ToxSci Advance Access originally published online on March 15, 2007
Toxicological Sciences 2007 98(1):39-42; doi:10.1093/toxsci/kfm056
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Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation Part IV: A Strategy in Genotoxicity Testing in Drug Development: Some Examples
* Servier Group, Drug Safety Assessment, 45403 Orléans-Gidy, France
Servier Group, Institut de Recherches Internationales Servier, 92400 Courbevoie, France
1 To whom correspondence should be addressed at Biologie Servier BP 43255, 45403 Fleury-les-Aubrais, France. Fax: +33 2 38 23 86 50. E-mail: elisabeth.lorge{at}fr.netgrs.com.
Received January 25, 2007; accepted March 7, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONTHE THREE-TEST BATTERYTHE IN VIVO MICRONUCLEUS...THE IN VITRO MAMMALIAN...ADDITIONAL TESTSSCREENING BATTERYCONCLUSIONREFERENCES |
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The minimal three-test battery of the International Conference on Harmonization guideline has been in use since 1997 for the development of new pharmaceuticals (ICH, 1997). After a 10-year experience of this core battery in regulatory genotoxicity testing, everywhere the time has come for reflection about what was learned from this experience. Different aspects of genotoxicity testing are currently being debated under different organizations (HESI, 2006; IWGT, 2007; Kirkland et al., 2007
). The main concerns are to develop relevant strategies and adequate complementary tests to the minimal battery, appropriate for each specific case to assess risk for humans when in vitro positive results or findings in rodent bioassays for carcinogenicity are found. In this article, an example of an in-house decision tree is shown, with some options which can contribute to the current reflections. Additionally, tools built for early genotoxicity are presented. Key Words: drug safety; genotoxicity strategy; genotoxicity battery.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONTHE THREE-TEST BATTERYTHE IN VIVO MICRONUCLEUS...THE IN VITRO MAMMALIAN...ADDITIONAL TESTSSCREENING BATTERYCONCLUSIONREFERENCES |
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Over decades of genotoxicity evaluation of candidates for pharmaceutical development, the number of unique in vitro positive compounds increased, without significantly changing either the way genotoxicity tests were conducted or the intended therapeutic axes. However, the concepts being developed now for discovering new active pharmaceuticals involve new cellular targets. This probably results in a diversification of the possible interactions of a chemical with the different cell functions, including interactions resulting in DNA damage. In the last few years, a number of publications about the non-DNA–reactive genotoxic compounds, i.e., indirect mutagens, have shown that these complex interactions may account for a number of in vitro genotoxic positive results on mammalian cells. This evolution is now a part of the current reflection on the improvement of genotoxicity tests, their adequacy, the possible sources of false-positive results, and the appropriate complementary tests to add to the minimal battery to finally correctly assess actual genotoxic activity presenting a potential hazard for humans. These reflections are conducted by the International Workshop on Genotoxicity Testing, European Center for the Validation of Alternative Methods, Health and Environmental Sciences Institute, and International Conference on Harmonization. This article shows how the knowledge on the limitations of genotoxicity tests is taken into account to improve the detection of actual genotoxic compounds within our company, to bring some contribution to the ongoing evolutions.
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THE THREE-TEST BATTERY |
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TOPABSTRACTINTRODUCTIONTHE THREE-TEST BATTERYTHE IN VIVO MICRONUCLEUS...THE IN VITRO MAMMALIAN...ADDITIONAL TESTSSCREENING BATTERYCONCLUSIONREFERENCES |
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To comply with the International Conference on Harmonization minimal battery (ICH, 1997), an Ames test including two Escherichia coli strains, a mouse lymphoma tk+/– assay and a rat bone marrow micronucleus assay are routinely performed (Fig. 1). We have chosen the mouse lymphoma assay rather than the chromosome aberration test because it is more simple, less time consuming, and presumably able to detect some aneugens. Moreover, the performances and limitations of both tests are similar (Kirkland et al., 2005
). Of course, this battery may be altered or complemented, depending on the questions raised during the conduct of the tests and according to the decision tree in use in our group (Fig. 1). Each compound is a specific case which will be solved adding different tests, depending on the theoretical mode of action which can be suggested on the basis of available genotoxicity results and taking into account the toxicity profile and chemical properties of the compound. Some options of this decision tree are described below.
