A mixture of: O,O-di(1-methylethyl)trithio-bis-thioformate; O,O-di(1-methylethyl)tetrathio-bis-thioformate; O,O-di(1-methylethyl)pentathio-bis-thioformate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiments carried out between 7 September 1987 and 11 January 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete or methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Di-isopropyl xanthogen polysulphide

IUPAC Name:

Di-isopropyl xanthogen polysulphide

Details on test material:

Identity: DIXT
Chemical name: Di-isopropyl xanthogen tetrasulphide (polysulphide).
Purity: 100% of total sulphur content as tetrasulphide
Appearance: Oily viscous liquid
Stability: Unstable in light
Solubility: Soluble in some organic solvents
Storage: Room temperature in the dark

Method

Target gene:

Not applicable.

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary (CHO) cells, strain K1-BH4 were obtained from BIBRA and stored in polypropylene ampoules at -196°C in 90% foetal calf serum abd 10% dimethylsulphoxide. The cells were routinely grown and subcultured in Hams F12 medium supplemented with 5% foetal calf serum at 37°C in a humid atmosphere containing 5% carbon dioxide in 175 cm2 plastic tissue culture flasks.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor inducted rat liver homogenate fraction (S9 mix)

Test concentrations with justification for top dose:

Preliminary toxicity test: 0.31, 0.63, 1.25, 2.5, 5, 10 and 20 µg/ml (with and without metabolic activation
Metaphase analysis:
With metabolic activation: 2, 10, 20 µg/ml
Without metabolic activation: 0.3, 1.5, 3.0 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DIXT was dissolved in ethanol immediately before use. The highest concentration which did not cause a precipitate in aqueous tissue culture medium was 20 µg/ml.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol and sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Mitomycin C was used as the positive control compound for the study in the absence of metabolic activation. It was prepared as a solution in sterile distilled water to give a final concentration of 0.4 µg/ml.

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol and sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Cyclophosphamide was used as the positive control compound for the study in the presence of metabolic activation. It was prepared as a solution in sterile distilled water to give a final concentration of 20 µg/ml.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

PRELIMINARY TOXICITY TEST:
A 50 ml culture of CHO cells was harvested as follows: The supernatant medium was removed and the cells washed in 0.9% sterile saline; 20 ml of 0.25% trypsin was then added for 30 seconds. The trypsin solution was removed and flask incubated at 37°C for 10 minutes. The cells were then resuspended in 20 ml Hams F12 + 5% FCS and diluted to give 1.0 x 10+5 cells/ml. Aliquots (5 ml) of cells were added to Nunc 25 cm2 tissue culture flasks and the cultures incubated at 37°C in a humid atmosphere containing 5% carbon dioxide.

After 24 hours 1.25 ml of S9 mix was added to one set of cultures followed by 62.5 µl of various dilutions of the test compound and of the solvent. To the second set of cultures (i.e. without S9 mix) 50 µl of dilutions of test compound and of the solvent were added. Final concentrations of test compound in both sets were 0.31, 0.63, 1.25, 2.5, 5, 10 and 20 µg/ml with single flasks for each concentration and duplicate flasks for the solvent control. The cultures without S9 mix were incubated in the presence of the test compound for 21 hours.

Four hours after the addition of the test compound to those cultures treated with S9 mix, the medium containing the S9 mix and test compound was carefully removed and replaced with fresh Hams F12 + 5%FCS. The cultures were returned to the incubator for a further 17 hours.

HARVESTING, FIXATION AND SLIDE PREPARATION:
Two hours before the end of the 21 hour incubation period, mitotic activity was arrested by the addition of colchicine to each culture at a final concentration of 0.25 µg/ml. After the incubation period the medium was removed and discarded. 4 ml of 0.25% trypsin solution was then added. After 45 seconds this was removed and placed in a plastic conical centrifuge tube. The flasks were then incubated for 10 minutes at 37°C after which the cells were resuspended in Hams F12 + 5% FCS. The cell suspensions were placed with the trypsin solution in the centrifuge tubes. These cell suspensions were then centrifuged for 10 minutes at 200 x 'g'. The supernatant was discarded and the cells resuspended in 2.5 ml 0.07 M KCl. After a 10 minute incubation at room temperature the cell suspensions were centrifuged for 5 minutes at 110 x 'g'. The supernatant was discarded and 4 ml of freshly prepared fixative (3 parts methanol: 1 part glacial acetic acid v/v) added. The cells were left in fixative for 2 - 3 hours, then the pellets resuspended by repeated aspiration through a 20 gauge needle, centrifuged at 200 x 'g' for 10 minutes, the supernatant discarded, and the cell pellet resuspended in about 0.5 ml of fresh fixative by repeated aspiration through a Pasteur pipette. Two drops of this cell suspension were dropped onto a cold, pre-cleaned microscope slide. The slides were left to air-dry at room temperature, then stained in 10% Giemsa. After air-drying they were mounted in DPX.

