2-methylcyclohexanone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2019-March 2020

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

Final

Deviations:

yes

Remarks:

Concerning: Experimental Performance Report: In the experiment with metabolic activation (for selection and plating efficiency) the cell culture medium was prepared without A. This deviation did not influence the quality or integrity of the present study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

2-methylcyclohexanone

EC Number:

209-513-6

EC Name:

2-methylcyclohexanone

Cas Number:

583-60-8

Molecular formula:

C7H12O

IUPAC Name:

2-methylcyclohexan-1-one

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

impurity 2

Reference substance name:

Unknown impurities

Molecular formula:

not applicable

IUPAC Name:

Unknown impurities

Test material form:

liquid

Details on test material:

Batch No.: 01246M0002, purity confirmed by analytical certificate

Specific details on test material used for the study:

Characterisation of the Test Item
The identity of the test item was inspected upon delivery at the test facility (e.g. test item name,
batch no. and additional data were compared with the label) based on the following specifications
provided by the sponsor. The following listed information applies to the sample as received.

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

(clone TK+/- 3.7.2C)

Details on mammalian cell type (if applicable):

These cells are characterised by their high proliferation rate (10 - 12 h
doubling time of the Eurofins Munich stock cultures) and their cloning efficiency, usually more than
50%. The cells obtain a near diploid karyotype (40 ± 2 chromosomes). They are heterozygous at the
Thymidine Kinase (TK) locus in order to detect mutation events at the TK-locus.
To prevent high backgrounds arising from spontaneous mutation, cells lacking TK can be eliminated
by culturing cells in RPMI 1640 supplemented with:
9.0 µg/mL hypoxanthine
15.0 µg/mL thymidine
22.5 µg/mL glycine
0.1 µg/mL methotrexate
The cells are resuspended in medium without methotrexate but with thymidine, hypoxanthine and
glycine for 1 - 3 days.
Large stock cultures of the cleansed L5178Y cell line are stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich. This allows the repeated use of the same cell batch in
experiments. Each cell batch is routinely checked for mycoplasma infection.
Thawed stock cultures are maintained in plastic culture flasks in RPMI 1640 complete medium and subcultured three times per week.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Homogenate
An advantage of using in vitro cell cultures is the accurate control of concentration and exposure
time of cells to the test item under study. However, due to the limited capacity of cells growing in
vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is
necessary [8]. Many substances only develop mutagenic potential when they are metabolized by the mammalian organism. Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix). Since the L5178Y cells used in this assay do not have the cytochrome-based P450 metabolic oxidation system, an exogenous metabolic activation system, the S9 microsomal fraction was added. The cells were exposed to the test substance both, in the presence and the absence of metabolic activation.
The S9 liver microsomal fraction was obtained from Trinova Biochem GmbH, Giessen, Germany.
Male Sprague Dawley rats were induced with phenobarbital / β-naphthoflavone.
The following quality control determinations were performed by Trinova Biochem GmbH:
a) Alkoxyresorufin-O-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene)
A stock of the supernatant containing the microsomes is frozen in aliquots of 5 mL and stored at
≤ -75 °C.
The protein concentration in the S9 preparation (Lot: 4146) was 38.5 mg/mL.

S9 Mix
An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to
result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the concentrations below:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-phosphate-buffer pH 7.4. During the experiment the S9 mix was stored on ice.
The final concentration of S9 mix in the cultures was 5%.

Test concentrations with justification for top dose:

The test item was investigated at the following concentrations: without and with metabolic activation:
0.2, 0.5, 1.0, 2.5, 5.0, 7.5 and 10 mM

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

RPMI 1640 medium supplemented with 5 % HS 100 U/100 µg/mL penicillin/streptomycin 1 mM sodium pyruvate 2 mM L-glutamine 25 mM HEPES 2.5 µg/mL amphotericin B

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

Experimental Design
Pre-Experiment for Toxicity
The toxicity of the test item was determined in a pre-experiment up to a maximum concentration of 10 mM. Eight concentrations [0.1, 0.2, 0.5, 1.0, 2.5, 5.0, 7.5 and 10 mM] were tested without and
with metabolic activation. The experimental conditions in these pre-experiments were the same as described below in the paragraph experimental performance. After a 2-day growth period the relative suspension growth (RSG) of the treated cell cultures was calculated according to the method of Clive and Spector.

