Registration Dossier
Registration Dossier
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EC number: 217-123-2 | CAS number: 1746-03-8
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test: The test item is not mutagenic in the bacterial reverse mutation assay.
Chromosome aberration test: The test item did not induce structural chromosome aberrations in Chinese Hamster lung cells in the presence and absence of metabolic activation.
HPRT test: The test item is not mutagenic in the in vitro mammalian cell gene mutation test performed in CHO cells.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 7 February 2017- 9 February 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted 21st July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- May 30, 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)
- Version / remarks:
- August 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use
- Version / remarks:
- June 2012
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Toxi-Coop ZRT, 8230 Balatonfüred, Arácsi út 97, Hungary
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: VPS9016001
- Expiration date of the lot/batch: 27.06.2018
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (15-25°C) - Target gene:
- The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations.
The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC). - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Additional strain / cell type characteristics:
- other: S.ty.mur. TA98,100,1537,1535 rfa (cell wall), uvrB (DNA-repair) mutation
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
- Test concentrations with justification for top dose:
- Selection of the concentrations was done on the basis of a solubility test and a concentration range finding test.
±S9 Mix: 5000; 1600; 500; 160; 50 and 16 μg/plate in Experiment I (Initial Mutation Test) and Experiment II (Confirmatory Mutation Test) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO; ultrapure water
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulfoxide (DMSO)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- without metabolic activation, TA1537, 50 µg
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- ultrapure water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- without metabolic activation, TA100 and TA1535, 2 µg
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- ultrapure water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- without metabolic activation, E.coli WP2 uvrA, 2 µL
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulfoxide (DMSO)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (2AA)
- Remarks:
- with metabolic activation, all of Salmonella strains (2 µg), E.coli strain (50 µg)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulfoxide (DMSO)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-1,2-phenylene-diamine (NPD)
- Remarks:
- without metabolic activation, TA98, 4 µg
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 hours in the dark at 37°C
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: revertant colony number
The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and induction of mutations with 3 plates each. - Evaluation criteria:
- The colony numbers on the untreated, vehicle and positive controls and the test item treated plates were determined by manual counting, the mean values, standard deviations and the mutation rates were calculated.
A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.
Criteria for a Negative Response:
A test item is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation. - Statistics:
- None
- Key result
- Species / strain:
- S. typhimurium TA 1535
- 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
- Key result
- Species / strain:
- S. typhimurium TA 1537
- 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
- Key result
- Species / strain:
- S. typhimurium TA 98
- 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
- Key result
- Species / strain:
- S. typhimurium TA 100
- 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
- Key result
- Species / strain:
- E. coli WP2 uvr A
- 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:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No. In the main experiments the pH of the test system varied between 6.21 and 7.45.
- Precipitation: No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration levels (±S9 Mix).
RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and induction of mutations with 3 plates each. In the Informatory Toxicity Test inhibitory effect of the test item was not observed. The colony and background lawn development was not affected in any case. All of the obtained slight revertant colony number decreases or increases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.
Slightly lower revertant colony numbers (within the biological variability range of the applied test system) were obtained in S. typhimurium TA98 at 5000, 1600, 160 μg/plate in the absence and presence (±S9 Mix) and at 500 μg/plate, in the presence of exogenous metabolic activation (+S9 Mix).
The revertant colony numbers were above the vehicle control data range (within the historical control data and biological variability range) in S. typhimurium TA100 at 5000 and 16 μg/plate (-S9 Mix) and in TA98 at 16 μg/plate (+S9 Mix). - Conclusions:
- The test item did not increase the frequency of revertant colonies in bacteria in the presence and absence of the metabolic activation system and thus is considered to be non-mutagenic in bacteria.
- Executive summary:
The mutagenic potential of the test item was investigated according OECD TG 471 in Salmonella typhimurium strains TA 98, TA100, TA 1535, TA 1537, and TA 1538 and in E. coli WP2 uvrA. A plate incorporation test and a pre-incubation test were conducted with and without metabolic activation using S9 fraction of Phenobarbital and β-naphthoflavone (BNF) induced rat liver. The following concentrations were examined in triplicate: ±S9 Mix: 5000, 1600, 500; 160; 50 and 16 μg/plate. Positive and negative (vehicle) controls were run concurrently.
No substantial increases were observed in revertant colony numbers following treatment with the test item, neither in the presence nor in the absence of metabolic activation. Sporadic increases in revertant colony numbers compared to the vehicle control remained within the corresponding historical control data ranges. Control plates without mutagen showed no increase in revertant colonies. The positive controls showed the expected, biological relevant increases in revertant colonies in all tests. No cytotoxicity was observed in either tested strains with or without metabolic activation.
