2-methylbutan-2-ol

Genetic toxicity: in vitro

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

Study period:

2011-11-22 to 2012-02-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-methylbutan-2-ol

EC Number:

200-908-9

EC Name:

2-methylbutan-2-ol

Cas Number:

75-85-4

Molecular formula:

C5H12O

IUPAC Name:

2-methylbutan-2-ol

Test material form:

other: liquid

Method

Target gene:

The object of this study was to determine wheter the test item or its metabolites can induce forward mutation at the hypoxanthine-guanine phosphoribosyltransferase enzyme locus (hprt) in cultured V79 chinese hamster cells.

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1 %)
- Properly maintained: yes (sub-cultured twice weekly)
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S-9 Mix (from induced rat liver)

Test concentrations with justification for top dose:

Experiment 1, 4-hours treatment without S-9-mix
27.5, 55.0, 110, 220, 440, 880 µg/mL
Experiment 1, 4-hours treatment with S-9-mix
27.5, 55.0, 110, 220, 440, 880 µg/mL
Experiment 2, 24-hours treatment without S-9-mix
27.5, 55.0, 110, 220, 440, 880 µg/mL
Experiment 2, 4-hours treatment with S-9-mix
27.5, 55.0, 110, 220, 440, 880 µg/mL

Highest test concentration corresponds to 10 µM/mL

Vehicle / solvent:

- Vehicle: DMSO
- Justification for choise of vehicle: suitable solvent

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

CULTURE MEDIUM: For seeding and treatment of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 µg/mL) and amphotericin B (1 %). For the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.

DURATION
- Preincubation period: After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µL/mL S9 mix
- Exposure duration: First experiment: 4 hours (+/- S9) and Second experiment: 4 hours (+ S9) and 24 hours (- S9)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days

SELECTION AGENT (mutation assays): 6-thioguanine


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

Linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

, but tested up to the limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: V79 cells of the chinese hamster

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

- There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.

- No precipitation of the test item was observed up to the maximum concentration of all experiments.

- No relevant cytotoxic effects occurred up to the maximum concentration of 880 µg/mL with and without metabolic activation.

- No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. The mutant frequency remained well within the historical range of solvent controls. An increase of the induction factor reaching the threshold of three times the mutation frequency of the corresponding solvent control was observed in the second culture of the second experiment without metabolic activation at 110 µg/mL. However, the isolated increase was judged as biologically irrelevant fluctuation as it was neither reproduced in the parallel culture under identical experimental conditions nor dose dependent as indicated by the lacking statistical significance.

- A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of < 0.05 was determined in any of the experimental groups.

- In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 10.5 up to 23.1 mutants per 10⁶ cells; the range of the groups treated with the test item was from 6.4 up to 33.0 mutants per 10⁶ cells.

- EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.