IIVS | Eye Irritation Test (EIT, OECD 492)
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Eye Irritation Test (EIT, OECD 492)

The Eye Irritation Test (EIT) is an OECD-approved in vitro non-animal test method for identifying chemicals and mixtures not requiring classification and labelling for eye irritation or serious eye damage. The test method utilizes an in vitro reconstructed human corneal epithelium (RhCE) model (EpiOcular™, MatTek Corp.), in an acute exposure assay to support international regulatory labeling requirements, according to the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS).

The Eye Irritation Test can be used to support European Cosmetics dossier and REACH registration requirements. Since the mechanistic basis for eye irritation involves corneal penetration and cellular/tissue damage, the measurement of cell viability is highly relevant. Therefore, the viability of the RhCE tissues is determined by the NAD(P)H-dependent microsomal enzyme reduction (and to a lesser extent by the succinate dehydrogenase reduction) of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Test materials that result in a relative tissue viability >60% do not require classification for eye irritation or serious eye damage.

Quick Facts
  • In vitro RhCE models are made from human-derived cells, which are cultured on specially designed cell culture inserts. The cells are cultured to form a multi-layered structure which closely models the corneal epithelium.
  • 3-D reconstructed epithelial tissues model the barrier properties of the native epithelium, thus providing a relevant platform for modeling the permeation kinetics of chemicals and ingredients.
  • Since test materials can be applied topically, tissue exposures occur just as they do in vivo.
  • IIVS participated in the original EIT test method validation by Cosmetics Europe, and supplied the test method SOPs.
  • The inclusion of relevant benchmark chemicals may be used to support the irritation assessment of tested materials.
Applications
  • The Eye Irritation Test (EIT) is ideal for meeting international regulatory requirements (European Cosmetics dossier and REACH registrations).
  • The assay is compatible with both water soluble and insoluble formulations.
  • In vitro RhCE models are suitable for testing liquids, creams, pastes, highly viscous materials, and solids.
  • IIVS has extensive experience handling challenging or novel physical forms and chemistries. We recommend contacting one of IIVS’ Study Directors to address your specific needs.
3D Tissue: Each tissue is comprised of human cells in a 3-dimensional multi-layered epithelium. Upon receipt, tissues are inspected and then transferred to pre-labeled plates containing pre-warmed medium. The plates are then placed in a humidified incubator to equilibrate the tissues.

Step-by-Step

Receipt of Tissues

  1. Dosing
  2. Rinsing
  3. Transfer to MTT
  4. Extraction in Isopropanol and Plate Reading
  5. Direct MTT Reduction and Killed Controls
Step 1: Receipt of Tissues

Each tissue is comprised of human cells in a 3-dimensional tissue structure.

Tissues are inspected for irregularities and then transferred to pre-labeled plates containing pre-warmed medium. The plates are then placed in a humidified incubator to equilibrate the tissues.

3D Tissue
Tissue Shipment
3D Tissue Incubation
Step 2: Dosing

After an incubation period, the test material, positive control or negative control is applied directly onto the tissue surface.

Both solid and liquid materials can be tested. Dosing preparation may be adjusted to accommodate specific physical test article characteristics or client needs.

Topical application of test material
Step 3: Rinsing

After a specified exposure time, the test material is rinsed from the tissue with a buffered saline solution. Specific protocols or tissue types may include additional rinsing procedures.

3D Rinsing
Step 4: Transfer to MTT

The tissues are transferred to MTT solution and incubated.

MTT is actively taken up by the tissues and subsequently reduced in the mitochondria of living cells. This chemical reaction produces a purple-colored formazan within the cells, causing the live tissues to turn deep purple in color.

Test materials that result in cell death will not produce this color change. The more toxic the test material, the less purple the tissue will be.

 

MTT addition

MTT addition

Step 5: Extraction in Isopropanol and Plate Reading

After the MTT incubation, the tissues are transferred from the MTT solution to isopropanol. The isopropanol extracts the purple-colored formazan from the tissues.

Aliquots of each extracted tissue are transferred to a 96-well plate to be read by the spectrophotometer. Absorbance readings from test material treated tissues are compared to negative control tissues. Changes in % cell viability relative to the negative controls are interpreted to evaluate the irritation potential of the test material.

MTT extraction
Optical density (OD) determination
Step 6: Direct MTT Reduction and Killed Controls

Prior to beginning an assay, each test material is pre-screened for direct MTT reduction.

The test material is added to MTT solution. A color change indicates its ability to directly reduce MTT. Freeze-killed control tissues may be utilized concurrently in the assay to determine the extent of direct MTT reduction (if any) by the test article.

MTT reduction test