Reaction mass of α,α,4-trimethyl-, (1S)-, 3-cyclohexene-1-methanol and α,α,4-trimethyl-, (1R)-, 3-cyclohexene-1-methanol and 1-methyl-4-(1-methylethylidene)-cyclohexanol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 March 2006 - 9 May 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study in compliance with OECD guideline 471 without any deviation.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(R)-α,α,4-trimethylcyclohex-3-ene-1-methanol

EC Number:

232-081-5

EC Name:

(R)-α,α,4-trimethylcyclohex-3-ene-1-methanol

Cas Number:

7785-53-7

Molecular formula:

C10H18O

IUPAC Name:

α,α-4-trimethyl-(1R)-3-cyclohexene-1-methanol

Constituent 2

Reference substance name:

p-menth-1-en-8-ol

EC Number:

233-986-8

EC Name:

p-menth-1-en-8-ol

Cas Number:

10482-56-1

Molecular formula:

C10H18O

IUPAC Name:

α,α-4-trimethyl-(1S)-3-cyclohexene-1-methanol

Constituent 3

Reference substance name:

1-methyl-4-(1-methylethylidene)cyclohexan-1-ol

EC Number:

209-584-3

EC Name:

1-methyl-4-(1-methylethylidene)cyclohexan-1-ol

Cas Number:

586-81-2

Molecular formula:

C10H18O

IUPAC Name:

1-methyl-4-(1-methylethylidene)-cyclohexanol

impurity 1

Reference substance name:

cis-4-isopropenyl-1-methylcyclohexanol

Cas Number:

7299-41-4

Molecular formula:

C10H18O

IUPAC Name:

cis-4-isopropenyl-1-methylcyclohexanol

impurity 2

Reference substance name:

4-(isopropyl)-1-methylcyclohex-3-en-1-ol

EC Number:

209-585-9

EC Name:

4-(isopropyl)-1-methylcyclohex-3-en-1-ol

Cas Number:

586-82-3

Molecular formula:

C10H18O

IUPAC Name:

4-isopropyl-1-methyl-3-cyclohexen-1-ol

impurity 3

Reference substance name:

1-methyl-4-[1-1-(1-methylethoxy)ethyl]-cyclohexene

Cas Number:

27153-55-5

Molecular formula:

C13H24O

IUPAC Name:

1-methyl-4-[1-1-(1-methylethoxy)ethyl]-cyclohexene

impurity 4

Reference substance name:

trans-1-methyl-4-(1-methylethenyl)-cyclohexanol

Cas Number:

7299-40-3

Molecular formula:

C10H18O

IUPAC Name:

trans-1-methyl-4-(1-methylethenyl)-cyclohexanol

impurity 5

Reference substance name:

(1S-endo)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-ol

EC Number:

208-135-9

EC Name:

(1S-endo)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-ol

Cas Number:

512-13-0

Molecular formula:

C10H18O

IUPAC Name:

1,3,3-trimethyl-(1S,2S,4R)-bicyclo[2.2.1]heptan-2-ol

impurity 6

Reference substance name:

1,3,3-trimethyl-(1R,2R,4S)-bicyclo[2.2.1]heptan-2-ol

Cas Number:

2217-02-9

Molecular formula:

C10H18O

IUPAC Name:

1,3,3-trimethyl-(1R,2R,4S)-bicyclo[2.2.1]heptan-2-ol

impurity 7

Reference substance name:

(1S-endo)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol

EC Number:

207-353-1

EC Name:

(1S-endo)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol

Cas Number:

464-45-9

Molecular formula:

C10H18O

IUPAC Name:

1,7,7-trimethyl-(1S,2R,4S)- bicyclo[2.2.1]heptan-2-ol

impurity 8

Reference substance name:

(1R,2S,4R)-borneol

EC Number:

207-352-6

EC Name:

(1R,2S,4R)-borneol

Cas Number:

464-43-7

Molecular formula:

C10H18O

IUPAC Name:

1,7,7-trimethyl-(1R,2S,4R)-bicyclo[2.2.1]heptan-2-ol

Test material form:

liquid

Details on test material:

Batch No.: 049807
Purity: 67.2% (sum of the three main constituents)
Name of test material (as cited in study report): TERPINEOL MULTICONSTITUENT
Physical state: colourless liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 30 November 2017

Method

Target gene:

Not applicable

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: bacterial broth medium
- Properly maintained: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

- Type and identity of media: bacterial broth medium
- Properly maintained: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 fraction from Sprague Dawley rats treated with beta-naphtoflavone and phenobarbital

Test concentrations with justification for top dose:

Cytotoxicity test: 50, 150, 500, 1500 and 5000 µg/plate
Main test: 19, 56, 167, 500 and 1500 µg/plate

Vehicle / solvent:

DMSO
- Justification for choice of solvent/vehicle: test substance not soluble in aqueous vehicles

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See table 1

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period (before treatment): 13 h at 37°C
- Preincubation period (with test substance) in the second main experiment with S9: 30 min
- Exposure duration: 48 h at 37 °C
- Expression time (cells in growth medium): 48 h at 37 °C

