(E)-1-(2,4,4-trimethyl-2-cyclohexen-1-yl)-2-buten-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:

25 April - 21 Aug 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted: 21 Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(E)-1-(2,4,4-trimethyl-2-cyclohexen-1-yl)-2-buten-1-one

EC Number:

254-663-8

EC Name:

(E)-1-(2,4,4-trimethyl-2-cyclohexen-1-yl)-2-buten-1-one

Cas Number:

39872-57-6

Molecular formula:

C13H20O

IUPAC Name:

(E)-1-(2,4,4-trimethyl-2-cyclohexen-1-yl)-2-buten-1-one

Method

Target gene:

his operon (for S. typhimurium strains)
trp operon (for E. coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment Ib (TA98): 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment 2: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Solvent/vehicle used: DMSO
- Justification for choice of solvent/ vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (Experiment 1 and 1b); preincubation (Experiment 2)

DURATION
- Preincubation period: 1 h
- Exposure duration: at least 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 (3 for TA 98) independent experiments

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

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

Mean values and standard deviations were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see "remarks on result"

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate with S9 mix in experiment 1 and from 1000 to 5000 μg/plate with S9 mix in experiment 2 in all strains used. In experiment 1b no precipitation in the overlay agar in the test tubes was observed. No precipitation of the test item occurred in the overlay agar on the incubated agar plates in all experiments.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as Experiment I (all strains tested). In strain TA98 in the pre-experiment no colonies were observed at 333 μg/plate and above and the decrease was steep and unexpected. Therefore, this part of the experiment was repeated (Exp 1b). Based on the outcome of experiment 1b the results of strain TA 98 without S9 mix in the pre-experiment 1 were judged to be invalid due to a technical error. Therefore this part of the experiment is not reported.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects, indicated by a reduced bacterial background lawn or decreased number of revertants (induction factor below 0.5), were observed in all strains used except srains TA 1535 and and WP2 uvrA in experiment I and II with and without S9 mix and in strain TA1537 in experiment I with and without metabolic activation. In Experiment 1 and 1b the strains TA1537, TA98 and TA100 showed reduced background growth. In Experiment 2 additionally strain TA1535 showed these quantitative signs of toxicity.

Remarks on result:

other: Cytotoxicity:

Remarks:

Exp 1: at 333 µg/plate and above for TA 1537 and TA 100 with and without metabolic activation, at 1000 µg/plate and above for TA 98 with metabolic activation; Exp 1b: at 1000 µg/plate for TA 98 without metabolic activation; Exp 2: at 333 µg/plate and above for TA 1537, TA 98 and TA 100 with and without metabolic activation, at 1000 µg/plate and above for TA 1535 without metabolic activation

Any other information on results incl. tables

Table 2. Results experiment 1

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ± SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		12 ± 3	11 ± 4	1)	184 ± 13	43 ± 5
	Untreated		9 ± 3	9 ± 5		200 ± 16	39 ± 7
	Test substance	3 µg	10 ± 5	14 ± 6		201 ± 30	37 ± 10
		10 µg	9 ± 3	9 ± 3		200 ± 8	38 ± 6
		33 µg	11 ± 3	10 ± 3		198 ± 8	37 ± 5
		100 µg	8 ± 2	8 ± 2		148 ± 11	40 ± 6
		333 µg	8 ± 3	14 ± 2R		38 ± 3R	44 ± 6
		1000 µg	11 ± 1	13 ± 2R		29 ± 5M R	36 ± 1
		2500 µg	10 ± 1	18 ± 6R		22 ± 8M R	48 ± 8
		5000 µg	9 ± 2	22 ± 6R		11 ± 1R M	38 ± 8
	NaN3	10 µg	1365 ± 40			2341 ± 189
	4-NOPD	10 µg
	4-NOPD	50 µg		70 ± 16
	MMS	2.0 µL					972 ± 44
With Activation	DMSO		11 ± 2	13 ± 5	32 ± 5	166 ± 11	47 ± 6
	Untreated		11 ± 4	16 ± 6	44 ± 7	181 ± 17	58 ± 2
	Test substance	3 µg	12 ± 0	13 ± 1	35 ± 14	152 ± 23	50 ± 8
		10 µg	9 ± 2	12 ± 5	37 ± 10	161 ± 16	54 ± 3
		33 µg	15 ± 1	13 ± 2	28 ± 3	163 ± 10	64 ± 5
		100 µg	10 ± 4	11 ± 2	33 ± 6	173 ± 17	61 ± 12
		333 µg	11 ± 2	11 ± 2R	26 ± 3	52 ± 10R	45 ± 3
		1000 µg	11 ± 2	19 ± 4R	26 ± 5R	31 ± 5R	44 ± 6
		2500 µg	9 ± 3	19 ± 4R	13 ± 2M R	30 ± 6M R	37 ± 3
		5000 µg	8 ± 1	22 ± 3R	9 ± 4M R	21 ± 6M R	41 ± 5
	2-AA	2.5 µg	491 ± 21	204 ± 47	4024 ± 726	4041 ± 138
	2-AA	10.0 µg					342 ± 13

