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EC number: 237-224-5 | CAS number: 13701-64-9
- 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:
- key study
- Study period:
- 4 December 2019 to 6 December 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 020
- Report date:
- 2020
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)
- Qualifier:
- according to guideline
- Guideline:
- other: Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006, 18 December 2006, of the European Parliament and of the Council on REACH
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- Reference substance 001
- Cas Number:
- 13701-64-9
- Test material form:
- solid
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: E00616-912
- Expiration date of the lot/batch: 18 September 2020
- Purity: 82 %
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: Human skin
- Details on animal used as source of test system:
- TEST SYSTEM:
- EpiDerm™ Reconstructed Human Epidermis Model Kit:
Supplier: MatTek In Vitro Life Sciences Laboratories
Date received: 03 December 2019
EpiDermTM Tissues (0.63cm2) lot number: 30838
Assay Medium lot number: 112819MJC
Upon receipt of the EpidermTM tissues, the sealed 24-well plate was stored in a refrigerator until use. - Justification for test system used:
- The purpose of this test was to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.
Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. Viable cells are able to reduce MTT to formazan whereas non-viable cells cannot. The results are used to make a prediction of the corrosivity potential of the test item (increased cytotoxicity is indicative of corrosion potential).
This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. - Vehicle:
- unchanged (no vehicle)
- Details on test system:
- PRE-TEST PROCEDURES:
- Assessment of Direct Test Item Reduction of MTT:
The MTT assay, a colourimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt by mitochondrial succinate dehydrogenase in viable cells. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem if at the time of the MTT test (after rinsing) there is still a sufficient amount of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.
To identify this possible interference, 25 mg of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control. If the MTT solution containing the test item turned blue/purple relative to the control, the test item was presumed to have reduced the MTT.
- Assessment of Colour Interference with the MTT Endpoint:
A test item may interfere with the MTT endpoint if it is coloured or if it becomes coloured when in wet or aqueous conditions. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed. 25 mg of test item was added to 300 µL of sterile water. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 60 minutes. A visual assessment of the colour was then made.
MAIN TEST:
- Pre-Incubation:
The assay medium was brought to room temperature before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labelled 6-well plates for both the 3-minute and 60-minute exposure periods. EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5 % CO2) for approximately 1 hour before dosing.
- Application of Test Item and Rinsing:
Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3-minute and 60-minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT-loading. Another 24-well plate was prepared for the MTT-loading. 300 µL of either pre-warmed assay medium (holding plate) or MTT medium (MTT-loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
After pre-incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3-minute exposure period was returned to the incubator, while the other was being dosed for the 60-minute exposure. For the 60-minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 25 mg of the test item and 50 µL of 8.0 N potassium hydroxide (positive control) were also applied to the corresponding tissues in turn. 25 µL of sterile water was added for wetting of the test item to increase tissue surface contact. The plate was returned to the incubator (37 °C, 5 % CO2) for the 60-minute exposure period.
When dosing for the 60-minute exposure period was complete, the same procedure was repeated for the 3-minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT-loading. The plate was incubated (37 °C, 5 % CO2) for 3 hours. Once the 60-minute exposure period was complete, the same rinsing and MTT-loading procedure was repeated.
After the 3-hour MTT incubation was complete, the tissue inserts were blotted and transferred to 24-well plates for formazan (reduced MTT) extraction. The formazan was extracted from the top and bottom of the tissue by completely immersing the tissue insert in 2 mL of isopropanol. The plate was covered with plate sealer, to prevent isopropanol evaporation, and stood overnight at room temperature, to allow extraction to proceed.
- Absorbance/Optical Density Measurements:
After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT-4500 microplate reader and LT-com analysis software. The microplate reader upper limit of accuracy for measured absorbance of MTT Formazan at 570 nm (OD570), filter band pass 10 nm, was determined to be at an optical density of 2.6.
DATA EVALUATION:
- Quantitative MTT Assessment (percentage tissue viability):
The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60-minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = [(mean OD570 of test item)/(mean OD570 of negative control)] x 100
ACCEPTANCE CRITERIA:
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved.
- Negative Control:
The absolute OD570 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD570 of the two negative control tissues should be >=0.8 and <=2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
- Positive Control:
Potassium hydroxide 8.0 N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60-minute positive control is <15 %.
- Coefficient of Variation:
In the range 20 and 100 % viability, the coefficient of variation between tissue replicates should be <=30 %. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied: 25 mg test item and 25 µL of sterile water was added for wetting of the test item to increase tissue surface contact
NEGATIVE CONTROL
- Amount(s) applied: 50 µL of sterile distilled water
POSITIVE CONTROL
- Amount(s) applied: 50 µL of 8.0 N Potassium Hydroxide - Duration of treatment / exposure:
- 3 and 60 minutes
- Number of replicates:
- 2
Results and discussion
In vitro
Resultsopen allclose all
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 3-Minute exposure period
- Value:
- 97
- Negative controls validity:
- valid
- Remarks:
- 100 %
- Positive controls validity:
- valid
- Remarks:
- 4.3 %
- Remarks on result:
- other: Non-corrosive
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 60-Minute exposure period
- Value:
- 104.5
- Negative controls validity:
- valid
- Remarks:
- 100 %
- Positive controls validity:
- valid
- Remarks:
- 3.8 %
- Remarks on result:
- other: Non-corrosive
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue/purple, this was taken to indicate the test item did not reduce MTT.
- Colour interference with MTT: The solution containing the test item did not become coloured, this was taken to indicate the test item did not have the potential to cause colour interference.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 2.162 for the 3-minute exposure period and 2.058 for the 60-minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 0.078 % relative to the negative control following the 60-minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100 % viability the coefficient of variation between the two tissue replicates of each treatment group did not exceed 30 %. The acceptance criterion was therefore satisfied.
Any other information on results incl. tables
Item | Exposure Period |
Mean OD570 of individual tissues |
Mean OD570 of duplicate tissues |
Standard Deviation |
Coefficient of Variation (%) |
Relative Mean Viability (%) |
Negative control |
3 minutes |
2.179 2.145 |
2.162 |
0.024 |
1.1 |
100 |
Negative control |
60 minutes |
1.984 2.131 |
2.058 |
0.104 |
5.1 |
100 |
Positive control |
3 minutes |
0.104 0.081 |
0.093 |
0.016 |
Not applicable |
4.3 |
Positive control | 60 minutes |
0.089 0.067 |
0.078 |
0.016 |
Not applicable |
3.8 |
Test item |
3 minutes |
2.022 2.171 |
2.097 |
0.105 |
5.0 |
97.0 |
Test item |
60 minutes |
2.104 2.196 |
2.150 |
0.065 |
3.0 |
104.5 |
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- The test item was considered to be non-corrosive to the skin.
- Executive summary:
The purpose of this test was to evaluate the corrosivity potential of the test item using the EpiDerm™ human skin model after treatment periods of 3 and 60 minutes.
Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. Viable cells are able to reduce MTT to formazan whereas non-viable cells cannot. The results are used to make a prediction of the corrosivity potential of the test item (increased cytotoxicity is indicative of corrosion potential).
Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).
The data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).
Negative control (3 minute exposure): 100 %
Positive control (3 minute exposure): 4.3 %
Test item (3 minute exposure): 97.0 %
Negative control (6 minute exposure): 100 %
Positive control (6 minute exposure): 3.8 %
Test item (6 minute exposure): 104.5 %
The acceptance criteria required for the test were satisfied and the test item was considered to be non-corrosive to the skin.
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