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EC number: 235-518-8 | CAS number: 12262-26-9 This substance is identified in the Colour Index by Colour Index Constitution Number, C.I. 53450.
- 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
Eye irritation
Administrative data
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- August 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
- Version / remarks:
- July, 2013
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- 2,5-cyclohexadien-1-one, 4-[[4-(phenylamino)phenyl]imino]-, reaction products with sodium sulfide (Na2(Sx)), leuco derivatives
- EC Number:
- 235-518-8
- EC Name:
- 2,5-cyclohexadien-1-one, 4-[[4-(phenylamino)phenyl]imino]-, reaction products with sodium sulfide (Na2(Sx)), leuco derivatives
- Cas Number:
- 12262-26-9
- Molecular formula:
- not applicable
- IUPAC Name:
- Reaction product of 2,5-cyclohexadien-1-one, 4-[[4-(phenylamino)phenyl]imino]- with polysulfide, leuco derivatives
- Test material form:
- solid
- Details on test material:
- Test item: Leuco Sulfur Blue 13
Appearance: Bluish black, solid
CAS No: 12262-26-9
Constituent 1
Test animals / tissue source
- Species:
- cattle
- Strain:
- not specified
- Details on test animals or tissues and environmental conditions:
- SOURCE OF COLLECTED EYES
- Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Characteristics of donor animals: at least 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were stored in HBSS containing 1 % (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- Indication of any antibiotics used: Yes
Test system
- Vehicle:
- physiological saline
- Controls:
- yes, concurrent vehicle
- yes, concurrent positive control
- Amount / concentration applied:
- The test item was tested as a 20 % suspension (w/v) in saline using sonication for 10 minutes. Prior to treatment of the corneae the pH-value of the test item solution or suspension was determined (pH 7.65).
TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: The test item was tested as a 20 % suspension (w/v) in saline using sonication for 10 minutes.
VEHICLE
- Amount applied: 0.75 mL
- Concentration: Saline (0.9 % NaCl in deionised water) - Duration of treatment / exposure:
- 240 minutes
- Number of animals or in vitro replicates:
- 3 corneae per group (test item, negative control, positive control)
- Details on study design:
- SELECTION AND PREPARATION OF CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. The corneae were directly used in the BCOP test on the same day.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OEDC guideline 437, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0).
QUALITY CHECK OF THE ISOLATED CORNEAS
The basal opacity of all corneae was recorded. Each corneae with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test item and the negative and positive controls.
NUMBER OF REPLICATES
3 corneae per group (test item, negative control, positive control)
SOLVENT CONTROL USED
Saline (0.9 % NaCl in deionised water)
POSITIVE CONTROL USED
10 % (w/v) benzalkonium chloride in 0.9 % (w/v) NaCl (saline)
APPLICATION DOSE AND EXPOSURE TIME
0.75 mL, 240 min
TREATMENT METHOD:
closed chamber
POST-INCUBATION PERIOD:
no
REMOVAL OF TEST SUBSTANCE
the test item or the control items, respectively, were each rinsed off from the according application sides with saline.
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: via opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France))
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:
DECISION CRITERIA
IVIS: In vitro Irritancy Score (according to OECD 437):
≤ 3 No Category (according to GHS)
> 3; ≤ 55 No prediction can be made
> 55 Serious eye damaging according to CLP/EPA/GHS (Cat 1)
Results and discussion
In vitro
Results
- Irritation parameter:
- in vitro irritation score
- Remarks:
- mean
- Run / experiment:
- 1-3
- Value:
- 1.7
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
Any other information on results incl. tables
Table 1: Results after 240 Minutes Treatment Time
Test Group |
Opacity value = Difference (t240-t0) of Opacity |
Permeability at 490 nm (OD490) |
IVIS |
Mean IVIS |
Proposedin vitroIrritancy Score |
Standard Deviation of in vitro Score |
||
|
|
Mean |
|
Mean |
|
|
|
|
Negative Control |
0 |
0.00 |
0.054 |
0.057 |
0.81 |
0.86 |
Not categorized |
0.04 |
0 |
0.059 |
0.89 |
||||||
0 |
0.058 |
0.87 |
||||||
Positive Control |
121.00* |
0.019* |
121.29 |
119.53 |
Category 1 |
2.91 |
||
116.00* |
0.011* |
116.17 |
||||||
121.00* |
0.009* |
121.14 |
||||||
Test Item |
2.00* |
-0.004* |
1.94 |
1.70 |
Not categorized |
0.57 |
||
1.00* |
0.003* |
1.05 |
||||||
2.00* |
0.007* |
2.11 |
*corrected values
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In an in vitro eye irritation assay (BCOP) according to OECD 437, the test item did not show eye irritating properties.
- Executive summary:
The eye irritating properties of the test item were assessed in a BCOP assay using fresh bovine corneae, according to OECD guideline 437.
After an initial opacity measurement of the fresh bovine corneae (t0), the 20 % (w/v) suspension in saline of the test item, the positive, and the negative controls were applied to the different corneae and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase, the test item as well as the positive and the negative controls were each rinsed off from the corneae and opacity was measured again (t240). After the opacity measurements, permeability of the corneae was determined by measuring the transfer of sodium fluorescein spectrophotometrically after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.
For the negative control (saline, 0.9 % NaCl) neither an increase of opacity nor permeability of the corneae was observed (mean IVIS =0.86). The positive control (10 % (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS =119.53) corresponding to a classification as serious eye damaging (UNGHS Category 1). All acceptability criteria were fulfilled.
The test item was tested as suspension. Relative to the negative control, the test item did not cause a relevant increase of the corneal opacity. The calculated mean IVIS was 1.70 (threshold for serious eye damage: IVIS > 55). According to the OECD guideline 437, the test item is not categorized (UN GHS No Category).
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