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7.0 AGGREGATE EXPOSURE/RISK CHARACTERIZATION ................................................................. 27

8.0 CUMULATIVE EXPOSURE/RISK CHARACTERIZATION ............................................................... 27

9.0 OCCUPATIONAL EXPOSURE/RISK CHARACTERIZATION ........................................................... 28

9.1 Short- and Intermediate-Term Handler Risk .................................................................... 28

9.2 Short- and Intermediate-Term Post-Application Risk ...................................................... 30

10.0 REFERENCES ...................................................................................................................... 31

APPENDIX A. TOXICOLOGY PROFILE AND EXECUTIVE SUMMARIES ............................................ 32

A.1Toxicology Data Requirements ........................................................................................ 32

A.2Toxicity Profiles ................................................................................................................ 33

A.3Hazard Identification and Endpoint Selection .................................................................. 44

A.4Executive Summaries........................................................................................................ 47

APPENDIX B. METABOLISM SUMMARY TABLE ............................................................................. 95

APPENDIX C. PHYSICAL/CHEMICAL PROPERTIES .......................................................................... 98

APPENDIX D. SUMMARY OF FLUENSULFONE RESIDUE DATA FOR CUCURBIT AND FRUITING 

VEGETABLES............................................................................................................................ 99

APPENDIX E. REVIEW OF HUMAN RESEARCH ............................................................................. 101

APPENDIX F. SUMMARY OF SUBMITTED STUDIES ....................................................................... 102
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1.0 Executive Summary 

Makhteshim-Agan of North America (MANA) has petitioned the Agency to register the 

nematicide fluensulfone for use on cucurbit vegetables (Crop Group 9) and fruiting vegetables 

(Crop Group 8-10) and to establish permanent tolerances 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-trifluoro-3-buten-1-yl)sulfonyl]thiazole} is the first nematicide 

in the fluoroalkenyl class and it is currently undergoing global joint review [review partners are 

the United States Environmental Protection Agency (U.S. EPA), the Pest Management 

Regulatory Agency (PMRA) of Canada and the Australian Pesticides and 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 application 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 transplanting, at a maximum rate of 3.5 lb a.i./A (3.84 

kg a.i./ha). Based on the pre-plant-incorporated nature of the use pattern, occupational exposures 

are expected for persons involved in handling and application activities but not for postapplication activities. Based on the use sites, there is the potential for dietary exposure to 

fluensulfone and/or its breakdown products via consumption of treated crops and from drinking 

water. There are no proposed or existing uses that would result in residential exposure including 

exposure from volatization. 

In mammals, exposure to fluensulfone results in effects on the hematopoietic system (decreased 

platelets, increased white blood cells, hematocrit, 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, neurotoxic effects were not observed in any other study, including a 

subchronic neurotoxicity study. Evidence of qualitative, but not quantitative, increased 

susceptibility of infants and children to the effects 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 quantitative susceptibility 

was observed in developmental toxicity studies in rats or rabbits. Dietary and inhalation studies 

in rats showed evidence of portal-of-entry effects in the forestomach, pharynx, epiglottis, and 

nasal cavity. Overall, the most sensitive endpoints for assessing human health risk are the 

increased pup-loss effects for acute dietary exposure; 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 portal-of-entry effects (histopathology of the 

epiglottis and nasal cavity) for inhalation exposures (short/intermediate term). For assessing 

occupational oral and dermal exposures, the standard inter- and intra-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 concentration for humans (RfC) supports reducing the 

interspecies factor to 3X; however, an additional 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 regulatory points of departure for the dermal and 
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inhalation routes of exposure, risk estimates from these two routes should not be combined. 

Based on the completeness of the toxicity data, the use of the most sensitive endpoints for risk 

assessment and the resulting low degree of concern regarding the observed qualitative 

susceptibility, and the conservatisms in the exposure assessments, HED is reducing the Food 

Quality Protection Act Safety Factor (FQPA SF) from 10X to 1X. 

Crop metabolism studies and environmental fate studies demonstrate that fluensulfone is rapidly 

and, in the case of crops, quantitatively broken down into smaller molecules. In crops, the 

predominant residues are two sulfonic acid metabolites, referred to as BSA and TSA1

. As a 

result, dietary exposures from treated 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, dechlorinated parent compound, a methyl-sulfone 

metabolite (MeS1

), and/or the sulfonic acid metabolites, 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 sulfonic acid metabolites are not of toxicological 

concern based on the available data from acute toxicity, 28-day oral toxicity, 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 BSA only due to lack of 

the parent compound in harvested crops and the 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 and 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 vegetables (Crop Group 9) and 0.70 ppm for fruiting 

vegetables (Crop Group 8-10). 

Dietary (food and water) risk estimates for fluensulfone 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 (aPAD) 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 there are no residential uses for fluensulfone, there 

are no residential exposure contributions to the aggregate risk estimates. Therefore, aggregate 

risk estimates are equivalent to the dietary risk 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 intermediate-term (1-6 months) exposures may occur. 

Provided the fluensulfone label is revised to specify additional 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 metabolite (5-chloro-thiazole-2-sulfonic acid; Company Code M3625); MeS refers to the methyl sulfone metabolite (5-chloro-2-methyl sulfonyl thiazole; Company Code M-3626). 
Fluensulfone Human Health Risk Assessment DP No. D403767 

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(PPE; e.g., PF5 respirator, engineering controls (enclosed-systems)), there are no risks of 

concern associated with mixing, loading, or applying fluensulfone via groundboom equipment. 

A potential dermal risk of concern was identified [margin of exposure (MOE) = 60, LOC = 100] 

for persons mixing/loading liquids for application via chemigation, even with maximum PPE 

[i.e., engineering controls (enclosed systems)]. The maximum single application rate to achieve 

an MOE of 100 for mixing/loading liquids for chemigation is 2.0 lb a.i./acre using engineering 

controls as the level of mitigation. Post-application exposures are expected to be negligible 

based on the pre-plant-incorporated nature of the use pattern and the mechanical equipment and 

precautions associated with transplanting operations. 

Aside from the potential occupational risk issue noted above, there are no human health risk 

concerns that would preclude granting the requested registrations for fluensulfone and 

establishing tolerances for residues at the recommended levels. 

Potential areas of environmental justice concerns were considered in this 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 studies are subject to ethics review, 

have received that review, and are compliant 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, the 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 analysis by reverse-phase HPLCMS/MS. Sufficient ion transitions are 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 Joint Meeting on Pesticide Residues. 
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At this time, the residue definition for enforcement is harmonized between the U.S. and 

Australia. For Canadian MRLs, PMRA is tentatively recommending the parent compound plus 

BSA as the residue definition for enforcement. The recommended regulatory levels for the 

commodities in this assessment are harmonized among 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 commodities 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 calculation 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 expressed as parent fluensulfone whereas the HED 

recommendations are based on residues of only the BSA metabolite, expressed as parent 

fluensulfone. Furthermore, the petitioner combined residue data from the representative 

commodities to obtain their proposed tolerances. In accordance with current policy, HED 

calculated separate tolerance levels for each representative commodity and 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 shirt, long pants, socks, shoes, and chemicalresistant gloves). Additional PPE (i.e., PF5 respirator) 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 application rate should be reduced (see Section 9.1). Note that even at 

the engineering-controls level of mitigation, there is still a potential risk from dermal exposure 

for workers involved in mixing and loading activities for chemigation. 
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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 room temperature but may be a liquid at warmer, 

environmentally relevant temperatures. The compound is moderately volatile; thus, there is 

some likelihood for exposure to fluensulfone in the vapor phase. The technical-grade material 

has a log KOW of 2.2 indicating a somewhat-fat-soluble compound, but not to the degree that 

bioaccumulation is of particular concern. A listing of the physical/chemical 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 (which 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-use product label is summarized in Table 3.3. 

Fluensulfone 480 EC may be applied as a pre-plant band/broadcast spray (incorporated by water 

or mechanical means) using ground or chemigation (i.e., drip irrigation), equipment. The 

proposed Restricted Entry Interval (REI) is 12 hours for all crops. 

The PPE statement on the proposed label requires applicators and other handlers to wear longsleeved shirt and long pants, socks, shoes, and chemical-resistant gloves, such as barrier laminate 

or Viton ≥ 14 mils. 
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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 pepper, 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 cucurbit vegetables (e.g., cucumbers, melons, squash) 

and fruiting vegetables (e.g., tomatoes, peppers, eggplant). The product is meant to be applied to 

soil prior to transplanting 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 sources of drinking water. From crops, the exposure will be to 

metabolites of fluensulfone (primarily BSA and TSA). In drinking water, the residues may 

include BSA and TSA as well as parent fluensulfone, dechlorinated fluensulfone, and the MeS 

metabolite. The distribution of residues between these compounds will be dependent on the 

source of the drinking water (surface 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 residential uses of fluensulfone and exposure via 

residential pathways is not expected. 

In an occupational setting, applicators may be exposed while handling the pesticide prior to 

application, as well as during application. The use of fluensulfone is application 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 operations are with mechanical equipment which 

minimizes contact with soil, HED does not expect there to be significant dermal or inhalation 

post-application exposures. 

3.5 Consideration of Environmental Justice 

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

human health risk assessment, in accordance with U.S. Executive Order 12898, "Federal Actions 
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to Address Environmental Justice in Minority Populations and Low-Income Populations," 

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

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

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

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

and activities in and around the home that involve pesticide use in a residential setting. 

Extensive data on food consumption patterns are compiled by the USDA under the National 

Health and Nutrition Survey/What We Eat in America (NHANES/WWEIA) and are used in 

pesticide risk assessments for all registered food uses of a pesticide. These data are analyzed and 

categorized by subgroups based on age and ethnic group. Additionally, OPP is able to assess 

dietary exposure to smaller, specialized subgroups and exposure assessments are performed 

when conditions or circumstances warrant. Whenever appropriate, non-dietary exposures based 

on home use of pesticide products and associated risks for adult applicators and for toddlers, 

youths, and adults entering or playing on treated areas post-application are evaluated. Further 

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

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

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

4.0 Hazard Characterization and Dose-Response Assessment 

4.1 Toxicology Studies Available for Analysis 

The toxicology database for fluensulfone is complete and acceptable for selecting toxicity 

endpoints and points of departure for risk assessment. The available toxicology studies include 

the following: (1) subchronic oral toxicity studies in rats, mice and dogs; (2) a 28-day dermal 

toxicity study in rats; (3) a 90-day inhalation study in rats; (4) developmental toxicity studies in 

rats and rabbits; (5) a 2-generation reproduction study in rats; (6) chronic toxicity studies in rats 

and dogs; (7) carcinogenicity studies in mice and rats; (8) a battery of genotoxicity studies; (9) 

four metabolism studies in rats; (10) a dermal penetration study in rats and an in vitro

comparative dermal absorption study with rat and human skin (testing an EC formulation of 

fluensulfone); (11) acute and subchronic neurotoxicity studies in rats; and (12) an 

immunotoxicity study in mice. 

For the BSA and TSA metabolites, toxicology studies include acute oral studies in rats, 28-day 

oral studies in rats, and bacterial and mammalian cell mutagenicity studies. 

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

In rats, fluensulfone is rapidly absorbed following oral dosing. The time to maximum plasma 

levels (tmax) 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 (tmax) was longer at 2-8 

hours at the low dose and 48 hours at the high dose. The difference in whole blood and plasma 

tmax is related to fluensulfone reacting with the 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 tested (5 and 500 mg/kg). Urine was the major 

route of excretion accounting for 75-88% of the administered dose (AD) with feces accounting 
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for 5-13% AD. Most of the AD (~80%) was excreted 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-dosing. The highest tissue levels 

were observed in the liver, kidney, and gastrointestinal tract. Identification of compounds in the 

urine (Table 4.2) showed no fluensulfone and indicates that fluensulfone cleaves at the sulfonyl 

bridge. The metabolic pathway begins with fluensulfone reacting with glutathione to displace 

butene sulfinic acid which is then oxidized to BSA. The glutathione adduct 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 glucuronic 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 14C-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 because 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 data demonstrated an inverse relationship between 

dosing concentration and percent 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 absorption values adjusted 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/cm2

; 

(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 absorbed plus absorbable) observed in vitro under the 

same exposure conditions. The DAF for humans is therefore 65.4%/1.6 = 40.9%. 

4.3 Toxicological Effects 
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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 doses. 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 observed at the LOAELs. Effects on the hematopoietic system are 

consistent with results from rat metabolism studies which demonstrated that fluensulfone can 

react with the free thiol moiety of globin, forming a covalent 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, DT50 = 6-9 days) 

compared to plasma (DT50 = 16-17 hours). 

Other effects commonly observed in mice, rats, and 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 clinical chemistry parameters included decreased 

plasma alanine amino transferase (ALAT, rats and dogs), increased cholesterol (rats), increased 

triglycerides (rats), increased phospholipids (rats), increased (mice) or decreased (dogs) 

bilirubin, decreased protein (dogs), decreased albumin and albumin/globulin ratio (dogs), 

decreased glucose (dogs), and increased TSH (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 effects on the kidney in male rats; however, 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 chronic dietary exposure in mice and rats. These 

effects consisted of an increased incidence and severity of lung interstitial inflammation in the 2-

year rat study and an increased incidence of bronchiolization (change from 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) and 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 increased incidence/severity of acanthosis/hyperkeratosis in the 28-day 

dermal study. The dermal effects were considered adaptive due to the moderate degree of 

acanthosis/hyperkeratosis observed. 