In conclusion, it can be stated that the test item is not mutagenic in these bacterial test systems either with or without metabolic activation at any dose level investigated.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 7 June 2017- 21 June 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 29 July 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 14 February 2017
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Version / remarks:
- August 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Toxi-Coop ZRT, 8230 Balatonfüred, Arácsi út 97, Hungary
- Type of assay:
- other: mammalian chromosome aberration assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: VPS9016001
- Expiration date of the lot/batch: 27.06.2018
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (15-25°C) - Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures)
- Suitability of cells: Stability of karyotype and morphology makes them suitable for gene toxicity assays with low background aberrations.
- Cell cycle length, doubling time or proliferation index: doubling time 12-14 h
- Modal number of chromosomes: diploid number, 2n=22
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 °C in a humidified atmosphere containing 5 % CO2. Medium: DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine and 1 % of Antibiotic-antimycotic solution (containing 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated fetal bovine serum (final concentration 10 %)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
- Test concentrations with justification for top dose:
- Dose levels were chosen mainly based on the cytotoxicity and the maximum recommended concentration. The maximum recommended concentration for lower -cytotoxic substances is 2000 μg/mL (based on the updated OECD Guideline 473 (2016)).
Experiment A with 3/20 h treatment/sampling time
without and with S9 mix 500, 1000 and 2000 μg/mL
Experiment A with 20/20 h treatment/sampling time
without S9 mix: 250, 500, 1000 and 2000 μg/mL
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 250, 500, 1000 and 2000 μg/mL
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 500, 1000 and 2000 μg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DME (Dulbecco’s Modified Eagle’s)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DME (Dulbecco’s Modified Eagle’s) medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with metabolic activation (study at a final concentration of 5.0 μg/mL)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DME (Dulbecco’s Modified Eagle’s) medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation (final concentrations of 0.4 and 1.0 μL/mL)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding: 5 x 10E5 cells/dish
DURATION
- Exposure duration: 3 hours and 20 hours
- Selection time: 2.5-3 hours prior to harvest
SPINDLE INHIBITOR: colchicine (0.2 μg/mL)
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation became plasma free) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies.
NUMBER OF CELLS EVALUATED: 300 metaphase cells containing 2 N ± 2 centromeres
DETERMINATION OF CYTOTOXICITY
- Method: cell counts, Relative Increase in Cell Counts (RICC) was calculated
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- OTHER: The changes in pH and osmolality of the test media were determined for every treatment. - Evaluation criteria:
- Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative because:
– 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 laboratory historical negative control data. - Statistics:
- For statistical analysis CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- 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:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: no precipitation in the medium at any concentration tested
- Solubility: The solubility was studied in a preliminary test. A clear solution was obtained up to a concentration of 50 mg/mL.
RANGE-FINDING/SCREENING STUDIES:
In order to determine the treatment concentrations of test item in the cytogenetic study a dose selection (cytotoxicity assay) was performed. Based on the cytotoxicity assay, the following concentrations were selected and evaluated in the main studies (Experiment A and B).
Experiment A with 3/20 h treatment/sampling time
without and with S9 mix 500, 1000 and 2000 μg/mL
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 250, 500, 1000 and 2000 μg/mL
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 250, 500, 1000 and 2000 μg/mL
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 500, 1000 and 2000 μg/mL - Conclusions:
- The test item did not induce structural chromosome aberrations in Chinese Hamster lung cells in the presence and absence of metabolic activation.
- Executive summary:
In a study according OECD TG 473 the ability of the test item to induce chromosome aberrations in V79 cells was investigated. The test item was dissolved in cell culture medium and cytotoxicity was tested with and without metabolic activation from S9 mix of phenobarbital and β-naphthoflavone induced rat liver. Based on these results, the following concentrations were analyzed in the main test:
Experiment A with 3/20 h treatment/sampling time without and with S9 mix 500, 1000 and 2000 μg/mL
Experiment A with 20/20 h treatment/sampling time without S9 mix: 250, 500, 1000 and 2000 μg/mL
Experiment B with 20/28 h treatment/sampling timewithout S9 mix: 250, 500, 1000 and 2000 μg/mL
Experiment B with 3/28 h treatment/sampling time with S9 mix: 500, 1000 and 2000 μg/mL
At least 300 well-spread metaphase cells were analyzed. There were no biologically significant increases in the number of cells showing structural chromosome aberrations, neither in the absence nor in the presence of metabolic activation. No statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberrations were inside the distribution of the laboratory historical negative control data. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in the presence or absence of metabolic activation. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. The concurrent positive controls Ethyl methanesulphonate and Cyclophosphamide caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.
In conclusion, the test item is considered as not clastogenic, neither in the presence nor absence of metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- March 27, 2017 - August 4, 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 29 July 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 30 May 2008
- Deviations:
- yes
- Remarks:
- There is a deviation from the guideline regarding the confirmation of negative results. Negative results were not confirmed.
