Octadecane-1,12-diol

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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)

Remarks:

Harlan Cytotest Cell Research GmbH (Harlan CCR), In den Leppsteinswiesen 19, 64380 Rossdorf, Germany

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Octadecane-1,12-diol

EC Number:

220-342-6

EC Name:

Octadecane-1,12-diol

Cas Number:

2726-73-0

Molecular formula:

C18H38O2

IUPAC Name:

octadecane-1,12-diol

Details on test material:

- Name of test material (as cited in study report): Sovermol 912
- Physical state: White, solid
- Storage condition of test material: At room temperature
- Analytical purity: 81.1 g/100 g
- Lot/batch No.: S30120009
- Expiration date of the lot/batch: 25 April 2015

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

experiment 1: 0.11, 0.22, 0.44, 0.88, 1.8, 3.5, 7.0, and 14.0 µg/mL without metabolic activation. 18.0, 35.0, 70.0, 140.0, 280.0, and 560.0 µg/mL with metabolic activation.
experiment 2: 0.13, 0.25, 0.50, 1.0, 1.5, 2.0, 3.0, and 4.0 µg/mL without metabolic activation. 0.52, 1.6, 4.7, 14.0, 28.0, and 42.0 µg/mL with metabolic activation.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

ethylmethane sulfonate without metabolic activation and 7,12-dimethylbenz(a)anthracene with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time: 7 days
- Selection time: approx. 8 days

SELECTION AGENT: 6-thioguanine

NUMBER OF REPLICATIONS: Two independent experiments and two cultures per experiment

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

CONCENTRATIONS ANALYZED
- Experiment 1: 0.22, 0.44, 0.88, 1.8, and 3.5 µg/mL (-S9); 35.0, 70.0, 140.0, 280.0, and 560.0 µg/mL (+S9).
Experiment 2: 0.25, 0.50, 1.0, 1.5, and 2.0 µg/mL (-S9); 1.6, 4.7, 14.0, 28.0, and 42.0 µg/mL (+S9).

Evaluation criteria:

Acceptability of the Assay:
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the numbers of mutant colonies per 1E6 cells found in the solvent controls fall within the laboratory historical control data range.
b) the positive control substances should produce a significant increase in mutant colony frequencies.
c) the cloning efficiency II (absolute value) of the solvent controls must exceed 50%. The data of this study comply with the above mentioned criteria.

Evaluation of Results:
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:

A 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 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:

cytotoxicity

Vehicle controls validity:

not valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRECIPITATION
Precipitation of the test item was observed in the pre-experiment at 27.6 μg/mL and above in the presence and absence of metabolic activation. In the first experiment precipitation was noted at 70.0 μg/mL and above with metabolic activation. In experiment II precipitation occurred at 14.0 μg/mL and above with metabolic activation.

CYTOTOXICITY
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both cultures occurred in experiment I at 1.8 μg/mL and above without metabolic activation and at 35.0 μg/mL and above with metabolic activation. In the experimental part with metabolic activation only the relative cell density was reduced at concentrations of 35 μg/mL and above, the relative cloning efficiency remained above 50% up to 280 μg/mL. In experiment II relevant cytotoxic effects as described above were noted at 1.5 μg/mL and above without metabolic activation and at 28.0 μg/mL with metabolic activation. The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered without metabolic activation.

GENOTOXICITY
No relevant and reproducible increase in mutant colony numbers/1E6 cells was observed in the main experiments up to the maximum concentration. The mutant frequency generally did not exceed the historical range of solvent controls. The induction factor exceeded the threshold of three times the corresponding solvent control in the first culture of the second experiment at 0.25, 0.5, and 1.0 μg/mL without metabolic activation. These effects however, were based upon a rather low solvent control of 8.2 mutant colonies/1E6 cells and thus, biologically irrelevant.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely determined in the second culture of the first experiment without metabolic activation. This trend however, was inverse going down versus increasing concentrations and consequently, irrelevant.

CONTROLS
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 8.2 up to 20.0 mutants per 1E6 cells; the range of the groups treated with the test item was from 0.7 up to 46.4 mutants per 1E6 cells. The highest value of 46.4 mutants per 1E6 cells slightly exceeded the historical range of solvent controls. However, this increase occurred two times at intermediate concentrations and was neither reproduced nor dose dependent as indicated by the lacking statistical significance.
EMS (150 μg/mL) and DMBA (2.2 μg/mL in experiment I, and 1.1 μg/mL in expeirment II) were used as positive controls and showed a distinct increase in induced mutant colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, under the conditions of this study the test substance is considered to be non-mutagenic.

Executive summary:

The study was conducted to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. This study was conducted according to OECD 476 and in compliance with GLP. The study was performed in two independent experiments, using identical experimental procedures. Cells were exposed for 4 hours with and without metabolic activation (Phenobarbital/β-naphthoflavone induced rat liver S9). The concentration range of the main experiments was limited by cytotoxicity and precipitation of the test item. The test item was dissolved in DMSO. In the first experiment concentrations of 0.22, 0.44, 0.88, 1.8, and 3.5 µg/mL test substance without metabolic and 35.0, 70.0, 140.0, 280, and 560.0 with metabolic activation were analyzed to determine the mutation rate. In the second experiment concentrations of 0.25, 0.5, 1.0, 1.5, and 2.0 µg/mL test substance without metabolic and 1.6, 4.7, 14.0, 28.0, and 42.0 with metabolic activation were analyzed. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (ethylmethane sulfonate and 7,12-dimethylbenz(a)anthracene), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. Under the conditions of this study the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, under the conditions of this study the test substance is considered to be non-mutagenic.