P25-17 New approach methods to study immunotoxicity of per- and polyfluoroalkylsubstances using peripheral blood mononuclear cells

In 2020, the EFSA CONTAM panel selected the reduced antibody response to vaccination in 1-year-old children as critical endpoint for the assessment of per- and polyfluoroalkylsubstances (PFASs). As a result, a tolerable weekly intake (TWI) of 4.4 ng/kg for the combined exposure to PFOA, PFNA, PFHxS and PFOS was established. However, mechanisms underlying this immunotoxicity of PFASs are largely unknown. Understanding these mechanisms is essential to support a causal link between PFASs exposure and immunotoxic effects observed in epidemiological studies. Additionally, mechanistic assays could aid in selecting relevant readouts for in vitro test systems, enabling the comparison of PFASs potencies and providing in vitro points of departure for next-generation risk assessment. Peripheral blood mononuclear cells (PBMCs) provide a valuable cell population to study immunotoxicity, as they comprise a relevant mixture of human immune cells. Additionally, since PBMCs are derived from human blood, also interindividual differences can be studied and characterized. The present study aims to develop new approach methods to assess immunotoxic effects of PFASs using PBMCs. Initially, the most effective strategy to stimulate B cells was investigated using five different types of stimulation, mimicking T cell-dependent and T cell-independent activation. B cell activation was subsequently evaluated by quantifying IgM and IgG release, as well as by evaluating B cell subsets, including class switched memory B cells, antibody secreting cells and plasma-blasts, along with their activation status using CD69 and CD83 marker expression through flow cytometry. Finally, effects of different PFASs on B cell activation were investigated. Results showed that PFASs caused concentration-dependent decreases in IgM and IgG secretion and (slight) reductions in B cell subsets and their activation. The extent of the observed effects varied amongst donors. Altogether, the current study provides insights into the effects of selected PFASs on B cells, which contributes to a better understanding of the reported immunotoxic effects of PFASs in vivo. In addition, these in vitro assays may serve as useful tools for evaluating the immunotoxic potential of other PFASs with limited or no available data.