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European Biopharmaceutical Review

Genotox Support

Genotoxicity testing can often be seen by chemists as a double-edged sword. While the goal is to have a set of toxicology results that confirms a compound is not genotoxic, there remains the possibility that such testing will result in an otherwise promising candidate being ‘killed’ due to adverse results. Testing for genetic toxicity is very important and is a regulatory requirement for all pharmaceuticals intended for human use. The ICH S2(R1) guidance covers genotoxicity testing and data interpretation in this area.

Damage to DNA and chromosome structure is widely acknowledged as having a very important role in the development of cancer in humans. The incidence of cancer can be increased by exposure to compounds that cause irreversible damage to the genome – such compounds are collectively classified as genotoxic carcinogens. The genetic toxicology field was established to determine the potential of a novel chemical entity to cause gene mutations, structural or numerical aberrations to chromosomes, as such compoundinduced damage would indicate a risk of carcinogenesis.

Testing Approaches

Genotoxicity that has relevance to tumourigenesis can occur through diverse mechanisms, but there are currently no regulator-approved tests available that detect all mechanisms of genotoxic hazard. As a consequence, current regulatory guidelines employ a testing battery approach, which includes in vitro tests for mutagenicity and cytogenetic damage, along with in vivo genotoxicity tests.

Over the last decade, several reviews have been conducted into the performance of the in vitro genotoxicity testing battery (1-3). Overall, these evaluations have highlighted that most rodent carcinogens are detected as positive in the current testing battery. The sensitivity was as high as 85.9% when a battery of Ames and in vitro micronucleus test were employed (1).

However, the evaluation showed that the majority of non-carcinogens also produced positive results in at least one of the battery tests. The in vitro mammalian assays, in particular, produced a very high number of these misleading positives. This poor specificity of the in vitro testing battery (as low as 32% for Ames and in vitro micronucleus test combination) potentially results in the abandonment of a development compound due to false conclusions of genotoxic hazard.

Regulatory Assays

In pharma, the ICH S2(R1) guidelines state that the genotoxicity testing battery should contain:
  •  A test for gene mutation in bacteria (Ames test)
  • A cytogenetic test for chromosomal damage (in vitro metaphase chromosome aberration test or in vitro micronucleus test), or an in vitro mouse lymphoma thymidine kinase (TK) gene mutation assay
  • An in vivo test for genotoxicity, generally a test for chromosomal damage using rodent haematopoietic cells, either for micronuclei or for chromosomal aberrations in metaphase cells
The guidelines also recommend either carrying out all three of the above battery elements; or performing the first and third, but with the addition of another in vivo end-point (4).

Key to the performance of safety assessment genotoxicity tests are the published Organisation for Economic Cooperation and Development (OECD) Guidelines for the Testing of Chemicals (5). These test-specific guidelines provide descriptions of internationally agreed methodologies that are referenced by cross-sector regulatory bodies, although the ICH S2(R1) guidance provides the definition of top-test concentration and assays that are acceptable for the genotoxicity hazard assessment of pharmaceuticals with respect to human health.

Ames Testing
The Ames test is a bacterial assay that detects mutagens. Four strains of the bacteria Salmonella typhimurium and one strain of Escherichia coli are typically used, which carry different defective (mutant) genes that prevent them from synthesising an essential amino acid – histidine in the case of S. typhimurium and tryptophan in E. coli. This means they can only grow on media that is supplemented with the relevant amino acid.

However, in the presence of a mutagenic chemical, the defective genes may be mutated back to the functional state, allowing the bacterium to grow in amino acid-depleted media because the revertant bacteria are able to synthesise what is missing. After a period of incubation, those plates which show growth of the revertant bacteria are indicative of the presence of a mutagen; the number of colonies grown being related to the concentration of the substance which allow a dose/response curve to be generated.

Different strains are used to detect different types of mutagen. Some of the strains have additional modifications, such as an induced defect in the lipopolysaccharide cell wall or a DNA excision repair mutation (uvrA or uvrB). These modifications increase the sensitivity of the test strains for the detection of mutagens.

The test is run both with and without the addition of exogenous metabolism – typically rat S9 fraction. This is an extract from a homogenate of rat liver after elevated levels of cytochrome P450 enzymes have been induced, by treatment with compounds such as Aroclor-1254 (a mixture of polychlorinated biphenyls). This allows potentially mutagenic Phase 1 metabolites of the test substance to be detected.

Micronucleus Testing (MNT)
This form of testing is based on an in vitro assay that detects numerical and structural chromosomal aberrations (7-8). A micronucleus (MN) is a small additional nucleus that is formed during the anaphase stage of mitosis or meiosis. MN may contain either whole chromosomes generated by compounds which cause mis-segregation (aneugens), or chromosome fragments with or without centromeres, generated by compounds which cause breakage (clastogens).

The test can be carried out using a variety of cell types, such as rodent and human cell lines, or primary human lymphocytes. Cells are dosed with the test compound and incubated. Analysis is again conducted with and without S9 metabolic activation.

