Registration Dossier
Registration Dossier
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EC number: 203-109-3 | CAS number: 103-41-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The test item was found to be negative in an Ames test, an in vitro mammalian gene mutation test and an in vitro micronucleus test. Hence, it can be concluded that the substance is not mutagenic according to these 3 in vitro tests.
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:
- 29.05.2015 - 19.06.2015
- 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:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hess. Ministerium fuer Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Salmonella typhimurium: histidine (his)
Escherichia coli: tryptophan (trp) - Species / strain / cell type:
- S. typhimurium TA 1537
- Details on mammalian cell type (if applicable):
- - Genotype: his C 3076; rfa-; uvrB-
- Type of mutations indicated: frame shift mutations - Species / strain / cell type:
- S. typhimurium TA 1535
- Details on mammalian cell type (if applicable):
- - Genotype: his G 46; rfa-; uvrB-
- Type of mutations indicated: base-pair substitutions - Species / strain / cell type:
- S. typhimurium TA 98
- Details on mammalian cell type (if applicable):
- - Genotype: his D 3052; rfa-; uvrB-; R-factor
- Type of mutations indicated: frame shift mutations - Species / strain / cell type:
- S. typhimurium TA 100
- Details on mammalian cell type (if applicable):
- - Genotype: his G 46; rfa-; uvrB-; R-factor
- Type of mutations indicated: base-pair substitutions - Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- - Genotype: trp-; uvrA-
- Type of mutations indicated: base-pair substitutions - Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital / ß-naphthoflavone
- Test concentrations with justification for top dose:
- Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (AA)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Two independent reverse mutation assays were performed. Experiment I was a plate incorporation assay. Since a negative result was obtained in this experiment, Experiment II was performed as a pre-incubation assay.
The test substance was mixed in a test tube and poured onto selective agar plates.
DURATION:
- Preincubation period: 37 °C for 60 minutes
- Expression time (cells in growth medium): 48 hours at 37 °C
NUMBER OF REPLICATIONS: 3
DATA RECORDING: colonies were counted using the Petri Viewer Mk2 with the software program Ames Study Manager (v.1.21).
ACCEPTABILITY OF THE ASSAY:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the historical data of the test facility
- the positive control substances should produce a significant increase in mutant colony frequencies
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5. - Evaluation criteria:
- A test item is considered mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant. - Statistics:
- According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- With S9 mix: only in experiment II, minor toxic effects were observed at 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- - The undissolved particles of precipitated test item had no influence on the data recording.
- Plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains.
- Only minor toxic effects were observed in experiment II in strain TA 100 with S9 mix at 5000 μg/plate.
- Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies. - Remarks on result:
- other: precipitation at 2500 µg/plate
- Conclusions:
- The test item was considered not genotoxic under the conditions of the current test.
- Executive summary:
In the current study the genotoxic potential of the test item was assessed according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The study was conducted according to OECD 471 and GLP.
The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: (a) Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate, (b) Experiment II: 33, 100, 333, 1000, 2500 and 5000 μg/plate.
The test item precipitated under some conditions, however, the undissolved particles had no influence on the data recording. Additionally, the plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix. No toxic effects occurred in any strain with and without metabolic activation. Only in experiment II in strain TA 100 with S9 mix minor toxic effects were observed at 5000 μg/plate.
No substantial increase in revertant colony numbers of any of the five tester strains was observed, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.
Therefore, under the experimental conditions of the current test the test item did not induce gene mutations and thus is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 26.07.2016 - 15.09.2016
- 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:
- 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hess. Ministerium fuer Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- HPRT (hypoxanthine-guanine phosphoribosyl transferase)
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Medium: MEM (minimal essential medium) supplemented with Hank’s salts, 10% Fetal Bovine Serum (except during 4 hour treatment), neomycin (5 μg/mL) and amphotericin B (1%). For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine.
- Culture conditions: 37°C in a 1.5% carbon dioxide atmosphere (98.5% air).
- Reason to use this cell line: The V79 cell line is chosen as it has been used successfully in in vitro experiments for many years. Especially the high proliferation rate and good cloning efficiency of untreated cells both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22. - Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital / ß-naphthoflavone
- Test concentrations with justification for top dose:
- 0.9, 2.7, 8.2, 24.7 and 74.0 µg/mL
Justification: The test concentrations were selected based on the pre- experiment. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hours
- Washing: 2x
- Fixation time and staining: 6 - 8 days after treatment
- Expression time: 7 days
STAINING: 10% methylene blue in 0.01% KOH solution.
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
DETERMINATION OF CYTOTOXICITY
- Reason: to determine the concentration range for the mutagenicity experiment.
- Test item concentrations: 0.3 μg/mL - 2000 μg/mL
- Analysis: the colony forming ability of approximately 500 single cells after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).
- OTHER:
Test Item Preparation: on the day of the experiment all formulations were prepared freshly before treatment and used within two hours of preparation.
ACCEPTANCE CRITERIA: The gene mutation assay is considered acceptable if it meets the following criteria:
a) the mean values of the numbers of mutant colonies per 10E6 cells found in the solvent controls of both parallel cultures remain within the 95% confidence interval of the laboratory historical control data range.
b) the positive control substances should produce a significant increase in mutant colony frequencies and remain within the historical control range of positive controls.
c) the cloning efficiency II (absolute value) of the solvent controls must exceed 50%.
The data of this study comply with the above mentioned criteria and Historical Data. - Rationale for test conditions:
- In the pre-experiment relevant cytotoxic effects were observed at 8.2 μg/mL and above without metabolic activation. Phase separation occurred at 24.7 μg/mL and above after 4 hours treatment with and without metabolic activation. The dose range of the main experiment was set according to data of the pre-experiment. The individual concentrations were spaced by a factor of 3.0 to cover soluble as well as insoluble concentrations. To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.
- Evaluation criteria:
- A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows: The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares). - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.
A t-Test was performed to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05. - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 24.7µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- The 95% confidence interval was slightly exceeded at 74.0 μg/mL in the first culture with metabolic activation (29.1 versus an upper limit of 28.7 mutant colonies/106 cells). This isolated increase was judged as irrelevant as it was not reproduced in culture II and there was no significant increase compared to the mutation frequency of the corresponding solvent control as indicated by a lacking statistical significance.
In the main experiment with and without S9 mix, the range of the solvent controls was from 10.2 up to 29.0 mutants per 10E6 cells; the range of the groups treated with the test item was from 8.2 up to 29.1 mutants per 106 cells.
The positive controls EMS (300 μg/mL) and DMBA (2.3 μg/mL) showed a distinct increase in induced mutant colonies. - Conclusions:
- Under the experimental conditions performed, the test item did not induce gene mutations at the HPRT locus in V79 cells, and thus, is considered to be non-mutagenic in this HPRT assay.
- Executive summary:
In the current in vitro study the potential of the test item to induce gene mutations was assessed at the HPRT locus using V79 cells of the Chinese hamster. The OECD guideline 476 was followed and the study was performed in compliance to GLP. This in vitro test is an assay for the detection of forward gene mutations in mammalian cells.
The V79 cells are exposed to the test item with and without exogenous metabolic activation for 4 hours. After which the descendants of the treated original population are monitored for the loss of functional HPRT enzyme.
The mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the surviving cells. Colonies are counted and the mutant frequencies are calculated from the number of mutant colonies corrected for cell survival.
The assay was performed in two independent experiments. The maximum test item concentration of the pre-experiment (2000 μg/mL) was chosen with respect to the current OECD Guideline 476. The concentration range of the main experiment was limited by phase separation of the test item.
The tested concentrations were: 0.9, 2.7, 8.2, 24.7 and 74.0 µg/mL.
No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment.
Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.
