Giridhar, J. and D. Acosta. Evaluation of Cytotoxicity Potential of Surfactants Using Primary Rat Keratinocyte Culture as an In Vitro Cutaneous Model. In Vitro Toxicol. 1993. 6(1): 33-46

Miranol HM 24% (lauroamphodiacetate) - 14350-96-0; Mirataine CBS (cocamidopropyl hydroxysultaine) - 98227-97-5; Triton X-100 - 9002-93-1

Primary rat keratinocytes were cultured from skin epidermis of neonatal rat pups by a trypsin digestion method. Two-three day old confluent cultures were treated with five surfactants, anionic, amphoteric and nonionic surfactants, in varying concentrations. Evaluation of cytotoxicity after surfactant treatment was measured by: (i) monitoring leakage of cytosolic enzyme lactate dehydrogenase (LDH) into the medium, (ii) mitochondrial reduction of 3-(4-5-dimethylthiazol-2yl-)-2,5 diphenyl tetrazolium bromide (MTT) and (iii) lysosomal uptake of the dye neutral red (2-amino-3-methyl-7-dimethyl-amino-phenazoniumchloride) (NR), at the end of a 1 hr treatment period and after 24 hrs immediately following treatment and removal of surfactants. Additionally, changes in cell morphology were assessed by light microscopy. Dose-response curves were generated using the ALLFIT curve fitting program and EC50 values were calculated. Results show a potential for causing cytotoxicity among the surfactants in the following way: nonionics = anionics > amphoterics. Triton X-100, a severely irritating nonionic surfactant in vivo, had a cytotoxicity potential equal to anionic surfactants and continued to show damage even after the 1 hr treatment period and subsequent removal of the surfactant. In contrast, the mild to moderately irritating amphoteric and anionic surfactants caused most of the damage during the 1 hr treatment period. LDH leakage was a good indicator of cell damage, and additionally the findings of the assays MTT and NR were comparable to the LDH leakage results. We conclude that primary rat keratinocytes serve adequately as an in vitro model in the screening of surfactants for skin irritancy potential.