An alternative test battery in detecting ocular irritancy of agrochemicals

Using animals in toxicological screening is a controversial issue. To get knowledge about eye irritation, recently only the in vivo Draize-test is accepted, which is one of the most criticized methods because of the injuries inflicted on the test animals. In recent years various in vitro methods have been developed to replace the primary irritation test. Most of these tests are in process of validation. The Draize rabbit eye test, or some modification of this test is essentially the only method for determining ocular irritation that is accepted by authorities. Several in vitro methods have been used to investigate the toxicity of potential eye irritants with the aim of replacing in vitro eye irritation testing. This study reports the results of an alternative approach for predicting irritation potential of agrochemicals. The approach was a two-stage test battery in vitro. The first stage was a cytotoxicity test, the MTT assay. The second stage was the HET-CAM test. The chick chorioallantoic membrane (CAM), being a connective tissue sheet with a visible blood supply, has been proposed as a substrate to identify the eye irritation potential of chemicals. During the HET-CAM test the chemicals are placed directly onto the chorioallantoic membrane. The changes of the vascular injury (haemorrhage, lysis or coagulation) are indications of the potential of the chemical to damage mucous membranes in vivo. MTT assay is a simple method to determine the viability of cells in the presence of a chemical. Cells are cultured with several concentrations of a substance or product, then the ratio of cell destruction is determined. LCC50 (concentration lethal for the 50 % of the cells) is assessed. This is in correlation of the eye irrigative potential of the chemical. In our studies comparative screening was performed with 6 agrochemicals to establish parallel data on alternative test battery (HET-CAM, MTT) and in vivo (Draize) results. In most cases, this study showed a good correlation between in vitro and in vivo data. By these results the present form of an alternative test battery (HET-CAM and MTT) can be proposed as a prescreen method of eye irritation tests.