ter the challenge trea tment with fluensulfone at 75% in PEG 300. Two of 5 control animals (40%) showed th e same skin reactions 24 hours after the challenge treatment. At 48 hours, 3 test animals (2 with a concurrent positive response, 1 with a negative response) were additionally observed with skin desquamation. No positive reactions
were observed in either the test group or control animals at th e site treated with only PEG 300.

Based on interpretation of challenge reactions, fluens ulfone technicalwas cons idered to be a skin sensitizer in the guinea pig.

Under OCS criteria for acute toxicity endpoint, the test material is c onsidered to be a sensitizer to the skin of guinea pigs (in a guinea pig maximization test).

870.2600 Dermal Sensitization – 480 EC

In a skin sensitization study conducted acco rding to the Maximization method, 10 Albino Dunkin Hartley [CRL:(HA)BR, SPF] guinea pigs were induced by in tradermal administration of MCW-2 480 EC at 15% in purified water and in parallel in an emulsi on of Freund's Complete Adjuvant (FCA)/physiological saline, followed ei ght days later by an epidermal exposure for 48
hours under occlusion with the undilu ted test substance. The 5 an imals of the control group were treated intradermally with purified water and FCA/physiological saline and epidermally with purified water under occlusion. Two weeks after epidermal induction, the test animals were challenged by epidermal application of MCW-2 480 EC at 1% in purif ied water and purified water alone under occlusive dressing. Control an imals received the same treatment. Cutaneous
reactions were evaluated at 24 and 48 hours after removal of the dressing.

All animals in the test group reacted positively after epidermal challenge with MCW-2 480 EC,
while no skin reactions were recorded in a ny of the control animals. MCW-2 480 EC was considered to be a potentia l dermal sensitizer.

MCW-2 480 EC is assigned category 1 (H317: ma y cause an allergic skin reaction) under GHS
CLP.

Under OCS criteria for acute toxi city endpoints, MCW-2 480 EC is considered to be a sensitizer to the skin of guinea pigs (in a guinea pig maximization test).

870.6200 Acute Neurotoxicity

In an acute neurotoxicity study (MRID 48574773, PMRA 2181226), groups of 10 male and 10
female 6-week old Wistar rats were administer ed single oral doses of 0 (control), 100, 400 and 1200 mg/kg bw of fluensulfone (MCW-2 tech., 98.65%, Batch No. 36372130-291-PF1) in polyethylene glycol (PEG) 300 and observed for 14 days.

Detailed clinical observations comprising of open field evalua tion of clinical signs and a functional observational battery (FOB) were pe rformed on days 1, 7 and 14. Motor activity was assessed over a period of 30 minutes after each FOB evaluation. At the end of the scheduled observation period, 5 rats/sex/g roup were perfusion-fixed in situ . Selected nervous system tissues were dissected, processed and examined histologically.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 58 of 107 One female dosed at 1200 mg/kg bw died w ithin 24 hours after dose administration of an
unknown cause.

Administration of a single oral ga vage dose of fluensulfone to Wi star rats at doses of 100, 400 and 1200 mg/kg bw produced reversible effects on motor activity and FOB parameters. Signs of general systemic toxicity in FOB parameters such as reduced activity and decreased rearing activity, reduced righting respons e, and salivation were obser ved only on day 1 (the day of dosing). Reduced activity and decreased rearing ac tivity in 2 or 3 males and 1 female, together with a reduced righting response in 4 males and 1 female were observed at 100 mg/kg bw although to a much lesser degree than at 400 mg /kg bw and 1200 mg/kg bw. Locomotor activity comprising total distance, center time and reari ng activity was dose dependently reduced on day 1 in males and females at 400 mg/kg bw and 1200 mg/kg bw and in females at 100 mg/kg bw. The locomotor effects in the 100 mg/kg group fema les included reductions for total distance, center time and rearing activity. At 1 to 4 hour s post-dose on day 1, a lower body temperature
was recorded in males and females at 400 mg/kg bw and 1200 mg/kg bw, which could be
attributed to an effect on the autonomous function.

Body weights were not affected by treatment. On day 8, the mean body weight gain was lower in males and females treated at 1200 mg/kg bw. F ood consumption was dose dependently reduced in males at 1200 mg/kg bw and in females at 400 and 1200 mg/kg on days 1 to 8 with statistical significance attained in females at 1200 mg/kg bw only.

No test item-related effects on absolute and rela tive brain weights were evident in males or females. There were no treatment-related findings in the nervous system during macroscopic and microscopic pathology examinations.

The LOAEL is 100 mg/kg bw in males and fema les, based on reduced locomotor activity in females, and decreased spontaneous activity, decr eased rearing and impaired righting response in both sexes at 100 mg/g bw. A NOAEL was not identif ied as effects were observed at the lowest
dose tested.

The acute neurotoxicity study in the rat is classified as acceptable/guideline (fully reliable) and satisfies the guideline requirement for an acu te neurotoxicity study (OPPTS 870.6200) in rats.

A.4.2 Subchronic Toxicity

870.3050 28-day Oral –Mouse

In a 4-week dietary study (MRID 48575024, PMRA 2181204), groups of 5 male and 5 female Crl:CD1 mice received fluensulfone (BYI 01921, MC W-2 technical, 97%) in their diet at 0, 100, 500 and 2000 ppm (equivalent to 0/0, 30/41, 101/155, and 375/353 mg/kg bw/day in males/females, respectively).

No mortality and no clinical signs occurred. Food intake was decreased at 500 ppm (28-33%) and 2000 ppm (34-63%) in both sexes. Water consum ption was slightly incr eased at 2000 ppm in females. Body weight loss was observed at 2000 pp m in the first week of treatment in both sexes. At the end of treatment the mean body wei ght difference from the controls was -13% in males at 2000 ppm.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 59 of 107 Effects on red blood cell parameters were dete cted at 2000 ppm in both sexes, and were generally more pronounced (and statis tically significant) in females (increases of erythrocyte and reticulocyte counts, concentrati on of hemoglobin, hematocrit values ). Platelets were reduced in males at 500 ppm (13%; not sta tistically significan t) and in females at 2000 ppm (20%; not statistically significant). Increased activities of plasma-ASAT and plasma-ALAT were recorded in both sexes at 2000 ppm and considered as corr elates of the observed microscopic and organ weight changes in the liver. Very slight increases in the activity of APh we re also noted in both sexes at 2000 ppm.

Liver weights (relative) were increased at 500 ppm and above in females. Histopathology revealed the following induced findi ngs at 2000 ppm in both sexes:

 A cytoplasmic alteration in the periportal region, characterized by a dense cytoplasmic structure, eosinophilia (females) or slight basophilia (males)

 Degenerative changes of the periportal zone which encompassed also single cell necrosis and in some of the females mitotic figures

 Slight increase in number and size of b ile ducts in the portal regions (bile duct hyperplasia) in one male and three females

The absolute kidney weights were decreased at 2000 ppm in males (20%), and absolute heart weights were decreased at 500 and 2000 ppm in males and at 2000 ppm in females (16-18%); relative weight in males did not show a dose re sponse. However, no further correlates for an influence on kidneys or the heart were detected.

The NOAEL of the study was considered to be 100 ppm for both sexes (30 and 41 mg/kg bw/day in male and female mice, respectively). Th e LOAEL for both sexes is 500 ppm (101 and 155 mg/kg bw/day in male and female mice, respec tively), based on decrea sed food consumption in both sexes and decreased platelets in males; based on the marginal changes at th is dose, it is
considered a threshold effect level.

This study is classified acceptable/guideline (fully reliable) and satisfies the requirement for a 28-day dietary study in the m ouse (OECD 407; OPPTS 870.3050).

870.3050 28-day Oral –Rat

In a 28-day oral toxicity study (MRID 48575023, PMRA 2181203), groups of 5 male and 5
female Wistar rats received fluensulfone (BYI 01921, MCW-2 technical, 98.4-98.9%, Lot No. NLL6692-3) in their diet at 0, 125, 500 and 2000 ppm (0/0, 10.4/12.2, 42.5/37.2, 152.0/165.9 mg/kg bw/day in males/females) for approxima tely four weeks. In addition to standard investigations, the clinical chemistry assessment included a determination of thyroid hormones (triiodothyronine, thyroxine, and thyroid stimulat ing hormone), liver samples were processed for enzyme determination, and immunotoxicity investigations were conducted. Clinical examinations included evaluation of sensory r eactivity to stimuli, open field assessments and grip strength at th e end of Week 4.

No mortality occurred. No treat ment-related clinical signs were noted during the study and no differences between the treated groups and cont rols were noted either during the weekly open field inspections or the sensory reactivity to st imuli of different types and assessment of gripFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 60 of 107 strength conducted durin g week 4. Mean food consumption wa s slightly decreased in male animals receiving 2000 ppm during the first few days of treatment and throughout the study for females receiving 500 and 2000 ppm of the test material. Water consumption was slightly increased in high dose males and females.

Body weight development was impeded in male and female rats receiving 2000 ppm, and to a lesser extent in males receiving 500 ppm (8%). Overall body weight gain was decreased in mid (11-12%) and high dose (19-27%) ma le and female animals.

Hematological examinations revealed an increase in leukocyte and lymphocyte counts in females in all dose groups. All changes were considered to be treatment-related although only changes in mid (17-18%) and high dose (27-29%) groups were c onsidered to be adverse. Slight reductions in hemoglobin levels were also noted in both sexes from the 500 and 200 ppm dose groups, while a slight reduction in m ean corpuscular hemoglobin conc entration (MCHC) was also observed in high dose females. Clinical chemistry revealed dose-related decreases in the activity of alanine aminotransferase (ALAT) in all treat ment groups of both sexes. Decreases were also observed triglycerides in mid (25%) and high (53%) dose males. Glutamate dehydrogenase (GLDH) levels were decreased in mid and high dose males and in high dose females, but these enzyme changes were not considered adverse. T hyroxine (T4) was slightly decreased in males (12%) and females (17%) receiving 2000 ppm of th e test material, possibly secondary to the effects on liver enzyme activities. Cholesterol was elevated in mid (17%) and high (39%) dose males and high dose females (14%). Total protei n was increased only slig htly (6%) in high dose males. Urinalysis did not indicate test compound-related effects.

Analysis of organ weight data s howed statistically significant increa ses in mean relative liver and kidney weights in male rats treated at 2000 ppm, and relative spleen weig ht in females of the same group. Absolute and relative prostate we ights were also decreased at the 2000 ppm dose level. Gross pathology revealed surface changes in the kidneys of males dosed at 500 ppm (1/5 animals) and 2000 ppm (5/5 animal s). Histopathology revealed incr eased severity of basophilic tubules as well as degeneration and hyaline inclusions in the pr oximal tubules in mid and high dose males. At 2000 ppm focal tubular necrosis wa s observed in one male rat. While consistent with male rat alpha-2-microglobulin accumulation pathology, this cause was not conclusively demonstrated. Histopathology of the liver as we ll as of spleen revealed no toxicological
correlates.

Monooxygenases (ECOD, EROD and ALD) were markedly decreased in all female dose groups.
EROD and ALD were decreased to a lesser extent in all male dose groups. Decreases in ALD and EROD in the low dose groups were so minimal th at they were not considered to be adverse. The phase II enzyme GS-T was clearly induced in all male dose groups and in females at 500 and 2000 ppm. The phase II enzyme epoxide hydro lase (EH) was induced in all male dose groups and mid and high dose females. Glucuronyl transferase (GLU-T) leve ls were increased in mid and high dose males and in high dose females. The changes in liver enzyme levels were considered to be adaptive. Liver cell prolifer ation examinations gave no indication of testcompound related alterations regarding this endpoint.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 61 of 107 Immunotoxicity examinations showed no substa nce induced effects on the splenic and bone marrow cell counts or in the antibody titers. A decrease in CD8 positive cells was observed in mid and high dose males and in high dose fe males but a dose response was not observed.

The NOAEL of the study is set at 125 ppm (equi valent to 10.4 and 12.2 mg/kg bw/day in male and female rats, respectively). The LOAEL in th is study is considered to be 500 ppm (equivalent to 42.5 and 37.2 mg/kg bw/day in male and fema le rats, respectively), based on reduced body weights in males, reduced body weight gain in both sexes, decreased food consumption in females, increased white blood cells in females, increased cholesterol in males, and renal pathology in males.

This study is classified as acceptable/guideline (fully reliable) and satisfies the guideline requirement for a 28-day dietary stud y in the rat (OECD 407; OPPTS 870.3050).

870.3100 90-day Oral –Mouse

In a 90-day dietary toxic ity study (MRID 48574753; PMRA 2181205), groups of 12 male and 12 female Crl:CD1 (ICR)BR mice received diets c ontaining 0, 60, 300 and 1500 ppm (equivalent to 0/0, 11/18, 51/68 and 228/252 mg/kg bw/day in male s/females, respectively) of fluensulfone (BYI 01921 technical, 97.0-97.8%, Batch No. N LL6692-7-5) for a period of 13 weeks. Additional satellite groups of 5 mice per sex were similarly treated at 0 or 1500 ppm for 4 weeks
and then terminated for determination of enzyme levels in liver tissue.

There were several mortalities during the study: 2, 2, 4 and 4 males and 3, 0, 0 and 5 females for the control and treated groups in order of ascending doses. Apart from the death of a single female of the highest dose group, which was al so the only animal in which clinical signs (piloerection, squatting position, poor general condition, and emaciation) were recorded, all mortalities were related to blood sampling.

At 1500 ppm food (males 22% and females 43% belo w controls; g/animal/d ay) and water intake (males 17% below controls; g/animal/day) were decreased. At 300 ppm food intake was decreased in females (15%). Body weight gain was significan tly reduced at 1500 ppm in both sexes during the first week of treatment, with a body weight loss recorded for females. At the end of treatment mean body weight s of animals in the high dose group differed from the control values by approximately -10% for both sexes. Cumulative gain was 33% and 60% less than controls for males and females, respectively.

Hematological investigations showed increa sed erythrocyte counts, and hemoglobin and hematocrit values at 300 ppm and above in males, increased mean corpuscular volume (ranging from 5-15% above controls) and decreased mean corpuscular hemoglobin concentration (5%) in males at 1500 ppm, and increased reticulocyte counts in both sexes (32-36%) treated at 1500 ppm. Leukocyte counts were increased in males treated at and above 300 ppm (34-49%). Platelet levels were reduced in males at 300 ppm (17 %) and above (23%) and in females at 1500 ppm (38%). Clinical chemistry showed increased co ncentrations of total bilirubin (45-77%) at 300 ppm and above in both sexes, and decreased urea concentrations in animals of both sexes treated at 1500 ppm (20-24%). The activitie s of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase were in creased at 1500 ppm in both sexe s (55-107%), often attaining statistical significance.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 62 of 107 No treatment-related macroscopic findings were noted at necropsy. At 1500 ppm liver weights were increased in males (relative) and females (absolute and relative). Additionally, due to the lower body weights, the weights of brain, heart, and epididymides were decreased in males at 1500 ppm. Histopathology revealed hepatocell ular hypertrophy, accompanied by degenerative changes mainly of the periporta l hepatocytes, minimal to slight pigment deposits, bile duct hyperplasia (observed in most animals) and pr oliferation of undiffere ntiated oval cells, and slightly reduced lipid conten t in both sexes treated at 1500 ppm. Hepatocellular hypertrophy, without any sign of liver cell damage, was also observed in females receiving 300 ppm. In the spleen, an increased incidence of animals with pigment deposition in th e red pulp was recorded in both sexes at 1500 ppm.

Determinations in liver tissues in satellite anim als sacrificed after 28 da ys of treatment showed no evidence for toxicologically si gnificant effects on the concentr ation of cytochrome P450 at 1500 ppm. In male mice only the monooxygenase 7- ethoxycoumarin deethylase was marginally increased. The phase II enzymes epoxide hydr olase and glutathione-S -transferase were marginally increased, whereas UDP-glucuronyltransferase was not affected.

In female mice the monooxygenases were not si gnificantly affected, whereas all phase II enzymes (epoxide hydrolase, glutathione-S-tra nsferase, UDP-glucuronyltransferase) were significantly induced at 1500 ppm.

The NOAEL of the study was 60 ppm (equivalent to 11 mg/kg bw/day for males and 18 mg/kg bw/day for females) based on increased bilirubin in both sexes, hematology effects in the males and reduced food consumption in females, at the LOAEL of 300 ppm (51 mg/kg bw/day for males and 68 mg/kg bw/day for females).

This study is classified acceptable/guideline (fully reliable) and satisfies the guideline requirement for a subchronic oral toxicity study in the mouse (OECD 408; OPPTS 870.3100).

870.3100 90-day Oral –Rat

In a 90-day dietary toxic ity study (MRID 48574754, PMRA 2181206), groups of 10 male and 10 female Wistar rats (Hsd Cpb:WU) receive d diets containing 0, 60, 120, 500 or 2000 ppm of fluensulfone (BYI 01921, MCW-2 technical, 97%, Batch No. NLL6692-7-5) for a period of 3 months. The mean daily intakes in order of ascending concentration were 0, 4.31, 8.26, 34.86 or 139.01 mg/kg bw/day in males and 0, 4.85, 11.68, 53.10 or 148.73 mg/kg bw/day in females of the main groups. Dosages were chosen on th e basis of a 28-day range-finding study (MRID 48575023, PMRA 2181203). Additional recovery groups were similarly treated at 0 or 2000 ppm (10/sex/dose) and then main tained without treatment for a recovery period of 28 days. Along with the standard guideline evaluations, all groups of anim als were assessed for changes
in functional observational batt ery (FOB) and motor activity (M A) at the end of the dosing period. Fluoride levels in ash of bone (left femur) and teeth lower left incisor) were examined in all animals of the main and recovery groups. Add itional groups of 5 males were treated for one week at 0 or 2000 ppm for investigation of alpha-2μ-globulin accumulation in kidneys.

No animals died during the study and no observations related to neurotoxicity were noted during the study.

Body weights were statistically significantly reduced by treatment in both males and females atFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 63 of 107 2000 ppm throughout the study (at study terminati on, 9% below controls for both sexes). Although no statistical difference occurred, mean body weights of males treated at 500 ppm during the study were also lower compared with the control group. Females of this dose group were not affected. Body weights of recovery animals were also stat istically significantly
decreased at the end of the r ecovery period (8-15% compared to 11-21% at the end of the treatment period). In the main group overall body weight gain was decreased in mid (9%) and high (15%) dose males and in high dose females ( 15%); recovery groups al so showed decreased body weight gain (17-20%). Food consumption wa s decreased in 2000 ppm females of the main group as well as in recovery females. Water intake was increased in males at 500 ppm and in males and females at 2000 ppm and in recovery animals.

At 2000 ppm there were some changes detected at hematological investig ations, most of which were reversible during the recovery period. In females the HQuick (thromboplastin time) was significantly decreased at 2000 ppm (6%). In the 2000 ppm dose gr oups, statistically significant decreases were observed in hemoglobin, MCH, and MCHC in males, and in hemoglobin and MCHC levels in females (2-7%). Reticulocyte counts were increased in high dose females (44%). Despite the low magnitude of the decrease s in MCHC, the changes were considered to be
treatment-related due to the st atistical significance and because such decreases have been observed in another long term rat study, as well as studies in other species.

Clinical laboratory tests gave evidence for e ffects on liver function in males at 120 ppm and above, and in females at 500 ppm and above, in cluding some effects indicating an adaptive response of the liver like a signifi cant increase of rela tive liver weight at th e end of the treatment period for both males and females treated at 2000 ppm, accompanied by hepatocellular hypertrophy at microscopic examination. Effect s on enzyme activities and hepatocellular hypertrophy were no longer record ed at the end of the recovery period. Significantly decreased
activities of alanine aminotrans ferase (ALAT) were observed in peripheral blood of males and females at 500 ppm and 2000 ppm. At the end of th e recovery period, ALAT levels were slightly increased compared to controls; however, these were not statistically significant differences. These changes were not considered adverse.