It can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 28.09.2016 - 23.11.2016
- 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:
- 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hess. Ministerium fuer Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- not applicable
- Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- - Experiment I: male donor (22 years old)
- Experiment II: male donor (31 years old)
- All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. - Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- cytochalasin B
- Metabolic activation:
- with and without
- Metabolic activation system:
- Mammalian Microsomal Fraction S9 Mix
- Test concentrations with justification for top dose:
- Experiment I 4 hours with and without S9 mix: 2.2, 3.9, 6.8, 11.8, 20.7, 36.3, 63.5, 111, 222, 667, 2000 µg/mL
Experiment II 20 hours without S9 mix: 0.9, 1.6, 2.8, 4.9, 8.5, 14.9, 26.1, 45.7, 80.0 µg/mL - Vehicle / solvent:
- - Solvent: DMSO
- Justification solvent: DMSO was chosen due to its solubility properties and its relative non-toxicity to the cell cultures. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- culture medium with 0.5 % DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: Demecolcin
- Details on test system and experimental conditions:
- CELL CULTURE CONDITIONS
- Culture medium: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) supplemented with 200 mM GlutaMAX™, penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA (3 µg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
- Temperature: 37 °C
- Humidity: 5.5 % CO2 in humidified air
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 48 hour with phytohemeagglutinine (PHA) to stimulate proliferation
- Exposure duration: 4 or 20 hours
- Washing: 2x
- Recovery period: 16 hours
- Harvesting: 40 hours after beginning of treatment
- Post-incubation: at 37 °C for 20 minutes
- Fixation time: 2 x 20 minutes
- Fixative: ice-cold mixture of methanol and glacial acetic acid (19 : 1 parts)
SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION: dropping the cell suspension in fresh fixative onto a clean microscope slide
NUMBER OF CELLS EVALUATED: at least 1000, except for the positive control in Experiment I in the absence of S9 mix, where only 500 binucleated cells per culture were evaluated.
- Frequency of micronucleated cells: reported as % micronucleated cells. To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture. The pre-test was performed with 11 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 40 hrs after start of the exposure. Concentrations ranging from 2.2 to 2000 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity.
OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): no
- OTHER:
- Test Item Preparation: Stock formulations of the test item and serial dilutions were made in DMSO. All formulations were prepared freshly before treatment and used within two hours of preparation. The osmolarity and pH were determined by using an osmometer or a pH meter.
ACCEPTANCE CRITERIA: The micronucleus assay will be considered acceptable if it meets the following criteria:
− The concurrent solvent control will normally be within the laboratory historical solvent control data range (95% confidence interval)
− The concurrent positive controls should induce responses that are compatible with the laboratory historical positive control data and produce a statistically significant increase
− Cell proliferation criteria in the solvent control are considered to be acceptable
− All experimental conditions were tested unless one exposure condition resulted in a clearly positive result
− The quality of the slides must allow the evaluation of an adequate number of cells and concentrations
− The criteria for the selection of top concentration are consistent with those described in OECD TG 487 - Rationale for test conditions:
- In the pre-test for toxicity, phase separation of the test item was observed at the end of treatment at 11.8 µg/mL and above in the absence of S9 mix and at 36.3 µg/mL and above in the presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I. Using a reduced Cytokinesis-block proliferation index (CBPI) as an indicator for toxicity in the pre-test, clear toxic effects were observed after 4 hours treatment with 63.5 µg/mL and above in the absence of S9 mix. Considering the toxicity and phase separation data of Experiment I, 80.0 µg/mL (without S9 mix) was chosen as top concentration in Experiment II.
- Evaluation criteria:
- A test item is considered to be clearly negative if, in all of the experimental conditions examined:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% confidence interval)
A test item is considered to be clearly positive if, in any of the experimental conditions examined:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data
There is no requirement for verification of a clear positive or negative response. In case the response is neither clearly negative nor clearly positive and/or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions (e.g. narrow concentration spacing, other metabolic activation conditions, i.e. S9 concentration or S9 origin) could be useful. However, results may remain questionable regardless of the number of times the experiment is repeated. If the data set will not allow a conclusion of positive or negative, the test item will therefore be concluded as equivocal. - Statistics:
- Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted 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: Human lymphocytes from healthy non-smoking donors not receiving medication
- Remarks:
- Experiment I
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- lymphocytes: Human lymphocytes from healthy non-smoking donors not receiving medication
- Remarks:
- Experiment II
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- highest dose tested
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- - Experiment I:
-- in the absence of S9 mix, 1 statistically significant increase in the number of micronucleate cells (1.55 %), which exceeded the historical control range (95% control limit: 0.07 – 1.15 %), was observed after treatment with 3.9 µg/mL of the test item. This can be declared as biologically irrelevant because the increase is not concentration-related.
-- in the presence of S9 mix: the solvent control and two evaluated concentrations (20.7 and 36.3 µg/mL) showed a slight increase in the number of micronucleate cells (1.35, 1.30 and 1.45 %, respectively) in comparison to the historical control data (95% control limit: 0.08 – 1.20 %). This can be declared as biological irrelevant, because the solvent control did not exceed the maximal limit of the laboratory historical solvent control data (max: 1.35 % micronucleate cells) and no statistical significance was observed.
