Reaction mass of 1-[rac-(1R,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol isomer 1 and 1-[rac-(1S,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was not mutagenic in the bacterial reverse mutation assay.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2000-11-08 to 2000-12-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: besides being histidine auxotrophic, all strains carry an additional mutation (loss of outer lipopolysaccharide barrier) which increases cell permeability

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix, Aroclor 1254 induced

Test concentrations with justification for top dose:

0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: distilled water and DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene

Remarks:

9-Aminoacridine, mitomycin C, and sodium azide were dissolved in distilled water. 2-Aminoanthracene, and 2-nitrofluorene were dissolved in DMSO.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);

DURATION
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3 parallel plates (2 independent experiments)

DETERMINATION OF CYTOTOXICITY
- Method: counting of the number of revertant colonies (his+ revertants)

OTHER EXAMINATIONS:
- Examination for the existente of a normal background lawn and/or precipitates and microscopically for microcolony growth

Evaluation criteria:

The number of spontaneous revertants observed using each of the five strains and the results with the positive controls were compared to historical control data. Differences between the number of revertants in the negative controls and the test plates were tested for significance.

Statistics:

Estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level was performed using a X2-test.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with S9 mix - at 5000 µg/plate; without S9 mix - at 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with and without S9 mix - at 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with S9 mix - at 5000 µg/plate; without S9 mix - at 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

with S9 mix - at 1500 µg/plate; without S9 mix - at 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with S9 mix - at 500 µg/plate; without S9 mix - at 150 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation of the test substance on the plates was observed at 5000 μg/plate. The test substance failed to induce a significant increase in the mutation frequency of the tester strains in the absence and presence of a metabolic activation system. The X2 test did not reveal a significant effect at any of the test points.

HISTORICAL CONTROL DATA
- historical overview of the revertant frequencies of the strains used of the years 1998 to 2000
- TA1535 -S9: 30 ± 14, +S9: 19 ± 5
- TA1537 -S9: 10 ± 4, +S9: 16 ± 5
- TA98 -S9: 29 ± 8, +S9: 40 ± 10
- TA100 -S9: 134 ± 31, +S9: 118 ± 26
- TA102 -S9: 279 ± 41, +S9: 307 ± 45

Table 1: Number of revertants per plate (mean of three plates), Experiment 1

Substance	conc. [µg/plate]	strain TA 98		Strain TA 100		strain TA 102		strain TA 1535		strain TA 1537
		-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
Control	0	18	24	133	129	309	284	22	10	10	11
Solvent control	0	17	24	123	122	250	268	23	10	9	13
Test item 1	5	20	25			251	292
Test item 2	15	15	21	102		259	295
Test item 3	50	17	23	92	126	205	299	28	11	6	14
Test item 4	150	17	28	101	119	193	270	16	10	7	14
Test item 5	500	16	21	100	95	171 T	204 T	10	9	6	11
Test item 6	1500	17	22	87 T	98	95 T	161 T	6 T	7	4 T	9
Test item 7	5000 P	8 T	15 T		92 T			5 T	6 T	4 T	4 T
Sodium azide NaN3	0.7			560				817
2-Nitrofluorene 2-NF	2.5	283
9-Aminoacridine 9-AA	50									583
Mitomycin C	0.15					959
2-aminoanthracene 2-AA	0.8 0.9 1.7		687		1035		747		207		231

-MA: absence of metabolic activation

+MA: presence of metabolic activation

P: precipitation of test item

T: bacteriotoxic

solvent control: DMSO, 50 µL/plate

Table 2: Number of revertants per plate (mean of three plates), Experiment 2

Substance	conc. [µg/plate]	strain TA 98		Strain TA 100		strain TA 102		strain TA 1535		strain TA 1537
		-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
Control	0	24	24	115	123	351	293	16	9	14	15
Solvent control	0	21	22	106	106	326	308	20	10	16	15
Test item 1	5			112		273	294	19		10
Test item 2	15			92	99	270	287	22		13	15
Test item 3	50	16	18	98	124	205	270	20	13	9	14
Test item 4	150	14	20	81	120	168 T	246	14	9	9	16
Test item 5	500	9	19	71 T	113	150 T	224 T	17	9	8	13
Test item 6	1500	10 T	19		83 T				6		7 T
Test item 7	5000 P	12 T	14 T						7
Sodium azide NaN3	0.7			317				789
2-Nitrofluorene 2-NF	2.5	355
9-Aminoacridine 9-AA	50									333
Mitomycin C	0.15					855
2-aminoanthracene 2-AA	0.8 0.9 1.7		453		735		383		141		197

-MA: absence of metabolic activation

+MA: presence of metabolic activation

P: precipitation of test item

T: bacteriotoxic

solvent control: DMSO, 50 µL/plate

Conclusions:

The test substance was not mutagenic in the bacterial reverse mutation assay.

Executive summary:

The genetic toxicity of the test item was tested according to OECD 471 and Regulation (EC) No. B13/14. The mutagenicity of the test substance was studied with five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98; TA100, and TA102). The investigations were carried using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system. The test substance was dissolved in DMSO and tested in concentrations 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in the presence and absence of S9 mix. The test substance was basteriotoxic towards all tested strain at different concentration levels in the presence and absence of S9 mix. Sodium azide, 2-nitrofluorene, 9-aminoacridine, mitomycin C, and 2-aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system.

In the tested concentration range, the test substance did not induce a significant increase in the mutation frequency of the tester strains with and without metabolic activation. In conclusion, these results indicate that the test substance under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The genetic toxicity of the test item was tested according to OECD 471 and Regulation (EC) No. B13/14. The mutagenicity of the test substance was studied with five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98; TA100, and TA102). The investigations were carried using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system. The test substance was dissolved in DMSO and tested in concentrations 5, 15, 50, 150, 500, 1500 and 5000µg/plate in the presence and absence of S9 mix. The test substance was basteriotoxic towards all tested strain at different concentration levels in the presence and absence of S9 mix.Sodium azide, 2-nitrofluorene, 9-aminoacridine, mitomycin C, and 2-aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system.

In the tested concentration range, the test substance did not induce a significant increase in the mutation frequency of the tester strains with and without metabolic activation. In conclusion, these results indicate that the test substance under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

Justification for classification or non-classification

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

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.