At the end of the treatment period concentrations of urea (22%) were significantly increased at 2000 ppm in males. In females, triglycerides were increased in all dose groups (statistically significant at 500 and 2000 ppm; 73% and 65%, respectively) and cholesterol was increased at 500 ppm (25%) and 2000 ppm (49%), while creatinin e was decreased at 2000 ppm. Statistically significant decreases in cholesterol and trigly cerides were observed in recovery males.

At 2000 ppm the concentration of calcium was slight ly but statistically signi ficantly increased in males of the main group and the concentrati on of phosphate was sta tistically significantly increased in females of the main group.

At 2000 ppm relative weights of kidneys were si gnificantly increased in both sexes (males: 21%, females: 14%), but not at the end of the recovery period. In this dose group changes of the surface of the kidneys were observed in 5 males at necropsy at the end of the treatment period. Histopathological investigations revealed hyaline droplet nephropa thy possibly due to alpha-2-μglobulin accumulation in males at 500 ppm and 2000 ppm. This is a phenomenon unique to the male rat and is regarded not to be of toxi cological relevance for humans. Hyaline droplet accumulation was reversible within the recovery period, while other tubular changes observed atFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 64 of 107 500 ppm and above after the treatment period were still discernible in 200 0 ppm males after the recovery period (i.e. basophilic cortical tubule s, tubulosclerosis, Schmorl-positive material in tubules, cystic tubules and mononucl ear infiltrates). In females, a slightly increased grade of Schmorl-positive pigment was seen in renal tubules at 2000 ppm. This change was reversible within the recovery period and did not cause any damage. The hist opathological inve stigations of the kidneys of males from the control and 2000 ppm dose groups using the Azan stain revealed an immunohistochemical reaction that was strong er and distributed more widely throughout the
renal cortex of animals dosed at 2000 ppm compared to controls.

The histopathological inve stigations of the kidne ys of males treated for one week with 2000 ppm
revealed no differences between control and 2000 ppm males using the Azan stain. With immunohistochemical staining for alpha-2μ-globuli n, the reaction was slightly more intense and more widely distributed in c ontrol than in treated males.

870.3150 90-day Oral –Dog

In a dietary study (MRID 48574755, PM RA 2181207), groups of 4 male and 4 female Beagle dogs received fluensulfone (MCW-2 tech., 96.67-96.78%, Batch No. 36372130-291-PF1) in their diets at 0, 5, 50 or 500 ppm (0/ 0, 0.2/02, 1.6/1.8 and 17.1/18.0 mg/kg bw/day in males/females) for 13 consecutive weeks. Simila r sized recovery groups for the control and the high dose were treated for 13 consecutive weeks and sacrificed after an additional 28-day period
without treatment.

There were no deaths or clinical signs related to treatment. At 500 ppm, body weight gains were reduced by 20% to 55% compared to controls in both sexes, while reductions in body weight of up to 17% were noted in females. Food consump tion was reduced in females at 50 and 500 ppm. Decreased plasma and liver ALAT activity were observed at 500 ppm as we ll as increased liver weight, Kupffer cell pigmentation in two males a nd one female, and an is olated incidence of hepatocellular hypertrophy in one female suggesting that the liver is a targ et organ. Both sexes dosed with the 500 ppm test diet exhibited increa ses in reticulocytes and decreases in bilirubin, albumin, and albumin/globulin ratio s. Thyroid stimulating hormone le vels were also elevated in both sexes from the 500 ppm dose group. Other effect s at 500 ppm included reduced hemoglobin and protein levels in males, a nd decreased glucose levels and t hymus weights in females.

Indications of phase I enzyme induction were seen at 50 and 500 ppm in females for 6-beta testosterone hydroxylase and of phase II enzymes at 500 ppm only for microsomal epoxide hydrolase. The results exclude a potent effect on liver enzymes by fluensulfone as the changes were considered biologically not re levant and likely to be a res ponse of the constant load on the
liver with a xenobiotic.

Other findings at 50 ppm included a slight decrease in plasma and hepatic ALAT activity in both sexes, a slight and transient decrease in bi lirubin in males, and slightly reduced food consumption in females. At 5 ppm, there was a slight reduction in he patic ALAT activity in females only.

All the effects observed at 5 and 50 ppm were slight and reversib le, and considered unlikely to represent an adverse effect; th erefore, the NOAEL was considered to be 50 ppm (equivalent to an achieved intake of 1.6 mg/kg/day in males and 1.8 mg/kg bw/day in females). The LOAEL is 500 ppm (equivalent to an achieved intake of 17.1 mg/kg bw/day in males and 18.0 mg/kgFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 65 of 107 bw/day in females) based on decreased plasma and liver ALAT, decreased body weight gains and decreased body weight in females, liver eff ects, hematological findings, altered clinical chemistry parameters, and increased TSH levels.

This 90-day oral toxicity study in the dog is classified as acceptable/guideline and satisfies the guideline requirement for a 90-day oral toxicity study (O ECD 409; OPPTS 870.3150) in dogs.

870.3200 21/28-day Dermal

In a repeated dose dermal toxicity st udy (MRID 48574758, PMRA 2181209), groups of 10 male and 10 female Wistar rats were dermally exposed for 6 hours each day for 5 consecutive days per week over a period of 4 weeks (28 days) to fluensulfone (MCW-2 tech., 94.44%, Lot No. 36372130-291-PFI) at doses of 0 (control), 80, 400 or 2000 mg/kg bw/day. Carboxymethylcellulose (0.5% CMC) was used as the vehicle and the exposed area of approximately 25 cm 2 (approximately 10% of the to tal body surface) was covered by a semiocclusive dressing.

No mortality occurred, and no clinical signs of toxicity were observed. During clinical observations, no test item-related effects were recorded at the treated skin sites. Increased
concentrations of fluoride in bones and teeth were noted in treated animal s of all dose groups but were not considered adverse, because no signs of dental fluorosis we re observed during the study, and no abnormality in bone structure wa s observed at microscopic examination.

Transient reductions in food consumption were no ted in males at 400 mg/kg bw/day and in both sexes at 2000 mg/kg bw/day. A slight reduction in body weight gain, driven by a decrease in body weight gain during week 1 of the study, was noted in males at the 2000 mg/kg bw/day dose level. In females dosed with 2000 mg/kg bw/day fl uensulfone, slight alterations in hematological (increased red cell volume di stribution width, decreased mean corpuscular hemoglobin concentration, and increased reticul ocytes) and clinical chemistry (increased cholesterol, reduced serum ALAT) parameters were observed. No ef fect on hepatic ALAT activity was observed.

The combination of the vehicle (CMC) with fluensulfone led to minimally increased incidence and/or severity of acanthosis/hyperkeratosis not ed upon histological examination of treated skin of animals dosed at 400 and 2000 mg/kg bw/day wh en compared with the vehicle alone. These dermal effects were considered indicative of a slight irritant potential of th e test item. Because of the moderate degrees of acanthosis/hyperkeratos is observed in this study, these changes were considered adaptive and likely to be reversible after cessation of treatment, and therefore not
adverse.

The NOAEL was determined to be 400 mg/kg bw/day, based on reduced body weight gain, increased prothrombin time and increased relative spleen weight in males and increased red cell distribution width, increased retic ulocytes and increased choles terol in females at 2000 mg/kg bw/day.

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

870.3465 90-day InhalationFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 66 of 107 In a 90-day inhalation toxicity study (MRI D 49067003; PMRA 2257061), groups of 10 male and 10 female Wistar Crl:WI(Han) rats were e xposed nose-only to fluensulfone (MCW-2 Technical, 96.8%, Batch No. 411-033-05) for 6 hours/day, 5 days/week for 13 consecutive weeks (resulting in 65 and 66 expos ure days for males and females, respectively) to target concentrations of 0, 0.04, 0.2 or 1.0 mg/L (MMAD ± GSD of 1.02 μm ± 1.59, 1.16 μm ± 1.71, and 1.19 μm ± 1.60 for increasing test concentrat ion). The achieved concentrations were 0.040, 0.204 and 0.983 mg/L for the 0.04, 0.2 and 1.0 mg/L target concentratio ns, respectively. A 14- day range-finding study was conducted on 5 ra ts/sex/dose at 0, 0.035, 0.35 or 3.5 mg/L (6 hours/day, 5 days/week) to identif y appropriate dose levels.
No mortality occurred during th e study. Piloerection in combina tion with hunched posture and/or blepharospasm was noted in a few animals of the mid- and high-concentration groups shortly after exposure on the first exposure day, but not thereafter. Reduced body temperatures were recorded in females from the mid- and high- concentration groups (decreases of 0.57 and 1.47°C, respectively, from the control mean) at the end of the 90-day exposure period; a reduction of 0.54°C was observed in males from the high c oncentration group but wa s not statistically significant. In male rats, body weight developm ent was affected from day 3 onwards at all concentrations. The terminal body weights were decreased by 7%, 12% and 14% at the low-, mid- and high-concentration, respectively. Th e exposed males showed weight loss during exposure days, followed by catch-up growth during the weekend (when they were not exposed). Cumulative weight gain in males was reduced by 17%, 29% and 36% (low, mid, high dose, respectively). Female body weights were decrea sed only at the high-concentration during the first half of the study and were comparable to the control afterwards; how ever cumulative gain was reduced by 16%. Feed consumption of the ex posed males from all groups and of females from the high concentration group was slightly lower compared to controls throughout the study. Water consumption was increased, concentration- dependently, in all concentration groups in
both sexes.
Hematology results showed a concentration-depende nt increase in prothrombin time in females at all exposure concentrations (7%, 10% and 19%) and in males from the high-concentration group only (16%), an increase in reticulocytes in males from the high-concentration group (26%), a decrease in platelet counts in females from the high-concentration group (9%), and slight decreases (2-3%) in mean corpuscular hem oglobin concentration in females from the midand high-concentration groups. Clinical chemistry values showed changes in alanine aminotransferase (ALAT; decreased 53%) and bili rubin (increased 53-56%) in both sexes at the high-concentration. ALAT was also decreased in males of the mid-concentration group (29%). In addition, males showed changes in glucose (dec reased 31%) and inorganic phosphate (increased 18%) at all concentrations, in pot assium (increased 13%) at the mid- and high-concentration, and in phospholipids (increased 17%) and alkaline ph osphatase (ALP; increased 15%) at the highconcentration. Females additionally showed increases in ALP at the mid- and highconcentrations (10-31%), and increases in trigly cerides (82%) and potassi um (10%) at the highconcentration.

The relative weights of the kidne ys (12-20%) and liver (7-19%) we re increased at the mid- and high concentrations in both sexe s. In females, the absolute weights of these organs were increased as well. In addition, males had decrease d thymus weights (absolute and relative) at all concentrations (15-45%), increased adrenal weig hts at the mid- and high concentrations, and increased relative heart weight at the high-concentration. The ch anges in kidney, liver, thymusFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 67 of 107 and adrenal weight showed a con centration-related response. Relativ e lung weights were slightly elevated in both sexes at the high concentra tion (9-15%). The other organ weight changes observed occurred in males only and were likely to be secondary to the lower body weight in this sex (higher relative weights of the brain at the mid dose and above and testes at the high dose, lower absolute weight of the spleen at the mid dose and above).
Microscopic examination revealed treatment-re lated changes in the epiglottis and the nose (mainly at the second level of the nasal cavity). No histopathological corr elates to the weight changes of liver, adrenals and thymus were observed. Females exhibited a slightly increased incidence of corticomedullary mineralization of the kidney at the high concentration when compared to controls (the kidney was not examined at lower concentrations), but no histopathological correlates to the change in kidney weight were noted in males.
The changes in the epiglottis were characterized by squamous metaplasia in all exposed animals, a concentration-dependent increase in the inciden ce of epithelial hyperplasi a (starting at the lowconcentration in males and the mid-concentration in females), and an increase in the presence of
focal mononuclear cell infiltrates in males (starting at the low-c oncentration). Alterations in the nasal cavity consisted of squamous epithelia l hyperplasia in males starting at the lowconcentration and in females starting at the mi d-concentration, squamous metaplasia in both sexes starting at the mid-concentration, squamous epithelial hyperkeratosis starting at the midconcentration in females and at the high-concentr ation in males, and an increased inflammatory load characterized by focal mononuclear cell inf iltrates (in both sexe s starting at the midconcentration) and focal polym orphonuclear leukocytic infilt ration (in females at the highconcentration).
Pale lower incisors at necropsy were observed in females at all exposure concentrations (2, 4 and 9 affected at the low, mid and high concentrations , respectively) and in ma les starting at the midconcentration (7 and 10 affected at the mid a nd high concentrations, re spectively), while pale upper incisors were seen in 10 males and 3 fema les at the high concen tration. Microscopically, remnants of enamel in the upper incisors (followi ng decalcification of samples) were recorded in both sexes at the highest concentration only (8 animals of each sex). Fluoride content of bone and teeth was not analyzed.
In conclusion, rats exposed to fluensulf one by inhalation (6 h ours/day, 5 days/week, 13 consecutive weeks) showed various treatment -related changes. Potentially adverse changes which were present at the lowest concentration consisted of:
- weight loss on exposure days in males
- prolonged prothrombin time in females
- decreased thymus weight in males (without histopatholog ical effects)
- histopathological changes in the epiglottis (squamous meta plasia, epithelial hyperplasia, focal mononuclear cell infiltrate) in bot h sexes and in the nasal cavity (squamous epithelial hyperplasia) in males.
For most of these changes, their magnitude/incidence increa sed with increasing exposure concentration. Squamous metaplasia in the epig lottis was present in all exposed animals.
The lowest concentration tested (actual c oncentration 0.040 mg/L) was a Lowest-ObservedAdverse-Effect-Concentration (LOAEC or LOAEL) based on decreased weight gain in males,Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 68 of 107 increased prothrombin time in females, decreased thymus weight in males and histopathology in the epiglottis in both sexes. A NOAEC (NOAEL) was not identified (<0.040 mg/L).
This study is classified as acceptable/guideline (fully reliable) and satisfies the guideline requirements for a subchronic inhalati on study in the rat (OECD 413, OPPTS 870.3465).

870.6200 90-day Neurotoxicity

In a subchronic dietary neurotoxicity study (MRID 48574774, PMRA 2181227), groups of 12 male and 12 female RccHan:WIST (SPF) rats received fluensulfone (MCW-2 tech., 95.7%, Batch No. 36372130-291-PF1) in their diet at co ncentrations of 0 (control), 100, 500 and 2500 ppm (equivalent to 0/0, 6/7, 31/ 34, and 153/162 mg/kg bw/day in males/females) for at least 90 days.

Functional observational batteries (FOBs) incl uding quantitative assessm ents of landing foot splay, sensory perception and muscle weakness (grip strength), were perfor med in all animals in a randomized order once prior to first ad ministration, and during weeks 2, 5, 9 and 13.
Locomotor activities were asse ssed after each FOB evaluation. At the end of the treatment period, 5 rats/sex/group were perfusion-fixed in situ . Selected nervous system tissues were dissected, processed and examined microscopically.

All animals scheduled for neurohistological ex amination survived the scheduled study period.
Treatment-related effects includ ed lower food consumption and body weight gain in males and females at 2500 ppm, as well as reduced body weight in males.

Detailed clinical observations and FOB evaluations of clinical symptoms revealed no treatmentrelated effects of fluensulfone. Locomotor activ ity (total distance, time in center arena and rearing counts) was significantly lower in male s at 2500 ppm during week 5. Males in this dose group also exhibited reduced grip strength at we ek 13, and a slight reduc tion in absolute brain weight. These treatment-related effects were attributed to sy stemic toxicity, and not to neurotoxicity. There were no treatmen t-related effects on neuropathology.

The NOAEL was 500 ppm (equivalent to 31 mg/kg bw/day for males and 34 mg/kg bw/day for females) and the LOAEL was 2500 ppm (equivale nt to 153 mg/kg bw/day for males and 162 mg/kg bw/day for females) based on decreased food consumption and body weight gain in both sexes and decreased body weight in males.

The subchronic neurotoxicity study in the rat is classified as acceptable/guideline (fully reliable) and satisfies the guideline requirement for a subchronic neur otoxicity study (OECD 424, OPPTS 870.6200b) in rats.

A.4.3 Chronic Toxicity

870.4100 Chronic Toxicity-Dog

In a 1 year oral toxicity study (MRID 48574756, PMRA 2181208), fl uensulfone (MCW-2 tech., 95.70-98.65%, Batch No. 36372130-291-PF1) was administered in the diet to groups of 4 male and 4 female Beagle dogs at 0, 5, 50 100 and 500 ppm (0.0, 0.1/0.1, 1.5/1.5, 3.1/3.3. 16.0/16.2 mg/kg bw/day in male/females) for a period of 52 weeks. Similar sized recovery groups for the control and the high dose were treated for 52 consecutive weeks and sacrificed after anFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 69 of 107 additional 8-week period without treatment.

Oral administration of fluensulfone to Beagle do gs in the diet at a dose of 500 ppm resulted in transient changes in consistenc y of feces, reductions in food intake in females and lower body weight gains, mean body weights and termin al body weights, effects on hematology and biochemistry parameters and on the liver.

A mild reduction in hemoglobin co ncentration and associated parame ters with reticulocytosis, as well as the presence of brownish pigment (likely hemosiderin) in the liver and increased platelets were observed. Changes in albumin and protein profile (reductions) were also noted. Several organ weight changes (increased kidney weight, reduced spleen we ight in females, and increased relative adrenal gland weight in males) were noted at 500 ppm without any pathological correlates. Females from the 500 ppm dose group e xhibited slight reductions in serum glucose levels as well. Decreases in plasma alanine aminotransferase activity were observed at 50, 100 and 500 ppm. An inhibition of hepatic alanine am inotransferase activity was evident after 52
weeks of treatment at the same dose levels. Thes e changes were not considered to be adverse since decreases of these enzymes had no impact on the health status of the animals. In addition, no effect was observed in animals previously treated at 500 ppm after the 8-week recovery period. The phase II enzyme glutat hione S-transferase was induced at 500 ppm in both sexes and after the recovery period the eff ect was still increased in males but of no statistical significance, or not present in the females. A slight inducti on of epoxide hydrolase was also evident in both sexes at 500 ppm. Slightly incr eased liver weight in females a nd minimal to slight degrees of diffuse hepatocellular hypertr ophy in both sexes were observed at 500 ppm. The findings were
still evident at the end of recove ry in a single female. The changes in consistency of feces were
also evident in a dose-relate d manner at the lower dose of 100 ppm. Also at 100 ppm, males demonstrated a slight reduction in partial thro mboplastin time, and females exhibited reduced body weights and associated organ weight changes in addition to slight reductions in serum bilirubin levels; at this dose level the e ffects were not considered adverse.

A dose-related increase of fluoride content was evident at doses of 50, 100 and 500 ppm in the ashes from bone and teeth, attain ing statistical significance only at the high dose level. No signs of dental fluorosis were observed during the study and no abnormality in bone structure was observed at microscopic examination and therefore the deposition of fluoride was not considered
adverse.

The NOAEL is 100 ppm (3.1/3.3 mg/kg bw/day, M/ F) based on reductions in body weights, changes in hematological and clinical chemistry parameters, and organ weights in both sexes at the LOAEL of 500 ppm (16.0/ 16.2 mg/kg bw/day, M/F).

This chronic study in the dog is classified as acceptable/guideline and satisfies the guideline requirement for a chronic oral st udy (OECD 452; OPPTS 870.4100) in dogs.

