Dimethyl disulphide

Neurotoxicity

Description of key information

GLP- and recognised test guideline-compliant studies of acute inhalation neurotoxicity (Nemec, 2005) and 90-day inhalation neurotoxicity (Nemec, 2006) in rats are available on dimethyl disulphide. The NOAEL for systemic toxicity and neurotoxicity of a single 6-hour whole body inhalation exposure to DMDS was 100 ppm. The NOAEL for systemic toxicity and neurotoxicity of DMDS via whole body inhalation exposure for 13 consecutive weeks was 20 ppm for males and 80 ppm for females.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on neurotoxicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

neurotoxicity

Remarks:

acute

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-07-27 to 2004-12-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.6200 (Neurotoxicity Screening Battery)

Deviations:

no

GLP compliance:

yes

Limit test:

no

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 : approximately 29 or 30 days old at receipt, 43-47 days old at the initiation of dose administration
- Weight at study initiation: 165.2 g to 227.4 g for males and from 132.7 g to 195.6 g for females
- Housing: three per cage by sex for at least 3 days to facilitate adaptation to the automatic watering system. Thereafter, all animals were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board
- Diet: PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002, ad libitum
- Water: Reverse
osmosis-treated (on-site) drinking water, ad libitum
- Acclimation period: 14-day

ENVIRONMENTAL CONDITIONS
- Temperature: (21.5°C to 21.7°C
- Humidity: 44.9% to 52.2%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

other: Whole-body inhalation exposure to vapours

Vehicle:

unchanged (no vehicle)

Details on exposure:

EXPOSURE METHODS
The exposures were conducted (in four replicates/sex; one replicate/day) in four 1.0-m3 stainless steel and glass whole-body exposure chambers. One chamber was dedicated for each group for the duration of the study.

EXPOSURE ATMOSPHERE GENERATION
Test article was metered at a known and constant rate from a reservoir to a glass vaporization column filled with various sized glass beads. The vaporization column was wrapped with heat tapes that were operated by an Omega temperature controller. Nitrogen was delivered to the bottom of the column using a flow meter. In these generators, the liquid test article was metered onto the heated glass beads and the resulting vapor was carried out of the column by the nitrogen flowing upward through the column. Concentrated vapors were piped from the vaporization column to the chamber inlet, where the concentration was diluted to the target level by the chamber ventilation airflow. If necessary, a portion of the test article was removed after the vaporization column by way of a siphon. The control group was exposed to clean, filtered air.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Actual exposure concentrations of the test article atmospheres were determined by gas chromatography (GC). Samples of the exposure atmospheres were automatically collected at approximately 35-minute intervals using a sample loop and a computer-controlled multiposition valve. The overall mean nominal concentrations for males were 120 ppm for the 100 ppm group, 240 ppm for the 200 ppm group and 885 ppm for the 750 ppm group. The overall mean nominal concentrations for females were 127 ppm for the 100 ppm group, 242 ppm for the 200 ppm group and 918 ppm for the 750 ppm group.
The overall mean analyzed concentrations were 103.1, 207.0 and 745.4 ppm for males and 102.5, 206.0 and 742.9 ppm for female for the 100, 200 and 750 ppm groups, respectively.

Duration of treatment / exposure:

6 hours

Frequency of treatment:

single exposure

Remarks:

Doses / Concentrations:
100, 200 and 750 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

12

Control animals:

yes, sham-exposed

Observations and clinical examinations performed and frequency:

CLINICAL OBSERVATIONS AND SURVIVAL
All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.
Clinical examinations were performed once daily on all animals. All significant findings were recorded. On the days that the FOB was conducted, no additional clinical signs were recorded. In addition, all animals were observed prior to exposure and at the midpoint.

BODY WEIGHTS
Individual body weights were recorded at least weekly, beginning approximately 1 week prior to test article administration (study day -7). Body weights were also recorded during FOB procedures and prior to euthanasia.

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
Functional observational battery (FOB) observations were recorded for all animals prior to the initiation of dose administration, at the time of peak effect on study day 0 and on study days 7 and 14. The peak effect of dimethyl disulfide was considered to be at the end of the exposure period.
All animals were observed for the following parameters as described below:
* HOME CAGE OBSERVATIONS
Posture, Convulsions/Tremors, Feces consistency, Biting, Palpebral (eyelid) closure
* HANDLING OBSERVATIONS
Ease of removal from cage, Lacrimation/Chromodacryorrhea, Piloerection, Palpebral closure, Eye prominence, Red/Crusty deposits, Ease of handling animal in hand, Salivation, Fur appearance, Respiratory rate/character, Mucous membranes/Eye/Skin color, Muscle tone
* OPEN FIELD OBSERVATIONS (EVALUATED OVER A 2-MINUTE OBSERVATION PERIOD)
Mobility, Rearing, Convulsions/Tremors, Grooming, Bizarre/Stereotypic behavior, Time to first step (seconds), Gait, Arousal, Urination/Defecation, Gait score, Backing
* SENSORY OBSERVATIONS
Approach response, Startle response, Pupil response, Forelimb extension, Air righting reflex, Touch response, Tail pinch response, Eyeblink response, Hindlimb extension, Olfactory orientation
* NEUROMUSCULAR OBSERVATIONS
Hindlimb extensor strength, Hindlimb foot splay, Grip strength-hind and forelimb, Rotarod performance.
* PHYSIOLOGICAL OBSERVATIONS
Catalepsy, Body temperature, Body weight

