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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
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: This study is classified as reliable with restrictions because it is comparable to the guideline with acceptable restrictions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1982

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
only 0.5E06 cells were plated for selection of mutants, individual culture data were not provided, and large vs small colonies were not determined
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
t-butyl mercaptan (2-methylpropane-2-thiol), CAS 75-66-1, purity not specified - assumed to be 100%

Method

Species / strain
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
L5178Y mouse lymphoma cells subline 3.7.2C
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:
61, 90, 135.0, 202, 300, 449, 670, or 1000 mg/ml. 1000 mg/ml limit of solubility.
Vehicle:
DMSO
Controls
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethanesulfonate (EMS, 620 µg/ml) without metabolic activation and 3-methylcholanthrene (3-MCA, 3 µg/ml) with metabolic activation
Details on test system and conditions:
TOXICITY EVALUATION:
The test compound dose levels were determined by a preliminary multidose range-finding study with the highest dose tested being selected to give approximately 50 - 90% inhibition of suspension cell growth depending on the solubility of the compound. T-Butyl mercaptan solubilized at approximately 100 mg/ml in dimethylsulfoxide. One-tenth ml of each test substance concentration and the solvent were added to 50 ml tubes containing 6.0 x 10(6) TK+/- cells in 10 ml F10P. Cells were exposed to the chemical for 4 hrs, washed, resuspended in 20 ml F10P and incubated for 2 days. After the first and second day of incubation, the cell counts and viability were determined by counting a sample of the cells using a hemocytometer in the presence of trypan blue. Following the first day of incubation, the viable cell count was adjusted to 3.0 x 10(5) cells/ml. Based on the cell counts, the relative toxicity of the chemical compared to the solvent control was determined. The maximum dose selected for the mutagenicity test was 1000 ug/ml because it represented the limits of solubility of the test material.

MUTAGENICITY ASSAY:
Each test concentration was prepared to contain the test dose in 0.1 ml volumes. Six million precleansed TK+/- cells in 6 ml of F10P were added to centrifuge tubes. An additional 4 ml of the S-9 mix were added to half of the tubes. Immediately thereafter, 0.1 ml of the 100x concentrations of the test chemical dilutions or the positive controls, and 0.1 ml of the solvent were added to the appropriate tubes. Each tube was mixed, gassed with a mixture of C02 and air, and incubated at 37± 0.5°C on a revolving roller drum for 4 hours. Following this incubation the tubes were centrifuged and the treatment solutions decanted. The cells were washed twice with F10P and resuspended in 20 ml F10P after the second wash. The tube cultures were then gassed and reincubated for a 2 day expression time. The cell cultures were readjusted to 3.0 x 10(5) cells/ml as necessary. At the end of the expression period, a sample of each of the cultures was centrifuged and the cells resuspended at 500,000 viable cells/ml in F10P. The concentrated cells were serially diluted and appropriate dilutions were plated in triplicate in cloning medium with and without TFT. Approximately 500,000 cells were plated on each of 3 selective medium plates containing 2 ug/ml TFT, and 100 cells were cloned on each of 3 non-selective plates for each test concentration and a control tube. The plates were incubated for 10-14 days. The mutant colonies (TK+/-) were counted on the selective TFT containing plates and the survivors (TK+/- and TK-/-) were counted on the non-selective medium plates. The daily cell counts were made using a microscope and hemocytometer; and the colony counts were obtained with an electronic colony counter. The number of mutants in the test and control groups was compared and their mutation frequencies determined, along with a determination of relative total survival based on the suspension cell growth and cloning efficiency.
Evaluation criteria:
Test substance considered positive if a dose-related response at two or more test concentrations (in the absence of severe toxicity) is at least two to three-fold higher than the mutation frequency of the solvent control.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Without metabolic activation: Survival = 10.6% at 670 µg/ml and 3.0% at 1000 µg/ml; with metabolic activation: Survival = 10.9% at 1000 µg/ml
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
Exposure to eight graded doses of the test material in the presence of metabolic activation did not increase the induction of forward mutations in L5178Y mouse lymphoma cells at the T/K locus. Exposure to the 202 and 1000 µg/ml dose levels of the test material in the absence of metabolic activation increased the induction of forward mutations in L5178Y mouse lymphoma cells at the T/K locus. Under these conditions, t-butyl mercaptan did exhibit a positive response. However, the induction of increases in mutant frequency was not dose responsive and, thus, did not satisfy the stated criteria for a positive response; therefore, t-buty mercaptan was not mutagenic under the conditions of this assay.

Any other information on results incl. tables

Treatment without S-9
µg/ml
%Survival
MF
Fold Increase
DMSO
--
100
3.4
1.0
EMS
620
78.0
14.8
4.4
TBM
1000
3.0
7.6
2.2
670
10.6
6.2
1.8
449
60.0
5.3
1.6
300
56.1
4.2
1.2
202
93.5
7.4
2.2
135
94.9
3.8
1.1
90
126.8
2.3
0.7
61
100.1
3.2
0.9
MF - mutation frequency [x10e-5]

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative
Executive summary:

An In vitro Mammalian Cell Gene Mutation (mouse lymphoma) assay was performed with 2 -methylpropane-2-thiol in a study similar to OECD Guideline 476. L5178Y TK (+/-) mouse lymphoma cells were exposed to eight concentrations of t-butyl mercaptan (61 to 10,000 µg/ml) in the presence and absence of a metabolic activation system. 2 -methylpropane-2-thiol did not result in mutagenic responses in this assay. Positive controls provided the appropriate responses. 

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