2-methylcyclohexyl acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Weight of evidence: In vitro gene mutation in bacteria: Based on the read-across approach from experimental results on analogue 4-tert-butylcyclohexyl acetate, cylcohexyl acetate and 2 -isopropyl-5-methylcyclohexanol (test methods similar to OECD 471), 2-methylcyclohexyl acetate was determined to be non-mutagenic.

Weight of evidence: in vitro cytogenicity in mammalian cells: Weight of evidence: Based on the read-across approach from experimental results on the analogue substance 2 -isopropyl-5-methylcyclohexanol (test method similar to OECD 473), 2-methylcyclohexyl acetate was determined to be negative for chromosome aberrations.

Key study: In vitro gene mutation in mammalian cells. Based on the read-across approach from experimental results on the analogue substance 2 -isopropyl-5-methylcyclohexanol (test method similar to OECD 476), 2-methylcyclohexyl acetate was determined to be non-mutagenic.

Weight of evidence: In vivo cytogenicity in mammalian cells. Based on the read-across approach from experimental results on the analogue substance 2 -isopropyl-5-methylcyclohexanol (test methods similar to OECD 474 and 475), 2-methylcyclohexyl acetate was determined to be negative for in-vivo chromosome aberrations and micronucleus tests.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(no data on controls)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

~0.0024-9.4 µg/plate.

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(with and without metabolic activation)

Cytotoxicity / choice of top concentrations:

not specified

No evidence of mutagenicity was observed at test conditions for 4-tert-butylcyclohexyl acetate.

Conclusions:

No evidence of mutagenicity was observed at test conditions for 4-tert-butylcyclohexyl acetate.

Executive summary:

An Ames test was conducted with Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 with and without metabolic S9 activation using 4-tert-butylcyclohexyl acetate at doses of 0.0024–9.4 µg/plate. No evidence of mutagenicity was observed.

Reference 2

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1978

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

unsuitable test system

Remarks:

Rec-assay in Bacillus subtilis in H17 and M45. Test method not reliable for full assessment of genetic toxicity in bacteria.

Principles of method if other than guideline:

A Rec assay using Bacillus subtilis type H17 and M45 with 19 µg/disc of cyclohexyl acetate in dimethyl sulfoxide was performed to detect DNA damaging activity by differences in growth inhibition zones (D (mm) = M45-H17).

GLP compliance:

no

Type of assay:

Bacillus subtilis recombination assay

Species / strain / cell type:

bacteria, other: Bacillus subtilis type H17 and M45

Metabolic activation:

not specified

Test concentrations with justification for top dose:

19 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide

Evaluation criteria:

The evaluation criteria for DNA damage activity was as follows:
Negative (-) when the differences in growth inhibition zones (D (mm) = M45-H17): D < 2 mm
Positive (+) when the differences in growth inhibition zones (D (mm) = M45-H17): D >= 2 mm
Positive (++) when the differences in growth inhibition zones (D (mm) = M45-H17): D >= 5 mm

Species / strain:

bacteria, other: Bacillus subtilis type H17 and M45

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Additional information on results:

Not mutagenic activity was observed since the difference between the inhibition zones (D) was < 2 mm.

No mutagenic activity was observed.

The results were as follows:

Maximal dose per disk	Inhibition zone (mm)			Conclusion
	M45	H17	D
29 µl	0.3	0	0.3	Negative

Conclusions:

No mutagenic activity was observed at test conditions for cyclohexyl acetate.

Executive summary:

No mutagenic activity was observed in Rec assay using Bacillus subtilis type H17 and M45 with 19 µg/disc of cyclohexyl acetate in dimethyl sulfoxide.

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1986

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Rec-assay in Bacillus subtilis in H17 and M45. Test method not reliable for full assessment of genetic toxicity in bacteria.

Principles of method if other than guideline:

A Rec assay using Bacillus subtilis type H17 and M45 with 20 µg/disc of cyclohexyl acetate in dimethyl sulfoxide was performed to detect DNA damaging activity by differences in growth inhibition zones (D (mm) = M45-H17).

GLP compliance:

no

Type of assay:

Bacillus subtilis recombination assay

Species / strain / cell type:

bacteria, other: Bacillus subtilis type H17 and M45

Metabolic activation:

not specified

Test concentrations with justification for top dose:

20 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide

Evaluation criteria:

The evaluation criteria for DNA damage activity was as follows:
Negative (-) when differences in growth inhibition zones (D (mm) = M45-H17): D < 4.
Positive (+) when differences in growth inhibition zones (D (mm) = M45-H17): 4 <= D < 8.
Positive (++) when diferences in growth inhibition zones (D (mm) = M45-H17): 8 <= D < 12.
Positive (++) when differences in growth inhibition zones (D (mm) = M45-H17): 8 <= D
Equivocal when no growth inhibition in both strains

Species / strain:

bacteria, other: Bacillus subtilis type H17 and M45

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Additional information on results:

No activity for DNA damage was observed since the difference between the inhibition zones (D) was < 4 mm.

No mutagenic activity was observed.

The results were as follows:

Maximal dose per disk	Inhibition zone (mm)			Conclusion
	M45	H17	D
20 µl	11	11	0	Negative

Conclusions:

No mutagenic activity was observed at test conditions for cyclohexyl acetate.

Executive summary:

No mutagenic activity was observed in Rec assay using Bacillus subtilis type H17 and M45 with 20 µg/disc of cyclohexyl acetate in dimethyl sulfoxide.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(no data on controls)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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:

S9

Test concentrations with justification for top dose:

33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA102, TA1535 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(with and without metabolic activation, in all doses)

Cytotoxicity / choice of top concentrations:

not specified

No evidence of mutagenicity was observed.

Conclusions:

No evidence of mutagenicity was observed at test conditions for 2-(1-methylpropyl)-1-vinylcyclohexyl acetate.

Executive summary:

No mutagenic activity was observed in an Ames test using the plate incorporation and the pre-incubation test conducted on Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 with and without S9 activation. The following concentrations were tested: 33, 100, 333, 1000, 2500 and 5000 µg/plate in dimethyl sulfoxide. This was a GLP study.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1982

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(3 strains tested)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA100, TA2637, TA98

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0.005, 0.01, 0.02, 0.05, 0.1, 0.2, and 0.5 mg/plate

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Remarks:

Dose: 100 µl/plate

Positive controls:

yes

Remarks:

TA100, TA 98 without S9 mix

Positive control substance:

other: AF2

Remarks:

Dose: 0.00002 mg/plate

Positive controls:

yes

Remarks:

TA2637 without S9 mix

Positive control substance:

9-aminoacridine

Remarks:

Dose: 0.2 mg/plate

Positive controls:

yes

Remarks:

TA100, TA2637, TA98 with S9 mix

Positive control substance:

other: 2-aminoanthracene

Remarks:

Dose: 0.05 mg/plate

Species / strain:

S. typhimurium, other: TA100, TA2637, TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(0.5 mg/plate for all strains +S9 and - S9; 0.2 mg/plate for TA100 -S9)

Vehicle controls validity:

valid

Positive controls validity:

valid

No increase in his+ revertant frequency at any concentration tested.

The results are included in the table below:

Compound	Dose mg/plate	TA100		TA2637		TA98
		-S9mix	+S9mix	-S9mix	+S9mix	-S9mix	+S9mix
dl-Menthol	0.005	109	121	42	38	36	27
	0.01	90	161	35	30	30	39
	0.02	104	165	32	29	36	44
	0.05	150	140	23	37	32	42
	0.1	96	159	20	32	16	29
	0.2	LE	82	7	10	9	28
	0.5	LE	LE	LE	LE	LE	LE
DMSO	100 µl	149	103	19	27	27	35
AF2	0.00002	1024	-	-	-	215	-
9-Aminoacridine	0.2	-	-	1316	-	-	-
2-Aminoanthracene	0.05	-	3458	-	369	-	2871

LE: Lethal effect in background lawn.

-: Not tested

Conclusions:

No evidence of mutagenicity was observed at test conditions for 2-Isopropyl-5-methylcyclohexanol.

Executive summary:

An Ames Test was carried out with 2 -isopropyl-5 -methylcyclohexanol for 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, and 0.5 mg/plate doses. Test item was not mutagenic when tested in Salmonella typhimurium strains TA100, TA2637 or TA98 with or without metabolic activation. Cytotoxicity was observed at 0.5 mg/plate for all strains and at 0.2 mg/plate for TA100 (-S9).

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(only 4 strains were tested)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA100, TA1535, TA97, and TA98

Metabolic activation:

with and without

Metabolic activation system:

S9 from liver of Aroclor-induced male Sprague-Dawley rat and male Syrian hamster

Test concentrations with justification for top dose:

3, 10, 33, 100, 166, 333, and 666 µg/plate

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

Positive controls:

yes

Positive control substance:

9-aminoacridine

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: Standard NTP protocol (preincubation). Test tube containing a suspension of each strain of Salmonella typhimurium plus S9 mix or plain buffer without S9, is incubated for 20 minutes at 37º C with the test chemical. Control cultures, with all the same ingredients except the test chemical, are also incubated. In addition, positive control cultures are prepared. After 20 minutes, agar is added to the cultures and the contents of the tubes are thoroughly mixed and poured onto the surface of Petri dishes containing standard bacterial culture medium. The plates are incubated, and bacterial colonies that do not require an excess of supplemental histidine appear and grow. These colonies are comprised of bacteria that have undergone reverse mutation to restore function of the histidine -manufacturing gene. The number of colonies is usually counted after 2 days.

