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EC number: 203-187-9 | CAS number: 104-23-4
- 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
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- 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
Ames test:
The test chemical did not iinduce gene mutation in the Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
In vitro chromosome aberration assay:
The test chemical did not induce chromosomal aberrations in the mammalian cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
In vitro mammalian chromosome aberration assay:
The test chemical did not induce mammalian cell gene mutation assay both in the presence and absence of S9 activation system in the mammalian cell line used and hence is not likely to be gene mutant in vitro.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- 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
- Remarks:
- Experimental data from various test chemicals
- Justification for type of information:
- WoE derived based on the experimental data from various test chemicals
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Principles of method if other than guideline:
- The test chemical was studied for its ability to induce mutations in strains of Salmonella typhimurium.
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine
- Species / strain / cell type:
- S. typhimurium, other: TA100, TA1535, TA97 and TA98
- Remarks:
- 1
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not specified
- Cytokinesis block (if used):
- No data
- Metabolic activation:
- with and without
- Metabolic activation system:
- 10% and 30% HLI and RLI S-9 (9,000 g supernatant) fractions were prepared from Aroclor 1254-induced, male Sprague- Dawley rat and male Syrian hamster livers
- Test concentrations with justification for top dose:
- 1. 0, 33, 100, 333, 1000 or 3333 µg/plate
2. 10-250 mg - Vehicle / solvent:
- 1./2.
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical is 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:
- 9-aminoacridine
- sodium azide
- other: 4-nitro-o-phenylenediamine (TA98; -S9) and 2-aminoanthracene (all strains; +S9)
- Remarks:
- 1
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- not specified
- Positive control substance:
- not specified
- Remarks:
- 2
- Details on test system and experimental conditions:
- 1. METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available
SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available
NUMBER OF REPLICATIONS: Triplicate
NUMBER OF CELLS EVALUATED: No data available
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available
OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available
OTHER: No data available
2. METHOD OF APPLICATION: Plate incorporation assay
DURATION
- Preincubation period: No data available
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available
SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available
NUMBER OF REPLICATIONS: No data available
NUMBER OF CELLS EVALUATED: No data available
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available
OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available
OTHER: No data available - Rationale for test conditions:
- No data
- Evaluation criteria:
- 1. Evaluations were made at both the individual trial and overall chemical levels.
Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or nonmutagenic (-), depending on the magnitude of the increase of his+ revertants, and the shape of the dose-response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “ +W,” if only a single dose was elevated over the control, or if the increase seen was not dose related. The distinctions between a questionable mutagenic response and a nonmutagenic or weak mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach two fold over background for a chemical to be judged mutagenic.
A chemical was judged mutagenic (+) or weakly mutagenic (+ W) if it produced a reproducible dose-related reponse over the solvent control in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials.
Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response. The chemicals were decoded by the chemical repository only after a determination had been made regarding their mutagenicity or nonmutagenicity.
2. Material which caused a 2-fold increase of revertants, as compared to the number of spontaneous revertants on the control plates, was denoted as mutagens. Those which reduced the number of revertants were considered inhibitory. - Statistics:
- 1. Mean ± SD
2. No data - Species / strain:
- S. typhimurium, other: TA100, TA1535, TA97 and TA98
- Remarks:
- 1
- 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, other: TA98, TA1537, TA100, TA1535
- Remarks:
- 2
- 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:
- not specified
- Additional information on results:
- 1. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available
RANGE-FINDING/SCREENING STUDIES: The test chemical was tested initially in a toxicity assay to determine the appropriate dose range for the mutagenicity assay. The toxicity assay was performed using TA100. Toxic concentrations were those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.
