Carbon disulphide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP, guideline study; four bacterial strains used instead of five

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

yes

Remarks:

only 4 bacterial strains tested

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

1985, amended 1987

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix- rat liver, Aroclor 1254 administered

Test concentrations with justification for top dose:

0.005%, 0.01%, 0.025%, 0.05%, 0.1% v/v

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Remarks:

no solvent/vehicle used

Positive controls:

yes

Positive control substance:

other: in the abscence and presence of metabolic activation: 2-aminoanthracene (TA1535), benzo[alpha]pyrene (TA1537, TA100, TA98); in the abscence of metabolic activation: sodium azide (TA1535, TA100), 9-aminoacridine (TA1537), 2-nitrofluorene (TA98);

Remarks:

dichloromethane in vapour phase (7.5% v/v) was included in each test without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS:3/ treatment

DETERMINATION OF CYTOTOXICITY

- Method: abscence or thinning of the background lawn of non-revertant colonies

EXPOSURE TO CS2:
Sets of solidified plates were placed, with lids removed, in stainless steel racks, designed to keep the plates separate and permit atmospheric circulation, inside stainless steel vessels. These vessels were then sealed and partially evacuated. Calculated volumes of carbon disulphide liquid were injected into the vessels via a septum and allowed to vaporize, producing atmospheres containing carbon disulphide at the nominal concentrations
mentioned above.Sterile air was admitted to the vessels in order to equilibrate the contents to atmospheric pressure, and the vessels with their contents were incubated at 37°C for 48 hours. After removal from the vessels, the plates were incubated for a further day in order to permit revertant
colonies to grow to a size large enough to be scored.

Evaluation criteria:

number of revertants/plate

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes, only at the highest concentration

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

No mutagenic activity of CS2 detected in the Ames test

Executive summary:

Carbon disulphide was examined for its mutagenic activity in four histidine-dependent auxotrophs ofSalmonella typhimurium, strains TA98, TA100, TA1535, TA1537. Agar plates, seeded with the tester strains, were exposed to the test material in vapour phase, in the abscence and presence of metabolic activation, in the following nominal concentrations: 0.005%, 0.01%, 0.025%, 0.05% and 0.1% v/v (nominal). These concentrations were selected following preliminary toxicity tests in strain TA 98. Carbon disulphide did not exhibit any mutagenic activity under the conditions of this test. No increases in revertants were obtained in any of the four tester strains following exposure to carbon disulphide at the concentrations tested. Inhibition of bacterial growth, observed as thinning of the background lawn of non-revertant cells and reduction in revertant colony numbers, occurred in all strains with carbon disulphide at a nominal concentration of 0.1%v/v. The positive and negative controls were valid.

The study was performed according to the OECD Guidelines for Testing of Chemicals No. 471 (1983) and US EPA (TSCA) Guideline § 798.5265 (1985, amended 1987).

 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-06-09 until 2010-08-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

first experiment: 4 hours treatment with and without metabolic activation
second experiment: 24 hours treatment without metabolic activation, 4 hours treatment with metaoblic activation

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase Locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: Clone 3.7.2C

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 23.8, 47,6, 95,1, 190,3, 380,5, 761 µg/mL
with S9 mix: 23.8, 47,6, 95,1, 190,3, 380,5, 761 µg/mL
Experiment II:
without S9 mix: 23.8, 47,6, 95,1, 190,3, 380,5, 761 µg/mL
with S9 mix: 23.8, 47,6, 95,1, 190,3, 380,5, 761 µg/mL

Following the expression phase of 48 hours the cultures at 23.8 µg/mL
(printed in bold letters) were not continued since a minimum of only four
concentrations is required by the guidelines.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: solubility properties

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without metabolic activation in experiment 1, 24 hours without metaoblic activation in experiment and 4 hours with metabolic activation in experiment 2
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL TFT

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5 x 10 exp. 6 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10 exp. 6 cells above the corresponding solvent control or negative control, respectively.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in negative
and/or vehicle con¬trols and the mutation rates of all negative and/or vehicle controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control
unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used
to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and
dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.

