4-[2-(Dimethylamino)-1-(1-hydroxycyclohexyl)ethyl]phenol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07/1988-03/1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Guideline followed was not stated. The detect mutations at the HFPRT locus, the CHO cells are subjected to a three-step procedure: exposure to the test sample, culturing for an appropriate phenotypic expression period after treatment and then growth in a selection medium containing the purine analogue 6-thioguanine. The wild type cells convert the purine analogue to toxic metabolites and are killed, while thioguanine and hence escape its lethal effect. The HGRPT mutants are quantitated by clonal growth in the selection medium.

GLP compliance:

yes

Type of assay:

other: CHO/HGPRT in vitro mammalian cell mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-[2-(dimethylamino)-1-(1-hydroxycyclohexyl)ethyl]phenol

EC Number:

700-516-2

Cas Number:

93413-62-8

Molecular formula:

C16H25NO2

IUPAC Name:

4-[2-(dimethylamino)-1-(1-hydroxycyclohexyl)ethyl]phenol

Method

Target gene:

HGPRT locus in CHO Cells

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

In the absence of S-9 at concentrations of 0.13, 0.4, 1.3, 4, 13, 40, 130, 400 μg/ml.
At concentrations of 25, 50, 100, 200, 400 μg/ml in parallel.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

1% DMSO

Positive controls:

yes

Positive control substance:

other: DMBA at 15 μg/ml

Remarks:

Activated Assay

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

1% DMSO

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

nonactivated mutation assay

Migrated to IUCLID6: ca. 235 μg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period:
- Exposure duration: 5 hours in the presence of S-9 and 16-18 hours in the absence of S-9
- Expression time (cells in growth medium): 7-10days
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 7-9days

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS:

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:

Evaluation criteria:

Cytotoxity was evaluated by the number of colonies present following 6-8days growth from cultures exposed to the substance for five hours in the presence of S-9 and for 16-18 hours in the absence of S-9. Survival frequency was determined by comparison to solvent controls. The number of mutant cells was determined by TG colonies after growth in selective media containing 6-thioguanine. The mutationfrequenvy was calculated by' dividing the total number of mutant colonies per duplicate culture by the total number of cells seeded per culture, corrected for the cloning efficiency and was expressed as mutants per 1000000 cloned cells.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test article was initially tested in a range-finding assay to select concentrations for the mutagenesis assay. Cells were exposed to concentrations ranging from 0.13 to400 µg/ml in half-logs in the nonactivated rang-finding assay. No toxicity, the highest concentration tested in the mutagenesis assay, 400 µg/ml, was based on the limited solubility of the test article in DMSO and the lack of solubility in the culture medium.
The results of the parallel cytotoxicity assays which accompanied the mutagenesis assay were similar to the result in the rang-finding assay. The test article was non-toxic at all concentrations.
In Experiment 1, the range mutation frequencies were not calculated due to the variation in the cloning efficiency at the time of selection. Due to a systematic technical error (presumably in dilution), several of the groups had cloning efficiencies which were greater than 100% (negative control and 25µg/ml). The remainder of the groups (positive control and 50µg/ml to 400µg/ml) had cloning efficiencies in the expected range, from 42% to 91%.
In order to analyze the mutation results, the number of total mutant colonies was compared for each group. The average number of mutants per 1000000 total cells was 19.5 for the negative control, and the range was 7 to 28. The average number of mutants for each test article concentration was similar to the negative control. There were 11.5, 14, 22, 14 and 23 mutants per 1000000 total cells at concentrations of 25 to 40 400µg/ml, respectively. Thus, the test article did not induce an increase in the total number of mutant colonies.
In Experiment 2, the cloning efficiencies at the time of selection were in the expected range and were used to calculate the mutation frequency. The average negative control mutation frequency was 13.6 ×10-6 and the range was 0.0 to 26.5×10-6. There of the average mutation frequencies from cells exposed to the test article were higher than the average negative control value. These were 27.2, 43.6, and 47.8×10-6 at 25 µg/ml, 50 µg/ml, and 400 µg/ml, respectively.
These values, however, are not an indication of mutagenesis for two reasons. First, there is no concentration-dependent increase; the mutation frequencies at 100 µg/ml and 200 µg/ml are equel to or lower than the negative control mutation frequency. Second, most of the individual mutation frequencies are within the range established by the negative control, 0 to 26.5×10-6. Only two values, 59.3 and 47.8 ×10-6 are outside this range, and these two higher frequency. Therefor, there is no convincing evidence that the test article induced mutations in Experiment 2.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test article was tested for its ability to induce mutations in CHO at the HGPRT locus in the presence and absence of S9. The test article was non- mutagenic in this study.

Executive summary:

CHO cells were exposed to the substance for five hours in the presence of S-9 and for 16-18 hours in the absence of S-9 at concentrations of 0.13-400μg/ml to evaluate its mutagenic potential as evidenced by its ability to induce forward mutations at the HGPRT locus. A slight reduction in cell survival occurred in the presence of S-9 at a concentration of 400μg/ml of the substance in the activated cytotoxicity assay. The test article was non-mutagenic in both the activated and non-activated mutation assay systems.