Inclusion of an extended treatment with recovery improves the results for the human peripheral blood lymphocyte micronucleus assay
Abstract The in vitro micronucleus (IVMN) test was endorsed for regulatory genotoxicity testing with adoption of the Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 487 in 2010. This included two equally acceptable options for extended treatment in the absence of metabolic activation: a treatment for 1.5–2.0 cell cycles with harvest at the end of treatment (Option A) or treatment for 1.5–2.0 cell cycles followed by recovery for 1.5–2.0 cell cycles prior to harvest (Option B). Although no preferences were discussed, TG 487 cautions that Option B may not be appropriate for stimulated lymphocytes where exponential growth may be declining at 96 h after phytohaemagglutinin (PHA) stimulation. Following revision of TG 487 in 2014 and 2016, emphasis has been placed on using Option A. Given the purpose of the IVMN assay is to determine both clastogenic and aneugenic potential, the authors believe the assay is compromised if an extended treatment with recovery is not included for sensitive detection of certain classes of chemical. In this study, average generation time (via bromodeoxyuridine incorporation) of human peripheral blood lymphocytes (HPBL) was measured up to 144 h after PHA stimulation. In addition, the HPBL micronucleus (MN) assay was performed using Option A and B treatment schedules. Cytotoxicity (replication index) and MN induction were determined following treatment with 14 chemicals. The data demonstrate that lymphocytes actively divide beyond 96 h after PHA stimulation. Furthermore, MN induction was only observed with some aneugenic chemicals and nucleoside analogues in HPBLs following extended treatment with a recovery period. For the majority of chemicals tested the magnitude of MN induction was generally greater and MN induction was observed across a wider concentration range following the Option B treatment schedule. In addition, steep concentration-related toxicity following treatment without recovery is more common, making selection of suitable concentrations (within regulatory toxicity limits) for MN analysis challenging.