Statistics:

Linear regression analysis (least squares) using SYSTAT 11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not effected
- Effects of osmolality: not increased
- Evaporation from medium: not examined
- Water solubility: --
- Precipitation: not observed
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES:

According to the results of the pre-test at least four adequate concentrations were chosen for the mutation experiment.
The highest concentration should be 10 mM, but not higher than 5 mg/mL, unless limited by the solubility or toxicity of the test item.
RSG (Relative Suspension Growth) or RTG (Relative Total Growth) values (main experiment) below 50 % are considered toxic. In case of toxic effects, the highest test item concentration of the main experiment should reduce the RSG or RTG value to approximately 10 - 20 %.
The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 6.0 µg/mL and 761 µg/mL were used. The highest concentration (equal to approximately 10 mM) in the pre-experiment was chosen with regard to the molecular weight (76.1 g/mol) of the test item. Relevant cytotoxic effects indicated by a relative suspension growth below 50% occurred at 380.5 µg/mL following 4 hour treatment with and without metabolic activation. Following 24 hour treatment without metabolic activation cytotoxicity was noted at 190.3 µg/mL and above.
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. No precipitation was observed by the unaided eye up to the maximum concentration. Therefore, the maximum concentration in experiments I and II was set to 761 µg/mL equal to approximately 10 mM as well. The lower concentrations were spaced by a factor of 2.
Both, pH value and osmolarity were determined in the pre-experiment at the maximum concentration of the test item and in the solvent control without metabolic activation. No relevant change in the osmolarity or pH value was observed (solvent control: 411 mOsm, pH 7.46 versus 441 mOsm and pH 7.47 at 761 µg/mL). The osmolarity was considerably above the physiological value of approximately 300 mOsm but that was based on ethanol used as solvent. The osmolarity is determined by freeze-point depression and ethanol interferes with this technique. Therefore, relative rather than absolute values should be considered.
To overcome problems with possible deviations in toxicity both main experiments were started with more than four concentrations.


COMPARISON WITH HISTORICAL CONTROL DATA: complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were observed at 761 µg/mL in the first experiment with metabolic activation and in the second experiment without metabolic activation.

Summary Table

			relative	mutant		relative	mutant
	conc. µg	S9	total	colonies/		total	colonies/
	per mL	mix	growth	10^6cells	threshold	growth	10^6cells	threshold
Column	1	2	3	4	5	6	7	8
Experiment I / 4 h treatment			culture I			culture II
Solv. control with ethanol		-	100.0	240	366	100.0	132	258
Pos. control with MMS	19.5	-	28.1	596	366	22.8	428	258
Test item	23.8	-	culture was not continued^#			culture was not continued^#
Test item	47.6	-	117.9	229	366	86.8	107	258
Test item	95.1	-	101.3	296	366	62.9	151	258
Test item	190.3	-	117.3	221	366	57.2	178	258
Test item	380.5	-	100.7	197	366	54.3	174	258
Test item	761.0	-	84.8	172	366	50.0	144	258
Solv. control with ethanol		+	100.0	197	323	100.0	200	326
Pos. control with CPA	3.0	+	30.5	396	323	94.2	291	326
Pos. control with CPA	4.5	+	43.1	264	323	44.4	414	326
Test item	23.8	+	culture was not continued^#			culture was not continued^#
Test item	47.6	+	75.9	170	323	101.0	272	326
Test item	95.1	+	103.3	116	323	90.4	212	326
Test item	190.3	+	60.9	173	323	142.5	161	326
Test item	380.5	+	58.2	182	323	135.7	154	326
Test item	761.0	+	31.6	182	323	44.8	173	326
Experiment II / 24 h treatment			culture I			culture II
Solv. control with ethanol		-	100.0	74	200	100.0	92	218
Pos. control with MMS	13.0	-	33.1	454	200	42.5	702	218
Test item	23.8	-	culture was not continued^#			culture was not continued^#
Test item	47.6	-	63.4	78	200	102.7	80	218
Test item	95.1	-	71.2	76	200	111.8	59	218
Test item	190.3	-	64.7	85	200	98.7	98	218
Test item	380.5	-	49.2	58	200	51.6	108	218
Test item	761.0	-	19.4	86	200	26.2	79	218
Experiment II / 4 h treatment			culture I			culture II
Solv. control with ethanol		+	100.0	84	210	100.0	64	190
Pos. control with CPA	3.0	+	51.7	433	210	40.1	303	190
Pos. control with CPA	4.5	+	31.1	423	210	26.6	544	190
Test item	23.8	+	culture was not continued^#			culture was not continued^#
Test item	47.6	+	116.2	90	210	122.0	51	190
Test item	95.1	+	101.1	72	210	69.3	58	190
Test item	190.3	+	118.1	97	210	71.4	86	190
Test item	380.5	+	84.4	75	210	55.5	71	190
Test item	761.0	+	57.6	100	210	46.5	86	190

