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EC number: 235-517-2 | CAS number: 12262-25-8 This substance is identified in the Colour Index by Colour Index Constitution Number, C.I. 53430.
- 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
Short-term toxicity to aquatic invertebrates
Administrative data
Link to relevant study record(s)
- Endpoint:
- short-term toxicity to aquatic invertebrates
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13 February - 24 May 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
- Version / remarks:
- 13th April 2004
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.2 (Acute Toxicity for Daphnia)
- Version / remarks:
- May 30, 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
- Version / remarks:
- April 1996
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, section 3.1.2 Media preparation methods, Direct addition. OECD Series on Testing and Assessment No. 23, Paris September 2000
- Version / remarks:
- September 2000
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- Expiry date: 01 December 2020
- Analytical monitoring:
- yes
- Details on sampling:
- ANALYSIS OF THE SAMPLES
For determination of the test item concentration, samples were taken in duplicates from the testing concentration and from the control at the start and end of each renewal period of the test. The first series of the samples taken at the start and at the end of each renewal were sent to individual analysis. The further samples taken were kept separately as a reserve until the date of the issue of the final report at a temperature below – 15 °C. - Vehicle:
- yes
- Details on test solutions:
- PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: As the test item is poorly soluble in deionized water as well in the test medium, preparation of test item solution was performed using the WAF method (according to OECD Series on Testing and Assessment No. 23). Appropriate amount of test item was suspended in the dilution water (ISO medium; see 4.4) in order to give the loading rate of 100 mg test item/L. The solution was handled by ultrasonic bath for 10 minutes thereafter stirred for a period of approximately 24 hours to achieve equilibrated concentration. The solution was then filtrated through a membrane filter (0.45 µm) to separate the possible non-dissolved test material.
- Controls: Negative control (ISO medium without addition of test item ), positive control (with the reference substance potassium dichromate) - Test organisms (species):
- Daphnia magna
- Details on test organisms:
- TEST ORGANISM
- Common name: Water flea
- Source: Laboraty culture; originally obtained from Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100-Gödöllő, Kotlán S. u. 3, Hungary
- Sex: Female
- Age of parental stock: Less than 24 h old at the beginning of the test.
- Feeding during test: Test animals were not fed during the exposure
ACCLIMATION
- Acclimation period: Test animals were bred under similar (or the same) conditions as that used during the exposure period (holding water, temperature, background colour etc.), therefore additional acclimatisation before the test was not necessary
- Acclimation conditions: Same as test
- Type and amount of food: The Daphnia were fed with concentrated algal suspension of Raphidocelis subcapitata during holding.
- Health during acclimation: Apparently healthy animals were used in this test with a known history (breeding method, pre-treatment). - Test type:
- semi-static
- Water media type:
- other: Reconstituted water (ISO Medium, according to OECD 202)
- Limit test:
- yes
- Total exposure duration:
- 48 h
- Remarks on exposure duration:
- Animals were exposed to the test concentration or included as control over a period of 48 hours.Daphnia were observed for immobility or mortality by visual observation after 24 and 48 hours of exposure.
- Post exposure observation period:
- Daphnia were observed for immobility or mortality by visual observation after 24 and 48 hours of exposure. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile.
- Hardness:
- 231.4 mg/L (as CaCO3)
- Test temperature:
- The test temperature was in the range of 20.1 - 20.6°C measured in the test vessels during the test. The additionally measured ambient temperature within the climate chamber was between 19.9 – 20.8°C.
- pH:
- The pH was in the range of 7.83 - 8.18 during the test.
- Dissolved oxygen:
- The dissolved oxygen concentration was in the range of 7.47 - 8.65 mg/L during the test.
- Salinity:
- Not specified
- Conductivity:
- Not specified
- Nominal and measured concentrations:
- Nominal: 100 mg/L
- Details on test conditions:
- TEST SYSTEM
- Test vessel: beakers
- Type: open
- Material, size: Glass, volume app. 50 mL
- Aeration: No
- Renewal rate of test solution: test solution was renewed once during the test (on day 1)
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4
TEST MEDIUM
- Preparation of dilution water: ISO Medium according to guideline, prepared with ultra-pure water
OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Complete darkness
EFFECT PARAMETERS MEASURED: Immobility after 24 and 48 hours of exposure. In addition to immobility, any abnormal behaviour or appearance was reported.
RANGE-FINDING STUDY
In order to select appropriate test concentrations for use in the definitive test, a non-GLP preliminary range-finding tests was conducted to determine the approximate toxicity of the test item. Due to the test item’s low solubility, preparation of test solutions was performed using the WAF method (according to OECD Series on Testing and Assessment No. 23) as follows: In the semi-static preliminary range-finding test the nominal test concentration of 100 mg/L was prepared and handled in ultrasonic bath for 10 minutes. The stock solution was mixed for a period of 24 hours to achieve an equilibrated concentration and then filtrated through a membrane filter (0.45 µm) to separate the possible non-dissolved test material. The test solution was renewed once during the test (on day 1). Untreated control ran parallel in the test.