LOCOMOTOR ACTIVITY
Observations were recorded for all animals prior to the initiation of dose administration, at the time of peak effect on study day 0 and on study days 7 and 14. The peak effect of dimethyl disulfide was considered to be at the end of the exposure period. Locomotor activity, recorded after the completion of the FOB, was measured automatically using the San Diego Instruments, Inc., Photobeam Activity System (San Diego Instruments, Inc., San Diego, California).

Sacrifice and (histo)pathology:

UNSCHEDULED DEATHS
A complete necropsy was conducted on the animal found dead. The necropsy included examination of the external surface, all orifices, and the cranial, thoracic, abdominal and pelvic cavities, including viscera.

NEUROPATHOLOGY - MACROSCOPIC EXAMINATION
On study day 15, all surviving animals were euthanized by carbon dioxide inhalation and then perfused in situ with a buffered 4.0% paraformaldehyde/1.4% glutaraldehyde solution. The central and peripheral nervous system tissues were dissected and preserved for six animals/sex in the control and 750 ppm groups. Fixed brain weight (excluding olfactory bulbs) and brain dimensions (length and width) were recorded for all animals. Any observable gross changes, abnormal coloration or lesions of the brain and spinal cord were recorded.

NEUROPATHOLOGY - MICROSCOPIC EXAMINATION
The following nerve tissues were prepared for a microscopic neuropathologic examination from six animals per sex in the control and 750 ppm groups:
Brain - olfactory bulbs, cerebral cortex (2 levels), hippocampus/dentate gyrus, basal ganglia, thalamus, hypothalamus, tectum, cerebral peduncles, central gray matter, cerebellum, pons and medulla oblongata
Spinal cord - at cervical swellings C3-C7 and at lumbar swellings T13-L4
Trigeminal ganglia/nervesa
Lumbar dorsal root ganglia at T13-L4(b
Lumbar dorsal root fibers at T13-L4(b
Lumbar ventral root fibers at T13-L4(b
Cervical dorsal root ganglia at C3-C7(b
Cervical dorsal root fibers at C3-C7(b
Cervical ventral root fibers at C3-C7(b
Sciatic nerves (mid-thigh region)(2)(c
Sciatic nerves (at sciatic notch)(2)(c
Sural nerves(2)(c
Tibial nerves(2)(c
Peroneal nerves(2)(c
Optic nervesa
Eyes(a
Skeletal muscle (gastrocnemius)
Other sites (if deemed necessary)
a - Both processed and evaluated microscopically.
b - Four to six tissues collected at necropsy; two evaluated microscopically.
c - One processed for microscopic examination.
(2) - Two sections (one cross and one longitudinal) of the tissue were evaluated fromn the right hind leg. The tissues from the left hind leg were collected and preserved for possible future evaluation.

Statistics:

Body weight, body weight change, continuous functional observational battery (FOB), ambulatory locomotor activity, and brain weight, length and width data were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test article-treated groups to the control group. Functional observational battery parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact Test.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Clinical biochemistry findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Details on results:

MORTALITY
One female in the 750 ppm group died during exposure on study day 0. A cause of death could not be determined at necropsy.

CLINICAL SIGNS
The only definite test article-related clinical finding was salivation. This finding was observed at the midpoint of exposure for 0, 4, 6 and 3 males and 0, 3, 6 and 6 females in the control, 100, 200 and 750 ppm groups, respectively. Salivation did not persist to the next day.

