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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
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 May 16th to June 19th, 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- July 28th, 2015
- GLP compliance:
- yes
Test material
- Reference substance name:
- Reaction mass of dodecane-1-thiol and tridodecyl trithiophosphite
- Molecular formula:
- Not applicable - Multiconstituent substance
- IUPAC Name:
- Reaction mass of dodecane-1-thiol and tridodecyl trithiophosphite
- Test material form:
- liquid
Constituent 1
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: adult donors.
- Source strain:
- not specified
- Details on animal used as source of test system:
- SOURCE ANIMAL
- Source:Human adult donors. - Justification for test system used:
- The test system EPISKIN™ is a reconstructed human epidermis (RhE) model, which in its overall design (the use of human derived epidermis keratinocytes as cell source and use of representative tissue and cytoarchitecture) closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis.
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin Small model- 0.38 cm^2
- Tissue batch number(s):17-EKIN-024 (alive tissues); 17-EKIN-005 (killed tissues)
- Shipping date: Monday 12 June 2017 (alive tissues); Monday 30 January 2017 (killed tissues)
- Delivery date: Tuesday 13 June 2017 (alive tissues); Tuesday 31 January 2017 (killed tissues)
Note: at arrival the test system was examinated and it resulted suitable for use (temperature indicator:pale grey; pH indicator: orange). Tissues are prepared for the test as follows:
# Alive tissues: at arrival, plates were opened under a sterile airflow and each insert, containing the epidermal tissue, was carefully taken out and placed in a 12-well plate in which each well had previously been filled with 2 ml/well SkinEthic Maintenance Medium. Culture plates were placed in the incubator at 37 °C, 5 % CO2 and saturated humidity for approximately 24 hours.
# Killed tissues: a sufficient number of epidermis units were placed in a 12-well plate in which each well had previously been filled with 2 ml/well sterile water for injection. Tissues were incubated for approximately 48 hours, then transferred into a new plate and stored at -20 °C. The day of the experiment, tissues were thawed at room temperature with 2 ml of maintenance medium.
PRELIMINARY TESTS:
Before the Main Assay, preliminary tests were carried out to evaluate the compatibility of the test item with the test system.
- Direct-MTT reduction: 2 ml of MTT ready-to-use solution (0.3 mg/ml) was incubated with 20 μl of test item at 37 °C, 5 % CO2 and saturated humidity for 3 hours, simulating test conditions. Observation of blue or purple appearance of the solution at the end of the incubation time was carried out.
- Colour interference with MTT: 20 μl of the test item was added to 180 µl of distilled water in at transparent tube and the resulting solution/suspension mixed by using a vortex for 15 minutes. Colouring of the solution/suspension at the end of the incubation time was evaluated by spectral analysis at 595 nm.
MAIN TEST
In the Main Assay, the test item was applied as supplied in three replicates at the treatment level of 20 µl/epidermis unit, each measuring 0.38 cm^2 (treatment level: 53 µl/cm2). Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 µl/epidermis unit. In order to verify if the test item results had to be corrected, the non specific colour (NSCliving) was evaluated using two alive treated tissues without MTT staining and compared with the D-PBS control. Moreover, non specific MTT reduction (NSMTT) was evaluated using two killed tissues and compared with negative control performed with alive tissues. Since the test item is able both to stain tissue and reduce MTT, to avoid a possible double correction for colour interference, a third control for Non Specific Colour in killed tissue (NSCkilled) was performed.
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature.
- Temperature of post-treatment incubation (if applicable): 37 °C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: at the end of the exposure, each tissue was rinsed with approximately 25 ml of sterile D-PBS, filling and empting the tissue insert. The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2 ml/well of maintenance medium.
- Observable damage in the tissue due to washing: not observed
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 ml/well of MTT ready-to-use solution
- Incubation time: 3 hours at 37 °C, 5 % CO2 and saturated humidity. At the end of the incubation period, tissues were placed on absorbent paper to dry. A total biopsy was carried out by means of a biopsy punch to allow biopsies of the same dimensions. The epidermis were separated from the collagen matrix and both placed in a microtube prefilled with 500 µl of acidic isopropanol. Tubes were preserved for approximately 3 days at 4 °C to allow formazan extraction. At the end of the extraction period, debris were eliminated by short centrifugation of the tubes (14000 rpm for 2 minutes) and aliquots of 200 µl from each sample were read in duplicate for their absorbance at 595 nm. Optical Density (OD) values were recorded. Six aliquots (200µl) of acidic isopropanol were analysed and used as blank. In order to ensure the spectrophotometer linear range , an MTT formazan calibration curve was performed.
- Wavelength: the absorbance was evaluated at 595 nm.
