Registration Dossier

Diss Factsheets

Toxicological information

Eye irritation

Currently viewing:

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2016-06-03 to 2016-11-25
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:
EC Number:
EC Name:
Cas Number:
Molecular formula:
digadolinium(3+) ion trioxalate
Test material form:
other: wet powder
Details on test material:
- Name of test material (as cited in study report): digadolinium oxalate
- CAS nr: 867-64-1
- Physical state: solid
- Appearance: wet white powder
Specific details on test material used for the study:
- No correction factor applied in this study
- The test item was used in its original form, as supplied by the Sponsor.

Test animals / tissue source

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, 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 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 consist 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.

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Amount(s) applied (volume or weight with unit): 51 mg
Duration of treatment / exposure:
6 hours
Observation period (in vivo):
Not applicable
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
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.
- Doses of test chemical and control substances used: 51 mg of test item, 50 µL of deinonised water for negative control, 50 µL methyl acetate for positive control
- 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.
- 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: MTT formazan precipicates are extracted using isopropanol and quantified using spectrophotometry. Formazan extraction was performed overnight at +2-8°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

Irritation parameter:
other: relative viability in %
Run / experiment:
mean of duplicate tissues
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: No

- Acceptance criteria met for negative control: Yes. Mean cOD for the two replicate tissues was 1.817 and 1.592.
- Acceptance criteria met for positive control: Yes. 22% viability was observed for both tissue replicates in the positive control, whereas 93 and 107 % 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 3% was obtained between the replicate tissues.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
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 mean viability in the treated tissues after MTT reduction was 104%, 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.