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EC number: 401-270-6 | CAS number: 122390-99-2
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Genetic toxicity: in vitro
Some information in this page has been claimed confidential.
Administrative data
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- migrated information: read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 2014-04-23 until 2014-07-14
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: according to OECD 476; conducted with a structural analogue
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 014
- Report date:
- 2014
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Principles of method if other than guideline:
- first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation - GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
Test material
- Test material form:
- solid: particulate/powder
- Remarks:
- migrated information: powder
- Details on test material:
- see below
Constituent 1
Method
- Target gene:
- HPRT
Species / strain
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
- Test concentrations with justification for top dose:
- Experiment I:
without metabolic activation: 250.0; 500.0; 1000; 2000; and 2500 µg/mL
with metabolic activation: 1875; 3750; 5500; 6500; and 7500 µg/mL
Experiment II:
without metabolic activation: 1875; 2750; 3750; 5500; and 6500 µg/mL
with metabolic activation: 1875; 3750; 5500; 6500; and 7500 µg/mL - Vehicle / solvent:
- Deionised water. The final concentration of deionised water in the culture medium was 10% (v/v).
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days
SELECTION AGENT (mutation assays): 6-Thioguanine
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: >1,5x10exp. 6
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concentrations a mutation frequency that is three times higher than the spontaneous mutation frequency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the corresponding solvent control data. - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. 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 and statistical significance were considered together.
Results and discussion
Test results
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- 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: Not effected (pH 7.35 in the solvent control versus pH 7.41 at 7520 µg/mL)
- Effects of osmolality: No relevant increase (286 mOsm in the solvent control versus 316 mOsm at 7520 µg/mL)
- Evaporation from medium: Not examined
- Water solubility: Soluble
- Precipitation:No precipitation of the test item was observed up to the maximum concentration in any of the experiments.
- Other confounding effects: None
RANGE-FINDING/SCREENING STUDIES:
According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20% relative survival or cell density at subcultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up to or beyond their limit of solubility. Precipitation should be evaluated at the beginning and at the end of treatment by the unaided eye.
The range finding pre-experiment was performed using a concentration range of 58.8 to 7520 µg/mL to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24-hours treatment) of metabolic activation. The highest applied concentration in the pre-test on toxicity (7520 µg/ml) was equal to 5 mg/mL of the pure substance.
Strong toxic effects occurred after 4 hours treatment at 3760 µg/mL and above without metabolic activation and at 7520 µg/mL with metabolic activation. Following 24 hours treatment without metabolic activation a strong toxic effect occurred at 7520 µg/mL indicated by a completely inhibited cell growth.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. No precipitation or phase separation was observed up to the maximum concentration with and without metabolic activation following 4 and 24 hours treamtment.
There was no relevant shift of the osmolarity and pH value of the medium even at the maximum concentration of the test item.
The dose range of the first experiment was set according to the data generated in the pre-experiment. The dose range of the second experiment was adjusted to data produced in the pre-experiment (without metabolic activation) and in the first experiment (with metabolic activation). The individual concentrations were generally spaced by a factor of 2.0. A narrower spacing was used at higher concentrations to cover the cytotoxic range more closely.
To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations.
COMPARISON WITH HISTORICAL CONTROL DATA: Complies
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects, indicated by a relative cloning efficiency I or a relative cell density at first subcultivation of less than 50% in both parallel cultures, occurred in the first experiment at 2000 µg/mL and above without metabolic activation and at 5500 µg/mL and above with metabolic activation following 4 hours treatment. In the second experiment relevant cytotoxic effects as described above were noted at 3750 µg/mL and above without metabolic activation and at 5500 µg/mL with metabolic activation. The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered with and without metabolic activation. - Remarks on result:
- other: strain/cell type: Chinese hamster lung fibroblasts (V79)
