Registration Dossier
Registration Dossier
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EC number: 460-490-0 | CAS number: 477218-42-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2004
negative, in vitro chromosome aberration test (with and without S-9 activation), OECD TG 473 - V79 Chinese Hamster Lung Chromosome Aberration Test, 2004
negative, in vitro gene mutation test in mammalian cells (with and without S-9 activation), OECD TG 476 - mouse lymphoma cell line locus - L5178Y thymidine kinase assay, 2010
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 06-05-2004 to 21-06-2004
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study performed under GLP. All relevant validity criteria were met.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- inspected: June 2001; signature: January 2002
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- histidine or tryptophan locus
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver S9
- Test concentrations with justification for top dose:
- Experiment 1 (plate incorporation method): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment 2 (pre-incubation method): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Salmonella strain TA1537 (with and without S9-mix): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Salmonella strain TA98 and E.coli strain WP2uvrA (with and without S9-mix): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Salmonella strains TA100 and TA1535 (with and without S9-mix): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Ethanol (purity > 99%)
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and relative nontoxicity to the bacteria.
- Other: No precipitation of the test item occurred up to the highest investigated dose. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 2-Aminoanthracene (2AA) ; 4-nitro-o-phenylenediamine (4NOPD)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Experiment 1. in medium; in agar (plate-incorporation) ; Experiment 2. in medium; in agar (pre-incubation)
DURATION
- Exposure duration:
Experiment 1. The appropriate bacterial strain culture, with or without S9-activation mix, as applicable, the test item formulation, solvent or appropriate positive control were incubated at 37°C for 4 hours (with shaking) prior to plating. All testing for this experiment was performed in triplicate. Concurrent negative controls were dosed using the standard plate incorporation method. All of the plates were incubated at 37°C for approximately 48 hours in the dark, and scored for the presence of revertant colonies using an automated colony counting system. Due to reduced background growth, some of the plates were counted manually.
Experiment 2. The procedure for incubation and duration was the same as in Experiment 1, with the exception: In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-incubation 2.0 ml overlay agar (45°C) was added to each tube. The mixture was poured on minimal agar plates.
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Rationale for test conditions:
- In accordance with the relevant guidelines.
- Evaluation criteria:
- There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance
4. Statistical analysis of data (as required or applicable).
5. Fold increase greater than two or three times the concurrent solvent control for respective tester strain
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
In instances of data prohibiting definitive judgement about test item activity are reported as equivocal. - Statistics:
- Not performed.
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See table 1 and 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- Interpretation of results:
Negative
Under the conditions of this study the test item was considered to be non-mutagenic in the presence and absence of S9 activation. - Executive summary:
The study was performed to the requirements of OECD Guideline 471 under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA102, TA1535, and TA1537 in both the presence and absence of S-9 mix. The test strains were treated with the test substance using the Ames plate incorporation method (Experiment 1) and pre incubation method (Experiment 2) at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (15% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 33 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. Six test item dose levels were again selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item the maximum recommended dose level of 5000 μg/plate. The vehicle (ethanol) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 -mix were validated. The maximum dose level of the test item in the first experiment (plate-incorporation) was selected as the maximum recommended dose level of 5000 μg/plate. For the second mutation test (pre-incubation) the toxic limit was employed as the maximum dose concentration. In the second experiment, reduced background growth was observed in strains TA1535, TA1537, and TA100 at 333 μg/plate and above with metabolic activation, in strains TA98 (with metabolic activation) and TA100 (without metabolic activation) at 1000 ug/plate and above, and in strain TA102 at 33 μg/plate and above with and without metabolic activation. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9 mix) or were within the expected ranges. It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA102, TA1535, and TA1537 in the presence and absence of S-9 mix.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 21-04-2004 to 06-08-2004
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study performed under GLP. All relevant validity criteria were met.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- inspected: June 2001; signature: January 2002
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not applicable (chromosome aberration test)
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Chinese Hamster Lung V79 cell line ; (source: Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt, D-64287 Darmstadt, Germany)
- Suitability of cells: The V79 cell line has been used successfully for many years in in-vitro experiments. Especially the high proliferation rate (clone V79/D3 doubling time determined ; cited in full study report) and a reasonable plating efficiency of untreated cells (as a rule more than 70 %) are both necessary for the appropriate performance of the study. The relevant guidelines recommend the use of this cell line.
- Normal cell cycle time (negative control): 12 hours (cited in full sturdy report)
For cell lines:
- Absence of Mycoplasma contamination: Before freezing each batch was screened for mycoplasm contamination and checked for karyotype stability
- Number of passages if applicable: One, but with an additional later sampling time (28 hours)
- Methods for maintenance in cell culture: Frozen. Thawed stock cultures were propagated at 37°C in 80 cm2 plastic flasks. Ca. 5 x10^5 cells per flask seeded into 15 mL of MEM with 10% fetal calf serum. Sub-cultured twice weekly. Incubated at 37°C in a humidified atmosphere with 1.5% CO2 (98.5% air).
