Methyl 2,6,6-trimethylcyclohex-2-ene-1-carboxylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2015

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:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP.

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Guideline stipulated by the Japanese Ministry of Health, Labour and Welfare, Ministry of Economy, Trade and Industry and Ministry of the Environment (revised March 31st, 2011)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected March 2013; signature: May 2013

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

Dose range finding study: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 ug/plate
Experiment 1: 52, 164, 512, 1600, 5000 ug/plate
Experiment 2: 275, 492, 878, 1568, 2800, 5000 ug/plate
Experiment 3 (strains TA1537, TA100): 17, 52, 164, 512, 1600, 5000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: A solubility test was performed. The test substance could not be dissolved in water. The test substance was soluble in dimethyl sulfoxide. Dimethyl sulfoxide was used in the assay.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: The plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli were counted. The revertant colonies were counted automatically with a Colony Counter.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth (bacterial background lawn) and reduction in the number of revertants

OTHER:
Dose range finding test on TA100 and WP2urvA with and without 5% (v/v) S9-mix; First mutation assay on TA1535, TA1537 and TA98 with and without 5% (v/v) S9-mix. To obtain more information about the possible mutagenicity of the test substance, a second mutation experiment was performed on all strains, in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the test substance was tested up to the dose level of 5000 μg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Evaluation criteria:

See 'Any other information on materials and methods' for details on evaluation of the assay and positive criteria.

Statistics:

No formal hypothesis testing was done. See 'Any other information on materials and methods' for details on the acceptability and evaluation criteria of the assay.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In strains TA100, TA1535 and TA1357, cytotoxicity was seen at the 1600 µg/plate or above (with or without S9-mix); TA98 strain at 2800 µg/plate (with S9-mix) or was tested to the limit dose.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of test substance on the plates was observed at the start of the incubation period and at 5000 µg/plate at the end of the incubation period. Except for the tester strains TA1535 (presence of S9-mix, end of the incubation period). The precipitation at the start of the incubation period was observed as oily droplets of the test substance. In experiment 3 on strains TA1537 and TA100 only: Precipitation of the test substance on the plates was observed at the start of the incubation period at concentrations of 1600 and 5000 μg/plate. At the end of the incubation period precipitate was only observed in tester strain TA100 at the top dose of 5000 μg/plate.

RANGE-FINDING/SCREENING STUDIES:
Test substance was tested in the tester strains TA100 and WP2uvrA with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate. To obtain more information on TA1537 and TA100 a third mutagenicity experiment was conducted at a concentration range of 17 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix. This was based on the results of the preceding tests.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative control values were within the laboratory historical control data ranges. The positive control values were not all within the laboratory historical control data ranges, specifically TA98 (first experiment, absence of S9-mix) and TA1537 (second experiment, absence of S9-mix). However, these responses were judged not to impact the reliability of the study. The strain-specific positive control values were at least three times the concurrent vehicle control group mean indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Historical control data from experiments was presented within the study report.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1 Dose range finding test: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with one strain of Salmonella typhimurium and one Escherichia coli strain
	TA100		WP2uvrA
Without S9-mix
Positive control	632	± 10	1450	± 32
Solvent control	104	± 15	32	± 6
1.7	116	± 14	35	± 9
5.4	112	± 5	36	± 12
17	118	± 13	34	± 5
52	118	± 15	39	± 6
164	103	± 4	38	± 10
512	110	± 14 n	28	± 4
1600	80	± 12 s NP	24	± 5 NP
5000	78	± 21 s SP	23	± 5 n SP
With S9-mix #1
Positive control	1553	± 167	196	± 18
Solvent control	112	± 5	34	± 13
1.7	115	± 14	38	± 4
5.4	103	± 11	39	± 11
17	109	± 14	43	± 7
52	101	± 13	42	± 15
164	92	± 7	39	± 4
512	101	± 7 n	37	± 3
1600	85	± 6 s NP	33	± 8 NP
5000	57	± 2 m SP	28	± 9 n SP

#1: Plate incorporation assay (5% S9)

NP: No precipitate

SP: Slight Precipitate

m: Bacterial background lawn moderately reduced

n: Normal bacterial background lawn

s: Bacterial background lawn slightly reduced

 

