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EC number: 213-448-9 | CAS number: 950-33-4
- 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
Endpoint summary
Administrative data
Description of key information
skin irritation (OECD 439): not irritating
eye irritation (MatTek’s EpiOcular (ET50) protocol): not irritating
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 17 - 22 Sep 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP Guideline Study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- adopted 26 July 2013
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: EpiDerm™ (Epi-200)
- Source strain:
- not specified
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 19674 Kit B
- Delivery date: 17 Sep 2014
- Date of initiation of testing: 17 Sep 2014
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 min in the incubator; thereafter at room temperature for 25 min in a sterile hood
REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least 3 times. Afterwards the inserts were once again rinsed with DPBS from the inside and the outside.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT was prepared on the day of testing using MTT-100 Assay Kit Components.
- Incubation time: 42 h
- Spectrophotometer: microplate reader (Versamax, Molecular Devices, Softmax Pro v.4.7.1)
- Wavelength: 570 ± 1 nm
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed by undertaking an MTT cell viability test.
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.5 h.
- Contamination: The cells used to produce the EpiDerm tissue were screened for the presence of viruses, bacteria, yeast and other fungi.
NUMBER OF REPLICATE TISSUES: 3
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Since the test substance did not directly reduce MTT, an additional test with freeze-killed tissues was not performed.
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 1 hour exposure is less than 50%. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount applied: 30 µL
NEGATIVE CONTROL
- Amount applied: 30 µL
POSITIVE CONTROL
- Amount applied: 30 µL - Duration of treatment / exposure:
- 60 min
- Duration of post-treatment incubation (if applicable):
- approximately 42 h
- Number of replicates:
- triplicates for each treatment and control group
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 60 min exposure
- Value:
- 101
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other:
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: The test substance was not considered to be a MTT reducer.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.742, 1.808 and 1.904) was in the range of ≥ 0.8 and ≤ 2.8 for 60 minutes treatment, thus showing the quality of tissues.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control to 6.3% thus confirming the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The standard deviations of the % variabilities of the test substance, the positive and negative controls were < 10%, thus ensuring the validity of the study. - Interpretation of results:
- other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
- Conclusions:
- In the skin irritation study in vitro, the treatment with the test substance resulted in a cell viability of 101% and thus the test substance does not possess any skin irritating potential.
Reference
Table 2. Results after treatment
Dose Group |
Treatment Interval |
Absorbance 570 nm Tissue 1* |
Absorbance 570 nm Tissue 2* |
Absorbance 570 nm Tissue 3* |
Mean Absorbance of 3 Tissues |
Rel. Absorbance [%] Tissue 1, 2 + 3** |
Relative Standard Deviation [%] |
Mean Rel. Absorbance [% of Negative Control]*** |
Negative Control |
60 min |
1.742 |
1.808 |
1.904 |
1.818 |
95.8; 99.5; 104.7 |
4.5 |
100.0 |
Positive Control |
60 min |
0.126 |
0.117 |
0.103 |
0.115 |
6.9; 6.4; 5.7 |
9.7 |
6.3 |
Test Item |
60 min |
1.889 |
1.740 |
1.877 |
1.835 |
103.9; 95.7; 103.2 |
4.5 |
101.0 |
* Mean of three replicate wells after blank correction
** relative absorbance per tissue [rounded values]: (100 x (absorbance tissue)) / (mean absorbance negative control))
*** relative absorbance per treatment group [rounded values]: (100 x mean absorbance test item/positive control) / (mean absorbance negative control)
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour. The mean relative absorbance value of the test item, corresponding to the cell viability, was not reduced (101.0%; threshold for irritancy: ≤ 50%), consequently, the test item was not irritant to skin.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 18 - 19 Sep 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: according to MatTek’s EpiOcular (ET50) protocol, equivalent to MatTek’s EpiOcular (EIT) protocol under current (2014) ECVAM validation and GLP. Study was perfomed before release of OECD 492 in July 2015.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
- Version / remarks:
- Jul 2015
- Deviations:
- yes
- Remarks:
- study was performed before publication of OECD 492; i.e. there are deviations in application of the test and control substance and in tissue viability measurements
- Principles of method if other than guideline:
- The study was performed according to MatTek’s EpiOcular (ET50) protocol, equivalent to MatTek’s EpiOcular (EIT) protocol under current ECVAM validation.