- Positive controls: Demecolcin (125 ng/mL), MMC (1.0 µg/mL) or CPA (17.5 µg/mL) showed distinct increases in cells with micronuclei. - Conclusions:
- The test item is considered to be unable to induce chromosome breaks and/or gain or loss in this test system and should be considered non-mutagenic.
- Executive summary:
In the current study the potential of the test item to induce micronuclei in human lymphocytes in vitro was assessed according to OECD 487 and in compliance to GLP.
The occurrence of micronuclei in interphase cells provides an indirect but easy and rapid measure of structural chromosomal damage and aneugenicity in cells that have undergone cell division during or after exposure to the test substance. Micronuclei arise from chromosomal fragments or whole chromosomes and are inducible by clastogens or agents affecting the spindle apparatus.
The induction of cytogenetic damage in human lymphocytes was assessed in two independent experiments and in each experimental group two parallel cultures were analyzed. The test item was dissolved in DMSO. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix.
The cells were stimulated for 48 hour with phytohemeagglutinine (PHA) to activate the proliferation, before exposure. After exposure, the cells were washed and left to recover for 16 hours before the cytokinesis was blocked by cytochalasin B for 20 minutes. The cells were prepared 40 hours after start of treatment with the test item.
The highest applied concentration in this study was 2000 µg/mL. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item phase separation in accordance with the OECD Guideline 487. The chosen treatment concentrations are in Experiment I (4 hours with and without S9 mix): 2.2, 3.9, 6.8, 11.8, 20.7, 36.3, 63.5, 111, 222, 667, 2000 µg/mL and in Experiment II (20 hours without S9 mix): 0.9, 1.6, 2.8, 4.9, 8.5, 14.9, 26.1, 45.7, 80.0 µg/mL. Phase separation of the test item in the culture medium was observed in Experiment I at 11.8 µg/mL and above in the absence of S9 mix and at 36.3 µg/mL and above in the presence of S9 mix at the end of treatment. In Experiment II no phase separation was observed.
Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage, except for the positive control in Experiment I in the absence of S9 mix, where only 500 binucleated cells per culture were evaluated. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis.
In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. In Experiment II cytotoxicity was observed at the highest applied concentration.
In the absence and presence of S9 mix, no relevant increase in the number of micronucleate cells was observed after treatment with the test item. However, in Experiment I in the absence of S9 mix, one statistically significant increase in the number of micronucleate cells (1.55 %), which exceeded the historical control range (95% control limit: 0.07 – 1.15 %), was observed after treatment with 3.9 µg/mL of the test item. This can be declared as biologically irrelevant because the increase is not concentration-related. In the presence of S9 mix, the solvent control and two evaluated concentrations (20.7 and 36.3 µg/mL) showed a slight increase in the number of micronucleate cells (1.35, 1.30 and 1.45 %, respectively) in comparison to the historical control data (95% control limit: 0.08 – 1.20 %). This is also biological irrelevant as the solvent control did not exceed the maximal limit of the laboratory historical solvent control data (max: 1.35 % micronucleate cells) and no statistical significance was observed. In both experiments, either Demecolcin (125 ng/mL), MMC (1.0 µg/mL) or CPA (17.5 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test item is considered to be unable to induce chromosome breaks and/or gain or loss in this test system and should be considered non-mutagenic.
Referenceopen allclose all
Summary of Results
Experiment I
Without Metabolic Activation |
|||||||||
Test Group |
Dose level (µg/plate) |
Revertant Colony Counts (mean±SD) |
|||||||
TA1535 |
TA1537 |
TA98 |
TA100 |
WP2uvrA |
|||||
DMSO |
n.a. |
9±1 |
12±4 |
22±3 |
167±7 |
42±9 |
|||
Untreated |
n.a. |
11±2 |
10±5 |
21±7 |
196±5 |
43±7 |
|||
Test item |
3 |
8±4 |
13±4 |
24±2 |
153±16 |
44±13 |
|||
|
10 |
10±4 |
14±2 |
26±6 |
160±8 |
44±10 |
|||
|
33 |
12±5 |
12±2 |
28±9 |
151±14 |
36±9 |
|||
|
100 |
11±3 |
16±3 |
23±7 |
164±17 |
42±5 |
|||
|
333 |
9±6 |
15±2 |
24±5 |
156±6 |
35±12 |
|||
|
1000 |
11±3 |
16±1 |
27±5 |
154±16 |
37±6 |
|||
|
2500 |
11±3P |
12±2PM |
28±4 |
153±4P |
44±7P |
|||
|
5000 |
7±2PM |
11±3PM |
22±2 |
169 ± 27P |
40±4P |
|||
NaN3 |
10 |
1017 ± 64
|
|
|
1646 ± 226
|
|
|||
4-NOPD |
10 |
|
|
229±36
|
|
|
|||
4-NOPD |
50 |
|
74±4
|
|
|
|
|||
MMS |
2.0 |
|
|
|
|
830±44
|
|||
|
|||||||||
With Metabolic Activation |
|||||||||
Test Group |
Dose level (µg/plate) |
Revertant Colony Counts (mean +/- SD) |
|||||||
TA1535 |
TA1537 |
TA98 |
TA100 |
WP2uvrA |
|||||
DMSO |
n.a. |
9±1 |
18±1 |
33±4 |
161±13 |
47±6 |
|||
Untreated |
n.a. |
12±2 |
18±5 |
39±9 |
170±16 |
56±2 |
|||
Test item |
3 |
9±3 |
20±4 |
36±5 |
154±12 |
55±8 |
|||
|
10 |
9±1 |
22±2 |
32±6 |
166±31 |
38±8 |
|||
|
33 |
11±2 |
16±4 |
28±3 |
138±19 |
48±5 |
|||
|
100 |
11±3 |
17±9 |
32±1 |
161±20 |
50±16 |
|||
|
333 |
12±0 |
20±3 |
33±4 |
141±8 |
47±7 |
|||
|
1000 |
14±2 |
19±5 |
34±7 |
132±16 |
32±10 |
|||
|
2500 |
12±6 |
17±5 |
34±4 |
126±4 |
26 ± 10 |
|||
|
5000 |
12±1P |
19±5P |
30±7P |
146±26P |
28 ± 9P |
|||
2-AA |
2.5 |
362±20 |
200 ± 11 |
4321 ± 308 |
4120 ± 59 |
|
|||
2-AA |
10 |
|
|
|
|
310 ± 27 |
|||
NaN3: sodium azide
2-AA: 2-aminoanthracene
4-NOPD: 4-nitro-o-phenylene-diamine
MMS: methyl methane sulfonate
P: Precipitate
M Manual count
Experiment II
Without Metabolic Activation |
|||||||||
Test Group |
Dose level (µg/plate) |
Revertant Colony Counts (mean±SD) |
|||||||
TA1535 |
TA1537 |
TA98 |
TA100 |
WP2uvrA |
|||||
DMSO |
n.a. |
10±3 |
8±0 |
22±3 |
127±12 |
40±7 |
|||
Untreated |
n.a. |
9±1 |
9±1 |
24±6 |
169±23 |
44±11 |
|||
Test item |
33 |
10±3 |
10±1 |
20±5 |
94±7 |
38±11 |
|||
|
100 |
8±1 |
9±3 |
20±1 |
97±8 |
35±5 |
|||
|
333 |
7±3 |
7±3 |
23±2 |
96±4 |
37±5 |
|||
|
1000 |
7±2 |
9±3 |
18±3 |
73±9 |
30±3 |
|||
|
2500 |
9±1P |
9±1P |
29±6P |
88±10P |
34 ± 4P |
|||
|
5000 |
8±1PM |
4±2PM |
18±2PM |
81 ± 6P M |
35 ± 11P |
|||
NaN3 |
10 |
1107 ± 17 |
|
|
1869 ± 141 |
|
|||
4-NOPD |
10 |
|
|
353±20 |
|
|
|||
4-NOPD |
50 |
|
82±6 |
|
|
|
|||
MMS |
2.0 |
|
|
|
|
533±64 |
|||
|
|||||||||
With Metabolic Activation |
|||||||||
Test Group |
Dose level (µg/plate) |
Revertant Colony Counts (mean +/- SD) |
|||||||
TA1535 |
TA1537 |
TA98 |
TA100 |
WP2uvrA |
|||||
DMSO |
n.a. |
11±1 |
9±2 |
25±5 |
108±11 |
48±7 |
|||
Untreated |
n.a. |
11±2 |
9±4 |
31±6 |
161±23 |
61±16 |
|||
Test item |
33 |
14±2 |
11±3 |
33±4 |
107±9 |
45±3 |
|||
|
100 |
9±3 |
8±1 |
27±2 |
115±11 |
50±9 |
|||
|
333 |
7±3 |
12±2 |
29±3 |
114±7 |
43±8 |
|||
|
1000 |
8±2 |
1±2 |
23±2 |
84±19 |
33±6 |
|||
|
2500 |
8±4P |
14±3P |
20±7P |
61±6P |
24±3P |
|||
|
5000 |
7±1P |
12±4P |
28±1P |
40±3PM |
26±5P |
|||
2-AA |
2.5 |
378±31 |
98 ± 14 |
4148 ± 434 |
3196 ± 225 |
|
|||
2-AA |
10 |
|
|
|
|
375 ± 60 |
|||
NaN3: sodium azide
2-AA: 2-aminoanthracene
4-NOPD: 4-nitro-o-phenylene-diamine
MMS: methyl methane sulfonate
P: Precipitate
M Manual count
Summary of Results
|
conc. (μg / mL) |
relative cloning efficiency I (%) |
relative cell density (%) |
relative adjusted cloning efficiency I (%) |
mutant colonies/ 10E6cells |
95% confidence interval |
relative cloning efficiency I (%) |
relative cell density (%) |
relative adjusted cloning efficiency I (%) |
mutant colonies/ 10E6cells |
95% confidence interval |
4 h treatment without S9 mix |
|
culture I |
culture II |
||||||||
Solvent control with DMSO |
|
100.0 |
100.0 |
100.0 |
29.0 |
0.2 - 29.7 |
100.0 |
100.0 |
100.0 |
17.4 |
0.2 - 29.7 |
Positive control (EMS) |
300.0 |
84.7 |
97.3 |
82.4 |
179.4 |
0.2 - 29.7 |
80.7 |
83.3 |
67.2 |
220.5 |
0.2 - 29.7 |
Test item |
0.9 |
92.6 |
97.5 |
90.3 |
12.9 |
0.2 - 29.7 |
85.4 |
90.5 |
77.3 |
15.3 |
0.2 - 29.7 |
|
2.7 |
93.1 |
105.4 |
98.1 |
12.7 |
0.2 - 29.7 |
74.1 |
83.1 |
61.6 |
12.8 |
0.2 - 29.7 |
|
8.2 |
95.1 |
97.5 |
92.7 |
22.0 |
0.2 - 29.7 |
80.7 |
85.1 |
68.6 |
13.8 |
0.2 - 29.7 |
|
24.7 * |
49.0 |
72.7 |
35.6 |
10.2 |
0.2 - 29.7 |
34.7 |
65.1 |
22.6 |
24.7 |
0.2 - 29.7 |
|
74.0 * |
28.3 |
61.1 |
17.3 |
8.2 |
0.2 - 29.7 |
23.9 |
75.2 |
17.9 |
12.7 |
0.2 - 29.7 |
4 h treatment with S9 mix |
|
culture I |
culture II |
||||||||
Solvent control with DMSO |
|
100.0 |
100.0 |
100.0 |
10.2 |
0.6 - 28.7 |
100.0 |
100.0 |
100.0 |
16.4 |
0.6 - 28.7 |
Positive control (DMBA) |
2.3 |
68.9 |
84.8 |
58.4 |
247.6 |
0.6 - 28.7 |
84.9 |
102.4 |
87.0 |
273.1 |
0.6 - 28.7 |
Test item |
0.9 |
67.4 |
95.8 |
64.5 |
20.9 |
0.6 - 28.7 |
85.9 |
93.3 |
80.1 |
17.1 |
0.6 - 28.7 |
|
2.7 |
65.9 |
95.0 |
62.6 |
15.2 |
0.6 - 28.7 |
87.3 |
81.5 |
71.1 |
23.2 |
0.6 - 28.7 |
|
8.2 |
60.3 |
94.3 |
56.8 |
23.6 |
0.6 - 28.7 |
84.9 |
98.1 |
83.3 |
22.0 |
0.6 - 28.7 |
|
24.7 * |
72.2 |
116.7 |
84.3 |
8.4 |
0.6 - 28.7 |
84.5 |
90.1 |
76.1 |
13.5 |
0.6 - 28.7 |
|
74.0 * |
74.4 |
77.8 |
57.9 |
29.1 |
0.6 - 28.7 |
70.1 |
104.5 |
73.3 |
12.2 |
0.6 - 28.7 |
* phase separation visible at the end of treatment
Summary of results
Test item |
Proliferation |
Cytostasis |
Micronucleated |
concentration |
index |
in %* |
cells |
in µg/mL |
CBPI |
|
in %** |
Experiment I Exposure period 4 hrs without S9 mix |
|||
Solvent control1# |
2.06 |
|
0.85 |
Positive control2## |
1.46 |
56.1 |
25.30S |
3.9# |
1.94 |
11.2 |
1.55S |
6.8# |
1.95 |
10.0 |
1.10 |
11.8PS # |
1.93 |
12.3 |
1.18 |
Experiment II Exposure period 20 hrs without S9 mix |
|||
Solvent control1 |
1.98 |
|
0.55 |
Positive control3 |
1.69 |
29.5 |
6.50S |
26.1 |
1.92 |
6.4 |
0.85 |
45.7 |
1.78 |
20.4 |
0.95 |
80.0 |
1.50 |
48.5 |
0.70 |
Experiment I Exposure period 4 hrs with S9 mix |
|||
Solvent control1 |
2.13 |
|
1.35 |
Positive control4 |
1.54 |
52.6 |
7.35S |
11.8 |
2.01 |
10.1 |
1.20 |
20.7 |
2.11 |
1.9 |
1.30 |
36.3PS |
2.07 |
5.1 |
1.45 |
* 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
## The number of micronucleated cells was determined in a sample of 1000 binucleated cells
PS Phase seperation occurred at the end of treatment
S The humber of micronucleated cells is statistically significantly higher than the corresponding control values
1 DMSO:
0.5 %
(v/v)
2 MMC: 1.0
µg/mL
3 Demecolcin: 125 ng/mL
4 CPA: 17.5 µg/mL
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
For this endpoint there are 3 studies available. All three assays have been performed under GLP.
