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European Biopharmaceutical Review

Early Identification

Although early screening for genetic toxicity has long been part of the drive to minimise drug candidate late-stage attrition cost, the lack of a single definitive assay, and the consequent need for a battery of tests, has precluded the development of a clear consensus with regard to screening strategy. However, new assays are providing researchers with more accurate information on the genotoxic liability of compounds, thus increasing the quality of compounds advanced and enabling the development of more cost-effective screening strategies.

Regulatory testing is focused on safety to ensure compounds with genotoxic properties are identified prior to pre-clinical development. Testing in a number of different assays is required because currently no single regulatory assay identifies all mechanisms of genotoxicity, and the accuracy of these has been low. Therefore, any screening strategy has traditionally been shaped by the ultimate regulatory requirements.

In an attempt to accurately predict the outcome of the regulatory tests and give an earlier indication of liability, ‘cutdown’ screening versions of these assays have been employed. However, these retain the undesirable properties and limitations of the full assays.

This approach is now being turned on its head due to the availability of more general screening assays, which give lower levels of false positive results, while providing superior sensitivity and breadth. The higher confidence in these new early screens allows the presumption that the compound is not genotoxic, and that subsequent positives in regulatory testing will be false or misleading. Mechanistic work can therefore begin to verify this hypothesis before expensive regulatory and in vivo work begins.

The Regulatory Environment

The role of pharmaceutical regulatory testing is to ensure the safety of those administered with clinical candidates, marketed drugs and a range of potential safety concerns that are covered by various governmental and cross territory mandatory requirements.

One area of toxicological concern is the potential for a drug substance to damage or interfere with DNA, thus causing genotoxicity. There are a number of different ways in which this may occur, either by causing direct damage leading to chemical modification, breakage or mutation, or through disruption of normal DNA processing and replication, which can lead to a change in chromosomal structure or the number of chromosomes. In turn, such damage or alteration of DNA may lead to changes that may be inherited by offspring or may lead to the generation of cancers (genotoxic carcinogenicity).

It is therefore important when assessing the regulatory safety of a pre-clinical drug candidate that any testing regime is capable of not only ensuring genotoxic chemicals are correctly identified, but that the tests employed are capable of detecting the different mechanisms by which genotoxicity occurs. In order to achieve this, the regulations require both in vitro and in vivo testing in cell-based assays. It is also important to consider the potential of a chemical to be metabolised to a genotoxic molecule.

A battery of tests is employed to provide adequate coverage of the different mechanisms of genotoxicity because there is no single regulatory test capable of detecting all of the mechanisms of genotoxic action. Separate tests are therefore required for the detection of mutagenicity and chromosomal damage. This article will concentrate on in vitro testing methods, which are of particular relevance to early screening strategies for improved drug discovery.

For the detection of mutagens, the regulations require testing in the Ames bacterial reverse mutation assay. This has been in use for many years, is well understood, and gives a good correlation for the detection of carcinogens. It is, however, an assay that is based on the detection of mutagens in bacteria, and hence not fully representative of the mammalian situation. There is also the possibility that bacterial-specific modes of action will lead to positive results that are not relevant to mammals.

A range of assays has been developed and incorporated to provide options for regulatory testing in order to assess the potential of a chemical to exhibit genotoxicity in mammalian cells. Of increasing importance in this regard is the micronucleus test (MNT).

Chromosomal damage is detected by the presence of micronuclei in the cytoplasm of interphase cells that originate from acentric fragments, or whole chromosomes that failed to segregate correctly during anaphase. This means the MNT is sensitive to both clastogenic and aneugenic mechanisms of genotoxicity.

