Validation Work

In Vivo Micronucleus

Sequani are currently validating the analysis of micronuclei in rat and mouse peripheral blood lymphocytes by flow cytometry, to be used on stand-alone or ‘bolt-on’ in vivo micronucleus assays.  This is advantageous as flow cytometry can analyse 20,000 cells in less than 1 minute per animal vs. 2,000 cells in 20 minutes per animal by manual microscopy.  The small sample size for peripheral blood lymphocytes also means that multiple samples can be taken throughout the time course of study.

Initial data is looking promising though further work is required:

• to confirm reproducibility of detecting a known clastogen (the proposed new ICH S2(R1) guideline (at Step 3, Consultation, of the ICH process) does not require concurrent positive controls on each study, therefore, a ‘library’ of positive control slides is being compiled, that would be scored on each study).

• to confirm sensitivity of flow cytometric analysis of rat peripheral blood lymphocytes (rats, but not mice, undergo splenic removal of micronuclei, hence flow cytometry needs to be sensitive enough to detect an increased micronucleated reticulocyte population.

• to build up an historical control database.

The inclusion of an in vivo micronucleus assessment on a repeat dose toxicology study (micronucleus ‘bolt-on’) has many advantages, such as: reduction in the numbers of animals used; reduction in test article usage; biological relevance of the micronucleus assessment in toxicology animals; concurrent toxicokinetic analysis for confirmation of systemic exposure.

In Vitro Micronucleus

The in vitro micronucleus assay has the same endpoint as in vivo micronucleus assay, in that breakage of chromatids or chromosomes can result in micronucleus formation if an acentric fragment is produced; therefore, this assay is appropriate for detecting clastogens.  Micronuclei can also result from the lagging of one or more whole chromosome(s) at anaphase and thus micronucleus tests have the potential to detect some aneuploidy inducers.  The assay concurrently measures cytotoxicity (using the cytokinesis-block proliferation index) and genotoxicity (measurement of the frequency of micronucleated binucleate cells). 

The in vitro micronucleus OECD draft guideline (487) is still under review.  Therefore, Sequani are currently validating a range of known non-mutagens, aneugens and clastogens (direct acting and those requiring metabolic activation) to confirm sensitivity, predictivity and reproducibility of the assay, prior to Sequani offering it as part of the ICH genotoxicology battery of tests. 

This assay will be invaluable as a high throughput screening tool, and is currently available as a screening assay for compounds in development; in conjunction with an Ames Screen, if required, for greater concordance with the regulatory genotoxicology battery of tests.