There was no evidence of immunotoxicity in the rat. 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 study, including a subchronic neurotoxicity study. 

Following dermal exposure for 28-days, systemic effects including increased reticulocytes, and 
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increased cholesterol were observed, but only at a very high dose (2000 mg/kg/day, which is 

twice the highest dose required by the guidelines for a dermal study). 

No evidence of increased quantitative or qualitative susceptibility was seen in developmental 

toxicity studies in rats and rabbits. In the rat developmental toxicity study, decreased fetal 

weight was observed in the presence 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, there was no evidence of 

quantitative susceptibility; evidence for qualitative susceptibility was manifested as decreased 

pup weight, decreased spleen weight, and an increase in post-natal pup loss [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 female 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/bronchiolar adenomas and carcinomas in the lung of mid- and high-dose 

female mice in the carcinogenicity study. All genotoxicity studies that were conducted on 

fluensulfone and three of its metabolites (thiazole sulfonic acid; TSA, butene sulfonic acid; BSA, 

and methyl sulfone; MeS) were negative, except for MeS with Salmonella typhimurium strain 

TA 100 in the absence of metabolic activation during 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 strains 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-grade material resulted in low 

toxicity classifications via the oral (Toxicity Category III), dermal (Toxicity Category III), and 

inhalation (Toxicity Category IV) routes of exposure. Fluensulfone technical-grade material was 

not an irritant to eyes (Toxicity Category IV) or skin (Toxicity 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 with 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 toxicity (Toxicity Category III). HED notes 

that all animals treated with BSA or TSA survived the entire study period and that the toxicity 

category for those metabolites is based on the dosing level rather than on actual LD50 estimates. 

Furthermore in the BSA and TSA acute toxicity studies, body weights of all animals were within 

the normal range, no macroscopic findings were recorded at necropsy, and no clinical signs were 

observed except for three animals which exhibited 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-related effects were observed at any 

dose (limit-dose = 1000 mg/kg/day). Based on the available data addressing toxicity of the BSA 

and TSA metabolites, the Agency has determined that they are not of toxicological concern. 
Fluensulfone Human Health Risk Assessment DP No. D403767 

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

The toxicology and the exposure databases for fluensulfone support a reduction of the required 

10X FQPA safety factor to 1X based on the following considerations: the completeness of the 

toxicity database including adequate studies to assess the potential susceptibility in the young; 

there is no indication of quantitative susceptibility in the developmental and reproductive toxicity 

studies, and there are no residual uncertainties concerning pre- or post-natal toxicity; the 

endpoints and doses chosen for risk assessment are protective of the qualitative susceptibility 

observed in the 2-generation reproduction study; acute and subchronic 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 required. The dietary assessment is based on reliable data and 

modeled drinking water residues, and will not underestimate exposure. Finally, there are no 

registered or requested uses for fluensulfone that would result in residential exposure. 

4.4.1 Completeness of the Toxicology Database 

The toxicology database is considered complete and is adequate for the purpose of assessing preand postnatal susceptibility. Acceptable/guideline developmental toxicity studies in rats and 

rabbits and a reproduction study in rats, as well as acute and subchronic neurotoxicity studies in 

rats are available for FQPA assessment. A developmental neurotoxicity study is not required. 

4.4.2 Evidence of Neurotoxicity 

Fluensulfone primarily targets the hematopoietic system, likely by interacting with the thiol 

groups in globin as observed in the rat metabolism study. Decreased locomotor activity in 

females, and decreased spontaneous activity, decreased rearing, and impaired righting response 

in both sexes were observed in the acute neurotoxicity study at the lowest dose tested. However, 

no other evidence for neurotoxicity was observed in the other studies in the toxicity database, 

including a subchronic neurotoxicity study. The doses and endpoints chosen for risk assessment 

are all protective of the effects seen in the acute 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 effects were observed in the acute neurotoxicity 

study (100 mg/kg). A developmental 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 susceptibility was seen in developmental toxicity 

studies in rats and rabbits. Offspring effects in those studies occurred in the presence of maternal 

toxicity and were not considered more severe than the parental effects. However, there was 

evidence of increased qualitative, but not quantitative, susceptibility of pups in the 2-generation 

reproduction study in rats. Maternal effects observed in that study were decreased body weight 

and body weight gain; at the same dose, effects in offspring were decreased pup weights, 

decreased spleen weight, and increased pup loss (PND 1-4). 
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Although there is evidence of increased qualitative susceptibility in the 2-generation 

reproduction study in rats, there are no residual uncertainties 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 and 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 tolerance-equivalent residue levels of the parent 

compound in foods, 100% crop treated, and modeled estimates of residues in drinking water. All 

of the exposure estimates are based on conservative assumptions and are not likely to 

underestimate risk. Furthermore, there are no proposed residential uses. 

4.5 Toxicity Endpoint and Point of Departure Selections 

4.5.1 Dose-Response Assessment 

The toxicity endpoints and points of departure are presented 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 selected endpoints from the available data to establish points of 

departure (PODs) for the risk assessment that are protective of the observed adverse effects 

across the entire database. Two studies with effects after one or two doses are available at 

similar LOAELs: pup mortality in the 2-generation reproduction toxicity study in rats (122 

mg/kg/day) and decreased motor activity in the 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) with a NOAEL of 16.2/23.0 mg/kg/day (M/F). The majority of 

the pup deaths occurred on post-natal day 2, although there were also a number that occurred on 

post-natal day 1. Therefore, the deaths may be attributable to a single dose. Effects observed in 

the ACN in rats also support the POD for acute dietary risk assessment. In this study, decreased 

motor activity in females and decreased spontaneous 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 curve, 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 study 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, based on a NOAEL of 3.1 mg/kg/day with decreased 
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body weight, increased mean hemoglobin concentration distribution width, increased relative and 

absolute reticulocyte counts in both sexes, decreased 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 bilirubin, albumin, and A/G ratio, and pigmented Kupffer 

cells in both sexes, decreased body weight in females, and decreased 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 dose spacing differences between 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 conducted with the technical 

material. Although that study is of the appropriate 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 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 inhalation exposure, 

the POD was chosen from the 90-day inhalation toxicity study in rats. In this study, decreased 

body weight, decreased thymus weight, and histopathological changes in the nasal cavity of 

males; prolonged prothrombin time in females; and histopathological changes in the epiglottis in 

both sexes were observed at the lowest dose tested (0.04 mg/L). Since a NOAEL could not be 

identified in this study, an additional 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 described in EPA’s reference concentration (RfC) 

guidance and OPP’s guidance on the application of the RfC methodology are suited for 

calculating human-equivalent concentrations (HECs) based on the inhalation toxicity NOAEL 

for use in margin of exposure (MOE) calculations. 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 respiratory 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 exposure of an animal to the predicted inhalation 

exposure for a human. For the 90-day inhalation study with fluensulfone, an RDDR for the 

extrathoracic region (based on effects observed in the nasal cavity) 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 relative to the human. Since the rat inhalation study was carried out for 6 

hr/day for 5 days/week, the only exposure adjustment was in the assumption of 8 hrs/day for 

human occupational exposures. The resulting HEC is 0.00429 mg/L. The level of concern 

(LOC) for this scenario is 300, which includes interspecies (3X), intraspecies (10X), and 
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LOAEL-to-NOAEL (10X) extrapolations. The interspecies factor was reduced from 10X to 3X 

due to the HEC calculation accounting for pharmacokinetic (not pharmacodynamic) interspecies 

differences. 

Endpoint for incidental oral exposure were not selected because there are no residential uses of 

fluensulfone. Long-term dermal and inhalation exposures are not expected; 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 for the dermal and inhalation risk assessments are 

different, these routes of exposure should not be combined in the risk assessment. 

4.5.3 Cancer Classification and Risk 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 increased 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/carcinogenicity 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 adenomas 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 considerations: (1) the 

tumors occurred in only one sex in one species; (2) fluensulfone and its metabolites are not 

mutagenic; (3) no carcinogenic response 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-times higher than the dose 

chosen for risk assessment (3.1 mg/kg/day). 

4.5.4 Summary of Points of Departure and Toxicity 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 

UFA= 10x 

UFH=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. 
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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

Chronic 

Dietary (All 

Populations) 

NOAEL = 3.1 

mg/kg/day 

UFA= 10x 

UFH=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 because 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: Suggestive 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 point that is derived from observed dose-response data and 

used to mark the beginning of extrapolation to determine risk associated with lower environmentally relevant human 

exposures. NOAEL = no observed adverse effect level. LOAEL = lowest observed adverse effect level. UF = 

uncertainty factor. UFA = extrapolation from animal to human (interspecies). UFH = potential variation in 

sensitivity among members of the human population (intraspecies). UFL = use of a LOAEL to extrapolate a 

NOAEL. FQPA SF = FQPA Safety Factor. PAD = population adjusted dose (a = acute, c = chronic). RfD = 

reference dose. MOE = margin of exposure. LOC = level of concern. 
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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% 

UFA=10x 

UFH=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) 

UFA=3x 

UFH=10x 

UFL=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: Suggestive evidence of carcinogenicity in humans based on lung tumors in 

female mice. The RfD is protective of potential cancer effects. 

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

mark the beginning of extrapolation to determine risk associated with lower environmentally relevant human exposures. NOAEL 

= no observed adverse effect level. LOAEL = lowest observed adverse effect level. UF = uncertainty factor. UFA = 

extrapolation from animal to human (interspecies). UFH = potential variation in sensitivity among members of the human 

population (intraspecies). UFL = use of a LOAEL to extrapolate a NOAEL. MOE = margin of exposure. LOC = level of 

concern. DAF = dermal absorption factor 

bHEC = 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. 
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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 toxicity studies in rats, tooth discoloration 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 discoloration or whitening of their teeth at the mid- and high-dose 

and females had similar effects at all doses. Other studies in the fluensulfone database did not 

identify similar effects but did show increased 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 subchronic oral studies, a 90-day neurotoxicity study in rats and a 2-

generation reproduction study in rats. These newer 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 chronic studies show any discoloration effects on teeth 

or histopathological effects on teeth or bones as would be expected if there was exposure to free 

fluoride. Increased fluoride levels in teeth and bone that is found in the subchronic and chronic 

studies does not necessarily indicate exposure to free fluoride because the analytical method for 

measuring fluoride levels includes fluorine bound in fluensulfone and its metabolites in its total 

of fluoride residues. The tooth discoloration 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 another 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. Nonetheless, the overall weight of the toxicity data as an indirect 

indicator of free fluoride exposure is not substantial. The inhalation study raises some possibility 

of free fluoride exposure, but this study carries the least weight for analyzing potential residues 

from exposure to fluensulfone in or on food due to the significantly different metabolic effects 

that can result from inhalation compared to oral exposures. 

Given that the tooth discoloration or whitening might be fluorosis caused by exposure to free 

fluoride and that fluensulfone contains several fluorine atoms, EPA investigated whether 

residues of fluensulfone in food might reasonably be expected to result in exposure to finite 

residues of free fluoride. Metabolism studies in plants and animals did not show that metabolism 

of fluensulfone resulted in the release of free fluoride as a metabolite. However, these studies 

are not definitive in that not all radio-labeled material in the studies was identified. More 

conclusive is a statistical analysis of the fluoride levels in treated crops, soil, and water 

performed by the petitioner. That analysis showed no statistically different levels of fluoride 

between treated and untreated samples. The indirect evidence suggesting the possibility of 

fluensulfone resulting in free fluoride exposure seen in the toxicity studies does not outweigh this 

direct examination of fluoride residues in food. Considering all of the direct and indirect 

information bearing on the potential for free fluoride exposure, and relying particularly on the 

statistical analysis of fluoride residues in crops, soil, and water, HED concludes that there is no 

reasonable expectation of exposure to finite residues of free fluoride resulting from exposure to 

fluensulfone residues.
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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 concern for the currently proposed uses. The studies 

were conducted at approximately a 1X application rate but with a shorter pre-plant interval (0 

days) than specified on the label (7 days). Mature and immature lettuce and potatoes and mature 

tomatoes were harvested and analyzed for residues. The only major residues in the harvested 

matrices were the BSA and TSA metabolites and, 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 the residue profile in the harvested 

crops. 

Rotational crop studies were conducted with radish, lettuce, and wheat 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 planting season. In the rotational 

crop studies, a low level 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 BSA were not quantifiable after the 120-day plant-back interval 

whereas residues of TSA persisted at quantifiable levels for at least one year. 

