Historical Overview

During the past 30 years, genetic toxicology testing has evolved technologically to play an important safety assessment role in the progression of chemical candidates through the drug discovery and development process.  Prior to application of the battery of regulatory tests, high-throughput screening assay methods are now used to reduce costs by terminating compounds with undesirable characteristics (mutagenic hazard or potential carcinogen).  With few exceptions, compounds found to be mutagenic in these assays are dropped from development, and clastogenic compounds result in unfavorable labeling, require disclosure in clinical trial consent forms, and can greatly impact the marketability of a new drug.  Furthermore, in vitro clastogenicity responses can delay drug development by requiring additional testing to determine the in vivo relevance, although these assays can at times be integrated into other in vivo toxicity studies to expedite the progression of drugs to clinical trials.  Thus, genetic toxicology testing at the drug discovery and optimization stages serves to quickly identify mutagenic compound so that they can be quickly dropped from development.

Genetic toxicology was the first branch of toxicology to fully embrace in vitro test methods, notably through the visionary work of Bruce Ames and coworkers with the development of the Salmonella typhimurium tester strains.  These prokaryotic assays demonstrated good correlation with rodent carcinogenicity results.  The Ames test is generally used as the first screening method to assess chemical genotoxicity.   Although it provides extensive information on DNA reactivity, the Ames assay is not suitable for detecting nongenotoxic carcinogens.  In time, in vitro assays were developed for the detection of gene mutations, chromosomal aberrations, and micronuclei formation.  The mouse lymphoma assay in particular has been developed to the point that  both gene mutations and chromosomal aberrations can be detected and quantified following exposure to test chemicals, when compared with known direct-acting mutagens and promutagens.

Current Perspectives

Assay Predictivity

The performance of a combination of the 3 most commonly used in vitro genotoxicty tests – the Ames, the mouse lymphoma, and the in vitro micronucleus or chromosomal aberration tests – have been evaluated for their ability to discriminate rodent carcinogens from non-carcinogens using a database of over 700 chemicals (Kirkland et al., 2005).  Based on the relative predictivity  measure (RP; the ratio of real:false positive results), that study demonstrated that positive results in all 3 tests indicated that a chemical is greater than 3 times more likely to be a rodent carcinogen than a non-carcinogen.  Similarly, negative results in all three tests indicated that a chemical is more than two times more likely to be a rodent non-carcinogen than a carcinogen.  But further evaluation of combinations of positive and negative results in this genotoxicity battery using the RP calculations indicated that it is not possible to predict outcome of a rodent carcinogenicity study when only 2/3 of the genotoxicity results are in agreement (Kirkland et al., 2006).

Assay Shortcomings

A basic if not critical shortcoming in all these mammalian in vitro assays is the lack of mammalian absorption, distribution, metabolism, and excretion (ADME) features.  As summarized in a recent European Centre for the Validation of Alternative Methods (ECVAM) workshop (Kirkland et al., 2007), cell lines used for genotoxicity testing have a number of deficiencies that may contribute to a high false-positive rate.  These include a lack of normal metabolism leading to reliance on exogenous metabolic activation systems (e.g., Aroclor-induced S9), impaired tumor protein 53 (p53) transcription factor function, and altered deoxyribonucleic acid (DNA) repair capacity.  Also the use of excessive test chemical concentrations to achieve an empirical correlation between genotoxicity and carcinogenicity can result in “promiscuous activation.”  Because these in vitro assays rely on such artificial activation systems, other enzymes that are relatively unimportant in vivo may take over the activation role, leading to the same or a different metabolite – hence, “promiscuous activation.”  Recently, a risk assessment method has been proposed that is dependent upon the availability of quantitative human and rodent ADME  data such that exposures to a metabolite of genotoxic concern can be estimated at the intended human efficacious dose and the maximum dose used in the 2-year rodent bioassay (Dobo et al., 2009).

