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EC number: 203-630-6 | CAS number: 108-93-0
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Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 21 December 2012 to 15 May 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 013
- Report date:
- 2013
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- from 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- from 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Version / remarks:
- from 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
Test material
- Reference substance name:
- Cyclohexanol
- EC Number:
- 203-630-6
- EC Name:
- Cyclohexanol
- Cas Number:
- 108-93-0
- Molecular formula:
- C6H12O
- IUPAC Name:
- cyclohexanol
- Test material form:
- not specified
- Details on test material:
- Supplier: Univar Benelux N.V.
Constituent 1
Method
- Target gene:
- Thymidine Kinase locus
Species / strain
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Complete culture medium: RPMI 1640 medium supplemented with 15 % horse serum, 100 U/ 100 µg/mL penicillin / streptomycin, 220 µg/mLsodium pyruvate and 0.5 - 0.75 U/mL amphotericin.Cloning medium: As for complete culture medium.Selective medium: Complete culture medium with the addition of 5 µg/mL TFT.- Properly maintained: Yes.- Periodically checked for Mycoplasma contamination: Yes- Periodically checked for karyotype stability:Yes- Periodically "cleansed" against high spontaneous background: YesPrior to mutagenicity testing, the amount of spontaneous mutants was reduced by growing the cells for one day in RPMI 1640-HAT medium supplemented with 1.0E-04 M hypoxanthine, 2.0E-07 M aminopterin and 1.6E-05M thymidine. The incubation of the cells in HAT-medium was followed by a recovery period of two days in RPMI 1640 medium containing 1.0E-04 M hypoxanthine and 1.6E-05 thymidine. After this incubation the cells were returned to normal RPMI 1640 medium. Large stocks of the cleansed L5178Y cell line are stored in liquid nitrogen in the cell back of Harlan CCR allowing the repeated use of the same cell culture batch in many experiments. Before freezing, each batch was screened for mycoplasma contamination and checked for karyotype stability.Thawed stock cultures are propagated in plastic flasks in RPMI 1640 complete culture medium. The cells are sub-cultured two times prior to treatment. The cell cultures were incubated at 37 ± 5 °C in humidified atmosphere with 4.5 % carbon dioxide and 95.5 % ambient air.
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 liver microsomal fraction
- Test concentrations with justification for top dose:
- - Experiment I, without S9 mix: 62.5, 125.0, 250.0, 500.0, 1000.0 µg/mL
- Experiment I, with S9 mix: 62.5, 125.0, 250.0, 500.0, 1000.0 µg/mL
- Experiment II, without S9 mix: 62.5, 125.0, 250.0, 500.0, 1000.0 µg/mL
- Experiment II, with S9 mix: 62.5, 125.0, 250.0, 500.0, 1000.0 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubilisation properties and its nontoxicity to the cells.
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: In medium
DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 - 15 days
SELECTION AGENT (mutation assays): RPMI 1640 medium supplemented with 15 % horse serum (HS), 100 U/100 µg/mL Penicillin/Streptomycin, 220 µg/mL Sodium-Pyruvate, and 0.5 – 0.75 U/mL Amphotericin B used as antifungal agent plus 5 µg/mL TFT.
NUMBER OF REPLICATIONS: 2NUMBER OF CELLS EVALUATED: 10^7
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, relative total growthIn the pre-test screen, 10^7 cells were exposed to each concentration of the test substance for four hours with and without metabolic activation. Following treatment the cells were washed twice by centrifugation (425 g, 10 mins) and re-suspended in “Saline G”. Subsequently the cells were re-suspended in 30 mL complete culture medium for a 2-day growth period. The cell density was determined immediately after treatment and at each day of the growth period and adjusted to 3E+05 cells/mL if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated at the end of the growth period.
- POSITIVE CONTROL: Methyl methane sulfonate in nutrient medium to a final concentration of 0.18 mM (19.5 µg) without metabolic activation. Cyclophosphamide in 0.9 % saline to a final concentration of 10.7 µM (3.0 µg/mL) and 16.1 µM (4.5 µg/mL) with metabolic activation. - Evaluation criteria:
- A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10^6 cells above the corresponding solvent control or negative control, respectively. A relevant increase of the mutation frequency should be dose-dependent. A mutagenic response is considered to be reproducible if it occurs in both parallel cultures. However, in the evaluation of the test results the historical variability of the mutation rates in negative and vehicle controls and the mutation rates of all negative and vehicle controls of this study are taken into consideration. Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled. Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated. A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 10^6 cells above the corresponding solvent control or negative control, respectively.A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation.
- Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using a statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.
Results and discussion
Test results
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Remarks:
- All strains/cell types tested
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant test substance related effect on pH was noted.
- Effects of osmolality: No relevant test substance related effect on osmolarity was noted.
- Precipitation: No precipitation of the test item was noted in experiment I and II with and without metabolic activation.
RANGE-FINDING/SCREENING STUDIES:
- A pre-test was performed at doses of 7.8, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0 and 1000.0 µg/mL in order to determine the concentration range of the mutagenicity experiments. Both pH value and osmolarity were determined at the maximum concentration of the test item and in the solvent control without metabolic activation. 10^7 cells were exposed to each concentration of the test item for 4 hours with and without metabolic activation.
COMPARISON WITH HISTORICAL CONTROL DATA:
- The mutation rates in the vehicle controls lie within the historical range.
OTHER:
- In both main experiments no precipitation was noted up to the maximum concentration with and without metabolic activation.No relevant cytotoxic effects indicated by a relative cloning efficiency 1 (survival) or a relative total growth of less than 50 % in both cultures occurred in experiment I and II with and without metabolic activation.No substantial and reproducible concentration dependent increase of the mutation frequency exceeding the threshold of 126 above the corresponding solvent control was observed in the main experiments up to the maximum concentration tested with and without metabolic activation. A linear regression analysis (least squares) was performed to asses a possible concentration dependent increase of mutant frequencies using SYSTAT® 11 statistics software. No significant concentration dependent trend of the mutation frequency indicated by a probability value of < 0.05 was determined in any of the experimental groups.In this study, the range of the solvent control values was from 64 up to 158 mutant colonies per 10^6 cells; the range of the acceptable group values treated with the test substance was from 29 up to 226 mutant colonies per 10^6 cells. The highest value of 226 slightly exceeded the historical range of solvent controls (42 – 216 colonies per 10^6 cells) but there was no concentration dependent increase as indicated by the lack of statistical significance and the mutation frequency in the parallel culture remained well within the historical range of solvent controls under identical conditions, hence these data are considered acceptable.The positive controls showed a distinct increase in induced total mutant colonies at acceptable levels of cytotoxicity.
Any other information on results incl. tables
Summary of results, Experiment I and II
|
|
|
Relative |
Relative |
Mutant |
|
Relative |
Relative |
Mutant |
|
|
Conc. µg per mL |
S9 mix |
cloning efficiency |
total growth |
colonies/ 106cells |
threshold |
cloning efficiency |
total growth |
colonies/ 106cells |
thresholds |
Column |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
Experiment I/ 4 h treatment |
Culture I |
Culture II |
||||||||
Solv. control with water |
|
- |
100.0 |
100.0 |
64 |
190 |
100.0 |
100.0 |
77 |
203 |
Pos. control with MMS |
19.5 |
- |
70.8 |
43.7 |
362 |
190 |
84.2 |
46.3 |
302 |
203 |
Test item |
31.3 |
- |
70.8 |
Culture was not continued # |
88.9 |
Culture was not continued # |
||||
Test item |
62.5 |
- |
67.0 |
163.9 |
29 |
190 |
96.0 |
107.2 |
82 |
203 |
Test item |
125.0 |
- |
51.5 |
111.3 |
53 |
190 |
92.3 |
126.3 |
56 |
203 |
Test item |
250.0 |
- |
85.4 |
132.1 |
54 |
190 |
70.2 |