SELECTION AGENT (mutation assays): minimum medium

NUMBER OF REPLICATIONS:
- cytotoxicity test: 1
- main test: 3

DETERMINATION OF CYTOTOXICITY
- Method: number of revertants

OTHER: 2 mL agar, 0.1 mL of test substance dilution, 0.1 mL of bacterial culture and 0.5 mL of S9 mix (when appropriate) were plated on solid minimum medium

Evaluation criteria:

- statistically significant ratio (revertants with test substance / revertants with solvent) higher than 2 for TA 98, TA 100 and TA 102, and higher than 3 for TA 1535 and TA 1537
- increase in the number of revertants linked with the increase in test substance concentration
- reproducible results

Statistics:

Dunett's test on revertants with test substance / revertants with solvent ratios.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate for all strains and slightly to strong at 1500 µg/plate depending on the strains

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

strong at 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation of the test substance was observed

RANGE-FINDING/SCREENING STUDIES: see table 4 for cytotoxicity results

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2: results of the first main experiment

Strain	Compound	Dose level (µg/plate)	S9 mix	Mean revertant colony counts	SD	Ratio treated/solvent	Individual revertant colony counts
TA 98	DMSO           2 nitrofluorene   DMSO           2-aminoanthracene	19 56 167 500 1500 1     19 56 167 500 1500 2	- - - - - - -   + + + + + + +	19 18 17 17 17 5 319   22 23 25 25 24 20 2653	4 2 3 3 4 5 46   5 6 7 4 7 4 150	0.9 0.9 0.9 0.9 0.3 *       1.0 1.1 1.1 1.1 0.9	23, 16, 19 19, 20, 16 14, 18, 20 19, 19, 14 13, 21, 17 3, 2, 11 320, 364, 273   27, 22, 18 16, 27, 25 29, 17, 29 30, 22, 23 18, 23, 32 24, 19, 17 2520, 2624, 2816
TA 100	DMSO           Sodium azide   DMSO           2-aminoant-racene	19 56 167 500 1500 1.5     19 56 167 500 1500 2	- - - - - - -   + + + + + + +	74 81 79 90 85 60 155   94 137 140 105 120 102 3397	6 6 16 12 12 17 15   12 3 3 3 14 9 411	1.1 1.0 1.2 1.1 0.8       1.5 * 1.5 * 1.1 1.3 * 1.1	71, 81, 70 74, 86, 82 65, 76, 97 79, 102, 89 95, 71, 88 79, 46, 55 141, 154, 171   102, 80, 101 135, 140, 136 140, 137, 142 106, 107, 102 105, 125, 131 107, 108, 92 3152, 3872, 3168
TA 102	DMSO           Mitomycine C   DMSO           Benzo(a)pyrene	19 56 167 500 1500 0.125     19 56 167 500 1500 4	- - - - - - -   + + + + + + +	192 193 225 125 73 0 1339   214 232 266 158 153 5 436	8 30 8 50 29 0 368   16 35 23 9 32 3 26	1.0 1.2 0.7 * 0.4 * 0.0 *       1.1 1.2 0.7 * 0.7 * 0.0 *	188, 186, 201 228, 172, 180 232, 216, 228 156, 68, 152 63, 51, 106 1060, 1200, 1756     208, 232, 201 212, 272, 212 288, 268, 242 158, 167, 149 176, 167, 116 3, 4, 8 464, 414, 429
TA 1535	DMSO           Azide sodium   DMSO           2-aminoanthracene	19 56 167 500 1500 1.5     19 56 167 500 1500 2	- - - - - - -   + + + + + + +	13 13 14 16 10 2 685   11 6 8 9 9 4 111	4 1 1 7 4 2 24   2 1 2 1 1 1 35	1.1 1.1 1.3 0.8 0.1 *     0.5 * 0.7 * 0.8 0.8 0.3 *	14, 8, 16 13, 13, 14 14,14, 15 9, 22, 17 5, 12, 12 0, 4, 1 658, 704, 694   11, 12, 9 6, 6, 5 7, 10, 6 8, 9, 9 8, 8, 10 3, 4, 4 149, 81, 103
TA 1537	DMSO           9-aminoacridine   DMSO           2-aminoanthracene	19 56 167 500 1500 40     19 56 167 500 1500 2	- - - - - - -   + + + + + + +	10 6 7 10 6 7 957   7 6 4 9 8 3 192	6 4 2 1 2 2 93   3 1 1 2 3 3 28	0.5 0.7 1.0 0.5 0.6       0.8 0.6 1.3 1.1 0.4 *	17, 5, 9 3, 4, 10 5, 7, 9 10, 10, 11 7, 4, 6 9, 6, 5 1002, 1018, 850   10, 5, 7 5, 6, 7 4, 4, 5 7, 11, 10 6, 7, 12 6, 2, 1 161, 202, 214

* statistically significant (p<0.05)