870.4100 Chronic Toxicity-Rat

In an oral combined chronic toxicity/onc ogenicity study in rats (MRID 48574765; PMRA 2181217), 4 groups of HANRcc Wist(SPF) rats (allo cation A, oncogenicity animals), 50 males and 50 females each, were treated for 104 weeks with 0, 30, 200 or 1200 ppm (0/0, 1.4/1.7, 9.6/11.6, and 57.7/69.3 mg/kg bw/day in males/female s) of fluensulfone (MCW-2 tech., 96.7%, Batch No. NLL6692-3) in their diets. A satellite gr oup (allocation B, chronic toxicity animals) ofFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 70 of 107 20 males and 20 females per group was similarly tr eated for 52 weeks at the same dose levels (0/0, 1.6/1.9, 11.0/13.1, 66.3/75.2 mg/kg bw/day in males/females).

Usual investigations were conducted on all animal s (viability/mortality, clinical signs, food/water consumption, body weights, ophthalmoscopic, hemato logy, clinical chemistry, urinalysis, etc.). In addition, a functional observational battery (F OB) and locomotor activity assessments were performed on allocation B animals at week 52. In 5 allocation B animals per group, a part of the liver was used for enzyme activity determinatio ns. After 52 and 104 weeks, the right femur and
the incisors of the maxilla of ten animals per sex and group were also taken for fluoride
determination.

Treatment of Wistar rats with fluensulfone at dietary con centrations of 30, 200, 1200 ppm for up to 104 weeks had no effects on survival, clinic al signs or ophthalmo scopic findings. Grip strength and the results of the functional obser vational battery were also not affected by treatment for 52 consecutive weeks. A slight decr ease in overall locomotor activity was noted in high dose males. No increase in palpable nodules or masses was observed at any dose level at either the interim and terminal sacrifices, and no increase in any type of neoplastic finding was
recorded at terminal sacrifice.

Food consumption in males treated with 1200 ppm wa s slightly lower during the first 4 weeks of treatment, but no further effects on food cons umption were recorded thereafter. Water consumption was also increased in high dose ma les and females. Absolute mean body weight and body weight gain were decreased by the tr eatment in a dose-dependent manner in males treated at 200 and 1200 ppm; however, the chan ges at 200 ppm were marginal (5-7%), although statistically significant for some weeks. At 1200 ppm, body weight and body weight gain were decreased by 11% and 15%, respectively, at study termination. In high dose females, body weight was decreased during the last 11 week s of the study (decreased by 6% at study termination) and body weight gain was reduced from week 2 onwards (decreased by 13%).

After 52 weeks a number of changes in hematology and biochemistry parameters were noted, mainly in animals treated with 1200 ppm. At 200 ppm and above, slightly decreased prothrombin time (5%) was noted in males. Dose-dependent decreases in neutroph il and white blood cell
counts were observed in all fema le dose groups. Clinical chemistr y findings in the low and mid dose groups included changes in ALAT, sodium, ch loride, calcium, phosphorus, total protein and globulin, in males and/or females. Clinical ch emistry and organ weight data revealed some effects on the liver, limited to the 1200 ppm group. In the absence of correlating histopathological findings these changes we re considered to be non-adverse metabolic adaptations. Other organ weight changes noted in high dose gr oups included increased kidney weights in males and females, and increased adre nal weights in males. No effects on adrenals were noted at histopathology. During the urinal ysis, increased ketone s were observed in 1200 ppm males and females, and increased leukocytes in high dose males.

After 104 weeks a few changes in hematology para meters, mainly in red blood cell parameters, were noted in animals treated with 1200 ppm. Decreased hemoglobin conc entration distribution width (HDW) was noted in females at 200 and 1200 ppm. Decreased mean corpuscular hemoglobin (MCH) was observed in high dose ma les, and decreases in mean corpuscular hemoglobin concentration (MCHC) were noted in all males at 78 weeks, as well as in 1200 ppm males and 200 and 1200 ppm females at 104 weeks. Neutrophil counts were also increased in high dose females, and eosinophil (EOS) levels were increased in high dose males.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 71 of 107 At necropsy after 52 weeks, no gross lesions th at could be attributed to treatment with
fluensulfone were observed. Mi croscopically, hyperkeratosis of the esophagus of minimal severity was observed in animals of both sexes treated with 1200 ppm, but no abnormality was noted in lungs.

At necropsy after 104 weeks, an increased number of foci in the lungs was recorded in the treated animals, compared to c ontrols, attaining sta tistical significance on ly in males at 1200 ppm. Enlarged livers were recorded in 4 females (versus one in the control group), as well as cysts in the livers of 8 females (versus 2 in the control group) and foci in the liver of 4 females (versus 0 in the control group) tr eated at 1200 ppm. In males, a s light increase in foci in the prostate was noted in mid and high dose groups (5 in each treatment group versus one in the control group). No correlative microscopic observations were noted in the prostate. Microscopically, an increased in cidence of hyperkeratosis in the esophagus was recorded in animals of both sexes treated at 200 and 1200 ppm. Since the severity grade was minimal in most cases, the hyperkeratosis was c onsidered to be of no toxicolo gical significan ce, and possibly from a slight irritating effect of fluensulfone. The incidence of chronic interstitial inflammation in the lungs was increased in males and females treated at 1200 ppm and in females treated at 200 ppm but was only statistica lly significant for males at 1200 ppm. This lesion was characterized by focal/multifocal changes consisting of interstitial or intra-alveolar inflammatory cells associated with hypertrophied reactive type II pneumocytes, associated with the presence of foamy intra-alveolar macrophages, which was also increased in mean severity grade. There were no treatment-related hyperplastic changes or tumors recorded. At 104 weeks, slight increases were observed in chronic nephropathy, tubular ba sophilia and mononuclear foci in the kidneys
and mononuclear infiltrates in the pharynx of females treated at 1200 ppm as compared to
controls.

Investigations of liver enzymes from liver tissues demonstrated that the phase II enzymes uridine diphosphoglucuronosyl-transferase ( UDPGT) and glutathione S-tran sferase (GST) were induced in 200 and 1200 ppm females and in 1200 ppm males, and epoxide hydrolase (mEH) was induced in all male and female dose groups. Thes e enzymes were more significantly induced in
females than males.

After 52 weeks of treatment, fluoride measuremen ts revealed a marked dose-dependent increase, several fold above controls, of the fluoride cont ent in the ashes from bones and teeth at 200 and 1200 ppm. Only very slight incr eases in fluoride content in b ones (up to 28% above controls) were noted in 30 ppm animals. No increase of th e fluoride content in the ashes from teeth was
recorded in animals tr eated with 30 ppm.

After 104 weeks of treatment, the fluoride content in the ashes from bones and teeth of all treated animals was markedly increased. At 30 ppm, si gnificant increases of 47-81% above controls
were seen in bones and non-sta tistically significant increases of 20-66% were seen in teeth. Changes in fluoride levels were not considered adverse as there were no associated structural signs of dental fluorosis (discoloration of the teeth) or skeletal fluorosis (mobility problems, changes in external appearance of bones, changes in bone histopathology) at any dose.

The NOAEL was established at 200 ppm (9.6/ 11.6 mg/kg/day, M/F) based on decreased body weight in males, hematology and clinical chem istry findings in both se xes, histopathological changes in the lungs of males, and histopathologi cal changes in the esophagus of both sexes seen at the LOAEL of 1200 ppm (57.7/69.9 mg/kg/day, M/F).Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 72 of 107 Fluensulfone showed no carcinogenic potential in rats. Dosing was considered adequate in both sexes based on the findings of decreased body wei ght and weight gain, slight hematological effects and microscopic findi ngs in the lung and esophagus.

This study is classified acceptable/guideline (fully reliable) and satisfies the guideline requirement (OPPTS 870.4300; OECD 453) for a comb ined oral chronic t oxicity/carcinogenicity study in the rat.

870.4200 Carcinogenicity-Mouse

In an oral oncogenicity study (MRID 48574766; PMRA 2181218), 4 groups of 50 male and 50 female CD-1 mice (allocation A) were trea ted for 78 weeks with 0, 30, 200 or 1200 ppm of fluensulfone (MCW-2 tech., 96.7-98.65%, Batch No. 36372130-291-PF1) in their feed. Additionally, 6 males and 6 fema les (allocation B) were ex posed to the same dietary concentrations and used for liver enzyme dete rmination after 13 weeks of treatment. The average daily intakes of test material were 0, 4.2, 27.6 or 152.3 mg/kg bw/day for males and 0, 6.4, 39.0 or 188.4 mg/kg bw/day for females.

After 13 weeks of treatment, fluensulfone caused a slight decrease of hepatic alanine aminotransferase (ALAT) activity in female s from the 1200 ppm dose group, but no change in ALAT activity was recorded afte r 78 weeks of treatment. In addition, after 13 weeks a weak increase of microsomal cytochrome P450 content was recorded, accompanied by a slight increase in the activity of CYP4A-1 and CYP1 A-1 in females only, and slight to moderate inductions of phase II enzymes uridine diphos phoglucuronosyl-transfe rase, glutathione Stransferase and epoxide hydrolase, mainly in fe males. Most of these changes occurred at 200 ppm and above, but were considered to be adap tive; none of these changes were considered
adverse.

Treatment of CD-1 mice with fluensulfone at dietary concen trations of 30, 200 or 1200 ppm for up to 78 weeks had no effect on survival, clinic al signs, or presence of palpable nodules and masses. Absolute food consumption (g/animal/day ) was statistically significantly reduced at most weighing intervals in both sexes at 1200 pp m. The overall mean for animals treated with 1200 ppm compared to controls wa s 17% lower in males and 24% lower in females. In males, relative food consumption (g/kg bw /day) was not affected by the treatment. However, in females, relative food consumption was stat istically significantly reduced at most weighing intervals at 1200 ppm and the overall mean over treatment wa s reduced by 21%. Cumulative body weight gain was decreased by 13% and 37% for male s at 200 and 1200 ppm, respectively, and by 30% for females at 1200 ppm.

Absolute mean body weight and body weight gain were statistically signi ficantly decreased in a dose-dependent manner in males at 200 and 1200 pp m (statistically significant from week 5 through 54 and throughout the study, respectively) , and in females at 1200 ppm (statistically significant from week 2 onward fo r most measurements). The mean body weights at the end of the treatment period were reduced by 6% and 17 % in the males of the mid and high dose groups, and by 12% in the females of the high dose group.

After 52 weeks, hematology revealed a slightly decreased absolute red blood cell counts in mice of both sexes at 1200 ppm (5% be low controls) and decreased to tal white blood cells count in females only, accompanied by reductions in absolute neutrophil, eosinophil, lymphocyte andFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 73 of 107 monocyte counts (19-25%). Total white blood cells and absolute neutrophil and eosinophil counts were also reduced in females at 200 ppm (19-43%). None of these changes was still present at terminal sacrifice af ter 78 weeks. Slight increases in serum ALAT and ASAT were noted in females from the 200 and 1200 ppm dose groups at week 78, and in creases in SDH were noted in both sexes at 200 and 1200 ppm at 78 w eeks (these parameters were not assessed at week 52).

Absolute and relative (to brain weight) prostate weights were observed in males in the 200 and 1200 ppm dose groups. Changes in other organ weights at 1200 pp m (increased relative kidney, adrenal gland, epididymal, testes, heart, and brai n weight in males; decreased absolute kidney weight in females, and increased relative liver we ight in females) were likely secondary to the
effect of fluensulfone exposure on body weight.

Non-neoplastic lesions consiste d of a statistically significan tly increased incidence of bronchiolization (i.e. a change fr om flattened epithelium to cuboidal epithelium) in the lung of males and females treated at 200 and 1200 ppm (f rom control to high dose respectively, 1, 0, 24 and 31 in males and 5, 7, 43 and 48 in female s; n = 50 all groups). Transmission electron microscopy (TEM) analysis revealed hypertrophy of the epithelium of the terminal bronchioles affecting mostly the non-ciliated Clara cells as well as the dire ctly surrounding ciliated cells.

The LOAEL was established at 200 ppm (27.35 a nd 38.96 mg/kg bw/day in males and females, respectively), based on decreased body weight/weight gain in males and an increased incidence of lung bronchiolization in males and females. The NOAEL was established at 30 ppm (4.2 and 6.4 mg/kg bw/day in males and females, respectively).

An increased incidence in nodules in the l ung of females treated at 200 and 1200 ppm was recorded at terminal necropsy. The incidence of affected animals was 1, 6, 11 and 13 in the control, 30, 200 and 1200 ppm groups, respectivel y. Microscopically, the incidence of neoplastic lesions in the lungs was increased in females treated at 200 and 1200 ppm. The neoplastic lesions consisted of st atistically significantly increased alveolar/bronchiolar adenomas in females treated at 200 and 1200 ppm, compared to control females (4, 8, 28 and 18%, control to high dose). The number of alveolar/bronchiol ar carcinomas in females treated at 1200 ppm did not attain statistical significance using the Fi sher’s exact test, while it was positive with the Peto trend test (4, 2, 2 and 8%, control to hi gh dose). The latency to tumor onset was also reduced for the carcinomas detected at 1200 ppm . The incidence of carcinoma, but not adenoma, was within historical control range. Combined tumor incidence was 8, 10, 30 and 26%, control to high dose (histo rical control values not provid ed). Males did not show a
treatment-related increase.

Dosing was considered adequate based on decreas ed body weight/weight gain and an increased incidence of lung bronchiol ization in both sexes.

This study is classified as acceptable/guideline and satisfies the guideline requirement for an oral carcinogenicity study in the mouse (OECD 451; OPPTS 870.4200).

870.4200 Carcinogenicity-Rat

See above, 870.4100 Chronic Toxicity-Rat

A.4.4 Developmental Toxicity and ReproductionFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 74 of 107 870.3700 Prenatal Developmental Toxicity-Rabbit

In a teratogenicity study (MRID 48574770, PMRA 2181224), 4 groups of 20 female Himalayan
rabbits were mated and then administered da ily doses of fluensulfone (MCW-2 tech., 96.67%, Batch No. 36372130-291-PF1) in 0.5% aqueous carboxymethylcellulose at 0, 2.5, 10 or 40 mg/kg bw at a dosing volume of 5 mL/kg bw on gestation days (GD) 6 through 27. Maternal evaluations during gestation incl uded clinical observat ions and changes in body weight gain and food consumption. All does were sa crificed on GD 28, at which time gross external and internal necropsies were performed. The gravid uterus wa s weighed and examined, and the foetuses were removed by caesarean section. Fetuses were removed from the uterus, sexed, weighed individually, examined for gross external abno rmalities, and sacrific ed by a subcutaneous injection of sodium pentobarbital. All fetuses were examined for visceral abnormalities. The
heads of half of the fetuses were serially sectioned and examine d. The other half of fetuses was
used for skeletal examination.

The total number of females with live fetuse s was 20, 19, 19 and 17, resp ectively, in order of ascending dosages. One female each at 2.5 and 40 mg/kg bw/day was not pregnant, and one female and two females respectively in groups tr eated at 10 and 40 mg/kg bw/day had resorption sites only.

All does survived until the scheduled necropsy, and no clinical signs related to treatment were
observed.

Mean body weight gain was slightly reduced in does treated at 40 mg/kg bw/day between GD 6 and 9 (control to high dose gains during this in terval were 13, 24, 10 a nd -29 g, respectively), attaining statistical significance on GD 7 and 8. This effect was accompanied by a reduction in food intake noted between GD 18 and 24 (-22.2% compared to the control group). When body weight gain was corrected for gravid uterine weight, does in the 10 and 40 mg/kg bw/day dose groups lost 29% and 39% more weight from GD 6 to 28 than control does, respectively. In the range-finding study, marked weight loss and d ecreased food consumpti on along with clinical signs were observed at 50 mg/kg bw/day from GD 6-12 and mortality was observed at 60 mg/kg bw/day in a dose toleration study; therefore, the transi ent weight change at 40 mg/kg bw/day was
considered treatment-related.

The relevant reproduction data ( post-implantation loss and the mean number of fetuses per doe) were not affected by treatment with the test it em at any dose level. No macroscopic findings
related to treatment were not ed at necropsy of the does.

Fetal data showed no treatment-related effect s on sex ratio. Fetal body weight was slightly reduced (7% below controls) at 40 mg/kg bw/day . Head sectioning, visceral examination and skeletal and cartilage examinations of fetuse s revealed only one finding related to treatment: incomplete ossification of digit 5 of the medial phalanx in both forelimbs. The maternal and developmental LOAEL is 40 mg/kg bw/day base d on the reduced body weight gain and effects on food consumption noted in maternal animals and the reduced fetal body weight and delayed ossification; the NOAEL for both maternal and deve lopmental toxicity was considered to be 10 mg/kg bw/day. Although maternal body weight effects were marginal at the LOAEL, the LOAEL is considered a threshold effect level, based on the significant toxicity observed in the range-finding studies at 50 and 60 mg/kg bw/day.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 75 of 107 The developmental toxicity study in the rabbit is classified as acceptable/guideline and satisfies the guideline requirement for a developmental toxicity study in the rabbit (OECD 414; OPPTS 870.3700).

870.3700 Prenatal Developmental Toxicity-Rat

In an oral teratogenicity study (MRI D 48575771, PMRA 2181223), groups of 30 Wistar Crl:WI(HAN) female rats were mated and then admi nistered daily dosages of fluensulfone (BYI 01921, MCW-2 tech., 96.1-97.0%, Batch No. NLL-6692- 7-5) in 2% Cremophor, 0.5% CMC, and deionized water at 8, 50, or 300 mg/kg bw/d ay at a dosing volume of 10 mL/kg bw/day on days 6-19 of gestation, inclusive. All dams were sacrificed on gestation day (GD) 20. All fetuses were evaluated for external anomalies. Approximate ly half of all fetuses from each litter were examined for visceral effects at necropsy and al so evaluated for cranial effects using the Wilson's technique. The remaining fetuses underwent an evaluation for general skeletal (including cartilage) development.

Maternal compound-related findings observed in dams during the treatment period at 300 mg/kg bw/day included body weight declines (up to 11% from control values; 6% when corrected for uterine weight) and body weight gain reducti on (35% during treatment when corrected for uterine weight), coupled with decreased food consumption and in creased absolute and relative liver and kidney weights. There were no other compound-relate d findings observed.

There were no reproductive effects attributed to the test compound nor we re there any significant differences in the litter size, the median percen t male fetuses, or plac ental weights in any group tested. One litter from the 300 mg/kg bw/day dos e group contained four dead fetuses, which may have been secondary to maternal body weight e ffects, since the dam showed weight loss and stopped eating during the last few days of treatment . A decline in female fetal weights of 8% was observed at 300 mg/kg bw/day and was considered to be secondary to maternal toxicity. No compound-related effects were observed on fetal exte rnal, visceral, or skeletal malformations or variations, or on the incidence of total malformations or variations.

Incidences of several skeletal variations, including incomple tely ossified parietal bone, interparietal bone and squamosal bone, as well as incidences of enlarged sagittal suture, anterior fontanelle, posterior fontanelle and squamosal suture were reduced compared to concurrent
controls on both a fetal and litte r basis in the 300 mg/kg bw/day dose group. These findings may represent a “more progressed” ossi fication of the fetal skeleton, pe rhaps related to the deposition of fluoride into bone during pre-natal development. Based on decreased body weight gain and food c onsumption in dams and on lower body weight of female pups observed at the maternal and developmental LOAELs of 300 mg/kg bw/day, the maternal and developmental NOAELs were 50 mg/kg bw/day.

This study is classified as acceptable/guideline (fully reliable) and satisfies the guideline requirement for a developmental toxicity study in the rat (OPPTS 870.3700; OECD 414).