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Species:
- human
- Strain:
- other: EpiOcular™
- Details on test animals or tissues and environmental conditions:
- TEST EYE MODEL
- Source: MatTek Corporation, Ashland, USA
- Lot No.: 19185
TEST METHOD
The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The test consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT, present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.
ADAPTATION TO CELL CULTURE CONDITIONS
At least 1 hour before dosing, EpiOcular™ tissues were removed from the refrigerator. Under sterile conditions using sterile forceps, the inserts were transferred into cell culture plates containing the pre-warmed assay medium. Additional cell cultures plates containing an adequate volume of medium were prepared as holding plates. The holding plates were pre-warmed in an incubator until use.
INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37 ± 1.5
- CO2 gas concentration (%): 5 ± 0.5 - Vehicle:
- unchanged (no vehicle)
- Controls:
- other: The negative control was deionised water and 0.3% Triton X-100 solution was used as positive control.
- Amount / concentration applied:
- TEST MATERIAL
- Amount applied: 100 µL
POSITIVE CONTROL SUBSTANCE
- Positive control substance: 0.3% Triton X-100 solution in deionised water
- Amount applied: 100 µL
NEGATIVE CONTROL SUBSTANCE
- Negative control substance: deionised water
- Amount applied: 100 µL - Duration of treatment / exposure:
- Test substance: 3, 30 and 60 min
Positive control: 15 and 45 min
Negative control: 60 min - Observation period (in vivo):
- not applicable
- Number of animals or in vitro replicates:
- not applicable
The test was performed in duplicates for each treatment and control group. - Details on study design:
- TEST SITE
- Area of exposure: 0.6 cm²
REMOVAL OF TEST SUBSTANCE
- Washing: The tissues were gently rinsed with PBS to remove any residual test material.
- Time after start of exposure: 3, 30 and 60 min (test substance); 15 and 45 min (positive control); 60 min (negative control)
- Post-treatment incubation period: at least 10 minutes but not longer than 20 minutes
CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed after the incubation period. Therefore, tissues were incubated in 300 µL freshly prepared MTT-reagent for 3 h at 37 ± 1.5 °C and 5 ± 0.5% CO2. After aspiration of the MTT reagent, tissues were rinsed three times with PBS. Extraction of the formazan product was carried out in 2 mL isopropanol for 18.5 h. The optical density was measured at 570 nm wave length in a microplate reader. - Irritation parameter:
- other: rel. absorbance (% of negative control) / mean value of 2 tissues
- Run / experiment:
- 3 min exposure
- Value:
- 112
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation parameter:
- other: rel. absorbance (% of negative control) / mean value of 2 tissues
- Run / experiment:
- 30 min exposure
- Value:
- 117.9
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation parameter:
- other: rel. absorbance (% of negative control) / mean value of 2 tissues
- Run / experiment:
- 60 min exposure
- Value:
- 94.8
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritant / corrosive response data:
- The relative absorbance values of the test item, corresponding to the cell viability, did not or did only irrelevantly decrease (94.8%) compared with the result of the negative control, consequently the test item was classified as non irritant.
Due to the lack of cytotoxicity, an ET50 value could not be calculated. - Interpretation of results:
- other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
- Conclusions:
- Under the conditions of the human cornea model test the test substance does not possess any eye irritating potential.
Reference
Table 2. Results after treatment with the test substance.
|
Treatment interval (min) |
Mean Absorbance of |
Rel. Absorbance |
Negative control |
60 |
1.1600 |
100.0 |
Positive control |
15 |
0.6920 |
59.7 |
45 |
0.3262 |
28.1 |
|
Test substance |
3 |
1.2988 |
112.0 |
30 |
1.3678 |
117.9 |
|
60 |
1.0992 |
94.8 |
* Mean of three replicate wells after blank correction
ET50 of the test item = could not be calculated since viability was not reduced below 50%
ET50 of the positive control = 24.2 min
Optical evaluation of the MTT-reducing capacity of the test substance after 1 h incubation with MTT-reagent did not show blue colour.
Both acceptability criteria of the assay were met:
The corrected mean OD values of the two tissues exposed to the negative control were ≥ 0.8 (1.2024 and 1.1175).
The ET50-value of the positive control is determined during the exposure period ≤ 30 min (24.2 min).
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for selection of skin irritation / corrosion endpoint:
There is only one study available.
Justification for selection of eye irritation endpoint:
There is only one study available.
Justification for classification or non-classification
The available data on skin and eye irritation do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.
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