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Summary Table
relative | relative | relative | mutant | relative | relative | relative | mutant | |||||
conc. | S9 | cloning | cell | cloning | colonies/ | induction | cloning | cell | cloning | colonies/ | induction | |
µg/mL | mix | efficiency I | density | efficiency II | 106cells | factor | efficiency I | density | efficiency II | 106cells | factor | |
% | % | % | % | % | % | |||||||
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
Experiment I / 4 h treatment | culture I | culture II | ||||||||||
Solvent control with water | - | 100.0 | 100.0 | 100.0 | 8.2 | 1.0 | 100.0 | 100.0 | 100.0 | 9.4 | 1.0 | |
Positive control (EMS) | 150.0 | - | 102.8 | 98.5 | 119.9 | 182.7 | 22.3 | 101.9 | 145.3 | 88.1 | 385.4 | 40.8 |
Test item | 250.0 | - | 102.6 | 90.1 | 139.8 | 4.6 | 0.6 | 105.3 | 101.0 | 106.6 | 6.0 | 0.6 |
Test item | 500.0 | - | 93.0 | 109.4 | 109.3 | 10.1 | 1.2 | 102.4 | 97.8 | 117.3 | 5.0 | 0.5 |
Test item | 1000.0 | - | 94.7 | 82.2 | 132.3 | 3.6 | 0.4 | 98.1 | 80.5 | 111.6 | 10.5 | 1.1 |
Test item | 2000.0 | - | 48.9 | 33.4 | 142.6 | 22.7 | 2.8 | 57.5 | 36.6 | 112.1 | 18.7 | 2.0 |
Test item | 2500.0 | - | 7.8 | 23.1 | 108.8 | 14.1 | 1.7 | 11.6 | 13.7 | 97.3 | 20.8 | 2.2 |
Test item | 3000.0 | - | culture was not continued# | culture was not continued# | ||||||||
Test item | 4000.0 | - | culture was not continued# | culture was not continued# | ||||||||
Solvent control with water | + | 100.0 | 100.0 | 100.0 | 11.4 | 1.0 | 100.0 | 100.0 | 100.0 | 16.6 | 1.0 | |
Positive control (DMBA) | 1.1 | + | 101.2 | 78.5 | 127.8 | 99.7 | 8.8 | 102.8 | 92.9 | 82.1 | 242.9 | 14.7 |
Test item | 468.75 | + | 99.1 | culture was not continued## | 101.5 | culture was not continued## | ||||||
Test item | 937.5 | + | 93.1 | culture was not continued## | 91.2 | culture was not continued## | ||||||
Test item | 1875.0 | + | 84.2 | 90.9 | 129.3 | 14.3 | 1.3 | 82.8 | 112.1 | 77.7 | 14.3 | 0.9 |
Test item | 3750.0 | + | 61.3 | 59.9 | 95.5 | 20.2 | 1.8 | 60.4 | 115.8 | 85.8 | 24.5 | 1.5 |
Test item | 5500.0 | + | 32.4 | 58.2 | 97.1 | 25.5 | 2.2 | 29.6 | 75.1 | 53.5 | 26.1 | 1.6 |
Test item | 6500.0 | + | 15.8 | 39.5 | 84.8 | 2.1 | 0.2 | 15.1 | 91.3 | 89.4 | 21.7 | 1.3 |
Test item | 7500.0 | + | 15.0 | 29.2 | 109.9 | 19.5 | 1.7 | 8.7 | 50.4 | 79.7 | 25.8 | 1.6 |
Experiment II / 24 h treatment | culture I | culture II | ||||||||||
Solvent control with water | - | 100.0 | 100.0 | 100.0 | 12.2 | 1.0 | 100.0 | 100.0 | 100.0 | 19.0 | 1.0 | |
Positive control (EMS) | 150.0 | - | 87.7 | 109.1 | 94.3 | 350.0 | 28.6 | 96.6 | 87.1 | 97.5 | 619.4 | 32.6 |
Test item | 468.75 | - | 86.8 | 97.5 | culture was not continued## | 105.1 | 83.0 | culture was not continued## | ||||
Test item | 937.5 | - | 88.9 | 83.5 | culture was not continued## | 99.9 | 75.9 | culture was not continued## | ||||
Test item | 1875.0 | - | 75.1 | 97.5 | 88.8 | 10.5 | 0.9 | 88.4 | 80.1 | 94.4 | 14.4 | 0.8 |
Test item | 2750.0 | - | 56.1 | 110.3 | 89.3 | 11.1 | 0.9 | 62.8 | 95.1 | 97.2 | 17.7 | 0.9 |
Test item | 3750.0 | - | 32.1 | 72.4 | 88.9 | 30.7 | 2.5 | 27.8 | 56.4 | 96.7 | 18.5 | 1.0 |
Test item | 5500.0 | - | 23.6 | 89.1 | 93.9 | 10.9 | 0.9 | 29.3 | 76.8 | 96.9 | 14.7 | 0.8 |
Test item | 6500.0 | - | 5.8 | 35.1 | 88.0 | 14.1 | 1.2 | 9.6 | 30.5 | 98.9 | 10.1 | 0.5 |
Test item | 7500.0 | - | 0.4 | 12.0 | culture was not continued# | 2.3 | 10.8 | culture was not continued# | ||||
Experiment II / 4 h treatment | ||||||||||||
Solvent control with water | + | 100.0 | 100.0 | 100.0 | 12.1 | 1.0 | 100.0 | 100.0 | 100.0 | 13.4 | 1.0 | |
Positive control (DMBA) | 2.2 | + | 80.8 | 89.3 | 94.4 | 226.6 | 18.7 | 91.1 | 91.7 | 71.5 | 334.7 | 25.0 |
Test item | 468.75 | + | 70.6 | culture was not continued## | 84.9 | culture was not continued## | ||||||
Test item | 937.5 | + | 72.8 | 73.3 | culture was not continued## | 81.3 | 79.3 | culture was not continued## | ||||
Test item | 1875.0 | + | 58.5 | 89.4 | 104.6 | 12.5 | 1.0 | 59.7 | 90.6 | 77.8 | 7.3 | 0.5 |
Test item | 3750.0 | + | 44.4 | 61.3 | 90.0 | 18.6 | 1.5 | 59.0 | 64.8 | 69.3 | 12.6 | 0.9 |
Test item | 5500.0 | + | 8.0 | 64.3 | 131.9 | 13.1 | 1.1 | 12.3 | 66.5 | 70.6 | 10.2 | 0.8 |
Test item | 6500.0 | + | 11.4 | 62.2 | 119.0 | 12.9 | 1.1 | 14.2 | 65.1 | 81.9 | 15.0 | 1.1 |
Test item | 7500.0 | + | 6.8 | 46.3 | 96.6 | 22.2 | 1.8 | 9.6 | 49.1 | 49.9 | 21.0 | 1.6 |
# culture was not continued due to exceedingly severe cytotoxic effects
## culture was not continued since a minimum of only four analysable concentrations is required
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay. - Executive summary:
In a mammalian cell gene mutation assay (HPRT), V79 cells of the Chinese hamster cultured in vitro were exposed to the test item in water at concentrations of 1875; 2750; 3750; 5500; and 6500 µg/mL (without metabolic activation) and 1875; 3750; 5500; 6500; and 7500 µg/mL (with metabolic activation) in the presence and absence of S9 mammalian metabolic activation.
The test item was tested up to cytotoxic concentrations at 3750 µg/mL and above without metabolic activation and at 5500 µg/mL with metabolic activation. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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