870.3800 Reproduction and Fertility Effects

In a two generation reproduction study, groups of 24 Wistar HanRcc:WIST(SPF) rats/sex/dose received fluensulfone [MCW-2 TECH (96.67-96.78% a.i., Batch No. 36372130-291-PF1)] inFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 76 of 107 their diets at concentrations of 0, 30, 250 or 1800 ppm. In addition to the standard evaluations performed in a guideline reproductive toxicity study, fluoride content of teeth (incisor) and bone (femur) was assessed in F1 parental animals and F2 offspring. The overall average daily compound intake (weighted for each period of th e study) was as follows: P adults: 0, 1.9, 15.4, or 114.2 mg/kg bw/day in males, 0, 2.7, 22.8 or 164.8 mg /kg bw/day in females; F1 adults: 0, 2.0, 17.0 or 129.7 mg/kg bw/day in males, 0, 2.9, 23.3 or 169.1 mg/kg bw/day in females; both generations combined: 0, 2.0, 16.2, or 122.0 mg/kg bw/day in males, 0, 2.8, 23.0, or 169.1 mg/kg bw/day in females). The premating averag e daily compound intake for both generations combined was 0, 2.2, 18.3 or 137 mg/kg bw/day in males and 0, 2.5, 20.3 or 149 mg/kg bw/day
in females.

Parental toxicity: High dose male and female body weights were reduced in both generations throughout the treatment period (at end of prem ating, 10-12% less for males and 4-7% less for females), although high dose animals of the F1 ge neration started the post-weaning period with lower body weights than controls that did not increase in ma gnitude over the dosing period. Cumulative premating body weight gain was reduced by 21% and 12% for P males and females, respectively, but was not decrease d in F1 animals. Body weight gain was also lower for P (8%) and F1 (10%) females during gestation, but not during lactation. In all other dose groups, body weight gain was either similar to controls or wa s increased to compensate for the initial losses.

Treatment with the test item at 1800 ppm resulted in a reduction of rela tive food consumption (g/kg bw/day) during the first w eek of dosing of the P genera tion. Due to lower body weights observed in the 1800 ppm dose group, absolute f ood consumption (g/animal/day) was reduced throughout most of the st udy at this dose.

Increased kidney and liver weights were seen in high dose animals of both generations and adrenal weights were reduced in high dose P females. Relative (to body weight) testes and epididymides weights were increased in the 180 0 ppm F1 males. Other organ weight changes were noted at the high dose that were consid ered to be secondary effects of a lowered body weight as organ to body weight ratios were not affected.

Centrilobular hypertrophy of the liver at minimal to slight severity (P males, F1 males and females) and reduced glycogen deposits (P males a nd females, F1 males) we re noted in parental animals dosed at 1800 ppm. The hypertrophy finding co rrelated with the incr ease in liver weight
and was considered to be of metabolic nature an d of adaptive character as there were no further indicators of liver injury. Glycogen deposits were reduced, likely due to the lower body weight. The incidence and severity of re nal tubular hyaline droplets were increased in males of P and F1- parental animals. The incidence and/or sever ity of renal tubular basophilia, mononuclear cell
infiltration and tubular casts we re also increased in males of both generations. These lesions
were considered to be the s econdary lesion resulting from hy aline droplets described above. Follicular cell hypertrophy of the th yroid gland was recorded at mi nimal to slight severity in males of the P and F1 generations.

Reproductive toxicity: There we re no treatment-related effect s on reproductive performance.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 77 of 107 Offspring toxicity: In both 1800 ppm F1 a nd F2 pups, pup body weights were reduced during lactation (10% below controls at PND 31). Post-natal pup losses before post-natal day 4 were observed in both the high dose P and F1 generati ons. From control to high dose, the percent loss was 0.4, 0.6, 1.4 and 2.6% (offspring from P mating) and 3.9, 3.1, 3.3 and 9.0% (offspring from F1 mating).

Spleen weights were reduced in both F1 and F2 high dose pups. Absolute and relative (to brain weight) thymus weights were reduced in the F1 pups and F2 pups, which were considered to be related to the lower body weight s of these animals. During the histopathological examination, the severity of glycogen deposits was observed to be reduced in the F2 male and female pup livers, and a decrease in incidence of glycoge n deposits was seen in the high dose female pups. This finding was likely associated with the lo wer body weights. The livers of F1 pups were not examined histologically.

Fluoride evaluation: In the F1 parental animal s, mean fluoride content was dose dependently higher in bone (femur) and in teeth (incisors) at 30, 250 and 1800 ppm. With increasing dose,
fluoride content of bones in males was increased by 100%, 494% and 1789% re lative to controls; in females the increase was 72%, 332% and 1094% . In teeth, increases in males were 32%, 305% and 1600% and in females were 58%, 329% a nd 1077%. In F2 pups, the mean content of fluoride was higher in teeth of males at 30 ppm and above (35%, 64% and 206% with increasing dose), in bones and teeth of females at 250 ppm (33%) and 1800 ppm (206% in bone, 145% in teeth), and in bones of males at 1800 ppm (389%). Since there were no e ffects of discoloration of the teeth or fragility of bones a nd teeth in either F1 adults or F2 pups, it is concluded that there was no evidence of fluorosis in this study.

The parental LOAEL in males and females is 1800 ppm (overall study intake of 122.0 mg/kg bw/day in males and 169.1 mg/kg bw/day in fema les; premating intake of 137 mg/kg bw/day in males and 149 mg/kg bw/day in females) ba sed on decreased body weight, body weight gain, food consumption and increased liver and kidn ey weights and histopathology changes in the liver and kidney. The parental NOAEL is 250 pp m (overall study intake of 16.2 mg/kg bw/day in males and 23.0 mg/kg bw/day in females; pr emating intake of 18.3 mg /kg bw/day in males and 20.3 mg/kg bw/day in females).

The reproductive NOAEL in males and females is 1800 ppm (overall st udy intake of 122.0 mg/kg bw/day in males and 169.1 mg/kg bw/day in females; premating intake of 137 mg/kg bw/day in males and 149 mg/kg bw/day in females). The reproductive LOAEL was not
established as no treatment-re lated effects on reproductive performance were observed.

The offspring LOAEL in males and females is 1800 ppm (overall study intake of 122.0 mg/kg bw/day in males and 169.1 mg/kg bw/day in fema les; premating intake of 37 mg/kg bw/day in males and 149 mg/kg bw/day in females) base d on decreased pup weights and spleen weights and an increase in post-natal pup loss between da ys 0 and 4 in the F1 and F2 offspring. The offspring NOAEL is 250 ppm (overall study in take of 16.2 mg/kg bw/day in males and 23.0 mg/kg bw/day in females; premating intake of 18.3 mg/kg bw/day in males and 20.3 mg/kg bw/day in females).Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 78 of 107 This study is classified as acceptable/guideline (fully reliable) and satisfies the guideline requirement (OPPTS 870.3800; OECD 416) for a tw o-generation reproductiv e toxicity study in
the rat.

A.4.5 Mutagenicity

Gene Mutation

870.5100, Bacterial Reverse Mutation
Fluensulfone
MRID 48575035 Acceptable/guideline Main test: 0, 16, 50, 158, 500, 1581 and 5000 μ g/plate

Repeat test (TA100 only): 0, 1000, 2000, 3000, 4000, 5000, and 6000

μ g/plate

Additional test (TA100 only): 0, 375, 750, 1300, 2600, and 5200 μ g/plate

Fluensulfone was not mutagenic either in the absence or in the presence of metabolic activation, up to a concentration of 5000 μg/plate, when tested on Salmonella typhimurium TA1535, TA1537, TA98 and TA102 strains. A weak positive response was obtained in Salmonella typhimurium strain TA100, both in the presence and in the absence of metabolic activation in two independent experiments(main and repeat, but was negative in the additional test) carried out up to 6000 μg/plate.

870.5100, Bacterial Reverse Mutation
Fluensulfone
MRID 48574760 Acceptable/guideline Experiment one (plate incorporation) : 0, 3, 10, 33, 100, 333, 1000, 2500,
5000 μ g/plate

Experiment two (pre-incubation): 0, 33, 100, 333, 1000, 2500, and 5000

μ g/plate

Fluensulfone was not mutagenic either in the absence or in the presence of metabolic activation, up to a concentration of 5000 μg/plate, when tested on Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and on
Escherichia coli strain WP2 uvrA, in two independent experiments, according to the Ames assay. 870.5100, Bacterial Reverse Mutation
Fluensulfone
MRID 48574761 Acceptable/guideline Experiment one (plate incorporati on): 0, 10.0, 31.6, 100, 316, 1000, 2500,
5000 μ g/plate

Experiment two (pre-incubation): 0, 3.16, 10.0, 31.6, 100, 316, 1000, 2500,
and 5000 μ g/plate

Fluensulfone was not mutagenic either in the absence or in the presence of metabolic activation, up to the concentration of 5000 μg/plate, when tested on Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and on
Escherichia coli strain WP2 uvrA, in two independent reverse mutation assay experiments (Ames Assay). 870.5100, Bacterial Reverse Mutation
TSA
MRID 48574784 Acceptable/guideline Experiment one (plate incorporation) : 0, 3, 10, 33, 100, 333, 1000, 2500,
5000 μ g/plate

Experiment two (pre-incubation): 0, 33, 100, 333, 1000, 2500, and 5000

μ g/plate

Under the conditions of this assay, thiazole sulfonic acid was not mutagenic either in the absence or in presence of metabolic activation, up to concentration of 5000 μg/plate when tested on Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and on Escherichia coli strain WP2 uvrA, in two independent experiments, according to Ames.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 79 of 107 870.5100, Bacterial Reverse Mutation
MS
MRID 48574788 Acceptable/guideline Experiment one (plate incorporation) : 0, 3, 10, 33, 100, 333, 1000, 2500,
5000 μ g/plate

Experiment two (pre-incubation): 0, 33, 100, 333, 1000, 2500, and 5000

μ g/plate

Experiment three (pre-incubation): 100, 333, 1000, 2500, and 5000 μ g/plate

Methyl sulfone did induce gene mutations in the genome of Salmonella typhimurium strain TA100 in the absence of metabolic activation. In strain WP2 uvrA a tendency towards higher mutation frequency was observed only in experiment II in the absence of metabolic activation but this was not reproduced in two other experiments. No induction of gene mutations was observed with any other strain. Therefore, methyl sulfone is considered to be weakly mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

870.5100, Bacterial Reverse Mutation
BSA
MRID 48574793 Acceptable/guideline Experiment one (plate incorporation) : 0, 3, 10, 33, 100, 333, 1000, 2500,
5000 μ g/plate

Experiment two (pre-incubation): 0, 33, 100, 333, 1000, 2500, and 5000

μ g/plate

Under the conditions of this assay, butene sulfonic acid was not mutagenic either in the absence or in the presence of metabolic activation, up to a concentration of 5000 μg/plate when tested on Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and on Escherichia coli strain WP2 uvrA, in two independent reverse mutation (Ames Assay) experiments.

870.5300, Mammalian Gene Mutation
Fluensulfone
MRID 48574763 Acceptable/guideline 0, 24, 32, 40, 48, 56, 64, 72 μ g/mL without metabolic activation;

0, 20, 30, 40, 50, 60, 70, 80 μ g/mL with metabolic activation

Fluensulfone was considered to be non-mutagenic in the V79/HPRT forward mutation assay, both with and without metabolic activation.

870.5300, Mammalian Gene Mutation
MS
MRID 48574789 Acceptable/guideline Experiment one: 0, 3.8, 7.5, 15, 30, 40, 50 and 60 μ g/mL without S9 mix (4 hours) 0, 37.5, 75, 150, 300, 600, 800 and 1000 μ g/mL with S9 mix (4 hours)

Experiment two: 0, 5.5, 10.9, 21.9, 43.8, 87.5, 131.3 and 175 μ g/mL without S9 mix (24 hours) 0, 87.5, 175, 350, 700, 800, 900 and 1000 μ g/mL with S9 mix (4 hours)

Under the conditions of this assay, methyl sulfone did not induce gene
mutations at the HPRT locus in V79 cells.

Cytogenetics

870.5395, In Vivo Micronucleus Assay
Fluensulfone
MRID 48574764 Acceptable/guideline 0, 75, 150, and 300 mg/kg/bw

Fluensulfone administered by intraperitoneal route twice up to 300 mg/kg bw showed no clastogenic effects in this in vivo test.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 80 of 107 870.5395, In Vivo Micronucleus Assay
TSA
MRID 48574786 Acceptable/guideline 0, 500, 1000, and 2000 mg/kg/bw

Under the conditions of this assay, thiazole sulfonic acid was considered to be non-genotoxic in this micronucleus assay. 870.5395, In Vivo Micronucleus Assay
MS
MRID 48574791 Acceptable/guideline 0, 125, 250, and 500 mg/kg/bw

Under the conditions of this assay, methyl sulfone was considered to be nongenotoxic in this micronucleus assay.

870.5395, In Vivo Micronucleus Assay
BSA
MRID 48574795 Acceptable/guideline 0, 500, 1000, and 2000 mg/kg/bw

Under the conditions of this assay, butene sulfonic acid was considered to be non-genotoxic in this micronucleus assay.

870.5375, Chromosomal Aberration
Fluensulfone
MRID 48574762 Acceptable/guideline 0, 62.5, 125, 250, 500, 1000 μ g/mL with and without metabolic activation for both the 4-hour and 24-hour exposures

Based on the results of this test, fluensulfone tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times without S9 and one exposure time with S9 revealed no indications of genotoxic properties with respect to chromosomal or chromatid damage.

870.5375, Chromosomal Aberration
TSA
MRID 48574785 Acceptable/guideline 0, 9.3, 18.5, 37.0, 74.1, 148.1, 296.3, 592.5, 1185.0, and 2370.0 μ g/mL

Under the conditions of this assay, thiazole sulfonic acid did not induce structural chromosomal aberrations and showed no clastogenic effects when
tested in Chinese hamster V79 cell line.

870.5375, Chromosomal Aberration
BSA
MRID 48574794 Acceptable/guideline 0, 8.3, 16.6, 33.3, 66.6, 133.2, 266.4, 532.8, 1065.5, and 2131.0 μ g/mL

Under the conditions of this assay, butene sulfonic acid did not induce structural chromosomal aberrations and showed no clastogenic effects when
tested in the Chinese hamster V79 cell line.

Other Genotoxicity

870.5550, Unscheduled DNA Synthesis
MS
MRID 48574790 Acceptable/guideline 0, 250 and 500 mg/kg bw

Under the conditions of this assay, methyl sulfone did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats.

A.4.6 Metabolism

870.7485 Metabolism-Rat (Single Oral Dose)

In a metabolism study (MRID 48574742, PMRA 2181184), the metabolism and excretion of [ 14 C]fluensulfone ([thiazole- 14 C]MCW-2, 98.7-99.3%, TJBIOS-NB21-33 and [butene14 C]MCW-2, 96.8-98.8%, TJBIOS-NB18-44), given as single oral doses at a high and a low dose level for each label, was determined in male and female rats.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 81 of 107 [ 14 C]Fluensulfone, prepared as a suspension in polyethylene glycol 300, was administered in single oral low (nominal dose of 5 mg/kg bw; actual doses of 4.6-5.4 mg/kg bw) or high (nominal dose of 500 mg/kg bw; actual doses of 450-483 mg/kg bw) doses to groups of 4 male
and 4 female Wistar rats. Rats were housed in individual glass metabolism cages equipped for
collection of 14 CO 2 , urine and feces over 120 hours, and were sacrificed thereaf ter. Metabolites in urine and feces were identified by comp arison to reference standards or by liquid chromatography with mass spectrometric detecti on (LC/MS). The radiochemical purity of the dose suspensions were measured by reverse phase HPLC before and after administration, and all measurements indicated a radiochemical purity of at least 96.8% and stab ility during the time of
dose administration.

Fluensulfone administered orally to rats is rapidly metabolized. The recovery of radioactivity for the low and high dose butene and thiazole labels was 91.3-97.3% of the admi nistered dose. The disposition and excretion of radiolabel for males and females were generally similar. There was no indication that the metabolic elimination proce sses were saturated by the 100-fold increase in dose level, as levels of radiolabel in urine, ca ge wash, feces, carcass, and gastro-intestinal (GI tract) were similar. Urine was the major route of excretion for both radiolabel positions and the only source of metabolites at ≥ 5% of the administered radiolabel for both radiolabel positions. The cage wash contained relatively high levels of radiolabeled metabolites and analysis showed that those metabolites in cage wash and urine were similar; thus indicating that the cage wash mainly recovered urine. Low amounts of thiazo le sulfonic acid were found in feces, but no other
fecal metabolites were ≥ 5% of the administered dose.

For the low-dose thiazole radiolabel, 84-86% of the administered radiolabel was excreted in urine (including in cage wash), 8-11% in feces, 2% remained in the carcass and GI tract, and 0.4-
0.6% was exhaled as CO 2 . The major urinary metabolite was a thiazole mercaptu rate (38-40% of the administered dose) with lesser levels of thiazole glucuronides ( 24-27%), and thiazole sulfonic acid (3-5%).

For the high-dose thiazole radiol abel, 85-88% of the administered radiolabel was excreted in urine (including cage wash), 5-7% in feces, 1% remained in the carca ss and GI tract, and approximately 0.07% was exhaled as CO 2 . The major urinary metabolite was a thiazole mercapturate (49-54% of the administered dose) w ith lesser levels of thiazole glucuronides (20- 22%), and thiazole sulfonic acid (1-3%).

For the low-dose butene radiolabel, 75-76% of the administered radiolabel was excreted in urine (including cage wash), 10-13% in feces, 2-3% remained in the car cass and GI tract, and 4% was
exhaled as CO 2 . The major urinary metabolite was a butene sulfinic acid (32-35% of administered dose) with lesser leve ls of butene sulfonic acid (4-5%).

For the high-dose butene radiolabel , 81-83% of the administered radi olabel was excreted in urine (including cage wash), 6-11% in feces, 1% remain ed in the carcass and GI tract, and 1-2% was
exhaled as CO 2 . The major urinary metabolite was a butene sulfinic acid (53-57% of administered dose) with lesser le vels of butene su lfonic acid (4%).

This metabolism study in the rat is classified as acceptable/guideline (f ully reliable) and, in
combination with other metabolis m studies conducted with fluens ulfone (Lab report number R23411/ MRID 48574741/ PMRA 2181185, R-23412/ MRID 48574743/ PMRA 2181186, and R23389/ MRID 48574744/ PMRA 2181187), satisfies the guideline requirement for a metabolism study (OPPTS 870.7485; OECD 417) in rats.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 82 of 107 870.7485 Metabolism-Rat (Repeat Oral Dose)

In a metabolism study (MRID 48574744, PMRA 21881187), the disposition and metabolism of [ 14 C]fluensulfone ([thiazole- 14 C]MCW-2, 98.5%, TJBIOS-NB21-33, Lot 2466-20) was studied in groups of 4 male and 4 female Wi star rats after 14 repeated da ily oral doses with unlabeled fluensulfone, followed by a single oral dose of [ 14 C]fluensulfone 1 day af ter the last unlabeled dose, with sacrifice 1 and 7 days later (4 rats/sex/dose/sacrifice time). The nominal daily doses were 5 mg/kg bw (actual daily mean 4.9-5.0 mg/kg bw). The study determined the distribution of

14 C derived from the test substanc e in selected tissues and identified the major metabolites of
fluensulfone in urine and feces.

Fluensulfone was administered as a su spension in PEG 300 by oral gavage.

The recovery of excreted radiol abel was 94-99% for individual ra ts. Radiolabel was found mainly in urine (71-83%), feces (9- 11%) and the cage wash (6-16%). One day after dosing with radiolabeled fluensulfone, an average of 0.6-0.8% of the administered radiolabel was in liver and 0.1% in kidney with 1.7-2.1% in the carcass (sel ected tissues removed) and 4.5-4.7% remained in the gastro-intestinal tract. By 7 days after treatment with radiolab eled fluensulfone, an average of
0.1-0.2% of the administered radiolabel remained in liver and 0.03% in kidney. There were no significant sex differences in either elimination or tissue residues.

Analysis of urine showed four major metabolites identified as thiazole sulfonic acid (TSA), glucuronide I, glucuronide II and thiazole mercapturate. Low levels of fluensulfone (0.1-0.2% of the administered radiolabel) were detected in f ecal extracts. Comparison to residues from rats treated with a single oral dose from anot her study (MRID 48574742, PMRA 2181184) showed similar residues in tissues, urine and feces in rats administered repeated doses in this study.

