4-cyclohexyl-4-methylpentan-2-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is not mutagenic in bacteria.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11.04.2016 - 19.05.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation 440/2008/EC, May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa- and uvrB-

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

- Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
- Experiment II:
-- Strain TA 1535: 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate
-- Strains TA 1537, TA 98 and TA 100: 1; 3; 10; 33; 100; 333; 1000 and 2500 μg/plate
-- Strain WP2 uvrA: 33; 100; 333; 1000; 2500 and 5000 μg/plate

- Justification: In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate and based on toxic effects observed the concentrations were adapted for experiment II. At least six concentrations were tested in experiment II with 5000 μg/plate as maximal concentration for strains TA 1535 and WP2 uvrA, and 2500 μg/plate for strains TA 1537, TA 98 and TA 100. The concentration range included two logarithmic decades.

Vehicle / solvent:

- Vehicle: DMSO
- Justification: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD; methyl methane sulfonate, MMS

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (Experiment I); preincubation (Experiment II)

DURATION
- Preincubation period: 1 h
- Exposure duration: at lease 48 h at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY:
- Method: clearing of the bacterial background lawn or reduction in the number of spontaneous revertants (below the induction factor of 0.5)

ACCEPTANCE CRITERIA: The assay is considered acceptable if the following criteria are met:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce an increase above 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
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

Rationale for test conditions:

These are according to the OECD guideline 471.

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:

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

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In Experiment I without S9: 1000 - 5000 µg/plate and with S9: 333 - 5000 µg/plate; in Experiment II: with and without S9: 333 - 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In Experiment I without S9: 100 - 5000 µg/plate and with S9: 333 - 5000 µg/plate; Experiment II without and with S9: 100 - 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In Experiment I without S9: 333 - 5000 µg/plate and with S9: 1000 - 5000 µg/plate; Experiment II without and with S9: 333 - 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In Experiment I without S9: 100 - 5000 µg/plate and with S9: 333 - 5000 µg/plate; In Experiment II with and without S9: 100 - 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

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 1000 to 5000 µg/plate. No precipitation of the test item was observed in the overlay agar on the incubated agar plates.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as Experiment I.

Detailed results

Experiment I

 

Without Metabolic Activation
Test Group	Dose level (µg/plate)	Revertant Colony Counts (mean±SD)
		TA1535		TA1537	TA98		TA100	WP2uvrA
DMSO	n.a.	11±2		8±1	29±4		126±12	32±4
Untreated	n.a.	11±5		12±4	22±1		123±11	40±4
Test item	3	12±3		8±1	25±7		113±8	39±4
	10	13±3		9±0	22±6		126±18	31±6
	33	11±5		10±3	25±3		112±6	31±3
	100	11±3		8±3R	24±7		63 ± 8R	34±6
	333	11±3		6 ± 2M R	17±1MR		52 ± 6R	38±11
	1000	9 ± 2M R		1 ± 1M R	8 ± 1M R		7 ± 1M R	45±6
	2500	9 ± 1M R		0 ± 0M R	4 ± 2M R		1 ± 1M R	40±6
	5000	8 ± 2M R		0 ± 0M R	1 ± 1M R		0 ± 0M R	38±4
NaN3	10	1220 ± 31					2137 ± 76
4-NOPD	10				373±28
4-NOPD	50			78±12
MMS	2.0 µL							995±9
With Metabolic Activation
Test Group	Dose level (µg/plate)	Revertant Colony Counts (mean +/- SD)
		TA1535	TA1537		TA98	TA100		WP2uvrA
DMSO	n.a.	10±1	8±1		30±4	96±3		42±4
Untreated	n.a.	15±3	10±4		39±4	102±11		46±3
Test item	3	12±3	10±2		30±6	94±11		43±4
	10	9±2	9±2		25±3	101±9		47±6
	33	10±2	9±3		31±6	115±19		44±4
	100	10±2	11±2		39±2	103±5		49±4
	333	12±2R	6 ± 2M R		30±5	52±4R		46±5
	1000	7±1R	3 ± 2M R		5 ± 2M R	0±0R		51±1
	2500	8 ± 1M R	0 ± 0M R		0 ± 0M R	0±0R		47±4
	5000	6 ± 1M R	0 ± 0M R		0 ± 1M R	0±0R		43±4
2-AA	2.5	403±19	203 ± 30		5296 ± 178	4451 ± 113
2-AA	10							331±16