- Cell cycle length, doubling time or proliferation index : clone V79/D3 doubling time = 12 hours, determined ; cited in full study report
- Modal number of chromosomes: The cells have a stable karyotype with a modal chromosome number of 22.
- Periodically checked for karyotype stability: Yes.
- Periodically ‘cleansed’ of spontaneous mutants: Yes. Sub-cultured twice weekly.
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: See above. Seeding of cultures: Exponentially growing stock cultures more than 50% confluent are treated with trypsinEDTA-solution at 37°C for approx. 5 minutes. Then the enzymatic treatment is stopped by adding complete culture medium and a single cell suspension is prepared. The trypsin concentration for all subculturing steps is 0.5 % (w/v) in Ca-Mg-free salt solution. Prior to the trypsin treatment the cells are rinsed with Ca-Mg-free salt solution. Cells were seeded into dishes which contained microscopic slides (at least 2 chambers per dish and test group). In each chamber 1 x 10^4 to 6 x10^4 cells were seeded with regard to the preparation time. The medium was MEM with 10% FCS. - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system: Phenobarbital/beta-Naphthoflavone induced rat liver S9 was used as the metabolic activation system.
- source of S9: Details available in the full study report ; Recognised supplier.
- method of preparation of S9 mix: induced by applications of 80 mg/kg b.w. Phenobarbital i.p. and beta-Naphthoflavone p.o. each on three consecutive days. The livers were prepared 24 hours after the last treatment.
- concentration or volume of S9 mix and S9 in the final culture medium: S9 fractions were produced by dilution of the liver homogenate with a KCI solution (1:3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at -80°C. Small numbers of ampules were kept at -20°C for up to one week.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Not stated ; referenced in accordance with Ames (therefore 1 – 2% S9 typical). Concurrent positive controls (EMS and CPA) were utilised within the study and gave positive responses indicating the suitability of the test system. - Test concentrations with justification for top dose:
- The maximum dose level was 3250 µg/mL, or 10 mM concentration, the maximum recommended dose level. During pre-tests, the test item was found to precipitate at 812.5 µg/mL. Clear toxic effects were observed after 4 hrs treatment with 25.4 µg/mL and above in the absence of S9 mix, and 406.3 µg/mL and above in the presence of S9 mix. Top concentrations for the main experiment were therefore chosen to be 50 µg/mL (S9-) and 500 µg/mL (S9+). Further repeats were necessary in the presence of S9 mix, increasing the final top test item concentrations up to 2500 µg/mL.
I. Preliminary toxicity test: 0 (control) , 25.4, 50.8, 101.6, 203.1, 406.3, 812.5, 1625.0, and 3250 μg/mL
Within groups:
i) 4-hours exposure to the test item without S9-mix, followed by a 14-hour recovery period in treatment-free media, 4(14)-hour exposure.
ii) 4-hours exposure to the test item with S9-mix, followed by a 14-hour recovery period in treatment-free media, 4(14)-hour exposure.
iii) 4(24)-hour exposure to the test item without S9-mix.
iv) 18-hour exposure to the test item with S9-mix.
v) 28-hour exposure to the test item with S9-mix.
II. Main Test:
EXP1
4(14)-hour without S9: 0, 62.5, 125.0, 250.0, 500.0, 1000.0, 1500.0, 2000.0, 2500.0 μg/mL
4(14)-hour with S9: 0, 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, 50.0 μg/mL
EXP2:
4(28)-hour with S9: 0, 125.0, 250.0, 500.0, 1000.0, 1500.0, 2000.0 μg/mL
18-hour without S9: 0, 0.6, 1.3, 2.5, 5.0, 10.0, 20.0 μg/mL
28-hour without S9: 0, 2.5, 5.0, 20.0 μg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen for its solubility properties and relative nontoxicity to the cell cultures in pre-test solubility checks. See above. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- ethanol in culture medium (0.5% v/v)
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Remarks:
- Full details on the positive controls is reported in the full study report.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Other:
Thawed stock cultures were propagated in 80 cm2 plastic flasks. Approximately 5x10^{5} cells were seeded into 15 mL MEM and 10% FCS.
DURATION
- Preincubation period: Not reported.
- Exposure duration: Definitive testing:
i) 4-hours exposure to the test item without S9-mix, followed by a 14-hour recovery period in treatment-free media, 4(14)-hour exposure.
ii) 4-hours exposure to the test item with S9-mix, followed by a 14-hour recovery period in treatment-free media, 4(14)-hour exposure.
iii) 4(24)-hour exposure to the test item without S9-mix.
iv) 18-hour exposure to the test item with S9-mix.
v) 28-hour exposure to the test item with S9-mix.