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

The test chemical is mutagenic to any particular strain of bacterium, if the number of histidine-independent colonies arising on those plates is significantly greater than the corresponding control plates.

Key result

Species / strain:

S. typhimurium, other: TA100, TA1535, TA97, and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

No increase seen in number of his+ revertants with or without any type/concentration of S9 at any of the concentrations tested. Strain:

Dose	No Activation		No Activation		10% HLI		30% HLI		10% RLI		30% RLI
	(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)
ug/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
Strain: TA100
0	121	9	161	6.6	113	7.2	167	3.8	133	7.4	172	6.1
1	122	9.6
3	127	7.3	158	8.3	131	8.8			147	6.9
10	112	5.3	142	6.2	118	7	134	20.6	141	2.6	169	8.1
33	108	4.1	142	4.1	123	4.5	155	13.5	136	1.8	173	5.7
100	107	16	127	2.2	125	8.8	150	18.5	144	4.6	165	8.2
166			115	9.7
333					98	9	129	2.6	117	10.4	132	14.9
666							112	6.9			126	16.7
Positive Control	455	47.7	425	17.1	1344	103.2	1074	17.1	486	10.2	319	23.8
Strain: TA1535
0	22	1.8	19	1.2	12	3.3	13	2	8	1.7	15	1.5
1	23	3.5
3	18	1.2	16	1.2	11	2.8			13	1.9
10	22	2.3	21	1.7	15	0.3	11	1.8	9	1.9	12	1.8
33	20	1	16	0.6	13	1	8	2	11	1.2	13	0.9
100	18	0.9	16	0.9	9	1.8	14	0.3	12	3.2	14	0.9
166			16	2.5
333					6	2.8	13	2.8	8	2.3	12	1.5
666							10	1.9			13	2
Positive Control	332	18.8	473	30.2	448	20.8	397	16	158	13.1	201	22
Strain: TA97
0	134	4	178	5.5	151	5	194	3.8	185	8.7	202	3.8
1	132	7.5
3	124	0.6	188	10.1	169	4.1			195	3.2
10	130	11.1	180	1.7	163	6.9	171	15.7	184	14.1	206	1.5
33	123	14	175	4.6	169	3.7	190	7.4	186	6.5	204	2.2
100	126	12.3	195	5.6	167	11.5	185	5.5	170	1.7	194	4.5
166			168	10.7
333					141	4	123s	7.2	145	10	139s	13.2
666							139s	5			144s	18.1
Positive Control	1229	27.9	1496	76.9	1519	14.7	1144	31.5	958	38.2	468	10.3
Strain: TA98
0	21	2.1	25	2.4	31	1.9	27	1.8	37	5.2	29	0.3
1	17	1.2
3	21	1.7	20	0.3	38	3			30	1.8
10	18	0.7	29	5.1	38	4.3	29	0.9	36	5.7	32	0.3
33	18	1.2	27	0.9	40	1.9	30	0.9	33	1.9	32	4.5
100	21	2.8	26	2.6	35	3	25	3.8	26	0.6	28	1.2
166			25	0.3
333					27	0.3	19	4.4	32	2.4	26	2.4
666							15s	1.8			18s	2.1
Positive Control	1437	25.8	1587	35.7	1367	25.7	510	17.6	297	21.7	233	18.5

Abbreviations:

RLI = induced male Sprague Dawley rat liver S9

HLI = induced male Syrian hamster liver S9

s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; T = Toxic; c = Contamination

Conclusions:

No evidence of mutagenicity was observed at test conditions for 2-Isopropyl-5-methylcyclohexanol.

Executive summary:

An Ames Test was carried out with 2 -isopropyl-5 -methylcyclohexanol for 0, 3, 10, 33, 100, 166, 333, and 666 µg/plate doses. Test item was not mutagenic when tested in Salmonella typhimurium strains TA100, TA1535, TA97, or TA98 with or without metabolic activation.

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(no data on controls)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94, and TA98

Details on mammalian cell type (if applicable):

All stains were provided by Dr B. N. Ames, University of California, Berkeley, USA.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from liver of PCB-induced Fischer rats

Test concentrations with justification for top dose:

6 different concentrations. Maximum dose = 5.0 mg/plate

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Remarks:

(Dimethylsulphoxide)

Details on test system and experimental conditions:

Overnight cell cultures were preincubated at 37 °C with the test chemical and S9 for 20 minutes prior to plating.
Six concentrations of the test chemical were tested in duplicate.
The number of revertants was scored after the plates were incubated for 2 days at 37 °C.

Evaluation criteria:

The result was considered positive if the number of colonies found was twice the number in the control.

Key result

Species / strain:

S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94, and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

No increase in the number of revertants.

Conclusions:

No evidence of mutagenicity was observed at test conditions for 2-Isopropyl-5-methylcyclohexanol.

Executive summary:

An Ames Test was carried out with 2 -isopropyl-5 -methylcyclohexanol for 6 different concentrations with a maximum dose of 5.0 mg/plate. Test item was not mutagenic when tested in Salmonella typhimurium strains TA92, TA1535, TA100, TA1537, TA94, or TA98 with metabolic activation.

Reference 8

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Heparinized peripheral blood samples were obtained from 12 male and 12 female adult human volunteers. Lymphocytes from whole blood were isolated by the Ficoll-gradient method. About 0.5-1.0 x 10E+06 isolated lymphocytes were cultured in RPM1 1640 medium supplemented with 2 mM-L-glutamine, 100 µg penicillin/ml, 100 µg streptomycin/ml, 10% foetal calf serum and 1% phytohaemagglutinin.

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:

0.1, 1.0, 10 mM

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO, with and without S9)

Positive controls:

yes

Remarks:

(1 x 10E-07 M)

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: All cultures were incubated in the dark at 37°C for 72 hr. Following 1 hr of exposure of the cells to colchicine (0.3 µg/ml), the slides were prepared. Chromosomal aberrations were scored in 100 metaphase cells from each donor according to the classification criteria suggested by Savage (1975).

OTHER: 10mM-menthol was chosen as the highest concentration because higher concentrations significantly affected the growth of human lymphocytes in phytohaemagglutinin-stimulated cultures.

Statistics:

Statistical significance tested by the chi-square test.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Menthol does not induce chromosomal aberrations.

Menthol-induced chromosomal aberrations in cultured human lymphocytes:

Experiment	No. of donors	Total no. of cells	Polyploid cells		Structural aberrations
			No.	No. per 100 cells	No.	No. per 100 cells
Solvent control (DMSO)	24	2621	7	0.27	46	1.76
Solvent control (DMSO + S9)	24	1551	5	0.20	51	2.00
Positive control (MMC)	5	646	6	0.93*	59	9.13*
0.1 mM - S9	24	2690	6	0.22^NS	51	1.90^NS
0.1 mM + S9	24	2564	7	0.27^NS	52	2.03^NS
1 mM – S9	24	2590	7	0.27^NS	56	2.16^NS
1 mM + S9	24	2525	6	0.24^NS	51	2.02^NS
10 mM – S9	24	2558	5	0.20^NS	54	2.11^NS
10 mM + S9	24	2538	5	0.20^NS	57	2.25^NS

NS: not significantly different from corresponding controls.

* P > 0.001

Conclusions:

2-Isopropyl-5-methylcyclohexanol did not induce chromosomal aberrations in human lymphocytes at test conditions.

Executive summary:

An in-vitro chromosome aberration test in human lymphocytes was carried out with 2 -isopropyl-5 -methylcyclohexanol for 0.1, 1.0, 10 mM with and without metabolic activation (S9). Solvent control was DMSO and positive control was MMC. Total structural aberrations (no./100 cells) for DMSO, DMSO+S9, MMC, 0.1 mM, 0.1 mM+S9, 1.0 mM, 1.0 mM+S9, 10 mM, and 10 mM+S9 was 1.76, 2.00, 9.13, 1.90, 2.03, 2.18, 2.02, 2.11 and 2.23, respectively. 2-Isopropyl-5-methylcyclohexanol did not induce chromosomal aberrations in human lymphocytes at test conditions.

Reference 9

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Heparinized peripheral blood samples were obtained from 12 male and 12 female adult human volunteers. Lymphocytes from whole blood were isolated by the Ficoll-gradient method. Lymphocytes from whole blood were isolated by the Ficoll-gradient method. About 0.5-1.0 x 10E+06 isolated lymphocytes were cultured in RPM1 1640 medium supplemented with 2 mM-L-glutamine, 100 µg penicillin/ml, 100 µg streptomycin/ml, 10% foetal calf serum and 1% phytohaemagglutinin. All cultures also contained 5-bromodeoxyuridine at a final concentration of 10 g M from the start of culture.

