Carbon disulphide

Endpoint summary

Description of key information

see discussion below.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Dose descriptor:

LOAEL

253 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well perfomed but older GLP study according to existing guideline at that time

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, MI
- Age at study initiation: 6 weeks
- Weight at study initiation: yes
- Housing: stainless steel and glass chambers, housed individually
- Diet: ad libitum
- Water: ad libitum
The animals were kept 14 days in quarantine before the initiation of the experiment. Only healthy animals were used.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-30
- Humidity (%): 35-84%
- Photoperiod (hrs dark / hrs light): 12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Reproduced from the original report:
The test article was vaporized in a 3-neck, round-bottom flask. The test article was metered to the vaporization flask using a FMI Lab Pump. The vapours were swept from the flask by a continuous supply of conditioned, compressed air and entered the chamber through a turret located at the top of the chamber. In the turret the vapours were mixed with chamber supply air. The metered flow of test article into the vaporization flask and total air through the chamber were adjusted to maintain the target concentration within the chamber. The test article delivery rate and total air flow through the chamber were used in calculating the nominal concentration within the chamber. Airflow was monitored continuously throughout the exposure by reading the pressure differential from a minihelic pressure gauge and recording the corresponding airflow from a prepared calibration graph showing airflow versus differential pressure. The graph was prepared by plotting various airflow readings from an Autotronic Controls Turbine Flow Meter at different differential pressure readings and fitting a line to the points. The negative pressure of each test chamber was maintained at 0.1 inches of water. The control chamber was maintained at a positive pressure of 0.02 inches of water. Negative and positive pressures were measured with minihelic pressure gauges.

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatographic chamber-monitoring system

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

6 hours/day; 5 days/week; at least 89 consecutive days

Frequency of treatment:

5 days/week

Remarks:

Target Concentrations:
0, 50, 300, 800 ppm

Remarks:

Concentrations:
0, 49.3, 297.1, 798.4 ppm (155.8, 938.8, 2522.9 mg/m3)
Basis:
analytical conc.

No. of animals per sex per dose:

15

Control animals:

yes

Details on study design:

The animals were sacrificed on day 90, 91, 92, 93 or 94 and 98 or 99. One female from each group was selected for viral serology. Five animals per group/sex were used for special neuropathologic studies.

Positive control:

no

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: twice per day

NEUROLOGICAL FUNCTION: observation of posture and gait, and facial muscular tone or symmetry; four neuromuscular reflexes were also evaluated

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined : Yes, weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before exposure and prior to necropsy
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No data
- Parameters examined: erythrocyte count, haematocrit, haemoglobin concentration, mean corpuscular haemoglobin, mean corpuscular volume
mean corpuscular haemoglobin concentration, total leukocyte count, differential leukocyte count, platelet count, prothrombin time

CLINICAL CHEMISTRY: Yes (serum)
- Animals fasted: No data
- Parameters examined: glutamic pyruvic transaminase, total bilirubin, urea nitrogen, glucose, glutamic oxaloactic transaminase, total protein, phosphorus, sodium, potassium, chloride, calcium, alkaline phosphatase and gamma-glutamyl transpeptidase

URINALYSIS: Yes
- Time schedule for collection of urine: prior necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (12 h)
- Parameters examined: volume, appearance, occult blood, specific gravity, protein, pH, ketone, and glucose

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

Special neuropathology study: 5 animals/sex/group. the right and left sciatic nerve and their branches were dissected together with specimens of the cervical and lumbar spinal cord and then placed in 4% glutaraldehyde. The left sural nerve and the large muscle branch of the left tibial nerve were osmicated then placed in cedarwood oil treated specimens were teased to seperate the individual fibers then mounted on glass slides. The teased nerve fibers were coverslipped and retained as permanent specimens. A minimum of 50 teased fibers per rat, approximately 25 per nerve, were prepared. Glutaraldehyde fixed specimens of the right sural nerve, the muscular branch of the right tibial nerve, and specimens of the spinal cord from the cervical and lumbar regions were osmicated, dehydrated, and embedded with Epon. Sections were created from the Epon specimens, stained with toluidine blue and examined under light microscope.