NUMBER OF REPLICATIONS: The study conducted two replicates (A and B) at each dose level and exposure duration groups.
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Exponentially growing stock cultures more than 50% confluent are treated with trypsinEDTA-solution at 37°C for approx. 5 minutes. Then the enzymatic treatment is stopped by adding complete culture medium and a single cell suspension is prepared. The trypsin concentration for all subculturing steps is 0.5 % (w/v) in Ca-Mg-free salt solution. Prior to the trypsin treatment the cells are rinsed with Ca-Mg-free salt solution. Cells were seeded into dishes which contained microscopic slides (at least 2 chambers per dish and test group). In each chamber 1 x 10^4 to 6 x10^4 cells were seeded with regard to the preparation time. The medium was MEM with 10% FCS.
- Test substance added in medium
NUMBER OF CELLS EVALUATED: A total of 500 cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes. - Evaluation criteria:
- Positive response criteria
A test item can be classified as genotoxic if:
(1) the number of induced structural chromosome aberrations is not in the range of historical control data (0.0- 4.0% aberrant cells, exclusive gaps).
(2) either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Negative response criteria
A test item can be classified as non-genotoxic if:
(1) the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the historical control data (0.0- 4.0 %aberrant cells, exclusive gaps).
(2) no significant increase of the number of structural chromosome aberrations is observed
(3) A test item can be classified as aneugenic if: the number of induced numerical aberrations is not in the range of the historical control data (0.0- 8.5% polyploid cells).
Statistical analysis is also performed (see: ‘Statistics’). Biological relevance of the results are to be considered first.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment. - Statistics:
- The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. Analysis of data from in vitro cytogenetic assays. In Statistical Evaluation of mutagenicity test data: UKEMS sub-committee on guidelines for mutagenicity testing. Report Part III (Ed: Kirkland, D.J.), Cambridge University Press (1989)
- 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:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test item was dosed into media
- Effects of osmolality: There was no significant change osmolality (did not decrease/increase by more than 50 mOsm) when the test item was dosed into media
- Evaporation from medium: Not reported.
- Water solubility: Not applicable.
- Precipitation: In the preliminary test: In the pre-experiment, precipitation of the test item in culture medium was observed after treatment with 812.5 μg/mL and above in the absence and the presence of S9 mix. Main test: Precipitate was observed at 1000 - 1500 μg/mL in experiments 1 and 2 with S9 mix.
- Other confounding effects: None reported.
RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 0 (control) , 25.4, 50.8, 101.6, 203.1, 406.3, 812.5, 1625.0, and 3250 μg/mL. The maximum dose was the maximum recommended dose level. The selection of the maximum dose level was based on toxicity (and/or precipitation) for the main test.
COMPARISON WITH HISTORICAL CONTROL DATA:
- All vehicle (ethanol) controls had frequencies of cells with aberrations within the range expected. (Within the Historic Control Data range presented in the full study report).
- All the positive control items induced statistically significant increases in the frequency of cells with aberrations. (Within the Historic Control Data range presented in the full study report).
ADDITIONAL INFORMATION ON CYTOTOXICITY: Not applicable. - Conclusions:
- Interpretation of results:
Negative
Under the conditions of this study, the test item was considered to be non-clastogenic to Chinese hamster lung V79 cells in vitro. - Executive summary:
The study was performed to the requirements of OECD TG 473 guideline under GLP conditions to assess the potential chromosomal mutagenicity of the test item, on Chinese hamster V79 cells in vitro. Duplicate cultures of V79 cells treated with the test item, were evaluated with 100 metaphase plates scored for structural chromosome aberrations. The maximum dose level was 3250 µg/mL, or 10 mM concentration, the maximum recommended dose level. Dose selection of the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. During pre-tests, the test item was found to precipitate at 812.5 µg/mL. Clear toxic effects were observed after 4 hrs treatment with 25.4 µg/mL and above in the absence of S9 mix, and 406.3 µg/mL and above in the presence of S9 mix. Top concentrations for the main experiment were therefore chosen to be 50 µg/mL (S9-) and 500 µg/mL (S9+). Further repeats were necessary in the presence of S9 mix, increasing the final top test item concentrations up to 2500 µg/mL. Toxic effects indicated by reduced cell numbers and/or mitotic indices of about or below 50% of control were observed in all experimental parts, except in the absence of S9 mix in experiment II at preparation interval 28 hrs. However, in this experimental section concentrations showing clear toxic effects were not scorable for cytogenetic damage. No biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item and any statistical significances were deemed as not biologically relevant. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. Under the conditions of this study, the test item was considered to be non-clastogenic to Chinese hamster lung V79 cells in vitro.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 09-09-2009 to 23-11-2009
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study performed under GLP. All relevant validity criteria were met.