This metabolism study in the rat is classified as acceptable/guideline (fully reliable) and, in
combination with other metabolism studies conduc ted with fluensulfone (Lab report number R23410/ MRID 48574742/ PMRA 2181184, R-23 411/ MRID 48574741/ PMRA 2181185, and R23412/ MRID 48574743/ PMRA 2181186), satisfies th e guideline requirement for a metabolism study (OPPTS 870.7485; OECD 417) in rats.

870.7485 Metabolism-Rat (Pharmacokinetics)

In a metabolism study (MRID 48574741, PMRA 2181185), groups of Wistar rats (8/sex/dose/radiolabel) received single nominal or al doses of either 5 mg/kg bw or 500 mg/kg bw
of fluensulfone radiolabeled with 14 C at two different pos itions ([thiazole- 14 C]MCW-2, 100%, TJBIOS-NB21-33 and [butene- 14 C]MCW-2, 96.5-97.0%, TJBIOS-NB18-44 and TJBIOS-NB21- 52), in PEG 300 to determine the pharmacokinetic parameters of fluensulfone. Blood was drawn at 11-12 sampling times via surgically impl anted jugular cannulae (up to 336 hours for the thiazole radiolabel and up to 192 hours for the bu tene radiolabel). The same sampling schedule was used for both low and high doses. Plasma was prepared from portions of the collected blood, and the total fluensulfone-derived 14 C in whole blood and plasma was measured by radiochemical techniques. The eight animals fo r each dose/sex were arranged in two groups, and blood was sampled from alternative groups during th e first 144 hours on test from thiazole label
and first 72 hours for the butene label. Therefor e, each blood or plasma concentration was the
mean from four animals.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 83 of 107 The following pharmacokinetic parameters were calculated: Maximum concentration (C max ), Time for maximum concentration (T max ), Time for decline to 1⁄2 T max (T 1/2 ), Time for decline to
1/10 T max (T 1/10 ), Time from dose to T 1/2 (DT 50 ), Time from dose to T 1/10 (DT 90 ), Elimination rate constant (k el ), Area under the curve (AUC), and Volume of distribution (V d (L)).

The absorption of fluensulfone-derived 14 C was rapid at both doses. The kinetic parameters for males and females were generally similar for the butene radiolabel, while for the thiazole label, male rats demonstrated a slightly higher ab sorption based on AUC values. There was no indication that the elimination processes were sa turated by the increase in dose from 5 mg/kg bw to 500 mg/kg bw. This is illustrated by the fact that the 100-fold concentration increase in dose
resulted in less than a 100-fold incr ease in the area under the curve (AUC).

Additional work was performed to determine the nature of the radiolabeled residues from [ 14 C]fluensulfone, and possible reaction with glutathione (GSH) and mercaptoethanol.

The amount of radiolabel associated with whol e blood increased relative to plasma with time (about 20-fold for thiazole label but only 2-fold for butene label). Fluensul fone reacts with the free thiol moiety of globin (prote in of hemoglobin) to form a c ovalent linkage to the thiazole group and displaces butene sulfinic acid. The rem oval of radiolabel from thiazole label whole
blood seems to follow the removal of derivatized hemoglobin with a DT 50 =148-216 hours (6-9 days) for the low dose.

This metabolism study in the rat is classified as acceptable/guideline (f ully reliable) and, in
combination with other metabolis m studies conducted with fluens ulfone (Lab report number R23410/ MRID 48574742/ PMRA 2181184, R-23412/ MRID 48574743/ PMRA 2181186, and R23389/ MRID 48574744/ PMRA 2181187), satisfies the guideline requirement for a metabolism study (OPPTS 870.7485; OECD 417) in rats.

870.7485 Metabolism-Rat (Tissue Distribution)

In a metabolism study (MRID 48574743, PMRA 2181186), the tissue distribution of [ 14 C]fluensulfone ([thiazole- 14 C]MCW-2, 99.0-99.2%, TJBIOS-NB21-33 and [butene14 C]MCW-2, 96.6-96.7%, TJBIOS-NB18-4) was studied in male and female Wistar rats after single oral doses in polyethylene glycol 300 (3 rats/sex/dose/radiolabel). Two nominal dose levels were used, 5 mg/kg bw (low dose) and 500 mg/kg bw (high dose) . The study was designed
to measure the distribution of test-substance-derived 14 C in selected tissues at three sacrifice intervals corresponding to their respectiv e pharmacokinetic parameters of C max (time to peak plasma concentration), DT 50 (time from dosing to 1/2 C max ), and DT 90 (time from dosing to 1/10
C max ) as determined from a pharmacokineti c study (Study R-23411/ MRID 48574741/ PMRA 2181185).

Residues were generally similar between males and females for each of the three sacrifice
intervals. For both labels and bot h dose levels, the gast rointestinal tract, li ver and kidney were among the tissues containing the highest radioactivity levels at C max for both sexes. Other tissues with high levels of radioactivity at C max (as a percentage of the ad ministered dose) included the pancreas (in males administered the low dose of th e thiazole label, in females administered the high dose of the thiazole label, and in both sexes administered the low dose of the butene label), the lung (in females administer ed the high dose of the thiazole label and in both sexes administered the low dose of the butene label), a nd the thyroid gland (in both sexes administeredFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 84 of 107 the high dose of the thiazole label). At DT 90 , the levels of radioactivity remaining in the tissues
accounted for ≤ 3% of the administered dose.

As predicted by the pharmacokinetic study, radioac tivity from the thiazo le label was slowly eliminated from red blood cells and organs asso ciated with their formation/storage (bone, bone marrow, spleen), consistent with the derivatizatio n of the hemoglobin cysteine thiol. The ratio of
residues at the C max compared to the DT 90 was near the expected 10% for plasma, but for the thiazole label (low and high doses) was >25% fo r hair and skin, heart, and lung. For the butene label (low and high doses), the ratio of residues at C max compared to DT 90 was >25% for numerous tissues/organs except t hose involved with the digestive system. The slow elimination of radioactivity with the thi azole and butene radiolabels from these tissues may reflect metabolism to 1- and 2- carbon fragments with re incorporation in natura l products (e.g. protein, fatty acids) with long turnover rates as previously found for goats and hens (MRID 48574801/ PMRA 2181244 and MRID 48574802/ PMRA 2181245).

This metabolism study in the rat is classified as acceptable/guideline (f ully reliable) and, in
combination with other metabolis m studies conducted with fluens ulfone (Lab report number R23410/ MRID 48574742/ PMRA 2181184, R-23411/ MRID 48574741/ PMRA 2181185, and R23389/ MRID 48574744/ PMRA 2181187), satisfies the guideline requirement for a metabolism study (OPPTS 870.7485; OECD 417) in rats.

A.4.7 Dermal penetration

870.7600 Dermal Absorption-Rat

The dermal absorption of fluensulfone formulat ed as MCW-2 480 EC was determined in rats (MRID 48574949; PMRA #2181332). MCW-2 480 EC (containing 14 C-labelled active ingredient) was applied to shorn rat skin at two target dose levels: a low dose (0.1 mg/cm 2 ) reflecting typical concentration recommended for the use in the fi eld (10 g a.i./L, diluted from the concentrate with water) and a high dose (4.8 mg/cm 2 ) reflecting the undiluted commercial product (480 g a.i./L). Each group consisted of 4 male Wistar rats. Collection periods were 0-10 hrs, 0-24 hrs, 0-72 hrs and 0-120 hrs for each dose level. Volatile test item was trapped by covering the application site with charcoal pape r during and after exposure. The exposure time of
10 hours was chosen as the worst case exposure period of a field worker.

At the end of the 10-hr exposure period, MCW- 2 480 EC was removed by washing with mild soap dilution. The dermal absorption of the te st item was determined. Furthermore, the amount remaining in/on the skin at the application site after washing a nd potentially penetrating through the skin was determined at 3 additional time points, 24, 72 and 120 hours after application of the test item in order to estimate the depletion of th e dose associated with the application site. The association of the remaining test item in/on the skin at the application site was investigated by skin stripping in or der to separate the stratum corneum from the epidermis; however, since cyanoacrylate adhesive was used in the first two tape st ripping of skin (which may affect the skin fractionation procedure), all tape s were included in the calcul ation of potentia lly absorbable dose. Urine and feces were collected up to 120 hours after application. Blood and plasma
residue levels were also measured up to 120 hours after application.

After dermal exposure to formulated [ 14 C]-MCW-2, 49-56% of the low dose and 16-20% of the high dose were systemically absorbed. Most of the remaining applied test item, i.e. 34-45% ofFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 85 of 107 the low dose and 76-78% of the high dose, could be dislodged from the a pplication site at the end of the exposure period. The radioactivity in skin strips I and II represented only ≤ 0.42% of the administered dose. The radioactivity remaini ng on/in skin application site was predominantly
located in the stratum corneum. Only very low amounts of radioactivity were found in lower skin levels, i.e. < 1% of the applied dose. W ithin 120 hours, there was no apparent increase in systemic absorption at both dose levels. However, radioactivity recovered at the application site ( stratum corneum and lower skin levels) decreased with time from 8.0 and 3.5% at 10 hours to 4.0 and 0.4% at 120 hours for the low and high dose, respectively, indicating that the residues in skin were slowly absorbed.

The systemically absorbed test item, a to tal of 53.50 and 19.57% for the low and high dose, respectively, was excreted mainly in urine. After 5 days, 46.58% of the low dose and 15.73% of the high dose were excreted with the urine. During the same period of time, 4.40% of the low dose and 1.86% of the high dose were excreted with the feces. A remarkable amount of the absorbed test item, i.e. 2.20% of the low dose a nd 0.66% of the high dose, was still remaining in the animals. The maximum potentially absorbable dose (systemic absorption + skin strips IIIVII + remaining treated skin; av erage of all time points) for the low and high dose groups is 64 (10 h) and 21 (120 h) percent of the administer ed dose, respectively. Ther e was also a relatively high amount of the total applied do se remaining in the cover and O -ring over the test site due to the volatility of the test material at skin temperature, but the occl usion of the exposed skin results in a higher total recovery a nd penetration than would occu r under typical human exposure conditions, thereby representing a “worst-case” scenario. In summary, MCW-2 formulated as MCW-2 480 EC penetrated at the low and high dos e level to a relatively high extent through rat skin. The calculated penetration rates did not increase in a proportional fashion with the dose
levels.

This study is classified as A cceptable/guideline (Fully Reliable) and satisfies the guideline requirement (OECD 427; OPPTS 870.7600) for an in vivo dermal absorption study in the rat.

A.4.8 Immunotoxicituy

870.7800 Immunotoxicity

In a study evaluating potential immunot oxicity (MRID 48574779, PMRA 2181189), groups of
10 female CD-1 mice received fluensulf one (MCW-2 technical, 96.7%, Batch No. 36372130- 291-PF1) in their diet at concentration of 0, 100, 500 or 2500/1500 ppm (equivalent to 0, 17, 86 and 204 mg/kg bw/day, respectively). After one w eek of treatment, one mouse in the high dose group died and signs of overt toxicity (decreas es in body weight, food and water consumption, clinical signs in a few animals) were noted in the other animals of this group. Following 3 days on untreated diet, the dietary co ncentration for the high dose gr oup was reduced to 1500 ppm and treatment continued for an additional 21 days . The treatment period for all the groups was extended to 32 days in order to complete the planned 28-day treatment period for all of the treated and negative control animals on study. Cy clophosphamide was used as positive control and was administered i.p. at 25 mg/kg bw/day dur ing days 27-31 to a group of 10 female mice.

On Day 27 a single intravenous dose of 0.2 mL of 1 x 10 8 sheep red blood cells (SRBC) in phosphate buffered saline was administered to all surviving animals. Five days after immunization (Day 32), all mice were weighed, eu thanized, serum samples were collected fromFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 86 of 107 each mouse for anti-SRBC IgM titers, and the spleen and thymus were weighed.

Apart from the effects observed when the highest dose animals were receiving 2500 ppm fluensulfone in their diet, treatment did not a ffect body weight or food or water consumption in mice at 100 and 500 ppm, and these parameters return ed to, or were similar to, vehicle control values in highest dose mice when treated at 1500 ppm.

No effects on absolute or relative spleen or thymus weights were noted in the fluensulfone treated animals. The mean anti-SRBC IgM values for the 100, 500, and 2500/1500 ppm groups were approximately 62 to 70% of control, but were not statistically significantly different from control and no dose-relationship wa s evident. A high inter-individual variability was noted in all the treatment groups as well as in the control group. Eval uation of individual animal data of this study did not show any trend or distribution th at would demonstrate si gnificant suppression of anti-SRBC IgM response. Absolute and relative spleen and thymus weights were statistically significantly reduced to approxima tely 75% of vehicle control values for the cyclophosphamide treated mice. Anti-SRBC IgM in positive control animals was also statistically significantly
reduced to 11% of the vehicle cont rol mean, thus confirming the sens itivity of the animal model.

The NOAEL was set at 500 ppm (86 mg/kg bw/day), based on clinical signs and body weight
effects noted at the LOAEL of 2500/1500 ppm (204 mg/kg bw/day).

This study is classified as accep table/guideline (fully reliable ) and satisfies the guideline requirement for an immunotoxic ity study (OPPTS 870.7800) in mice.

A.4.9 Non-Guideline studies

28-day Oral –Dog

In a 4-week dietary study (MRID 48574752, PMRA 2181202), groups of 2 male and 2 female Beagle dogs received fluensulfone (MCW-2 tech., 96.67%, Lot No. 36372130-291-PF1) in their diet at 0, 50, 200 and 900 ppm (0, 1.9, 7.3, 31 mg /kg bw/day in males; 0, 2.0, 7.9, 30 mg/kg bw/day in females) for 29 days.

Special investigations included thyroid hormone levels in bl ood, alanine aminotransferase activity in liver tissue and meta bolic enzyme activity in liver.

No mortality occurred and no clin ical signs were noted during th e study. Body weight stasis in males and body weight loss in females was noted at 900 ppm during the treatment period when compared to the pretreatment values or the control group. No treatment-related changes were recorded in ophthalmologic examination, hema tological or urinalysis parameters.

Decreased alanine aminotransferase activity in the blood was observed in all treated groups and decreased protein and albumin levels and albumin /globulin ratios were noted in the females at 900 ppm. A tendency of an incr ease in thyroid stimulating horm one (TSH) values was observed in male and female dogs at 900 ppm when co mpared to the control values. Generally, no differences in triiodothyronine (T 3) or thyroxine (T4) levels we re observed in the treated groups when compared to the control values.

Increased absolute and relative liver and thyroid weight s were recorded in the males at 900 ppm; however, there was no associated macroscopic or microscopic histopathol ogy. A slight decreaseFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 87 of 107 in absolute heart weight wa s observed in both sexes from the 900 ppm dose group, which was considered to be a secondary effect to the reduced growth in these animals.

Fluensulfone caused a slight inhi bition of hepatic alanine aminot ransferase (ALAT) activity in both sexes at 900 ppm. While some hepatic enzyme s were elevated in treated animals, the overall results of liver enzyme determinations exclude a potent effect on liver enzymes by
fluensulfone.

A NOAEL and LOAEL were not established for th is study as it is a rang e-finding study. Adverse effects were noted at 900 ppm (31 and 30 mg/kg bw/day in male and female dogs, respectively), in the form of slight changes in body weight and organ weight (liver and t hyroid) at 900 ppm. No adverse effects were noted at 200 ppm (7.3 a nd 7.9 mg/kg bw/day in male and female dogs, respectively).

This subchronic toxicity study in the dog is accep table/non-guideline (relia ble with restrictions) as it is a range-finding study.

14-day Effect on ALAT in Dogs

The purpose of this study (MRID 48574776, PMRA 2181190) was to assess other possible modes of action by an investigati on of alanine aminotransferase (ALAT) activity, concentrations of pyridoxal 5’-phosphate a nd ALAT1-protein and ALAT1– messenger RNA expression in Beagle dogs, treated for 2 weeks with flue nsulfone (MCW-2 tech., 96.7%, Batch No. 36372130- 291-PF1) by the oral (feeding) route.

Two groups of 4 male dogs each received pelleted diet, either without test item (control animals - group 1), or with 500 ppm fluensulf one (treated animals - group 2). The mean test item intake in the animals receiving fluensulfone in the diet was 21.1 mg/kg bw/day.

During pre-test, and on days 7 and 14 of treatment, blood samples were collected for quantification of the enzyme activity of th e liver enzymes (leakage markers) aspartate aminotransferase (ASAT), alanine aminotrans ferase (ALAT), sorbito l dehydrogenase (SDH).

Plasma concentrations of the ALAT co-factor, pyridoxal 5’-phosphate, were determined before commencement of treatment and on days 7 a nd 14 of treatment with fluensulfone

Following completion of the treatment period, sample s of liver tissue were taken for mechanistic investigations, includin g investigations of ALAT1 mRNA e xpression by PCR, ALAT1 protein expression by Western Blot, ALAT activity in liver tissue, and liver concentrations of pyridoxal 5’-phosphate.

All animals survived the scheduled treatment peri od. No clinical signs were considered to be
related to treatment and all anim als appeared in good general h ealth throughout the study. There were no effects on food consumption or body weight . There were no macros copic or microscopic findings that could be attributed to the treatment.

Decreased plasma ALAT was observed after 2 week s at 500 ppm. No changes were observed in
ASAT or SDH.

ALAT1 mRNA levels were 3.36 time s higher in the treated group co mpared to the control group. Hepatic ALAT1 protein levels were similar in th e control and treated gr oups. After 2 weeks of treatment at 500 ppm, mean hepa tic ALAT activity was significan tly decreased to 13.6% of theFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 88 of 107 control group mean. A slight decr ease in the concentrations of Pyridoxal 5’-phosphate in plasma was recorded, whereas slightly higher hepatic Py ridoxal 5’-phosphate concentrations (attaining statistical significance) were observed in fluens ulfone-treated dogs than in control dogs. The amplitude of the effects appears small and does not correlate with the effect pattern (ALAT activity was reduced in both plasma and liver). Th us, it is considered unlikely to be the primary cause of the marked reduction in ALAT activity.

Based on the results of this st udy, the reduction of th e activity of alanine aminotransferase activity by fluensulfone is not mediated by

i) a reduction of ALAT1 mRNA expression

ii) a reduction of ALAT 1 protein expression

iii) a decrease of the cofact or Pyridoxal 5’-phosphate

Other mechanisms, such as alteration of post- translational modificati on might be the cause for the transiently decreased act ivity of ALAT upon fl uensulfone treatment. The increase in ALAT1 mRNA might be indicativ e of a counter-regulation to the decrease in ALAT activity.

This special study is classified as acceptable/non-guideline (reliable with rest rictions) as it does not satisfy any guideline requirement.

Determination of ALAT in Untreated Dogs

In this study (MRID 48574778, PMRA 2181188), liver samples from adult untreated male and female dogs were collected between June 2009 and September 2009 in order to provide a historical database for hepatic alanine aminotransferase (ALAT) activity. ALAT activities were measured in homogenates prepar ed from the respective livers.

In male and female control animals a mean ± S.D. activity of 119.6 ± 23.6 U/g Liver (Range 71.3 to 174.7 U/g Liver, 23 animals) and 126.4 ± 30.6 U/g Liver (Range 88.7 to 186.7 U/g Liver, 14 animals), was obtained, respectively.

This special study is acce ptable/nonguideline (reliable with re strictions) as it does not satisfy any guideline requirement.

In Vitro Mode of Action for Decrease of ALAT in Dog Microsomes

In this special study (MRID 48574777, PMRA 2 181191), dog liver homogenate from untreated dogs was incubated with fluensulfone (M CW-2 tech., 96.7%, Batch No. 36372130-291-PF1) for different time periods in order to investigate a possible direct inhibition of ALAT activity by
fluensulfone and/or its metabolite s,. LC-MS/MS determination in the incubation mixtures clearly
demonstrated an effective metabolism of fluensulfone.

