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EC number: 203-375-0 | CAS number: 106-22-9
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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
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: study according to OECD guideline and GLP
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 010
- Report date:
- 2010
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
Test material
- Reference substance name:
- reaction mass of geraniol and nerol
- IUPAC Name:
- reaction mass of geraniol and nerol
Constituent 1
Method
- Target gene:
- hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells
Species / strain
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- substrain K3
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 rat
- Test concentrations with justification for top dose:
- 1st Experiment
without S9 mix (4-hour exposure period)
0; 6.3; 12.5; 25.0; 50.0; 100.0; 150.0; 200.0 μg/mL
with S9 mix (4-hour exposure period)
0; 6.3; 12.5; 25.0; 50.0; 100.0; 150.0; 200.0 μg/mL
2nd Experiment
without S9 mix (24-hour exposure period)
0; 25.0; 50.0; 100.0; 150.0; 175.0; 200.0 μg/mL
with S9 mix (4-hour exposure period)
0; 12.5; 25.0; 50.0; 100.0; 150.0; 175.0; 200.0 μg/mL - Vehicle / solvent:
- Due to the insolubility of the test substance in water, dimethylsulfoxide (DMSO) was selected
as vehicle, which has been demonstrated to be suitable in the CHO/HPRT assay and for
which historical control data are available.
The final concentration of the vehicle DMSO in the culture medium was 1% (v/v).
Controlsopen allclose all
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation
- Positive controls:
- yes
- Positive control substance:
- other: methylcholanthrene
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium: Ham's F12 + 10%FCS
DURATION
- Preincubation period: 1 week, elimination of spontaneous HPRT-deficient mutants by pretreatment with "HAT" medium
- Exposure duration: 4 h and 24 h
- Expression time (cells in growth medium): about one week
- Selection time (if incubation with a selection agent): about one week
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days
SELECTION AGENT (mutation assays): Hypoxanthine-free Ham's F12 medium supplemented with 6-thioguanine (10 μg/mL), 1% (v/v) L-glutamine (200 mM), and 10% (v/v) fetal calf serum (FCS)
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: six flasks from every treatment group
NUMBER OF CELLS EVALUATED:3*10^5 cells seeded per flask at beginning of selection period
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; - Evaluation criteria:
- Cytotoxicity
The cloning efficiency (CE, %) was calculated for each test group as follows:
total number of colonies in the test group
CEabsolute = ————————————————————— x 100
total number of seeded cells in the test group
CEabsolute of the test group
CErelative = —————————————— x 100
CEabsolute of the vehicle/negative control
The number of colonies in every flask was counted and recorded. Using the formula above the values of absolute cloning efficiencies were
calculated. Based on these values the relative cloning efficiencies of the test groups were calculated and given in percentage compared with the
respective CEabsolute value of the corresponding vehicle/negative control (vehicle/negative control = 100%).
Mutant frequency
The number of colonies in every flask was counted and recorded. The sum of the mutant colony counts within each test group was subsequently normalized to 10^6 cells seeded.
The uncorrected mutant frequency (MFuncorr.) per 10^6 cells was calculated for each test group as follows:
total number of mutant colonies
MFuncorr. = —————————————–— x 106
number of seeded cells
The uncorrected mutant frequency was corrected with the absolute cloning efficiency 2 for each test group to get the corrected mutant frequency (MFcorr.):
MFuncorr.
MFcorr. = —–——–— x 100
CE2 absolute - Statistics:
- Due to the clearly negative findings, a statistical evaluation was not carried out.
Results and discussion
Test results
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at highest conc. of 200 µg/ml each
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- MUTANT FREQUENCY
- no relevant increase in the number of mutant colonies either without S9 mix or after addition of S9 in both experiments (4 and 24 hours treatment)
- positive control substances EMS (without S9 mix; 300 μg/mL) and MCA (with S9 mix; 20 μg/mL) induced clearly increased mutant frequencies
CYTOTOXICITY
- Cytotoxic effects indicated by clearly reduced cloning efficiencies of below 20% of control were observed in both experiments in the absence and presence of S9 mix in the highest applied concentration of 200 μg/mL each.
CELL MORPHOLOGY
- In both experiments, in the absence and presence of S9 mix after 4 and 24 hours treatment the morphology and attachment of the cells was adversely influenced at least at the highest applied concentration.
TREATMENT CONDITIONS
- Osmolarity and pH values were not influenced by test substance treatment.
- In this study, in the absence and the presence of S9 mix no precipitation in culture medium was observed up to the highest applied test substance concentration.
