2-methylcyclohexyl acetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1991

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Menthol

EC Number:

201-939-0

EC Name:

Menthol

Cas Number:

89-78-1

IUPAC Name:

2-isopropyl-5-methylcyclohexanol

Details on test material:

- Name of test material (as cited in study report): dl-menthol
- Molecular formula (if other than submission substance): C10H20O
- Molecular weight (if other than submission substance): 156.26

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 from liver of Aroclor-induced male Sprague-Dawley rat

Test concentrations with justification for top dose:

+ and -S9: 0, 12.5, 25, 50, 75, 100, 150, 200, and 300 ug/ml

Vehicle / solvent:

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

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

(ethanol)

Controls 2

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

(without metabolic activation)

Controls 3

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

(with metabolic activation)

Details on test system and experimental conditions:

Two trials (3 replicates/concentration) were conducted for each: non-activated cultures and activated cultures. RPMI 1640 medium used for growth, expression and cloning. Ethanol used as vehicle control.

METHOD OF APPLICATION: Mouse lymphoma L5178Y TK+/- cells were maintained at 37° C as suspension cultures in Fischer's medium supplemented with 2 mM l-glutamine, 110 ug/mL sodium pyruvate, 0.05% luronic F68, antibiotics, and heatinactivated horse serum; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring trifluorothymidine (TFT) resistant cells, subcultures were exposed once to medium containing thymidine, hypoxanthine, methotrexate, and glycine for one day; to thymidine, hypoxanthine, and glycine for one day; and to normal medium for 3 to 5 days. For cloning, horse serum content was increased and Noble agar was added.
All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Treated cultures contained 6 x 10 6 cells in 10 mL of medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with the test chemical continued for 4 hours, at which time the medium plus chemical was removed and the cells were resuspended in 20 mL of fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, 3 x 106 cells were plated in medium and soft agar supplemented with TFT for selection of TFT-resistant cells (TK-/-) and in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37 C. in 5% CO2 for 10 to 12 days. At the end of incubation, colonies were counted with an automated counter. The test was initially performed without S9. If a clearly positive response was not obtained, the test was repeated using freshly prepared S9 from the livers of either Aroclor 1254-induced or non-induced male Fischer 344 rats.

Evaluation criteria:

Trend and peak responses had to be significant (P < 0.05) for a chemical to be considered capable of inducing TFT resistance; a single significant response led to a "questionable" conclusion, and the absence of both a trend and a peak response resulted in a "negative" call.

Statistics:

All data were evaluated statistically for trend and peak responses.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at >= 200 µg/ml)

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Test substance was cytotoxic at 200 µg/ml and higher, with or without S9.

Without S9,

Trial 1 at 0, 12.5, 25, 50, 100, and 150 µg/ml: 37, 36, 27, 38, 29, and 33 mutant cells/10E+06 cells

Trial 2 at 0, 25, 50, 75, 100, and 150 µg/ml: 27, 30, 24, 19, 28, and 29 mutant cells/10E+06 cells

With S9,

Trial 1 at 0, 25, 50, 75, 100, 150, and 200 µg/ml: 35, 42, 43, 45, 33, 33, and 35 mutant cells/10E+06 cells

Trial 2 at 0, 25, 50, 75, 100, and 150 µg/ml: 48, 54, 58, 53, 64, and 37 mutant cells/10E+06 cells

 

Nonactivation Trial: 1 Experiment Call: Negative
		Conc.	Cloning	Relative Total	Mutant Colonies	Mutant Frequency	AVG Mutant Frequency
		µg/mL	Efficiency	Growth
Vehicle Control	Ethanol	0	77	96	65	28	37
			96	94	134	47
			77	100	75	32
			78	110	95	41
Test Chemical	DL-menthol	12.5	68	91	83	41	36
			70	95	76	36
			72	99	68	31
		25	68	27	51	25	27
			83	108	83	33
			87	78	59	23
		50	74	71	74	33	38
			56	88	78	46
			69	56	73	35
		100	100	67	87	29	29
			74	65	62	28
			97	68	88	30
		150	68	25	59	29	33
			83	24	91	37
			LETHAL
		200	LETHAL
			LETHAL
			LETHAL
Positive Control	Methyl Methane Sulfonate	5	40	34	480	400	488*
			33#	29	467	472
			37	16	659	591

