Registration Dossier
Registration Dossier
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EC number: 203-273-6 | CAS number: 105-13-5
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Data is from guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Principles of method if other than guideline:
- Pre-incubation method
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 100
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- S. typhimurium TA 1535
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- S. typhimurium TA 102
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- S. typhimurium TA 1537
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- S. typhimurium TA 98
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Sprague-Dawley rat liver S9 mix
- Test concentrations with justification for top dose:
- 100,333, 1000, 2500 and 5000µg/plate
- Vehicle / solvent:
- Dimethyl sulphoxide
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulphoxide
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- without S9 activation Migrated to IUCLID6: 3µg/plate for TA100 and 5µg/plate for TA1535
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulphoxide
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- without S9 activation Migrated to IUCLID6: 80µg/plate for TA1537
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulphoxide
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- without S9 activation Migrated to IUCLID6: 0.5µg/plate for TA102
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulphoxide
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- without S9 activation Migrated to IUCLID6: 0.2µg/plate for TA98
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulphoxide
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene (2AA): 1µg/plate for TA100; 2µg/plate for TA1535 and TA 1537
- Remarks:
- with S9 activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulphoxide
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- with S9 activation Migrated to IUCLID6: 5µg/plate for TA98
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulphoxide
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 1,8-Dihydroxyanthraquinone (DAN): 10µg/plate for TA102
- Remarks:
- with S9 activation
- Details on test system and experimental conditions:
- Pre- incubation method:
The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1 (100-5000µg/plate). The experimental method followed the pre – incubation modification where 0.1 ml of bacterial culture, 0.1 ml of vehicle or test material formulation and 0.5 ml of S9-mix or phosphate buffer were mixed for 20 min at 37˚C prior to the addition of 2ml of molten, trace histidine supplemented, top agar and plating onto the surface of Vogel- Bonner Minimal Agar plates. - Evaluation criteria:
- The test material may be considered positive in this test system if the following criteria are met :
The test material should have induced a reproducible, dose related and statistically(Dunnett’s method of linear regression) significant increase in the revertant count in at least one strain of bacteria. - Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not valid
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
Test substance was considered to be non mutagenic in the Pre-incubation assay- Executive summary:
Introduction:The method was designed to meet the requirements of the OECD Guidelines for testing of the Chemicals No. 471 “Bacterial Reverse Mutation Test”, Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Methods:Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment (plate incorporation) was determined in a preliminary toxicity assay and was 100 to 5000µg/plate. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. The pre-incubation modification was employed for the second experiment.
Results:The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.
Conclusion:The test material was considered to be non-mutagenic under the conditions of this test
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
- Justification for type of information:
- Data is from handbook or collection of data.
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- other: As given below
- Principles of method if other than guideline:
- Weight of evidence prepared from various publication discribed below
1,This in vitro assay was performed to assess the potential of test substance to induce structural / numerical chromosomal aberrations in one experiment (phase I). The induction of cytogenetic damage in human lymphocytes was assessed with and without metabolic activation. Due to the negative result in phase I, a second experiment (phase II) was performed.
2,Chromosomal aberration study was performed to determine the mutagenic nature of the test chemical - GLP compliance:
- not specified
- Type of assay:
- other: In vitro mammalian chromosome aberration assay
- Target gene:
- No data
- Species / strain / cell type:
- lymphocytes: human peripheral blood lymphocytes
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Human blood
- Suitability of cells: No data
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable:Age: 25-30 years age
- Whether whole blood or separated lymphocytes were used if applicable: Separated lymphocytes were used
- Number of passages if applicable: No data
- Methods for maintenance in cell culture if applicable: No data
- Modal number of chromosomes: No data
- Normal (negative control) cell cycle time: No data
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Blood cultures were set up in medium containing RPMI-1640, Fetal Bovine Serum, Phytohaemagglutinin, Heparin solution, Whole Blood and Antibiotic Solution
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically 'cleansed' against high spontaneous background: No data - Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- other: Chinese hamster fibroblast cell line CHL
- Additional strain / cell type characteristics:
- not specified
- Cytokinesis block (if used):
- No data
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 metabolic activation system
- Test concentrations with justification for top dose:
- 1,0.00 (NC), 0.00025 (T1), 0.0005 (T2) and 0.001 (T3) mg/mL
2,At three different doses with 0.5 mg/mL being the maximum dose concentration - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO - Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Untreated negative controls:
- yes
- Remarks:
- Untreated cells served as negative control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Medium
- True negative controls:
- not specified
- Positive controls:
- not specified
- Positive control substance:
- not specified
- Details on test system and experimental conditions:
- 1,METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks
DURATION
- Preincubation period: No data
- Exposure duration: Phase 1: 4 hrs (with and without metabolic activation system)
Phase 2: 4 hrs (with metabolic activation system) and 24 hrs (without metabolic activation system)
- Expression time (cells in growth medium): Phase 1: 20 hrs (with and without metabolic activation system)
Phase 2: 20 hrs (with metabolic activation system)
- Selection time (if incubation with a selection agent):No data
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs
SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa stain in phosphate buffer
NUMBER OF REPLICATIONS: No data
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cultures were incubated at 37 ± 2 °C for duration (exposure period) as mentioned. For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The labelled slides were dried over a slide warmer at 50°C and labelled. At least one slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant.
NUMBER OF CELLS EVALUATED: A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation.
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Mitotic index
- Any supplementary information relevant to cytotoxicity: Cytotoxicity was assessed at the concentrations of 0.00 (NC), 0.00025 (T7), 0.0005 (T8) and 0.001 (T9) mg/mL of culture media.
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data
- OTHER: No data
2,METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: No data
- Exposure duration: 24 and 48 hrs
- Expression time (cells in growth medium): 24 and 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data
SELECTION AGENT (mutation assays): Giemsa solution (1.5%, pH 6.8)
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): No data
NUMBER OF REPLICATIONS: No data
NUMBER OF CELLS EVALUATED: 100 well spread metaphases
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: No data
- Other: No data
OTHER: No data - Evaluation criteria:
- A test item can be classified as clastogenic if:
At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
If the increase is dose-related
Any of the results are outside the historical negative control range
A test item can be classified as non – clastogenic if:
None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
If there is no dose-related increase
All results are within the historical negative control range
Statistical significance was confirmed by means of the non-parametric Mann Whitney Test. However, both biological and statistical significance should be considered together.
If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed. - Statistics:
- Statistical significance at the p < 0.05 was evaluated by means of the non-parametric Mann-Whitney test
- Species / strain:
- lymphocytes: Human perpheral blood lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- In the cytotoxicity experiment III the highest test concentration 0.001 (T9) mg/ mL of culture media show 41.8 % reduction in absence of metabolic activation and 42.18% in the presence of metabolic activation indicates slight cytotoxicity of test item
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Species / strain:
- other: Chinese hamster fibroblast cell line CHL
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- not specified
- Additional information on results:
- 1,TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of test item in culture medium was assessed at 0 h and 4 h after incubation at 37 ± 2 °C. Significant change in pH was not observed at 0 h and 4 h when compared with negative controls.
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: There was no precipitation observed at 0.0625 mg/mL concentration
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data
RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item a cytotoxicity assay was performed both in the presence and absence of metabolic activation system. Three test concentrations (0.00025, 0.0005 and 0.001 mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with vehicle control. The procedure for conducting cytotoxicity was the same as main experiment phase I up to the scoring of the mitotic index, except slide coding.
Before conducting the chromosomal aberration study, Methyl-2-napthyl ether (CAS no. 93-04-9) was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.016 (T1), 0.0312 (T2) and 0.0625 (T3) mg/mL at initial cytotoxicity experiment (cytotxicity experiment I). All the tested concentrations at intial cytotoxicity experiment were cytotoxic. A second cytotoxicity experiment (cytotoxicity experiment II) was conducted with 0.002 (T4), 0.004 (T5) and 0.008 (T6) mg/mL of culture media. In second cytotoxicity experiment all tested concentrations were cytotoxic.
Hence one more cytotoxicity experiment (cytotoxic experiment III) was conducted with further lower concentrations of 0.00025 (T7), 0.0005 (T8) and 0.001 (T9) mg/mL of culture media. In the absence of S9 mix, the mean mitotic index observed was 10.03 (NC), 9.95 (VC), 8.69 (T7), 6.54 (T8), 5.79 (T9) and 8.54 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.05 (NC), 9.94 (VC), 8.84 (T7), 6.55 (T8), 5.74 (T9) and 8.55 (PC).
In the cytotoxicity experiment III the highest test concentration 0.001 (T9) mg/ mL of culture media show 41.8 % reduction in absence of metabolic activation and 42.18% in the presence of metabolic activation indicates slight cytotoxicity of test item. Hence 0.001 was selected as highest concentaration for main study considering the selection of test concentrations upto cytotoxicity. The mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation.
Hence the concentrations selected for the main study are 0.00025, 0.0005 and 0.001 mg/mL. The main study was performed in two independent phases;
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: Please refer table remarks section
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data - Remarks on result:
- other: No mutagenic potential
- Conclusions:
- Test substance is non-clastogenic at the highest tested concentration both in the presence and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
- Executive summary:
Data for the various test chemicals was reviewed to determine the mutagenic nature of test substance. The studies are as mentioned below:
This study was conducted to determine the chromosomal aberration induction potential of test substance in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29thJuly 2016 “ In Vitro Mammalian Chromosome Aberration Test. The experiment was conducted using human peripheral blood lymphocytes. Blood was drawn from a healthy volunteer, by venous puncture using heparinised syringe. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in DMSO and used at dose level of 0, 0.00025, 0.0005 and 0.001 mg/mL.in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system(1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations (0.5, 1 and 2mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with negative control. The medium of the proliferatingblood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 ± 2 °C for duration (exposure period). For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. At least two slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation.300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pluverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis. Methyl-2-napthyl ether (CAS no. 93-04-9) is non-clastogenic at the highest tested concentration of 0.001mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
Chromosomal aberration study was performed to determine the mutagenic nature of the test chemical. The cells were exposed to the test material at three different doses with 0.5 mg/mL being the maximum concentration for 48hr. Colcemid (final concn 0.2µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. The incidence of polyploid cells for 48hr after treatment was 0.0%. The incidence of cells with structural chromosomal aberrations at 0.4mg/ml for 48hr after treatment was 47.0%. Also positive at 0.3 mg/ml at 24hr (11.0 %) and at 48hr (30.0%). D20 was 0.25 mg/ml (the dose at which structural aberrations were detected in 20 % of theMetaphases observed). TR value (the frequency of cells with exchange-type aberrations per unit dose(mg/ml) ) was 0.35. The test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL in the absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant.
Referenceopen allclose all
Table 1 Spontaneous Mutation Rates (Concurrent Negative Control)
