Di-tert-butyl trisulphide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Di-tert-butyl trisulphide

EC Number:

224-226-6

EC Name:

Di-tert-butyl trisulphide

Cas Number:

4253-90-1

Molecular formula:

C8H18S3

IUPAC Name:

di-tert-butyltrisulfane

Method

Species / strain

Species / strain / cell type:

other: Human Lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

. for treatment:
- 3, 10, 30, 100, 300, 500 µg/ml with and without S9 mix.
As very strong toxicity was seen at 300 and 500 µg/ml and as the requested toxicity was not obtained in this experiment for the lower dose-levels without S9 mix, it was repeated using the following dose-levels: 50, 100, 150, 200, 250 µg/ml.
. for chromosome aberrations scoring:
- without S9 mix: 150, 200, 250 µg/ml
- with S9 mix: 3, 10, 30 µg/ml.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9 mix, mitomycin C: 0.2 µg/ml. With S9 mix, cyclophosphamide: 50 µg/ml.

Details on test system and experimental conditions:

No preliminary cytotoxicity test was performed. Dose-levels were selected on the basis of pH, osmolality and solubility. A wide-range of treatment-levels was used and dose-levels for scoring of chromosome aberrations were selected on the basis of cytotoxicity indicated by reduction of mitotic index (MI), when possible.

For each culture (two cultures/dose level):, heparinised whole blood was added to culture medium containing a mitogen (phytohaemogglutinin) and incubated at 37°C in a humidified atmosphere of 5%CO2 / 95% air, for 48 hours.

Treatment was as follows:
. without S9 mix, cells were exposed continuously to the test or control substances.
. with S9 mix, cells were exposed to the test or control substances for three hours and then rinsed.
Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later. One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/ml) to block cells at the metaphase-stage of mitosis.

After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring.

Evaluation criteria:

Acceptance criteria
The study was considered valid since the following criteria were met:
. the frequency of celas with structural chromosome aberrations in the vehicle controls was within the range of our historical data,
. the frequency of cells with structural chromosome aberrations in the positive controls was significantly higher than that of the controls and within the range of our historical data.

Evaluation criteria
A statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, might be also taken into account in the evaluation of the findings.

Statistics:

For each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. The comparison was performed using the X² test, in which p = 0.05 was used as the lowest level of significance.

Results and discussion

Test results

Species / strain:

other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 300 µg/ml without S9, >= 100 µg/ml with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TPS 44 was poorly soluble in the vehicle (DMSO), the limit of solubility being approximately 100 mg/ml.

Consequently, with a maximum dose-volume of 100 µl/5.5 ml culture medium, the dose levels were for treatment:
- 3, 10, 30, 100, 300, 500 µg/ml.

A moderate emulsion was observed in the culture medium at 500 µg/ml.

The top dose-level for scoring chromosome aberrations was selected according to criteria specified in the international regulations: since the test substance was toxic, the top doselevel was based on the level of toxicity, i.e. when possible a reduction greater than 50% of mitotic index.

Without S9 mix, marked cytoxicity was noted after treatment:
- at 500 µg/ml, haemolysis was observed and, therefore, no harvest was performed;
- at 300 µg/ml, the mitotic index was in mean 19 and 22% of control cultures, for the first and the second harvest times respectively. At lower dose-levels, no toxicity was seen.

As the request degree of cytotoxicity was not achieved, this experiment was consequently considered as a preliminary toxicity test (only mitotic index was scored). A new experiment was performed at the following treatment-levels:
- 50, 100, 150, 200, 250 pg/ml.
However, no marked reduction of the mitotic index was noted.

The highest dose-level of this new experiment (250 µg/ml) being very closed to the previous dose-level of the preliminary toxicity test (300 µg/ml) which induced marked toxicity (approximately 80% reduction of the mitotic index), the requested toxicity was considered as satisfactory. The slides corresponding to the three highest dose-levels of the experiment were consequently scored for chromosome aberrations.

With S9 mix, cytotoxicity of the test substance was shown by the reduction of the mitotic index of treated cultures when compared to control cultures:
. at 100, 300 and 500 µg/ml, the mitotic index was between 0 and 12% of the control, for the two harvest times.
at 30 µg/ml, it was 47% or 56% of the control for the first or second harvest times respectively,
at 10 and 3 µg/ml, it was quite equivalent to the control.

Therefore, chromosome aberrations were scored on the slides corresponding to the following dose-levels: 3, 10, 30 pg/ml.

The test substance did not induce chromosome aberrations both with and without S9 mix for the two harvest times.

The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria and within the range of our historical data for both harvest times.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

TPS 44 did not induce chromosome aberrations in cultured human lymphocytes.

Executive summary:

The potential of di-tert-butyl polysulfides (TPS44) to induce chromosome aberrations was evaluated in cultured human lymphocytes. TPS 44 was tested, both with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. No preliminary cytotoxicity test was performed. Dose-levels were selected on the basis of pH, osmolality and solubility. A wide-range of treatment-levels was used and dose-levels for scoring of chromosome aberrations were selected on the basis of cytotoxicity indicated by reduction of mitotic index (MI), when possible. For each culture, heparinised whole blood was added to culture medium containing a mitogen (phytohaemogglutinin) and incubated at 37°C in a humidified atmosphere of 5% CO2 / 95% air, for 48 hours. Without S9 mix, cells were exposed continuously to the test or control substance(mitomycin C: 0.2 µg/ml), with S9 mix, cells were exposed to the test or control substance (cyclophosphamide: 50 µg/ml) for three hours and then rinsed. Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later. One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/ml) to block cells at the metaphase-stage of mitosis. After hypotonic treatment (KC1 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring. TPS 44 was dissolved in dimethylsulfoxide (DMSO). The dose-levels of TPS44 scored for chromosomal aberrations (two cultures/dose level) were 150, 200, 250 µg/ml, without S9mix and 3, 10, 30 µg/ml, with S9mix. The frequency of cells with structural chromosome aberrations in the vehicle and positive controls was as specified in the acceptance criteria and within the range of the historical data. TPS 44 did not induce any significant increase in the frequency of cells with structural chromosome aberrations, with and without S9 mix for the two harvest times. TPS44 did not induce chromosome aberrations in cultured human lymphocytes.