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REACH

Dimethyl disulphide

EC number: 210-871-0 | CAS number: 624-92-0

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

Acute oral, dermal and inhalation toxicity studies were available on DMDS. The oral LD50 was 190 mg/kg bw in rats (Shapiro 1985), the dermal LD0 was higher than 2000 mg/kg bw in rats (Yasso, 2015) and the 4h-LD50 in rats was 1310 ppm, equivalent to 5.05 mg/L (Kirkpatrick, 2005).

In acute inhalation studies in which rats were exposed to DMDS vapours, functional damage in the upper respiratory tract (degeneration of transitional and olfactory nasal epithelium) was observed.

In acute dermal studies, DMDS induces transient depression of CNS function at non-lethal doses.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

remark : GLP guideline study but the DMDS tested in this study did not correspond to the specifications of DMDS proposed for registration (high level of methyl mercaptan).
RAC concludes that the presence of methyl mercaptan as an impurity at such a low concentration does not affect the acute oral toxicity profile of DMDS ; the study is considered valid.

Qualifier:

according to guideline

Guideline:

other: EPA 40 CFR 163.81-1

Deviations:

GLP compliance:

yes

Test type:

standard acute method

Limit test:

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS:
- Source: Royalhart Colony, Newhampton, NY
- Age: no data
- Weight at study initiation: 163-269 g
- Adaptation period: 14 days
- Fasting before treatment: 18 hours
- Adaptation period : 14 days

HOUSING : The animals were housed individually in suspended stailess steel wire-bottomed cages

FOOD and WATER
- Food: Purina rat chow ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature : 68-72°F
- Relative humidity : no data
- Light/dark cycle : 12h/12h
- Ventilation : no data

Route of administration:

oral: gavage

Vehicle:

other: 3% CMC (carboxymethyl cellulose)

Details on oral exposure:

- Volume administered: no data

Doses:

125, 188, 250, 375 and 500 mg/kg

No. of animals per sex per dose:

Control animals:

Details on study design:

Clinical signs, mortality and body weight gain were checked for a period of up to 14 days following the single administration of the test item. All animals were subjected to necropsy.

Statistics:

Litchfield-Wilcoxon method of probit analysis.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

190 mg/kg bw

95% CL:

150 - 240

Remarks on result:

other: Post-exposure clinical signs: sedation, hypotonia, dyspnea, piloerection and coma

Mortality:

Group        Dose                Mortality         Mortality %
g/kg   Male     Female
1                0.125                0/5        1/5                10
2                0.188                5/5        1/5                60
3                0.250                3/5        4/5                70
4                0.375                5/5        5/5                100
5                0.50                 5/5        5/5                100

Clinical signs:

other: Dose Level 0.125 g/kg: One female died approximately 2-1/2 hours after administration of the test material. The second female did not exhibit any movement on the day of dosing, but appeared to be recovered by the next morning, and did not exhibit any sign

Gross pathology:

Dose Level : 0.125 g/kg - Group 1: The necropsy of the 1 mortality showed evidence of pulmonary hemorrhage and an enlarged spleen. Necropsy of the survivons was unrevealing. All organs and tissues appeared normal.

Dose Level 0.188 g/kg: Necropsy of the mortalities on the day of dosing revealed evidence of pulmonary hemorrhage and one animal exhibited an enlarged spleen. Necropsy of the survivons was unreveallng. All organs and tissues appeared normal.

Dose Level 0.250 g/kg: Necropsy of the mortalities revealed evidence of pulmonary hemorrhage. One male exhibited gastro-intestinal hemorrhage and an enlarged spleen. Necropsy of survivors was unreveallng. All organs and tissues appeared normal.

Dose Level 0.375 g/kg: The necropsy revealed evidence of pulmonary hemorrhage in all but 2 rats. One male exhibited an enlarged spleen, and 1 female exhibited a gastro-intestinal hemorrhage. One male and 1 female did not exhibit any abnormal organs or tissues.

Dose Level 0.50 g/kg: 5 All animals died approximately 2-1/2 hours after administration of the test material. The necropsy revealed evidence of pulmonary hemorrhage in all animalis. One male and 1 female exhibited enlarged spleens, and 2 females exhibited gastro-intestinal hemorrhages.

Interpretation of results:

Category 3 based on GHS criteria

Conclusions:

Under these experimental conditions, the oral LD50 of DMDS was 190 (150 -240) mg/kg in female and male rats.

Executive summary:

The Acute oral toxicity of DIMETHYL DISULFIDE was evaluated in male and female Wistar rats according to EPA 40 CFR 163.81-1and in compliance with principles of Good Laboratory Practices. Animals were treated with dose levels of 125, 188, 250, 375 and 500 mg/kg and then observed for 14 days for mortality, clinical signs and effect on body weight.

According to the dose levels, mortality was 10, 60, 70, 100 and 100% respectively.

Sedation, hypotonia, dyspnea, piloerection and coma, appeared just after the administration and disappeared after 24 hours. No effect was noted on the body weight gain of the surviving rats. Haemorragic stomachs was observed at the macroscopic examination of the rats dead on the first day (250 and 500 mg/kg)

Under these experimental conditions, the oral LD50 of DMDS was 190 (150 -240) mg/kg in female and male rats.

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: LD50
Value:: 190 mg/kg bw
Quality of whole database:: Shapiro studies are considered to be reliable

Acute toxicity: via inhalation route

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 2nd 2004 to July 16th 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Deviations:

GLP compliance:

yes

Remarks:

U.S. EPA Good Laboratory Practice Standards (40 CFR Part 160) and OECD Principles of Good Laboratory Practice

Test type:

standard acute method

Limit test:

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS:
- Source: Charles River Laboratories, Raleigh, North Carolina, USA
- Age: 8 to 12 weeks
- Weight at study initiation: 254 g to 307 g for males and from 226 g to 260 g for females
- Acclimatation: for a minimum of 7 days.

HOUSING
Individual suspended wire-mesh cages

FOOD and WATER
- Food: PMI Nutrition International, LLC, Certified Rodent LabDiet 5002, ad libitum
- Water: ad libitum municipal water, delivered by an automatic watering system

ENVIRONMENTAL CONDITIONS
- Temperature : 21.5°C to 21.9°C
- Relative humidity : 33.0% to 48.3%
- Light/dark cycle : 12h/12h
- Ventilation : filtered, not recycled air

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

The acute inhalation toxicity of dimethyl disulfide (DMDS) was evaluated in a 4-hour, single-exposure study in rats. DMDS was initially administered to a single group of five male and five female Sprague-Dawley albino rats via whole-body vapor exposure at concentrations of 847, 1188, 1308 and 1650 ppm. Exposures were conducted in an approximately 165-L plexiglas whole-body exposure chamber, which allowed all animals to be easily seen and observed during exposure. Animals were housed individually during exposure. Food and water were withheld during the exposures. Exposure atmosphere conditions (temperature, relative humidity and oxygen content) were recorded approximately hourly during each exposure.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

Analyzed exposure concentrations were obtained using a GC. Samples of the exposure atmospheres were collected at least once every 30 minutes.

Duration of exposure:

4 h

Concentrations:

847, 1188, 1308 and 1650 ppm (3.26, 4.57, 5.04 and 6.35 mg/L)

No. of animals per sex per dose:

Control animals:

Details on study design:

Mortality, clinical observations for pharmacotoxic signs and body weight changes were evaluated over a 14-day observation period. Detailed clinical observations (to determined peak effect of test article exposure) were conducted immediately following each exposure and at 1, 2, 4, 6 and 8 hours post-exposure. All animals were subjected to a gross necropsy.

Statistics:

LC50 values (in ppm) and slopes (with 95% confidence limits) were calculated by the method of Litchfield and Wilcoxon.57,

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

1 310 ppm

95% CL:

1 167 - 1 471

Exp. duration:

4 h

Remarks on result:

other: (5.05 (4.49-5.66) mg/l) Clinical signs consisted of rales, decreased defecation and dried material on various body surfaces, including the mouth and nose.

Mortality:

847 ppm : 0/10
1188 ppm : 4/10
1308 ppm : 4/10
1650 ppm : 9/10

All deaths were noted during exposure or immediately following exposure

Clinical signs:

other: see below

Body weight:

Slight body weight losses (2 to 7 grams) were noted for one or more females each in the 847, 1188, 1308 ppm groups during the study. There were no other remarkable body weight changes. All surviving animals met or surpassed their initial (study day 0) body weight by study day 14.

Gross pathology:

Dark red discoloration of the lungs was noted for animals found dead during or immediately following exposure in the 1308 and 1650 ppm groups. One male in the 1308 ppm group also was noted with lungs that did not fully collapse at necropsy. There were no gross findings for any of the found dead animals in the 1188 ppm group or for the surviving animals at the scheduled necropsy.

Other findings:

details on clinical signs : Toxicologically relevant pharmacotoxic signs consisted of rales in the 1188 and 1650 ppm groups, decreased defecation in the 1188 ppm group and dried material on various body surfaces, including the mouth and nose for the surviving 1650 ppm female. Detailed clinical observations immediately following exposure consisted of salivation in the 1188, 1308 and 1650 ppm group males and/or females, tremors in the 847 and 1308 ppm group females, low arousal in the 847, 1188 and 1308 ppm group females, ataxia and impaired mobility in the 1188 ppm group females, hunched gait in the 1308 and 1650 ppm group females, and increased difficulty in removing from cage/handling in females in all groups. Over the course of the 8-hour observation interval, females in all groups had tremors, low arousal levels and were harder to remove from their cages and handle. Hunched gait was noted in the 1188 and/or 1308 ppm groups throughout the 8-hour period.

Total Incidence of Clinical Observations Immediately Following Exposure

(Number of Occurrences/Number of Animals)

	DMDS (ppm)
	Males				Females
	847	1188	1308	1650	847	1188	1308	1650
Number of Rats	5	2	3	0	5	4	3	1
Salivation (slight)	-	1/2	1/3	-	-	-	1/3	-
Salivation (severe)	-	-	-	-	-	3/4	2/3	1/1
Tremors (slight - 1.5 mm)	-	-	-	-	1/5	-	1/3	-
Ease of Removal (moderately difficult)	-	-	-	-	-	-	1/3	-
Ease of Handling (moderately low)	-	-	-	-	4/5	4/4	3/3	1/1
Low Arousal	-	-	-	-	1/5	1/4	1/3	-
Hunched Gait	-	-	-	-	-	-	3/3	1/1
Ataxia	-	-	-	-	-	1/4	-	-
Impaired Mobility (moderately)	-	-	-	-	-	1/4	-	-

- = No observations at this interval.

Interpretation of results:

Category 3 based on GHS criteria

Conclusions:

The LC50 of dimethyl disulfide was 1310 ppm (with 95% confidence limits of 1167-1471 ppm) when male and female albino rats were exposed whole-body to a vapor of the test article for a single, 4-hour period. The time of peak effect of DMDS was considered to be immediately following to 1 hour post-exposure.

Executive summary:

The acute inhalation toxicity of dimethyl disulfide (DMDS) was evaluated in 4-hour, single-exposure study in rats performed in compliance with the EPA/OPPTS Guideline 870.1300 (1998) and the OECD Guidelines # 403 (1981). DMDS was initially administered to a single group of five male and five female Crl:CD(SD) albino rats via whole-body vapor exposure at concentrations of 847, 1188, 1308 and 1650 ppm. Mortality, clinical observations for pharmacotoxic signs and body weight changes were evaluated over a 14-day observation period. Detailed clinical observations (to determined peak effect of test article exposure) were conducted immediately following each exposure and at 1, 2, 4, 6 and 8 hours post-exposure. All animals were subjected to a gross necropsy.

Mortality was 0/0, 4/10, 4/10 and 9/10 animals for the 847, 1188, 1308 and 1650 ppm groups, respectively. All deaths were noted during exposure or immediately following exposure. Toxicologically relevant pharmacotoxic signs consisted of rales in the 1188 and 1650 ppm groups, decreased defecation in the 1188 ppm group and dried material on various body surfaces, including the mouth and nose for the surviving 1650 ppm female. Detailed clinical observations immediately following exposure consisted of salivation in the 1188, 1308 and 1650 ppm group males and/or females, tremors in the 847 and 1308 ppm group females, low arousal in the 847, 1188 and 1308 ppm group females, ataxia and impaired mobility in the 1188 ppm group females, hunched gait in the 1308 and 1650 ppm group females, and increased difficulty in removing from cage/handling in females in all groups. Over the course of the 8-hour observation interval, females in all groups had tremors, low arousal levels and were harder to remove from their cages and handle. Hunched gait was noted in the 1188 and/or 1308 ppm groups throughout the 8-hour period.

Based on the results of this study, the LC50 of dimethyl disulfide was 1310 ppm (with 95% confidence limits of 1167-1471 ppm) when male and female albino rats were exposed whole-body to a vapor of the test article for a single, 4-hour period. The time of peak effect of DMDS was considered to be immediately following to 1 hour post-exposure.

Reference 2

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: Toxicological Test Methods of Pesticide Registration, National Standard of the People’s Republic of China, Document Number GB15670-1995.

Deviations:

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

yes

Remarks:

2-h exposure

GLP compliance:

yes (incl. QA statement)

Test type:

traditional method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, (Raleigh, NC)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 8 weeks old
- Weight at study initiation: 239 g to 262 g for males and from 180 g to 192 g for females.
- Fasting period before study: no
- Housing: individually in suspended wire-mesh cages
- Diet (ad libitum): PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (block),
- Water (ad libitum) Reverse osmosis-treated water
- Acclimation period: a minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.4 to 21.9
- Humidity (%): 39.4 to 48.7
- Air changes (per hr): minimum of 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

clean air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel and glass whole-body exposure chamber
- Exposure chamber volume: 0.5 m3
- Method of holding animals in test chamber:wire mesh battery containing separate cages
- Source and rate of air: at least 12 air changes per hour
- Method of conditioning air:in-house supply air source utilizing HEPA-filtration and activated charcoal to pre-treat room air.
- System of generating vapor: Vapors of the test substance were generated using a 500-mL bubbler-type vaporization system (gas washing bottle, Ace Glass, Inc.; Vineland, NJ) located within a laboratory hood that was exhausted through the facility exhaust system. Nitrogen was metered into the inlet stem of the gas washing bottle containing an appropriate quantity of test substance and bubbled through a fritted disc and the liquid test substance to produce concentrated vapors of the test substance. Nitrogen was metered to the gas washing bottle using a regulator (model no. 8802K, Coilhose Pneumatics Inc.; East Brunswick, NJ) and controlled using a rotameter-type flowmeter (Cole-Parmer; Vernon Hills, IL). The test substance vapors were piped to the exposure chamber inlet and diluted to the desired exposure concentration by mixing with the chamber supply air.
- Method of particle size determination: not appropriate for vapor
- Treatment of exhaust air: redundant exhaust blowers preceded by activated-charcoal and HEPA-filtration units
- Temperature, humidity, pressure in air chamber: Temperature, relative humidity, and ventilation rate within the chamber were recorded at approximately 60-minute intervals during the exposure.

TEST ATMOSPHERE
- Brief description of analytical method used: GC/FID
- Samples taken from breathing zone: yes

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

2 h

Remarks on duration:

Duration specific of the Chinese guideline

Concentrations:

533 ppm (target) 525 ppm (analysed)

No. of animals per sex per dose:

Control animals:

Details on study design:

MORTALITY
Each animal was observed for mortality approximately hourly throughout the exposure and twice daily for 14 days (once in the morning and once in the afternoon, except on the day of scheduled necropsy).

CLINICAL OBSERVATIONS
During exposure those animals visible through chamber windows were observed for pharmacotoxic signs at least 2 times, approximately hourly, throughout the exposure. A separate protocol was used to record observations during exposure noted outside the above specified intervals.
Each animal was observed for clinical signs of toxicity immediately following exposure on study day 0 and once daily thereafter for 14 days. Observations were recorded each day and included, but were not limited to: changes in the skin and fur, eyes and mucous membranes, and also
changes to the respiratory, circulatory, autonomic and central nervous systems, somatomotor activity, and behavior pattern.

