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Diss Factsheets

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-08 to 2018-03-19
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
Adopted, 28 July 2015
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Zirconium oxide, hafnium and ytterbium doped
EC Number:
Molecular formula:
Zirconium oxide, hafnium and ytterbium doped
Test material form:
Details on test material:
- Nam in test report: Ytterbium stabilized Zirconium and Hafnium Oxide
- EC No.: 945-888-9
- Batch No.: 7170502
- Physical state: solid
- Colour: white
- Purity: > 95%
- Expiry date: 26 October 2022
- Storage conditions: room temperature, in a tightly closed container in a dry place

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
- Justification of the test method: This in vitro method is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS “No Category”) and consequently require no further testing within a tiered testing strategy in contrast from those requiring classification and labelling (UN GHS categories 1 and 2). Therefore, it can be used for regulatory purposes as an initial step in the bottom-up approach or as one of the last steps in a top-down approach to test eye irritation/corrosion potential. It is not intended to differentiate between UN GHS “Category 1” (serious eye damage) and UN GHS “Category 2” (eye irritation) which would require additional testing. Ocular irritation potential is predicted by the relative viability of the tissue after a single exposure to the test substance. Relative viability is determined by measuring the MTT dye to formazan conversion by the EpiOcular™ tissue construct after topical exposure to the test substance.

- Description of the cell system used: The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium that is morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Amount(s) applied (volume or weight with unit): 50 mg
Duration of treatment / exposure:
6 h
Duration of post- treatment incubation (in vitro):
18 h
Number of animals or in vitro replicates:
2 tissues per dose group. 3 dose groups.
Details on study design:
- RhCE tissue construct used, including batch number : EpiOcular™ tissues on agarose (Lot No.: 27018, 27019)
- Pre-experiments:
- To check the non-specific MTT-reducing capability of the test item 50 mg of the test item was mixed per 1 mL MTT medium and incubated for 3 h in a humidified incubator at 37+/- 1 °C, 5.0% CO2 / 95% air. The part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues if the mean relative tissue viability of the test item treated tissues (TM) is above the 60% threshold value.
- For quantitative correction of results, two killed tissues were treated with 50 mg of the test item (KT) and one tissue was treated with 50 µL of the negative control (Aqua dest.; KU). NSMTT was calculated relative to the negative control of living tissues (NK) according to the following formula:
NSMTT [%] = [(ODKT - ODKU)/ODNK] * 100

- To check the colouring potential of the test item, 50 mg of the test item was mixed per 1 mL Aqua dest. or per 2 mL isopropanol each in a 6-well plate. The water solution was incubated for at least 1 h in a humidified incubator at 37 +/- 1 °C, 5.0% CO2 / 95% air. The isopropanol solution was shaken on a plate shaker for 2 to 3 h. After the respective incubation periods, 2 x 200 µL aliquots per test solution were transferred into a 96-well plate as well as using 200 µL Aqua dest. and isopropanol as respective blanks and OD was measured in a range of 570 ± 30 nm without reference wavelength in a plate spectrophotometer.

- For test items which act as non-specific MTT-reducers and show non-specific colouring of living tissues, a third control for non-specific colouring in killed tissues (NSCkilled) was performed to avoid a possible double-correction for colour interference. Therefore, two killed tissues were treated with 50 mg of the test item (TKT). For the MTT-staining of the test item treated with tissues, incubation was performed in medium without MTT. The non-specific colour of additional killed tissues (NSCkilled) was then calculated according to the following formula:
NSCkilled [%] = [ODTKT/ODNK]*100
- The true tissue viabilitywas then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSCliving plus NSCkilled.

- Experimental Procedure:
- Upon receipt of the EpiOcular™, the tissues were equilibrated in the 24-well shipment plate to room temperature for about 15 min. The EpiOcular™ tissues were then transferred into 6-well plates containing 1 mL pre-warmed assay medium per well and incubated for 1 h at 37 +/- 1 °C, 5.0% CO2 / 95% air. Then the tissues were transferred into new 6-well plates containing 1 mL fresh assay medium per well and pre-incubated in a humidified incubator at 37 +/- 1 °C, 5.0% CO2 / 95% air for 16 - 24 h.
- After the overnight incubation the tissues were pre-treated with 20 µL of DPBS and incubated for 30 ± 2 min in a humidified incubator at 37 +/- 1 °C, 5.0% CO2 / 95% air to mimic the wet conditions of the human eye.
- Afterwards, the tissues were treated with each dose group in duplicate, starting with the negative and positive control. Then the 6-well plate(s) were incubated for 6 ± 0.25 h at 37 ± 1 °C, 5.0% CO2 / 95% air until the 6 ± 0.25 h of the first dosed tissue was over. At the end of the exposure period the test item and control substances were removed by extensively rinsing the tissue with DPBS. Excess DPBS was removed by decanting the insert and blotting bottom with blotting paper. After rinsing the inserts were transferred to and immersed in a prepared 12-well “post-soak plate“, containing 2 mL fresh pre-warmed assay medium per well and incubated for 25 ± 2 min at room temperature. Afterwards, the inserts were removed from the assay medium, the medium was decanted off the tissue and the tissues were blotted on blotting paper. The inserts were transferred to a new 6-well plate (post-treatment plate) containing 1 mL pre-warmed assay medium. The tissues were incubated for 18 ± 0.25 h at 37 ± 1°C, 5.0% CO2 / 95% air.
- After this incubation period excess medium was removed by blotting bottom on absorbent paper before the inserts were transferred in a prepared 24-well “MTT assay plate” containing 0.3 mL pre-warmed MTT medium and further incubated for 3 h ± 10 min at 37 ± 1 °C, 5.0% CO2 / 95% air.
- After the 3 h MTT incubation period the inserts were removed, the bottom of the inserts blotted on blotting paper, and then transferred into new 6-well “extraction plates“, containing 2 mL of isopropanol to extract the formazan only from the bottom of the tissues to avoid possible contamination of test material. The extraction plates were sealed to inhibit isopropanol evaporation. Extraction was carried out immediately by shaking on an orbital plate shaker for 2 - 3 h at room temperature. At the end of the extraction period the tissues were not pierced to avoid contamination of the extract with remaining test item.
Then the inserts were discarded and the extracts were mixed three times using a pipette. If any visible cell/tissue fragments were in suspension, extracts were centrifuged to eliminate the fragments and avoid further possible interference with the absorbance readings.
- For each tissue 2 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer using isopropanol as a blank.

Results and discussion

In vitro

Irritation parameter:
other: mean relative tissue viability
> 0.6
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- Visible damage on test system: None

- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
In this study under the given conditions the test item showed no irritant effects. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category”
Executive summary:

In a primary eye irritation study conducted according to OECD guideline 492, 50 mg of zirconium oxide, hafnium and ytterbium doped (purity >99.5%) was applied topically on EpiOcular tissue, a reconstituted three-dimensional human corneal epithelium model for 6 h and a post-treatment period of 18 h compared to that of the concurrent negative control. Irritation was scored by the method of mean relative tissue viability.

 The mean relative tissue viability (% negative control) was > 60% (60.2%). As the results were within the borderline values (60 ± 5%), a second experiment was performed to confirm the first results. The mean relative tissue viability (% negative control) of the second experiment was > 60% (99.2%) and the test item did not show irritant effects. Based on these results, the test item can be classified as a non-irritant according to the UN GHS Criteria.