2-methylcyclohexanone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three different in vitro studies were performed for the following endpoints: in vitro gene mutation study in bacteria (OECD 471), in vitro cytogenicity / micronucleus study (OECD 487), and in vitro gene mutation study in mammalian cells (OECD 490).  The substance was found being negative for mutagenicity. Therefore, currently there is no concern on mutagenic effects caused by the substance.

Three main conclusions for the mentioned above endpoints:

1. OECD471 - In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, 2-MCH did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, 2-MCH is considered to be non-mutagenic in this bacterial reverse mutation assay.

2. OECD478 - In addition, it can be stated that during the study described and under the experimental conditions reported, 2-MCH did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells. Therefore, 2-MCH is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

3. OECD490 - Finally, 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.

Link to relevant study records

Reference

Reference 1

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)

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

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.

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

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.

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

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.

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

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March-November 2019

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

Final

Deviations:

yes

Remarks:

4.4. Schedule, study plan, p. 6 Study Plan: Arrival of the Test Item: 06 March 2019 Report: Arrival of the Test Item: 06 March 2019, 05 April 2019 Reason: Delivery of a new test material, due to precipitation in the bottle delivered on 06 March 2019.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Negative controls (cell culture medium) were treated in the same way as all concentration groups.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

An advantage of using cell cultures is the accurate control of the 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.
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).
The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced
with phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route. The preparation was performed according to Ames et al. The following quality control determinations are performed:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and
benzo[a]pyrene
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL and stored at ≤ -75°C.
The protein concentration in the S9 preparation (Lot: 220219) was 43 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 is 5%.

Test concentrations with justification for top dose:

All concentrations were determined in the pre-experiment. The following concentrations were tested with and without S9 mix:
0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 7.5 and 10 mM
The concentration of 10 mM was considered to be the highest test concentration used in this test
system following the recommendation of the corresponding OECD testing guideline 487

Vehicle / solvent:

Solvent Control: DMSO 1% v/v or ethanol 0.5% v/v in cell culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Remarks:

Complete Culture Medium
10 % fetal bovine serum (FBS)
100 U/100 µg/mL penicillin/streptomycin solution
2 mM L-glutamine
2.5 µg/mL amphotericin
25 mM HEPES
Treatment Medium (short-term exposure)
Complete culture medium without FBS.

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Pre-Experiment for Toxicity
A pre-experiment was conducted under identical conditions as described for the main experiment I.
The following concentrations were tested with and without S9 mix:
0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 7.5 and 10 mM
The concentration of 10 mM was considered to be the highest test concentration used in this test system following the recommendation of the corresponding OECD testing guideline 487.
Exposure Concentrations
Duplicate cultures were treated at each concentration. The following concentrations were used in the
main experiments:
Experiment I:
without metabolic activation: 0.25, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.25, 1.50 and 2.0 mM
with metabolic activation: 0.5, 1.0, 2.5, 5.0, 7.5 and 10 mM
Experiment II:
without metabolic activation: 0.05, 0.10, 0.25, 0.50, 0.60, 0.70, 0.80 and 1.0 mM
The following concentrations were selected for the microscopic analyses of micronuclei frequencies:
Experiment I (short-term exposure 4 h):
without and with metabolic activation: 0.7, 0.9, 1.0 and 1.5 mM
with metabolic activation: 2.5, 5.0 and 10 mM
Experiment II (long-term exposure 24 h):
without metabolic activation: 0.10, 0.25, 0.50 and 0.60 mM