Studies with laying hens and lactating goats showed uptake and subsequent elimination of 

radiolabeled fluensulfone. In hens, radioactive 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. Radioactive residues in eggs were not identified; 

most residues were aqueous phases in the extraction schemes. In liver, 20-30% of the 

radioactivity was associated with proteins and/or amino acids and approximately 3% was 

identified as TSA. Fluensulfone was a major 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 identified residues were trifluorobutene 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 glucose, lactose, proteins/amino acids, and 

triglycerides/fatty acids and the radioactivity appears to be due to incorporation of the 

radiolabeled carbon. 
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5.1.2 Summary of Environmental Degradation 

In environmental fate studies, fluensulfone showed different degradation outcomes depending on 

the environmental system. Fluensulfone is stable to hydrolysis under environmental conditions 

(pH 4-9) but is prone to photolysis (t1/2 of 0.6-0.7 days in water and t1/2 of 39 days in soil). In 

soils, the rate of degradation is dependent on the aerobic state of the system and some parent 

compound persisted in both the aerobic and anaerobic soil metabolism studies. In an aerobic soil 

metabolism study, major degradation products were BSA, TSA, and MeS. In an anaerobic soil, 

fluensulfone degraded slowly (t1/2 of 397 days) and the major degradates were BSA and TSA. In 

aquatic systems, the time to 50% dissipation (DT50) was less dependent on the aerobic state, with 

estimates of 39-42 days under aerobic conditions and 26-28 days under anaerobic conditions. 

Under anaerobic conditions, the major degradates were deschloro-fluensulfone, MeS, BSA, and 

trifluorobutene sulfinic acid. DT50 estimates for the TSA and MeS metabolites under aerobic 

soil conditions are 421 and 33 days, respectively. In field dissipation studies, DT50 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 after application for BSA and TSA and 28 days after 

application for MeS. 

Fluensulfone is classified as “moderately mobile” 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 different metabolic outcomes following 

application to environmental systems in comparison 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, deschloro-fluensulfone, deschloro-thiazole sulfonic acid, and 

trifluorobutene sulfinic acid. 

In rat, poultry, and ruminant studies, 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. The major residues in at least one animal matrix 

were fluensulfone (poultry fat), MeS (ruminant urine), trifluorobutene sulfinic acid (rat and 

ruminant urine), thiomercapturate (rat urine), and thiazole glucaronides I and II (rat urine). 

The commonality of the residues in the target crop, rotational crop, and soil 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 data 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 Subcommittee (ROCKS) has recommended that 

enforcement for residues of fluensulfone be determined by analysis of the BSA metabolite only 
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and that risk assessment be based on parent compound as well as MeS and deschlorofluensulfone, with the latter two for drinking water only (Table 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 Assessment 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-ene-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 metabolism studies. At levels 

expected in foods, the TSA and BSA metabolites are considered to be relatively non-toxic. 

Enforcement of tolerances should be via measurement of residues of BSA only. The parent 

compound alone is a poor indicator of misuse due to the lack of quantifiable residues. The 

metabolites TSA and BSA both appear at quantifiable levels in the crop field trials and are good 

candidates for marker compounds. The TSA metabolite 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 indicator of misuse given its 

persistence. For tolerance enforcement, residues of BSA, expressed 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 deschloro-fluensulfone in the risk assessment. Although there 

are several major metabolites, acute toxicity data for the sulfonic acid metabolites (TSA and 

BSA) and MeS indicate that the acids, specifically TSA and BSA, are significantly less toxic 

than the parent fluensulfone and that MeS has 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 significantly increases the likelihood of rapid body 
Fluensulfone Human Health Risk Assessment DP No. D403767 

Page 24 of 107 

excretion. First, the sulfonic acid moiety significantly decreases lipophilicity relative to the 

parent chemical and thus the likelihood for bioaccumulation (predicted Log P = -1.06 versus 1.96 

for the parent chemical). Second, the sulfonic acid functional group is predicted 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 metabolites being detoxification products. In contrast, 

MeS and deschloro-fluensulfone are expected to be more similar to parent fluensulfone in terms 

of excretion by the body. Therefore, only fluensulfone, deschloro-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, establishing 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 fruiting-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 result from application of fluensulfone according to the proposed 

use patterns, and the residue data are supported by adequate data from analytical method 

validation and storage stability studies. For regulatory enforcement, analysis of residues can be 

accomplished through standard analytical techniques, and residues do not show any trends for 

dissipation during frozen storage. 

Quantifiable residues of parent fluensulfone are not expected from application patterns addressed 

by the field trials, including the pre-plant application listed on the proposed label, as well as preplant + post-transplant applications. In biological environments, fluensulfone undergoes rather 

rapid cleavage at the sulfonyl moiety via glutathione conjugation to yield the BSA and TSA 

breakdown products. This, combined with its early season use profile, results in the nearly 

complete absence of parent compound in any residue studies to date. Quantifiable residues of 

the metabolites BSA and TSA are likely to occur and appear at similar levels across all of the 

examined crops (Appendix D); although there is a rather high degree 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 higher residues than application by 

drip irrigation; however, there are too few data points from the drip irrigation trials for a 

meaningful statistical comparison. In melon, residues of BSA 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 uses have higher application rates, limited field 

rotational crop data may be required. Also, if there are future uses on crops that are significant 

livestock feedstuffs, then livestock feeding studies may be necessary. 

In the submitted tomato processing study, all analytes (fluensulfone, BSA, TSA, MeS) were 

<0.01 ppm in all commodities. As a result, the residue data cannot be used to derive processing 
Fluensulfone Human Health Risk Assessment DP No. D403767 

Page 25 of 107 

factors for tomato commodities. In a separate study provided by the Australian extension of 

Makhteshim-Agan (Farmoz), quantifiable residues of BSA occurred in the raw commodity 

making it possible to calculate the following processing factors for tomato. Typically, HED 

makes recommendations regarding tolerances for residues 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 the 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 analyses 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 analyses 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 (location 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 (EFED) provided HED with modeled estimates of 

fluensulfone residues in drinking water (D411774, J. Lin, 1 August 2013 and D417634, J. Lin, 

30 January 2014). The initial estimates (D411774) 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-GROW). Of those estimates, the highest 

estimated drinking water concentrations (EDWCs) were from PRZM-GW and resulted in dietary 

risk estimates of concern. Refined ground water EDWCs (D417634) were 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 and, 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-EXAMS; D411774) 3.57 11.8 0.173 

Ground water (Tier I: PRZM-GW; D417634) 3.57 77.6 52.5 
Fluensulfone Human Health Risk 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 fluensulfone, 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 + drinking water) and risk estimates were 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, youth 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 acute 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 the “all infants” population subgroup and occupied 7.4% of the aPAD 

and 9.5% of the cPAD. 

Fluensulfone is classified as having “suggestive evidence of carcinogenic potential.” Risk 

assessments based on the chronic RfD are considered 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 (95th 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 fluensulfone that are defined as residential uses; 

therefore, a quantitative residential exposure assessment was not conducted. 
Fluensulfone Human Health Risk Assessment DP No. D403767 

Page 27 of 107 

6.1 Residential Bystander Post-Application Inhalation Exposure 

Volatilization of pesticides may be a source of post-application inhalation exposure to 

individuals nearby pesticide applications. The agency sought expert advice and input on issues 

related to volatilization of pesticides from its Federal Insecticide, Fungicide, and Rodenticide Act 

Scientific Advisory Panel (SAP) in December 2009, and received the SAP’s final report on 

March 2, 2010 (http://www.epa.gov/scipoly/SAP/meetings/2009/120109meeting.html). The 

agency has evaluated the SAP report and has developed a Volatilization Screening Tool and a 

subsequent Volatilization Screening Analysis 

(http://www.regulations.gov/#!docketDetail;D=EPA-HQ-OPP-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. 

6.2 Spray Drift 

Spray drift is a potential source of exposure to those nearby pesticide applications. This is 

particularly the case with aerial application, but, to a lesser extent, spray drift can also be a 

potential source of exposure from the ground 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 developed as a result of a Data Call-In issued by 

EPA), EPA Regional Offices and State Lead Agencies for pesticide regulation and other parties 

to develop the best spray drift management practices (see the agency’s Spray Drift website for 

more information). The agency has also developed 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 pesticide will be considered concurrently. The 

approach is outlined in the revised (2012) Standard Operating Procedures for Residential Risk 

Assessment (SOPs) - Residential Exposure Assessment Standard Operating Procedures Addenda 

1: Consideration of Spray Drift. This document outlines the quantification 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 the risks themselves can be aggregated. When 

aggregating exposures and risks from various sources, HED considers both the route and 

duration of exposure. 

As previously noted, there are no residential uses for fluensulfone at this time; therefore, dietary 

(food and water) exposure is the only pathway appropriate for assessment. Aggregate risk 

estimates are equivalent to the dietary risk estimates discussed in Section 5 and are below HED’s 

level of concern. 

8.0 Cumulative Exposure/Risk Characterization 
Fluensulfone Human Health Risk Assessment DP No. D403767 

Page 28 of 107 

Unlike other pesticides for which EPA has followed 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 appear 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 the cumulative effects of such chemicals, see the 

policy statements released by EPA’s Office of Pesticide Programs concerning common 

mechanism determinations and procedures for cumulating effects from substances found to have 

a common mechanism on EPA’s website at http://www.epa.gov/pesticides/cumulative/. 

9.0 Occupational Exposure/Risk Characterization 

Figueroa, Z. 18 July 2014, D403814 

There is a potential for occupational exposure associated with handler activities (i.e., mixing, 

loading, and applying). Based on the use pattern 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-term (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 other 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 assumptions 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 proposed labels range from 1.75-3.50 lb a.i./A, and 

were used to assess the handler exposure scenarios. Refer to Table 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 the same for dermal and inhalation exposure. 

Therefore, the quantitative short-term handler assessment represents both exposure durations and 

is protective of intermediate-term exposure. All inhalation occupational handler exposure risk 

estimates were not of concern (MOEs > 300) using some level of personal protective equipment 

(PPE; i.e., no respirator or PF5 respirator). Furthermore, dermal risk estimates were also not of 

concern when using some level of PPE [e.g., chemical-resistant gloves, engineering-controls 

(enclosed-systems)] with one exception. The mixing/loading liquids for chemigation scenario 

resulted in a potential dermal risk of concern (MOE=60) using engineering-controls (Table 9.1). 

The maximum single application rate to achieve an MOE of 100 for mixing/loading liquids for 
Fluensulfone Human Health Risk Assessment DP No. D403767 

Page 29 of 107 

chemigation is 2.0 lb a.i./acre using engineering controls as level of mitigation. At the request of 

the registration division, Table 9.1 includes an application rate of 2.5 lb ai/A in addition to the 

minimum and maximum labeled use rates. 

There are handler dermal and inhalation risks of concern at the label-specified PPE (i.e., longsleeved shirt, long pants, socks, shoes, and chemical-resistant gloves). Additional PPE (i.e., PF5 

respirator) and levels of mitigation (i.e., engineering 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 Intermediate-Term Occupational Handler Non-Cancer Exposure and Risk Estimates for Fluensulfone.

Exposure 

Scenario 

Mitigation 

Levela

Unit Exposureb

(μg/lb ai) 

Application 

Ratec 

Area 

Treatedd

Dermal Inhalation 

Dermal Inhalation Dosee

(mg/kg/day)

MOEf Doseg

(mg/kg/day)

MOEh

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 Dermal 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) / Dermal 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/dayM/L or 0.244 mg/kg/dayA) / Inhalation Daily Dose (mg/kg/day). LOC = 300. 
Fluensulfone Human Health Risk Assessment DP No. D403767 

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9.2 Short- and Intermediate-Term Post-Application Risk 

Inhalation 

There are multiple potential sources of post-application inhalation exposure to individuals 

performing post-application activities in previously treated fields. These potential sources 

include volatilization of pesticides and resuspension of dusts and/or particulates that contain 

pesticides. The agency sought expert advice and input on issues related to volatilization of 

pesticides from its Federal Insecticide, Fungicide, and Rodenticide Act Scientific Advisory Panel 

(SAP) in December 2009, and received the SAP’s final report on March 2, 2010 

(http://www.epa.gov/scipoly/SAP/meetings/2009/120109meeting.html). The agency has 

evaluated the SAP report and has developed a Volatilization 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 evaluate the available post-application inhalation 

exposure data generated by the Agricultural 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 exposure into the agency's risk assessments. 

Although a quantitative occupational post-application inhalation exposure assessment was not 

performed, an inhalation exposure assessment was performed for occupational/commercial 

handlers. Handler exposure resulting from application of pesticides outdoors is likely to result in 

higher exposure than post-application exposure. Therefore, it is expected that these handler 

inhalation exposure estimates would be protective of most occupational post-application 

inhalation exposure scenarios. 

Dermal 

Occupational quantitative dermal post-application risk estimates for agricultural workers 

following treatments to agricultural crops were not assessed because fluensulfone is applied 

directly to the soil as a pre-plant/pre-emergent broadcast 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 bearing residues is minimal). 

Furthermore, there is a potential for workers to contact treated soil during mechanically assisted 

cucurbit and fruiting vegetables transplanting activities. However, this event is likely to result in 

negligible exposures because transplanting 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, which 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 punched in the tops of the raised beds. This 

agricultural machinery also minimize the contact potential with treated soil. Even though 
Fluensulfone Human Health Risk Assessment DP No. D403767 

Page 31 of 107 

fluensulfone is stable under relevant laboratory conditions, the parent compound has half-life of 

approximately 10 days in field dissipation studies, which further decreases the potential for 

occupational exposures with treated soil. These conclusions are appropriate for the proposed 

use-pattern of fluensulfone. Therefore, dermal post-application exposures are considered 

negligible and a quantitative dermal post-application exposure assessment was not conducted. 

