Toxicity assessment of historical aqueous film-forming foams (AFFFs) using cell-based assays

Aqueous film-forming foam (AFFF) has historically contained high concentrations of long-chain per-and polyfluoroalkyl substances (PFAS), which have been linked with adverse health outcomes. However, the toxicity of historical AFFFs remains largely unknown, presenting uncertainties in their risk assessment. This study assessed the toxicity of historical AFFFs by exposing human liver cells (HepG2) to various dilutions of 3M Light Water AFFF or Ansulite AFFF (0.001%, 0.002%, 0.005%, 0.009%, 0.019%, 0.038%, 0.075%, 0.15%, and 0.3%) for 24 h. The effects of the two AFFF formulations on the cell viability, intracellular reactive oxygen species (ROS) production, Nrf2-ARE activity, and DNA damage were assessed by CellTiter 96® Aq_ueous One Solution Cell Proliferation Assay (MTS kit), dichlorofluorescein diacetate assay, luciferase assay, and alkaline Comet assay, respectively. The results revealed that the two brands of AFFFs tested were toxic to HepG2 cells at dilutions lower than the recommended 3% application formulation. Specifically, exposure to 3M Light Water AFFF or Ansulite AFFF induced a dilution-dependent decrease in cell viability, increased intracellular ROS production, and increased Nrf2-ARE activity. However, except for the highest concentration (lowest dilution) of 3M Light Water AFFF tested (0.038%.), both 3M Light Water AFFF and Ansulite AFFF did not significantly induce cellular DNA damage. Overall, 3M Light Water AFFF was more toxic than Ansulite AFFF. The findings from this study provided valuable in vitro toxicity data that may better inform the health risk assessment of these historical AFFFs.