BODY WEIGHTS
Body weights were obtained prior to exposure on study day 0 and on post-exposure days 1, 3, 7, and 14.

NECROPSY
Animals at the scheduled necropsy were euthanized by carbon dioxide (CO2) inhalation followed by exsanguination. The major organ systems of the cranial, thoracic, and abdominal cavities were examined for all animals. No tissue or organs were retained and the carcasses were discarded after necropsy.

Statistics:

None

Sex:

male/female

Dose descriptor:

LC0

Effect level:

>= 525 other: ppm (analytical)

Exp. duration:

2 h

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

None of the animals died during exposure or during the 14-day post-exposure observation period.

Clinical signs:

other: Clinical observations noted prior to the 1-hour scheduled observation period during exposure consisted of salivation for all animals and moderate clear discharge of the eye(s) for 2 males and 4 females. Clinical observations noted at the 1-hour observatio

Body weight:

All males and 4 females lost weight (3 to 10 grams) from study day 0 to 1. All animals surpassed their initial (study day 0) body weight by study day 14.

Gross pathology:

There were no macroscopic findings for animals at the scheduled necropsy.

Interpretation of results:

study cannot be used for classification

Conclusions:

No deaths occurred in this study. The LC0 and LC50 of dimethyl disulfide TC were greater than 525 ppm when male and female Crl:CD(SD) albino rats were exposed to vapors of the test substance as a single, 2-hour, whole-body exposure.

Executive summary:

The acute inhalation toxicity of dimethyl disulfide TC was determined when administered for a single, 2-hour, whole-body inhalation exposure to rats. The protocol was designed to be in compliance with the Toxicological Test Methods of Pesticide Registration, National Standard of the People’s Republic of China, Document Number GB15670-1995 and is comparable to the OECD TG 403. The acute inhalation toxicity of dimethyl disulfide TC was evaluated in this 2-hour, single-exposure study in rats. The test substance was administered to 1 group of 5 male and 5 female Crl:CD(SD) albino rats for 2 hours via whole-body inhalation exposure at a vapor concentration of 533 ppm (2000 mg/m3). The mean analyzed concentration for the 2-hour exposure was 525 ppm. Mortality, clinical observations, body weights, and body weight changes were evaluated over a 14-day post-exposure observation period. Necropsies were conducted on all animals.

None of the animals died during exposure or during the 14-day post-exposure observation period. Clinical observations noted during exposure consisted of slight to moderate clear discharge from the eye(s), partial closure of the eye(s), slight clear nasal discharge, salivation, and wet clear material around the mouth. Clinical observations immediately following exposure consisted of dried clear material around the mouth and ventral neck for 1 female. There were no significant clinical observations for the animals during the 14-day post-exposure observation period and all animals were considered clinically normal by study day 1. All males and 4 females lost weight from study day 0 to 1. All animals surpassed their initial (study day 0) body weight by study day 14. There were no macroscopic findings noted for animals at the scheduled necropsy.

Reference 3

Endpoint:: acute toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: key study
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:: no guideline followed
Principles of method if other than guideline:: Evaluation of the potential toxic effects of dimethyl disulphide (DMDS) on tissues of the upper respiratory tract (URT) when administered 24 hours as a vapor via whole-body inhalation to rats.
GLP compliance:: yes
Test type:: other: Single 24-hour inhalation toxicity study
Limit test:: no
Species:: rat
Strain:: Sprague-Dawley
Sex:: male
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, USA
- Age at study initiation: 9 weeks old
- Weight at study initiation: 277 g to 346 g
- Housing: individually in clean, stainless-steel, wire-mesh cages suspended above cage-board
- Diet (ad libitum): PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002
- Water (ad libitum): Reverse osmosis treated (on site) drinking water
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature : 21.5°C to 21.8°C
- Humidity: 40.6% to 44.0%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:: inhalation: vapour
Type of inhalation exposure:: whole body
Vehicle:: other: unchanged (no vehicle)
Details on inhalation exposure:: Animal exposures were conducted in 2000-L stainless-steel and glass whole-body exposure chambers. The exposure systems were operated under dynamic conditions from a HEPA- and charcoal-filtered air source. The air flow rate through the chamber was such that there were 12-15 air changes per hour. Mean chamber temperature and relative humidity were set to be 22 ± 2°C and 30-70%, respectively. These parameters were monitored continuously and recorded approximately every 30-40 minutes throughout the exposures. Oxygen content of the exposure atmospheres was measured during the method development phase of the study, while operating at the above targeted conditions, and was at least 19%.

Vapors of the test substance were generated by bubbling metered nitrogen through gas washing bottles containing appropriate aliquots of the test substance. Prior to entering the exposure chambers, the vapors of the test substance were mixed with dilution air to achieve the desired exposure levels.
Analytical verification of test atmosphere concentrations:: yes
Remarks:: Analyzed concentrations of the test substance in each exposure chamber were determined by a specific on-line gas chromatographic method and were measured approximately every 30-40 minutes throughout the exposure period.
Duration of exposure:: 24 h
Concentrations:: 5, 9, 12.5, and 18 ppm (19, 34, 48 and 69 mg/m3)
No. of animals per sex per dose:: 10
Control animals:: yes
Details on study design:: One day following the 24-hour exposure, all animals were euthanized. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed prior to randomization and prior to the scheduled necropsy. Individual body weights were recorded during pretest and prior to the study day 0 exposures. A final, non-fasted body weight was also recorded prior to the scheduled necropsy. Complete necropsies were conducted on all animals, and selected organs were weighed (kidneys, lungs (prior to inflation with fixative) and liver) at the scheduled necropsy. Microscopic examination was performed on nasal tissues and gross lesions from all animals in all exposure groups. Nasal sectioning was performed using the method of Morgan in which 6 nasal sections were produced using specifically defined landmarks for sectioning (Mery et al., 1994). All 6 nasal levels were examined microscopically.
Statistics:: All statistical tests were performed using appropriate computing devices or programs. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. In addition, percent difference from the control group is presented for body weights and organ weights. Due to the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ by ±1 in the last significant figure.
Body weight, body weight change, and organ weight data were subjected to a parametric one way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance treated groups to the control group.
: Key result
Sex:: male
Dose descriptor:: other: NOAEC for nasal irritation
Effect level:: 12.5 ppm
Exp. duration:: 24 h
Remarks on result:: other: 48 mg/m3
Mortality:: All animals survived to the scheduled necropsy.
Clinical signs:: other: There were no test substance related clinical observations.
Body weight:: Body weights were unaffected by test substance exposure.
Gross pathology:: There were no test substance-related macroscopic observations noted in animals at the scheduled necropsy. Organ weights were unaffected by test substance exposure.
Other findings:: MICROSCOPIC EXAMINATION
Test substance exposure-related degeneration of the olfactory epithelium was observed in Nasal Levels II-VI at exposure concentrations of 12.5 and 18 ppm, and in Nasal Levels III, IV, and V at an exposure concentration of 9 ppm. Degeneration of the olfactory epithelium was also observed in a single nasal level for 2 of 10 control group animals. The degeneration in the affected nasal sections in the 9 ppm group animals consisted of single or very few discrete, extremely small foci that consisted of cellular vacuolation and individual cell pyknosis, without sloughing. Changes in the 12.5 ppm group were also discrete areas comprising a very small percentage of the olfactory epithelium. These changes would be completely reversible and without clinical consequences and were not considered adverse. Degeneration of the olfactory epithelium was observed in 3 or more nasal levels in all 10 animals and was considered adverse in the 18 ppm group. This conclusion was based on the extent and severity of degeneration including the amount of damaged epithelium with sloughing of sensory and sustentacular cells. The lesions in this group were considered reversible. Slightly higher incidences of inflammation in the olfactory and respiratory epithelium were noted at a concentration of 18 ppm in Nasal Levels III-V. There were no test substance-related degenerative changes in the respiratory, transitional or squamous epithelium.
Interpretation of results:: study cannot be used for classification
Conclusions:: Dimethyl disulphide (DMDS) vapors administered via a 24-hour whole-body inhalation to Crl:CD(SD) rats resulted in test substance exposure related degeneration of the olfactory epithelium at exposure levels of 9, 12.5, and 18 ppm and a slight increase in inflammation of the respiratory and olfactory epithelia at 18 ppm. All findings of olfactory epithelial degeneration in the 9 and 12.5 ppm group animals were graded as minimal and considered non-adverse based on the very limited extent and severity of the lesions and the finding of degeneration in 2 control animals. Although all of the changes in the test substance-exposed groups were considered reversible, degeneration of the olfactory epithelium was considered adverse for the 18 ppm group based on the extent and severity of the lesions. Therefore, the no observed adverse-effect concentration (NOAEC) for 24-hour whole-body exposure of DMDS to Crl:CD(SD) rats was 12.5 ppm.
Executive summary:: Dimethyl disulphide (DMDS) was administered as a 24-hour, whole-body exposure to 4 groups (Groups 2-5) of Crl:CD(SD) rats. Target exposure concentrations were 5, 9, 12.5, and 18 ppm (19, 34, 48 and 69 mg/m3) for Groups 2, 3, 4, and 5, respectively. A concurrent control group (Group 1) was exposed to filtered air on a comparable regimen. The control group (Group 1) and Groups 2-5 each consisted of 10 males. One day following the 24-hour exposure, all animals were euthanized. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed prior to randomization and prior to the scheduled necropsy. Individual body weights were recorded during pretest and prior to the study day 0 exposures. A final, non-fasted body weight was also recorded prior to the scheduled necropsy. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy. Selected tissues were examined microscopically from all animals. All animals survived to the scheduled necropsy. No clinical or macroscopic findings attributed to test substance exposure were noted at any exposure concentration. Body weight was unaffected by test substance exposure. Test substance exposure-related degeneration of the olfactory epithelium was observed in Nasal Levels II-VI at exposure concentrations of 12.5 and 18 ppm, and in Nasal Levels III, IV, and V at an exposure concentration of 9 ppm. Degeneration of the olfactory epithelium was also observed in a single nasal level for 2 of 10 control group animals. The degeneration in the affected nasal sections in the 9 ppm group animals consisted of single or very few discrete, extremely small foci that consisted of cellular vacuolation and individual cell pyknosis, without sloughing. Changes in the 12.5 ppm group were also discrete areas comprising a very small percentage of the olfactory epithelium. These changes would be completely reversible and without clinical consequences and were not considered adverse. Degeneration of the olfactory epithelium was observed in 3 or more nasal levels in all 10 animals and was considered adverse in the 18 ppm group. This conclusion was based on the extent and severity of degeneration including the amount of damaged epithelium with sloughing of sensory and sustentacular cells. The lesions in this group were considered reversible. Slightly higher incidences of inflammation in the olfactory and respiratory epithelium were noted at a concentration of 18 ppm in Nasal Levels III-V. There were no test substance-related degenerative changes in the respiratory, transitional or squamous epithelium. The no-observed-adverse-effect concentration (NOAEC) for 24-hour whole-body exposure of DMDS to Crl:CD(SD) rats was 12.5 ppm (48 mg/m3).

Reference 4

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 January 2008 to 27 May 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

Evaluation of the potential toxic effects of dimethyl disulphide (DMDS) on tissues of the upper respiratory tract (URT) when administered 6 hours/day as a vapor via whole-body inhalation to rats for 1 day or 5 consecutive days.

GLP compliance:

yes

Test type:

other: A 5-day inhalation toxicity study with microscopic examination of the upper respiratory tract

Limit test:

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA
- Age at study initiation: 8-9 weeks old
- Weight at study initiation: 240 g to 305 g for males and from 178 g to 234 g for females.
- Fasting period before study: none
- Housing: individually in clean, stainless steel, wire-mesh cages suspended above cage-board.
- Diet (ad libitum): PMI Nutrition International, LLC, Certified Rodent
- Water (ad libitum): reverse osmosis-treated (on-site) drinking water,
- Acclimation period: at least 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2 to 21.8
- Humidity (%): 38.3 to 45.8
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless-steel and glass whole-body exposure chambers
- Exposure chamber volume: 1000 L
- Method of holding animals in test chamber: animals were individually housed in stainless steel wire-mesh caging suspended over cageboard during the exposures.
- Source and rate of air: HEPA-and charcoal-filtered air at a flow of 12-15 air changes per hour.
- Method of conditioning air:
- System of generating vapors: vapors of DMDS were generated by bubbling metered nitrogen through gas-washing bottles containing appropriate aliquots of the test article. Prior to entering the test chambers, the vapors of the test article were mixed with dilution air to achieve the desired exposure levels.
- Treatment of exhaust air:
- Temperature, humidity, pressure in air chamber: 20°C and 24°C, 30% to 70%, approximately 12 to 15 air changes per hour (200 to 250 LPM) at a slight negative pressure.

TEST ATMOSPHERE
- Brief description of analytical method used: analyzed concentrations of the test article in each exposure chamber were determined by a specific on-line gas chromatographic method and were measured approximately every 35 minutes throughout the exposure period. The calibration of the gas chromatograph was considered acceptable if the r2 value for the prime calibration curve was = 0.98 and the individual points of the calibration were
within 10% of their target concentrations.
- Samples taken from breathing zone: no

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

6 h

Concentrations:

50, 150, 300 and 600 ppm (192, 577, 1154 and 2318 mg/m3)

No. of animals per sex per dose:

The control group (Group 1) and Groups 2-5 each consisted of 20 animals/sex.
Following one 6-hour exposure, 10 animals/sex/group were euthanized (approximately 24 hours following the single exposure); the remaining 10 animals/sex/group were exposed for an additional 4 days (total of 5 consecutive exposure days) and were euthanized approximately 24 hours following the final exposure.

Control animals:

yes

Details on study design:

CLINICAL OBSERVATIONS AND SURVIVAL
All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.
During the exposure phase, clinical examinations were performed twice daily, prior to and following each exposure.

BODY WEIGHTS
Individual body weights were recorded during the pretest period, beginning approximately 1 week prior to test article exposure (study day -7 for males) and prior to the first, third and fifth exposures (study days 0, 2, and 4, respectively). Final body weights (fasted) were recorded prior to the scheduled necropsies.

MACROSCOPIC EXAMINATION
A complete necropsy was conducted on all animals at the scheduled necropsies.
The following tissues and organs were collected and placed in 10% neutral-buffered formalin (except as noted):
Kidneys (2)
Larynx
Liver (sections of 2 lobes)
Lungs (including bronchi, fixed by inflation with fixative)
Nasal tissues*
Trachea
Gross lesions (when possible)
* = Following fixation in formalin, nasal tissues were decalcified and sections of 6 nasal levels were prepared for microscopic examination.

ORGAN WEIGHTS
The following organs were weighed from all animals at the scheduled necropsies: kidneys, liver and lungs (prior to inflation with fixative)

MICROSCOPIC EXAMINATION
Microscopic examination was performed on nasal tissues and gross lesions from all animals in all exposure groups. Nasal sectioning was performed using the method of Morgan in which 6 nasal sections were produced using specifically defined landmarks for sectioning (Mery et al., 1994b). All 6 nasal levels were examined microscopically.

Statistics:

Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test article-treated group to the control group by sex.
Body weight, body weight change and organ weight data were subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test article-treated groups to the control group.

Key result

Sex:

male/female

Dose descriptor:

other: BMD10% for nasal irritation (severity 2)

Effect level:

19 ppm

95% CL:

>= 9.3 - <= 28.7

Exp. duration:

6 h

Remarks on result:

other: 70 (53 - 110) mg/m3. Use of severity 2 would be consistent with a determination that minimal effects are not adverse.

Mortality:

All animals survived to the scheduled necropsies.

Clinical signs:

other: There were no test article-related clinical observations.

