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EC number: 239-816-9 | CAS number: 15721-78-5
- 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 assay:
The test chemical did not induce gene mutation in Salmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA 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 mammalian chromosome aberration study:
The test chemical did not induce chromosome aberration in mammalian cell line 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 mammalian cell gene mutation assay:
With S9 metabolic activation system:
In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.025, 0.05, 0.1 or 0.5 mM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. The results showed an indication of gene toxicity when treated with 0.5 mM when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentration of 0, 0.025, 0.05 or 0.1 mM does not cause genetic mutation(s) in the presence of metabolic activation, whereas treatment with 0.5 mM may, when CHO cells are exposed to the test chemical in the presence of metabolic activation.
Without S9 metabolic activation system:
In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.025, 0.05, 0.01 or 0.5 mM and without S9-induced metabolic activation. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. Independently of treatment concentration, the results showed no evidence of gene toxicity when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentration of 0, 0.025, 0.05, 0.01 or 0.5 mM does not cause genetic mutation(s) in the absence of metabolic activation.
Overall conclusion
At time of exposure, the test chemical was added in the absence or presence of S9 liver microsomal fraction. The test chemical was added to each applicable well to give a final concentration of 0, 0.025, 0.05, 0.1 or 0.5 mM. Negative controls, solvent/vehicle controls and positive control substance(s) were also included in each experiment. pH and osmolality was not determined in the gene mutation test. The positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity except when treated with 0.5 mM in the presence of 4% S9 liver microsomal fraction. Thus, the results show evidence of genotoxicity when CHO cells are exposed to the test chemical in the highest tested concentrations, i.e. at 0.5 mM. It was concluded that the test chemical does not give rise to gene mutations when used at concentrations of ≤ 0.1 mM in organisms with a fully functioning metabolic activation, whereas concentrations > 0.1 mM may induce gene mutations. However, when treated with test chemical in the absence of S9 liver microsomal fraction, no evidence of occurring gene mutations were detected. Hence, test chemical does not give rise to gene mutations in organisms who has no or a non-functional metabolic activation at the above mentioned concentrations. When the mutation frequency was determined, a frequency of 3.67 x 10-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction, and a frequency of -1.92 x 10-4was observed for 0.5 mM in the presence of S9 liver microsomal fraction. Since no other tested concentration of the test chemical and in the absence or presence of S9 liver microsomal fraction resulted in colonies, it was concluded that the test chemical does not give rise to gene mutations when CHO cells are exposedin vitroto the test chemical at ≤ 2.5 mM for 3 hrs.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- 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 from various test chemicals
- Justification for type of information:
- Data for the target chemical is summarized based on the data from various test chemicals
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- 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:
- bacterial reverse mutation assay
- Target gene:
- Histidine
- 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
- Species / strain / cell type:
- E. coli WP2 uvr A
- 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:
- S9 prepared by enzyme induction of male Sprague-Dawley male rats of 7 weeks of age by combination administration of phenobarbital (PB) and 5, 6-benzoflavone (BF)
- Test concentrations with justification for top dose:
- 1. Trial 1.1:
Without S9: 0, 1.56, 3.13, 6.25, 12.5, 25 or 50 µg/plate
With S9: 0, 6.25, 12.5, 25, 50, 100 or 200 µg/plate
Trial 1.2:
With and without S9: 0, 125, 250, 500, 1000 or 2000 µg/plate
Trial 2.1:
Without S9: 0, 1.56, 3.13, 6.25, 12.5, 25 or 50 µg/plate
With S9: 0, 6.25, 12.5, 25, 50, 100 or 200 µg/plate
Trial 2.2:
With and without S9: 0, 125, 250, 500, 1000 or 2000 µg/plate
2. 0, 15.6, 31.3, 62.5, 125, 250, 500 or 1000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO - Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- other: AF-2 (2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide) and 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: No data
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data
SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data
NUMBER OF REPLICATIONS: No data
NUMBER OF CELLS EVALUATED: No data
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data
OTHER: No data - Rationale for test conditions:
- No data
- Evaluation criteria:
- 1. When the number of revertive mutant colonies on the flat plate containing the test substance increases more than twice as compared with that of the negative control and the reproducibility or dose dependency is recognized in the increase, the test substance It was decided to have mutagenicity (positive) in the system.
