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EC number: 278-130-4 | CAS number: 75214-63-0
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Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From January 30th to February 15th, 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted 29 July, 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Test material
- Reference substance name:
- Basic Orange 064
- IUPAC Name:
- Basic Orange 064
Constituent 1
Method
Species / strain
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures).
- Cells: Sub-line (K1) of Chinese hamster ovary cell line CHO. The CHO cell line was originally derived from the ovary of a female Chinese hamster (Kao and Puck, 1967). The CHO K1 is a sub-line of CHO cell line.
- Methods for maintenance in cell culture if applicable: the cell stocks are kept in liquid nitrogen. For each experiment the cells were thawed rapidly, the cells diluted in Ham's F12 medium containing 10 % foetal bovine serum and incubated at ca. 37 °C in a humidified atmosphere of 5 % CO2 in air. Growing cells were subcultured in an appropriate number of flasks.
- Culturing: the CHO K1 cells for the study were grown in Ham's F12 medium (F12-10) supplemented with 1 % Antibiotic-antimycotic solution (containing 10000 U/ml penicillin, 10 mg/ml streptomycin and 25 µg/ml amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %).
MEDIA USED
- Periodically checked for Mycoplasma contamination: each batch of frozen cells was purged of HPRT mutants and was free for mycoplasma infections, tested by Central Agricultural Office, National Animal Health Institute, Budapest, Hungary.
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 mix
- Test concentrations with justification for top dose:
- MAIN EXPERIMENT: 250, 500, 1000 and 2000 µg/ml, with and without metabolic activation.
PRELIMINARY EXPERIMENT for CYTOTOXICITY: 125, 250, 500, 1000, 1500 and 2000 µg/ml, with and without metabolic activation. - Vehicle / solvent:
- - Solvent used: DMSO
- Justification for choice of solvent/vehicle: the solvent is compatible with the survival of the CHO cells and the S9 activity and was chosen based on the results of the preliminary Solubility Test. The suitability was confirmed with the available laboratory’s historical database.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- PROCEDURES
- Preparation of the dishes: on the day of treatment the culture medium of exponentially growing cell cultures were replaced with medium (F12-5) containing the test item.
- Treatment: a 5-hour treatment in the presence and absence of S9-mix was performed.
- Number of cells: 5 × 10^6 cells were each placed in sterile dishes.
- Incubation conditions: dishes were incubated for approximately 24 hours before treatment at 37 °C in a humidified atmosphere of 5 % CO2.
- Post-exposure incubation: following the exposure period the cells were washed with F12-5 medium and incubated in fresh F12-10 medium for 19 hours.
- Count: after the 19-hour incubation period, cells were washed twice with F12-10 medium and suspended by treatment with trypsin-EDTA solution and counted using a Bürker chamber.
- Precipitation check: solubility of the test item in the cultures was assessed by the naked eye, at the beginning and end of treatment.
- Adjustement of cell number: in samples where sufficient cells survived, cell number was adjusted to 10^5 cells/ml. Throughout the expression period, cells were transferred to dishes for growth or diluted to be plated for survival.
REPLICATES: duplicate cultures were used at each test item concentration, for negative (solvent) controls and the positive controls for treatment without and with S9-mix.
PLATING SURVIVAL
Following adjustment of the cultures to 2 × 10^5 cells/ml, samples from these cultures were diluted to 40 cells/ml.
A total of 5 ml (200 cells/dish) of the final concentration of each culture was plated into 3 parallel dishes (diameter is approx. 60 mm).
The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 6 days for growing colonies.
Then, colonies were fixed with methanol, stained with Giemsa and counted. Survivals were assessed by comparing the cloning efficiency of the test item treated groups to the negative (solvent) control.
EXPRESSION OF MUTANT PHENOTYPE: during the phenotypic expression period the cultures were subcultured. Aliquots of approximately 2×10^6 cells were taken on days 1, 3, 6 and evaluated on day 8.
SELECTION OF THE MUTANT PHENOTYPE: at the end of the expression period, cultures from each dose level were adjusted to 2 × 10^5 cells / dish (4 × five dishes) in selection medium (hypoxanthine Ham's F12-SEL medium) containing 3.4 µg/ml of thioguanine (6-TG).
PLATING OF VIABILITY: at the end of the expression period cell number in the samples was adjusted to 2 × 10^5 cells/ml. Cells were plated in 3 parallel dishes (diameter is approx. 60 mm) for a viability test as described in “Plating for Survival“ section for the survival test.
