Registration Dossier
Registration Dossier
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EC number: 915-671-3 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Gene mutation in bacteria (OECD 471, Ames test): negative in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA with and without metabolic activation
Cytogenicity in mammalian cells in vitro (OECD 487, Micronucleus test): negative in cultured peripheral human lymphocytes with and without metabolic activation
Gene mutation in mammalian cells in vitro (OECD 476, Mouse lymphoma assay): negative in mouse lymphoma L5178Y cells with and without metabolic activation
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:
- 2008-05-30 to 2008-06-19
- 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 21 Jul 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- adopted 08 Jun 2000
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon for S. typhimurium strains and trp operon for E. coli strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and ß-naphthoflavone
- Test concentrations with justification for top dose:
- Experiment 1: 333, 1000, 3330 and 5000 µg/plate for all strains with and without metabolic activation.
Experiment 2:
10, 33, 100, 333 and 666 µg/plate for TA1535, TA98 and TA100 with and without metabolic activation,
3, 10, 33, 100 and 333 µg/plate for TA1537 with and without metabolic activation,
33, 100, 333, 1000 and 3330 µg/plate for WP2 uvrA with and without metabolic activation.
Experiment 3: 100, 333, 1000 and 3330 µg/plate for TA98 with and without metabolic activation
A dose range finding test was conducted in S. typhimurium strain TA100 and in E. coli strain WP2 uvrA using the concentrations 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the presence and absence of metabolic activation (S9 mix). In tester strain TA100, toxicity was observed at dose levels of 333 µg/plate and above in the presence and absence of S9 mix. In tester strain WP2 uvrA cytotoxicity was observed at 3330 µg/plate and above in the absence of S9 mix and at 5000 µg/plate in the presence of S9 mix. The test was reported as a part of the first experiment of the present study. The highest concentration was the level at which the test substance inhibited bacterial growth or the test substance exhibited limited solubility. - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
The test item doses were corrected for the purity, a correction factor of 1.13 was used. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- methylmethanesulfonate
- other: 2-aminoanthracene (2AA), +S9, 1 µg/plate in DMSO for TA98 and TA100, 2.5 µg/plate in DMSO for TA1535, TA1537 and TA100, 5 µg/plate in DMSO for TA1537 and 10 µg/plate in DMSO for WP2 uvrA
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 h at 37.0 ± 1.0 °C
NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. - Evaluation criteria:
- A test substance is considered negative (not mutagenic) in the test if:
a) the total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 and WP2 uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2 uvrA is greater than three (3) times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment. - Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- ≥ 333 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- ≥ 333 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- ≥ 1000 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- ≥ 333 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- 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:
- cytotoxicity
- Remarks:
- at 3330 µg/plate without metabolic activation and at 5000 µg/plate with metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS:
- Precipitation and time of the determination: Precipitation was noted at 3330 and 5000 µg/plate at the end of the incubation period.
RANGE-FINDING/SCREENING STUDIES:
A dose range finding test was conducted in S. typhimurium strain TA100 and in E. coli strain WP2 uvrA using the concentrations 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the presence and absence of metabolic activation (S9 mix). In tester strain TA100, cytotoxicity was observed at dose levels of 333 µg/plate and above in the presence and absence of S9 mix. In tester strain WP2 uvrA cytotoxicity was observed at 3330 µg/plate and above in the absence of S9 mix and at 5000 µg/plate in the presence of S9 mix. The test was reported as a part of the first experiment of the present study.
HISTORICAL CONTROL DATA: please refer to Table No. 2 under “Any other information on results incl. tables” - Conclusions:
- Interpretation of results: negative with and without metabolic activation
The test substance did not show mutagenic activity in Salmonella typhimurium TA1535, TA1537, TA98 or TA100 or in Escherichia coli WP2 uvrA with and without metabolic activation. - Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2013-11-04 to 2013-12-09
- 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:
- adopted 21 Jul 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted 30 May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Version / remarks:
- adopted Aug 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- mouse lymphoma thymidine kinase locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit in Brighton, UK.
- Suitability of cells: The L5178Y cell line has been used successfully in in vitro experiments for many years. L5178Y cells are characterized by a high proliferation rate and cloning efficiency of untreated cells of usually more than 50% both necessary for the appropriate performance of the study.
For cell lines:
- Absence of Mycoplasma contamination: Yes, each batch was screened for mycoplasm contamination.
- Methods for maintenance in cell culture: Thawed stock cultures were propagated in plastic flasks in RPMI 1640 complete culture medium. The cells were subcultured two times prior to treatment. The cell cultures were incubated at 37 ± 1.5 °C in a humidified atmosphere with 4.5 % carbon dioxide and 95.5% ambient air.
- Cell doubling time: 10 - 12 h
- Modal number of chromosomes: The cells have a near diploid (40 ± 2) chromosome number.
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes (by treatment with HAT medium)
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Roswell Park Memorial Institute (RPMI) 1640 complete culture medium. The cells were incubated at 37 ± 1.5 °C in a humidified atmosphere with 4.5% CO2 and 95.5% ambient air. - Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital / β-naphthoflavone
- Test concentrations with justification for top dose:
- Experiment 1:
Without S9 mix: 0.03*, 0.06*, 0.1*, 0.3*, 1.0*, 2.0, 3.0 and 4.0 µg/mL (4 h)
With S9 mix: 0.3, 0.5, 1.0*, 2.0*, 4.0*, 8.0*, 12.0* and 16.0 µg/mL (4 h)
Experiment 2:
Without S9 mix: 0.02*, 0.03*, 0.06*, 0.13*, 0.25*, 0.5, 1.0 and 2.0 µg/mL (24 h)
With S9 mix: 0.3, 0.5, 1.0, 2.0*, 4.0*, 8.0*, 10.0* and 12.0* µg/mL (4 h)
* selected for mutation rate analysis
The dose range of the first main experiment was set according to data generated in a pre-experiment, where cells were exposed for 4 h to test item concentrations in the range of 15.6 to 2000 µg/mL in the presence of S9 mix, and for 4 and 24 h in the absence of S9 mix. Strong cytotoxic effects were observed from the lowest to the highest concentration following 4 and 24 h treatment with and without metabolic activation. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 h) before the test item was removed. Precipitation was observed by the unaided eye at 250.0 µg/mL and above following 4 and 24 h treatment with and without metabolic activation. - Vehicle / solvent:
- - Vehicle/solvent used: DMSO (final concentration 0.5% (v/v))
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1x 10E7 for 4 h exposure and 3x 10E6 for 24 h exposure in 80 cm^2 flasks
- Test substance added in medium
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: two independent experiments
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration:
Experiment I: 4 h exposure with and without S9 mix
Experiment II: 4 h exposure with S9 mix and 24 h exposure without S9 mix
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 48 h
- Selection time (if incubation with a selective agent): 10 - 15 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12 - 17 days
- Method used: Microwell plates. Colonies were counted manually.
- Selection agent: 5 µg/mL trifluorothymidine (TFT). For selection, cells from each experimental group were seeded into two microtiter plates (4 x 10E3 cells per plate) with selection medium containing TFT and incubated for 10 - 15 days at 37 ± 1.5 °C.
- Method to enumerate numbers of viable and mutants cells: Cloning efficiency
- Criteria for small (slow growing) and large (fast growing) colonies: Colonies were counted manually. In accordance with their size the colonies were classified into two groups. The colony size distribution was determined in the controls and at all concentrations of the test item. Criteria to determine colony size were the absolute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the optical density of the large ones).
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative cloning efficiency - Evaluation criteria:
- A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10E+6 cells above the corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
A test item is considered equivocal in this assay if the threshold is reproducibly exceeded but the increase of the mutation frequency is not dose dependent.
However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10% of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 10E+6 cells above the corresponding solvent control.
A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation. - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT(R)11 (SYSTAT Software, Inc.) 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 was judged as significant whenever the p-value (probability value) was below 0.05. However, both, biological relevance and statistical significance was considered together.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in Exp. I at 12.0 µg/mL + S9 and at ≥ 0.3 µg/mL – S9; in Exp. II at ≥ 10.0 µg/mL + S9 and at ≥ 0.13 µg/mL -S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: No relevant shift of the osmolarity or pH value was observed.
- Precipitation: Precipitation was observed by the unaided eye at 250.0 µg/mL and above following 4 and 24 hours treatment with and without metabolic activation.
RANGE-FINDING/SCREENING STUDIES:
A pre-test was performed in order to determine the concentration range of the mutagenicity
experiments. The cells were exposed to each concentration of the test item for 4 and 24 h without and 4 h with metabolic activation. Test item concentrations in the range of 15.6 to 2000.0 µg/mL were used. Strong cytotoxic effects were observed from the lowest to the highest concentration following 4 and 24 h treatment with and without metabolic activation. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 h) before the test item was removed. Precipitation was observed by the unaided eye at 250.0 µg/mL and above following 4 and 24 h treatment with and without metabolic activation.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects defined as a decline of the relative total growth (RTG) in at least one of the parallel cultures to values below 50% of the corresponding solvent control were noted at 12.0 µg/mL in the first experiment with and at 0.3 µg/mL and above without metabolic activation. In the second experiment cytotoxic effects as described above occurred at 10.0 µg/mL and above with and at 0.13 µg/mL and above without metabolic activation.
ADDITIONAL INFORMATION ON RESULTS:
No substantial and reproducible increase of the mutation frequency was noted in both experiments with and without metabolic activation. The threshold of 126 above the mutationfrequency of the solvent control was solely exceeded at the maximum concentration of the first experiment, culture I with metabolic activation (297 compared to 265 colonies per 10E6cells). Another increase of the mutation frequency reaching but not exceeding the threshold was notedat 10.0 µg/mL in the second culture of the second experiment with metabolic activation. None ofthe increases described above was reproduced in the parallel culture under identical conditions. Furthermore, the increase above the threshold detected at 12.0 µg/mL in the first culture of thefirst experiment with metabolic activation occurred at a relative total growth of just 6.6 and thus below the recommended limit of 10%. However, the data are covered by the IWGT exceptioncriteria and thus valid as the RTG showed no cytotoxic level at the next lower concentration of 8.0 µg/mL. - Conclusions:
- Interpretation of results: negative with and without metabolic activation
Under the conditions of the test the test item is not mutagenic in mammalian cells in vitro with and without metabolic activation. - Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2013-10-30 to 2014-01-16
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- adopted 22 Jul 2010
- Deviations:
- yes
- Remarks:
- Both recovery phase and harvest time were slightly modified comparing to the current recommendation given in the OECD Guideline 487
- Qualifier:
- according to guideline
- Guideline:
- other: EU Method B.49 (In vitro mammalian cell micronucleus test)
- Version / remarks:
- adopted 06 Jul 2012
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes: cultured peripheral human lymphocytes
- Details on mammalian cell type (if applicable):
- CELLS USED
- Cell proliferation: Blood samples were drawn from healthy non-smoking donors not receiving medication.
- Sex, age and number of blood donors if applicable:
- Donor for pre-experiment: 33-year old female
- Donor for experiment I: 29-year old female
- Donor for experiment IIA and IIB: 34-year old female
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Methods for maintenance in cell culture if applicable: Blood cultures were established by preparing an 11% mixture of whole blood in medium within 30 h after blood collection.
MEDIA USED
- Type and identity of media: DMEM/Ham’s F12, mixture (1:1) supplemented with 200 mM GlutaMAX, 100 U/mL / 100 µg/mL penicillin / streptomycin, 3 µg/mL of the mitogen phytohaemagglutinine (PHA), 10% fetal bovine serum (FBS), 10 mM HEPEPS and 125 U.S.P.-U/mL heparin. During exposure to the test substance (4 h treatment), medium was used without FBS supplementation. Cultures were maintained at 37°C in a humidified atmosphere of 5.5% CO2 in air.
- Properly maintained: yes - Cytokinesis block (if used):
- Cytochalasin B (4 µg/mL)
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital / ß-naphthoflavone
- Test concentrations with justification for top dose:
- Preliminary experiment:
For 4 h treatment with and without S9 mix: 22.7, 39.8, 69.9, 121.9, 213.2, 373.2, 653.1, 1142.9, 2000.0 and 3500.0 µg/mL
Experiment I:
For 4 h treatment without S9 mix: 0.02, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.1, 6.3#, 12.5#, 25.0# and 50.0 µg/mL
For 4 h treatment with S9 mix: 0.1, 0.2, 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25.0#, 50.0#, 100.0# and 200.0 µg/mL
Experiment IIA*:
For 20 h treatment without S9 mix: 0.013, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.1, 6.3, 12.5 and 25.0 µg/mL
For 4 h treatment with S9 mix: 0.8, 1.6, 3.1, 6.3, 12.5, 25.0#, 50.0#, 75.0#, 100.0, 125.0, 150.0 and 200.0 µg/mL
Experiment IIB: For 20 h treatment without S9 mix: 0.1, 0.2, 0.4, 0.8, 1.6, 3.1#, 6.3#, 12.5#, 25.0, 50.0, 75.0, 100.0, 125.0, 150.0 and 200.0 µg/mL
* was repeated due to the lack of cytotoxicity, not analysed
# selected for analysis - Vehicle / solvent:
- - Vehicle/solvent used: DMSO (final concentration 1.0% (v/v))
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: Demecolcin, -S9, 75.0 ng/mL in water for continuous treatment
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 and 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 40 h
ACTIN POLYMERISATION INHIBITOR (cytogenetic assays): cytochalasin B, 4 µg/mL
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: Duplicates in two independent experiments
NUMBER OF CELLS EVALUATED: At least 1000 binucleated cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity was described as % cytostasis. For details please refer to “Any other information on material and methods incl. tables”.
DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI) - Evaluation criteria:
- A test item can be classified as clastogenic and aneugenic if:
-The number of micronucleated cells is not in the range of the historical laboratory's control data and
-Either a concentration-related increase in three test groups or a statistically significant increase in the number of micronucleated cells is observed.
If the above mentioned criteria for the test item are not clearly met, the test item will be classified as equivocal or a confirmatory experiment may be performed. However, results may remain questionable regardless of the number of times the experiment is repeated. An increase in the number of micronucleated mononucleate cells may indicate that the test item has aneugenic potential. - Statistics:
- Statistical significance was confirmed by using the Chi-squared test (α < 0.05) for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
- Key result
- Species / strain:
- lymphocytes: cultured peripheral human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Statistically significant increases in the number of micronucleated cells was observed for some concentrations, all values were within the range of the laboratory's historical control data.
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Exp. I (+S9): cytotoxicity at 100.0 µg/mL (66.3% cytostasis)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: No relevant influence on osmolality or pH value was observed.
- Precipitation: At the end of treatment, visible precipitation of the test item was observed in Experiment I at ≥ 12.5 µg/mL (-S9 mix) and at ≥ 100.0 µg/mL (+S9 mix), in Experiment IIA at 200.0 µg/mL (+S9 mix) and in Experiment IIB at 200.0 µg/mL (-S9 mix).
RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. The test was conducted with 10 concentrations in the range of 22.7 to 3500.0 µg/mL, in the presence and absence of S9 mix. Precipitation was observed at the end of treatment at ≥ 373.2 µg/mL in the absence of S9 mix and at ≥ 653.1 µg/mL in the presence of S9 mix.
HISTORICAL CONTROL DATA: Please refer to Table no. 2 under “Any other information on results incl. tables”.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment I and IIB in the absence of S9 mix and in Experiment IIA in the presence of S9 mix concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. In Experiment I in the presence of S9 mix clear cytotoxicity was observed at the highest evaluated concentration (66.3 % cytostasis). - Conclusions:
- Interpretation of results: negative with and without metabolic activation
Under the experimental conditions of the in vitro micronucleus test the test substance did not induce micronuclei in cultivated peripheral human lymphocytes with and without metabolic activation.
Referenceopen allclose all
Table 1: Experimental results
| Experiment 1: Standard plate test (SPT) | ||||||||||
| Strain | TA 1535 | TA 1537 | TA 98 | TA 100 | WP2 uvrA | |||||
| Metabolic activation | without S9 | with S9 | without S9 | with S9 | without S9 | with S9 | without S9 | with S9 | without S9 | with S9 |
| Vehicle control | ||||||||||
| DMSO mean | 14 | 10 | 5 | 4 | 18 | 28 | 121 | 118 | 29 | 30 |
| ± SD | ± 2 | ± 1 | ± 2 | ± 1 | ± 6 | ± 3 | ± 1 | ± 6 | ± 8 | ± 3 |
| Test item [µg/plate] | ||||||||||
| 3 mean | 136 | 131 | 31 | 35 | ||||||
| ± SD | ± 8 | ± 5 | ± 2 | ± 10 | ||||||
| 10 mean | 10 | 9 | 5 | 4 | 24 | 27 | 148 | 116 | 28 | 31 |
| ± SD | ± 4 | ± 3 | ± 3 | ± 2 | ± 5 | ± 3 | ± 2 | ± 8 | ± 8 | ± 9 |
| 33 mean | 11 | 7 | 3 | 3 | 32 | 28 | 135 | 117 | 27 | 28 |
| ± SD | ± 2 | ± 4 | ± 1 | ± 0 | ± 5 | ± 3 | ± 14 | ± 3 | ± 5 | ± 6 |
| 100 mean | 8 | 9 | 4 | 5 | 22 | 27 | 126 | 125 | 27 | 35 |
| ± SD | ± 3 | ± 3 | ± 2 | ± 1 | ± 6 | ± 6 | ± 5 | ± 6 | ± 6 | ± 6 |
| 333 mean | 5 s | 5 s | 0 s | 2 s | 18 | 21 | 70 s | 70 s | 27 | 29 |
| ± SD | ± 1 | ± 3 | ± 0 | ± 2 | ± 6 | ± 4 | ± 14 | ± 3 | ± 3 | ± 3 |
| 1000 mean | MC e | MC e | 0 a | 0 a | MC e | 6 m | MC e | MC e | 18 | 19 |
| ± SD | ± 0 | ± 0 | ± 3 | ± 4 | ± 8 | |||||
| 3330 mean SP | 0 a | 0 a | 0 a | 0 a | 0 a | 0 a | 0 a | 0 a | MC e | 9 |
| ± SD | ± 0 | ± 0 | ± 0 | ± 0 | ± 0 | ± 0 | ± 0 | ± 0 | ± 5 | |
| 5000 mean SP | 0 a | 0 a | MC e | 29 s | ||||||
| ± SD | ± 0 | ± 0 | ± 10 | |||||||
| Positive control | ||||||||||
| §mean | 960 | 408 | 234 | 312 | 999 | 923 | 1013 | 1196 | 1037 | 581 |
| ± SD | ± 16 | ± 42 | ± 40 | ± 22 | ± 60 | ± 51 | ± 40 | ± 123 | ± 56 | ± 85 |
| Experiment 2: Standard plate test (SPT) | ||||||||||
| Strain | TA 1535 | TA 1537 | TA 98 | TA 100 | WP2 uvrA | |||||
| Metabolic activation | without S9 | with S9 | without S9 | with S9 | without S9 | with S9 | without S9 | with S9 | without S9 | with S9 |
| Vehicle control | ||||||||||
| DMSO mean | 7 | 9 | 9 | 7 | 26 | 29 | 125 | 93 | 30 | 33 |
| ± SD | ± 2 | ± 2 | ± 4 | ± 2 | ± 4 | ± 3 | ± 13 | ± 14 | ± 5 | ± 5 |
| Test item | ||||||||||
| 3 mean | 8 | |||||||||
| ± SD | ± 3 | ± | ||||||||
| 10 mean | 9 | 8 | 8 | 7 | 20 | 27 | 124 | 71 | ||
| ± SD | ± 4 | ± 4 | ± 4 | ± 2 | ± 4 | ± 4 | ± 7 | ± 8 | ||
| 33 mean | 10 | 7 | 5 | 8 | 21 | 29 | 133 | 74 | 30 | 22 |
| ± SD | ± 3 | ± 3 | ± 2 | ± 3 | ± 4 | ± 4 | ± 23 | ± 3 | ± 5 | ± 1 |
| 100 mean | 6 | 5 | 6 | 6 | 25 | 28 | 121 | 72 | 34 | 32 |
| ± SD | ± 2 | ± 1 | ± 3 | ± 1 | ± 7 | ± 6 | ± 6 | ± 12 | ± 9 | ± 4 |
| 333 mean | 4 s | 5 s | 0 s | 5 | 17 | 29 | 39 m | 55 m | 34 | 23 |
| ± SD | ± 1 | ± 2 | ± 1 | ± 1 | ± 1 | ± 7 | ± 9 | ± 9 | ± 1 | ± 8 |
| 666 mean | MC e | MC e | 3 s | 14 | 21 | MC e | MC e | |||
| ± SD | ± 1 | ± 2 | ± 1 | |||||||
| 1000 mean | 30 | 21 | ||||||||
| ± SD | ± 10 | ± 8 | ||||||||
| 3330 mean SP | MC e | 12 | ||||||||
| ± SD | ± 5 | |||||||||
| Positive control | ||||||||||
| §mean | 859 | 185 | 314 | 236 | 993 | 389 | 677 | 573 | 1152 | 264 |
| ± SD | ± 10 | ± 12 | ± 42 | ± 108 | ± 40 | ± 114 | ± 58 | ± 161 | ± 28 | ± 67 |
| Experiment 3: Standard plate test (SPT) | ||||||||||
| Strain | TA 1535 | TA 1537 | TA 98 | TA 100 | WP2 uvrA | |||||
| Metabolic activation | without S9 | with S9 | without S9 | with S9 | without S9 | with S9 | without S9 | with S9 | without S9 | with S9 |
| Vehicle control | ||||||||||
| DMSO mean | 25 | 28 | ||||||||
| ± SD | ± 3 | ± 3 | ||||||||
| Test item | ||||||||||
| 100 mean | 25 | 28 | ||||||||
| ± SD | ± 3 | ± 3 | ||||||||
| 333 mean | 16 | 27 | ||||||||
| ± SD | ± 2 | ± 3 | ||||||||
| 1000 mean | MC a | 6 m | ||||||||
| ± SD | ± 2 | |||||||||
| 3330 mean SP | 0 e | 0 a | ||||||||
| ± SD | ± 0 | ± 0 | ||||||||
| Positive control | ||||||||||
| §mean | 1156 | 662 | ||||||||
| ± SD | ± 10 | ± 65 | ||||||||
| §= information on respective positive control is reported in the Method section above (Controls, positive control substances) | ||||||||||
| b= thinning of the background lawn of non-revertant cells was observed | ||||||||||
| MC: Microcolonies | ||||||||||
| e: Bacterial background lawn extremely reduced | ||||||||||
| a: Bacterial background lawn absent | ||||||||||
| m: Bacterial background lawn moderately reduced | ||||||||||
| s: Bacterial background lawn slightly reduced | ||||||||||
| SP: slight precipitate | ||||||||||
Table 2: Historical control data
| Strain | TA 1535 | TA 1537 | TA 98 | TA 100 | WP2 uvrA | |||||
| Metabolic activation | without S9 | with S9 | without S9 | with S9 | without S9 | with S9 | without S9 | with S9 | without S9 | with S9 |
| Vehicle control (DMSO) | ||||||||||
| HCD [range] mean ± SD |
3 - 28 12 ± 14 |
3 - 29 12 ± 14 |
3 - 19 7 ± 9 |
3 - 21 7 ± 10 |
12 - 45 21 ± 19 |
12 - 51 26 ± 21 |
63 - 194 121 ± 85 |
60 - 195 107 ± 94 |
4 - 39 17 ± 19 |
4 - 44 17 ± 20 |
| Positive controls | ||||||||||
| SA | 2AA | 9AC | 2AA | NF | 2AA | MMS | 2AA | 4-NQO | 2AA | |
| HCD [range] mean ± SD |
375 - 1923 1119 ± 688 |
58 - 538 185 ± 227 |
79 - 927 335 ± 417 |
57 - 833 353 ± 418 |
286 - 1790 1072 ± 648 |
171 - 1703 698 ± 935 |
452 - 1593 1074 ± 526 |
223 - 2061 1098 ± 1021 |
67 - 1387 755 ± 805 |
56 - 760 262 ± 272 |
| SA: sodium azide; 2AA: 2 -aminoanthracene; 9AC: 9 -aminoacridine; NF: 2 -nitrofluorene; MMS: methylmethanesulfonate; 4 -NQO: 4 -nitroquinoline-N-oxide | ||||||||||
Table 1: Experimental results
| Concentration [µg/mL] |
± S9 mix | RTG | mutant colonies/ 10E6 cells | threshold | RTG | mutant colonies/ 10E6 cells | threshold | |
| Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| Experiment I / 4 h treatment | culture I | culture II | ||||||
| Solv. control with DMSO | - | 100 | 75 | 201 | 100 | 133 | 259 | |
| Pos. control with MMS | 19.5 | - | 10.5 | 1024 | 201 | 14.3 | 599 | 259 |
| Test item | 0.03 | - | 113 | 54 | 201 | 90.6 | 101 | 259 |
| Test item | 0.06 | - | 84.7 | 65 | 201 | 69.9 | 127 | 259 |
| Test item | 0.1 | - | 91.7 | 43 | 201 | 66 | 107 | 259 |
| Test item | 0.3 | - | 29.3 | 68 | 201 | 29.6 | 92 | 259 |
| Test item | 1 | - | 6.7 | 162 | 201 | 24.6 | 148 | 259 |
| Test item | 2 | - | culture was not continued# | culture was not continued# | ||||
| Test item | 3 | - | culture was not continued# | culture was not continued# | ||||
| Test item | 4 | - | culture was not continued# | culture was not continued# | ||||
| Solv. control with DMSO | + | 100 | 139 | 265 | 100 | 92 | 218 | |
| Pos. control with CPA | 3 | + | 41.4 | 458 | 265 | 62.2 | 190 | 218 |
| Pos. control with CPA | 4.5 | + | 15.6 | 960 | 265 | 22.7 | 324 | 218 |
| Test item | 0.3 | + | culture was not continued# | culture was not continued# | ||||
| Test item | 0.5 | + | culture was not continued# | culture was not continued# | ||||
| Test item | 1 | + | 58.6 | 189 | 265 | 70 | 126 | 218 |
| Test item | 2 | + | 56.3 | 159 | 265 | 85.7 | 103 | 218 |
| Test item | 4 | + | 78.8 | 104 | 265 | 92.2 | 86 | 218 |
| Test item | 8 | + | 86.1 | 53 | 265 | 61.3 | 117 | 218 |
| Test item | 12 | + | 6.6 | 297 | 265 | 3.3 | 148 | 218 |
| Test item | 16 | + | culture was not continued## | culture was not continued## | ||||
| Experiment II / 24 h treatment | culture I | culture II | ||||||
| Solv. control with DMSO | - | 100 | 69 | 195 | 100 | 84 | 210 | |
| Pos. control with MMS | 13 | - | 9.8 | 763 | 195 | 10.8 | 518 | 210 |
| Test item | 0.02 | - | 92.9 | 121 | 195 | 91.9 | 111 | 210 |
| Test item | 0.03 | - | 89.1 | 105 | 195 | 97.8 | 75 | 210 |
| Test item | 0.06 | - | 66.2 | 140 | 195 | 73.7 | 91 | 210 |
| Test item | 0.13 | - | 50 | 96 | 195 | 43 | 93 | 210 |
| Test item | 0.25 | - | 12.6 | 130 | 195 | 7.7 | 168 | 210 |
| Test item | 0.5 | - | culture was not continued## | culture was not continued## | ||||
| Test item | 1 | - | culture was not continued## | culture was not continued## | ||||
| Test item | 2 | - | culture was not continued## | culture was not continued## | ||||
| Experiment II / 4 h treatment | culture I | culture II | ||||||
| Solv. control with DMSO | + | 100 | 134 | 260 | 100 | 141 | 267 | |
| Pos. control with CPA | 3 | + | 40.9 | 253 | 260 | 49 | 481 | 267 |
| Pos. control with CPA | 4.5 | + | 24 | 573 | 260 | 36.5 | 676 | 267 |
| Test item | 0.3 | + | culture was not continued# | culture was not continued# | ||||
| Test item | 0.5 | + | culture was not continued# | culture was not continued# | ||||
| Test item | 1 | + | culture was not continued# | culture was not continued# | ||||
| Test item | 2 | + | 123.8 | 65 | 260 | 109.5 | 175 | 267 |
| Test item | 4 | + | 89 | 71 | 260 | 176.7 | 135 | 267 |
| Test item | 8 | + | 54.4 | 151 | 260 | 156.2 | 143 | 267 |
| Test item | 10 | + | 12.3 | 208 | 260 | 47.1 | 267 | 267 |
| Test item | 12 | + | 11.7 | 236 | 260 | 49.2 | 259 | 267 |
| RTG: relative total growth threshold = number of mutant colonies per 10E 6 cells of each solvent control plus 126 # culture was not continued due to exceedingly severe cytotoxic effects ## culture was not continued since only a minimum of four concentrations is required by the guidelines |
||||||||
Table No. 1: Experimental results of the in vitro micronucleus test in human lymphocytes
| Exp. | Preparation interval | Test item concentration | Proliferation index | Cytostasis in %* | Micronucleated cells |
| in µg/mL | CBPI | in %** | |||
| Exposure period 4 hrs without S9 mix | |||||
| I | 40 hrs | Solvent control1*** | 1.92 | 0.5 | |
| Positive control2 | 1.36 | 60.6 | 20.60S | ||
| 6.3*** | 1.95 | n.c. | 0.8 | ||
| 12.5P*** | 1.84 | 8.6 | 1.60S | ||
| 25.0P*** | 1.71 | 22.8 | 1.25S | ||
| Exposure period 20 hrs without S9 mix | |||||
| IIB | 40 hrs | Solvent control1 | 1.76 | 0.45 | |
| Positive control3 | 1.53 | 30.3 | 3.20S | ||
| 3.1 | 1.79 | n.c. | 0.35 | ||
| 6.3 | 1.65 | 14.9 | 0.45 | ||
| 12.5 | 1.76 | 0.7 | 0.65 | ||
| Exposure period 4 hrs with S9 mix | |||||
| I | 40 hrs | Solvent control1 | 1.92 | 0.1 | |
| Positive control4 | 1.55 | 40.1 | 2.45S | ||
| 25 | 1.94 | n.c. | 0.35 | ||
| 50 | 1.7 | 24.7 | 0.60S | ||
| 100.0P | 1.31 | 66.3 | 0.90S | ||
| Exposure period 4 hrs with S9 mix | |||||
| IIA | 40 hrs | Solvent control1 | 2.19 | 0.4 | |
| Positive control5 | 1.85 | 28.2 | 2.25S | ||
| 25 | 2.17 | 1.3 | 0.5 | ||
| 50 | 2.14 | 4.1 | 0.35 | ||
| 75 | 1.99 | 16.3 | 0.95S | ||
* For the positive control groups and the test item treatment groups the values are related to the solvent controls
** The number of micronucleated cells was determined in a sample of 2000 binucleated cells
*** The number of micronucleated cells was determined in a sample of 4000 binucleated cells
P: Precipitation occurred at the end of treatment
S: The number of micronucleated cells is statistically significantly higher than corresponding control values
n.c.: Not calculated as the CBPI is equal to or higher than the solvent control value
1. DMSO, 1.0 %(v/v)
2. MMC, 2.0 µg/mL
3. Demecolcin, 75.0 ng/mL
4. CPA, 17.5 µg/mL
5. CPA, 15.0 µg/mL
Table No. 2: Historical control data (2009 - 2012):
| Without metabolic activation | ||||
| Micronucleated cells (%) | ||||
| Concentration | No. of experiments | |||
| Range | Mean | Standard deviation | ||
| Solvent control (pulse treatment) | ||||
| Aqueous solv.1 | 35 | 0.20 – 1.30 | 0.38 | ±0.28 |
| Organic solv.2 | 26 | 0.15 – 1.40 | 0.64 | ±0.39 |
| Total | 61 | 0.15 – 1.40 | 0.61 | ±0.33 |
| Solvent control (continuous treatment) | ||||
| Aqueous solv.1 | 34 | 0.05 – 1.45 | 0.39 | ±0.27 |
| Organic solv.2 | 26 | 0.10 – 1.35 | 0.4 | ±0.27 |
| Total | 60 | 0.05 – 1.45 | 0.39 | ±0.27 |
| Total#3 | 10 | 0.10 – 0.80 | 0.43 | ±0.21 |
| Positive control (pulse treatment) | ||||
| Mitomycin C 1.0 – 3.0 µg/mL | 26 | 3.60 – 25.10 | 11.27 | ±5.67 |
| Positive control (continuous treatment) | ||||
| Demecolcin 50 – 175 ng/mL | 28 | 1.40 – 6.10 | 3.12 | ±1.22 |
| Demecolcin#50 – 175 ng/mL | 11 | 1.30 – 5.45 | 2.79 | ±1.18 |
| With metabolic activation | ||||
| Micronucleated cells (%) | ||||
| Concentration | No. of experiments | |||
| Range | Mean | Standard deviation | ||
| Negative control (pulse treatment) | ||||
| Aqueous solv.1 | 70 | 0.15 – 1.70 | 0.66 | ±0.35 |
| Organic solv.2 | 43 | 0.20 – 1.65 | 0.7 | ±0.36 |
| Total | 113 | 0.15 – 1.70 | 0.68 | ±0.35 |
| Positive control (pulse treatment) | ||||
| CPA | 47 | 2.20 – 11.05 | 5.19 | ±2.22 |
| 7.5 – 15.0 µg/mL | ||||
1 Aqueous solvents: DMEM/Ham’s F12, deionised water (10 %v/v)
2 Organic solvents: DMSO (0.5 or 1.0%), acetone, ethanol and THF (0.5%)
3 Aqueous and organic solvents
# Micronucleated mononucleate cells
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
In vitro gene mutation in bacteria
The potential of the test item to cause gene mutations in bacteria was evaluated in an Ames test performed according to OECD guideline 471 and in compliance with GLP (2008-0222-DGM). Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, and E. coli strain WP2 uvrA were exposed to the test item, vehicle (DMSO) and positive controls in the presence and absence of metabolic activation (S9 mix). Two independent experiments were performed using the plate incorporation method using test item concentrations in the range of 3 to 5000 µg/plate. Concentrations were selected based on the result of a preliminary test performed with TA100 and WP2 uvrA; the test was used as a part of the first experiment of the main study. In all experiments, the tester strains were exposed for 48 h at 37 ± 1 °C with and without S9 mix. Following exposure, the background lawn of bacterial growth was assessed and the mean number of revertant colonies was determined.
Precipitation was noted in all experiments at concentrations of 3330 and 5000 µg/plate at the end of the incubation period. Cytotoxicity was noted for TA98 at ≥ 1000 µg/plate with and without metabolic activation, for TA100, TA1535 and TA1537 at ≥ 333 µg/plate with and without metabolic activation. For WP2 uvrA, cytotoxicity was observed at 3330 µg/plate without metabolic activation and at 5000 µg/plate with metabolic activation.
Treatment with the test item did not induce a biologically relevant increase in the number of revertant colonies for any tester strain in the presence or in the absence of metabolic activation. The vehicle and positive controls demonstrated the sensitivity and validity of the test system. Based on the experimental results, the test item is not considered to be mutagenic in bacterial cells with and without metabolic activation.
