Cyclohexylbenzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Cyclohexylbenzene was negative in all 3 available genotox tests: Ames, gene mutation (HPRT) and chromosome aberration.

Link to relevant study records

Reference

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: Recent and very well documented study with complete information in accordance with OECD and GLP guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

There were 8 deviations that had been report but this deviations are not considered to have had an adverse impact on the integrity of the study.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO cells were maintained in Ham's F1 medium supplemented with L-glutamine and 5% heat-inactivated and dialyzed fetal bovine serium (F12CM5) under standard conditions (37+/-1°C in a humidified atmosphere of 5+/-1% CO2 in air). Hypoxanthine-free F12CM5 (Hx F12CM5) was used for mutant selection. Medium for selection of mutants also contained 10 µM TG. All media contained antimycotics and antibiotics.

To reduce the frequency of spontaneous HPRT mutations prior to use in an assay, the cells were cleansed in medium supplemented with hypoxanthine, aminoterpin and thymidine (HAT), prior to freezing. Cells used in the mutation assay did not exceed four passages from the frozen stock.

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Preliminary test:
3.13, 6.25, 12.5, 25.0, 50.0, 100, 200, 400, 800, 1600 µg/mL with and without S9

Definitive mutagenicity assay:
6.25, 12.5, 25.0, 50.0, 100, 125 µg/mL with S9
0.781, 1.56, 3.13, 6.25, 12.5, 15.0, 20.0 µg/mL without S9

Independent confirmatory assay:
12.5, 25.0, 50.0, 60.0, 80.0, 100, 125 µg/mL with S9
1.56, 3.13, 6.25, 12.5, 15.0, 20.0, 25.0 µg/mL without S9

Vehicle / solvent:

Test article was diluted for use in dimethyl sulfoxide (DMSO; CAS No. 67-68-5; lot No. MKBF8188V with a purity of 99.98% and an expiration of July 2014; Lot No.SHBC3749V purity of 99.92% and an expiration of April 2016) obtained from Sigma-Aldricht.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

The assay was conducted by exposing CHO cells to appropriate concentrations of the test articleas well as the concurrent positive and vehicule controls, in the presence and absence of an exogenous metabolic activation system.

Evaluation criteria:

The test article was considered to have produced a positive response if it induced a statistically significant and dose-dependant increase in mutant
frequency (p<0.05) representing an increase of >15TG mutant/1000 000 clonable cells over the concurrent vehicule controls.
If only one criterion was met, the result was considered equivocal. If neither criterion is met, the results were considered to be negative.

Statistics:

Statistical analyses were performed using the method of See and Irr (1981), with a significance established at the 0.05 level.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 125.0µg/ml with S9 and 25.0µg/ml without S9

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Definitive mutagenicity assay

The average adjusted relative survivals at concentrations of 100 µg/mL with S9 and 20.0 µg/mL without S9 were 76.9 and 16.2%, respectively. No significant increase in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p > 0.05). In contrast, the positive controls induced significant increases in mutant frequency (p < 0.01).

Independent confirmatory assay:

The average adjusted relative survivals at concentrations of 125µg/mL with S9 and 25.0 µg/mL without S9 were 9.7 and 21.9%, respectively. No significant increase in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p > 0.05). In contrast, the positive controls induced significant increases in mutant frequency (p < 0.01).

Conclusions:

Interpretation of results (migrated information):
negative

The results indicate that phenylcyclohexane was negative in the In vitro Mammialan Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate Cultures under the conditions of the test and acording to the criteria of the test protocol.

Executive summary:

This study evaluates the ability of phenylcyclohexane to induce forwards mutation at the HPRT locus of CHO cells in presence and absence of S9 (an exogenous metabolic activation system), and in presence of 6 -TG (6- thioguanine).

Seven or eight dose levels were tested based on the cytotoxicity profile of the test article but only five concentrations were carried through the entire assay and evaluation. All test and control articles and concentrations were evaluated in duplicate cultures. Cells were treated for 5 hours in the presence and absence of S9, by addition of the test and control (Benzo(a)pyrene and ethyl methanesulfate) article formulations to the treatment medium. At the end of the experiment the cells were fixed and counted. Mutant frequency were expressed as the number of TG mutant/1 000 000 clonable cells.

