Disulfiram

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Aug 1985 - 1 May 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopten in 2016

Deviations:

yes

Remarks:

For details on guideline deviations, please refer to "Principles of method if other than guideline".

Principles of method if other than guideline:

Deviations from current OECD guideline 476 (adopted 2016)
- No information on chromosome number and mycoplasma contamination checks
- No information on cell cycle length
- Chosen concentrations were too low as the relative cell survival did not reach 20%
- No Historical Control Data provided

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Thiram

EC Number:

205-286-2

EC Name:

Thiram

Cas Number:

137-26-8

Molecular formula:

C6H12N2S4

IUPAC Name:

tetramethylthiuram disulfide

Method

Target gene:

hypoxanthine guanine phosporibosyl transferase (HPRT) locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Department Toxicology, Agricultural University of Wageningen, The Netherlands
- Suitability of cells: standard cells for the respective assay

For cell lines:
- Methods for maintenance in cell culture: cell density was kept below 4x10^6 cells per 75 cm² flask

- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media: Ham's F-10 medium (without thymidine and hypoxanthine) supplemented with 10% newborn calf serum, L-glutamine (2 mM), and penicillin/streptomycin/cefoxtamin (50 U/mL, 50 µg/mL and 2 µg/mL, respectively)
- Exposure medium: F-10 culture medium buffered with 20 mM Hepes in the absence of serum
- CO2 concentration: 5% CO2
- humidity level: humid atmosphere (95%)
- Temperature: 37 °C

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver homogenate (S9) was prepared previously. The S9/cofactor mixture consisted of

1.02 mg MgCl2, 2.46 mg KCl, 1.7 mg glucose-6-phosphate, 3.4 mg NADP, 4 µmol HEPES and 0.5 mL S9.

0.2 mL of the S9/cofactor mixture was added to 1 mL of medium for cytotoxicity or mutagenicity testing.

Test concentrations with justification for top dose:

1, 3.3, 5.6 or 10 µg /mL without S9 mix
10, 18, 33 or 56 µg/mL in the presence of S9 mix for 2 h

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9-mix: Ethylmethane sulfonate (EMS); 6 mM With S9-mix: Dimethylnitrosamine (DMN); 4 and 8 mM

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 1 culture per dose
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 6 x 10^6 (exposure), 10^6 (expression)
- Test substance added in cell culture medium as described above (serum-free, buffered with Hepes)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2 h (with and without metabolic activation)
- Harvest time after the end of treatment (sampling/recovery times): 14 - 17 days

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days, during this time, the cells were subcultured every 2 or 3 days to maintain exponential growth
After the expression period, in total 10^6 cells were plated into 10 petri dishes with selective medium.
- Selective agent: 10 µM 6-thioguanine, 7 - 10 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 10^6 cells in total for mutant selection after the expression period, 2 x 200 cells of each dose were plated in cloning medium directly after exposure to assess cell survival (cloning efficiency)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency (CE)
CE: (Average No. of colonies on cloning plates)/200 *100%

Mutant Frequency per 10^5 survivors: (100/CE)*(Total number of mutant colonies on selective plates/number of seeded cells)

Rationale for test conditions:

A pre-test was performed assessing the cytotoxicity of the cells. 3x10^6 cells were treated in the presence (0.001 - 10 µg/mL) or absence (0.01 - 100 µg/mL) of S9 mix.

Evaluation criteria:

A mutant assay was considered acceptable if the following criteria were met:
- The absolute colony forming efficiency of the solvent control is 60%
- At least 3/4 doses of the test substance have an acceptable number of surviving cells (10^6) analysed for HPRT mutations.
- The spontanous mutant frequency in the untreated or solvent treated control is <3 per 10^5 survivors.
- The positive controls induced significant (at least 3-fold) increases in mutant frequencies.

A test substance is considered mutagenic if
- It induces a mutant frequency that is at least 3 x times higher than the spontaneous mutant frequency of the untreated control.
- The results show a dose response relationship and are reproducible in an independently repeated test.

A test substance is considered negative (nonmutagenic) if
- None of the test concentrations induce a mutant frequency that is at least 3 x times higher than the spontaneous mutant frequency of the untreated control.
- The results are reproducible in an independently repeated test.

Statistics:

No statistical evaluation was performed.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Chosen concentrations were too low as the cell survival did not reach 20%

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: A concentration of 10 µg/mL,without metabolic activation, reduced cloning efficiency by approximatively 50%. In the presence of S9 mix, cytotoxicity occurred only from 30 µg/mL. Summarized results can be found in Attachment 1 in the attached background material.

STUDY RESULTS
- Genotoxicity: No statistically significant increases in mutant frequency or dose– response were observed.
- Concurrent vehicle negative and positive control data: The solvent and positive control induced satisfactory mutagenicicty rates, indicating the adequacy of the experimental conditions.
- Results from cytotoxicity measurements: the test item was observed to be significantly cytotoxic at the level of 10 µg/mL in the absence of S9 mix (CE was reduced by approcx. 55 - 75%). In the presence of S9 mix, the cloning efficiency was only reduced by 25 - 50%, even though the test item concentrations were higher (up to 56 µg/mL).

For summarized results, please refer to Attachment 1 in the attached background material.

HISTORICAL CONTROL DATA: not provided

Applicant's summary and conclusion

Conclusions:

The present study was conducted in compliance with GLP and EPA OPP 84-2, and similar to OECD test guideline 476. Under the conditions of the study, no statistically significant increase in mutant frequency was observed for any dose level in the absence or presence of metabolic activation. No significant dose-response was observed either. Therefore, the test substance is concluded not to be mutagenic in CHO cells in vitro.