A screening approach for the evaluation of tobacco-free 'modern oral' nicotine products using Real Time Cell Analysis

Abstract

In many regulated industries there is an increasing pressure to provide timely and robust risk assessment data to support product launches. Real-time cell analysis (RTCA) is a tool that allows for the fast and relatively labour-free cytotoxic assessment of test compounds, compared to traditional methods. Here, we propose an application for the RTCA platform to provide a screening approach, to evaluate the cytotoxic potential of tobacco-free nicotine pouches, also termed modern oral product (MOP), to determine the contribution of differing nicotine strengths (4-11 mg) and a range of available flavour types from multiple markets, on overall product toxicity. Aqueous extracts were prepared for all products using 1 pouch in 20 mL cell culture media and applied to the cell system for 24 h. Test extract nicotine concentrations reflected the increases in product nicotine strength; however, these changes were not present in the same magnitude in the cytotoxicity data obtained from both primary human gingival fibroblasts (HGF) and an NCI-H292 human bronchial epithelial continuous cell line. Furthermore, across the range of flavours and product nicotine strengths tested, H292 cells whilst not the target organ for oral product use, accurately predicted the results seen in HGFs and could be considered a useful surrogate for fast screening studies. H292 cells are more easily cultured and for longer periods, offering a more compatible test system. In conclusion, the data demonstrate the utility of the RTCA platform for the quick assessment of a large range of product variants. Furthermore, for a cytotoxicity measure with this test product, the simple H292 cell line can predict outcomes in the more complex HGF and provide useful pre-clinical cytotoxicity screening data to inform the risk assessment of MOPs and the relative contribution of flavourings, nicotine and other components.

Keywords: AqE, Aqueous extract; CRP, 1.1 CORESTA Reference Product 1.1; Cytotoxicity; H292, Human bronchial epithelial cells; HGF, Human gingival fibroblasts; In vitro; LDH, Lactate dehydrogenase assay; MOP, Modern oral product; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NRU, Neutral red uptake assay; Nicotine; RTCA; RTCA, Real Time Cell Analysis; Risk assessment; Tobacco-free modern oral tobacco; Tobacco-free nicotine pouches.

Graphical abstract

Fig. 1

Graphical representation of the experimental method using Real-Time Cell Analysis for the assessment of Modern Oral Products. A. shows xCELLigence with docking plate. B. example of an impedance measure C. example of RT trace data w/ phases growth. D. shows sample preparation for MOP and snus.

Fig. 2

A schematic representation of a proposed MOPs in vitro assessment strategy.

Fig. 3

Recovered nicotine concentration in all extracts used in this study across all variants. Solid line represents regression; dotted line is 95 % confidence intervals.

Fig. 4

Cytotoxicity in H292 vs HGF cells as determined by RTCA for 3 different flavours (Berry Frost 4 mg nicotine (BF04), Polar Mint 4 mg nicotine (PM04), Tropical Breeze 6 mg nicotine (TB06)). Data are global mean ± SD (average cell viability across concentration range tested) from 3 independent experiments. Upper dotted line represents HGF averaged response and lower dotter line represents H292 averaged response.

Fig. 5

Samples of the same flavour (Tropical Breeze) in H292 as determined by RTCA for 3 different nicotine strengths. Data are mean ± SD from 3 independent experiments. Dotted line represents the averaged response.

Fig. 6

A. Cytotoxic assessment of LYFT flavour variants, commercial comparator and snus reference product (CRP1.1). Data are mean viability at highest concentration tested ± SD from 3 independent experiments (CRP1.1 response was adjusted for osmolarity). B. Osmolarity measures for all products tested, dotted lines indicate the physiological range of 280-400 mOsm.