Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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 α-3,3-trimethylcyclohexanemethanol multiconstituent. 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. 

Link to relevant study records
Reference
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:
11 February - 28 March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
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
Target gene:
None
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):
- 80, 160, 300, 625, 1250, 2500 and 5000 μg/plate, with and without S9-mix

Experiment 3 (plate-incorporation method without S9 mix; preincubation method with S9 mix):
TA 102: 20, 40, 80, 160, 300, 625 and 1250 μg/plate, without S9-mix
TA 98, TA 100 and TA 1535: 20, 40, 80, 160, 300, 625 and 1250 μg/plate, with S9-mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that α-3,3-trimethylcyclohexanemethanol 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 article stock solutions were prepared by formulating α-3,3-trimethylcyclohexanemethanol multiconstituent under subdued lighting in DMSO, with the aid of vortex mixing, 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 4.5 hours of initial formulation.
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
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, 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) or manually for accuracy or where confounding factors such as bubbles or splits in the agar affected the accuracy of the automated counter (manual counts for Experiment 2 treatments of strain TA98 in the presence of S-9 at 625 μg/plate were conducted to confirm the accuracy of the automated counts). The background lawn was inspected for signs of toxicity.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic if:
1. 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
2. Any observed response is reproducible under the same treatment conditions.
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).
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: No precipitation was observed on the test plates following incubation.
- 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 in the form of a slight thinning of the background bacterial lawn, with or without a concurrent marked reduction in revertant numbers was observed at 500 and/or 1600 μg/plate in all strains in the absence and presence of S-9. A complete killing of the test bacteria was observed at 5000 μg/plate in all strains in the absence and presence of S-9.
- Experiment 2: 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 625 μg/plate and above in strains TA100, TA1535 and TA102 in the absence of S-9, at 2500 μg/plate and above in strain TA1537 in the absence of S-9 and at 1250 μg/plate and above in strain TA98 in the absence of S-9 and all strains in the presence of S-9. Complete killing of the test bacteria was observed at a single replicate at 1250 μg/plate in strain TA102 in the presence of S-9. In addition, decreases in revertant numbers were observed at 300 μg/plate in strains TA98 and TA1537 in the presence of S-9.
- Experiment 3: Following the treatment, evidence of toxicity ranging from a diminution of the background bacterial lawn, to a complete killing of the test bacteria was observed at 625 μg/plate and above in strain TA102 in the absence of S-9 and at 160 μg/plate and above in strains TA98, TA100 and TA1535 in the presence of S-9.

None

Conclusions:
The test item is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98, TA100 and TA102) strains.
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 α-3,3-trimethylcyclohexanemethanol multiconstituent at the following concentrations: 

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)

- 80, 160, 300, 625, 1250, 2500 and 5000 μg/plate, with and without S9-mix

Experiment 3 (plate-incorporation method without S9 mix; preincubation method with S9 mix)

TA 102: 20, 40, 80, 160, 300, 625 and 1250 μg/plate, without S9-mix

TA 98, TA 100 and TA 1535: 20, 40, 80, 160, 300, 625 and 1250 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.

 

No precipitation was observed on the test plates following incubation. In Experiment 1, following the treatment, evidence of toxicity was observed at 500 and/or 1600 μg/plate and above in all strains in the absence and presence of S-9. In Experiment 2, following treatment, evidence of toxicity was observed from 625 and/or 1250 and/or 2500 μg/plate in all strains in the absence and presence of S-9. In addition, a decrease in revertant numbers was observed at 300 μg/plate in strains TA98 and TA1537 in the presence of S-9. It should be noted that, due to the level of toxicity observed, only four concentrations could be scored for strain TA102 in the absence of S-9 and strains TA98, TA100 and TA1535 in the presence of S-9. In Experiment 3, following the treatment, evidence of toxicity was observed at 625 μg/plate and above in strain TA102 in the absence of S-9 and at 160 μg/plate and above in strains TA98, TA100 and TA1535 in the presence of S-9.

 

Vehicle and positive control treatments were included for all strains. The mean numbers of revertant colonies all 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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

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 α-3,3-trimethylcyclohexanemethanol multiconstituent at the following concentrations: 

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)

- 80, 160, 300, 625, 1250, 2500 and 5000 μg/plate, with and without S9-mix

Experiment 3(plate-incorporation method without S9 mix; preincubation method with S9 mix)

TA 102: 20, 40, 80, 160, 300, 625 and 1250 μg/plate, without S9-mix

TA 98, TA 100 and TA 1535: 20, 40, 80, 160, 300, 625 and 1250 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.

 

No precipitation was observed on the test plates following incubation. In Experiment 1, following the treatment, evidence of toxicity was observed at 500 and/or 1600 μg/plate and above in all strains in the absence and presence of S-9. In Experiment 2, following treatment, evidence of toxicity was observed from 625 and/or 1250 and/or 2500 μg/plate in all strains in the absence and presence of S-9. In addition, a decrease in revertant numbers was observed at 300 μg/plate in strains TA98 and TA1537 in the presence of S-9. It should be noted that, due to the level of toxicity observed, only four concentrations could be scored for strain TA102 in the absence of S-9 and strains TA98, TA100 and TA1535 in the presence of S-9. In Experiment 3, following the treatment, evidence of toxicity was observed at 625 μg/plate and above in strain TA102 in the absence of S-9 and at 160 μg/plate and above in strains TA98, TA100 and TA1535 in the presence of S-9.

 

Vehicle and positive control treatments were included for all strains. The mean numbers of revertant colonies all 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.

Justification for classification or non-classification

In a reverse gene mutation assay in bacteria, performed according to OECD Guideline 471 and in compliance with GLP, 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.