NEUTRAL RED ASSAY USING NORMAL RABBIT CORNEAL EPITHELIAL CELLS GROWN IN SERUM-FREE MEDIUM AS AN ALTERNATIVE TO THE DRAIZE IRRITATION TEST

HISASHI TORISHIMA1, RYOHEI YAMAMOTO1 and MASAMI WATANABE2
1Biochemical Department, Technical Research Laboratory, Kurabo Industries Ltd., 14-5 Shimokida, Neyagwa, Osaka 572, Japan; 2School of Pharmaceutical Science, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki 852, Japan

Regular article :AATEX3(1):29-36

Abstract
Neutral red assay using normal rabbit corneal epithelial (NRCE) cells were examined as an in vitro alternative to the Draize ocular irritation test. The cells were cultured in serum-free medium to reduce interference with serum components, especially serum proteins.
It was confirmed that the amount of neutral red incorporated into the cells was proportional to the number of viable cells, and the incorporation of the dye was affected by the concentration of calcium ion in the medium. Seventeen detergents were compared for the NR50 values obtained by this method to Draize ocular irritation test scores. The correlation coefficient between the NR50 value and the Draize rank was -0.694, and that between the NR50 and the DS20, a Draize score equal to 20 units from a possible total of 110, was 0.644.
By using neutral red assay, the sensitivity of chemicals was compared NRCE cells with established cells (SIRC) grown in serum-containing medium. NRCE cells cultured in serum-free medium were more sensitive to the cytotoxicity of chemicals than SIRC cells in serum containing medium. The higher sensitivity of the NRCE cells was mainly due to the use of serum-free medium, and also due to the higher sensitivity of normal cells to irritants tested.



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