COMPARISON WITH HISTORICAL CONTROL DATA: Yes
ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available
2. No data available - Remarks on result:
- other: No mutagenic potential
- Conclusions:
- The test chemical did not iinduce gene mutation in the Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
- Executive summary:
Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:
The test chemical was studied for its ability to induce mutations in strains of Salmonella typhimurium. The test compound was dissolved in DMSO and was tested at concentration of 0, 33, 100, 333, 1000 or 3333 µg/plate using Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of 10 % and 30 % rat and hamster liver S9 metabolic activation system. Preincubation assay was performed with a preicubation for 20 mins. The plates were observed for histidine independence after 2 days incubation period. Concurrent solvent and positive controls were included in the study. The test chemical is not mutagenic to the Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
Salmonella/ mammalian-microsome test was performed to evaluate the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98, TA1537, TA100, TA1535 with and without S9 metabolic activation system. The test material was dissolved in DMSO and used at dose levels from 10-250 mg. DMSO was used as the solvent control. The 2 ml of liquid top agar was cooled to 45°C and 0.1 ml of a broth culture of microorganism and test substance in volumes of ≤ 0.4 ml of DMSO was added prior to placing on minimal agar plates. After 48 h incubation at 37°C, the colonies which reverted to the prototroph were counted and compared to counts on the control plate (containing no test substance) to demonstrate mutagenicity or toxicity. Materials which caused a 2-fold increase of revertants, as compared to the number of spontaneous revertants on the control plates, were denoted as mutagens. Those which reduced the number of revertants were considered inhibitory. The test chemical did not induce gene mutation in Salmonella typhimurium TA98, TA1537, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence it is negative for gene mutation in vitro.
Based on the observations made, the test chemical did not induce gene mutation in the Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- 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
- Remarks:
- Experimental data from various test chemicals
- Justification for type of information:
- Data for the target chemical is summarized based on the various test chemicals
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- other: Refer below principle
- Principles of method if other than guideline:
- WoE derived based on the experimental data from various test chemicals
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- No data
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Remarks:
- CHO-WBL / 1
- Details on mammalian cell type (if applicable):
- No data
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- mammalian cell line, other: Chinese hamster V79 cells
- Remarks:
- 2
- Details on mammalian cell type (if applicable):
- No data
- Additional strain / cell type characteristics:
- not specified
- Cytokinesis block (if used):
- No data
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fractions (livers of Aroclor 1254-treated male Sprague-Dawley rats.)
- Test concentrations with justification for top dose:
- 1. -S9 (Harvest time: 10 hrs): 0, 2500, 3850, 5000 µg/mL
+S9 (Harvest time: 12 hrs): 0, 2500, 3850, 5000 µg/mL
2. Experiment IA: 128.1, 256.3, 2050.0 and 4100.0 μg/ml without S9-mix
Experiment IA: 128.1, 256.3, 1025.0 and 2050.0 μg/ml with S9-mix
Experiment IB: 31.3, 62.5, and 125.0 without S9-mix
Experiment IIA: 128.1, 256.3 and 512.5 μg/ml without S9-mix
Experiment IIA: 128.1, 256.3, 512.5 and 1025.0 μg/ml with S9-mix
Experiment IIB: 100.0, 150.0, 200.0, 250.0, 300.0 and 350.0 μg/ml without S9-mix
Experiment IIB: 100.0, 200.0, 400.0, 600.0 and 800.0 μg/ml with S9-mix - Vehicle / solvent:
- 1. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical is soluble in DMSO
2. - Vehicle(s)/solvent(s) used: Deionized water
- Justification for choice of solvent/vehicle: The test chemical is soluble in Deionized water - Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Remarks:
- 1
- Untreated negative controls:
- yes
- Remarks:
- as per OECD
- Negative solvent / vehicle controls:
- not specified
- True negative controls:
- not specified
- Positive controls:
- yes
- Remarks:
- as per OECD
- Positive control substance:
- not specified
- Remarks:
- 2
- Details on test system and experimental conditions:
- 1. METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: No data
- Exposure duration:
- S9: 8 hrs
+ S9: 2 hrs
- Expression time (cells in growth medium): 8 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): -S9: 10 hrs, +S9: 12 hrs
SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: No data
NUMBER OF CELLS EVALUATED: One hundred to 200 cells from each of the three highest scorable doses were analyzed
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available
OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available
OTHER: No data available
2. METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: No data available
- Exposure duration:
Experiment I (without & with S9-mix): 4 hr
Experiment II (with S9-mix): 4 hr
Experiment II (without S9-mix): 20 hrs
- Expression time (cells in growth medium): No data available
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available
SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available
NUMBER OF REPLICATIONS: No data available
NUMBER OF CELLS EVALUATED: No data available
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: For assessment of cytotoxicity a XTT test was performed
OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available
OTHER: On the basis of pre-test (range finding study) and the occurrence of precipitation of test chemical, 4100 μg/ml (≈ 10 mM the prescribed maximum concentration) was chosen as top concentration in experiment IA. To corroborate the data of this experiment in the absence of S9-mix, a confirmatory experiment (experiment IB) was performed with a top dose of 500 μg/ml. Dose selection in experiment IIA was influenced by toxicity and precipitation observed in experiment I. Due to the steep dose toxicity curve, a repeat experiment (experiment IIB) was performed with narrower dilution steps to proof if genotoxicity observed at highly toxic concentrations far below the 40% of control level was an artificial finding.
Harvest time was 24 h or 48 h (experiment II with S9-mix only) after the beginning of culture.
For assessment of cytotoxicity a XTT test was additionally carried out in parallel to the main micronucleus test. - Rationale for test conditions:
- No data
- Evaluation criteria:
- 1. All aberrations were individually classified (e.g., chromatid breaks, chromosome breaks, triradials, etc.). These data were combined as the percent of cells with simple (deletions), complex (exchanges), and total (simple, complex and other) aberrations. Only the total percent cells with aberrations was considered in the statistical evaluation. Gaps and endoreduplications were recorded but were not included in the statistical analyses.
2. The cell line was observed for micronucleated cells - Statistics:
- 1. Trend test.
2. No data - Species / strain:
- Chinese hamster Ovary (CHO)
- Remarks:
- CHO-WBL / 1
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- mammalian cell line, other: Chinese hamster V79 cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- 1. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 7.0 – 7.5
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data
RANGE-FINDING/SCREENING STUDIES: Chemicals were tested up to 5 mg/ml or as limited by solubility and/or toxicity. Solubility tests were conducted to determine dose range and choice of solvent (water, dimethyl sulfoxide, acetone, or ethanol, in that order of preference). In the assays for chromosomal aberrations, the top dose (TD) was based on toxicity, solubility, or the upper testing limit (5 mg/ml). The doses used were generally the TD, 0.75 TD, 0.50 TD, 0.25 TD, 0.1 TD, 0.075 TD, 0.05 TD, and 0.025 TD. The highest three doses with a sufficient number of cells were analyzed for chromosomal aberrations
COMPARISON WITH HISTORICAL CONTROL DATA: No data
ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
2. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available
RANGE-FINDING/SCREENING STUDIES: A pretest on cell growth inhibition (XTT assay) with 4 h treatment was performed in order to determine the toxicity of test substance, the solubility during exposure and thus the test concentrations for the main micronucleus test.
COMPARISON WITH HISTORICAL CONTROL DATA: Yes, In experiment IIA, in the absence and the presence of S9-mix, a statistically significant increase in the number of micronucleated cells exceeding the range of the historical control data was observed at the highest doses (512.5 and 1025 μg/ml, respectively). These concentrations were strongly cytotoxic indicated as by cell numbers of 7.9% and 12.9% of control, respectively.
Due to the steep dose-toxicity curve a repeat experiment, designated experiment IIB, was performed with narrower dilution steps to prove if the genotoxicity observed could have been an artefact induced by general test item toxicity. In the absence of S9-mix, at a cytotoxic level of about 40% of control the number of micronucleated cells (2.05% and 2.00%) slightly exceeded the historical control data range (0.0 – 1.8% micronucleated cells). Therefore, the test item was regarded as non-genotoxic in the absence of metabolic activation.