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Version / remarks:
- August 1998
- Deviations:
- yes
- Remarks:
- There is a deviation from the guideline regarding the confirmation of negative results. Negative results were not confirmed.
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Toxi-Coop ZRT, 8230 Balatonfüred, Arácsi út 97, Hungary
- Type of assay:
- in vitro mammalian cell transformation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: VPS9016001
- Expiration date of the lot/batch: 27.06.2018
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (15-25°C) - Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Remarks:
- Sub-line (KI)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures)
MEDIA USED
- Type and identity of media including CO2 concentration:
Ham's F12 medium containing 10 % fetal bovine serum supplemented with 1 % of Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B); incubated at 37°C in humidified atmosphere of 5 % CO2 in air; during treatment with test item, solvent (negative control) and positive controls, the serum content was reduced to 5 %
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
- Test concentrations with justification for top dose:
- 125 µg/mL, 250 µg/mL, 500 µg/mL, 1000 µg/mL, 2000 µg/mL (without S9 fraction)
125 µg/mL, 250 µg/mL, 500 µg/mL, 1000 µg/mL (with S9 fraction)
Concentration levels were chosen mainly based on the cytotoxicity and the maximum recommended concentration. The maximum recommended concentration for lower cytotoxic substances is 2000 μg/mL (based on the updated OECD Guideline 476 (2016)). The concentrations selected covered a range from maximum (19-20% relative survival) to little or no cytotoxicity as recommended by Guideline. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Ham's F12 medium
- Justification for choice of solvent/vehicle: A clear solution was obtained after dissolving the test item up to a concentration of 50 mg/mL. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Ham's F12 medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Remarks:
- with metabolic activation (20 μg/mL)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Ham's F12 medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation (1.0 μL/mL)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in cell culture medium
- Cell density at seeding: 5 x10E6 cells/dish
DURATION
- Exposure duration: 5 hours
- Expression time: 19 h
- Selection time: 8 days
SELECTION AGENT: selection medium (hypoxanthine Ham's F12-SEL medium) containing 3.4 μg/mL of thioguanine (6-TG)
STAIN: stained with Giemsa
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 200 cells/dish for C.E. (Cloning Efficiency)
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - 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.
Test item is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria were fulfilled, the test item is considered clearly negative because:
- 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 compatible the distribution of the historical negative control data (based 95% control limit).
- The test item is then considered unable to induce gene mutations in cultured mammalian cells in this test system. - Statistics:
- Statistical analysis was done with SPSS PC+ software. The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis one-way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test. - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Remarks:
- Sub-line (KI)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: not observed
RANGE-FINDING/SCREENING STUDIES:
Toxicity was determined by comparing the colony forming ability of the treated groups to the negative (solvent) control. Results were noted as percentage of cells in relation to the negative control. Based upon the toxicity data of the pre-test the following concentrations were chosen for the main mutation assay:
5-hour treatment period without S9-mix:
125, 250, 500, 1000 and 2000 μg/mL
5-hour treatment period with S9-mix:
125, 250, 500, 750 and 1000 μg/mL - Conclusions:
- The test item is not mutagenic in a in vitro mammalian cell gene mutation test performed in CHO-K1 cells.
- Executive summary:
In the HPRT test conducted according to the OECD Guideline 476, CHO-K1 cells were treated with test item concentrations (using cell culture medium as vehicle) ranging from 125 to 2000 µg/mL in the main study, with and without metabolic activation. Concentration levels were chosen mainly based on the cytotoxicity and the maximum recommended concentration.
Ethylmethanesulphonate (1 µL/mL; without S9 fraction) and 7,12-dimethylbenzanthracene (20 µg/mL; with S9 fraction) were used as positive controls. Phenotypic expression was evaluated up to 8 days following exposure.
The test substance did not induce mutation at the hprt locus of CHO-K1cells when tested under the conditions employed in this study. There were no statistically and biologically significant differences between treatment groups compared to the concurrent and historical control groups and no dose-response relationships were noted. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in the osmolality of the test system were noted at the different dose levels tested. The measured pH of treatment solution was similar compared to the control values. 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.
In conclusion, the test item was not mutagenic in the in vitro mammalian cell gene mutation test performed in CHO-K1 cells.
