[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-01-13 to 2021-01-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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:

2019-06-18

Deviations:

yes

Remarks:

In the pre-test for Colour Interference the absorption of the test item water solution in the range of 570 nm was not evaluated by OD measurement. No historical data of pos. and neg. control are available.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

titanium, chromium, iron, zinc oxide

Molecular formula:

x(Zn, Ti, Cr, Fe)3O4 * yTiO2 * z(Fe, Cr, Zn, Ti)3O5

IUPAC Name:

titanium, chromium, iron, zinc oxide

Test material form:

solid: particulate/powder

Details on test material:

- Chemical description: Chromium, iron, titanium and zinc spinel and rutile
- Physical state: Solid, brown powder, odourless
- Crystal Structure: Spinel and rutile
- Storage condition of test material: Kept dry in closed containers
- Purity : ca 98%

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

JUSTIFICATION OF THE TEST METHODS AND CONSIDERATIONS REGARDING APPLICABILITY:
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”) without further testing within a tiered testing strategy 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.

RhCE TISSUE CONSTRUCT USED: EpiOcular™ (Lot No.:31770; OCL-200-EIT and MTT-100 assy kit; source: MatTek Corporation (82105 Bratislava, Slovakia))
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 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

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): 50 mg of the neat test substance was applied to the tissue surface. All tissues were pre-wetted with 20 µL DPBS prior application and incubated for 30 min at 37.2 °C.

Duration of treatment / exposure:

6 hours

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

about 18 hours

Number of animals or in vitro replicates:

Number of EpiOcular tissues per group: duplicates

Details on study design:

PRE-TEST FOR MTT INTERFERENCE
Approximately 50 mg of test substance or 50 μL deionized water (control) was added to 1 mL aliquot of MTT medium. After ~3 hours of incubation (37.2 - 37.4 °C, 5% CO2), the mixtures were evaluated for change in coloration to purple/blue.

PRE-TEST FOR COLOUR INTERFERENCE
Approximately 50 mg of test substance was added to both 1 mL of deionized water and 2 mL of isopropanol. After 60 minutes of incubation (37.2 - 37.4 °C, 5% CO2) in deionized water, or 3 hours at ambient temperature in isopropanol. The water solution was evaluated for the presence and intensity of staining/coloration. For the isopropanol solution the absorption in the range of 570±20 nm was evaluated by OD measurement.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE AND COLOUR INTERFERENCE
Since the test item/ water and test item/ isopropanol solutions did not change colour and the test item did not interfere with MTT, no additional tissues were necessary in the main experiment.

DETAILS OF THE TEST PROCEDURE USED
- Pre-warming:
EpiOcularTM tissues were equilibrated at room temperature for 15 min. The inserts with the tissues were transferred into 6-well-plates containing 1.0 ml assay medium and incubated for 60 minutes (37.2 °C, 5 % CO2). Afterwards, the medium was changed and a further pre-incubation for approximately 16 hours (37.0 - 37.3 °C, 5 % CO2) follows.
-Pre-treatment:
After pre-warming and prior to application of the test item respectively the controls, all tissues were pre-wetted with 20 µL DPBS and incubated for 30 min (37.2 °C, 5 % CO2).
- Treatment/Exposure:
Concurrent negative and positive control were applied at a volume of 50 µL and for the test item 50 mg to the tissue surface and incubated for 6 h in assay medium (37.1 - 37.3 °C, 5 % CO2)
- Rinsing:
Afterwards all tissues were rinsed 3 times in 100 - 150 mL DPBS.
- Post-exposure immersion:
The inserts were transferred to new 12-well plates containing 5 ml assay medium and incubated for 35 min at room temperature and allowed to soak.
Post-exposer incubation:
The tissues were transferred into a 6-well plate with 1 ml assay medium and were incubated for a for about 18 h (37.3 °C, 5 % CO2).
- MTT Assay:
The tissues were placed into the 24-well plate containing 300 µL of MTT (1 mg/ mL) solution and were incubated for 3 hours (37.2 °C, 5 % CO2).
The inserts were transferred to 6-well plates containing 1 mL of isopropanol in each well. Samples were shaken for 2 hours at room temperature on a plate shaker. A volume of 1 mL of isopropanol was added to the extraction solution and the solution was mixed in each well. Duplicate 200 μL aliquots of each were transferred into a 96-well plate. Isopropanol was used as blanks.
-Measurement:
Optical density (OD) was read in a 96-well plate spectrophotometer using a wavelength of 570 nm without using a reference filter.

TEST ACCEPTANCE CRITERIA
The test meets acceptance criteria:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.5
- mean relative tissue viability of the positive control is < 50%
- relative tissue viability difference of replicate tissues is < 20%

DESCRIPTION OF DATA EVALUATION
Optical density (OD) was read in a 96-well plate spectrophotometer using a wavelength of 570 nm without using a reference filter.
Blank corrected OD values were calculated by subtracting the OD of the blank wells from the OD of all other measured wells. The mean OD value of each pair of aliquots was then calculated for each tissue.
The mean corrected OD for treated viable tissues was calculated. The resulting mean OD for the negative control treated tissue corresponds to 100% viability for the assay.

Viability [%] = corrected test article OD / corrected mean negative control OD x 100

PREDICTION MODEL
If the test item-treated tissue viability is > 60% after exposure and post-exposure incubation relative to the negative control treated tissue viability, the test item is identified as not requiring classification and labelling according to UN GHS (No Category).
If the test item-treated tissue viability is ≤ 60% after exposure and post-exposure incubation relative to negative control treated tissue viability, the test is identified as irritant to eyes and requires classification and labelling according to UN GHS (Category 2).

Results and discussion

In vitro

Results

Irritation parameter:

mean percent tissue viability 

Value:

80

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

TEST FOR MTT INTERFERENCE
In the pre-experiment for MTT interference no significant colour change was noted, so the test item did not reduce MTT to Formazan salt.

TEST FOR COLOUR INTERFERENCE
In the pre-experiment for colour interference the test item did not change colour when mixed with deionised water or isopropanol. The connected OD of the test item isopropanol solution was well below 0.08.

ACCEPTANCE OF RESULTS:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.8 (1.850)
- mean relative tissue viability of the positive control is < 50% (47.5%)
- relative tissue viability difference of replicate tissues is < 20% (9.21 - 14.47)

Any other information on results incl. tables

Optical density and viability results I 

Treatment Group	Tissue No.	Raw data OD 570 nm		Blank Corrected data OD 570 nm		Mean OD of aliquots	% of viability
		Aliq. 1	Aliq. 2	Aliq. 1	Aliq. 2
Blank		-	-	-	-	0.0866	-
Negative Control	1	1.8371	1.8662	1.751	1.780	1.765	95.4
	2	2.0065	2.0375	1.920	1.951	1.935	104.6
Positive Control	1	0.8543	0.8567	0.768	0.770	0.769	41.6
	2	1.0846	1.0658	0.998	0.979	0.989	53.4
Test Item	1	1.6925	1.7077	1.606	1.621	1.614	87.2
	2	1.4227	1.4419	1.336	1.355	1.346	72.7

Optical density and viability results II

Substance	Average Optical Density reading	SD (Optical Density)	Average % Viability	SD (Viability)
Test Item	1.480	0.268	80.0	14.47
Negative Control (deionized water)	1.850	0.170	100.0	9.21
Positive Control (methyl acetate)	0.879	0.220	47.5	11.87

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in this study and under the experimental conditions reported, Chromium, iron, titanium and zinc spinel and rutile is non-irritant to eyes and does not require classification and labelling for eye irritation or serious eye damage according to UN GHS and EU CLP.