BODY WEIGHT AND WEIGHT GAIN
Mean body weight gain in the 750 ppm group males and females was lower (statistically significant at p<0.01 for the males) than the control group mean for study days 0 to 7. As a result, mean body weights for study days 7 and 14 and the cumulative body weight gain (study days 0 to 14) in both sexes at 750 ppm were lower (statistically significant at p<0.05 or p<0.01 for the males) than the control group values.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
* HOME CAGE OBSERVATIONS
A test article-related change in palpebral closure was observed in the 750 ppm group males and females at the time of peak effect on study day 0. The numbers of males and females in the 750 ppm group with drooping eyelids (half-closed) were statistically significantly (p<0.05) increased when compared to the control group. The percentages of animals affected in the 750 ppm group were markedly increased when compared to the WIL FOB historical control data. In the current study, 50% of the males and 36% of the females in the 750 ppm group had drooping eyelids (half-closed), while only 4% of the males and 1% of the females in the historical control data had this finding. Similar increases were not observed in the 750 ppm group at the study day 7 and 14 evaluations.
* HANDLING OBSERVATIONS
The numbers of females in the 200 and 750 ppm groups with red deposits around the mouth at the time of peak effect on study day 0 were statistically significantly (p<0.05) higher than the control group; this finding was attributed to the test article. In addition, a change in fur appearance (slightly soiled) was observed for one male in the 750 ppm group and for one, two and three females in the 100, 200 and 750 ppm groups, respectively. This finding was related to test article exposure.
* OPEN FIELD OBSERVATIONS
Mean grooming and urination counts in the 750 ppm group males and females were significantly (p<0.05) increased when compared to the control group at the time of peak effect on study day 0. The unusually high mean urination count in the females was at least due in part to urination being counted differently for the first two female replicates compared to the remainder of the study. On these 2 days, individual urine drops were counted without taking into account animal movement during urination. Similar increases were not observed in this group at the study day 7 and 14 evaluations.
Tremors were observed for 2/12 and 2/11 females in the 200 and 750 ppm groups, respectively. Even though the differences from the control group were not statistically significant, this finding has not been observed in the WIL FOB historical control data for females. Therefore, the tremors were considered to be related to dimethyl disulfide exposure.
* SENSORY OBSERVATIONS
Sensory observations were unaffected by test article administration.
* NEUROMUSCULAR OBSERVATIONS
Neuromuscular observations were unaffected by test article administration.
* PHYSIOLOGICAL OBSERVATIONS
Mean body temperatures were decreased in the 750 ppm group males and females at the time of peak effect on study day 0, despite being equal to or slightly greater than at pretest. The difference from the control group was statistically significant at p<0.05 for the females. Similar decreases were not observed in this group at the study day 7 and 14 evaluations.
* LOCOMOTOR ACTIVITY
The only test article-related effect on locomotor activity consisted of decreased mean ambulatory and total motor activity values in the 750 ppm group males and females at the time of peak effect on study day 0.

BRAIN WEIGHTS AND MEASUREMENTS
Mean brain weights and measurements were unaffected by exposure to dimethyl disulfide.

MICROSCOPIC EXAMINATION
No test article-related microscopic lesions were observed in any of the central or peripheral nervous system tissues examined from six animals/sex in the 750 ppm group.

Dose descriptor:

NOAEC

Remarks:

neurotoxicity

Effect level:

100 ppm (analytical)

Sex:

male/female

Basis for effect level:

other: treatment related effects at 200 ppm consisting of salivation during exposure in males and females and slightly soiled fur, red deposits around the mouth and tremors in females.

Remarks on result:

other:

Conclusions:

Based on the results of the FOB and locomotor activity evaluations, the following domains were affected by a single 6-hour exposure of dimethyl disulfide at 750 ppm: autonomic, CNS activity, CNS excitability and physiological. The no-observed-adverse-effect concentration (NOAEC) for systemic toxicity and neurotoxicity of a single whole body inhalation exposure of dimethyl disulfide to rats was 100 ppm.

Executive summary:

An acute neurotoxicity study via the inhalation route was conducted with DMDS, the study was performed following the OECD guideline # 424. In that study, four groups of 12 male and 12 female Sprague-Dawley rats were exposed whole body for 6 hours to either clean filtered air or DMDS vapor atmospheres of 100, 200 or 750 ppm. All animals were observed twice daily for mortality and moribundity, clinical examinations were performed daily except on days when the functional observational battery (FOB) was performed and body weights were recorded weekly. FOB and locomotor activity evaluations were performed on all animals prior to the initiation of exposure, at the time of peak effect following exposure and on study days 7 and 14. Following perfusion, all animals were necropsied and brain weights and brain dimensions were measured. Neuropathologic evaluations were performed on central and peripheral nervous system tissues for control and 750 ppm groups.

There were no test article-related clinical findings in the test article-treated groups. No dimethyl disulfide-related effects were apparent between treated and control group animals on sensorimotor and neuromuscular parameters when FOB (functional observational battery) evaluations were performed at the time of peak effect on study day 0 and on study days 7 and 14. Fixed brain weights and brain dimensions for perfused animals were unaffected. No test article-related neuropathological lesions were observed upon microscopic examination of 6 animals per sex in the 750 ppm group.