Statistics:

Parametric data analyzed with ANOVA, statistically significant differences with Turkey's or Scheffe's Test of Multiple Comparison, Kruskal-Wallis ANOVA and Test Of Multiple Comparison for non-parametric data

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):

effects observed, treatment-related

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: no mortality; crusty muzzle and ataxia noted primarily at 800 ppm, other observations noted in few animals: yellow/brown stained fur, swollen eye, red and swollen ear, poor coat quality, red stained fur, muscle tremors, crusty eye and nose

NEUROLOGICAL FUNCTION: ataxia was observed at 800 ppm

BODY WEIGHT AND WEIGHT GAIN: statistically significant lower body weights after 1st week at 800 ppm for both sexes (Fig. 5&6, attachment below), and after the 5th week for males exposed to 300 ppm (already observed before but not statistically significant)

FOOD CONSUMPTION: significant decrease in males exposed to 800 ppm; lower response in females, significant decrase only at week 1 and 3

OPHTHALMOSCOPIC EXAMINATION: no significant abnormality observed

HAEMATOLOGY: in males a clear erythorocyte count depression was detected at 800 ppm, accompanied by a slight, though significant, increase in the mean corpuscular volume, the mean corpuscular haemoglobin volume and concentration; this discrepancy cannot be explained. An increase in the mean corpuscular haemoglobin volume and inhemoglobin levels was also measured at 300 ppm. Changes in lymphocytes, eosinophils and platelet levels were detected at 800 ppm in males, as well as in females, except for the platelets levels (see Tables 2&3 for details, attachment).

CLINICAL CHEMISTRY (serum): statistically significant changes were observed only in animals exposed to the highest concentration (Tables 2&3); calcium depression (8.8 and 13.5%, for males and females, respectively), elevated transaminases in both sexes suggesting mild liver damage, urea nitrogen increased in males (27.8%), total protein decreased in females (10.2%), potassium levels decreased in males.

URINALYSIS: urine volume decreased in females at 300 and 800 ppm, while only at the highest (although a 40% decrease, still not statistically significant) for males; urine specific gravity was increased at the highest concentration for both sexes (Tables 2&3, attachment), traces of ketones and blood detected in urine samples.

ORGAN WEIGHTS: brain weights significantly decreased at the 2 highest concentrations for females and at all concentrations for males, spleen and liver weights depressed at 800 ppm in males and ovarian weights also decreased at the same dose level in females (see details in Tables 4&5, attachment). All male and female organ/bw ratios (at 800 ppm) were increased, except for the spleen in males and spleen and brain in females. Organ/brain ratios can be seen in Tables 4&5 (attachment).


HISTOPATHOLOGY: axonal swelling of nerve fibers of the ventral and lateral funiculi of the spinal cord at 800 ppm for both sexes, segmental degeneration of fibers in the sciatic nerve in few animals of the high dose group, slight increase in iron positive pigmentaion of the spleen in high dose males and females

Dose descriptor:

NOAEC

Effect level:

50 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: only a slight reduction in absolute brain weight in males but not in relative brain weight

Dose descriptor:

NOAEC

Effect level:

300 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: only a slight reduction in absolute brain weight but not in relative brain weight

Critical effects observed:

not specified

SPECIAL NEUROPATHOLOGICAL EXAMINATIONS: axonal swelling of the muscular nerve in all 5 animals exposed to the high concentration, as well as of the sural nerve in all males and 3/5 females (Table 1, attachment). One female rat exposed to the 2nd concentration exhibited a similar lesion in a single fiber of the sural nerve. Axonal swelling was observed in the muscular and sural nerves, as well as in the lumbar and cervical cord fibers in both sexes exposed to 800 ppm.

Conclusions:

The study yielded a NOAEC of 50 ppm, a slight reduction was seen in absolute brain weight but not in relative brain weight of males. At 300 ppm, decreased male body weights, as well as some hematological changes were seen. Clinical observations, neurological function disorders, changes in hematological and urine examinations, decreased organ weights, increased organ/bw & organ/brain weight ratios, as well as neuropathological manifestations were detected at 800 ppm.

