Evaluation of the human epidermal keratinocyte neutral red release and neutral red uptake assay using the first 10...

Two methodologies used in vitro to estimate cytotoxicity in cell culture systems were compared: these were the neutral red uptake assay (NRU), which is used to measure toxicity caused by an extended (48-hr) exposure to the test material, and the neutral red release assay (NRR), which is used to measure toxicity caused by a short-term (1-min) exposure to the test material. Both methodologies used the normal human epidermal keratinocyte (NHEK)-based NeutralRed Bioassay supplied by Clonetics Corporation (San Diego, CA, USA). 10 materials (paracetamol, acetylsalicylic acid, ferrous sulphate, diazepam, amitriptyline, digoxin, ethylene glycol, methanol, ethanol and isopropanol), which are part of the Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) panel, were tested. NRU(50) values for the 10 compounds covered more than an eight-log range from 0.004 mum (digoxin) to 1.0 x 10(6) mum (methanol). Because of solubility limits, NRR(50) values for diazepam, digoxin, ferrous sulphate and paracetamol could not be determined. NRR(50) values for the remaining six compounds covered approximately a three-log range from 3.2 x 10(3) to 7.1 x 10(6) mum. When compared with documented values for either the human acute oral lethal dose or the human acute lethal blood concentration, the NRU assay was found to be much more useful in predicting human acute toxicity than was the NRR assay.