Test article-related effects in the 750 ppm group included the death of one female during exposure, salivation during exposure in 3/12 males and 6/12 females and decreased mean body weight gain in both sexes for study days 0 to 7. FOB parameters that were affected were observed at the time of peak effect on study day 0 and did not persist to the study day 7 and 14 evaluations. Findings observed at 750 ppm included increased numbers of animals with a change in palpebral closure (drooping eyelids in 6/12 males and 4/12 females) during the home cage observations, slightly soiled fur (1/12 males and 3/12 females) during the handling observations, increased grooming (1.0 for males and 1.4 for females compared to control values of 0.0 and 0.2, respectively) and urination counts (2.5 for males and 4.0 for females compared to control values of 0.4 and 0.3, respectively) during the open field observations and decreased body temperature (38.1° for both sexes compared to control values of 38.5° for males and 38.6° for females) and body weight (166.3 g for males and 138.9 g for females compared to control values of 180.2 g and 149.0 g, respectively) during the physiological observations. Findings observed in the females at 750 ppm consisted of red deposits around the mouth (5/11 females) during the handling observations and tremors (2/11 females) during the open field observations. Ambulatory and total motor activity counts over 60 minutes were reduced in both sexes at 750 ppm on study day 0 only.

Test article-related effects in the 200 ppm group consisted of salivation during exposure (6/12 males and 6/12 females) and slightly soiled fur (2/12 females), red deposits around the mouth (7/12 females) and tremors (2/12 females).

The only test article-related findings in the 100 ppm group were salivation during exposure (4/12 males and 3/12 females) and slightly soiled fur (1/12 females).

Several of the aforementioned findings (drooping eyelids, salivation, increased grooming counts, red deposits and soiled fur) were most likely a result of the slightly irritating properties of dimethyl disulfide. Therefore, these test article-related findings were not considered to be indicative of systemic toxicity.

The Lowest Observed Adverse Effet Concentration (LOAEC) was 200 ppm based on treatment related effects consisting of salivation during exposure in males and females and slightly soiled fur, red deposits around the mouth and tremors in females. The No Observed Adverse Effect Concentration (NOAEC) for systemic toxicity and neurotoxicity was 100 ppm.

Reference 2

Endpoint:

neurotoxicity

Remarks:

subchronic

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-07-12 to 2006-02-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.6200 (Neurotoxicity Screening Battery)

Deviations:

no

GLP compliance:

yes

Limit test:

no

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: 31 days old at receipt
- Weight at study initiation: 179 g to 234 g for males and from 147 g to 207 g for females
- Housing: individually in clean, stainless steel, wire-mesh cages suspended above cage-board
- Diet: PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002, ad libitum
- Water: Reverse osmosis-treated (on-site) drinking water, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21.3°C to 21.5°C
- Humidity: 40.5% to 55.6%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

other: whole-body inhalation to vapours

Vehicle:

unchanged (no vehicle)

Details on exposure:

EXPOSURE METHODS
Animal exposures were conducted in four 2.0-m3 stainless-steel and glass whole-body exposure chambers. One chamber was dedicated to each group for the duration of the study.

EXPOSURE ATMOSPHERE GENERATION
Vapors of the test article were generated using a bubbler type (gas washing bottle) vaporization system. The test article vapors were then directed to the exposure chamber inlet where vapor concentration was reduced to the desired level by mixing with the chamber ventilation air. The control group was exposed to clean, filtered air.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Actual exposure concentrations of the test article atmospheres were determined by gas chromatography (GC). The overall mean analyzed concentrations were 5.0 ± 0.14 ppm for the 5 ppm group, 20.4 ± 0.56 ppm for the 20 ppm group and 80.0 ± 1.81 ppm for the 80 ppm group.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours per day, 7 days per week

Remarks:

Doses / Concentrations:
5, 20 and 80 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

12

Control animals:

yes, sham-exposed

Observations and clinical examinations performed and frequency:

CLINICAL OBSERVATIONS AND SURVIVAL
All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity. Clinical examinations were performed prior to exposure, at the midpoint of exposure and approximately 1 hour after completion of exposure.

BODY WEIGHTS
Individual body weights were recorded at least weekly, beginning 1 week prior to test article exposure (study week -1).

FOOD CONSUMPTION
Individual food consumption was recorded weekly, beginning 1 week prior to test article exposure (study week -1 to 0).

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY [FOB]
Functional observational battery (FOB) assessments were recorded for all animals prior to the initiation of test article exposure and during study weeks 3, 7 and 12. Testing was performed around the time of peak effect (at the completion of exposure).
All animals were observed for the following parameters as described below:
* HOME CAGE OBSERVATIONS
Posture, Convulsions/Tremors, Feces consistency, Biting, Palpebral (eyelid) closure
* HANDLING OBSERVATIONS
Ease of removal from cage, Lacrimation/Chromodacryorrhea, Piloerection, Palpebral closure, Eye prominence, Red/Crusty deposits, Ease of handling animal in hand, Salivation, Fur appearance, Respiratory rate/character, Mucous membranes/Eye/Skin color, Muscle tone
* OPEN FIELD OBSERVATIONS (EVALUATED OVER A 2-MINUTE OBSERVATION PERIOD)
Mobility, Rearing, Convulsions/Tremors, Grooming, Bizarre/Stereotypic behavior, Time to first step (seconds), Gait, Arousal, Urination/Defecation, Gait score, Backing
* SENSORY OBSERVATIONS
Approach response, Startle response, Pupil response, Forelimb extension, Air righting reflex, Touch response, Tail pinch response, Eyeblink response, Hindlimb extension, Olfactory orientation
* NEUROMUSCULAR OBSERVATIONS
Hindlimb extensor strength, Hindlimb foot splay, Grip strength-hind and forelimb, Rotarod performance.
* PHYSIOLOGICAL OBSERVATIONS
Catalepsy, Body temperature, Body weight

