Homosalate

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: OECD guideline study performed under GLP without significant deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - 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:

Homosalate

EC Number:

204-260-8

EC Name:

Homosalate

Cas Number:

118-56-9

Molecular formula:

C16H22O3

IUPAC Name:

3,3,5-trimethylcyclohexyl salicylate

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Techni-cal University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/-naphthoflavone induced rat liver S9 was used as the metabolic activation system.

Test concentrations with justification for top dose:

Based on the results of the pre-experiment, the main experiments were started with 6 to 8 concentrations. A series of concentrations spaced by a factor of 2 was placed into the lower range. Narrower spacing was used at high concentrations without metabolic activa-tion to cover the toxic range more closely.
Experiment I:
4 hours without metabolic activation: 0.08, 0.15, 0.3, 0.6, 1.2 µg/ml
4 hours with metabolic activation: 20.0, 40.0, 80.0, 160.0, 640.0 µg/ml
Experiment II:
24 hours without metabolic activation: 1.3, 2.5, 5.0, 10.0, 20.0 µg/ml
4 hours with metabolic activation: 20.0, 40.0, 320.0, 640.0 µg/ml

In experiment I cultures at the three highest concentrations without metabolic activation were not continued due to exceedingly severe cytotoxicity. The cultures at 320 µg/mL with metabolic activation were not continued for the same reason. In experiment II the cultures at the two highest concentrations without metabolic activation and the cultures at 80 and 160 µg/mL with metabolic activation were not continued based on exceedingly severe cytotoxicity. In experiment II the cultures at the lowest concentration without metabolic activation were not continued as a minimum of only four analysable concentrations is required by the guidelines.

Vehicle / solvent:

Ethanol was used as solvent

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

ethylmethanesulphonate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

Culture Medium
For seeding and treatment of the cell cultures the complete culture medium was MEM (minimal essential medium) supplemented with Hank’s salts, 10% Fetal Bovine Serum (except during 4 hour treatment), 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.
Seeding
Two to three days after sub-cultivation of the V79 cell stock culture, rinsing with PBS buffer containing 200 mg/L EDTA (ethylene diamine tetraacetic acid) occurred..
Following rinsing, the cells were trypsinized at 37 °C for 5 minutes. The trypsin concentration for all sub-culturing steps was 0.2% in PBS.
Then, the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared.
From this cell suspension, approximately 1.5x10E6 cells were seeded in a plastic culture flask for further mutation rate and cloning efficiency II (viability) analysis. Independently, twice 5x10E2 cells were seeded in two plastic culture flasks for further survival (Cloning Efficiency I) analysis.
The cells were grown for 24 hours prior to treatment with the test item, to enable attach-ment of the cells to the bottles.
The PBS is composed as follows (per litre):
NaCl 8000 mg
KCl 200 mg
KH2PO4 200 mg
Na2HPO4 150 mg
Treatment
After the seeding phase, the complete medium was replaced with serum-free medium containing the test item, either with or without 50 µl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel.
In the first experiment the treatment period was 4 hours with and without metabolic activa-tion. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
After the relevant treatment periods, the cultures were washed twice with "saline G" and replaced with complete medium.
The "saline G" solution had the following constituents (per litre):
NaCl 8000 mg
KCl 400 mg
Glucose 1100 mg
Na2HPO4x2H2O 192 mg
KH2PO4 150 mg
The pH was adjusted to 7.2
Cloning efficiency I (Survival) determination:
The colonies contained in the two flasks used to determine the cloning efficiency I (survival) were fixed and stained approx. 7 days after end of treatment. The colonies were stained with 10% methylene blue in 0.01% KOH solution.
The stained colonies with more than 50 cells were counted. When in doubt the colony size was checked with a microscope preparation.
Mutation and Cloning efficiency II (Viability) determination:
Three or four days after end of treatment with the test item, 1.5x10E6 cells from the single flask used for mutation and CE II (viability) analysis were sub-cultivated in a single 175 cm² flask containing 30 mL medium. Following the overall expression time of 7 days after end of treatment, the cells were selected as follow:
- Five 80 cm² cell culture flasks were seeded, with about 3 - 5x10E5 cells each, in medium containing 6-TG to determine mutation.
- Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (CE II).
All seven subcultures per concentration including controls were then incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. Finally, the colonies were stained with 10% methylene blue in 0.01% KOH solution.The stained colonies with more than 50 cells were counted. When in doubt the colony size was checked with a preparation microscope.

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:

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.
experimental group p-value
experiment I, culture I without S9 mix 0.067
experiment I, culture II without S9 mix 0.776
experiment I, culture I with S9 mix 0.139
experiment I, culture II with S9 mix 0.718
experiment II, culture I without S9 mix 0.147
experiment II, culture II without S9 mix 0.045 (S)
experiment II, culture I with S9 mix 0.868
experiment II, culture II with S9 mix 0.164
(S) = Significant trend

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1.2 µg/mL and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 20 µg/mL and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Phase separation was observed in the experimental parts with metabolic activation at 640 µg/mL in the first and at 320 and 640 µg/mL in the second experiment.
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% occurred without metabolic activation at 1.2 µg/mL and above in experiment I and at 20.0 µg/mL and above in experiment II. In the experimental parts with metabolic activation exceedingly severe cytotoxic effects precluding analysis occurred at intermediate concen-trations just below the phase separating concentrations. In experiment I no data on mutagenicity were generated at 160 µg/mL due to exceedingly severe cytotoxicity, whereas no cytotoxicity was noted at the next higher concentration of 320 µg/mL showing phase separation. In the second experiment with metabolic activation the concentrations of 80 and 160 µg/mL were too toxic for analysis. Relevant but tolerable cytotoxic effects were noted at the next lower concentration of 40 µg/mL and at the two higher concentrations of 320 and 640 µg/mL showing phase separation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I or relative cell density was covered with and without metabolic activation.
No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutant frequency did not exceed the historical range of solvent controls.
The induction factor exceeded the threshold of three times the mutation frequency of the solvent control in the first experiment without metabolic activation at 0.1 µg/mL in culture II. However, this increase was judged as biologically irrelevant as it was based on the rather low solvent control of just 3.9 colonies per 106 cells and the absolute value of the mutation frequency (17.7 mutant colonies/106 cells) remained well within the historical range of solvent controls (2.8 - 43.5 mutant colonies/106 cells).
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 detected in the second culture of the second experiment without metabolic activation. Since the mutation frequency neither exceeded the historical range of solvent controls nor the threshold as indicated above, the statistical results were considered as biologically irrelevant.
In both experiments of this study, with and without S9 mix, the range of the solvent controls was from 3.9 up to 17.7 mutants per 106 cells; the range of the groups treated with the test item was from 0.7 up to 24.5 mutants per 106 cells.
The cloning efficiency II of the solvent control for the experiment I culture II with metabolic activation (40%) fell short of the acceptable limit of >50%. The data are valid however, as the cloning efficiency II of the culture I (70%) and the mean value of both duplicate cultures (55%) was acceptable.
EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
A summary table of results is attached (see file 1486001ResultsSummaryTable.pdf)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

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, Neo Heliopan HMS is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of homosalate to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The study is comprised of a pre-experiment and two independent main experiments. In the pre-experiment the cell cultures were treated with the test item for 4 hours with metabolic activation and for 4 and 24 hours without metabolic activation. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The maximum concentration of the pre-experiment (2700 µg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxic effects and phase separation. Ethanol was used as solvent.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in either of the main experiments.

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.