IIVS, Non Animal Testing, Alternative Test Methods, In Vitro Testing | Ocular Screening
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Ocular Screening

The ocular irritation potential of formulations, products, ingredients, and chemicals can be evaluated using in vitro reconstructed human corneal epithelium (RhCE) models, such as the EpiOcular™ (MatTek Corp.) and SkinEthic HCE (EPISKIN) organotypic 3-D tissue constructs.  Whether evaluating ultra-mild cosmetics and personal care products, or rank ordering the irritation potential of candidate formulations and ingredients, we can provide custom Ocular Screening protocols to best meet your testing goals.

The Ocular Screening protocols use a time-to-toxicity procedure to determine the test material exposure time that induces a 50% reduction in viability, relative to the negative control (ET50 or t50).  This value can be used to evaluate the degree of irritancy of the test material (non/minimal, mild, moderate, severe/extreme).

For regulatory requirements or GHS classification needs, we recommend performing the Eye Irritation Test.

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 formulations can be applied topically at full formulation strength, tissue exposures occur just as they do in vivo.
  • The inclusion of benchmark chemicals or formulations can greatly improve the irritation assessment of tested materials.
  • The Ocular Screening protocols are ideal for testing materials ranging from very mild to moderate eye irritants.
  • The assay is compatible with both water soluble and insoluble raw materials, as well as mixtures and formulations.
  • In vitro RhCE models are suitable for testing liquids, creams, pastes, highly viscous materials, and solids.
  • Cytokine expression analyses may be included to further discriminate among extremely mild formulations (suitable for products intended for sensitive individuals).
  • Based upon an eye irritation testing strategy developed at IIVS, a custom EpiOcular protocol is approved for determination of eye irritation potential of anti-microbial cleaning products in the U.S. EPA’s Office of Pesticide Programs under the U.S. EPA classification and labeling system (US EPA, 2013).

Specialized protocols may be prepared as requested through consultation with the Study Director.

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.


  1. Receipt of Tissues
  2. Dosing
  3. Rinsing
  4. Transfer to MTT
  5. Extraction in Isopropanol and Plate Reading
  6. 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
Topical application of test material

Topical application of test material

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.

Step 3: Rinsing
3D Rinsing

3D 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.

Step 4: Transfer to MTT
MTT addition

MTT addition

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.

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
MTT reduction test

MTT reduction test

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.