Executive summary:

In a subchronic inhalation toxicity study, carbon disulfide was administered to male and female Fischer 344 rats as vapour by inhalation at the following nominal concentrations: 0, 50, 300, and 800 ppm (analytical: 0, 153.7, 925.9, and 2488.3 mg/m3-49.3, 297.1, 798.4 ppm), for 6 hours/day, 5 days/week for at least 89 consecutive days. No mortality was observed among the animals during the experiment. No substance related clinical signs were observed except for ataxia (highest concentration). Depression in body weights was seen in males at 300 and 800 ppm and only at 800 ppm in females. Some hematological changes and clinical changes were measured, mainly at 800 ppm. The elevated transaminases in the serum indicate liver damage, although no histologic lesions were noted. The increase in urea nitrogen was probably related to reduced water consumption, as suggested by the diminished urine volumes and elevated urine specific gravity. Calcium changes might be associated with the histologic lesions that were observed in the kidneys. The histopathological examinations revealed axonal swelling of nerve fibers of the spinal cord, and the muscular and sural nerve at 800 ppm for both sexes, as well as segmental degeneration of fibers in the sciatic nerve in few animals of the high dose group, and positive iron pigmentation in both sexes exposed to 800 ppm. The study yielded a NOAEC of 50 ppm for males (only a slight reduction in absolute brain weight but not in relative brain weight), and of 300 ppm for females for the same reason.

This subchronic inhalation toxicity study in the Fischer 344 rats is similar to the guideline requirement for a subchronic inhalation study OECD 413.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

February 1982 to May 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well performed but older GLP study according to existing guideline at that time

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

other: B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, MI
- Housing: stainless steel and glass chambers, housed individually
- Diet: ad libitum
- Water: ad libitum
The animals were kept in a 14 day quarantine before the initiation of the experiment. Only healthy animals were used.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-30
- Humidity (%): 35-84%
- Photoperiod (hrs dark / hrs light): 12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Reproduced from the original report:
The test article was vaporized in a 3-neck, round-bottom flask. The test article was metered to the vaporization flask using a FMI Lab Pump. The vapours were swept from the flask by a continous supply of conditioned, compressed air and entered the chamber through a turret located at the top of the chamber. In the turret the vapours were mixed with chamber supply air. The metered flow of test article into the vaporization flask and total airo through the chamber were adjusted to maintain the target concentration within the chamber. The test article delivery rate and total air flow through the chamber were used in calculating the nominal concentration within the chamber. Airflow was monitored continuously throughout the exposure by reading the pressure differential from a minihelic pressure gauge and recording the corresponding airflow from a prepared calibration graph showing airflow versus differential pressure. The graph was prepared by plotting various airflow readings from an Autotronic Controls Turbine Flow Meter at different differential pressure readings and fitting a line to the points. The negative pressure of each test chamber was maintained at 0.1 inches of water. The control chamber was maintained at a positive pressure of 0.02 inches of water. Negative and positive pressures were measured with minihelic pressure gauges.

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatographic chamber-monitoring system

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

6 hours/day, 5 days/week, for at least 89 consecutive days

Frequency of treatment:

5 days/week

Remarks:

Target concentrations:
0, 50, 300, 800 ppm

Remarks:

Concentrations:
0, 49.3, 297.1, 798.4 ppm (155.8, 938.8, 2522.9 mg/m3)
Basis:
analytical conc.

No. of animals per sex per dose:

10 males and 12 females

Control animals:

yes

Details on study design:

Animals were sacrificed on day 90, 91, 92, 93 or 94 and 98. Ten male and female per group were designated for clinical pathology, gross and histopathology, while two female animals were used for viral serology.

Positive control:

no

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice daily; detailed examinations were performed weekly

NEUROLOGICAL FUNCTION: observation of posture and gait, and facial muscular tone or symmetry; four neuromuscular reflexes were also evaluated

BODY WEIGHT: Yes
- Time schedule for examinations: before exposure and weekly until the end of the experiment

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes, weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior exposure and within 7 dasy before necropsy
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No data
- Parameters examined: erythrocyte count, haematocrit, haemoglobin concentration, mean corpuscular haemoglobin, mean corpuscular volume
mean corpuscular haemoglobin concentration, total leukocyte count, differential leukocyte count, platelet count, prothrombin time

CLINICAL CHEMISTRY: Yes (serum)
- Animals fasted: No data
- Parameters examined: glutamic pyruvic transaminase, total bilirubin, urea nitrogen, glucose, glutamic oxaloactic transaminase, total protein, phosphorus, sodium, potassium, chloride, calcium, alkaline phosphatase and gamma-glutamyl transpeptidase

