In the context of the 6th Amendment of the European Directive on Cosmetics, several cosmetic companies concentrate their basic research on the development of the best adapted battery of in vitro tests able to be incorporated in the ocular risk assessment process. Consequently, the European Cosmetic Toiletry and Perfumery Association (COLIPA) has initiated an international multicentric study with the main purpose to validate available alternatives in vitro methods for assessing the eye irritation potential of cosmetic raw materials and formulations. The alternative methods assessed in this validation study were chosen since all of these tests had already been used and continue to be conducted in the risk assessment process. The different endpoints of these assays are mainly biological parameters except for the biochemical assay named EYTEX(TM). In this article, the defined prediction models and the different protocols used in the COLIPA study are described. Then, the EYTEX assay results are presented and discussed in details in order to understand the failure of this assay during this validation study. The relevance and the reliability of the EYTEX assay were particularly low in two laboratories, whereas one laboratory presented acceptable data with a low compatibility with tested samples. These results underline the problem of the complex qualification process of this assay, since sometimes the same sample has been qualified with different protocols in the three laboratories. This validation study also demonstrates that, in the case of EYTEX assay, the criteria used to establish a prediction model have not been rigorous enough. For instance, the mixture of all the EYTEX protocols is not suitable for the establishment of a well-adapted prediction model. Furthermore, a clearer definition of limitations of the EYTEX assay seems to be necessary to better harmonize the qualification procedure in the three laboratories. The COLIPA validation process clearly demonstrated that the EYTEX assay was first, not suitable for the assessment of the eye irritation potential of surfactants and formulations based on surfactants, and secondly not ready for a validation study requiring the establishment of adequate and well defined mathematical prediction models. However, internal comparative studies with specific benchmarks on emulsions containing a low percentage of surfactants may be more adaptable to this type of assay.