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 international validation trial of in vitro alternatives to the Draize eye irritation test. In a blind trial, 55 substances were tested at four laboratories. Following testing, a prediction of the in vivo Draize modified maximum average score (MMAS) for each substance was made by each laboratory using a prediction model relating mean NR(50) value (concentration causing 50% reduction in NRU from that of untreated control cells) to MMAS. Following statistical analysis of the results and breaking of the code, assessment of the results and further analysis was carried out by the participating laboratories. This paper presents the conclusions with regard to the NRU assay. The initial trial analysis indicated that the interlaboratory reproducibility of results of the NRU assay was good. However, there was a poor correlation between 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 limits 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 than the individual assays which, like NRU, have been shown to be poor predictors of eye irritation.