In vitro gene mutation in mammalian cells
The test item was investigated for the mutagenic potential in an in vitro mouse lymphoma assay performed according to OECD guideline 476(version adopted in 1997)and in compliance with GLP (2013-0396-DGM). In two independent experiments using duplicate cultures, gene mutations in the thymidine kinase (TK) locus were analysed in L5178Y TK +/- cells in the presence and absence of metabolic activation (S9 mix). Concentration levels were chosen based on the results of a pre-experiment, where cells were exposed to test item concentrations in the range of 15.6 to 2000 µg/mL; for 4 h in the presence of S9 mix, and for 4 and 24 h in the absence of S9 mix.
In the first experiment, the cells were exposed to concentrations in the range of 0.03 to 4.0 µg/mL for 4 h in the absence of S9 mix and to concentrations in the range of 0.3 to 16.0 µg/mL for 4 h in the presence of S9 mix. In the second experiment, cells were exposed for 24 h without S9 mix to concentrations in the range of 0.02 to 2.0 µg/mL, and were exposed for 4 h with S9 mix to concentrations in the range of 0.3 to 12.0 µg/mL. In each experiment, an appropriate vehicle (DMSO) and positive controls (methylmethane sulfonate without S9 and cyclophosphamide with S9) were included. After 4 or 24 h of exposure, the cells were incubated for 48 h to allow expression of the mutant phenotype, followed by a selection period of 10-15 days during which the cells were incubated inmedium containing trifluorothymidine (TFT).
Toxicity was observed in the first experiment at 12.0 µg/mL with S9 mix and at ≥ 0.3 µg/mL without S9 mix, and in the second experiment at ≥ 10.0 µg/mL with S9 mix and at ≥ 0.13 µg/mL without S9 mix. Precipitation was observed by the unaided eye at 250.0 µg/mL and above following 4 and 24 hours treatment with and without metabolic activation. Mutation frequencies (MF) in the first experiment (4 h exposure) was determined for concentrations in the range of 0.03 to 1.0 µg/mL in the absence of S9 mix and for concentrations in the range of 1.0 to 12.0 in the presence of S9 mix, respectively. In the second experiment, concentration ranges investigated for MF were 0.02 to 0.25 µg/mL in the absence of S9 mix (24 h exposure) and 2.0 to 12.0 in the presence of S9 mix (4 hexposure), respectively. None of the experimental conditions showed a substantial and reproducible increase in mutation frequencies (MF) in the presence or in the absence of metabolic activation. Solvent controls remained within the range of historical control data, while the positive compounds markedly increased mutation frequencies, thus demonstrating the sensitivity and validity of the test system. Based on these results and under the experimental conditions reported, the test item is considered to be non-mutagenic in mammalian cells in vitro.
In vitro cytogenicity in mammalian cells
The potential of the test item to induce micronuclei in mammalian cells in vitro was assessed in a micronucleus test in cultured human peripheral lymphocytes. The test was conducted according to OECD guideline 487 (version adopted in 2010) and in compliance with GLP (2013-0398-DGM). Concentration levels were chosen based on the results of a preliminary experiment, in which cells were exposed for 4 h to test item concentrations in the range of 22.7 to 3500 µg/mL in the presence and absence of metabolic activation (S9 mix). In the main test, two independent experiments using individual donors were performed using two parallel cultures per condition. In the first experiment, the human lymphocytes were treated for 4 h at concentrations in the range of 0.1 to 200.0 µg/mL in the presence of metabolic activation and at concentrations in the range of 0.02 to 50.0 µg/mL in the absence of metabolic activation. The second experiment was performed with concentrations in the range of 0.8 to 200.0 µg/mL for 4 h in the presence of S9 mix and with concentrations in the range of 0.013 to 25.0 µg/mL for 20 h in the absence of S9 mix. Due to the lack of cytotoxicity, the second experiment with 20 h exposure in the absence of S9 mix was repeated using concentrations in the range of 0.1 to 200.0 µg/mL. The cells were prepared 40 h after start of treatment. At least 2000 binucleated cells (1000 per culture) were evaluated for the presence of micronuclei and cytogenetic damage. In the first experiment (4 h exposure), concentrations in the range of 6.3 to 25.0 µg/mL without S9 mix and in the range of 25.0 to 100.0 µg/mL with S9 mix, respectively, were evaluated for the presence of micronuclei. In the second experiment, the scoring was performed for concentrations in the range of 3.1 to 12.5 µg/mL without S9 mix (24 h exposure, repeated experiment only) and concentrations in the range of 25.0 to 75.0 µg/mL with S9 mix (4 h exposure), respectively.
Treatment with the test item showed no biologically relevant, reproducible increase in the frequency of micronucleated cells in the presence or in the absence of metabolic activation. In both experiments, a number of test concentrations with and without metabolic activation showed statistically significant increases in the number of micronucleated cells, however, none of the observations was consistent and all fluctuations remained within the range of historical control data.
Mutant frequencies of vehicle controls (DMSO) remained within the range of historical negative control data. Concurrent positive controls (cyclophosphamide (+S9), mitomycin C and demecolcine (-S9)) induced a statistically significant increase in micronuclei frequencies when compared to vehicle controls and the response was within the range of historic positive control data.
Under the experimental conditions of the study, the test item showed no clastogenic or aneugenic potential.
Justification for classification or non-classification
The available data on genetic toxicity in vitro do not meet the criteria for classification according to Regulation No. (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.
No classification for genetic toxicity is warranted according to the criteria of the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
