[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 Mar - 7 Apr 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt und Verkehr Baden-Württemberg, Stuttgart, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

EC Number:

216-365-6

EC Name:

[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

Cas Number:

1565-76-0

Molecular formula:

C11H22O

IUPAC Name:

[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

Method

Target gene:

his operon

Species / strain

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

Experiment 1
15, 50, 150, 500 and 1500 µg/plate without metabolic activation for TA1535, TA102, TA98 and TA1537
50, 150, 500, 1500 and 5000 µg/plate without metabolic activation for TA100
15, 50, 150, 500, 1500 and 5000 µg/plate with metabolic activation for all strains

Experiment 2
15, 50, 150, 500 and 1500 µg/plate without metabolic activation for TA100
50, 150, 500, 1500 and 5000 µg/plate without metabolic activation for TA1535, TA102, TA98 and TA1537
15, 50, 150, 500, 1500 and 5000 µg/plate with metabolic activation for TA98
50, 150, 500, 1500 and 5000 µg/plate with metabolic activation for TA100, TA1535, TA102 and TA1537

Vehicle / solvent:

- Vehicle/solvent used: ethanol

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-aminoanthracene (2-AA)

Remarks:

+S9: 2-AA (0.8 µg/plate, TA98, TA100; 0.9 µg/plate, TA102, TA1535; 1.7 µg/plate, TA1537); -S9: NaN3 (0.7 µg/plate, TA100, TA1535); 2-NF (2.5 µg/plate, TA98); 9-AA (50 µg/plate, TA1537); Mitomycin C (0.15 µg/plate, TA102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies and/or diminution of the background lawn

Statistics:

X²-test was used to estimate the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level. Mean values and standard deviation were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation occurred up to the highest investigated dose.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and positive control data were between the minimum and maximum value of the historical control data of the laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9 mix the test substance was bacteriotoxic in the strains TA98, TA100, and TA1537 at 5000 µg/plate. In the presence of S9 mix the test substance was bacteriotoxic in the strains TA98 and TA100 at 5000 µg/plate.

Any other information on results incl. tables

Table 1. Test results of experiment 1 (plate incorporation).

With or without S9 mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA100	TA1535	TA102	TA98	TA1537
–	0	92 ± 7	26 ± 7	328 ± 28	24 ± 3	11 ± 4
–	0 (EtOH)	76 ± 8	21 ± 4	326 ± 12	29 ± 4	12 ± 3
–	15	-	20 ± 4	289 ± 22	22 ± 5	8 ± 3
–	50	70 ± 8	22 ± 6	247 ± 19	32 ± 3	9 ± 2
–	150	66 ± 5	21 ± 5	331 ± 8	30 ± 3	8 ± 5
–	500	66 ± 10	20 ± 4	326 ± 11	31 ± 5	5 ± 2
–	1500	74 ± 12	18 ± 7	311 ± 38	28 ± 5	9 ± 4
–	5000	46 ± 6 T	-	-	-	-
Positive controls, –S9 mix	Name	NaN3	NaN3	MMC	2-NF	9-AA
	Concentrations (μg/plate)	0.7	0.7	0.15	2.5	50
	Mean No. of colonies/plate (average of 3 ± SD)	429 ± 70	835 ± 19	967 ± 67	724 ± 73	200 ± 11
+	0	92 ± 11	12 ± 2	241 ± 29	19 ± 4	12 ± 3
+	0 (EtOH)	94 ± 4	31 ± 3	248 ± 4	18 ± 2	12 ± 3
+	50	95 ± 6	29 ± 7	257 ± 39	24 ± 3	12 ± 4
+	150	94 ± 8	27 ± 1	268 ± 21	19 ± 5	10 ± 4
+	500	90 ± 4	28 ± 4	261 ± 37	20 ± 3	11 ± 2
+	1500	86 ± 10	26 ± 5	254 ± 10	17 ± 2	12 ± 4
+	5000	72 ± 10 T	24 ± 5	227 ± 26	11 ± 5 T	11 ± 3
Positive controls, +S9 mix	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	0.8	0.9	0.9	0.8	1.7
	Mean No. of colonies/plate (average of 3 ± SD)	593 ± 57	198 ± 24	535 ± 25	532 ± 57	108 ± 5