Restricted Entry Interval 

Fluensulfone is classified as Toxicity Category III via the dermal route and Toxicity Category IV 

for skin and eye irritation potential. It is a skin sensitizer. Since a quantitative dermal postapplication exposure assessment was not conducted, the REI specified on the proposed label is 

based on the acute toxicity of fluensulfone. 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 Protection 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 for Use of the New Active Ingredient on 

Fruiting Vegetables (Crop Group 8-10) and Cucurbit Vegetables (Crop Group 9). 

Summary of Analytical Chemistry and Residue Data. 

Doherty, M. 5 June 2014, D418204. Fluensuflone Acute and Chronic Aggregate Dietary (Food 

and Drinking Water) Exposure and Risk Assessments for the First Food Use Section 3 

Registration Action on Fruiting and Cucurbit Vegetables. 

Figueroa, Z. 18 July 2014, D403814. Fluensulfone. Occupational and Residential Exposure 

and Risk Assessment to Support the Registration 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. Fluensulfone: Refined Drinking Water Exposure 

Assessment for Proposed New Registration for Uses on Cucurbits and Fruiting 

Vegetables Based on Ground Water Sources. 

Kitchens, B. 2014, Fluensulfone OECD Monograph 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 Risk Assessment DP No. D403767 

Page 32 of 107 

Appendix A. Toxicology Profile 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 

yes 
Fluensulfone Human Health Risk 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 LD50 = 671 III 

870.1100 Acute oral - rat 48574746 LD50 > 300 mg/kg 

but < 2000 mg/kg 

III 

870.1200 Acute dermal - rat 48575035 LD50 > 2000 mg/kg III 

870.1300 Acute inhalation - rat 48574748 LC50 > 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 - Fluensulfone EC480 Formulation

Guideline No. Study Type MRID(s) Results Toxicity Category 

870.1100 Acute oral - rat 48574924 LD50 > 2000 mg/kg III 

870.1100 Acute oral - rat 48574918 LD50 > 300 mg/kg 

but < 2000 mg/kg 

III 

870.1200 Acute dermal - rat 48574962 LD50 > 2000 mg/kg III 

870.1300 Acute inhalation - rat 48574920 LC50 > 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 Profile - Fluensulfone Metabolites

Metabolite Study Type MRID(s) Results Toxicity Category 

TSA Acute oral - rat 48574783 LD50 > 2000 mg/kg III 

MeS Acute oral - rat 48574787 LD50 > 300 mg/kg 

but < 2000 mg/kg 

II 

BSA Acute oral - rat 48574792 LC50 > 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 Risk 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): 50 
Fluensulfone Human Health Risk 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-

generation 

reproductionrat 

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 in 
Fluensulfone Human Health Risk 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 Negative 
Fluensulfone Human Health Risk 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 activation 
Fluensulfone Human Health Risk 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 Negative 
Fluensulfone Human Health Risk 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 Negative 
Fluensulfone Human Health Risk 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-Acute 

neurotoxicityrat 

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-day 

neurotoxicityrat 

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 

([thiazole14C]MCW-2 

and [butene14C]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 ( 

[butene14C]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 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 (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 thiazole mercapturate. 

870.7485 2010-

pharmacokineti

cs, single oralmg/kg/day = 

5 and 500 

48574741 The absorption of fluensulfone-derived 14C 

was rapid at both doses. The kinetic 

parameters for males and females were 
Fluensulfone Human Health Risk Assessment DP No. D403767 

Page 41 of 107 

Toxicity Profile - Fluensulfone 

Guideline Study Type Doses Tested/Study 

Classification 

MRID Results 

rat ([thiazole14C]MCW-2 

and [butene14C]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 

([thiazole14C]MCW-2 

and [butene14C]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 Cmax 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 Cmax

compared to the DT90 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 Cmax compared to DT90 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 [14C] 

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/cm2 = 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 the 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 Risk 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 or 

cytosolic 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/cm2 = 

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 Risk Assessment DP No. D403767 

Page 43 of 107 

Toxicity Profile - Fluensulfone 

Guideline Study Type Doses Tested/Study 

Classification 

MRID Results 

(MCW-2 

[Ring-U-14C]) 

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 Risk Assessment DP No. D403767 

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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 reproductive 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 extrapolation, 10x intraspecies variability, 1x FQPA 

Safety Factor) 

 Acute RfD = 

100 ( )

16 .2 / /

UF

mg kg day = 0.16 mg/kg/day 

Comments about Study/Endpoint/Uncertainty Factor: 

The endpoint of concern in this study is protective 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 consideration 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 decreased body weights at the LOAEL of 16 mg/kg/day 

observed in both the chronic and subchronic studies. The higher NOAEL from the chronic dog 

study was used as the point of departure since the differences in doses are an artifact of dose 

selection. 

Uncertainty Factor: 100x (10x interspecies extrapolation, 10x intraspecies variability) 
Fluensulfone Human Health Risk Assessment DP No. D403767 

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 Chronic RfD = 

100 ( )

3.1 / /

UF

mg kg day = 0.03 mg/kg/day 

Comments about Study/Endpoint/Uncertainty Factor: The endpoint of concern in the chronic 

and subchronic studies is protective of all populations and appropriate for the exposure duration 

of concern. The 90-day and chronic dog toxicity studies exhibit similar toxic effects occurring at 

the same LOAEL (16-17 mg/kg/day) with no progression in toxicity over time. The NOAEL of 

3.1 mg/kg is also protective of the effects observed 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 exposure 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 data demonstrated an inverse relationship between 

dosing concentration and percent 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 absorption values adjusted 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/cm2

; 

(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 absorbed 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 decreased body weights at the LOAEL of 16 mg/kg/day 
Fluensulfone Human Health Risk Assessment DP No. D403767 

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observed in both the chronic and sub-chronic studies. The higher NOAEL from the chronic dog 

study was used as the point of departure since the differences in doses are an artifact of dose 

selection. 

Uncertainty Factor: 100x (10x interspecies extrapolation, 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 appropriate for short- and intermediate-term 

exposures because of the timing of the effects (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 conducted with the technical 

material. Although that study is of the appropriate 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 toxicity 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 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 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 toxicity 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 females, 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 Risk Assessment DP No. D403767 

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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 equivalent concentration in mg/L (0.00429); CF 

= conversion factor for breathing rate in L/hr-kg bw (11.8); AF = activity factor specific to the 

exposure scenario (0.6, 1.2, and 2.1 for low, medium, 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 intraspecies 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 the only study available of the appropriate route and duration. An 

additional uncertainty factor of 10x for the extrapolation 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 (SPF) 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 and 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 the 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 commonly 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 tech. was calculated to be 300 mg/kg bw < LD50 (female 

rat) < 2000 mg/kg bw (LD50 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 oral 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 for acute toxicity endpoints, the test material is considered to be 

of low acute oral toxicity.
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870.1100 Acute Oral Toxicity –TGAI (2010) 

In an acute oral toxicity study performed according to the Acute 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 animals 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 animals treated at 1500 mg/kg 

bw. One animal treated at 2000 mg/kg bw as well 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 was recorded at necropsy of either decedent 

or surviving rats. 

Using the Probit analysis according to Finney, the oral LD50 of fluensulfone was calculated to be 

671 mg/kg bw. 

According to the acute toxicity classification criteria for the PMRA, MCW-2 tech. is moderately 

acutely toxic via the oral route based on the LD50 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 classification criteria, MCW-2 tech. falls under Category III 

for acute oral toxicity (signal word “Caution”). Under OCS criteria for acute 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 class 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 LD50 Females: 300 mg/kg bw < LD50 < 2000 mg/kg bw (LD50 cut off = 1000 mg/kg bw) 

According to the acute toxicity classification criteria for the PMRA, MCW-2 480 EC is of Slight 

Toxicity via the oral route of exposure based on the LD50 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 toxicity endpoints, the test material 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 had to be killed in extremis 28 hours following 

dosing. All other animals in this dose group survived to study termination. Clinical signs of 

toxicity observed in some or all of the animals in the high dose group not surviving to study 

termination include: poor coordination, ventral recumbancy, colourless urine, cloudy eyes, 

hypothermia, yellowish discoloured ears, cyanosis, and no reflexes. Clinical signs of toxicity 
Fluensulfone Human Health Risk Assessment DP No. D403767 

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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, including: 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 noted on body weight and no treatment related 

macroscopic observations. 

This acute oral study is classified 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 according to the Acute Toxic Class method, one group 

of three, followed by another group of three female 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 severe clinical signs on test day 1 including swaying gait, dragging 

of front and/or rear limbs, decreased activity, ruffled 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 recorded for rats of the strain and age used in 

this study. 

No macroscopic findings were noted at necropsy. 

The LD50 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 (SPF) rats, were treated with fluensulfone 

metabolite thiazole sulfonic acid (MCW-2 metabolite #3625) by oral gavage at a dosage of 2000 

mg/kg bw. Purified water was used as the vehicle and dosing volume was 10 mL/kg bw. 

All animals survived until the end of the study period. No clinical signs were observed during the 

course of the study with the exception of 3 animals, in which slightly ruffled fur was observed 2 

hours after administration, persisting up to the 3-hour observation in 2 animals. 
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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 macroscopic findings were 

recorded at necropsy. 

The oral LD50 of thiazole sulfonic acid was greater than 2000 mg/kg bw. Based on PMRA’s 

acute toxicity classification system, thiazole sulfonic acid (metabolite #3625) is considered to be 

of low acute toxicity via the oral route. Based on the EPA classification criteria, thiazole sulfonic 

acid falls under Category III for acute oral toxicity. Under OCS criteria for acute toxicity 

endpoints, thiazole sulfonic acid is considered to be of low acute oral toxicity. 

The acute oral toxicity of fluensulfone metabolite #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 (SPF) 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 overt 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 included swaying gait, sedation, 

hunched posture, labored breathing, tightly shut eyes 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 gained 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 the 6 animals dosed at 300 mg/kg bw. 

The acute median lethal dose of methyl sulfone was 300 mg/kg bw < LD50 < 2000 mg/kg bw 

(LD50 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 criteria, methyl sulfone falls under Category II for acute oral 

toxicity. Under OCS criteria for acute toxicity endpoints, methyl sulfone is considered to be of 

low to moderate acute oral toxicity. 

The acute oral toxicity of fluensulfone metabolite #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 (SPF) rats, were treated with fluensulfone 

metabolite butene sulfonic acid (MCW-2 metabolite #3627) by oral gavage at a dosage of 2000 

mg/kg bw. Purified water was used as the vehicle and dosing volume was 10 mL/kg bw. 

All animals survived until the end of the study period. No clinical signs were observed during the 

course of the study. The body weight of the animals was within the range commonly recorded 
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for rats of this strain and age (all animals gained weight over the course of the study) and no 

macroscopic findings were recorded at necropsy. 

The LD50 of butene sulfonic acid was greater than 2000 mg/kg bw. Based on PMRA’s acute 

toxicity classification system, butene sulfonic acid (metabolite #3627) is considered to be of low 

acute toxicity via the oral route. Based on the EPA classification criteria, butene sulfonic acid 

falls under Category III for acute oral 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 metabolite #3627 is lower than that of fluensulfone.

870.1200 Acute Dermal Toxicity – TGAI 

In an acute dermal toxicity study, 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 study, 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. Otherwise, the body weights of the 

animals were within the range commonly recorded for rats of this strain and age. No 

macroscopic findings were observed at necropsy. 

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

According to the acute toxicity classification criteria for PMRA, MCW-2 tech. (fluensulfone) is 

of low acute toxicity via the dermal route (based on the dermal LD50 value for males and 

females). Based on the EPA classification criteria, MCW-2 tech. falls under Category III for 

acute dermal toxicity (signal word “Caution”). Under OCS criteria for acute 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 five female RccHan:WIST (SPF) rats were treated 

with MCW-2 480 EC at 2000 mg/kg bw by dermal 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 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. 

There were no deaths, and no clinical signs were observed. Very slight to well-defined erythema 

was observed at the application site 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 was within the range commonly recorded for rats 

of the strain and age used in the study. No abnormality was detected at necropsy. 
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The dermal LD50 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 labeling under GHS CLP. 

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

‘Caution’. According to the acute toxicity classification criteria for PMRA, MCW-2 480 EC is of 

low acute toxicity via the dermal route (based on the dermal LD50 value for males and females). 

Under OCS criteria for acute toxicity 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 during 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 were 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 were noted at necropsy. 

The 4-hour LC50 of fluensulfone was estimated to be greater than 5.1 mg/L air (chemically 

determined mean aerosol concentration). 

According to the acute toxicity classification criteria for the PMRA, MCW-2 tech. (fluensulfone) 

is of low acute toxicity via the inhalation route (based on the LC50 value in male and female rats). 

Based on the EPA classification system, MCW-2 tech. falls under Category IV for acute inhalation 

toxicity. Under OCS criteria for acute toxicity endpoints, the test material 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 concentration of 6.0 mg/L air (MMAD = 1.8 μm, 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 the study. Salivation and ruffled fur were observed in all animals 

during or after exposure. Decreased activity was recorded in all males and two females at the end 

of the exposure period and/or one hour after exposure. Rales were recorded in two males one 

hour after the end of exposure. There were no clinical signs in any animals 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 female failed to regain the initial body weight by 

study termination. 
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No abnormality was detected at necropsy. 