In order to accurately determine the proportion of micronuclei-containing cells, it is essential that cells are proliferating. One way to measure this is to use a cytokinesis blocking agent, such as Cytochalasin B, which inhibits cytoplasmic division resulting in the formation of binucleate (or multinucleate) cells. The cytokinesisblock proliferation index is used to quantify proliferation and cytostasis/cytotoxicity. Bi-nucleate cells blocked at cytokinesis indicate a proliferating culture.

It may be appropriate to conduct studies in the absence of cytochalasin B – either due to known interactions of cytochalasin B with test article, or to the specific cell type used – in what is commonly referred to as the mononucleated micronucleus test. In this method, proliferation is ensured by using cell counts to determine the relative population doubling or relative increase in cell counts, with between 1.5 and 2 cell cycles required in the untreated control population.

Scoring of these cells for the presence of micronuclei can be carried out using either a manual microscopic method, or automatically with validated flow cytometry or image analysis methods.

Mouse Lymphoma Testing
The mouse lymphoma assay is a mammalian forward mutation assay based on mutations at the TK locus, conducted using the mouse lymphoma cell line L5178Y, which is heterozygous at the TK locus (9). The assay is based on the resistance to the incorporation of the cytotoxic pyrimidine analogue trifluorothymidine (TFT). Forward mutations to the TK locus, resulting in a change for TK+/- to TK-/-, result in resistance of mutant cells to TFT incorporation and thus survival. The frequency of induced mutation is calculated from colony formation under selective conditions, against the spontaneous background.

In Vivo Testing

The ICH S2(R1) guidance for pharmaceuticals intended for human use requires that at least one in vivo assay is carried out, and which produces a negative result. A cytogenetic endpoint in haematopoietic cells is generally the initial in vivo test, with secondary testing usually conducted in the liver for DNA damage. For the haematopoietic cytogenetic damage end-point, two approaches are commonly employed.

The first test involves animals, usually rodents, which are exposed to the test substance. Either bone marrow or peripheral blood is collected at the appropriate time, and samples are prepared, stained and scored manually for micronuclei, as in the in vitro assay.

The second test also uses rodents, which are exposed to the test substance. They are treated with a metaphase-arresting agent, and bone marrow is extracted. Samples are then prepared and strained, and the frequency of asymmetrical structural chromosome aberrations are counted.

Screening Assays

Companies can save significant time and cost by early identifi cation of compounds that are likely to show genotoxic liability in the regulatory assays. This does not automatically mean halting their development. Modifying the structure, or de-risking, are options if the genotoxicity is identified early enough.

The most obvious predictor of genotoxicity would be to run the regulatory assays early in the process, but they are expensive, time consuming and require large amounts of test compound. As a result, other assays are often used that are either ‘cut-down’ versions of the regulatory assays or, more recently, panmechanistic high-throughput assays that have been optimised for use in screening. Although the results of screening assays in general provide good agreement with their regulatory equivalent, it must be kept in mind that this is not always 100% accurate.

Screening versions of the regulatory assay methods have been developed that are designed to be predictive of the regulatory assay result, but require significantly less time and compound to conduct (10-12).

Unlike the regulatory Ames assay which uses agar plates, Ames II and Ames microplate format (MPF) are fluctuation tests conducted in microtitre plates. They are based on revertant growth in histidine or tryptophan-deficient medium, with a colorimetric readout based on a lowering of the pH, indicating mutant wells.

The primary difference between the two assays lies in the strains of bacteria used. While Ames MPF uses the tester strains recommended in the OECD guidelines, the Ames II assay uses TA98 and TAmix, which is a mixture of six histidine-mutant Salmonella strains.

Assays such as the Microflow® Micronucleus test use a dual-staining methodology to restrict scoring of micronucleus frequency to viable cells (10). Initially, cells are stained with a light-activated, fluorescent, nucleic acid-binding cell permeability stain, with only apoptotic/necrotic cells stained. The cells are then lysed and a second DNA stain is applied. Cells which have dual fluorescence are gated out and only those which fluoresce in the colour of the second stain are counted. Micronucleated events exhibit a fl uorescence intensity that is 10-100 times lower than nucleated events, allowing the two to be distinguished and scored by flow cytometry.

In recent years, assays have been developed which are sensitive to all mechanisms of genotoxicity. Several of these assays are based on reading a reporter for the expression of the human GADD45a gene in the p53-competent human lymphoblastoid TK6 cell line. The GADD45 gene has a central role in the cell’s genotoxic stress response. By using a reporter such as green fl uorescent protein, up-regulation of the GADD45a gene can be detected and measured using fl uorescence (11). An alternative assay, based on the same GADD45a gene, has also been developed that uses a Gaussia luciferase reporter to give a luminescent output that removes sensitivity to autofl uorescent compounds.

These assays have the advantage of having both high sensitivity and specificity, require very little compound (typically 10mg) and provide a result in 48 hours (12-14). Because they respond to all known mechanisms of genotoxicity and are highly predictive of in vivo genotoxicity results, they are increasingly being used as an alternative to the screening Ames and micronucleus tests.

