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EC number: 276-770-9 | CAS number: 72705-24-9
- 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:
- 2016-01-18 to 2016-06-29
- 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 guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- 26 Sep 2014
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Regulation (EC) No 440/2008, In vitro Mammalian Cell Micronucleus Test, No B.49
- Version / remarks:
- Commission Regulation (EC) No 640/2012 of 06 July 2012
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
Test material
- Reference substance name:
- Ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate
- EC Number:
- 276-770-9
- EC Name:
- Ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate
- Cas Number:
- 72705-24-9
- Molecular formula:
- C26H24N4O9S3.H3N.Na
- IUPAC Name:
- ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate
- Test material form:
- solid
Constituent 1
- Specific details on test material used for the study:
- Batch identification: #13298465
Physical state, appearance: Solid, yellow to orange
Storage conditions: Room temperature
Expiry date: January 2019
Method
Species / strain
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Suitability of cells: Yes, V79 cell line has shown its suitability to detect aneugenic effects in the Micronucleus test in vitro either in the absence and presence of CytB
- Cell cycle length, doubling time or proliferation index: high proliferation rate (doubling time of about 12 - 14 hours),
MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with
− 10% (v/v) fetal calf serum (FCS)
− 1% (v/v) penicillin/streptomycin (10000 IU / 10000 μg/mL)
− 1% (v/v) amphotericine B (250 μg/mL)
Cells were grown with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity and subcultured
twice weekly
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix induced with phenobarbital and β-naphthoflavone
- Test concentrations with justification for top dose:
- 156.3 μg/mL, 312.5 μg/mL, 625.0 μg/mL, 1250.0 μg/mL, 2500.0 μg/mL, 5000.0 μg/mL
Based on the observations and toxicity data of a previously performed pretest of a HPRT study (top concentration: 2400 μg/mL; data not shown) and with regard to the composition of the test substance 5000 μg/mL test substacne was used as top concentration in both experiments of this cytogenetic study. - Vehicle / solvent:
- - Vehicle used: Culture medium (Minimal Essential Medium: MEM)
- Justification for choice of vehicle: Due to the good solubility of the test substance in water, culture medium (Minimal Essential Medium: MEM) was selected as vehicle.
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- culture medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
Exposure time: Without S9-mix: Exp.1: 4 hrs; Exp. 2: 24 hrs // With S9-mix: Exp.1 and Exp. 2: 4 hrs
Recovery time : Without S9-mix: Exp.1: 20 hrs // With S9-mix: Exp.1: 20 hrs, Exp. 2: 40 hrs
Harvest time: Without S9-mix: Exp.1 and Exp. 2: 24 hrs // With S9-mix: Exp.1: 24 hrs, Exp. 2: 44 hrs
SPINDLE INHIBITOR: Actin polymerisation inhibitor cytochalasin B
STAIN: 4’,6-diamidino-2-phenylindole dihydrochloride and propidium iodide
NUMBER OF REPLICATIONS: 2 per concentration
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
5x10E4 cells per slide were centrifuged at 600 rpm for 7 minutes onto labeled slides using a Cytospin centrifuge. At least two slides per flask were prepared.
After drying, the slides were fixed in 90% (v/v) methanol for 10 minutes. Before scoring, the slides were stained with a mixture of 4’,6-diamidino-2-phenylindole dihydrochloride and propidium iodide at a concentration of 0.25 μg/mL each.
NUMBER OF CELLS EVALUATED: At least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, were evaluated for the occurrence of micronuclei.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
− The diameter of the micronucleus is less than 1/3 of the main nucleus.
− The micronucleus and main nucleus retain the same color.
− The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
− Only binucleated cells clearly surrounded by a membrane were scored.
DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth (CBPI), cell morphology (microscopically)
OTHER EXAMINATIONS:
pH value: was measured at least for the top concentration and for the vehicle control with and without S9 mix.
Osmolality: was measured at least for the top concentration and for the vehicle control with and without S9 mix
Solubility: Test substance precipitation was checked immediately after start of treatment of the test cultures (macroscopically) and at the end of treatment (macroscopically / microscopically). - Evaluation criteria:
- Acceptance criteria
The in vitro micronucleus assay is considered valid if the following criteria are met:
• The quality of the slides allowed the evaluation of a sufficient number of analyzable cells both in the control groups (vehicle/positive) and in at least three exposed test groups.
• Sufficient cell proliferation was demonstrated in the vehicle control.
• The number of cells containing micronuclei in the vehicle control was within the range of our laboratory’s historical negative control data (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
• The positive control substances both with and without S9 mix induced a distinct, statistically significant increase in the number of micronucleated cells in the expected range.
Assessment criteria
A test substance is considered to be clearly positive if the following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of our laboratory’s historical negative control data (95% control limit).
A test substance is considered to be clearly negative if the following criterion is met:
• Neither a statistically significant nor dose-related increase in the number of cells containing
micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit). - Statistics:
- The statistical evaluation of the data was carried out using an appropriate statistical analysis. The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test, BASF SE).
If the results of this test were statistically significant compared with the respective vehicle control (p ≤ 0.05), labels (S) have been printed in the tables.
