In vitro cell-transforming capacity of micro- and nanoplastics derived from 3D-printing waste

The increasing use of plastic polymers in 3D printing applications may lead to human exposure to micro- and nanoplastics (MNPLs), raising concerns regarding adverse health consequences such as cancer induction. Little attention has been given to MNPLs originated at the end of the life cycle of 3D-printed objects because of the mechanical and environmental degradation of plastic waste. This study assessed the carcinogenic potential of secondary MNPLs generated through cryomilling of 3D objects using the validated in vitro Bhas 42 cell transformation assay (CTA). Three-dimensional objects were printed using four types of polycarbonate (PC)- and polypropylene (PP)-modified thermoplastic filaments, undoped and doped with single-walled carbon nanotubes (PC-CNT) and silver nanoparticles (PP-Ag), respectively. MNPLs (< 5 µm) generated following a three-step top-down process were thoroughly characterized. Bhas 42 cells were treated once (initiation assay) or repeatedly (promotion assay) with several concentrations of MNPLs (3.125-100 µg/mL) mimicking realistic exposure conditions, and transformed foci formation was evaluated after 21 days. Furthermore, cellular internalization and the mRNA expression of seven genes previously recognized as part of a predictive early cell transformation signature were also evaluated. Despite being internalized, none of the particles was able to initiate or promote in vitro cell transformation, regardless of doping with nanomaterials. Alternatively, all the particles significantly increased and decreased the mRNA expression of Prl2c3 and Timp4, respectively, under promotion conditions, indicating early changes that occur before the formation of transformed foci. These findings suggest that the test MNPLs could have a tumorigenic potential despite not showing morphological changes in Bhas 42 cells.