Evaluation criteria:

The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 106 cells and
- a concentration-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or
chromosomal aberrations.
Statistical methods might be used as an aid in evaluation of the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF or the
trend of the test is negative.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Pre-Experiment for Toxicity, without metabolic activation.

Test Group	Concentration [mM]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	SG^a	RSG^b[%]
C1	0	384000	1010000	1300000	13.1	100.0
C2		405000	1040000	1320000	13.7
1	0.1	406000	1020000	1520000	15.5	115.5
2	0.2	445000	1150000	1270000	14.6	108.8
3	0.5	445000	1000000	1440000	14.4	107.2
4	1.0	386000	1030000	1410000	14.5	108.1
5	2.5	350000	906000	1510000	13.7	101.9
6	5.0	405000	980000	1390000	13.6	101.4
7	7.5	345000	897000	1500000	13.5	100.2
8	10	349000	933000	1490000	13.9	103.5

C: Negative control
a: Suspension Growth, SG = [((value 24 h x 30) / 1x10⁷) x ((value 48 h x 20) / (value 24 h* x 20))];
* : for value 24 h > 3x105 then value 24 h = 3x105
b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]

Table 2. Pre-Experiment for Toxicity, with metabolic activation

Test Group	Concentration [mM]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	SG^a	RSG^b[%]
C1	0	337000	914000	1420000	13.0	100.0
C2		359000	978000	1230000	12.0
1	0.1	332000	867000	1370000	11.9	95.0
2	0.2	361000	961000	1400000	13.5	107.6
3	0.5	338000	908000	1210000	11.0	87.9
4	1.0	362000	959000	1260000	12.1	96.6
5	2.5	332000	959000	1420000	12.3	98.6
6	5.0	331000	937000	1490000	14.0	111.7
7^#	7.5	138000	358000	1410000	5.0	40.4
8^#	10	152000	382000	1480000	5.7	45.2

C: Negative control
a: Suspension Growth, SG = [((value 24 h x 30) / 1x10⁷) x ((value 48 h x 20) / (value 24 h* x 20))];
* : for value 24 h > 3x10⁵ then value 24 h = 3x10⁵
b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100] #
: for these concentrations only half of the cell number was used for treatment, but since this test was only used for toxicity determination, this was concluded as acceptable.

Table 3. Main Experiment - Toxicity Data, without metabolic activation

Test Group	Concentration [mM]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	SG^a	RSG^b[%]	RCE^c [%]	RTG^d [%]
C1	0	392000	1150000	1060000	12.2	100.0	100.0	100.0
C2		407000	1110000	1110000	12.3
2	0.2	406000	851000	1100000	9.4	76.4	97.2	74.2
3	0.5	389000	787000	1200000	9.4	77.1	84.1	64.8
4	1.0	413000	802000	1090000	8.7	71.3	70.4	50.2
5	2.5	353000	750000	1120000	8.4	68.5	84.1	57.7
6	5.0	351000	692000	1110000	7.7	62.7	85.4	53.5
7	7.5	343000	610000	1160000	7.1	57.7	92.5	53.4
8	10	326000	601000	1140000	6.9	55.9	82.8	46.3
EMS	300 µg/mL	387000	803000	1050000	8.4	68.8	56.0	38.5
MMS	10 µg/mL	418000	896000	907000	8.1	66.3	49.3	32.7

C: Negative control
a: Suspension Growth, SG = [((value 24 h x 30) / 1x10⁷) x ((value 48 h x 20) / (value 24 h* x 20))];
* : for value 24 h > 3x10⁵ then value 24 h = 3x10⁵
b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]
c: Relative Cloning Efficiency, RCE = [(CE test group / CE of corresponding controls) x 100], Cloning Efficiency, CE = ((-ln (((96 - (mean Plate 1, Plate 2)) / 96)) / 1.6) x 100)
d: Relative Total Growth, RTG = (RSG x RCE) / 100
EMS: Ethylmethanesulfonate
MMS: Methylmethanesulfonate