In the presence of S9-mix, at cytotoxic test item levels and associated with precipitation from doses equal or exceeding 200 μg/ml, the number of micronucleated cells (2.68% and 2.33%) slightly exceeded the range of the historical control data (0.0 – 1.8% micronucleated cells). Due to the high value of the respective solvent control (1.80% micronucleated cells), these two slight increases have to be regarded as biologically irrelevant.
The observations of experiment IIA in the absence and the presence of metabolic activation were not confirmed in the repeat experiment IIB with narrower dilution steps. Therefore, it has to be considered that the findings in both parts of experiment IIA were artefacts induced by general test item toxicity.
Other: In all experiments clear toxic effects indicated by reduced cell numbers below 40% of control were observed at least at the highest concentrations scored after treatment with test chemical except in experiment IB in the absence of S9-mix. In experiment IA, in the absence of S9-mix, a statistically significant but non-dose-related increase in the rate of micronucleated cells was observed at the lowest and highest dose. The values of highest dose were at the laboratory’s control data range (0.0 – 1.8% micronucleated cells). Concerning the lowest dose, in the confirmatory experiment IB this finding was not confirmed. Consequently, the positive finding was considered not biologically relevant. In experiment IA, in the presence of S9-mix no biologically relevant increase in the percentage of micronucleated cells was observed after treatment with the test item.
ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available - Remarks on result:
- other: No mutagenic potential
- Conclusions:
- The test chemical did not induce chromosomal aberrations in the mammalian cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
- Executive summary:
Data from various test chemicals was reviewed to determine the mutagenic nature of the test chemicals. The studies are as mentioned below:
In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using CHO-WBL cells in the presence and absence of exogeneous metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels upto 5 mg/mL. In the chromosome aberration assay without activation, cells were exposed to the test chemical for 8 hr. The test chemical was washed off, and the cells were treated with 0.1µg/ml Colcemid for 2-2.5 hr. With metabolic activation, the cells were exposed to the test chemical plus the metabolic activation mixture for 2 hr, washed, incubated for 8 hr, and then treated with Colcemid for 2-2.5 hr. A delayed harvest was used in the aberration assay in most instances when cell cycle delay was observed in the SCE assay. In these tests the cell growth period was extended to about 20 hr. Cells were harvested. Air-dried slides were coded and stained with Giemsa. One hundred to 200 cells from each of the three highest scorable doses were analyzed and the chromosomal aberrations were scored. The test chemical did not induce chromosomal aberrations when tested to toxicity. Precipitate was evident at doses of 250µg/ml and above. Based on the observations made, the test chemical did not induce chromosome aberrations in the CHO-WBL cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
In vitro mammalian chromosome aberration test was carried out using Chinese hamster V79 cells to determine the mutagenic nature of the test chemical.
