4-methylcyclohexanone

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:

21 June 2016 to 08 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

The most widely used assays for detecting gene mutations are those using bacteria. They are relatively simple and rapid to perform, and give reliable data on the ability of an agent to interact with DNA and produce mutations.
Reverse mutation assays determine the frequency with which an agent reverses or suppresses the effect of the forward mutation. The genetic target presented to an agent is therefore small, specific and selective. Several bacterial strains, or a single strain with multiple markers are necessary to assure reliable detection of mutagens that may be specific to one tester strain or locus. The reversion of bacteria from growth-dependence on a particular amino acid to growth in the absence of that amino acid (reversion from auxotrophy to prototrophy) is the most widely used marker.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-methylcyclohexanone

EC Number:

209-665-3

EC Name:

4-methylcyclohexanone

Cas Number:

589-92-4

Molecular formula:

C7H12O

IUPAC Name:

4-methylcyclohexan-1-one

Test material form:

liquid

Details on test material:

- Other: Colourless liquid

Method

Target gene:

The S. typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system measures his- → his+ and trp- → trp+ reversions, respectively. The S. typhimurium and Escherichia coli strains are constructed to differentiate between base pair (TA1535, TA100, WP2 uvrA pKM101, and WP2 pKM101) and frameshift (TA1537, TA98) mutations.

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

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Expriment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
In the pre-experiment, the concentration range of the test substance was 3 - 5000 μg/plate. The pre-experiment is reported as experiment I. Since no cytotoxic effects were observed in experiment I, 5000 μg/plate was chosen as the maximal concentration for experiment II.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Controls 3

True negative controls:

no

Controls 4

Positive controls:

yes

Positive control substance:

sodium azide

Controls 5

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Controls 6

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Controls 7

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene 2.5 µg/plate in DMSO for TA1535, TA1537, TA98 and TA100; 10 µg/plate in DMSO for WP2 uvrA (pKM101) and WP2 (pKM101) with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION:in agar (plate incorporation - Expt. I); preincubation - Expt. II

DURATION
- Preincubation period: 60 mins
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test substance is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.
A concentration 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 concentration 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:

Not applicable

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

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:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Summary of results Experiment I without S9 - Revertant Colony Counts (Mean ±SD)

Group	Concn. (per plate)	TA1535	TA1537	TA98	TA100	WP2 pKM101	WP2 uvrA pKM101
DMSO	-	11 ± 3	8 ± 1	35 ± 4	186 ± 13	217 ± 17	350 ± 14
Untreated	-	7 ± 3	14 ± 5	36 ± 5	199 ± 13	224 ± 21	362 ± 23
CA5110	3 µg	10 ± 4	8 ± 2	25 ± 3	169 ± 11	188 ± 5	336 ± 14
CA5110	10 µg	13 ± 3	9 ± 2	24 ± 7	167 ± 19	199 ± 12	331 ± 19
CA5110	33 µg	10 ± 1	11 ± 4	25 ± 5	165 ± 13	198 ± 10	311 ± 17
CA5110	100 µg	13 ± 3	8 ± 3	26 ± 9	163 ± 5	201 ± 9	337 ± 24
CA5110	333 µg	12 ± 5	8 ± 2	29 ± 8	178 ± 9	192 ± 9	337 ± 8
CA5110	1000 µg	10 ± 2	11 ± 5	35 ± 7	178 ± 10	197 ± 7	316 ± 15
CA5110	2500 µg	7 ± 2	9 ± 2	35 ± 2	176 ± 6	191 ± 17	323 ± 23
CA5110	5000 µg	8 ± 1	13 ± 5	39 ± 3	91 ± 9	166 ± 9	331 ± 7
NaN3	10 µg	1060 ± 26	-	-	1693 ± 110	-	-
4-NOPD	10 µg	-	-	426 ± 37	-	-	-
4-NOPD	50 µg	-	63 ± 10	-	-	-	-
MMS	2.0 µL	-	-	-	-	3807 ± 415	3555 ± 69

Table 2: Summary of results Experiment I with S9 - Revertant Colony Counts (Mean ±SD)

