Cyclohexanol, 4-(1,1-dimethylethyl)-, trans-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD guideline No. 471; GLP compliant): negative

Link to relevant study records

Reference

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 2008-03-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study reliable without restrictions. Minor deviations from the guideline were: - the stability of the test material were not stated.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

yes

Remarks:

please refer to "Rationale for reliability incl. deficiencies" above

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

yes

Remarks:

please refer to "Rationale for reliability incl. deficiencies" above

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2007-10-15

Type of assay:

bacterial reverse mutation assay

Target gene:

not applicable

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 -mix (for further information please to "Any other information on materials and methods incl. tables")

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment I:
- TA100, TA1535, TA102, TA1537, and TA98 (with S9-mix):5, 15, 50, 150, 500, 1500 and 5000 µg/plate
- TA100, TA1535, TA102, TA1537, and TA98 (without S9-mix):5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment II:
- TA100 andTA1535 (with and without S9-mix): 15, 50, 150, 500 and 1500 µg/plate
- TA102 and TA98 (with and without S9-mix): 15, 50, 150, 500, 1500 and 5000 µg/plate
- TA1537 (with and without S9-mix): 5, 15, 50, 150 and 500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: the test material was fully soluble in dimethyl sulphoxide at 50 mg/mL solubility checks performed in-house. Sterile distilled water was not evaluated as a potential vehicle in this test system due to information supplied by the sponsor. Dimethyl sulphoxide was therefore selected as the vehicle.

A solvent treatment group was used as the vehicle control and test in parallel with the test material.

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 for TA102

Remarks:

with metabolic activation; strain TA102

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 for TA98

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 2µg/plate for TA1535 and TA1537

Remarks:

with metabolic activation; strains TA100, TA1535 and TA 1537

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 for TA98

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 for TA102

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 for TA1537

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 TA 1535 Migrated to IUCLID6: 3 µg/plate for TA 100 and 5 µg/plate for TA1535

Details on test system and experimental conditions:

A) DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The test was performed by mixing 0.1 mL of bacterial culture (TA100), 2 mL of molten, trace histidine supplemented, top agar, 0.1 mL of test material formulation, 0.5 mL of S9-mix or phosphate buffer and overlaying into sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). Ten doses of the test material and a vehicle control (dimethyl sulphoxide) were tested.
In addition, 0.1 mL of the maximum concentration of the test material and 2 mL of molten, trace histidine supplemented, top agar was overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effect on the growth of the bacterial background lawn.

B) MAIN STUDY:

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

DURATION
- Exposure duration: all of the plates were incubated at 37°C for approximately 48 hours

NUMBER OF REPLICATIONS: 3

OTHER:
- a second experiment (Experiment II) was performed using methodology as described for Experiment 1, using fresh bacterial cultures, test material and control solutions. The test material dose range was amended slightly, based on the results from 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

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxic at and above 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no 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: A light, oily precipitate was observed in the preliminary toxicity test only at 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
The test material was toxic at and above 1500 µg/plate to the strain of Salmonella used (TA100). The test material formulation and the S9-mix used in this experiment 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 a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both experiments at various dose levels. Weakened bacterial background lawns were initially noted to TA1537 at 500 µg/plate and to the remaining Salmonella strains at 1500 µg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test material varied between strain type, experiment number and exposures with or without S9. The test material was tested up to either the maximum recommended dose level of 5000 µg/plate or the toxic limit depending on bacterial strain type and experiment number.

Conclusions:

The test material was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The Ames test (2008) performed according to OCED 471 was rated reliable without restrictions and used as a key study. The results indicate that the test substance under the experimental conditions described, is not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA 102 in the presence and absence of a metabolising system.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

All available reliable studies showed no genetic toxicity for the test substance. Thus, no classification is required according to Regulation 1272/2008 (CLP), as amended for the seventeenth time in Regulation (EU) 2021/849.