Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

The overall goal of the eye irritation test is to classify and label substances and mixtures according to their eye irritation potential. This procedure utilizes a three-dimensional reconstructed cornea like tissue model. Upon arrival, the test kit is pre incubated overnight in the assay medium the following day, the tissues are hydrated, followed by the topical application of the positive and negative controls and the test chemicals of interest onto the surfaces of the tissues In duplicate.

After a fixed period of exposure test chemicals are removed by washing. The tissues are post incubated in fresh assay medium and the toxicity of the chemicals is then evaluated by MTT assay. Ultimately, the irritating potential of the test articles can be assessed by comparing the relative viability of the test article treated tissues to the negative control treated tissues.

The main advantage of this technique of existing methods like rapid trace irritation tests, are that it does not involve laboratory animals. It utilizes in vitro deconstructed cornal like tissue model ocular, which comprises of normal human cells. It's applicable to all type of substances and mixtures and can discriminate between chemicals that induce ocular irritation or serious eye damage from materials.

Non irritants that do not require classification and labeling. The reconstructed human cornea like epithelial tissue or epi. Ocular is prepared in inserts with a porous membrane through which the nutrients passed to the cells.

Reconstructed tissue forms a non keratinized epithelium that models the cornea epithelium with progressively stratified but not cornified cells. Upon receipt of the human cornea like epithelial kit equilibrate the tissues in the 24 well shipping container to room temperature After 15 minutes, open the bag containing the 24 well tissue plate under sterile conditions and inspect all tissues for air bubbles between the agros gel and inserts.Cut. Open the tape.

Remove the cover with a sterile gauze and inspect the tissue surface. Next aliquot one milliliter of 37 degree Celsius kit assay medium into the wells of pre-labeled six well plates. Then use sterile forceps to gently blot the tissue containing inserts on sterile gauze to remove any remaining shipping aros and place them into individual wells of the six well plates.

After one hour, replace the medium with one milliliter of fresh 37 degree Celsius assay medium, and incubate the tissues under standard culture conditions overnight. The next day I pet 20 microliters of calcium and magnesium free DPBS onto the apical surface of each tissue. If the DPBS does not spread across the tissues, gently hold the insert with forceps and tap on the plate to ensure that the saline wets the entire tissue surface.

Incubate the plates for 30 minutes. Then to treat the tissues with liquid test articles, apply 50 microliters of the negative control, the positive control and the appropriate test articles in duplicate according to the timetable. Take care to ensure that the treatments cover the entire tissue surfaces and then incubate the inserts for 30 minutes to treat the tissues with solid test articles.

First, transfer the inserts onto a sterile surface. Keep the plate covered to prevent contamination with airborne solid particles. Then using a leveled spoon topically apply 50 milligrams of the test article onto the duplicate tissues according to the timetable taking care that the treatment covers the entire tissue surface.

Alternatively, use a prefilled one milliliter syringe with the head cut off. To place the powders directly onto the culture. Press the plunger to apply the powder immediately after applying the solid test articles, return the inserts to their six well plates and incubate the tissues for six hours to rinse the tissues.

After treatment, fill 3 150 milliliter beakers per test article with 100 milliliters of DPBS at the end of the exposure incubation. Grasp the upper edge of the inserts with curved forceps to remove them from the wells. Then using forceps, pick up two inserts from the same treatment by their upper edges and decant the treatments onto a clean absorbent material.

Next, dip the inserts into the first beaker of DPBS washing the tissues with a circular swirling motion for two seconds. Then lift the inserts so that they are mostly filled with DPBS and decant the liquid back into the beaker. Repeat the wash two times in the first beaker.

Then rinse the inserts in the second and third beakers of DPB S3 times each in the same way after the last wash. Rotate each insert to an approximately 45 degree angle with the open end facing down and touch the upper lip to the absorbent material to decant any remaining DPBS. Then immediately immerse the tissues in five milliliters of room temperature assay medium.

In a pre-labeled 12 well plate at the end of the post soak immersion period, decant the assay medium and blot the inserts onto an absorbent material. Then transfer the inserts into a new six well plate containing one milliliter of warm assay medium per well, and place the plates in the incubator to start the MTT viability assay. Transfer the inserts into a 24 well plate containing 0.3 milliliters of freshly prepared MTT solution.

Dab the inserts on the absorbent material prior to transfer. Release any air bubbles trapped underneath the inserts and return the tissues to the incubator after three hours, lot the bottoms of the inserts on the absorbent material. Then for the submerged extraction of non colorant liquid test articles, immerse the inserts into a pre-labeled 24 well plate containing two milliliters of the appropriate extraction solution per well for a nons submerged extraction of solid or liquid colorants.

Transfer the inserts into a pre-labeled six well plate containing one milliliter of extraction solution per well. Then seal the plates with a heat sealer or paraform to inhibit evaporation and extract on an orbital shaker for two to three hours at room temperature with gentle shaking at the end of the submerged extraction. Pierce the tissues and then decant the liquid within each insert back into the, well then discard the inserts with the tissues.

Mix the extract solution in each well and transfer two 200 microliter aliquots of each sample into the appropriate wells of a pre-labeled 96 Well plate as illustrated in the schematic at the end of the nons submerged extraction. Discard the inserts and tissues without piercing and add one milliliter of extracting solution to each. Well mix the extracting solution in the wells and transfer two 200 microliter aliquots from each sample into the appropriate wells of a pre-labeled 96 well plate as just demonstrated.

Finally, read the optical density of the samples in a 96 well spectrophotometer at a 570 nanometer wavelength without using a reference filter and enter the results into a spreadsheet to calculate the respective tissue liabilities. Account for corrections due to test chemical interference as indicated in the text protocol here, representative eye irritation test results conducted with 10 test articles and negative and positive controls are shown in this experiment. A mean optical density of 1.31 for the negative control corresponding to a 100%tissue viability was observed with a relative tissue viability of 31.2%For the positive control test articles 1, 2, 4, 7 and eight exhibited tissue variabilities of greater than 60%classifying these articles as non irritants test articles 3, 5, 6, 9 and 10.

On the other hand, exhibited tissue variabilities of less than or equal to 60%resulting in their classification as irritants. The difference in tissue viability between duplicate tissues in this experiment was less than 20%for all of the articles tested with the exception of test article two. Therefore, the results for all of the test articles except test article two were considered qualified as they met all of the eye irritation test acceptance criteria.

After watching this video, you should have a good understanding of how it should perform the DICATION test, utilizing in vitro reconstructive human corneal like tissue model four, hazard identification labeling of chemical. This procedure has been implemented in the EOC D test guidelines as a part of the tier testing strategy.