threshold = number of mutant colonies per 10⁶cells of each solvent control plus 126

#    culture was not continued since a minimum of four concentrations is required by the guidelines

 

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Executive summary:

The study was performed to investigate the potential of Carbon disulfide to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

This study was conducted according to the procedures indicated by the following internationally accepted guidelines and recommendations:

Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, February 1998,
adopted July 21, 1997, Guideline No. 476 "In vitro Mammalian Cell Gene Mutation Test“

Commission Regulation (EC) No. 440/2008 B.17: ”Mutagenicity – In vitro Mammalian Cell Gene Mutation Test“, dated May 30, 2008.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

The main experiments were evaluated at the following concentrations:

Experiment I and II:

without S9 mix: 47.6; 95.1; 190.3; 380.5; 761.0 µg/mL
with S9 mix:     47.6; 95.1; 190.3; 380.5; 761.0 µg/mL

The test medium was checked for precipitation visible to the naked eye at the end of the 4 hours treatment just before the test item was removed. No precipitation meeting the criteria mentioned above was noted in the pre-experiment and in both main experiments.

Relevant cytotoxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were observed at 761 µg/mL in the first experiment with metabolic activation and in the second experiment without metabolic activation.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments up to the maximum concentration with and without metabolic activation. The threshold of 126 above the corresponding solvent control was not reached or exceeded.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTATâ11 statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In this study the range of the solvent controls was from 64 up to 240 mutant colonies per 10⁶cells; the range of the groups treated with the test item was from 51 up to 296 mutant colonies per 10⁶cells.The highest solvent control value (240 colonies per 10⁶cells) exceeded the recommended
50 – 170 x 10⁶control range as stated in the acceptability criteria of this report.The corresponding control value of the parallel culture however, was fully acceptable. Even the solvent controls of both cultures of the first experiment with metabolic activation slightly exceeded the threshold of 170 per 10⁶cells (197 and 200).The data are acceptable however, since the range of 50-200 colonies per 10⁶cells recommended by the IWGT in 2003 was covered. The cloning efficiency of the solvent control of the second culture of the first experiment and of the first culture of the second experiment without metabolic activation exceeded the upper limit of 120%. The data are acceptable however, since the parallel culture remained within the recommended range and the absolute values of the cloning efficiency were used to calculate the mutation frequency. The total suspension growth exceeded the upper limit of 32 in the first culture of the first experiment with metabolic activation.The data are acceptable since again, the parallel culture remained within the acceptable range and the absolute value of the total suspension growth was used to calculate the relative total growth.

MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large induced colonies with at least one of the concentrations used.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

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

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

Remarks:

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

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

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.

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).

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

CS2 has been tested for its genotoxicity in a series of in-vitro tests and in-vivo tests. The main results are summarized in the table below.