- 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
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- Study predated the publication of the OECD TG 490 (2015) guideline.
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- inspected: October 2008 ; signature: March 2009
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Specific details on test material used for the study:
- RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: Not applicable.
- Specific activity: Not applicable.
- Locations of the label: Not applicable.
- Expiration date of radiochemical substance: Not applicable.
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from light.
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Stability not indicated by sponsor. Soluble at 3000 μg/mL in DMSO.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item emulsified in DMSO. Serial dilutions prepared from stock.
- Preliminary purification step (if any): Not applicable.
- Final dilution of a dissolved solid, stock liquid or gel: Liquid test item emulsified in stock prepared at 3000 μg/mL
- Final preparation of a solid: Not applicable.
FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid formulations in vehicle, of the test item.
OTHER SPECIFICS: Not applicable - Target gene:
- Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Recognised supplier (documented in full study report).
- Suitability of cells: The L5178Y TK+/- 3.7.2c mouse lymphoma cell line has been used successfully in in vitro experiments for many years and is guideline specified (OECD TG 490 (2015)). The test system has been extensively validated since the conduct of the study.
- Cell cycle length, doubling time or proliferation index: Doubling time ca. 12 hours. Determined under normal growth conditions.
- Sex, age and number of blood donors if applicable: Not applicable.
- Whether whole blood or separated lymphocytes were used if applicable: Not applicable.
- Number of passages if applicable: Not applicable.
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: The karyotype for the cell line has been published and the modal chromosome number is 40 (ref: OECD TG 490 (2015))
- Normal (negative control) cell cycle time: Doubling time ca. 12 hours.
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: stocks of cells are stored in liquid nitrogen. Cells were routinely cultured in RPMI 1640 medium with horse serum (15%, or 3% during 4-hour treatment) and supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (220 μg/mL), Amphotericin (0.5-0.75%) (giving R10 media) at 37 °C with 4.5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly.
- Properly maintained: Yes.
- Periodically checked for Mycoplasma contamination: Yes. Master stocks of cells were tested and found to be free of mycoplasma.
- Periodically checked for karyotype stability: Yes.
- Periodically 'cleansed' against high spontaneous background: Yes. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (16 μM),
Hypoxanthine (0.1 mM), and Aminopterin (0.2 mM). For the following 48 hours the cells were cultured in RPMI 1640 medium containing Hypoxanthine (0.1 mM) and Thymidine (16 μM). - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix. S9-mix was prepared by mixing S9, NADP (4 mM), glucose-6-phosphate (5 mM), KCl (33 mM) and MgCl2 (8 mM) in 100 mM sodium orthophosphate buffer. 5% final concentration of S9 was added to the subsequent testing and control cultures.
- Test concentrations with justification for top dose:
- The maximum recommended dose level was 3000 µg/mL, on the basis of the molecular weight of the test item. Phase separation occurred at 750 µg/mL in the absence of, and 1500 µg/mL in the presence of, metabolic activation. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991). A preliminary toxicity test dose range 2.9 to 188.0 µg/mL was selected based on precipitate observed during solubility testing. Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments.
I. Preliminary toxicity test:-
Within two exposure groups:
i) 4-hours exposure to the test item without S9-mix: 0 (control), 2.9, 5.9, 11.8, 23.5, 47.0, 70.5, and 94.0 μg/mL
ii) 4-hours exposure to the test item with S9-mix (5%): 0 (control), 5.9, 11.8, 23.5, 47.0, 94.0, 141.0, and 188.0 μg/mL
In experiment I, the cultures at 70.5 and 94.0 µg/mL without metabolic activation, and 141 and 188 µg/mL with metabolic activation, were not continued due to exceedingly strong toxic effects following the 48h expression phase.
II. Main Test:
4-hour without S9: 0 (control), 1.1, 2.2, 4.4, 8.8, 17.6, 35.3, and 70.5 μg/ml
4-hour with S9: 0 (control), 4.4, 8.8, 17.6, 35.3, 94.0, 117.3, and 141.0 μg/ml
24-hour without S9: 0 (control), 50, 70, 90, and 100 μg/mL
The cultures were then incubated for 24h, and sub-cultured subject to acceptable limits of mean cell count. Following a further 24h the cultures were counted and discarded, giving total of 48h for expression period.
In experiment II the cultures at 1.1 and 2.2 µg/mL without, and 4.4 and 8.8 µg/mL with metabolic activation, were not continued since a minimum of only four analysable concentrations is required by the guidelines. The cultures of the concentrations of 80, 90, and 100 µg/mL without metabolic activation in experiment II were not continued due to exceedingly strong toxic effects. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells. The maximum recommended dose level was 3000 µg/mL, on the basis of the molecular weight of the test item. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991). A preliminary toxicity test dose range 2.9 to 188.0 µg/mL was selected based on high levels of precipitate observed during solubility testing. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Remarks:
- Full details of the positive control substances, is available in the full study report
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation with test item.
