4-tert-butylcyclohexanol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Apr - 24 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Freie und Hansestadt Hamburg, Behörde für Soziales, Familie, Gesundheit und Verbraucherschutz, Hamburg, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-tert-butylcyclohexanol

EC Number:

202-676-4

EC Name:

4-tert-butylcyclohexanol

Cas Number:

98-52-2

Molecular formula:

C10H20O

IUPAC Name:

4-tert-butylcyclohexan-1-ol

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

1.0, 3.16, 10.0, 31.6, 100, and 316 µg/plate with and without metabolic activation

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cyclophosphamide

methylmethanesulfonate

other: 2-amino anthracene

Details on test system and experimental conditions:

Experiment 1:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 - 72 h

Experiment 2:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

Experiment 1+2:
NUMBER OF REPLICATIONS: 3 plates per condition per experiment

DETERMINATION OF CYTOTOXICITY
- Method: clearing or diminution of the background lawn, reduction in the number of spontaneous revertants, degree of survival of the treated cultures

Evaluation criteria:

The test item is considered positive in the Ames test if
- at one or more concentrations the number of revertants is reproducibly increased (in comparison to the solvent control; 2-fold in TA 98, TA 100, and TA 102), 3-fold in TA 1535 and TA 1537) in at least one strain with or without metabolic activation.
- a concentration-related increase of the revertants is observed.
- positive results have to be reporducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the solvent control and/or a scarce background lawn.

Statistics:

Mann and Whitney test (p <= 0.05)
Spearman's rank correlation coefficient

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 316 µg/plate (highest concentration) in both experiments in all strains with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The test substance was examined in a preliminary cytotoxicity test (plate incorporation test with and without S9 mix) in TA 100 using ten concentrations ranging from 0.316 to 5000 µg/plate. Cytotoxicity was noted at concentrations of 316 µg/plate and higher, thus, 316 µg/plate was chosen as the highest concentration in the main test.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

In the plate incorporation assay with metablic activation, a more than 3-fold increase in the number of revertants was observed for TA 1537 at a concentration of 3.16 µg/plate (7.3 ± 2.3 revertants) compared to the vehicle control (2.0 ± 1.0 revertants). However, this increase was not dose-dependent and was not reproducible using the preincubation method. Furthermore, compared to the mean number of revertants of the historical control data (7.8 ± 1.9 revertants) for strain TA 1537 in the presence of metabolic activation, the increase in the number of revertants for the test substance was no longer evident.

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In the reverse mutation assay in bacteria the test substance did not induce biologically significant increases in the number of revertants und is thus considered non-mutagenic in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 under the test conditions chosen.