Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 21st to July 17th, 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:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 28th, 2015
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

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-028 (alive tissues) and 17-EKIN-014 (killed tissues)
- Delivery date: Tuesday 11 June 2017 (alive tissues); Tuesday 4 April 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 ±2 mg 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 ± 2 mg 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 mg/epidermis unit, each measuring 0.38 cm^2 (treatment level: 53 mg/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 (200 µl) 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 of negative control, positive control and test item applied on alive tissues with MTT. 2 replicates of negative control applied on killed tissues. Regarding 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 alive 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 mg/epidermis unit of solid test item for each replicate (total number of replicates for alive 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 alive 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 alive 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 mg/epidermis unit of liquid test item for each replicate (total number of replicates for killed tissues or for alive 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 hour recovery period by incubation at 37 °C, 5% CO2 and saturated humidity.
Number of replicates:
3 replicates for negative control, positive control and test item applied on alive tissues with MTT.
2 replicates for negative control applied on killed tissues. Regarding 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. 106
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
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 was 106 % when compared to negative control. The NSCliving value was 1 %, while the NSMTT was -2%. Based on these results, only the OD-blank background subtraction must be performed on mean cell viability of test item and therefore 106 % is the definitive value. Acceptable intra-replicate variability was obtained (SD of % viability = 8.9 lower than 18). The blank, negative and positive controls gave acceptable results and the study was accepted as valid. The test item Tetraphosphorus trisulphide 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. A dark purple suspension, with dark purple precipitate, was noted in the MTT solution at the end of the incubation period, 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 light yellow solution 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 1.395, 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 0.038, 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 = 4 % - lower than the maximum acceptable value 40 %) and variability (SD of % viability for three replicates equal to 0.8 - lower than the maximum acceptable value 18 %).
- Acceptance criteria met for variability between replicate measurements: the mean cell viability of test item treated tissues was 106 % when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 8.9 -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 (NSCliving) was 1 % and NSMTT value was - 2 %.

Applicant's summary and conclusion

Interpretation of results:
other: not classified as skin irritant according to the CLP Regulation (EC n.1272/2008).
Conclusions:
Tetraphosphorus trisulphide 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 Tetraphosphorus trisulphide 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 Guideline for testing of chemicals 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. Dark purple suspension with dark purple precipitate was noted in the MTT solution at the end of the incubation period, 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 light yellow solution 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 1.395, 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 mg/epidermis unit, each measuring 0.38 cm^2 (treatment level: 53 mg/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.

In the Main Assay, the negative control gave the expected baseline value (Optical Density values of the three replicates 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 (4% of cell viability when compared to the negative control) and variability (SD of % viability equal to 0.8). Based on the stated criteria (mean viability 40% and SD of % viability 18), the assay was regarded as valid.

The NSCliving value was 1%, while the NSMTT was -2 %, thus only the OD-blank background subtraction was performed.

The test item did not induce cell death in any replicate, the mean cell viability after the blank subtraction was 106 % when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 8.9 (lower than 18, as stated in the Study Protocol).

Based on the results obtained, the test item Tetraphosphorus trisulphide is classified as not irritant to the skin.