Table 2 Experiment 1: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium
	TA1535		TA1537		TA98
Without S9-mix
Positive control	857	± 44	586	± 27	1379	± 128
Solvent control	17	± 2	6	± 2	19	± 8
52	21	± 2	15	± 8	17	± 6
164	27	± 9	9	± 1	16	± 3
512	23	± 7	7	± 1	16	± 2 n
1600	15	± 7 n NP	10	± 7 n NP	15	± 6 s NP
5000	-	e SP MC	10	± 8 m SP	16	± 6 m SP
With S9-mix #1
Positive control	273	± 19	424	± 41	1161	± 58
Solvent control	22	± 14	6	± 2	27	± 11
52	45	± 16	10	± 4	37	± 2
164	36	± 6	12	± 6	31	± 5
512	31	± 13 n	11	± 4	29	± 8
1600	32	± 8 m	7	± 4 n NP	19	± 6 n NP
5000		e NP MC	5	± 2 m SP	18	± 2 s SP

#1: Plate incorporation assay (5% S9)

MC: Microcolonies

NP: No precipitate

SP: Slight Precipitate

e: Bacterial background lawn extremely reduced

m: Bacterial background lawn moderately reduced

n: Normal bacterial background lawn

s: Bacterial background lawn slightly reduced

 

Table 3 Experiment 2: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA1535		TA1537		TA98		TA100		WP2uvrA
Without S9-mix
Positive control	939	± 74	1565	± 137	793	± 268	800	± 42	1242	± 62
Solvent control	16	± 3	7	± 6	27	± 2	99	± 7	28	± 10
275	13	± 5	-	-	-	-	-	-	-	-
492	10	± 1	3	± 2 n	20	± 3	73	± 13 s	21	± 2
878	7	± 2	3	± 2 s	16	± 10	51	± 14 m	23	± 2
1568	5	± 1 n	3	± 3 m	11	± 1 n	47	± 12 m	20	± 6
2800	7	± 2 s NP		e NP MC	15	± 1 s NP	52	± 6 m NP	25	± 10 NP
5000	5	± 2 m SP		e NP MC	9	± 1 s SP		e SP MC	27	± 4 n SP
With S9-mix #1
Positive control	295	± 30	342	± 40	406	± 54	1279	± 105	175	± 23
Solvent control	12	± 3	10	± 2	32	± 2	110	± 18	31	± 4
275	14	± 5	-	-	-	-	-	-	-	-
492	11	± 1	12	± 7 n	30	± 6	91	± 19 n	28	± 3
878	9	± 4 n	11	± 4 s	23	± 3 n	73	± 9 s	30	± 7
1568	6	± 4 m	3	± 2 m	17	± 3 s	60	± 6 m	31	± 13
2800		e MC	2	± 1 m NP	15	± 6 m NP	60	± 4 m NP	29	± 3 NP
5000		e SP MC		e NP MC	11	± 1 m SP		e SP MC	26	± 4 n SP

#1: Plate incorporation assay (10% S9)

MC: Microcolonies

MP: Moderate Precipitate

NP: No precipitate

SP: Slight Precipitate

e: Bacterial background lawn extremely reduced

m: Bacterial background lawn moderately reduced

n: Normal bacterial background lawn

s: Bacterial background lawn slightly reduced

- : Not tested

Table 4 Experiment 3: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA1537		TA100
Without S9-mix
Positive control	1070	± 57	848	± 61
Solvent control	9	± 3	91	± 6
17	11	± 4	106	± 12
52	10	± 6	105	± 12
164	13	± 4	95	± 8
512	9	± 4	87	± 7 n
1600	4	± 1 n		e NP MC
5000		e SP MC		e SP MC
With S9-mix #1
Positive control	368	± 17	1287	± 52
Solvent control	11	± 4	107	± 8
17	15	± 8	102	± 12
52	14	± 3	98	± 6 #2
164	10	± 3	91	± 22
512	11	± 1	81	± 11 n
1600	6	± 4 n	85	± 14 s NP
5000		e NP MC		e SP MC

