2-methylpropane-2-thiol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Deviations:

yes

Remarks:

cytotoxicity data and individual culture SCE data were not presented in the report

GLP compliance:

yes

Type of assay:

sister chromatid exchange assay in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

2-methylpropane-2-thiol (CAS # 75-66-1)

IUPAC Name:

2-methylpropane-2-thiol (CAS # 75-66-1)

Details on test material:

t-butyl mercaptan (2-methylpropane-2-thiol), purity not stated but assumed to be 100%

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S-9 liver microsomes from male Sprague Dawley rats treated with a single intraperitoneal injection of Aroclor 1254 at a dose of 500 mg/kg 5 days before sacrifice.

Test concentrations with justification for top dose:

With and without metabolic activation:
Experiment 1: mg/ml
Experiment 2: mg/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: Ethylmethanesulfonate (EMS, 400 µg/ml) without metabolic activation and CP (1.4 µg/ml) with metabolic activation

Details on test system and experimental conditions:

TOXICITY EVALUATION:
The test compound dose levels were determined by a preliminary multidose-range finding study with the highest concentration of the chemical tested depending upon its solubility. t-Butyl mercaptan solubilized at approximately 270 mg/ml in dimethylsulfoxide. The toxicity was determined by testing a wide range of concentrations for relative inhibition of the growth of the cells following a 4-hr exposure. Twenty-four hrs after exposure, cell counts were determined. Based on the cell counts, the relative toxicity of the chemical doses compared to the solvent control was determined. The maximum dose selected for the mutagenicity test was approximately 1350 ug/ml because it exhibited growth inhibition.

SCE ASSAY:
Cells were treated in an exponential stage of growth by setting up cultures with 2 to 5E05 cells per 25 cm2 flask, 24 hours prior to treatment. Cells were exposed to the chemical for 2 hours, washed twice and then 5-bromodeoxyuridine (BrdU) was added to each culture. All cultures were sampled a minimum of 24 hours after addition of BrdU to ensure completion of two full cell cycles. Duplicate cultures were set up for each dose level and all controls. Twenty-four hours after the above initiation of the cultures, the cells were treated with the test chemical in the presence of an S-9 rat liver activation system for 2 hours and washed twice in a balanced salt solution. The cells were then sampled and treated as described above. Two hours after, colcemid (0.2 ug/ml) was added to each tube and metaphases were collected by mitotic shake-off. The cells were swollen in a 0.075M KCL hypotonic, and washed three times in an acetic alcohol fixative. Slides were prepared and stained. Fifty cells in the metaphase stage of mitosis were scored at each dose level for the number of sister chromatid exchanges (SCE).

Evaluation criteria:

Test substance considered positive if a statistically significant increase in SCEs and a concomitant two-fold increase in SCEs relative to solvent control occurs.

Statistics:

Statistical analysis conducted - details not provided.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1350 µg/ml exhibited growth inhibition

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Following exposure to five graded doses of t-Butyl Mercaptan, in the presence of metabolic activation, a statistically significant increase in the number of SCEs per cell (10.88 vs 7.58 in the DMSO control [1.4-fold] at 1350 µg/ml and 10.66 vs 7.58 [1.4-fold] at 450 µg/ml) and SCEs per chromosome (0.55 vs 0.37 [1.5-fold] at 1350 µg/ml and 0.53 vs 0.37 [1.4-fold] at 450 µg/ml); but no significant increase was seen in the remaining dose levels, and no dose level showed a two-fold increase in SCEs. The increases in SCEs ranged from 0.9 to 1.2-fold in the absence of metabolic activation and 1.0 to 1.5-fold in the presence of activation. Therefore t-butyl mercaptan is not considered to be positive in this test system.

Applicant's summary and conclusion

Conclusions:

An in vitro sister chromatid exchange assay in a similar way to OECD TG 476 and cin compliance with GLP, concluded 2-methylpropane-2-thiol to be negative in both the presence and the absence of metabolic activation.

Executive summary:

An in vitro Sister Chromatid Exchange (CHO cells) assay was performed with 2 -methylpropane-2-thiol in a study similar to OECD Guideline 476. CHO cells were exposed to five concentrations of 2 -methylpropane-2-thiol (up to 1350 µg/ml) in the presence and absence of a metabolic activation system. In the presence of metabolic activation, a statistically significant increase in the number of SCEs per cell (10.88 [1.4-fold] at 1350 µg/ml and 10.66 [1.4-fold] at 450 µg/ml vs 7.58 in the DMSO control) and SCEs per chromosome (0.55 [1.5-fold] at 1350 µg/ml and 0.53 [1.4-fold] at 450 µg/ml vs 0.37 in control) was observed; but no significant increase was seen in the remaining dose levels, and no dose level showed a two-fold increase in SCEs. The increases in SCEs ranged from 0.9 to 1.2-fold in the absence of metabolic activation and 1.0 to 1.5-fold in the presence of activation. Therefore 2 -methylpropane-2-thiol is not considered to be positive in this test system. 

The study received a Klimisch Score of 2 and is classified as reliable with restrictions because it is comparable to a guideline study.