NUMBER OF REPLICATE TISSUES: 3 replicates for negative control, positive control and test item applied on live tissues with MTT. 2 replicates for negative control applied on killed tissues. For the 3 additional controls (NSMTTkilled to evaluate direct MTT reduction; NSCliving to evaluate test item colouring potential; NSCkilled to avoid a double correction) 2 replicates of test item applied on killed tissues and 2 replicates of test item applied on live tissues without MTT were performed.
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
In the preliminary test the test item showed its ability of reducing MTT per se. For this reason non specific MTT reduction (NSMTT) was evaluated using two killed tissues and compared with negative control performed with alive tissues.
PREDICTION MODEL / DECISION CRITERIA
-The test substance is considered to be non-corrosive and non-irritant to skin if the viability after an exposure period of 15 minutes followed by 42 ± 1 hour recovery period is greater than 50%.
# For direct MTT interacting test items, non specific MTT reduction calculation (NSMTT) relative to the Negative Control is evaluated as follows:
NSMTT = 100 x (OD treated killed tissues - OD non-treated killed tissues) / OD negative control living tissues
If the NSMTT ≤ 5 % only blank subtraction is carried out.
If 5% < NSMTT ≤ 50 % blank and appropriate background subtraction is carried out.
If NSMTT> 50 % the test item is not suitable for this test method.
# For colouring test items, Non Specific Colour (NSCliving) relative to the D-PBS Control is evaluated as follows:
NSCliving = 100 x (OD test item not incubated with MTT)/ OD negative control living tissues
If the NSCliving ≤ 5 % only blank subtraction is carried out.
If 5% < NSCliving ≤ 50 % blank and appropriate background subtraction is carried out.
If NSCliving > 50 % the test item is not suitable for this test method.
# For test item able both to stain tissue and reduce MTT, a third control for Non Specific Colour in killed tissues:
NSCkilled = 100 x (OD test item treated killed tissues not incubated with MTT)/ OD negative control living tissues
If the [(NSMTT + NSCliving) - NSCkilled] ≤ 5 % this value is not considered for the final calculation.
If 5% < [(NSMTT + NSCliving) - NSCkilled] ≤ 50 % blank and appropriate background subtraction is carried out.
If [(NSMTT + NSCliving) - NSCkilled] > 50 % results should be taken with caution.
ACCEPTANCE CRITERIA
The assay was considered valid if the following criteria were met:
– Blank controls: mean OD value < 0.1.
– Negative controls: mean OD value ≥ 0.6 and ≤ 1.5, SD of % viability ≤ 18.
– Positive controls: mean viability expressed as percentage of the negative control ≤ 40% and SD of % viability ≤ 18.
– Test item: SD of % viability ≤ 18. - Control samples:
- other: Positive control: 5% (w/v) sodium dodecyl sulphate (SDS) solution; negative control: Dulbecco’s phosphate buffered saline (D-PBS; 3 additional controls because the test item can directly reduce MTT and it has colouring potential.
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit):20 µl/epidermis unit of liquid test item for each replicate (total number of replicates for live tissues with MTT: 3).
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 20 µl/epidermis unit of Dulbecco’s phosphate buffered saline (D-PBS) (total number of replicates for live tissues: 3; total number of replicates for killed tissues: 2).
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 20 µl/epidermis unit of 5%(w/v) sodium dodecyl sulphate (SDS) solution for each replicate (total number of replicates for live tissues: 3).
- Concentration (if solution): 5 %(w/v) sodium dodecyl sulphate (SDS) solution, obtained by1:1 dilution in sterile water of a sterile commercial 10 %(w/v) SDS solution in water.
3 ADDITIONAL CONTROLS (NSMTTkilled to evaluate direct MTT reduction; NSCliving to evaluate test item colouring potential; NSCkilled to avoid a double correction)
- Amount(s) applied (volume or weight with unit): 20 µl/epidermis unit of liquid test item for each replicate (total number of replicates for killed tissues or for live tissues without MTT: 2). - Duration of treatment / exposure:
- 15 ± 0.5 minutes in a ventilated cabinet at room temperature.
- Duration of post-treatment incubation (if applicable):
- 42 ± 1 hours with incubation at 37°C, 5% CO2 and saturated humidity.
- Number of replicates:
- 3 replicates for negative control, positive control and test item applied on live tissues with MTT.
2 replicates for negative control applied on killed tissues. For the 3 additional controls (NSMTTkilled to evaluate direct MTT reduction; NSCliving to evaluate test item colouring potential; NSCkilled to avoid a double correction) 2 replicates of test item applied on killed tissues and 2 replicates of test item applied on live tissues without MTT were performed.
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Mean of 3 replicates (B1, B2, B3) for test item (for each replicate the reading of absorbance was performed in duplicate) with appropriate background subtractions.
- Value:
- ca. 73
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- - RESULTS OF MAIN TEST:
The mean cell viability of test item treated tissues, after the subtractions of NSMTT, was 73 % when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 5.0 (lower than 18). Based on the results obtained, the test item Trilauryl trithiophosphite is classified as not irritant to the skin.