#1: Plate incorporation assay (10% S9)

#2: Mean of two plates, one plate could not be determined

MC: Microcolonies

NP: No precipitate

SP: Slight Precipitate

a: Bacterial background lawn absent

e: Bacterial background lawn extremely reduced

m: Bacterial background lawn moderately reduced

n: Normal bacterial background lawn

s: Bacterial background lawn slightly reduced

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study the test substance is not considered to be mutagenic. Negative control values were within laboratory historical control data. The strain-specific positive control values were at least three times the concurrent vehicle control group mean indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Executive summary:

The study was performed to OECD TG 471, EU Method B.13/14, EPA OPPTS 870.5100 and the Japan Guidelines for Screening Mutagenicity of Chemicals in accordance with GLP; to evaluate the mutagenic activity of the test substance in the Salmonella typhimurium and the Escherichia coli in a reverse mutation assay (with independent repeat). In the dose range finding test, the test substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test substance precipitated on the plates at the top dose of 5000 μg/plate. In tester strain TA100, toxicity was observed at dose levels of 1600 and 5000 μg/plate in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose range finding test were reported as part of the first mutation assay. Based on the results of the dose range finding test, the test substance was tested in the first mutation assay at a concentration range of 52 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test substance precipitated on the plates at the top dose of 5000 μg/plate. The test substance was tested up to or beyond a precipitating dose level. Toxicity was observed in all three tester strains in the absence and presence of S9-mix. In a follow-up experiment of the assay with additional parameters, the test substance was tested at a concentration range of 275 to 5000 µg/plate in tester strain TA1535 and at a concentration range of 492 to 5000 µg/plate in tester strains TA1537, TA98, TA100 and WP2uvrA in the absence and presence of 10% (v/v) S9-mix. The test substance precipitated on the plates at the top dose of 5000 μg/plate. Toxicity was observed in all tester strains in the absence and presence of S9-mix. Except tester strain WP2uvrA, where no toxicity is observed. The negative control values were within the laboratory historical control data ranges. The positive control values were not all within the laboratory historical control data ranges, specifically TA98 (first experiment, absence of S9-mix) and TA1537 (second experiment, absence of S9-mix). However, these responses were judged not to impact the reliability of the study. The strain-specific positive control values were at least three times the concurrent vehicle control group mean indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the conditions of this study, it is concluded that that the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

OECD TG 471, 2015 - The study was performed to OECD TG 471, EU Method B.13/14, EPA OPPTS 870.5100 and the Japan Guidelines for Screening Mutagenicity of Chemicals in accordance with GLP; to evaluate the mutagenic activity of the test substance in the Salmonella typhimurium and the Escherichia coli in a reverse mutation assay (with independent repeat). In the dose range finding test, the test substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test substance precipitated on the plates at the top dose of 5000 μg/plate. In tester strain TA100, toxicity was observed at dose levels of 1600 and 5000 μg/plate in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose range finding test were reported as part of the first mutation assay. Based on the results of the dose range finding test, the test substance was tested in the first mutation assay at a concentration range of 52 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test substance precipitated on the plates at the top dose of 5000 μg/plate. The test substance was tested up to or beyond a precipitating dose level. Toxicity was observed in all three tester strains in the absence and presence of S9-mix. In a follow-up experiment of the assay with additional parameters, the test substance was tested at a concentration range of 275 to 5000 µg/plate in tester strain TA1535 and at a concentration range of 492 to 5000 µg/plate in tester strains TA1537, TA98, TA100 and WP2uvrA in the absence and presence of 10% (v/v) S9-mix. The test substance precipitated on the plates at the top dose of 5000 μg/plate. Toxicity was observed in all tester strains in the absence and presence of S9-mix. Except tester strain WP2uvrA, where no toxicity is observed. The negative control values were within the laboratory historical control data ranges. The positive control values were not all within the laboratory historical control data ranges, specifically TA98 (first experiment, absence of S9-mix) and TA1537 (second experiment, absence of S9-mix). However, these responses were judged not to impact the reliability of the study. The strain-specific positive control values were at least three times the concurrent vehicle control group mean indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the conditions of this study, it is concluded that that the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity