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

1. Preparation of RhCE Tissues for Treatment – Day 0

1. Upon receipt of the commercial human cornea-like epithelial (RhCE) kit, check all kit components for integrity (for kit details see Standard Assay Kit Components (Table 1) and Equipment and Materials required to perform the EIT assay (Table 2). On the day of receipt, equilibrate tissues (in its 24-well shipping container) to RT for 15 min.

Table 1: Standard Assay Kit Components.  

Table 2: Equipment and materials required to perform the EIT.

2. Under sterile conditions, open the plastic bag containing the 24-well plate with the RhCE tissues and remove the sterile gauze. Inspect all tissues for air bubbles between the agarose gel and insert. Do not use cultures with air bubbles under the insert covering >50% of the insert area, defective tissues, or tissues which are completely covered with liquid.
3. Label the 6-well plates with the test article or control codes and exposure times. Aliquot 1.0 ml of Assay Medium (provided with the kit), pre-warmed to approximately 37 °C, into the wells of pre-labeled 6-well plates.
4. Use sterile forceps to remove each insert containing the RhCE tissue and place the insert in the labeled 6-well plate. During this step, remove any remaining shipping agarose that adheres to the outer sides of the insert by gentle blotting on sterile filter paper. Release any air bubbles trapped underneath the inserts.
5. Pre-incubate the RhCE tissues in the 6-well plates to standard culture conditions (SCC, humidified atmosphere with 5 ± 1% CO₂ at 37 ± 1 °C) for 1 hr.
6. After 1 hr, replace the Assay Medium with 1.0 ml of fresh Assay Medium prewarmed to 37 °C and incubate the RhCE tissues at SCC conditions (overnight = O/N) (16-24 hr).

2. Pre-treatment – Day 1

1. After the O/N incubation, apply 20 µl of Ca²⁺ Mg²⁺-free-Dulbecco’s Phosphate Buffered Saline (DPBS, provided) using an appropriate pipetting device. If the DPBS does not spread across the tissues, gently tap the insert on the plate to assure that the DPBS wets the entire tissue surface.
2. Incubate the RhCE tissues at SCC for 30 ± 2 min.
   Note: This step is necessary for tissue hydration and to mimic in vivo conditions.

3. Test Material Exposure Procedures 

1. Apply each test article and controls to duplicate RhCE tissues (n = 2). The test article dosing procedure is different for liquids and solids. Topically apply 50 µl of liquid test articles using a pipette. The exposure time for liquids is 30 min. Apply 50 mg of solid test articles using a leveled spoonful (calibrated to hold 50 mg of sodium chloride). The exposure time for solids is 6 hr.
   Note: Liquids are defined as fluid substances (e.g., liquids, gels, and creams) that can be applied utilizing a pipetting device. Solids are defined as non-fluid substances (e.g., powders, resinous or waxy materials) that cannot be applied using a pipette.
   1. If the physical state of test articles is not easy to determine, place the vials with test article in a water bath for 15 min (37 °C). Follow EIT protocol for liquids for those test articles that liquefy at 37 °C.
   2. Use a positive displacement pipette for particularly viscous materials.
2. Dose the negative control and positive controls first and then dose the test articles.
   1. Apply 50 µl of the negative control (NC) and the positive control (PC) to the RhCE tissues using a standard pipette. The NC is sterile de-ionized water; the PC is methyl acetate (CAS# 79-20-9). Apply the NC and PC for 30 min when testing liquid test articles and for 6 hr when testing solid test articles.

4. Test Article Exposure – Day 1

1. For the treatment of liquid test articles, follow the timing schedule given in Table 3. Leave 1 min intervals between applications of each test article to ensure equal exposure for all tissues.
   1. After the 30 ± 2 min DPBS pre-treatment, topically apply 50 µl of NC and PC, and each liquid test article topically onto the RhCE tissues using an appropriate pipetting device.
   2. Apply 50 µl of the liquid test article directly onto the tissue to cover the upper surface. Cut off the narrow point of the pipette tip to widen the orifice for viscous materials. For very viscous materials, apply the test article to a dosing device (a flat headed cylinder of diameter slightly less than the inner diameter of the tissue insert or a plastic pushpin), invert the dosing device and place it onto the tissue so that the test article evenly contacts the tissue surface.
   3. If test article does not spread across the tissue, gently tap the insert to make sure that it spreads on the entire tissue surface. Mechanical spreading of the test articles (e.g., with a pipette tip) is not recommended since it may damage the tissues.
   4. Incubate the tissues at SCC for 30 ± 2 min.
2. For the treatment of solid test articles – Day 1, follow the timing schedule given in Table 4. Leave 2 min intervals between applications of each test article to ensure equal exposure for all tissues.
   1. After the 30 ± 2 min DPBS pre-treatment, apply 50 µl of NC and PC topically on the RhCE tissues using an appropriate pipetting device.
   2. For solid test article application, remove the inserts (n = 2) from the well and place them onto a sterile surface (e.g., the lid of a multiwell plate) to avoid a test article spilling into the medium.
   3. Using a leveled spoon, topically apply approximately 50 mg of the test article onto the tissue surface; make sure that the surface of the tissue is completely covered by the test article. If the test article does not spread across the tissue, shake the insert gently from side to side to ensure that the tissue is completely covered by the test article. Mechanical spreading of the test articles (e.g., with a pipette tip) is not recommended since it may damage the tissues.
      1. If needed, grind crystalline powders with a mortar and pestle to guarantee better contact between the test article and the tissue.
      2. Alternatively, place powders directly onto the tissue culture inside the insert by using a 1 ml syringe with its head cut off. Stuff powders into the syringe when the plunger is drawn back and then apply by pressing the plunger down.
      3. If the outer wall of the insert is contaminated e.g., by powders, wipe the particles off with a sterile gauze.
   4. After dosing, return the tissues to the 6-well plates containing culture medium and incubate at SCC for 6 h ± 15 min.

5. Rinsing

1. Prepare a set of three clean beakers (150 ml capacity) per test article and fill each of them with 100 ml of DPBS. For each test article, utilize a different set of three beakers.
2. At the end of the 30 ± 2 min exposure for liquid materials or 6 hr ± 15 min exposure for solid materials, remove and discard the dosing device if it was used.
3. Lift the inserts containing the RhCE tissue out of the medium by grasping the upper edge of the plastic 'collar' with fine forceps. Use curved forceps to facilitate handling and decanting. Rinse the tissues two at a time by holding the duplicate inserts together by their collars using forceps. Be careful not to damage the tissues with the forceps.
4. Decant the test articles or controls from the tissue surface onto a clean absorbent material (paper towel, gauze, etc.)
5. Dip the inserts into the first beaker of DPBS, swirl in a circular motion in the DPBS for approximately 2 sec, lift the inserts so that they are mostly filled with DPBS, and decant the liquid back into the beaker. Repeat this process three times in the first beaker.
6. Rinse the inserts in the second and third beakers of DPBS three times each in the same manner.
7. Decant any liquid remaining in the insert onto the absorbent material. Rotate the insert to an approximate 45° angle (open end down) and touch the upper lip to the absorbent material.
   Note: If it is not possible to remove all of the visible test material, no further rinsing should be done to avoid tissue damage due to excessive handling.

6. Post-soak

1. After rinsing, immediately immerse tissues in 5 ml of Assay Medium previously warmed to RT in a pre-labeled 12-well plate.
2. Incubate the tissues for 12 ± 2 min for liquid materials or 25 ± 2 min for solid materials immersed at RT to facilitate removal of any residual test article.

7. Post-incubation

1. At the end of the Post-Soak immersion period, decant the Assay Medium from the tissues and blot the inserts onto an absorbent material.
2. Transfer the inserts into the pre-labeled 6-well plate containing 1 ml of warm Assay Medium.
   1. Incubate the tissues for 120 ± 15 min at SCC for liquid test materials.
   2. Incubate the tissues for 18 ± 0.25 hr at SCC for solid test materials.

