Mammalian cell genotoxicity – Micronucleus test

Aim: Assessment of genotoxic potential in an in vitro mammalian cell screening assay.

Background information: Test items showing genotoxic activity are considered to have the potential to be human carcinogens and/or mutagens, this being an important liability for drug development. Micronuclei (MN) are small chromatin containing bodies that represent fragments of chromosomes or whole chromosomes that are not incorporated into the daughter cells nuclei during the cell division process and remain as small nuclei in the cell cytoplasm. Test items that are clastogens (cause DNA breaks) or aneugens (interfere with the mitotic spindle) can increase the frequency of micronucleus formation in cells undergoing cell division, this being considered and indication of genotoxic damage (OECD 487, 2016).

In this assay, the induction of micronuclei in CHO cells after test item exposure is assessed by flow cytometry analysis.

Cell type: CHO cells

Experimental procedure

  • Top concentration: 1 mM or 500 µg/mL (whichever lower), unless limited by solubility/toxicity
  • 4 h exposure period; 37ºC, 5% CO2; 24 h expression period
  • Positive controls:
  • – S9: Mitomycin C (0.4 µg/mL)
  • + S9: Cyclophosphamide (4 µg/mL)

Delivered results

  • Cytotoxicity: flow cytometry nuclei to beads ratio
  • Genotoxicity: flow cytometry frequency of MN per (‰)
  • Positive when > 2-fold increase in MN frequency & <50% cytotoxicity