Ocular irritation assay


Protocol principle

The purpose of this study (prevalidated for ECVAM by a multicentric study -Pfizer / Novartis / Johnson & Johnson-) is to apply the test compound on the surface of triplicate in vitro reconstituted human ocular epithelia (HCE), and to determine quantitatively the cell viability after 10 minutes.

A prediction model of ocular irritation allows the classification of the test product.

Detailed assay procedure

Test method

30 l or 30 mg of the test compound are deposited on the surface of each of4 in vitro reconstituted human corneal tissues (size 0.5 cm), which are then incubated at room temperature for 10 minutes. Negative and positive control compounds are applied on triplicate parallel tissues (saline solution and 1% SDS). Cell viability is evaluated quantitatively using the MTT test.

Evaluation of cell viability (MTT test)

For each test condition, triplicate cultures are placed on 300 l of 0,5 mg/ml MTT and incubated 3 hours at 37 C 5% CO. Extraction is performed in 1.5 ml of isopropanol at room temperature, for a minimum of 2 hours. Optical density is measured at 570 nm. Results are expressed as percentage of viability compared to negative control : % Viability = [OD (570nm) test compound / OD (570nm ) negative control)] x 100

Prediction model

HCE viability classification prediction model: NI (viability ≥ 60%), I (viability < 60%)
The product is classified as an irritant (according in vivo classifications) if the percentage of viability compared to the negative control obtained for the test product is < 60%.


Prevalidation of a new in vitro reconstituted human cornea model to assess the eye irritating potential of chemicals.
Van Goethem F., Adriaens E., Alepee N., Straube F., De Wever B., Cappadoro M., Catoire S., Hansen E., Wolf A., and Vanparys P. Toxicol. In Vitro 2004. In press.

Three-dimensional construct of the human corneal epithelium for in vitro toxicology. D.H. Nguyen, R.W. Beuerman, B. De Wever and M. Rosdy. In: Alternative Toxicological Methods. Eds. Harry Salem and Sidney Katz, CRC Press, 147-159, 2003.