An NC3Rs project grant awarded to Professor Richard Walmsley, from the University of Manchester, has been used to develop a human cell-based assay for genotoxicity, now taken up by over 100 companies worldwide.
Principal Investigator: Richard Walmsley, Professor of Genetics
Organisations: University of Manchester and Gentronix Limited
Award: £133,012 in 2006, over 12 months
Title: Development of a new human cell genotoxicity assay to reduce the use of live animals in drug development
Read more about Professor Walmsley's research
Genotoxicity assays have a high incidence of false positive results
It is a legal requirement that new substances such as pharmaceuticals, chemicals and consumer products are screened for their potential to cause cancer by genotoxic and non-genotoxic mechanisms. The standard regulatory test battery includes an assessment of genotoxicity in bacterial and mammalian cells in vitro. The assays are highly sensitive but have poor specificity and consequently many substances (historically over 50%) are erroneously identified as potential carcinogens (so-called false or misleading positives).
A positive result in an in vitro test can trigger extensive mechanistic studies, usually in rats and mice, to confirm the genotoxic potential of the substance and assess the relevance of the result in terms of exposure. False positives therefore drive the use of animals. In 2006, Professor Richard Walmsley, University of Manchester, was awarded NC3Rs funding to support the development of a mammalian cell-based genotoxicity assay with high specificity and a low rate of false positive findings.
Development of a new human cell-based assay
Professor Walmsley had previously developed the GreenScreen HC assay. This is a human cell-based reporter assay which exploits elements of the human GADD45a gene coupled to the gene encoding green fluorescent protein (GFP). Expression of GADD45a is up-regulated in response to genotoxic stress, and DNA damage therefore leads to increased cellular fluorescence through the production of GFP, which can be detected spectrophotometrically.
With NC3Rs funding, Professor Walmsley has been able to further develop and test the GreenScreen HC assay. This has included validation of a flow cytometric method, which allowed the development of the S9 version of the assay that tests for compounds requiring metabolic activation to become genotoxic. There are now published GreenScreen HC validation data for 161 compounds with in vivo genotoxicity data and 129 compounds with carcinogenicity data.
A high specificity genotoxicity assay reduces the use of animals
The GreenScreen HC assay is unique in two respects. Firstly, it detects all mechanistic classes of genotoxicants, including those which cause mutations, chromosome breakages and abnormal chromosome numbers. It therefore provides a single test for genotoxic hazard screening. This is in contrast to current regulatory assays, such as the Ames test and mouse lymphoma assay, which only identify mutagens and hence the requirement for a test battery approach. Secondly, in addition to its high sensitivity (89%) to genotoxic carcinogens, GreenScreen HC also has a high specificity (95%) - a much lower false positive rate than other routinely used tests.
For each positive identified in a genotoxicity assay, up to 200 animals are used for follow-up investigation. The improved specificity of the GreenScreen HC assay has had a significant impact with around 500,000 fewer animals used globally each year for genotoxicity studies in the pharmaceutical and chemical sectors.
The assay has a 96-well microplate format suitable for high-throughput screening. Four compounds are tested at nine serial dilutions per plate, and results are available within 48 hours. Small amounts of compound are required and this allows the assay to be used to make internal decisions early in development to eliminate genotoxic substances, thus preventing animals being used unnecessarily for other studies. To realise the full potential of GreenScreen HC, however, particularly in terms of reducing animal use, it needs to be approved for regulatory purposes. Formal validation and acceptance by regulatory authorities is essential. Currently the only genotoxicity assays that have this status are the bacterial and mammalian assays, which have lower specificity than GreenScreen HC. In April 2013 Gentronix submitted a case for retrospective validation to the European Centre for the Validation of Alternative Methods.
Global uptake across the pharmaceutical and chemical sectors
The GreenScreen HC assay has been commercialised through the spin-out company Gentronix, which was founded by Professor Walmsley with the support of the University of Manchester. The assay has been used by around 100 companies worldwide in diverse sectors including pharmaceuticals, agrochemicals and consumer products. Close to 12,000 compounds have now been assayed. Gentronix has also introduced the BlueScreen HC, with the GADD45a gene coupled to the Gaussia luciferase reporter gene. Using the Gentronix assay as part of an in vitro screening cascade, GlaxoSmithKline has effectively removed attrition due to candidate drug genotoxicity, saving approximately 300 animals per year.
There have been five publications from the NC3Rs-funded research.
This case study was published in a review of our research portfolio in November 2013.