Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2016-05-11 to 2016-05-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
zirconium(IV) oxide, stabilised with erbium(III) oxide, gadolinium(III) oxide and yttrium(III) oxide
EC Number:
946-001-8
Molecular formula:
(Er2O3)w (Gd2O3)x (Y2O3)y (ZrO2)z: w/w% Er2O3 >= 1 <= 8 w/w% Gd2O3 >= 1 <= 8 w/w% Y2O3 >= 1 <= 8 w/w% ZrO2 >= 76 <= 97
IUPAC Name:
zirconium(IV) oxide, stabilised with erbium(III) oxide, gadolinium(III) oxide and yttrium(III) oxide
Test material form:
solid: particulate/powder
Details on test material:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PEGYZ-23
- Expiration date of the lot/batch: 31 March 2021
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 ºC, below 70% relative humidity), protected from light.

OTHER SPECIFICS
Description: pink powder (the colour was determined by visual inspection upon arrival at test facility)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personal health and safety. Possibly irritative.
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: not applicable, the test item was applied in its original form, no formulation was required

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none, the test item was applied in its original form, no formulation was required

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified (adult)
Source strain:
other: not applicable
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
- Model used: EPISKIN Small Model
- Manufacturer: SkinEthic, France
- Batch No.: 16-EKIN-019
- Expiry Date: 2016-05-16
- This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been 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. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
- EPISKIN (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.

KIT CONTENTS
- Units: EPISKIN (SM) plate containing 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.
- Plate: 12-well assay plate
- Punch: EPISKIN (SM) biopsy punch for easy sampling of epidermis.
- Medium: A flask of sterile “Maintenance Medium” (Batch No.: 16 MAIN3 031; Exp. Date: 04 May 2016) and a flask of sterile “Assay Medium” (Batch No.: 16 ESSC 017; Exp. Date: 04 May 2016)
- The kits were found to be in good order at reception.

STORAGE CONDITIONS
- The EPISKIN TM (SM) kits were kept in their packaging at 37°C; the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8°C until the initiation of the test.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Disks of EPISKIN (SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature (24.8-25.8°C). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light.
- All incubations were carried out in a humid atmosphere (> 95%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C. Temperature and humidity were continuously monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variations to these conditions were evaluated and maintained in the raw data.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the 15 minutes incubation time, the EPISKIN (SM) units were removed and rinsed thoroughly with Phosphate Buffered Saline (PBS) to remove any remaining material from the epidermal surface as much as possible. To remove the test item that was stuck on the surface of the epidermis, additional rinsing was used. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis). After rinsing the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours (+/- 5 min)
- Spectrophotometer: plate reader
- Wavelength: 570 nm
- After the 42 hours incubation, all EPISKIN (SM) units (except of two 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 EPISKIN (SM) units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light.

FORMAZAN EXTRACTION
- Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04 N HCl (1.8 mL of 12 N HCl acid was diluted in 500 mL isopropanol, or similar ratio was applied). The solution was prepared on the day of use.
- After the incubation with MTT, 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.

CELL VIABILITY MEASUREMENTS
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: 240 72800, Serial Number: 0920-14, Date of calibration: 02 September 2014, calibration is valid until September 2016) at the required wavelength on each day before use.

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test item is considered to be non-irritant to skin if the mean relative viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post incubation is more than 50% of the mean viability of the negative controls.




Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 20 mg (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)

NEGATIVE CONTROL (PBS)
- Amount applied: 50 µL

POSITIVE CONTROL (5% SDS)
- Amount applied: 50 µL
Duration of treatment / exposure:
15 +/- 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours +/- 1 hour
Number of replicates:
Three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay. Furthermore, as the test item was coloured, two additional test item-treated tissues were used for the non-specific OD evaluation.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
relative mean tissue viability compared to the negative control tissues
Value:
86.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
relative viability of each test item sample: 77.7, 80.3, 101.2%
Other effects / acceptance of results:
Additional controls:
As no colour change (yellow colour) was observed after three hours of incubation of the test items in MTT working solution, the test materials did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability could be excluded.

As the test item was coloured, two additional test item-treated tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of tissues was 0.027. Non Specific Colour % was calculated as 3.2%. This value was below 5%, therefore additional data calculation was not necessary.

Validity results:
- The mean OD value of the three negative control tissues was in the recommended range (0.836). Standard deviation of the viability results for negative control samples was 4.9.
- The positive control treated tissues showed 7.4% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.3.
- The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 12.9.
- 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.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Following exposure to erbium gadolinium yttrium zirconium oxide, the mean relative viability was 86.4% compared to the negative control value. This is above the threshold of 50%, therefore the test item was considered being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.