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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:
weight of evidence
Study period:
FROM 01 NOV 2021 to 26 NOV 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

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:
Adopted June 18, 2019.
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Cerium gadolinium oxide
EC Number:
953-470-2
Molecular formula:
Gd2CeO5
IUPAC Name:
Cerium gadolinium oxide
Test material form:
solid: nanoform
Specific details on test material used for the study:
The test item was a white powder, which is an appropriate form to test solids in this type of test.

Test animals / tissue source

Species:
human
Strain:
not specified
Details on test animals or tissues and environmental conditions:
- Justification of the test method : in the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications.
- Description of the cell system used: the EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek).
- RhCE tissue used: OCL-200-EIT MatTek Corporation, Lot: 32878. Keratocyte strain 4F1188.
- Analysis for tissue functionality:
1)Tissue viability: MTT QC Assay, 1 hour, n=3; Result = 1.797+/- 0.121 for OD (540-570 nm) acceptance criteria of [ 1.1-3.0]
2) Barrier function: ET-50 assay, 100 µL 0.3% Triton X-100, 3 time points, n=2, MTT Assay. Result = 18.81 min for ET-50 acceptance criteria of [12.2 - 37.5 min].
3) Sterility: long term antibiotic and antimycotic free culture. Result = sterile for acceptance criteria of [no contamination].

- Environmental conditions: all incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 56 - 92%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.1 - 36.9°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
At least 50 mg (73.4 mg) of the solid test item (powder) was added into the 6-well plates on top of the tissues.
Duration of treatment / exposure:
6 hours +/- 15 minutes.
Duration of post- treatment incubation (in vitro):
After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minutes immersion incubation at room temperature (Post-Soak).
After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C
Number of animals or in vitro replicates:
2 replicates (tissues) for each of the test item, the negative control and the positive control.
Details on study design:
Details of the test procedure used:
- On the day of receipt, the tissues were equilibrated (in its 24-well shipping container) to room temperature. Subsequently, tissues were transferred to 6-well plates and incubated for 20 ± 4 hours at 37°C in 1.0 mL fresh pre-warmed Assay Medium, which was refreshed after approximately 1 hour. Assay Medium was supplied by MatTek Corporation, Ashland, USA. Before the assay was started, the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+ Free D-PBS. The tissues were incubated at standard culture conditions for a minimum of 30 minutes.
- No correction was made for the purity/composition of the test item. The solid test item (73.4 mg) was applied and spread into the 6-well plates on top of the tissues, to match their size. After the exposure period with the test item (6 hours ± 15 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+ free D-PBS (brought to room temperature) to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minutes immersion incubation at room temperature (Post-Soak). After the Post-Soak period, cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C.

- Doses of control substances used:
1) negative control: 2 tissues were treated with 50 µL Milli-Q water.
2) positive control: 2 tissues were treated with 50 µL Methyl Acetate.

- Test for the Interference of the Test Item with the MTT Endpoint: the test item had been tested previously for possible direct MTT reduction and color interference in the Skin irritation test using EpiSkinTM as a skin model (see Skin Irr. in vitro KS V1 2021JACO - Study No. 20302180). The solutions were not turned blue / purple nor a blue / purple precipitate was observed in presence of MTT, therefore it was concluded that the test item did not interfere with the MTT endpoint. The Optical Density (OD) for the test item solution was ≤ 0.08, therefore it was concluded that the test item did not interact with the MTT measurement.
- Description of the method used to quantify MTT formazan: after incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL). The tissues were incubated for 180 ± 10 minutes at 37°C. After incubation with MTT-medium, the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labeled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol was flowing into the insert. Formazan was extracted with 2 mL isopropanol for 2 - 3 hours at room temperature with gentle shaking.
- Quantification of MTT formazan: The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Evaluation criteria: cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item. The test chemical is identified as not requiring classification and labeling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is > 60%. In this case no further testing in other test methods is required. The test chemical is identified as “no prediction can be made” if the mean percent tissue viability after exposure and postexposure incubation is ≤ to 60%.

- Positive and negative control means and acceptance ranges based on historical data:
Absorption OD 570:
1) negative control : Range [ 0.648 - 2.190], mean = 1.595, SD = 0.261 (n=76).
2) positive control : Range [ 0.027 - 0.720], mean = 0.372, SD = 0.172 (n=76).
Viability % :
Positive control: Range [ 1.72 - 44.55], mean = 23.55, SD = 10.40 (n=76).
- Acceptable variability between tissue replicates for negative and positive controls: the absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5. and the mean relative tissue viability of the positive control should be <50% relative to the negative control.
- Acceptable variability between tissue replicates for the test chemical: the difference between the % tissue viabilities of the two identically treated replicates should be <20.

Results and discussion

In vitro

Results
Irritation parameter:
mean percent tissue viability 
Run / experiment:
One run.
Value:
56
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
Positive controls validity:
valid
Remarks:
The positive control had a mean cell viability after 6 hours ± 15 minutes exposure of 20%.
Remarks on result:
not determinable
Other effects / acceptance of results:
The difference between the percentage of viability of two tissues treated identically was 18%, indicating that the test system functioned properly.

Any other information on results incl. tables

Results of the experiment are detailed below:


Table 1: Mean Absorption in the EpiOcular™ Test with the test item


 






























 



A


(OD570)



B


(OD570)



Mean +/- SD


(OD570)



Negative control



1.385



1.656



1.521 +/-  0.192



Test item



0.823



0.870



0.847 +/-  0.033



Positive control



0.258



0.344



0.301 + /- 0.061



OD = optical density


SD = Standard deviation


Duplicate exposures are indicated by A and B.


In this table the values are corrected for background absorption (0.0433). Isopropanol was used to measure the background absorption.


 


Table 2: Mean Tissue Viability in the EpiOcular™ Test with the test item


 


























 



Mean tissue viability


(percentage of control)



Difference between two tissues


(percentage)



Negative control



100



18



Test item



56



3.1



Positive control



20



5.7



 

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 56%. Since the mean relative tissue viability for the test item was below or equal to 60% after 6 hours ± 15 minutes treatment, the test item is considered to be potentially irritant or corrosive to the eye.
Conclusions:
In conclusion, the test item was identified as no prediction can be made regarding the classification in the EpiOcular™ test under the experimental conditions described for this study.
Executive summary:

The objective of this study was to evaluate the eye hazard potential of Cerium gadolinium oxide. For this purpose Cerium gadolinium oxide was topically applied on the Reconstructed Human EpiOcular™ Model. The study procedures were based on the OECD test guideline n° 492 and the study was performed according to GLP.


The test item (73.4 mg) was applied as such directly on top of 2 EpiOcular tissues for 6 hours ± 15 minutes. Positive (Methyl Acetate) and negative (Sterile Milli-Q water) controls were added in this study (2 tissues each) using the same experimental conditions than the test item.


After exposure the cornea epithelial construct was thoroughly rinsed to remove the test substances and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect using a MTT assay.


The positive control  (Methyl Acetate) had a mean cell viability of 20% after 6 hours ± 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically was 18%, indicating that the test system functioned properly.


Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 56%. Since the mean relative tissue viability for the test item was below or equal to 60% after 6 hours ± 15 minutes treatment, the test item was considered to be potentially irritant or corrosive to the eye.


In conclusion, the test item was identified as no prediction can be made regarding the classification in the EpiOcular™ test under the experimental conditions described for this study.