- Cell density at seeding (if applicable): Preliminary Test: 4-hour exposure: 3 x10^5 cells/mL ; 24-hour exposure: 3 x10^5 cells/mL ; Definitive Test: 4-hour exposure: 3 x10^5 cells/mL; 24-hour exposure: 3 x10^5 cells/mL
DURATION
- Preincubation period: Preliminary Test: 4-hour exposure: unspecified ; 24-hour exposure: unspecified ; Definitive Test: 4-hour exposure: unspecified ; 24-hour exposure: unspecified.
- Exposure duration: Treatment was for 4 hours or 24 hours, respectively at 37 °C in an incubator with a humidified atmosphere of 4.5% CO2 in 95%-humidified air.
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): Not applicable.
- Fixation time (start of exposure up to fixation or harvest of cells): Microtitre plates were scored manually after 10-15 days incubation at 37 °C with 4.5% CO2 in 95%-humidified air, post exposure (total time 12 to 17 days from exposure start).
SELECTION AGENT (mutation assays): 5-Trifluorothymidine
SPINDLE INHIBITOR (cytogenetic assays): Not applicable.
STAIN (for cytogenetic assays): Not applicable.
NUMBER OF REPLICATIONS: Not specified.
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Microtitre plates were scored manually after 10-15 days incubation at 37 °C with 4.5% CO2 in 95%-humidified air, post exposure.
NUMBER OF CELLS EVALUATED: 4000 cells/well were seeded with selection agent. Cells were also diluted and plated (2 cells/well) for viability (%V) analysis. Scoring of plates daily for %RSG and %V to obtain RTG. Mutation scoring of plates was ultimately performed for the presence of mutant colonies; large and small colonies analyses was additionally conducted.
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Not applicable.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Not applicable.
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG) and cloning efficiency via viability (%)
- Any supplementary information relevant to cytotoxicity: percentage relative suspension growth (%RSG) (post exposure toxicity during the expression period); viability (%) from non-selective medium cultures.
OTHER EXAMINATIONS:
- Determination of polyploidy: Not applicable.
- Determination of endoreplication: Not applicable.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): Not applicable. - Rationale for test conditions:
- See tables. The rationale for selection of concentrations was based on preliminary toxicity testing and number of cultures was in accordance with the guideline (duplicate). In the absence of precipitation: optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved.
It was noted: Optimum toxicity was achieved in the in the 24-hour exposure group in the absence of metabolic activation. Phase separation of the test item was observed at 750 µg/mL in the 4-hour exposure groups in both the absence of metabolic activation, and 1500 µg/mL in the presence of metabolic activation. Phase separation was observed above 750 µg/mL in the 24-hour exposure group in the absence of metabolic activation. Therefore, the maximum dose levels in the Mutagenicity Test were limited by the onset of test item precipitate in all three of the exposure groups (as recommended by the OECD 490 (2015) guideline). - Evaluation criteria:
- An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10^-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered
unable to induce mutations in this test system.
Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance. - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT 11 statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: Precipitate of the test item was observed at 750 µg/mL in the 4-hour exposure groups in the absence of metabolic activation, and 1500µg/mL in the presence of metabolic activation. It was observed at 750 µg/mL in the 24-hour exposure group.
- Conclusions:
- Interpretation of results:
Negative
Under the conditions of this study, the test item was considered to be non-mutagenic at the TK +/- locus to mouse L5178Y lymphoma cells in vitro. - Executive summary:
The study was performed to the requirements of OECD TG 476 and EU Method B.17 under GLP conditions, to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. Following a preliminary cytotoxicity test at 2.9 to 188.0 μg/mL concentration in DMSO vehicle (selection was based on precipitation), a definitive test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item. Exposures were conducted using a 4-hour exposure with and without metabolic activation (5% S9), and a 24 -hour exposure without metabolic activation. For the exposure periods respectively conducted: 4 -hours with S9 (5%): 4.4, 8.8, 17.6, 35.3, 94.0, 117.3, and 141.0 µg/mL and without S9: 1.1, 2.2, 4.4, 8.8, 17.6, 35.3, and 70.5 µg/mL and 24-hours without S9: 50, 70, 80, 90, and 100 µg/mL. Together with vehicle (DMSO) and positive controls. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. Precipitate of the test item was observed at 750 and 1500 µg/mL in the 4 -hour exposure groups in the absence and presence of metabolic activation, respectively; and at and above 750 µg/mL in the 24 -hour exposure group in the absence of metabolic activation, at the end of the exposure periods. Therefore, following the recommendations of the guidelines, the maximum dose levels in the subsequent Mutagenicity Test were limited by the onset of test item precipitate. The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups. Vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolism activation system. The test item did not induce any increases in mutant frequency above the Global Evaluation Factor (GEF). Under the conditions of this study, the test item was considered to be non-mutagenic at the TK +/- locus to mouse L5178Y lymphoma cells in vitro.