Results and discussion
Test results
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Water solubility: Good
- Precipitation: No
GENOTOXICITY
The single statistical significance observed in the 1st Experiment without metabolic activation in an intermediate concentration of 2500 μg/mL has to be regarded as biologically irrelevant under the circumstance that this value was clearly within the historical negative control data range.
CYTOKINESIS BLOCK: CytB blocks
- Distribution of mono-, bi- and multi-nucleated cells: 1x No. mononucleate cells, 2 x No. binucleate cells, 3 x No. multinucleate cells
HISTORICAL CONTROL DATA
- Positive historical control data:
Cyclophosphamide (CPP): range: 2.4 – 13.8% micronucleated cells; mean: 4.8; SD: 2.0; 95% Lower Control Limit: 0.8; 95% Upper Control Limit: 8.8
Ethyl methanesulfonate: range: 2.0-6.4 % micronucleated cells; mean: 2.9; SD:0.7; 95% Lower Control Limit: 1.5; 95% Upper Control Limit: 4.3
- Negative (vehicle) historical control data:
range: 0.1 - 1.5% micronucleated cells; mean 0.5; SD: 0.2; 95% Lower Control Limit: 0.0; 95% Upper Control Limit: 1.0
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI, Relative population doubling (RPD) for the time period before addition of CytB
no cytotoxicity indicated by reduced RPD of below 50% of control, no reduced proliferative activity, cell morphology/attachment was not adversely influenced (grade > 2) at any dose tested for the occurrence of micronuclei.
Any other information on results incl. tables
Table 1: Summary table - experimental parts without S9 mix
Exp. |
Exposure/ Preparation interval |
Test groups [μg/mL] |
S9-mix |
Genotoxicity Micronucleated cells** [%] |
Proliferation index cytostasis (CBPI) [%] |
Relative population doubling (RPD) [%] |
1 |
4/24 hrs |
Negative control |
- |
0.3 |
0.0 |
100.00 |
|
|
156.3 |
- |
n.d. |
n.d. |
116.7 |
|
|
312.5 |
- |
n.d. |
n.d. |
122.8 |
|
|
625.0 |
- |
n.d. |
n.d. |
123.0 |
|
|
1250.0 |
- |
0.4 |
3.0 |
119.5 |
|
|
2500.0 |
- |
1.0 (s) |
3.8 |
115.3 |
|
|
5000.0 |
- |
0.3 (s) |
5.7 |
122.8 |
|
|
Positive control (1) |
- |
1.5 (s) |
8.1 |
112.5 |
|
|
Positive control (2) |
- |
2.4 (s) |
9.0 |
112.8 |
2 |
24/24 hrs |
Negative control |
- |
0.7 |
0.0 |
100.00 |
|
|
312.5 |
- |
n.d. |
n.d. |
103.3 |
|
|
625.0 |
- |
n.d. |
n.d. |
97.3 |
|
|
1250.0 |
- |
0.6 |
14.4 |
107.9 |
|
|
2500.0 |
- |
0.7 |
14.6 |
108.6 |
|
|
5000.0 |
- |
0.3 |
18.2 |
101.3 |
|
|
Positive control (1) |
- |
2.9 (s) |
24.0 |
101.2 |
** Relative number of binucleated cells with micronuclei per 2000 cells scored per test group
S Frequency statistically significant higher than corresponding control values (p≤0.05)
n.d. Not determined
(1) EMS 500 μg/mL
(2) EMS 600 μg/mL
Table 2: Summary table - experimental parts with S9 mix
Exp. |
Exposure/ Preparation interval |
Test groups [μg/mL] |
S9-mix |
Genotoxicity Micronucleated cells** [%] |
Proliferation index cytostasis (CBPI) [%] |
Relative population doubling (RPD) [%] |
1 |
4/24 hrs |
Negative control |
+ |
0.5 |
0.0 |
100.0 |
|
|
156.3 |
+ |
n.d. |
n.d. |
99.1 |
|
|
312.5 |
+ |
n.d. |
n.d. |
108.6 |
|
|
625.0 |
+ |
n.d. |
n.d. |
106.8 |
|
|
1250.0 |
+ |
0.4 |
0.3 |
104.2 |
|
|
2500.0 |
+ |
0.5 |
3.4 |
108.8 |
|
|
5000.0 |
+ |
0.4 |
0.1 |
111.6 |
|
|
Positive control (1) |
+ |
2.3 (s) |
33.2 |
105.1 |
2 |
4/44 hrs |
Negative control |
+ |
0.5 |
0.0 |
100.0 |
|
|
312.5 |
+ |
n.d. |
n.d. |
94.1 |
|
|
625.0 |
+ |
n.d. |
n.d. |
90.1 |
|
|
1250.0 |
+ |
0.6 |
- 0.6 |
88.3 |
|
|
2500.0 |
+ |
0.7 |
- 3.1 |
93.1 |
|
|
5000.0 |
+ |
0.7 |
0.9 |
87.1 |
|
|
Positive control (1) |
+ |
3.8 (s) |
-11.0 |
86.6 |
** Relative number of binucleated cells with micronuclei per 2000 cells scored per test group
(s) Frequency statistically significant higher than corresponding control values (p≤0.05)
n.d. Not determined
(1) Cyclophosphamide: CPP 0.5 μg/mL
Applicant's summary and conclusion
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