8. MTT Viability Assay – Day 1 (Protocol for Liquids) and Day 2 (Protocol for Solids) 

1. Perform the MTT assay after the Post-Incubation of 120 ± 15 min for liquids and 18 ± 0.25 hr for solids, respectively.
2. Prepare 1.0 mg/ml MTT solution and aliquot 0.3 ml of the solution into each well of a pre-labeled 24-well plate.
   1. Use the commercial MTT kit (Table 5):
   2. 2 hr before use, thaw the MTT concentrate at RT. Combine 2 ml of the MTT concentrate and 8 ml of the MTT diluent to produce 1.0 mg/ml MTT solution.
   3. Store the MTT solution at 4 °C in the dark until use. Do not store the MTT solution for more than 1 day.
3. At the end of the Post Incubation, remove each insert from the 6-well plate and gently blot on an absorbent material.
4. Place the inserts into the 24-well plate containing 0.3 ml of MTT solution. Release any air bubbles trapped underneath the inserts. Incubate the plate for 180 ± 10 min at SCC.
5. MTT extraction
   1. After 180 ± 10 min incubation in the MTT solution, remove each insert from the 24-well plate and blot the bottom of the insert on an absorbent material.
   2. For non-colorant liquid test articles (submerged extraction): Transfer the inserts into a pre-labeled 24-well plate containing 2.0 ml of an extractant solution (isopropanol) so that it submerges the insert.
   3. For solids and liquid colorants (non-submerged extraction to avoid contamination of the extractant solution): Transfer the inserts into a pre-labeled 6-well plate containing 1.0 ml of the extractant solution (isopropanol) so that it does not submerge the insert.
      Note: Perform the same non-submerged extraction for the corresponding negative and positive controls.
6. Seal the plates (e.g., with parafilm between the plate cover and upper edge of the wells or with a standard plate sealer). Place the plates on an orbital plate shaker and shake for 2 to 3 hr at RT to extract the MTT.
   1. Alternatively, perform the extraction O/N at 2-8 °C in the dark without shaking.
7. For non-colorant liquid test articles (submerged extraction): At the end of the extraction period, decant the liquid from each insert back into the well and discard the inserts with the RhCE tissues.
   1. Mix the extract solution and transfer two 200 µl aliquots into the appropriate wells of a pre-labeled 96-well plate according to the plate configuration (Figure 2).
8. For solids and liquid colorants (non-submerged extraction): At the end of the extraction period, discard the tissues (make sure not to pierce the tissues).
   1. Add 1.0 ml of the extractant solution into each well of the 24-well plate containing the solution extracted from the tissues. Mix the extractant solution and transfer two 200 µl aliquots into the appropriate wells of a pre-labeled 96-well plate according to the plate configuration (Figure 2).
9. Determine the optical density (OD) of the extracted samples at a single wavelength between 550 and 590 nm (should be consistent within a laboratory) on a plate reader or a spectrophotometer.
   1. In case of turbid extract solutions caused by insoluble solids, centrifuge the solutions prior to measuring the OD (cool down the centrifuge to 4 °C to avoid evaporation). In case rinsing does not remove the test article (TA) and the TA interferes with MTT reduction, additional controls must be used. Please refer to a detailed SOP to correct for MTT reduction¹⁶.
   2. In case a TA is shown to have, or to develop color which can interact with the MTT measurement, an additional test must be performed to determine the amount of color bound to, and subsequently extracted from the tissues. Please refer to a detailed SOP to correct for colored test articles¹⁶.

9. Calculations for Tissue Viability Test (Table 6 and Figure 3) 

1. General calculations
   1. Calculate the mean OD value of the blank control wells (OD Blk) for each experiment.
   2. Subtract OD Blk from each OD value of the same experiment (Blk corrected data).
   3. Calculate the mean value of the two aliquots for each tissue (= corrected OD).
2. Calculate the percent viability of each of the two replicate tissues for each control and test article relative to the average negative control (100% control).
   Viability (%) = [corrected OD treated tissues / corrected OD negative control] x 100%
3. Calculate the difference of the viability (the viability difference between two replicate tissues).
4. Calculate the mean test article viability (TA viability) and classify the test article according to the prediction model.

10. Prediction Model (Figure 3)

1. If the TA-treated tissue viability is >60.0 relative to NC-treated tissue viability, label the test article as non-irritant (NI) (GHS No Category).
2. If the TA-treated tissue viability is   ≤ 60.0 relative to NC-treated tissue viability, label the test article as irritant (I) (GHS Categories 1 and 2).
   Note: The EIT test results are considered qualified if:
   • the EIT NC OD >0.8 and <2.5;
   • the EIT PC tissue viability (%, relative to NC) is ≤50.0%;
   • the difference between the two replicate tissues (NC, PC, and test article) is <20.0%.