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:

0.1, 1.0, 10 mM

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO, with and without S9).

Positive controls:

yes

Remarks:

(1 x 10E-08 M)

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

Lymphocytes were obtained from healthy donors (12 of each sex) and cultured at approximately 0.5-1.0E6 isolated cells. All cultures contained BrdU and metaphase preparations were stained with Hoechst-Giemsa stain. From each culture, a minimum of 25 2nd-division cells were scored and the mean no. of SCEs/cell were compared with solvent controls.

OTHER: 10mM-menthol was chosen as the highest concentration because higher concentrations significantly affected the growth of human lymphocytes in phytohaemagglutinin-stimulated cultures.

Statistics:

The mean SCE/cell values between menthol- and solvent-treated cultures were compared for statistical significance by Student's t-test.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Menthol does not induce SCEs in human chromosomes.

Menthol-induced SCEs in cultured human lymphocytes:

Experiment	No. of donors	Total no. of cells	No. of SCE/cell
			Mean ±SEM	Range
Solvent control (DMSO)	24	624	7.90±0.45	0-19
Solvent control (DMSO + S9)	24	624	8.00±0.69	0.20
Positive control (MMC)	5	135	21.46±3.02*	6-49
0.1 mM - S9	24	636	7.39±0.47^NS	0-20
0.1 mM + S9	24	640	8.25±0.56^NS	0-19
1 mM – S9	24	657	8.00±0.47^NS	0-22
1 mM + S9	24	648	8.10±0.41^NS	0.22
10 mM – S9	24	650	8.50±0.32^NS	0.19
10 mM + S9	24	655	8.60±0.48^NS	0.16

NS = Not significantly different from corresponding controls (Student's t-test).

*P > 0.001

Conclusions:

2-Isopropyl-5-methylcyclohexanol did not affect the frequency of SCEs in human lymphocytes at test conditions.

Executive summary:

An in-vitro Sister Chromatid Exchange test in human lymphocytes was carried out with 2 -isopropyl-5 -methylcyclohexanol for 0.1, 1.0, 10 mM with and without metabolic activation (S9). Solvent control was DMSO and positive control was mitomycin C (MMC). Total no. of SCEs/cell (mean) for DMSO, DMSO+S9, MMC, 0.1 mM, 0.1 mM+S9, 1.0 mM, 1.0 mM+S9, 10 mM, and 10 mM+S9 was 7.90, 8.00, 21.46, 7.39, 8.25, 8.00, 8.10, 8.50, and 8.60, respectively. 2-Isopropyl-5-methylcyclohexanol did not affect the frequency of SCEs in human lymphocytes.

Reference 10

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Principles of method if other than guideline:

Method: Sister Chromatid Exchange (Galloway et al. 1985, 1987)

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

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:

Trial 1: 0, 5, 16.7, or 50 ug/ml
Trial 2: 0, 2.5, 5, 10, or 25 ug/ml
Trial 3: 0, 16.7, 50, or 167 ug/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl Sulfoxide

Negative solvent / vehicle controls:

yes

Remarks:

(dimethyl sulfoxide)

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

(0.001 and 0.01 µg/mL)(without metabolic activation)

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

(0.4 and 2 µg/mL)(with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: In the SCE test without S9, CHO cells were incubated with the test chemical for 26 hours in supplemented McCoy’s 5A medium. Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation. After 26 hours, the medium containing the chemical was removed and replaced with fresh medium plus BrdU and Colcemid, and incubation was continued for 2 hours. Cells were then harvested by mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa. In the SCE test with S9, cells were incubated with the test chemical, serum-free medium, and S9 for 2 hours. The medium was then removed and replaced with medium containing serum and BrdU and no test item. Incubation proceeded for an additional 26 hours, with Colcemid present for the final 2 hours. Harvesting and staining were the same as for cells treated without S9. Up to 50 second-division metaphase cells were scored for frequency of SCEs per cell from each dose level.

Evaluation criteria:

An SCE frequency 20% above the concurrent solvent control value was chosen as a statistically conservative positive response. The probability of this level of difference occurring by chance at one dose point is less than 0.01; the probability for such a chance occurrence at two dose points is less than 0.001. An increase of 20% or greater at any single dose was considered weak evidence of activity; increases at two or more doses indicated that the trial was positive. A statistically significant trend (P<0.005) in the absence of any responses reaching 20% above background led to a call of equivocal.

Statistics:

Statistical analyses were conducted to assess the presence of a dose-response (trend test) and the significance of the individual dose points compared to the vehicle control.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

other: questionable (trial 2, without metabolic activation)

Trial 1 (-S9) at 0, 5, 16.7, and 50 µg/ml: 7.72, 9.34. 8.06, and 7.94.

Trial 2 (-S9) at 0, 2.5, 5, 10, and 25 µg/ml: 8.36, 7.78, 9.02, 9.75, and 10.04.

Trial 3 (+S9) at 0, 16.7, 50, and 167 µg/ml: 8.42, 8.10, 8.74, and 8.48.

In trial 1, a less than 20% increase in SCEs was reported at the low dose only. In trial 2, none of the concentrations showed an increase over 20%, but the test was considered equivocal based on a positive trend. No effects were reported in the presence of S9 in trial 3.

Trial #:1   Activation: No Activation   Date: 07/17/1985   Trial Call: Negative
		Dose µg/mL	Number cells examined	Number of chromosome examined	Total Number SCEs	SCE / Chrom.	SCE / Cell	Hours in BRDU	% Increase over solvent control
Vehicle Control	Dimethyl Sulfoxide		50	1020	386	0.378	7.72	26.0	0.000
Test Chemical	DL-menthol	5	50	1029	467	0.454	9.34	26.0	19.926
		16.7	50	1022	403	0.394	8.06	26.0	4.200
		50	50	1023	397	0.388	7.94	26.0	2.548
		167	0	0	0	0.000	0.00	26.0	N/A
Positive Control	Mitomycin-C	0.001	50	1017	649	0.638	12.98	26.0	68.631
		0.01	5	100	178	1.780	35.60	26.0	370.363
	Trend				-0.348
	Probability				0.636

                              

 

Trial #:2   Activation: No Activation   Date: 08/07/1985   Trial Call: Questionable
		Dose µg/mL	Number cells examined	Number of chromosome examined	Total Number SCEs	SCE / Chrom.	SCE / Cell	Hours in BRDU	% Increase over solvent control
Vehicle Control	Dimethyl Sulfoxide		50	1022	418	0.409	8.36	26.0	0.000
Test Chemical	DL-menthol	2.5	50	1016	389	0.383	7.78	26.0	-6.388
		5	50	1025	451	0.440	9.02	26.0	7.579
		10	50	1000	468	0.468	9.36	26.0	14.425
		25	50	1026	502	0.489	10.04	26.0	19.627
Positive Control	Mitomycin-C	0.001	50	1027	544	0.530	10.88	26.0	29.510
		0.01	5	105	158	1.505	31.60	26.0	267.911
	Trend				3.722
	Probability				0.000

               

 

Trial #:1   Activation: Induced Rat Liver S9   Date: 07/17/1985   Trial Call: Negative
		Dose µg/mL	Number cells examined	Number of chromosome examined	Total Number SCEs	SCE / Chrom.	SCE / Cell	Hours in BRDU	% Increase over solvent control
Vehicle Control	Dimethyl Sulfoxide		50	1011	421	0.416	8.42	26.0	0.000
Test Chemical	DL-menthol	16.7	50	1020	405	0.397	8.10	26.0	-4.649
		50	50	1004	437	0.435	8.74	26.0	4.524
		167	50	1028	423	0.411	8.46	26.0	-1.186
		500	0	0	0	0.000	0.00	26.0	N/A
Positive Control	Mitomycin-C	0.4	50	1035	663	0.641	13.26	26.0	53.830
		2	5	97	198	2.041	39.60	26.0	390.188
	Trend				0.237
	Probability				0.406

 

Conclusions:

2-Isopropyl-5-methylcyclohexanol was determined to be negative in the SCE assay.

Executive summary:

An in-vitro Sister Chromatid Exchange test in Chinese hamster ovary cells was carried out with 2 -isopropyl-5 -methylcyclohexanol concentrations of: Trial 1: 0, 5, 16.7, or 50 µg/ml; Trial 2: 0, 2.5, 5, 10, or 25 µg/ml; and Trial 3: 0, 16.7, 50, or 167 µg/ml. Positive controls ere mitomycin C and cyclophosphamide. SCE/cell no were observed to be: Trial 1 (-S9) at 0, 5, 16.7, and 50 ug/ml: 7.72, 9.34. 8.06, and 7.94; Trial 2 (-S9) at 0, 2.5, 5, 10, and 25 ug/ml: 8.36, 7.78, 9.02, 9.75, and 10.04; and Trial 3 (+S9) at 0, 16.7, 50, and 167 ug/ml: 8.42, 8.10, 8.74, and 8.48. In trial 1, a less than 20% increase in SCEs was reported at the low dose only. In trial 2, none of the concentrations showed an increase over 20%, but the test was considered equivocal based on a positive trend. No effects were reported in the presence of S9 in trial 3. 2-Isopropyl-5-methylcyclohexanol did not induce SCE in Chinese hamster ovary cells.