Body weight:

Test article-related effects on body weight and/or body weight gain were noted for males and females at all exposure levels. During study days 0-2, lower mean body weight gains or mean body weight losses were noted in the 50, 150, 300 and 600 ppm group males and females; the differences from the control group were generally statistically significant (p<0.01). During study days 2-4, mean body weight gains in these same groups were significantly (p<0.01) lower for the males but similar to the control group values for the females. As a result of the body weight gain deficits, mean body weights in the 50, 150, 300 and 600 ppm group males on study days 2 and 4 were significantly (p<0.05 or p<0.01) lower than the control group values; the differences on study day 4 (8.8% to 15.4% lower) were greater than those noted for study day 2. Mean female body weights on study days 2 and 4 were similar to (50 and 150 ppm groups) or lower than (300 and 600 ppm groups) the control group values; the differences at 300 and 600 ppm (6.7% to 8.1% lower) were generally significant (p<0.01 or p<0.05).

There were no test article-related changes in mean final body or organ weights at the interim necropsy.

At the primary necropsy, mean final body weights were statistically significantly (p<0.01) lower in all test article-exposed male groups. These mean body weight changes in males showed a dose relationship at the highest 2 exposure levels (differences of 9.5%, 9.5%, 10.2% and 16.4% in the 50, 150, 300 and 600 ppm groups, respectively, compared to the control group mean) and were considered test article-related in all groups. Mean final body weights in test article-exposed female groups were not significantly different from the control group mean.

Gross pathology:

There were no test article-related macroscopic findings at the scheduled necropsies.

Other findings:

- Organ weights: In females at the primary necropsy, mean absolute lung and mean lung/body weights were higher in the 300 ppm group (7.8% and 14.9%, respectively) and the 600 ppm group (21.6% and 29.1%, respectively) when compared to the control group values. Except for the mean absolute lung weight in the 300 ppm group, these changes were statistically significant (p<0.01) and were considered test article-related. In males, the mean lung/final body weights were statistically significantly (p<0.01) higher in the 300 and 600 ppm groups (15.6% and 23.8%, respectively), but the mean absolute lung weights in these groups were similar to the control group. The relationship of lung/final body weight changes in males to test article exposure cannot be determined because the changes were potentially confounded by significantly lower mean final body weights in the test article- exposed male groups.

Other statistically significant organ weight differences were observed in males at the primary necropsy but were considered secondary to lower mean final body weights; these differences were lower mean kidney weights in the 150 and 600 ppm groups, higher mean kidney/final body weight in the 600 ppm group and lower mean liver weights in all test article-exposed groups. In females, statistically significantly higher mean kidney/body weight for the 600 ppm group at the primary necropsy was considered secondary to slightly lower but not statistically significant mean body weight in that group, since the mean absolute kidney weight for the 600 ppm group females was not different than that of the control group.

- Histopathology: (for tables see the attached document)
INTERIM NECROPSY (1-DAY EXPOSURE)
After a single 6-hour whole-body exposure to dimethyl disulfide at concentrations of 50, 150, 300 or 600 ppm, test article-related changes were observed in 3 types of epithelia (transitional, respiratory and olfactory epithelia) and in all 6 levels; changes in squamous epithelium were suggestive of an effect of exposure. Olfactory epithelium, which was consistently present in Levels III-VI, and respiratory epithelium, which was consistently present in Levels II-VI, showed decreasing lesion severity with progressively more posterior levels. Transitional epithelium, which was consistently present only in th e more anterior nasal levels (Levels I and II), generally did not show an anterior-posterior gradient of lesion severity. Degenerative changes at a particular level, except for Level I, were generally dose- related in incidence and/or severity.
Minimal acute inflammation of the squamous epithelium was observed in Level I in all test article-exposed groups and was characterized by focal to multifocal infiltrates of primarily eosinophils and neutrophils in the mucosa/submucosa, sometimes accompanied by mucosal edema, thinning, erosion and/or ulceration. Incidences are shown in Text Table 3. The incidences of this finding were not related to exposure concentration, but because the inflammation was observed only in test article-exposed groups, it was considered a probable effect of test article exposure. No test article exposure-related changes in squamous epithelium were noted in Nasal Level II.
Test article exposure-related changes in the transitional epithelium were observed in Nasal Levels I and II and are presented in Text Table 4. Degeneration of the transitional epithelium was characterized by epithelial cell flattening, vacuolation, individualization, pyknosis, erosion and/or ulceration and was observed in all test article-exposed groups in both Nasal Levels I and II. The severity of degeneration was dose-related in Nasal Level II. In Nasal Level I, the incidences and severities were dose-related in females but not in males, possibly because of the inconsistent presence of transitional epithelium in this level.
Inflammation of the transitional epithelium was also observed in Levels I and II, and was characterized by edema and polymorphonuclear infiltrates within the mucosa and/or submucosa, with occasional polymorphonuclear luminal exudates when ulcers were present. In Nasal Level II, all animals from the 150 ppm and higher groups, as well as most males and half of the females from the 50 ppm group, were affected. The severity of acute inflammation was dose-related in males in Level II, while in females in Level II, the severity was higher in the 150 ppm and higher exposure groups than in the 50 ppm group but did not show a clear dose response. In Level I, all test article-exposed groups except males exposed to 150 ppm were affected, but there was no dose response in incidence or severity, possibly because of the inconsistent presence of transitional epithelium in this level, as previously mentioned. Test article exposure-related changes in the respiratory epithelium were observed in all 6 nasal levels and are presented in Text Table 5.
Degeneration of the respiratory epithelium was characterized by flattening, loss of cilia, vacuolation and/or pyknosis of the epithelial cells. Mild degeneration was observed in a few of the 300 and 600 ppm group males and 600 ppm group females in Nasal Level I. Degeneration was observed most frequently in Levels II and III at higher exposure concentrations, with the highest incidence and severity at 600 ppm in these levels. The incidence and severity of respiratory epithelial degeneration decreased in the more caudal levels; in Level IV it was only observed at 600 ppm, and it was not observed at all in Levels V or VI.
An increased incidence of acute inflammation of the respiratory epithelium was noted in males and females in at least 1 nasal level in all test article exposure groups. The test article exposure groups affected varied with the nasal level. The incidence and severity of inflammation at a given exposure concentration of test article generally decreased in nasal levels caudal to Nasal Level III. Additionally, inflammation was observed farther caudally at higher exposure concentrations. For instance, in males, acute inflammation of the respiratory epithelium was observed in Level II at 50 ppm, in Levels II and III at 150 ppm, and in Levels II-IV at 300 and 600 ppm. Incidences of inflammation in male Nasal Levels V and VI were similar to control incidences. In females, respiratory epithelial inflammation extended to Nasal Level V at 150, 300 and 600 ppm and to Nasal Level VI at 600 ppm.
Test article exposure-related degeneration and/or inflammation of the olfactory epithelium were observed in Nasal Levels II-VI, as presented in Text Tables 6 and 7. Degeneration of the olfactory epithelium was characterized by sloughing of sensory and sustentacular cells that resulted in only a thin layer of basal cells remaining. Less severe degeneration was noted as focal or multifocal pyknosi s and/or vacuolation of sensory epithelium resulting in an irregular surface and decreased nuclear density from sensory cell loss. Bowman’s glands and nerve bundles in the lamina propria were not affected at any test article exposure concentration. At all levels except Level II, olfactory epithelial degeneration showed a dose-response relationship in incidence and/or severity at all test article exposure concentrations. Additionally, at a particular exposure, the incidence and/or severity of degeneration generally decreased with progressively more caudal sections. Higher exposure concentrations generally caused more degeneration farther caudally than lower exposure concentrations.
Acute inflammation of the olfactory epithelium, observed in Levels II-VI, was noted in at least 1 nasal level at all test article exposure levels. Inflammation generally showed a dose response in incidence and/or severity with higher exposure concentration for a given nasal level, and occurred with lower incidence and severity with progressively farther caudal sections at a given exposure concentration.

PRIMARY NECROPSY (5-DAY EXPOSURE)
After 5 consecutive days of 6-hour/day whole-body exposure to dimethyl disulfide at concentrations of 50, 150, 300 or 600 ppm, test article-related changes were observed in all 4 epithelial cell types and in all 6 nasal levels. As at the interim necropsy, olfactory and respiratory epithelia showed decreasing incidence and/or severity of degeneration and inflammation with progressively more caudal level s at a given exposure concentration. Squamous and transitional epithelia generally did not show an anteriorposterior gradient of lesion severity. All 4 types of epithelia showed evidence of regeneration/ hyperplasia despite continued exposure to the test article, with an anteriorposterior gradient of incidence/severity for olfactory and respiratory epithelia.
Changes in squamous epithelium were observed in Levels I and II and are presented in Text Table 8. Minimal to mild acute inflammation of the squamous epithelium was observed sporadically in Nasal Level I. At this level, the highest incidence in both males and females was in the 300 ppm exposure group. Despite the lack of a dose response, inflammation was considered to be test article-related because it was not observed in the control group.
Hyperplasia of the squamous epithelium was observed as thickening of the epithelial cell layer. In females, it was noted in all test article-exposed groups in Nasal Level I. In males, squamous epithelial hyperplasia was limited to the 300 and 600 ppm exposure concentrations in Nasal Levels I and II. There was a general increase in incidence/severity with exposure concentration except at the highest exposure level in females.
Test article exposure-related changes in the transitional epithelium were observed in Nasal Levels I and II and are presented in Text Table 9.
Test article-related changes in the transitional epithelium consisted of degeneration, hyperplasia and inflammation. Degeneration was observed in all test article-exposed male and female groups in Nasal Level II. At this level, compared to the study day 1 necropsy, the overall severity of degeneration was decreased for all test article-exposed groups and the incidences of degeneration were decreased for the 50 and 150 ppm groups, possibly because of the presence of transitional epithelial hyperplasia at study day 5.
Hyperplasia of the transitional epithelium was characterized by increased thickness of the epithelial layer, nuclear disorganization, and/or loss of apical cytoplasm, often with flattening of the adluminal cells. Hyperplasia was observed in all test article exposure groups in Nasal Level II, but was less consistent in Nasal Level I, possibly because transitional epithelium was inconsistently present in that level.
Inflammation of the transitional epithelium was also observed in Nasal Levels I and II. The incidence in all test article-exposed female groups and 150, 300 and 600 ppm male groups was increased compared to controls in at least 1 level. Inflammation was most consistently observed in Nasal Level
II. At the primary necropsy, the severity of transitional epithelial inflammation was generally higher in the more caudal level (Level 2) compared to the interim necropsy for all exposure concentration
s. One 300 ppm group male had osteitis of the nasoturbinate in Nasal Level II that resulted from inflammation and ulceration of the overlying transitional epithelium and exposure of the bone.
Test article exposure-related changes in the respiratory epithelium after 5 consecutive days of whole body exposure to DMDS consisted of degeneration, hyperplasia and acute inflammation. Incidences of selected changes are presented in Text Table 10.
Unlike at the interim necropsy, in which respiratory epithelial degeneration was observed at 50 ppm and higher exposure concentrations, at the primary necropsy respiratory epithelial degeneration was observed only at the 600 ppm exposure concentration. There were no consistent changes in the incidences or severities of degeneration at 600 ppm when the interim and primary necropsies were compared, although degeneration was observed farther caudal (in Nasal Level V) in females at study day 5 compared to study day 1. The apparent lack of degeneration at lower exposure concentrations at the primary necropsy may be secondary to the presence of respiratory epithelial hyperplasia.
Hyperplasia of the respiratory epithelium was characterized by increased thickness of the epithelial layer, nuclear disorganization, and/or loss of cilia and apical cytoplasm, often with flattening of the adluminal cells. Hyperplasia was inconsistent in Nasal Level II, possibly because of the variable presence of respiratory vs. transitional epithelium in this level. Hyperplasia was consistently observed in Nasal Level III at exposure concentrations of 50 ppm and higher, and generally showed a dose response relationship in incidence and/or severity in this nasal level. Respiratory epithelial hyperplasia was not observed caudal to Nasal Level III at 50 ppm and was observed in Nasal Level IV, but not Levels V or VI, at 150 ppm. At the two highest exposure levels, respiratory epithelial hyperplasia extended to Nasal Level VI, although the incidence and severity decreased in the 300 ppm exposure concentration group in these caudal levels.
Acute inflammation of the respiratory epithelium was observed at the 300 ppm and higher exposure concentrations in males and the 150 ppm and higher exposure concentrations in females; findings are presented in Text Table 11, below. There was a gradient of higher to lower incidences of inflammation with more caudal sections at a given exposure concentration. When compared to the 1-day exposure groups, test article exposure-related inflammation in the 5-day exposure groups was observed only at higher exposure concentrations for a given nasal level, but generally occurred at similar incidences and severities at these concentrations.
Test article exposure-related degeneration, regeneration and/or inflammation of the olfactory epithelium were observed in Nasal Levels II-VI; findings are presented in Text Tables 12, 13 and 14. Whole body exposure to the test article for 5 days resulted in olfactory epithelial degeneration in Nasal Levels II-VI at all exposure concentrations.
At 50 ppm, the incidences of degeneration were higher after 5 days of exposure in Nasal Levels II and III in males and in all nasal levels in females. Also in females, degeneration was observed farther caudal in the 50 and 150 ppm 5-day exposure concentrations than was observed at these concentrations after the single exposure. For both males and females, the severity of degeneration in the more anterior nasal levels (Nasal Levels II, III and IV) was generally less severe for a given exposure concentration after 5 days of exposure than after a single day exposure, primarily because of olfactory epithelial regeneration.
Minimal to mild olfactory epithelial regeneration was present with degeneration in all test article-exposed groups of males and females in Nasal Levels II-VI. Regeneration consisted of areas of 1 or 2 layers of disorganized basophilic cells overlying the previously desquamated basal cell layer. Areas of regeneration were sometimes observed adjacent to areas of acute degeneration. Incidences of regeneration in Nasal Levels II-V were >80% and frequently near 100% for exposure concentrations of 150 ppm and higher, and decreased in incidence and severity in Nasal Level VI. At 50 ppm, incidences of olfactory epithelial regeneration became progressively lower caudal to Nasal Level III, but extended to Nasal Level VI.
Acute inflammation of the olfactory epithelium was observed in Levels III-VI at similar incidences and severities as were noted at the study day 1 interim necropsy. At a given nasal level and exposure concentration, there were no consistent differences in the incidence or severity of inflammation between animals exposed to DMDS for 1 or 5 days. As at the interim necropsy, the incidence of inflammation of the olfactory epithelium tended to increase with higher exposure concentration at a given nasal level, and occurred with lower incidence and severity in the caudal nasal levels at a given exposure; findings are presented in Text Table 14.
In addition to changes in epithelia, whole-body exposure to DMDS for 5 consecutive days resulted in changes in the bony trabeculae of the nasal turbinates. Fibro-osseous proliferation of the bony trabeculae of the naso-, maxillo-, and/or ethmo-turbinates in Nasal Levels II-IV was observed in at least 1 nasal level in the 150 ppm and higher group males and 300 ppm and higher group females. Fibro-osseous proliferation was characterized by the presence of lightly basophilic new bone and increased fibroblast-like cells near the periosteal surface; findings are presented in Text Table 15. The incidence and severity of this lesion in Nasal Level II were related to exposure concentration, and showed an anterior-posterior decrease at a given exposure concentration.
There were no other test article-related microscopic changes observed. All other microscopic observations were considered spontaneous or incidental, with no relationship to text article exposure.

- Potential target organs: nasal epithelium

INTERIM NECROPSY (1-DAY EXPOSURE)

Severity grade	Severity score
minimal	1
mild	2
moderate	3
severe	4

Text Table 3. Incidences of Acute Inflammation in Squamous Epithelium,

Study Day 1 Interim Necropsy

	Males					Females
Target Exposure Level (ppm):
	0	50	150	300	600	0	50	150	300	600
Squamous epithelium, acute inflammation
Nasal Level Ia	10	10	10	10	10	10	10	10	10	10
Minimal	0	2	2	4	2	0	1	2	3	1
Mean score	0	0.2	0.2	0.4	0.2	0	0.1	0.2	0.3	0.1

a= Number of tissues examined from each group.

