In vitro and in silico genetic toxicity screening of flavor compounds and other ingredients in tobacco products with emphasis on ENDS

J Appl Toxicol. 2020 Nov;40(11):1566-1587. doi: 10.1002/jat.4020. Epub 2020 Jul 13.


Electronic nicotine delivery systems (ENDS) are regulated tobacco products and often contain flavor compounds. Given the concern of increased use and the appeal of ENDS by young people, evaluating the potential of flavors to induce DNA damage is important for health hazard identification. In this study, alternative methods were used as prioritization tools to study the genotoxic mode of action (MoA) of 150 flavor compounds. In particular, clastogen-sensitive (γH2AX and p53) and aneugen-sensitive (p-H3 and polyploidy) biomarkers of DNA damage in human TK6 cells were aggregated through a supervised three-pronged ensemble machine learning prediction model to prioritize chemicals based on genotoxicity. In addition, in silico quantitative structure-activity relationship (QSAR) models were used to predict genotoxicity and carcinogenic potential. The in vitro assay identified 25 flavors as positive for genotoxicity: 15 clastogenic, eight aneugenic and two with a mixed MoA (clastogenic and aneugenic). Twenty-three of these 25 flavors predicted to induce DNA damage in vitro are documented in public literature to be in e-liquid or in the aerosols produced by ENDS products with youth-appealing flavors and names. QSAR models predicted 46 (31%) of 150 compounds having at least one positive call for mutagenicity, clastogenicity or rodent carcinogenicity, 49 (33%) compounds were predicted negative for all three endpoints, and remaining compounds had no prediction call. The parallel use of these predictive technologies to elucidate MoAs for potential genetic damage, hold utility as a screening strategy. This study is the first high-content and high-throughput genotoxicity screening study with an emphasis on flavors in ENDS products.

Keywords: DNA damage; QSAR; biomarkers; computational toxicology; electronic nicotine delivery systems; flavor; in vitro; in vitro genotoxicity; tobacco products.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Biomarkers / metabolism
  • Cell Line
  • Consumer Product Safety
  • DNA Damage*
  • Electronic Nicotine Delivery Systems*
  • Flavoring Agents / chemistry
  • Flavoring Agents / toxicity*
  • Flow Cytometry
  • Histones / metabolism
  • Humans
  • Machine Learning*
  • Mice
  • Models, Molecular*
  • Mutagenicity Tests*
  • Phosphorylation
  • Quantitative Structure-Activity Relationship
  • Rats
  • Risk Assessment
  • Tumor Suppressor Protein p53 / metabolism


  • Biomarkers
  • Flavoring Agents
  • H2AX protein, human
  • Histones
  • TP53 protein, human
  • Tumor Suppressor Protein p53