N-butyl acetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study; but as read-across from supporting substance maximum reliability is 2 Read-across hypothesis: for details please see read-across report in IUCLID section 13

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Butan-1-ol

EC Number:

200-751-6

EC Name:

Butan-1-ol

Cas Number:

71-36-3

Molecular formula:

C4H10O

IUPAC Name:

butan-1-ol

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

Method

Target gene:

HPRT

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:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

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:

DURATION
- Exposure duration: Experiment I: 4 h; Experiment II: 24 h

SELECTION AGENT (mutation assays): 6-TG (6-thioguanine)
NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 50

DETERMINATION OF CYTOTOXICITY
- 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 fre-quencies.
- 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.

Statistics:

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

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

see attached document for details of results

Read-across justification: for details please see read-across report in IUCLID section 13

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the experimental conditions reported the test item butan-1-ol did not induce gene mutations at the HPRT locus in V79 cells.

Executive summary:

A gene mutation assay (HPRT test) was performed with butan-1-ol in Chinese hamster lung fibroblasts (V79). The test item was dissolved in DMSO and added to the cell culture at concentration of 23.1, 46.3, 92.5, 185.0, 370.0 and 740.0 µg/mL in both experiments. Exposure duration was 4 h in the first experiment (in the presence and absence of a metabolic activating system) and 24 hours (in the absence of a metabolic activating system) or 4 hours (in the absence of a metabolic activating system) in the second experiment. No gene mutations at the HPRT locus in V79 cells were induced under the conditions tested (Harlan, 2010).