LOCOMOTOR ACTIVITY
Locomotor activity was assessed for all animals prior to the initiation of test article exposure and during study weeks 3, 7 and 12. Locomotor activity, recorded after the completion of the FOB, was measured automatically using the San Diego Instruments, Inc., Photobeam Activity System (San Diego Instruments, Inc., San Diego, California).

Sacrifice and (histo)pathology:

NEUROPATHOLOGY
* MACROSCOPIC EXAMINATION
At the end of the exposure period (study week 13), all animals were anesthetized by an intraperitoneal injection of sodium pentobarbital and then perfused in situ with a buffered 4% paraformaldehyde/1.4% glutaraldehyde solution. The central and peripheral nervous system tissues were dissected and preserved. Fixed brain weight (excluding olfactory bulbs) and brain dimensions (length and width) were recorded. Any observable gross changes, abnormal coloration or lesions of the brain and spinal cord were recorded. Six animals/sex/group from the control and 80 ppm groups were selected for microscopic neuropathology examinations. After the central nervous system tissues were collected, the head was collected and placed in 10% neutral-buffered formalin to preserve the nasal cavity, paranasal sinuses, oral cavity, nasopharynx, middle ear, lacrimal glands and Zymbal’s glands.

SLIDE PREPARATION AND MICROSCOPIC EXAMINATION
The following nervous system tissues were prepared for microscopic neuropathologic examination from 6 animals/sex in the control and 80 ppm groups:
Brain - olfactory bulbs, cerebral cortex (2 levels), hippocampus/dentate gyrus, basal ganglia, thalamus, hypothalamus, tectum, cerebral peduncles, central gray matter, cerebellum, pons and medulla oblongata
Spinal cord - at cervical swellings C3-C7 and at lumbar swellings T13-L4
Trigeminal ganglia/nerves (a
Lumbar dorsal root ganglia at T13-L4*
Lumbar dorsal root fibers at T13-L4*
Lumbar ventral root fibers at T13-L4*
Cervical dorsal root ganglia at C3-C7*
Cervical dorsal root fibers at C3-C7*
Cervical ventral root fibers at C3-C7*
Sciatic nerves (mid-thigh region) (2)+
Sciatic nerves (at sciatic notch) (2)+
Sural nerves (2)+
Tibial nerves (2)+
Peroneal nerves (2)+
Optic nervesa
Eyes (a
Skeletal muscle - gastrocnemius
Other sites - if deemed necessary
a) Both tissues were processed and evaluated microscopically.
* 4-6 tissues were collected following euthanasia; 2 were evaluated
microscopically.
+ 1 nerve processed for microscopic examination.
(2) 2 sections (1 cross and 1 longitudinal) of the tissue were evaluated from the right hind leg. The tissues from the left hind leg were collected and
preserved for possible future evaluation.

NASAL PATHOLOGY
* MACROSCOPIC EXAMINATION
The heads were collected from 6 animals/sex/group (not selected for neuropathological evaluation) from all groups and placed into 10% neutral-buffered formalin to preserve the nasal cavity, paranasal sinuses, oral cavity, nasopharynx, middle ear, lacrimal glands and Zymbal’s glands.
* MICROSCOPIC EXAMINATION
The nasal tissues were prepared for qualitative histopathological examination by embedding in paraffin, sectioning and staining with hematoxylin and eosin. The nasal tissues (Levels I, II, III and IV) were prepared and evaluated according to Young, 1981.

Statistics:

Body weight, body weight change, food consumption, continuous functional observational battery (FOB), ambulatory locomotor activity, and brain weight, length and width data were subjected to a parametric 1-way analysis of variance (ANOVA) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test article-treated groups to the control group.
Functional observational battery parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact Test.

All RANOVA statistical analyses for total locomotor activity counts were conducted by BioSTAT Consultants, Inc. using SAS version 8.2 software (SAS Institute, Inc., 1999- 2001).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Clinical biochemistry findings:

not examined

Behaviour (functional findings):

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Details on results:

CLINICAL OBSERVATIONS AND SURVIVAL
All animals survived to the scheduled euthanasia. There were no test article-related clinical findings during the detailed physical examinations, at the midpoint of exposure or at the time of peak effect (within 1 hour after exposure).