URINALYSIS: Yes
- Time schedule for collection of urine: prior necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (12 h)
- Parameters examined: volume, appearance, occult blood, specific gravity, protein, pH, ketone, and glucose

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Parametric data analyzed with ANOVA, statistically significant differences with Turkey's or Scheffe's Test of Multiple Comparison, Kruskal-Wallis ANOVA and Test Of Multiple Comparison for non-parametric data

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):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: 4 animals exposed to 800 ppm died (2 males day 92, 2 females day 87); no substance related clinical signs

NEUROLOGICAL FUNCTION: 1 animal from each group (50 and 300 ppm) and 4 animals from the highest concentration group did not respond to artificial light stimulus

BODY WEIGHT AND WEIGHT GAIN: CS2-treatment related depression of body weight gain in male and female mice at the highest exposure level for both sexes (Table 1, Fig. 5&6, attachment).

FOOD CONSUMPTION: only significant decrease observed at week 13 at the highest concentration for females

OPHTHALMOSCOPIC EXAMINATION: no significant abnormalities observed

HAEMATOLOGY: red blood cells, haemoglobin and haematocrit were significantly reduced at the highest concentration in both sexes; leucocytes counts were elevated in the two lowest concentrations in males, but not at the highest one; probably this was not a substance related effect (Tables 2&3)

CLINICAL CHEMISTRY (serum): total serum protein was diminished in both sexes at the highest concentration; urea nitrogen was reduced in females exposed to the highest concentration, but based on the authors it was non conclusive (Tables 2&3)

URINALYSIS: no statistical significant changes were detected (Tables 2&3)

ORGAN WEIGHTS: brain weight depression at the high dose level for both males and females, as well as in kidneys and testicular of males and ovaries of females; heart/bw and heart/brain weight ratios were increased at the high concentration level in both sexes, as well as the ratios kidney/brain and ovary/bw in females (Tables 4&5)

HISTOPATHOLOGY: segmental degeneration in peripheral nerves at the high dose group, axonal swelling in ventral and lateral funiculi of the thoracic and lumbar spinal cord at 800 ppm, renal lesions diagnosed as nephropathy at 800 ppm, iron positive pigmentation (haemosiderin) observed in macrophages of the red pulp of the spleen in high dose animals.

Dose descriptor:

NOAEC

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no significant treatment-related changes observed

Dose descriptor:

LOAEC

Effect level:

800 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

The study yielded a NOAEC of 300 ppm. No statistically significant effects were observed at 50 and 300 ppm for both sexes. At 800 ppm significant decreases in body weight, in hematological and clinical chemistry parameters, as well as in several absolute organ weights were detected. Increases of organ/bw and organ/brain ratios, as well as histopathological changes of the neurons, spleen and kidneys were seen.

Executive summary:

In a subchronic inhalation toxicity study, carbon disulfide was administered to male and female B6C3F1 mice as vapour by inhalation at the following targeted concentrations: 0, 50, 300, and 800 ppm (analytical: 0, 154, 926, and 2488 mg/m3- 49.3, 297.1, 798.4 ppm), for 6 h/day, 5 days/week for at least 89 consecutive days. Four animals exposed to the high dose level died (2 males day 92, 2 females day 87) during the experiment. Depression of body weight gain was observed in male and female mice exposed to the highest concentration. Significant decreases in erythrocyte numbers, total haemoglobin and haematocrit were detected at the high concentration groups, which seems to be in accordance with the increased levels of iron positive pigment in the spleens. Nephropathy was diagnosed in the majority of males exposed to 800 ppm. Segmental degeneration in peripheral nerves were detected in animals exposed to 800 ppm, as well as axonal swelling in the thoracic and lumbar spinal cord. The NOAEC for the present study is 300 ppm, based on all observations performed.

This subchronic inhalation toxicity study in the B6C3F1 mice is similar to the guideline requirement for a subchronicinhalation study OECD 413.

