Cyclohexylbenzene

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test 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

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexylbenzene

EC Number:

212-572-0

EC Name:

Cyclohexylbenzene

Cas Number:

827-52-1

Molecular formula:

C12H16

IUPAC Name:

cyclohexylbenzene

Test material form:

other: liquid

Details on test material:

- Material: clear, colorless liquid
- Analytical purity: 99.4%
- Lot/batch No.: B9107
- Expiry date: Jan 2015
- Storage condition of test material: room temperature, protected from light

Method

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus

Species / strain

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.

Controls

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.

Results and discussion

Test results

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'.

Any other information on results incl. tables

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).

Applicant's summary and conclusion

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.