Use of human derived cell lines to increase the biological relevance of cigarette condensate, non-tobacco materials and e-liquids testing
The in vitro testing strategy of tobacco products is mainly based on the testing of extracts from cigarette smoke condensates. An accepted industry wide assay battery has been developed over recent years which delivers reliable results for the toxicological assessment of tobacco products. The testing battery comprises the neutral red uptake (NRU) assay, the in vitro Micronucleus (IVM), and the Ames test. This combination of tests evaluates relevant biological endpoints and is also used for the toxicological evaluation of non-tobacco materials (NTM) such as ingredients and, more recently, e-liquids. All methods are based on the corresponding OECD guidelines which leave some choices regarding the specific bacteria strains used in Ames test and cell lines utilized in NRU and IVM assays. To increase relevance for toxicity assessment, human cell lines were established whenever possible to measure cytotoxicity and genotoxicity of smoke extracts, non-tobacco materials and e-liquids. Human liver (HepG2) and bronchial cells (BEAS-2B) were used in the NRU assay where both demonstrated high sensitivity against cigarette condensate and delivered good results for e-liquids. The IVM assay was performed with the human lymphoblastoid cell line TK6 as extension to the V79 rodent cells. According to literature false positive effects are significantly reduced when using TK6 in comparison to widely used rodent cell lines. The assay battery was completed by mutagenicity testing using the Ames test. Here, representative results using different human cell lines with cigarette smoke condensate and e-liquids in the NRU and IVM tests are shown. Results of cigarette smoke condensate induced bacterial mutagenicity are shown for the two Salmonella typhimurium strains TA 98 and TA 100.
In summary this poster shows representative results that demonstrate the general applicability of IB’s biological in vitro testing for the assessment of next generation products (NGPs).