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EC number: 270-393-3 | CAS number: 68427-35-0
- 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
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- Endpoint summary
- Stability
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 17 July 2016 to 03 August 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 2008
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- 2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3-yl]benzoxazole-5-sulphonamide
- EC Number:
- 270-393-3
- EC Name:
- 2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3-yl]benzoxazole-5-sulphonamide
- Cas Number:
- 68427-35-0
- Molecular formula:
- C20H19N3O5S
- IUPAC Name:
- 2-[7-(diethylamino)-2-oxo-2H-chromen-3-yl]-1,3-benzoxazole-5-sulfonamide
- Test material form:
- solid: particulate/powder
- Details on test material:
- - Appearance: Yellowish orange powder
- Storage conditions of test material: At room temperature
- Stable under storage conditions until: 30 November 2020 (expiry date)
Constituent 1
Method
- Target gene:
- - Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Properly maintained: Yes. The Salmonella typhimurium strains are regularly checked to confirm their histidine requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV sensitivity and the number of spontaneous revertants. Stock cultures of the strains were stored in liquid nitrogen (-196 °C).
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Properly maintained: Yes. The strain is regularly checked to confirm the tryptophan requirement, UV-sensitivity and the number of spontaneous revertants. Stock cultures were stored in liquid nitrogen (-196 °C).
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix (rat liver S9-mix induced by Aroclor 1254)
- Test concentrations with justification for top dose:
- - Dose range finding study (TA100 and WP2uvrA only): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (absence and presence of S9-mix)
- Experiment 1 (TA1535, TA1537 and TA98): 5.4, 17, 52, 164, 512 and 1600 μg/plate (absence and presence of S9-mix)
- Experiment 2 (all strains): 86, 154, 275, 492, 878 and 1568 μg/plate (absence and presence of S9-mix) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed. The test material could not be dissolved in water. A homogeneous suspension of 50 mg/mL was obtained in dimethyl sulfoxide. At concentrations of 16 mg/mL and lower the test material was dissolved in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves to obtain an homogeneous suspension (Dose range finding test) or until the test material had completely dissolved (mutation experiments). The lower test concentrations were prepared by subsequent dilutions in DMSO.
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 2-nitrofluorene
- sodium azide
- methylmethanesulfonate
- other: ICR-191; 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DOSE RANGE FINDING TEST/ MUTATION ASSAY
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5 % (v/v) S9-mix and reported as part of the first mutation experiment.
MUTATION ASSAY
At least five different doses (increasing with approximately half-log steps) of the test material were tested in each strain both in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the test material was tested both in the absence and presence of 10 % (v/v) S9-mix in all tester strains.
Top agar in top agar tubes was melted by heating to 45 ± 2 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test material in DMSO and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.
NUMBER OF REPLICATIONS: Testing was performed in triplicate
COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually. Evidence of test material precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. - Evaluation criteria:
- ACCEPTABILITY OF THE ASSAY
The assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at the testing facility.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5 % of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
DATA EVALUATION
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment. - Statistics:
- No formal hypothesis testing was done.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium, other: TA1535, TA1537 and TA100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- DOSE RANGE FINDING TEST/FIRST MUTATION EXPERIMENT
- Precipitate: In the dose range finding test, precipitation of the test material on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards. At the end of the incubation period, precipitation was observed at 164 μg/plate and above (absence of S9-mix) and at 512 and 1600 μg/plate and upwards (TA100 and WP2uvrA, respectively). In the first experiment, precipitation of the test material on the plates was observed at the start and at the end of the incubation period at concentrations of 512 μg/plate and upwards.
- Toxicity: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In strain TA98 (absence of S9-mix) a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the lowest dose level tested. However, since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test material. It is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies.
- Mutagenicity: In the presence of S9-mix, the test material induced an up to 3.8-dose related increase in tester strain TA98. In all other tester strains, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested.
MUTATION EXPERIMENT 2
- Precipitate: Precipitation of the test material on the plates was observed at the start of the incubation period at the concentration of 492 μg/plate. At the end of the incubation period, precipitation was observed at 1568 μg/plate in the absence of S9-mix, except in tester strain TA100. In the presence of S9-mix, precipitation was only observed in tester strain TA1535 at 1568 μg/plate.
- Toxicity: The bacterial background lawn was reduced in all tester strains in the presence of S9-mix at the highest tested concentration. In addition, a reduction in the bacterial background lawn was observed in tester strain TA100 in the absence of S9-mix and a biologically relevant decrease in the number of revertants was observed in tester strain TA1537 in the absence of S9-mix.
- Mutagenicity: In the presence of S9-mix, the test material induced an up to 4.4-dose related increase in tester strain TA98. In all other tester strains, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested.
DISCUSSION
In the presence of S9-mix, the test material induced dose related increases in tester strain TA98. The increases observed were above the laboratory historical control data range, in two independently repeated experiments and were up to 4.4-fold the concurrent vehicle controls and therefore considered to be biologically relevant.
In the other tester strains (TA1535, TA1537, TA100 and WP2uvrA), the test material did not induce a significant dose-related increase in the number of revertant colonies in any experiment.
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Any other information on results incl. tables
Table 1: Dose Range-finder and Experiment 1 (Plate incorporation assay 5 % S9)
+/- S9 Mix |
Concentration (µg/plate) |
Mean number of colonies/plate |
||||
Base-pair Substitution Type |
Frameshift Type |
|||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||
- |
PC DMSO 1.7 5.4 17 52 164 512 1600 5000 |
711 76 83 68 77 92 NP 90 SP 88 SP 71 MP 68 n MP |
815 9 - 6 7 12 15 NP 10 SP 10 n MP - |
1367 24 25 24 28 25 NP 26 SP 19 SP 21 MP 13 n MP |
1227 10 - 6 14 10 13 NP 11 SP 16 n MP - |
1187 4 - 4 4 6 7 NP 6 SP 4 n MP - |
+ |
PC DMSO 1.7 5.4 17 52 164 512 1600 5000 |
1003 75 60 73 71 80 79 NP 82 SP 79 MP 72 n MP |
201 12 - 8 7 5 11 NP 14 SP 9 n MP - |
390 32 34 32 38 33 33 34 NP 43 SP 19 n MP |
743 22 - 14 18 23 48 NP 58 SP 84 n MP - |
362 6 - 5 11 7 12 NP 7 SP 14 n MP - |
Mean number of revertant colonies/3 replicate plates
PC = Positive control
MP = Moderate Precipitate
NP = No precipitate
SP = Slight Precipitate
n = Normal bacterial background lawn
Table 2: Experiment 2 (Plate incorporation assay 10 % S9)
+/- S9 Mix |
Concentration (µg/plate) |
Mean number of colonies/plate |
||||
Base-pair Substitution Type |
Frameshift Type |
|||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||
- |
PC DMSO 86 154 275 492 878 1568 |
1049 98 128 115 133 105 103 n 122 s NP |
947 18 17 11 14 18 13 NP 12 n SP |
1494 23 20 22 28 21 23 NP 21 n SP |
1466 16 15 17 21 14 14 NP 16 n SP |
1029 5 7 5 3 4 4 NP 1 n SP |
+ |
PC DMSO 86 154 275 492 878 1568 |
1062 97 111 114 117 112 108 n 111 s NP |
138 14 12 16 16 18 16 n NP 12 s SP |
435 32 30 29 26 22 n 37 s 33 s NP |
475 20 24 38 34 66 n 70 s 88 s NP |
480 10 7 9 6 6 12 n 13 s NP |
Mean number of revertant colonies/3 replicate plates
PC = Positive control
NP = No precipitate
SP = Slight Precipitate
n = Normal bacterial background lawn
s = Bacterial background lawn slightly reduced
Applicant's summary and conclusion
- Conclusions:
- Under the conditions of this study, it is concluded that the test material is mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations with metabolic activation in one strain.
- Executive summary:
The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.
The test material was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).
In the dose range finding test, the test material was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test material was tested up to or beyond a precipitating dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the first mutation assay.
Based on the results of the dose range finding test, the test material was tested in the first mutation assay at a concentration range of 5.4 to 1600 μg/plate in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test material was precipitated on the plates at concentrations of 512 and 1600 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
In a follow-up experiment of the assay with additional parameters, the test material was tested at a concentration range of 86 to 1568 μg/plate in the absence and presence of 10 % (v/v) S9-mix in all strains. Precipitation of the test material on the plates was observed at 1568 μg/plate in the absence of S9-mix, except in tester strain TA100 and in the presence of S9-mix in tester strain TA1535 only. In the presence of S9-mix, the bacterial background lawn was reduced in all tester strains at the highest tested concentration. In the absence of S9-mix, a reduction in the bacterial background lawn was observed in tester strain TA100 and a biologically relevant decrease in the number of revertants was observed in tester strain TA1537.
In the presence of S9-mix, the test material induced dose related increases in tester strain TA98. The increases observed were above the laboratory historical control data range, in two independently repeated experiments and were up to 4.4-fold the concurrent vehicle controls and therefore considered to be biologically relevant. In the other tester strains (TA1535, TA1537, TA100 and WP2uvrA), the test material did not induce a significant dose-related increase in the number of revertant colonies in any experiment.
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Under the conditions of the study, it is concluded that the test material is mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations with metabolic activation in one strain.
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