Abstract: Objective To explore the reliability and sensitivity of an in vitro PIG-A gene mutation assay based on TK6 cells, and establish an experimental system for evaluating N-nitrosamine impurities. Methods Immunomagnetic bead separation was used to eliminate the spontaneous mutation rate of the PIG-A gene in TK6 cells. As required by the in vitro metabolic activation for mutagenicity assessment of nitrosamine impurities, the assay was performed with hamster liver S9 metabolic activation. DMSO served as the negative control, and NDEA (31.25-2 000 μg·mL^-1) as the positive control. High-throughput flow cytometry was used to detect changes in the CD55 and CD59 phenotypes of TK6 cells. Under the established conditions, two nitrosamine impurities related to pharmaceutical structures were tested: 1-cyclopentyl-4-nitrosopiperazine (CPNP, 50-300 μg·mL^-1) and N-nitrosobumetanide (250-2 000 μg·mL^-1). Results The spontaneous mutation frequency decreased to 0.007 2% after the final elimination. In the absence of metabolic activation, no significant differences were observed in any of the NDEA groups compared with the negative control. In the presence of hamster S9, the PIG-A gene mutation frequency was significantly increased at NDEA concentrations of 500 μg·mL^-1 and above. Under S9 activation, the mutation frequency was significantly elevated in the 150 and 300 μg·mL^-1 CPNP groups, but no significant differences were found in any of the N-nitrosobumetanide groups. Without metabolic activation, neither CPNP nor N-nitrosobumetanide showed significant differences at any dose compared with the negative control. Conclusion The in vitro PIG-A gene mutation assay based on TK6 cells can effectively detect the mutagenic risk of N-nitrosamine impurities. In the future, it can be combined with next-generation sequencing technology to conduct genotoxicity evaluation of nitrosamine compounds.

Key words: N-nitrosamine Impurities, PIG-A Gene Mutation Assay, TK6 Cells, Metabolic Activation