- OTHER EFFECTS:
- Direct-MTT reduction: before the main assay, the test item was evaluated for the ability of reducing MTT per se. At the end of the incubation period with the MTT, a violet gray solution with purple precipitate was observed, indicating that the test item could direct interact with MTT.
- Colour interference with MTT: before the main assay, the test item was evaluated for the ability of colouring water per se. A blank suspension was observed; spectral analysis of the test item in water, to evaluate the ability of the test chemical to absorb light at 595 nm, was performed. The value obtained for the Optical Density (OD) was 2.282, indicating that the test item has a potential interfering ability.
Based on the results obtained, additional controls were added in the Main Assay for the evaluation of Non Specific Colouring potential (NSCliving) and Non Specific MTT reduction (NSMTT). Since the test item was able both to stain tissue and reduce MTT, to avoid a possible double correction for colour interference, a third control for Non Specific Colour in killed tissue (NSCkilled) was performed.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for blank controls: the mean Optical Density of Blank Controls was 0.036, lower than the maximum acceptable value (0.1).
- Acceptance criteria met for negative control: the negative control gave the expected baseline value (mean Optical Density value equal or higher than 0.6) and variability (Standard Deviation of the % viability lower or equal to 18).
- Acceptance criteria met for positive control: the positive control caused the expected cell death (Mean of cell viability of the three replicates = 6 % - lower than the maximum acceptable value 40 %) and variability (SD of % viability for three replicates equal to 2.1 - lower than the maximum acceptable value 18 %).
- Acceptance criteria met for variability between replicate measurements: the mean cell viability of test item treated tissues, after the subtractions of NSMTT, was 73 % when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 5.0 -lower than the maximum acceptable value (18 %).
Based on the stated criteria, mean viability, expressed as percentage of the negative control, lower or equal to 40 % and standard deviation of % viability equal or lower than 18, the study was accepted as valid.
Note: The colouring interference (NSC) was 1 % and NSMTT value was 34 %.
Probably due to a technical oversight, an unreliable high NSMTT value was obtained for one of the killed tissues, while the OD value of the replicate tissue was coherent with the OD values obtained for killed tissues treated with the test item in the previous experiment. Therefore, these data were used for NSMTT evaluation and the calculated value was 34%. Based on these results, the appropriate background subtraction was performed. The final value mean cell viability for test item was 73%.
Applicant's summary and conclusion
- Interpretation of results:
- other: not classified as skin irritant, according to the CLP Regulation (EC n.1272/2008).
- Conclusions:
- The test item is identified as not irritant to the skin according to the CLP Regulation (EC n.1272/2008).
- Executive summary:
The potential of the test item to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD TG 439. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42±1 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability.
Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se. At the end of the incubation period with the MTT, a violet gray solution with purple precipitate was obtained, indicating that the test item could direct interact with MTT. In a second step, the test item was assayed for the ability of colouring water per se. A blank suspension was observed; spectral analysis of the test item in water, to evaluate the ability of the test chemical to absorb light at 595 nm, was performed. The value obtained for the Optical Density (OD) was 2.282, indicating that the test item has a potential interfering ability. Based on these results, additional controls were added in the Main Assay.
In the Main Assay, the test item was applied as supplied in three replicates at the treatment level of 20 µl/epidermis unit, each measuring 0.38 cm^2 (treatment level: 53 µl/cm^2). Positive and negative controls [a 5 % (w/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 µl/epidermis unit. In order to verify if the test item results had to be corrected, the non specific colour (NSCliving) was evaluated using two alive treated tissues without MTT staining and compared with the D-PBS control. Moreover, non specific MTT reduction (NSMTT) was evaluated using two killed tissues and compared with negative control performed with alive tissues. Since the test item is able both to stain tissue and reduce MTT, to avoid a possible double correction for colour interference, a third control for Non Specific Colour in killed tissue (NSCkilled) was performed.
In the Main Assay, the negative control gave the expected baseline value (mean Optical Density values equal or higher than 0.6) and variability [Standard Deviation (SD) of % viability lower or equal to 18], in agreement with the guideline indications. According to the method, the negative control mean value is considered the baseline value of the experiment and thus represents 100 % of cell viability.
The positive control caused the expected cell death (6 % of cell viability when compared to the negative control) and variability (SD of % viability equal to 2.1). Based on the stated criteria (mean viability 40% and SD of % viability 18), the assay was regarded as valid.
The colouring interference(NSC) was 1 %, while the non specific MTT reduction( NSMTT) was 34 %. Based on these results, the appropriate back ground subtraction was performed.
The test item did not induce cell death in any replicate, the mean cell viability of the test item treated tissues after overall subtractions was 73 % when compared to the negative control.Intrareplicate variability was acceptable with a SD of%viability value equal to 5.0 (lowerthan 18).
Based on the results obtained, the test item is classified as not irritant to the skin.
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