Text Table 4. Incidences of Changes in Transitional Epithelium,

Study Day 1 Interim Necropsy

	Males					Females
Target Exposure Level (ppm):	0	50	150	300	600	0	50	150	300	600
Transitional epithelium, degeneration
Nasal Level Ia	10	10	10	10	10	10	10	10	10	10
Total incidence	0	1	1	4	1	0	1	3	8	9
Minimal	0	1	1	2	0	0	1	3	4	3
Mild	0	0	0	1	0	0	0	0	4	4
Moderate	0	0	0	1	1	0	0	0	0	1
Severe	0	0	0	0	0	0	0	0	0	1
Mean score	0	0.1	0.1	0.7	0.3	0	0.1	0.3	1.2	1.8
Nasal Level IIa	10	10	10	10	10	10	10	10	10	10
Total incidence	0	9	10	10	0	0	3	10	10	10
Minimal	0	7	2	0	0	0	3	0	0	0
mild	0	2	2	0	0	0	0	2	0	0
Moderate	0	0	3	3	0	0	0	4	5	2
Severe	0	0	3	7	10	0	0	4	5	8
Mean score	0	1.1	2.7	3.7	4.0	0	0.3	3.2	3.5	3.8
Transitional epithelium, acute inflammation
Nasal Level Ia	10	10	10	10	10	10	10	10	10	10
Total incidence	0	2	0	4	1	0	6	2	8	9
Minimal	0	2	0	4	1	0	6	0	7	6
Mild	0	0	0	0	0	0	0	2	1	3
Mean score	0	0.2	0	0.4	0.1	0	0.6	0.4	0.9	1.2
Nasal Level IIa	10	10	10	10	10	10	10	10	10	10
Total incidence	3	9	10	10	10	0	5	10	10	10
Minimal	3	9	7	4	0	0	5	6	9	6
Mild	0	0	3	6	10	0	0	4	1	4
Mean score	0	0.9	1.3	1.6	2.0	0	0.5	1.4	1.1	1.4

a= Number of tissues examined from each group.

Text Table 5. Incidences of Changes in Respiratory Epithelium,
Study Day 1 Interim Necropsy

	Males					Females
Target Exposure Level (ppm):	0	50	150	300	600	0	50	150	300	600
Respiratory epithelium, degeneration
Nasal Level I^a	10	10	10	10	10	10	10	10	10	10
total incidence (%)	0 (0)	0 (0)	0 (0)	2 (20)	1 (10)	0 (0)	0 (0)	0 (0)	0 (0)	3 (30)
mild	0	0	0	2	1	0	0	0	0	3
Mean score	0	0	0	0.4	0.2	0	0	0	0	0.6
Nasal Level II^a	10	10	10	10	10	10	10	10	10	10
total incidence (%)	0 (0)	0 (0)	1 (10)	2 (20)	7 (70)	0 (0)	1 (10)	0 (0)	1 (10)	3 (30)
minimal	0	0	1	2	6	0	1	0	1	3
mild	0	0	0	0	1	0	0	0	0	0
Mean score	0	0	0.1	0.2	0.8	0	0.1	0	0.1	0.3
Nasal Level III^a	10	7	8	7	10	9	9	8	8	8
total incidence (%)	0 (0)	0 (0)	1 (13)	1 (14)	5 (50)	0 (0)	0 (0)	1 (13)	0 (0)	5 (63)
minimal	0	0	1	0	1	0	0	1	0	3
mild	0	0	0	1	4	0	0	0	0	2
Mean score	0	0	0.1	0.3	0.9	0	0	0.1	0	0.9
Nasal Level IV^a	8	6	5	6	6	5	2	7	7	6
total incidence (%)	0 (0)	0 (0)	0 (0)	0 (0)	2 (33)	0 (0)	0 (0)	0 (0)	0 (0)	3 (50)
minimal	0	0	0	0	2	0	0	0	0	3
Mean score	0	0	0	0	0.3	0	0	0	0	0.5
Respiratory epithelium, acute inflammation
Nasal Level I^a	10	10	10	10	10	10	10	10	10	10
total incidence (%)	1 (10)	0 (0)	0 (0)	2 (20)	1 (10)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
minimal	2	0	0	2	1	0	0	0	0	0
Mean score	0	0	0	0.2	0.1	0	0	0	0	0
Nasal Level II^a	10	10	10	10	10	10	10	10	10	10
total incidence (%)	3 (30)	6 (60)	8 (80)	8 (80)	10 (100)	0 (0)	2 (20)	6 (60)	7 (70)	4 (40)
minimal	3	5	8	6	8	0	2	6	7	4
mild	0	1	0	2	2	0	0	0	0	0
Mean score	0	0.7	0.8	1.0	1.2	0	0.2	0.6	0.7	0.4
Nasal Level III^a	10	7	8	7	10	9	9	8	8	8
total incidence (%)	1 (10)	0 (0)	5 (63)	6 (86)	7 (70)	0 (0)	0 (0)	2 (25)	6 (75)	7 (88)
minimal	1	0	5	6	5	0	0	2	6	6
mild	0	0	0	0	2	0	0	0	0	1
Mean score	0	0	0.6	0.8	0.9	0	0	0.3	0.8	1.0
Nasal Level IV^a	8	6	5	6	6	5	2	7	7	6
total incidence (%)	3 (38)	0 (0)	2 (40)	5 (83)	5 (83)	0 (0)	1 (50)	3 (43)	4 (57)	6 (100)
minimal	3	0	2	5	4	0	1	3	4	5
mild	0	0	0	0	1	0	0	0	0	1
Mean score	0	0	0.4	0.9	1.0	0	0.5	0.4	0.6	1.2
Nasal Level V^a	9	8	10	8	10	8	10	10	10	10
total incidence (%)	2 (22)	0 (0)	1 (10)	2 (25)	3 (30)	0 (0)	0 (0)	1 (10)	1 (10)	4 (40)
minimal	2	0	1	2	3	0	0	1	1	4
Mean score	0	0	0.1	0.3	0.3	0	0	0.1	0.1	0.4
Nasal Level VI^a	9	10	9	10	9	9	10	9	10	9
total incidence (%)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (11)
minimal	0	0	0	0	0	0	0	0	0	1
Mean score	0	0	0	0	0	0	0	0	0	0.1

^a= Number of tissues examined from each group.

Text Table 6. Incidences of Degeneration of Olfactory Epithelium,
Study Day 1 Interim Necropsy

	Males					Females
Target Exposure Level (ppm):	0	50	150	300	600	0	50	150	300	600
Olfactory epithelium, degeneration
Nasal Level II^a	10	10	10	10	10	10	10	10	10	10
total incidence	0	6	10	9	10	0	2	10	10	9
minimal	0	3	1	0	2	0	0	1	0	1
mild	0	2	1	1	0	0	0	1	0	0
moderate	0	1	1	3	4	0	1	1	1	1
severe	0	0	7	5	4	0	1	7	9	7
Mean score	0	1.0	3.4	3.1	3.0	0	0.7	3.4	3.9	3.2
Nasal Level III^a	10	7	8	7	10	9	9	8	8	8
total incidence	0	6	8	7	10	0	2	8	8	8
minimal	0	1	0	0	0	0	1	1	0	0
mild	0	4	1	0	0	0	1	3	1	0
moderate	0	1	6	3	4	0	0	4	7	5
severe	0	0	1	4	6	0	0	0	0	3
Mean score	0	1.4	3.0	3.6	3.6	0	0.3	2.4	2.9	3.4
Nasal Level IV^a	8	6	5	6	6	5	2	7	7	6
total incidence	0	4	5	6	6	0	1	7	7	6
minimal	0	4	2	0	0	0	0	4	0	0
mild	0	0	2	4	0	0	1	3	3	1
moderate	0	0	1	2	4	0	0	0	4	4
severe	0	0	0	0	2	0	0	0	0	1
Mean score	0	0.7	1.4	2.3	3.3	0	1.0	1.4	2.6	3.0
Nasal Level V^a	9	8	10	8	10	8	10	10	10	10
total incidence	0	5	8	8	10	0	0	10	10	10
minimal	0	5	8	5	0	0	0	10	3	0
mild	0	0	0	3	5	0	0	0	7	3
moderate	0	0	0	0	5	0	0	0	0	6
severe	0	0	0	0	0	0	0	0	0	1
Mean score	0	0.6	0.8	1.4	2.5	0	0	1.0	1.7	2.8
Nasal Level VI^a	9	10	9	10	9	9	10	9	10	9
total incidence	0	2	4	9	9	0	0	0	3	7
minimal	0	2	4	5	4	0	0	0	3	3
mild	0	0	0	4	4	0	0	0	0	3
moderate	0	0	0	0	1	0	0	0	0	1
Mean score	0	0.2	0.4	1.3	1.6	0	0	0	0.3	1.4

^a= Number of tissues examined from each group

Text Table 7. Incidences of Acute Inflammation of Olfactory Epithelium,
Study Day 1 Interim Necropsy

	Males					Females
Target Exposure Level (ppm):	0	50	150	300	600	0	50	150	300	600
Olfactory epithelium, acute inflammation
Nasal Level II^a	10	10	10	10	10	10	10	10	10	10
total incidence	0	1	5	1	3	0	1	2	4	2
minimal	0	1	5	1	3	0	1	2	4	2
Mean score	0	0.1	0.5	0.1	0.3	0	0.1	0.2	0.4	0.2
Nasal Level III^a	10	7	8	7	10	9	9	8	8	8
total incidence	0	1	4	5	8	0	0	2	8	6
minimal	0	1	3	4	6	0	0	2	8	6
mild	0	0	1	1	2	0	0	0	0	0
Mean score	0	0.1	0.6	0.9	1.0	0	0	0.2	1.0	0.8
Nasal Level IV^a	8	6	5	6	6	5	2	7	7	6
total incidence	0	0	3	4	6	0	0	2	7	6
minimal	0	0	3	3	4	0	0	2	7	6
mild	0	0	0	1	2	0	0	0	0	0
Mean score	0	0	0.6	0.8	1.3	0	0	0.3	1.0	1.0
Nasal Level V^a	9	8	10	8	10	8	10	10	10	10
total incidence	0	0	0	3	7	0	0	0	3	4
minimal	0	0	0	3	6	0	0	0	3	4
mild	0	0	0	0	1	0	0	0	0	0
Mean score	0	0	0	0.4	0.8	0	0	0	0.3	0.4
Nasal Level VI^a	9	10	9	10	9	9	10	9	10	9
total incidence	0	0	0	0	1	0	0	0	0	0
minimal	0	0	0	0	1	0	0	0	0	0
Mean score	0	0	0	0	0.1	0	0	0	0	0

^a= Number of tissues examined from each group.

FINAL NECROPSY (5-DAY EXPOSURE)

Text Table8. Incidences of Changes in Squamous Epithelium,
Study Day 5 Primary Necropsy

	Males					Females
Target Exposure Level (ppm):	0	50	150	300	600	0	50	150	300	600
Squamous epithelium, acute inflammation
Nasal Level I^a	10	10	10	10	10	10	10	10	10	10
total incidence	0	1	0	4	1	0	0	0	6	1
minimal	0	1	0	4	1	0	0	0	4	1
mild	0	0	0	0	0	0	0	0	2	0
Mean score	0	0.1	0	0.4	0.1	0	0	0	0.8	0.1
Nasal Level II^a	10	10	10	10	10	10	10	10	10	10
total incidence	0	0	0	0	1	0	0	0	0	0
minimal	0	0	0	0	1	0	0	0	0	0
Mean score	0	0	0	0	0.1	0	0	0	0	0
Squamous epithelium, hyperplasia
Nasal Level I^a	10	10	10	10	10	10	10	10	10	10
total incidence	0	0	0	5	1	0	2	6	9	5
minimal	0	0	0	3	1	0	2	6	3	3
mild	0	0	0	2	0	0	0	0	6	2
Mean score	0	0	0	0.7	0.1	0	0.2	0.6	1.5	0.7
Nasal Level II^a	10	10	10	10	10	10	10	10	10	10
total incidence	0	0	0	1	2	0	0	0	0	0
minimal	0	0	0	0	1	0	0	0	0	0
mild	0	0	0	1	1	0	0	0	0	0
Mean score	0	0	0	0.2	0.3	0	0	0	0	0

^a= Number of tissues examined from each group.

Text Table 9. Incidences of Changes in Transitional Epithelium,
Study Day 5 Primary Necropsy

	Males					Females
Target Exposure Level (ppm):	0	50	150	300	600	0	50	150	300	600
Transitional epithelium, degeneration
Nasal Level II^a	10	10	10	10	10	10	10	10	10	10
total incidence	0	4	2	10	9	0	3	1	10	10
minimal	0	4	2	3	3	0	2	1	5	2
mild	0	0	0	7	6	0	1	0	5	8
Mean score	0	0.4	0.2	1.7	1.5	0	0.4	0.1	1.5	1.8
Transitional epithelium, acute inflammation
Nasal Level I^a	10	10	10	10	10	10	10	10	10	10
total incidence	0	0	0	2	1	0	1	0	1	1
minimal	0	0	0	2	1	0	1	0	1	1
Mean score	0	0	0	0.2	0.1	0	0.1	0	0.1	0.1
Nasal Level II^a	10	10	10	10	10	10	10	10	10	10
total incidence	3	3	7	9	9	0	2	5	10	10
minimal	3	3	7	3	3	0	2	5	4	3
mild	0	0	0	6	6	0	0	0	6	7
Mean score	0	0.3	0.7	1.5	1.5	0	0.2	0.5	1.6	1.7
Transitional epithelium, hyperplasia
Nasal Level I^a	10	10	10	10	10	10	10	10	10	10
total incidence	0	0	0	2	1	0	4	5	5	0
minimal	0	0	0	0	1	0	4	1	5	0
mild	0	0	0	2	0	0	0	4	0	0
Mean score	0	0	0	0.4	0.1	0	0.4	0.9	0.5	0
Nasal Level II^a	10	10	10	10	10	10	10	10	10	10
total incidence	0	9	10	10	9	0	8	10	10	10
minimal	0	3	0	0	0	0	5	0	1	1
mild	0	6	10	10	9	0	3	10	9	9
Mean score	0	1.5	2.0	2.0	1.8	0	1.1	2.0	1.9	1.9

^a= Number of tissues examined from each group.