Reference 3

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well performed but older GLP study according to existing guideline at that time

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

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 Breeding Laboratories, Inc., Portage, MI
- Age at study initiation: 6 weeks
- Weight at study initiation: yes
- Housing: stainless steel and glass chambers, housed individually
- Diet: ad libitum
- Water: ad libitum
The animals were kept in a 14 day quarantine before the initiation of the experiment. Only healthy animals were used.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-30
- Humidity (%): 35-84%
- Photoperiod (hrs dark / hrs light): 12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Reproduced from the original report:
The test article was vaporized in a 3-neck, round-bottom flask. The test article was metered to the vaporization flask using a FMI Lab Pump. The vapours were swept from the flask by a continuous supply of conditioned, compressed air and entered the chamber through a turret located at the top of the chamber. In the turret the vapours were mixed with chamber supply air. The metered flow of test article into the vaporization flask and total air through the chamber were adjusted to maintain the target concentration within the chamber. The test article delivery rate and total air flow through the chamber were used in calculating the nominal concentration within the chamber. Airflow was monitored continuously throughout the exposure by reading the pressure differential from a minihelic pressure gauge and recording the corresponding airflow from a prepared calibration graph showing airflow versus differential pressure. The graph was prepared by plotting various airflow readings from an Autotronic Controls Turbine Flow Meter at different differential pressure readings and fitting a line to the points. The negative pressure of each test chamber was maintained at 0.1 inches of water. The control chamber was maintained at a positive pressure of 0.02 inches of water. Negative and positive pressures were measured with minihelic pressure gauges.

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatographic chamber-monitoring system

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

6 hours/day, 5 days/week, for at least 89 consecutive days

Frequency of treatment:

5 days/week

Remarks:

Target concentrations:
0, 50, 300, 800 ppm

Remarks:

Concentrations:
0, 49.3, 297.1, 798.4 ppm (155.8, 938.8, 2522.9 mg/m3)
Basis:
analytical conc.

No. of animals per sex per dose:

15

Control animals:

yes

Details on study design:

The animals were sacrifcsed on day 90, 91, 92, 93 or 94 and 98 or 99. One female from each group was selected for viral serology. Five animals per group/sex were used for special neuropathologic studies.

Positive control:

no

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: twice per day

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined : Yes, weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before exposure and prior to necropsy
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No data
- Parameters examined: erythrocyte count, haematocrit, haemoglobin concentration, mean corpuscular haemoglobin, mean corpuscular volume
mean corpuscular haemoglobin concentration, total leukocyte count, differential leukocyte count, platelet count, prothrombin time

CLINICAL CHEMISTRY: Yes (serum)
- Animals fasted: No data
- Parameters examined: glutamic pyruvic transaminase, total bilirubin, urea nitrogen, glucose, glutamic oxaloactic transaminase, total protein, phosphorus, sodium, potassium, chloride, calcium. alkaline phosphatase and gamma-glutamyl transpeptidase

URINALYSIS: Yes
- Time schedule for collection of urine: prior necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (12 h)
- Parameters examined: volume, appearance, occult blood, specific gravity, protein, pH, ketone, and glucose

NEUROLOGICAL FUNCTION: observation of posture and gait, and facial muscular tone or symmetry; four neuromuscular reflexes were also evaluated

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Other examinations:

Special neuropathology study: 5 animals/sex/group; after animal perfusion, the right and left sciatic nerve and their branches were dissected together with specimens of the cervical and lumbar spinal cord and then placed in 4% glutaraldehyde. The left sural nerve and the large muscle branch of the left tibial nerve were osmicated then placed in cedarwood oil treated specimens were teased to seperate the individual fibers then mounted on glass slides. The teased nerve fibers were coverslipped and retained as permanent specimens. A minimum of 50 teased fibers per rat, approximately 25 per nerve, were prepared. Glutaraldehyde fixed specimens of the right sural nerve, the muscular branch of the right tibial nerve, and specimens of the spinal cord from the cervical and lumbar regions were osmicated, dehydrated, and embedded with Epon. Sections were created from the Epon specimens, stained with toluidine blue and examined under light microscope.

Statistics:

Parametric data analyzed with ANOVA, statistically significant differences with Turkey's or Scheffe's Test of Multiple Comparison, Kruskal-Wallis ANOVA and Test Of Multiple Comparison for non-parametric data

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):

effects observed, treatment-related

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: one male exposed to the highest concentration level was found dead on day 41; observations included crusty eye and nose, yellow/brown stained fur, lethargy, emaciation and poor coat quality.

NEUROLOGICAL FUNCTION: ataxia was noted the highest concentration exposed rats, while some animals (6/29) exhibited slight foot drag. No other abnormalities were observed.