Table 3: results of the second main experiment

Strain	Compound	Dose level (µg/plate)	S9 mix	Mean revertant colony counts	SD	Ratio treated/solvent	Individual revertant colony counts
TA 98	DMSO           nitro-2-fluorene   DMSO           2-aminoanthracene	19 56 167 500 1500 1.5     19 56 167 500 1500 1	- - - - - - -   + + + + + + +	22 22 15 22 16 6 360   21 28 28 27 13 2 1229	5 4 2 2 3 2 60   9 6 2 9 6 4 85	1.0 0.7 * 1.0 0.7 0.3 *       1.3 1.3 1.3 0.6 0.1 *	23, 16, 26 24, 24, 17 16, 15, 13 23, 20, 24 19, 13, 15 8, 5, 6 316, 428, 336   30, 13, 20 33, 22, 28 29, 29, 26 35, 27, 18 20, 8, 11 0, 0, 7 1140, 1308, 1240
TA 100	DMSO           Sodium azide   DMSO           2-aminoanthracene	19 56 167 500 1500 1.5     19 56 167 500 1500 1	- - - - - - -   + + + + + + +	90 80 67 90 78 54 201   94 92 97 129 82 36 808	8 8 7 12 18 1 20   3 8 19 23 16 49 78	0.9 0.7 * 1.0 0.9 0.6 *       1.0 1.0 1.4 0.9 0.4 *	92, 82, 97 72, 87, 80 72, 59, 70 91, 78, 101 59, 80, 95 54, 54, 55 223, 185, 195   92, 93, 97 98, 83, 95 114, 100, 77 102, 138, 146 64, 95, 86 0, 16, 92 812, 884, 728
TA 102	DMSO           Mitomycine C   DMSO           Benzo(a)pyrene	19 56 167 500 1500 0.125     19 56 167 500 1500 2	- - - - - - -   + + + + + + +	180 205 159 154 105 4 1131   107 137 110 98 93 0 214	47 33 15 10 8 4 110   10 4 19 14 19 0 18	1.1 0.9 0.9 0.6 * 0.0 *       1.3 * 1.0 0.9 0.9 0.0 *	228, 178, 134 228, 168, 220 175, 147, 154 150, 147, 165 95, 110, 109 0, 8, 4 1192, 1004, 1196   100, 118, 103 142, 136, 134 93, 131, 106 113, 85, 95 76, 88, 114 0, 0, 0 218, 229, 194
TA 1535	DMSO           Azide sodium   DMSO           2-aminoanthracene	19 56 167 500 1500 1.5     19 56 167 500 1500 1	- - - - - - -   + + + + + + +	13 16 18 13 11 6 647   9 11 10 5 3 0 218	2 2 2 1 3 2 50   3 2 3 2 2 0 12	1.2 1.3 1.0 0.8 0.4 *     1.2 1.1 0.6 0.3 * 0.0 *	15, 12, 13 15, 15, 18 19, 18, 16 12, 13, 13 11, 8, 14 7, 7, 3 670, 590, 682   6, 10, 12 10, 11, 13 13, 9, 8 7, 5, 4 2, 5, 1 0, 0, 0 205, 229, 219
TA 1537	DMSO           9-aminoacridine   DMSO           2-aminoanthracene	19 56 167 500 1500 40     19 56 167 500 1500 1	- - - - - - -   + + + + + + +	10 7 10 6 7 3 336   6 9 7 7 9 0 45	1 4 3 4 4 3 118   2 0 1 2 14 0 6	0.7 1.0 0.6 0.6 0.3 *       1.4 1.2 1.1 1.5 0.0 *	11, 11, 9 12, 4, 5 13, 10, 8 11, 4, 4 3, 10, 7 0, 3, 6 468, 242, 298   5, 8, 6 9, 9, 9 7, 8, 7 5, 7, 8 26, 2, 0 0, 0, 0 38, 49, 48

* statistically significant (p<0.05)

Applicant's summary and conclusion

Conclusions:

Terpineol multiconstituent was found not to be mutagenic in TA 98, TA 100, TA 102, TA 1535 and TA 1537 with and without metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to OECD Guideline 471 and in compliance with GLP, strains of S. typhimurium (TA 98, TA 100, TA 102, TA 1535 and TA 1537) were exposed to Terpineol multiconstituent at concentrations of 50, 150, 500, 1500 and 5000 µg/plate in both the absence and presence of S9 metabolic activation according to the direct plate incorporation method for a preliminary cytotoxicity test. In the main test, two experiments were performed at 19, 56, 167, 500 and 1500 µg/plate (up to cytotoxic concentrations) in both the absence and presence of S9. These experiments were performed according to the direct plate incorporation method except for the second experiment with S9 which was performed with a pre incubation period of 30 min with the test substance and S9 mix.

The positive controls induced the appropriate responses in the corresponding strains. Terpineol multiconstituent showed no biologically significant increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of S9 mix.

This study is classified as acceptable and satisfies the requirement for bacterial reverse gene mutation endpoint.

Therefore, Terpineol multiconstituent is not considered as mutagenic in this bacterial system according to CLP Regulation (EC) No 1272/2008.