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EC number: 944-884-4 | 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
Skin irritation / corrosion
Administrative data
- Endpoint:
- skin corrosion: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- April 23rd to 27th, 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:
- adopted 28th July 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Deviations:
- no
- Principles of method if other than guideline:
- Additional information was taken from:
- ECVAM international validation study on in vitro tests for acute skin irritation: “Report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Function Test” (Altern Lab Anim. 2007 Dec; 35 (6): 559-601).
- Protocol for In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT), Rev. 07/11/2014, MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, Bratislava - Slovakia - GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- Calcium fluoride hydroxide phosphate (5 : 0.6-0.8 : 0.2-0.4 : 3)
- EC Number:
- 944-884-4
- Molecular formula:
- Ca5(PO4)3(F)0.6-0.8(OH)0.2-0.4
- IUPAC Name:
- Calcium fluoride hydroxide phosphate (5 : 0.6-0.8 : 0.2-0.4 : 3)
Constituent 1
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200-SIT
- Tissue batch number(s): 28600
- Delivery date: 24. Apr. 2018
CHEMICALS AND MEDIA
- MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (=MTT), which can be reduced to a blue formazan. A MTT stock solution of 5 mg/mL in DPBS buffer was prepared and stored in aliquots of 2 ml in the freezer (– 20 ± 5 °C). 2 ml of the stock solution were thawed and diluted with 8 ml of medium. This MTTsolution with the resulting concentration of 1 mg/ml was used in the test. For the pre-test (testing the ability of direct MTT reduction), the stock solution was thawed and diluted with serum-free MEM directly before use. For the main test, the stock solution was thawed and diluted with assay medium directly before use.
- MEM with Phenol Red for Pre-Test Serum-free MEM (Minimum Essential Medium), procured by Life Technologies GmbH, batch no.: 1880322
- Assay Medium Serum-free DMEM (Dulbecco’s Modified Eagle’s Medium), procured by MatTek In Vitro Life Science Laboratories, batch no.: 041918TMA.
- DPBS-buffer: solution for the rinsing of the tissues and solvent for MTT concentrate, also used as negative control. A subset was procured by MatTek In Vitro Life Science Laboratories; the other subset was prepared by the laboratory.
Composition of the subset from MatTek In Vitro Life Science Laboratories (batch no.: 092817MGKA): KCl 0.2 g, KH2PO4 0.2 g, NaCl 8.0 g, Na2HPO4 * 7H2O 2.16 g, H2O ad 1 l.
Composition of the subset from the laboratory (batch no.: 20171114): KCl 0.2 g, KH2PO4 0.2 g, NaCl 8.0 g, Na2HPO4 * 2H2O 1.44 g, H2O ad 1 l.
The buffer which was procured by MatTek Corporation was used as negative control and for rinsing the test item from the tissues. The buffer which was prepared by the laboratory was used as solvent for MTT concentrate and for rinsing the outside of the inserts at the end of the incubation time with MTT.
ASSESSMENT OF COLOURED OR STAINING TEST ITEMS: it was tested whether the test item develops a colour without MTT addition. 26.8 mg test item were given in a test tube with 0.3 ml H2O demin. and incubated at 37 ± 1°C and 5.0 ± 0.5 % CO2 and 80-100 % relative humidity for 1 hour.
The resulting solution was colourless, therefore no binding capacity had to be tested.
ASSESSMENT OF DIRECT REDUCTION OF MTT BY THE TEST ITEM: the test item was tested for the ability of direct MTT reduction. To test for this ability, 25.6 mg test item were added to 1 ml of MTT solution and the mixture was incubated in the dark at 37 ± 1 °C and 5.0 ± 0.5 % CO2 for 1 hour. Untreated MTT medium was used as control. The MTT solution did not change its colour within 1 hour. Therefore, direct MTT reduction by the test item had not taken place and no data correction was necessary.
PRE-INCUBATION OF TISSUES
All working steps were performed under sterile conditions. For each treatment group (negative control, test item and positive control) a 6-well-plate was prepared with 0.9 ml assay medium in 3 of the 6 wells (upper row). The tissues were inspected for viability. Then, the tissues were transferred into the wells, which contain medium by using sterile forceps and placed into the incubator at 37 ± 1 °C and 5 ± 0.5 % CO2 and 80-100 % relative humidity for 1 hour. After 1 hour pre-incubation, the other 3 wells of each plate (lower row) were filled with fresh assay medium (0.9 ml). Every tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator at 37 ± 1 °C and 5.0 ± 0.5 % CO2 and 80-100 % relative humidity for 18 hours and 55 min.
TREATMENT
One plate (3 tissues) was used as negative control; each tissue was treated with 30 μl DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue surface. One plate (3 tissues) was used as positive control; each tissue was treated with 30 μl 5 % SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface. One plate (3 tissues) was used for treatment with the test item: the tissues were wetted with 25 μl DPBS buffer before applying the test item and spreading it to match the tissue size.
Tissues were dosed in 1-minute-intervals. 25 min after dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ± 1 °C and 5.0 ± 0.5 % CO2 and 80-100 % relative humidity. 1 hour after the first application, the inserts were removed from the plates in 1-minute intervals using sterile forceps and rinsed immediately. After rinsing thoroughly with DPBS, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 ml). The surface of the inserts was then carefully dried with a sterile cotton tipped swab. Then, the tissues were set in the incubator for 23 hours 25 minutes at 37 ± 1°C and 5.0 ± 0.5 % CO2 and 80-100 % relative humidity.
MEDIUM RENEWAL
After post-incubation, the tissues were removed from the incubator and shaken for 5 minutes (120 rpm). 0.9 ml assay medium were filled in the lower row of the 6-wellplate. Then the inserts were transferred into the lower row of the 6-well-plate and set into the incubator for 19 hours 20 minutes for post-incubation at 37 ± 1 °C and 5.0 ± 0.5 % CO2 and 80-100 % relative humidity.
MTT Assay
After a total incubation time of 42 hours 45 minutes, a 24-well-plate was prepared with 300 μl freshly prepared MTT-solution (1 mg/ml) in each well. The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 hours at 37 ± 1 °C and 5.0 ± 0.5 % CO2 and 80-100 % relative humidity. After this time, the MTT-solution was aspirated and replaced by DPBS buffer. This was then aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 ml isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken (120 rpm) for 2 hours at room temperature. After 2 hours, the inserts were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 μl solution (each) were pipetted into a 96-wellplate which was read in a plate spectrophotometer at 570 nm.
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive or irritant to skin if the viability after the exposure is less than or equal to 50 %
- The test substance is considered to be non-irritant to skin if the viability after the exposure is greater than 50 % - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amounts applied: tissue 1 - 25.2 mg, tissue 2 - 25.8 mg, tissue 3 - 25.0 mg.
NEGATIVE CONTROL
- Amount applied: 30 μl
POSITIVE CONTROL
- Amount applied: 30 μl - Duration of treatment / exposure:
- 1 hour
- Duration of post-treatment incubation (if applicable):
- 23 hours 25 minutes
- Number of replicates:
- 3
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 1
- Value:
- 123.5
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- The mean value of relative tissue viability of the test item was increased to 123.5 % after the treatment. This value is above the threshold for skin irritation (50 %). Therefore, the test item is considered as non-irritant to skin
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: OD for negative control 1.2. validity criteria: ≥ 0.8 and ≤ 2.8
- Acceptance criteria met for positive control: % tissue viability of positive control SDS: 4.1 %, validity criteria: ≤ 20 % of negative control
- Acceptance criteria met for variability between replicate measurements: 9.8 % (negative control), 0.2 % (positive control), 3.0 % (test item), validity criteria: ≤ 18 %
- Values for negative control and for positive control were within the range of historical data of the test facility
Any other information on results incl. tables
As blank, the optical density of isopropanol was measured in 8 wells of the 96-well-plate.
Replicate | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | Mean |
Absorbance | 0.039 | 0.038 | 0.039 | 0.039 | 0.037 | 0.037 | 0.039 | 0.038 | 0.038 |
The absorbance values of negative control, test item and positive control are given in the following table
Designation | Measurement | Negative Control |
Test substance | Positive control |
Tissue 1 | 1 | 1.239 | 1.498 | 0.083 |
2 | 1.259 | 1.584 | 0.084 | |
Tissue 2 | 1 | 1.321 | 1.481 | 0.088 |
2 | 1.340 | 1.534 | 0.089 | |
Tissue 3 | 1 | 1.099 | 1.448 | 0.089 |
2 | 1.103 | 1.491 | 0.088 |
From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol The mean of the three tissues was also calculated.
Designation | Negative control | Test substance | Positive control |
Mean – blank (tissue 1) | 1.211 | 1.503 | 0.046 |
Mean – blank (tissue 2) | 1.293 | 1.470 | 0.051 |
Mean – blank (tissue 3) | 1.063 | 1.432 | 0.051 |
Mean of the three tissues | 1.189 | 1.468 | 0.049 |
Comparison of Tissue Viability: for the test item and the positive control, the following percentage values of tissue viability were calculated in comparison to the negative control
Designation | Test substance | Positive control |
% Tissue viability (tissue 1) | 126.4 % | 3.9 % |
% Tissue viability (tissue 2) | 123.6 % | 4.3 % |
% Tissue viability (tissue 3) | 120.4 % | 4.3 % |
% Tissue viability (mean) | 123.5 % | 4.1 % |
± SD of mean tissue viability (%) | 3.0 % | 0.2 % |
Applicant's summary and conclusion
- Interpretation of results:
- other: not classified for skin irritation according to the CLP Regulation (EC) No.1272/2008
- Conclusions:
- Non irritant to skin
- Executive summary:
The potential of the substance to evoke skin irritation was evaluated in-vitro in a reconstructed human epidermis (RhE) test method according to the OECD Guideline 439 and EU Method B.46. Three tissues of the human skin model EpiDermTM were treated with the substance for 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2). DPBS-buffer was used as negative control and 5 % SDS solution was used as positive control.
After treatment with the negative control, the mean absorbance value was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.2. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 4.1 % (required: ≤ 20 %). The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤ 18 %).
After the treatment with the test item, the mean value of relative tissue viability was increased to 123.5 %. This value is above the threshold for skin irritation potential (50 %). Test items that induce values above the threshold of 50% are considered non-irritant to skin.
Therefore, the substance is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.
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