4-(2-butenylidene)-3,5,5-trimethylcyclohex-2-en-1-one

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:

27 May - 01 Jul 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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 Regulation (EC) No 440/2008 of 30 May 2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, U.K.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-(2-butenylidene)-3,5,5-trimethylcyclohex-2-en-1-one

EC Number:

236-187-2

EC Name:

4-(2-butenylidene)-3,5,5-trimethylcyclohex-2-en-1-one

Cas Number:

13215-88-8

Molecular formula:

Not applicable, UVCB substance.

IUPAC Name:

(4E)-4-[(2E)-but-2-en-1-ylidene]-3,5,5-trimethylcyclohex-2-en-1-one

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:

cofactor supplemented post-mitochondrial fraction (S9 mix) prepared from rats treated with phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Pre-experiment: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate with and without metabolic activation in TA 100
Experiment 1: 15, 50, 150, 500, 1500, 5000 µg/plate with and without metabolic activation in all strains
Experiment 2: 5, 15, 50, 150, 500, 1500, 5000 µg/plate with and without metabolic activation in all strains

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: In solubility checks the test material was fully soluble in DMSO at 50 mg/mL.

Controls

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

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: experiment 1: plate incubation method; experiment 2: pre-incubation method for the test groups and vehicle controls, positive and negative controls were dosed using standard plate incubation method

DURATION
- Preincubation period: 20 min (experiment 2)
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: clearing of the bacterial background lawn

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 UKEMS (Kirkland ( 1989) Sub-committee on Guidelines for mutagenicity Testing, Report-Part III, Cambridge University Press) 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.

Acceptance criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
Spontaneous revertants of tester strain cultures for vehicle and negative control should be in the range of historical control data. The appropriate characteristics for each tester strain have been confirmed (eg rfs cell-wall-mutation, pKM101 plasmid R-factor etc.). All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per mL. Each mean positive control value should be at least two times the respective vehicle control for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and integrity of the S9-mix. There should be a minimum of four non-toxic test material dose levels. There should be no evidence of excessive contamination.

Statistics:

Mean value and standard deviations were calculated.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: +/-S9: ≥ 1500 µg/plate; Exp. 2: -S9: ≥ 500 µg/plate, +S9: ≥ 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: -S9: ≥ 1500 µg/plate, +S9: 5000 µg/plate; Exp. 2: -S9: ≥ 500 µg/plate, +S9: ≥ 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: +/-S9: ≥ 1500 µg/plate; Exp. 2: -S9: ≥ 500 µg/plate, +S9: ≥ 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: +/-S9: ≥ 1500 µg/plate; Exp. 2: -S9: ≥ 500 µg/plate, +S9: ≥ 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: +/-S9: 5000 µg/plate; Exp. 2: -S9: ≥ 500 µg/plate, +S9: ≥ 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test substance precipitate was observed on the plates at any concentrations tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES: In order to select appropriate dose levels for use of the maine test, a preliminary test was carried out to determine the toxicity of the test material. Ten doses of the test material and a vehicle control (DMSO) were tested in TA 100 using the plate incubation method. The test material was initially toxic at and above 1500 µg/plate.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment 1 the test substance caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains both in absence and presence of S9-mix, initially from 1500 µg/plate. In Experiment 2 the test material exhibited toxicity as weakened background lawns to all of the bacterial strains, initially from 500 µg/plate and 1500 µg/plate in the absence and presence of S9-mix, respectively.

Any other information on results incl. tables

Table 1. Test results of experiment 1 (plate incorporation method)

