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EC number: 228-779-4 | CAS number: 6358-57-2
- 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 June 19th to June 21th, 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
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
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Version / remarks:
- 2012
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- Acid Red 111
- IUPAC Name:
- Acid Red 111
Constituent 1
In vitro test system
- Test system:
- isolated skin discs
- Source species:
- human
- Cell type:
- other: Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
- Justification for test system used:
- The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purpose of distinguishing between skin irritating and no-skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- -Pre-incubation: the "maintenance medium" was pre-warmed to 37°C. The appropriate number of an assay plate wells were filled with the pre-warmed medium (2 ml per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37±1°C in an incubator with 5±1 % CO2, >/=95 % humidified atmosphere.
-Application: three replicates were used for the test item and positive and negative controls respectively. Test item: the epidermal surface was first moistened with 5 µl deionised water in order to improve further contact between powder and epidermis. Subsequently, approximately 10 mg of the test item was applied evenly to the epidermal surface. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necesssary. Positive and negative control: a volume of 10 µl positive control (SDS 5 % aq.) or negative control (1x PBS) was applied on the skin surface by using a suitable pipette. Chemicals were gently spread with the pipette trip in order to cover evenly all the epidermal surface if necessary. Additional controls for dyes and chemicals able to colour the tissue: in addition to the normal procedure, two additional test item treated tissues were used for the non-specific OD evaluation (NSCliving).
-Exposure: the plates with the treated epidermis units were incubated for the exposure time of 15 minutes (±0.5 min) at room temperature.
-Rinsing: after the incubation time, the EpiSkin SM units were removed and rinsed thoroughly with approximately 25 ml PBS 1x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).
-Post-incubation: after rinsing the units were placed into the plate wells with fresh pre-warmed "maintenance medium" (2ml/well) below them and then incubated for 42 hours (±1 h) at 37±1°C in an incubator with 5±1 % CO2 , >/= 95 % humidified atmosphere.
-MTT test after 42 hours incubation: after the 42 hours (±1 h) incubation, the EpiSkin Sm units were transferred into the MTT solution filled wells ( 2 ml of 0.3 mg/ml MTT per well) and then incubated for 3 hours (± 5 min) at 37±1°C in an incubator with 5±1 % CO2 protected from light, >/=95 % himidified atmosphere.
-Formazan extraction: at the end of incubation with MTT a formazan extraction was undertaken: a disk of epidermis was cut from the units (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µl acidified isopropanol (one tube corresponding to one well of the tissue culture plate). The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated for approximately four hours at room temperature protected from light with gentle agitation (ca. 150 rpm) for formazan extraction. At the middle and at the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.
-Cell viability measurements: following the formazan extraction, 2x200 µl sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the OD (Absorbance/Optical Density) of the samples in a 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC) at 570 nm (±10 nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752) using acidified isopropanol solution as the blank (6×200 µl). - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- 10 mg
- Duration of treatment / exposure:
- 15 minutes ±0.5 min
- Duration of post-treatment incubation (if applicable):
- Epidermis units were incubated for 42 hours (±1 h)
- Number of replicates:
- three
Results and discussion
In vitro
Resultsopen allclose all
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Replicate 1
- Value:
- 92
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Replicate 2
- Value:
- 97
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Replicate 3
- Value:
- 93
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- The mean OD value of the three negative controls tissues was 0.951. The mean OD value obtained for the positive control was 0.204 and this result corresponds to 21 % viability when compared to the reults obtained from the negative control. Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.
Possible direct MTT reduction with test item: no colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore addittional controls and data calculations were not necessary. A false estimation of viability can be precluded.
Colouring potential of test item: the test item has an intrinsic colour red. If the test item is not white, off white, almost white and/or colorless the addtional controls are automatically used and based on the OD results of additional controls the Non Specific Colour % (NSCliving %) is determined. This step prevents the false viability, which can possibly be caused by the stained surface of the tissues by the test item. Two addtional test item-treated tissues were used for the non.specific OD evaluation. The mean OD (measured at 570 nm) of these tissues was determined as 0.060. The NSCliving % was calculated as 6.3 %. Therefore, additional data calculation was necessary.
Any other information on results incl. tables
OD values and viability percentages of the controls
Substance | Optical Density (OD) | Viability (%) | |
Negative Control: | 1 | 1.002 | 105 |
1x PBS | 2 | 0.899 | 94 |
3 | 0.952 | 100 | |
mean | 0.951 | 100 | |
standard deviation (SD) | 5.46 | ||
Positive Control: | 1 | 0.238 | 25 |
SDS (5 % aq.) | 2 | 0.197 | 21 |
3 | 0.178 | 19 | |
mean | 0.204 | 21 | |
standard deviation (SD) | 3.2 |
OD values and viability percentages of the test item (including corrected values)
Test Item | Optical Density (OD) | TODTT | Viability (%) | Relative Viability (%) | |
Acid Red 111 | 1 | 0.871 | 0.811 | 92 | 85 |
2 | 0.921 | 0.861 | 97 | 90 | |
3 | 0.885 | 0.824 | 93 | 87 | |
mean | 0.892 | 0.832 | 94 | 87 | |
standard deviation (SD) | 2.68 | 2.68 |
TODTT: OD true MTT metabolic conversion for treated tissues
OD values and NSCliving% of additional control
Additional colour control | Optical Density (OD) | Non Specific Colour %(NSCliving %) | |
Acid Red 111 (test item treated tissues without MTT incubation) |
1 | 0.052 | 6.3 |
2 | 0.068 | ||
mean | 0.06 |
The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (</=) to 50 % of the negative control.
Applicant's summary and conclusion
- Interpretation of results:
- other: According to CLP Regulation (EC) 1272/2008 criteria the test item do not need to be classified.
- Conclusions:
- In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean corrected value:87 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non.irritant to skin. Positive and negative controls showed the expected ODs, cell viability values were within acceptable limits and standard deviations of all calculated viability values (positive and negative control, test item) were below 18. The experiment was considered to be valid.
The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilisied testing conditions. The test item is considered to be non-irritant to skin and is therefore not classified.
According to CLP Regulation (EC) 1272/2008 criteria the test item do not need to be classified. - Executive summary:
EpiSkin SM test of the test item has been performed to predict its irritation potential by measurements of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 28 July 2015.
Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes (±0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1 °C for 42 hours (±1 h) in an incubator with 5±1 % CO2, >/= 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (±5 min) with MTT solution at 37±1°C in 5±1 % CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.
The test item has an intrisic colour (red), therefore two additional test item treated tissues were used for the non-specific OD evaluation (NSCliving).
SDS (5 % aq.) and 1x PBS treated (three units/positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.
The test chemical is identified as requiring classification and labelling according to UN GHS (category 2 or category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (</=) to 50 % of the negative control.
In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean corrected value: 87 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.
Positive and negative controls showed the expected ODs, cell viability values were within acceptable limits and standars deviations of all calculated viability values (positive and negative control, test item) were below 18. The experiment was considered to be valid.
The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item is considered to be non-irritant to skin and is therefore not classified.
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