Experimental Performance
Seeding of the Cultures
Three or four day-old stock cultures (in exponential growth), more than 50% confluent, were rinsed
with Ca-Mg-free PBS solution prior to the trypsin treatment. Cells subsequently were trypsinised with a solution of 0.05% trypsin in Ca-Mg-free PBS at 37°C for 5 min. By adding complete culture
medium the detachment was stopped and a single cell suspension was prepared.
Experiment I
Exponentially growing V79 cells were seeded into 25 cm2 cell culture flasks (two flasks per test
group). Approx. 50 000 cells were seeded per cell culture flask, containing 5 mL complete culture
medium (minimum essential medium supplemented with 10% FBS). After an attachment period of approx. 48 h, the complete culture medium was removed and subsequently the test item was added to the treatment medium in appropriate concentrations. The cells were incubated with the test item for 4 h in presence or absence of metabolic activation. At the end of the incubation, the treatment medium was removed and the cells were washed twice with PBS. Subsequently, the cells were incubated in complete culture medium + 1.5 µg/mL cytochalasin B for 20 h at 37 °C.
Experiment II
If negative or equivocal results are obtained, they should be confirmed using continuous treatment (long-term treatment) without metabolic activation. Approx. 50 000 exponentially growing V79 cells were seeded in 25 cm2 cell culture flasks in absence of metabolic activation. After an attachment period of approx. 48 h the test item was added in complete culture medium. 1 h later 1.5 µg/mL cytochalasin B were added and the cells were incubated for 23 h at 37 °C. At the end of the treatment the cell culture medium was removed and the cells were prepared for microscopic analysis.
Number of Cultures
Duplicate cultures were performed at each concentration level except for the pre-experiment.
Preparation of the Cultures
At the end of the cultivation, the complete culture medium was removed. Subsequently, cells were
trypsinated and resuspended in about 9 ml complete culture medium. The cultures were transferred into tubes and incubated with hypotonic solution (0.4% KCl) for some minutes at room temperature.
After the treatment with the hypotonic solution the cells were fixed with methanol + glacial acetic acid (3+1). The cells were resuspended gently and the suspension was dropped onto clean glass slides. Consecutively, the cells were dried on a heating plate. Finally, the cells were stained with acridine orange solution.
Analysis of Micronuclei
All slides, including those of positive and negative controls were independently coded before
microscopic analysis. For each experimental point, at least 2000 binucleated cells per concentration (1000 binucleated cells per slide) were analysed for micronuclei according to the criteria of Fenech, i.e. clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmic bridges. Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered.
Cytokinesis Block Proliferation Index
As an assessment of the cytotoxicity, a cytokinesis block proliferation index (CBPI) was determined from 500 cells according to the following formula:
CBPI=((c1 x 1) + (c2 x 2) + (cx x 3))/n
c1: mononucleate cells
c2: binucleate cells
cx: multinucleate cells
n: total number of cells
The CBPI can be used to calculate the % cytostasis, which indicates the inhibition of cell growth of treated cultures in comparison to control cultures:
% Cytostasis= 100 – 100 x ((CBPIT – 1) / (CBPIC – 1))
CBPIT: Cytokinesis Block proliferation index of treated cultures
CBPIC: Cytokinesis Block proliferation index of control cultures

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment II

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 0.25 mM a cytostasis of 35% and at 0.60 mM a cytostasis of 61% was noted.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment I

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment I

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1.5 mM a cytostasis of 55% was noted

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Pre-Experiment for Toxicity
According to the used guideline the highest recommended concentration is 10 mM. The test item was dissolved in cell culture medium. No precipitate of the test item was noted. The highest dose group evaluated in the pre-experiment was 7.5 mM without and 10 mM with metabolic activation.
The cytokinesis block proliferation index (CBPI) was used to calculate the cytostasis (cytostasis [%] = 100 - CBPI relative [%]). Cytostasis was used to describe cytotoxicity. The concentrations evaluated in the main experiment were based on the results obtained in the pre-experiment.

Table 1. Summary: Experiment I and II, without metabolic activation

	Dose Group	Concentration [mM]	Number of cells evaluated	Cytostasis [%]	Relative Cell Growth [%]	Micronucleated Cells Frequency [%]	Historical Control Limits Negative Control	P	Statistically Significant Increasea
Exp. I 4 h treatment, 24 h fixation interval	C	0	2000	0	100	0.65	0.37% -1.37%	/	/
	4	0.7	2000	14	86	1.00		-	-
	6	0.9	2000	42	58	0.90		-	-
	7	1.0	2000	45	55	1.25		-	-
	9	1.5	2000	55	45	1.20		-	-
	MMS	25 µg/mL	3000	8	92	1.98		-	+
	Colc	2.0 µg/mL	2000	8	92	3.25		-	+

 

	Dose Group	Concentration [mM]	Number of cells evaluated	Cytostasis [%]	Relative Cell Growth [%]	Micronucleated Cells Frequency [%]	Historical Control Limits Negative Control	P	Statistically Significant Increasea
Exp. II 24 h treatment, 24 h fixation interval	C	0	2000	0	100	0.80	0.37% -1.37%	/	/
	4	0.10	2000	14	86	0.95		-	-
	6	0.25	2000	35	65	1.00		-	-
	7	0.5	2000	27	73	0.75		-	-
	9	0.6	2000	61	39	1.20		-	-
	MMS	25 µg/mL	2000	32	68	8.10		-	+
	Colc	0.08 µg/mL	2000	12	88	5.25		-	+

Table 2: Summary: Experiment I, with metabolic activation

	Dose Group	Concentration [mM]	Number of cells evaluated	Cytostasis [%]	Relative Cell Growth [%]	Micronucleated Cells Frequency [%]	Historical Control Limits Negative Control	P	Statistically Significant Increasea
Exp. I      4 h treatment, 24 h fixation interval	C	0	4000	0	100	0.78	0.42% -1.64%	/	/
	3	2.5	2000	20	80	1.15		-	-
	4	5.0	4000	30	70	1.23		-	+
	6	10	4000	27	73	1.23		-	+
	CPA	2.5 µg/mL	2000	48	52	5.65		-	+