1) experiment not valid; no results reported

NaN3: sodium azide

2AA: 2-Aminoanthracene

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

R: reduced background growth

M: manual count

Table 3: Results experiment 1b

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean  ±SD)
			TA 98
Without Activation	DMSO		24 ± 1
	Untreated		19 ± 3
	Test substance	3 µg	27 ± 2
		10 µg	20 ± 3
		33 µg	25 ± 7
		100 µg	20 ± 5
		333 µg	22 ± 6
		1000 µg	18 ± 4R
		2500 µg	19 ± 2M R
		5000 µg	21 ± 6M R
	4-NOPD	10 µg	487 ± 16

4-NOPD: 4-nitro-o-phenylene-diamine

R: reduced background growth

M: manual count

Table 4. Results experiment 2

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		12 ± 2	10 ± 1	22 ± 7	139 ± 9	40 ± 7
	Untreated		13 ± 3	8 ± 3	29 ± 7	161 ± 10	47 ± 5
	Test substance	3 µg			25 ± 3	131 ± 13
		10 µg			25 ± 3	133 ± 3
		33 µg	12 ± 3	7 ± 3	22 ± 6	134 ± 18	40 ± 3
		100 µg	8 ± 2	9 ± 2	15 ± 1	63 ± 17	38 ± 10
		333 µg	10 ± 3	8 ± 1R	21 ± 8R	31 ± 9R	37 ± 4
		1000 µg	9 ± 3R	3 ± 2R M	11 ± 1M R	14 ± 3M R	27 ± 5
		2000 µg		4 ± 1M R
		2500 µg	10 ± 5R		6 ± 2M R	6 ± 1M R	40 ± 3
		3000 µg		2 ± 1M R
		4000 µg		0 ± 1M R
		5000 µg	11 ± 5R	0 ± 0M R	4 ± 2M R	0 ± 1M R	38 ± 6
	NaN3	10 µg	1334 ± 26			2333 ± 91
	4-NOPD	10 µg			301 ± 18
	4-NOPD	50 µg		99 ± 27
	MMS	2.0 µL					603 ± 35
With Activation	DMSO		13 ± 2	10 ± 2	38 ± 4	160 ± 12	53 ± 5
	Untreated		12 ± 3	10 ± 3	41 ± 5	174 ± 26	56 ± 3
	Test substance	3 µg			32 ± 4	148 ± 3
		10 µg			43 ± 3	161 ± 9
		33 µg	12 ± 2	11 ± 4	33 ± 4	156 ± 6	48 ± 8
		100 µg	10 ± 2	10 ± 4	39 ± 2	106 ± 16	57 ± 7
		333 µg	11 ± 3	12 ± 5R	33 ± 8R	35 ± 5R	57 ± 15
		1000 µg	9 ± 3	10 ± 1M R	10 ± 2M R	10 ± 2M R	49 ± 6
		2000 µg		10 ± 3M R
		2500 µg	8 ± 1		6 ± 2M R	12 ± 1M R	46 ± 5
		3000 µg		9 ± 2M R
		4000 µg		7 ± 3M R
		5000 µg	10 ± 6	5 ± 1M R	2 ± 0M R	2 ± 1M R	45 ± 4
	2-AA	2.5 µg	403 ± 86	141 ± 28	4973 ± 717	3765 ± 212
	2-AA	10.0 µg					518 ± 29

NaN3: sodium azide

2AA: 2-Aminoanthracene

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

R: reduced background growth

M: manual count

Applicant's summary and conclusion

Conclusions:

Under the conditions of the conducted test the substance was not mutagenic in any of the five bacterial strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation.