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EC number: 228-794-6 | CAS number: 6359-10-0
- 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
Skin irritation / corrosion
Administrative data
- Endpoint:
- skin corrosion: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- 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 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
- Version / remarks:
- July 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EU Method B.40Bis (In Vitro Skin Corrosion: Human Skin Model Test)
- Version / remarks:
- February 2017
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- Disodium 2-(11-oxido-3-oxo-3H-dibenzo[c,h]xanthen-7-yl)benzoate
- EC Number:
- 228-794-6
- EC Name:
- Disodium 2-(11-oxido-3-oxo-3H-dibenzo[c,h]xanthen-7-yl)benzoate
- Cas Number:
- 6359-10-0
- Molecular formula:
- C28H14Na2O5
- IUPAC Name:
- disodium 2-(11-oxido-3-oxo-3H-dibenzo[c,h]xanthen-7-yl)benzoate
- Details on test material:
- - Physical appearance: Black powder
- Aggregate State at RT: Solid
- Bacht No./ Lot. No.: 5811022884
- Date of expiry: June 2019
Constituent 1
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Justification for test system used:
- The Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SCT) was selected as test system to assess the skin corrosion potential of the test item as it represents a recommended in vitro test system according to OECD Guideline No. 431.
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200-SCT) obtianed from MatTek In Vitro Life Science Laboratories, s.r.o, MlynskéNivy 73, 821 05, Bratislava II, Slovak Republic.
- Tissue batch number(s): 28644
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing with sterile PBS (filling and emptying of insert 20 times in a constant soft stream of PBS)
- Observable damage in the tissue due to washing: No
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Wavelength: 750 nm
NUMBER OF REPLICATE TISSUES: 2
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item is considered as MTT reducer as it formed dark black colour solution with MTT. Hence, additional live and freeze killed tissue controls were maintained with and without MTT in duplicates. All assay procedures were performed as for the viable tissue but controls without MTT tissue were incubated with medium instead of MTT solution during the MTT incubation step.
- Method of calculation used:
Calculations for Data Correction Procedure for Colored Test Item:
OD = OD colored tissue (MTT assay) – OD colored tissue (no MTT assay)
In 3 minutes application, as the extract from tissues treated by colored substance (test item detected in step 1) has an OD <5 % of the negative control treated tissue, correction of the OD was not considered.
Correction of results were considered for 1 hour application as the OD of extract from tissues treated by colored substance was >5 % of the negative control treated tissue.
Check for direct MTT reducing substances:
To check whether test item directly reduces MTT or not, 25 mg of the test item was added to 1 mL of the MTT medium in a 6-well plate and incubated in the incubator (37±1 °C, 5±1 % CO2) for 60 minutes. Untreated MTT medium was used as control. Post incubation, MTT solution containing test item turned to dark blue color, hence considered as reducer of MTT. As test item reduced MTT, a functional check using freeze-killed tissue controls (killed controls = KC) was performed in one definitive assay to evaluate whether the test item is not binding to the tissue and leading to a false MTT reduction signal. All assay procedures are performed as for the viable tissue.
Calculations for Data Correction Procedure for MTT reducers:
True viability = Viability of treated tissue – Interference from test chemical
= OD tvt – OD kt
where OD kt = (mean OD tkt – mean OD ukt)
tvt = treated viable tissue
kt = killed tissues
tkt = treated killed tissue
ukt = untreated killed tissue (NC treated tissue)
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied: 25 mg
NEGATIVE CONTROL
- Amount(s) applied: 50 µL
POSITIVE CONTROL
- Amount(s) applied: 50 µL - Duration of treatment / exposure:
- 3 min or 1 h
- Number of replicates:
- 2
Results and discussion
In vitro
Resultsopen allclose all
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 3 minutes exposure (mean value of tissue 1 and 2)
- Value:
- 108.87
- Vehicle controls validity:
- not examined
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 1 hour exposure (mean value of tissue 1 and 2)
- Value:
- 13.44
- Vehicle controls validity:
- not examined
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- - OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: Yes
- Colour interference with MTT: Yes. The test item is considered as MTT reducer as it formed dark black colour solution with MTT.
