3-Cyclohexen-1-ol, 4-methyl-1-(1-methylethyl)-, (1R)-

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 - 22 Dec 2000

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

June 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Niedersächsisches Umweltministerium, Hannover, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(1R)-1-isopropyl-4-methyl-cyclohex-3-en-1-ol

Cas Number:

20126-76-5

Molecular formula:

C10H18O

IUPAC Name:

(1R)-1-isopropyl-4-methyl-cyclohex-3-en-1-ol

Method

Target gene:

his operon

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 5

Species / strain / cell type:

S. typhimurium, other: TA 97a

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats treated with phenobarbital (i.p.) and β-naphthoflavone (oral)

Test concentrations with justification for top dose:

Experiment 1:
16, 50, 160, 500 and 1600 µg/plate with and without metabolic activation

Experiment 2:
16, 50, 160, 500 and 1600 µg/plate with and without metabolic activation in TA 97a
50, 160, 500, 1600 and 5000 µg/plate with and without metabolic activation in TA 98 and TA 102
16, 50, 160, 500 and 1600 µg/plate without metabolic activation in TA 100 and TA 1535
50, 160, 500, 1600 and 5000 µg/plate with metabolic activation in TA 100 and TA 1535

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: ICR 191 acridine mutagen dihydrochloride (ICR 191), 2-aminoanthracene (2-AA), 4-nitro-1,2-phenylenediamine (4-NOPD), nitrofurantoine (NF), cumene hydroperoxide (CUHP), dantron

Remarks:

+S9: 2-AA (2.0 µg/plate, TA97a, TA98, TA100 and TA1535); dantron (30 µg/plate, TA102); -S9: ICR 191 (0.5 µg/plate, TA97a); NaN3 (0.25 µg/plate, TA1535); 4-NOPD (0.5 µg/plate, TA98); NF (0.2 µL/plate, TA100); CUHP (100 µg/plate, TA102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: at least 48 h

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduced bacterial background lawn

Evaluation criteria:

Since a reduced background lawn is regarded to be a cytotoxic effect, plates with reduced background Iawn were not included into evaluation procedures.
The test substance is considered as mutagenic if there is a concentration effect relationship and the induction rate is ≥ 2.

Statistics:

Mean values and standard deviations 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:

cytotoxicity

Remarks:

Exp. 1: -S9: at 1600 µg/plate; Exp. 2: -S9: at 1600 µg/plate, +S9: at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 2: -S9 and +S9: at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: -S9: at 1600 µg/plate; Exp. 2: -S9: at 1600 µg/plate, +S9: at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 2: -S9 and +S9: at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium, other: TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1 and 2: -S9 and +S9: at 1600 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

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 ± SD)
		Base-pair substitution type			Frameshift type
		TA1535	TA100	TA102	TA97a	TA98
-	0 (DMSO)	24 ± 6.1	151 ± 42.2	427 ± 73.1	391 ± 9.9	34 ± 0.6
-	16	17 ± 4.4	143 ± 6.1	465 ± 65.9	441 ± 72.6	26 ± 7.8
-	50	19 ± 7.5	141 ± 5.9	400 ± 24.0	422 ± 7.1	28 ± 4.0
-	160	8 ± 3.0	145 ± 12.9	435 ± 54.0	386 ± 11.7	28 ± 1.7
-	500	14 ± 2.0	119 ± 20.2	388 ± 14.5	354 ± 15.6	27 ± 3.2
-	1600	t	t	177 ± 18.7	t	18 ± 8.7
Positive controls, –S9	Name	NaN3	NF	CUHP	ICR 191	4-NOPD
	Concentration [μg/plate]	0.25	0.2	100	0.5	0.5
	Induction rate	9.0	2.4	> 2.8	> 3.1	3.6
+	0 (DMSO)	14 ± 2.1	130 ± 2.1	538 ± 57.5	315 ± 66.2	37 ± 4.5
+	16	8 ± 4.0	136 ± 2.0	454 ± 49.2	371 ± 23.4	34 ± 2.6
+	50	12 ± 4.0	137 ± 11.0	406 ± 38.2	347 ± 27.2	35 ± 2.1
+	160	10 ± 5.5	140 ± 15.5	443 ± 46.3	358 ± 62.4	35 ± 7.6
+	500	11 ± 2.5	127 ± 8.4	439 ± 41.6	262 ± 86.7	39 ± 1.2
+	1600	10 ± 5.0	75 ± 9.1	337 ± 23.8	t	51 ± 14.6
Positive controls, +S9	Name	2-AA	2-AA	Dantron	2-AA	2-AA
	Concentration [μg/plate]	2.0	2.0	30	2.0	2.0
	Induction rate	9.8	> 9.3	> 2.2	> 3.8	> 32.1

