Use of the 3T3 cell-neutral red uptake assay for irritants as an alternative to the rabbit (Draize) test

An in vitro procedure based on neutral red uptake by mouse fibroblasts in culture (Borenfreund & Puerner, Toxicology Lett. 1985, 24, 119) is being used to screen raw materials, chemical intermediates and drug products submitted for eye-irritancy testing. The relative toxicity data determined for the compounds by this method generally show good agreement with published Draize test results. 3T3 cells cultured to 75% confluency are incubated overnight with dilutions of the toxicant and then rinsed and incubated for 3 hr with culture medium containing 50 μg neutral red/ml. The medium is discarded, the cells are rinsed with 5% glutaraldehyde and the dye in the cells is extracted by addition of 150 μl of 1% acetic acid, 50% ethanol. The optical density of the solutions is determined in an automatic plate reader and the highest tolerated dose of toxicant is calculated at 90% of maximum dye uptake.