2. When the increase increased more than 2 times and the increase was found to be reproducible or dose dependent, it was decided that the test substance has mutagenicity (positive) in this test system. - Statistics:
- No data
- Species / strain:
- S. typhimurium, other: TA98, TA1535, TA1537, TA100
- 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:
- E. coli WP2 uvr A
- 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
- Additional information on results:
- 1. 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
- Other confounding effects: No data
RANGE-FINDING/SCREENING STUDIES: When the test was carried out in the range of 50 to 5000 μg / plate, antibacterial activity was strongly observed in the direct test and metabolic activation test of all test bacteria. Antimicrobial activity was found more strongly than WP 2 in S. typhimurium test organisms, and was also remarkable in the direct test. For this reason,
additional tests were conducted on 4 tested bacteria of S. typhimurium at a dose of 20 to 200 μg / plate for TA 100 and TA 98, at a dose of 10 to 100 μg / plate for TA 1535 and TA 1537 only for direct test. Antimicrobial activity was observed in 2 to 3 dose groups of high dose in any of the test organisms.
From the above results, the maximum dose in this study was 50 μg / plate in the direct test of S. typhimurium 4 test bacteria, 200 μg / plate in the metabolic activation test, both direct test and metabolic activation test for WP 2 2000 μg / plate, 4 doses of S. typhimurium, 6 doses and 5 doses of WP 2 were established at a common ratio of 2.
COMPARISON WITH HISTORICAL CONTROL DATA: No data
ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
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
- Other confounding effects: No data
RANGE-FINDING/SCREENING STUDIES: When the test was carried out with the common ratio in the range of 50.0 to 5000 μg / plate being about 3, it was found that WP 2 uvrA was 1500 μg / plate or more in the S9 mix-free test and the antibacterial activity was observed in other cases at 500 μg /plate or more It was done. In the S9 mix addition test, antibacterial activity was confirmed at TA 98 and WP 2 uvr A above 1500 μg / plate, and others at doses above 500 μg / plate.
COMPARISON WITH HISTORICAL CONTROL DATA: No data
ADDITIONAL INFORMATION ON CYTOTOXICITY: No data - Remarks on result:
- other: No muatgenic potential
- Conclusions:
- The test chemical did not induce gene mutation in Salmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA 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:
Bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA in the presence and absence of S9 metabolic cativation system. The test chemical was dissolved in DMSO and used at dose level of 0, 1.56, 3.13, 6.25, 12.5, 25 or 50µg/plate without S9 and 0, 6.25, 12.5, 25, 50, 100 or 200µg/plate with S9 in trial 1.1 And 2.1 and at dose level of 0, 125, 250, 500, 1000 or 2000µg/plate in trial 1.2 and 2.2 with and without S9. Concurrent solvent and positive controls were also included in the study.The test chemical did not induce gene mutation inSalmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA 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 another study, bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA in the presence and absence of S9 metabolic cativation system. The test chemical was dissolved in DMSO and used at dose level of 0, 15.6, 31.3, 62.5, 125, 250, 500 or 1000µg/plates with and without S9. Concurrent solvent and positive controls were also included in the study.The test chemical did not induce gene mutation inSalmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
Based on the data available, the test chemical did not induce gene mutation in Salmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA 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
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
- Remarks:
- Experimenal data from various test chemicals
- Justification for type of information:
- Data for the target chemical is summarized based on the data from various test chemicals
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- 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 chromosome aberration study
- Target gene:
- No data
- Species / strain / cell type:
- mammalian cell line, other: Chinese hamster cultured cells (CHL)
- Remarks:
- 1
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Eagle MEM culture medium supplemented with 10% fetal bovine serum
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data - Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- lymphocytes: Rat
- 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 mix
- Test concentrations with justification for top dose:
- 1. 0, 4, 8 or 16 µg/mL
2. -S9 (20-hour cultures): 1.0 – 125 µg/mL
+S9 (20-hour cultures): 1.88 –120 µg/mL
+S9 (30-hour cultures): 1.88 – 60 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO - Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Remarks:
- 1
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Remarks:
- 2
- Details on test system and experimental conditions:
- 1. METHOD OF APPLICATION: in medium
Cells at the start of the experiment: 20000 cells
DURATION
- Preincubation period: No data
- Exposure duration: Direct method: 24 and 48 hrs
Short term treatment method with S9: 6 hrs
- Expression time (cells in growth medium):
Direct method: 24 and 48 hrs
Short term treatment method with S9: 18 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data
SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: No data
NUMBER OF CELLS EVALUATED: For structural abnormalities, 200 metaphase cells per group and 800 division metastatic cells for multiplicative cells were analyzed.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data
OTHER: No data
2. METHOD OF APPLICATION: in medium - Rationale for test conditions:
- No data
- Evaluation criteria:
- 1. The presence or absence of structural abnormality such as chromosome type or chromosome type gap, The presence or absence of cells (polyploid) was also observed.
