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EC number: 219-708-8 | CAS number: 2503-73-3
- 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
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 03.05.-01.07.2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Deviations:
- yes
- Remarks:
- See any other information...
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
Test material
- Reference substance name:
- Tetrasodium 2-[[4-[[4-[[1-hydroxy-6-(phenylamino)-3-sulphonato-2-naphthyl]azo]-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate
- EC Number:
- 219-708-8
- EC Name:
- Tetrasodium 2-[[4-[[4-[[1-hydroxy-6-(phenylamino)-3-sulphonato-2-naphthyl]azo]-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate
- Cas Number:
- 2503-73-3
- Molecular formula:
- C42H29N7O13S4.4Na
- IUPAC Name:
- tetrasodium 2-[[4-[[4-[[1-hydroxy-6-(phenylamino)-3-sulphonato-2-naphthyl]azo]-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate
- Reference substance name:
- Sodium chloride
- EC Number:
- 231-598-3
- EC Name:
- Sodium chloride
- Cas Number:
- 7647-14-5
- Molecular formula:
- ClNa
- IUPAC Name:
- sodium chloride
- Test material form:
- solid: particulate/powder
- Details on test material:
- - Name of test material (as cited in study report): Direct Blue 78- Physical state: solid, powder- Analytical purity: 95% (w/w)- Impurities (identity and concentrations): NaCl (CAS: 7647-14-5) 10% (w/w)- Lot/batch No.: 7013/207- Expiration date of the lot/batch: unlisted- Storage condition of test material: The test substance should be stored in dry room in dark in closed container at the room temperature.
Constituent 1
impurity 1
Method
Species / strain
- Species / strain / cell type:
- mammalian cell line, other:
- Remarks:
- peripheral blood lymphocytes mammalian cell line
- Details on mammalian cell type (if applicable):
- The human peripheral blood lymphocytes used for testing were obtained from healthy non smokingdonors (up to 35 years of age). Peripheral blood (heparinized) is taken from donors in certified medicallaboratory (MeDiLa) in the morning and as soon as possible transported into the test facility.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction of rat liver homogenate and mixture of cofactors
Controls
- Untreated negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- other: Colchicine
- Details on test system and experimental conditions:
- Principle of test is the detection of binucleated cells with micronuclei, which are induced by the test substance in human peripheral blood lymphocytes. Lymphocytes are cultured in growth medium and test substance is added to them. Cell cycle is then stopped by cytochalasin B, cultures are sampled and microscopic preparations are prepared. Preparations are then analysed by microscope. Genotoxicity is indicated by increased incidence of binucleated cells with micronuclei.Experiments with and without metabolic activation with short treatment (3 hours) are done at first. If both experiments with the short treatments are negative or equivocal, subsequently, extended exposure treatment without metabolic activation is performed.
Results and discussion
Test results
- Key result
- Species / strain:
- mammalian cell line, other: peripheral blood lymphocytes
- Metabolic activation:
- with and 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:
- Results overviewResults of experiments are summarized in the tables in the Annexes 1 (cytotoxicity) and 2 (genotoxicity). The tables contain the dose applied per culture in µg/mL (concentrations of test substance were applied to cultures at a volume of 50 µl), amount of S9 per culture in µl, number of mononucleated, binucleated and multinucleated cells, CBPI index and % cytotoxicity, numbers of binucleated cells with micronuclei and average numbers of binucleated cells with micronuclei in 1000 cells, number of micronuclei and average number of micronuclei in 1000 cells, parameter Mt / Mc, i.e. ratio of number of binucleated cells with micronuclei at tested dose (Mt) to number of binucleated cells with micronuclei at negative control (Mc, UTC or S9-mix). Numbers of binucleated cells with micronuclei in negative and positive controls were compared with historical controls in our laboratory. The current ranges are given in Annex 3 (Table 7 and Table 8). Values of negative and positive controls in this study are within the ranges of historical data, so that test system responds adequately and the experiment is acceptable.The cytotoxic effect was characterized as % of cytotoxicity. Results of the cytotoxic effect are given in the Table 1 - 3 (Annex 1). All of test concentrations did not show the cytotoxicity higher than 55±5 % in the time of exposure 3 hours. The second experiment with the prolonged exposition without activation (23 hours) gave in three tested concentrations (2000, 1000 and 500 µg/mL) high cytotoxicity (small and dark cells) therefore microscopic slides could not be analyzed for cytotoxicity and genotoxicity. The concentrations 250 µg/mL was evaluated only for cytotoxicity, but could not be used for genotoxicity evaluation because of poor appearance of microscopic slides (small and dark cells). On the basis of these results, the third experiment without metabolic activation with extended exposure and lower concentrations 125, 62.5 and 31.25 µg/mL had to be done. In the third experiment with the prolonged exposition without activation (23 hours), all of tested concentrations did not show the cytotoxicity higher than 55±5 %. Therefore the concentration of 125 µg/mL was selected as the highest one for the analysis of genotoxic effect.
