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Toxicological information

Genetic toxicity: in vitro

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Administrative data

in vitro gene mutation study in mammalian cells
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): n-Butanol
- Molecular weight (if other than submission substance): 74.12 g/mol
- Physical state: colourless, clear liquid
- Analytical purity: 99.9 corr. area-%
- Lot/batch No.: Tk10_20091019
- Expiration date of the lot/batch: October 19, 2010
- Stability in solvent: Not indicated by the sponsor
- Storage condition of test material: At room temperature


Target gene:
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix from phenobarbital/β-naphthoflavone induced rat liver
Test concentrations with justification for top dose:
Experiment I (4 h treatment)
23.1 - 740 µg/mL (with and without metabolic activation)

Experiment II
23.1 - 740 µg/mL (24 h; without metabolic activation)
23.1 - 740 µg/mL (4 h; with metabolic activation)
(740 μg/mL equals to approximately 10 mM, the recommended limit dose of OECD TG 476)
Vehicle / solvent:
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: with metabolic activation: DMBA (1.1 μg/mL = 4.3 μM); without metabolic activation: EMS (0.150 mg/mL = 1.2 mM)
Details on test system and experimental conditions:
- Exposure duration: Experiment I: 4 h; Experiment II: 24 h

SELECTION AGENT (mutation assays): 6-TG (6-thioguanine)


- Method: cloning efficiency
Cloning efficiency I (survival): cloning efficiency determined immediately after treatment to measure toxicity.
Cloning efficiency II (viability): cloning efficiency determined after the expression period to measure viability of the cells without selective agent.

cloning efficiency I (survival, absolute): mean number of colonies per flask divided by the number of cells seeded
cloning efficiency I (survival, relative): (mean number of colonies per flask divided by the mean number of colonies per flask of the corresponding control) × 100
cell density % of control: (cell density at 1st subcultivation divided by the cell density at 1st subcultivation of the corresponding control) × 100
cloning efficiency II (viability, absolute): mean number of colonies per flask divided by the number of cells seeded
cloning efficiency II (viability, relative): cloning efficiency II absolute divided by the cloning efficiency II absolute of the corresponding control × 100
cells survived (after plating in TG containing medium): number of cells seeded × cloning efficiency II absolute
mutant colonies / 10e6 cells: mean number of mutant colonies per flask found after plating in TG medium × 10e6 divided by the number of cells survived
induction factor: mutant colonies per 10e6 cells / mutant colonies per 10e6 cells of the corresponding solvent control
Evaluation criteria:
The gene mutation assay is considered acceptable if:
- the numbers of mutant colonies per 10e6 cells found in the solvent controls fall within the laboratory historical control data range.
- the positive control substances must produce a significant increase in mutant colony frequencies.
- the cloning efficiency II (absolute value) of the solvent controls must exceed 50 %.

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 correspon-ding solvent control data. If there is by chance a low spontaneous mutation rate in the range normally found (3.0 – 33.2 mutants per 106 cells) 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.
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. 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 were considered together.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
Untreated negative controls validity:
not applicable
Positive controls validity:
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Any other information on results incl. tables

see attached document for details

Applicant's summary and conclusion