2. The cell line was observed for chromosome aberration - Statistics:
- 1. With respect to the frequency of occurrence of chromosomal abnormal cells, a significant difference test between the solvent control group and the test substance treated group and between the solvent control group and the positive control group was carried out by Fisher's "Exact probability test" method. According to the criteria of Ishikan et al, the frequency of chromosomal abnormalities is negative, less than 5% negative, less than 10% false positive, less than 10% Was regarded as positive.
2. No data - Species / strain:
- mammalian cell line, other: Chinese hamster cultured cells (CHL)
- 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:
- lymphocytes: Rat
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- With metabolic activation: 60.0 µg/ml and more dose (20- and 30-hour cultures) Without metabolic activation: 30.0 µg/ml and more dose
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- 1. 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
- Other confounding effects: No data
RANGE-FINDING/SCREENING STUDIES: To determine the treatment concentration of the test substance used for the chromosomal aberration test, the influence of the test substance on cell proliferation was investigated.
The proliferation inhibitory effect of the test substance on CHL cells was evaluated by measuring the proliferation level of each group using a monolayer cultured cell densitometer and using the ratio of cell proliferation to the solvent control group of the test substance treated group as an indicator. As a result, the concentration showing growth inhibition of about 50% in the direct method calculated from the regression line equation was 16 μg / ml, and in the metabolic activation method it was 40 μg / ml
COMPARISON WITH HISTORICAL CONTROL DATA: No data
ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
2. No data - Remarks on result:
- other: No mutagenic potential
- Conclusions:
- The test chemical did not induce chromosome aberration in mammalian cell line 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 test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:
In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster cultured cells (CHL) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 4, 8 or 16µg/mL. The doses for the main study were based on data from preliminary dose range finding study. Concurrent solvent and positive control plates were also included in the study. The cells were exposed to the test chemical for 24 or 48 hrs in the direct method and for 6 hrs in the short term treatment method. Two hours before the end of the culture, colcemid was added to the culture solution to a final concentration of about 0.1 μg / ml. Chromosome specimens were prepared according to a conventional method. Giemsa stained six slide specimens were prepared for each petri dish. The presence or absence of structural abnormality such as chromosome type or chromosome type gap, the presence or absence of cells (polyploid) was also observed.The test chemical did not induce chromosome aberration inChinese hamster cultured cells (CHL) 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 another vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using rat lymphocytes (CHL) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 1.0 – 125 µg/mL -S9 (20-hour cultures), 1.88 –120 µg/mL +S9 (20-hour cultures) and 1.88 – 60 µg/mL +S9 (30-hour cultures). Concurrent solvent and positive control plates were also included in the study. Based on the observations made,the test chemical did not induce chromosome aberration inrat lymphocytes in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
Based on the data available, the test chemical did not induce chromosome aberration in mammalian cell line 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 gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- The study was conducted in accordance with the Good Laboratory Practice Principles as Published by OECD in 1998, No 1 ENV/MC/CHEM(98)17
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Principles of method if other than guideline:
- In vitro mammalian cell gene mutation assa ywas performed to determine the mutagenic nature of the test chemical
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.
This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.
Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days. - Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable - Additional strain / cell type characteristics:
- other: Hypodiploid, modal No. 20
- Cytokinesis block (if used):
- No data
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats
- Test concentrations with justification for top dose:
- 0, 0.025, 0.05, 0.1 or 0.5 mM
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The test chemical was dissolved in DMSO - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Remarks:
- N-ethyl-N-nitrosourea (ENU) was the positive control substance in the tests done without S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: In medium with pre-incubation
DURATION
-Pre-incubation: One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.
- Exposure duration: 3 hours
-Expression time: 7 days
- Selection time: 14 days
- Fixation time: 7 days (harvest of cells)
SELECTION AGENT (mutation assays): 6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Crystal violet
NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.
NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated
DETERMINATION OF CYTOTOXICITY
- Cytotoxicity test: After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.
OTHER EXAMINATIONS: Not applicable
- Determination of polyploidy:
- Determination of endoreplication:
- Other: - Rationale for test conditions:
- No data
- Evaluation criteria:
- The cell line was observed for gene mutation at the HGPRT locus
- Statistics:
- No data
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- 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: Completed without S9 metabolic activation. A range of test concentrations (0, 0.01, 0,05, 0.1, 0.5, 1, 5, 10 or 50mM) was applied 24 hours after seeding to single cultures in fresh medium in 96-well plates. The cell population (control and treated cells) were assessed 24 and 48 hours after treatment using the colorimetric assayMTTand the BCA assay to assess cell viability and total protein concentration, respectively. From the basis of these results, the test concentrations of the chemical was chosen to be included in the gene toxicity test. Since no cytotoxicity was evident at the tested concentrations in this preliminary dose-finding test further testing concentrations were adapted to have a maximum test concentration of 0.5 mM. Since the test chemical was dissolved in DMSO, higher concentrations of the test chemical than the concentration mentioned above would result in a toxic effect of DMSO. The test chemical could only be dissolved in 100% DMSO.
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:
- 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:
- When CHO cells were exposed to the test chemical in the concentration of 0, 0.025, 0.05, 0.01 or 0.5 mM the results did not show any evidence of gene toxicity without metabolic activation. CHO cells when exposed to the test chemical in the presence of metabolic activation at the concentration of 0, 0.025, 0.05 or 0.1 mM did not show any evidence of gene toxicity. However, treatment with 0.5 mM showed an indication of genetic toxicity.
- Executive summary:
With S9 metabolic activation system:
In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.025, 0.05, 0.1 or 0.5 mM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. The results showed an indication of gene toxicity when treated with 0.5 mM when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentration of 0, 0.025, 0.05 or 0.1 mM does not cause genetic mutation(s) in the presence of metabolic activation, whereas treatment with 0.5 mM may, when CHO cells are exposed to the test chemical in the presence of metabolic activation.
Without S9 metabolic activation system:
In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.025, 0.05, 0.01 or 0.5 mM and without S9-induced metabolic activation. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. Independently of treatment concentration, the results showed no evidence of gene toxicity when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentration of 0, 0.025, 0.05, 0.01 or 0.5 mM does not cause genetic mutation(s) in the absence of metabolic activation.
Overall conclusion
At time of exposure, the test chemical was added in the absence or presence of S9 liver microsomal fraction. The test chemical was added to each applicable well to give a final concentration of 0, 0.025, 0.05, 0.1 or 0.5 mM. Negative controls, solvent/vehicle controls and positive control substance(s) were also included in each experiment. pH and osmolality was not determined in the gene mutation test. The positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity except when treated with 0.5 mM in the presence of 4% S9 liver microsomal fraction. Thus, the results show evidence of genotoxicity when CHO cells are exposed to the test chemical in the highest tested concentrations, i.e. at 0.5 mM. It was concluded that the test chemical does not give rise to gene mutations when used at concentrations of ≤ 0.1 mM in organisms with a fully functioning metabolic activation, whereas concentrations > 0.1 mM may induce gene mutations. However, when treated with test chemical in the absence of S9 liver microsomal fraction, no evidence of occurring gene mutations were detected. Hence, test chemical does not give rise to gene mutations in organisms who has no or a non-functional metabolic activation at the above mentioned concentrations. When the mutation frequency was determined, a frequency of 3.67 x 10-4 was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction, and a frequency of -1.92 x 10-4 was observed for 0.5 mM in the presence of S9 liver microsomal fraction. Since no other tested concentration of the test chemical and in the absence or presence of S9 liver microsomal fraction resulted in colonies, it was concluded that the test chemical does not give rise to gene mutations when CHO cells are exposed in vitro to the test chemical at ≤ 2.5 mM for 3 hrs.