 

 

Nonactivation Trial: 2 Experiment Call: Negative
		Conc.	Cloning	Relative Total	Mutant Colonies	Mutant Frequency	AVG Mutant Frequency
		µg/mL	Efficiency	Growth
Vehicle Control	Ethanol	0	61	82	45	24	27
			97#	105	80	27
			98	103	77	26
			94	110	86	30
Test Chemical	DL-menthol	25	73	69	73	33	30
			64	56	71	37
			76	84	44	19
		50	81	55	71	29	24
			83	73	43	17
			78	71	60	26
		75	102	54	64	21	19
			91	63	54	20
			82	28	39	16
		100	85	43	73	29	28
			81	34	73	30
			88	54	70	26
		150	91	21	95	35	29
			81	30	60	25
			89	29	76	29
Positive Control	Methyl Methane Sulfonate	5	65	38	602	310	274*
			67	43	523	260
			77	53	576	251

 

 

Induced S9 Trial: 1 Experiment Call: Negative
		Conc.	Cloning	Relative Total	Mutant Colonies	Mutant Frequency	AVG Mutant Frequency
		µg/mL	Efficiency	Growth
Vehicle Control	Ethanol	0	102	112	91	30	35
			81	108	88	36
			71	95	62	29
			63	84	88	47
Test Chemical	DL-menthol	25	73	92	74	34	42
			95	93	108	38
			58	65	93	53
		50	73	71	111	50	43
			71	76	88	41
			76	94	88	39
		75	76	68	109	48	45
			79	73	93	39
			72	80	102	47
		100	75	82	87	39	33
			89	81	93	35
			110	89	84	26
		150	75	71	78	35	33
			79	66	74	31
			107	144	104	32
		200	95	52	122	43	35
			96	47	79	27
			LETHAL
		300	LETHAL
			LETHAL
			LETHAL
Positive Control	3-Methylcho-lanthrene	2.5	105	71	390	124	145*
			84	315	315	125
			84	468	438	186

 

 

Induced S9 Trial: 2 Experiment Call: Negative
		Conc.	Cloning	Relative Total	Mutant Colonies	Mutant Frequency	AVG Mutant Frequency
		µg/mL	Efficiency	Growth
Vehicle Control	Ethanol	0	110	115	124	38	46
			122r	115	97	27
			84	96	136	54
			84	88	113	45
Test Chemical	DL-menthol	25	98	78	162	55	54
			100	62	159	53
			101	79	165	54
		50	109	56	174	53	58
			84	60	146	58
			104	66	197	63
		75	122r	65	194	53	53
			99	60	187	63
			83	52	106	43
		100	118	44	237	67	64
			104	70	160	51
			113	51	249	73
		150	115	46	125	36	37
			110	64	125	38
			111	46	126	38
		200	LETHAL
			LETHAL
			LETHAL
Positive Control	3-Methylcho-lanthrene	2.5	66	32	398	201	271*
			64	38	579	303
			73	33	676	310

               

 

Footnotes:

Asterisks(*) indicate significant responses.

r = rejected value due to quality control criteria

# = reduced sample size because of the loss of one culture dish due to contamination                                   

                                                                                                             

Applicant's summary and conclusion

Conclusions:

2-Isopropyl-5-methylcyclohexanol did not increase mutation frequency in mouse lymphoma cells at test conditions.

Executive summary:

A Forward mutation assay was performed in mouse lymphoma cells with 2 -isopropyl-5 -methylcyclohexanol at a concentration of 0, 12.5, 25, 50, 75, 100, 150, 200 and 300 µg/ml, with and without S9 metabolic activation. Two trials (3 replicates/concentration) were conducted for each: non-activated cultures and activated cultures. RPMI 1640 medium was used for growth, expression and cloning. Ethanol was used as vehicle control. Test substance was cytotoxic at 200 ug/ml and higher with or without S9. 2-Isopropyl-5-methylcyclohexanol did not increase mutation frequency in mouse lymphoma cells at test conditions.