Other notable genotoxicity testing methods are available for use in the drug discovery and lead-optimization process.  The comet assay is a microgel electrophoresis technique for detecting DNA damage – in vitro and in vivo- at the level of a single cell.  When used in vivo, DNA lesions can be measured in any organ, regardless of the extent of mitotic activity and under normal ADME conditions.  The conventional mouse micronucleus test in the hematopoietic system is a simple method to assess the in vivo clastogenicity of chemicals if the chemical reaches the hematopoietic system.  When multiple organs in the mouse were analyzed following exposure to 208 chemicals, the comparison of comet assay results and carcinogenicity suggested that the comet assay was more capable than the mouse micronucleus assay of detecting rodent carcinogens (Sasaki et al., 2000).

Regulatory Guidance

At present, the ICH/FDA Guidance Document S2(R1) outlines two GLP genotoxicity testing assay options.  Option 1 requires completion of: (1) a test for gene mutation in bacteria., (2) a cytogenetic test for chromosomal damage (choice of three), and (3) an in vivo test for chromosome damage using rodent hematopoietic cells (either micronuclei or chromosomal aberrations in metaphase cells).  Option 2 combines (1) the highly predictive gene mutation assay in bacteria with (2) an in vivo assessment in 2 tissues (e.g., micronuclei using rodent hematopoietic cells plus a second in vivo assay, such as the liver unscheduled DNA synthesis (UDS) assay, transgenic mouse assay, comet assay, etc.  Thus, the ICH guidance allows  for the registration of pharmaceuticals without the submission of data from in vitro mammalian genotoxicity tests (e.g., the in vitro micronucleus test, chromosomal aberrations, mouse lymphoma assay).  This is important because some authors (Matthews et al., 2006) have indicated that 2 of the tests in the FDA battery show good correlation for carcinogenicity prediction (Ames and in vivo micronucleus) and 2 tests show poor correlation (mouse lymphoma and in vitro chromosomal aberrations).

High-Throughput Screens

With the trend towards the application of early pre-screening, high-throughput methods to eliminate potential mutagens/clastogens prior to application of the more resource-intensive and time-consuming regulatory testing methods, many pharmaceutical companies are using these screening methods early in the discovery/lead optimization process.  Examples of modified or high-throughput methods for early screening include: (1) computer-assisted (in silico) structural activity relationship (SAR) methods for predictive toxicity screening, (2) modified assays such as the in vitro assessment of micronucleus induction in Chinese hamster ovary (CHO) cells, the Ames II assay (TA98 and TA Mix), the in vitro comet assay, or well-based (e.g., 96- or 384-well format) modifications of the yeast deletion (DEL) assay, or (3) proprietary assays such as Vitotox™ (mutagenicity), RadarScreen® (clastogenicity), and GreenScreen® HC (genotoxicity).

About the Author:

David Amacher is a senior investigative and biochemical toxicologist with extensive experience in the safety evaluation of human and animal health products.  Dr. Amacher is a Diplomate of the American Board of Toxicology, a Fellow of the National Academy of Clinical Biochemistry, and serves as an Assistant Research Professor of Toxicology and Adjunct Professor in the Graduate School of the University of Connecticut.  His professional affiliations include memberships in the American Society for Pharmacology and Experimental Therapeutics, Society of Toxicology, American Society of Biochemistry and Molecular Biology, International Society for the Study of Xenobiotics, American Association of Clinical Chemistry, and the American College of Toxicology.


Dobo KL, Obach RS, Luffer-Atlas D, et al.  A strategy for the risk assessment of human genotoxic metabolites.  Chem Res Toxicol. 2009;22(2):348-56.

International Conference for Harmonization Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use.  S2(R1), 6 March 2008.

Kirkland D, Aardema M, Henderson L, et al.  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 [published erratum appears in Mutation Research 2005;588(1):70].

Kirkland D; Aardema M; Müller L; et al.  Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens II. Further analysis of mammalian cell results, relative predictivity and tumour profiles.  Mutation Research 2006;608(1):29-42.

Kirkland D; Pfuhler S; Tweats D; et al.  How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-up animal tests: Report of an ECVAM Workshop.  Mutation Research 2007;628(1):31-55.

Matthews EJ, Kruhlak NL, Cimino MC, et al.  An analysis of genetic toxicity, reproductive and developmental toxicity, and carcinogenicity data: I.  Identification of carcinogens using surrogate endpoints.  Regul. Toxicol. Pharmacol. 2006;44(2):83-96.

