Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 12, 2017 - December 14, 2017
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 guidelineopen allclose all
Qualifier:
according to guideline
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:
October 9, 2017
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; For use with MatTek Corporation's Reconstructed Human EpiOcular Model; MatTek Corporation
Version / remarks:
June 29, 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: DB-ALM Protocol No. 164: Ocular Irritation Assay for Chemicals using EpiOcular™ EIT
Version / remarks:
September 14, 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Titanium zirconium oxide
Molecular formula:
TixZryOz (x = 0.985 - 0.995, y = 1-x, z = 1.65 - 1.75)
IUPAC Name:
Titanium zirconium oxide
Test material form:
solid: bulk

Test animals / tissue source

Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability :
The reconstructed human comea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye Epiocular™-model closely mimics the biochemical and physiological properties ofthe human eye, i.e. the comea.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The Epiocular™-model is a nonkeratinized epithelium prepared from normal human keratinocytes. lt models the cornea epithelium with progressively stratified, but not cornified cells. lt is used for eye irritation testing.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 mg

The test item was applied neat to the tissues.
Duration of treatment / exposure:
6 hours +/- 15 minutes
Duration of post- treatment incubation (in vitro):
18 hours +/- 15 min
Number of animals or in vitro replicates:
two tissues
Details on study design:
- Details of the test procedure used
- RhCE tissue construct used, including batch number: EpiOcular™ Tissue (OCL-200, OCL-212), Lot No. 27017
- Doses of test chemical and control substances used: 50 mg test item, 50 µL negative control, 50 µL positive control
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 6 hours (+/- 15 min) at 37°C, 25 min (+/- 2 min) at room temperature, 18 hours (+/- 15 min) at 37°C
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: A pre-test for direct MTT-reducing capacity of the test item was conducted and the test item was checked for its colorant properties.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2 tissues (test item, positive control, negative control), 2 tissues to assess direct MTT reduction and staining test items, respectively
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm, ELx800, BioTek Instruments GmbH
- Description of the method used to quantify MTT formazan: To extract the MTT, the plates was placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. The extract solution was mixed and 2 x 200 μL were transfe1Ted into a 96-well plate. The OD was read using a spectrophotometer at 570 nm wavelength.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: If the test item-treated tissue viability is >60.0% relative to negative control-treated tissue viability, the test item is labeled non-irritant (UN GHS No Category). If the test item-treated tissue viability is less than or equal to 60.0% relative to negative control-treated tissue viability, the test item is labeled irritant (UN GHS Category 1 or Category 2).

- Acceptability of the Test:
- The negative control OD is >0.8 and <2.5
- The mean relative viability of the positive control is below 50% of the negative control viability.
- The difference of viability between the two relating tissues of a single chemical is <20% in the same run (for positive and negative control tissues and tissues of single chemicals).


Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: Cell viability (%)
Run / experiment:
Tissue 1
Value:
93
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Cell viability (%)
Run / experiment:
Tissue 2
Value:
107
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- 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
Conclusions:
Under the conditions ofthe present study, the test item did not show an eye hazard potential. The test item is labeled non-irritant (UN GHS: No Category).
Executive summary:

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model.

The test item was applied topically to a reconstructed human cornea-like epithelium model (Epiocular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential. Duplicates of the Epiocular™-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 μL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues.

After treatment with the negative control (sterile deionized water) the mean OD was 1.509 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 47.0% (study acceptance criterion: <50%). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 85.7% and, thus, higher than 60%, i.e. according to OECD 492 the test item is labeled non-irritant (UN GHS: No Category).