In the first study the genotoxic potential of the test item was assessed according to OECD 471 in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested in Experiment I at 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate and in Experiment II at 33, 100, 333, 1000, 2500 and 5000 μg/plate.
The test item precipitated under some conditions, however, the undissolved particles had no influence on the data recording. Plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix and no toxic effects occurred in any strain with and without metabolic activation. No substantial increase in revertant colony numbers in the presence nor absence of S9 mix was observed. Therefore, the test item did not induce gene mutations in bacteria and is considered non-mutagenic in the Ames test.
In the second study the potential of the test item to induce micronuclei in human lymphocytes in vitro was assessed according to OECD 487. The test item was dissoved in DMSO and two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix and the tested concentrations were 2.2, 3.9, 6.8, 11.8, 20.7, 36.3, 63.5, 111, 222, 667, 2000 µg/mL. In Experiment II, the exposure period was 20 hours without S9 mix and the tested concentrations were 0.9, 1.6, 2.8, 4.9, 8.5, 14.9, 26.1, 45.7, 80.0 µg/mL.
The cells were stimulated with phytohemeagglutinine (PHA) to activate the proliferation, before exposure and after exposure, washed and left to recover before the cytokinesis was blocked by cytochalasin B. The cells were prepared 40 hours after start of treatment with the test item. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage, except for the positive control in Experiment I in the absence of S9 mix, where only 500 binucleated cells per culture were evaluated. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis.
In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. In Experiment II cytotoxicity was observed at the highest applied concentration.
No relevant increase in the number of micronucleate cells was observed after treatment with the test item and in both experiments the positive controls showed a clear induction in micronuclei.
The test item is considered to not induce chromosome breaks and/or gain or loss in this test system and thus non-mutagenic.
In the third study the potential of the test item to induce gene mutations at the HPRT locus was assessed according to OECD 476 in V79 Chinese hamster cells. The cells were exposed to the test item for 4 hours with and without exogenous metabolic activation. The mutant frequency is calculated from the number of mutant colonies corrected for cell survival.
The assay was performed in two independent experiments and the tested concentrations were 0.9, 2.7, 8.2, 24.7 and 74.0 µg/mL.
No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.
The test item did not induce gene mutations at the HPRT locus in V79 cells and is considered non-mutagenic.
In conclusion, the test item does not induce gene mutations in bacteria and mammalian cells nor causes chromosome breaks and should therefore be considered non-mutagenic.
Justification for classification or non-classification
According to the criteria set in the Regulation EC No 1272/2008 in section 3.5 the test item is not to be considered as genotoxic/mutagenic.
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.