- Test concentration: 100 mg/L
- Results used to determine the conditions for the definitive study: Yes. Based on the results of the non-GLP Preliminary Range-Finding Tests, the main test was conducted as a limit test using the WAF method (according to OECD Series on Testing and Assessment No. 23) including a loading rate of 100 mg test item/L and a concurrent control group. - Reference substance (positive control):
- yes
- Key result
- Duration:
- 48 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 1.27 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat. (dissolved fraction)
- Basis for effect:
- mobility
- Key result
- Duration:
- 48 h
- Dose descriptor:
- LOEC
- Effect conc.:
- > 1.27 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat. (dissolved fraction)
- Basis for effect:
- mobility
- Key result
- Duration:
- 48 h
- Dose descriptor:
- EC50
- Effect conc.:
- > 100 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mobility
- Details on results:
- Validity of the Study
Immobilisation was not observed in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was in the range of 7.47 - 8.65 mg/L (more than 3 mg/L in all cases). All validity criteria were within acceptable limits and therefore the study can be considered as valid.
Analytical Results
The concentration of the test item was analytically determined based on total product at the start and at the end of each renewal period of the experiment.
The test item was not detected in the untreated control group (i.e. signal intensities measured for the control samples were less than 20 % of the analytical quantification limit (LOQ).
In the treated group the mean measured test item concentration was 1.62 mg/L at the start and 0.931 mg/L at the end of the first renewal period (corresponding to 57 % of the initial measured test concentration) while it was measured to be 1.55 mg/L at the start and 1.10 mg/L at the end of the second renewal period (corresponding to 71 % of the initial measured test concentration).
The exposure concentration was calculated as the geometric mean of the start and end values and determined to be 1.27 mg/L. This concentration was considered as the saturation concentration in the test medium, based on water-accommodated fractions of the test item (equivalent to 100 mg/L nominal concentration).
Biological Results and Performance of the Test
Testing units were kept in a climate chamber at a temperature of 19.9 – 20.8 °C, while temperature in the test glasses ranged between 20.1 - 20.6 °C. The pH was measured as 7.83 - 8.18 and the dissolved oxygen concentration between 7.47 - 8.65 mg/L during the study. The test was carried out in total darkness. There was no immobilisation observed in twenty daphnids exposed to either test item treated or control group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected. - Reported statistics and error estimates:
- Statistical analysis to detect significant differences between control and treated test group on immobilization was assessed by analysis of variance (ANOVA) and appropriate statistical procedure (e.g. by Dunnett’s Test, α = 0.05).
- Validity criteria fulfilled:
- yes
- Conclusions:
- Based on the results of this study, the test item Leuco Sulfur Blue 9 had no toxic effect up to aquatic saturation (i.e. limit test concentration) on Daphnia magna; the 48h LOEC is higher than the solubility level of the test item in the test medium, which corresponds to a mean measured concentration of 1.27 mg/L (based on total product). The NOEC value is equal to the mean measured test item concentration of 1.27 mg/L (based on total product).
- Executive summary:
Acute toxicity of the test item Leuco Sulfur Blue 9 was assessed on Daphnia magna in a 48-hour Acute Immobilisation Test. Young Daphnia were exposed to aqueous test media containing the test item for 48 hours in a semi-static test. A limit test was performed in which the test organisms were exposed to aqueous test media containing the test item for 48 hours at the limit of its solubility in the test medium (i.e. saturation) plus a control in order to demonstrate that the test item has no influence on the mobility of Daphnia up to at least the saturation concentration (equivalent to 100 mg/L nominal concentration, prepared as water-accommodated fraction (WAF)).
The quantification of the test item Leuco Sulfur Blue 9 was performed by a previously validated analytical method by the analytical laboratory of TOXI-COOP ZRT. Samples were taken from the test concentration and the control at the start and at the end of each renewal period of the experiment and analysed by UV/VIS spectrophotometric method. The mean determined exposure concentration was 1.27 mg/L (based on total product) which was calculated as the geometric mean of the measured start and end concentrations of both renewal periods.
Healthy, young female daphnids < 24 hours of age and with known origin, history and acclimatisation conditions were used. Twenty Daphnia (divided into 4 replicates) were tested each, exposed to the saturated test concentration and in the control. Each test vessel contained approximately 80 mL test medium. Fully characterized (content, physico-chemical characteristics) ISO Medium was used as test medium.
The immobilisation of the test animals was recorded 24 and 48 hours after test start. Environmental conditions were recorded at the start and at the end of each renewal period. All measured values remained within the acceptable ranges.
There was no immobilisation in twenty daphnids exposed to either test item treated or control group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected. All validity criteria were met and therefore the study can be considered as valid.
