Neutral red uptake for starting doses for acute oral toxicity

The in vitro Neutral Red Uptake (NRU) test method is a cell-based approach that assesses the cellular uptake of a dye in the presence of a particular substance. The protocols use normal human epidermal keratinocytes (NHK) and BALB/c 3T3 mouse fibroblasts (3T3). NHK cells are primary cells while 3T3 cells are an inmortal line.

The method is used to assess basal cytotoxicity quantified through IC50 values (the concentration of test substance needed to decrease cell viability by 50% in the cell culture) by spectrophotometric measurments of the cellular uptake of NR dye.

Assays that assess this type of toxicity look at how chemicals exert effects by disrupting structures and functions that are universal to all cells (e.g. cell membrane integrity, energy production and metabolism, transportation of molecules, secretion of molecules).This is opposed to assays that assess the effects of chemicals on specific molecular targets (such as receptors, ion channels etc.), lacking in the cells used in this method.

Due to the fact that substances that produce injury and death are ultimately exerted at the cellular level, in vitro cytotoxiciicty assays may be also useful for the prediction of acute lethaly potency.

The NRU IC50 concentration of a test substance in either 3T3 or NHK cells could be used to estimate the rodent LD50 value as a mean for predicting the starting doses for rodent acute oral toxicity studies.