No inhibition of the ALAT ac tivity was observed following inc ubation of dog liver homogenates
with fluensulfone. Based on the results of th is study, the reduction of ALAT activity by fluensulfone is not considered to be caused by a direct inhibition of ALAT by the test item or its
metabolites.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 89 of 107 This special study is classified as acceptable/non-guideline (reliable with rest rictions) as it does not satisfy any guideline requirement.

Mode of Action for Decrease of ALAT in Dogs

In a special mechanistic study (MRID 48574775, PMRA 2181192), the direct inhibition of liver alanine-aminotransferase (ALAT) activity by flue nsulfone was evaluated. In a previous study (Harlan Laboratory Study B89087, MRID 48574752, PMRA 2181202), treatment of dogs for 28 days with 50, 200 and 900 ppm fluensulf one (MCW-2 tech., 96.67%, Batch No. 36372130-PF1) in the diet resulted in a dose-dependent decr ease of mean alanine-aminotransferase (ALAT) activity to maximally 15-24% in serum and to 55-64% in liver homogenates. To check if the decrease of ALAT activity might be due to a di rect inhibition of the enzyme by MCW-2 or one of its metabolites, or by an inhibition of ALAT gene or protein expression in the liver, serum and liver cytosolic fractions of control and hi gh dose dogs obtained in the 28-day study were
used.

Serum and liver cytosolic fractions of control animals were spiked with respective samples from dogs treated at 900 ppm (the highest dose level te sted). ALAT activity was determined to show whether serum or cytosolic fractions from fluens ulfone-treated animals, considered to contain excess of fluensulfone or relevant metabolites, are able to inhibit the activity of ALAT. In addition, the amount of ALAT in serum and cytoso lic fractions was determined for both control and fluensulfone-treated dogs usi ng western blotting to see whethe r fluensulfone suppresses gene expression at the protein level.

No inhibition of the ALAT activity by cytosol or serum from treated animals was observed. In cytosol and serum samples of control dogs, spiked with samples obtained from dogs treated at 900 ppm, the observed activities corresponded exactlyto the theoretically calculated activities.

Immunoblotting revealed very similar levels of ALAT in serum and cytosolic fractions of controls and treated dogs. However, as specifici ty of the used antibody was not tested against dog ALAT, the results in dicating the unlikely decrease of th e expression of ALAT protein upon
treatment with fluensulfone were considered only provisional and n eed to be evaluated with care.

It is noted that it is not known how much, if any, fluensulfone or its metabolites was contained in the serum and liver cytosol fractions taken from tr eated animals and used in this study. Levels of
fluensulfone or its metabolites were not determined in serum or liver cytosol.

This special study is classified as acceptable/non-guideline (reliable with rest rictions) as it does not satisfy any guideline requirement.

Mechanistic Lung Toxicity in Mice

This mechanistic toxicity study (MRID 48574767, PMRA 2181220) was conducted with the purpose to analyze local cell prolif eration in the lungs as a basi s for assessment of the human relevance of observed lung tumors in mice.

Groups of 10 CD-1 female mice were treated via the diet with 0 ppm or 1200 ppm fluensulfone (MCW-2 tech., 96.17%, Batch No. 36372130-291-PF1) for 3 days or 7 days. An additional 10Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 90 of 107 females each were administered 1305 ppm Isoniazid (positive control, ≥ 99%, Batch No. 060M0090) for 3 or 7 days. Approximately 14 and 2 hours prior to necropsy, all animals received an intraperitoneal inj ection of BrdU for determinatio n of cell proliferation. Lungs, brain, and a piece of the small intestine were collected from all animals at necropsy. The weights of the lungs and brain were recorde d. Lungs and intestine were processed for the determination of BrdU positive cells and analyzed histopathologically.

There were no mortalities and no clinical signs. Decreased food consumption was recorded in females treated with 1200 ppm fluensulfone fr om day 1 to day 2 when compared to the respective controls. Afterwards the food consumption of these animals returned to a level comparable to the respective controls. Animals treated wi th 1305 ppm Isoniazid showed increased food consumption from days 5 to 8 when compared to the respectiv e controls. A slight loss of body weight was recorded in animals treated with 1305 ppm Isoniazid and 1200 ppm fluensulfone on day 4. However, a minimal lo ss of body weight was also recorded in the respective controls. A slight loss of body weight was also recorded in animals treated for 7 days. Analysis of organ weight data showed no effect of treatment either with fluensulfone or
Isoniazid.

Labelling in small intestine demonstrated correct administration of BrdU. In animals treated for 3 days, a marked increase in BrdU positive cells was observed in the epithelium of the bronchioles in the lungs of animals treated with 1305 ppm Isoniazid and 1200 ppm fluensulfone. There was no significant difference in the severity and incidence of the change between the two groups
treated with Isoniazid and fluensul fone. In animals treated for 7 days, no difference in the BrdU incorporation in the lung was observed among the groups.

Based on the results, female mice reacted to trea tment with both fluensulfone and Isoniazid with increased cell proliferation that was detected by BrdU incorpor ation. The reaction to treatment was transient, with an effect at an early time point (after 3 days) that was not visible at the later time point (after 7 days).

This special study is classified as acceptable/non-guideline (reliable with rest rictions) as it does not satisfy any guideline requirement.

Comparative Biotransformation in Human and Mice Microsomes

The objective of this study (MRID 48574768, PMRA 2181219) was to compare the metabolic activity of lung microsomes of mice and humans towards fluens ulfone (MCW-2, 98.6%, Batch No. 031H0722), and to determine the contribution of Cyp 2f2 (mice specific) and CYP 2E1/Cyp 2e1 (expressed in humans and mice) to the metabolism of MCW-2, by co-incubation with and
without the selective in hibitors, 4-methyl pyrazole (CYP 2E 1/Cyp 2e1) and 5-phenyl-1-pentyne (Cyp 2f2).

The metabolic activity was determined by incubati on of lung microsomes with fluensulfone in the presence of an NADH-rege nerating system, and analytical determination of remaining (unmetabolized) fluensulfone by LC-MS/MS at different time points. Two concentrations of fluensulfone were tested (2 a nd 20 μM); however, the highest con centration was found to be too high (only a small percentage was metabolized) a nd the results obtained were considered only as confirmatory. The metabolism was further investig ated by addition of inhi bitors of CYP 2E1 and CYP 2f2 (a mouse-specific isof orm). Lung microsomes were prepared from 12 female and 12Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 91 of 107 male untreated mice, and their activity towards fluensulfone was compared with that of lung microsomes pooled from at least 10 different human non-smokers.

The metabolic capability of the microsomal prepar ations was confirmed by determination of the
turnover of a standard substrat e, chlorzoxazone, under conditions similar to those used for
fluensulfone.

The detected activity towards chlorzoxazone wa s approximately 450-fold lower in human lung microsomes compared to mouse lung microsomes , and thus in good correla tion with the activity differences reported in the literature.

With human lung microsomes, no metabolism of fl uensulfone was detectab le. In contrast, in mice female and male lung preparations only appr oximately 10% of the applied fluensulfone (2

μ M) remained after 120 minutes of incubation.

The negative control without NADH regenerating system showed no me tabolic activity in humans as well as in both mice genders showin g the validity of the assay and indicating an enzymatic degradation of fluensulfone.

The inhibition of (mouse-specif ic) Cyp 2f2 (with 5-phenyl-1-pentyne) had a more pronounced effect on the degradation of fluensulfone than inhibition of CYP 2E1 (with 4-methyl pyrazole) which was essentially not effective, although the metabolic activity towards fluensulfone was not abolished completely.

Under the conditions of this study, no metabolic activity towards fluensulfone was detectable after incubation with human lung microsomes of non-smokers. In contrast, fluensulfone was extensively metabolized by lung microsomes of female and male mice, and the mice-specific isoenzyme Cyp 2f2 appears to play a ma jor role in the degradation process.

This special study is classified as acceptable/non-guideline (reliable with rest rictions) as it does not satisfy any guideline requirement.

Comparative Dermal Absorption

The comparative percutaneous penetration of fluensulfone, formulated as MCW-2 480 EC (emulsifiable concentrate), was investigated in vitro using split-thickness skin membranes from rat and human skin (MRID 48574950; PMRA 2181330).

The skin membranes were set up in flow-through diffusion cells and 14 C-fluensulfone, formulated as MCW-2 480 EC, was applied onto the skin membranes at two dose levels: a low dose (0.10 mg/cm 2 ) reflecting a typical concentration reco mmended for the use in the field as spray dilution, i.e. 10 g a.i./L (achieved by dilutio n of the commercial product with water), and a high dose (4.8 mg/cm 2 ) which represents the concentration of active ingredient in the undiluted commercial product, i.e. 480 g a.i./L. The expos ure of the test item was performed under nonoccluded conditions over an exposure time of 24 hours. During the exposure period the receptor fluid (ethanol/water 1:1 v/v) was collected in hourly in tervals between 0-6 hours and thereafter in
2 hours intervals until the end of the experiments. Volatilized test item was trapped by coveringFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 92 of 107 the donor cell with charcoal pape r during exposure. At the end of the experiment, the remaining

14 C-fluensulfone was removed from the skin surface by rinsing. The skin membranes were
removed from the diffusion cell and consecutively st ripped until the stratum corneum was removed.

The test item was stable on the skin membranes during the exposure period of 24 hours; analysis of skin membrane rinse pools revealed essentially unchanged fluensulfone.

The totally absorbed test item was calculated based on the amount that penetrated through the skin membrane (perfusate), the amount measured in tape strips (excluding the first two tape strips), and the amount measured in the remainin g skin membrane layers after tape stripping. The total absorption was calculated to be 92.11% and 81.26% (low and high dose), and 62.46% and 4.65% (low and high dose) for rat and human skin membranes, respectively.

Based on percentages of absorption, the human/rat ratio for use in risk assessment is 1/1.5 for the low dose level and 1/1.8 for the high dose level.

This study is classified as accepta ble/nonguideline (fully reliable).

Acute Neurotoxicity –Time to Peak Effect

In a preliminary acute neurotoxicity study (MRID 48574772, PMRA 2181225) designed to determine the time to peak effect and set dose le vels for a subsequent study, groups of 3 male
and 3 female Wistar rats were administered single oral doses of 0 (control), 100, 500, 1000 and 2000 mg/kg bw of fluensulfone (MCW-2 tech., 98.65%, Batch No. 36372130-291-PF1) in polyethylene glycol (PEG) 300, and we re observed for 8 days post-dosing.

Detailed clinical observations were performed on day 1 at 0.5 hours after dose administration and at hourly intervals for 8 hours, and on days 2 to 8. Body weights and food consumption were measured once during acclimatization and on days 1 and 8. At the end of the observation period, all surviving animals were sacrifi ced and assessed by gross pathology.

All animals of the high dose group (2000 mg/kg bw ) survived the 8-hour observation period but died spontaneously within 24 hour s after dose administration after they had developed signs of overt toxicity. All animals of the remaining dose groups survived to term inal sacrifice without showing any signs of toxicity.

Effects observed at 2000 mg/kg bw included a transient occurrence of salivation within 0.5 to 2 hours after dose administration followed by som nolence, decreased activity, prostration, and
ruffled fur of variable duration between 2 and 8 hours after dose administration, with a peak effect observed on the day of dosing in animal s of both sexes at approximately 5 hours. In addition, in 2 males dosed at 2000 mg/kg bw th e push-off reflex was absent. Due to the premature death of all males and females dos ed at 2000 mg/kg bw it was not possible to investigate any effect of the test item on body weight and food consumption in this high dose group. Slight body weight reductions were obser ved in males and females dosed with 1000 mg/kg bw. Reduced body weight gain was noted in males from the 500 mg/kg bw dose group, and in both sexes from the 1000 mg/kg bw dose group. Slight decreases in food consumption
were observed in females from the 500 and 1000 mg/kg bw dose groups. No macroscopic findings were recorded in any anim als of any dose groups at necropsy.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 93 of 107 A peak systemic effect was observed on the day of dosing in animals of both sexes treated at 2000 mg/kg bw approximately 5 hours after administra tion, but this dose level had exceeded the maximal tolerated dose (MTD).

This study is a non-guideline study and is ther efore classified as acceptable/non-guideline (reliable with restrictions) as a prelim inary acute neurotoxicity study in rats.

28-day oral Rat -BSA

In a subchronic toxicity study (MRID 49387602, PMRA 2407952), 3,4,4-trif luoro-but-3-ene-1- sulfonic acid (BSA; 93%) was administ ered to 3 Wistar rats/sex/dose in the diet at dose levels of 0, 100, 500, 1000, or 10,000 ppm (0/0, 6.4/8.6, 30/39, 82/ 120, or 732/1024 mg/kg bw/day in males/females; doses were corrected for analytic al recovery of BSA from the test diet) for 28 days. Examinations included clinical signs of toxicity, body weight, food consumption, hematology and clinical chemistry parameters, organ weight measuremen ts, gross pathology, and limited histopathology examinations ( liver, kidney, lung, and thymus).

Aside from marginal (<25%) decreases in mean se rum aspartate aminotransferase levels in both
sexes and alanine aminotransferase levels in ma les at the high dose of 10,000 ppm, no treatmentrelated effects were observed in this study up to the highest dose test ed. A NOAEL and LOAEL
were not established as this was a dose range-finding study with limited investigations.

This subchronic toxicity study in the rat is acceptable as a range-finding study, but does not satisfy the guideline requirement (OECD 407; OPPTS 870.3050) for a rep eat-dose oral study in rats due to the small group sizes and limited he matology, clinical chem istry and histopathology examinations. However, the number of parameters measured is sufficient to indicate that BSA is significantly less toxic than fluensulfone in rats.

28-day oral Rat –TSA

In a subchronic toxicity study (MRID 49387601, PMRA 2402076), 5-chlorthiazole-2-sulfonic acid (99.38% a.i.) was administered to 3 Wistar rats/sex/dose in the diet at dose levels of 0, 120, 500, 1200, or 12,000 ppm (0/0, 10/12, 41/43, 113/123, or 1194/1779 mg/kg bw/day in males/females) for 28 days. Examinations includ ed clinical signs of toxicity, body weight, food consumption, hematology and clinical chemistry parameters, organ weight measurements, gross pathology, and limited histopathology examina tions (liver, kidney, lung, and thymus).

No treatment-related effects were observed in this study up to the hi ghest dose tested. A
NOAEL and LOAEL were not established as this was a dose range-finding study with limited investigations.

This subchronic toxicity study in the rat is acceptable as a range-finding study, but does not satisfy the guideline requirement (OECD 407; OPPTS 870.3050) for a rep eat-dose oral study in rats due to the small group sizes and limited he matology, clinical chem istry and histopathology examinations. However, the number of parameters measured is sufficient to indicate that TSA is significantly less toxic than fluensulfone in rats.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 94 of 107Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 95 of 107 Appendix B. Metabolism Summary Table

Table B1. Summary of Metabolites and Degradates for Fluensulfone Chemical Name (other names in parenthesis)
and Structure Matrix Percent TRR (PPM) Matrices - Major Residue (>10% TRR) Matrices - Minor Residue (<10%TRR)

Fluensulfone 5-chloro-2-(3,4,4-
trifluoro-but-3-ene-1- sulfonyl)-thiazole Tomato -- --
Potato -- 1.1 / 3.1 Lettuce -- 0.2 / 0.4 (immature only) Rot. Crops
- Lettuce - Radish (Top) - Radish (Root) - Wheat (Grain) - Wheat (Forage) - Wheat (Hay) - Wheat (Straw) --

--

--

--

--

--

-- 30 DAT 120 DAT 360 DAT 5.1 (0.02) -- 4.0 (0.002) 3.1 (0.02) 5.1 (0.02) 3.0 (0.001) 1.5 (0.002) -- --

-- -- -- 1.1 (0.03) 2.9 (0.02) -- 0.4 (0.02) 1.1 (0.004) -- 0.2 (0.004) -- --
Ruminant -- Poultry -- 20.9 (0.009) omental fat 54.7 (0.04) subcutaneous fat

Rat -- --

TSA M-3625 [M1+M3]
Thiazole Sulfonic Acid
5-chloro-thiazole-2-
sulfonic acid Tomato 45.4 (0.12) + [14.5 (0.04)] Potato 65.3 (0.31) + [3.4 (0.02)] Lettuce 70.6 (4.34) + [0.4 (0.02)] Rot. Crops
- Lettuce

- Radish (Top)

- Radish (Root)

- Wheat (Grain)

- Wheat (Forage)

- Wheat (Hay)

- Wheat (Straw) 30 DAT 120 DAT 360 DAT 41.9 (0.24) 55.6 (0.18) 82.3 (0.28) [20.4 (0.11) 27.2 (0.09) 1.2 (0.004)]

89.8 (4.7) 92.5 (1.6) 89.4 (2.6) 2.8 (0.14) 4.2 (0.07) --

86.6 (0.69) 81.2 (0.36) 90.0 (0.34) 0.5 (0.004) 1.1 (0.005) 1.1 (0.004)

33.4 (0.12) 57.8 (0.17) 57.0 (0.20) [18.1 (0.07) 6.7 (0.019) 11.1 (0.039)]

75.1 (12.5) 76.0 (2.3) 82.3 (2.7) [ 4.1 (0.68) 3.5 (0.10) 1.4 (0.05)]

57.7 (15.6) 66.2 (6.2) 53.3 (5.8) [ 3.3 (0.91) 4.2 (0.40) 5.8 (0.62)]

57.7 (10.7) 62.4 (2.2) 76.3 (5.1) [ 3.5 (0.63) 0.9 (0.03) 6.8 (0.46)] 30 DAT 120 DAT 360 DAT

Ruminant -- -- Poultry -- 2.7 (0.02) liver Rat -- 1.5% AD (urine, 48574742)

BSA M-3627 [ F1+F2]
Butene Sulfonic Acid 3,4,4-trifluoro-but-3-
ene-1-sulfonic acid Tomato 41.6 (0.22) + [26.5 (0.14)] Potato 25.8 (0.04) + [17.8 (0.03)] Lettuce 37.6 (0.49) + [7.3 (0.09)] Rot. Crops
- Lettuce

- Radish (Top)

- Radish (Root) 30 DAT 120 DAT 360 DAT 8.8 (0.02) -- -- [5.4 (0.01) 35.6 (0.02) 11.8 (0.002)]

65.8 (0.32) 70.2 (0.33) -- [3.4 (0.02) 0.4 (0.002) 5.1 (0.002)

40.0 (0.06) 39.4 (0.04) -- [6.2 (0.009) 2.3 (0.002) 8.3 (0.001) 30 DAT 120 DAT 360 DAT S N Cl

S

O O F

F F

OH S N Cl

S

O O

O H S

O O F

F FFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 96 of 107 Table B1. Summary of Metabolites and Degradates for Fluensulfone Chemical Name (other names in parenthesis)
and Structure Matrix Percent TRR (PPM) Matrices - Major Residue (>10% TRR) Matrices - Minor Residue (<10%TRR)

- Wheat (Grain)

- Wheat (Forage)

- Wheat (Hay)

- Wheat (Straw) -- -- -- [4.0 (0.007) 18.6 (0.01) 10.5 (0.004)]

54.4 (1.4) 59.9 (0.43) 3.2 (0.002) [3.6 (0.09) 3.6 (0.03) 15.0 (0.009)]

27.2 (1.2) 25.5 (0.10) -- [1.3 (0.06) 8.8 (0.03) 16.4 (0.02)

14.1 (0.27) 10.2 (0.03) 7.0 (0.01) [9.8 (0.19) 31.4 (0.10) 9.7 (0.02)
Ruminant -- -- Poultry -- -- Rat -- 4.6% AD (Urine, 48574742)

