Abstract
The development of reduced-risk products aims to provide alternatives to cigarettes that present less risk of harm for adult smokers. Responsible use of flavoring substances in these products may fulfill an important role in product acceptance. While most flavoring substances used in such products are also used by the food industry and are considered safe when ingested, their impact when inhaled may require further assessment. To aid in such an assessment, a three-step approach combining real-time cellular analysis, phenotypic high-content screening assays, and gene expression analysis was developed and tested in normal human bronchial epithelial cells with 28 flavoring substances commonly used in e-liquid formulations, dissolved individually or as a mixture in a base solution composed of propylene glycol, vegetable glycerin, and 0.6% nicotine. By employing this approach, we identified individual flavoring substances that potentially contribute greatly to the overall mixture effect (citronellol and alpha-pinene). By assessing modified mixtures, we showed that, although cytotoxic effects were found when assessed individually, alpha-pinene did not contribute to the overall mixture cytotoxicity. Most of the cytotoxic effect appeared to be attributable to citronellol, with the remaining substances contributing due to synergistic effects. We developed and used different scoring methods (Tox-Score, Phenotypic Score, and Biological Impact Factor/Network Perturbation Amplitude), ultimately enabling a ranking based on cytotoxicity, phenotypic outcome, and molecular network perturbations. This case study highlights the benefits of testing both individual flavoring substances and mixtures for e-liquid flavor assessment and emphasized the importance of data sharing for the benefit of consumer safety.
Highlights
As an alternative to cigarettes, a large number of electronic nicotine delivery systems (ENDS), including electronic cigarettes (e-cigarettes), have been developed and launched on the market
As previously shown [4], both propylene glycol (PG) and vegetable glycerol (VG) mostly exert their effect by inducing a dosedependent increase in osmolality, which appeared to be the main cause of cytotoxicity (Fig. S5)
The approach described in this paper is based on three pillars: (1) real-time cellular analysis (RTCA), (2) evaluation of a panel of phenotypic highcontent screening (HCS) endpoints, and (3) gene expression analysis
Summary
As an alternative to cigarettes, a large number of electronic nicotine delivery systems (ENDS), including electronic cigarettes (e-cigarettes), have been developed and launched on the market. These systems heat a solution (e-liquid) to generate an aerosol, which is inhaled by the user. A combination of different flavorings substances characterizing each unique product is added to complete the formulation. Many of these flavor additives, usually found in food, have been extensively assessed and are “generally recognized as safe” (GRAS) [1]. GRASTM certification by the Flavor Extracts Manufacturers
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