Referenceopen allclose all
Table 1: Number of revertants per plate (mean of three plates), Experiment 1
strain TA 98 |
strain TA 100 |
Strain TA 1535 |
strain TA 1537 |
E. coli WP2 uvrA |
||||||
conc. [µg] |
-MA |
+MA |
-MA |
+MA |
-MA |
+MA |
-MA |
+MA |
-MA |
+MA |
Untreated control |
19.0 |
23.3 |
81.7 |
101.0 |
11.7 |
13.7 |
10.3 |
8.0 |
25.7 |
31.3 |
DMSO Control |
15.0 |
23.3 |
– |
99.0 |
– |
10.7 |
7.7 |
8.7 |
– |
28.7 |
Water control |
23.3 |
24.0 |
76.0 |
110.0 |
11.7 |
11.7 |
8.3 |
8.7 |
23.7 |
30.0 |
5000 |
24.7 |
21.0 |
90.7 |
80.0 |
12.0 |
10.0 |
9.7 |
6.3 |
25.0 |
30.7 |
1600 |
23.7 |
20.0 |
92.0 |
114.7 |
11.0 |
11.0 |
9.7 |
8.3 |
31.3 |
28.3 |
500 |
25.0 |
23.0 |
90.7 |
105.3 |
10.3 |
13.0 |
8.0 |
10.3 |
26.0 |
39.3 |
160 |
16.7 |
23.0 |
88.0 |
108.0 |
10.7 |
10.0 |
9.7 |
7.3 |
35.0 |
33.7 |
16 |
23.0 |
24.3 |
84.7 |
99.3 |
12.0 |
13.7 |
9.0 |
7.7 |
28.3 |
40.7 |
NPD (4µg) |
17.7 |
24.7 |
90.0 |
110.0 |
9.7 |
12.7 |
9.0 |
10.0 |
27.0 |
27.3 |
SAZ (2µg) |
344.0 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
9AA (50 µg) |
– |
– |
1072.0 |
– |
1037.3 |
– |
– |
– |
– |
– |
MMS (2µL) |
– |
– |
– |
– |
– |
– |
450.0 |
– |
– |
– |
2AA (2µg) |
– |
– |
– |
– |
– |
– |
– |
– |
810.7 |
– |
2AA (50 µg) |
– |
1597.3 |
– |
3014.7 |
– |
171.3 |
– |
153.3 |
– |
– |
Table 2: Number of revertants per plate (mean of three plates), Experiment 2
strain TA 98 |
strain TA 100 |
Strain TA 1535 |
strain TA 1537 |
E. coli WP2 uvrA |
||||||
conc. [µg] |
-MA |
+MA |
-MA |
+MA |
-MA |
+MA |
-MA |
+MA |
-MA |
+MA |
Untreated control |
15.7 |
28.7 |
81.7 |
106.7 |
11.7 |
11.7 |
8.0 |
9.0 |
30.0 |
33.7 |
DMSO Control |
17.0 |
25.3 |
– |
111.3 |
– |
13.3 |
8.3 |
8.3 |
– |
38.7 |
Water control |
19.3 |
30.0 |
82.3 |
104.0 |
12.0 |
14.3 |
8.0 |
8.7 |
34.3 |
51.0 |
5000 |
15.0 |
17.3 |
81.7 |
99.3 |
11.3 |
17.7 |
5.7 |
9.7 |
30.3 |
41.3 |
1600 |
17.7 |
22.3 |
90.3 |
93.0 |
11.3 |
10.0 |
9.3 |
9.3 |
35.3 |
49.3 |
500 |
18.0 |
25.3 |
120.3 |
90.0 |
10.7 |
12.0 |
6.7 |
9.7 |
40.0 |
49.0 |
160 |
19.3 |
21.3 |
90.3 |
94.7 |
12.7 |
11.7 |
10.3 |
12.0 |
34.3 |
37.7 |
50 |
19.7 |
22.3 |
94.3 |
90.3 |
10.3 |
12.3 |
9.3 |
10.7 |
33.0 |
38.3 |
16 |
16.3 |
24.0 |
90.3 |
102.7 |
10.7 |
19.3 |
9.0 |
10.7 |
24.7 |
36.7 |
NPD (4µg) |
187.3 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
SAZ (2µg) |
– |
– |
607.3 |
– |
984.0 |
– |
– |
– |
– |
– |
9AA (50 µg) |
– |
– |
– |
– |
– |
– |
170.7 |
– |
– |
– |
MMS (2µL) |
– |
– |
– |
– |
– |
– |
– |
– |
898.7 |
– |
2AA (2µg) |
– |
2496.0 |
– |
2546.7 |
– |
229.3 |
– |
333.0 |
– |
– |
2AA (50 µg) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
280.0 |
Table 1: Mean percentages of cells with structural chromosome aberration(s), Experiment A