MeS
M-3626 Thiazole Methyl
Sulfone
5-chloro- 2- (methylsulfonyl) Tomato -- --

Potato -- --

Lettuce -- -- Rot. Crops
- Lettuce - Radish (Top) - Radish (Root) - Wheat (Grain) - Wheat (Forage) - Wheat (Hay) - Wheat (Straw) 30 DAT 120 DAT 360 DAT

-- -- --

-- -- --

-- -- --

-- -- --

-- -- --

-- -- --

-- -- -- 30 DAT 120 DAT 360 DAT

-- -- --

-- -- --

-- -- --

-- -- --

-- -- --

-- -- --

-- -- -- Ruminant 24.7 (Urine) -- Poultry -- -- Rat -- Minor (feces, amount not specified, 48574744)
Butene Sulfinic Acid

O H S O F

F F Ruminant 65.6 (Urine) Rat 64.7% AD (Urine, 48574742)

Thiazole Mercapturate

S N Cl

S

O OH NH O Rat 62.5% AD (Urine, 48574742)

MW-327-I and MW327-II
Thiazole Glucuronides ( α and β isomers) Rat 21.5% AD (Urine, 48574742)* S N Cl

S

O OFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 97 of 107 Table B1. Summary of Metabolites and Degradates for Fluensulfone Chemical Name (other names in parenthesis)
and Structure Matrix Percent TRR (PPM) Matrices - Major Residue (>10% TRR) Matrices - Minor Residue (<10%TRR) All crop studies were conducted at an application rate of 4 kg a.i./ha (3.5 7 lb a.i./A), which is the 1X rate. Tomato (48574798): Harvest 87 DAT Potato (48574797): Harvest 70 (immature) and 106 (mature) DAT Lettuce (48574799): Harvest 49 (immature) and 64 (mature) DAT Poultry (48574801): 9.8 mg/kg feed for 7 days; relative rate is unknown. Tissue collection 20-22 hours after last dose. Ruminant (48574802): 10.5 mg/kg feed for 5 days; relative ra te is unknown. Tissue collecti on 20-22 hours after last dose. Rat (48574742): Single dose of 500 mg/kg body weight to females. Non-carcass samples collec ted 0-120 hrs after dosing. Tissue collection 120 hrs after dosing. Rat (48574744): Multiple doses of 5 mg/kg body weight. Non- carcass samples collected 0-168 hrs after dosing. Tissue collection 24 or 168 hrs after dosing. * Combined MW-327-I and MW-327-II. The two glucuronides were f ound in a nearly 1:1 ratio in ma le rats treated at the same dose in the same study (12% AD for I vs. 10% AD for II).Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 98 of 107 Appendix C. Physical/Chemical Properties

Table C-1. Physicochemical Properties of Technical-Grade Fluensulfone
Parameter Value Reference Molecular weight (g/mole) 291.7 EPA/PMRA joint review of fluensulfone technical-grade active ingredient. Monograph Volume 3, Parts
B1 and B2. B. Kitchens.
December 2013. Melting point/range (°C) 34.8 ± 0.5 pH 5.11 (1% solution at 21°C) Density (g/cm 3 ) 1.876 Solubility (g/L at 20°C) Water 0.5453

Acetone 350.49
Dichloromethane 306.14 Ethyl acetate 350.76 n-Heptane 19.01
Methanol 359.29
n-Octanol 90.42 Xylene 356.18 Vapor pressure at 25°C (Pa) 3.0x10 -2 Dissociation constant (pK
a ) No acidic or basic functional groups Octanol/water partition coefficient Log(K O W ) 2.2Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 99 of 107 Appendix D. Summary of Fluensulfone Re sidue Data for Cucurbit and Fruiting Vegetables

Table D-1. Summary of Fluensulfone Residue Field Trial Results. Crop Use Pattern Analyte n Residue Levels (mg/kg) A

min max HAFT Med Mean SD

Bell Pepper Broadcast Spray 7 days Pre-Transplant 3.82-4.10 kg a.i./ha Fluensulfone 7 <0.01 <0.01 <0.01 <0.01 <0.01 0
MeS 7 <0.01 0.014 0.013 <0.01 0.01 0.001
TSA 7 0.027 0.483 0.461 0.169 0.216 0.155
BSA B 7 0.027 (0.041) 0.239 (0.367) 0.232 (0.356) 0.063 (0.097) 0.083 (0.127) 0.068 (0.104) Drip Irrigation 7 days Pre-Transplant 4.00-4.01 kg a.i./ha Fluensulfone 2 <0.01 <0.01 <0.01 <0.01 <0.01 0
MeS 2 <0.01 <0.01 <0.01 <0.01 <0.01 0
TSA 2 0.067 0.118 0.116 0.095 0.095 0.03
BSA 2 0.062 (0.095) 0.077 (0.118) 0.073 (0.112) 0.07 (0.107) 0.07 (0.107) 0.004 (0.006) Broadcast Spray 7 days Pre-Transplant + Drip Irrigation 40 days Post-Transplant 5.85-8.07 g a.i./ha Fluensulfone 8 <0.01 <0.01 <0.01 <0.01 <0.01 0
MeS 8 <0.01 0.017 0.017 <0.01 0.011 0.002
TSA 8 0.027 1.285 1.171 0.25 0.447 0.467
BSA 8 0.055 (0.084) 0.379 (0.581) 0.376 (0.577) 0.151 (0.232) 0.177 (0.272) 0.096 (0.147)

Non-Bell Pepper Broadcast Spray 7 days Pre-Transplant 3.97-4.10 kg a.i./ha Fluensulfone 4 <0.01 <0.01 <0.01 <0.01 <0.01 0
MeS 4 <0.01 0.012 0.012 <0.01 0.01 0.001
TSA 4 0.033 0.274 0.249 0.139 0.141 0.097
BSA 4 0.031 (0.048) 0.201 (0.308) 0.184 (0.282) 0.088 (0.135) 0.10 (0.153) 0.072 (0.110) Broadcast Spray 7 days Pre-Transplant + Drip Irrigation 40 days Post-Transplant 5.93-6.11 kg a.i./ha Fluensulfone 4 <0.01 <0.01 <0.01 <0.01 <0.01 0
MeS 4 <0.01 0.024 0.022 0.011 0.013 0.006
TSA 4 0.076 0.389 0.378 0.16 0.196 0.127
BSA 4 0.084 (0.129) 0.392 (0.601) 0.376 (0.577) 0.261 (0.400) 0.247 (0.379) 0.12 (0.184)

Tomato Broadcast Spray 7 days Pre-Transplant 3.64-4.12 kg a.i./ha Fluensulfone 20 <0.01 <0.01 <0.01 <0.01 <0.01 0
MeS 20 <0.01 <0.01 <0.01 <0.01 <0.01 0
TSA 20 <0.01 0.374 0.332 0.076 0.095 0.094
BSA 20 <0.01 (<0.01) 0.275 (0.422) 0.273 (0.419) 0.069 (0.106) 0.09 (0.138) 0.089 (0.137) Drip Irrigation 7 days Pre-Transplant 4.00 kg a.i./ha Fluensulfone 2 <0.01 <0.01 <0.01 <0.01 <0.01 0
MeS 2 <0.01 <0.01 <0.01 <0.01 <0.01 0
TSA 2 <0.01 0.106 0.096 0.053 0.053 0.06
BSA 2 0.024 (0.037) 0.087 (0.133) 0.078 (0.120) 0.053 (0.081) 0.053 (0.081) 0.035 (0.054) Drip Irrigation 3 days Pre-Transplant 3.99-4.01 kg a.i./ha Fluensulfone 15 <0.01 <0.01 <0.01 <0.01 <0.01 0
MeS 0 -- -- -- -- -- --
TSA 15 <0.01 0.043 0.04 0.026 0.023 0.009
BSA 15 <0.01 (<0.01) 0.044 (0.067) 0.044 (0.067) 0.012 (0.018) 0.015 (0.023) 0.009 (0.014) Cantaloupe Broadcast Spray 7 days Pre-Transplant 3.85-4.11 kg a.i./ha Fluensulfone 8 <0.01 <0.01 <0.01 <0.01 <0.01 0
MeS 8 <0.01 <0.01 <0.01 <0.01 <0.01 0
TSA 8 <0.01 0.673 0.555 0.069 0.149 0.192
BSA 8 <0.01 (<0.01) 0.065 (0.100) 0.064 (0.098) 0.023 (0.035) 0.028 (0.043) 0.02 (0.031)
Fluensulfone 8 <0.01 <0.01 <0.01 <0.01 <0.01 0Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 100 of 107 Table D-1. Summary of Fluensulfone Residue Field Trial Results. Crop Use Pattern Analyte n Residue Levels (mg/kg) A

min max HAFT Med Mean SD Drip Irrigation 7 days Pre-Transplant 3.93-3.94 kg a.i./ha MeS 2 <0.01 <0.01 <0.01 <0.01 <0.01 0
TSA 8 <0.01 0.31 0.31 0.04 0.086 0.109
BSA 8 <0.01 (<0.01) 0.20 (0.307) 0.20 (0.307) 0.02 (0.031) 0.058 (0.089) 0.071 (0.109) Drip Irrigation 3 days Pre-Transplant 4.00-4.01 kg a.i./ha Fluensulfone 9 <0.01 <0.01 <0.01 <0.01 <0.01 0
MeS 0 -- -- -- -- -- --
TSA 9 0.031 0.215 0.206 0.1 0.103 0.062
BSA 9 <0.01 (<0.01) 0.117 (0.179) 0.109 (0.167) 0.046 (0.071) 0.046 (0.071) 0.039 (0.060)

Cucumber Broadcast Spray 7 days Pre-Transplant 3.72-4.11 kg a.i./ha Fluensulfone 7 <0.01 <0.01 <0.01 <0.01 <0.01 0
MeS 7 <0.01 0.079 0.075 0.023 0.032 0.028
TSA 7 <0.01 0.524 0.515 0.083 0.201 0.209
BSA 7 <0.01 (<0.01) 0.215 (0.330) 0.17 (0.261) 0.063 (0.097) 0.069 (0.106) 0.067 (0.103) Drip Irrigation 7 days Pre-Transplant 4.00 kg a.i./ha Fluensulfone 2 <0.01 <0.01 <0.01 <0.01 <0.01 0
MeS 2 <0.01 0.042 0.039 0.024 0.024 0.02
TSA 2 <0.01 0.718 0.609 0.31 0.31 0.424
BSA 2 <0.01 (<0.01) 0.353 (0.542) 0.219 (0.336) 0.114 (0.175) 0.114 (0.175) 0.147 (0.225) Drip Irrigation 3 days Pre-Transplant 3.99-4.01 kg a.i./ha Fluensulfone 15 <0.01 <0.01 <0.01 <0.01 <0.01 0
MeS 0 -- -- -- -- -- --
TSA 15 0.013 0.09 0.077 0.044 0.043 0.017
BSA 15 <0.01 (<0.01) 0.071 (0.109) 0.06 (0.092) 0.012 (0.018) 0.02 (0.031) 0.014 (0.021)

Summer Squash Broadcast Spray 7 days Pre-Transplant 3.80-4.14 kg a.i./ha Fluensulfone 8 <0.01 0.017 <0.014 <0.01 0.01 0.001
MeS 8 <0.01 0.05 0.048 0.013 0.02 0.014
TSA 8 0.028 0.715 0.708 0.182 0.242 0.241
BSA 8 <0.01 (<0.01) 0.256 (0.393) 0.247 (0.378) 0.134 (0.206) 0.124 (0.190) 0.096 (0.147) Drip Irrigation 7 days Pre-Transplant 4.00 kg a.i./ha Fluensulfone 2 <0.01 <0.01 <0.01 <0.01 <0.01 0
MeS 2 <0.01 <0.01 <0.01 <0.01 <0.01 0
TSA 2 0.038 0.127 0.103 0.076 0.076 0.038
BSA 2 0.029 (0.044) 0.062 (0.095) 0.06 (0.092) 0.052 (0.080) 0.052 (0.080) 0.011 (0.017) Use patterns in bold type are the proposed GAPs.

A Values reported as < 0.01 mg/kg were assumed to be 0.01 mg/kg for purposes of calculating HAFT, median, average, and standard deviation. Values for min and ma x reflect all samples; all ot her values reflect per-trial averages. HAFT = Highes t Average Field Trial.

B Parenthetical values for BSA are fluensulfone-equivalents ([BSA] × 1.534).Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 101 of 107 Appendix E. Review of Human Research

This risk assessment relies in part on data from studies in which adult human subjects were intentionally exposed to a pesticide or other chemical. Thes e data, which include studies from PHED 1.1 and the AHETF database are (1) subjec t to ethics review pursuant to 40 CFR 26, (2) have received that review, and (3) are compliant w ith applicable ethics requirements. For certain studies, the ethics review ma y have included review by the Human Studies Review Board. Descriptions of data sources, as well as gui dance on their use, can be found at the Agency
website 2

.

2 http://www.epa.gov/pesticides/science/handler-exposure-data.html and http://www.epa.gov/pesticides/science/post-app-exposure-data.htmlFluensulfone Human Health Ri sk Assessment DP No. D403767