EXPERIMENT 1
S9-Mix	Without
Test substance (µg/plate)	TA 100	TA 1535	TA 102	TA 98	TA 1537
SC	77 ± 8.5	11 ± 0.6	237 ± 16.7	23 ± 4.6	15 ± 8.3
15	80 ± 5.8	10 ± 0.6	232 ± 27.2	23 ± 1.7	15 ± 0.6
50	79 ± 10	11 ± 2.6	240 ± 30.1	17 ± 5.9	13 ± 5.5
150	71 ± 2.3	10 ± 1.5	225 ± 11.7	22 ± 8.7	13 ± 3.2
500	89 ± 6.1	14 ± 2.6	229 ± 10.5	18 ± 4.6	9 ± 4.4
1500	74 ± 13.3 S	8 ± 3.6 S	197 ± 12.6	7 ± 2.1 S	5 ± 2.3 S
5000	0 ± 0.0 T	0 ± 0.0 T	30 ± 17.4 S	0 ± 0.0 T	0 ± 0.0 T
ENNG	386 ± 47.9	103 ± 10.0
MMC			933 ± 199.2
4NQO				155 ± 12.1
9AA					1003 ± 210.7
S9-Mix	With
Test substance (µg/plate)	TA 100	TA 1535	TA 102	TA 98	TA 1537
SC	85 ± 0.6	11 ± 0.6	261 ± 23.6	23 ± 9.9	10 ± 1.0
15	75 ± 10.1	10 ± 1.7	283 ± 6.2	26 ± 7.2	15 ± 4.2
50	86 ± 3.2	10 ± 1.2	258 ± 27.6	25 ± 7.1	8 ± 3.6
150	78 ± 6.0	9 ± 1.0	244 ± 31.6	31 ± 1.5	11 ± 2.1
500	77 ± 2.9	8 ± 0.0	249 ± 9.8	28 ± 2.6	14 ± 2.9
1500	53 ± 4.4 S	8 ± 1.7 S	37 ± 12.7 S	21 ± 3.8 S	7 ± 6.4
5000	0 ± 0.0 T	0 ± 0.0 T		0 ± 0.0 T	0 ± 0.0 T
2AA	1994 ± 80.3	92 ± 7.9			148 ± 31.5
DAN			916 ± 34.2
BP				279 ± 36.4
SC = Solvent Control (DMSO) S = Sparse bacterial background lawn T = Toxic, no bacterial background lawn 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 2. Test results of experiment 2 (pre-incubation method)

EXPERIMENT 2
S9-Mix	Without
Test substance (µg/plate)	TA 100	TA 1535	TA 102	TA 98	TA 1537
SC	84 ± 7.4	16 ± 3.1	222 ± 16.7	15 ± 2.9	12 ± 3.8
5	77 ± 9.5	15 ± 1.0	237 ± 4.5	16 ± 4.5	8 ± 1.5
15	70 ± 6.9	16 ± 2.6	230 ± 9.3	18 ± 1.2	8 ± 2.5
50	69 ± 15.6	18 ± 1.5	230 ± 8.4	17 ± 2.5	9 ± 4.5
150	61 ± 4.5	18 ± 1.2	208 ± 7.1	16 ± 2.1	12 ± 3.5
500	0 ± 0.0 V	14 ± 5.2 S	118 ± 8.5 S	0 ± 0.0 V	0 ± 0.0 V
1500	0 ± 0.0 T	0 ± 0.0 T	68 ± 39.2 S	0 ± 0.0 T	0 ± 0.0 T
5000	0 ± 0.0 T	0 ± 0.0 T	0 ± 0.0 T	0 ± 0.0 T	0 ± 0.0 T
ENNG	466 ± 47.0	284 ± 67.3
MMC			699 ± 12.1
4NQO				91 ± 4.7
9AA					1109 ± 144.2
S9-Mix	With
Test substance (µg/plate)	TA 100	TA 1535	TA 102	TA 98	TA 1537
SC	77 ± 10.5	12 ± 1.7	244 ± 14.2	22 ± 6.9	15 ± 4.4
5	73 ± 9.7	8 ± 2.5	255 ± 8.6	19 ± 4.6	11 ± 0.6
15	66 ± 16.5	15 ± 4.6	264 ± 13.3	24 ± 4.6	9 ± 5.2
50	71 ± 3.8	9 ± 2.0	266 ± 4.5	21 ± 4.5	9 ± 2.5
150	76 ± 13.3	13 ± 4.2	281 ± 11.1	27 ± 9.3	8 ± 4.4
500	69 ± 10.5	10 ± 1.0	262 ± 17.6	24 ± 6.4	9 ± 4.5
1500	0 ± 0.0 V	0 ± 0.0 T	199 ± 1.2	0 ± 0.0 V	4 ± 1.5 V
5000	0 ± 0.0 T	0 ± 0.0 T	0 ± 0.0 V	0 ± 0.0 T	0 ± 0.0 T
2AA	2559 ± 78.3	286 ± 38.7			246 ± 16.4
DAN			1532 ± 138.5
BP				680 ± 9.8
SC = Solvent Control (DMSO) S = Sparse bacterial background lawn T = Toxic, no bacterial background lawn V = Very weak bacterial background lawn 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 test 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 up to the limit dose of 5000 µg/plate.

Executive summary:

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2009). In this study the 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 up to 5000 µg/plate.