Text Table 10. Incidences of Changes in Respiratory Epithelium,
Study Day 5 Primary Necropsy

	Males					Females
Target Exposure Level (ppm):	0	50	150	300	600	0	50	150	300	600
Respiratory epithelium, degeneration
Nasal Level II^a	10	10	10	10	10	10	10	10	10	10
total incidence (%)	0 (0)	0 (0)	0 (0)	0 (0)	1 (10)	0 (0)	0 (0)	0 (0)	0 (0)	3 (30)
minimal	0	0	0	0	0	0	0	0	0	3
mild	0	0	0	0	1	0	0	0	0	0
Mean score	0	0	0	0	0.2	0	0	0	0	0.3
Nasal Level III^a	10	10	10	10	10	10	8	10	9	10
total incidence (%)	0 (0)	0 (0)	0 (0)	0 (0)	2 (20)	0 (0)	0 (0)	0 (0)	0 (0)	7 (70)
minimal	0	0	0	0	2	0	0	0	0	6
mild	0	0	0	0	0	0	0	0	0	1
Mean score	0	0	0	0	0.2	0	0	0	0	0.8
Nasal Level IV^a	8	7	6	6	9	6	7	4	7	4
total incidence (%)	0 (0)	0 (0)	0 (0)	0 (0)	1 (11)	0 (0)	0 (0)	0 (0)	0 (0)	3 (75)
minimal	0	0	0	0	1	0	0	0	0	3
Mean score	0	0	0	0	0.1	0	0	0	0	0.8
Nasal Level V^a	9	8	8	10	10	10	9	10	9	10
total incidence (%)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	2 (20)
minimal	0	0	0	0	0	0	0	0	0	1
mild	0	0	0	0	0	0	0	0	0	1
Mean score	0	0	0	0	0	0	0	0	0	0.3
Respiratory epithelium, hyperplasia
Nasal Level II^a	10	10	10	10	10	10	10	10	10	10
total incidence (%)	0 (0)	1 (10)	1 (10)	5 (50)	8 (80)	0 (0)	0 (0)	0 (0)	4 (40)	6 (60)
minimal	0	1	1	4	5	0	0	0	4	6
mild	0	0	0	1	3	0	0	0	0	0
Mean score	0	0.1	0.1	0.6	1.1	0	0	0	0.4	0.6
Nasal Level III^a	10	10	10	10	10	10	8	10	9	10
total incidence (%)	0 (0)	2 (20)	8 (80)	10 (100)	9 (90)	0 (0)	3 (38)	6 (60)	9 (100)	10 (100)
minimal	0	2	7	5	0	0	3	5	6	4
mild	0	0	1	5	9	0	0	1	3	6
Mean score	0	0.2	0.9	1.5	1.8	0	0.4	0.7	1.3	1.6
Nasal Level IV^a	8	7	6	6	9	6	7	4	7	4
total incidence (%)	0 (0)	0 (0)	0 (0)	5 (83)	8 (89)	0 (0)	0 (0)	2 (50)	6 (86)	4 (100)
minimal	0	0	0	5	0	0	0	2	4	1
mild	0	0	0	0	8	0	0	0	2	3
Mean score	0	0	0	0.5	1.8	0	0	0.5	1.1	1.8
Nasal Level V^a	9	8	8	10	10	10	9	10	9	10
total incidence (%)	0 (0)	0 (0)	0 (0)	10 (100)	10 (100)	0 (0)	0 (0)	0 (0)	6 (67)	10 (100)
minimal	0	0	0	9	1	0	0	0	5	5
mild	0	0	0	1	9	0	0	0	1	5
Mean score	0	0	0	1.1	1.9	0	0	0	0.8	1.5
Nasal Level VI^a	10	9	10	9	8	10	10	10	8	10
total incidence (%)	0 (0)	0 (0)	0 (0)	1 (11)	8 (100)	0 (0)	0 (0)	0 (0)	2 (25)	9 (90)
minimal	0	0	0	1	1	0	0	0	2	6
Mild	0	0	0	0	7	0	0	0	0	3
Mean score	0	0	0	0.1	1.9	0	0	0	0.3	1.2

^a= Number of tissues examined from each group.

Text Table 11. Incidences of Acute Inflammation in Respiratory Epithelium,
Study Day 5 Primary Necropsy

	Males					Females
Target Exposure Level (ppm):	0	50	150	300	600	0	50	150	300	600
Respiratory epithelium, acute inflammation
Nasal Level II^a	10	10	10	10	10	10	10	10	10	10
total incidence (%)	2 (20)	5 (50)	4 (40)	3 (30)	5 (50)	1 (10)	0 (0)	0 (0)	3 (30)	6 (60)
minimal	2	5	4	3	5	1	0	0	3	5
mild	0	0	0	0	0	0	0	0	0	1
Mean score	0	0.5	0.4	0.3	0.5	0	0	0	0.3	0.7
Nasal Level III^a	10	10	10	10	10	10	8	10	9	10
total incidence (%)	1 (10)	0 (0)	1 (10)	5 (50)	9 (90)	0 (0)	0 (0)	1 (10)	5 (56)	8 (80)
minimal	1	0	1	5	6	0	0	1	4	5
mild	0	0	0	0	3	0	0	0	1	3
Mean score	0	0	0.1	0.5	1.2	0	0	0.1	0.7	1.1
Nasal Level IV^a	8	7	6	6	9	6	7	4	7	4
total incidence (%)	1 (13)	0 (0)	0 (0)	1 (17)	7 (78)	0 (0)	0 (0)	0 (0)	1 (14)	3 (75)
minimal	1	0	0	1	5	0	0	0	1	2
mild	0	0	0	0	2	0	0	0	0	1
Mean score	0	0	0	0.2	1.0	0	0	0	0.1	1.0
Nasal Level V^a	9	8	8	10	10	10	9	10	9	10
total incidence (%)	0 (0)	0 (0)	0 (0)	0 (0)	2 (20)	0 (0)	0 (0)	0 (0)	0 (0)	3 (30)
minimal	0	0	0	0	2	0	0	0	0	3
Mean score	0	0	0	0	0.2	0	0	0	0	0.3
Nasal Level VI^a	10	9	10	9	8	10	10	10	8	10
total incidence (%)	0 (0)	0 (0)	0 (0)	0 (0)	1 (13)	0 (0)	0 (0)	0 (0)	0 (0)	2 (20)
minimal	0	0	0	0	1	0	0	0	0	2
Mean score	0	0	0	0	0.1	0	0	0	0	0.2

^a= Number of tissues examined from each group.

Text Table 12. Incidences of Degeneration of Olfactory Epithelium,
Study Day 5 Primary Necropsy

	Males					Females
Target Exposure Level (ppm):	0	50	150	300	600	0	50	150	300	600
Olfactory epithelium, degeneration
Nasal Level II^a	10	10	10	10	10	10	10	10	10	10
total incidence	0	9	10	9	8	0	9	10	10	10
minimal	0	5	1	0	1	0	6	0	0	0
mild	0	4	2	1	0	0	3	4	2	1
moderate	0	0	7	8	7	0	0	6	8	9
Mean score	0	1.3	2.6	2.6	2.2	0	1.2	2.6	2.8	2.9
Nasal Level III^a	10	10	10	10	10	10	8	10	9	10
total incidence	0	10	10	10	9	0	8	10	9	10
minimal	0	2	0	0	0	0	5	0	0	0
mild	0	8	0	0	0	0	1	2	0	0
moderate	0	0	10	10	9	0	2	8	9	10
Mean score	0	1.8	3.0	3.0	2.7	0	1.6	2.8	3.0	3.0
Nasal Level IV^a	8	7	6	6	9	6	7	4	7	4
total incidence	1	7	6	6	8	0	4	4	7	4
minimal	1	4	0	0	0	0	2	0	0	0
mild	0	3	5	1	0	0	2	2	2	0
moderate	0	0	1	5	8	0	0	2	5	4
Mean score	0	1.4	2.2	2.8	2.7	0	0.9	2.5	2.7	3.0
Nasal Level V^a	9	8	8	10	10	10	9	10	9	10
total incidence	0	6	8	10	10	0	3	10	9	10
minimal	0	6	5	1	2	0	3	9	2	0
mild	0	0	3	6	5	0	0	1	5	5
moderate	0	0	0	3	3	0	0	0	2	5
Mean score	0	0.8	1.4	2.2	2.1	0	0.3	1.1	2.0	2.5
Nasal Level VI^a	10	9	10	9	8	10	10	10	8	10
total incidence	0	2	6	8	7	0	2	4	6	8
minimal	0	2	2	4	3	0	2	4	4	6
mild	0	0	3	4	4	0	0	0	1	2
moderate	0	0	1	0	0	0	0	0	1	0
Mean score	0	0.2	1.1	1.3	1.4	0	0.2	0.4	1.1	1.0

^a= Number of tissues examined from each group.

Text Table 13. Incidences of Regeneration of Olfactory Epithelium,
Study Day 5 Primary Necropsy

	Males					Females
Target Exposure Level (ppm):	0	50	150	300	600	0	50	150	300	600

Olfactory epithelium, regeneration
Nasal Level II^a	10	10	10	10	10	10	10	10	10	10
total incidence (%)	0 (0)	8 (80)	10 (100)	9 (90)	8 (80)	0 (0)	8 (80)	10 (100)	10 (100)	10 (100)
minimal	0	5	3	1	3	0	6	3	1	2
mild	0	3	7	8	5	0	2	7	9	8
Mean score	0	1.1	1.7	1.7	1.3	0	1.0	1.7	1.9	1.8
Nasal Level III^a	10	10	10	10	10	10	8	10	9	10
total incidence (%)	0 (0)	9 (90)	10 (100)	10 (100)	9 (90)	0 (0)	4 (50)	10 (100)	9 (100)	10 (100)
minimal	0	4	0	0	0	0	1	0	0	3
mild	0	5	10	10	9	0	3	10	9	7
Mean score	0	1.4	2.0	2.0	1.8	0	0.9	2.0	2.0	1.7
Nasal Level IV^a	8	7	6	6	9	6	7	4	7	4
total incidence (%)	0 (0)	4 (57)	6 (100)	6 (100)	8 (89)	0 (0)	3 (43)	4 (100)	7 (100)	4 (100)
minimal	0	3	2	0	1	0	2	0	3	0
mild	0	1	4	6	7	0	1	4	4	4
Mean score	0	0.7	1.7	2.0	1.7	0	0.6	2.0	1.6	2.0
Nasal Level V^a	9	8	8	10	10	10	9	10	9	10
total incidence (%)	0 (0)	2 (25)	8 (100)	10 (100)	10 (100)	0 (0)	2 (22)	10 (100)	9 (100)	10 (100)
minimal	0	2	8	6	7	0	2	10	7	2
mild	0	0	0	4	3	0	0	0	2	8
Mean score	0	0.3	1.0	1.4	1.3	0	0.2	1.0	1.2	1.8
Nasal Level VI^a	10	9	10	9	8	10	10	10	8	10
total incidence (%)	0 (0)	2 (22)	4 (40)	7 (78)	6 (75)	0 (0)	1 (10)	1 (10)	4 (50)	7 (70)
minimal	0	2	4	6	6	0	1	1	4	7
mild	0	0	0	1	0	0	0	0	0	0
Mean score	0	0.2	0.4	0.9	0.8	0	0.1	0.1	0.5	0.7

^a= Number of tissues examined from each group.

Text Table 14. Incidences of Acute Inflammation in Olfactory Epithelium,
Study Day 5 Primary Necropsy

	Males					Females
Target Exposure Level (ppm):	0	50	150	300	600	0	50	150	300	600
Olfactory epithelium, acute inflammation
Nasal Level II^a	10	10	10	10	10	10	10	10	10	10
total incidence (%)	0 (0)	0 (0)	2 (20)	2 (20)	6 (60)	0 (0)	0 (0)	1 (10)	6 (60)	3 (30)
minimal	0	0	2	2	5	0	0	1	6	3
mild	0	0	0	0	1	0	0	0	0	0
Mean score	0	0	0.2	0.2	0.7	0	0	0.1	0.6	0.3
Nasal Level III^a	10	10	10	10	10	10	8	10	9	10
total incidence (%)	0 (0)	0 (0)	7 (70)	9 (90)	6 (60)	0 (0)	0 (0)	4 (40)	7 (78)	9 (90)
minimal	0	0	7	8	4	0	0	4	7	9
mild	0	0	0	1	2	0	0	0	0	0
Mean score	0	0	0.7	1.0	0.8	0	0	0.4	0.8	0.9
Nasal Level IV^a	8	7	6	6	9	6	7	4	7	4
total incidence (%)	0 (0)	1 (14)	1 (17)	2 (33)	8 (89)	0 (0)	0 (0)	1 (25)	2 (29)	3 (75)
minimal	0	1	1	2	5	0	0	1	2	1
mild	0	0	0	0	3	0	0	0	0	2
Mean score	0	0.1	0.2	0.3	1.2	0	0	0.3	0.3	1.3
Nasal Level V^a	9	8	8	10	10	10	9	10	9	10
total incidence (%)	0 (0)	0 (0)	0 (0)	6 (60)	4 (40)	0 (0)	0 (0)	1 (10)	4 (44)	7 (70)
minimal	0	0	0	6	3	0	0	1	4	5
mild	0	0	0	0	1	0	0	0	0	2
Mean score	0	0	0	0.6	0.5	0	0	0.1	0.4	0.9
Nasal Level VI^a	10	9	10	9	8	10	10	10	8	10
total incidence (%)	0 (0)	0 (0)	1 (10)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (10)
minimal	0	0	1	0	0	0	0	0	0	1
Mean score	0	0	0.1	0	0	0	0	0	0	0.1

^a= Number of tissues examined from each group.

Text Table 15. Incidences of Fibro-Osseous Proliferation,
Study Day 5 Primary Necropsy

	Males					Females
Target Exposure Level (ppm):	0	50	150	300	600	0	50	150	300	600
Fibro-osseous proliferation
Nasal Level II^a	10	10	10	10	10	10	10	10	10	10
total incidence	0	0	3	7	9	0	0	0	10	10
minimal	0	0	3	5	4	0	0	0	8	4
mild	0	0	0	2	5	0	0	0	2	6
Mean score	0	0	0.3	0.9	1.4	0	0	0	1.2	1.6
Nasal Level III^a	10	10	10	10	10	10	8	10	9	10
total incidence	0	0	0	0	2	0	0	0	0	2
minimal	0	0	0	0	2	0	0	0	0	2
Mean score	0	0	0	0	0.2	0	0	0	0	0.2
Nasal Level IV^a	8	7	6	6	9	6	7	4	7	4
total incidence	0	0	0	0	1	0	0	0	1	0
minimal	0	0	0	0	1	0	0	0	1	0
Mean score	0	0	0	0	0.1	0	0	0	0.1	0

^a= Number of tissues examined from each group.

COMPARISON OF MEAN SCORES AT THE 1-DAY INTERIM SACRIFICE AND AT THE 5-DAY FINAL SACRIFICE.

At the 5-day final sacrifice, values in green are lower than at the 1-day interim sacrifice and values in red are higher than at the 1-day interim sacrifice.

	Males					Females
Target Exposure Level (ppm):
	0	50	150	300	600	0	50	150	300	600
Squamous epithelium, acute inflammation
Nasal Level Ia
Day 1	0	0.2	0.2	0.4	0.2	0	0.1	0.2	0.3	0.1
Day 5	0	0.1	0	0.4	0.1	0	0	0	0.8	0.1
Transitional epithelium, degeneration
Nasal Level II
Day 1	0	1.1	2.7	3.7	4.0	0	0.3	3.2	3.5	3.8
Day 5	0	0.4	0.2	1.7	1.5	0	0.4	0.1	1.5	1.8
Transitional epithelium, acute inflammation
Nasal Level I
Day 1	0	0.2	0	0.4	0.1	0	0.6	0.4	0.9	1.2
Day 5	0	0	0	0.2	0.1	0	0.1	0	0.1	0.1
Nasal Level II
Day 1	0	0.9	1.3	1.6	2.0	0	0.5	1.4	1.1	1.4
Day 5	0	0.3	0.7	1.5	1.5	0	0.2	0.5	1.6	1.7
Respiratory epithelium, degeneration
Nasal Level II
Day 1	0	0	0.1	0.2	0.8	0	0.1	0	0.1	0.3
Day 5	0	0	0	0	0.2	0	0	0	0	0.3
Nasal Level III
Day 1	0	0	0.1	0.3	0.9	0	0	0.1	0	0.9
Day 5	0	0	0	0	0.2	0	0	0	0	0.8
Nasal Level IV
Day 1	0	0	0	0	0.3	0	0	0	0	0.5
Day 5	0	0	0	0	0.1	0	0	0	0	0.8
Respiratory epithelium, acute inflammation
Nasal Level II
Day 1	0	0.7	0.8	1.0	1.2	0	0.2	0.6	0.7	0.4
Day 5	0	0.5	0.4	0.3	0.5	0	0	0	0.3	0.7
Nasal Level III
Day 1	0	0	0.6	0.8	0.9	0	0	0.3	0.8	1.0
Day 5	0	0	0.1	0.5	1.2	0	0	0.1	0.7	1.1
Nasal Level IV
Day 1	0	0	0.4	0.9	1.0	0	0.5	0.4	0.6	1.2
Day 5	0	0	0	0.2	1.0	0	0	0	0.1	1.0
Olfactory epithelium, degeneration
Nasal Level II
Day 1	0	1.0	3.4	3.1	3.0	0	0.7	3.4	3.9	3.2
Day 5	0	1.3	2.6	2.6	2.2	0	1.2	2.6	2.8	2.9
Nasal Level III
Day 1	0	1.4	3.0	3.6	3.6	0	0.3	2.4	2.9	3.4
Day 5	0	1.8	3.0	3.0	2.7	0	1.6	2.8	3.0	3.0
Nasal Level IV
Day 1	0	0.7	1.4	2.3	3.3	0	1.0	1.4	2.6	3.0
Day 5	0	1.4	2.2	2.8	2.7	0	0.9	2.5	2.7	3.0
Nasal Level V
Day 1	0	0.6	0.8	1.4	2.5	0	0	1.0	1.7	2.8
Day 5	0	0.8	1.4	2.2	2.1	0	0.3	1.1	2.0	2.5
Nasal Level VI
Day 1	0	0.2	0.4	1.3	1.6	0	0	0	0.3	1.4
Day 5	0	0.2	1.1	1.3	1.4	0	0.2	0.4	1.1	1.0
Olfactory epithelium, acute inflammation
Nasal Level II
Day 1	0	0.1	0.5	0.1	0.3	0	0.1	0.2	0.4	0.2
Day 5	0	0	0.2	0.2	0.7	0	0	0.1	0.6	0.3
Nasal Level III
Day 1	0	0.1	0.6	0.9	1.0	0	0	0.2	1.0	0.8
Day 5	0	0	0.7	1.0	0.8	0	0	0.4	0.8	0.9
Nasal Level IV
Day 1	0	0	0.6	0.8	1.3	0	0	0.3	1.0	1.0
Day 5	0	0.1	0.2	0.3	1.2	0	0	0.3	0.3	1.3
Nasal Level V
Day 1	0	0	0	0.4	0.8	0	0	0	0.3	0.4
Day 5	0	0	0	0.6	0.5	0	0	0.1	0.4	0.9
Nasal Level VI
Day 1	0	0	0	0	0.1	0	0	0	0	0
Day 5	0	0	0.1	0	0	0	0	0	0	0.1

Interpretation of results:

study cannot be used for classification

Conclusions:

The no-observed-adverse-effect level (NOAEL) for dimethyl disulfide (DMDS) vapors administered via whole-body inhalation to Crl:CD(SD) rats for 1 or 5 consecutive days was less than 50 ppm. A BMD10% of 19 ppm (with a lower limit at 95% of 9.3 ppm) was calculated for the nasal irritation.

