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Applications Of The Neutral Red Cytotoxicity Assay To In Vitro Toxicology

Harvey Babich, Ellen Borenfreund

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A concerted effort is currently in progress to develop alternatives to the use of live animals for the acute toxicity testing of xenobiotics. To this end, the neutral red in vitro cytotoxicity assay was developed which, although initially based on the use of mammalian cells in culture, has also been adapted for ecotoxicity studies using fish cells in culture. The neutral red assay is based on the binding of neutral red, a weakly cationic supravital dye, to the lysosomal matrix of viable cells after their incubation with toxic agents. Spectrophotometric quantitation of the extracted dye with a scanning microtitre well reader at 540nm was found to be linear with the number of surviving, viable cells. The assay has been used to determine the relative acute cytotoxicities of a broad spectrum of chemical test agents, to establish structure-toxicity relationships for series of related chemicals, to study metabolism-mediated cytotoxicity, to evaluate interactions between combinations of test agents, to evaluate differential and selective toxicities of cancer chemotherapeutics and other pharmaceuticals, and to study temperature-toxicity interactions.