NaN3: Sodium azide

2-NF: 2-nitrofluorene

9-AA: 9-aminoacridine

MMC: Mitomycin C

2-AA: 2-aminoanthracene

EtOH: Ethanol

T: bacteriotoxic

Table 2. Test results of experiment 2 (plate incorporation).

With or without S9 mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA100	TA1535	TA102	TA98	TA1537
–	0	80 ± 5	14 ± 5	316 ± 23	52 ± 7	13 ± 3
–	0 (EtOH)	77 ± 6	8 ± 2	294 ± 11	37 ± 4	9 ± 3
–	15	71 ± 7	-	-	-	-
–	50	63 ± 3	8 ± 4	311 ± 35	38 ± 5	7 ± 3
–	150	72 ± 7	7 ± 2	256 ± 21	29 ± 6	10 ± 3
–	500	68 ± 10	5 ± 3	253 ± 21	19 ± 3	7 ± 3
–	1500	66 ± 114	4 ± 2	266 ± 16	18 ± 7	6 ± 2
–	5000		6 ± 4	221 ± 5	17 ± 3 T	4 ± 3 T
Positive controls, –S9 mix	Name	NaN3	NaN3	MMC	2-NF	9-AA
	Concentrations (μg/plate)	0.7	0.7	0.15	2.5	50
	Mean No. of colonies/plate (average of 3 ± SD)	625 ± 52	451 ± 37	807 ± 21	589 ± 45	115 ± 10
+	0	97 ± 8	11 ± 3	279 ± 48	24 ± 2	17 ± 4
+	0 (EtOH)	84 ± 3	18 ± 3	314 ± 6	27 ± 1	12 ± 3
+	15				26 ± 2
+	50	87 ± 12	17 ± 3	313 ± 14	23 ± 2	9 ± 4
+	150	73 ± 2	15 ± 2	346 ± 29	27 ± 5	13 ± 3
+	500	78 ± 8	18 ± 6	287 ± 10	19 ± 3	14 ± 4
+	1500	82 ± 4	16 ± 5	288 ± 11	19 ± 3	12 ± 4
+	5000	67 ± 4	13 ± 4	249 ± 41	19 ± 3	11 ± 2
Positive controls, +S9 mix	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	0.8	0.9	0.9	0.8	1.7
	Mean No. of colonies/plate (average of 3 ± SD)	334 ± 64	145 ± 7	613 ± 24	727 ± 42	114 ± 17

NaN3: Sodium azide

2-NF: 2-nitrofluorene

9-AA: 9-aminoacridine

MMC: Mitomycin C

2-AA: 2-aminoanthracene

EtOH: Ethanol

T: bacteriotoxic

Applicant's summary and conclusion

Conclusions:

Under the conditions of the Ames Assay the substance was not mutagenic in any of the five strains (TA100, TA1535, TA102, TA98 and TA1537) tested up to the limit dose with and without metabolic activation.

Executive summary:

The mutagenicity of the test item was studied with five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100, and TA102). The investigations were carried using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system.

The test item was dissolved in ethanol and tested in concentrations of 15 to 5000 µg per plate in the absence and presence of S9. In the absence of S9-mix test item was bacteriotoxic towards the strains TA98, TA100, and TA1537 at 5000 µg/plate. In the presence of S9-mix the test item was bacteriotoxic towards the strains TA98 and TA100 at 5000µg/plate. Precipitation of the test compound an the plates was not observed.

Sodium azide, 2-nitrofluorene, 9-aminoacridine, mitomycin C, and 2-aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system.

In the concentration range investigated, the test item did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system.