Any other information on results incl. tables
Cytotoxic effect
Table No. 1: Evaluation of cytotoxic effect without metabolic activation-3h exposure
- MA I | ||||||
Culture No. | Treatment/Test substance concentration | Number of MNC | Number of BNC | Number of MTNC | CBPI | Cytotoxicity (%) |
1 | UTC | 756 | 282 | 39 | 1.334 | 0.0 |
2 | 2000 μg/mL | 928 | 182 | 9 | 1.179 | 46.5 |
3 | 1000 μg/mL | 691 | 386 | 26 | 1.397 | -18.8 |
4 | 500 μg/mL | 811 | 348 | 55 | 1.377 | -12.9 |
5 | 250 μg/mL | 607 | 422 | 77 | 1.521 | -55.8 |
6 | 125 μg/mL | 707 | 290 | 56 | 1.382 | -14.2 |
13 | Colchicine | 857 | 176 | 76 | 1.296 | 11.5 |
Table No. 2: Evaluation of cytotoxic effect with metabolic activation (S9-mix) -3h exposure
+MA I | ||||||
Culture No. | Treatment/Test substance concentration | Number of MNC | Number of BNC | Number of MTNC | CBPI | Cytotoxicity (%) |
7 | S9-mix | 925 | 298 | 45 | 1.306 | 0.0 |
8 | 2000mg/mL + S9-mix | 923 | 204 | 16 | 1.206 | 32.5 |
9 | 1000mg/mL + S9-mix | 939 | 183 | 20 | 1.195 | 36.2 |
10 | 500mg/mL + S9-mix | 828 | 187 | 28 | 1.233 | 23.9 |
11 | 250mg/mL + S9-mix | 764 | 267 | 69 | 1.368 | -20.3 |
12 | 125mg/mL + S9-mix | 724 | 406 | 67 | 1.451 | -47.4 |
14 | Cyclophosphamide + S9-mix | 630 | 453 | 35 | 1.468 | -52.9 |
1 | UTC | 756 | 282 | 39 | 1.334 | -9.2 |
Table No. 3: Evaluation of cytotoxic effect without metabolic activation -23h exposure
- MA III | ||||||
Culture No. | Treatment/Test substance concentration | Number of MNC | Number of BNC | Number of MTNC | CBPI | Cytotoxicity (%) |
15 | UTC | 528 | 426 | 61 | 1.54 | 0.0 |
17 | 125 μg/mL | 642 | 338 | 54 | 1.43 | 20.1 |
18 | 62.5 μg/mL | 607 | 382 | 50 | 1.46 | 14.1 |
19 | 31.25 μg/mL | 603 | 497 | 47 | 1.52 | 4.6 |
16 | Colchicine | 823 | 256 | 67 | 1.34 | 37.0 |
Genotoxic effect
Table No. 4: Evaluation of genotoxicity without metabolic activation-3h exposure
Culture No. | Treatment/Test substance concentration | Number of binucleated cells with MN | Number of MN | Average number of BN cells with MN per 1000 binucleated cells | Average number of MN per 1000 binucleated cells | Mt/Mc |
1 | UTC | 22 | 24 | 11 | 12 | 1.00 |
2 | 2000mg/mL | 42 | 45 | 21 | 22.5 | 1.91 |
3 | 1000mg/mL | 15 | 17 | 7.5 | 8.5 | 0.68 |
4 | 500mg/mL | 35 | 40 | 17.5 | 20 | 1.59 |
13 | Colchicine | 273 | 323 | 136.5 | 161.5 | 12.41 |
Table No. 5:Evaluation ofgenotoxicitywith metabolic activation (S9-mix) -3h exposure
Culture No. | Treatment/Test substance concentration | Number of binucleated cells with MN | Number of MN | Average number of BN cells with MN per 1000 binucleated cells | Average number of MN per 1000 binucleated cells | Mt/Mc |
7 | S9-mix | 23 | 24 | 11.5 | 12 | 1.00 |
8 | 2000 mg/mL + S9-mix | 20 | 22 | 10 | 11 | 0.87 |
9 | 1000mg/mL + S9-mix | 20 | 21 | 10 | 10.5 | 0.87 |
10 | 500mg/mL + S9-mix | 24 | 25 | 12 | 12.5 | 1.04 |
1 | UTC | 22 | 24 | 11 | 12 | 0.96 |
14 | Cyclophosphamide + S9-mix | 58 | 64 | 29 | 32 | 2.52 |
Table No. 6: Evaluation of genotoxicity without metabolic activation- 23h exposure
Culture No. | Treatment/Test substance concentration | Number of binucleated cells with MN | Number of MN | Average number of binucleated cells with MN per 1000 binucleated cells | Average number of MN per 1000 binucleated cells | Mt/Mc |
15 | UTC | 17 | 18 | 8.5 | 9 | 1.00 |
17 | 125mg/mL | 20 | 21 | 10 | 10.5 | 1.18 |
18 | 62.5mg/mL | 22 | 23 | 11 | 11.5 | 1.29 |
19 | 31.25mg/mL | 26 | 29 | 13 | 14.5 | 1.53 |
16 | Colchicine | 50 | 55 | 25 | 27.5 | 2.94 |
Applicant's summary and conclusion
- Conclusions:
- Under the experimental design described above, the test substance, Direct Blue 78, had no genotoxic effects in the micronucleus test performed in human peripheral blood lymphocytes in experiments both without and with metabolic activation.The result of micronucleus test was negative, test substance is then considered not able to induce chromosome breaks and/or chromosome gain or loss in this test system.
- Executive summary:
In Vitro Mammalian Cell Micronucleus Test assayed genotoxicity of the test substance, Direct Blue 78. The test was performed according to OECD Test Guideline No. 487 -In Vitro Mammalian Cell MicronucleusTest, Adopted 26thSeptember, 2014.
The human peripheral blood lymphocytes from healthy donors were used for testing. The test substance was suspended in RPMI medium and assayed in five concentrations 31.25-2000 µg/mL, which were applied to cultures in volume of 50 mL.
Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.
Under the experimental design described above, the test substance, Direct Blue 78,had no genotoxic effects in the micronucleus test performed in human peripheral blood lymphocytesin experiments both without and with metabolic activation.
The result of micronucleus test was negative, test substance is then considered not able to induce chromosome breaks and/or chromosome gain or loss in this test system.
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