Carbon disulphide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Carbon disulphide

EC Number:

200-843-6

EC Name:

Carbon disulphide

Cas Number:

75-15-0

Molecular formula:

CS2

IUPAC Name:

methanedithione

Test material form:

liquid: volatile

Details on test material:

Batch 4680300
Complies to product sepcification: >99.5%

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (UK)
- Age at study initiation: 4-5 weeks
- Weight at study initiation: 16.1-26 g
- Assigned to test groups randomly: yes
- Housing: high density polypropylene cages with stainless steel taps
- Diet: ad libitum
- Water: supplied via a polythene bottle and sipper tube
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2
- Humidity (%): 55 ±15 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light):12

Administration / exposure

Route of administration:

inhalation: vapour

Vehicle:

no vehicle used

Details on exposure:

TYPE OF INHALATION EXPOSURE: snout only

GENERATION OF TEST ATMOSPHERE
Test atmospheres were generated using controlled fluid feed from a gasthight syringe driven by a syringe pump to an all glas evaporator generator maintained at ca. 50 deg. C. Dry oil-free comprssed air was passed through the vapour generator at a flow rate of ca. 5 L/min. Further dilution of the concentrated test substance vapour to provide a total flow of ca. 28 L/min was perfomed immediately before delivery of vapour to each chamber. The atmospheres were removed at a constant rate of 30 L/min and exhaust air vented to atmosphere after first passing through charcoal filters.

TEST ATMOSPHERE CONTROL
The concentrations was determined prior to exposure of the animals and then once each h during exposure. Atmosphere samples were taken using a gas sampling loop and the concentrations were determined using a GC fitted with a flame photometric detector.

EXPOSURE OF THE ANIMALS
Animals were exposed to the test material by snout-only inhalation. Prior to exposure of animals, the test material atmospheres were generated for each exposure chamber and samples analysed. Each mouse was placed in an individual polymethyl methacrylate restraining tube so that only the snout protruded. Each restraining tube was marked with the animal and group numbers. The restraining tubes were attached to the appropriate chamber so that the snout of each mouse projected into the lumen of the chamber. When the pre-exposure observations were complete, the syringe pump was switched on and the exposure timed for six hours following a 4.5 minute equilibration period, the theoretical time required for the concentration of vapour to reach 90% of its final value under the conditions of exposure employed (Silver and Arsenal, 1946). After six hours, the test atmosphere supply was switched off and the mice removed from the restraining tubes for examination.

Duration of treatment / exposure:

6 h

Frequency of treatment:

once

Post exposure period:

24 or 48 h

Doses / concentrations

Doses / concentrations 1

Remarks:

Target Concentrations:
467, 1558, 4675 mg/m3 (150, 500, 1500 ppm)

Doses / concentrations 2

Remarks:

Analytical concentrations
157.4 +/- 8.7 ppm, 502.3 +/- 67.5 ppm, 1557.5 +/- 71.8 ppm

No. of animals per sex per dose:

10 (5 for positive control)

Control animals:

yes, sham-exposed

Positive control(s):

chlorambucil
- Route of administration: oral gavage
- Doses / concentrations: 30 mg/kg

Examinations

Tissues and cell types examined:

bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: based on the preliminary toxicity testing
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): samples were taken 24 and 48 h after treatment
DETAILS OF SLIDE PREPARATION: Animals were killed by cervical dislocation following carbon dioxide inhalation. Femurs from each animal were rapidly dissected out and cleaned of adherent tissue. The epiphyses were cut off to obtain access to the marrow canal. Marrow cells were flushed out with 2.5 ml foetal calf serum using a syringe and needle. The recovered cells were centrifuged at 1000 rpm for five minutes. The bulk of the supernatant fluid was discarded and the cell pellet resuspended in the remaining fluid. Single drops of the cell suspension were transferred to clean, dry slides, two or three smears (for the preliminary toxicity test or main micronucleus test respectively) prepared, and the slides left to air-dry. Following fixation in methanol for ten minutes, they were stained manually in 5% Giemsa stain (in Sorensen's buffer: pH 6.8), washed in buffer, air-dried, cleared for five minutes in xylene and made permanent using DPX mountant.

METHOD OF ANALYSIS: The slides were examined under the light microscope. At high magnification (x 1000, oil immersion) a total of at least 2000 erythrocytes per animal were examined. Each erythrocyte scored was classed as polychromatic or mature. Each erythrocyte scored was also examined for the presence or absence of micronuclei. Thereafter, the frequencies of micronucleated cells per 1000 erythrocytes were calculated. The ratio of polychromatic to mature cells was also determined (indicating the rythm of cell division). The frequency of micronuclei in polychromatic cells provides an index of induced genetic damage.

Evaluation criteria:

Positive for clastogenicity was a statistically and biologically significant increase in micronucleated polychromatic cells, compared to vehicle control, in at least one treatment group; particularly if supported by evidence of a dose-related response.

Statistics:

Mann-Whitney U procedure (Mann and Whitney, 1942), two-tailed test, one-tailed test.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 45, 150, 450 and 1500 ppm
All animals exposed to carbon disulphide at 1500 ppm were unconscious at the end of the exposure period, but regained consciousness the following day.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
Mean values of treated groups and control groups, in the incidence of polychromatic micronucleated cells was not significant, at both termination times (Table 2, attachment). Similarly, no significant differences were seen, among the two sexes, in the frequences of micronucleated polychromatic cells (Table 3, attachment).

Applicant's summary and conclusion

Conclusions:

No evidence of induced chromosomal or other damage leading to the formation of micronuclei in erythrocytes of the bone marrow was detected after exposure of the animals to CS2 via inhalation.

Executive summary:

The effect of carbon disulphide on chromosome structure in the bone marrow erythrocytes of mice was examined. The animals (males and females) were exposed via inhalation snout-only, for 6 h to the following concentrations: 0, 467, 1558, 4675 mg/m3 (0, 150, 500, 1500 ppm). The exposure concentrations were based on a preliminary toxicity test. Chlorambucil (30 mg/kg bw) was used as a positive control, adminstered via the oral route. Animals were sacrifised and examined 24 and 48 h after exposure. No evidence of induced chromosomal or other damage leading to the formation of micronuclei in erythrocytes of the bone marrow was detected, uder the present test condition, after exposure of the animals to CS2 via inhalation. Mice exposed at 1500 ppm, however, showed a small increase in the ratio of polychromatic/mature cells, which may indicate disturbance of erythropoiesis.

Carbon disulphide was tested for induction of micronuclei in the bone marrow ertythrocytes of mice according to the OECD Guidelines 474 (1983).