Reaction mass of 2-(cis-4-methylcyclohexyl)propan-2-ol and 2-(trans-4-methylcyclohexyl)propan-2-ol and cis-1-methyl-4-(propan-2-yl)cyclohexanol and trans-1-methyl-4-(propan-2-yl)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:

weight of evidence

Study period:

18 February - 07 April 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

2014

Report date:

2014

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:

cis-α,α,4-trimethylcyclohexanemethanol

EC Number:

230-795-1

EC Name:

cis-α,α,4-trimethylcyclohexanemethanol

Cas Number:

7322-63-6

Molecular formula:

C10H20O

IUPAC Name:

cis-2-(4-methylcyclohexyl)propan-2-ol

Constituent 2

Reference substance name:

trans-α,α,4-trimethylcyclohexanemethanol

EC Number:

225-844-9

EC Name:

trans-α,α,4-trimethylcyclohexanemethanol

Cas Number:

5114-00-1

Molecular formula:

C10H20O

IUPAC Name:

trans-2-(4-methylcyclohexyl)propan-2-ol

Constituent 3

Reference substance name:

cis-4-isopropyl-1-methylcyclohexanol

Cas Number:

3901-95-9

Molecular formula:

C10H20O

IUPAC Name:

cis-4-isopropyl-1-methylcyclohexanol

Constituent 4

Reference substance name:

trans-4-isopropyl-1-methylcyclohexanol

Cas Number:

3901-93-7

Molecular formula:

C10H20O

IUPAC Name:

trans-4-isopropyl-1-methylcyclohexanol

impurity 1

Reference substance name:

Non identified impurities

Molecular formula:

Not applicable

IUPAC Name:

Non identified impurities

Test material form:

liquid

Details on test material:

Batch No. MP8 du 29/10/2013
Purity: 98.8% (sum of the 4 main constituents)
Name of the test item (as cited in the study report): DIHYDROTERPINEOL MULTICONSTITUENT
IUPAC Name of the test item: Reaction mass of cis-2-(4-methylcyclohexyl) propan-2-ol and trans-2-(4-methylcyclohexyl) propan-2-ol and
cis- 4-isopropyl-1-methylcyclohexanol and trans- 4-isopropyl-1-methylcyclohexanol
Synonym: Reaction mass of cis-α,α-4-trimethyl-cyclohexanemethanol and trans-α,α-4-trimethyl-cyclohexanemethanol and cis-1-methyl-4-(1-methylethyl)-cyclohexanol and trans-1-methyl-4-(1-methylethyl)-cyclohexanol
Physical state: colourless – slightly amber liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 28 October 2015

Method

Target gene:

None

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10 % S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1 (plate-incorporation method):
- TA1535, TA1537, TA98, TA100 and TA102: 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, with and without S9-mix

Experiment 2 (plate-incorporation method without S9 mix; preincubation method with S9 mix):
- TA1535, TA1537, TA98, TA100 and TA102: 8.192, 20.48, 51.20, 128, 320, 800 and 2000 μg/plate, without S9-mix
- TA100, TA1537 and TA102: 8.192, 20.48, 51.20, 128, 320, 800 and 2000 μg/plate, with S9-mix
- TA98 and TA1535: 20.48, 51.20, 128, 320, 800, 2000 and 5000 μg/plate, with S9-mix

Experiment 3 (preincubation method):
- TA98 and TA1535: 3.277, 8.192, 20.48, 51.2, 128, 320 and 800 μg/plate, with S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Dihydroterpineol multiconstituent was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to at least 100 mg/mL.Therefore, DMSO was selected as vehicle.
- Test substance preparation: Test substance stock solutions were prepared by formulating Dihydroterpineol multiconstituent under subdued lighting in DMSO, to give the maximum required treatment concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 3.5 h of initial formulation.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM:
Strains TA98, TA1535 and TA1537 were originally obtained from the UK NCTC. Strains TA100 and TA102 were derived from cultures originally obtained from Covance Laboratories Inc., USA.

METHOD OF APPLICATION: In agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes at 37 ± 1 °C, with shaking
- Incubation period: Plates were inverted and incubated at 37 ± 1 °C in the dark for 3 days in both direct plate and preincubation methods.

