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Diss Factsheets
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EC number: - | CAS number: -
- 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
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Skin irritation / corrosion
Administrative data
- Endpoint:
- skin corrosion: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2 December 2008 - 5 December 2008
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 009
- Report date:
- 2009
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Test material form:
- other: liquid (not specified)
- Details on test material:
- Description: dark brown liquid
Storage conditions: room temperature in the dark
Constituent 1
Test animals
- Species:
- other: Reconstructed human epidermis model
- Strain:
- other: human-derived epidermal keratinocytes
- Details on test animals or test system and environmental conditions:
- The EPISKIN model is a three-dimensional reconstituted human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
EPISKIN Model Kit
Supplier: SkinEthic Laboratories, Nice, France
Test system
- Type of coverage:
- other: not applicable
- Preparation of test site:
- other: not applicable
- Vehicle:
- unchanged (no vehicle)
- Amount / concentration applied:
- 50 µL.
- Duration of treatment / exposure:
- Exposure periods of 3, 60 and 240 minutes.
- Observation period:
- Not applicable.
- Number of animals:
- Not applicable.
- Details on study design:
- PRE-TEST
Assessment of Direct Test Material Reduction of MTT
MTT Dye Metabolism, Cell Viability Assay:
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 purple formazan dye by mitochondrial succinate dehydrogenase in viable cells.
Assessment of Direct Test Material Reduction of MTT
One limitation of the assay is possible interference of the test material with MTT. A test materialmay directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test material is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test material 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.
Test for Direct MTT Reduction:
The test material was checked for the ability to directly reduce MTT according to the procedure below:
50 µL of the test material was added to 2.2 mL of a 0.3 mg/mL MTT solution (v/v) freshly prepared in assay medium. The solution was incubated in the dark at room temperature for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test material turns blue/purple relative to the control, the test material was presumed to have reduced the MTT.
PRE-INCUBATION (Day 0: tissue arrival):
2.2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into two wells of the first column of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (2 units per plate). A different 12-well plate was used for each test material, control and time point. The tissues were incubated at 37 °C, 5 % CO2 in air for at least 24 hours.
MAIN TEST
APPLICATION OF TEST MATERIAL AND RINSING (Day 1)
2.2 mL of assay medium, warmed to approximately 37 °C, was pipetted into 2 wells of the second and third columns of the 12-well plate. The tissues were transferred into the second column.
Duplicate tissues were treated with the test material for exposure periods of 3, 60 and 240 minutes. Duplicate tissues were treated with the positive and negative control items for an exposure period of 240 minutes. 50 µL of the test material was applied topically to the corresponding tissues ensuring uniform coverage of the tissues. 100 µL of 0.9 % w/v sodium chloride solution was added for wetting of the test material. Duplicate tissues, treated with 50 µL of 0.9 % w/v sodium chloride solution served as negative controls. Duplicate tissues, treated with 50 µL of glacial acetic acid served as positive controls. The treated tissues were kept in a biological safety cabinet at room temperature for the appropriate exposure period.
At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test material. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed.
2.2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into 2 wells of the fourth column of each 12 well plate. The tissues were transferred into the MTT filled wells. The tissues were incubated for 3 hours ± 5 minutes at room temperature in a biological safety cabinet ensuring that the plates were protected from light. At the end of the 3 hour incubation period each tissue was placed onto absorbent paper to dry. The tissues were examined and the degree of MTT staining evaluated (qualitative evaluation of cell viability).
Following qualitative evaluation of tissue viability, a total biopsy of the epidermis was taken using the EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labelled 1.5 mL micro tubes containing 850 µL of acidified isopropanol. Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals out of the MTT-loaded tissues.
ABSORBANCE/OPTICAL DENSITY MEASUREMENTS (Day 2)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.
For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as 'blanks'. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.
INTERPRETATION OF RESULTS
Quantitative MTT Assessment (percentage tissue viability)
The corrosivity potential of the test material was predicted from the relative mean tissue viabilities obtained after the 3, 60 and 240-minute treatments, compared to the mean of the negative control tissues (n = 2) treated with 0.9 % w/v sodium chloride solution. The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD540 of test material / mean OD540 of negative control) x 100
Classification of corrosivity potential was based on relative viabilities for each exposure time according to the prediction model:
Treatment Time Relative Mean Tissue Viability Prediction EU Risk Phrase UN Packing Group
(minutes) (percentage of negative control)
3 <35 Corrosive R35 I
3/60 ≥35 / <35 Corrosive R34 II
60/240 ≥35 / <35 Corrosive R34 III
240 ≥35 Non-Corrosive No label Non-Corrosive
QUALITY CRITERIA
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Positive Control:
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was 0 to 20 % relative to the negative control treated tissues following the 240-minute exposure period.
Results and discussion
In vivo
- Irritant / corrosive response data:
- Test Material, Positive Control Material and Negative Control Material:
Individual and Mean OD540 Values and Viabilities for the Negative Control, Positive Control Material and Test Material are given in Table 1.
The relative mean viability of the test material treated tissues was as follows:
240 minutes exposure: 17.2 %
60 minutes exposure: 76.3 %
3 minutes exposure: 107.7 %
The qualitative evaluation of tissue viability is given in Table 2.
Following the 3 and 60-minutes exposure periods the test material treated tissues appeared blue which was considered to be indicative of viable tissue. Following the 240-minute exposure period the test material treated tissues appeared blue/white which was considered to be indicative of semi-viable tissue. - Other effects:
- Direct MTT Reduction
The MTT solution containing the test material did not turn blue/purple. This was taken to indicate the test material did not reduce MTT.
Quality Criteria
The relative mean tissue viability for the positive control treated tissues was 4.1 % relative to the negative control treated tissues following the 240-minute exposure period. The positive control acceptance criterion was therefore satisfied.
Any other information on results incl. tables
Table 1 Individual and Mean OD540 Values and Viabilities
Material |
Exposure Period (minutes) |
Mean OD540 of Duplicate Tissues |
Relative Mean % Viability |
Negative control |
240 |
0.169 |
100* |
Positive control |
240 |
0.007 |
4.1 |
Test material |
240 |
0.029 |
17.2 |
60 |
0.129 |
76.3 |
|
3 |
0.182 |
107.7 |
*The mean viability of the control tissues is set at 100 %.
Table 2 Qualitative Evaluation of Tissue Viability (MTT Uptake Visual Assessment)
Material |
Exposure Period (minutes) |
Tissue 1 |
Tissue 2 |
Negative control |
240 |
- |
- |
Positive control |
240 |
++ |
++ |
Test material |
240 |
+ |
+ |
60 |
- |
- |
|
3 |
- |
- |
- = Blue tissue (viable)
+ = Blue/white tissue (semi-viable)
++ = Tissue completely white (dead)
Applicant's summary and conclusion
- Interpretation of results:
- corrosive
- Remarks:
- Migrated information Criteria used for interpretation of results: EU
- Conclusions:
- The test material was considered to have the potential to be corrosive to the skin and accredited the EU risk phrase R34 and UN packing group III.
- Executive summary:
The potential corrosivity of the test material was investigated in vitro in accordance with the standardised guideline OECD 431 using the EPISKIN reconstituted human epidermis model. Tissues were treated with the test material for periods of 3, 60 and 240 minutes and the relative mean viability of the tissues assessed.
The relative mean viability of the test material treated tissues was as follows:
240 minutes exposure: 17.2 %
60 minutes exposure: 76.3 %
3 minutes exposure: 107.7 %
The test material was considered to have the potential to be corrosive to the skin and accredited the EU risk phrase R34 and UN packing group III.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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