Reference 11

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

(negative control not specified, no replicates)

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

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:

Trial 1: 100, 150, or 200 µg/ml
Trial 2: 50, 125, or 250 µg/ml

Vehicle / solvent:

Dimethyl sulfoxide

Untreated negative controls:

yes

Remarks:

(not specified)

Negative solvent / vehicle controls:

yes

Remarks:

(dimehtyl sulfoxide)

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

(0.15 and 0.5 µg/mL) (without metabolic activation)

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

(7.5 and 37.5 µg/mL) (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: In the test without S9, cells were incubated in McCoy’s 5A medium with test item for 8-12 hours. Colcemid was added and incubation continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the test with S9, cells were treated with test item and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 10 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9.
Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). Up to 200 first-division metaphase cells were scored at each dose level. The classes of aberrations include simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

Evaluation criteria:

For a single trial, a statistically significant (P>=0.05) difference for one dose point and a significant trend (P>=0.015) were considered weak evidence for a positive response; significant differences for two or more doses indicated the trial was positive. A strong trend (P < 0.003) with a single significant dose level was designated weak positive, to indicate a high level of induced aberrations. A strongly positive trend (P < 0.003), in the absence of a statistically-significant increase at any one dose point, led to an equivocal call.
The trial calls were based on a consideration of the statistical analyses as well as the biological information available to the reviewers.

Statistics:

Chromosomal aberration data are presented as percentages of cells with aberrations. To arrive at a statistical call for a trial, analyses were conducted on both the dose-response (trend) and the significance of the individual dose points compared to the vehicle control.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Total percent cells with aberrations:

Trial 1 at 0, 100, 150, or 200 µg/ml: 4, 2, 2, or 4

Trial 2 at 0, 50, 125, or 250 µg/ml: 4, 2, 1, or 4.

No genotoxic effects were observed.

Trial #:1   Activation: No Activation   Date: 09/11/1985   Harvest Time: 10.5 hrs   Trial Call: Negative
		Dose (µg/mL)	Total Cells Examined	Total Aberrations No. of Abs.	Abs Per Cell	% Cells With Abs.	Complex Aberrations No. of Abs.	Abs Per Cell	% Cells With Abs.	Simple Aberrations	Abs Per Cell	% Cells With Abs.	Other Abs.	Abs Per Cell	% Cells With Abs.
Abs: Aberrations
Vehicle Control	Negative (Not Specified)		200	1	0.005	1.0	0	0.000	0.0	1	0.005	1.0	0	0.000	0.0
Vehicle Control	Dimethyl Sulfoxide		200	12	0.060	4.0	1	0.005	1.0	11	0.055	4.0	0	0.000	0.0
Test Chemical	DL-menthol	100.5	200	4	0.020	2.0	0	0.000	0.0	4	0.020	2.0	0	0.000	0.0
		150	200	4	0.020	2.0	4	0.020	2.0	0	0.000	0.0	0	0.000	0.0
		200	200	8	0.040	4.0	0	0.000	0.0	8	0.040	4.0	0	0.000	0.0
Positive Control	Mitomycin-C	0.15	200	26	0.130	10.0	16	0.080	6.0	10	0.050	5.0	0	0.000	0.0
		0.5	25	10	0.400	32.0	3	0.120	12.0	6	0.240	20.0	1	0.040	4.0
	Trend			-0.060			-0.212			-0.561
	Probability			0.524			0.584			0.713

 

Trial #:2   Activation: Induced Rat Liver S9   Date: 09/11/1985   Harvest Time: 12.5 hrs   Trial Call: Negative
		Dose (µg/mL)	Total Cells Examined	Total Aberrations No. of Abs.	Abs Per Cell	% Cells With Abs.	Complex Aberrations No. of Abs.	Abs Per Cell	% Cells With Abs.	Simple Aberrations	Abs Per Cell	% Cells With Abs.	Other Abs.	Abs Per Cell	% Cells With Abs.
Abs: Aberrations
Vehicle Control	Negative (Not Specified)		200	3	0.015	2.0	0	0.000	0.0	3	0.015	2.0	0	0.000	0.0
Vehicle Control	Dimethyl Sulfoxide		200	8	0.040	4.0	2	0.010	1.0	6	0.030	3.0	0	0.000	0.0
Test Chemical	DL-menthol	50	200	4	0.020	2.0	2	0.010	1.0	2	0.010	1.0	0	0.000	0.0
		123.7	200	2	0.010	1.0	0	0.000	0.0	2	0.010	1.0	0	0.000	0.0
		250	200	8	0.040	4.0	2	0.010	1.0	6	0.030	3.0	0	0.000	0.0
Positive Control	Mitomycin-C	7.5	100	14	0.140	14.0	7	0.070	7.0	7	0.070	7.0	0	0.000	0.0
		37.5	25	12	0.480	40.0	5	0.200	20.0	7	0.280	24.0	0	0.000	0.0
	Trend			-0.275			-0.430			-0.056
	Probability			0.608			0.666			0.522

 

Abs. aberrations.

Conclusions:

2-Isopropyl-5-methylcyclohexanol did not induce chromosomal aberrations in Chinese hamster ovary cells at study conditions.

Executive summary:

A in vitro Chromosome Aberration test in Chinese hamster ovary cells was carried out with 2 -isopropyl-5 -methylcyclohexanol concentrations of: Trial 1: 100, 150, or 200 µg/ml and Trial 2: 50, 125, or 250 µg/ml. Positive controls were mitomycin C and cyclophosphamide. Total percent cells with aberrations observed were: Trial 1 at 0, 100, 150, or 200 µg/ml: 4, 2, 2, or 4 and Trial 2 at 0, 50, 125, or 250 µg/ml: 4, 2, 1, or 4. No genotoxic effects were observed. 2-Isopropyl-5-methylcyclohexanol did not induce chromosomal aberrations in Chinese hamster ovary cells.

Reference 12

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1983

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

(only without metabolic activation, no data on positive controls)

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The cell line was originally established from the lung of a newborn female at the Cancer Research Institute (Tokyo) and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum. The modal chromosome number was 25 and the doubling time was approximately 15 hours.

Metabolic activation:

without

Test concentrations with justification for top dose:

Each sample was exposed to three different concentrations.
Max. Concentration = 0.2 mg/ml

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Remarks:

(ethanol)

Details on test system and experimental conditions:

Colcemid was added 2 hours before harvesting. Cells were trypsinized, suspended in a hypotonic KCl solution (0.075 M) for 13 min at room temperature, centrifuged, fixed with acetic acid-methanol (1:3 v/v) and applied to slides. After drying, preparations were stained with Giemsa solution (1.5% at pH 6.8).

DURATION
- Exposure duration: 24 and 48 hours

NUMBER OF CELLS EVALUATED: 100 well-spread metaphases were microscopically observed

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Structural chromosomal aberrations: chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others.

Evaluation criteria:

Test chemicals were considered positive if the incidence of aberrations was greater than 10%, equivocal if between 5.0 and 9.9%, and negative if less than 4.9%.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

The incidence of polyploid cells at 48 hours was observed to be 0%.

The incidence of cells with structural aberrations at 48 hours after treatment was observed to be 4%.

Conclusions:

2-Isopropyl-5-methylcyclohexanol did not induce chromosomal aberrations in Chinese hamster fibroblasts at test conditions.

Executive summary:

A chromosomal aberration test was performed in Chinese hamster fibroblast with 2 -isopropyl-5 -methylcyclohexanol at a maximum concentration of 2.0 mg/plate. No metabolic activation was applied and the negative controls were untreated cells and vehicle-treated cells. The incidence of polyploid cells at 48 hours was observed to be 0%. The incidence of cells with structural aberrations at 48 hours after treatment was observed to be 4%. 2-Isopropyl-5-methylcyclohexanol did not induce chromosomal aberrations in Chinese hamster fibroblasts at test conditions.

Reference 13

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

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

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

Negative solvent / vehicle controls:

yes

Remarks:

(ethanol)

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

(without metabolic activation)

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.

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

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                                   

                                                                                                             

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.

Reference 14

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
See attached the reporting format and read-across rationale.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(with and without metabolic activation)

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

based on a read-across from an analogue substance

Conclusions:

Based on the read-across approach from experimental data on the analogue 4-tert-butylcyclohexyl acetate, 2-methylcyclohexyl acetate was determined to be non-mutagenic.

Executive summary:

An Ames test was conducted on the analogue substance 4-tert-butylcyclohexyl acetate with Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 with and without metabolic S9 activation at doses up to 9.4 µg/plate. No evidence of mutagenicity was observed. Based on these results, the read-across approach was applied and 2 -methylcyclohexyl acetate was determined to be non-mutagenic.