BODY WEIGHTS
Test article-related statistically significant (p<0.05 or p<0.01) lower mean body weight gains in the 20 and 80 ppm group males were noted during study week 0 to 1; these differences generally persisted through study week 8 for the 80 ppm group males.
Consequently, statistically significantly (p<0.01) lower mean body weights and mean cumulative body weight gains were noted for the 80 ppm group males compared to the control group from study week 1 through the end of the exposure period. At the end of the exposure period (study week 13), the mean body weight of the 80 ppm group males was 16% lower than the control group.

A statistically significant (p<0.01) test article-related lower mean body weight gain was also noted in the 80 ppm group females during study week 0 to 1. Lower, usually statistically significant (p<0.05 or p<0.01), mean cumulative body weight gains were observed in the 80 ppm group females throughout the remainder of the study primarily due to this lower mean body weight gain during study week 0 to 1. Mean body weights in this group were slightly although not statistically significantly lower compared to the control group throughout the study. At the end of the exposure period (study week 13), the mean body weight of the 80 ppm group females was 5% lower than the control group.

FOOD CONSUMPTION
Test article-related lower mean food consumption, evaluated as g/animal/day, was noted in the 80 ppm group males and females beginning study week 0 to 1. Lower mean food consumption persisted for the duration of the study in the males only, although not all intervals were statistically significant (p<0.05 or p<0.01). These lower mean food consumption values correlated with the lower body weight gains noted throughout the study in the males and during the first week of exposure in the females.

FUNCTIONAL OBSERVATIONAL BATTERY [FOB]
Home cage, handling, open field, sensory , neuromuscular and physiological (excepted the body weight) observation were unaffected by test article exposure.

LOCOMOTOR ACTIVITY
There were no test article-related effects on total or ambulatory motor activity counts in either sex during the study week 3 and 7 evaluations. Within session repeated measures analyses of variance were conducted across the subintervals of each test session for total activity counts, and linear trend analyses were conducted for overall interval means (representing the entire 60-minute session activity) during each test session. A test article-related significant decrease in overall total counts (p=0.05) was obtained for males in the 80 ppm group (35% reduction in total session activity) during the study week 12 evaluation compared to the mean control group values. These differences resulted from reduced (not statistically significant) activity levels throughout the 60-minute session.
There were no effects on ambulatory motor activity in the 80 ppm group males during the study week 12 evaluation.
There were no effects of the test article on habituation at any exposure level.

PATHOLOGY
* BRAIN WEIGHTS AND MEASUREMENTS
The increased mean brain weight relative to body weight observed in males at 80 ppm was statistically significant (p<0.01) but were considered to be a result of a test article-related decrease in terminal body weight. The mean absolute brain weight was not different from the control group.

MICROSCOPIC EXAMINATION
Test article-related degeneration of the olfactory epithelium on nasal Level II was observed with minimal to moderate severity in all 80 ppm and most 20 ppm group rats. The degree of degeneration on nasal Level II was more severe at the exposure level of 80 ppm. Olfactory epithelial degeneration was also found on Levels III and IV. This degeneration was characterized by a loss of the adluminal cytoplasmic layer of the sustentacular cells, typically with an overall thinning of the olfactory epithelium and disorganization of the underlying layers of olfactory neurons. The degeneration on nasal Levels III and IV occurred with a much higher incidence in the 80 ppm group. The only microscopic finding in the 5 ppm group consisted of minimal degeneration of the olfactory nasal epithelium on nasal Level III in 1/6 males. This was not considered to be test article-related because the findings occurred in a single animal, were of minimal severity and epithelial degeneration was not observed on any other nasal Levels in this animal. On Level II, degeneration of the olfactory epithelium was most prominent on the dorsal arches, while on Levels III and IV, the alteration typically affected the medial aspects of turbinates in the dorsal meatus. Selected histopathological findings are presented in Table 1.

There were no changes in brain tissues (including olfactory bulbs), cranial, spinal or peripheral nerves, spinal nerve roots, eyes, or muscle associated with exposure to test article.

Dose descriptor:

NOAEC

Remarks:

systemic toxicity and neurotoxicity

Effect level:

20 ppm (analytical)

Sex:

male

Basis for effect level:

other: Lower body weight gains, lower food consumption and lower total session motor activity counts at 80 ppm.

Remarks on result:

other:

Dose descriptor:

NOAEC

Remarks:

systemic toxicity and neurotoxicity

Effect level:

80 ppm (analytical)

Sex:

female

Basis for effect level:

other: No adverse effect at 80 ppm.

Remarks on result:

other:

Dose descriptor:

NOAEC

Remarks:

local contact (nasal) irritation

Effect level:

5 ppm (analytical)

Sex:

male/female

Basis for effect level:

other: Mild to moderate degeneration of the nasal olfactory epithelium at 20 and 80 ppm.