Number of Revertant (Mean number of colonies per plate) |
||||
Base pair substitution type |
Frameshift type |
|||
TA100 |
TA1535 |
TA102 |
TA98 |
TA1537 |
110 |
19 |
414 |
18 |
9 |
96 (105) |
23 (20) |
319 (357) |
17 (18) |
16 (14) |
110 |
18 |
337 |
20 |
18 |
Table 2 Test Result: Without Metabolic Activation Pre-incubation method
Test Period |
From: 07 October 2003 |
To: 10 October 2003 |
|||||||||
With or without S9-Mix |
Test substance concentration (µg/plate) |
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||||
TA100 |
TA1535 |
TA102 |
TA98 |
TA1537 |
|||||||
- |
0 |
112 105 106 |
(108) 3.8# |
29 34 30 |
(31) 2.6 |
267 253 260 |
(260) 7.0 |
18 12 18 |
(16) 3.5 |
9 6 25 |
(13) 10.2 |
- |
100
|
95 100 89 |
(95) 5.5 |
31 39 29 |
(33) 5.3 |
296 230 269 |
(265) 33.2 |
12 9 16 |
(12) 3.5 |
12 16 15 |
(14) 2.1 |
- |
333 |
100 95 96 |
(97) 2.6 |
29 26 33 |
(29) 3.5 |
242 259 266 |
(256) 12.3 |
18 18 20 |
(19) 1.2 |
8 3 11 |
(7) 4.0 |
- |
1000 |
96 115 92 |
(101) 12.3 |
31 39 35 |
(35) 4.0 |
269 277 287 |
(278) 9.0 |
13 13 9 |
(12) 2.3 |
9 10 7 |
(9) 1.5 |
- |
2500 |
108 97 117 |
(107) 10.0 |
38 33 46 |
(39) 6.6 |
264 303 260 |
(276) 23.8 |
12 17 8 |
(12) 4.5 |
7 9 17 |
(11) 5.3 |
- |
5000 |
112 79 105 |
(99) 17.4 |
18 45 26 |
(30) 13.9 |
222 263 235 |
(240) 21.0 |
15 9 12 |
(12) 3.0 |
8 5 10 |
(8) 2.5 |
Positive control |
Name Concentration (µg/plate) |
ENNG |
ENNG |
MMC |
4NQO |
9AA |
|||||
3 |
5 |
0.5 |
0.2 |
80 |
|||||||
S9-Mix - |
No. colonies per plate |
440 589 394 |
(474) 101.9 |
189 223 203 |
(205) 17.1 |
1854 1920 1884 |
(1886) 33.0 |
137 123 140 |
(133) 9.1 |
1645 1414 999 |
(1353) 327.3 |
EENG N-ethyl-N’-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
MMC Mitomycin C
C Contaminated
# Standard deviation
Table 3 Test Result: With Metabolic Activation Pre-incubation method
Test Period |
From: 07 October 2003 |
To: 10 October 2003 |
|||||||||
With or without S9-Mix |
Test substance concentration (µg/plate) |
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||||
TA100 |
TA1535 |
TA102 |
TA98 |
TA1537 |
|||||||
+ |
0 |
104 76 89 |
(90) 14.0# |
19 17 18 |
(18) 1.0 |
298 300 295 |
(298) 2.5 |
31 34 35 |
(33) 2.1 |
12 16 8 |
(12) 4.0 |
+ |
100
|
96 109 109 |
(105) 7.5 |
17 8 9 |
(11) 4.9 |
262 260 279 |
(267) 10.4 |
29 30 28 |
(29) 1.0 |
15 20 15 |
(17) 2.9 |
+ |
333 |
95 113 105 |
(104) 9.0 |
17 8 9 |
(20) 4.2 |
301 275 256 |
(277) 22.6 |
34 26 39 |
(33) 6.6 |
16 22 12 |
(17) 5.0 |
+ |
1000 |
105 121 93 |
(106) 14.0 |
21 10 17 |
(16) 5.6 |
265 260 318 |
(297) 19.4 |
29 40 35 |
(35) 5.5 |
16 12 18 |
(15) 3.1 |
+ |
2500 |
108 87 93 |
(96) 10.8 |
11 9 12 |
(11) 1.5 |
280 292 318 |
(297) 19.4 |
25 28 33 |
(29) 4.0 |
12 15 16 |
(14) 2.1 |
+ |
5000 |
104 117 98 |
(106) 9.7 |
8 11 19 |
(13) 5.7 |
300 296 290 |
(295) 5.0 |
34 33 21 |
(29) 7.2 |
20 17 12 |
(16) 4.0 |
Positive control |
Name Concentration (µg/plate) |
2AA |
2AA |
DAN |
BP |
2AA |
|||||
1 |
2 |
10 |
5 |
2 |
|||||||
S9-Mix + |
No. colonies per plate |
1305 1579 1506 |
(1463) 141.9 |
257 262 283 |
(267) 13.8 |
888 1091 788 |
(922) 154.4 |
306 226 231 |
(254) 44.8 |
427 567 455 |
(483) 74.1 |
2AA 2-Aminoanthracene
BP Benzo(a)pyrene
DAN 1,8-Dihydroxyanthraquinone
# Standard deviation
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Additional information from genetic toxicity in vitro:
AMES test
Introduction:The method was designed to meet the requirements of the OECD Guidelines for testing of the Chemicals No. 471 “Bacterial Reverse Mutation Test”, Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Methods:Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment (plate incorporation) was determined in a preliminary toxicity assay and was 100 to 5000µg/plate. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. The pre-incubation modification was employed for the second experiment.