NUMBER OF REPLICATIONS:
- Vehicle and positive controls were included in quintuplicate and triplicate plates, respectively.
- Treatment (test item) groups were included in triplicate plates

DETERMINATION OF CYTOTOXICITY
- Method: The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response. Where mutation data from fewer than five treatment concentrations was obtained, an evaluation of the mutation data for the study as a whole was made. If the mutation data for any strain treatment was considered insufficient to provide a thorough and robust assessment of mutagenicity then additional testing was conducted.

OTHER:
- Strain characteristics: The inocula were taken from master plates or vials of frozen cultures, which had been checked for strain characteristics (histidine dependence, rfa character, uvrB character and resistance to ampicillin or ampicillin plus tetracycline). Checks were carried out according to Maron and Ames, 1983 and De Serres and Shelby, 1979.
- Colony counting: Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments). The background lawn was inspected for signs of toxicity.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control values.
The test article was considered positive in this assay if the above criterion was met.
The test article was considered negative in this assay if the above criterion was not met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and between experiments.

Statistics:

The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate). However, adequate interpretation of biological relevance was of critical importance (OECD, 1997; ICH S2(R1), 2011).

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: None
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA: Mean vehicle control counts fell within the laboratory’s historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiment 1: Following the treatment, evidence of toxicity ranging from a diminution of the background bacterial lawn, with or without a concurrent marked reduction in revertant numbers, to a complete killing of the test bacteria was observed at 500 μg/plate and/or 1600 μg/plate and above in all strains in the absence and presence of S-9.
- Experiment 2: Following the treatment, evidence of toxicity ranging from a slight thinning of the background bacterial lawn, to a complete killing of the test bacteria was observed at 800 μg/plate and/or at 2000 μg/plate and/or 5000 μg/plate in the absence and presence of S-9 in all strains. In addition, complete toxicity was observed at 320 μg/plate on a single plate for strain TA1537 in the presence of S-9 only. Since mutation data were only available for four concentrations in the presence of S-9 for strains TA98 and TA1535 due to toxicity, a further Experiment (Experiment 3) was performed.
- Experiment 3: Following the treatment, evidence of toxicity ranging from a slight thinning of the background bacterial lawn to a complete killing of the test bacteria was observed at 320 μg/plate and above in both strains.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

The test item is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98, TA100 and TA102) strains in a study conducted according to OECD Guideline 471.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102) were exposed to test item dihydroterpineol multiconstituent at the concentrations below. 

Experiment 1 (plate-incorporation method)

- TA1535, TA1537, TA98, TA100 and TA102: 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, with and without S9-mix 

Experiment 2 (plate-incorporation method without S9 mix; preincubation method with S9 mix)

- TA1535, TA1537, TA98, TA100 and TA102: 8.192, 20.48, 51.20, 128, 320, 800 and 2000 μg/plate, without S9-mix

- TA100, TA1537 and TA102: 8.192, 20.48, 51.20, 128, 320, 800 and 2000 μg/plate, with S9-mix

- TA98 and TA1535: 20.48, 51.20, 128, 320, 800, 2000 and 5000 μg/plate, with S9-mix 

Experiment 3 (preincubation method)

- TA98 and TA1535: 3.277, 8.192, 20.48, 51.2, 128, 320 and 800 μg/plate, with S9-mix 

Metabolic activation system used in this test is 10% S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254. Vehicle and positive control groups were also included in mutagenicity tests.

 

In Experiment 1, following the treatment, evidence of toxicity was observed at 500 μg/plate and/or 1600 μg/plate and above in all strains in the absence and presence of S-9. In Experiment 2, evidence of toxicity was observed at 800 μg/plate and/or at 2000 μg/plate and/or at 5000 μg/plate in the absence and presence of S-9 in all strains. In addition, complete toxicity was observed at 320 μg/plate on a single plate for strain TA1537 in the presence of S-9 only. Since mutation data were only available for four concentrations for strains TA98 and TA1535 in the presence of S-9 due to toxicity, a further experiment (Experiment 3) was performed. In Experiment 3, evidence of toxicity was observed at 320 μg/plate and above in both strains.

 

The mean numbers of revertant colonies fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation. 

 

Therefore, the test item is not considered as mutagenic in this bacterial system.