Executive summary:

The objective of this study was to evaluate the potential toxic effects of dimethyl disulfide (DMDS) on tissues of the upper respiratory tract (URT) when administered as a vapor via whole-body inhalation to rats for 1 day or 5 consecutive days.

Toxicity of dimethyl disulfide (DMDS) vapors administered via 6-hour whole-body inhalation to Crl:CD(SD) rats for 1 or 5 days was observed at exposure levels of 50, 150, 300 and 600 ppm as evidenced by body weight deficits, histopathologic changes in the nasal epithelia (acute inflammation, degeneration and/or hyperplasia) and turbinate bones (fibro-osseous proliferation at 150 ppm and higher, probably secondary to inflammation), and organ weight changes in the lung (300 and 600 ppm females at the primary necropsy only). Regeneration of the olfactory epithelium despite continued exposure indicated possible recovery after cessation of exposure. The no-observed-adverse-effect level (NOAEL) for dimethyl disulfide (DMDS) vapors administered via whole-body inhalation to Crl:CD(SD) rats for 1 or 5 consecutive days was less than 50 ppm. A BMD10% of 19 ppm (with a lower limit at 95% of 9.3 ppm) was calculated for the nasal irritation.

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: LC50
Value:: 5 046 mg/m³ air
Physical form:: inhalation: vapour
Quality of whole database:: GLP guideline study

Acute toxicity: via dermal route

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: National Standard of the People’s Republic of China Toxicological Test Methods of Pesticides for Registration (GB 15670-1995)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

yes

Remarks:

4 rats/sex

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Raleigh NC
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
- Weight at study initiation: 307 – 340 g for males and 216 - 236 g for females.
- Fasting period before study: no
- Housing: individually housed in suspended stainless steel wire bottom cages
- Diet: ad libitum fresh PMI Rat Chow (Diet #5012)
- Water: ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- temperature controlled
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: The clipped area began at the shoulders and extended to the hipbone and half way down the flank of each side of the animal
- % coverage: 10
- Type of wrap if used: porous dressing to retain the gauze dressing (semi-occlusive) and porous non-irritating tape

REMOVAL OF TEST SUBSTANCE
- Washing (if done): room temperature distilled water
- Time after start of exposure: 4h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.54-0.60 ml/rat
- Constant volume or concentration used: no

Duration of exposure:

4 hours

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

Control animals:

Details on study design:

Animals were observed at 4 hours post-dosing on Day 0 and once daily thereafter for 14 days. Observations included, but were not limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypies or bizarre behavior (e.g., self-mutilation, walking backwards). All animals were observed twice daily for mortality on Days 1 through 14.
Body weights were recorded pretest, Day 7 and at termination.
All animals were humanely sacrificed using CO2 following study termination and were examined for gross pathology. Additional signs were described.

Statistics:

None

Sex:

male/female

Dose descriptor:

LD0

Effect level:

>= 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

All four males and four females survived, following the 4-hour dermal exposure at 2000 mg/kg.

Clinical signs:

other: Abnormal physical signs including wetness of the anogenital area was observed.

Gross pathology:

The gross necropsy revealed no observable abnormalities.

Interpretation of results:

GHS criteria not met

Conclusions:

The dermal LD0 of Dimethyl disulfide is greater than 2000 mg/kg in rats.

Executive summary:

The dermal toxicity of Dimethyl Disulfide was evaluated in a study performed following the National Standard of the People’s Republic of China Toxicological Test Methods of Pesticides for Registration (GB 15670-1995), which is similar to the OECD TG 402. This study was conducted in accordance with current Good Laboratory Practice Regulations of the US EPA. Four healthy male and four healthy female Sprague Dawley rats were dosed dermally with Dimethyl disulfide at 2000 mg/kg of body weight. The test article was kept in contact with the skin for 4 hours at which time the wrappings were removed. Animals were observed at 4 hours post-dosing on Day 0 and once daily thereafter for 14 days. Observations included, but were not limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypies or bizarre behavior (e.g., self-mutilation, walking backwards). All animals were observed twice daily for mortality on Days 1 through 14. Body weights were recorded pretest, Day 7 and at termination. All animals were examined for gross pathology.

All four males and four females survived, following the 4-hour dermal exposure at 2000 mg/kg. Abnormal physical signs including wetness of the anogenital area was observed. All eight animals gained body weight by study termination. The gross necropsy revealed no observable abnormalities.

The dermal LD0 of Dimethyl disulfide is greater than 2000 mg/kg in rats.

Reference 2

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Deviations:

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Ace Animals, Boyertown, PA, USA
- Age at study initiation: 8-9-week old
- Weight at study initiation: 216 - 264 g for males and 202 - 231 g for females
- Fasting period before study: no data
- Housing: individually in suspended wire mesh cages
- Diet (e.g. ad libitum): PMI Rat Chow (Diet #5012)
- Water (e.g. ad libitum): tap water
- Acclimation period: at least one wek

ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: dorsal area of the trunk
- % coverage: approximately 10% of the body surfac
- Type of wrap if used: 4 ply porous gauze dressing measuring 10 x 15 cm wrapped with plastic in a semi-occlusive manner and was secured with non-irritating tape

REMOVAL OF TEST SUBSTANCE
- Washing (if done): washed with distilled water
- Time after start of exposure: 24 hours

Duration of exposure:

24 hours

Doses:

5000 mg/kg

No. of animals per sex per dose:

Control animals:

Details on study design:

- Duration of observation period following administration: 14 days

- Frequency of observations and weighing:
The test sites were scored for dermal irritation at 24 hours postdose and on days 7 and 14 using the numerical Draize scoring. The skin was also evaluated for ulceration and necrosis or any evidence of tissue destruction. Additional signs were described.
The animals were observed 1, 2 and 4 hours postdose and once daily for 14 days for toxicity and pharmacological effects. The animals were observed twice daily for 14 days for mortality.
Body weights were recorded pretest, weekly and at termination.

- Necropsy of survivors performed: yes
All animals were humanely sacrificed using CO2 following study termination and were examined for gross pathology. Abnormal tissues were preserved in 10% neutral buffered formalin for possible future histological examination.

Statistics:

Not appropriate for a limit test

Sex:

male/female

Dose descriptor:

LD0

Effect level:

> 5 000 mg/kg bw

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

All ten animals survived the 5000 mg/kg dermal application.

Clinical signs:

other: Instances of wetness and soiling of the anogenital area, chromorhinorrhea, sagging eyelids, emaciated appearance, few feces, lethargy, ataxia, wet red substance on anogenital area and nose/mouth area were noted during the study.

Gross pathology:

Necropsy revealed abnormalities of the treated skin and thymus. Seven animals were normal at necropsy.

Other findings:

Dermal Observations:
Dermal effects ranged from absent to very slight on Day 1, absent to severe on Day 7 and absent to very slight on Day 14.

Interpretation of results:

GHS criteria not met

Conclusions:

The dermal LD50 of Dimethyl Disulfide 94:5:1 is greater than 5000 mg/kg of body weight.

Executive summary:

The acute dermal toxicity of Dimethyl Disulphide 94:5:1 was evaluated in rats according to OPPTS 870.1200(August 1998) guideline. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations. Five male and female Wistar Albino rats were dosed dermally with Dimethyl Disulfide 94:5:1 at 5000 mg/kg of body weight. The test article was kept in contact with the skin for 24 hours. Clinical signs, skin reactions, mortality and body weight gain were checked for a period of up to 14 days following the single administration. All animals were subjected to necropsy.

All ten animals survived the 5000 mg/kg dermal application. Instances of wetness and soiling of the anogenital area, chromorhinorrhea, sagging eyelids, emaciated appearance, few feces, lethargy, ataxia, wet red substance on anogenital and nose/mouth areas were noted during the study. Dermal effects ranged from absent to very slight on Day 1, absent to severe on Day 7 and absent to very slight on Day 14. Two animals lost weight from Day 0 to Day 7, but gained weight overall by study termination. All other body weight changes are normal. Necropsy revealed abnormalities of the treated skin and thymus. Seven animals were normal at necropsy. The dermal LD₀of Dimethyl Disulfide 94:5:1 is greater than 5000 mg/kg of body weight.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: LD0
Value:: 2 000 mg/kg bw
Quality of whole database:: GLP guideline study

Additional information

ACUTE TOXICITY ORAL

Oral study / Key study (Shapiro 1985a)

The Acute oral toxicity of DMDS was evaluated in male and female Wistar rats according to EPA 40 CFR 163.81-1 and in compliance with principles of Good Laboratory Practices. Animals were treated with dose levels of 125, 188, 250, 375 and 500 mg/kg and then observed for 14 days for mortality, clinical signs and effect on body weight.

According to the dose levels, mortality was 10, 60, 70, 100 and 100% respectively. Sedation, hypotonia, dyspnea, piloerection and coma, appeared just after the administration and disappeared after 24 hours. No effect was noted on the body weight gain of the surviving rats. Haemorragic stomachs was observed at the macroscopic examination of the rats dead on the first day (250 and 500 mg/kg)

Under these experimental conditions, the oral LD50 of DMDS was 190 (150 -240) mg/kg in female and male rats.

Oral study / Supporting study 1 (Yasso 2016).

The oral toxicity of Dimethyl Disulfide was evaluated in a study performed following the National Standard of the People’s Republic of China Toxicological Test Methods of Pesticides for Registration (GB 15670-1995), which is similar to the OECD TG 401. This study was conducted in accordance with current Good Laboratory Practices regulations of the US EPA. Four Groups consisting of four male and four female Sprague Dawley rats were dosed orally with Dimethyl disulfide at a dose level of 50, 400, 500 and 1000 mg/kg. The rats were observed at 15 minutes, 1, 2 and 4 hours post-dose and once daily thereafter for 14 days. Observations included, but were not limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypies or bizarre behavior (e.g. self-mutilation, walking backwards). All animals were observed twice daily for mortality from Day 1 to Day 14. Body weights were recorded immediately pretest, every three days, at death and at termination in the survivors. All animals were examined for gross pathology. Abnormal tissues were preserved in 10% neutral buffered formalin for possible future histological examination.

No mortality was observed at 50 mg/kg. 2/4 males died at 400 mg/kg (no died female), 3/ 4 males died at 500 mg/kg (no died female). All 4/4 males and 4/4 females died at 1000 mg/kg. At lethal dose levels, abnormal physical signs including wetness of the nose/mouth area, anogenital areas,dyspnea, piloerection, prostration, flaccid muscle tone, lethargy, negative righting reflex, piloerection, lacrimationcoma, and/or lethargywere observed prior to death. Among the survivors, chromorhinorrhea, wetness and red staining of the anogenital area, piloerection, prostration, flaccid muscle tone, lethargy, lacrimation, negative righting reflex, wetness of the nose/mouth area, sagging eyelids, and ataxia were observed. Terminal body weight loss was observed among the animals that died. All survivors gained weight by study termination. Among the animals that died, the gross necropsy revealed wetness of the nose/mouth area, red staining and wetness of the anogenital area, red areas on the lungs, red and pale areas on the intestines,pale and dark areas on the liver.bladder contents red, and/or stomach distended with gas. Among the survivors, the gross necropsy revealed no observable abnormalities.

Using the Litchfield-Wilcoxon (graphical) Method, the estimated oral LD50 and 95% confidence limits were 415 (207 to 833) mg/kg for male rats and 750 (362 to 1552) mg/kg for female rats.

Oral study / Supporting study 2 (Pelcot, 2010).

The acute oral toxicity of dimethyl disulphide (purity 99.88%) was evaluated in rats according to OECD No. 423 (17th December 2001) and Council Regulation (EC) No. 440/2008, B.1tris(30 May 2008) guidelines. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations. Dimethyl disulphide was administered in corn oil by oral route (gavage), under a volume of 10 mL/kg to 6 fasted female Sprague-Dawley rats. Clinical signs, mortality and body weight gain were checked for a period of up to 14 days following the single administration. All animals were subjected to necropsy. No deaths occurred. Hypoactivity, dyspnea and hypersalivation were noted in all animals 10 minutes after treatment. Hypoactivity persisted in 4/6 animals until 4 hours after treatment. In addition, piloerection and dyspnea were observed in one animal 3 hours after treatment and in another one 4 hours after treatment. When compared to historical control animals, a lower body weight gain was noted in 3/6 animals between day 1 and day 8, persisting in one of them between day 8 and day 15. The body weight gain of the other animals was not affected by treatment with the test item. The oral LD0 of dimethyl disulphide was higher than 300 mg/kg in rats.

Oral study / Supporting study 3 (Lombard, 1986)

The acute oral toxicity of dimethyl disulphide (DMDS, purity 99.31%) was evaluated in male and female Sprague-Dawley rats according to EU Method B.1. Animals were treated with dose levels of 100, 170, 290, 350, 500 and 5300 mg/kg (5 ml/kg) and then observed for 14 days for mortality, clinical signs and effect on body weight. No mortality was observed at the dose levels of 100, 170 and 350 mg/kg. At 290 mg/kg the mortality was 30% and 100% at 500 and 5300 mg/kg. Sedation, hypotonia, dyspnea, piloerection and coma, appeared just after the administration and disappeared after 24 hours. No effect was noted on the body weight gain of the surviving rats. Haemorragic stomachs was observed at the macroscopic examination of the rats dead on the first day (290 and 500 mg/kg).

Under these experimental conditions, the oral LD50 of DMDS is between 290 and 500 mg/kg in female and male rats and an approximate LD50 of 385 mg/kg was calculated according to the standard probit method.

Oral study / Supporting study 4 (Gilotti 2006)

The acute oral toxicity of dimethyl disulphide was evaluated in rats according to OECD No. 423 (2001) guideline. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations. Dimethyl disulphide EC (94% DMDS with emulsifier and odorant) was administered undiluted by oral route (gavage) to 3 female Wistar rats. Clinical signs, mortality and body weight gain were checked for a period of up to 6 days following the single administration. All animals were subjected to necropsy.