BODY WEIGHT AND WEIGHT GAIN: statistically significant lower body weights after 1st week at the highest concentrations for both sexes, as well as at weeks 4 and 6 at the 2nd concentration for males (Fig. 5&6, attachment below)

FOOD CONSUMPTION: significant decrease in males exposed to the highest concentration; in females, significant decrease at the same exposure concentration only after the 1st week

OPHTHALMOSCOPIC EXAMINATION: no significant abnormality observed

HAEMATOLOGY: changes in leukocyte counts measured in males at the two first concentration levels (Tables 2&3, attachment). In females, haemoglobin and hematocrit were decreased in the first group; mean corpuscular volume, as well as mean corpuscular haemoglobin levels were slightly increased (still significant) in the last two groups in females.

CLINICAL CHEMISTRY (serum): statistically significant decreases were observed in potassium and calcium levels, in males exposed to the highest concentration (Tables 2&3); still within the normal range for rats. In females exposed to the highest concentration, a decrease in serum calcium levels and an increase in alkaline phosphatase was detected.

URINALYSIS: no significant changes observed (Tables 2&3)

ORGAN WEIGHTS: brain weights decreased significantly at 800 ppm for both sexes, and additionally at 300 ppm for females.
Kidney weights depressed at 800 ppm in males. All organ/bw ratios were increased significantly in males of the high-concentration exposure group, while for females it was only heart and liver ratios. Similarly, heart and liver/brain weight ratios were also increased in females at the same concentration level, as well as at 300 ppm only for the liver (see details in Tables 4&5, attachment)

GROSS PATHOLOGY

HISTOPATHOLOGY: axonal swelling of nerve fibers of the ventral and lateral funiculi of the spinal cord at the highest concentration for both sexes, with most commonly affected the thoracic cord, followed by lumbar and cervical cords.Segmental degeneration of fibers in the sciatic or tibial nerve in some animals (6/10 per sex) of the high dose group. A slight increase in iron positive pigmentation was observed in macrophages of the spleen in high dose males and females

Dose descriptor:

NOAEC

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects at this level

Dose descriptor:

LOAEC

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight decrease in males, mean corpuscular hemoglobin increased and serum alkaline phosphatase decreased in females, brain weight decrease and the liver/brain weight ratio increase in females

Critical effects observed:

not specified

SPECIAL NEUROPATHOLOGICAL EXAMINATIONS: axonal swelling of the muscular nerve in all 5 animals/sex exposed to the high concentration, as well as of the sural nerve in all males and 4/5 females (Table 1, attachment). In Epon sections axonal swelling was observed in the muscular and sural nerves, as well as in the lumbar and cervical cord fibers at the high-concentration group. Similar lessions were seen in the lumbar cord of one male exposed to the 2nd concentration level.

Conclusions:

Body weight (decrease) in males, a few hematological parameters (increased) and alkaline phosphatase (decreased) in females appeared significantly different after exposure to 300 ppm. At the same concentration, the brain weight decreased and the liver/brain wight ratio of increased, but only for females. Histopathological findings were significant and CS2 -treatment related only at 800 ppm. Moreover, potassium and calcium serum levels declined at 800 ppm.

Executive summary:

In a subchronic inhalation toxicity study, carbon disulfide was administered to male and female Sprague-Dawley rats as vapour by inhalation at the following nominal concentrations: 0, 50, 300, and 800 ppm (analytical: 0, 154, 926, and 2488 mg/m3 - 49.3, 297.1, 798.4 ppm), for 6 hours/day, 5 days/week for at least 89 consecutive days. Substance related body weight depression was observed at the highest concentration exposure groups. Hematology findings did not reveal definite adverse effects that could be attributed to CS2. Serum calcium decreases, although within the normal range, were probably related to CS2 exposure. Brain weight depression was seen in males (at 800 ppm) and females (at 300 and 800 ppm) and was considered exposure related. Axonal swelling of the muscular nerve, as well as of the sural nerve was observed in the highest concentration group. In Epon sections axonal swelling was observed in the muscular and sural nerves, as well as in the lumbar and cervical cord fibers at the high-concentration group. Similar lessions were seen in the lumbar cord of one male exposed to the 300 ppm . The study yielded a NOAEC of 50 ppm.