Group	Concn. (per plate)	TA1535	TA1537	TA98	TA100	WP2 pKM101	WP2 uvrA pKM101
DMSO	-	12 ± 4	14 ± 3	34 ± 12	164 ± 17	230 ± 13	393 ± 10
Untreated	-	15 ± 4	15 ± 2	47 ± 6	192 ± 21	259 ± 20	436 ± 10
CA5110	3 µg	11 ± 1	16 ± 5	36 ± 4	153 ± 23	229 ± 22	404 ± 9
CA5110	10 µg	9 ± 3	18 ± 3	41 ± 4	142 ± 27	224 ± 16	401 ± 23
CA5110	33 µg	13 ± 3	12 ± 2	40 ± 6	122 ± 16	195 ± 27	379 ± 24
CA5110	100 µg	13 ± 3	13 ± 3	42 ± 6	150 ± 13	214 ± 18	396 ± 15
CA5110	333 µg	12 ± 0	15 ± 3	45 ± 10	163 ± 20	225 ± 35	402 ± 21
CA5110	1000 µg	10 ± 3	17 ± 6	45 ± 3	171 ± 9	208 ± 4	371 ± 5
CA5110	2500 µg	14 ± 3	17 ± 3	45 ± 8	175 ± 11	198 ± 15	345 ± 20
CA5110	5000 µg	12 ± 3	14 ± 2	37 ± 7	137 ± 8	170 ± 21	359 ± 26
2-AA	2.5 µg	381 ± 25	189 ± 12	4418 ± 246	4830 ± 406	-	-
2-AA	10.0 µg	-	-	-	-	1082 ± 26	2285 ± 128

Table 3: Summary of results Experiment II without S9 - Revertant Colony Counts (Mean ±SD)

Group	Concn. (per plate)	TA1535	TA1537	TA98	TA100	WP2 pKM101	WP2 uvrA pKM101
DMSO		11 ± 5	12 ± 2	21 ± 5	158 ± 17	201 ± 30	350 ± 9
Untreated		14 ± 3	13 ± 4	28 ± 10	205 ± 23	241 ± 21	369 ± 10
CA5110	33 µg	16 ± 5	8 ± 3	20 ± 5	157 ± 5	209 ± 22	338 ± 10
CA5110	100 µg	12 ± 3	8 ± 3	23 ± 2	139 ± 11	189 ± 10	355 ± 15
CA5110	333 µg	16 ± 3	10 ± 1	25 ± 5	137 ± 16	200 ± 4	331 ± 18
CA5110	1000 µg	17 ± 4	9 ± 1	30 ± 2	120 ± 15	211 ± 5	351 ± 11
CA5110	2500 µg	16 ± 3	10 ± 4	34 ± 3	123 ± 8	199 ± 8	330 ± 7
CA5110	5000 µg	14 ± 3	7 ± 2	20 ± 2	68 ± 10	110 ± 20	303 ± 25
NaN3	10 µg	1053 ± 27			2256 ± 119
4-NOPD	10 µg			334 ± 33
4-NOPD	50 µg		81 ± 3
MMS	2.0 µL					3232 ± 332	2779 ± 377

 

Table 4: Summary of results Experiment II - with S9 - Revertant Colony Counts (Mean ±SD)

Group	Concn. (per plate)	TA1535	TA1537	TA98	TA100	WP2 pKM101	WP2 uvrA pKM101
DMSO		15 ± 1	15 ± 6	45 ± 6	129 ± 14	249 ± 26	394 ± 10
Untreated		12 ± 2	19 ± 3	37 ± 4	216 ± 12	296 ± 20	483 ± 3
CA5110	33 µg	13 ± 2	11 ± 3	39 ± 9	144 ± 19	240 ± 38	396 ± 20
CA5110	100 µg	15 ± 3	13 ± 3	36 ± 8	141 ± 19	245 ± 18	417 ± 16
CA5110	333 µg	16 ± 6	16 ± 6	44 ± 15	135 ± 18	241 ± 29	407 ± 5
CA5110	1000 µg	11 ± 1	12 ± 4	38 ± 9	113 ± 3	239 ± 14	400 ± 22
CA5110	2500 µg	15 ± 1	15 ± 5	43 ± 11	87 ± 7	183 ± 8	363 ± 6
CA5110	5000 µg	17 ± 3	10 ± 2	32 ± 10	53 ± 4	157 ± 2	341 ± 27
2 AA	2.5 µg	380 ± 45	159 ± 16	3865 ± 655	4594 ± 579
2 AA	10.0 µg					1055 ± 88	2035 ± 113

NaN3 sodium azide 2-AA 2-aminoanthracene 4-NOPD 4-nitro-o-phenylene-diamine MMS methyl methane sulfonate

 

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli strains WP2 uvrA pKM101 and WP2 pKM101.

The plates incubated with the test substance showed normal background growth up to 5000 μg/plate in all strains with and without metabolic activation.

In experiment II, cytotoxicity (evident as a reduction in the number of revertants (below the indication factor of 0.5)) was observed in strain TA100 at 5000 μg/plate both with and wihout metabolic activation. No other cytotoxic effects were observed in any other strain both with and without metabolic activation.

No increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any concentration, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations and all mutation rates were within the range of normal biological variability.

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

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.