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EC number: 269-284-3 | CAS number: 68214-04-0
- 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
- 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 cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 05 April 2017 to 28 June 2017
- 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 487 (In vitro Mammalian Cell Micronucleus Test)
- Qualifier:
- according to guideline
- Guideline:
- other: Commission Regulation (EU) No. 640/2012 of 06. July 2012, amending Regulation (EC) No. 440/2008, EU Method B.49: “In Vitro Mammalian Cell Micronucleus Test”
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
Test material
- Reference substance name:
- Hexasodium 4,4'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino]]bis[5-hydroxy-6-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate]
- EC Number:
- 269-284-3
- EC Name:
- Hexasodium 4,4'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino]]bis[5-hydroxy-6-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate]
- Cas Number:
- 68214-04-0
- Molecular formula:
- C44H24Cl2N14Na6O20S6
- IUPAC Name:
- hexasodium 4,4'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino]]bis[5-hydroxy-6-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate]
- Test material form:
- solid: particulate/powder
- Details on test material:
- Appearance Red powder
Storage at room temperature (20 ± 5°C).
Constituent 1
Method
- Target gene:
- NA
Species / strain
- Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- CELLS USED
- Suitability of cells: according to the guideline
- Cell cycle length, doubling time or proliferation index: not indicated
- Sex, age and number of blood donors if applicable: exp 1 1 male, age 35 (healthy, non-smoking) exp 2 1 female age 20 (healthy, non-smoking)
- Whether whole blood or separated lymphocytes were used if applicable: lymphocytes
- Culturing:within 24 hours after collection for 72 h (exp 1) and 47.5 h (exp 2) hours at 37 ± 1 °C in a humidified atmosphere with 5.0 ±0.5 % CO2. in RPMI 1640 (+ heparinized blood)
- Normal (negative control) cell cycle time: no data
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 at 37 ± 1 °C in a humidified atmosphere with 5.0 ±0.5 % CO2.
- Cytokinesis block (if used):
- yes, cytochalasin B
- Metabolic activation:
- with and without
- Metabolic activation system:
- from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intra-peritoneally
- Test concentrations with justification for top dose:
- Main test (exp 1 and 2): 0, 1.25, 25 and 5 mg/mL with (exp 1) and without metabolic activation
Cytotoxicity test (exp 1 and 2) 0.16, 0.31, 0.63, 1.25, 2.5 and 5.0 mg/mL with (exp 1) and without metabolic activation - Vehicle / solvent:
- Minimal Culture Medium (MCM) (Penicillin/Streptomycin 1%/Phytohaemagglutinin solution 2%)
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: Colchicin
- Details on test system and experimental conditions:
- DURATION Exp 1
- Exposure duration: 4 h in at RMPI medium with and without S9 at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2
- Expression time: 19 h in presence of Cytochalasin B at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2
- Fixation time (start of exposure up to fixation or harvest of cells): 23 hours after exposure start
DURATION Exp 2
- Exposure duration: 23.5 h in at RMPI medium without S9 at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2
- Expression time: NA
CYTOKINESIS INHIBITOR: Cytochalasin B
STAIN: Giemsa
NUMBER OF REPLICATIONS: 2/concentration and controls
NUMBER OF CELLS EVALUATED: 1000 binucleated lymphocytes/ replicate with microscope (40-100X)
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: only in cells with sufficiently distinguishable cytoplasmic boundaries and clearly visible cytoplasm
Readable binucleated cells are identified by the following criteria:
• The cell must have two main nuclei
• The two main nuclei must each have an intact and well-defined membrane
• The two main nuclei must be contained within the cytoplasm
• The cell must be visible in its entirety in the field
• The area around the cell must not contain micronucleus-like debris
• The cytoplasmic boundary should be intact and distinguishable from the boundaries of adjacent cells
Micronuclei (MN) are identified by the following criteria:
• The diameter of the MN must not exceed 1/3rd of each of the two main nuclei diameter
• The micronuclei can touch but must not overlap the two main nuclei
• Micronuclei should be large enough to discern morphological characteristics
• Micronuclei should possess a generally rounded shape with a clearly defined outline
• Micronuclei should be similar in color to the nuclei
• Should lie in the same focal plane as the cell
• Micronuclei must not be linked to the nuclei by a nucleoplasmic bridge
• Micronuclei must be within cytoplasmic boundary
• Micronuclei must be non-refractive (staining)
DETERMINATION OF CYTOTOXICITY: Cytokinesis-Block Proliferation Index in 500 cells/replicate - Rationale for test conditions:
- Based on the outcome of the cytotoxicity test. No precipitate was observed in exp 1 at any of the concentration at 2.5 and 5 mg/L cytotoxicity was 0% without metabolic ativation and 1.7-2.5% with metabolic activation (expressed as decrease compared to solvent control CBPI), No precipitate was observed in exp 1 at any of the concentration at 2.5 and 5 mg/L cytotoxicity was 8.3-16.1%% without metabolic ativation (expressed as decrease compared to solvent control CBPI)
- Evaluation criteria:
- The genotoxicity assay is considered acceptable if it meets the following criteria:
• All experimental conditions are tested (short exposure with and without metabolic acti-vation, extended exposure without metabolic activation) unless a positive result is achieved in any experiment.
• In each experiment, an adequate number of cells is analysable both in the controls and in at least 3 test item concentrations.
