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.

Safety assessment of the substance calcium tert‐butylphosphonate for use in food contact materials

“…The Panel noted that the experiment with extended treatment did not follow the protocol recommended in the OECD TG 487, as a 24‐h incubation with the test article was performed without CytB, which was added in the subsequent 24‐h recovery period in order to avoid any possible interaction between CytB and the test article. This modified protocol was in line with more recent recommendations for the conduct of the micronucleus test with cultured human lymphocytes (Whitwell et al., 2019 ) and the EFSA recommendations for the conduct of the in vitro micronucleus assay with nanomaterials because of the known inhibition of endocytosis by CytB (EFSA Scientific Committee, 2021b ). The application of this modified protocol was considered acceptable for the testing of calcium tert ‐butylphosphonate also in view of the low solubility, with formation of precipitate, of the test item in culture medium.…”

The genotoxicological assessment of a tobacco heating product relative to cigarette smoke using the in vitro micronucleus assay

“…It was only with the application of an extended recovery/expression period that responses in TK6 and CHO cells became evident. The addition of the extended exposure/recovery period has also been recently shown to be advantageous for increasing the IVMN assay sensitivity [ 46 ] and could help with test article discrimination within category. However, these studies did not investigate alternative contemporary approaches such as high content screening, as investigated here.…”

Flavouring Group Evaluation 413 (FGE.413): Naringenin

“…In this extended treatment, cytoB was added only at the end of the 24‐h treatment and then incubated for further 24 h. The Panel noted that the extended treatment exposure conditions differed from the suggested cell treatment schedule in OECD TG 487 (OECD, 2016a ). However, the Panel considered that the protocol applied for the extended treatment could potentially enhance the sensitivity of the MN test (Whitwell et al., 2019a ). Therefore, the Panel did not consider this aspect as a limitation.…”