One of three animals survived the 501 mg/kg oral dose to Day 6 at which time the animal was sacrificed. Two animals died prior to the 0.5 hour observation period. Necropsy results of these animals revealed abnormalities of the lungs, liver, stomach and intestines. The sacrificed animal was noted with chromorhinorrhea at 0.5 hour postdose and bloated abdomen and alopecia on forelimbs on Day 6. All other observations in the survivor were normal.

The oral LD50 of dimethyl disulphide was lower than 501 mg/kg in rats.

Oral study / Supporting study 5 (Gilotti 2007)

The acute oral toxicity of dimethyl disulphide was evaluated in rats according to OECD No. 423 (2001) guideline. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations. Five healthy female Wistar albino rats were dosed orally with Dimethyl Disulfide (94% DMDS with emulsifier and odorant) at 500 mg/kg. All five rats dosed at 500 mg/kg died by the 2 hour observation period. Five females and five males were dosed at 56 mg/kg. Animals were observed 0.5, 1, 2 and 4 hours postdose and once daily for 14 days for mortality, toxicity and pharmacological effects. Body weights were recorded immediately pretest, weekly and at termination. All animals were examined for gross pathology. Abnormal tissues were preserved in 10% neutral buffered formalin for possible future histological examination. At 500 mq/kg, no animals survived. Death occurred within 70 minutes of dosing. Predeath signs included wetness of the nose/mouth area, flaccid muscle tone, prostration, negative righting reflex and coma. Necropsy results revealed abnormalities of the lungs, thymus and liver, as well as wetness of the nose/mouth area. At 56 mg/kg, ten of ten animals survived. Instances of wetness of the nose/mouth area, lethargy, sagging eyelids, negative righting reflex, ataxia and wetness of the anogenital area were noted in the males within 2 hours postdose. Otherwise, the males appeared normal for the duration of the study. Females appeared normal throughout the study. Body weight changes were normal. All animals appeared normal at necropsy.

The LD50 of Dimethyl Disulfide is greater than 56 mg/kg but less than 500 mg/kg.

ACUTE TOXICITY INHALATION

Inhalation study / Key study 1 (Kirkpatrick, 2005)

In a key study, the acute inhalation toxicity of dimethyl disulphide (DMDS, purity 99.72%) was evaluated in single 4-hour, whole-body exposure study in male and female Sprague-Dawley rats performed in compliance with the EPA/OPPTS Guideline 870.1300 (1998) and the OECD Guidelines #403. The animals were exposed to concentrations of 847, 1188, 1308 and 1650 ppm then observed for 14 days for mortality, clinical signs, effect on body weight and then necropsied. Mortality following 4 hour whole body exposure to DMDS was 0 animals at 847 ppm, 4/10 at 1188 ppm, 4/10 at 1308 ppm and 9/10 at 1650 ppm. All deaths occurred during exposure or immediately following exposure. Toxicologically relevant pharmacotoxic signs consisted of rales in the 1188 and 1650 ppm groups, decreased defecation in the 1188 ppm group and dried material on various body surfaces, including the mouth and nose for the surviving 1650 ppm female. Detailed clinical observations immediately following exposure consisted of salivation in the 1188, 1308 and 1650 ppm group males and/or females, tremors in the 847 and 1308 ppm group females, low arousal in the 847, 1188 and 1308 ppm group females, ataxia and impaired mobility in the 1188 ppm group females, hunched gait in the 1308 and 1650 ppm group females, and increased difficulty in removing from cage/handling in females in all groups. Over the course of the 8-hour observation interval, females in all groups had tremors, low arousal levels and were harder to remove from their cages and handle. Hunched gait was noted in the 1188 and/or 1308 ppm groups throughout the 8-hour period. Slight body weight losses (2 to 7 grams) were noted for one or more females each in the 847, 1188, 1308 ppm groups during the study. All surviving animals met or surpassed their initial (study day 0) body weight by study day 14. Dark red discolouration of the lungs was noted for animals found dead during or immediately following exposure in the 1308 and 1650 ppm groups. One male in the 1308 ppm group also had lungs that did not fully collapse at necropsy. There were no gross findings for any of the mortalities in the 1188 ppm group or for the surviving animals at the scheduled necropsy.

Based on the results of this study, the LC50 of DMDS was 1310 ppm (with 95% confidence limits of 1167-1471 ppm) equivalent to 5.05 mg/L (with 95% confidence limits of 4.49-5.66 mg/l).

Inhalation study / Key study 2 (Kirkpatrick, 2009)

In a key study, dimethyl disulphide (purity 99.5%) vapors were administered via a 24-hour whole-body inhalation to 4 groups of 10 male Crl:CD(SD) rats. Target exposure concentrations were 5, 9, 12.5, and 18 ppm (19, 34, 48 and 69 mg/m3) and a concurrent control group was exposed to filtered air on a comparable regimen. One day following the 24-hour exposure, all animals were euthanized. All animals were observed daily for clinical signs and mortality whilst detailed physical examinations were performed prior to the scheduled necropsy. Individual body weights were recorded prior to the study day 0 exposures and prior to the scheduled necropsy. Complete necropsies were conducted on all animals, and kidneys, liver and lungs (prior to inflation) were weighed at the scheduled necropsy. Nasal tissues at 6 levels were examined microscopically from all animals. All animals survived to the scheduled necropsy. No clinical, body weight or macroscopic findings attributed to test substance exposure were noted at any exposure concentration. Test substance exposure-related degeneration of the olfactory epithelium was observed in Nasal Levels II-VI at exposure concentrations of 12.5 and 18 ppm, and in Nasal Levels III, IV, and V at an exposure concentration of 9 ppm. Degeneration of the olfactory epithelium was also observed in a single nasal level for 2 of 10 control group animals. The degeneration in the affected nasal sections in the 9 ppm group animals consisted of single or very few discrete, extremely small foci that consisted of cellular vacuolation and individual cell pyknosis, without sloughing. Changes in the 12.5 ppm group were also discrete areas comprising a very small percentage of the olfactory epithelium. These changes would be completely reversible and without clinical consequences and were not considered adverse. Degeneration of the olfactory epithelium was observed in 3 or more nasal levels in all 10 animals and was considered adverse in the 18 ppm group. This conclusion was based on the extent and severity of degeneration including the amount of damaged epithelium with sloughing of sensory and sustentacular cells. The lesions in this group were considered reversible. Slightly higher incidences of inflammation in the olfactory and respiratory epithelium were noted at a concentration of 18 ppm in Nasal Levels III-V. There were no test substance-related degenerative changes in the respiratory, transitional or squamous epithelium. The no-observed-adverse-effect concentration (NOAEC) for 24-hour whole-body exposure of DMDS to Crl:CD(SD) rats was 12.5 ppm equivalent to 48 mg/m3.

Inhalation study / Key study 3 (Kirkpatrick, 2008)

In a key study, the potential toxic effects of dimethyl disulfide (DMDS, purity 99.9%) on tissues of the upper respiratory tract (URT) was evaluated when administered as a vapour via 6-hour whole-body inhalation to rats for 1 day or 5 consecutive days at target concentrations of 0, 50, 150, 300 and 600 ppm (192, 577, 1154 and 2318 mg/m3). Clinical examinations were performed daily and detailed physical examinations were performed prior to necropsy. Individual body weights were recorded pretest, prior to the first, third and fifth exposures and prior to necropsy. Ten animals/sex/group were killed approximately 24 hours after a single exposure whilst the remaining 10 animals/sex/group were killed approximately 24 hours after the 5thexposure. Complete necropsies were conducted on all animals, selected organs were weighed and selected tissues were examined microscopically from all animals. No clinical findings attributed to test article exposure were noted at any exposure concentration. Lower mean body weight gains or mean body weight losses were noted in males of all DMDS-exposed groups throughout the study and in females of all groups during study days 0-2. These deficits in body weight gain resulted in lower mean body weights for males in all groups and females in the 300 and 600 ppm groups on study days 2 and 4. Test article-related pathology effects included histopathologic changes in the nasal epithelia and turbinate bones and organ weight changes in the lung. After a single exposure, acute inflammation and degeneration of the transitional and olfactory epithelia and acute inflammation of the respiratory epithelium were noted at all DMDS exposure concentrations, and degeneration of the respiratory epithelium was noted at concentrations of 150 ppm and higher. These changes were generally exposure concentration-related at a given nasal level, and changes in the respiratory and olfactory epithelia generally lessened in incidence and severity in more caudal nasal sections. After 5 consecutive days of exposure, mean absolute lung and lung/body weights were higher in the 300 and 600 ppm group females. Hyperplasia of the squamous epithelium was noted in =300 ppm group males and all DMDS-exposed group females. Hyperplasia of the transitional and respiratory epithelia and degeneration and regeneration of the olfactory epithelium were noted at all test article exposure concentrations in both sexes. Degeneration and acute inflammation of the transitional and respiratory epithelia, and acute inflammation of squamous and olfactory epithelia, were more variable after the 5-day exposure; DMDS-related effects were noted as acute inflammation of the squamous epithelium (50, 300 and 600 ppm group males and =300 ppm group females), transitional epithelium (=150 ppm group males and all test article exposure concentrations in females), respiratory epithelium (=300 ppm group males and =150 ppm group females) and olfactory epithelium (=50 ppm group males and =150 ppm group females), and degeneration of the respiratory epithelium (=600 ppm). Fibro-osseous proliferation of the bones of the nasal turbinates was observed at exposure concentrations of 150 ppm and higher in males and 300 ppm and higher in females; this finding may be secondary to inflammation rather than a direct effect of DMDS. The presence of regeneration of the olfactory epithelium despite continued exposure suggested that the olfactory epithelial degeneration may be reversible once exposure is discontinued. Based on the results of this study, toxicity of DMDS vapours administered via 6-hour whole-body inhalation to Crl:CD(SD) rats for 1 or 5 days was observed at exposure levels of 50, 150, 300 and 600 ppm as evidenced by body weight deficits, histopathologic changes in the nasal epithelia (acute inflammation, degeneration and/or hyperplasia) and turbinate bones (fibro-osseous proliferation at 150 ppm and higher, probably secondary to inflammation), and organ weight changes in the lung (300 and 600 ppm females after 5 consecutive days treatment only). Regeneration of the olfactory epithelium despite continued exposure indicated possible recovery after cessation of exposure. The no-observed-adverse-effect concentration (NOAEC) for DMDS vapours administered via whole-body inhalation to Crl:CD(SD) rats for 1 or 5 consecutive days was less than 50 ppm. A BMD 10% of 19 ppm (73 mg/m3) (with a lower limit at 95% of 9.3 ppm (35.8 mg/m3)) was calculated for the nasal irritation (Haber et al., 2008).

Inhalation study / Supporting study (Weinberg, 2016)

No deaths occurred in this study. The LC0 and LC50 of dimethyl disulfide TC were greater than 525 ppm when male and female Crl:CD(SD) albino rats were exposed to vapors of the test substance as a single, 2-hour, whole-body exposure.

ACUTE TOXICITY DERMAL

Dermal study / Key study (Yasso, 2015)

The dermal LD0 of Dimethyl disulfide is greater than 2000 mg/kg in rats.

Dermal study / Supporting study (Gilotti, 2007)

Supporting information is provided by an acute dermal toxicity study in rats performed with a dimethyl disulphide formulation and according to OPPTS 870.1200 (August 1998) guideline and GLP. In this study, five male and female Wistar Albino rats were dosed dermally with dimethyl disulphide (94% DMDS technical grade (purity 99.2%) with 5% emulsifier and 1% odorant) at 5000 mg/kg of body weight. The test article was kept in contact with the skin for 24 hours. Clinical signs, skin reactions, mortality and body weight gain were checked for a period of up to 14 days following the single administration. All animals were subjected to necropsy. All ten animals survived the 5000 mg/kg dermal application.Instances of wetness and soiling of the anogenital area, chromorhinorrhea, sagging eyelids, emaciatedappearance, few feces, lethargy, ataxia, wet red substance on anogenital and nose/mouth areas were noted during the study.Dermal effects ranged from absent to very slight on Day 1, absent to severe on Day 7 and absent to veryslight on Day 14.Two animals lost weight from Day 0 to Day 7, but gained weight overall by study termination. All other bodyweight changes are normal. Necropsy revealed abnormalities of the treated skin and thymus. Seven animals were normal at necropsy.

The dermal LD0 of dimethyl disulphide is greater than 5000 mg/kg of body weight.

OTHER ROUTE OF EXPOSURE

Three groups of two male and two female rats were given the test article as a single dose on Day 1 by intravenous injection at dose levels of 1, 5 and 50 mg/kg (Dreher 2005). Dimethyl disulphide was dispersed in vehicle (Intralipid 20) and administered at a dose volume of 2 mL/kg. Animals surviving treatment were killed on Day 8. Necropsies were only performed on animals that died during the observation period.

Both males dosed at 50 mg/kg died within 10 minutes of dosing. No other mortalities were noted. Clinical signs of reaction to treatment noted in animals dosed at 50 mg/kg were prone posture, limpness, cyanosis, dyspnoea, lethargy and hunched posture. Recovery of the females, as judged by external appearance and behaviour, was complete by one hour after dosing. No clinical signs were noted in animals dosed at 1 mg/kg or 5 mg/kg except for an isolated incident of bloated abdomen noted in on Day 3 in one male dosed at 1 mg/kg. All surviving rats achieved body weight gains during the study period. Macroscopic changes noted at necropsy of animals that died during the study were mottled and dark liver and enlarged atria of the heart. Dimethyl disulphide, was found to be well-tolerated in female rats at the dose levels administered, but was not tolerated in male rats at 50 mg/kg.

Justification for classification or non-classification

Acute oral toxicity:

Based on the oral LD50 of DMDS was 190 mg/kg in rats, and in accordance with Regulation (EC) No 1272/2008, DMDS is classified as Acute Tox. 3 (Hazard statement: H301; Toxic if swallowed).

Justification of RAC : In the study of Lombard (1986) the LD50 values were within the range of the classification criteria for acute oral toxicity category 4 (300 < LD50 ≤ 2000 mg/kg bw). In the Pelcot study (2010) the LD50 was above 300 mg/kg bw. In the study by Gilotti (2006) only a dose of 501 mg/kg bw was tested (mortality was 2/3 animals), fitting within the range of values for category 4, but a more severe classification category could not be excluded. In the second Gilotti study (2007), the oral LD50 was above 56 and below 500 mg/kg bw. Based on the results of both Gilotti studies, the LD50 was within the range of values for acute toxicity category 3 (50 < LD50 ≤ 300 mg/kg bw/day) or 4 (300 < LD50 ≤ 2000 mg/kg bw/day), therefore these studies were inconclusive.
In two studies by Shapiro (1985a and 1985b), the LD50 values were within a range of classification criteria for the acute toxicity category 3 (50 < LD50 ≤ 300 mg/kg bw/day). These studies were conducted with DMDS (purity 98%) contaminated with up to 1% methyl mercaptan (EC no.: 200-822-1). The current CLH of methyl mercaptan for acute toxicity in Annex VI Table 3.1 (CLP) is Acute Tox. 3*; H331, but it is not classified for acute oral toxicity. In addition, methyl mercaptan has been predicted to be one of the major metabolites of DMDS. Therefore, RAC concludes that the presence of methyl mercaptan as an impurity at such a low concentration does not affect the acute oral toxicity profile of DMDS, and the Shapiro studies are considered valid.
In the additional study (Yasso, 2015), the LD50 for male and female rats were found within the range for classification in acute toxicity category 4 (300 < LD50 ≤ 2000 mg/kg bw/day).
Taking into account the results of above studies, RAC considers that classification of DMDS in category 3 for acute oral toxicity is warranted. The lowest LD50 of 190 mg/kg bw determined in Shapiro (1985a) study for male and female rats is accepted by RAC as the ATE value for determining acute oral toxicity of mixtures containing DMDS.

