Optimization of Cytokinesis-Block Micronucleus Assay for Clastogenic Risk Assessment of Silver-Containing Nanomaterials

Nanomaterials have been extensively incorporated and are commonly used in everyday life. Rising human exposure to risks has highlighted the carcinogenic hazards from prolonged exposure, prompting the development of “Nanogenotoxicology” as a distinct academic field. However, conventional in vitro micronucleus assay systems cannot ensure adequate contact between nanomaterials and cellular genetic material, which prevents an accurate assessment of the clastogenic risk posed by nanomaterials. In this study, the p53-competent human cell line TK6 and the karyotypically stable conventional cell line CHL were used to conduct a cytokinesis-block micronucleus assay (CBMN), and the cytoskeleton inhibitor cytochalasin B was added to the culture system after nanomaterial exposure. Ag40 (40 nm silver nanoparticles) and polystyrene microspheres were employed as the nanoscale positive and negative controls, respectively. Prior to this study, hyperspectral imaging and inductively coupled plasma mass spectrometry were used to verify that the cellular association of Ag40 was not markedly affected by the addition of rat liver S9 mixture and cytochalasin B. Our results showed that, under exposure periods of 4 h (with or without rat liver S9 mixture), 24 h, 48 h and 72 h, Ag40 at concentrations ranging from 5 to 40 µg/mL significantly increased the micronucleus frequency up to 6%, while polystyrene microspheres showed negative results. Therefore, Ag40 and polystyrene microspheres were identified as the respective nanoscale positive and negative controls for CBMN. The established method was further validated in two independent laboratories using a nano-silver burn wound dressing, and both chemical and nanoscale controls were included. The methodology developed in the present study provided a foundational basis for the development of the standard YY/T 1897-2023 in China.