The in vitro ToxTracker and Aneugen Clastogen Evaluation extension assay as a tool in the assessment of relative genotoxic potential of e-liquids and their aerosols

This study evaluated the utility of the ToxTracker assay—a high-throughput, mechanism-based genotoxicity screening tool—for assessing the relative safety of electronic vapour products (EVPs) compared to conventional cigarettes. The researchers sought to determine if the assay could provide mechanistic insights into (geno)toxicity, such as DNA damage, oxidative stress, and protein damage, for complex mixtures like e-liquids and aerosols. The study tested various tobacco-flavoured e-liquids (with and without nicotine) and their corresponding aerosols, using Kentucky 1R6F reference cigarette smoke as a comparative benchmark. The researchers also validated the assay's performance when using different delivery vehicles (DMSO, neat e-liquid base, and PBS-bubbled aerosol) to ensure that the test medium itself did not interfere with the results.

The study found that EVP-derived samples (both aerosols and neat e-liquids) showed significantly reduced biological activity compared to 1R6F cigarette smoke. While cigarette smoke induced strong responses in DNA damage and oxidative stress reporters, the EVP aerosol samples did not trigger any of the six ToxTracker GFP reporters, even at high concentrations. Neat e-liquids did induce oxidative stress markers at higher concentrations, but this was attributed to increased osmolarity in the cell culture medium rather than direct genotoxicity. Furthermore, the nicotine content of the e-liquids did not drive the observed responses, as pure nicotine only induced stress at supraphysiological levels far exceeding those found in the test articles.

As the market for smoking alternatives grows, there is a critical need for rapid, sensitive, and mechanistic screening tools to supplement traditional regulatory assays like the Ames test. This study demonstrates that the ToxTracker assay is a robust, informative platform for assessing the relative risk of compositionally complex products. By successfully distinguishing between the genotoxic potential of cigarette smoke and the lack thereof in EVP aerosols, the assay provides a valuable, high-throughput method for the safety assessment of next-generation nicotine delivery systems.