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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:
From May 31st to June 16th, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

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:
Adopted 28 July 2015
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
human
Details on test animals or tissues and environmental conditions:
IN VITRO TEST METHODS
- Justification of the test method and considerations regarding applicability: the measurement of viability of the EpiOcular™ RhCE tissue construct after topical exposure to a test chemical to discriminate chemicals not requiring classification for serious eye damage/eye irritancy (UN GHS No Category) from those requiring classification and labeling (UN GHS Categories 1 and 2) is based on the assumption that all chemicals inducing serious eye damage or irritation will induce cytotoxicity in the corneal epithelium and/or conjunctiva.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: cell system used consists of a 0.6 cm2 reconstructed non-keratinized cornea epithelium from primary human keratinocytes. EpiOcular™ tissue models the corneaepithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The tissue is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top to the tissue so that the epithelium surface is in direct contact with air.Tissues are tested by supplier for the absence of HIV-1, hepatitis C and B. They are certified bacteria, yeast and other fungi free.

PREPARATION OF EPIOCULAR TISSUE
The tissue construct was first controlled to demonstrate that the batch meets defined production release criteria (viability and barrier function).

After arrival, the tissues were equilibrated at room temperature on the agarose gel for about 15 minutes. Then, 6 RhCE were transferred into 1 new 6-well plates pre-filled with 1 ml of pre-warmed at 37 °C EpiOcular™ Assay Medium. The absence of air bubbles was checked by observing underneath the well of the plate. The plate was then incubated for 1 hour at 37°C ± 1°C, in a humidified atmosphere of 5 ± 1 % CO2 in air (standard culture conditions). After 1 hour, the EpiOcular™ Assay Medium was replaced by 1 ml of fresh medium pre-warmed at 37 °C and the tissues were incubated overnight at standard culture conditions.

PRE-TREATMENT OF TISSUE CONSTRUCT
Before starting the test, RhCE surface was pre-wetted with 20 μl of Ca2+/Mg2+ free -D-PBS and incubated in the dark at 37 ± 1°C, in a humidified atmosphere of
5 ± 1 % CO2 in air, for 30 ± 2 minutes. After this pre-treatment the tissues are exposed to the test chemical and control substances.

QUALITY CONTROL OF THE RhCE TISSUE CONSTRUCT
The certificate of analysis of the RhCE inserts employed in this study shows that all acceptance criteria for barrier functionality and viability are met.
Viability: the tissues treated with the negative control should show OD values between 0.8 and 2.5. Result: 1.836 ± 0.282.
Barrier functionality: the RhCE tissue construct should form a functional barrier with sufficient robustness to resist rapid penetration of cytotoxic benchmark substance, as estimated by e.g. ET50 (exposure time required to reduce tissue viability by 50 %). Acceptability criteria: 12.2 min < ET50 < 37.5 min. Result: 20.48 min.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 μl
Duration of treatment / exposure:
30 ± 2 minutes
Duration of post- treatment incubation (in vitro):
post-exposure incubation: 120 ± 15 minutes
Number of animals or in vitro replicates:
Two replicates
Details on study design:
PRELIMINARY ASSAY
- Coloring interference: the coloring properties of the test substance in contact with water was assessed prior to proceed to the analysis with RhCE. 50 μl of test substance were added to 1 ml of water in a 6-well plate, in duplicate. The plate was incubated in the dark at 37 ± 1 °C, in a humidified atmosphere of 5 ± 1 % CO2 in air, for 1 hour. After incubation, visual evaluation of coloring properties of the test substance was performed.
- Indication of controls used for direct MTT-reducers: the non-specific reduction of MTT by the substance under test conditions was assessed prior to proceed to the analysis with RhCE. In a 6-well plate 4 wells were filled with 1.0 ml of MTT 1 mg/ml solution in Dulbecco’s modified Eagle Medium (DMEM). 50 μl of sterile ddH2O, as a negative control was added to two wells and carefully mixed. Then 50 μl of test substance was added to two wells and carefully mixed. The plate was incubated in the dark at 37 ± 1 °C, in a humidified atmosphere of 5 ± 1 % CO2 in air, for 180 ± 10 min. After incubation, visual scoring of MTT interaction was performed.