Reference 15

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Based on the metabolic fate and OECD QSAR Application Toolbox, where the substances share analogue structural alerts (see attached rationale), the toxicity values of cyclohexyl acetate and 2-methylcyclohexyl acetate are comparable.

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
See attached the reporting format and read-across rationale.

Reason / purpose for cross-reference:

read-across source

Species / strain:

bacteria, other: Bacillus subtilis type H17 and M45

Metabolic activation:

not applicable

Genotoxicity:

negative

Remarks:

(with and without metabolic activation)

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

based on a read-across from an analogue substance

Additional information on results:

Based on experimental results on the analogue cyclohexyl acetate (no mutagenic activity in Bacillus subtilis at 19 µg/plate since the inhibition zone was < 2mm), the read-across approach was applied and 2-methylcyclohexyl acetate was determined to be non-mutagenic.

Conclusions:

Based on the read-across approach from experimental data on the analogue cyclohexyl acetate, 2-methylcyclohexyl acetate was determined to be non-mutagenic.

Executive summary:

Based on the experimental results on the analogue cyclohexyl acetate (no mutagenic activity observed in a Rec assay using Bacillus subtilis type H17 and M45 at 19 µg/disc), the read-across approach was applied and 2 -methylcyclohexyl acetate was determined to be non-mutagenic.

Reference 16

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Based on the metabolic fate and OECD QSAR Application Toolbox, where the substances share analogue structural alerts (see attached rationale), the toxicity values of cyclohexyl acetate and 2-methylcyclohexyl acetate are comparable.

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
See attached the reporting format and read-across rationale.

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

Read-across approach from experimental results on the analogue substance cyclohexyl acetate.

Species / strain:

bacteria, other: Bacillus subtilis type H17 and M45

Metabolic activation:

not applicable

Genotoxicity:

negative

Remarks:

(with and without metabolic activation)

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

based on a read-across from an analogue substance

Conclusions:

Based on the read-across approach from experimental data on the analogue cyclohexyl acetate, 2-methylcyclohexyl acetate was determined to be non-mutagenic.

Executive summary:

Based on the experimental results on the analogue cyclohexyl acetate (no mutagenic activity observed in a Rec assay using Bacillus subtilis type H17 and M45 at 20 µg/disc), the read-across approach was applied and 2 -methylcyclohexyl acetate was determined to be non-mutagenic.

Reference 17

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
See attached the reporting format and read-across rationale.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA102, TA1535 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(with and without metabolic activation)

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Basec on read-across structural analogue

Conclusions:

Based on the read-across approach from experimental data on the analogue 2-(1-methylpropyl)-1-vinylcyclohexyl acetate, 2-methylcyclohexyl acetate was determined to be non-mutagenic.

Executive summary:

An Ames test was performed using the plate incorporation and the pre-incubation method on the analogue substance 2-(1-methylpropyl)-1-vinylcyclohexyl acetate. No mutagenic activity was observed on Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 with and without S9 activation up to 5000 µg/plate in dimethyl sulfoxide. Based on these results the read-across approach was applied and 2 -methylcyclohexyl acetate was determined to be non-mutagenic.

Reference 18

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
See attached the reporting format and read-across rationale.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium, other: TA100, TA2637, TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

( at estimated concentration of 0.5 mg/plate for all strains +S9 and -S9; 0.2 mg/plate for TA100 -S9)

Vehicle controls validity:

valid

Remarks:

Based on read-across structural analogue

Positive controls validity:

valid

Conclusions:

Based on the read-across approach from experimental data on the analogue 2 -isopropyl-5-methylcyclohexanol, 2-methylcyclohexyl acetate was determined to be non-mutagenic.

Executive summary:

An Ames Test was carried out with the analogue substance 2 -isopropyl-5-methylcyclohexanol in Salmonella typhimurium strains TA100, TA2637 and TA98 up to 0.5 mg/plate with and without metabolic activation. No mutagenic activity was observed. Cytotoxicity was observed at 0.5 mg/plate for all strains and at 0.2 mg/plate for TA100 (-S9). Based on these results, the read-across approach was applied and 2 -methylcyclohexyl acetate was determined to be non-mutagenic.

Reference 19

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
See attached the reporting format and read-across rationale.

Reason / purpose for cross-reference:

reference to other study

Key result

Species / strain:

S. typhimurium, other: TA100, TA1535, TA97, and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Based on Read-Across from structural analogue

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on the read-across approach from experimental data on the analogue 2-isopropyl-5-methylcyclohexanol, 2-methylcyclohexyl acetate was determined to be non-mutagenic.

Executive summary:

An Ames Test was carried out with the analogue substance 2 -isopropyl-5 -methylcyclohexanol in Salmonella typhimurium strains TA100, TA1535, TA97, or TA98 up to 666 µg/plate doses with and without metabolic activation. No mutagenic activity was observed under test conditions. Based on these results, the read-across was applied and 2 -methylcyclohexyl acetate was determined to be non-mutagenic.

Reference 20

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
See attached the reporting format and read-across rationale.

Reason / purpose for cross-reference:

reference to other study

Key result

Species / strain:

S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94, and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Based on Read-Across analogue

Vehicle controls validity:

valid

Conclusions:

Based on the read-across approach from experimental data on the analogue 2-isopropyl-5-methylcyclohexanol, 2-methylcyclohexyl acetate was determined to be non-mutagenic.

Executive summary:

An Ames Test was carried out with the analogue substance 2 -isopropyl-5 -methylcyclohexanol in Salmonella typhimurium strains TA92, TA1535, TA100, TA1537, TA94, or TA98 up to 5.0 mg/plate with metabolic activation. No mutagenic activity was observed. Based on these results, the read-across approach was applied and 2 -methylcyclohexyl acetate was determined to be non-mutagenic.

Reference 21

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
See attached the reporting format and read-across rationale.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Based on read-acriss structural analogue

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on the read-across approach from experimental data on the analogue 2-isopropyl-5-methylcyclohexanol, 2-methylcyclohexyl acetate was determined to be negative for chromosome aberrations.

Executive summary:

An in-vitro chromosome aberration test in human lymphocytes was carried out on the analogue substance 2 -isopropyl-5 -methylcyclohexanol up to 10 mM with and without metabolic activation (S9). Test item did not induce chromosomal aberrations in human lymphocytes at test conditions. Based on these results, the read-across approach was applied and 2 -methylcyclohexyl acetate was determined to be negative for chromosome aberrations.

Reference 22

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
See attached the reporting format and read-across rationale.

Reason / purpose for cross-reference:

reference to other study

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Based on Read-Across analogue

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on the read-across approach from experimental data on the analogue 2-isopropyl-5-methylcyclohexanol, 2-methylcyclohexyl acetate was determined to be negative for sister chromatid exchange in mammalian cells.

Executive summary:

An in-vitro Sister Chromatid Exchange test in human lymphocytes was carried out on the analogue substance 2 -isopropyl-5 -methylcyclohexanol up to 10 mM with and without metabolic activation (S9). Test item did not affect the frequency of SCEs in human lymphocytes. Based on these results, the read-across approach was applied and 2 -methylcyclohexyl acetate was determined to be negative for SCE in mammalian cells.

Reference 23

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
See attached the reporting format and read-across rationale.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Based on Read-Across structural analogue

Vehicle controls validity:

valid

Positive controls validity:

other: other: questionable (trial 2, without metabolic activation)

Conclusions:

Based on the read-across approach from experimental data on the analogue 2-isopropyl-5-methylcyclohexanol, 2-methylcyclohexyl acetate was determined to be negative for sister chromatid exchange in mammalian cells.

Executive summary:

An in-vitro Sister Chromatid Exchange test in Chinese hamster ovary cells was carried out on the analogue substance 2 -isopropyl-5 -methylcyclohexanol up to 50 µg/ml in Trial 1 (-S9), up to 25 µg/ml in Trial 2 (-S9) and up to 167 µg/ml in Trial 3 (+S9). In trial 1, 19.9% increase in SCEs was reported at the low dose only. In trial 2, none of the concentrations showed an increase over 20%, but the test was considered equivocal based on a positive trend. No effects were reported in the presence of S9 in trial 3. Based on these results, the read-across was applied and 2 -methylcyclohexyl acetate was determined negative for SCE in Chinese hamster ovary cells.

Reference 24

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
See attached the reporting format and read-across rationale.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Based on Read-Across structural analogue

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on the read-across approach from experimental data on the analogue 2-isopropyl-5-methylcyclohexanol, 2-methylcyclohexyl acetate was determined to be negative for chromosome aberrations.

Executive summary:

A in vitro Chromosome Aberration test in Chinese hamster ovary cells was carried out on the analogue substance 2 -isopropyl-5 -methylcyclohexanol up to 200 µg/ml in Trial 1 (-S9) and up to 250 µg/ml in Trial 2 (+S9). No genotoxic effects were observed. Based on these results, the read-across was applied and 2 -methylcyclohexyl acetate was determined negative for chromosomal aberrations in Chinese hamster ovary cells.

Reference 25

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
See attached the reporting format and read-across rationale.

