2-Cyclohexen-1-one, 2-methyl-5-propyl-

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:

2013-12-09 to 2014-01-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997-07-21

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2013-04-11

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-methyl-5-propylcyclohex-2-en-1-one

EC Number:

807-489-7

Cas Number:

1447712-18-6

Molecular formula:

C10H16O1

IUPAC Name:

2-methyl-5-propylcyclohex-2-en-1-one

Test material form:

other: liquid

Details on test material:

- Physical state: liquid, clear pale yellow
- Storage condition of test material: at room temperature

Method

Target gene:

TA1537: his C 3076
TA98: his D 3052
TA1535 & TA100: his G 46
E. coli WP2 uvrA: trp-

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:

Phenobarbital/β-naphthoflavone induced rat liver S9 were used as the metabolic activation system. Protein concentration of the S9 prepartion was 37.8 mg/mL.

Test concentrations with justification for top dose:

Pre-experiment/Experiment 1 (with and without S9 mix): 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate
Experiment II (without S9 mix): 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate
Experiment II (with S9 mix): 1, 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) 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

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Positive control without metabolic activation: dissolved in deionised water; concentration: 10 μg/plate (strains TA 1535, TA 100)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

Positive control without metabolic activation: dissolved in DMSO; concentration: 10 μg/plate (strain TA 98); 50 μg/plate (strain TA 1537)

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Positive control without metabolic activation: dissolved in deionised water; concentration: 2 μL/plate (strain WP2 uvrA)

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Positive control with metabolic activation: dissolved in DMSO; concentration: 2.5 μg/plate (strains TA 1535, TA 1537, TA 98, TA 100); 10.0 μg/plate in WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation; pre-experiment/Experiment 1) and preincubation (Experiment 2)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: at least 48 hours at 37°C

NUMBER OF REPLICATIONS: for each strain and dose level including the controls, three plates were used.

The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager (v.1.21). Due to reduced background growth, the colonies were partly counted manually.

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. Whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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 in the presence of metabolic activation. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 μg/plate in the presence of metabolic activation in experiment I. The undissolved particles had no influence on the data recording.

CYTOTOXICITY:
- reduced background growth was observed at higher concentrations in all experimental parts with and without metabolic activation (please refer to the table 1 in the field "Any other information on results incl. tables" below).
- toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred at higher concentrations in all experimental parts with and without metabolic activation (please refer to the table 2 in the field "Any other information on results incl. tables" below).


MAIN EXPERIMENTS:
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

Please also refer to table 3 and 4 in the field "Any other information on results incl. tables" below.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Reduced background growth was observed at the following concentration (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	2500, 5000	2500, 5000	333, 5000	1000 - 5000
TA 1537	1000 - 5000	2500, 5000	333 - 5000	1000 - 5000
TA 98	2500, 5000	2500, 5000	333 - 5000	1000 - 5000
TA 100	2500, 5000	1000, 5000	333 - 5000	1000 - 5000
WP2 uvrA	5000	2500, 5000	1000 - 5000	2500 - 5000

Table 2: Toxic effects (µg/plate)

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	2500, 5000	2500, 5000	2500, 5000	1000 - 5000
TA 1537	1000 - 5000	2500, 5000	2500, 5000	1000 - 5000
TA 98	2500, 5000	2500, 5000	2500, 5000	1000 - 5000
TA 100	2500, 5000	2500, 5000	1000 - 5000	1000 - 5000
WP2 uvrA	5000	2500, 5000	5000	5000

Table 3: Summary of experiment I

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		21 ± 1	10 ± 5	24 ± 2	88 ± 5	48 ± 5
	Untreated		15 ± 6	14 ± 3	26 ± 5	108 ± 15	44 ± 7
	PI 27137	3 µg	15 ± 3	9 ± 4	24 ± 2	97 ± 13	43 ± 1
		10 µg	15 ± 3	9 ± 3	26 ± 3	99 ± 10	46 ± 8
		33 µg	18 ± 6	9 ± 4	23 ± 3	89 ± 11	44 ± 9
		100 µg	23 ± 4	8 ± 2	22 ± 2	94 ± 6	46 ± 5
		333 µg	18 ± 5	10 ± 2	25 ± 3	71 ± 7	41 ± 10
		1000 µg	23 ± 1	3 ± 1^MR	12 ± 4	62 ± 4	24 ± 3
		2500 µg	8 ± 2^MR	2 ± 1^MR	5 ± 1^MR	9 ± 2^MR	22 ± 4
		5000 µg	1 ± 1^MR	1 ± 1^MR	1 ± 1^MR	1 ± 1^MR	14 ± 4^MR
	NaN3	10 µg	2506 ± 413			2065 ± 15
	4-NOPD	10 µg			271 ± 12
	4-NOPD	50 µg		69 ± 9
	MMS	2.0 µL					1157 ± 102
With activation	DMSO		15 ± 7	17 ± 4	31 ± 3	102 ± 4	50 ± 3
	Untreated		21 ± 6	21 ± 9	38 ± 6	113 ± 2	47 ± 6
	PI 27137	3 µg	14 ± 2	17 ± 3	38 ± 12	96 ± 11	44 ± 7
		10 µg	16 ± 4	16 ± 4	30 ± 4	94 ± 2	49 ± 9
		33 µg	14 ± 2	13 ± 3	25 ± 3	95 ± 3	48 ± 4
		100 µg	14 ± 5	12 ± 5	26 ± 3	105 ± 1	45 ±4
		333 µg	11 ± 3	16 ± 8	36 ± 10	93 ± 2	45 ± 7
		1000 µg	15 ± 6	14 ± 4	29 ± 7	63 ± 11^R	26 ± 5
		2500 µg	1 ± 1^MR	1 ± 1^R	3 ± 1^MR	0 ± 0^MR	11 ± 3^MR
		5000 µg	0 ± 1^MRP	0 ± 0^MRP	0 ± 0^PMR	0 ± 0^PMR	0 ± 0^PMR
	2-AA	2.5 µg	407 ± 35	369 ± 3	2325 ± 578	3608 ± 319
	2-AA	10.0 µg					287 ±58