Different conc. of test chemical was used in the various experiment. They are-
Experiment IA:128.1, 256.3, 2050.0 and 4100.0 μg/ml without S9-mix
Experiment IA:128.1, 256.3, 1025.0 and 2050.0 μg/ml with S9-mix
Experiment IB:31.3, 62.5, and 125.0 without S9-mix
Experiment IIA:128.1, 256.3 and 512.5 μg/ml without S9-mix
Experiment IIA:128.1, 256.3, 512.5 and 1025.0 μg/ml with S9-mix
Experiment IIB:100.0, 150.0, 200.0, 250.0, 300.0 and 350.0 μg/ml without S9-mix
Experiment IIB:100.0, 200.0, 400.0, 600.0 and 800.0 μg/ml with S9-mix
Deionised water was used as a vehicle. Liver S9 fraction from phenobarbital/β-naphthoflavone-induced rats was used as exogenous metabolic activation system. A pretest on cell growth inhibition (XTT assay) with 4 h treatment was performed in order to determine the toxicity of test substance, the solubility during exposure and thus the test concentrations for the main micronucleus test. On the basis of pre-test (range finding study) and the occurrence of precipitation of test chemical, 4100 μg/ml (≈ 10 mM the prescribed maximum concentration) was chosen as top concentration in experiment IA. To corroborate the data of this experiment in the absence of S9-mix, a confirmatory experiment (experiment IB) was performed with a top dose of 500 μg/ml. Dose selection in experiment IIA was influenced by Basic Brown 17 toxicity and precipitation observed in experiment I. Due to the steep dose toxicity curve, a repeat experiment (experiment IIB) was performed with narrower dilution steps to proof if genotoxicity observed at highly toxic concentrations far below the 40% of control level was an artificial finding. The treatment period in the main test was 4 h in experiment I (without and with S9-mix) and in experiment II (with S9-mix) or 20 h in experiment II (without S9-mix). Harvest time was 24 h or 48 h (experiment II with S9-mix only) after the beginning of culture. For assessment of cytotoxicity a XTT test was additionally carried out in parallel to the main micronucleus test. Under the experimental conditions, the test chemical did not induce an increase in micronucleated cells in the V79 cell line in the presence and absence of S9 metabolic activation system and thus, the test was considered to be negative.
Based on the data available for the various test chemicals, the test chemical did not induce chromosomal aberrations in the mammalian cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
- Endpoint:
- in vitro gene mutation 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
- Remarks:
- Experimental data from various test chemicals
- Justification for type of information:
- Data for the target chemical is summarized based on various test chemicals
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- other: Refer below principle
- Principles of method if other than guideline:
- WoE derived based on the experimental data from various test chemicals
- GLP compliance:
- not specified
- Type of assay:
- other: In vitro mammalian cell gene mutation assay
- Target gene:
- 1. Thymidine kinase
2. hprt - Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Remarks:
- L5178Y TK+/- 3.7.C / 1
- Details on mammalian cell type (if applicable):
- - Type and identity of media:
The cells were grown in Fischer’s medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68.
- Properly maintained: No data available
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available - Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Remarks:
- 2
- Details on mammalian cell type (if applicable):
- No data
- Additional strain / cell type characteristics:
- not specified
- Cytokinesis block (if used):
- No data
- Metabolic activation:
- with and without
- Metabolic activation system:
- Liver S9 prepared from Aroclor 1254-induced male Sprague- Dawley rats.
- Test concentrations with justification for top dose:
- 1. 1642-3680 µg/mL
2. 0, 5, 19, 20, 50 and 100 μg/ml without S9-mix.
0, 100, 300, 1000, 3000 and 6000 μg/plate with S9-mix - Vehicle / solvent:
- 1. No data available
2. - Vehicle(s)/solvent(s) used: Phosphate buffered saline
- Justification for choice of solvent/vehicle: The test chemical was soluble in Phosphate buffered saline - Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- not specified
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- methylmethanesulfonate
- other: 3-methylcholanthrene at 1.86 × 10-5 M (or dimethylbenz[a]- anthracene at 0.5-4 µg/mL) for the test with metabolic activation.
- Remarks:
- 1
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- not specified
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- not specified
- Remarks:
- 2
- Details on test system and experimental conditions:
- 1. METHOD OF APPLICATION: in medium
Cells at start: 6000000 cells
DURATION
- Preincubation period: No data available
- Exposure duration: 4 h
- Expression time (cells in growth medium):48 h
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available
SELECTION AGENT (mutation assays): 1×106 cells/plate for mutant selection
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available
NUMBER OF REPLICATIONS: Duplicate
NUMBER OF CELLS EVALUATED: 1 X 106 cells/plate for mutant selection and 200
cells/plate for viable count determinations
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: The rate of cell growth was determined for each of the treated cultures
OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available
OTHER: No data available
2. METHOD OF APPLICATION: In medium
DURATION
- Preincubation period: No data available
- Exposure duration: 2 h treatment with S9-mix or 20 h without S9-mix
- Expression time (cells in growth medium): 5 days
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): No data available
SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available
NUMBER OF REPLICATIONS: Triplicate
NUMBER OF CELLS EVALUATED: No data
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available
OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available
OTHER: Cells were treated for 2h with S9-mix or for 20h without S9-mix followed by an expression period of 5 days to fix the DNA damage into stable hprt mutations. - Rationale for test conditions:
- No data
- Evaluation criteria:
- 1. Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency was previously reported as representing a positive effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.
2. The cell line was observed for gene mutation at the hprt locus - Statistics:
- No data
- Species / strain:
- mouse lymphoma L5178Y cells
- Remarks:
- TK+/- 3.7.C / 1
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Remarks:
- 2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- valid
- Positive controls validity:
- not specified
- Additional information on results:
- 1. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available
RANGE-FINDING/SCREENING STUDIES: No data available
COMPARISON WITH HISTORICAL CONTROL DATA: No data available
ADDITIONAL INFORMATION ON CYTOTOXICITY: The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity.
2. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data
RANGE-FINDING/SCREENING STUDIES: Test concentrations were based on the results of an initial toxicity test measuring cell survival 5 days after treatment.
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: No data
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data - Remarks on result:
- other: No mutagenic potential
- Conclusions:
- The test chemical did not induce mammalian cell gene mutation assay both in the presence and absence of S9 activation system in the mammalian cell line used and hence is not likely to be gene mutant in vitro.
- Executive summary:
Data available for the various test chemicals were reviewed to determine the mutagenic nature. The studies are as mentioned below:
The gene mutation study was conducted according toL5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature ofthe test chemical. The cells at a concentration of 6 X 105/mL (6 X106cells total) were exposed for 4 h to a range of concentrations from 1642-3680 µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. The test chemical did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.
In an in vitro mammalian cell gene mutation assay, the test chemical was exposed to the V79 cells in the concentration of 0, 5, 19, 20, 50 and 100 μg/ml without S9-mix and 0, 100, 300, 1000, 3000 and 6000 μg/plate with S9-mix in phosphate buffered saline without and with S9-mix. Test concentrations were based on the results of an initial toxicity test measuring cell survival 5 days after treatment. In the main tests, cells were treated for 2h with S9-mix or for 20h without S9-mix followed by an expression period of 5 days to fix the DNA damage into stable hprt mutations. Data on toxicity were restricted to those on cloning efficiency after the selection period. Negative and positive controls were included. The data on clonings efficiency after the selection period did not indicate strong cytotoxicity; the required 10-20% survival after the highest dose was not reached at any concentration. In both experiments a biologically relevant and dose dependent increase in the mutant frequency was not observed, neither in the presence nor in the absence of metabolic activation. Occasionally, an increase in mutant frequency was found; these were not reproducible and considered not biologically relevant. Since, biologically relevant and concentration dependent increase in the mutant frequency was not observed, neither in the presence nor in the absence of metabolic activation, hence, the test chemical was considered to be negative for mammalian cell gene mutation assay in vitro (with and without) when tested on V79 cells.
Based on the observations made, the test chemical did not induce mammalian cell gene mutation assay both in the presence and absence of S9 activation system in the mammalian cell line used and hence is not likely to be gene mutant in vitro.
