Reaction mass of 1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol and 1-[(1S*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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:

(Commission Directive 2000/32/EC)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol and 1-[(1S*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol

EC Number:

947-716-8

Molecular formula:

C15H30O

IUPAC Name:

Reaction mass of 1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol and 1-[(1S*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-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:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbitone/ß-naphthoflavone

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 with and without metabolic activation in TA100

Experiment 1 and 2:
0, 50, 150, 500, 1500, 5000 µg/plate with and without metabolic activation in all strains

Vehicle / solvent:

- Vehicle/solvent used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water at 50 mg/mL but was fully soluble in dimethyl sulphoxide at the same concentration in solubility checks performed in-house.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

mitomycin C

other: 2-Aminoanthracene (2AA); 1,8-Dihydroxyanthraquinone (DAN)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for experiment 1 and 2

DURATION
- Exposure duration: approx. 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Rationale for test conditions:

The test conditions were chosen according to OECD 471.

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 was considerd first, statistical methods, as recommended by the UKEMS (Kirkland D.J., Statistical Evaluation of Mutagenicity Test Data. UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III, Cambridge University Press, 1989) were also used as an aid to evaluation, however, statistical significance was not the only determining factor for a positive response. A test material was considered non-mutagenic (negative) in the test system if the above criteria were not met.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

greasy precipitate ≥ 1500 µg/plate

Vehicle controls validity:

valid

Untreated 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:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

greasy precipitate ≥ 1500 µg/plate

Vehicle controls validity:

valid

Untreated 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:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

greasy precipitate ≥ 1500 µg/plate

Vehicle controls validity:

valid

Untreated 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:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

greasy precipitate ≥ 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

greasy precipitate ≥ 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A greasy precipitate was observed at and above 1500 μg/plate, this did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: The dose range of 50 to 5000 µg/plate for both main experiments was determined in a preliminary toxicity assay in S. typhimurium strain TA 100.

HISTORICAL CONTROL DATA: Positive and vehicle controls were within the normal range of historical data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level.

Any other information on results incl. tables

Spontaneous mutation rates for the negative controls were considered to be acceptable. The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial stains, with any dose of the test substance, either with or without metabolic activation.

Table 2. Results of Experiment 1

EXPERIMENT 1
S9-Mix	Without
Test substance (µg/plate)	TA 100	TA 1535	TA 102	TA 98	TA 1537
SC	114 ± 7.4	23 ± 4.0	332 ± 14.1	21 ± 5.6	16 ± 3.5
50	109 ± 19.5	22 ± 0.6	293 ± 39.7	24 ± 6.7	13 ± 3.0
150	99 ± 3.8	21 ± 4.6	256 ± 41.6	22 ± 6.8	15 ± 3.6
500	96 ± 17.7	21 ± 4.7	265 ± 9.3	19 ± 5.5	12 ± 2.3
1500	105 ± 22.7 P	20 ± 4.4 P	242 ± 34.1 P	20 ± 2.1 P	13 ± 4.6 P
5000	85 ± 11.0 P	23 ± 1.2 P	264 ± 33.0 P	21 ± 1.2 P	12 ± 2.3 P
ENNG	479 ± 50.8	382 ± 25.7
MMC			1739 ± 6.5
4NQO				275 ± 16.8
9AA					756 ± 230.1
S9-Mix	With
Test substance (µg/plate)	TA 100	TA 1535	TA 102	TA 98	TA 1537
SC	88 ± 6.4	14 ± 0.0	328 ± 14.2	32 ± 5.0	19 ± 3.5
50	93 ± 6.5	14 ± 1.7	336 ± 30.1	32 ± 1.7	18 ± 4.7
150	89 ± 18.6	10 ± 2.3	285 ± 23.1	30 ± 5.5	20 ± 2.1
500	88 ± 12.5	13 ± 4.2	308 ±19.0	28 ± 5.5	14 ± 6.7
1500	83 ± 7.6 P	10 ± 2.3 P	296 ± 27.1 P	23 ± 5.9 P	11 ± 0.6 P
5000	89 ± 9.6 P	13 ± 2.5 P	286 ± 11.0 P	26 ± 2.3 P	12 ± 3.5 P
2AA	2762 ± 93.6	174 ± 42.2			210 ± 19.6
DAN			1010 ± 101.7
BP				158 ± 36.7
SC = Solvent Control (DMSO) P = Precipitate Positive Controls: ENNG: N-ethyl-N´-nitro-N-nitrosoguanidine; MMC: Mitomycin C; 4NQO: 4-Nitroquinoline-1-oxide; 9-AA: 9-aminoacridine; 2AA: 2-Aminoanthracene; DAN: 1,8-Dihydroxyanthraquinone; BP: Benzo(a)pyrene

