Assessing the cytotoxicity of e-liquids without the use of the neutral red uptake assay
As part of the pre-clinical assessment performed for consumer products in the tobacco industry, an in vitro assessment of a tobacco product’s cytotoxicity was performed using the neutral red uptake (NRU) assay. Since 2004, the CORESTA In Vitro Task Force has recommended the performance of the NRU for cigarette smoke condensate testing. The objective of this study was to assess the cytotoxicity of (-)-nicotine and (-)-nicotine-containing e liquids in the NRU assay. Balb/c 3T3 cells were exposed to (-)-nicotine for 24 hours according to the INVITTOX Protocol 3a (1990). Following this exposure, the viability of the cells was determined spectroscopically by measuring the absorbance of the dye Neutral Red, which had been taken up into the lysosomes of the cells. The test was performed as part of a Good Laboratory Practice study. It was found that the robust relationship between absolute cell number (assessed by an electronic cell counter) and the amount of Neutral Red stored by a given population of treated cells was lost when (-)-nicotine was the test item. Disruption of lysosomal volume by (-)-nicotine appeared to underlie the loss of data integrity in the NRU assay. In the course of a search for an orthologous high-throughput method to assess the cytotoxicity of (-)-nicotine and e liquids, several alternative in vitro approaches were evaluated. The most consistent and robust approach that emerged was the use of the tetrazolium salt WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt], which produces a water-soluble formazan dye, in direct proportion to the number of living cells, upon reduction in the presence of an electron. A validation study was performed to characterise the performance of the WST-8 assay over several days and with different laboratory technicians performing the test.