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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 November 2017 - 13 March 2018
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:
yes
Remarks:
Minor deviations that were not considered to have compromised the validity or integrity of the study.
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): yttrium trifluoride
- Physical state: solid
- Appearance: white powder
- Further information on test material confidential.
Specific details on test material used for the study:
- No correction factor was applied in this study.
- 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 (tissues)
Details on test animals or tissues and environmental conditions:
- Source: MatTek, Bratislava, Slovak Republic
- Expiry date: The EpiOcular tissues were used within 72 hours of their production.
- Selection: At receipt, the tissues were inspected for obvious defects as they could have been rejected based on blistering, excess fluid or air bubbles below the tissue insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used.
- Storage conditions: At receipt, the living EpiOcular tissues were stored on their day of arrival, at 37°C, 5% CO2, in a humidified incubator.
- Description of the cell system used: EpiOcular living tissue consists of an airlifted, living, mult ilayered ocular tissue construction (surface 0.60 cm²), reconstructed from normal (non-transformed) human-derived keratinocytes. This is a non-keratinised epithelium which models the corneal 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 organotype 3D cornea-like model. The 3D tissue consists of highly organised cell layers similar to those found in the cornea. The model features a normal ultra-structure and is functionally equivalent to human in vivo tissue.

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
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 at 37°C, then soaked in assay medium for 25 minutes at room temperature, blotted, and then incubated for 18 hours at 37°C.
- 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 and any direct MTT reducing properties, no additional controls were run during the main test.
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan: MTT formazan precipicates are extracted using isopropanol and quantified using spectrophotometry. Formazan extraction was performed overnight at +2 to 8°C and protected from light. 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

Results
Irritation parameter:
other: relative viability in %
Run / experiment:
mean of duplicate tissues
Value:
90
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:
OTHER EFFECTS:
- Visible damage on test system:
None
- 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 were observed for the two tissue replicates in the positive control, whereas 99 and 101% viability were 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 11% was obtained between the replicate tissues.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test substance was tested for its eye irritation potential in a Reconstructed Human Cornea-like Epithelium assay performed according to OECD guideline 492. Since the mean relative viability in the treated tissues after MTT reduction was 90%, which is higher than the cut-off level of 60%, the test results meet the criteria for a non-irritant response. Therefore, the test substance is not to be classified for eye irritation under the CLP Regulation.