This subchronic inhalation toxicity study in the Fischer 344 rats is similar to the guideline requirement for a subchronic inhalation study OECD 413.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

158 mg/m³

Study duration:

subchronic

Experimental exposure time per week (hours/week):

30

Species:

rat

Quality of whole database:

Well performed GLP study with 3 species (Fischer 344 and SD rats, and mice); NOAEC 50 ppm (158 mg/m3)

System:

other: nervous system and cardiovascular

Additional information

Inhalation

Inhalation is the most relevant route of exposure to CS2. The three 90 -day inhalation studies of Toxigenics, which are summarized in the present section, provide information on the possible toxic effects likely to arise from repeated exposure to CS2, assessing the major endpoints, and are used as the key studies. Each study was performed under almost identical conditions; they differed as regards the test animals: 2 strains of rats (Fischer 344 and Sprague-Dawley) and one strain of mice (B6C3F1). Design and methods were similar to those prescribed in applicable guidelines. Animals were exposed to CS2 vapours at 0, 50, 300, and 800 ppm, for 6 h/d, 5 d/w for at least 89 consecutive days.

Fischer rats: The study yielded a NOAEC of 50 ppm, as there was only a slight decrease in absolute brain weight but not in relative brain weight in males. At 300 ppm, decreased male body weights, as well as some hematological changes were seen. Clinical observations, neurological function disorders, changes in the outcome of hematological and urine examinations, decreased organ weights, increased organ/bw & organ/brain weight ratios, as well as neuropathological manifestations were detected at 800 ppm.

Mice B6C3F1: The study yielded a NOAEC of 300 ppm. No statistically significant effects were observed at 50 and 300 ppm for both sexes. At 800 ppm significant decreases in body weight, changes of hematological and clinical chemistry parameters, as well as of several absolute organ weights were detected. Increases of organ/bw and organ/brain ratios, as well as histopathological changes of the neurons, spleen and kidneys were seen.

Sprague- Dawley rats: Body weight (decrease) in males, a few hematological parameters (increased) and serum alkaline phosphatase (decreased) in females appeared significantly different after exposure to 300 ppm. At the same concentration, the brain weight decreased and the liver/brain weight ratio increased, but only for females. Histopathological findings (nerve axonal swelling) were significant and CS2 -treatment related only at 800 ppm.

 

In addition to the Toxigenics study, a large number of non-standard studies was identified based on the most recent and elaborate reviews, which adress the toxicity of CS2 after subchronic or/and chronic exposure. Due to the huge amount of data, since CS2 toxicity has been thoroughly investigated for many years, it was not possible to include all of them as separate endpoint study records. Parts of the reviews are reproduced as such, as a separate endpoint study record. Besides this, a selection of some important publications was made based on the reviews, in order to provide a complete and up-to-date picture of the repeated dose toxicity of the substance. Robust summaries were prepared for some of the publications, while others were summarized based on information collected from reviews and on the published abstract. Most of these studies have focused on particular endpoints associated with effects observed in exposed humans; they do not encompass the standard set of endpoints that is investigated in a standard inhalation repeated-dose toxicity study. Therefore, these studies are not summarized in this section of Chapter 7, but in Section 7.9 Specific investigations. For instance, publications that are dealing with effects of CS2 on the nervous system can be found in Section 7.9.1 Neurotoxicity, while studies adressing cardiovascular toxicity are summarized in Section 7.9.4.

As was already observed in the standard repeated-dose toxicity studies, the publications show that the nervous system is an important and sensitive target of CS2 toxicity. The most complete neurotoxicity study was conducted in a collaboration of the US National Institue for Environmental Health Sciences (NIEHS), the US Environmental Protection Agency (EPA) and the Duke University Medical Center. Male and female F344 rats were exposed to CS2 at 0, 50, 500, or 800 ppm for 6 h/d, 5 d/wk for 2, 4, 8, or 13 weeks. Within 2 weeks of exposure to either 500 or 800 ppm, the expression of the nerve-growth factor receptor mRNA in the sciatic nerve was increased in all animals, implying changes in the reactions between the axon and Schwann cells; this expression increased during further exposure (Toews et al. 1998). Starting from 2-4 weeks of exposure, the histopathological examination of the spinal cord revealed neurofilamentous cross-linking at 50 ppm (although significant only at 13 weeks; Valentine et al., 1997 & 1998), and axonal swelling was not detected at this level. In erythrocytes, covalent modification of globin was observed at all CS2 concentrations, which was paralled by spectrin crosslinking (Valentine et al. 1998). Neuromotor dysfunction was observed, mainly manifesting itself in the hindlimbs, evident as early as 2 weeks of exposure at 800 ppm, and at 500 ppm, after longer exposure (Moser et al. 1998). Axonal swelling, axonal degeneration, and electrophysiologic alterations in the peripheral nerves or the spinal cord occurred at the two highest concentrations in later stages (from 8 weeks on) of the study (Herr et al. 1998; Sills et al. 1998; Valentine et al. 1998). A full histopathologic examination showed no lesions related to CS2 exposure in the heart, brain, aorta, lung, trachea, liver, kidneys, nasal cavity, testes, epididymis, ovaries, oviducts, uterus and vagina (Sills et al., 1998).