TEST SYSTEM
- Details of the test procedure used: the inserts were exposed to test substance under standard conditions. After treatment the inserts were rinsed with Ca2+/Mg2+-free Dulbecco's Phosphate Buffered Saline (DPBS) at room temperature. This rinsing step was followed by a post-exposure immersion in 5 ml EpiOcular Assay medium and a further post-exposure incubation. Finally, cell viability was evaluated.
- RhCE tissue construct used, including batch number: EpiOcular™ OCL-200-EIT, consisting of at least 3 viable layers of cells and a non-keratinized surface. Batch: 23714.
- Doses of test chemical and control substances used: 50 μl of test chemical, 50 μl of negative control (sterile and deionised), 50 μl of positive control (methyl acetate).
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
exposure for 30 ± 2 minutes at 37 ± 1°C, in a humidified atmosphere of 5 ± 1 % CO2 in air
post-exposure immersion: for 12 ± 2 minutes at room temperature, in order to remove any test chemical absorbed into the tissue.
post-exposure incubation: for 120 ± 15 minutes at 37°C ± 1°C, in a humidified atmosphere of 5 ± 1 % CO2 in air.
- MTT stock solution: a 5 mg/ml MTT stock solution was prepared in D-PBS Ca2+/Mg2-free and diluted 1:5 in EpiOcular™ Assay medium to obtain a 1 mg/ml solution.
- Description of the method used to quantify MTT formazan: RhCE inserts were transferred into MTT plate with 0.3 ml of MTT solution in each well. The plate was incubated for 180 ± 10 min at 37°C ± 1°C, in a humidified atmosphere 5 ± 1 % CO2 in air. At the end of the incubation, RhCE inserts were transferred into a new 24 well plate with 2 ml of isopropanol in each well. The plate was sealed and incubated overnight at 2 - 8 °C in the dark, with no agitation. At the end of the incubation period, each tissue was pierced with a needle, rinsed accurately in the extraction solution of the corresponding well and then removed. A 96 well plate was prepared for the MTT absorbance measurement. 200 μl of each solution extracted with isopropanol was placed into the corresponding well of the 96 well plate, in duplicate. In position H1, H2 and H3 of the 96 - well plate, 200 μl of isopropanol were added as blank.
- Wavelength used for quantifying MTT formazan: Optical Density (OD) at 570 ± 8 nm was measured.
- Number of tissue replicates used per test chemical and controls: two tissues per test substance and (positive and negative) controls.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: the percentage tissue viability cut-off value distinguishing classified from non-classified test chemicals is 60 %.

ACCEPTANCE CRITERIA
- Mean OD570 of negative control should be in the range 0.8 and 2.5 a.u.
- The viability of the positive control should be < 50 %
- Difference of Viability of negative and positive controls and test substance expressed in % should be < 20 %

Results and discussion

In vitro

Results
Irritation parameter:
other: tissue viability
Value:
21.58
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: SD 0.30 %
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS
- Acceptance criteria met for negative control: mean OD570 of negative control was 2.34, thus it was within the acceptance range of 0.8 and 2.5.
- Acceptance criteria met for positive control: mean viability of positive control was 41.03 %, thus it was within the acceptance range (< 50 % relative to the negative control viability which was 99.79 %).
- The difference of viability between tissue replicates is less than 20 % in all cases:
difference of viability between tissue replicates of test substace 0.42 %.
difference of viability between tissue replicates of negative control 0.43 %.
difference of viability between tissue replicates of positive control 1.71 %.

Applicant's summary and conclusion

Interpretation of results:
other: eye irritant or serious eye damage
Conclusions:
Under these conditions, the test substance resulted to potentially induce eye itrritation or serious eye damage.
Executive summary:

The in-vitro eye irritation potential of the substance was assessed according to the Guideline OECD 492. For this purpose, a (RhCE) Reconstructed Human EpiOcular™ Model was used. Prior to the test, the reconstructed tissue was evaluated for its viability and barrier function. The direct reduction ability of MTT and the light interference of the test chemical to MTT absorption was also evaluated prior to the test. The test chemical, positive and negative control substances were tested in duplicate. After a pre-treatment of the tissue, it was exposed to the chemical for 30 ± 2 min at standard conditions, after which it was rinsed with Ca2 +/Mg2 +-free DPBS at room temperature. This rinsing step was followed by a 12 ± 2 minutes post - exposure immersion in fresh medium at room temperature and a 120 ± 15 minutes post-exposure incubation in fresh medium at standard culture conditions, prior to performing the MTT assay (180 ± 10 minutes at standard conditions).

The mean viability of the test substance is 21.58 %.