The acute inhalation LC50 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 labeling 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 inhalation route based on the LC50 value in male and female rats. 

Under OCS criteria for acute toxicity 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 fluensulfone (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 any 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. (fluensulfone) is considered non-irritating to the 

rabbit eye according to the PMRA’s classification scheme, and falls under Category IV for 

primary eye irritation according to the EPA’s classification criteria. Under OCS criteria for acute 

toxicity endpoints, the test material 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. Animals were observed for 10 days. Eye reactions were 

assessed according to the Draize scale. 

Slight to marked reddening and chemosis of the conjunctivae, and 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 post dose. This animal also had delayed/reduced 

light reflex at the 24 hour observation point. One animal showed transient corneal opacity 

affecting less than one quarter of the area at 48 and 72 hours post treatment. All scores were zero 
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by 7 days post treatment, except for slight reddening 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 corresponds to the following label signal words and 

hazard statement: “Warning – Eye Irritant” for the Canadian label. 

According to the US EPA criteria, the test material would be assigned to category III, with signal 

word “Caution.” 

Under OCS criteria for acute toxicity endpoints, MCW-2 480 EC is considered to be a moderate 

irritant to the eye of rabbits. 

This study is classified as acceptable (fully reliable). This study satisfies the guideline 

requirement for a primary eye irritation study (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 rabbits. Scoring of irritation effects was 

performed approximately 1, 24, 48 and 72 hours, as well as 7, 10 and 14 days after instillation. 

The instillation of MCW-2 480 EC into the eye resulted in slight corneal opacity, slight to 

moderate reddening and slight to marked chemosis 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 days (2 animals) or 14 days (the 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 treatment. No abnormal findings were observed for the 

iris light reflex and no corrosion was observed at any of the examinations. No staining of the 

treated eyes by the test item was observed. No clinical signs 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: causes serious eye irritation) under GHS CLP. 

As corneal effects had resolved, and no positive grades 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 signal words and hazard statement: “Warning – Eye 

Irritant” for the Canadian label. 

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

870.2500 Primary Dermal Irritation – TGAI 

In a primary skin irritation study, 0.5 g of fluensulfone (MCW-2 tech., 96.78% a.i.) moistened 

with ~0.5 mL purified water was applied to the intact left flank 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 remaining test substance. The mean score was 

calculated across 3 scoring times (24, 48 and 72 hours 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, respectively 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 slight signs of irritation, such as erythema. This effect was reversible and 

was no longer evident 7 days after treatment, the end of the observation period for all animals. 

The test item caused no staining of the treated 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) 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 classification criteria, MCW-2 tech. falls under 

Category IV for primary skin irritation. Under OCS criteria for acute toxicity endpoints, the test 

material is considered to be a slight 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 cm2

 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 corrosive effects were noted on the treated 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 determined to be 3.4. The Maximum Irritation Score 

(MIS) was calculated to be 3.7, which was 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 following label signal words and hazard statement: 

“Warning – Skin Irritant” on the Canadian label. According to the US EPA criteria, the test 
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material would be assigned to category IV. Under OCS criteria for acute toxicity endpoints, 

MCW-2 480 EC is considered to be a moderate irritant to the skin of rabbits. 

This study is classified as acceptable (fully reliable). 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 study, 0.5 mL of undiluted MCW-2 480 EC was applied to the intact 

left flank of each of three young female New Zealand 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 erythema 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. All skin reactions had reverted 14 days after 

treatment, the end of the observation period for all animals. 

The test item did not cause any staining of the treated 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 statement: “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 according 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, following 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 intradermally with PEG 300 and FCA/physiological 

saline and epidermally with PEG 300 under occlusion. Two weeks after epidermal induction, the 

test animals were challenged by epidermal application 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 and 48 hours after removal of the dressing. 
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Ten and 6 out of 10 test animals (100% and 60%, respectively) showed discrete/patchy erythema 

at 24 and 48 hours, respectively after the challenge treatment with fluensulfone at 75% in PEG 

300. Two of 5 control animals (40%) showed the 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 the site treated with only PEG 300. 

Based on interpretation of challenge reactions, fluensulfone technicalwas considered to be a skin 

sensitizer in the guinea pig. 

Under OCS criteria for acute toxicity endpoint, the test material is considered 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 according to the Maximization method, 10 Albino 

Dunkin Hartley [CRL:(HA)BR, SPF] guinea pigs were induced by intradermal administration of 

MCW-2 480 EC at 15% in purified water and in parallel in an emulsion of Freund's Complete 

Adjuvant (FCA)/physiological saline, followed eight days later by an epidermal exposure for 48 

hours under occlusion with the undiluted test substance. The 5 animals 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 purified water and purified 

water alone under occlusive dressing. Control animals 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 any of the control animals. MCW-2 480 EC was 

considered to be a potential dermal sensitizer. 

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

CLP. 

Under OCS criteria for acute toxicity 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 administered 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 evaluation of clinical signs and a 

functional observational battery (FOB) were performed 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/group were perfusion-fixed in situ. Selected nervous system tissues 

were dissected, processed and examined histologically. 
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One female dosed at 1200 mg/kg bw died within 24 hours after dose administration of an 

unknown cause. 

Administration of a single oral gavage dose of fluensulfone to Wistar 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 response, and salivation were observed only on day 1 (the day of 

dosing). Reduced activity and decreased rearing activity 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 rearing 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 females included reductions for total distance, 

center time and rearing activity. At 1 to 4 hours 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. Food 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 relative 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 females, based on reduced locomotor activity in 

females, and decreased spontaneous activity, decreased rearing and impaired righting response in 

both sexes at 100 mg/g bw. A NOAEL was not identified 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 acute 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, MCW-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 consumption was slightly increased at 2000 ppm in 

females. Body weight loss was observed at 2000 ppm in the first week of treatment in both 

sexes. At the end of treatment the mean body weight difference from the controls was -13% in 

males at 2000 ppm. 
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Effects on red blood cell parameters were detected at 2000 ppm in both sexes, and were 

generally more pronounced (and statistically significant) in females (increases of erythrocyte and 

reticulocyte counts, concentration of hemoglobin, hematocrit values). Platelets were reduced in 

males at 500 ppm (13%; not statistically significant) 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 correlates of the observed microscopic and organ 

weight changes in the liver. Very slight increases in the activity of APh were 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 findings 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 bile 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 response. 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). The LOAEL for both sexes is 500 ppm (101 and 155 

mg/kg bw/day in male and female mice, respectively), based on decreased food consumption in 

both sexes and decreased platelets in males; based on the marginal changes at this 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 mouse (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 approximately four weeks. In addition to standard 

investigations, the clinical chemistry assessment included a determination of thyroid hormones 

(triiodothyronine, thyroxine, and thyroid stimulating hormone), liver samples were processed for 

enzyme determination, and immunotoxicity investigations were conducted. Clinical 

examinations included evaluation of sensory reactivity to stimuli, open field assessments and 

grip strength at the end of Week 4. 

No mortality occurred. No treatment-related clinical signs were noted during the study and no 

differences between the treated groups and controls were noted either during the weekly open 

field inspections or the sensory reactivity to stimuli of different types and assessment of grip 
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strength conducted during week 4. Mean food consumption was 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%) male 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 considered 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 mean corpuscular hemoglobin concentration (MCHC) was also 

observed in high dose females. Clinical chemistry revealed dose-related decreases in the activity 

of alanine aminotransferase (ALAT) in all treatment 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. Thyroxine (T4) was slightly decreased in males 

(12%) and females (17%) receiving 2000 ppm of the 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 protein was increased only slightly (6%) in high dose 

males. Urinalysis did not indicate test compound-related effects. 

Analysis of organ weight data showed statistically significant increases in mean relative liver and 

kidney weights in male rats treated at 2000 ppm, and relative spleen weight in females of the 

same group. Absolute and relative prostate weights 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 animals). Histopathology revealed increased severity of basophilic 

tubules as well as degeneration and hyaline inclusions in the proximal tubules in mid and high 

dose males. At 2000 ppm focal tubular necrosis was 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 well 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 that 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 hydrolase (EH) was induced in all male dose 

groups and mid and high dose females. Glucuronyl transferase (GLU-T) levels 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 proliferation examinations gave no indication of testcompound related alterations regarding this endpoint. 
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Immunotoxicity examinations showed no substance 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 females but a dose response was not observed. 

The NOAEL of the study is set at 125 ppm (equivalent to 10.4 and 12.2 mg/kg bw/day in male 

and female rats, respectively). The LOAEL in this study is considered to be 500 ppm (equivalent 

to 42.5 and 37.2 mg/kg bw/day in male and female 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 study in the rat (OECD 407; OPPTS 870.3050). 

870.3100 90-day Oral –Mouse

In a 90-day dietary toxicity study (MRID 48574753; PMRA 2181205), groups of 12 male and 12 

female Crl:CD1 (ICR)BR mice received diets containing 0, 60, 300 and 1500 ppm (equivalent to 

0/0, 11/18, 51/68 and 228/252 mg/kg bw/day in males/females, respectively) of fluensulfone 

(BYI 01921 technical, 97.0-97.8%, Batch No. NLL6692-7-5) 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 also 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% below controls; g/animal/day) 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 significantly 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 weights 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 increased 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 concentrations 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 activities of aspartate aminotransferase, alanine aminotransferase 

and alkaline phosphatase were increased at 1500 ppm in both sexes (55-107%), often attaining 

statistical significance. 
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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 hepatocellular hypertrophy, accompanied by degenerative 

changes mainly of the periportal hepatocytes, minimal to slight pigment deposits, bile duct 

hyperplasia (observed in most animals) and proliferation of undifferentiated oval cells, and 

slightly reduced lipid content 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 the red pulp was recorded 

in both sexes at 1500 ppm. 

Determinations in liver tissues in satellite animals sacrificed after 28 days of treatment showed 

no evidence for toxicologically significant effects on the concentration of cytochrome P450 at 

1500 ppm. In male mice only the monooxygenase 7-ethoxycoumarin deethylase was marginally 

increased. The phase II enzymes epoxide hydrolase and glutathione-S-transferase were 

marginally increased, whereas UDP-glucuronyltransferase was not affected. 

In female mice the monooxygenases were not significantly affected, whereas all phase II 

enzymes (epoxide hydrolase, glutathione-S-transferase, 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 toxicity study (MRID 48574754, PMRA 2181206), groups of 10 male and 10 

female Wistar rats (Hsd Cpb:WU) received 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 the 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 maintained without treatment for a recovery period of 28 days. 

Along with the standard guideline evaluations, all groups of animals were assessed for changes 

in functional observational battery (FOB) and motor activity (MA) 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. Additional 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 at 
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2000 ppm throughout the study (at study termination, 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 statistically significantly 

decreased at the end of the recovery 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 also showed decreased 

body weight gain (17-20%). Food consumption was 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 investigations, 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 groups, 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 decreases in MCHC, the changes were considered to be 

treatment-related due to the statistical 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 effects on liver function in males at 120 ppm and 

above, and in females at 500 ppm and above, including some effects indicating an adaptive 

response of the liver like a significant increase of relative liver weight at the end of the treatment 

period for both males and females treated at 2000 ppm, accompanied by hepatocellular 

hypertrophy at microscopic examination. Effects on enzyme activities and hepatocellular 

hypertrophy were no longer recorded at the end of the recovery period. Significantly decreased 

activities of alanine aminotransferase (ALAT) were observed in peripheral blood of males and 

females at 500 ppm and 2000 ppm. At the end of the 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 creatinine was decreased at 2000 ppm. Statistically 

significant decreases in cholesterol and triglycerides were observed in recovery males. 

At 2000 ppm the concentration of calcium was slightly but statistically significantly increased in 

males of the main group and the concentration of phosphate was statistically significantly 

increased in females of the main group. 

At 2000 ppm relative weights of kidneys were significantly 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 nephropathy 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 toxicological relevance for humans. Hyaline droplet 

accumulation was reversible within the recovery period, while other tubular changes observed at 
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500 ppm and above after the treatment period were still discernible in 2000 ppm males after the 

recovery period (i.e. basophilic cortical tubules, tubulosclerosis, Schmorl-positive material in 

tubules, cystic tubules and mononuclear 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 histopathological investigations of 

the kidneys of males from the control and 2000 ppm dose groups using the Azan stain revealed 

an immunohistochemical reaction that was stronger and distributed more widely throughout the 

renal cortex of animals dosed at 2000 ppm compared to controls. 

The histopathological investigations of the kidneys 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-globulin, the reaction was slightly more intense and 

more widely distributed in control than in treated males.

870.3150 90-day Oral –Dog 

In a dietary study (MRID 48574755, PMRA 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. Similar 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 consumption was reduced in females at 50 and 500 ppm. 

Decreased plasma and liver ALAT activity were observed at 500 ppm as well as increased liver 

weight, Kupffer cell pigmentation in two males and one female, and an isolated incidence of 

hepatocellular hypertrophy in one female suggesting that the liver is a target organ. Both sexes 

dosed with the 500 ppm test diet exhibited increases in reticulocytes and decreases in bilirubin, 

albumin, and albumin/globulin ratios. Thyroid stimulating hormone levels were also elevated in 

both sexes from the 500 ppm dose group. Other effects at 500 ppm included reduced hemoglobin 

and protein levels in males, and decreased glucose levels and thymus 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 relevant and likely to be a response 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 bilirubin in males, and slightly reduced food 

consumption in females. At 5 ppm, there was a slight reduction in hepatic ALAT activity in 

females only. 