Reason / purpose for cross-reference:

reference to other study

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Based on read-across structural analogue

Vehicle controls validity:

valid

Conclusions:

Based on the read-across approach from experimental data on the analogue 2-isopropyl-5-methylcyclohexanol, 2-methylcyclohexyl acetate was determined to be negative for chromosome aberrations.

Executive summary:

A chromosomal aberration test was performed in Chinese hamster fibroblast with the analogue substance 2 -isopropyl-5 -methylcyclohexanol at a maximum concentration of 2.0 mg/plate without metabolic activation. The incidence of polyploid cells at 48 hours was observed to be 0%. The incidence of cells with structural aberrations at 48 hours after treatment was observed to be 4%. Based on these results, the read-across approach was applied and 2 -methylcyclohexyl acetate was determined to be negative for chromosomal aberrations in Chinese hamster fibroblasts at test conditions.

Reference 26

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
See attached the reporting format and read-across rationale.

Reason / purpose for cross-reference:

read-across source

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Based on Read-across structural Analogue

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at an estimated concentration of >= 200 µg/ml

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Based on experimental results on the analogue 2-isopropyl-5-methylcyclohexanol (no increase of mutation frequency in mouse lymphoma cells up to 200 µg/ml with and without S9 metabolic activation), the read-across approach was applied and 2-methylcyclohexyl acetate was determined to be negative for chromosome aberrations.

Conclusions:

Based on the read-across approach from experimental data on the analogue 2-isopropyl-5-methylcyclohexanol, 2-methylcyclohexyl acetate was determined to be non-mutagenic for in-vitro mammalian cells.

Executive summary:

A Forward mutation assay was performed in mouse lymphoma cells with the analogue substance 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. 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. Based on these results, the read-across approach was applied and 2 -methylcyclohexyl acetate was determined to be non-mutagenic for in-vitro mammalian cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Weight of evidence: A bone marrow and peripheral blood micronucleous test was performed by NTP (1993) on the analogue 2 -isopropyl-5 -methylcyclohexanol. Mice were intraperitoneally exposure to 0, 250, 500 and 1000 mg/kg bw/day (3 injections, 24 hours apart). Based on the mean MN-PCE per 1000 PCE per group observed, the analogue 2-isopropyl-5-methylcyclohexanol was considered to be negative under the test conditions. Based on these results, the read-across approach was applied and the test item 2 -methylcyclohexyl acetate was also determined to be negative.

Weight of evidence: An acute and a subacute chromosome aberration tests were performed by the Food and Drug Administration (USA, 1975) with the analogue substance 2 -isopropyl-5 -methylcycloehxanol. In the acute test, rats were exposure to a single oral dose or 5 consecutive doses (24 hours apart) of 1.45, 14.5, or 145 mg/kg bw (test 1) and 500 or 3000 mg/kg bw (acute) and 1500 mg/kg bw (subacute) (test 2). After bone marrows were removed and analysed the analogue 2-Isopropyl-5-methylcyclohexanol was determined not to produce detectable significant aberration of the metaphase chromosomes of rats. Based on these results, the read-across was applied and 2 -methylcyclohexyl acetate was determined to be negative for the in-vivo cytogenicity.

Weight of evidence: Moreover, an in-vivo chromosome aberration assay was performed by NTP (1993) with analogue 2 -isopropyl-5 -methylcyclohexanol on bone marrow. The test item was administered to mice (8 mice per group) by intraperitoneal injection at 225, 450 and 900 mg/kg. After 17 or 36 hours, 50 cells per animal were analyzed and the mean total number of aberrations and the mean percentage of cells with aberrations (excluding gaps) were calculated. The results were determined to be equivocal. Based on these results, the read-across approach was applied and the in-vivo chromosome aberration test for 2 -methylcyclohexyl acetate was also determined to be equivocal.

Supporting study: Furthermore, in the study by NTP (1987), an in-vivo sister chromatid exchange assay on bone marrow was performed on the analogue substance 2 -isopropyl-5 -methylcyclohexanol. Mice were exposed by intraperitoneal injection to 225, 450 and 900 mg/kg test item. After 23 hours, 25 cells per animal were analysed and 2 -isopropyl-5 -methylcyclohexanol was determined not to induce SCE in bone marrow under test conditions. Based on these results, the read-across approach was applied and 2 -methylcyclohexyl acetate was also determined to be negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

November 14, 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No data on GLP. Equivalent or similar to OECD 474.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

(only males were tested)

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: National Toxicology Program production facility at Taconic Farms
- Age at study initiation: 9-14 weeks
- Weight at study initiation: within a 2 g range of a mean weight between 25 and 33 g.

Route of administration:

intraperitoneal

Vehicle:

Yes.

Details on exposure:

All treatments were by intraperitoneal (IP) injection at a volume of 0.4 ml per mouse.

Duration of treatment / exposure:

3 days.

Frequency of treatment:

Daily

Post exposure period:

24 hours after the last treatment mice were killed.

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5-6 mice.

Control animals:

yes, concurrent vehicle

Positive control(s):

Dimethylbenzanthracene (12.5 mg/kg bw).

Tissues and cell types examined:

Bone marrow and perypheral blood: erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The selection of the maximum dose was based on either mortality, administration characteristics (ability to be administered as a homogeneous suspension in corn oil or dissolved in PBS), depression in the percentage of bone marrow PCE (no less than 15% of the erythrocytes), or on the arbitrary maximum dose of 2000 mg/kg/day.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Mice were euthanized with CO2 24 hr after the third treatment.

DETAILS OF SLIDE PREPARATION: Bone marrow smears (two slides per mouse) were prepared, fixed in absolute methanol, and stained with acridine orange.

METHOD OF ANALYSIS: Slides were evaluated at 1000X magnification for the number of MN-PCE among 2000 PCE and for the percentage of PCE among 200 erythrocytes. MN-PCE = Micronucleus in polychromatic erythrocytes.

OTHER: As part of these bone marrow micronucleus tests measuring induction of chromosomal changes after short-term exposures to potentially mutagenic agents, peripheral blood were taken. 2000 PCE are analyzed for frequency of micronucleated cells, but 1000 erythrocytes are scored for determination of %PCE, since the percentage of PCE in blood is only around 3-5% in a healthy animal.

Evaluation criteria:

Data are presented as the mean number of micronucleated cells per 1000 cells for each treatment group. A positive trend test is one in which the P value is equal to or less than 0.025.

Statistics:

The data were analyzed using the Micronucleus Assay Data Management and Statistical software package (version 1.4), which was designed specifically for in vivo micronucleus data. The level of significance was set at an alpha level of 0.05. To determine whether a specific treatment resulted in a significant increase in MN-PCE, the number of MN-PCE were pooled within each dose group and analyzed by a one-tailed trend test. In the software package used, the trend test incorporates a variance inflation factor to account for excess animal variability. In the event that the increase in the dose response curve is non-monotonic, the software program allows for the data to be analyzed for a significant positive trend after data at the highest dose only has been excluded. However, in this event, the alpha level is adjusted to 0.01 to protect against false positives. The %PCE data were analyzed by an analysis of variance (ANOVA) test based on pooled data. Pairwise comparisons between each group and the concurrent solvent control group was by an unadjusted one-tailed Pearson chi-squared test which incorporated the calculated variance inflation factor for the study.

Key result

Sex:

male

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Bone marrow:

Start Date	Sample Collection Time		Sex	Cell	Method Used	Dosing Regimen	Route	Trend Test P-Value
11/14/1988	24 Hours		Male	PCE	Slide Scoring	IP/IJ x 3, 72 Hours	Intraperit. Injection	0.374
					Dose (mg/kg)	Number of Animals Scored	Mean MN-PCE/1000 PCE ± SEM	Pairwise P
Vehicle Control		Corn Oil			0	5	2.90 ± 0.43
Test Chemical		DL-menthol			250	5	3.60 ± 0.58	0.1922
					500	5	2.20 ± 0.34	0.8368
					1000	3	3.67 ± 0.60	0.2025
Positive Control		Dimethylbenzanthracene			12.5	5	9.60 ± 0.87	< 0.0001

Peripheral blood:

Start Date	Sample Collection Time		Sex	Cell	Method Used	Dosing Regimen	Route	Trend Test P-Value
11/14/1988	24 Hours		Male	PCE	Slide Scoring	IP/IJ x 3, 72 Hours	Intraperit. Injection	0.164
					Dose (mg/kg)	Number of Animals Scored	Mean MN-PCE/1000 PCE ± SEM	Pairwise P
Vehicle Control		Corn Oil			0	5	2.50 ± 0.69
Test Chemical		DL-menthol			250	5	2.80 ± 0.72	0.3399
					500	5	2.40 ± 0.37	0.5569
					1000	3	3.50 ± 1.04	0.1264
Positive Control		Dimethylbenzanthracene			12.5	5	8.10 ± 0.83	< 0.0001

Conclusions:

2-isopropyl-5-methylcyclohexanol was considered to be negative in the mammalian erythrocyte micronucleus test under the test conditions.