Table 4. Main Experiment - Mutagenicity Data, without metabolic activation

Test Group	Concentration [mM]	Cloning Efficiency (CE)			Mutagenicity Data
		Plate 1^e	Plate 2^e	CE^f[%]	Number of cultures / 96 wells					MF^g [mutants / 10^6 cells]	IMF^h [mutants / 10^6cells]
					Plate 1^e	Plate 2^e	Plate 3^e	Plate 4^e	Mean
C1	0	79	76	102.9	14	15	12	16	14.3	78.1	/
C2		79	76	116.0	12	9	12	9	10.5	50.0	/
2	0.2	79	78	106.4	11	11	6	15	10.8	56.1	-7.9
3	0.5	73	75	92.1	11	6	14	11	10.5	63.2	-0.9
4	1.0	67	69	77.0	14	7	13	10	11.0	79.3	15.3
5	2.5	74	74	92.1	14	10	7	10	10.3	61.5	-2.5
6	5.0	71	78	93.5	13	9	14	12	12.0	71.5	7.5
7	7.5	81	73	101.2	12	7	11	20	12.5	69.7	5.6
8	10	69	78	90.7	10	11	8	9	9.5	57.5	-6.6
EMS	300 µg/mL	56	64	61.3	63	60	65	59	61.8	842.6	778.6
MMS	10 µg/mL	59	52	53.9	34	40	38	37	37.3	455.8	391.7

C: Negative control
e: Number of cultures with cell growth.
f: Cloning Efficiency, CE = ((-ln (((96 - (mean Plate 1, Plate 2)) / 96)) / 1.6) x 100)
g: Mutant frequency,
MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800
h: Induced mutant frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls
EMS: Ethylmethanesulfonate
MMS: Methylmethanesulfonate

Table 5. Main Experiment - Colony Sizing, without metabolic activation

Test Group*	Concentration [mM]	Wells with at least 1 colony	Large colonies	Small colonies	% small colonies
C1	0	57	38	19	33.3
C2		42	27	15	35.7
EMS	300 µg/mL	247	208	39	15.8
MMS	10 µg/mL	149	59	90	60.4

C: Negative control
EMS: Ethylmethanesulfonate
MMS: Methylmethanesulfonate
*: Based on the non-mutagenic effects of the test item, an assessment of clastogenicity was not feasible.

Table 6. Main Experiment - Toxicity Data, with metabolic activation

Test Group	Concentration [mM]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	SG^a	RSG^b [%]	RCE^c [%]	RTG^d [%]
C1	0	301000	826000	1440000	11.9	100.0	100.0	100.0
C2		288000	826000	1420000	11.7
2	0.2	325000	896000	1380000	12.4	104.7	103.8	108.7
3	0.5	288000	860000	1420000	12.2	103.4	103.8	107.4
4	1.0	305000	914000	1450000	13.3	112.2	112.3	126.0
5	2.5	310000	849000	1330000	11.3	95.6	117.8	112.7
6	5.0	290000	830000	1400000	11.6	98.4	121.8	119.8
7	7.5	269000	815000	1420000	11.6	98.0	117.8	115.5
8	10	286000	794000	1430000	11.4	96.1	123.9	119.1
B[a]P	2.5 µg/mL	266000	563000	1420000	8.0	67.7	86.9	58.8

a: Suspension Growth, SG = [((value 24 h x 30) / 1x10⁷) x ((value 48 h x 20) / (value 24 h* x 20))];
* : for value 24 h > 3x10⁵ then value 24 h = 3x10⁵
b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]
c: Relative Cloning Efficiency, RCE = [(CE test group / CE of corresponding controls) x 100]
Cloning Efficiency, CE = ((-ln (((96 - (mean Plate 1, Plate 2)) / 96)) / 1.6) x 100)
d: Relative Total Growth, RTG = (RSG x RCE) / 100
B[a]P: Benzo[a]pyrene

Table 7. Main Experiment - Mutagenicity Data, with metabolic activation

Test Group	Concentration [mM]	Cloning Efficiency (CE)			Mutagenicity Data
		Plate 1^e	Plate 2^e	CE^f[%]	Number of cultures / 96 wells					MF^g [mutants / 10^6 cells]	IMF^h [mutants / 10^6cells]
					Plate 1^e	Plate 2^e	Plate 3^e	Plate 4^e	Mean
C1	0	73	70	85.4	11	8	13	10	10.5	68.0	/
C2		73	73	89.3	11	8	14	9	10.5	65.1	/
2	0.2	68	78	89.3	16	7	18	7	12.0	75.8	9.3
3	0.5	76	71	90.7	13	10	7	8	9.5	57.7	-8.9
4	1.0	82	70	98.0	11	11	10	13	11.3	63.6	-2.9
5	2.5	79	76	102.9	16	8	13	12	12.3	66.6	0.1
6	5.0	79	78	106.4	14	13	11	11	12.3	64.2	-2.3
7	7.5	81	74	102.9	9	9	12	14	11.0	59.3	-7.2
8	10	78	80	108.2	13	14	17	16	15.0	78.6	12.1
B[a]P	2.5 µg/mL	61	74	75.9	69	72	66	73	70.0	864.1	797.5