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
Gene mutation in vitro:
Data available for the test chemical was reviewed to determine the mutagenic nature of the test chemical. The studies are mentioned below:
Ames test:
Bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA in the presence and absence of S9 metabolic cativation system. The test chemical was dissolved in DMSO and used at dose level of 0, 1.56, 3.13, 6.25, 12.5, 25 or 50µg/plate without S9 and 0, 6.25, 12.5, 25, 50, 100 or 200µg/plate with S9 in trial 1.1 And 2.1 and at dose level of 0, 125, 250, 500, 1000 or 2000µg/plate in trial 1.2 and 2.2 with and without S9. Concurrent solvent and positive controls were also included in the study.The test chemical did not induce gene mutation inSalmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA 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 another study, bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA in the presence and absence of S9 metabolic cativation system. The test chemical was dissolved in DMSO and used at dose level of 0, 15.6, 31.3, 62.5, 125, 250, 500 or 1000µg/plates with and without S9. Concurrent solvent and positive controls were also included in the study.The test chemical did not induce gene mutation inSalmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA 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 mammalian chromosome aberration study:
In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster cultured cells (CHL) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 4, 8 or 16µg/mL. The doses for the main study were based on data from preliminary dose range finding study. Concurrent solvent and positive control plates were also included in the study. The cells were exposed to the test chemical for 24 or 48 hrs in the direct method and for 6 hrs in the short term treatment method. Two hours before the end of the culture, colcemid was added to the culture solution to a final concentration of about 0.1 μg / ml. Chromosome specimens were prepared according to a conventional method. Giemsa stained six slide specimens were prepared for each petri dish. The presence or absence of structural abnormality such as chromosome type or chromosome type gap, the presence or absence of cells (polyploid) was also observed.The test chemical did not induce chromosome aberration inChinese hamster cultured cells (CHL) 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 another vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using rat lymphocytes (CHL) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 1.0 – 125 µg/mL -S9 (20-hour cultures), 1.88 –120 µg/mL +S9 (20-hour cultures) and 1.88 – 60 µg/mL +S9 (30-hour cultures). Concurrent solvent and positive control plates were also included in the study. Based on the observations made,the test chemical did not induce chromosome aberration inrat lymphocytes 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 mammalian cell gene mutation assay:
With S9 metabolic activation system:
In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.025, 0.05, 0.1 or 0.5 mM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. The results showed an indication of gene toxicity when treated with 0.5 mM when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentration of 0, 0.025, 0.05 or 0.1 mM does not cause genetic mutation(s) in the presence of metabolic activation, whereas treatment with 0.5 mM may, when CHO cells are exposed to the test chemical in the presence of metabolic activation.
Without S9 metabolic activation system:
In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.025, 0.05, 0.01 or 0.5 mM and without S9-induced metabolic activation. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. Independently of treatment concentration, the results showed no evidence of gene toxicity when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentration of 0, 0.025, 0.05, 0.01 or 0.5 mM does not cause genetic mutation(s) in the absence of metabolic activation.
Overall conclusion
At time of exposure, the test chemical was added in the absence or presence of S9 liver microsomal fraction. The test chemical was added to each applicable well to give a final concentration of 0, 0.025, 0.05, 0.1 or 0.5 mM. Negative controls, solvent/vehicle controls and positive control substance(s) were also included in each experiment. pH and osmolality was not determined in the gene mutation test. The positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity except when treated with 0.5 mM in the presence of 4% S9 liver microsomal fraction. Thus, the results show evidence of genotoxicity when CHO cells are exposed to the test chemical in the highest tested concentrations, i.e. at 0.5 mM. It was concluded that the test chemical does not give rise to gene mutations when used at concentrations of ≤ 0.1 mM in organisms with a fully functioning metabolic activation, whereas concentrations > 0.1 mM may induce gene mutations. However, when treated with test chemical in the absence of S9 liver microsomal fraction, no evidence of occurring gene mutations were detected. Hence, test chemical does not give rise to gene mutations in organisms who has no or a non-functional metabolic activation at the above mentioned concentrations. When the mutation frequency was determined, a frequency of 3.67 x 10-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction, and a frequency of -1.92 x 10-4was observed for 0.5 mM in the presence of S9 liver microsomal fraction. Since no other tested concentration of the test chemical and in the absence or presence of S9 liver microsomal fraction resulted in colonies, it was concluded that the test chemical does not give rise to gene mutations when CHO cells are exposedin vitroto the test chemical at ≤ 2.5 mM for 3 hrs.
Based on the data available and applying the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. 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 data available and applying the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. 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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