A mixture of cis- and trans-cyclohexadec-8-en-1-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-06-25 to 2021-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of cis- and trans-cyclohexadec-8-en-1-one

EC Number:

401-700-2

EC Name:

A mixture of cis- and trans-cyclohexadec-8-en-1-one

Cas Number:

3100-36-5

Molecular formula:

C16H28O

IUPAC Name:

(8E)-cyclohexadec-8-en-1-one; (8Z)-cyclohexadec-8-en-1-one

Method

Target gene:

HPRT gene

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. The use of this cell is recommended due to its high proliferation rate, a good cloning efficiency and a stable karyotype with a modal chromosome number of 22.
- Normal cell cycle time: 12 - 16 hours doubling time in stock cultures

For cell lines:
- Absence of Mycoplasma contamination: Each master cell stock is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency.
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37 °C in 75 cm^2 plastic flasks. About 2-3×10^6 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly.
- Doubling time: 12 - 16 hours
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: Yes
- Periodically ‘cleansed’ of spontaneous mutants: Yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
Medium: MEM containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%) for maintainance and seeding (complete culture medium), Complete culture medium without FBS during treatment, Complete culture medium supplemented with 11 μg/mL 6-thioguanine during selection.
CO2 concentration: 1.5 % CO2 (98.5 % air)
Humidity level: Humidified atmosphere
Temperature: 37 °C

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Phenobarbital/β-naphthoflavone induced rat liver
- Method of preparation of S9 mix: S9 was mixed with MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- Concentration or volume of S9 mix and S9 in the final culture medium: The final protein concentration was 0.75 mg/mL in the cultures.
- Quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

Pre-test for toxicity (with and without S9): 15.9, 31.9, 63.8, 127.6, 255.1, 510.3, 1020.5 and 2041.0 µg/mL

Main experiment (with and without S9): 0.95, 1.9, 3.8, 7.5, 11.3, 15.0, 22.5, 30.0, 45.0 µg/mL. Concentrations of 0.95, 1.9, 3.8, 7.5 µg/mL were used for mutation rate analysis in the experiment without metabolic activation and concentrations of 3.8, 7.5, 11.3, 15.0 and 22.5 µg/mL were used for mutation rate analysis in the experiment with metabolic activation.

Vehicle / solvent:

- Vehicle used: DMSO (test item, positive control with metabolic activation (DMBA)), cell culture medium (positive control without metabolic activation (EMS))

- Justification for choice of solvent/vehicle: Solubility properties, no toxicity and no influence on the outcome of the study at concentrations used.

- Justification for percentage of solvent in the final culture medium:

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

With metabolic activation
DMBA: 2.3 μg/mL = 8.9 μM in DMSO

Without metabolic activation
300 μg/mL = 2.4 mM in cell culture medium

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: One pre-experiment for toxicity and one main experiment

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 0.7 to 1.2×10^7 cells were seeded for the main experiment. After treatment, at least 2.0×10^6 cells per experimental point were subcultivated. Two additional flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (RS). Three or four days after the first sub-cultivation, at least 2.0×10^6 cells per experimental point were again sub-cultivated. Following the expression time of approximately 7 days, 4 - 5×10^5 cells were seeded in cell culture flasks. Two additional flasks were seeded with approx. 500 cells to determine the viability.
- Test substance added in suspension

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours (with and without metabolic activation)
- Harvest time after the end of treatment: 8 ± 2 days after the end of treatment (colonies used to determine the relative survival), 9 ± 2 days after the end of the 7-day expression time (colonies used to determine mutation frequency), 8 days after the end of the 7-day expression time (colonies used for evaluation of viability)

FOR GENE MUTATION:
- Expression time: Approx. 7 days
- Selection time: 9 ± 2 days
- Fixation time: 14 - 18 days
- Selective agent used: Cells were exposed to 6-thioguanine (11 µg/mL) for 9 ± 2 days for selection.
- Method to enumerate numbers of viable and mutants cells: Colonies were stained with 10% methylene blue in 0.01% KOH solution. Colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cloning efficiency, relative survival (RS)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Mutant colonies per 10^6 cells

Rationale for test conditions:

The concentration range of the main experiment was limited by phase separation and cytotoxicity of the test item. In the main experiment in the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentration. The next higher concentration tested, however, which was separated by a factor smaller than requested by the guideline, could not be evaluated for mutagenicity due to strong toxic effects. In the presence of S9 mix, moderate cytotoxicity was observed at the highest evaluated concentration, which showed phase separation.

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

Statistics:

The statistical analysis was performed on the mean values of culture I and II for the main experiments.

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mean 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.

A t-test was performed only for the positive controls since all mean mutant frequencies of the groups treated with the test item were well within the 95% confidence interval of our laboratory’s historical negative control data.

However, both, biological and statistical significance will be considered together.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH-value was determined in culture medium of the solvent control (pH 6.99) and of the maximum concentration (pH 6.95) in the pre-experiment without metabolic activation.
- Data on osmolality: The osmolarity was determined in culture medium of the solvent control (471mOsm) and of the maximum concentration (453 mOsm) in the pre-experiment without metabolic activation.
- Precipitation and time of the determination: Phase separation occurred at 15.0 μg/mL and above (without metabolic activation) after four hours treatment. Phase separation occurred at 22.5 μg/mL and above (with metabolic activation) after four hours treatment.

RANGE-FINDING/SCREENING STUDIES: Test item concentrations between 15.9 μg/mL and 2041 μg/mL were used in the pre-experiment with and without metabolic activation following 4 hours treatment. Phase separation occurred at 127.6 μg/mL and above with and without metabolic activation. Cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed at 63.8 μg/mL and above with and without metabolic activation. There was no relevant shift of osmolarity and pH of the medium even at the maximum concentration of the test item measured in the pre-experiment.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative survival (RS) and cloning efficiency : See "Attached background material"

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: 0.7 to 1.2×10^7 (number of cells treated), 2.0×10^6 (number of cells subcultivated)
o Number of cells plated in selective and non-selective medium: 4 - 5×10^5 cells
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency: See "Attached background material"

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material"

Applicant's summary and conclusion

Conclusions:

In conclusion it can be stated that under the experimental conditions reported, the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Executive summary:

This Gene Mutation Assay in Chinese Hamster V79 Cells in vitro (V79/HPRT) according to OECD guideline 476 and GLP 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.

The treatment period was 4 hours with and without metabolic activation.

The maximum test item concentration of the pre-experiment (2041 μg/mL) was chosen with respect to the OECD guideline 476 regarding the purity of the test item. The concentration range of the main experiment was limited by phase separation and cytotoxicity of the test item.

In the main experiment, in the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentration. The next higher concentration tested, however, which was separated by a factor smaller than requested by the guideline, could not be evaluated for mutagenicity due to strong toxic effects. In the presence of S9 mix, moderate cytotoxicity was observed at the highest evaluated concentration, which showed phase separation.

No substantial and dose dependent increase of the mutation frequency was observed in the main experiment.

The tested concentrations are shown below:

Exposure period	S9 mix	concentrations in μg/mL
		Main experiment
4 hours	-	0.95	1.9	3.8	7.5	11.3	15.0^PS	22.5^PS	30.0^PS	45.0^PS
4 hours	+	0.95	1.9	3.8	7.5	11.3	15.0	22.5^PS	30.0^PS	45.0^PS

PS = Phase Separation

Appropriate reference mutagens, 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.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, the test item is considered to be non-mutagenic in this HPRT assay.