NaN3: sodium azide

4-NOPD: 4-nitro-1,2-phenylene-diamine

2-AA: 2-aminoanthracene

ICR 191: ICR 191 acridine mutagen dihydrochloride

NF: nitrofurantoine

CUHP: cumene hydroperoxide

t: toxic (reduced background lawn; not counted)

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 ± SD)
		Base-pair substitution type			Frameshift type
		TA1535	TA100	TA102	TA97a	TA98
-	0 (DMSO)	15 ± 4.7	118 ± 8.3	354 ± 34.8	368 ± 72.3	18 ± 3.0
-	16	11 ± 1.5	103 ± 14.2	-	467 ± 47.3	-
-	50	8 ± 4.0	122 ± 25.3	338 ± 33.2	419 ± 7.1	30 ± 3.6
-	160	8 ± 2.0	110 ± 4.6	315 ± 18.3	396 ± 17.1	28 ± 2.6
-	500	15 ± 6.6	101 ± 15.0	312 ± 24.2	392 ± 100.1	28 ± 5.7
-	1600	t	t	151 ± 22.6	t	36 ± 3.6
-	5000	-	-	t	-	t
Positive controls, –S9	Name	NaN3	NF	CUHP	ICR 191	4-NOPD
	Concentration [μg/plate]	0.25	0.2	100	0.5	0.5
	Induction rate	9.9	3.2	> 3.4	> 3.3	5.3
+	0 (DMSO)	13 ± 3.5	134 ± 4.9	488 ± 107.0	338 ± 38.8	34 ± 6.1
+	16	-	-	-	368 ± 31.4	-
+	50	10 ± 3.2	107 ± 22.5	279 ± 26.0	354 ± 11.1	32 ± 4.0
+	160	11 ± 3.5	97 ± 11.1	314 ± 16.4	311 ± 21.2	35 ± 0.6
+	500	9 ± 2.1	105 ± 22.3	294 ± 8.3	329 ± 12.0	34 ± 6.1
+	1600	8 ± 2.0	75 ± 4.2	192 ± 27.2	t	50 ± 7.0
+	5000	t	t	t	-	t
Positive controls, +S9	Name	2-AA	2-AA	Dantron	2-AA	2-AA
	Concentration [μg/plate]	2.0	2.0	30	2.0	2.0
	Induction rate	6.3	> 8.9	> 2.5	> 3.6	> 35.3

NaN3: sodium azide

4-NOPD: 4-nitro-1,2-phenylene-diamine

2-AA: 2-aminoanthracene

ICR 191: ICR 191 acridine mutagen dihydrochloride

NF: nitrofurantoine

CUHP: cumene hydroperoxide

t: toxic (reduced background lawn; not counted)

Applicant's summary and conclusion

Conclusions:

Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA 97a, TA 98, TA 100, TA 102 and TA 1535) tested with and without metabolic activation up to cytotoxic concentrations or 5000 µg/plate.

Executive summary:

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2001). In this study the substance was not mutagenic in any of the five strains (TA 97a, TA 98, TA100, TA 102 and TA 1535) tested with and without metabolic activation up to cytotoxic concentrations or 5000 µg/plate.