Remarks on result:

other:

Table 1: Selected Test Article-Related Histopathological Findings

Incidence and Severity of Degeneration of the Olfactory Epithelium in Nasal Levels I through IV

Exposure Level (ppm)		Males				Females
		0	5	20	80	0	5	20	80
Young Nasal Level II a		6	6	6	6	6	6	6	6
Degen, Olfactory. Epithelium		0	0	6	6	0	0	4	6
	Minimal	0	0	6	3	0	0	4	0
	Mild	0	0	0	2	0	0	0	1
	Moderate	0	0	0	1	0	0	0	5
Young Nasal Level III a		6	6	6	6	6	6	6	6
Degen, Olfactory. Epithelium		0	1	1	5	0	0	0	6
	Minimal	0	1	1	1	0	0	0	0
	Mild	0	0	0	2	0	0	0	4
	Moderate	0	0	0	2	0	0	0	2
Young Nasal Level IV a		6	6	6	6	6	6	6	6
Degen, Olfactory. Epithelium		0	0	1	4	0	0	0	4
	Minimal	0	0	1	1	0	0	0	2
	Mild	0	0	0	2	0	0	0	2
	Moderate	0	0	0	1	0	0	0	0

a = Number of tissues examined from each group.

Degen = Degeneration

Conclusions:

Systemic toxicity of DMDS administered by whole-body inhalation exposure to Crl:CD(SD) rats for 13 weeks was observed at an exposure level of 80 ppm in males as evidenced by lower body weight gains and lower food consumption through the first 8 weeks of the exposure period. Lower total session motor activity counts were noted in the 80 ppm group males during study week 12. Local contact (nasal) irritation was observed at exposure levels of 20 and 80 ppm in males and females as a dose-dependent mild to moderate degeneration of the nasal olfactory epithelium.

Executive summary:

A subchronic neurotoxicity study via the inhalation route was conducted with dimethyl disulphide (DMDS) following the OECD guideline # 424. Four groups of 12 male and 12 female Crl:CD(SD)BR were exposed to either clean filtered or DMDS vapor atmospheres or 5, 20 or 80 ppm for 6 hours daily in whole-body inhalation chambers for 13 consecutive weeks. All animals were observed twice daily for mortality and moribundity and clinical examinations were perfortned daily. Body weights and food consumption were recorded weekly. Functional observational battery and locomotor activity assessments were evaluated prior to initiation of exposure and at the time of peak effect (one hour post exposure) during study weeks 3, 7 and 12. Brain weights and dimensions were determined and neuropathologic evaluations were performed. In addition, a microscopic examination of nasal tissues was performed.

DMDS-related, dose-dependent findings in the 20 and 80 ppm group males and females included lower food consumption and corresponding lower body weight gains during study week 0 to 1. Thereafter, body weight gain and food consumption for the 20 and 80 ppm group females and 20 ppm group males returned to levels comparable to the control group beginning during study week 1 to 2, but lower food consumption and body weight gains were sustained in the 80 ppm group males for the duration of the study. As a result, body weights of the males were reduced compared to the control group and cumulative body weight gains were reduced for the duration of the study for both sexes.

Lower total motor activity counts were observed in the 80 ppm group males during all sub-intervals of the study week 12 evaluation. Habituation patterns for these animals were unremarkable. Neuropathologic parameters affected by test article exposure included lower brain length in the males. Minimal to moderate degeneration of the olfactory epithelium on nasal Level II was observed in all 80 ppm males and females. In general, the olfactory epithelium of the females was more severely affected. Minimal to moderate degeneration of the olfactory epithelium was noted on nasal Level II in 6/6 males and 4/6 females in the 20 ppm group. Olfactory epithelial degeneration, characterized by a loss of the adluminal cytoplasmic layer of the sustentacular cells, was also found on Levels III and IV in the 80 ppm group. Minimal olfactory epithelial degeneration on Levels III and IV was noted in 1/6 males in the 20 ppm group. The only test article-related finding in the 5 ppm group consisted of minimal degeneration of the olfactory nasal epithelium on nasal Level III in 1/6 males. On Level II, degeneration of the olfactory epithelium was most noticeable on the dorsal arches, while on Levels III and IV, the alteration typically affected the medial aspects of turbinates in the dorsal meatus.

The no-observed-adverse-effect concentration (NOAEC) for systemic toxicity and neurotoxicity of DMDS via whole-body inhalation exposure to Crl:CD(SD) rats for 13 consecutive weeks was 20 ppm for males and 80 ppm for females. The NOAEL for local contact (nasal) irritation of DMDS via whole-body inhalation exposure for 13 consecutive weeks was 5 ppm for both males and females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

