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EC number: 448-300-4 | CAS number: 88642-03-9
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
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- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
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- Endpoint summary
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial reverse mutation assay in vitro (Ames, OECD 471): negative
Chromosome Aberration assay in vitro (OECD 473): negative
Gene Mutation Assay in Chinese Hamster V 79 Cells in vitro (OECD476): negative
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:
- 2005-10-02
- 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:
- 2000
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9 : Phenobarbital/ß-Naphthoflavone induced rat liver
- method of preparation of S9 mix: prepared from 8 - 12 weeks old male Wistar Hanlbm rats (approx. 220 - 320 g) induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D-22335 Hamburg) and ß-Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCI solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at -80° C. Small numbers of the ampoules can be kept at -20°C for up to one week. The protein concentration in the S9 preparation was 34.5 mg/mL (lot no. R 300404) in the pre-experiment and experiment I (strains TA 98 and TA 100), and 32.8 mg/mL (lot no. R 151004) in experiment I and II.
- concentration or volume of S9 mix and S9 in the final culture medium: Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCI
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al. - Test concentrations with justification for top dose:
- Pre-Experiment: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment I: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II, Ila: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Concentrations applied in the main experiment (I, II and IIa) werer based on the results of the Pre-Experiment. - Vehicle / solvent:
- - Vehicle used: ethanol
- 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, 2-AA
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation, experiment I) and preincubation (experiment II and IIa)
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 60 min
- Exposure duration/duration of treatment: 48 h
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
- Rationale for test conditions:
- according to the test guidelines
- Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) 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:
- A statistical analysis of the data is not required.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: The test item precipitated weakly at different concentrations in the overlay agar. The undissolved particles of the test item had no influence on the data recording.
Ames test:
- Signs of toxicity : cytotoxicity was observed
HISTORICAL CONTROL DATA
- Positive historical control data: Please refer to "Any other information on results"
- Negative (solvent/vehicle) historical control data: Please refer to "Any other information on results" - Conclusions:
- In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
- Executive summary:
This study was performed to investigate the potential of the test item to induce gene mutations 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 TA 102, according to OECD 471. The assay was performed in two independent experiments both with and without liver microsomal activation. An additional experiment was performed as pre-incubation with strain TA 98 without S9, only (reported as exp. IIa). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:
Pre-Experiment: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment 1: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II, Ila: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plateThe plates incubated with the test item showed reduced background background growth in almost all strains with and without S9 mix. Toxic effects, evident as a reduction in the number of revertants, occurred in strains TA 100 (with S9 mix) and TA 102 (with and without S9 mix) in experiment I and in strains TA 98 (IIa), 100, and TA 102 without S9 mix and in strains TA 1537 and TA 102 with S9 mix in experiment II. A seemingly dose dependent increase in revertant colony numbers was observed following treatment with the test item in strain TA 98 without metabolic activation in experiment II. The number of colonies reached or exceeded the threshold of twice the number of the corresponding solvent control at concentrations of 2500 µg/plate and above in the absence of metabolic activation. To verify the results an additional experiment was performed as pre-incubation test with TA 98 without S9 mix (exp. IIa). In this experiment an increase of the revertant colonies was not observed. However, reduced background growth was observed from 333 µg/plate up to 5000 µg/plate. Therefore, the observed large number of very small colonies in experiment II were judged to be based upon toxicity rather than indicating a possible mutagenic potential. A major reduction of the background growth results in less bacteria competing for the traces of histidine introduced by the top agar. The traces of histidine are sufficient to allow the surviving bacteria to grow into very small colonies until the histidine is depleted. All other strains did not show any mutagenic effect of the test item up to the maximal concentration applied. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2007-09-17
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 2000
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- lymphocytes: from healthy human donors
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: blood samples from human donors
- Suitability of cells: according to guideline
- Normal cell cycle time:
For lymphocytes:
- Sex, age and number of blood donors: blood samples were obtained from healthy donors not receiving medication:
Experiment I: two donors: 44-year-old male and 45 year-old female;
Experiment II 47-year old female)
- whole blood was used
- Whether blood from different donors were pooled or not: no
- mitogen: phytohaemagglutinin (PHA)
MEDIA USED
- Type and composition of media:
DMEM:F12 (Dulbecco's modified eagle medium/ Ham's F12 medium; mixture 1:1, containing 10% FCS (fetal calf serum), antibiotic solution contains 10,000 U/mL penicillin and 10,000 µg/mL streptomycin. The medium was supplemented with Phytohemagglutinin (PHA, final concentration 3 µg/mL, SEROMED), the anticoagulant heparin (25,000 U.S.P.-U/mL) and HEPES (final concentration 10 mM) - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9 : phenobarbital/betanaphthoflavone induced rat liver (8-12 weeks old male Wistar rats)
- method of preparation of S9 mix: The S9 fractions were produced by dilution of the liver homogenate with a KCI solution (1+3) followed by centrifugation at 9000 g
- concentration or volume of S9 mix and S9 in the final culture medium : final protein concentration of 0.75 g/mL in cultures - Test concentrations with justification for top dose:
- Please refer to table 1 bleow. Test concentrations in the main test were chosend based on the results of the cytotoxicity pre-test.
Using reduced mitotic indices as an indicator for cytotoxicity in Experiment I, clearly reduced mitotic indices were observed at 285.2 µg/mL after 4 hrs treatment in the absence of S9 mix. In the presence of S9 mix, no clear cytotoxicity could be observed up to the highest applied concentration. After continuous treatment in the absence of S9 mix, cytotoxicity was observed at 49 µg/mL and above. Therefore, 93.1 µg/mL were chosen as top treatment concentration in Experiment II, in the absence of S9 mix. In the presence of S9 mix, 2675.0 µg/mL were chosen as top treatment concentration with respect to the results obtained in Experiment I. - Vehicle / solvent:
- - Vehicle/solvent used: ethanol
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 3
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h, 22 h and 46 h (without S9
FOR CHROMOSOME ABERRATION:
- Spindle inhibitor: colcemid
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): fixation of cells with methanol glacial acetic acid; staining according to Flurescent and Giemsa technique
- Number of cells spread and analysed per concentration: at least two slides per experimental group were prepared
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment II, in the presence of metabolic activation, where only 50 metaphase plates were scored due to strong clastogenic effects. Only metaphases with 46 ± 1 centromer regions were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in 250 metaphase cells (% polyploid metaphases) was scored.
- Rationale for test conditions:
- according to the guidelines
- Evaluation criteria:
- A test item is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Statistical significance was confirmed by means of the Fisher's exact test (11) (p < 0.05). 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.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 0.8 % polyploid cells). - Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- Under the experimental conditions of this study, the test item did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro. Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test when tested up to cytotoxic and/or precipitating concentrations in the absence and presence of metabolic activation.
- Executive summary:
The test item, dissolved in ethanol, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments according to OECD 473 TG. The following study design was performed:
Without S9
With S9
Exp I
Exp II
Exp I
Exp II
Exposure period
4 hrs
22 hrs
46 hrs
4 hrs
4 hrs
Recovery
18 hrs
-
-
18 hrs
42 hrs
Preparation interval
22 hrs
22 hrs
46 hrs
22 hrs
46 hrs
In each experimental group two parallel cultures were analysed at three to four test concentrations. Per culture 100 metaphase plates were scored for structural chromosomal aberrations, except for the positive control in Experiment II, in the presence of metabolic activation, where only 50 metaphase plates were scored due to strong clastogenic effects. The highest applied concentration in Experiment I (2675.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiments was performed considering the toxicity data and the occurrence of test item precipitation in accordance to OECD Guideline 473. In Experiment I, in the absence of S9 mix, after pulse treatment, and in Experiment II, in the absence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment I, after continuous treatment in the absence of S9 mix, and in Experiment II, in the presence of S9 mix, clear cytotoxic effects could be observed at the highest evaluated concentration. In Experiment I, in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration being far in excess of test item precipitation. In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment II, in the presence of S9 mix, a single significant increase in the number of aberrant cells was observed. This value was in the range of the laboratory's historical control data and thus regarded as being without biological relevance. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro. Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test when tested up to cytotoxic and/or precipitating concentrations in the absence and presence of metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2019-01-31 to 2019-04-17
- 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 using the Hprt and xprt genes)
- Version / remarks:
- 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Version / remarks:
- 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Japanese Guidelines: “Kanpoan No. 287 – Environment Protection Agency“ “Eisei No. 127 – Ministry of Health & Welfare“ “Heisei 09/10/31 Kikyoku No. 2 – Ministry of International Trade & Industry“
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- hprt locus
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- For cell lines:
- Absence of Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 µg/mL), 10% FBS, and amphotericin B (1%). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 (98.5% air). - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9 : Phenobarbital/beta-naphthoflavone induced rat liver S9
- concentration or volume of S9 mix and S9 in the final culture medium: final protein concentration of 0.75 mg/mL in the cultures
- quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test - Test concentrations with justification for top dose:
- Relevant cytotoxic effects, indicated by a relative cloning efficiency of 50% or below were observed at 15.7 µg/mL, and above without metabolic activation and at 125.0 µg/mL and above with metabolic activation. To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.
Concentrations main test:
Exposure period 4 h, -S9
0.2; 0.5; 0.9; 1.9; 3.8; 7.5; 15.0; 30.0 µg/mL
Exposure period 4 h, +S9
1.9; 3.8; 7.5; 15.0; 30.0; 60.0; 120.0; 240.0 µg/mL - Vehicle / solvent:
- - Vehicle/solvent used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
- Justification for percentage of solvent in the final culture medium: final concentration of ethanol in the culture medium was 0.5% (v/v) as recommended by the guidelines - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : one
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: Approximately 0.7 to 1.2 x 10^7
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 24 h
- Exposure duration/duration of treatment: 4h
FOR GENE MUTATION:
- Expression time: 6 days
- Selection time: 8 -11 days
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency; relative survival (RS)
- Evaluation criteria:
- A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and therefore the test chemical response will be concluded to be equivocal. - Statistics:
- A linear regression analysis (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependency 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.
Linear regression:
Experimental group: -S9: p-value (calculated for the mean mutant frequencies of culture I and II): 0.083
Experimental group: +S9: p-value (calculated for the mean mutant frequencies of culture I and II): 0.127
A t-test was not performed since the 95% confidence interval was not exceeded at any experimental point.
However, both, biological and statistical significance was considered together. - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 7.5 µg/mL and above (-S9); at 60.0 µg/mL and above (+S9)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: solvent control pH 7.13; test item (1000 µg/mL) pH 7.11
- Data on osmolality: solvent control: 413 mOsm; test item (1000 µg/mL): 391 mOsm
- Possibility of evaporation from medium: no
- Precipitation and time of the determination: precipitation was observed in the main test at 15 (-S9) and 60 (+S9) µg/mL, respectively - Conclusions:
- The test item was found to be not mutagenic under the conditions of this study.
- Executive summary:
The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment (1000.0 µg/mL) was based on the solubility properties of the test item. The concentration range of the main experiment was limited by precipitation of the test item. No substantial and 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. In conclusion 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.
Referenceopen allclose all
Table 1 Summary of Results Pre-Experiment and Experiment I
Metabolic Activation |
Test Item |
Dose Level (µg/plate) |
Revertant Colony Counts (Mean ± SD) |
|
TA 98 |
TA 100 |
|||
Without Activation |
Ethanol |
|
36 ± 6 |
92 ± 14 |
Untreated |
|
26 ± 6 |
99 ± 6 |
|
Aurelione |
3 µg |
26 ± 3 |
91 ± 25 |
|
10 µg |
28 ± 4 |
83 ± 3 |
||
33 µg |
29 ± 7 |
74 ± 12 |
||
100 µg |
31 ± 7 |
86 ± 12 |
||
333 µg |
33 ± 6 |
70 ± 6 |
||
1000 µg |
33 ± 4 |
78 ± 11 |
||
2500 µg |
34 ± 7 |
79 ± 5 |
||
5000 µg |
31 ± 3 |
64 ± 7 |
||
4-NOPD |
10 µg |
376 ± 5 |
|
|
Sodium azide |
10 µg |
|
2211 ± 80 |
|
With Activation |
Ethanol |
|
49 ± 3 |
118 ± 10 |
Untreated |
|
37 ± 6 |
114 ± 5 |
|
Aurelione |
3 µg |
49 ± 6 |
93 ± 11 |
|
10 µg |
44 ± 6 |
104 ± 6 |
||
33 µg |
51 ± 5 |
112 ± 30 |
||
100 µg |
41 ± 4 |
124 ± 3 |
||
333 µg |
43 ± 9 |
96 ± 9 |
||
1000 µg |
37 ± 7 |
44 ± 2 |
||
2500 µg |
30 ± 5 |
45 ± 5 |
||
5000 µg |
32 ± 8 |
48 ± 3 |
||
2-AA |
2.5 µg |
2505 ± 191 |
2282 ± 179 |
4-NOPD 4-nitro-o-phenylene-diamine
2-AA 2-aminoanthracene
Table 2 Summary of Results Experiment I
Metabolic Activation |
Test Item |
Dose Level (µg/plate) |
Revertant Colony Counts (Mean ± SD) |
||
TA 1535 |
TA 1537 |
TA 102 |
|||
Without Activation |
Ethanol |
|
30±7 |
17±6 |
450±15 |
Untreated |
|
31±2 |
12±3 |
416±27 |
|
Aurelione |
33 µg |
33±8 |
20±7 |
439±24 |
|
|
100 µg |
31±9 |
15±6 |
453±28 |
|
|
333 µg |
39±6 |
8±3 |
365±62 |
|
|
1000 µg |
32±5 |
13±5 |
241±47 |
|
|
2500 µg |
30±15 |
10±2 |
167±25 |
|
|
5000 µg |
21±4 |
18±3 |
156±23 |
|
Sodium azide |
10 µg |
1228±152 |
|
|
|
4-NOPD |
50 µg |
|
94±12 |
|
|
MMS |
4.0 µL |
|
|
5696±196 |
|
With Activation |
Ethanol |
|
33±8 |
17±3 |
554±37 |
Untreated |
|
32±2 |
17±5 |
482±69 |
|
Aurelione |
33 µg |
35±8 |
18±1 |
520±76 |
|
|
100 µg |
32±4 |
18±3 |
529±69 |
|
|
333 µg |
32±2 |
9±4 |
498±10 |
|
|
1000 µg |
27±6 |
9±1 |
263±13 |
|
|
2500 µg |
30±12 |
14±3 |
189±13 |
|
|
5000 µg |
28±3 |
9±3 |
165±18 |
|
2-AA |
2.5 µg |
270±12 |
219±33 |
|
|
2-AA |
10.0 µg |
|
|
4004±238 |
4-NOPD 4-nitro-o-phenylene-diamine
MMS methyl methane sulfonate
2-AA 2-aminoanthracene
Table 3 Summary of Results Experiment II
Metabolic Activation |
Test Item |
Dose Level (µg/plate) |
Revertant Colony Counts (Mean ± SD) |
|||||
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
TA 102 |
||||
II |
IIa |
|||||||
Without Activation |
Ethanol |
|
24 ± 2 |
11 ± 1 |
34 ± 3 |
21 ± 2 |
132 ± 4 |
301 ± 3 |
Untreated |
|
22 ± 6 |
12 ± 3 |
35 ± 11 |
27 ± 6 |
144 ± 22 |
292 ± 14 |
|
Aurelione |
10 µg |
24 ± 8 |
9 ± 2 |
31 ± 4 |
26 ± 4 |
125 ± 14 |
325 ± 32 |
|
33 µg |
29 ± 6 |
11 ± 2 |
32 ± 12 |
24 ± 2 |
122 ± 7 |
327 ± 20 |
||
100 µg |
24 ± 7 |
8 ± 1 |
32 ± 6 |
21 ± 4 |
113 ± 13 |
305 ± 19 |
||
333 µg |
31 ± 2 |
11 ± 3 |
43 ± 5 |
7 ± 5 |
77 ± 5 |
243 ± 48 |
||
1000 µg |
23 ± 4 |
15 ± 8 |
52 ± 8 |
14 ± 5 |
72 ± 13 |
140 ± 3 |
||
2500 µg |
30 ± 6 |
9 ± 3 |
101 ± 13 |
n.a. |
63 ± 12 |
84 ± 9 |
||
5000 µg |
27 ± 9 |
12 ± 2 |
80 ± 4 |
n.a. |
56 ± 1 |
26 ± 5 |
||
Sodium azide |
10 µg |
1291 ± 46 |
|
|
|
1773 ± 102 |
|
|
4-NOPD |
10 µg |
|
|
458 ± 61 |
|
|
|
|
4-NOPD |
50 µg |
|
100 ± 7 |
|
354 ± 14 |
|
|
|
MMS |
4.0 µL |
|
|
|
|
|
1593 ± 130 |
|
With Activation |
Ethanol |
|
36 ± 7 |
31 ± 8 |
50 ± 1 |
|
151 ± 10 |
317 ± 16 |
Untreated |
|
35 ± 4 |
36 ± 5 |
60 ± 2 |
|
160 ± 17 |
402 ± 5 |
|
Aurelione |
10 µg |
37 ± 7 |
34 ± 3 |
53 ± 6 |
|
138 ± 3 |
360 ± 12 |
|
33 µg |
30 ± 3 |
31 ± 4 |
58 ± 10 |
|
142 ± 16 |
213 ± 32 |
||
100 µg |
31 ± 2 |
33 ± 3 |
49 ± 3 |
|
129 ± 5 |
141 ± 22 |
||
333 µg |
39 ± 3 |
41 ± 5 |
61 ± 2 |
|
114 ± 12 |
57 ± 13 |
||
1000 µg |
37 ± 8 |
31 ± 2 |
56 ± 6 |
|
85 ± 17 |
32 ± 13 |
||
2500 µg |
35 ± 5 |
4 ± 1 |
35 ± 5 |
|
98 ± 20 |
19 ± 7 |
||
5000 µg |
27 ± 4 |
6 ± 2 |
33 ± 3 |
|
77 ± 8 |
16 ± 2 |
||
2-AA |
2.5 µg |
376 ± 34 |
166 ± 19 |
1417 ± 81 |
|
1462 ± 127 |
|
|
2-AA |
10.0 µg |
|
|
|
|
|
1628 ± 128 |
4-NOPD 4-nitro-o-phenylene-diamine
MMS methyl methane sulfonate
2-AA 2-aminoanthracene
n.a. not analysable due to reduced background growth
Table 4 Historical Control Data
Strain |
|
Without S9 mix |
With S9 mix |
||||||
Mean |
SD |
Min |
Max |
Mean |
SD |
Min |
Max |
||
TA 1535 |
Solvent control Negative control Positive control |
20 18 3042 |
6 5 756 |
9 10 1003 |
30 29 3618 |
18 18 357 |
10 10 111 |
7 9 172 |
39 38 476 |
TA 1537 |
Solvent control Negative control Positive control |
11 12 97 |
7 6 21 |
4 5 52 |
29 29 191 |
18 18 141 |
9 6 47 |
6 8 94 |
31 29 380 |
TA 98 |
Solvent control Negative control Positive control |
24 26 379 |
9 10 98 |
14 15 137 |
58 52 976 |
37 43 1239 |
13 15 510 |
21 17 229 |
57 64 5466 |
TA 100 |
Solvent control Negative control Positive control |
121 141 2089 |
29 23 408 |
91 101 1262 |
198 189 2872 |
149 147 921 |
36 43 346 |
109 13 546 |
281 254 2589 |
TA 102 |
Solvent control Negative control Positive control |
338 326 2764 |
77 53 1479 |
242 242 1220 |
430 390 5593 |
426 450 2104 |
70 60 752 |
332 280 872 |
514 556 3052 |
Mean = mean value of revertants/plate
SD = standard deviation
Min = minimal value
Max = maximal value
Table 2 Summary of results of the chromosomal aberration study
experiment |
Preparation interval |
Test item concentration µg/mL |
Polyploid cells in % |
Mitotic indices in % of control |
Incl. gaps |
Aberrant cells in % excl. gaps |
With exchanges |
Exposure period 4 hrs without S9 |
|||||||
I |
22 hrs |
Solvent control |
0.0 |
100.0 |
1.0 |
1.0 |
1.0 |
|
|
Positive control |
0.0 |
85.5 |
20.5 |
18.0 |
5.5 |
|
|
53.2 |
0.2 |
113.9 |
2.0 |
1.5 |
0.0 |
|
|
93.1 |
0.0 |
108.0 |
1.5 |
1.5 |
0.0 |
|
|
163.0 |
0.4 |
97.6 |
0.5 |
0.5 |
0.0 |
Exposure period 22 hrs without S9 |
|||||||
I |
22 hrs |
Solvent control |
0.0 |
100.0 |
4.0 |
3.5 |
0.0 |
|
|
Positive control |
0.2 |
31.1 |
21.0 |
20.5 |
8.0 |
|
|
16.0 |
0.2 |
83.9 |
2.5 |
2.0 |
0.0 |
|
|
28.0 |
0.4 |
85.0 |
1.0 |
1.0 |
0.0 |
|
|
49.0 |
0.0 |
30.7 |
2.5 |
2.5 |
0.0 |
Exposure period 46 hrs without S9 |
|||||||
II |
46 hrs |
Solvent control |
0.2 |
100.0 |
0.5 |
0.5 |
0.0 |
|
|
Positive control |
0.0 |
108.0 |
9.5 |
9.0 |
1.0 |
|
|
9.9 |
0.2 |
118.2 |
1.5 |
1.5 |
0.0 |
|
|
17.4 |
0.2 |
93.0 |
0.5 |
0.5 |
0.0 |
|
|
30.4 |
0.2 |
79.3 |
2.0 |
2.0 |
0.0 |
Exposure period 4 hrs with S9 |
|||||||
I |
22 hrs |
Solvent control |
0.2 |
100.0 |
1.0 |
1.0 |
0.0 |
|
|
Positive control |
0.0 |
40.4 |
11.0 |
9.0 |
1.5 |
|
|
53.2 |
0.0 |
86.8 |
2.0 |
1.5 |
0.0 |
|
|
93.1 |
0.4 |
76.9 |
1.0 |
1.0 |
0.0 |
|
|
163.0 |
0.0 |
76.0 |
3.0 |
2.5 |
0.0 |
II |
46 hrs |
Solvent control |
0.0 |
100.0 |
0.5 |
0.5 |
0.0 |
|
|
Positive control |
0.0 |
26.2 |
37.0 |
37.0 |
10.0 |
|
|
93.1 |
0.0 |
89.5 |
1.0 |
0.5 |
0.0 |
|
|
163.0 |
0.0 |
86.9 |
2.0 |
1.5 |
0.0 |
|
|
1528.6 |
0.0 |
88.5 |
4.5 |
3.5 |
0.0 |
|
|
2675.0 |
0.2 |
34.0 |
2.5 |
1.5 |
0.0 |
Table 1 Summary of results
|
Concentration µg/mL |
precipitation |
S9 |
Relative cloning efficiency |
Relative cell density |
Rel. adjusted cloning efficiency |
Mutant colonies/10^6 cells |
95 % confidence interval |
Main Experiment 4 h treatment |
Mean values of culture I and II |
|||||||
Solvent control with ethanol |
|
|
- |
100.0 |
100.0 |
100.0 |
11.1 |
2.8 – 30.9 |
Positive control (EMS) |
300.0 |
|
- |
93.2 |
113.1 |
106.1 |
150.9 |
2.8 – 30.9 |
Test item |
0.2 |
|
- |
96.2 |
109.6 |
107.6 |
# |
|
0.5 |
|
- |
78.8 |
107.1 |
84.7 |
# |
||
0.9 |
|
- |
64.4 |
92.3 |
59.8 |
14.0 |
2.8 – 30.9 |
|
1.9 |
|
- |
63.2 |
110.0 |
72.3 |
10.2 |
2.8 – 30.9 |
|
3.8 |
|
- |
60.5 |
109.5 |
68.2 |
13.4 |
2.8 – 30.9 |
|
7.5 |
|
- |
24.8 |
104.3 |
24.4 |
9.6 |
2.8 – 30.9 |
|
15.0 |
P |
- |
4.4 |
29.3 |
1.2 |
22.7 |
2.8 – 30.9 |
|
30.0 |
P |
- |
Culture was discontinued due to exceedingly severe cytotoxicity |
|||||
Solvent control with ethanol |
|
|
+ |
100.0 |
100.0 |
100.0 |
13.0 |
3.1 – 30.7 |
Positive control (EMS) |
2.3 |
|
+ |
93.6 |
96.0 |
89.8 |
125.6 |
3.1 – 30.7 |
Test item |
1.9 |
|
+ |
99.3 |
107.1 |
106.0 |
# |
|
3.8 |
|
+ |
91.8 |
114.1 |
104.7 |
11.9 |
3.1 – 30.7 |
|
7.5 |
|
+ |
91.2 |
97.6 |
88.9 |
8.7 |
3.1 – 30.7 |
|
15.0 |
|
+ |
75.7 |
107.8 |
83.8 |
9.9 |
3.1 – 30.7 |
|
30.0 |
|
+ |
74.9 |
98.5 |
73.7 |
11.7 |
3.1 – 30.7 |
|
60.0 |
P |
+ |
44.1 |
13.1 |
5.8 |
16.5 |
3.1 – 30.7 |
|
120.0 |
P |
+ |
Culture was discontinued due to exceedingly severe cytotoxicity |
|||||
240.0 |
P |
+ |
Culture was discontinued due to exceedingly severe cytotoxicity |
# culture was not continued as a minimum of only four analyzable concentrations are required
Table 2 Historical control values
2014 - 2018 |
||
Number of mutant colonies per 106 cells |
||
without metabolic activation (4 hours treatment time) |
||
|
Positive control |
Solvent control |
Range: |
53.9 – 872.3 |
3.4 – 41.0 |
Mean value: |
223.3 |
16.9 |
Standard deviation: |
101.1 |
7.0 |
95% confidence interval |
-- |
2.8 – 30.9 |
Number of studies: |
199 |
199 |
with metabolic activation (4 hours treatment time) |
||
|
Positive control |
Solvent control |
Range: |
55.6 – 739.9 |
2.4 – 40.4 |
Mean value: |
188.0 |
16.9 |
Standard deviation: |
98.6 |
6.9 |
95% confidence interval |
-- |
3.1 – 30.7 |
Number of studies: |
193 |
193 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Bacterial reverse mutation assay (Ames), OECD 471
This study was performed to investigate the potential of the test item to induce gene mutations 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 TA 102, according to OECD 471. The assay was performed in two independent experiments both with and without liver microsomal activation. An additional experiment was performed as pre-incubation with strain TA 98 without S9, only (reported as exp. IIa). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:
Pre-Experiment: 3; 10; 33; 100; 333; 1000;
2500; and 5000 µg/plate
Experiment 1: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II, Ila: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
The plates incubated with the test item showed reduced background growth in almost all strains with and without S9 mix. Toxic effects, evident as a reduction in the number of revertants, occurred in strains TA 100 (with S9 mix) and TA 102 (with and without S9 mix) in experiment I and in strains TA 98 (IIa), 100, and TA 102 without S9 mix and in strains TA 1537 and TA 102 with S9 mix in experiment II. A seemingly dose dependent increase in revertant colony numbers was observed following treatment with the test item in strain TA 98 without metabolic activation in experiment II. The number of colonies reached or exceeded the threshold of twice the number of the corresponding solvent control at concentrations of 2500 µg/plate and above in the absence of metabolic activation. To verify the results an additional experiment was performed as pre-incubation test with TA 98 without S9 mix (exp. IIa). In this experiment an increase of the revertant colonies was not observed. However, reduced background growth was observed from 333 µg/plate up to 5000 µg/plate. Therefore, the observed large number of very small colonies in experiment II were judged to be based upon toxicity rather than indicating a possible mutagenic potential. A major reduction of the background growth results in less bacteria competing for the traces of histidine introduced by the top agar. The traces of histidine are sufficient to allow the surviving bacteria to grow into very small colonies until the histidine is depleted. All other strains did not show any mutagenic effect of the test item up to the maximal concentration applied. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
Chromosome Aberration test in vitro, OECD 473
The test item, dissolved in ethanol, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments according to OECD 473 TG. The following study design was performed:
|
Without S9 |
With S9 |
|||
Exp I |
Exp II |
Exp I |
Exp II |
||
Exposure period |
4 hrs |
22 hrs |
46 hrs |
4 hrs |
4 hrs |
Recovery |
18 hrs |
- |
- |
18 hrs |
42 hrs |
Preparation interval |
22 hrs |
22 hrs |
46 hrs |
22 hrs |
46 hrs |
In each experimental group two parallel cultures were analysed at three to four test concentrations. Per culture 100 metaphase plates were scored for structural chromosomal aberrations, except for the positive control in Experiment II, in the presence of metabolic activation, where only 50 metaphase plates were scored due to strong clastogenic effects. The highest applied concentration in Experiment I (2675.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiments was performed considering the toxicity data and the occurrence of test item precipitation in accordance to OECD Guideline 473. In Experiment I, in the absence of S9 mix, after pulse treatment, and in Experiment II, in the absence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment I, after continuous treatment in the absence of S9 mix, and in Experiment II, in the presence of S9 mix, clear cytotoxic effects could be observed at the highest evaluated concentration. In Experiment I, in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration being far in excess of test item precipitation. In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment II, in the presence of S9 mix, a single significant increase in the number of aberrant cells was observed. This value was in the range of the laboratory's historical control data and thus regarded as being without biological relevance. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro. Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test when tested up to cytotoxic and/or precipitating concentrations in the absence and presence of metabolic activation.
Gene Mutation Assay in Chinese Hamster V 79 Cells in vitro, OECD 476
The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.The treatment period was 4 hours with and without metabolic activation(S9).The maximum test item concentration of the pre-experiment (1000.0 µg/mL) was based onthe solubility properties of the test item. The concentration range of the main experiment was limited by precipitation of the test item. No substantial and dose dependent increase of the mutation frequency was observed in themain experiment.
Doses
applied in the gene mutation assay
(concentrations
given in bold letters were chosen for the mutation rate analysis)
exposure period |
S9 mix |
concentrations in µg/mL |
|||||||
|
|
experiment I |
|||||||
4 hours |
- |
0.2 |
0.5 |
0.9 |
1.9 |
3.8 |
7.5 |
15.0 P |
30.0 P |
4 hours |
+ |
1.9 |
3.8 |
7.5 |
15.0 |
30.0 |
60.0P |
120.0P |
240.0P |
P = precipitation visible at the end of treatment
Appropriate reference mutagens, used as positive controls (ethylmethane sulfonate (-S9), and 7,12 -dimethylbenz(a)anthracene (+S9)), induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.
In conclusion 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.
Justification for classification or non-classification
Classification,
Labelling, and Packaging Regulation (EC) No 1272/2008
The
available experimental test data are reliable and suitable for
classification purposes under Regulation (EC) No 1272/2008. Based on
available data on genotoxicity, the test item does not require
classifcation as mutagen according to Regulation (EC) No 1272/2008
(CLP), as amended for the tenth time in Regulation (EU) No 2017/776.
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