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THE IN VIVO MICRONUCLEUS ASSAY AND THEIN VIVO CHROMOSOME ABERRATION ASSAY |
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TOPABSTRACTINTRODUCTIONTHE THREE-TEST BATTERYTHE IN VIVO MICRONUCLEUS...THE IN VITRO MAMMALIAN...ADDITIONAL TESTSSCREENING BATTERYCONCLUSIONREFERENCES |
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It is now well established that a chemically induced decrease in body temperature, bleeding, or hemolysis, leading to accelerated erythropoiesis, may artifactually increase the spontaneous level of micronuclei (Hamada et al., 2001
). When such a positive response is found in an in vivo micronucleus assay with a compound proved to act on erythropoiesis, an in vivo chromosome aberration study can be included as a part of the demonstration that the micronuclei were not induced via a clastogenic mode of action. However, when an effect on erythropoiesis or body temperature is demonstrated or suspected before the three-test genotoxicity battery is completed, it would be more appropriate to perform an in vivo chromosome aberration test on bone marrow rather than an in vivo micronucleus test to adequately evaluate the actual genetic potential of the compound (Fig. 1). The in vivo chromosome aberration test can be used equally to the in vivo micronucleus assay as both are considered equivalent by authorities (ICH, 1997). |
THE IN VITRO MAMMALIAN ASSAYS |
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TOPABSTRACTINTRODUCTIONTHE THREE-TEST BATTERYTHE IN VIVO MICRONUCLEUS...THE IN VITRO MAMMALIAN...ADDITIONAL TESTSSCREENING BATTERYCONCLUSIONREFERENCES |
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In our laboratory, 20% of the Ames negative compounds are found positive in at least one in vitro mammalian genotoxicity test (tk+/– mouse lymphoma assay, in vitro chromosome aberration assay, or in vitro micronucleus assay). These observations meet the evaluation recently made on the compared performances of the in vitro genotoxicity tests on mammalian cells and the Ames test, showing a high specificity for the Ames test and a low specificity for in vitro mammalian genotoxicity tests, as well as the role of bad conditions of cell culture (Kirkland et al., 2005).
Extreme pH and osmolality have been known for a long time to induce artifactual effects such as chromosome breakage (Brusick, 1986; Kirkland and Mueller, 2000). Therefore, as long as in-house genotoxicity studies were made, pH and osmolality are systematically checked. No concentration inducing a higher variation of pH than 0.5 or a higher variation of osmolality than 50 mOsm/kg compared to the culture medium will be tested in any of the in vitro mammalian cell systems (tk+/– mouse lymphoma assay, in vitro chromosome aberration, or in vitro micronucleus assay). So, cells are exposed to the compound under physiological conditions and the culture conditions are not responsible for false-positive responses.
High cytotoxicity, required for the highest tested concentrations, may also account for some irrelevant in vitro positive findings. When the surviving 10% of the cell population is exposed to 90% of dying cells releasing reactive oxygen species in the medium, it is not surprising that DNA of surviving cells is damaged, especially when the time of exposure is extended to 24 h. Such positive responses are regularly seen, quantitatively and qualitatively correlated with high cytotoxicity in the tk+/– mouse lymphoma assay. Caution has to be paid to mouse lymphoma L5178Y cells which produce a dysfunctional p53 protein (Storer et al., 1997) and, therefore, may be theoretically more susceptible than other cells to genotoxic damages. As recommended in the FDA guidance (FDA, 2006), an in vitro chromosome aberration assay or an in vitro micronucleus assay is performed when a positive effect is found on tk+/– mouse lymphoma assay, either on mouse lymphoma L5178Y cells or on primary cultures of human lymphocytes, depending on the cell type or the target which may be critical for the genotoxic mode of action. Additionally, these systems will give more information about clastogenicity or aneuploidy. Of course, after sources of artifactual responses are eliminated, some of the positive compounds in in vitro mammalian genotoxicity tests and negative in the Ames test will probably be true mutagens. First, the Ames test is not so good at detecting genotoxins that induce large DNA deletions because if the target sequence in the histidine or tryptophan gene of the tester bacteria is deleted, reversion to auxotrophy (histidine or tryptophan independence) cannot occur. Second, compounds that are negative in the Ames test but positive in in vitro mammalian cell genotoxicity tests have the potential to be non-DNA–reactive molecules that act on other targets of mammalian cells. Thus, some of the compounds specifically positive in in vitro mammalian systems could be true non-DNA–reactive mutagens. In these cases, a threshold can be determined to assess risk for humans (Kirkland and Mueller, 2000; Mueller and Kasper, 2000). However, more work has to be done in this field since very few examples of substances demonstrated to induce indirect genotoxic effects.