The assay can be conducted in either the absence or presence of cytochalasin B, with micronuclei assessed from either mononucleate cells where the population is within 1.5-2 population doublings (non-cytochalasin B blocked) or binucleate cells restricted to 1 population doubling by the cytokinesis blocking action of cytochalasin B. Micronuclei are typically scored by microscopy, with upwards of 1,000 cells scored per culture, per compound dose. The in vitro MNT is covered by The Organisation for Economic Co-operation and Development (OECD) guideline 487 and is included in The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidance as part of the in vitro genotoxicity regulatory battery of tests.

A major drawback of the in vitro battery approach is that, while it ensured high detection of genotoxins (true positives), this was accompanied by unacceptable levels of false positives. This meant that non-toxic compounds were inappropriately identified as toxic, resulting in pre-clinical development delays and the need for more comprehensive additional testing.

The ICH regulations were amended in 2011 to reflect changes to the existing battery, which reduced false positives, by lowering the top concentration for testing from 10mmol to 1mmol. A second option, allowing in vitro Ames testing followed by direct in vivo testing with measurement of two end points, was also implemented.

Early Genotoxicity Testing

Historically, genotoxicity testing was carried out in the later stages of pre-clinical development leading to unacceptably high late-stage attrition after the investment of considerable R&D spend. In order to improve the productivity of drug discovery, increasing numbers of companies have adopted earlier testing for genotoxicity.

Initially, early testing methods were based on the regulatory battery of assays to provide coverage of the different modes of genotoxic action. However, these also had to take into consideration the increased number of compounds needing to be screened as a consequence of moving the tests to an earlier stage in the discovery process.

This has been achieved for both the Ames assay – with the use of Ames II, mini-Ames and Ames microplate format (MPF) as examples – and various mammalian cell-based assays, such as the MNT assay. Dependant on the assay format, the screening assay either closely follows the regulatory assay – for example, Ames MPF and mini-Ames use the OECD-required bacterial strains – or have achieved similar results using modified approaches – such as Ames II, which uses different strains.

The role of automation and detection methods has also had an impact. The regulatory MNT uses microscopic scoring of micronuclei, and is operator-dependant and time-consuming, whereas the introduction of flow cytometric scoring of micronuclei – such as the MicroFlow approach – has improved the scoring aspect of this assay. It has also been reported recently that the assay has now been validated in 96-well plate format, further increasing its utility for early screening (1).

Many organisations have implemented one of the screening Ames assays as a front-line filter for genotoxicity. While this provides an acceptable approach for the detection of mutagens, it is not comprehensive and additional mammalian cell based testing is still required. Irrespective of the testing strategy adopted, it remains an issue that these screening assays retain the drawbacks of the full regulatory assays, particularly in respect of the unacceptable levels of false positives that require further evaluation.

Separating Early Screening from Regulatory Assays

In looking to gain greater productivity in drug discovery, it is becoming increasingly common for more comprehensive toxicological profiling to be conducted earlier in the discovery phase. This enables a more appropriate selection of lead compounds for further elaboration and, where choice of chemical lead is limited, allows for closer monitoring of compound liabilities as medicinal chemists look to ‘breed-out’ undesirable properties while increasing potency and other key attributes. This all helps to achieve identification of development candidates more quickly and cost-effectively.

This has previously been a challenge for genotoxicity due to the lack of an assay strategy that combined the positive attributes of higher throughput screening with the need for an assay system capable of both identifying a broad range of genotoxic modes of action and providing low false positives. The introduction of reporter-based assays has changed this situation dramatically.

The GADD45a gene is central to the maintenance of genomic integrity and is up-regulated in response to DNA stress, as well as providing a method for the detection of genotoxic activity across all of the known mechanisms of action.

This property has been exploited in the GreenScreen HC and BlueScreen HC assays, and both are conducted in human-derived TK6 cells which are p53 competent. A reporter construct that incorporates the gene for either green fluorescent protein (GFP) or Gaussia Luciferase (G.Luc) is merged into the cells in a specific position, ensuring a dose-dependant genotoxic-specific readout in response to increasing concentrations of a genotoxin.