• The micronucleus induction of the solvent and positive controls is compatible with the historical laboratory control data or the literature data.
• The positive control shows a statistically significant increase of binucleated cells with micronuclei compared with the concurrent solvent control.
• The criteria for cell proliferation and for the selection of concentrations are fulfilled.
Classification
The test item is considered to have no genotoxic effects if:
• Neither a statistically significant nor a concentration-related increase of the number of micronucleate cells in the evaluated test concentrations is observed.
• The obtained results lie within the range of the historical laboratory control data for sol-vent controls.
The test item is considered to have genotoxic effects if:
• At least one test concentration shows a statistically significant increase of micronucle-ate cells compared to the concurrent solvent control.
• In at least one experimental condition a dose-related increase of micronucleate cells can be observed.
• Any of the results lies outside the range of the historical laboratory control data for sol-vent controls.
- Statistics:
- Fisher’s exact test and chi-square-test
Results and discussion
Test results
- Key result
- Species / strain:
- lymphocytes: human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
Any other information on results incl. tables
Genotoxicity Results Experiment 1
Treatment |
Average CBPI |
Cytotoxicity (%) |
Total No. of BINC examined |
Total No. of MBNC |
% MBNC |
Without metabolic activation |
|||||
Solvent control MCM |
1.69 |
- |
2000 |
7 |
0.35 |
Solvent control 0.9% NaCl 0.5% v/v |
1.73 |
- |
2000 |
3 |
0.15 |
Positive control MMC 0.3 µg/mL |
1.47 |
14.8 |
2000 |
98 |
4.90** |
Test item 5 mg/mL |
1.65 |
2.5 |
2000 |
3 |
0.15 |
Test item 2.5 mg/mL |
1.66 |
1.7 |
2000 |
3 |
0.15 |
Test item 1.25 mg/mL |
1.69 |
-0.2 |
2000 |
3 |
0.15 |
With metabolic activation |
|||||
Solvent control MCM |
1.68 |
- |
2000 |
5 |
0.25 |
Solvent control 0.9% NaCl 0.5% v/v |
1.75 |
- |
2000 |
2 |
0.10 |
Positive control CPA 30 µg/mL |
1.32 |
24.6 |
2000 |
41 |
2.05** |
Test item 5 mg/mL |
1.75 |
-3.8 |
2000 |
7 |
0.35 |
Test item 2.5 mg/mL |
1.75 |
-4.0 |
2000 |
3 |
0.15 |
Test item 1.25 mg/mL |
1.68 |
0.4 |
2000 |
4 |
0.20 |
Asterisks indicate statistically significant differences to solvent control, with * p < 0.05, ** p < 0.01
BINC Binucleated cell
CBPI Cytokinesis-block proliferation index
MBNC Binucleated cell with micronucleus/i
Results Experiment 2
Treatment |
Average CBPI |
Cytotoxicity (%) |
Total No. of BINC examined |
Total No. of MBNC |
% MBNC |
Solvent control MCM |
1.883 |
- |
2000 |
10 |
0.50 |
Solvent control 0.9% NaCl 0.5% v/v |
1.911 |
- |
2004 |
8 |
0.40 |
Positive control MMC 0.3 µg/mL |
1.619 |
15.3 |
2000 |
56 |
2.80** |
Positive control Colchicine 0.035 µg/mL |
1.147 |
40.0 |
2000 |
68 |
3.40** |
Test item 5 mg/mL |
1.580 |
16.1 |
2016 |
13 |
0.64 |
Test item 2.5 mg/mL |
1.726 |
8.3 |
2000 |
17 |
0.85 |
Test item 1.25 mg/mL |
1.707 |
9.3 |
2000 |
22 |
1.10* |
Asterisks indicate statistically significant differences to solvent control, with * p < 0.05, ** p < 0.01
BINC Binucleated cell
CBPI Cytokinesis-block proliferation index
MBNC Binucleated cell with micronucleus/i
Applicant's summary and conclusion
- Conclusions:
- Under the experimental conditions of the test the substance did not show genotoxic activity in this in vitro test for the induction of micronuclei.
- Executive summary:
A study according to OECD 487 was performed to assess the genotoxic potential of the substance to induce formation of micronuclei in human lymphocytes in vitro in presence and absence of metabolic activation. Human lymphocytes were exposed during 4 hours or 23 hours to solvent control (serum free medium), substance and positive controls in duplicate. The proportion of cells containing micronuclei was determined with a microscope after Giemsa staining.
The following schedule was followed
Procedure
Exp. 1
Exp. 2*
Metabolic activation
Without S9 mix
With S9 mix
Without S9 mix
Exposure period
4 h
4 h
23 h
Expression time in
growth medium
19 h
19 h
-
Culture harvest time
23 h
23 h
23 h
Concentrations selected for scoring of micronuclei [mg/mL]
5, 2.5, 1.25
5, 2.5, 1.25
5, 2.5, 1.25
*extended exposure
No cytotoxic effect was detected in all tested concentrations. Therefore the three highest test item concentrations were evaluated for genotoxicity.
The substance did not induce significant and biologically relevant increases in the number of binucleated cells containing micronuclei with and without metabolic activation. in the two experiments performed.
Under the experimental conditions of the test the substance did not show genotoxic activity in this in vitro test for the induction of micronuclei.
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