C: Negative Control (Culture medium)
a: statistical significant increase compared to negative control (χ² test , p<0.05). +: significant; -: not significant
MMS: Methylmethanesulfonate, Positive Control (without metabolic activation)
Colc: Colchicine, Positive Control (without metabolic activation)
CPA: Cyclophosphamide, Positive Control (with metabolic activation)
CBPI: Cytokinesis Block Proliferation Index, CBPI = ((c1 x 1) + (c2 x 2) + (cx x 3))/n
Relative Cell Growth: 100 x ((CBPI Test conc – 1) / (CBPI control -1))
c1: mononucleate cells
c2: binucleate cells
cx: multinucleate cells
n: total number of cells
P: Precipitation

Cytostasis [%] = 100- Relative Cell Growth [%]

*: the cytostasis is defined 0, when the relative cell growth exceeds 100%.

If cytotoxicity is observed the highest concentration evaluated should not exceed the limit of 55% ± 5% cytotoxicity according to the OECD Guideline 487. Higher levels of cytotoxicity may induce chromosome damage as a secondary effect of cytotoxicity. The other concentrations evaluated should exhibit intermediate and little or no toxicity. However, OECD 487 does not define the limit for discriminating between cytotoxic and non-cytotoxic effects. According to laboratory experience this limit is a value of the relative cell growth of 70% compared to the negative/solvent control which corresponds to 30% of cytostasis.
In experiment I without metabolic activation no increase of the cytostasis above 30% was noted at 0.7 mM. At 0.9 mM a cytostasis of 42%, at 1.0 mM a cytostasis of 45% and at 1.5 mM a cytostasis of 55% was noted. In experiment I with metabolic activation no increase of the cytostasis above 30% was observed.
In experiment II without metabolic activation no increase of the cytostasis above 30% was noted at 0.10 mM and at 0.50 mM. At 0.25 mM a cytostasis of 35% and at 0.60 mM a cytostasis of 61% was noted.
In the main experiment I with and without metabolic activation no biologically relevant increase of the micronucleus frequency was noted after treatment with the test item. No statistically significant enhancement (p<0.05) of cells with micronuclei was noted in the concentration groups of the test item evaluated in experiment I and II without metabolic activation.
In experiment I with metabolic activation a statistically significant increase (p = 0.0431) of cells with micronuclei was noted at 5.0 and 10 mM. However, since the corresponding numbers of micronucleated cells were within the historical control limits of the negative control and no concentration-related increase was observed, this effect was considered as not biologically relevant.
The χ² Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. No statistically significant increase in the frequency of micronucleated cells under the experimental conditions of the study was observed in experiment I and II. Methylmethanesulfonate (MMS, 25 µg/mL) and cyclophosphamide (CPA, 2.5 µg/mL) were used as clastogenic controls. Colchicine (0.08 and 2.0 µg/mL) was used as aneugenic control. All induced distinct and statistically significant increases of the micronucleus frequency. This demonstrates the validity of the assay.

Conclusions:

In conclusion, it can be stated that during the study described and under the experimental conditions reported, 2-MCH did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells.
Therefore, 2-MCH is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

Executive summary:

In order to investigate 2-MCH for a possible potential to induce micronuclei in Chinese hamster V79 cells an in vitro micronucleus assay was carried out. The following study design was performed:

	Without S9		With S9
	Exp. I	Exp. II	Exp. I
Exposure period	4 h	24 h	4 h
Cytochalasin B exposure	20 h	23 h	20 h
Preparation interval	24 h	24 h	24 h
Total culture period*	72 h	72 h	72 h

*Exposure started 48 h after culture initiation

The selection of the concentrations was based on data from the pre-experiment. In the first main experiment without and with metabolic activation 1.5 mM and 10 mM test item, respectively, and in experiment II 0.60 mM test item was selected as the highest concentration for microscopic
evaluation.
The following concentrations were evaluated for micronuclei frequencies:
Experiment I (short-term exposure 4 h):
without and with metabolic activation: 0.7, 0.9, 1.0 and 1.5 mM
with metabolic activation: 2.5, 5.0 and 10 mM
Experiment II (long-term exposure 24 h):
without metabolic activation: 0.10, 0.25, 0.50 and 0.60 mM.

No precipitate of the test item in the cultures at the end of treatment was noted in any concentration group evaluated in experiment I and II.

