Performance of the neutral red uptake assay in the COLIPA international validation study on alternatives to the rabbit eye irritation test

The neutral red uptake (NRU) assay was included as part of the COLIPA inter national validation trial of in vitro alternatives to the Draize eye irrita tion test. In a blind trial, 55 substances were tested at four laboratories . Following testing, a prediction of the in vivo Draize modified maximum av erage score (MMAS) for each substance was made by each laboratory using a p rediction model relating mean NR50 value (concentration causing 50% reducti on in NRU from that of untreated control cells) to MMAS. Following statisti cal analysis of the results and breaking of the code, assessment of the res ults and further analysis was carried out by the participating laboratories . This paper presents the conclusions with regard to the NRU assay. The ini tial trial analysis indicated that the interlaboratory reproducibility of r esults of the NRU assay was good. However, there was a poor correlation bet ween observed and predicted MMAS (using the proposed prediction model) when all the test substances were analysed together (r = 0.246). Data analysis of subsets of substances indicated that the best predictions were for pure surfactants only (r = 0.843) although this data did not fit within the limi ts of the prediction model. The NRU assay therefore appears to have limited use as a complete Draize replacement. A further examination of the COLIPA trial data may identify combinations of assays which may be more useful tha n the individual assays which, like NRU, have been shown to be poor predict ors of eye irritation. (C) 1999 Elsevier Science Ltd. All rights reserved.