Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-11-16 to 2017-12-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
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:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Tungsten zirconium hydroxide oxide
- Physical state: solid
- Appearance: white powder
- Further information on test material confidential
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None, the test item was used in its original form.

Test animals / tissue source

Species:
human
Strain:
other: Reconstructed human Cornea-like Epithelium
Details on test animals or tissues and environmental conditions:
- Source: MatTek, Bratislava, Slovak Republic.
- Selection: At receipt, the tissues were inspected for obvious defects prior to use.
- Storage conditions: At receipt, the tissues were equilibrated (in the 24-well shipping container) to room temperature for at least 15 minutes. Each tissue was inspected as specified in an internal procedure (a check of the batch number and of any information relating to the characterisation and integrity of the test system was performed and recorded in CiToxLAB France files). They were then removed from the 24-well shipping containers and placed in a 6-well plate containing 1 mL of pre-warmed assay medium. Tissues were then incubated for 1 hour at 37°C (± 2°C), 5% CO2 in a humidified incubator. After the pre-incubation period, the assay medium was removed and replaced by 1 mL of fresh assay medium before incubation overnight (16-24h) at 37°C (± 2°C), 5% CO2 in a humidified incubator.
- Description: The EpiOcularTM model consists of an airlifted, living, multilayered ocular tissue construction (surface 0.60 cm²), reconstructed from normal (non-transformed) human-derived keratinocytes. This is a non-keratinised epithelium which models the cornea epithelium with progressively stratified, but not cornified cells. The cells are cultured in proprietary serum-free culture media, which induces corneal differentiation and the formation of the organotypic 3D cornea-like model. The 3D tissue consists of highly organised cell layers similar to that found in the cornea. The model features a normal ultra-structure and is functionally equivalent to human in vivo tissue.
- Expiry date: The EpiOcular tissues were used within 72 hours of their production.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 51 mg (± 1 mg)

NEGATIVE CONTROL:
- Amount(s) applied (volume or weight with unit): 50 μL

POSITIVE CONTROL:
- Amount(s) applied (volume or weight with unit): 50 μL
Duration of treatment / exposure:
6 hours (± 15 minutes)
Duration of post- treatment incubation (in vitro):
18 hours (± 15 minutes)
Number of animals or in vitro replicates:
2 per test group (test item, negative control and positive control)
Details on study design:
Details of the test procedure used:
- RhCE tissue construct used, including batch number: EpiOcular tissue, MatTek, Bratislava, Slovak Republic. Batch number documented in a certificate of analysis archived in the study files.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: Exposure for 6 hours (± 15 minutes) at 37°C (± 2°C). The rinsed tissues were transferred to new wells of a pre-labeled 12-well plate containing 5 mL of assay medium pre-warmed at room temperature and then incubated for 25 minutes (± 2 minutes) at room temperature. This incubation in assay medium was intended to remove any test article from the tissue. However, residual amounts of test item were still noted on both test item treated tissues. At the end of the post-soak immersion, each insert of test item, negative and positive controls was blotted on absorbent material and transferred to an appropriate well of the pre-labeled 6-well plate containing 1 mL of assay medium. The tissues were then incubated for 18 hours (± 15 minutes) at 37°C (± 2°C), 5% CO2 in a humidified incubator.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: As the test item was found in the preliminary test not to have any colouring potential or any direct MTT-reducing properties, no additional controls were run during the main test.
- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan: Following the post-treatment incubation, a volume of 0.3 mL of a freshly prepared MTT solution (1.0 mg/mL) was added into new wells of pre-labeled 24-well plates. At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent paper. The tissues were then transferred to the MTT pre-filled wells and incubated for 3 hours (± 10 minutes) at 37°C (± 2°C), 5% CO2 in a humidified incubator. At the end of the 3-hour incubation period, the underside of each tissue was blotted on absorbent paper to dry. Each tissue was examined with the naked eye and the degree of MTT staining was evaluated. Again, at this step, residual amounts of test item were observed on both test item-treated tissues.
As the test item was a solid, the inserts of each test item, negative control and positive control treated tissues were transferred to a 6-well plate containing 2 mL of isopropanol in each well so that no isopropanol was flowing into the insert. This avoided any potential contamination of the isopropanol solution with any test item that may have remained on the tissue. Plates were surrounded with parafilm to prevent evaporation. Formazan extraction was performed overnight at +2-8°C and protected from light. At the end of the formazan extraction period, the plates were placed under orbital shaking at room temperature for 11 minutes. Since the test item was a solid, the tissues (test item, negative and positive control treated tissues) were not pierced. The extract solution was mixed using a pipette and two 200 µL aliquots were transferred to the appropriate wells of a pre-labeled 96-well plate. One 96-well plate was used for the negative and positive controls (placed at the opposite end of the plate), and a separate 96-well plate was used for test item treated tissues. For each 96-well plate, the average Optical Density value (OD) of 4 wells containing 200 µL of isopropanol only was used as the blank. The OD was measured at 570 nm using a plate reader.
- Acceptable variability between tissue replicates for positive and negative controls: Negative control acceptance criteria: mean cOD between 0.8 and 2.5. Positive control acceptance criteria: relative mean viability of the positive control is < 50% of the relative mean viability of the negative control.
- Acceptable variability between tissue replicates for the test chemical: Acceptable if the difference of viability between the two tissue replicates is < 20%.
- Data interpretation: A test substance is predicted as ocular irritant, if the mean relative tissue viability (%) of two tissues exposed to the test item is ≤ 60%.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: relative viability in %
Run / experiment:
1 / mean of 2
Value:
99
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
individual values of % viability: 99-100
Irritation parameter:
other: optical density
Run / experiment:
1 / mean of 2
Value:
1.64
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: individual values: 1.628 - 1.652
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

- Test for direct MTT reduction with the test item:
The MTT solution containing the test item did not turn blue/purple when compared with the negative control. The test item was therefore considered not to have direct MTT reducing properties. As a result, no additional controls were performed on freeze-dead tissues in parallel to the main test.

- Test for the detection of the colouring potential of the test item:
During this test, as both water and isopropanol solutions containing the test item did not change colour, the test item was found not to have a colouring potential. As a result, no additional controls were used in parallel to the main test.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. Mean cOD for the two replicate tissues was 1.636 and 1.662.
- Acceptance criteria met for positive control: Yes. 27 and 19% viability was observed for both tissue replicates in the positive control, whereas 99 and 100% viability was observed for the two tissue replicates in the negative control. The relative mean viability in the positive control is hence < 50% of that in the negative control.
- Acceptable variability between replicate tissues treated with test item: Yes. A difference of only 2%, 8% and 1% was obtained between the replicate tissues of the negative control, positive control and test item, respectively.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of the study, the test item is considered to be non-irritant to Reconstructed human Cornea-like Epithelium.
The substance does not need to be classified according to the CLP Regulation.