Applicant assessment indicates: the study would be considered equivalent or similar to OECD TG 490 (2015) published after the conduct of the study.
Referenceopen allclose all
Table 1 : Test Results: Experiment 1 with and without metabolic activation and results of concurrent positive controls
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||
Base-pair substitution strains |
Frameshift strains |
|||||
TA100 |
TA102 |
TA1535 |
TA98 |
TA1537 |
||
Solvent Control |
150 (151) 139 13.1 165 |
111 (129) 122 21.8 153 |
18 (16) 21 6.2 9 |
14 (16) 19 2.6 15 |
6 (8) 7 3.2 12 |
|
33 µg |
137 (136) 140 4.6 131 |
99 (104) 96 10.8 116 |
18 (18) 14 4.0 22 |
15 (14) 16 3.2 10 |
6 (5) 3 1.5 5 |
|
100 µg |
141 (137) 132 4.7 139 |
71 (94) 111 20.5 99 |
15 (14) 12 1.7 15 |
17 (17) 15 1.5 18 |
8 (8) 7 0.6 8 |
|
333 µg |
122 (132) 136 9.1 139 |
112 (111) 108 2.3 112 |
19 (17) 16 2.1 15 |
16 (16) 18 2.5 13 |
7 (8) 6 2.1 10 |
|
1000 µg |
143 (134) 137 11.4 121 |
72 (86) 96 12.5 90 |
12 (14) 11 3.8 18 |
17 (16) 15 1.2 17 |
6 (6) 7 0.6 6 |
|
2500 µg |
135 (136) 143 7.0 129 |
117 (108) 114 13.7 92 |
13 (15) 19 3.8 12 |
14 (14) 12 2.0 16 |
5 (4) 2 2.1 6 |
|
5000 µg |
140 (144) 149 4.5 144 |
98 (103) 112 7.8 99 |
21 (19) 16 2.5 19 |
14 (12) 11 2.1 10 |
3 (4) 5 1.2 3 |
|
Positive controls S9-Mix (-) |
Name |
Sodium Azide |
methyl methane sulfonate |
Sodium Azide |
4-nitro-o-phenylene-idamine |
4-nitro-o-phenylene-idamine |
Dose Level |
10.0 µg/plate |
4.0 µg/plate |
10.0 µg/plate |
10.0 µg/plate |
50.0 µg/plate |
|
No. of Revertants |
632 (574) 537 50.7 554 |
530 (530) 486 44.0 574 |
296 (318) 312 26.1 347 |
124 (135) 145 10.5 136 |
68 (67) 58 8.1 74 |
|
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||
Base-pair substitution strains |
Frameshift strains |
|||||
TA100 |
TA102 |
TA1535 |
TA98 |
TA1537 |
||
Solvent Control |
166 (167) 170 3.1 164 |
120 (136) 141 13.8 146 |
20 (18) 18 2.0 16 |
17 (17) 16 1.5 19 |
16 14 13 1.7 13 |
|
33 µg |
140 (154) 163 12.3 159 |
104 (122) 118 19.8 143 |
24 (20) 15 4.6 21 |
12 (13) 13 1.5 15 |
21 (15) 8 6.5 15 |
|
100 µg |
148 (149) 152 2.6 147 |
111 (121) 128 8.9 124 |
22 (22) 24 2.5 19 |
14 (16) 18 2.1 17 |
10 (13) 21 6.7 9 |
|
333 µg |
138 (132) 121 9.8 138 |
115 (120) 132 10.8 112 |
19 (21) 23 2.0 21 |
17 (17) 14 3.0 20 |
10 (10) 10 0.6 9 |
|
1000 µg |
131 (118) 114 11.2 110 |
67 (78) 79 10.1 87 |
14 (16) 17 1.7 17 |
20 (17) 14 3.0 17 |
15 (12) 11 2.3 11 |
|
2500 µg |
102 (106) 116 9.1 99 |
61 (53) 63 16.2 34 |
5 (7) 5 2.9 10 |
21 (20) 20 0.6 20 |
16 (9) 6 6.1 5 |
|
5000 µg |
80 (90) 93 8.5 96 |
20 (24) 23 4.0 28 |
4 (2) 1 1.7 1 |
14 (16) 19 2.5 16 |
3 (3) 2 0.6 3 |
|
Positive controls S9-Mix (+) |
Name |
2-aminoanthracene |
2-aminoanthracene |
2-aminoanthracene |
2-aminoanthracene |
2-aminoanthracene |
Dose Level |
2.5 µg/plate |
10.0 µg/plate |
2.5 µg/plate |
2.5 µg/plate |
2.5 µg/plate |
|
No. of Revertants |
1224 (1179) 1206 63.0 1107 |
757 (821) 840 56.9 866 |