Type of test and quality	Result
In vitro
Gene mutations in bacteria (GLP study); adequate	Negative
Gene mutations in mammalian tissue-culture cells (GLP study); adequate	Negative
Cytogenetic effects in primary human lymphocytes; adequate	Weak and doubtful effects (gaps) for chromosomal aberrations – judged as negative Small but significant and dose dependent increase of sister chromatid exchanges - positive
Unscheduled DNA synthesis in tissue-culture cells; low purity (86%) of the test compound	Inconclusive; it cannot be excluded that the weak, doubtful positive response is caused by impurities
Cytogenetic effects in human sperm; absence of data on purity	Positive; it cannot be excluded that the positive response is caused by impurities
In vivo
Cytogenetic effects in rat bone marrow (inhalation); low purity (86%) of the test compound	Negative
Micronuclei in mouse bone-marrow erythrocytes (inhalation; GLP study); adequate	Negative
Host-mediated assay with mice (host) and Salmonella typhimurium TA98 (indicator) (inhalation); doubtful adequacy due to lack of positive control, low purity of test compound (86%) and low survival at the highest concentration in the males	Negative
Drosophila melanogaster sex-linked recessive lethal assay (vapour-phase exposure); low purity (86%) of the test compound	Inconclusive; it cannot be excluded that the weak, doubtful positive response is caused by impurities
Sperm-head abnormality test with rats and mice (inhalation); doubtful adequacy because of the low purity (86%) of the test compound and the weak response obtained with the positive control	Negative
Dominant-lethal test with rats (inhalation); low purity (86%) of the test compound	Negative
Chromosomal aberrations in female rats and their fetuses after treatment during preganancy (inhalation); quote from the Priority Substance List Assessment Report of Health Canada and Environment Canada.Carbon disulfide induced chromosomal aberrations and polyploid cells in the bone marrow of female rats and in rat embryos exposed on days 10–13 of gestation. It is difficult to assess the validity of these findings, as the reporting was brief (e.g., the statistical significance was often not indicated) and the effective dose was not reported, except to indicate that it was one-tenth of the LD50.	Positive; doubt as regards the validity due to lack of information.
Chromosomal aberrations in the oocytes and pronuclei zygotes (inhalation)	Positive; doubtful as regards the validity due to lack of information; only published in the form of a meeting abstract
Sperm-head abnormality test with rats	Positive; doubts as regards the validity due to lack of sufficient experimental details

 

CS2 was found to be negative in fully adequate standard GLP in vitro tests for gene mutations with bacteria and mammalian tissue culture cells.

However, an in vitro test for chromosomal aberration test with primary human lymphocytes suggested a weak positive effect based on gaps, while in the same cells a significant and dose-dependent increase of sister chromatid exchanges was found. A weak positive effect was also found in a non-standard unscheduled DNA systhesis test in an established human cell line. The latter test was not fully adequate, because the carbon disulfide tested has a low purity, which points to the possibility that the effect was caused by an impurity. The positive effects obtained in these tests depended on the presence of metabolic activation with S9 fractions. An in vitro test with human sperm cells yielded a positive result; however, there is no information about the purity of the test compound.    

The in-vivo data show a negative and fully adequate GLP test for micronuclei in mouse bone marrow and negative tests for dominant-lethals with rats, sperm-head abnormalities with rats and mice, chromosomal aberrations in bone marrow with rats and a host-mediated assay with mice as hosts and Salmonella typhimurium TA98 as indicator. Three in vivo studies showed positive results. The study by Vasil’eva (1982) used as endpoint the induction of chromosomal aberrations in bone marrow of female rats (dams) and their fetuses after inhalation of carbon sulfide. It is hard to judge the validity of this study, and in the reviews by internationally recognized bodies it is regarded doubtful. The same holds for the study of Bao et al. (1996), in which chromosomal aberrations were observed in oocytes and prenuclei zygotes of exposed adult female mice. The sperm head abnormality test of Kumar et al., (1999)

suggests genotoxic potential of CS2 but only sperm shape was evaluated. Nonetheless, the method is not validated, and the publication does not give sufficient experimental details.

In conclusion, with regard to the database on the genotoxicity of carbon disulfide, although in three studies a positive result was found, it can be concluded that there are clear doubts regarding their validity due to a lack of information on essential methodological details and or the fact they follow a design that has not been validated. In contrast there are 3 GLP studie reports (Ames test, MLA, and an in vivo MN test) showing clear negative results.

Justification for classification or non-classification

In conclusion, 3 GLP study reports for standard toxicity testing (Ames test - OECD 471, MLA - OECD 476, and an in vivo MN test - OECD 474) show clear negative results, as such no classification is needed for genotoxicity.