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Data available for the various test chemicals was reviewed to determine the mutagenic nature. The studies are as mentioned below:
Ames assay:
The test chemical was studied for its ability to induce mutations in strains of Salmonella typhimurium. The test compound was dissolved in DMSO and was tested at concentration of 0, 33, 100, 333, 1000 or 3333 µg/plate using Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of 10 % and 30 % rat and hamster liver S9 metabolic activation system. Preincubation assay was performed with a preicubation for 20 mins. The plates were observed for histidine independence after 2 days incubation period. Concurrent solvent and positive controls were included in the study. The test chemical is not mutagenic to the Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
In another study Salmonella/ mammalian-microsome test was performed to evaluate the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98, TA1537, TA100, TA1535 with and without S9 metabolic activation system. The test material was dissolved in DMSO and used at dose levels from 10-250 mg. DMSO was used as the solvent control. The 2 ml of liquid top agar was cooled to 45°C and 0.1 ml of a broth culture of microorganism and test substance in volumes of ≤ 0.4 ml of DMSO was added prior to placing on minimal agar plates. After 48 h incubation at 37°C, the colonies which reverted to the prototroph were counted and compared to counts on the control plate (containing no test substance) to demonstrate mutagenicity or toxicity. Materials which caused a 2-fold increase of revertants, as compared to the number of spontaneous revertants on the control plates, were denoted as mutagens. Those which reduced the number of revertants were considered inhibitory. The test chemical did not induce gene mutation in Salmonella typhimurium TA98, TA1537, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence it is negative for gene mutation in vitro.
In vitro chromosome aberration assay:
In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using CHO-WBL cells in the presence and absence of exogeneous metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels upto 5 mg/mL. In the chromosome aberration assay without activation, cells were exposed to the test chemical for 8 hr. The test chemical was washed off, and the cells were treated with 0.1µg/ml Colcemid for 2-2.5 hr. With metabolic activation, the cells were exposed to the test chemical plus the metabolic activation mixture for 2 hr, washed, incubated for 8 hr, and then treated with Colcemid for 2-2.5 hr. A delayed harvest was used in the aberration assay in most instances when cell cycle delay was observed in the SCE assay. In these tests the cell growth period was extended to about 20 hr. Cells were harvested. Air-dried slides were coded and stained with Giemsa. One hundred to 200 cells from each of the three highest scorable doses were analyzed and the chromosomal aberrations were scored. The test chemical did not induce chromosomal aberrations when tested to toxicity. Precipitate was evident at doses of 250µg/ml and above. Based on the observations made, the test chemical did not induce chromosome aberrations in the CHO-WBL cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
In another vitro mammalian chromosome aberration test was carried out using Chinese hamster V79 cells to determine the mutagenic nature of the test chemical.
Different conc. of test chemical was used in the various experiment. They are-
Experiment IA:128.1, 256.3, 2050.0 and 4100.0 μg/ml without S9-mix
Experiment IA:128.1, 256.3, 1025.0 and 2050.0 μg/ml with S9-mix
Experiment IB:31.3, 62.5, and 125.0 without S9-mix
Experiment IIA:128.1, 256.3 and 512.5 μg/ml without S9-mix
Experiment IIA:128.1, 256.3, 512.5 and 1025.0 μg/ml with S9-mix
Experiment IIB:100.0, 150.0, 200.0, 250.0, 300.0 and 350.0 μg/ml without S9-mix
Experiment IIB:100.0, 200.0, 400.0, 600.0 and 800.0 μg/ml with S9-mix
Deionised water was used as a vehicle. Liver S9 fraction from phenobarbital/β-naphthoflavone-induced rats was used as exogenous metabolic activation system. A pretest on cell growth inhibition (XTT assay) with 4 h treatment was performed in order to determine the toxicity of test substance, the solubility during exposure and thus the test concentrations for the main micronucleus test. On the basis of pre-test (range finding study) and the occurrence of precipitation of test chemical, 4100 μg/ml (≈ 10 mM the prescribed maximum concentration) was chosen as top concentration in experiment IA. To corroborate the data of this experiment in the absence of S9-mix, a confirmatory experiment (experiment IB) was performed with a top dose of 500 μg/ml. Dose selection in experiment IIA was influenced by Basic Brown 17 toxicity and precipitation observed in experiment I. Due to the steep dose toxicity curve, a repeat experiment (experiment IIB) was performed with narrower dilution steps to proof if genotoxicity observed at highly toxic concentrations far below the 40% of control level was an artificial finding. The treatment period in the main test was 4 h in experiment I (without and with S9-mix) and in experiment II (with S9-mix) or 20 h in experiment II (without S9-mix). Harvest time was 24 h or 48 h (experiment II with S9-mix only) after the beginning of culture. For assessment of cytotoxicity a XTT test was additionally carried out in parallel to the main micronucleus test. Under the experimental conditions, the test chemical did not induce an increase in micronucleated cells in the V79 cell line in the presence and absence of S9 metabolic activation system and thus, the test was considered to be negative.