Reference
Results of the Preliminary Range-finding Tests
Nominal concentrations |
Preliminary |
|
Untreated |
100 |
|
Number of treated animals |
10 |
10 |
Number of immobilised animals (at 48h) |
0 |
0 |
pH-values Measured during the Experiment
Test Group |
Replicate |
1strenewal period |
2ndrenewal period |
|||
0 h |
24 h |
24 h |
48 h |
|||
Control |
1 |
8.07 |
8.13 |
7.83 |
8.12 |
|
2 |
8.16 |
8.12 |
||||
3 |
8.17 |
8.12 |
||||
4 |
8.18 |
8.11 |
||||
Saturated test |
1 |
8.13 |
8.12 |
7.80 |
8.08 |
|
2 |
8.13 |
8.06 |
||||
3 |
8.12 |
8.06 |
||||
4 |
8.12 |
8.07 |
Dissolved Oxygen Concentrations Measured during the Experiment
Test Group |
Replicate |
1strenewal period |
2ndrenewal period |
|||
0 h |
24 h |
24 h |
48 h |
|||
Control |
1 |
8.51 |
8.20 |
8.33 |
8.60 |
|
2 |
8.20 |
8.42 |
||||
3 |
8.18 |
8.39 |
||||
4 |
8.22 |
8.51 |
||||
Saturated test |
1 |
7.47 |
8.16 |
8.65 |
8.47 |
|
2 |
8.27 |
8.38 |
||||
3 |
8.11 |
8.32 |
||||
4 |
8.29 |
8.34 |
Temperatures Measured during the Experiment
Test Group |
Replicate |
1strenewal period |
2ndrenewal period |
|||
0 h |
24 h |
24 h |
48 h |
|||
Control |
1 |
20.6 |
20.4 |
20.5 |
20.2 |
|
2 |
20.4 |
20.2 |
||||
3 |
20.5 |
20.1 |
||||
4 |
20.4 |
20.3 |
||||
Saturated test |
1 |
20.6 |
20.5 |
20.5 |
20.3 |
|
2 |
20.5 |
20.1 |
||||
3 |
20.4 |
20.2 |
||||
4 |
20.3 |
20.3 |
Immobilization
of the Test Animals
Test Group |
Replicate |
Number of |
Number of immobilised animals |
|
24 h |
48 h |
|||
Control |
1 |
5 |
0 |
0 |
2 |
5 |
0 |
0 |
|
3 |
5 |
0 |
0 |
|
4 |
5 |
0 |
0 |
|
Saturated test |
1 |
5 |
0 |
0 |
2 |
5 |
0 |
0 |
|
3 |
5 |
0 |
0 |
|
4 |
5 |
0 |
0 |
Description of key information
In a Daphnia sp. Acute Immobilisation Test according to OECD Guideline 202 with a WAF, the test item Leuco Sulfur Blue 9 had no toxic effect up to aquatic saturation (i.e. limit test concentration); the 48h LOEC is higher than the solubility level of the test item in the test medium, which corresponds to a mean measured concentration of 1.27 mg/L (based on total product). The NOEC value is equal to the mean measured test item concentration of 1.27 mg/L (based on total product).
Key value for chemical safety assessment
Additional information
Acute toxicity of the test item Leuco Sulfur Blue 9 was assessed on Daphnia magna in a 48-hour Acute Immobilisation Test. Young Daphnia were exposed to aqueous test media containing the test item for 48 hours in a semi-static test. A limit test was performed in which the test organisms were exposed to aqueous test media containing the test item for 48 hours at the limit of its solubility in the test medium (i.e. saturation) plus a control in order to demonstrate that the test item has no influence on the mobility of Daphnia up to at least the saturation concentration (equivalent to 100 mg/L nominal concentration, prepared as water-accommodated fraction (WAF)).
The quantification of the test item Leuco Sulfur Blue 9 was performed by a previously validated analytical method by the analytical laboratory of TOXI-COOP ZRT. Samples were taken from the test concentration and the control at the start and at the end of each renewal period of the experiment and analysed by UV/VIS spectrophotometric method. The mean determined exposure concentration was 1.27 mg/L (based on total product) which was calculated as the geometric mean of the measured start and end concentrations of both renewal periods.
Healthy, young female daphnids < 24 hours of age and with known origin, history and acclimatisation conditions were used. Twenty Daphnia (divided into 4 replicates) were tested each, exposed to the saturated test concentration and in the control. Each test vessel contained approximately 80 mL test medium. Fully characterized (content, physico-chemical characteristics) ISO Medium was used as test medium.
The immobilisation of the test animals was recorded 24 and 48 hours after test start. Environmental conditions were recorded at the start and at the end of each renewal period. All measured values remained within the acceptable ranges.
There was no immobilisation in twenty daphnids exposed to either test item treated or control group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected. All validity criteria were met and therefore the study can be considered as valid.
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