GFP and Luciferase-based detection assays are well known in drug discovery for the elucidation of biochemical processes. Furthermore, these types of assays are amenable to automation and the development of higher throughput assay formats. The GFP and G.Luc versions of the GADD45a reporter assay are both conducted in 96-well format and the luciferase assay has also been miniaturised further to 384 well format. Additionally in the 96-well formats, both assays are compatible with S9 mix for the determination of metabolic activation.

This means that options for early genotoxicity testing that can accommodate earlier positioning in the drug discovery process and the higher screening requirements of early profiling now exist. However, in order for these assays to be of use in drug discovery they must possess the requisite sensitivities – the ability of the assay to correctly identify true positives (low false negative rate) – and specificities – (the ability to correctly identify negative compounds (low false positive rate). Both the GFP and G.Luc-based GADD45a assays have been reported in peer-reviewed literature showing sensitivity at 87.5 per cent, 80 per cent and specificity at 95.5 per cent, 98 per cent respectively; these properties show good concordance with other in vitro genotoxicity assays (1-3).

The GADD45a reporter assays are therefore appropriate for the early identification of genotoxic hazards, which gives further options for early screening and mechanistic follow-up strategies. The most direct screening strategy places the GADD45a assay as the primary screen. Genotoxic compounds can then be removed (irrespective of mechanism) at this stage. For negative compounds in the GADD45a assay, further testing with more traditional assays can be conducted as required, either to give a direct indication of regulatory assay output or as a confirmation of non-genotoxicity.

An alternative, particularly in relation to mutagenicity (acknowledging the bacterial nature of the Ames assay), is to run a screening Ames and GADD45a assay in parallel, giving an indication of the Ames assay output and coverage of mutagenicity, and other mechanisms of action from the GADD45a assay.

For pharmaceutical companies with extensive drug discovery operations it is more appropriate to install the 384-well version of the G.Luc assay as the primary screen, following up with metabolic activation studies and other confirmatory testing as appropriate after having identified the majority of genotoxic compounds in the primary profiling BlueScreen.

Fundamentally, these approaches capitalise on the early identification of toxicity issues and their removal, meaning that fewer toxic compounds are progressed, more effort is directed towards compounds with a more robust profile and better compounds are advanced through regulatory testing and into further development and clinical assessment.


The detection of compounds that damage DNA, with the potential to go on to cause cancer or heritable mutation, is a key regulatory concern. This toxicity can therefore lead to late-stage project failure costing time and money. Strategies for the early identification of genotoxicity can improve attrition by moving it earlier on in the discovery process, meaning better compounds with improved properties are generated faster. This, combined with the introduction of new superior technology with more appropriate properties for higher throughput profiling, are creating new options and fl exibility to address toxicities before they become a regulatory concern.


1. Bryce SM et al, Flow cytometric 96-well microplate-based in vitro micronucleus assay with human TK6 cells: protocol optimization and transferability assessment, Environ Mol Mutagen 54(3): pp180-194, 2013

2. Hastwell PW et al, Analysis of 75 marketed pharmaceuticals using the GADD45a-GFP 'GreenScreen HC' genotoxicity assay, Mutagenesis 24(5): pp455-463, 2009

3. Hughes C et al, Development of a high-throughput gaussia luciferase reporter assay for the activation of the GADD45a gene by mutagens, promutagens, clastogens, and aneugens, J Biomol Screen 17(10): pp1,302-1,315, 2012


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Steve Beasley has over 30 years’ experience gained from global pharmaceutical, biotechnology and chemical companies in both research and commercial fields. He was previously Chief Operating Offi cer for De Novo Pharmaceuticals, where he was responsible for all research and commercial activity, and most recently he was Chief Executive of the knowledge-base organisation, bioKneX. Steve joined Gentronix in 2007 and is responsible for the commercialisation of its technology and services. He is a Chartered Chemist and Member of the Royal Society of Chemistry and Institute of Directors.
Steve Beasley
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