Concentration |
S9 mix |
Treatment |
Harvesting time |
Mean aberrant cells/150 cells |
|
incl. gaps |
excl. gaps |
||||
Solvent control |
- |
3 h |
20 h |
6 |
2 |
Vinylphosphonic acid |
|||||
500µg/mL |
- |
3 h |
20 h |
7 |
2 |
1000µg/mL |
- |
3 h |
20 h |
6 |
3 |
2000µg/mL |
- |
3 h |
20 h |
6 |
3 |
Pos. Control (EMS) |
- |
3 h |
20 h |
39** |
33** |
Solvent control |
+ |
3 h |
20 h |
6 |
3 |
Vinylphosphonic acid |
|||||
500µg/mL |
+ |
3 h |
20 h |
5 |
3 |
1000µg/mL |
+ |
3 h |
20 h |
8 |
3 |
2000µg/mL |
+ |
3 h |
20 h |
8 |
3 |
Pos. Control (Cycl.) |
+ |
3 h |
20 h |
48** |
40** |
Positive control (-S9): Ethyl methanesulfonate (1.0µL/mL), Positive control (+S9): Cyclophosphamide (5.0µg/mL) **: p<0.01
Table 2: Mean percentages of cells with structural chromosome aberration(s), Experiment B
Concentration |
S9 mix |
Treatment |
Harvesting time |
Mean aberrant cells/150cells |
|
incl. gaps |
excl. gaps |
||||
Solvent control |
- |
20 h |
20 h |
5 |
3 |
Vinylphosphonic acid |
|||||
250 µg/mL |
- |
20 h |
20 h |
7 |
4 |
500µg/mL |
- |
20 h |
20 h |
6 |
2 |
1000µg/mL |
- |
20 h |
20 h |
5 |
3 |
2000µg/mL |
- |
20 h |
20 h |
6 |
4 |
Pos. Control (EMS) |
- |
20 h |
20 h |
46** |
39** |
Solvent control |
- |
20 h |
28 h |
6 |
3 |
Vinylphosphonic acid |
|||||
250 µg/mL |
- |
20 h |
28 h |
7 |
4 |
500 µg/mL |
- |
20 h |
28 h |
6 |
3 |
1000 µg/mL |
- |
20 h |
28 h |
7 |
3 |
2000 µg/mL |
- |
20 h |
28 h |
7 |
4 |
Pos. Control (EMS) |
- |
20 h |
28 h |
41** |
36** |
Solvent control |
+ |
3 h |
28 h |
6 |
3 |
500 µg/mL |
+ |
3 h |
28 h |
5 |
4 |
1000 µg/mL |
+ |
3 h |
28 h |
6 |
4 |
2000 µg/mL |
+ |
3 h |
28 h |
7 |
3 |
Pos. Control (Cycl.) |
+ |
3 h |
28 h |
44* |
35** |
Positive control (-S9): Ethyl methanesulfonate (0.4µL/mL), Positive control (+S9): Cyclophosphamide (5.0 µg/mL) **: p<0.01
Table 3: Number of polyploid cells and endoreduplicated cells, Experiment A
Concentration |
S9 mix |
Treatment |
Harvesting time |
Polyploid Cells (mean) |
Endoredup-lication (mean) |
Solvent control |
- |
3 h |
20 h |
0.0 |
0.0 |
Vinylphosphonic acid |
|||||
500µg/mL |
- |
3 h |
20 h |
0.0 |
0.0 |
1000 µg/mL |
- |
3 h |
20 h |
0.0 |
0.0 |
2000 µg/mL |
- |
3 h |
20 h |
0.0 |
0.0 |
Pos. Control (EMS)* |
- |
3 h |
20 h |
0.0 |
0.0 |
Solvent control |
+ |
3 h |
20 h |
0.0 |
0.0 |
Vinylphosphonic acid |
|||||
500 µg/mL |
+ |
3 h |
20 h |
0.0 |
0.0 |
1000 µg/mL |
+ |
3 h |
20 h |
0.0 |
0.0 |
2000 µg/mL |
+ |
3 h |
20 h |
0.0 |
0.0 |
Pos. Control (Cycl.)** |
+ |
3 h |
20 h |
0.0 |
0.0 |
* :Ethyl methanesulfonate (1.0µL/mL), ** :Cyclophosphamide (5.0µg/mL)
The number of polyploid and endoreduplicated cells was determined in 150 cells of each test group.
