Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 July 2017 - 15 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Willing New Material Technology Co.,Ltd.; 23161211201
- Expiration date of the lot/batch: 10 December 2017
- Purity: 97%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 ºC)

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Adult donors
Justification for test system used:
The EPISKINTM (SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
EPISKINTM (SM) (Manufacturer: SkinEthic, France, Batch No.: 17-EKIN-032, Expiry
Date: 14 August 2017) is a three-dimensional human epidermis model. The EPISKINTM (SM) plate contains up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.

EPISKINTM (SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EPISKINTM (SM) test kits used in the present study) and are documented in Appendix 2.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (23.2-27.6°C).
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the 15 minutes incubation time, the EPISKINTM (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).

- Observable damage in the tissue due to washing: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solution (prepared on 08 August 2017) was stored in refrigerator (2-8°C) protected from light. It was diluted with pre-warmed (37°C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.

After the 42 hours incubation, all EPISKINTM (SM) units (except the two living colour control units) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred EPISKINTM (SM) units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light, in a >95% humidified atmosphere.

After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate). The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.

The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 24072800, Serial Number: 0920-14, Date of calibration: 22 August 2016, calibration is valid until August 2018) at the required wavelength on each day before use.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Check-method for possible direct MTT reduction with test item
10 mg of test item was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in a shaking water bath for 3 hours protected from light, and then any colour change was recorded:
-Test items which do not react with MTT: yellow
-Test items reacting with MTT: blue or purple
After three hours incubation, yellow colour of the mixture was detected in the test tube. Thus, the test item did not react with MTT and therefore the use of additional controls was not necessary.

Check-method to detect the colouring potential of test-items

Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol* (simulating a tissue humid environment).
*Note: Water is the environment during exposure, isopropanol is the extracting solution.
Therefore, in addition to the normal procedure, two additional test item-treated living tissues were used for the non specific OD evaluation. This tissue followed the same test item application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item that may be present in the test disks. OD reading was conducted following the same conditions as for the other tissues.


PREDICTION MODEL / DECISION CRITERIA
The test item considered to be irritant to skin (Category 2), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control. If mean relative viability after 15 minutes exposure and 42 hours post incubation is greater than or equal (≥) to 50% of the negative control, the substance is not classified.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
As the test item was solid, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis and then 10 mg of the test item was applied evenly to the epidermal surface.

NEGATIVE CONTROL
50 μL of negative control (PBS) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).

POSITIVE CONTROL
50 μL positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
Duration of treatment / exposure:
15 minutes (± 0.5 min)
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
TiBTD
Value:
93.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: SD (%)
Remarks:
4.6
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Positive Control
Value:
8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: SD (%)
Remarks:
2.3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Negative Control
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: OD (%)
Remarks:
3.8
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None
- Direct-MTT reduction: As no colour change was observed after three hours of incubation of the test item in MTT solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary to exclude the false estimation of viability.

- Colour interference with MTT: As the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of tissues were 0.008, Non Specific Colour % was calculated as 1.1% (see Table 1). This value was below 5%, therefore additional data calculation was not necessary.

ACCEPTANCE OF RESULTS:
After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.

The mean OD value of the three negative control tissues (0.751) was in the recommended range of 0.6 and 1.5. Standard deviation of the viability results for negative control samples was 3.8%.

The positive control treated tissues showed 8.0% viability, which is in the recommended range of 0 – 40%, demonstrating the proper performance of the assay.

The standard deviation of the viability results for positive control samples was 2.3%.

The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 4.6%.

The mean OD value of the blank samples (acidified isopropanol) was 0.047.

All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Historical control data are presented in Appendix 3.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this in vitro EPISKINTM (SM) model test with TiBTD, the results indicate that the test item is a non-irritant to skin.
Executive summary:

In an in vitro skin irritation assay in a human epidermal model EPISKINTM (SM) (17_053-043B), water-moistened reconstructed human epidermis tissue was exposed to 10 mg of TiBTD (97%) for 15 minutes (± 0.5 min). PBS was used for the negative control and 5% SDS was used for the positive control. After removal of the test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and a 2 hour isopropyl alcohol extraction period followed. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

The colour of the test substance did not interfere with the endpoint. The test substance is not directly MTT reducing. The average viability of tissues treated by the test substance TiBTD was 93.1 % of negative control average value i.e. viability was > 50 %. The average viability of tissues treated by the positive control (5% SDS) was 2.3 % of negative control average value. According to these results, the test substance is not irritating.

This in vitro skin irritation study in the human epidermal model EPISKINTM (SM) is acceptable and satisfies the guideline requirement for an OECD 439 study.