All the effects observed at 5 and 50 ppm were slight and reversible, and considered unlikely to 

represent an adverse effect; therefore, 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/kg 
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bw/day in females) based on decreased plasma and liver ALAT, decreased body weight gains 

and decreased body weight in females, liver effects, 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 (OECD 409; OPPTS 870.3150) in dogs.

870.3200 21/28-day Dermal 

In a repeated dose dermal toxicity study (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 cm2

 (approximately 10% of the total 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 animals of all dose groups but 

were not considered adverse, because no signs of dental fluorosis were observed during the 

study, and no abnormality in bone structure was observed at microscopic examination. 

Transient reductions in food consumption were noted 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 fluensulfone, slight alterations in hematological 

(increased red cell volume distribution width, decreased mean corpuscular hemoglobin 

concentration, and increased reticulocytes) and clinical chemistry (increased cholesterol, reduced 

serum ALAT) parameters were observed. No effect 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 noted upon histological examination of treated skin 

of animals dosed at 400 and 2000 mg/kg bw/day when compared with the vehicle alone. These 

dermal effects were considered indicative of a slight irritant potential of the test item. Because of 

the moderate degrees of acanthosis/hyperkeratosis 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 reticulocytes and increased cholesterol 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 toxicity study (OPPTS 870.3200/OECD 410) in rats. 

870.3465 90-day Inhalation 
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In a 90-day inhalation toxicity study (MRID 49067003; PMRA 2257061), groups of 10 male and 

10 female Wistar Crl:WI(Han) rats were exposed 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 exposure 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 concentration). 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 concentrations, respectively. A 14-

day range-finding study was conducted on 5 rats/sex/dose at 0, 0.035, 0.35 or 3.5 mg/L (6 

hours/day, 5 days/week) to identify appropriate dose levels. 

No mortality occurred during the study. Piloerection in combination 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 concentration group but was not statistically 

significant. In male rats, body weight development 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. The 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 decreased only at the high-concentration during the 

first half of the study and were comparable to the control afterwards; however cumulative gain 

was reduced by 16%. Feed consumption of the exposed 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-dependent 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 hemoglobin concentration in females from the midand high-concentration groups. Clinical chemistry values showed changes in alanine 

aminotransferase (ALAT; decreased 53%) and bilirubin (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 (decreased 31%) and inorganic phosphate (increased 

18%) at all concentrations, in potassium (increased 13%) at the mid- and high-concentration, and 

in phospholipids (increased 17%) and alkaline phosphatase (ALP; increased 15%) at the highconcentration. Females additionally showed increases in ALP at the mid- and highconcentrations (10-31%), and increases in triglycerides (82%) and potassium (10%) at the highconcentration. 

The relative weights of the kidneys (12-20%) and liver (7-19%) were increased at the mid- and 

high concentrations in both sexes. In females, the absolute weights of these organs were 

increased as well. In addition, males had decreased thymus weights (absolute and relative) at all 

concentrations (15-45%), increased adrenal weights at the mid- and high concentrations, and 

increased relative heart weight at the high-concentration. The changes in kidney, liver, thymus 
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and adrenal weight showed a concentration-related response. Relative lung weights were slightly 

elevated in both sexes at the high concentration (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-related changes in the epiglottis and the nose 

(mainly at the second level of the nasal cavity). No histopathological correlates 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 incidence of epithelial hyperplasia (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-concentration). Alterations in the 

nasal cavity consisted of squamous epithelial hyperplasia in males starting at the lowconcentration and in females starting at the mid-concentration, squamous metaplasia in both 

sexes starting at the mid-concentration, squamous epithelial hyperkeratosis starting at the midconcentration in females and at the high-concentration in males, and an increased inflammatory 

load characterized by focal mononuclear cell infiltrates (in both sexes starting at the midconcentration) and focal polymorphonuclear leukocytic infiltration (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 males starting at the midconcentration (7 and 10 affected at the mid and high concentrations, respectively), while pale 

upper incisors were seen in 10 males and 3 females at the high concentration. Microscopically, 

remnants of enamel in the upper incisors (following 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 fluensulfone by inhalation (6 hours/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 histopathological effects) 

 - histopathological changes in the epiglottis (squamous metaplasia, epithelial hyperplasia, 

 focal mononuclear cell infiltrate) in both sexes and in the nasal cavity (squamous 

epithelial hyperplasia) in males. 

For most of these changes, their magnitude/incidence increased with increasing exposure 

concentration. Squamous metaplasia in the epiglottis was present in all exposed animals. 

The lowest concentration tested (actual concentration 0.040 mg/L) was a Lowest-ObservedAdverse-Effect-Concentration (LOAEC or LOAEL) based on decreased weight gain in males, 
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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 inhalation 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 concentrations 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) including quantitative assessments of landing foot 

splay, sensory perception and muscle weakness (grip strength), were performed in all animals in 

a randomized order once prior to first administration, and during weeks 2, 5, 9 and 13. 

Locomotor activities were assessed 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 examination survived the scheduled study period. 

Treatment-related effects included 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 activity (total distance, time in center arena and 

rearing counts) was significantly lower in males at 2500 ppm during week 5. Males in this dose 

group also exhibited reduced grip strength at week 13, and a slight reduction in absolute brain 

weight. These treatment-related effects were attributed to systemic toxicity, and not to 

neurotoxicity. There were no treatment-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 (equivalent 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 neurotoxicity 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), fluensulfone (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 an 
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additional 8-week period without treatment. 

Oral administration of fluensulfone to Beagle dogs in the diet at a dose of 500 ppm resulted in 

transient changes in consistency of feces, reductions in food intake in females and lower body 

weight gains, mean body weights and terminal body weights, effects on hematology and 

biochemistry parameters and on the liver. 

A mild reduction in hemoglobin concentration and associated parameters 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 weight 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 exhibited 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 aminotransferase activity was evident after 52 

weeks of treatment at the same dose levels. These 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 glutathione S-transferase was induced at 500 ppm in both sexes and 

after the recovery period the effect was still increased in males but of no statistical significance, 

or not present in the females. A slight induction of epoxide hydrolase was also evident in both 

sexes at 500 ppm. Slightly increased liver weight in females and minimal to slight degrees of 

diffuse hepatocellular hypertrophy in both sexes were observed at 500 ppm. The findings were 

still evident at the end of recovery in a single female. The changes in consistency of feces were 

also evident in a dose-related manner at the lower dose of 100 ppm. Also at 100 ppm, males 

demonstrated a slight reduction in partial thromboplastin 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 effects 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, attaining 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 study (OECD 452; OPPTS 870.4100) in dogs.

 870.4100 Chronic Toxicity-Rat 

In an oral combined chronic toxicity/oncogenicity study in rats (MRID 48574765; PMRA 

2181217), 4 groups of HANRcc Wist(SPF) rats (allocation 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/females) of fluensulfone (MCW-2 tech., 96.7%, 

Batch No. NLL6692-3) in their diets. A satellite group (allocation B, chronic toxicity animals) of 
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20 males and 20 females per group was similarly treated 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 animals (viability/mortality, clinical signs, food/water 

consumption, body weights, ophthalmoscopic, hematology, clinical chemistry, urinalysis, etc.). 

In addition, a functional observational battery (FOB) 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 determinations. 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 concentrations of 30, 200, 1200 ppm for up 

to 104 weeks had no effects on survival, clinical signs or ophthalmoscopic findings. Grip 

strength and the results of the functional observational battery were also not affected by 

treatment for 52 consecutive weeks. A slight decrease 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 was slightly lower during the first 4 weeks of 

treatment, but no further effects on food consumption were recorded thereafter. Water 

consumption was also increased in high dose males and females. Absolute mean body weight 

and body weight gain were decreased by the treatment in a dose-dependent manner in males 

treated at 200 and 1200 ppm; however, the changes 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 weeks 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 neutrophil and white blood cell 

counts were observed in all female dose groups. Clinical chemistry findings in the low and mid 

dose groups included changes in ALAT, sodium, chloride, calcium, phosphorus, total protein and 

globulin, in males and/or females. Clinical chemistry 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 were considered to be non-adverse metabolic 

adaptations. Other organ weight changes noted in high dose groups included increased kidney 

weights in males and females, and increased adrenal weights in males. No effects on adrenals 

were noted at histopathology. During the urinalysis, increased ketones were observed in 1200 

ppm males and females, and increased leukocytes in high dose males. 

After 104 weeks a few changes in hematology parameters, mainly in red blood cell parameters, 

were noted in animals treated with 1200 ppm. Decreased hemoglobin concentration distribution 

width (HDW) was noted in females at 200 and 1200 ppm. Decreased mean corpuscular 

hemoglobin (MCH) was observed in high dose males, 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. 
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At necropsy after 52 weeks, no gross lesions that could be attributed to treatment with 

fluensulfone were observed. Microscopically, 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 controls, attaining statistical significance only 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) treated at 1200 ppm. In males, a slight 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 incidence 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 considered to be of no toxicological significance, 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 statistically 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 basophilia 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-transferase (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. These enzymes were more significantly induced in 

females than males. 

After 52 weeks of treatment, fluoride measurements revealed a marked dose-dependent increase, 

several fold above controls, of the fluoride content in the ashes from bones and teeth at 200 and 

1200 ppm. Only very slight increases in fluoride content in bones (up to 28% above controls) 

were noted in 30 ppm animals. No increase of the fluoride content in the ashes from teeth was 

recorded in animals treated 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, significant increases of 47-81% above controls 

were seen in bones and non-statistically 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 chemistry findings in both sexes, histopathological 

changes in the lungs of males, and histopathological changes in the esophagus of both sexes seen 

at the LOAEL of 1200 ppm (57.7/69.9 mg/kg/day, M/F). 
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Fluensulfone showed no carcinogenic potential in rats. Dosing was considered adequate in both 

sexes based on the findings of decreased body weight and weight gain, slight hematological 

effects and microscopic findings 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 combined oral chronic toxicity/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 treated 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 females (allocation B) were exposed to the same dietary 

concentrations and used for liver enzyme determination 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 females from the 1200 ppm dose group, but no change in 

ALAT activity was recorded after 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 CYP1A-1 in females only, and slight to moderate 

inductions of phase II enzymes uridine diphosphoglucuronosyl-transferase, glutathione Stransferase and epoxide hydrolase, mainly in females. Most of these changes occurred at 200 

ppm and above, but were considered to be adaptive; none of these changes were considered 

adverse. 

Treatment of CD-1 mice with fluensulfone at dietary concentrations of 30, 200 or 1200 ppm for 

up to 78 weeks had no effect on survival, clinical 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 ppm. The overall mean for animals treated with 

1200 ppm compared to controls was 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 statistically significantly reduced at most weighing intervals at 

1200 ppm and the overall mean over treatment was reduced by 21%. Cumulative body weight 

gain was decreased by 13% and 37% for males at 200 and 1200 ppm, respectively, and by 30% 

for females at 1200 ppm. 

Absolute mean body weight and body weight gain were statistically significantly decreased in a 

dose-dependent manner in males at 200 and 1200 ppm (statistically significant from week 5 

through 54 and throughout the study, respectively), and in females at 1200 ppm (statistically 

significant from week 2 onward for 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% below controls) and decreased total white blood cells count in 

females only, accompanied by reductions in absolute neutrophil, eosinophil, lymphocyte and 
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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 after 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 increases in SDH were 

noted in both sexes at 200 and 1200 ppm at 78 weeks (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 ppm (increased relative kidney, 

adrenal gland, epididymal, testes, heart, and brain weight in males; decreased absolute kidney 

weight in females, and increased relative liver weight in females) were likely secondary to the 

effect of fluensulfone exposure on body weight. 

Non-neoplastic lesions consisted of a statistically significantly increased incidence of 

bronchiolization (i.e. a change from flattened epithelium to cuboidal epithelium) in the lung of 

males and females treated at 200 and 1200 ppm (from control to high dose respectively, 1, 0, 24 

and 31 in males and 5, 7, 43 and 48 in females; 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 directly surrounding ciliated cells. 

The LOAEL was established at 200 ppm (27.35 and 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 lung 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, respectively. Microscopically, the incidence of 

neoplastic lesions in the lungs was increased in females treated at 200 and 1200 ppm. The 

neoplastic lesions consisted of statistically 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/bronchiolar carcinomas in females treated at 1200 ppm 

did not attain statistical significance using the Fisher’s exact test, while it was positive with the 

Peto trend test (4, 2, 2 and 8%, control to high 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 (historical control values not provided). Males did not show a 

treatment-related increase. 

Dosing was considered adequate based on decreased body weight/weight gain and an increased 

incidence of lung bronchiolization 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 Reproduction 
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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 daily 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 included clinical observations and changes in body weight gain and 

food consumption. All does were sacrificed on GD 28, at which time gross external and internal 

necropsies were performed. The gravid uterus was weighed and examined, and the foetuses were 

removed by caesarean section. Fetuses were removed from the uterus, sexed, weighed 

individually, examined for gross external abnormalities, and sacrificed 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 examined. The other half of fetuses was 

used for skeletal examination. 