Page 102 of 107 Appendix F. Summary of Submitted Studies

MRID No. Citation
48574731 Marin, J. (2010) Method Validation of an Analytical Method for the Determination of MCW-2 and its Metabolites in Plant Matrices. Project Number: R/ 23334/OCR, 1977W. Unpublished study prepared by PTRL West, Inc. 138p.
48574732 Witte, A. (2011) Validation of an Analytical Method for the Determination of Residues of MCW-2 Metabolite #3626 (Methylsulfone) in Water Containing Plant Commodities: Final Report. Project Number: R/28495/OCR, 11M04006/01/VMPL. Unpublished study prepared by CIP Chemisches Institut Pforzheim GmbH. 32p.
48574733 Witte, A. (2011) Validation of an Analytical Method for the Determination of Residues of MCW-2 and Three Metabolites in Plant Commodities (Acidic, Dry and Fatty): Final Report. Project Number: R/23489/OCR, 10M03036/01/VMPL, 11M03036/01/VMPL. Unpublished study prepared by CIP Chemisches Institut Pforzheim GmbH. 113p.
48574734 Bacher, R. (2011) Independent Laboratory Validation (ILV) of an Analytical Method for the Determination of MCW-2 and Three Metabolites in Plant Commodities. Project Number: R/27478/OCR, P/2243/G. Unpublished study prepared by PTRL Europe Gmbh. 139p.
48574735 Witte, A. (2011) Validation of an Analytical Method for the Determination of Residues of MCW-2 and Three Metabolites in Food Stuff of Animal Origin: Final Report. Project Number: R/28512/OCR, 11M03036/01/VMAT. Unpublished study prepared by CIP Chemisches Institut Pforzheim GmbH. 166p.
48574736 Marin, J. (2010) Method Validation of an Analytical Method for the Determination of Fluensulfone and its Metabolites in Soil. Project Number: R/2 3339/OCR, 2049W. Unpublis hed study prepared by PTRL West, Inc. 167p.
48574737 Marin, J. (2011) Method Validation of the Analytical Method for the Determination of Fluensulfone and its Metabolites in Water. Project Number: R/ 23385/OCR, 1870W. Unpublished study prepared by PTRL West, Inc. 193p.
48574797 Quistad, G. ; Bautista, A. (2011) A Metabolism Study with [Carbon 14]MCW-2 (2 Radiolabels) Using Potatoes. Project Number: OR/25459/OCR, 1785W. Unpublished study prepared by PTRL West, Inc., and Excel Research Services, Inc. 146p.
48574798 Quistad, G.; Bautista, A. (2011) A Metabolism Study with [Carbon 14]Fluensulfone (MCW-2) (2 Radiolabels) Using Tomatoes. Project Number: R/25456/OCR, 1787W. Unpublished study prepared by PTRL West, Inc., and Excel Re search Services, Inc. 166p.
48574799 Bautista, A.; La Mar, J.; Quistad, G. (2011) A Metabolism Study with [Carbon 14]MCW-2 (2 Radiolabels) Using Lettuce. Project Number: R/25455/OCR, 1786W. Unpublished study prepared by PTRL West, Inc., and Excel Re search Services, Inc. 230p.
48574801 LaMar, J.; Quistad, G. (2010) A Metabolism Study with [Carbon 14] MCW-2 (2 Radiolabels) in Laying Hens. Project Number: OR/25454/OCR, 1793W. Unpublished study prepared by PTRL West, Inc., and Genesis Midwest Laboratories. 331p.
48574802 LaMar, J.; Quistad, G. (2010) The Metabolism of [Carbon 14] MCW-2 (2 Radiolabels) in the Lactating Goat. Project Number: OR/25458/OCR, 1792W. Unpublished study prepared by PTRL West, Inc., and Genesis Midwest Laboratories. 429p.
48574803 Korpalski, S. (2011) Magnitude of the Residue of MCW-2 on Fruiting Vegetables: Final Report. Project Number: R/23481/OCR, 09/01858. Unpublished study prepared by Eurofins Agroscience Services, Inc. 379p.
48574804 Jones, G. (2011) Magnitude of the Residue of Fluensulfone in Fruiting Vegetables and Processed Commodities: Final Report. Project Number : R/23487/OCR, AA100707. Unpublished study prepared by American Agricultural Services, Inc. (AASI) , PTRL We st, Inc., and GLP Technologies. 665p.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 103 of 107 MRID No. Citation
48574804 Jones, G. (2011) Magnitude of the Residue of Fluensulfone in Fruiting Vegetables and Processed Commodities: Final Report. Project Number : R/23487/OCR, AA100707. Unpublished study prepared by American Agricultural Services, Inc. (AASI) , PTRL We st, Inc., and GLP Technologies. 665p. 48574805 Jones, G. (2011) Magnitude of the Residue of Fluensulfone in Cucurbit Vegetables: Final Report. Project Number: R/23488/OCR, AA100708. Unpublished study prepared by American Agricultural Services, Inc. (AASI) and PTRL West, Inc. 460p.
48574806 Korpalski, S. (2011) Magnitude of the Residue of MCW-2 on Cucurbit Vegetables: Final Report. Project Number: R/23482/OCR, 09/01859. Unpublished study prepared by Eurofins Agroscience Services, Inc. 312p.
48574820 Quistad, G.; Bautista, A.; Kovatchev, A. et al. (2011) A Confined Rotational Crop Study with [Carbon 14]MCW-2 (2 Radiolabels) Using Ra dish, Lettuce, and Wheat. Project Number: OR/25457/OCR, 1788W. Unpublished study prepared by PTRL West, Inc., Excel Research Services, Inc. 434p.
48575013 Ballard, T. (2012) FDA PAM Multiresidue Meth od (MRM) Testing for Fluensulfone as Two Sulfonic Acid Metabolites. Project Number: R/29565/OCR, 11 /0015. Unpublished study prepared by En-Cas Analytical Laboratories. 73p.
48575014 Bacher, R. (2012) Independent Laboratory Validation (ILV) of an Analytical Method for the Determination of MCW-2 and Three Metabolites in Foodstuff of Animal Origin. Project Number: R/29562/OCR, P/2455/G. Unpublished study prepared by PTRL Europe Gmbh. 245p.
48574741 Quuistad, G. ; Kovatchev, A. ; Mar, J. (2010) The Pharmacokinetics of [Carbon 14]MCW-2 in the Rat Upon Administration of Single Oral High and Low Doses. Project Number: R/23411/OCR, 1790W, 10174G/X01G. Unpublished study prepared by PTRL West, Inc. 233p.
48574742 Quistad, G.; Kovatchev, A.; Mar, J. (2011) The Metabolism and Excretion of [Carbon 14]MCW-2 in the Rat Upon Administration of Single Oral High and Low Doses. Project Number: R/23410/OCR, 1789W, 10B0104G/X01G. Unpublished study prepared by PTRL West, Inc., and Pacific BioLabs. 351p.
48574743 Quaistad, G. ; Kovatchev, A. (2011) The Tissue Distribution of [Carbon 14]MCW-2 in the Rat Upon Administration of Single Oral High and Low Doses. Project Number: R/23412/OCR, 1791W. Unpublished study prepared by PTRL West, Inc., and Pacific BioLabs. 397p.
48574744 Quistad, G.; Kovatchev, A.; LaMar, J. (2011) The Metabolism, Excretion, and Tissue Distribution of [Thiazole-(Carbon 14)]Fluensulfone (MCW-2) in the Rat Upon Administration of Repeated Oral Doses (5mg/kg). Project Number: R/23389/OCR 2134W, 10L0405G/X01G. Unpublished study prepared by PTRL West, Inc., and Pacific BioLabs. 247p.
48574745 Haferkorn, R. (2010) Acute Oral Toxicity Study of MCW-2 Technical in Rats. Project Number: R/23373/OCR, 24555. Unpublished study prepared by Laboratory of Pharmacology & Toxicology. 33p.
48574746 Simon, C. (2009) MCW-2 Tech: Acute Oral Toxicity Study in Rats: Final Report. Project Number: R/23417/OCR, C14933. Unpublished study prepared by Harlan Laboratories, Ltd. 31p.
48574747 Simon, C. (2009) MCW-2 Tech: Acute Dermal Toxicity Study in Rats: Final Report. Project Number: R/23418/OCR, C14944. Unpublished study prepared by Harlan Laboratories, Ltd. 26p.
48574748 Pothmann, D. (2009) MCW-2 Tech: 4-Hour Acute Inhalation Toxicity Study in Rats: Final Report. Project Number: R/23419/OCR, C14955. Unpublished study prepared by Harlan Laboratories, Ltd. 47p. 48574749 Simon, C. (2009) MCW-2 Tech: Primary Skin Irritation Study in Rabbits (4-Hour Semi-Occlusive Application). Project Number: R/23420/OCR, C14966. Unpublished study prepared by Harlan Laboratories, Ltd. 35p.
48574750 Simon, C. (2009) MCW-2 Tech: Primary Eye Irritation Study in Rabbits: Final Report. Project Number: R/23421/OCR, C14977. Unpublished study prepared by Harlan Laboratories, Ltd. 35p.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 104 of 107 MRID No. Citation
48574751 Simon, C. (2009) MCW-2 Tech: Contact Hypersensitivity in Albino Guinea Pigs, Maximization-Test. Project Number: R/23422/OCR, C14988. Unpublished study prepared by Harlan Laboratories, Ltd. 52p.
48574752 Braun, L. (2009) 28-Day Range-finding Oral (Feeding) Toxicity Study in the Beagle Dog: Final Report. Project Number: R/23349/OCR, B89087. Unpublished study prepared by Harlan Laboratories, Ltd. 424p.
48574753 Krotlinger, F.; Ruhl-fehlert, C. (2003) BYI 01921: Study for Subchronic Oral Toxicity in Mice (Feeding Study for 13 Weeks). Project Number: R/23151/OCR, T7071801, M/103258/01/1. Unpublished study prepared by Bayer HealthCare AG. 397p.
48574754 Schladt, L.; Ruhl-fehlert, C. (2003) BYI 01921: Study on Subchronic Toxicity in Wistar Rats (Dietary Administration for 3 Months with a Subsequent Recovery Period over 1 Month). Project Number: R/23150/OCR, T0071363, AT00814. Unpublished study prepared by Bayer HealthCare AG. 785p.
48574755 Braun, L. (2009) MCW-2 Tech: 90-Day Oral (Feeding) Toxicity Study in the Beagle Dog: Final Report. Project Number: R/23350/OCR, C15552. Unpublished study prepared by Harlan Laboratories, Ltd. 764p.
48574756 Braun, L. (2011) MCW-2: Tech 52-Week Oral (Feeding) Toxicity Study in the Beagle Dog with an 8- Week Recovery Period: Final Report. Study Prepared By Harlan Laboratories, Ltd. 1294p. Project Number: R/23351/OCR C57031. Unpublished study prepared by Harlan Laboratories Ltd.. 1294p.
48574759 Herbold, B. (2002) BYI 01921: Salmonella/Microsome Test Plate Incorporation Method. Project Number: R/23135/OCR, T/7070901, PH/32046. Unpublished study prepared by Bayer HealthCare AG. 47p. 48574760 Sokolowski, A. (2008) Salmonella Typhimuriu m and Escherichia Coli Reve rse Mutation Assay with MCW-2 Tech. Project Number: R/23352/OCR, 1176200. Unpublished study prepared by Cytotest Cell Research Gmbh and Co. 31p.
48574761 Donath, C. (2011) Reverse Mutation Assay Using Bacteria (Salmonella typhimurium and Escherichia coli) with MCW-2 Tech: Final Report. Project Number: R/23356/OCR, 110567. Unpublished study prepared by BSL Bioservice Scientific Laboratories Gmbh. 67p.
48574762 Flugge, R. (2010) In vitro Assessment of the Clastogenic Activity of MCW-2 Technical in Cultured Human Peripheral Lymphocytes. Project Number: R/23372/OCR, 24556. Unpublished study prepared by Laboratory of Pharmacology & Toxicology. 46p.
48574763 Herbold, B. (2003) BYI 01921: V79/HPRT-Test in vitro for the Detection of Induced Forward Mutations. Project Number: R/23147/OCR, T/4071484, AT00357. Unpublished study prepared by Bayer HealthCare AG. 38p.
48574764 Herbold, B. (2003) BYI 01921: Micronucleus Test on the Male Mouse. Project Number: R/23143/OCR, T/3071483, AT00208. Unpublished study prepared by Bayer HealthCare AG. 45p.
48574765 Kaiser, S. (2011) MCW-2 Tech: 104-Weeks Combined Chronic Toxicity and Oncogenicity (Feeding) Study in the Rat: Final Report. Project Number: R/23353/OCR, B80188. Unpublished study prepared by Harlan Laboratories, Ltd. 3678p.
48574766 Kaiser, S. (2011) MCW-2 Tech: 78-Weeks Oncogenicity (Feeding) Study in CD-1 Mice: Final Report. Project Number: R/23354/OCR, B80190. Unpublished study prepared by Harlan Laboratories, Ltd. 2616p.
48574769 Ceccatelli, R. (2011 ) MCW-2 Tech: Two-Generation Reproductio n Toxicity Study in the Han Wistar Rat. Project Number: R/23355/OCR, B92654. Unpublished study prepared by Harlan Laboratories, Ltd. 2230p.
48574770 Ceccatelli, R. (2009) MCW-2 Tech: Prenatal Developmental Toxicity Study in the Himalayan Rabbit: Final Report. Project Number: R/23357/OCR, B92687. Unpublished study prepared by Harlan Laboratories, Ltd. 361p.
48574771 Young, A. (2003) Technical Grady BYI 01921: A Prenatal Developmental Toxicity Study in the Wistar Rat. Project Number: R/23155/OCR, 02/T12/LD, M/111438/01/1. Unpublished study prepared by Bayer CropScience. 418p.Fluensulfone Human Health Ri sk Assessment DP No. D403767

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48574773 Sommer, E. (2010) MCW-2 Tech: Acute Oral Neurotoxicity (Gavage) Study in Rats: Final Report. Project Number: R/23375/OCR, C56895. Unpublished study prepared by Harlan Laboratories, Ltd. 982p.
48574774 Sommer, E. (2011) MCW-2 Tech: 13-Week Neurotoxicity (Feeding) Study in Rats: Final Report. Project Number: R/23336/OCR, C95347. Unpublished study prepared by Harlan Laboratories, Ltd. 999p.
48574779 Struve, P. (2011) Immunotoxicity Evaluation of MCW-2 in 28-Dietary Study in CD-1 Female Mice Evaluation of Anti-Sheep Red Blood Cell(SRBC) Response: Final Report. Project Number: R/23383/OCR, BRT/20100420. Unpublished study prepared by Burleson Research Technologies, Inc. (BRT). 173p.
48574780 Sommer, E. (2011) MCW-2 TECH: 4-Week Dermal Toxicity Study in Rats: Final Report. Project Number: R/23348/OCR, D09837. Unpublished study prepared by Harlan Laboratories, Ltd. 551p.
48574783 Arcelin, G. (2010) MCW-2: Metabolite #3625: Acute Oral Toxicity Study in Rats: Final Report. Project Number: R/23470/OCR, C81241. Unpublished study prepared by Harlan Laboratories, Ltd. 59p. 48574784 Sokolowski, A. (2010) Salmonella typhimurium and Escherichia coli Reverse Mutation Assay with MCW-2 Metabolite 3625: Final Report. Project Number: R/23469/OCR, 1317202. Unpublished study prepared by Cytotest Cell Research Gmbh and Co. 56p.
48574785 Hall, C. (2010) In vitro Chromosome Aberra tion Test in Chinese Hamster V79 Cells with MCW-2 Metabolite 3625: Final Report. Project Numb er: R/23471/OCR, 1345801. Unpublished study prepared by Cytotest Cell Research Gmbh and Co. 57p.
48574786 Merker, M. (2011) Micronucleus Assay in B one Marrow Cells of the Rat with MCW-2 Metabolite #3625: Final Report. Project Number: R/23472/OCR, 1345802. Unpublished study prepared by Cytotest Cell Research Gmbh and Co. 55p.
48574787 Mallaun, M. (2010) MCW-2 Metabolite 3626: Acute Oral Toxicity Study in Rats: Final Report. Project Number: R/23474/OCR, C81252. Unpublished study prepared by Harlan Laboratories, Ltd. 67p. 48574788 Sokolowski, A. (2010) Salmonella typhimurium and Escherichia coli Reverse Mutation Assay with MCW-2 Metabolite 3626: Final Report. Project Number: R/23473/OCR, 1317203. Unpublished study prepared by Cytotest Cell Research Gmbh and Co. 58p.
48574789 Hall, C. (2011) Gene Mututation Assay in Ch inese Hamster V79 Cell in vitro (V79 / HPRT) with MCW-2 Metabolites 3626: Final Report. Project Number: R/23464/OCR, 1388400. Unpublished study prepared by Cytotest Cell Research Gmbh and Co. 63p.
48574790 Merker, M. (2011) In vivo Unscheduled DNA Synthesis in Rat Hepatocytes with MCW-2 Metabolite # 3626: Final Report. Project Number: R/23475/OCR, 1345901. Unpublished study prepared by Cytotest Cell Research Gmbh and Co. 68p.
48574791 Merker, M. (2011) Micronucleus Assay in B one Marrow Cells of the Rat with MCW-2 Metabolite #3626: Final Report. Project Number: R/23476/OCR, 1345902. Unpublished study prepared by Cytotest Cell Research Gmbh and Co. 56p.
48574792 Arcelin, G. (2010) MCW-2 Metabolite 3627 - Ac ute Oral Toxicity Study in Rats: Final Report. Project Number: R/23466/OCR, C81230. Unpublished study prepared by Harlan Laboratories, Ltd. 64p.
48574793 Sokolowski, A. (2010) Salmonella typhimurium and Escherichia coli Reverse Mutation Assay with MCW-2 Metabolite 3627: Final Report. Project Number: R/23465/OCR, 1317201. Unpublished study prepared by Cytotest Cell Research Gmbh and Co. 59p.
48574794 Hall, C. (2010) In vitro Chromosome Aberra tion Test in Chinese Hamster V79 Cells with MCW-2 Metabolite 3627: Final Report. Project Numb er: R/23467/OCR, 1328701. Unpublished study prepared by Cytotest Cell Research Gmbh and Co. 66p.Fluensulfone Human Health Ri sk Assessment DP No. D403767

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48574795 Merker, M. (2010) Micronucleus Assay in B one Marrow Cells of the Rat with MCW-2 Metabolite # 3627: Final Report. Project Number: R/23468/OCR, 1328702. Unpublished study prepared by Cytotest Cell Research Gmbh and Co. 55p.
48574918 Arcelin, G. (2009) MCW-2 480 EC: Acute Oral Toxicity Study in Rats: Final Report. Project Number: R/23425/OCR, C45364. Unpublished study prepared by Harlan Laboratories, Ltd. 57p.
48574919 Arcelin, G. (2009) MCW-2 480 EC: Acute Dermal Toxicity Study in Rats: Final Report. Project Number: R/23426/OCR, C45342. Unpublished study prepared by Harlan Laboratories, Ltd. 52p.
48574920 Pothmann, D. (2009) MCW-2 480 EC: 4-Hour Acute Inhalation Toxicity Study in the Wistar Rat: Final Report. Project Number: R/23430/OCR, C45454. Unpublished study prepared by Harlan Laboratories, Ltd. 63p.
48574921 Arcelin, G. (2009) MCW-2 480 EC: Primary Skin Irritation Study in Rabbits (4-Hour Semi-Occlusive Application): Final Report. Project Number: R/23428/OCR, C45375. Unpublished study prepared by Harlan Laboratories, Ltd. 59p.
48574922 Arcelin, G. (2009) MCW-2 480 EC: Primary Eye Irritation Study in Rabbits: Final Report. Project Number: R/23427/OCR, C45397. Unpublished study prepared by Harlan Laboratories, Ltd. 58p.
48574923 Arcelin, G. (2009) MCW-2 480 EC: Contact Hy persensitivity in Albino Guinea Pigs, MaximizationTest: Final Report. Project Number: R/23429/OCR, C45421. Unpublished study prepared by Harlan Laboratories, Ltd. 78p.
48574924 Mallaun, M. (2009) MCW-2 480 EC: Acute Oral Toxicity Study in Rats: Final Report. Project Number: R/27473/OCR, D23934. Unpublished study prepared by Harlan Laboratories, Ltd. 53p.
48574925 Mallaun, M. (2011) MCW-2 480 EC: Primary Skin Irritation Study in Rabbits (4-Hour SemiOcclusive Application): Final Report. Project Number: R/27474/OCR, D23945. Unpublished study prepared by Harlan Laboratories, Ltd. 59p.
48574926 Mallaun, M. (2011) MCW-2 480 EC: Primary Eye Irritation Study in Rabbits: Final Report. Project Number: R/27475/OCR, D23956. Unpublished study prepared by Harlan Laboratories, Ltd. 61p.
48574949 Hassler, S. (2011) MCW-2: Study on the Dermal Penetration of (Carbon 14)-MCW-2 Formulated as MCW-2 480 EC in Rats: Final Report. Project Number: R/23344/OCR, C56873. Unpublished study prepared by Harlan Laboratories, Ltd. 89p.
48574950 Hassler, S. (2011) MCW-2: Percutaneous Penetration of (Carbon 14)-MCW-2 Formulated as MCW-2 480 EC through Rat and Human Split-thickness Skin Membranes (in vitro): Final Report. Project Number: R/23332/OCR, C56884. Unpublished study prepared by Harlan Laboratories, Ltd. 96p.
48575023 Temerowski, M. (2002) BYI 01921: Study for Subacute Oral Toxicity in Rats (Feeding Study Over 4 Weeks). Project Number: PH/32327/OCR, T/6069704, 32327/MO/02/012047. Unpublished study prepared by Bayer Ag Inst. of Toxicology. 245p.
48575024 Krotlinger, F. (2003) BYI 01921: Subacute Oral Toxicity Study in Mice (4 Weeks Administration by Diet). Project Number: AT00816/OCR, T2071608, MO/03/015427. Unpublished study prepared by Bayer HealthCare AG. 200p.
48574739 Jaitz, L. (2011) Validation of a Residue Analytical Method for the Determination of Fluensulfone in Air: Final Report. Project Number: R/28477, D27275/OCR. Unpublished study prepared by Harlan Laboratories, Ltd. 61p.
48574767 Kaiser, S. (2011) MCW-2 Tech: 3- and 7-Day Oral (Feeding) Mechanistic Lung Toxicity Study in Mice: Final Report. Project Number: R/27479/OCR, D26983. Unpublished study prepared by Harlan Laboratories, Ltd. 243p.
48574768 Jeager, M. (2011) Comparative Biotransforma tion of MCW-2 in Human and Mice Lung Microsomes. Project Number: R/23346/OCR, 1388500. Unpublished study prepared by Cytotest Cell Research Gmbh and Co. 82p.
48574772 Sommer, E. (2009) MCW-2 Tech: Acute Oral Neurotoxicity Peak-Effect Study in Rats: Final Report. Project Number: R/23376/OCR, C57413. Unpublished study prepared by Harlan Laboratories, Ltd. 178p.Fluensulfone Human Health Ri sk Assessment DP No. D403767

Page 107 of 107 MRID No. Citation
48574775 Sagelsdorff, P. (2009) MCW2-Tech: Mode-of-Action Investigations on Inhibition of AlanineAminotransferase: (in Dogs): Final Report. Project Number: R/23424/OCR, C15686. Unpublished study prepared by Harlan Laboratories, Ltd. 36p.
48574776 Braun, L. (2011) MCW-2 Tech: 2-Week Oral (Feeding) Mode of Action (Effects on Alanine Amino Transferase Activites - ALAT) Study in the B eagle Dog: Final Report. Project Number: R/23457/OCR, C59044. Unpublished study prepared by Harlan Laboratories, Ltd. 267p.
48574777 Sagelsdorff, P. (2010) MCW-2 Tech: Mode of Action Study on Alanine Aminotransferase (ALAT) in vitro in Dog Liver Homogenate: Final Report. Project Number: R/23458/OCR, C58796. Unpublished study prepared by Harlan Laboratories, Ltd. 39p.
48574778 Sagelsdorff, P. (2009) Determination of Hepatic Alanine Aminotransferase (ALAT) in Untreated Dogs: Final Report. Project Number: R/23459/OCR, C57896. Unpublished study prepared by Harlan Laboratories, Ltd. 26p.
48574809 Chevallier, E. (2011) Magnitude of Residue of MCW-2 and Its Major Metabolites in Melon Raw Agricultural Commodity After One Application of MCW-2 480EC - 8 Trials - Spain, Italy and Greece - 2010: Final Report. Project Number: R/23486/OCR, BPL10/237/CL. Unpublished study prepared by Biotek Agriculture. 123p.
48575022 Barker, W. (2012) Independent Laboratory Validation (ILV) of the PTRL Method 2061 W Entitled
"Determination of MCW-2 and Metabolites in Plan t Matrices". Project Number: R/29563/OCR, 11/0009. Unpublished study prepared by En-Cas Analytical Laboratories. 231p.
48575028 Gordon, E. (2011) Weight of Evidence Evaluation of Fluensulfone Immunotoxicity. Project Number: R/28510/OCR, MANA/FL/2015. Unpublished study prepared by Elliot Gordon Consulting, LLC. 30p.
48575029 Strupp, C. (2011) The Toxicological Significance of Lower Alanine-Aminotransferase (ALAT) Activity following Dietary Exposure in Animal Studies. Project Number: R/28511/OCR. Unpublished study prepared by Ellio t Gordon Consulting, LLC. 31p.
48575041 Strupp, C.; Banas, D.; Cohen, S. et al. (2012) Relationship of Metabolism and Cell Proliferation to the Mode of Action of Fluensulfone-Induced Mouse Lung Tumors: Analysis of their Human Relevance Using the IPCS Framework. Project Number: MANA/CS/04/2012/OCR. Unpublished study prepared by Makhteshim Agan Holding B.V. 50p.
49330201 Polakoff, M. (2012) Position Paper: The Use of Fluensulfone on Fruiting Vegetables and Cucurbits Will Not Increase Fluoride Exposure. Unpublished st udy prepared by Exponent International, Ltd. 214p.

49402701 Burn, R and Winner, S. (2012) To determ ine fluensulfone residues in fruiting vegetables (tomato & processed fracti ons, capsicum, rockmelon, cucumber and zucchini) following one applic ation of MCW-2 applied 7 da ys prior to transplanting. Study Number FOZ1001. Unpublished paper pr epared by AgriSolutions Australia. 203 p.