Executive summary:

In a micronucleous test (bone marrow and peripheral blood), groups of 5-6 mice were intraperitoneally injected on 3 consecutive days with 1X, 0.5X and 0.25X of the test chemical where X was the maximum dose determined in the range-finding experiment. The dose concentrations were 0, 250, 500 and 1000 mg/kg bw/day. A positive control and solvent control were also used. 24 hours after the last treatment, mice were killed, bone marrow and peripheral blood samples removed and slides were prepared. For each mouse, the number of MN-PCE in 2000 PCE and the percent PCE in 200 erythrocytes were determined and the mean MN-PCE per 1000 PCE per group were calculated. 2-isopropyl-5-methylcyclohexanol was considered to be negative in the mammalian erythrocyte micronucleus test under the test conditions.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1975

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

(only males were tested, no data on controls)

Principles of method if other than guideline:

Cytogenetic assay - acute study

GLP compliance:

no

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

other: albino

Sex:

male

Route of administration:

oral: gavage

Vehicle:

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

Frequency of treatment:

Single oral dose

Post exposure period:

Rats were killed at 6, 24 and 48 hours.

Dose / conc.:

1.45 mg/kg bw/day (actual dose received)

Dose / conc.:

14.5 mg/kg bw/day (actual dose received)

Dose / conc.:

145 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

3 000 mg/kg bw/day (actual dose received)

Control animals:

yes, concurrent vehicle

Positive control(s):

Triethylene melanine (0.3 mg/kg).

Details of tissue and slide preparation:

4 hours after administration and 2 hours prior to termination, rats were intraperitoneally injected with 4 mg colcemid/kg bw. Bone marrow was removed and slides were prepared and analysed.

Key result

Sex:

male

Genotoxicity:

negative

The compound produced no detectable significant aberration of the bone marrow metaphase chromosomes of rats when administered orally up to 3000 mg/kg bw.

Conclusions:

2-Isopropyl-5-methylcyclohexanol did not induce chromosomal aberrations at test conditions.

Executive summary:

A chromosome aberration test was performed in albino male rats with 2 -isopropyl-5 -methylcycloehxanol at 1.45, 14.5, or 145 mg/kg bw (test 1) and 500 or 3000 mg/kg bw (test 2). The groups of rats were exposed to the test item by a single oral dose. The rats were killed after 6, 24 and 58 hours. 4 hours after administration and 2 hours prior to termination, rats were intraperitoneally injected with 4 mg colcemid/kg bw. Bone marrow was removed and slides were prepared and analysed. 2-Isopropyl-5-methylcyclohexanol did not induce chromosomal aberrations at test conditions.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1975

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

(only males were tested, no data on controls)

Principles of method if other than guideline:

Cytogenetic assay - Subacute study

GLP compliance:

no

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

other: albino

Sex:

male

Route of administration:

oral: gavage

Vehicle:

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

Duration of treatment / exposure:

5 days.

Frequency of treatment:

Five doses 24 hours apart

Post exposure period:

Rats were killed 6 hours after last dose

Dose / conc.:

1.45 mg/kg bw/day (actual dose received)

Dose / conc.:

14.5 mg/kg bw/day (actual dose received)

Dose / conc.:

145 mg/kg bw/day (actual dose received)

Dose / conc.:

1 500 mg/kg bw/day (actual dose received)

Control animals:

yes, concurrent vehicle

Positive control(s):

Triethylene melamine (0.3 mg/kg).

Details of tissue and slide preparation:

4 hours after administration and 2 hours prior to termination, rats were intraperitoneally injected with 4 mg colcemid/kg bw. Bone marrow was removed and slides were prepared and analysed.

Sex:

male

Genotoxicity:

negative

The compound produced no detectable significant aberration of the bone marrow metaphase chromosomes of rats when administered orally up to 1500 mg/kg bw.

Conclusions:

2-Isopropyl-5-methylcyclohexanol did not induce chromosomal aberrations at test conditions.

Executive summary:

A chromosome aberration test was performed in albino male rats with 2 -isopropyl-5 -methylcycloehxanol at 1.45, 14.5, or 145 mg/kg bw (test 1) and 1500 mg/kg bw (test 2). The groups of rats were exposed to 5 consecutive doses, 24 hours apart and were killed 6 hours after last dose. 4 hours after administration and 2 hours prior to termination, rats were intraperitoneally injected with 4 mg colcemid/kg bw. Bone marrow was removed and slides were prepared and analysed. 2-Isopropyl-5-methylcyclohexanol did not induce chromosomal aberrations at test conditions.

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1989-1993

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

(only males, mitotic index not given, 50 cells analyzed per animal)

GLP compliance:

not specified

Type of assay:

chromosome aberration assay

Species:

mouse

Strain:

B6C3F1

Sex:

male

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Details on exposure:

Injection volume = 0.4 ml.

Duration of treatment / exposure:

17 and 36 h (see details on any other information on results section).

Frequency of treatment:

Single treatment.

Dose / conc.:

225 mg/kg diet

Dose / conc.:

450 mg/kg diet

Dose / conc.:

900 mg/kg diet

No. of animals per sex per dose:

8 male mice per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Dimethylbenzanthracene
- Route of administration: intraperitoneal
- Doses / concentrations: from 15 to 100 mg/kg (see any other information on results section for details).

Tissues and cell types examined:

Tissue: Bone marrow
Number cells examined: 50 cells per animal.

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The mice were subcutaneously implanted with a BrdUrd tablet 18 hrs before the scheduled harvest. The use of BrdU allowed selection of the appropriate cell population for scoring. Two hrs prior to sacrifice, the mice received an IP injection of colchicine in saline to arrest cells in metaphase and facilitate visualization of chromosomes. The animals were killed 17 or 36 hrs after test chemical administration (18 hrs after BrdU dosing). One or both femurs were removed and the marrow was flushed out with phosphate-buffered saline (pH 7.0).

DETAILS OF SLIDE PREPARATION: Cells were treated with a hypotonic salt solution, fixed, and dropped onto chilled slides. After a 24 hr drying period, the slides were stained with Giemsa and scored.

METHOD OF ANALYSIS: Fifty first-division metaphase cells were scored from each of eight animals per treatment. The types of aberrations observed (gaps, breaks, rearrangements, and chromatid vs. chromosome) were recorded separately for each animal.

Evaluation criteria:

The mean total number of aberrations and the mean percentage of cells with aberrations (excluding gaps) were determined for each treatment group and significance was set at P =0.025.

Statistics:

The values for percent cells with aberrations were analyzed by a one-tailed trend test.

Sex:

male

Genotoxicity:

other: Equivocal

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Summary of Chromosome Aberrations:

 

Test Type	Start Date	Sample Time (h)	Species	Sex	Tissue	Protocol	Trend Test P
CA	05/12/1992	36	Mice	M	Bone Marrow	IP/IJ x 1, 36hours	0.0191
Test Group		Dose	n	Percent Cells w/ Aberrations (Mean ± SEM)			Pairwise P
Vehicle Control	Corn Oil	0	8	1.00 ± 0.38
Test Chemical	DL-menthol	225	8	1.25 ± 0.65			0.3687
		450	8	1.50 ± 0.63			0.2622
		900	8	2.75 ± 0.75			0.0340
Positive Control	Dimethylbenzanthracene	25	8	21.50 ± 2.35			0.0000

 

 

Test Type	Start Date	Sample Time (h)	Species	Sex	Tissue	Protocol	Trend Test P
CA	02/09/1993	36	Mice	M	Bone Marrow	IP/IJ x 1, 36hours	0.0319
Test Group		Dose	n	Percent Cells w/ Aberrations (Mean ± SEM)			Pairwise P
Vehicle Control	Corn Oil	0	8	3.75 ± 0.59
Test Chemical	DL-menthol	225	8	2.75 ± 0.65			0.7874
		450	8	3.25 ± 1.06			0.6498
		900	7	6.00 ± 0.87			0.0752
Positive Control	Dimethylbenzanthracene	25	8	24.50 ± 1.72			0

 

 

Test Type	Start Date	Sample Time (h)	Species	Sex	Tissue	Protocol	Trend Test P
CA	08/15/91	17	Mice	M	Bone Marrow	IP/IJ x 1, 17hours	0.9794
Test Group		Dose	n	Percent Cells w/ Aberrations (Mean ± SEM)			Pairwise P
Vehicle Control	Corn Oil	0	7	4.57 ± 1.43
Test Chemical	DL-menthol	450	8	2.50 ± 0.73			0.9391
		675	8	2.75 ± 0.65			0.9092
		900	8	2.00 ± 0.85			0.9770
Positive Control	Dimethylbenzanthracene	50	8	8.50 ± 1.76			0.0157

Footnotes:

IP/IJ: Intraperitoneal injection

n: Number of animals analyzed                                                     

     

Conclusions:

An in-vivo chromosome aberration on bone marrow study performed with 2-isopropyl-5-methylcyclohexanol was determined to be equivocal under test conditions.

