Preparation and stability characterization of flavour ingredients in E-liquids for preclinical assessment of electronic nicotine delivering system products: a case study of 38 flavour ingredients in a single mixture.

Abstract

E-vapour products generate aerosols typically containing nicotine, flavour ingredients, and aerosol formers (propylene glycol and vegetable glycerine). While many flavour ingredients are 'generally recognized as safe (GRAS)' for oral use in the food industry, there exist knowledge gaps on their effects when delivered by the inhalation route. Due to the large number of available ingredients and potential combinations used to create e-liquids, toxicological and analytical evaluation of each flavour ingredient is impractical. Moreover, chemical characterization requires analytical methods to be developed and validated to measure key ingredients, as well as stability assessments to demonstrate that these test materials were stable during the testing period, which is equally challenging. In this study, we present a pragmatic approach of preparing 'preblends' prior to making a test formulation, containing 38 flavour ingredients as an example case, ahead of preclinical toxicity testing. We used the preblends to simplify the preparation and the characterization of test formulations, establishing the stability criteria for the subsequent toxicity testing. We prepared preblends by dividing the 38 flavour ingredients into five preblend groups based on structural moiety, solubility, and chemical reactivity. These preblends were mixed to make two different 'final' test formulations (containing all 38 flavour ingredients with and without nicotine). We evaluated the stability of the preblends and the two test formulations prior to the subsequent in vivo inhalation studies. Based on the analytical assessment, all the preblends were stable up to 4 weeks at 0°C-4°C. When all preblends were mixed, the test formulation was stable up to 3 days in the presence of nicotine and 10 days without nicotine when stored at 0°C-4°C. These stability results were used to set the frequency of preparing the test formulations for the in vivo inhalation studies, ensuring stability o