Sasaki YF, Sekihashi K, Izumiyama F., et al.  The comet assay with multiple mouse organs: comparison of comet assay results and carcinogenicity with 208 chemicals selected from the IARC Monographs and U.S. NTP Carcinogenicity Database.  CRC Crit. Rev. Toxicol. 2000;30(6):629-799.

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11 Responses to “Genetic Toxicology: Historical and Current Perspectives”

  1. Bob van Lier Says:

    Nice overview of current genetox status. Unfortunately, none of the links supplied in the reference section work. I just get an error 404. Nothing found. Would you please update post with corrected links?

  2. cdavenport Says:

    Links are now corrected and should be functional! Thanks for the head’s up, Bob!


  3. Vijay Kale Says:

    Very good overview. Thanks.

  4. Upendra Says:

    Interesting overview. Unfortunately, slide set are available. Would you please update post with slide set?

  5. Nick Billinton Says:

    If I may offer a small update: GreenScreen HC is mentioned in the above piece but the slides refer to GreenScreen GC. These are different assays – the former is the more recent human TK6 cell line-based reporter assay with a superior sensitivity / specificity combination. The latter is a yeast-based assay – easy to perform and with high specificity. You can find links to peer-reviewed literature on both of these assays from Gentronix’s website.

  6. Mallikarjun Bidarimath Says:

    Thanks for the great knowledge and good review..

  7. Bob Mueller Says:

    Cynthia, This is an excellent review and the links are working as stated. You and Tigertox are a great resource to our profession. Many thanks,


  8. Younus Says:

    Dear Cynthia, Thanks for your efforts in sharing the information of genotoxicity test, could you please extend your efforts in suggesting the permissible limits of genotoxic substances.

  9. cdavenport Says:


    If you are still unable to open the link to the slide set, please try the SlideShare link directly: http://www.slideshare.net/TigerTox/fundamentals-of-genetic-toxicology-in-the-pharmaceutical-industry-sept-2010-5245345. Thanks for joining into the discussion!

  10. cdavenport Says:


    Good question! There are 2 current guidelines available. The more stringent one by the EMA (2007) and a more pragmatic staged-approach proposed by the FDA (DRAFT, 2008).

    Guideline on the Limits of Genotoxic Impurities: http://bit.ly/EMAGenotoxicImpuritiesFINAL2007

    Genotoxic and Carcinogenic Impurities in Drug Substances and Products: Recommended Approaches: http://bit.ly/FDAGenotoxicImpurityDRAFTDec2008

    The FDA DRAFT guidance is intended as an adjunct to the ICH guidances for industry Q3A(R2) Impurities in New Drug Substances, Q3B(R2) Impurities in New Drug Products, and Q3C(R3) Impurities: Residual Solvents that deal with the topic of impurities in a more general fashion. The FDA DRAFT guidance provides specific recommendations regarding the safety qualification of impurities with known or suspected genotoxic or carcinogenic potential while the ICH guidances provide only general direction.

    Other useful articles on this topic include:

    Question & Answers on the CHMP Guideline on the Limits of Genotoxic Impurities:

    Success and Accuracy in Genotoxicity and Carcinogenicity Testing: An FDA Perspective. Podcast:

    Chemical and Engineering News:
    Genotoxic Impurities: http://bit.ly/CandENGenotoxicImpuritiesSept2010
    Detecting Low Level Impurities: http://bit.ly/CandENDetectingLowLevelImpuritiesSept2010

    Assessment of Genotoxic Impurities in Small Molecule Drug Candidates. Wisler J, Black K. Northern California SOT Meeting 6 May 2010:

    And last but not least, an upcoming conference to directly address your question:

    Genotoxicity & Carcinogenicity Testing 2010 : http://bit.ly/akFVci
    18 – 20 October, 2010, Le Meridien Piccadilly, London

  11. Carol R. Tometsko Says:

    Great overview. Can’t imagine how our paths
    have not crossed. Our Litron folks are regularly at EMS and SOT and our expertise is genetox assays using flow cytometry: In Vivo & In Vitro micronucleus assessments (MicroFlow®) and In vivo Pig-a mutation analyses (Presently the organizers for an international validation relative to Pig-a). /crt

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