Table 3. Results of Experiment 2

EXPERIMENT 2
S9-Mix	Without
Test substance (µg/plate)	TA 100	TA 1535	TA 102	TA 98	TA 1537
SC	92 ± 13.9	25 ± 5.3	293 ± 33.0	21 ± 1.0	13 ± 5.9
50	98 ± 17.1	19 ± 5.2	321 ± 17.6	14 ± 5.7	13 ± 3.8
150	103 ± 5.3	19 ± 2.6	284 ± 8.5	11 ± 3.5	11 ± 2.6
500	91 ± 13.1	9 ± 3.5	272 ± 16.2	12 ± 4.0	8 ± 1.5
1500	78 ± 7.4 P	12 ± 4.2 P	288 ± 22.5 P	9 ± 0.6 P	11 ± 1.5 P
5000	83 ± 14.2 P	14 ± 5.1 P	274 ± 7.5 P	8 ± 2.0 P	17 ± 2.3 P
ENNG	673 ± 76.4	859 ± 123.0
MMC			1341 ± 95.8
4NQO				128 ± 28.4
9AA					323 ± 32.5
S9-Mix	With
Test substance (µg/plate)	TA 100	TA 1535	TA 102	TA 98	TA 1537
SC	84 ± 12.7	11 ± 2.1	310 ± 30.7	15 ± 4.2	11 ± 3.5
50	84 ± 9.8	17 ± 2.0	315 ± 10.1	17 ± 1.2	10 ± 3.1
150	81 ± 4.0	9 ± 4.0	283 ± 16.5	20 ± 6.1	7 ± 2.1
500	61 ± 7.8	11 ± 0.0	291 ± 20.4	17 ± 5.1	8 ± 1.0
1500	57 ± 5.5 P	10 ± 4.2 P	268 ± 12.1 P	14 ± 2.6 P	11 ± 2.9 P
5000	59 ± 7.0 P	10 ± 2.3 P	272 ± 12.6 P	14 ± 3.6 P	10 ± 3.6 P
2AA	1536 ± 162.9	174 ± 52.5			568 ± 46.2
DAN			733 ± 22.6
BP				173 ± 17.7
SC = Solvent Control (DMSO) P = Precipitate Positive Controls: ENNG: N-ethyl-N´-nitro-N-nitrosoguanidine; MMC: Mitomycin C; 4NQO: 4-Nitroquinoline-1-oxide; 9-AA: 9-aminoacridine; 2AA: 2-Aminoanthracene; DAN: 1,8-Dihydroxyanthraquinone; BP: Benzo(a)pyrene

Applicant's summary and conclusion

Conclusions:

Under the conditions of the Ames assay the test substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) tested with and without metabolic activation.

Executive summary:

A bacterial gene mutation assay (Ames) with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2016). In two independent experiments, the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 were exposed to the test substance using the plate incorporation method. Based on the results of a pre-experiment, test substance concentrations of 50 to 5000 µg/plate were selected for the incubation with and without metabolic activation in both experiments. Precipitation occurred at and above 1500 µg/plate. The test substance was not bacteriotoxic at any dose and strain. In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tested strains in the presence and absence of metabolic activation. The vehicle and positive control data were in the range of the historical control data of the laboratory. Under the conditions of this experiment, the test substance did not show mutagenicity in the selected S. typhimurium strains in the presence and absence of metabolic activation.