Any other information on results incl. tables
Summary of results
Exp. |
Exposure period |
Test groups |
S9 mix |
Prec.* |
Genotoxicity** MFcorr. |
Cytotoxicity*** |
|
CE1[%] |
CE2[%] |
||||||
1 |
4 hrs |
Vehicle control1 |
- |
- |
8.17 |
100.0 |
100.0 |
|
|
6.3 μg/mL |
- |
- |
(-) |
109.5 |
(-) |
|
|
12.5 μg/mL |
- |
- |
1.67 |
108.9 |
104.0 |
|
|
25.0 μg/mL |
- |
- |
2.95 |
114.7 |
105.4 |
|
|
50.0 μg/mL |
- |
- |
1.69 |
102.4 |
97.1 |
|
|
100.0 μg/mL |
- |
- |
2.65 |
99.9 |
102.7 |
|
|
150.0 μg/mL |
- |
- |
1.45 |
42.3 |
93.9 |
|
|
200.0 μg/mL |
- |
- |
- |
0.0 |
- |
|
|
Positive control2 |
- |
- |
164.63 |
112.5 |
77.8 |
2 |
24 hrs |
Vehicle control1 |
- |
- |
3.01 |
100.0 |
100.0 |
|
|
25.0 μg/mL |
- |
- |
3.24 |
105.8 |
99.8 |
|
|
50.0 μg/mL |
- |
- |
5.89 |
88.6 |
98.0 |
|
|
100.0 μg/mL |
- |
- |
1.82 |
72.7 |
87.1 |
|
|
150.0 μg/mL |
- |
- |
7.35 |
49.7 |
83.1 |
|
|
175.0 μg/mL |
- |
- |
0.69 |
30.6 |
97.1 |
|
|
200.0 μg/mL |
- |
- |
- |
1.6 |
- |
|
|
Positive control2 |
- |
- |
682.83 |
55.2 |
59.8 |
1 |
4 hrs |
Vehicle control1 |
+ |
- |
3.59 |
100.0 |
100.0 |
|
|
6.3 μg/mL |
+ |
- |
(-) |
91.0 |
(-) |
|
|
12.5 μg/mL |
+ |
- |
(-) |
95.3 |
(-) |
|
|
25.0 μg/mL |
+ |
- |
1.02 |
86.3 |
108.2 |
|
|
50.0 μg/mL |
+ |
- |
7.31 |
71.7 |
106.2 |
|
|
100.0 μg/mL |
+ |
- |
2.04 |
94.9 |
105.2 |
|
|
150.0 μg/mL |
+ |
- |
4.35 |
71.4 |
100.8 |
|
|
200.0 μg/mL |
+ |
- |
3.60 |
5.0 |
118.6 |
|
|
Positive control3 |
+ |
- |
75.54 |
96.7 |
102.1 |
2 |
4 hrs |
Vehicle control1 |
+ |
- |
7.11 |
100.0 |
100.0 |
|
|
12.5 μg/mL |
+ |
- |
(-) |
83.5 |
(-) |
|
|
25.0 μg/mL |
+ |
- |
3.53 |
85.3 |
90.3 |
|
|
50.0 μg/mL |
+ |
- |
4.04 |
79.2 |
87.6 |
|
|
100.0 μg/mL |
+ |
- |
2.42 |
100.7 |
90.8 |
|
|
150.0 μg/mL |
+ |
- |
7.77 |
76.9 |
88.2 |
|
|
175.0 μg/mL |
+ |
- |
3.86 |
34.0 |
94.0 |
|
|
200.0 μg/mL |
+ |
- |
- |
0.0 |
- |
|
|
Positive control3 |
+ |
- |
157.17 |
86.2 |
96.5 |
* Precipitation in culture medium at the end of exposure period
** Mutant frequency MFcorr.: number of mutant colonies per 10^6 cells corrected with the CE2 value
*** Cloning efficiency related to the respective negative/vehicle control
- Due to strong cytotoxicity the cultures were not continued
(-) Culture was not continued since a minimum of four concentrations is required by the guidelines
1DMSO 1% (v/v) 2EMS 300 μg/mL 3MCA 20 μg/mL
According to the results of the present in vitro study, the test substance did not lead to a relevant increase in the number of mutant colonies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each
other. The mutant frequencies at any concentration were within the range of the concurrent vehicle control values and within the range of our historical negative control data.
The mutation frequencies of the vehicle control groups were within our historical negative control data range including all vehicles used in our laboratory and, thus, fulfilled the acceptance criteria of this study.
The increase in the frequencies of mutant colonies induced by the positive control substances EMS and MCA clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative
According to the authors, under the experimental conditions chosen here, the conclusion is drawn that the test substance is not a mutagenic substance in the HPRT locus assay using CHO cells in the absence and the presence of metabolic activation.
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