FIXATION AND STAINING OF COLONIES: after the selection period, the colonies were fixed with methanol for five minutes, stained with Giemsa and counted for either mutant selection or cloning efficiency determination.
MEASUREMENT PF pH AND OSMOLALITY
The pH and osmolality of the negative (solvent) control and test item solutions were determined in Pre-test on Toxicity (Concentration selection) and the Main Mutation Assay.
DETERMINATION OF CYTOTOXICITY
Treatment concentrations for the mutation assay were selected on the basis of the result of Pre-test on Toxicity. During the Pre-test on Toxicity (cytotoxicity assay), the cultures (more than 50 % confluent) was trypsinised and cell suspensions were prepared in Ham's F12-10 medium. Cells was seeded into petri dishes (tissue cultures quality: TC sterile) at 5 × 10^6 cells each and incubated with culture medium.
After 24 hours the cells were treated with the suitable concentrations of the test item in absence or in presence of S9 mix (50 µld at 37 °C in a humidified atmosphere of 5 % CO2 in air for 6 days for colony growing.
Colonies were then fixed with methanol and was stained with Giemsa and the colonies were counted. In order to determine cytotoxicity, survivals were assessed by comparing the colony forming ability of the treated groups to the negative (solvent) control. Precipitation of the test item in the final culture medium was visually examined at beginning and end of the treatments. In addition, pH and osmolality was considered for dose level selection. The results of pre-test on cell toxicity were used for dose selection of the test item concentrations used in the Main Mutation Assay.
PRELIMINARY SOLUBILITY TEST
A non GLP Preliminary Solubility Test was performed suspending the test item in DMSO. A homogeneous suspension was obtained up to a concentration of 100 mg/ml.
METABOLIC ACTIVATION FRACTION
The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH (Rathenau Strasse 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA). The protein concentrations of the S9 batch used in the experiments were 33.7, 33.8, 35.7 and 37.8 mg/ml.
The S9 Mix (with Rat Liver S9)
Before adding to the culture medium the S9 Mix will be kept in an ice bath.
S9 mix preparation (concentration in the mix): HEPES 0.2 ml/ml, KCl 0.1 ml/ml, MgCl2 0.1 ml/ml, NADP 0.1 ml/ml, D-Glucose-6-phosphate (Monosodium salt) 0.1 ml/ml, F12 medium 0.1 ml/ml and S9 0.3 ml/ml.
ACCEPTANCE CRITERIA
The assay was considered valid as all the following criteria were met:
- The mutant frequency of concurrent negative controls is within the 95 % control limits of the distribution of the laboratory’s historical negative control database.
- The positive control chemicals induced a statistically significant and biologically relevant increase in mutant frequency compared to the concurrent negative control. The increases are compatible with the laboratory historical positive control database.
- Adequate number of cells and concentrations were analysable.
- Two experimental conditions with and without metabolic activation were tested.
- The highest concentration is adequate.
- The cloning efficiency of the negative controls is between the range of 60 % to 140 % on Day 1 and 70 % to 130 % on Day 8. - Evaluation criteria:
- Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- any of the results are outside the distribution of the laboratory historical negative control data (based 95 % control limit),
- the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
all results are inside the distribution of the historical negative control data (based 95 % control limit). - Statistics:
- Statistical Analysis was performed with SPSS PC+ software for the following data:
- mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
- mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups.
- The lower and upper 95 % confidence intervals of historical control were calculated with C-chart.
Results and discussion
Test results
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- In both experimental parts, there were no statistically significant increases in mutation frequency when compared to the concurrent solvent control at any concentration tested in the absence and presence of metabolic activation. In the absence and in the presence of S9 mix, in the cultures at any concentrations tested the mean mutation frequency a little bit exceeded the 95 % confidence interval of the historical control data. Statistically significant differences to the historical control data was observed at concentration 2000 µg/ml in 1 out of 4 cultures. These findings were not considered to be biologically relevant since no dose-response relationships were noted, all values were within the normal range of mutation frequency and no statistical difference to the concurrent controls were observed.
CYTOTOXICITY
On Day 1, there was no cytotoxicity observed at the tested concentrations with the test item in absence of metabolic activation (S9 mix) and low cytotoxicity observed at the tested concentrations with the test item in presence of metabolic activation (S9 mix) when compared to the negative (solvent) controls, confirming the response seen in the dose selection cytotoxicity assays.