In experiment I without metabolic activation the micronucleated cell frequency of the negative control (0.65%) was within the historical control limits of the negative control (0.37% – 1.37%). The mean values of micronucleated cells found after treatment with the test item were 1.00% (0.7 mM), 0.90% (0.9 mM), 1.25% (1.0 mM) and 1.20% (1.5 mM). The numbers of micronucleated cells were within the historical control limits of the negative control and did not show a biologically relevant increase compared to the concurrent negative control.
In experiment I with metabolic activation the micronucleated cell frequency of the negative control (0.78%) was within the historical control limits of the negative control (0.42% – 1.64%).
The mean values of micronucleated cells found after treatment with the test item were 1.15% (2.5 mM), 1.23% (5.0 mM) and 1.23% (10 mM). The numbers of micronucleated cells were within the historical control limits of the negative control and did not show a biologically relevant increase compared to the concurrent negative control.
In experiment II without metabolic activation the micronucleated cell frequency of the negative control (0.80%) was within the historical control limits of the negative control (0.37% – 1.37%). The mean values of micronucleated cells found after treatment with the test item were 0.95% (0.10 mM), 1.00% (0.25 mM), 0.75% (0.50 mM) and 1.20% (0.60 mM). The numbers of micronucleated cells were within the historical control limits of the negative control and did not show a biologically relevant increase compared to the concurrent negative control.
No statistically significant enhancement (p<0.05) of cells with micronuclei was noted in the concentration groups of the test item evaluated in experiment I and II without metabolic activation.
In experiment I with metabolic activation a statistically significant increase (p = 0.0431) of cells with micronuclei was noted at 5.0 and 10 mM. However, since the corresponding numbers of micronucleated cells were within the historical control limits of the negative control and no concentration-related increase was observed, this effect was considered as not biologically relevant.
The χ² Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. No statistically significant increase in the frequency of micronucleated cells under the experimental conditions of the study was observed in experiment I and II.
MMS (25 µg/mL) and CPA (2.5 µg/mL) were used as clastogenic controls and colchicine as aneugenic controls (0.08 and 2.0 µg/mL). They induced distinct and statistically significant increases of the micronucleus frequency. This demonstrates the validity of the assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April - July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The OECD Guideline for Testing of Chemicals, Section 4, No. 471 – Bacterial Reverse Mutation Test - recommends using a combination of S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA102.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Chemical Name: 2-Methylcyclohexanone (2-MCH), CAS No.: 583-60-8

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

The bacteria most commonly used in these reverse mutation assays do not possess the enzyme system which, in mammals, is known to convert pro-mutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system was added in the form of mammalian microsome enzyme activation mixture.
S9 Homogenate: The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route. The following quality control determinations are performed:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL and stored at ≤ -75 °C. The protein concentration in the S9 preparation (Lot: 220219 and Lot: 210918) was 43 mg/mL and 35.7 mg/mL, respectively and was adjusted to 30 mg/mL.
Preparation of S9 Mix: The S9 mix preparation was performed according to Ames et al.
100 mM of sodium-ortho-phosphate-buffer, pH 7.4, was ice-cold added to the following pre-weighed sterilised reagents to give final concentrations in the S9 mix of: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, and 4 mM NADP. This solution was mixed with the liver 9000 x g supernatant fluid in the following proportion: co-factor solution 9.5 parts and liver preparation 0.5 parts. During the experiment the S9 mix was stored on ice.
S9 Mix Substitution Buffer: The S9 mix substitution buffer was used in the study as a replacement for S9 mix, without metabolic activation (-S9).
Phosphate-buffer (0.2 M) contains per litre of purified water: 0.2 M NaH2PO4 x H2O 120 mL and 0.2 M Na2HPO4 880 mL.
The two solutions were mixed and the pH was adjusted to 7.4. Sterilisation was performed for 20 min at 121 °C in an autoclave. This 0.2 M phosphate-buffer was mixed with 0.15 M KCl solution (sterile) in the following proportion: 0.2 M phosphate-buffer 9.5 parts, 0.15 M KCl solution 0.5 parts. This S9 mix substitution buffer was stored at 4 °C or use.

Test concentrations with justification for top dose:

In the pre-experiment with TA98 and TA100 eight doses were tested from 3.16 µg/plate up to 5000 µg/plate.
Based on the results from the pre-experiment, 5 doses were selected for the main experiment I and II as follows: 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate. 5.0 µL/plate was selected as the maximum concentration according to the results of the pre-experiment and as is the maximum concentration recommended by the guideline.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

10 µg/plate, Tester Strains: S. typhimurium: TA100, TA1535 without S9)

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine (99-56-9)

Remarks:

10 µg/plate for TA98, 40 µg/plate for TA1537, Tester Strains: S. typhimurium: TA98, TA1537 (without S9)

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

1 µL/plate, Tester Strain: S. typhimurium: TA102 (without S9)

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (613-13-8)

Remarks:

2.5 µg/plate; 10 µg/plate for TA102, Tester Strains: S. typhimurium: TA98, TA100, TA1535, TA1537 and TA102 (with S9)