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ADDITIONAL TESTS |
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TOPABSTRACTINTRODUCTIONTHE THREE-TEST BATTERYTHE IN VIVO MICRONUCLEUS...THE IN VITRO MAMMALIAN...ADDITIONAL TESTSSCREENING BATTERYCONCLUSIONREFERENCES |
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To help to assess the relevance of in vitro positive results, understand the mode of action of the compound, and finally evaluate the risk for humans, additional tests may be useful (Fig. 1). Alternative in vitro chromosome aberration test or micronucleus assay on other cell types than L5178Y cells will give indications about the possible involvement of cell specificity on an in vitro positive response, as well as possible aneugenic effects. In vivo experiments, i.e., micronucleus assays after long-term exposure or in relevant target organs, i.e., in most cases in liver for pharmaceuticals, added to toxicokinetic and pharmacokinetic data, will attest to the predictive value of in vitro positive results for in vivo situation. The in vivo comet assay is also found very helpful for the assessment of in vivo genotoxicity on the appropriate organs detected as targets in other toxicity studies. Finally, short-term carcinogenicity tests (p53, rasH2) will increase confidence in in vivo negative results before the standard rodent bioassays had started for the assessment of carcinogenicity.
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SCREENING BATTERY |
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TOPABSTRACTINTRODUCTIONTHE THREE-TEST BATTERYTHE IN VIVO MICRONUCLEUS...THE IN VITRO MAMMALIAN...ADDITIONAL TESTSSCREENING BATTERYCONCLUSIONREFERENCES |
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Screening tools were built to assess genotoxic effects as early in pharmaceutical development as possible and help the choice of new candidates. An early evaluation of in-house compounds in a high-throughput Ames II test for screening purposes (Fluckiger-Isler et al., 2004
) is routinely performed. Since 2000, all the compounds detected as positive in the screening Ames IITM test and tested in the regulatory Ames test were confirmed positive in the regulatory Ames test. The positive compounds detected in any of these Ames tests were also positive in the tk+/– mouse lymphoma assay, the in vitro chromosome aberration assay, and/or the in vitro micronucleus assay. However, as shown above, some negative compounds in the Ames test were positive in at least one in vitro mammalian cell assay for genotoxicity. Therefore, the Ames II test was complemented with a miniaturized in vitro micronucleus assay on mouse lymphoma L5178Y cells (Nesslany and Marzin, 1999) for evaluating all the Ames negative compounds in a screening battery using only 45 mg of compound for both tests. These miniaturized tests are also used for assessing genotoxicity of impurities, precursors, or metabolites (e.g., when positive findings are restricted to the presence of a metabolic activation system) as these tests need small amounts of compounds. |
CONCLUSION |
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TOPABSTRACTINTRODUCTIONTHE THREE-TEST BATTERYTHE IN VIVO MICRONUCLEUS...THE IN VITRO MAMMALIAN...ADDITIONAL TESTSSCREENING BATTERYCONCLUSIONREFERENCES |
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A decision tree was developed here with some examples of strategic choices made either to avoid producing irrelevant results, using, e.g., the in vivo chromosome aberration test instead of the in vivo micronucleus test or to better assess the relevance of positive results obtained on mammalian cell systems. The options given in this decision tree were useful in our experience to better understand the relevance of positive findings in genotoxicity testing. Other solutions could have also been suitable and other concerns could have been addressed, especially the modes of action of indirect genotoxic compounds. Hopefully, this return on experience will help progress in the definition of adequate strategies to improve genotoxicity assessment of pharmaceutical compounds, when various aspects of genotoxicity testing are at that time being debated under different organizations.
In parallel, a screening battery, using minimal amounts of compound, provides crucial information to pharmacologists at the very early steps of development so that new nongenotoxic backups can be found and less compounds are dropped during their preclinical development.
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REFERENCES |
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Brusick D. Genotoxic effects in cultured mammalian cells produced by low pH treatment conditions and increased ion concentrations. Environ Mutagen (1986) 8:879–886.[Web of Science][Medline]
Food and Drug Administration (FDA). Food and Drug Administration—U.S. Department of Health and Human Services (2006). Guidance for Industry and Review Staff—Recommended Approaches to Integration of Genetic Toxicology Study Results (2006) Food and Drug Administration. http://www.fda.gov.
Fluckiger-Isler S, Baumeister M, Braun K, Gervais V, Hasler-Nguyen N, Reimann R, Van Gompel J, Wunderlich HG, Engelhardt G. Assessment of the performance of the Ames II assay: A collaborative study with 19 coded compounds. Mutat. Res. (2004) 558:181–197.[Web of Science][Medline]
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Kirkland D, Pfuhler S, Tweats D, Aardema M, Corvi R, Darroudi F, Elhajouji A, Glatt H, Hastwell P, Hayashi M, et al. How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-ip animal tests: Report of an ECVAM workshop. Mutat. Res. (2007) 628:31–55. doi:10.1016/j.mrgentox.2006.11.008.[Web of Science][Medline]
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