DEMONSTRATION OF TECHNICAL PROFICIENCY:
The technical proficiency of the test method was established by using proficieney chemicals under Bioneeds Study No.: BIO-GT 1000. according to OECD test Guideline No. 431.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
Any other information on results incl. tables
Table 1: Summary of optical density (OD) and viability
3 Minutes Exposure
Treatment |
|
OD |
Viability (%) |
CV (%) |
Classification |
Negative Control (Sterile water) |
Mean |
1.105 |
100.0 |
8.68 |
NC |
±SD |
0.096 |
12.2 |
|||
n |
2 |
2 |
2 |
||
Positive Control (Glacial acetic acid) |
Mean |
0.083 |
7.5 |
11.55 |
C (Category 1A) |
±SD |
0.010 |
1.2 |
|||
n |
2 |
2 |
2 |
||
Test Item |
Mean |
1.203 |
108.87 |
- |
NC |
±SD |
- |
- |
|||
n |
2 |
2 |
OD (killed tissue) |
= |
Mean OD of treated killed tissue |
- |
Mean OD of untreated killed tissue |
0.161 |
= |
0.205 |
- |
0.045 |
|
||||
True Viability |
= |
OD of treated viable tissue |
- |
OD of killed tissue |
1.042 |
= |
1.203 |
- |
0.161 |
1 Hour Exposure
Treatment |
|
OD |
Viability (%) |
CV (%) |
Classification |
Negative Control (Sterile water) |
Mean |
1.838 |
100.00 |
0.40 |
NC |
±SD |
0.007 |
0.6 |
|||
n |
2 |
2 |
2 |
||
Positive Control (Glacial acetic acid) |
Mean |
0.065 |
3.6 |
1.08 |
C (Category 1A) |
±SD |
0.001 |
0.1 |
|||
n |
2 |
2 |
2 |
||
Test Item |
Mean |
0.197 |
7.40 |
- |
C |
±SD |
- |
- |
|||
n |
2 |
2 |
NC = Non Corrosive; C = Corrosive; n = No. of tissues; SD = Standard Deviation; CV = Coefficient of variation
Data Correction for Colored substance |
= |
OD of Colored tissue (with MTT) |
- |
OD of Colored tissue (Without MTT) |
1.070 |
= |
1.197 |
- |
0.127 |
|
|
|
|
|
OD (killed tissue) |
= |
Mean OD of treated killed tissue |
- |
Mean OD of untreated killed tissue |
0.822 |
= |
0.890 |
- |
0.067 |
|
|
|
|
|
True Viability |
= |
OD of treated viable tissue (MTT Assay) |
- |
OD of killed tissue |
0.147 |
= |
1.203 |
- |
0.161 |
True viability (%) = 13.44%
Acceptance Criteria:
1. The assay met the acceptance criterion as
the mean OD750 of the negative control tissues are 1.105 (after 3
minutes exposure) and 1.838 (after 1 hour exposure), which are in the
range of ≥0.8 and ≤2.8.
2. The assay met the acceptance criterion as the mean viability of
positive control tissues is 7.5 % (after 3 minutes exposure) and 3.6 %
(after 1 hour exposure) which were ≤15 % of the
negative control tissues and the SD of the three tissues replicates is
0.0 to 12.2.
3. The assay met the acceptance criterion as the OD of the extraction
solvent was ≤0.1.
4. Two tissue replicates per exposure time was in range between 0.40 and
11.55 % viability the coefficient of variation (CV).
Applicant's summary and conclusion
- Interpretation of results:
- Category 1 (corrosive) based on GHS criteria
- Conclusions:
- Based on the results obtained under the conditions of this study, the test item is considered as corrosive to reconstructed Human Epidermis (RhE) in accordance with UN GHS category 1, as the mean percentage tissue viability was greater than 50 % after 3 minutes exposure and less than 15 % after 1 hour exposure of the negative control.
- Executive summary:
In an in vitro skin corrosion assay (RhE) according to OECD Guideline 431, the potential of the test item to induce skin corrosion in an in vitro human skin model was investigated. The test item developed dark blue colour when dissolved in distilled water and isopropanol. The test item is considered as MTT reducer as it formed dark black colour solution with MTT. Hence, additional live and freeze killed tissue controls were maintained with and without MTT in duplicates. All assay procedures were performed as for the viable tissue but controls without MTT tissues were incubated with medium instead of MTT solution during the MTT incubation step.
Exposure to the test item was performed for 1 hour and 3 minutes separately. Tissues were treated with 25 mg test item or 50 µL of positive control (glacial acetic acid). At the end of treatment time, tissue inserts were rinsed with sterile PBS to remove any residual test item. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm. Viabilily of tissues was calculated.
For 3 minutes exposure, percentage viability of negative control, positive control and test item was 100±12.2, 7.5±1.2 and 108.87, respectively. Correction procedure for colorant control was not considered as the mean OD of extract from tissues treated by colored test item was less than 5 % of negative control. As the percentage viability of test item was greater than 50 % of the negative control, the test item is considered as non-corrosive, whereas the percentage viability of positive control (PC) is less than 15 % of negative control and clearly represents the irritation potential of positive control.
For 1 hour exposure, percentage viability of negative control, positive control and test item was 100±0.6, 3.6±0.1 and 13.44. The percentage viability of the test item was less than 50 % of negative control. Therefore, the test item is considered as corrosive. The percentage viability of positive control (PC) is less than 15 % of negative control clearly represents the irritation potential of positive control.
Based on the results obtained under the conditions of this study, the test item is considered as corrosive to Reconstructed Human Epidermis (RhE) in accordance with UN GHS category 1, as the mean percentage tissue viability was greater than 50 % after 3 minutes exposure and less than 15 % after 1 hour exposure of the negative control.
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