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

yes

Positive controls:

no

Remarks:

A. dest., Eurofins Munich, Lot No. 190412, 190423, 190517

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Remarks:

DMSO, AppliChem Lot No. 0001446873, Honeywell Lot No. G2170

Details on test system and experimental conditions:

Bacteria
Five strains of S. typhimurium with the following characteristics were used:
TA98: his D 3052; rfa-; uvrB-; R-factor: frame shift mutations
TA100: his G 46; rfa-; uvrB-; R-factor: base-pair substitutions
TA1535: his G 46; rfa-; uvrB-: base-pair substitutions
TA1537: his C 3076; rfa-; uvrB-: frame shift mutations
TA102: his G 428 (pAQ1); rfa-; R-factor: base-pair substitutions
Tester strains TA98, TA1535 and TA102 were obtained from MOLTOX, INC., NC 28607, USA. Tester strains TA100 and TA1537 were obtained from Xenometrix AG, Switzerland. They were stored as stock cultures in ampoules with nutrient broth (OXOID) supplemented with DMSO (approx. 8% v/v) over liquid nitrogen.
All Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. They contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain of the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene coding for a protein of the DNA nucleotide excision repair system resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chl) and biotin (bio) genes (bacteria require biotin for growth).
The tester strains TA98, TA100 and TA102 contain the R-factor plasmid, pkM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non R-factor parent strains. pkM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system which is normally present in these organisms.
The properties of the S. typhimurium strains with regard to membrane permeability, ampicillin- and tetracycline-resistance as well as normal spontaneous mutation rates are checked regularly according to Ames et al.. In this way it is ensured that the experimental conditions set up by Ames are fulfilled.
Preparation of Bacteria: Samples of each tester strain were grown by culturing for 12 h at 37 °C in Nutrient Broth to the late exponential or early stationary phase of growth (approx. 109 cells/mL). The nutrient medium consists per litre of purified water: 8 g Nutrient Broth and 5 g NaCl.
A solution of 125 µL ampicillin (10 mg/mL) (TA98, TA100, TA102) was added in order to retain the phenotypic characteristics of the strain.
Agar Plates: The Vogel-Bonner Medium E agar plates with 2% glucose used in the Ames Test were prepared by Eurofins Munich. Quality controls were performed. Vogel-Bonner-salts contain per litre of purified water: 10 g MgSO4 x 7 H2O, 100 g citric acid, 175 g NaNH4HPO4 x 4 H2O, and 500 g K2HPO4. Sterilisation was performed for 20 min at 121 °C in an autoclave. Vogel-Bonner Medium E agar plates contain per litre of purified water: 15 g Agar Agar, 20 mL Vogel-Bonner salts, and 50 mL glucose-solution (40%). Sterilisation was performed for 20 min at 121 °C in an autoclave.
Overlay Agar: The overlay agar contains per litre of purified water: 7.0 g Agar Agar, 6.0 g NaCl, 10.5 mg L-histidine x HCl x H2O, and 12.2 mg biotin. Sterilisation was performed for 20 min at 121 °C in an autoclave.
Mammalian Microsomal Fraction S9 Mix: The bacteria most commonly used in these reverse mutation assays do not possess the enzyme system which, in mammals, is known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system was added in the form of mammalian microsome enzyme activation mixture.
S9 Homogenate: The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route. The following quality control determinations are performed: a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene, b) Sterility Test.
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL and stored at ≤-75 °C.
The protein concentration in the S9 preparation (Lot: 220219 and Lot: 210918) was 43 mg/mL and 35.7 mg/mL, respectively and was adjusted to 30 mg/mL.
Preparation of S9 Mix: The S9 mix preparation was performed according to Ames et al..
100 mM of sodium-ortho-phosphate-buffer, pH 7.4, was ice-cold added to the following pre-weighed sterilised reagents to give final concentrations in the S9 mix of: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, and 4 mM NADP.
This solution was mixed with the liver 9000 x g supernatant fluid in the following proportion: co-factor solution 9.5 parts, liver preparation 0.5 parts. During the experiment the S9 mix was stored on ice.
S9 Mix Substitution Buffer: The S9 mix substitution buffer was used in the study as a replacement for S9 mix, without metabolic activation (-S9). Phosphate-buffer (0.2 M) contains per litre of purified water: 0.2 M NaH2PO4 x H2O - 120 mL, 0.2 M Na2HPO4 - 880 mL. The two solutions were mixed and the pH was adjusted to 7.4. Sterilisation was performed for 20 min at 121 °C in an autoclave. This 0.2 M phosphate-buffer was mixed with 0.15 M KCl solution (sterile) in the following proportion: 0.2 M phosphate-buffer - 9.5 parts, 0.15 M KCl solution - 0.5 parts. This S9 mix substitution buffer was stored at 4 °C.