Executive summary:

An in-vivo chromosome aberration assay was performed with analogue 2 -isopropyl-5 -methylcyclohexanol on bone marrow. The test item was administered to mice (8 mice per group) by intraperitoneal injection at 225, 450 and 900 mg/kg. After 17 or 36 hours, 50 cells per animal were analyzed. Both solvent control (corn oil) and positive control (dimethylbenzanthracene) were also tested.

The mean total number of aberrations and the mean percentage of cells with aberrations (excluding gaps) were calculated. The results were determined to be equivocal under test conditions.

Reference 5

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

10/08/1987

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Principles of method if other than guideline:

An in-vivo sister chromatid exchange assay (non-standard protocol) was performed with dl-menthol. The test item was administered to mice (4 mice per group) by intraperitoneal injection at 225, 450 and 900 mg/kg. After 23 hours, 25 cells per animal the bone marrow were analyzed. Both solvent control (corn oil) and positive control (dimethylbenzanthracene) were also observed. Only 1 trial was performed.

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay

Species:

mouse

Strain:

B6C3F1

Sex:

male

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Duration of treatment / exposure:

23 h

Frequency of treatment:

Single treatment

Dose / conc.:

225 mg/kg diet

Dose / conc.:

450 mg/kg diet

Dose / conc.:

900 mg/kg diet

No. of animals per sex per dose:

4 male mice per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Dimethylbenzanthracene
- Route of administration: intreperitoneal
- Doses / concentrations: 2.5 mg/kg

Tissues and cell types examined:

Tissue: Bone marrow
Number cells examined: 25 cells per animal.

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Detailed Sister Chromatid Exchange Data (Trial 1)

Test Group		Dose	Animal	No.	SCE / Cell
		(mg/kg)	Number	Cells	Mean	±	SEM
Solvent	Corn Oil	0	6663	25	4.91	±	.528
			6664	25	3.41	±	.427
			6665	25	3.65	±	.325
			6666	25	4.34	±	.353
Average					4.08	±	.341
Test Chemical	dl-menthol	225	6648	25	3.31	±	.415
			6649	25	3.85	±	.393
			6650	25	3.50	±	.318
			6651	25	4.57	±	.355
Average					3.81	±	.277
Test Chemical	dl-menthol	450	6653	25	4.54	±	.389
			6655	25	2.26	±	.275
			6656	25	3.65	±	.380
			6657	25	3.14	±	.346
Average					3.40	±	.478
Test Chemical	dl-menthol	900	6658	25	4.81	±	.494
			6659	25	6.08	±	.665
			6660	25	2.72	±	.268
			6662	25	4.34	±	.523
Average					4.48	±	.694
Positive Control	Dimethyl-benzanthracene	2.5	6668	25	4.94	±	.421
			6669	25	4.69	±	.409
			6670	25	5.86	±	.505
			6671	25	6.85	±	.548
Average					5.59	±	.490

* indicates an incomplete evaluation, either an unconfirmed positive response (no replicate trial) or a negative response seen at only one of two harvest times (standard or extended)

Conclusions:

An in-vivo sister chromatid exchange on bone marrow study performed with dl-menthol was determined to be negative under test conditions.

Executive summary:

An in-vivo sister chromatid exchange assay on bone marrow(non-standard protocol) was performed with dl-menthol. The test item was administered to mice (4 mice per group) by intraperitoneal injection at 225, 450 and 900 mg/kg. After 23 hours, 25 cells per animal were analyzed. Both solvent control (corn oil) and positive control (dimethylbenzanthracene) where also observed. Only 1 trial was performed. 2 -isopropyl-5 -methylcyclohexyl acetate was determined not to induce SCE in bone marrow under test conditions.

Reference 6

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
See attached the reporting format and read-across rationale.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Based on Read-Across structural Analogue

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on the read-across approach from experimental data on the analogue 2-isopropyl-5-methylcyclohexanol, 2-methylcyclohexyl acetate was determined to be negative for the mammalian erythrocyte micronucleus test.

Executive summary:

A mouse bone marrow and peripheral blood micronucleous test was performed on the analogue 2 -isopropyl-5 -methylcyclohexanol. Groups of 5 -6 mice were intraperitoneally exposure to 0, 250, 500 and 1000 mg/kg bw/day (3 injections, 3 consecutive days). 24 hours after the last treatment, mice were killed, bone marrow and peripheral blood samples removed and slides were prepared. For each mouse, the number of MN-PCE in 2000 PCE and the percent PCE in 200 erythrocytes were determined and the mean MN-PCE per 1000 PCE per group were calculated. The analogue 2-isopropyl-5-methylcyclohexanol was considered to be negative under the test conditions. Based on these results, the read-across approach was applied and the test item 2 -methylcyclohexyl acetate was determined to be negative for the mammalian erythrocyte micronucleus test.

Reference 7

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
See attached the reporting format and read-across rationale.

Reason / purpose for cross-reference:

reference to other study

Sex:

male

Genotoxicity:

negative

Remarks:

Based on Read-Across structural analogue

Conclusions:

Based on the read-across approach from experimental data on the analogue 2-isopropyl-5-methylcyclohexanol, 2-methylcyclohexyl acetate was determined was determined to be negative for the in-vivo chromosome aberration test.

Executive summary:

A chromosome aberration test was performed in albino male rats with the analogue substance 2 -isopropyl-5 -methylcyclohexanol at 1.45, 14.5, or 145 mg/kg bw (test 1) and 500 or 3000 mg/kg bw (test 2). The groups of rats were exposed to the analogue a single oral dose. After bone marrow was removed and analysed the analogue 2-Isopropyl-5-methylcyclohexanol produced no detectable significant aberration of the bone marrow metaphase chromosomes of rats when administered orally up to 3000 mg/kg bw. Based on these results the read-across was applied and 2 -methylcyclohexyl acetate was determined to be negative for the in-vivo chromosome aberration test.

Reference 8

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
See attached the reporting format and read-across rationale.

Reason / purpose for cross-reference:

reference to other study

Type of assay:

chromosome aberration assay

Sex:

male

Genotoxicity:

negative

Remarks:

Based on read-across structural analogue

Conclusions:

Based on the read-across approach from experimental data on the analogue 2-isopropyl-5-methylcyclohexanol, 2-methylcyclohexyl acetate was determined was determined to be negative for the in-vivo chromosome aberration test.

Executive summary:

A chromosome aberration test was performed in albino male rats with the analogue substance 2 -isopropyl-5 -methylcyclohexanol at 1.45, 14.5, or 145 mg/kg bw (test 1) and 1500 mg/kg bw (test 2). The groups of rats were orally exposed to 5 consecutive doses (24h apart). After bone marrow was removed and analysed, the analogue 2-Isopropyl-5-methylcyclohexanol produced no detectable significant aberration of the bone marrow metaphase chromosomes of rats when administered orally up to 1500 mg/kg bw. Based on these results the read-across was applied and 2 -methylcyclohexyl acetate was determined to be negative for the in-vivo chromosome aberration test.

Reference 9

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
See attached the reporting format and read-across rationale.

Reason / purpose for cross-reference:

reference to other study

Sex:

male

Genotoxicity:

other: equivocal

Remarks:

Based on read-across structural analogue

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on the read-across approach from experimental data on the analogue 2-isopropyl-5-methylcyclohexanol, the results on the in-vivo bone marrow chromosome aberration assay for 2-methylcyclohexyl acetate were considered to be equivocal.

Executive summary:

An in-vivo chromosome aberration assay was performed with analogue 2 -isopropyl-5 -methylcyclohexanol on bone marrow. The test item was administered to mice (8 mice per group) by intraperitoneal injection at 225, 450 and 900 mg/kg. After 17 or 36 hours, 50 cells per animal were analyzed. The mean total number of aberrations and the mean percentage of cells with aberrations (excluding gaps) were calculated. The results were determined to be equivocal under test conditions. Based on these results, the read-across approach was applied and the in-vivo chromosome aberration test for 2 -methylcyclohexyl acetate was also determined to be equivocal.

Reference 10

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
See attached the reporting format and read-across rationale.

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Read-across approach from experimental results on the analogue substance 2-isopropyl-5-methylcyclohexanol.

GLP compliance:

no

Type of assay:

sister chromatid exchange assay

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Remarks:

Based on Read-Across structural analogue

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on the read-across approach from experimental data on the analogue 2-isopropyl-5-methylcyclohexanol, 2-methylcyclohexyl acetate was determined to be negative for the in-vivo sister chromatid exchange assay.

Executive summary:

An in-vivo sister chromatid exchange assay on bone marrow was performed on the analogue substance 2 -isopropyl-5 -methylcyclohexanol. The analogue substance was administered to mice (4 mice per group) by intraperitoneal injection at 225, 450 and 900 mg/kg. After 23 hours, 25 cells per animal were analyzed and 2 -isopropyl-5 -methylcyclohexanol was determined not to induce SCE in bone marrow under test conditions. Based on these results, the read-across approach was applied and 2 -methylcyclohexyl acetate was determined to be negative for in-vivo sister chromatid exchange assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data on genetic toxicity, 2 -methylcyclohexyl acetate is not classified for genetic toxicity in accordance with CLP Regulation (EC) no. 1272/2008.