C: Negative control
e: Number of cultures with cell growth.
f: Cloning Efficiency, CE = ((-ln (((96 - (mean Plate 1, Plate 2)) / 96)) / 1.6) x 100)
g: Mutant frequency,
MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800
h: Induced mutant frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls
B[a]P: Benzo[a]pyrene

Table 8. Main Experiment - Colony Sizing, with metabolic activation

Test Group*	Concentration [mM]	Wells with at least 1 colony	Large colonies	Small colonies	% small colonies
C1	0	42	33	9	21.4
C2		42	31	11	26.2
B[a]P	2.5 µg/mL	280	168	112	40.0

C: Negative control
B[a]P: Benzo[a]pyrene
*: Based on the non-mutagenic effects of the test item, an assessment of clastogenicity was not feasible.

Applicant's summary and conclusion

Conclusions:

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item 2-MCH is considered to be non-mutagenic in this in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Executive summary:

Summary Results
The test item 2-MCH was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay.
The selection of the concentrations used in the main experiment was based on data from the pre-experiment. The test item was investigated at the following concentrations:
without and with metabolic activation:
0.2, 0.5, 1.0, 2.5, 5.0, 7.5 and 10 mM
No precipitation of the test item was noted in the experiment.
No growth inhibition was observed with metabolic activation. Growth inhibition was observed in the experiment without metabolic activation: The relative total
growth (RTG) was 46.3% for the highest concentration evaluated.
No biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of thenegative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was not exceeded by the induced mutant frequency at any concentration.
No concentration-related increase was observed.
EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of
small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

Summary: Main Experiment, without and with metabolic activation

	Test Group	Conc. [mM]	RCE^a [%]	RTG^b [%]	MF^c[mutants/ 10^6 cells]	IMFd [mutants/ 10^6 cells]	GEF^eexceeded	Statistical Significant Increase^f	Precipitate
Exp without S9	C1	0	100.0	100.0	64.1	/	/	/	-
	C2					/	/	/	-
	2	0.2	97.2	74.2	56.1	-7.9	-		-
	3	0.5	84.1	64.8	63.2	-0.9	-	-	-
	4	1.0	70.4	50.2	79.3	15.3	-	-	-
	5	2.5	84.1	57.7	61.5	-2.5	-	-	-
	6	5.0	85.4	53.5	71.5	7.5	-	-	-
	7	7.5	92.5	53.4	69.7	5.6	-	-	-
	8	10	82.8	46.3	57.5	-6.6	-	-	-
	EMS	300 µg/mL	56.0	38.5	842.6	778.6	+	+	-
	MMS	10 µg/mL	49.3	32.7	455.8	391.7	+	+	-
Exp with S9	C1	0	100.0	100.0	66.5	/	/	/	-
	C2					/	/	/	-
	2	0.2	103.8	108.7	74.6	8.1	-	-	-
	3	0.5	103.8	107.4	57.7	-8.9	-	-	-
	4	1.0	112.3	126.0	63.6	-2.9	-	-	-
	5	2.5	117.8	112.7	66.6	0.1	-	-	-
	6	5.0	121.8	119.8	64.2	-2.3	-	-	-
	7	7.5	117.8	115.5	59.3	-7.2	-	-	-
	8	10	123.9	119.1	78.6	12.1	-	-	-
	B[a]P	2.5 µg/mL	86.9	58.8	864.1	797.5	+	+	-

C: Negative Controls
a: Relative Cloning Efficiency, RCE = [(CE _{test group} / CE _{of corresponding controls}) x 100]
Cloning Efficiency, CE = ((-ln (((96 - (mean Plate 1, Plate 2)) / 96)) / 1.6) x 100)
b: Relative Total Growth, RTG = (RSG x RCE) / 100
c: Mutant Frequency,
MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800
d: Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls
e: Global Evaluation Factor, GEF (126 mutants/106 cells); +: GEF exceeded, -: GEF not exceeded
f: statistical significant increase in mutant frequency compared to negative controls (Mann Whitney test, p<0.05).
+: significant; -: not significant
EMS: Ethylmethanesulfonate
MMS: Methylmethanesulfonate
B[a]P: Benzo[a]pyrene