Table 4: Number of polyploid cells and endoreduplicated cells, Experiment B
Concentration |
S9 mix |
Treatment |
Harvesting time |
Polyploid Cells (mean) |
Endoredup-lication (mean) |
Solvent control |
- |
20 h |
20 h |
0.0 |
0.0 |
Vinylphosphonic acid |
|||||
250 µg/mL |
- |
20 h |
20 h |
0.0 |
0.0 |
500 µg/mL |
- |
20 h |
20 h |
0.0 |
0.0 |
1000 µg/mL |
- |
20 h |
20 h |
0.0 |
0.0 |
2000 µg/mL |
- |
20 h |
20 h |
0.0 |
0.0 |
Pos. Control (EMS) |
- |
20 h |
20 h |
0.0 |
0.0 |
Solvent control |
- |
20 h |
28 h |
0.0 |
0.0 |
Vinylphosphonic acid |
|||||
250 µg/mL |
- |
20 h |
28 h |
0.0 |
0.0 |
500 µg/mL |
- |
20 h |
28 h |
0.0 |
0.0 |
1000 µg/mL |
- |
20 h |
28 h |
0.0 |
0.0 |
2000 µg/mL |
- |
20 h |
28 h |
0.0 |
0.0 |
Pos. Control (EMS) |
- |
20 h |
28 h |
0.0 |
0.0 |
Solvent control |
+ |
3 h |
28 h |
0.0 |
0.0 |
500 µg/mL |
+ |
3 h |
28 h |
0.0 |
0.0 |
1000 µg/mL |
+ |
3 h |
28 h |
0.0 |
0.0 |
2000 µg/mL |
+ |
3 h |
28 h |
0.0 |
0.0 |
Pos. Control (Cycl.)** |
+ |
3 h |
28 h |
0.0 |
0.0 |
Ethyl methanesulfonate (0.4µL/mL), **Cyclophosphamide (5.0µg/mL)
The number of polyploid and endoreduplicated cells was determined in 150 cells of each test group.
Table 1: CHO/HPRT mutagenesis assay results, 5-hour treatment without S9-mix
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
TOTAL |
ABSOLUTE |
MUTANT |
|||
MEAN COLONY |
PERCENT |
|||||||
Solvent control a |
202.7 |
± |
2.08 |
100 |
100 |
7 |
100 |
7.00 |
Pos. control |
56.3 |
± |
1.15 |
28 |
64 |
991 |
64 |
1548.44** |
TEST ITEM |
|
|||||||
125 µg/mL a |
201,3 |
± |
3,21 |
99 |
101 |
6 |
101 |
5.94 |
250 µg/mL a |
190,7 |
± |
1,15 |
94 |
100 |
7 |
100 |
7.00 |
500 µg/mL a |
181,0 |
± |
1,00 |
89 |
99 |
5 |
99 |
5.05 |
1000 µg/mL a |
172,7 |
± |
1,15 |
85 |
98 |
6 |
98 |
6.12 |
2000 µg/mL a |
169,0 |
± |
1,00 |
83 |
97 |
7 |
97 |
7.22 |
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
TOTAL |
ABSOLUTE |
MUTANT |
|||
MEAN COLONY |
PERCENT |
|||||||
Solvent control b |
201.3 |
± |
1.53 |
100 |
100 |
6 |
100 |
6.00 |
Pos. control |
56.0 |
± |
1.00 |
28 |
64 |
978 |
64 |
1528.13** |
TEST ITEM |
|
|||||||
125 µg/mL b |
193,3 |
± |
0,58 |
96 |
100 |
5 |
100 |
5.00 |
250 µg/mL b |
186,3 |
± |
2,52 |
93 |
99 |
7 |
99 |
7.07 |
500 µg/mL b |
178,7 |
± |
1,15 |
89 |
99 |
7 |
99 |
7.07 |
1000 µg/mL b |
172,0 |
± |
0,00 |
85 |
99 |
6 |
99 |
6.06 |
2000 µg/mL b |
165,7 |
± |
0,58 |
82 |
98 |
6 |
98 |
6.12 |
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
TOTAL |
ABSOLUTE |
MUTANT |
|||
MEAN COLONY |
PERCENT |
|||||||
Solvent control c |
201.0 |
± |
2.65 |
100 |
100 |
6 |
101 |
5.94 |
Pos. control |
53.7 |
± |
1.15 |
27 |
62 |
1006 |
63 |
1596.83** |
TEST ITEM |
|
|||||||
125 µg/mL c |
193,3 |
± |
1,53 |
96 |
100 |
6 |
101 |
5.94 |
250 µg/mL c |
187,3 |
± |
1,53 |
93 |
99 |
7 |
100 |
7.00 |
500 µg/mL c |
178,3 |
± |
2,08 |
89 |
98 |
5 |
99 |
5.05 |
1000 µg/mL c |
176,3 |
± |
1,53 |
88 |
97 |
6 |
98 |
6.12 |
2000g/mL c |
166,7 |
± |
2,31 |
83 |
98 |
7 |
99 |
7.07 |
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
TOTAL |
ABSOLUTE |
MUTANT |
|||
MEAN COLONY |
PERCENT |
|||||||
Solvent control d |
202.3 |
± |
2.89 |
100 |
100 |
6 |
100 |
6.00 |
Pos. control |
53.7 |
± |
1.53 |
27 |
61 |
1005 |
62 |
1620.97** |
TEST ITEM |
|
|||||||
125 µg/mL d |
193,3 |
± |
1,53 |
96 |
99 |
5 |
100 |
5.00 |
250 µg/mL d |
186,3 |
± |
1,15 |
92 |
99 |
6 |
100 |
6.00 |
500 µg/mL d |
179,3 |
± |
3,06 |
89 |
99 |
7 |
99 |
7.07 |
1000 µg/mL d |
175,0 |
± |
1,00 |
86 |
99 |
5 |
99 |
5.05 |
2000 µg/mL d |
167,0 |
± |
2,65 |
83 |
98 |
7 |
99 |
7.07 |
a = parallel of first culture ,b = parallel of first culture, c = parallel of second culture, d = parallel of second culture.