106.6 |
77 |
203 |
Test item |
500.0 |
- |
50.0 |
108.3 |
69 |
190 |
96.0 |
130.1 |
57 |
203 |
Test item |
1000.0 |
- |
61.4 |
88.9 |
88 |
190 |
88.9 |
73.1 |
97 |
203 |
|
|
|
|
|
|
|
|
|
|
|
Solv. control with water |
|
|
100.0 |
100.0 |
81 |
207 |
100.0 |
100.0 |
74 |
200 |
Pos, control with CPA |
3.0 |
+ |
29.4 |
27.1 |
665 |
207 |
18.6 |
26.6 |
928 |
200 |
Pos, control with CPA |
4.5 |
+ |
25.5 |
14.4 |
1295 |
207 |
23.4 |
14.9 |
1355 |
200 |
Test item |
31.3 |
+ |
124.2 |
Culture was not continued # |
72.8 |
Culture was not continued # |
||||
Test item |
62.5 |
+ |
91.6 |
80.8 |
95 |
207 |
97.8 |
94.0 |
103 |
200 |
Test item |
125.0 |
+ |
104.6 |
137.8 |
92 |
207 |
75.3 |
101.4 |
87 |
200 |
Test item |
250.0 |
+ |
103.0 |
84.8 |
109 |
207 |
80.6 |
116.4 |
88 |
200 |
Test item |
500.0 |
+ |
82.9 |
54.4 |
142 |
207 |
74.0 |
103.6 |
56 |
200 |
Test item |
1000.0 |
+ |
74.0 |
127.1 |
115 |
207 |
88.4 |
105.3 |
90 |
200 |
Experiment II / 4 h treatment |
Culture I |
Culture II |
||||||||
Solv. control with water |
|
- |
100.0 |
100.0 |
117 |
243 |
100.0 |
100.0 |
88 |
214 |
Pos. control with MMS |
19.5 |
- |
73.4 |
33.9 |
447 |
243 |
69.3 |
27.4 |
576 |
214 |
Test item |
31.3 |
- |
74.7 |
Culture was not continued # |
89.6 |
Culture was not continued # |
||||
Test item |
62.5 |
- |
69.7 |
121.3 |
104 |
243 |
79.7 |
96.0 |
59 |
214 |
Test item |
125.0 |
- |
77.5 |
151.2 |
114 |
243 |
76.0 |
86.3 |
74 |
214 |
Test item |
250.0 |
- |
68.5 |
133.4 |
97 |
243 |
81.0 |
68.3 |
97 |
214 |
Test item |
500.0 |
- |
76.1 |
142.1 |
87 |
243 |
86.6 |
60.3 |
79 |
214 |
Test item |
1000.0 |
- |
70.9 |
133.6 |
106 |
243 |
77.2 |
51.0 |
125 |
214 |
|
|
|
|
|
|
|
|
|
|
|
Solv. control with water |
|
|
100.0 |
100.0 |
150 |
276 |
100.0 |
100.0 |
158 |
284 |
Pos, control with CPA |
3.0 |
+ |
51.0 |
29.2 |
744 |
276 |
41.7 |
34.8 |
1434 |
284 |
Pos, control with CPA |
4.5 |
+ |
21.3 |
4.1 |
1652 |
276 |
12.8 |
4.7 |
3153 |
284 |
Test item |
31.3 |
+ |
96.6 |
Culture was not continued # |
98.1 |
Culture was not continued # |
||||
Test item |
62.5 |
+ |
126.2 |
100.5 |
106 |
276 |
118.9 |
155.1 |
146 |
284 |
Test item |
125.0 |
+ |
90.4 |
80.6 |
146 |
276 |
110.9 |
127.9 |
208 |
284 |
Test item |
250.0 |
+ |
95.0 |
133.4 |
106 |
276 |
87.9 |
155.1 |
181 |
284 |
Test item |
500.0 |
+ |
86.2 |
91.1 |
92 |
276 |
927 |
77.7 |
150 |
284 |
Test item |
1000.0 |
+ |
67.8 |
62.2 |
129 |
276 |
86.4 |
83.2 |
226 |
284 |
threshold = number of mutant colonies per 10^6 cells of each solvent control plus 126
# culture was not continued since a minimum of only four analysable concentrations is required
Applicant's summary and conclusion
- Conclusions:
- The test material did not induce mutations in the absence and presence of metabolic activation.
- Executive summary:
The mutagenic potential of the test material was assessed according to OECD Guideline 476. This GLP-study was performed to investigate the potential of the test item to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in three independent experiments, using two parallel cultures each. Experiments I and II were performed with and without liver microsomal activation and a treatment period of 4 hours.
The concentration range of the main experiments went up to approximately 10 mM. No cytotoxic effects occurred in the main experiments up to the maximum concentration in the absence and presence of metabolic activation. No substantial and reproducible dose dependent increase in mutant colony numbers was observed up to the maximum concentrations tested with and without metabolic activation. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.
In conclusion, it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, the test item is considered to be non mutagenic in this mouse lymphoma assay.
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