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EC number: 268-684-5 | CAS number: 68133-40-4
- 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 irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From January 18 to 26, 2018
- 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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Version / remarks:
- 2009
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- Reactive Yellow 084
- IUPAC Name:
- Reactive Yellow 084
- Test material form:
- solid: particulate/powder
Constituent 1
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Details on test system:
- The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Bratislava, Slovakia) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. The EpiDerm™ system is manufactured according to defined quality assurance procedures.
The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped as kits, containing tissues on shipping agarose together with the necessary amount of culture media.
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 25 min at room temperature, 35 min at 37 ±1 °C
- Temperature of post-treatment incubation: 37 ±1 °C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: two, the first after 1 h of exposure; the second, after 25 h and 7 min of post-incubation period
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Libra S22
- Wavelength: 570 nm
- Blank: isopropyl alcohol
- Filter bandwidth: 2-3 nm
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: one
PREDICTION MODEL / DECISION CRITERIA
The relative cell viability is calculated for each tissue as % of the mean of the negative control tissues viability, which is set at 100 %.
The cut-off values for the prediction of irritation are given below; these values are stated in OECD guideline 439, par. 36:
- in case test chemical is found to be non-corrosive (e.g., based on TG 430, 431 or 435), and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50 %, the test chemical is considered to be irritant to skin in accordance with UN GHS Category 2.
- test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.
A single testing run composed of three replicate tissues should be sufficient for a test chemical when the classification is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent viability equal to 50 ± 5 %, a second run should be considered, as well as a third one in case of discordant results between the first two runs.
ASSAY ACCEPTANCE CRITERIA
Negative control
The absolute OD of the negative control (NC) tissues (treated with sterile PBS) in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD570 of the NC tissues is ≥ 0.8 and ≤ 2.8. OD570 historical negative control range is 1.455-2.347.
Positive control
A 5 % SDS (in H2O) solution is used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be tested in each assay, but not more than one PC is required per testing day. Viability of the positive control should be within 95±1 % confidence interval of the historical data. The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20 %. The 95% confidence interval of historical positive control is 0-0.190.
Standard deviation (SD)
Each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, so the variability of tissue replicates should be acceptably low. The assay meets the acceptance criterion if the SD calculated from individual % tissue viabilities of the 3 identically treated replicates is < 18 %.
When any of the acceptance criteria is not met, the experiment has to be repeated. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- yes, concurrent MTT non-specific colour control
- Amount/concentration applied:
- 25 mg of test substance was dosed directly on previously moistened tissue. The substance was spread on the entire tissue surface. A single experiment, composed of three replicate tissues, was run.
- Duration of treatment / exposure:
- On the day of receipt, EpiDerm tissues were conditioned by incubation to release transport stress related compounds and debris. After 60 minutes of pre-incubation, medium was replaced by a fresh one and pre-incubation continued for other 20 hours 25 minutes.
After pre-incubations, tissues were topically exposed to the test chemicals for 60±1 minutes (25 minutes at room temperature and the remaining 35 minutes at culture conditions). Three tissues were used for test substance, three for positive (PC) and three for negative (NC) controls. Tissues were then thoroughly rinsed with PBS. Inserts were then transferred to fresh medium. - Duration of post-treatment incubation (if applicable):
- After 25 hours and 7 minutes of post-incubation period, the medium was replaced by fresh one. Tissues were incubated for another 18 hours, 37 minutes.
- Number of replicates:
- 3 tissues per test substance, 3 tissues per positive control, 3 tissues per negative control.
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 1
- Value:
- 84.8
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- - OTHER EFFECTS
- Direct-MTT reduction: 25 mg of the test substance was added to 1.0 ml of MTT medium. Solution was incubated for 1 hour at culture conditions. After incubation, the medium changed colour towards red. Test substance did not reduce MTT directly.
- Colour interference with MTT: test substance is insoluble in isopropyl alcohol – its colour was not changed after shaking with the test substance.
Mean value of blank (isopropyl alcohol) was 0.046. Mean value of the test substance isopropyl alcohol extract was also 0.046. The difference was 0.0 what is < 0.08, so colorant control tissues were not employed.
ACCEPTANCE OF RESULTS:
The mean OD570 of the NC tissue was 1.963 which meets the acceptance criteria of ≥ 0.8 and ≤ 2.8.
The mean viability of PC tissues expressed as % of negative control tissues was 2.6 % which meets the acceptance criterion of ≤ 20 %.
The SD calculated from individual % tissue viabilities of 3 identically treated replicates was 0.1 % for positive control, 2.2 % for negative control and 5.7 % for test substance, i.e. < 18 % in all cases.
All study acceptance criteria were fulfilled.
Any other information on results incl. tables
OD570 values in MTT test, standard deviation % and relative viabilities
treatment | OD570 | mean | SD | mean viability (% NC) | |||
1 | 2 | 3 | |||||
NC | PBS | 2.021 | 1.950 | 1.917 | 1.963 | 0.043 | 100 |
% | 103.0 | 99.4 | 97.7 | 100.0 | 2.2 | ||
sample | test substance | 1.802 | 1.527 | 1.663 | 1.664 | 0.112 | 84.8 |
% | 91.8 | 77.8 | 84.7 | 84.8 | 5.7 | ||
PC | 5 % SDS | 0.047 | 0.051 | 0.053 | 0.05 | 0.002 | 2.6 |
% | 2.4 | 2.6 | 2.7 | 2.6 | 0.1 |
Applicant's summary and conclusion
- Interpretation of results:
- other: not irritant according to the CLP Regulation (EC 1272/2008)
- Conclusions:
- Not irritant.
- Executive summary:
Method
Test substance was assayed for in vitro skin irritation in the human epidermal model EpiDermTM. The test was performed according to OECD guideline 439.
Direct MTT reduction and colour interference were assessed.
In main experiment after pre-incubation of tissues, 25 mg of test substance was placed directly on previously moistened tissues and spread on the entire tissue surface. Exposure length was 60 minutes. Three tissues were used for test substance as well as for positive and negative controls.
After removal of test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD570) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of mean viability of negative control tissues.
Results
No direct MTT reduction or colour interference were found.
Under the above-described experimental design mean viability of treated tissues was 84.8 %, i.e. viability was >50 %.
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