abs.C.E. = Absolute Cloning Efficiency
EMS=Ethyl methanesulfonate
** = p < 0.01 to the concurrent negative control and to the historical control
Table 2: CHO/HPRT mutagenesis assay results, 5-hour treatment with S9-mix
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
TOTAL |
ABSOLUTE |
MUTANT |
|||
MEAN COLONY |
PERCENT |
|||||||
Solvent control a |
199.3 |
± |
1.15 |
100 |
100 |
7 |
101 |
6.93 |
Pos. control |
121.3 |
± |
3.21 |
61 |
78 |
594 |
79 |
751.90** |
TEST ITEM |
|
|||||||
125 µg/mL a |
195,0 |
± |
0,00 |
98 |
99 |
6 |
100 |
6.00 |
250 µg/mL a |
182,3 |
± |
1,53 |
91 |
99 |
7 |
100 |
7.00 |
500 µg/mL a |
133,3 |
± |
3,06 |
67 |
99 |
8 |
100 |
8.00 |
750 µg/mL a |
90,0 |
± |
2,00 |
45 |
97 |
5 |
98 |
5.10 |
1000 µg/mL a |
39,7 |
± |
0,58 |
20 |
97 |
7 |
98 |
7.14 |
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
TOTAL |
ABSOLUTE |
MUTANT |
|||
MEAN COLONY |
PERCENT |
|||||||
Solvent control b |
200.0 |
± |
0.00 |
100 |
100 |
6 |
100 |
6.00 |
Pos. control |
121.0 |
± |
1.00 |
61 |
78 |
598 |
78 |
766.67** |
TEST ITEM |
|
|||||||
125 µg/mL b |
195,0 |
± |
1,00 |
98 |
99 |
6 |
100 |
6.00 |
250 µg/mL b |
180,0 |
± |
1,00 |
90 |
100 |
7 |
100 |
7.00 |
500 µg/mL b |
135,0 |
± |
1,00 |
68 |
99 |
6 |
99 |
6.06 |
750 µg/mL b |
90,3 |
± |
0,58 |
45 |
98 |
7 |
98 |
7.14 |
1000 µg/mL b |
38,7 |
± |
0,58 |
19 |
98 |
7 |
98 |
7.14 |
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
TOTAL |
ABSOLUTE |
MUTANT |
|||
MEAN COLONY |
PERCENT |
|||||||
Solvent control c |
199.3 |
± |
2.08 |
100 |
100 |
6 |
101 |
5.94 |
Pos. control |
120.3 |
± |
1.53 |
60 |
77 |
591 |
77 |
767.53** |
TEST ITEM |
|
|||||||
125 µg/mL c |
197,0 |
± |
1,00 |
99 |
99 |
7 |
100 |
7.00 |
250 µg/mL c |
176,0 |
± |
1,00 |
88 |
99 |
7 |
100 |
7.00 |
500 µg/mL c |
138,0 |
± |
1,00 |
69 |
98 |
5 |
99 |
5.05 |
750 µg/mL c |
95,0 |
± |
1,00 |
48 |
98 |
7 |
98 |
7.14 |
1000 µg/mL c |
40,7 |
± |
0,58 |
20 |
98 |
6 |
99 |
6.06 |
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
TOTAL |
ABSOLUTE |
MUTANT |
|||
MEAN COLONY |
PERCENT |
|||||||
Solvent control d |
200.0 |
± |
2.65 |
100 |
100 |
7 |
100 |
7.00 |
Pos. control |
119.0 |
± |
1.0 |
60 |
77 |
595 |
77 |
772.73** |
TEST ITEM |
|
|||||||
125 µg/mL d |
196,3 |
± |
1,15 |
98 |
99 |
7 |
99 |
7.07 |
250 µg/mL d |
176,0 |
± |
0,00 |
88 |
99 |
6 |
99 |
6.06 |
500 µg/mL d |
137,3 |
± |
0,58 |
69 |
98 |
7 |
99 |
7.07 |
750 µg/mL d |
95,0 |
± |
1,00 |
48 |
98 |
6 |
99 |
6.06 |
1000 µg/mL d |
38,0 |
± |
1,00 |
19 |
98 |
6 |
99 |
6.06 |
a = parallel of first culture,b = parallel of first culture, c = parallel of second culture, d = parallel of second culture
abs.C.E. = Absolute Cloning Efficiency
DMBA=7,12-Dimethyl benzanthracene
** = p < 0.01 to the concurrent negative control and to the historical control
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Ames test
The mutagenic potential of the test item was investigated according OECD TG 471 in Salmonella typhimurium strains TA 98, TA100, TA 1535, TA 1537, and TA 1538 and in E. coli WP2 uvrA. A plate incorporation test and a pre-incubation test were conducted with and without metabolic activation using S9 fraction of Phenobarbital and β-naphthoflavone (BNF) induced rat liver. The following concentrations were examined in triplicate: ±S9 Mix: 5000, 1600, 500; 160; 50 and 16 μg/plate. Positive and negative (vehicle) controls were run concurrently. No substantial increases were observed in revertant colony numbers following treatment with the test item, neither in the presence nor in the absence of metabolic activation. Sporadic increases in revertant colony numbers compared to the vehicle control remained within the corresponding historical control data ranges. Control plates without mutagen showed no increase in revertant colonies. The positive controls showed the expected, biological relevant increases in revertant colonies in all tests. No cytotoxicity was observed in either tested strains with or without metabolic activation. In conclusion, it can be stated that the test item is not mutagenic in these bacterial test systems either with or without metabolic activation at any dose level investigated.