320 (297) 302 25.9 269 |
147 (14) 162 15.0 132 |
201 (179) 177 21.5 158 |
|
Table 2 : Test Results: Experiment 2 with and without metabolic activation and results of concurrent positive controls
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||
Base-pair substitution strains |
Frameshift strains |
|||||
TA100 |
TA102 |
TA1535 |
TA98 |
TA1537 |
||
Solvent Control |
81 (83) 89 5.3 79 |
142 (139) 141 4.9 133 |
23 (19) 16 3.6 18 |
27 (31) 38 5.9 29 |
6 (6) 7 1.0 5 |
|
33 µg |
85 (78) 74 5.9 76 |
94 (90) 81 7.8 95 |
15 (13) 14 2.6 10 |
18 (21) 25 3.5 21 |
4 (4) 3 1.0 5 |
|
100 µg |
84 (69) 60 5.9 63 |
88 (93) 99 5.7 91 |
12 (14) 16 2.1 15 |
23 (32) 20 1.5 21 |
3 (4) 6 1.5 4 |
|
333 µg |
73 (74) 79 5.0 69 |
38 (35) 39 6.1 28 |
9 (10) 11 1.2 9 |
25 (24) 20 3.6 27 |
5 (6) 4 2.1 8 |
|
1000 µg |
54 (57) 67 8.5 51 |
27 (23) 17 5.3 25 |
13 (12) 3 2.3 13 |
21 (22) 24 1.5 22 |
8 (8) 8 0.6 7 |
|
2500 µg |
48 (49) 54 7.0 46 |
18 (18) 20 2.5 15 |
8 (9) 9 1.5 11 |
25 (24) 28 5.1 18 |
8 (5) 4 2.3 4 |
|
5000 µg |
38 (29) 26 7.6 24 |
17 (15) 9 4.9 18 |
7 (9) 10 1.7 10 |
23 (25) 26 2.1 27 |
2 (3) 3 1.0 4 |
|
Positive controls S9-Mix (-) |
Name |
Sodium Azide |
methyl methane sulfonate |
Sodium Azide |
4-nitro-o-phenylene-idamine |
4-nitro-o-phenylene-idamine |
Dose Level |
10.0 µg/plate |
4.0 µg/plate |
10.0 µg/plate |
10.0 µg/plate |
50.0 µg/plate |
|
No. of Revertants |
559 (580) 595 18.7 586 |
801 (794) 798 10.2 782 |
1266 (1244) 1201 37.0 1264 |
297 (300) 306 5.5 296 |
103 (102) 106 4.6 97 |
|
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||
Base-pair substitution strains |
Frameshift strains |
|||||
TA100 |
TA102 |
TA1535 |
TA98 |
TA1537 |
||
Solvent Control |
101 (167) 108 5.1 98 |
161 (162) 159 3.6 166 |
24 (21) 19 2.6 20 |
29 (28) 26 1.5 28 |
11 (9) 7 2.1 10 |
|
33 µg |
114 (107) 116 13.9 91 |
101 (105) 119 12.5 95 |
13 (13) 12 1.5 15 |
28 (29) 30 1.0 29 |
11 (9) 6 2.5 9 |
|
100 µg |
86 (78) 76 7.6 71 |
99 (97) 104 7.6 89 |
12 (14) 16 2.1 13 |
22 (21) 23 3.2 17 |
11 (9) 6 2.9 11 |
|
333 µg |
57 (46) 41 9.9 39 |
29 (30) 34 4.0 26 |
15 (14) 16 2.6 11 |
25 (28) 31 3.1 27 |
7 (5) 2 3.5 n.a. |
|
1000 µg |
65 (57) 55 7.6 50 |
21 (19) 17 2.1 18 |
6 (10) 9 4.6 15 |
33 (31) 30 1.5 31 |
2 (5) 7 2.9 7 |
|
2500 µg |
51 (45) 45 6.5 38 |
10 (14) 18 5.7 n.a. |
8 (8) 5 3.0 11 |
7 (9) 11 2.1 10 |
4 (2) 0 2.8 n.a. |
|
5000 µg |
28 (35) 37 6.7 41 |
9 (9) n.a. 0.7 8 |
5 (5) 6 0.6 5 |
14 (11) 7 4.9 n.a. |
1 (1) 1 0.0 1 |
|
Positive controls S9-Mix (+) |
Name |
2-aminoanthracene |
2-aminoanthracene |
2-aminoanthracene |
2-aminoanthracene |
2-aminoanthracene |
Dose Level |
2.5 µg/plate |
10.0 µg/plate |
2.5 µg/plate |
2.5 µg/plate |
2.5 µg/plate |
|
No. of Revertants |
599 (684) 768 84.5 685 |
864 (786) 795 83.4 698 |
182 (181) 170 10.5 191 |
655 (585) 548 60.9 551 |
129 (124) 118 5.7 126 |
|
n.a. = not analysable (no distinction possible between colonies and reduced background growth).