Cardiovascular effects after repeated inhalation exposure have also been reported. According to Antov et al. (1985), rats exposed to levels of 16 ppm and higher exhibited concentration-related structural and functional changes (distention of the lumen, attenuation of myocardial vessels, irregular thickening of the aorta wall, as well as microscopic histological changes). However, the study does not provide sufficient experimental details. Information on the analytical purity of CS2 is not given. Exposure of rats to levels between 74 to 545 ppm for 6-15 months, resulted in lipid disturbances in the vascular walls and in the blood serum, probably contributing in the process of atheroma formation (Wronska-Nofer et al., 1973, 1980). Short-term exposures of rats to relatively high levels (642 ppm) caused altered catecholamine levels in the brain and adrenals, and most often increases in dopamine and its metabolites (Caroldi et al., 1984).

The key study performed by Toxigenics in Fischer 344 rats, yields an overall NOAEC of 50 ppm.  

Oral

No standard oral repeated dose toxicity studies were located in the literature. Some non-standard studies were identified based on the most recent and elaborate reviews, which addressed the toxicity of CS2 on particular systems and/or organs. The procedure that was followed is the same as described above for the endpoint of repeated-dose inhalation. The studies selected are summarized under section 7.9.

The recently carried out oral EOGRTS (see section 7.8) yielded a NOAEL of 12 mg/kg bw based on slight reductions in body and brain weights and slight to moderate retinal atrophy at 120 mg/kg bw.

Decreased body weight has been observed in rats following oral exposure to carbon disulfide for a short term (4 weeks) exposure period to 253 mg/kg bw (Hoffman et al., 1990; Klapperstück et al., 1991). Additionally, in the same studies, CS2 had a cardiodepressive effect (electrophysiological and mechanical parameters), increased the blood pressure and caused electrocardiograph alterations indicative of myocardial ischemia, following administration of epinephrine or norepinephrine (Hoffman et al., 1990; Klapperstück et al., 1991). Body weight depression, and neurological deficits, i.e. abnormal gait, were observed in the two studies of Song et al. (2006a & b), after oral administration of 300 mg/kg bw for 12 w. These effects were accompanied by significant alterations in the cytoskeletal proteins of the cerebral cortex and spinal cord homogenates, suggesting that they might be implicated with the manifested neuropathy.

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: heart

Repeated dose toxicity: inhalation - systemic effects (target organ) neurologic: central nervous system

Justification for classification or non-classification

The results of the studies concerned with repeated-dose toxicity of carbon disulfide in experimental animals (see sections 7.5.2 and 7.9.1 and 7.9.2) combined with the effects of the compound on the nervous and cardiovascular system point to the need for classification for specific target organ toxicity (STOT) repeated exposure (RE). STOT RE cat. 1 would apply if significant toxicity would be seen at vapour levels <= 200 mg/m3 (63.5 ppm); however, the general NOAEC is 50 ppm and significant effects following inhalation exposure are seen at levels (substantially) higher than 63.5 ppm. Based on these studies, classification as STOT RE 2 (significant effects between 200 -1000 mg/m3; or 63.5 - 318 ppm) would be more appropriate.

Also STOT RE cat. 1 would apply if significant toxicity would be seen at oral levels <= 10 mg/kg bw. However, a recently carried out EOGRTS (see section 7.8.1) showed a NOAEL of 12 mg/kg bw, as only limited effects were seen at the next higher level tested of 120 mg/kg bw. Based on these data, classification as STOT RE 2 (significant effects between 10 and 100 mg/m3) would also not apply.