The following evaluation criteria were adopted: If the test article induced an increase of more than 15 TG mutant/ 1 000 000 clonable cells in compare with the vehicle controls, the test article was considered to have produced a positive response.

The results of this experiment showed no significant increase in mutant frequency, as compared to the concurrent vehicule controls at any of the evaluated concentrations with and without S9, whereas the positive control did induce a significant increases in mutant frequency. These results were confirmed in an independent confirmatory assay.

In conclusion, the results indicate that phenylcyclohexane does not induce mutagenicity on CHO/HPRT cells under the applied test conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In total 3 studies are available that assess the in vitro genotoxicity of cyclohexylbenzene. Cyclohexylbenzene did not induce genotoxic responses in any of the tests.

Bacterial reverse mutation

The first study (Bowles, 2001) is a well-documented Ames test that is carried out according to GLP and OECD guidelines. In this test,Salmonella typhimuriumstrains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2 uvrA were treated with the test material using the Ames plate incorporation method at up to 6 dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors).   The test material caused a visible reduction in the growth of the bacterial background lawn to all of the Salmonella tester strains, initially at 500 and 1500 μg/plate, without and with S9 -mix respectively. No toxicity was observed in E. coli tester strain WP2 uvrA. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Chromosome aberration

The second study (Wright, 2001) assessed the potential chromosomal mutagenicity of cyclohexylbenzene on the metaphase chromosomes of the Chinese Hamster Lung (CHL) cell line according to the requirements of the Japanese New Chemical Substance Law (METI), OECD 473 and the updated Annex V B10 Method. Duplicate cultures of CHL cells were treated with the test material at several dose levels, together with vehicle and positive controls. Five exposure groups were used: Experiment 1 included a 6(18)-hour exposure, both with and without the addition of an induced rat liver homogenate metabolising system, Experiment 2 included a 24 -h continuous exposure, a 48 h continuous exposure and a repeat of the 6(18) h exposure with metabolic activation. The dose levels evaluated in the main experiments were selected from a range of dose levels based on the results of a preliminary toxicity test and were in the range of 3.125 to 25 μg/ml for the 6(18)-h exposure without S9, 6.25 to 100 μg/ml for the 24 and 48 h treatments. The vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for the CHL cell line. All the positive control chemicals induced highly significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any significant increases in the frequency of cells with aberrations in any of the exposure groups. The test material was shown to be toxic to CHL cells in vitro and optimal levels of toxicity were achieved in all exposure groups.

Gene mutation

The third study (Stankoweski, 2013) evaluates the ability of cyclohexylbenzene to induce forwards mutation at the HPRT locus of CHO cells in presence and absence of S9 (an exogenous metabolic activation system), and in presence of 6 -TG (6- thioguanine). Seven or eight dose levels were tested based on the cytotoxicity profile of the test article but only five concentrations were carried through the entire assay and evaluation. All test and control articles and concentrations were evaluated in duplicate cultures. Cells were treated for 5 hours in the presence and absence of S9, by addition of the test and control (Benzo(a)pyrene and ethyl methanesulfate) article formulations to the treatment medium. At the end of the experiment the cells were fixed and counted. Mutant frequency were expressed as the number of TG mutant/1 000 000 clonable cells. The following evaluation criteria were adopted: If the test article induced an increase of more than 15 TG mutant/ 1 000 000 clonable cells in compare with the vehicle controls, the test article was considered to have produced a positive response. The results of this experiment showed no significant increase in mutant frequency, as compared to the concurrent vehicule controls at any of the evaluated concentrations with and without S9, whereas the positive control did induce a significant increases in mutant frequency. These results were confirmed in an independent confirmatory assay. In conclusion, the results indicate that phenylcyclohexane does not induce mutagenicity on CHO/HPRT cells under the applied test conditions.

Justification for selection of genetic toxicity endpoint
For the endpoint on genetic toxicity, 3 types of tests are required: Ames, gene mutation and chromosome aberration. Therefore, all 3 studies available in this endpoint are of equal importance.

Justification for classification or non-classification

As no genotoxic effects are observed in the available studies addressing the different endpoint of genetic toxicity, classification for genetic toxicity under EU Regulation No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures (CLP) and/or under Council Directive 67/548/EEC on the Classification, Packaging and Labelling of Dangerous substances (DSD) is not required.