NaN3: sodium azide

2-AA: 2-aminoanthracene

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

MMS: methyl methane sulfonate

R: Reduced background growth

M: Manual count

Experiment II

 

Without Metabolic Activation
Test Group	Dose level (µg/plate)	Revertant Colony Counts (mean±SD)
		TA1535	TA1537	TA98	TA100	WP2uvrA
DMSO	n.a.	11±5	10±2	28±3	142±9	36±5
Untreated	n.a.	11±4	13±4	26±8	180±3	30±4
Test item	1		11±1	30±8	140±10
	3		11±2	25±9	142±4
	10	10±2	10±2	30±3	141±11
	33	11±5	8±4	29±2	101±4	36±1
	100	12±2	9±3^R	20±9	60 ± 10^R	34 ± 6
	333	7±2R	3 ± 1M R	14±1M R	42 ± 5 M R	39 ± 10
	1000	8 ± 1M R	0 ± 1M R	11±3M R	36 ± 12 M R	32 ± 4
	2500	5 ± 2M R	0 ± 0M R	2 ± 3M R	9 ± 8 M R	36 ± 1
	5000	1 ± 1M R				35 ± 5
NaN3	10	1234 ± 25			2181 ± 111
4-NOPD	10			438±19
4-NOPD	50		86±11
MMS	2.0 µL					648 ± 29
With Metabolic Activation
Test Group	Dose level (µg/plate)	Revertant Colony Counts (mean +/- SD)
		TA1535	TA1537	TA98	TA100	WP2uvrA
DMSO	n.a.	17±6	17±3	41±5	151 ± 8	43±3
Untreated	n.a.	20±1	16±5	42±5	188 ± 2	53±12
Test item	1		18±4	41±7	149±15
	3		15±4	43±4	142±30
	10	16±1	19±3	33±4	139 ± 6
	33	17±3	13±5	38±12	133±14	47±3
	100	12±3	17±1R	41±6	50±13R	45±11
	333	8±3R	9 ± 2M R	7 ± 3M R	46 ± 4R	48±8
	1000	8 ± 2M R	2 ± 1M R	4 ± 1M R	3 ± 4M R	34±5
	2500	5 ± 1M R	1 ± 1M R	0 ± 1M R	1±1R	32±5
	5000	2 ± 1M R				36 ± 8
2-AA	2.5	347±14	209±28	5931 ± 232	4856 ± 250
2-AA	10					399 ± 57

 

NaN3: sodium azide

2-AA: 2-aminoanthracene

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

MMS: methyl methane sulfonate

R: Reduced background growth

M: Manual count

Conclusions:

The test item is not mutagenic.

Executive summary:

In the current study the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) was assessed according to OECD 471 and GLP. The Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA were used.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

In the Experiment I the test item was tested at 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate. In Experiment II the test item was tested in Strain TA 1535 at 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate; in Strains TA 1537, TA 98 and TA 100 at 1; 3; 10; 33; 100; 333; 1000 and 2500 μg/plate; and in Strain WP2uvrA at 33; 100; 333; 1000; 2500 and 5000 μg/plate. The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 μg/plate. No precipitation of the test item was observed in the overlay agar on the incubated agar plates.

The plates incubated with the test item showed reduced background growth in all strains, except for strain WP2uvrA. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains, except for strain WP2uvrA. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment 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.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used and is considered to be non-mutagenic in the reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

For this endpoint there is one study available in which the potential of the test item to induce gene mutations was assessed according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) according to OECD 471 and GLP. The Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA were used with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. In the Experiment I the test item was tested at 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate. In Experiment II the test item was tested in Strain TA 1535 at 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate; in Strains TA 1537, TA 98 and TA 100 at 1; 3; 10; 33; 100; 333; 1000 and 2500 μg/plate; and in Strain WP2uvrA at 33; 100; 333; 1000; 2500 and 5000 μg/plate.

The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 μg/plate. No precipitation of the test item was observed in the overlay agar on the incubated agar plates.

Toxic effects, evident as a reduction in the number of revertants, occurred in all strains, except for strain WP2uvrA. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

The test item did not induce gene mutations and is considered to be non-mutagenic in the reverse mutation assay.

Justification for classification or non-classification

The test item did not induce gene mutations in a reverse mutation assay (Ames test) and is therefore considered to be non-mutagenic.