Micronucleus assay use in genetic toxicology

Micronucleus assays have been extensively used in genotoxicity assessment for decades. Tests were for many years conducted in vivo in the bone marrow or peripheral blood cells, but it eventually got traction in vitro as well. Adoption of OECD test guideline 487 in 2010 made the in vitro micronucleus assay part of the available battery of in vitro studies for regulatory use. In a few years, it has largely replaced the in vitro chromosome aberration assay due to the ability to detect aneugenicity in addition to clastogens. In fact, combined with the Ames OECD 471 test, the in vitro micronucleus is in some cases considered sufficient for genotoxicity assessment of test compounds.

Gentronix move into micronucleus testing

Gentronix invented the Greenscreen and the Bluescreen, and originally started the business as a kit provider for these assays, but then realised that there was a market in performing contract testing, particularly for clients that did not operate their own labs. 

As we looked to grow as a company, it then made sense to look at other genotoxicity assays, and of course, ones relevant to our client’s needs. The in vitro micronucleus assay fit the bill, and we dipped our toes in the water with the flow cytometric format of the assay in TK6 cells. This works very well for most compounds and required only 15 mg for ICH top test item concentrations, or 50 mg for ten mM tests (as would be required for an OECD 487 study if there was no cytotoxicity or solubility limiting).

The next step of Gentronix’s growth was to start to offer Good laboratory practice (GLP) testing services, which we began to work up as we left the Manchester University campus and relocated to Alderley Park. This included a significant recruitment drive, and we hired several members of staff that had previously worked on the Alderley Park site for AstraZeneca or Syngenta. We set up the Ames lab first with the in vitro micronucleus assay going online to GLP soon after, using human peripheral blood lymphocytes which are primary human cells.  

Following our GLP lymphocyte offering, we started to work on using TK6 cells, primarily for screening but also for GLP studies. In 2015/2016, we setup the TK6 in vitro micronucleus assay in 96-well plates, allowing us to score the same number of cells as a conventional GLP study, but using significantly less test item.  

Adding mode of action data

The latest evolution of our in vitro micronucleus service offering is incorporating fluorescence in situ hybridisation (FISH). FISH is used to differentiate micronuclei with and without centromeres, and an increase in centric micronuclei is indicative of aneuploidy; a decrease is indicative of clastogenicity. Being able to determine whether micronuclei are likely to result from clastogenicity or aneuploidy can help inform project risk, confirm whether effects are related to the chemical’s intended mode of action, or help select risk assessment options.  

We setup FISH originally as an addition to our 96-well screening in vitro micronucleus format, essentially using extra wells when setting up the micronucleus study, allowing us to make slides for FISH and score them only if the study was positive for micronuclei. This way, we had the reassurance that the FISH slides were from the same experiment (reducing potential variability) and reducing the time to results.

Increasingly there is a focus on determining whether positive in vitro micronucleus GLP studies are associated with increases or decreases in centric micronuclei. EFSA has made this more of a focus in their draft guidance document on aneugenicity for example, so it made sense to include FISH in our GLP offering.  

Gentronix offers the following in vitro micronucleus options to suit a range of purposes and stages of product development:

  • OECD 487 GLP studies in TK6 cells with the addition of FISH as an option – scored by microscopy
  • OECD 487 GLP studies in isolated human lymphocytes – scored by microscopy
  • 96-well studies in TK6 cells with the addition of FISH as an option – scored by microscopy
  • MicroFlow® in TK6 cells – scored by flow cytometry

If you would like to know more about our in vitro micronucleus services or would like a quote, please contact us at info@gentronix.co.uk.