Combined in vitro tests as an alternative to in vivo eye irritation tests

Accurate methods that test the eye irritation potential of chemicals, which do not involve the use of animals, are needed to meet new regulatory standards. We evaluated the applicability and predictive capacity of five in vitro tests for eye irritation: the Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) assay; the Chorioallantoic Membrane-Trypan Blue Staining (CAM-TBS) assay; the Fluorescein Leakage Test (FLT); the 3T3-Neutral Red Uptake (3T3-NRU) cytotoxicity assay; and the red blood cell (RBC) haemolysis assay. A panel of 16 chemicals (some at multiple concentrations) was assessed by using the five tests, and the results were compared with historical in vivo Draize test data. The results showed rank correlation and class concordance between the five alternative methods and the Draize test for the 16 chemicals. These in vitro assays had good predictive capacity, reproducibility and reliability when compared to the Draize test. The best relationship was between the HET-CAM, CAM-TBS and FLT results, and the modified maximum average score(s) (MMAS). A prediction model (PM) was developed, based on the maximum possible correlation between the MMAS and the HET-CAM, CAM-TBS and FLT results. The PM had a good predictive capacity when compared to the results of animal tests, indicating its potential value for the in vitro screening of chemicals for eye irritation effects.