Results:The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.
Conclusion:The test material was considered to be non-mutagenic under the conditions of this test
Test substance gives negative results for gene mutation in Ames test conducted on S. typhimurium TA 100 without metabolic activation.
Chromosomal aberration study
Chromosomal aberration study was performed to determine the mutagenic nature of 70 -80% structurally and functionally similar test chemical. The cells were exposed to the test material at three different doses with 0.5 mg/mL being the maximum concentration for48hr. Colcemid (final concn 0.2µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. The incidence of polyploid cells for 48hr after treatment was 0.0%. The incidence of cells with structural chromosomal aberrations at 0.4mg/ml for 48hr after treatment was 47.0%. Also positive at 0.3 mg/ml at 24hr (11.0 %) and at 48hr (30.0%). D20 was 0.25 mg/ml (the dose at which structural aberrations were detected in 20 % of theMetaphases observed). TR value (the frequency of cells with exchange-type aberrations per unit dose(mg/ml) ) was 0.35. The test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL in the absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant.
Sister chromatid exchanges test was performed to determine the mutagenic nature of 70 -80% structurally and functionally similar test compound. The test chemical was studied at a dose level of 160-1600µg/mL using Chinese hamster ovary cells (CHO-W-B1) both in the presence and absence of S9 metabolic activation system. 5-Bromodeoxyuridine (BrdUrd; 10 pM) was added 2 hr after addition of the test chemical (without S9) or immediately after the S9 mix plus chemical had been removed. The chemical treatment periods were appoximately 25 hr without S9 and 2 hr with S9. The total incubation time with BrdUrd was 25-26 hr, with colcemid (0.1µg/ml) present during the final 2-3 hr. Immediately before the cells were harvested, the cell monolayers were examined, and the degree of confluence and availability of mitotic cells were noted. Cells were collected by mitotic shake-off at doses up to the maximum considered likely to yield sufficient metaphase cells for analysis; supernatant medium was returned to appropriate flasks so that subsequent harvests could bemade from the same cultures if necessary. Because all mitotic cells were removed in the initial harvest, cells collected during subsequent harvests had come into mitosis during the period between harvests and thus had been exposed to colcemid for an average of 4 hr. After 1-3 min treatment with hypotonic solution (75 mM KCl), cells were fixed in 3: 1 methano1: glacial acetic acid (V/V). For a preliminary assessment of cell cycle delay, test slides were prepared from cells treated at the highest dose levels to see if later harvests were necessary. These test slides were stained with “dilute” Hoeschst 33258 (0.5µg/ml in Sorensen’s buffer, pH 6.8) and examined by fluorescence microscopy to assess cell cycle kinetics. In control cultures, almost all cells completed two cycles in BrdUrd (M2 cells) in 25-26 hr, whereas, in treated cultures, cell cycle delay was common. In cases of severe delay, additional harvests were made from the same cultures at a later time to obtain sufficient second metaphase (M2) cells for SCE analysis. After staining for 10 min in “concentrated” Hoechst 33258 (5µg/ml in pH 6.8 buffer) and exposure to “black light” at 55 to 60°C for about 5 min, slides were stained in Giemsa. All slides were coded, and 50 cells per dose were scored from the three highest doses at which sufficient M2 cells were available. When cell cycle delay was noted, cell kinetics were recorded by classifying each of 100 metaphases as M1, M1+, or M2, i.e., having completed one (M1), two (M2), or between one and two (M1 +) cell cycles in BrdUrd. Delay was noted at the top dose with S9 (5 mg/ml), and the culture was harvested 2 hr later than the controls. There were increases in aberrations, but these were statistically significant only without S9. The test compound did not induce an increase in the number of Sister chromatid exchanges in the Chinese hamster ovary cells (CHO-W-B1) in the presence of S9 metabolic activation system but it induced sister chromatid exchanges in the absence of S9 metabolic activation system.
Justification for classification or non-classification
Substance will not be classified in Genetic toxicity category as per the C & L criteria set by EU.
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