The total number of females with live fetuses was 20, 19, 19 and 17, respectively, 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 treated 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 interval were 13, 24, 10 and -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 decreased food consumption 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 transient 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 item at any dose level. No macroscopic findings 

related to treatment were noted at necropsy of the does. 

Fetal data showed no treatment-related effects 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 fetuses 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 based 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 developmental 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. 
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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 (MRID 48575771, PMRA 2181223), groups of 30 Wistar 

Crl:WI(HAN) female rats were mated and then administered 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/day 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. Approximately half of all fetuses from each litter were 

examined for visceral effects at necropsy and also 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 reduction (35% during treatment when corrected for 

uterine weight), coupled with decreased food consumption and increased absolute and relative 

liver and kidney weights. There were no other compound-related findings observed. 

There were no reproductive effects attributed to the test compound nor were there any significant 

differences in the litter size, the median percent male fetuses, or placental weights in any group 

tested. One litter from the 300 mg/kg bw/day dose group contained four dead fetuses, which may 

have been secondary to maternal body weight effects, 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 external, visceral, or skeletal malformations or 

variations, or on the incidence of total malformations or variations. 

Incidences of several skeletal variations, including incompletely 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 litter basis in the 300 mg/kg bw/day dose group. These findings may 

represent a “more progressed” ossification of the fetal skeleton, perhaps related to the deposition 

of fluoride into bone during pre-natal development. 

Based on decreased body weight gain and food consumption 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)] in 
Fluensulfone Human Health Risk Assessment DP No. D403767 

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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 the 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 average 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 premating, 10-12% less for males and 4-7% less for 

females), although high dose animals of the F1 generation started the post-weaning period with 

lower body weights than controls that did not increase in magnitude over the dosing period. 

Cumulative premating body weight gain was reduced by 21% and 12% for P males and females, 

respectively, but was not decreased 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 was increased to compensate for the initial losses. 

Treatment with the test item at 1800 ppm resulted in a reduction of relative food consumption 

(g/kg bw/day) during the first week of dosing of the P generation. Due to lower body weights 

observed in the 1800 ppm dose group, absolute food consumption (g/animal/day) was reduced 

throughout most of the study 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 1800 ppm F1 males. Other organ weight changes 

were noted at the high dose that were considered 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 and females, F1 males) were noted in parental 

animals dosed at 1800 ppm. The hypertrophy finding correlated with the increase in liver weight 

and was considered to be of metabolic nature and 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 renal tubular hyaline droplets were increased in males of P and F1-

parental animals. The incidence and/or severity of renal tubular basophilia, mononuclear cell 

infiltration and tubular casts were also increased in males of both generations. These lesions 

were considered to be the secondary lesion resulting from hyaline droplets described above. 

Follicular cell hypertrophy of the thyroid gland was recorded at minimal to slight severity in 

males of the P and F1 generations. 

Reproductive toxicity: There were no treatment-related effects on reproductive performance. 
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Offspring toxicity: In both 1800 ppm F1 and 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 generations. 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 weights 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 glycogen deposits was seen in the high dose female pups. 

This finding was likely associated with the lower body weights. The livers of F1 pups were not 

examined histologically. 

 

Fluoride evaluation: In the F1 parental animals, 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% relative 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% and 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 effects of discoloration 

of the teeth or fragility of bones and 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 females; premating intake of 137 mg/kg bw/day in 

males and 149 mg/kg bw/day in females) based on decreased body weight, body weight gain, 

food consumption and increased liver and kidney weights and histopathology changes in the 

liver and kidney. The parental NOAEL is 250 ppm (overall study intake 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). 

The reproductive NOAEL 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 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-related 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 females; premating intake of 37 mg/kg bw/day in 

males and 149 mg/kg bw/day in females) based on decreased pup weights and spleen weights 

and an increase in post-natal pup loss between days 0 and 4 in the F1 and F2 offspring. The 

offspring NOAEL is 250 ppm (overall study intake 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). 
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This study is classified as acceptable/guideline (fully reliable) and satisfies the guideline 

requirement (OPPTS 870.3800; OECD 416) for a two-generation reproductive 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 μg/plate 

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

μg/plate 

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

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

μg/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 incorporation): 0, 10.0, 31.6, 100, 316, 1000, 2500, 

5000 μg/plate 

Experiment two (pre-incubation): 0, 3.16, 10.0, 31.6, 100, 316, 1000, 2500, 

and 5000 μg/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 μg/plate 

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

μg/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. 
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870.5100, Bacterial Reverse Mutation 

MS 

MRID 48574788 

Acceptable/guideline 

Experiment one (plate incorporation): 0, 3, 10, 33, 100, 333, 1000, 2500, 

5000 μg/plate 

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

μg/plate 

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

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

μg/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 μg/mL without metabolic activation; 

0, 20, 30, 40, 50, 60, 70, 80 μg/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 μg/mL without S9 mix (4 

hours) 

0, 37.5, 75, 150, 300, 600, 800 and 1000 μg/mL with S9 mix (4 hours) 

Experiment two: 0, 5.5, 10.9, 21.9, 43.8, 87.5, 131.3 and 175 μg/mL without 

S9 mix (24 hours) 

0, 87.5, 175, 350, 700, 800, 900 and 1000 μg/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. 
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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 μg/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 μg/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 μg/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 

[

14C]fluensulfone ([thiazole-14C]MCW-2, 98.7-99.3%, TJBIOS-NB21-33 and [butene14C]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. 
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[

14C]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 14CO2, urine and feces over 120 hours, and were sacrificed thereafter. Metabolites 

in urine and feces were identified by comparison to reference standards or by liquid 

chromatography with mass spectrometric detection (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 stability 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 administered dose. The 

disposition and excretion of radiolabel for males and females were generally similar. There was 

no indication that the metabolic elimination processes were saturated by the 100-fold increase in 

dose level, as levels of radiolabel in urine, cage 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 thiazole 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 CO2. The major urinary metabolite was a thiazole mercapturate (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 radiolabel, 85-88% of the administered radiolabel was excreted in 

urine (including cage wash), 5-7% in feces, 1% remained in the carcass and GI tract, and 

approximately 0.07% was exhaled as CO2. The major urinary metabolite was a thiazole 

mercapturate (49-54% of the administered dose) with 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 carcass and GI tract, and 4% was 

exhaled as CO2. The major urinary metabolite was a butene sulfinic acid (32-35% of 

administered dose) with lesser levels of butene sulfonic acid (4-5%). 

For the high-dose butene radiolabel, 81-83% of the administered radiolabel was excreted in urine 

(including cage wash), 6-11% in feces, 1% remained in the carcass and GI tract, and 1-2% was 

exhaled as CO2. The major urinary metabolite was a butene sulfinic acid (53-57% of 

administered dose) with lesser levels of butene sulfonic acid (4%). 

This metabolism study in the rat is classified as acceptable/guideline (fully reliable) and, in 

combination with other metabolism studies conducted with fluensulfone (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. 
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870.7485 Metabolism-Rat (Repeat Oral Dose) 

In a metabolism study (MRID 48574744, PMRA 21881187), the disposition and metabolism of 

[

14C]fluensulfone ([thiazole-14C]MCW-2, 98.5%, TJBIOS-NB21-33, Lot 2466-20) was studied in 

groups of 4 male and 4 female Wistar rats after 14 repeated daily oral doses with unlabeled 

fluensulfone, followed by a single oral dose of [14C]fluensulfone 1 day after 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 

14C derived from the test substance in selected tissues and identified the major metabolites of 

fluensulfone in urine and feces. 

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

The recovery of excreted radiolabel was 94-99% for individual rats. 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 (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 thiazole mercapturate. Low levels of fluensulfone (0.1-0.2% of 

the administered radiolabel) were detected in fecal extracts. Comparison to residues from rats 

treated with a single oral dose from another 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 conducted with fluensulfone (Lab report number R23410/ MRID 48574742/ PMRA 2181184, R-23411/ MRID 48574741/ PMRA 2181185, and R23412/ MRID 48574743/ PMRA 2181186), satisfies the 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 oral doses of either 5 mg/kg bw or 500 mg/kg bw 

of fluensulfone radiolabeled with 14C at two different positions ([thiazole-14C]MCW-2, 100%, 

TJBIOS-NB21-33 and [butene-14C]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 implanted jugular cannulae (up to 336 hours for the 

thiazole radiolabel and up to 192 hours for the butene 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 14C in whole blood and plasma was measured by 

radiochemical techniques. The eight animals for each dose/sex were arranged in two groups, and 

blood was sampled from alternative groups during the first 144 hours on test from thiazole label 

and first 72 hours for the butene label. Therefore, each blood or plasma concentration was the 

mean from four animals. 
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The following pharmacokinetic parameters were calculated: Maximum concentration (Cmax), 

Time for maximum concentration (Tmax), Time for decline to 1⁄2 Tmax (T1/2), Time for decline to 

1/10 Tmax (T1/10), Time from dose to T1/2 (DT50), Time from dose to T1/10 (DT90), Elimination rate 

constant (kel), Area under the curve (AUC), and Volume of distribution (Vd(L)). 

The absorption of fluensulfone-derived 14C 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 absorption based on AUC values. There was no 

indication that the elimination processes were saturated 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 increase in the area under the curve (AUC). 

Additional work was performed to determine the nature of the radiolabeled residues from 

[

14C]fluensulfone, and possible reaction with glutathione (GSH) and mercaptoethanol. 

The amount of radiolabel associated with whole blood increased relative to plasma with time 

(about 20-fold for thiazole label but only 2-fold for butene label). 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. The removal of radiolabel from thiazole label whole 

blood seems to follow the removal of derivatized hemoglobin with a DT50 =148-216 hours (6-9 

days) for the low dose. 

This metabolism study in the rat is classified as acceptable/guideline (fully reliable) and, in 

combination with other metabolism studies conducted with fluensulfone (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 

[

14C]fluensulfone ([thiazole-14C]MCW-2, 99.0-99.2%, TJBIOS-NB21-33 and [butene14C]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 14C in selected tissues at three sacrifice 

intervals corresponding to their respective pharmacokinetic parameters of Cmax (time to peak 

plasma concentration), DT50 (time from dosing to 1/2 Cmax), and DT90 (time from dosing to 1/10 

Cmax) as determined from a pharmacokinetic 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 both dose levels, the gastrointestinal tract, liver and kidney were 

among the tissues containing the highest radioactivity levels at Cmax for both sexes. Other tissues 

with high levels of radioactivity at Cmax (as a percentage of the administered dose) included the 

pancreas (in males administered the low dose of the 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 administered the high dose of the thiazole label and in both sexes 

administered the low dose of the butene label), and the thyroid gland (in both sexes administered 
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the high dose of the thiazole label). At DT90, the levels of radioactivity remaining in the tissues 

accounted for ≤ 3% of the administered dose. 

As predicted by the pharmacokinetic study, radioactivity from the thiazole label was slowly 

eliminated from red blood cells and organs associated with their formation/storage (bone, bone 

marrow, spleen), consistent with the derivatization of the hemoglobin cysteine thiol. The ratio of 

residues at the Cmax compared to the DT90 was near the expected 10% for plasma, but for the 

thiazole label (low and high doses) was >25% for hair and skin, heart, and lung. For the butene 

label (low and high doses), the ratio of residues at Cmax compared to DT90 was >25% for 

numerous tissues/organs except those involved with the digestive system. The slow elimination 

of radioactivity with the thiazole and butene radiolabels from these tissues may reflect 

metabolism to 1- and 2- carbon fragments with reincorporation in natural 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 (fully reliable) and, in 

combination with other metabolism studies conducted with fluensulfone (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 formulated as MCW-2 480 EC was determined in rats 

(MRID 48574949; PMRA #2181332). MCW-2 480 EC (containing 14C-labelled active 

ingredient) was applied to shorn rat skin at two target dose levels: a low dose (0.1 mg/cm2

) 

reflecting typical concentration recommended for the use in the field (10 g a.i./L, diluted from 

the concentrate with water) and a high dose (4.8 mg/cm2) 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 paper 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 test item was determined. Furthermore, the amount 

remaining in/on the skin at the application site after washing and 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 the 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 order to separate the stratum corneum from the epidermis; however, since 

cyanoacrylate adhesive was used in the first two tape stripping of skin (which may affect the skin 

fractionation procedure), all tapes were included in the calculation of potentially 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 [14C]-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% of 
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the low dose and 76-78% of the high dose, could be dislodged from the application 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 remaining 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. Within 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 total 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 and 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; average of all time points) for the low and high dose groups is 64 

(10 h) and 21 (120 h) percent of the administered dose, respectively. There was also a relatively 

high amount of the total applied dose remaining in the cover and O-ring over the test site due to 

the volatility of the test material at skin temperature, but the occlusion of the exposed skin results 

in a higher total recovery and penetration than would occur 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 dose 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 Acceptable/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 immunotoxicity (MRID 48574779, PMRA 2181189), groups of 

10 female CD-1 mice received fluensulfone (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 week of treatment, one mouse in the high dose 

group died and signs of overt toxicity (decreases 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 concentration for the high dose group 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. Cyclophosphamide was used as positive control 

and was administered i.p. at 25 mg/kg bw/day during days 27-31 to a group of 10 female mice. 