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Key study : OECD TG 471, 2004 : The study was performed to the requirements of OECD Guideline 471 under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA102, TA1535, and TA1537 in both the presence and absence of S-9 mix. The test strains were treated with the test substance using the Ames plate incorporation method (Experiment 1) and pre incubation method (Experiment 2) at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (15% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 33 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. Six test item dose levels were again selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item the maximum recommended dose level of 5000 μg/plate. The vehicle (ethanol) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 -mix were validated. The maximum dose level of the test item in the first experiment (plate-incorporation) was selected as the maximum recommended dose level of 5000 μg/plate. For the second mutation test (pre-incubation) the toxic limit was employed as the maximum dose concentration. In the second experiment, reduced background growth was observed in strains TA1535, TA1537, and TA100 at 333 μg/plate and above with metabolic activation, in strains TA98 (with metabolic activation) and TA100 (without metabolic activation) at 1000 ug/plate and above, and in strain TA102 at 33 μg/plate and above with and without metabolic activation. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9 mix) or were within the expected ranges. It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA102, TA1535, and TA1537 in the presence and absence of S-9 mix.
Key study : OECD TG 473, 2004 : The study was performed to the requirements of OECD TG 473 guideline under GLP conditions to assess the potential chromosomal mutagenicity of the test item, on Chinese hamster V79 cells in vitro. Duplicate cultures of V79 cells treated with the test item, were evaluated with 100 metaphase plates scored for structural chromosome aberrations. The maximum dose level was 3250 µg/mL, or 10 mM concentration, the maximum recommended dose level. Dose selection of the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. During pre-tests, the test item was found to precipitate at 812.5 µg/mL. Clear toxic effects were observed after 4 hrs treatment with 25.4 µg/mL and above in the absence of S9 mix, and 406.3 µg/mL and above in the presence of S9 mix. Top concentrations for the main experiment were therefore chosen to be 50 µg/mL (S9-) and 500 µg/mL (S9+). Further repeats were necessary in the presence of S9 mix, increasing the final top test item concentrations up to 2500 µg/mL. Toxic effects indicated by reduced cell numbers and/or mitotic indices of about or below 50% of control were observed in all experimental parts, except in the absence of S9 mix in experiment II at preparation interval 28 hrs. However, in this experimental section concentrations showing clear toxic effects were not scorable for cytogenetic damage. No biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item and any statistical significances were deemed as not biologically relevant. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. Under the conditions of this study, the test item was considered to be non-clastogenic to Chinese hamster lung V79 cells in vitro.
Key study : OECD TG 476, 2010 : The study was performed to the requirements of OECD TG 476 and EU Method B.17 under GLP conditions, to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. Following a preliminary cytotoxicity test at 2.9 to 188.0 μg/mL concentration in DMSO vehicle (selection was based on precipitation), a definitive test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item. Exposures were conducted using a 4-hour exposure with and without metabolic activation (5% S9), and a 24 -hour exposure without metabolic activation. For the exposure periods respectively conducted: 4 -hours with S9 (5%): 4.4, 8.8, 17.6, 35.3, 94.0, 117.3, and 141.0 µg/mL and without S9: 1.1, 2.2, 4.4, 8.8, 17.6, 35.3, and 70.5 µg/mL and 24-hours without S9: 50, 70, 80, 90, and 100 µg/mL. Together with vehicle (DMSO) and positive controls. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. Precipitate of the test item was observed at 750 and 1500 µg/mL in the 4 -hour exposure groups in the absence and presence of metabolic activation, respectively; and at and above 750 µg/mL in the 24 -hour exposure group in the absence of metabolic activation, at the end of the exposure periods. Therefore, following the recommendations of the guidelines, the maximum dose levels in the subsequent Mutagenicity Test were limited by the onset of test item precipitate. The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups. Vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolism activation system. The test item did not induce any increases in mutant frequency above the Global Evaluation Factor (GEF). Under the conditions of this study, the test item was considered to be non-mutagenic at the TK +/- locus to mouse L5178Y lymphoma cells in vitro.
Applicant assessment indicates: the study would be considered equivalent or similar to OECD TG 490 (2015) published after the conduct of the study.
Justification for classification or non-classification
The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity
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