Acute inhalation toxicity:

The LC50 of DMDS was 1310 ppm (with 95% confidence limits of 1167-1471 ppm) equivalent to 5.05 mg/L (with 95% confidence limits of 4.49-5.66 mg/l). On the basis of this study and in accordance with Regulation (EC) No 1272/2008 DMDS is classified as Acute Tox. 3 (Hazard statement: H331; Toxic if inhaled).

Acute dermal toxicity:

Based on the percutaneous LD0 greater than 2000 mg/kg for both male and female rats, DMDS shall not be classified with respect to acute dermal toxicity according to the Regulation (EC) No 1272/2008.

Specific Target Organ Toxicity (STOT SE) / Evaluation and conclusion from RAC

Based on the available data, the following classifications are applied to DMDS according to the Regulation EC n°1272/2008 : STOT SE 1 (H370 : Causes damage to upper respiratory tract, inhalation) and STOT SE 3 (H336 : May cause drowsiness or dizziness).

Oral route
The observation of animals after a single application of DMDS by gavage indicates that the substance is irritating to conjunctivae, intestinal and respiratory mucous membranes and induces transient depression of the central nervous system, leading in some cases to narcosis and coma.
In the acute toxicity study of Yasso (2015) in rats, DMDS at a non-lethal dose of 50 mg/kg bw induced ataxia, prostration and sagging eyelids, indicating a depressive effect on central nervous system. At 400 mg/kg, prostration, flaccid muscle tone, negative righting reflex, sagging eyelids, ataxia and lethargy were noted in surviving and non-surviving rats. Wetness of the nose/mouth area, lacrimation, irregular breathing, chromorhinorrhea, and red staining of the anogenital area were observed after oral administration of DMDS indicating irritation of conjunctivae, intestinal, nasal and respiratory mucous membranes.
DMDS after single oral administration at dose of 300 mg/kg bw to rats (Pelcot, 2010) was not lethal, but induced hypoactivity, dyspnoea and hypersalivation in all animals 10 minutes after treatment. Hypoactivity persisted in 4/6 animals until 4 hours after treatment. In addition, piloerection and dyspnoea were observed in one animal 3 hours after treatment and in another 4 hours after treatment.
In the acute oral toxicity study (Lombard, 1986) DMDS at doses of 100 and 170 mg/kg bw induced sedation, dyspnea and piloerection between 5 min and 4h post treatment, but not mortality. These symptoms were quickly reversible since no clinical signs were observed from D2 to D15 after treatment. At 290 mg/kg bw sedation, dyspnea, hypotonia, piloerection and mortality (30% of animals) were noted between 5 min and 4h post treatment, but no clinical signs were reported from D2 to D15 after treatment in surviving animals. At 350 mg/kg bw DMDS induced sedation between 5 min and 3h post treatment, but no mortality. No clinical signs were reported from D2 to D15.
At 500 mg/kg sedation, hypotonia, dyspnea and mortality (100%) were observed between 5 and 30 min post exposure.
In the acute oral toxicity study (Gilotti, 2007) DMDS at 56 mg/kg bw, non-lethal to rats, induced wetness of the nose/mouth area, lethargy, sagging eyelids, negative righting reflex, ataxia and wetness of the anogenital area in the males within 2 hours post-dosing. Otherwise, the males appeared normal for the full duration of the study. Females appeared normal throughout the study.
In the acute oral toxicity study (Shapiro, 1985) DMDS at 125 mg/kg bw caused clear reversible behavioural changes in surviving female rats: females did not exhibit any movement on the day of dosing, but appeared to have recovered by the next morning, and did not show any signs of toxicity later on. After a single oral administration of DMDS at 188 mg/kg bw the 4 surviving female rats exhibited signs of excessive salivation and lethargy. The rats exhibited signs of lethargy until day 5 post-dosing. From day 6 through 14 the rats did not exhibit any signs of toxicity.

In summary, these acute toxicity studies indicate that DMDS at non-lethal oral doses induces transient, reversible depression of CNS function from reduced locomotor activity to narcosis and causes irritation of conjunctivae, intestinal and respiratory mucous membranes.

Inhalation route
After 4-hour inhalation exposures at 1188 ppm and 1650 ppm, which were lethal for rats, (Kirkpatrick, 2005a), DMDS induced rales, which were not heard at a lower, non-lethal concentration of 847 ppm. No microscopic examination was conducted, but at necropsy dark discoloration of the lungs was noted in animals found dead during or immediately following exposure at 1308 and 1650 ppm. These observations indicate that DMDS was irritating to the respiratory system, but since these effects were only observed at lethal concentrations they do not fulfil the classification criteria for STOT SE 3 for respiratory tract irritation. Behavioural alterations consisting of ataxia, impaired mobility and low arousal were noted immediately after exposure in a few exposed females, but not in males.
Single inhalation exposure to DMDS for 6 hours at 150 ppm (0.577 mg/L) and higher (300-600 ppm (1.154-2.318 mg/L)) caused acute inflammation and degeneration of the transitional and olfactory epithelia and acute inflammation of the respiratory epithelium (Kirkpatrick, 2008). The reversibility of these changes was not studied according to the CLH report.
A 5-day exposure for 6 hours/day (Kirkpatrick, 2008) caused an increase in mean absolute and relative lung weight of female rats at 300 and 600 ppm (1.154 and 2.318 mg/L). Hyperplasia of the squamous epithelium was noted in ≥300 ppm (1.154 mg/L) group males and in all DMDS-exposed group females. Hyperplasia of the transitional and respiratory epithelia and degeneration and regeneration of the olfactory epithelium were reported in the CLH report at all test article exposure concentrations in both sexes. Fibro-osseous proliferation of the bones of the nasal turbinates was observed at exposure concentrations of 150 ppm (0.577 mg/L) and higher (1.154 and 2.318 mg/L) in males and at 300 ppm and higher (1.154 and 2.318 mg/l) in females. This finding was considered as secondary to inflammation rather than a direct effect of DMDS (Kirkpatrick, 2008). The NOAEC for DMDS vapours administered via whole-body inhalation to rats for 6 hours for 1 or 5 consecutive days was less than 50 ppm. A BMD10 of 19 ppm (73 mg/m3) (with a lower limit at 95% confidence of 9.3 ppm (35.8 mg/m3)) was calculated for the nasal irritation based on the findings in this study (Haber et al., 2008).
In a key study (Kirkpatrick, 2009), rats were exposed for 24 hours to DMDS vapour at 5, 9, 12.5, and 18 ppm (19, 34, 48 and 69 mg/m3) and a concurrent control group was exposed to filtered air on a comparable regimen. One day after the 24-hour exposure, all animals were euthanised. All animals were observed daily for clinical signs and mortality whilst detailed physical examinations were performed prior to the scheduled necropsy. No clinical, body weight or macroscopic findings attributed to test substance exposure were noted at any exposure concentration. Complete necropsies were conducted on all animals, and kidneys, liver and lungs (prior to inflation) were weighed at the scheduled necropsy. Nasal tissues at 6 levels were examined microscopically from all animals. All animals survived to the scheduled necropsy.
Test substance exposure-related degeneration of the olfactory epithelium was observed in nasal levels II-VI at exposure concentrations of 12.5 and 18 ppm, and in nasal levels III, IV and V at an exposure concentration of 9 ppm.
The degeneration in the affected nasal sections in the 9 ppm group animals consisted of single or very few discrete, extremely small foci with cellular vacuolation and individual cell pyknosis, without sloughing. Changes in the 12.5 ppm group were also discrete areas comprising a very small percentage of the olfactory epithelium.

Degeneration of the olfactory epithelium was observed in 3 or more nasal levels in all 10 animals and was considered adverse in the 18 ppm (69 mg/m3) group. This conclusion was based on the extent and severity of degeneration including the amount of damaged epithelium with sloughing of sensory and sustentacular cells. Slightly higher incidences of inflammation in the olfactory and respiratory epithelium were noted at 18 ppm (69 mg/m3) in nasal levels III-V. The NOAEC for 24-hour whole-body exposure of DMDS to Crl: CD(SD) rats was 12.5 ppm equivalent to 48 mg/m3.
The results indicated that a single 24-hour whole body inhalation exposure to DMDS at concentrations of 0,019, 0.034, 0.048 and 0.069 mg/L (5, 9, 12.5, and 18 ppm ) induced degeneration of mostly olfactory nasal epithelium at concentrations 0.034, 0.048 and 0.069 mg/L. The study authors noted that there were no test substance-related degenerative changes in the respiratory transitional or squamous epithelium.
In summary, the results of the above studies indicate that a single 24-hour inhalation exposure to non-lethal low DMDS concentrations (0.034, 0.048 and 0.069 mg/L) induced damage of nasal olfactory epithelium (Kirkpatrick, 2009), and much higher but still non-lethal concentrations of 150 - 600 ppm (0.577 mg/L - 2.318 mg/L) for 6 hours caused acute inflammation and degeneration of the transitional and olfactory epithelia and acute inflammation of the respiratory epithelium (Kirkpatrick, 2008).
RAC considers that degeneration of transitional and olfactory nasal epithelium represents significant morphological changes indicating functional damage in the upper respiratory tract, possibly in the form of loss of sense of smell. Olfactory cell degeneration after single exposure to DMDS provides the evidence of appreciable cell death in olfactory epithelium, which may lead to atrophy of this epithelium, which was in fact observed after repeated inhalation exposure to vapour of DMDS (Collins, 1992). RAC concludes that the severity of the observed nasal epithelium changes after single inhalation exposure meets the criteria for STOT SE 1 and 2, listed in point 3.8.2.1.7.3 of Annex I to the CLP Regulation. They were observed at concentrations ≤ 10 mg/L/4h, being thus within the guidance value range for category 1. RAC is of the opinion that DMDS warrants classification as STOT SE 1; H370 (Causes damage to upper respiratory tract (inhalation).

Dermal route

The acute dermal toxicity studies provide evidence that DMDS at non-lethal doses induces transient depression of CNS function.
In the acute dermal toxicity study in rats (Gilotti, 2007) at non-lethal dose of 5000 mg/kg bw, the following clinical signs were noted; instances of wetness and soiling of the anogenital area, chromorhinorrhea, sagging eyelids, emaciated appearance, few faeces, lethargy, ataxia, wet red substance on the anogenital area and the nose/mouth area.
In the second acute dermal toxicity study (Shapiro, 1986) at a non-lethal dose of 2000 mg/kg bw in rabbits within 5 minutes of test material application and for approximately 4 hours thereafter, the following symptoms were observed; heavy breathing, loss of righting reflex, spontaneous spasms, pupillary dilation and constriction, unwillingness to stand, lethargy, excessive salivation and flared nostrils. By 24 hours after dosing, the rabbits appeared active and healthy. Necropsy observations revealed dark foci on all lobes and surfaces of the lungs, pale purple or cloudy discoloration of the spleen accompanied by rough texture and edges in most rabbits. Slight haemorrhage, dark foci and indentation were noted on the surface of the kidneys of a few animals.
In the acute dermal toxicity study in rabbits (Hazleton, 1985) at a non-lethal dose of 2000 mg/kg bw, apathy and prostration were noted in most of the animals between 15 minutes and 3 hours after the application of the substance. An increase in the spontaneous activity was noted for some animals on the first day of treatment. The behaviour of the animals during the remainder of the observation period was considered normal. No macroscopic lesions were observed at sacrifice.
In the acute dermal toxicity study in rabbits (Haynes, 1988), at a non-lethal dose of 2000 mg/kg bw, the following treatment-related observations were recorded; constriction of the pupils in one male, inflammation of iris in 2 females, nasal discharge in 3 males and 4 females and slow breathing in 2 males shortly after dosing. All animals had recovered within 1 hour of dosing.
In addition, transient narcotic effects were observed in rabbits exposed for 28 days or 13 days by dermal route at 106.3 or 1063 mg/kg bw/day, respectively. The effect disappeared by the end of each daily exposure and was not linked with permanent damage of brain or peripheral nervous tissues since no adverse effects were detected in brain, spinal cord or sciatic nerve of animals in histopathological examination.
The observed effects fulfil the CLP criteria for STOT SE 3; H336. Since the observed narcotic effects were transient in nature, disappearing by the end of daily exposure, they shall not be considered to support classification for Category 1 or 2 specific target organ toxicity single exposure (3.8.2.2.2 of Annex I to the CLP Regulation).

Taking into account observations made during acute and repeated dose toxicity studies, RAC considers that DMDS warrants classification as STOT SE 3; H336 (May cause drowsiness or dizziness).

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Parameter	Mean Concentration, ppm (% of Target)
Parameter	Group 2 (5 ppm)	Group 3 (9 ppm)	Group 4 (12.5 ppm)	Group 5 (18 ppm)
Nominal Concentrations	5.4 (108)	9.7 (108)	13.0 (104)	18.3 (102)
Analyzed Exposure Concentrations	5.0 (100)	8.9 (99)	12.6 (101)	18.4 (102)

Number of Nasal Levels with Degeneration of Olfactory Epithelium Scheduled Necropsy (Study Day 2)
	Males
Target Exposure Level (ppm):	0	5	9	12.5	18

Olfactory epithelium, degeneration^a	2	0	4	6	10
One level	2	0	3	1	0
Two levels	0	0	0	1	0
Three levels	0	0	1	2	2
Four levels	0	0	0	1	7
Five levels	0	0	0	1	1

Incidences of Degeneration of Olfactory Epithelium, Scheduled Necropsy (Study Day 2)
	Males
Target Exposure Level (ppm):	0	5	9	12.5	18

Olfactory epithelium, degeneration
Nasal Level II^a	10	10	10	10	10
Total incidence	0	0	0	2	5
minimal	-	-	-	2	2
mild	-	-	-	0	2
moderate	-	-	-	0	1

Nasal Level III^a	10	10	10	10	10
Total incidence	1	0	2	2	10
minimal	1	-	2	2	5
mild	0	-	0	0	2
moderate	0	-	0	0	3

Nasal Level IV^a	10	10	10	10	10
Total incidence	1	0	2	5	10
minimal	1	-	2	5	3
mild	0	-	0	0	7

Nasal Level V^a	10	10	10	10	10
Total incidence	0	0	2	6	10
minimal	-	-	2	6	9
mild	-	-	0	0	1

Nasal Level VI^a	10	10	10	10	10
Total incidence	0	0	0	3	4
minimal	-	-	-	3	3
mild	-	-	-	0	1

Incidences of Inflammation of Olfactory and Respiratory Epithelia in Selected Nasal Levels Scheduled Necropsy (Study Day 2)
	Males
Target Exposure Level (ppm):	0	5	9	12.5	18

Nasal Level II^a	10	10	10	10	10
Olfactory epithelium, inflammation, minimal	0	0	0	0	1
Respiratory epithelium, inflammation, minimal	1	0	0	0	0

Nasal Level III^a	10	10	10	10	10
Olfactory epithelium, inflammation, minimal	0	0	0	2	4
Respiratory epithelium, inflammation, minimal	1	1	1	1	4

Nasal Level IV^a	10	10	10	10	10
Olfactory epithelium, inflammation, minimal	2	0	3	3	5
Respiratory epithelium, inflammation, minimal	0	1	1	2	3

Nasal Level V^a	10	10	10	10	10
Olfactory epithelium, inflammation, minimal	1	0	0	0	3
Respiratory epithelium, inflammation, minimal	1	1	0	0	6

Nasal Level VI^a	10	10	10	10	10
Olfactory epithelium, inflammation, minimal	0	0	1	0	1
Respiratory epithelium, inflammation, minimal	0	0	1	0	0

Registration Dossier

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Diss Factsheets

Dimethyl disulphide

Endpoint summary

Administrative data

Description of key information

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint conclusion

Acute toxicity: via inhalation route

Link to relevant study records

Referenceopen allclose all

Reference 1

Males

Females

Reference 2

Reference 3

Reference 4

Males

Females

Males

Females

Males

Females

Endpoint conclusion

Acute toxicity: via dermal route

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

Endpoint conclusion

Additional information

Justification for classification or non-classification

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