[[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile

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:

Experimental start date 30 May 2017 Experimental completion date 23 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian forward mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile

EC Number:

258-964-5

EC Name:

[[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile

Cas Number:

54079-53-7

Molecular formula:

C27H31N3O

IUPAC Name:

2-[(4-{[2-(4-cyclohexylphenoxy)ethyl](ethyl)amino}-2-methylphenyl)methylidene]propanedinitrile

Test material form:

solid: particulate/powder

Remarks:

orange

Specific details on test material used for the study:

Appearance: Solid powder, orange
Expiry / Retest Date: 17 February 2021
Storage Conditions: At room temperature
Stability in Solvent: Stable in THF for at least 24 h at room temperature (Envigo Study BN22GC, Stability and Homogeneity)

Method

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) gene locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.

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 Envigo CRS GmbH allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants may be reduced by treatment with HAT-medium. Each master cell stock 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.

Thawed stock cultures were propagated at 37 °C in 75 cm2 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.

All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.

For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL), 10% FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. 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 (98.5 % air).

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-experiment toxicity test:
With and without S9-mix: 7.8, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0, 1000.0 μg
The pre-experiment was performed in the presence and absence of metabolic activation. Test item concentrations between 7.8 μg/mL and 1000 μL/mL were used. The highest concentration was chosen based on the solubility properties of the test item.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) before the test item was removed. Precipitation occurred at 62.5 μL/mL and above in the presence and absence of metabolic activation.

Main experiment:
With and without S9-mix: 3.9, 7.8, 15.6, 31.3, 62.5, 125.0, 250.0 μg/mL
The concentrations used in the main experiment were selected based on the data of the pre-experiment. The maximum concentration was 250.0 μg/mL. The individual concentrations were spaced by a factor of 2.
To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.

Vehicle / solvent:

The vehicle was THF. The final concentration of THF in the culture medium was 0.5 %. The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Tetrahydrofuran (0.5%)

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without S9-mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Tetrahydrofuran (0.5%)

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

Pre-Test on Toxicity
A pre-test was performed in order to determine the toxicity of the test item. In addition the pH and osmolarity were measured (2). The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment.
In this pre-test approximately 1.5 million cells were seeded in 25 cm² flasks 24 hours prior to treatment. After approximately 24 hours the test item was added and the treatment proceeds for 4 hours (duplicate cultures per concentration level). Immediately after treatment the test item was removed by rinsing with PBS. Subsequently, the cells were trypsinized and suspended in complete culture medium. After an appropriate dilution the cell density was determined with a cell counter. Toxicity of the test item is evident as a reduction of the cell density compared to a corresponding solvent control. A cell density of approximately 1.5 million cells in 25 cm² flasks is about the same as approximately 10 million cells seeded in 175 cm² bottles 24 hours prior to treatment with the main experiment.

Experimental Performance

Seeding
Two to four days after sub-cultivation stock cultures were trypsinized at 37 °C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in saline.
Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2×10^7 were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.

Treatment
After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μL/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with PBS.
Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0×10^6 cells per experimental point (concentration series plus controls) were subcultured in 175 cm² flasks containing 30 mL medium.

Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (cloning efficiency I) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
The colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment as described below.
Three or four days after first sub-cultivation approximately 2.0×10^6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium.
Following the expression time of 7 days five 75 cm² cell culture flasks were seeded with about 4 to 5×10^5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II).
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the mean values of the numbers of mutant colonies per 10^6 cells found in the solvent controls of both parallel cultures remain within the 95% confidence interval of the laboratory historical control data range.
b) the positive control substances should produce a significant increase in mutant colony frequencies and remain within the historical control range of positive controls.
c) Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
d) An adequate number of cells and concentrations (at least four test item concentrations) are analysable even for the cultures treated at concentrations that cause 90% cytotoxicity during treatment.
e) The criteria for the selection of the top concentration are fulfilled.

Evaluation criteria:

Evaluation of Results
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 (based 95% control limits).
In cases when the response is neither clearly negative nor clearly positive as described above, or in order to judge the biological relevance of a result, the data should be evaluated by expert judgement or further investigations.

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated 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 using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. Again a t-test is judged as significant if 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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item Macrolex Gelb 6G was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation.
Precipitation visible at the end of treatment was noted in Experiment I at 125 μg/mL and above with and without metabolic activation. In Experiment IA precipitation occurred at 62.5 μg/mL and above.
In Experiment I no relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred up to the maximum concentration with and without metabolic activation.
The 95% confidence interval was exceeded at 62.5 and 125.0 μg/mL in the first culture, and at 7.8, 15.6, 31.3, and 125.0 μg/mL in the second culture in the absence of metabolic activation (35.0, 31.4, 32.9, 33.9, 32.0, and 40.1 versus an upper limit of 30.2 mutant colonies/10^6 cells). In the presence of metabolic activation the confidence interval was exceeded at 15.6 μg/mL 34.0 versus an upper limit of 29.4 mutant colonies/10^6 cells).
A t-test evaluating the data of both parallel cultures showed a significant response at only one single data point exceeding the 95% confidence limit. The linear regression analysis was positive in the first culture without metabolic activation indicating a concentration related effect. Therefore, the experimental part without metabolic activation was repeated (Experiment IA).
In experiment IA no relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred up to the maximum concentration without metabolic activation.
The 95% confidence interval was exceeded in culture I at 3.9 μg/mL without metabolic activation (32.7 versus an upper limit of 30.2 mutant colonies/10^6 cells), and in culture II at 15.6 μg/mL (32.6 versus an upper limit of 30.2 mutant colonies/10^6 cells). The t-test evaluating the data of both parallel cultures showed a negative response. Furthermore, the linear regression analysis was negative.
In the main experiments with and without S9 mix the range of the solvent controls was from 19.2 up to 34.0 mutants per 10^6 cells; the range of the groups treated with the test item was from 12.2 up to 40.1 mutants per 10^6 cells.
The highest solvent control value of 34.0 mutants per 10^6 cells exceeded the 95% confidence interval in the first culture of experiment IA, but remained well within the historical range of solvent controls (3.4 – 41.0 mutant colonies/10^6 cells). The mutation frequency of the parallel culture (23.6 mutant colonies/10^6 cells) and the mean mutant frequency of both parallel cultures (28.8) was fully acceptable. In Experiment I with metabolic activation the solvent control value of 31.7 mutants per 10^6 cells exceeded the 95% confidence interval in the second culture, but remained well within the historical range of solvent controls (2.4 – 39.2 mutant colonies/10^6 cells). The mutation frequency of the parallel culture (19.2 mutant colonies/10^6 cells) and the mean mutant frequency of both parallel cultures (25.5) was fully acceptable.
No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration.
EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

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, Macrolex Gelb 6G is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of Macrolex Gelb 6G 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 in Experiment I and 4 hours without metabolic activation in Experiment IA.

The maximum test item concentration of the pre-experiment (1000 μg/mL) was chosen by the limited solubility of the test item.

No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration.

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.

Conclusion

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, Macrolex Gelb 6G is considered to be non-mutagenic in this HPRT assay.