In vitro human lymphocyte micronucleus assay - protocol optimisation

UKEMS 2024 -- The in vitro micronucleus (IVMN) test was endorsed for regulatory genotoxicity testing with the adoption of OECD test guideline (TG) 487 in 2010, updated in 2016 and corrected on 04 July 2023. This guideline includes recommendations for treatment of p53 competent human lymphocytes. TG 487 also allows sampling or recovery times to be extended up to a further 1.5 to 2.0 normal cell cycle lengths if a test chemical affects the cell cycling time, especially for p53 competent cells. However, it is not specified if the extended sampling or recovery times should be performed +/- Cyto-B. The merits of including a recovery period for the extended treatment arm, -S-9, have been previously investigated and discussed by Whitwell et al., 2019. These current investigations [international collaboration by 6 different laboratories coordinated by iGUM (industry subgroup of the German speaking EMGS)] concentrate on the experimental arm with pulse treatments. The standard 4-hour treatment with 1.5 to 2.0 cell cycle recovery in the presence of Cyto-B was compared against a 4-hour treatment with 2.5 to 3 cell cycle recovery, with Cyto B present for the last 1.5 to 2.0 cell cycles. The objective of this study was to compare the MN response using a variety of known clastogens and nucleoside analogues using the same short treatment length but with two differing recovery periods.