Key to positive Controls          

NaN3: sodium azide

2-AA: 2-aminoanthracene

4-NOPD: 4-nitro-o-phylene-diamine

MMS: methyl methane sulfonate

 

Key to Plate Postfix Codes

M: Manual count

R: Reduced background growth

P: Precipitate

Table 4: Summary of experiment II

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		10 ± 2	9 ± 1	21 ± 4	88 ± 12	43 ± 2
	Untreated		11 ± 3	10 ± 4	23 ± 5	115 ± 16	42 ± 5
	PI 27137	3 µg	10 ± 4	9 ± 1	33 ± 7	101 ± 12	51 ± 3
		10 µg	13 ± 7	10 ± 2	25 ± 4	90 ± 6	49 ± 6
		33 µg	12 ± 2	8 ± 2	16 ± 3	95 ± 18	51 ± 12
		100 µg	11 ± 1	13 ± 3	23 ± 3	107 ± 12	43 ± 5
		333 µg	7 ± 1^MR	3 ± 1^MR	13 ± 3^MR	63 ± 13^R	29 ± 6
		1000 µg	6 ± 1^MR	13 ± 2^RM	23 ± 6^MR	21 ± 2^MR	24 ± 4^R
		2500 µg	4 ± 1^MR	0 ± 0^MR	4 ± 1^MR	0 ± 0^MR	23 ± 7^R
		5000 µg	2 ± 1^MR	0 ± 0^MR	0 ± 0^MR	0 ± 0^MR	5 ± 1^MR
	NaN3	10 µg	2968 ± 19			2435 ± 30
	4-NOPD	10 µg			306 ± 20
	4-NOPD	50 µg		59 ± 2
	MMS	2.0 µL					669 ± 40
With activation	DMSO		14 ± 3	21 ± 6	45 ± 4	109 ± 16	46 ± 5
	Untreated		12 ± 5	18 ± 3	59 ± 10	124 ± 6	56 ± 12
	PI 27137		6 ± 1	18 ± 4	35 ± 1	123 ± 14	70 ± 13
		3 µg	11 ± 5	23 ± 4	51 ± 4	101 ± 21	55 ± 12
		10 µg	10 ± 4	15 ± 1	48 ± 7	123 ± 4	58 ± 12
		33 µg	9 ± 4	16 ± 3	35 ± 7	115 ± 8	56 ± 9
		100 µg	14 ± 6	14 ± 1	26 ± 3	116 ± 15	39 ± 9
		333 µg	14 ± 5	22 ± 6	48 ± 3	83 ± 20	41 ± 5
		1000 µg	0 ± 0^MR	3 ± 1^MR	4 ± 1^MR	25 ± 2^MR	31 ± 2
		2500 µg	0 ± 0^R	0 ± 0^MR	0 ± 0^MR	0 ± 0^MR	23 ± 4^R
		5000 µg	0 ± 0^R	0 ± 0^MR	0 ± 0^MR	0 ± 0^MR	0 ± 0^R
	2-AA	2.5 µg	436 ± 34	342 ± 14	3217 ± 173	3801 ± 192
	2-AA	10.0 µg					265 ± 17

Key to positive Controls          

NaN3: sodium azide

2-AA: 2-aminoanthracene

4-NOPD: 4-nitro-o-phylene-diamine

MMS: methyl methane sulfonate

 

Key to Plate Postfix Codes

M: Manual count

R: Reduced background growth

For further results please also refer to the field "Attached background material" below.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
According to Directive 67/548 /EEC and its subsequent amendments and according to Regulation (EC) No 1272/2008 and subsequent regulations, the test substance should not be considered to have a mutagenic potential, and hence no classification or labelling is required.

Executive summary:

A bacterial reverse mutation assay was performed with the test item according to the OECD guideline 471 (1997) using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 as well as E. coli WP2 uvr A. The test was carried out using the plate incorporation method and the preincubation method with plating in triplicate with and without metabolic activation. The test item was tested at the following concentrations in both experiments: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate. Additionally, a negative control and positive controls were run concurrently.

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate in the presence of metabolic activation. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 μg/plate in the presence of metabolic activation in the plate incorporation test. The undissolved particles had no influence on the data recording.

Reduced background growth was observed at higher concentrations in all experimental parts with and without metabolic activation.

Toxic effects, evident as a reduction of the number of revertants (below an induction factor of 0.5), occurred at higher concentrations in all experimental parts with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

In conclusion, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.