77 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP guideline study

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Acute neurotoxicity
Four groups of 12 male and 12 female Sprague-Dawley rats were exposed whole body for 6 hours to either clean filtered air or DMDS vapour atmospheres of 100, 200 or 750 ppm (Nemec, 2005b). All animals were observed twice daily for mortality and moribidity, clinical examinations were performed daily except on days when the functional observational battery (FOB) was performed and body weights were recorded weekly. FOB and locomotor activity evaluations were performed on all animals prior to the initiation of exposure, at the time of peak effect following exposure and on study days 7 and 14. Following perfusion, all animals were necropsied and brain weights and brain dimensions were measured. Neuropathologic evaluations were performed on central and peripheral nervous system tissues for control and 750 ppm groups. 
No DMDS-related effects were apparent between treated and control group animals on sensorimotor and neuromuscular parameters when FOB (functional observational battery) evaluations were performed at the time of peak effect on study day 0 and on study days 7 and 14. Fixed brain weights and brain dimensions for perfused animals were unaffected. No DMDS-related neuropathological lesions were observed upon microscopic examination of 6 animals per sex in the 750 ppm group. 
DMDS-related effects in the 750 ppm group included the death of one female during exposure, salivation during exposure and decreased mean body weight gain in both sexes for study days 0 to 7. FOB parameters that were affected were observed at the time of peak effect on study day 0 and did not persist to the study day 7 and 14 evaluations. Findings observed at 750 ppm included increased numbers of animals with a change in palpebral closure (drooping eyelids) during the home cage observations, slightly soiled fur during the handling observations, increased grooming and urination counts during the open field observations and decreased body temperature and body weight during the physiological observations. Findings observed in the females at 750 ppm consisted of red deposits around the mouth during the handling observations and tremors during the open field observations. Ambulatory and total motor activity counts over 60 minutes were reduced in both sexes at 750 ppm on study day 0 only.
Test article-related effects in the 200 ppm group consisted of salivation during exposure and slightly soiled fur, red deposits around the mouth and tremors. The only test article-related findings in the 100 ppm group were salivation during exposure and slightly soiled fur. 
Several of the aforementioned findings (drooping eyelids, salivation, increased grooming counts, red deposits and soiled fur) were most likely a result of the slightly irritating properties of DMDS. Therefore, these test article-related findings were not considered to be indicative of systemic toxicity. The Lowest Observed Adverse Effect Concentration (LOAEC) was 200 ppm based on treatment related effects consisting of salivation during exposure in males and females and slightly soiled fur, red deposits around the mouth and tremors in females. The No Observed Adverse Effect Concentration (NOAEC) for systemic toxicity and neurotoxicity was 100 ppm.

Subchronic neurotoxicity
The objective of the study was to evaluate the potential neurotoxic effects of DMDS when administered by whole-body inhalation exposure for 13 weeks, using a neurotoxicity screening battery consisting of functional observational battery, locomotor activity and neuropathological assessments (Nemec, 2006c).
Lower mean body weight gain in the 80 ppm group males corresponded with lower mean food consumption recorded through study week 8. Thereafter, body weight gains were comparable to the control group, but body weights and cumulative body weight gains remained persistently and significantly lower due to the lower body weight gains early in the study. Lower mean body weight gain in the 20 ppm group males and 80 ppm group females was observed at the initiation of exposure (study week 0 to 1), but body weight gains in these groups were comparable to the control group during the remainder of the study suggesting a recovery from these effects.
DMDS-related effects on FOB parameters at the time of anticipated peak effect (within 1 hour of the end of exposure) included effects on the physiological domain (as defined by Moser, 1991a) of the 80 ppm group males during study weeks 3, 7 and 12 as evidenced by lower mean body weights. Because these lower mean body weights correlated with lower mean food consumption observed throughout the study and no other physiological parameters or other functional domains were affected, the lower body weights were considered to be an expression of systemic toxicity rather than neurotoxicity.
During the locomotor activity assessment, lower, though not statistically significant by repeated measure analysis of variance tests, mean total session activity counts were observed in the 80 ppm group males. Overall habituation patterns in these animals were unaffected. The motor activity assessment during study week 12 was the only assessment where any test article-related effects were observed. These same animals exhibited a substantial body weight effect that resulted in slower overall growth than the control or other test article-exposed groups.
Histopathologic examination noted degeneration of the nasal olfactory epithelium on nasal Levels II, III and/or IV in the 20 and 80 ppm groups. The severity was graded minimal to moderate with a greater degree and incidence of degeneration generally being noted at higher exposure levels. This was considered to be indicative of DMDS-related local contact (nasal) irritation. No other remarkable nasal alterations were noted during microscopic examination. No changes were noted on microscopic examination of the peripheral or central nervous system tissues. No remarkable findings were noted in gross pathology or brain weights or measurements.
Therefore, the no-observed-adverse-effect concentration (NOAEC) for systemic toxicity and neurotoxicity of DMDS via whole-body inhalation exposure to Crl:CD(SD) rats for 13 consecutive weeks was 20 ppm for males and 80 ppm for females. The NOAEC for local contact (nasal) irritation of DMDS via whole-body inhalation exposure for 13 consecutive weeks was 5 ppm for both males and females.

Justification for classification or non-classification

From the results of the acute and subchronic neurotoxicity studies and in accordance with Regulation (EC) No 1272/2008, DMDS does not warrant classification with regard to Specific Target Organ Toxicity – Single or Repeated Exposure.