Understanding Positives

Once a positive result has been generated, there are considerations of the data that should be made, as well as various courses of action available. Early screening allows the selection of compounds without genotoxic liability, or further chemical development to separate genotoxic liability from useful pharmacology. Positive results at a later stage in development are potentially disastrous, leading to the abandonment of candidate molecules, unless a successful safety case can be made.

While a positive result most likely reflects detrimental interactions with DNA or structures involved in replication and repair, a single in vitro result may not be relevant to the intended use of the compound. If a positive result is generated in a single test, the most appropriate course of action is to determine if the compound is positive in other genotoxicity assays, or whether this result is a test-specific genotoxin – for example, a positive result in the Ames test via a bacterial specific mechanism would be unlikely to produce micronuclei in rodent cells. A review of any in silico prediction data is useful at this stage, as it will inform whether a positive genotoxicity result was anticipated and what the mechanism may be.

The synthetic route and potential impurities should be reviewed to determine whether observed genotoxicity is actually associated with the desired chemical. If genotoxic impurities are suspected, re-testing should be conducted with improved purity stocks, or alternative synthetic routes should be considered. The dose at which a positive genotoxicity result is observed can also provide a basis for de-risking of the genotoxicity hazard. Increasingly, statistically-based threshold models are being used to demonstrate non-linear dose responses for genotoxicity. If the therapeutic dose can be shown to be below the lowest observed effect level or inflection point, then it is possible for a genotoxic compound (or a compound containing a genotoxic impurity) to be considered safe for human exposure (15). Furthermore, the threshold of toxicological concern can be employed in managing the safety of a genotoxic compound: genotoxin is considered acceptable if human exposure to the test compound is lower than 1.5μg per day.

Ultimately, discovery of genotoxic liability early in compound development will allow the most effective response, provide the opportunity to design out the liability, or provide safety assessment departments with sufficient notice to implement a management strategy to handle the associated genotoxicity.


1. Kirkland D, Aardema M, Henderson L and Müller L, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens: I. Sensitivity, specificity and relative predictivity, Mutation Research Genetic Toxicology and Environmental Mutagenesis 584(1-2): pp1-256, 2005
2. Matthews EJ et al, An analysis of genetic toxicity, reproductive and developmental toxicity, and carcinogenicity data: I. Identification of carcinogens using surrogate endpoints, Regulatory Toxicology and Pharmacology 44(2): pp83-96, 2006
3. Kirkland D, A core in vitro genotoxicity battery comprising the Ames test plus the in vitro micronucleus test is sufficient to detect rodent carcinogens and in vivo genotoxins, Mutat Res 18:721(1): pp27-73, 2011
4. Visit: Web_Site/ICH_Products/Guidelines/ Safety/S2_R1/Step4/S2R1_Step4.pdf
5. Visit: www.oecd-ilibrary. org/environment/oecdguidelines- for-the-testing-ofchemicals- section-4-healtheffects_ 20745788
6. Gatehouse D, Bacterial mutagenic assays: Test methods, Genetic Toxicology, Springer, 2012
7. Parry JM and Sors A, The detection and assessment of the aneugenic potential of environmental chemicals: the European Community aneuploidy project, Mutat Res 287: pp3-15, 1993
8. Kirsch-Volders M, Towards a validation of the micronucleus test, Mutat Res 392: pp1-4, 1997
9. Clive D and Spector JA, Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells, Mutat Res 31: pp17-29, 1975
10. Visit: in_vitro_micronucleus.html
11. Chalfie M et al, Green fluorescent protein as a marker for gene expression, Science 263: pp735-888, 1994
12. Hastwell PW et al, High specificity and high sensitivity genotoxicity assessment in a human cell line: Validation of the GreenScreen HC GADD45a-GFP genotoxicity assay, Mutat Res 607: pp160-175, 2006
13. Birrell L et al, GADD45a-GFP GreenScreen HC assay results for the ECVAM recommended lists of genotoxic and non-genotoxic chemicals for assessment of new genotoxicity tests, Mutat Res 695: pp87-95, 2010
14. 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 Biomolecular Screening 17(10): pp1,302-1,315, 2012
15. Lutz WK, The Viracept (nelfinavir)- ethyl methanesulfonate case: A threshold risk assessment for human exposure to a genotoxic drug contamination? Toxicol Lett 190(3): pp239-242, 2009

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Andrew Eaton is Commercial Services Manager for Gentronix Ltd and has over 30 years of experience in a range of industries, including inorganic and organic mass spectrometry, medical devices, wound care, pharma and biotech. He is a chemist by training and has a PhD in Mass Spectrometry from the University of Durham.

Matthew Tate is the Technical Manager at Gentronix Ltd. He has a PhD in Biotechnology from the University of Manchester, and over the past eight years has been extensively involved in the development of genotoxicity testing services at Gentronix.

Andrew Eaton
Matthew Tate
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