Ethyl 2,6,6-trimethylcyclohexa-1,3-ene-1-carboxylate

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:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

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

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 2,6,6-trimethylcyclohexa-1,3-ene-1-carboxylate

EC Number:

252-335-9

EC Name:

Ethyl 2,6,6-trimethylcyclohexa-1,3-ene-1-carboxylate

Cas Number:

35044-59-8

Molecular formula:

C12H18O2

IUPAC Name:

ethyl 2,6,6-trimethylcyclohexa-1,3-diene-1-carboxylate

Details on test material:

- Physical state: Liquid
- Storage condition of test material: At room temperature protected from light
- Other: Colourless liquid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain 2

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: 154, 275, 492, 878, 1568, 2800, 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 and ethanol. Dimethyl sulfoxide is was used in the assay.

Controls

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.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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:

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

Remarks:

In strains TA98, TA100, TA1535 and TA1357, cytotoxicity was seen at the 2800 µg/plate (without S9-mix) or above; and/or at 5000 µg/plate limit dose level (with S9-mix).

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 at concentrations of 1600 and 5000 µg/plate and at 5000 µg/plate at the end of the incubation period. Except for the tester strains TA1535 and TA1537 (absence of S9-mix, end of the incubation period), where also precipitate was observed at 1600 µg/plate. The precipitation at the start of the incubation period was observed as oily droplets of the test substance.

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.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and strain-specific positive control values were within the laboratory historical control data ranges 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'.

Any other information on results incl. tables

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	788	± 57	1335	± 34
Solvent control	110	± 10	25	± 1
1.7	102	± 17	23	± 7
5.4	90	± 3	30	± 9
17	106	± 10	27	± 4
52	108	± 18	22	± 2
164	94	± 6	36	± 9
512	101	± 18	35	± 9
1600	83	± 11	23	± 7 NP
5000	64	± 11 m SP	23	± 7 n SP
With S9-mix #1
Positive control	1148	± 149	196	± 18
Solvent control	99	± 5	26	± 3
1.7	114	± 2	28	± 5
5.4	105	± 15	31	± 7
17	99	± 27	35	± 9
52	100	± 17	26	± 9
164	91	± 3	33	± 9
512	94	± 3 n	31	± 6
1600	90	± 1 s NP	33	± 15
5000	68	± 18 m SP	24	± 5 n SP

#1: Plate incorporation assay (5% S9)

NP: No precipitate

SP: Slight Precipitate

n: Normal bacterial background lawn

s: Slightly reduced background lawn

m: Moderately reduced background lawn

 

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	909	± 73	687	± 40	1237	± 114
Solvent control	15	± 4	9	± 4	26	± 2
52	14	± 6	8	± 4	15	± 5
164	16	± 3	6	± 3	16	± 4
512	13	± 3	3	± 2	15	± 4
1600	17	± 5	7	± 3	17	± 6
5000	20	± 2 n 2 NP	7	± 3 n 2 NP	29	± 6 n 2 NP
With S9-mix #1
Positive control	288	± 10	622	± 104	1335	± 129
Solvent control	16	± 7	8	± 0	30	± 7
52	18	± 6	10	± 3	22	± 2
164	15	± 4	8	± 4	23	± 5
512	17	± 5	6	± 2	19	± 5
1600	18	± 4	5	± 2	21	± 5
5000	23	± 1 n 2 NP	12	± 7 n 2 NP	23	± 10 n 2 NP

#1: Plate incorporation assay (5% S9)

MC: Microcolonies

NP: No Precipitate

SP: Slight Precipitate

n: Normal bacterial background lawn

s: Slightly reduced background lawn

m: Moderately reduced background lawn

e: Extremely reduced background lawn

 

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	870	± 26	482	± 42	1008	± 20	852	± 35	1050	± 51
Solvent control	18	± 2	8	± 3	14	± 2	91	± 8	36	± 4
492	16	± 6	5	± 2	23	± 6	79	± 13	30	± 5
878	21	± 5	7	± 0	17	± 11	98	± 5	29	± 5
1568	24	± 7	8	± 4	19	± 6	88	± 11	34	± 5
2800	17	± 3	10	± 10	22	± 7	90	± 12	30	± 2
5000	19	± 4 n 2 NP	7	± 5 n 2 NP	19	± 6 n 2 NP	96	± 6 n 2 NP	32	± 7 n 2 NP
With S9-mix #1
Positive control	262	± 32	526	± 33	653	± 60	1670	± 165	313	± 17
Solvent control	25	± 1	11	± 3	35	± 9	99	± 13	39	± 9
492	24	± 5	6	± 2	29	± 5	39	± 6	86	± 9
878	23	± 3	9	± 4	34	± 11	30	± 4	73	± 5
1568	22	± 10	8	± 4	29	± 4	80	± 11	37	± 5
2800	27	± 10	9	± 8	32	± 9	74	± 6	27	± 9
5000	21	± 6 n 2 NP	7	± 2 n 2 NP	28	± 5 n 2 NP	79	± 24 n 2 NP	46	± 11 n 2 NP

#1: Plate incorporation assay (10% S9)

#2: Oily droplets of the test substance were observed

NP: No precipitate

n: Normal bacterial background lawn

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

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with the Escherichia coli strain WP2uvrA
With S9-mix #1
Positive control	402	± 17
Solvent control	37	± 14
492	22	± 4
878	27	± 5
1560	26	± 8
2800	17	± 7
5000	21	± 6 n 2 NP

#1: Plate incorporation assay (10% S9)

#2: Oily droplets of the test substance were observed

NP: No precipitate

n: Normal bacterial background lawn

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study the test substance is not considered to be mutagenic. Negative and strain specific positive control values were within laboratory historical control data.

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 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 154 to 5000 µg/plate in tester strain TA1535, at 492 to 5000 µg/plate in tester strains TA1537, TA100 and WP2uvrA and at 275 to 5000 µg/plate in strain TA98 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 tester strains TA1535, TA1537 and TA100 in the absence and presence of S9-mix. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges 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.