OSMOLALITY AND pH
The osmolality values of test item solutions did not show any significant alterations compared to the concurrent control groups in the Pre-test on Toxicity and Main Mutation Assay. The pH values of test item solutions showed a dose associated decrease in the acceptable range compared to the concurrent control groups in the Pre-test on Toxicity and Main Mutation Assay.
SOLUBILITY
A clear solution was obtained up to a concentration of 100 mg/ml. For examined test item concentrations no precipitation in the medium was noted.
CONTROLS
The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with ethyl methanesulfonate (1.0 µl/ml) and 7,12-Dimethyl benz[a]anthracene (20 µg/ml). The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies performed at this laboratory. Thus, the study is considered valid.
Any other information on results incl. tables
Preliminary experiment for cytotoxicity (5 hours treatment)
Test group | S9-mix | Treatment/time/ hour | Number of colonies/200cells/dish | Mean | Relativeasurvival (%) | ||
Untreated Control | – | - | 206 | 201 | 203 | 203.3 | 101 |
Solvent Control (DMSO) | – | 5 | 202 | 200 | 204 | 202.0 | 100 |
Test item, 125µg/ml | – | 5 | 203 | 199 | 203 | 201.7 | 100 |
Test item, 250µg/ml | – | 5 | 205 | 202 | 203 | 203.3 | 101 |
Test item, 500µg/ml | – | 5 | 199 | 203 | 204 | 202.0 | 100 |
Test item, 1000µg/ml | – | 5 | 203 | 202 | 204 | 203.0 | 100 |
Test item, 1500µg/ml | – | 5 | 194 | 200 | 197 | 197.0 | 98 |
Test item, 2000µg/ml | – | 5 | 194 | 199 | 198 | 197.0 | 98 |
Untreated Control | + | - | 201 | 201 | 203 | 201.7 | 103 |
Solvent Control (DMO) | + | 5 | 195 | 196 | 196 | 195.7 | 100 |
Test item, 125µg/ml | + | 5 | 191 | 190 | 195 | 192.0 | 98 |
Test item, 250µg/ml | + | 5 | 194 | 192 | 192 | 192.7 | 98 |
Test item, 500µg/ml | + | 5 | 152 | 158 | 157 | 155.7 | 80 |
Test item, 1000µg/ml | + | 5 | 135 | 139 | 140 | 138.0 | 71 |
Test item, 1500µg/ml | + | 5 | 128 | 132 | 127 | 129.0 | 66 |
Test item, 2000µg/ml | + | 5 | 124 | 126 | 122 | 124.0 | 63 |
* Relative to Solvent Control
Applicant's summary and conclusion
- Conclusions:
- The substance tested up to the maximum recommended concentration with and without metabolic activation system over a 5-hour treatment period did not induce statistically and biologically significant increases in mutant frequency over the background (negative solvent control).
Thus, the test item is considered as being non-mutagenic in this system. - Executive summary:
The test item was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver. Mutation Assay was performed at the following concentrations and treatment intervals: mutation assay 5-hour treatment period with and without S9-mix at 250, 500, 1000 and 2000 µg/ml.
In the performed Mutation Assay the concentration levels were chosen mainly based on the maximum recommended concentration. The maximum recommended concentration for soluble, lower -cytotoxic substances is 2000 µg/ml (based on the updated OECD guideline 476 (2016)).
Phenotypic expression was evaluated up to 8 days following exposure.
In both experimental parts, there were no statistically significant increases in mutation frequency when compared to the concurrent solvent control at any concentration tested in the absence and presence of metabolic activation. In the absence and in the presence of S9 mix, in the cultures at any concentrations tested the mean mutation frequency a little bit exceeded the 95 % confidence interval of the historical control data. Statistically significant differences to the historical control data was observed at concentration 2000 µg/ml in 1 out of 4 cultures. These findings were not considered to be biologically relevant since no dose-response relationships were noted, all values were within the normal range of mutation frequency and no statistical difference to the concurrent controls were observed.
The osmolality values of test item solutions did not show any significant alterations compared to the concurrent control groups in the Pre-test on Toxicity and Main Mutation Assay. The pH values of test item solutions showed a dose associated decrease in the acceptable range compared to the concurrent control groups.
The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.
Conclusion
The substance tested up to the maximum recommended concentration with and without metabolic activation system over a 5-hour treatment period did not induce statistically and biologically significant increases in mutant frequency over the background (negative solvent control).
Thus, the test item is considered as being non-mutagenic in this system.
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