Cyclohexanepentanol, .beta.-methyl-

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:

2008-02-20 to 2008-04-12 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

See "Principles of method if other than guideline"

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See "Principles of method if other than guideline"

Principles of method if other than guideline:

GLP guideline study reliable without restrictions. Only minor deviations from the guideline: No standard deviation presented for untreated-negative controls.

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2007-10-15

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(R,S)-5-cyclohexyl-2-methyl-pentan-1-ol

EC Number:

700-146-1

Cas Number:

1141487-54-8

Molecular formula:

C12H24O

IUPAC Name:

(R,S)-5-cyclohexyl-2-methyl-pentan-1-ol

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Preliminary test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment I:
- TA100, TA1535 & TA1537 (without S9): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate
- TA102 & TA98 (without S9): 1.5, 5, 15, 50, 150, 500 µg/plate
- TA100, TA1535 & TA1537 (with S9): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate
- TA102 & TA98 (with S9): 5, 15, 50, 150, 500, 1500 µg/plate
Experiment II:
- TA100, TA1535 & TA1537 (without S9): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate
- TA102 (without S9): 5, 15, 50, 150, 500, 1500, 5000 µg/plate
- TA98 (without S9): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate
- TA100, TA1535 & TA1537 (with S9): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate
- TA102 & TA98 (with S9): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: information that the test material was insoluble in water, but fully soluble in DMSO (solubility checks performed in-house confirmed solubility at 50 mg/mL).

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without metabolic activation; strains TA100 and TA1535 Migrated to IUCLID6: 3 µg/plate for TA 100 and 5 µg/plate for TA1535

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation; strain TA1537 Migrated to IUCLID6: 80 µg/plate

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation; strain TA102 Migrated to IUCLID6: 0.5 µg/plate

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation; strain TA98 Migrated to IUCLID6: 0.2 µg/plate

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; 1 µg/plate for TA100 and 2 µg/plate for TA1535 and TA1537

Remarks:

with metabolic activation; strain TA100, TA1535 and TA1537

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation; strain TA98 Migrated to IUCLID6: 5 µg/plate

Controls 7

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 1,8-dihydroxyanthraquinone; 10 µg/plate

Remarks:

with metabolic activation; strain TA102

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (direct plate incorporation method)

DURATION
- Exposure duration: approximately 48 hours incubation at 37°C

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: (Experiment I and II)
- The frequency of revertant colonies was assessed using a Domino colony counter.

DETERMINATION OF CYTOTOXICITY
- Method:relative total growth:
A preliminary test was carried out to determine the toxicity of the test material. Ten doses of the test material and a vehicle control were tested. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn (using a Dominga colony counter).

OTHER:
- A second experiment (Experiment II) was performed in the same way as described for Experiment I, using fresh bacterial cultures, test material and control solutions. The test material dose range was slightly amended, based on the results of Experiment I.

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

not mandatory for this test system

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: not soluble in water (information provided by the sponsor); therefore DMSO was used as a vehicle
- Precipitation: precipitation was observed in the preliminary study at 5000 µg/plate. In the main experiments an oily precipitate was observed at 5000 µg/plate, but this did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: the test material was initially toxic to the strain used (TA100) at and above 150 µg/plate. The test material formulation and the S9-mix were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA: A historical profile of vehicle and positive control values is available.

ADDITIONAL INFORMATION ON CYTOTOXICITY: the test material caused visible reduction in the growth of the bacterial background lawn of all Salmonella strains in experiment I and II at various levels. For the bacterial strains dosed in the absence of S9, weakened lawns were initially noted from 15 µg/plate in Experiment I and from 50 µg/plate in Experiment II. In the presence of S9, weakened lawns were initially noted at 50 µg/plate in Experiment I and from 150 µg/plate in Experiment II. The sensitivity of the bacterial tester strains to the toxicity of the test material varied between strain type, exposures with and without S9 and experiment number. Therefore, the test substance was tested up to either the maximum recommended level of 5000 µg/plate or the toxic limit depending on experiment number.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

The test material was considered to be non-mutagenic under the conditions of the test. No toxicological significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material. According to Regulation (EC) No 1272/2008 as amendet, the test substance is not considered to have a mutagenic potential, and hence no classification or labelling is required.