On Day 27 a single intravenous dose of 0.2 mL of 1 x 108 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, euthanized, serum samples were collected from 
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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 affect body weight or food or water consumption in 

mice at 100 and 500 ppm, and these parameters returned 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 was evident. A high inter-individual variability was noted in all 

the treatment groups as well as in the control group. Evaluation of individual animal data of this 

study did not show any trend or distribution that would demonstrate significant suppression of 

anti-SRBC IgM response. Absolute and relative spleen and thymus weights were statistically 

significantly reduced to approximately 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 control mean, thus confirming the sensitivity 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 acceptable/guideline (fully reliable) and satisfies the guideline 

requirement for an immunotoxicity 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 blood, alanine aminotransferase 

activity in liver tissue and metabolic enzyme activity in liver. 

No mortality occurred and no clinical signs were noted during the 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, hematological 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 increase in thyroid stimulating hormone (TSH) values was observed 

in male and female dogs at 900 ppm when compared to the control values. Generally, no 

differences in triiodothyronine (T3) or thyroxine (T4) levels were observed in the treated groups 

when compared to the control values. 

Increased absolute and relative liver and thyroid weights were recorded in the males at 900 ppm; 

however, there was no associated macroscopic or microscopic histopathology. A slight decrease 
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in absolute heart weight was 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 inhibition of hepatic alanine aminotransferase (ALAT) activity in 

both sexes at 900 ppm. While some hepatic enzymes 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 this study as it is a range-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 thyroid) at 900 ppm. No 

adverse effects were noted at 200 ppm (7.3 and 7.9 mg/kg bw/day in male and female dogs, 

respectively). 

This subchronic toxicity study in the dog is acceptable/non-guideline (reliable 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 investigation of alanine aminotransferase (ALAT) activity, concentrations 

of pyridoxal 5’-phosphate and ALAT1-protein and ALAT1–messenger RNA expression in 

Beagle dogs, treated for 2 weeks with fluensulfone (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 fluensulfone (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 the liver enzymes (leakage markers) aspartate 

aminotransferase (ASAT), alanine aminotransferase (ALAT), sorbitol dehydrogenase (SDH). 

Plasma concentrations of the ALAT co-factor, pyridoxal 5’-phosphate, were determined before 

commencement of treatment and on days 7 and 14 of treatment with fluensulfone 

Following completion of the treatment period, samples of liver tissue were taken for mechanistic 

investigations, including investigations of ALAT1 mRNA expression 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 period. No clinical signs were considered to be 

related to treatment and all animals appeared in good general health throughout the study. There 

were no effects on food consumption or body weight. There were no macroscopic or microscopic 

findings that could be attributed to the treatment. 

Decreased plasma ALAT was observed after 2 weeks at 500 ppm. No changes were observed in 

ASAT or SDH. 

ALAT1 mRNA levels were 3.36 times higher in the treated group compared to the control group. 

Hepatic ALAT1 protein levels were similar in the control and treated groups. After 2 weeks of 

treatment at 500 ppm, mean hepatic ALAT activity was significantly decreased to 13.6% of the 
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control group mean. A slight decrease in the concentrations of Pyridoxal 5’-phosphate in plasma 

was recorded, whereas slightly higher hepatic Pyridoxal 5’-phosphate concentrations (attaining 

statistical significance) were observed in fluensulfone-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). Thus, it is considered unlikely to be the primary 

cause of the marked reduction in ALAT activity. 

Based on the results of this study, the reduction of the activity of alanine aminotransferase 

activity by fluensulfone is not mediated by 

 i) a reduction of ALAT1 mRNA expression 

 ii) a reduction of ALAT1 protein expression 

 iii) a decrease of the cofactor Pyridoxal 5’-phosphate 

Other mechanisms, such as alteration of post-translational modification might be the cause 

for the transiently decreased activity of ALAT upon fluensulfone treatment. The increase in 

ALAT1 mRNA might be indicative of a counter-regulation to the decrease in ALAT 

activity. 

This special study is classified as acceptable/non-guideline (reliable with restrictions) 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 prepared 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 acceptable/nonguideline (reliable with restrictions) 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 2181191), dog liver homogenate from untreated 

dogs was incubated with fluensulfone (MCW-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 metabolites,. LC-MS/MS determination in the incubation mixtures clearly 

demonstrated an effective metabolism of fluensulfone. 

No inhibition of the ALAT activity was observed following incubation of dog liver homogenates 

with fluensulfone. Based on the results of this 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. 
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This special study is classified as acceptable/non-guideline (reliable with restrictions) 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 fluensulfone 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 fluensulfone (MCW-2 tech., 96.67%, Batch No. 36372130-PF1) 

in the diet resulted in a dose-dependent decrease 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 direct 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 high 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 tested). ALAT activity was determined to show 

whether serum or cytosolic fractions from fluensulfone-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 cytosolic fractions was determined for both control 

and fluensulfone-treated dogs using western blotting to see whether 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 exactly to the theoretically calculated activities. 

Immunoblotting revealed very similar levels of ALAT in serum and cytosolic fractions of 

controls and treated dogs. However, as specificity of the used antibody was not tested against 

dog ALAT, the results indicating the unlikely decrease of the expression of ALAT protein upon 

treatment with fluensulfone were considered only provisional and need 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 treated 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 restrictions) 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 proliferation in the lungs as a basis 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 10 
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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 injection of BrdU for determination 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 recorded. 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 from 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 with 1305 ppm Isoniazid showed 

increased food consumption from days 5 to 8 when compared to the respective 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 loss 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 fluensulfone. 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 treatment with both fluensulfone and Isoniazid with 

increased cell proliferation that was detected by BrdU incorporation. 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 restrictions) 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 fluensulfone (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 inhibitors, 4-methyl pyrazole (CYP 2E1/Cyp 2e1) and 5-phenyl-1-pentyne 

(Cyp 2f2). 

The metabolic activity was determined by incubation of lung microsomes with fluensulfone in 

the presence of an NADH-regenerating system, and analytical determination of remaining 

(unmetabolized) fluensulfone by LC-MS/MS at different time points. Two concentrations of 

fluensulfone were tested (2 and 20 μM); however, the highest concentration was found to be too 

high (only a small percentage was metabolized) and the results obtained were considered only as 

confirmatory. The metabolism was further investigated by addition of inhibitors of CYP 2E1 and 

CYP 2f2 (a mouse-specific isoform). Lung microsomes were prepared from 12 female and 12 
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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 preparations was confirmed by determination of the 

turnover of a standard substrate, chlorzoxazone, under conditions similar to those used for 

fluensulfone. 

The detected activity towards chlorzoxazone was approximately 450-fold lower in human lung 

microsomes compared to mouse lung microsomes, and thus in good correlation with the activity 

differences reported in the literature. 

With human lung microsomes, no metabolism of fluensulfone was detectable. In contrast, in 

mice female and male lung preparations only approximately 10% of the applied fluensulfone (2 

μM) remained after 120 minutes of incubation. 

The negative control without NADH regenerating system showed no metabolic activity in 

humans as well as in both mice genders showing the validity of the assay and indicating an 

enzymatic degradation of fluensulfone. 

The inhibition of (mouse-specific) 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 major role in the degradation process. 

This special study is classified as acceptable/non-guideline (reliable with restrictions) 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 14C-fluensulfone, 

formulated as MCW-2 480 EC, was applied onto the skin membranes at two dose levels: a low 

dose (0.10 mg/cm2

) reflecting a typical concentration recommended for the use in the field as 

spray dilution, i.e. 10 g a.i./L (achieved by dilution of the commercial product with water), and a 

high dose (4.8 mg/cm2

) which represents the concentration of active ingredient in the undiluted 

commercial product, i.e. 480 g a.i./L. The exposure 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 intervals between 0-6 hours and thereafter in 

2 hours intervals until the end of the experiments. Volatilized test item was trapped by covering 
Fluensulfone Human Health Risk Assessment DP No. D403767 

Page 92 of 107 

the donor cell with charcoal paper during exposure. At the end of the experiment, the remaining 

14C-fluensulfone was removed from the skin surface by rinsing. The skin membranes were 

removed from the diffusion cell and consecutively stripped 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 remaining 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 acceptable/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 levels 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 were 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 sacrificed 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 hours after dose administration after they had developed signs of 

overt toxicity. All animals of the remaining dose groups survived to terminal 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 somnolence, 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 animals of both sexes at approximately 5 hours. In 

addition, in 2 males dosed at 2000 mg/kg bw the push-off reflex was absent. Due to the 

premature death of all males and females dosed 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 observed 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 animals of any dose groups at necropsy. 
Fluensulfone Human Health Risk Assessment DP No. D403767 

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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 administration, but this dose level had exceeded the 

maximal tolerated dose (MTD). 

This study is a non-guideline study and is therefore classified as acceptable/non-guideline 

(reliable with restrictions) as a preliminary acute neurotoxicity study in rats. 

 28-day oral Rat -BSA 

In a subchronic toxicity study (MRID 49387602, PMRA 2407952), 3,4,4-trifluoro-but-3-ene-1-

sulfonic acid (BSA; 93%) was administered 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 analytical 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 measurements, gross pathology, and 

limited histopathology examinations (liver, kidney, lung, and thymus). 

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 treatmentrelated effects were observed in this study up to the highest 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 repeat-dose oral study in 

rats due to the small group sizes and limited hematology, clinical chemistry 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 included clinical signs of toxicity, body weight, food 

consumption, hematology and clinical chemistry parameters, organ weight measurements, gross 

pathology, and limited histopathology examinations (liver, kidney, lung, and thymus). 

No treatment-related effects were observed in this study up to the highest 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 repeat-dose oral study in 

rats due to the small group sizes and limited hematology, clinical chemistry 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 Risk Assessment DP No. D403767 

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Fluensulfone Human Health Risk 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

HO

S

O

O F

F

F
Fluensulfone Human Health Risk 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 

HO

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

O
Fluensulfone Human Health Risk 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 (3.57 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 rate is unknown. Tissue collection 20-22 hours after last dose. 

Rat (48574742): Single dose of 500 mg/kg body weight to females. Non-carcass samples collected 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 found in a nearly 1:1 ratio in male rats treated at the same 

dose in the same study (12% AD for I vs. 10% AD for II). 
Fluensulfone Human Health Risk Assessment DP No. D403767 

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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/cm3) 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 (pKa) No acidic or basic functional groups

Octanol/water partition coefficient Log(KOW) 2.2 

 
Fluensulfone Human Health Risk Assessment DP No. D403767 

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Appendix D. Summary of Fluensulfone Residue 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 0 
Fluensulfone Human Health Risk Assessment DP No. D403767 

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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 max reflect all samples; all other values reflect per-trial 

averages. HAFT = Highest Average Field Trial. B Parenthetical values for BSA are fluensulfone-equivalents ([BSA] × 1.534). 
Fluensulfone Human Health Risk 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. These data, which include studies from 

PHED 1.1 and the AHETF database are (1) subject to ethics review pursuant to 40 CFR 26, (2) 

have received that review, and (3) are compliant with applicable ethics requirements. For certain 

studies, the ethics review may have included review by the Human Studies Review Board. 

Descriptions of data sources, as well as guidance on their use, can be found at the Agency 

website2

. 

 

 

2 http://www.epa.gov/pesticides/science/handler-exposure-data.html and 

http://www.epa.gov/pesticides/science/post-app-exposure-data.html
Fluensulfone Human Health Risk 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/23339/OCR, 2049W. Unpublished 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 Research 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 Research 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 West, Inc., and GLP Technologies. 

665p. 
Fluensulfone Human Health Risk 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 West, 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 Radish, 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 Method (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 Risk 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 Typhimurium and Escherichia Coli Reverse 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 Reproduction 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 Risk Assessment DP No. D403767 

Page 105 of 107 

MRID No. Citation 

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 Aberration Test in Chinese Hamster V79 Cells with MCW-2 

Metabolite 3625: Final Report. Project Number: R/23471/OCR, 1345801. Unpublished study 

prepared by Cytotest Cell Research Gmbh and Co. 57p. 

48574786 Merker, M. (2011) Micronucleus Assay in Bone 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 Chinese 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 Bone 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 - Acute 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 Aberration Test in Chinese Hamster V79 Cells with MCW-2 

Metabolite 3627: Final Report. Project Number: R/23467/OCR, 1328701. Unpublished study 

prepared by Cytotest Cell Research Gmbh and Co. 66p. 
Fluensulfone Human Health Risk Assessment DP No. D403767 

Page 106 of 107 

MRID No. Citation 

48574795 Merker, M. (2010) Micronucleus Assay in Bone 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 Hypersensitivity 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 Biotransformation 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 Risk 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 Beagle 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 Plant 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 Elliot 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 study prepared by Exponent International, Ltd. 

214p. 

49402701 Burn, R and Winner, S. (2012) To determine fluensulfone residues in fruiting 

vegetables (tomato & processed fractions, capsicum, rockmelon, cucumber and 

zucchini) following one application of MCW-2 applied 7 days prior to transplanting. 

Study Number FOZ1001. Unpublished paper prepared by AgriSolutions Australia. 

203 p.