MICROSCOPICAL EXAMINATION FOR MITOTIC INDEX:
The prepared slides were examined at a magnification of x160. The proportion of metaphase figures in each culture was recorded. From these results the EC50 was estimated (the EC 50 is that concentration of test substance expected to cause a 50% reduction in the mitotic index). Where possible, this concentration, or the maximum achievable concentration, was used as the top dose in the main study. The intermediate and low doses were 50% and 10% of the top concentration.

METAPHASE ANALYSIS:
All cultures were treated with colchicine, harvested, fixed and slides prepared as described in harvesting, fixation and slide preparation section. The slides were stained in 10% Giemsa, mounted in DPX and coded. Metaphase spreads were identified using a magnification of x160 and examined at a magnification of x1000 using an oil immersion objective. Approximately 100 metaphase figures were examined where possible from each culture, with normally a maximum of 25 from each slide.

Evaluation criteria:

The proportion of metaphase figures in each culture was recorded. From these results the EC50 was estimated (the EC 50 is that concentration of test substance expected to cause a 50% reduction in the mitotic index).

Statistics:

Fisher's test.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST:
The mitotic indices of CHO cells treated with various concentrations of DIXT are shown in Table 1 (see attached background material).

In the absence of metabolic activation, DIXT caused a dose-related decrease in the mitotic index. It was estimated that DIXT at a concentration of 3 µg/ml would cause a decrease in the mitotic index of 50% when compared with the solvent control. 3, 1.5 and 0.3 µg/ml were consequently selected as dose levels for the metaphase analysis.

In the presence of metabolic activation, the maximum achievable concentration, 20 µg/ml , of DIXT caused no decrease in the mitotic index when compared with the solvent control. 20, 10 and 2 µg/ml were selected as concentrations for the metaphase analysis.

METAPHASE ANALYSIS:
The effects of DIXT on cultured CHO cells are shown in Table 2 (see attached background material) in which the number and type of chromosomal aberrations are recorded.

Both positive control compounds, mitomycin C (0.4 µg/ml) and cyclophosphamide (20 µg/ml) caused statistically highly significant increases in the proporation of metaphase figures containing aberrations when compared with the relevant solvent controls.

In both the absence and presence of metabolic activation, DIXT caused no statistically signficant increases in chromosomal aberrations at any dose level, when compared with the solvent control.

Remarks on result:

other: other: CHO

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

See attached background material for:

Table 1: Preliminary toxicity test - mitotic indices of cultured CHO cells treated with DIXT.

Table 2: Effect of DIXT on the chromosomes of cultured CHO cells

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

DIXT has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

Executive summary:

DIXT was tested in vitro to determine whether it could cause chromosomal aberrations in a mammalian cell line derived from Chinese hamster ovary tissue. The cells were routinely grown and subcultured in tissue culture medium at 37 °C in a humid atmosphere containing 5% carbon dioxide. They were incubated with the test compound both with and without supplementary metabolic activation (rat S9 mix).

A preliminary toxicity test was carried out to assess the effect of the compound on the mitotic index of cultured CHO cells. The results of this test indicated that a top dose level of 3 µg/ml should be used for the metaphase analysis in the absence of metabolic activation and 20 µg/ml in its presence.

In both the absence and presence of metabolic activation, DIXT caused no statistically significant increases in the level of chromosomal aberrations at any dose level, when compared with the solvent control.

Both positive control compounds showed statistically highly significant increases in the number of chromosomal aberrations when compared with solvent controls. This demonstrates the sensitivity of this test system and the efficacy of the S9 mix.

It is concluded that DIXT has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

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