In vitro mammalian chromosome aberration assay:
The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of the test chemical. The cells at a concentration of 6 X 105/mL (6 X106cells total) were exposed for 4 h to a range of concentrations from 1642-3680 µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. The test chemical did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.
In yet another in vitro mammalian cell gene mutation assay, the test chemical was exposed to the V79 cells in the concentration of 0, 5, 19, 20, 50 and 100 μg/ml without S9-mix and 0, 100, 300, 1000, 3000 and 6000 μg/plate with S9-mix in phosphate buffered saline without and with S9-mix. Test concentrations were based on the results of an initial toxicity test measuring cell survival 5 days after treatment. In the main tests, cells were treated for 2h with S9-mix or for 20h without S9-mix followed by an expression period of 5 days to fix the DNA damage into stable hprt mutations. Data on toxicity were restricted to those on cloning efficiency after the selection period. Negative and positive controls were included. The data on clonings efficiency after the selection period did not indicate strong cytotoxicity; the required 10-20% survival after the highest dose was not reached at any concentration. In both experiments a biologically relevant and dose dependent increase in the mutant frequency was not observed, neither in the presence nor in the absence of metabolic activation. Occasionally, an increase in mutant frequency was found; these were not reproducible and considered not biologically relevant. Since, biologically relevant and concentration dependent increase in the mutant frequency was not observed, neither in the presence nor in the absence of metabolic activation, hence, the test chemical was considered to be negative for mammalian cell gene mutation assay in vitro (with and without) when tested on V79 cells.
In vitro mammalian cell transformation assay:
In vitro transformation study in mammalian cells was performed to evaluate the mutagenic nature of the test chemical. The study was performed using BHK21/C13 in the presence of aroclor 1245 rat liver induced S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 0.025, 0.25, 2.5, 25 or 250µg/mL. The methods employed when testing a compound for potential carcinogenicity using growth of mammalian cells in semi-solid agar. The experiments were conducted in small groups using duplicate plates at each dose level, and on each occasion 4-dimethylaminoazobenzene and its 3-methyl analogue were used as the appropriate chemical-class-positive and -negative controls. A positive result is recorded when the transformation frequency per 106survivors at the LD50 exceeds 5X the control frequency. Based on the observations made, thetest chemical does not exhibit in vitro mammalian cell transformation frequency in the presence of S9 metabolic activation system and thus proves to be non-mutagenic and non-carcinogenic. There are at least 2 possible explanations for the non-carcinogenicity of the test chemical.First, it could be suggested that the addition of a sulphonic-acid group to the test chemical will reduce its lipid solubility and thereby prevent, or inhibit, its transport in vivo to critical intracellular sites. Second, it is possible that the marked negative inductive effect exerted by the sulphonic-acid group might critically affect both the electronic resonance of the NMe2 group with the aromatic system and the metabolism of the molecule, which would lead to the test chemical being inactive both in vivo or in vitro.
Based on the observations made and applying the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. This is further supported by the negatiive in vitro cell tranformation ability of the test chemical. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
Justification for classification or non-classification
Based on the observations made and applying the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. This is further supported by the negatiive in vitro cell tranformation ability of the test chemical. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
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