Evaluation criteria:

Evaluation of Cytotoxicity: Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or "B", respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
Evaluation of Mutagenicity: The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation). A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

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:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 1.0 µL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 5.0 µL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

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:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

Table 1: Results Pre-Experiment

Substance	Dose (µg/plate)	TA98		TA100
		Mutation Factor [toxicity]*		Mutation Factor [toxicity]*
		without S9	with S9	without S9	with S9
Solvent Control (DMSO)		1.0	1.0	1.0	1.0
4-NOPD	10.0	13.8	-	-	-
NaN3	10.0	-	-	6.8	-
2-AA	2.50	-	56.9	-	17.2
Test Item	3.16	1.4	0.8	1.0	0.9
	10.0	1.0	0.9	1.0	1.0
	31.6	1.1	0.9	0.9	0.9
	100	1.2	0.9	0.9	1.0
	316	1.1	0.9	0.8	0.9
	1000	0.8	0.9	1.0	0.9
	2500	1.3	1.1	1.1	1.1
	5000	1.3	0.9	0.9	1.1

* [toxicity parameter]: B = Background lawn reduced; N = No background lawn

 

Experiment I (Plate-incorporation Test)
Table 2: Results Experiment I
Tester Strain: TA98 1                                                                                                                                                                                          Experiment: 1

 

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A.dest.		36 31 35	34	2.6	44 37 45	42	4.4	1.0	0.9
DMSO		43 29 30	34	7.8	42 44 48	45	3.1	1.0	1.0
Test Item	0.0316 µL	42 33 35	37	4.7	38 32 52	41	10.3	1.1	0.9
Test Item	0.100 µL	34 51 36	40	9.3	34 47 44	42	6.8	1.2	0.9
Test Item	0.316 µL	31 49 37	39	9.2	43 43 32	39	6.4	1.1	0.9
Test Item	1.0 µL	27 25 27	26	1.2	45 45 35	42	5.8	0.8	0.9
Test Item	2.5 µL	68 29 31	43	22.0	55 41 46	47	7.1	1.3	1.1
Test Item	5.0 µL	49 34 53	45	10.0	33 45 47	42	7.6	1.3	0.9
4-NOPD	10 µg	454 480 475	470	13.8	/ / /	/	/	13.8	/
2-AA	2.5 µg	/ / /	/	/	2993 2086 2547	2542	453.5	/	56.9

SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn

Mutation factor =mean revertants (test item)/mean revertants (vehicle control)

Tester Strain: TA100                                                                                         Experiment: 1

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A.dest.		112 125 100	112	12.5	108 123 120	117	7.9	1.1	1.0
DMSO		98 102 100	100	2.0	116 111 116	114	2.9	1.0	1.0
Test Item	0.0316 µL	79 99 80	86	11.3	111 100 98	103	7.0	0.9	0.9
Test Item	0.100 µL	101 94 74	90	14.0	83 111 137	110	27.0	0.9	1.0
Test Item	0.316 µL	78 90 83	84	6.0	96 112 112	107	9.2	0.8	0.9
Test Item	1.0 µL	107 93 114	105	10.7	96 105 93	98	6.2	1.0	0.9
Test Item	2.5 µL	110 111 102	108	4.9	135 110 130	125	13.2	1.1	1.1
Test Item	5.0 µL	83 82 101	89	10.7	110 130 120	123	6.1	0.9	1.1
NaN3	10 µg	620 675 736	677	58.0	/ / /	/	/	6.8	/
2-AA	2.5 µg	/ / /	/	/	1632 2032 2235	1966	306.8	/	17.2

SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn

Mutation factor =mean revertants (test item)/mean revertants (vehicle control)

Tester Strain: TA1535                                                               Experiment: 1

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A.dest.		14 13 16	14	1.5	11 12 14	12	1.5	0.9	1.1
DMSO		19 17 11	16	4.2	9 14 11	11	2.5	1.0	1.0
Test Item	0.0316 µL	16 10 11	12	3.2	11 12 8	10	2.1	0.8	0.9
Test Item	0.100 µL	14 9 10	11	2.6	10 17 20	16	5.1	0.7	1.4
Test Item	0.316 µL	13 13 7	11	3.5	18 13 16	16	2.5	0.7	1.4
Test Item	1.0 µL	12 26 14	17	7.6	14 16 12	14	2.0	1.1	1.2
Test Item	2.5 µL	13 17 14	15	2.1	22 14 13	16	4.9	0.9	1.4
Test Item	5.0 µL	11 13 11	12	1.2	9 17 23	16	7.0	0.7	1.4
NaN3	10 µg	1026 1011 951	996	39.7	/ / /	/	/	63.6	/
2-AA	2.5 µg	/ / /	/	/	117 147 114	126	18.2	/	11.1

SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn

Mutation factor =mean revertants (test item)/mean revertants (vehicle control)

Tester Strain: TA1537                                                   Experiment: 1

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A.dest.		16 13 20	16	3.5	22 23 20	22	1.5	1.2	1.0
DMSO		10 16 14	13	3.1	18 23 22	21	2.6	1.0	1.0
Test Item	0.0316 µL	16 20 10	15	5.0	25 22 16	21	4.6	1.2	1.0
Test Item	0.100 µL	16 24 13	18	5.7	21 16 18	18	2.5	1.3	0.9
Test Item	0.316 µL	12 16 20	16	4.0	17 13 15	15	2.0	1.2	0.7
Test Item	1.0 µL	24 29 29	27	2.9	19 16 15	17	2.1	2.1	0.8
Test Item	2.5 µL	22 20 17	20	2.5	29 14 15	19	8.4	1.5	0.9
Test Item	5.0 µL	17 12 28	19	8.2	24 25 27	25	1.5	1.4	1.2
4-NOPD	10 µg	33 34 42	36	4.9	/ / /	/	/	2.7	/
2-AA	2.5 µg	/ / /	/	/	94 77 81	84	8.9	/	4.0

SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn

Mutation factor =mean revertants (test item) / mean revertants (vehicle control)

Tester Strain: TA102                                                        Experiment: 1

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A.dest.		456 455 481	464	14.7	512 505 487	501	12.9	1.0	1.1
DMSO		471 473 458	467	8.1	469 459 462	463	5.1	1.0	1.0
Test Item	0.0316 µL	60 103 252	138	100.8	482 436 487	468	28.1	0.3	1.0
Test Item	0.100 µL	/ 258 358	308	70.7	498 507 485	497	11.1	0.7	1.1
Test Item	0.316 µL	411 411 487	436	43.9	525 538 501	521	18.8	0.9	1.1
Test Item	1.0 µL	502 475 467	481	18.3	526 548 494	523	27.2	1.0	1.1
Test Item	2.5 µL	539 466 469	491	41.3	481 511 517	503	19.3	1.1	1.1
Test Item	5.0 µL	460 461 447	456	7.8	580 538 542	553	23.2	1.0	1.2
MMS	1 µg	1168 1232 1231	1210	36.7	/ / /	/	/	2.6	/
2-AA	10 µg	/ / /	/	/	1002 1073 1182	1086	90.7	/	2.3

SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn

Mutation factor =mean revertants (test item)/mean revertants (vehicle control)

Experiment II (Pre-incubation Test)

Table 3: Results Experiment II
Tester Strain: TA98                                               Experiment: 2

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A.dest.		46 35 39	40	5.6	36 30 39	35	4.6	1.1	1.2
DMSO		55 19 34	36	18.1	24 32 32	29	4.6	1.0	1.0
Test Item	0.0316 µL	23 29 32	28	4.6	31 41 43	38	6.4	0.8	1.3
Test Item	0.100 µL	17 27 45	30	14.2	48 40 46	45	4.2	0.8	1.5
Test Item	0.316 µL	34 28 34	32	3.5	38 49 40	42	5.9	0.9	1.4
Test Item	1.0 µL	21 B 30 B 30 B	27	5.2	57 52 49	53	4.0	0.8	1.8
Test Item	2.5 µL	7 B 26 B 14 B	16	9.6	64 44 57	55	10.1	0.4	1.9
Test Item	5.0 µL	0 N 0 N 0 N	0	0.0	0 B 8 B 2 B	3	4.2	0.0	0.1
4-NOPD	10 µg	477 433 377	429	50.1	/ / /	/	/	11.9	/
2-AA	2.5 µg	/ / /	/	/	968 903 1466	1112	308.0	/	37.9

SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn

Mutation factor =mean revertants (test item)/mean revertants (vehicle control)

Tester Strain: TA100                                                        Experiment: 2

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A.dest.		98 100 92	97	4.2	110 112 97	106	8.1	1.2	1.0
DMSO		71 82 92	82	10.5	101 97 110	103	6.7	1.0	1.0
Test Item	0.0316 µL	79 71 77	76	4.2	92 99 88	93	5.6	0.9	0.9
Test Item	0.100 µL	61 109 89	86	24.1	87 82 86	85	2.6	1.1	0.8
Test Item	0.316 µL	72 60 67	66	6.0	76 74 90	80	8.7	0.8	0.8
Test Item	1.0 µL	84 74 62	73	11.0	86 88 82	85	3.1	0.9	0.8
Test Item	2.5 µL	48 B 45 B 0 B	31	26.9	92 97 99	96	3.6	0.4	0.9
Test Item	5.0 µL	10 B 21 B 0 B	10	10.5	57 B 92 B 78 B	76	17.6	0.1	0.7
NaN3	10 µg	526 589 703	606	89.7	/ / /	/	/	7.4	/
2-AA	2.5 µg	/ / /	/	/	448 443 661	517	124.4	/	5.0

SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn

Mutation factor =mean revertants (test item)/mean revertants (vehicle control)

Tester Strain: TA1535                                                                  Experiment: 2

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A.dest.		15 16 6	12	5.5	14 5 8	9	4.6	0.8	0.6
DMSO		13 16 20	16	3.5	10 17 15	14	3.6	1.0	1.0
Test Item	0.0316 µL	12 10 9	10	1.5	15 7 12	11	4.0	0.6	0.8
Test Item	0.100 µL	11 10 11	11	0.6	8 12 12	11	2.3	0.7	0.8
Test Item	0.316 µL	6 8 10	8	2.0	8 8 9	8	0.6	0.5	0.6
Test Item	1.0 µL	14 8 7	10	3.8	12 17 12	14	2.9	0.6	1.0
Test Item	2.5 µL	6 B 4 B 9 B	6	2.5	17 11 12	13	3.2	0.4	1.0
Test Item	5.0 µL	0 B 0 B 0 B	0	0.0	5 B 4 B 7 B	5	1.5	0.0	0.4
NaN3	10 µg	1369 1344 1264	1326	54.8	/ / /	/	/	81.2	/
2-AA	2.5 µg	/ / /	/	/	61 83 97	80	18.1	/	5.7

SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn

Mutation factor =mean revertants (test item)/mean revertants (vehicle control)

Tester Strain: TA1537                                                                Experiment: 2

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A.dest.		14 16 15	15	1.0	19 16 22	19	3.0	1.2	1.2
DMSO		6 17 14	12	5.7	14 13 20	16	3.8	1.0	1.0
Test Item	0.0316 µL	17 12 5	11	6.0	10 12 14	12	2.0	0.9	0.8
Test Item	0.100 µL	12 8 12	11	2.3	22 14 23	20	4.9	0.9	1.3
Test Item	0.316 µL	14 9 7	10	3.6	13 9 21	14	6.1	0.8	0.9
Test Item	1.0 µL	14 16 12	14	2.0	21 19 11	17	5.3	1.1	1.1
Test Item	2.5 µL	9 B 13 B 16 B	13	3.5	19 10 22	17	6.2	1.0	1.1
Test Item	5.0 µL	13 B 7 B 15 B	12	4.2	8 B 9 B 8 B	8	0.6	0.9	0.5
4-NOPD	40 µg	110 134 134	126	13.9	/ / /	/	/	10.2	/
2-AA	2.5 µg	/ / /	/	/	85 85 82	84	1.7	/	5.4

SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn

Mutation factor =mean revertants (test item)/mean revertants (vehicle control)

Tester Strain: TA102                                                               Experiment: 2

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A.dest.		421 379 370	390	27.2	444 443 450	446	3.8	1.0	1.0
DMSO		385 399 417	400	16.0	412 439 448	433	18.7	1.0	1.0
Test Item	0.0316 µL	340 306 303	316	20.6	389 384 406	393	11.5	0.8	0.9
Test Item	0.100 µL	417 351 368	379	34.3	443 419 458	440	19.7	0.9	1.0
Test Item	0.316 µL	420 447 456	441	18.7	411 359 394	388	26.5	1.1	0.9
Test Item	1.0 µL	361 363 410	378	27.7	343 333 356	344	11.5	0.9	0.8
Test Item	2.5 µL	315 277 296	296	19.0	317 329 312	319	8.7	0.7	0.7
Test Item	5.0 µL	288 B 333 B 265 B	295	34.6	356 395 411	387	28.3	0.7	0.9
MMS	1 µg	1630 1522 1396	1516	117.1	/ / /	/	/	3.8	/
2-AA	10 µg	/ / /	/	/	598 1216 1204	1006	353.4	/	2.3

SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn

Mutation factor =mean revertants (test item)/mean revertants (vehicle control)

 

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, 2-MCH did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, 2-MCH is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In order to investigate the potential of 2-MCH for its ability to induce gene mutations the plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed with the Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102. In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments: 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate.

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiment I

In experiment II toxic effects of the test item were noted at concentrations of 1.0 µL/plate and higher (without metabolic activation) and at a concentration of 5.0 µL/plate (with metabolic activation), depending on the particular tester strain.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with 2-MCH at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

All criteria of validity were met.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

As the substance was found being negative in three independent in vitro genotoxicity studies (OECD471, OECD478, OECD490), 2-methycyclohexanone is not subject to classification according to Regulation EC No. 1272/2008, Annex 1, section 3.5.