In the supporting Ames study, the test item was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium. The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. The test substance was dissolved in Aqua bidest. and a dose range of 6 different doses from 4 microgram/plate to 5000 microgram/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similiar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies. 5000 microgram/plate was chosen as top dose level for the mutagenicity study. In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with the test substance did not result in relevant increases in the number of revertant colonies.
A further supporting Ames study is available which was conducted with the test item at lower purity (technical grade). The study was conducted similar to the above mentioned study. The test item was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium. The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. The test substance was dissolved in Aqua bidest. and a dose range of 6 different doses from 4 microgram/plate to 5000 microgram/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similiar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies. 5000 microgram/plate was chosen as top dose level for the mutagenicity study. In the absence and in the presence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains.
Summarizing, it can be stated that the test substance is not mutagenic in these bacterial test systems neither with nor without exogenous metabolic activation at the dose levels investigated.
Chromosome Aberration test
In a study according OECD TG 473 the ability of the test item to induce chromosome aberrations in V79 cells was investigated. The test item was dissolved in cell culture medium and cytotoxicity was tested with and without metabolic activation from S9 mix of phenobarbital and β-naphthoflavone induced rat liver. Based on these results, the following concentrations were analyzed in the main test:
Experiment A with 3/20 h treatment/sampling time without and with S9 mix 500, 1000 and 2000 μg/mL
Experiment A with 20/20 h treatment/sampling time without S9 mix: 250, 500, 1000 and 2000 μg/mL
Experiment B with 20/28 h treatment/sampling timewithout S9 mix: 250, 500, 1000 and 2000 μg/mL
Experiment B with 3/28 h treatment/sampling time with S9 mix: 500, 1000 and 2000 μg/mL
At least 300 well-spread metaphase cells were analyzed. There were no biologically significant increases in the number of cells showing structural chromosome aberrations, neither in the absence nor in the presence of metabolic activation. No statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberrations were inside the distribution of the laboratory historical negative control data. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in the presence or absence of metabolic activation. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. The concurrent positive controls ethyl methanesulphonate and cyclophosphamide caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid. In conclusion, the test item is considered as not clastogenic, neither in the presence nor absence of metabolic activation.
HPRT test
In the HPRT test conducted according to the OECD Guideline 476, CHO-K1 cells were treated with test item concentrations (using cell culture medium as vehicle) ranging from 125 to 2000 µg/mL in the main study, with and without metabolic activation. Concentration levels were chosen mainly based on the cytotoxicity and the maximum recommended concentration. Ethylmethanesulphonate (1 µL/mL; without S9 fraction) and 7,12-dimethylbenzanthracene (20 µg/mL; with S9 fraction) were used as positive controls. Phenotypic expression was evaluated up to 8 days following exposure. The test substance did not induce mutation at the hprt locus of CHO-K1cells when tested under the conditions employed in this study. There were no statistically and biologically significant differences between treatment groups compared to the concurrent and historical control groups and no dose-response relationships were noted. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in the osmolality of the test system were noted at the different dose levels tested. The measured pH of treatment solution was similar compared to the control values. 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. In conclusion, the test item was not mutagenic in the in vitro mammalian cell gene mutation test performed in CHO-K1 cells.
Justification for classification or non-classification
Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/ 776.
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