An overestimation of acids and alkalines in our system might be caused by the fact that any pH buffering mucous- and/or tearfilm-producing component, which is present in vivo, is missing. DOI. Areas of metabolically active or inactive cells are quantitatively analyzed on cryosection images with ImageJ software analysis tools. By incorporating the total tissue thickness, the relative MTT-DOI (rMTT-DOI) was calculated. Using the rMTT-DOI and human reconstructed cornea equivalents, we developed a prediction model based on suitable viability cut-off values. We tested 25 chemicals that cover the whole range of eye irritation potential based on the globally harmonized system of classification and labelling of chemicals (GHS). Principally, the MTT-DOI test method allows distinguishing between the cytotoxic effects of the different chemicals in accordance with all 3 GHS categories for eye irritation. Although the prediction model is slightly over-predictive with respect to non-irritants, it promises to be highly valuable to discriminate between severe irritants (Cat. 1), and mild to moderate irritants (Cat. 2). We also tested 3D conjunctiva models with the aim to specifically address conjunctiva-damaging substances. Using the MTT-DOI method in this model delivers comparable results as the cornea model, but does not add additional information. However, the MTT-DOI method using reconstructed cornea models already provided good predictability that was superior to the already existing establishedinvitro/exvivomethods. == Introduction Mulberroside C == To date, the rabbit Draize eye irritation test[1]is still the only OECD-approved test for the prediction of all three GHS categories for eye irritation in one single test system[2]. In the past, a number of ex vivo and in vitro methods have been developed in order to replace the Draize test. For example, tests either based on isolated animal eyes, like the Bovine Corneal Opacity and Permeation (BCOP) test and the Isolated Chicken Eye (ICE) test[3],[4],[5],[6], or cell-based assays[7],[8],[9],[10],[11],[12]have been described. Furthermore, approaches have been published that take advantage of the reactions evoked by chemicals in incubated hen eggs[7]or invertebrates[13],[14]. Also a test system based on the perturbation and denaturation of corneal proteins which is supposed to mimic the disruptive effect of ocular irritants[15]and various 3D cornea epithelial models[16],[17],[18],[19],[20],[21],[22]have been developed. Currently some methods have gained regulatory acceptance for selected GHS categories. For example, both the BCOP and the ICE test method have been implemented at OECD level to screen for corrosives and severe eye irritants (Cat. 1) on the one hand and for nonclassified chemicals on the other hand[23],[24]. In the European Union, the HET-CAM (Hens Egg Test Chorioallantoic Membrane) and the Isolated Rabbit Eye (IRE) test have also been accepted for the identification of severe eye irritants[25]. In addition, the Cytosensor Microphysiometer test method has gained validation status for the identification of severe irritants (limited to water-soluble materials) and non-classified substances (limited to water-soluble surfactants and surfactant- containing Mulberroside C mixtures) Mulberroside C and is now the subject of a draft OECD guideline[26]. All available methods reveal their strengths preferably in the HERPUD1 accurate identification of either severe eye-irritants or non-irritants. Test methods which reliably distinguish the mild/moderate irritatants (Cat. 2) from Cat. 1 and the non-irritants (No Cat.) directly, are not yet available. Therefore many of these test methods are intended to be used only within the framework of an integrated testing strategy, either in a top-down or in a bottom-up approach[8],[27],[28]. According to an expert group[27], only methods based on ocular tissues comprising both, the epithelium and the stroma, are thought to allow discrimination between all three GHS categories. This perception is based on studies by Jester and Maurer[29],[30],[31],[32],[33],[34]who showed that the surface area and depth of initial corneal injury (DOI) in epithelium and stroma of rabbit eyes strongly correlate with the eye-irritating potential of chemicals which had been topically applied to the eye. The conclusions of the authors are based on in vivo rabbit studies using live/dead assay in combination with the low Mulberroside C volume eye test (LVET) as well as on ex vivo studies on isolated rabbit eyes[33]. In the presented study we aimed to establish an in vitro test method which reliably predicts the eye-irritation potential of chemicals for all three GHS categories within one test. For this purpose.