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Repeated dose toxicity: inhalation

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sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well perfomed but older GLP study according to existing guideline at that time

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
Limit test:

Test material

Constituent 1
Reference substance name:
Carbon disulfide
Carbon disulfide
Constituent 2
Chemical structure
Reference substance name:
Carbon disulphide
EC Number:
EC Name:
Carbon disulphide
Cas Number:
Molecular formula:

Test animals

Fischer 344
Details on test animals or test system and environmental conditions:
- 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.

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

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Details on inhalation exposure:
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.

- Brief description of analytical method used: gas chromatographic chamber-monitoring system
Analytical verification of doses or concentrations:
Duration of treatment / exposure:
6 hours/day; 5 days/week; at least 89 consecutive days
Frequency of treatment:
5 days/week
Doses / concentrationsopen allclose all
Target Concentrations:
0, 50, 300, 800 ppm

0, 49.3, 297.1, 798.4 ppm (155.8, 938.8, 2522.9 mg/m3)
analytical conc.
No. of animals per sex per dose:
Control animals:
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:


Observations and examinations performed and frequency:
- Time schedule: twice per day

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

- Time schedule for examinations: weekly

- Food consumption for each animal determined : Yes, weekly

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

- 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

- 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

- 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:
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.
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

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
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

Effect levels

open allclose all
Dose descriptor:
Effect level:
50 ppm
Based on:
test mat.
Basis for effect level:
other: only a slight reduction in absolute brain weight in males but not in relative brain weight
Dose descriptor:
Effect level:
300 ppm
Based on:
test mat.
Basis for effect level:
other: only a slight reduction in absolute brain weight but not in relative brain weight

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.