Toxicol. Sci. 2007 96: 1
Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation
We are pleased to announce a new initiative for the Forum Section of Toxicological Sciences, which is designed to include discussions and commentaries providing an interface between toxicology and public policy. A series of articles focused on the theme "Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation" has been formulated. Clearly, this is an important and controversial topic. Gene and chromosomal mutations have been implicated in adverse health consequences in humans including cancer and birth defects. Hence, it is incumbent upon the scientific and regulatory community that the best available scientific tools be employed not only to identify the inherent mutagenic hazard but also to mitigate the potential risk posed by such agents to humans.
Currently, genetic toxicology tests are routinely used to predict carcinogenic potential of new chemical entities during the discovery process as well as to screen the chemicals that are already in commerce. These assays are also important measures for determining whether exposure to agent may induce heritable mutations. In general, results from the tests are viewed in simple binary terms, i.e., "yes" or "no" for the induction of the effect being investigated with little consideration for the dose-response or the definition of a no-observed-effect level. A positive result in one or more of the standard genetic toxicology assays may cause a test chemical to be dropped from further development, to be labeled as a mutagen or carcinogen if it is a marketed product in certain geographies, or to be evaluated exclusively with a linear, nonthreshold approach in risk assessment.
The interpretation of the results of genetic toxicity tests is frequently called into question because the assays are usually conducted at high concentrations (up to 10mM) or at excessive cytotoxic levels (e.g., up to 90% inhibition in cell growth) in cell lines that do not have normal DNA repair pathways. Furthermore, data accumulated over the past few decades have shown that the sensitivity and specificity of the widely used genetic toxicology tests, especially the in vitro mammalian cell culture-based assays, to predict animal carcinogens are somewhat disappointing. As a result, there continues to be ongoing dialogue, discussion, and debate over the most appropriate tests for assessing the mutagenic potential of xenobiotics, the best way to interpret positive results obtained in the tests, and the most meaningful way to apply these results to a comprehensive assessment of human risk.
The goal of the Forum Series is to facilitate the communication of ideas and promote discussion and new thinking in order to enhance the scientific basis for evaluating the mutagenic risk that chemicals might pose for people. Over the next several months, an invited article will appear in successive issues of the journal featuring state-of-the-art assessments of mutagenicity test performance, strategies for evaluating and interpreting mutagenicity test results, and perspectives and challenges concerning the best approaches to screening mutagenic potential. Contributors have been selected in order to encompass diverse opinions and experiences. It is also our intention to conclude the series with an Editorial addressing our perspective on the state of the science of genetic toxicology and the impact of the series on identifying important issues for further resolution and refinement. We hope that content of these articles will be informative, provocative, and useful to the broad readership of the journal.
In the future, we would like to have additional series of articles in the Forum Section addressed to different, topical themes. Your suggestions for these are welcome.
NOTES
Editors‘ Note: In light of the fact that some of the papers in the series will focus on a comparison of the performance of different assays for mutagenic potential, rather than an emphasis on individual chemicals per se, the use of code letters/numbers, rather than chemical names and structures, will be permitted. This is an exception from the journal‘s policy that requires the use of chemical names/structures for all research papers, but we recognize that this flexibility is necessary in order to obtain the most current, critical assessments of test performance.
Toxicol. Sci. 2007 96: 16-20
Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation Part I: Early Screening for Potential Human Mutagens
Results of genetic toxicology tests are used by FDA‘s Center for Drug Evaluation and Research as a surrogate for carcinogenicity data during the drug development process. Mammalian in vitro assays have a high frequency of positive results which can impede or derail the drug development process. To reduce the risk of such delays, most pharmaceutical companies conduct early non-GLP (good laboratory practices) studies to eliminate drug candidate with mutagenic or clastogenic activity. Early screens include in silico structure activity assessments and various iterations of the ultimate regulatory mandated GLP studies.
Toxicol. Sci. 2007 96: 214-217
Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation Part II: Performances of the In Vitro Micronucleus Test Compared to the Mouse Lymphoma Assay and the In Vitro Chromosome Aberration Assay
The in vitro micronucleus test is commonly used in the early stages of pharmaceutical development as a predictive tool for the regulatory mouse lymphoma assay or in vitro chromosome aberration test. The accumulated data from this assay leads to the suggestion that it could be used as an alternative to the chromosome aberration test or the mouse lymphoma assay in the regulatory genotoxicity battery. In this paper, we present the results of the in vitro micronucleus test on L5178Y mouse lymphoma cells with 25 compounds from Servier research and have compared these results to those obtained in the genotoxicity regulatory battery. All the negative compounds were also negative in the in vitro micronucleus assay. Among the 14 positive compounds, two of them, positive in the mouse lymphoma assay, were found negative in the in vitro micronucleus test. However, this apparent discordance was likely to be due to cytotoxicity- or high concentration–related false positive responses in the mouse lymphoma assay. In addition, we confirmed that the in vitro micronucleus assay is useful for detecting aneugens, especially, when cells in metaphasis and multinucleated cells are also scored and when cells are allowed to recover after the long treatment. On this series of compounds, the in vitro micronucleus assay showed high sensitivity and possibly a better specificity than the mouse lymphoma assay. Thus, the in vitro micronucleus assay was shown to be at least as adequate as the mouse lymphoma assay or the in vitro chromosome aberration test to be used in the standard genotoxicity battery.
Toxicol. Sci. 2007 97: 21-26
Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation Part III: The Comet Assay as an Alternative to In Vitro Clastogenicity Tests for Early Drug Candidate Selection
Early screening of drug candidates for genotoxicity typically includes an analysis for mutagenicity in bacteria and for clastogenicity in cultured mammalian cells. In addition, in recent years, an early assessment of photogenotoxicity potential has become increasingly important. Also, for screening purposes, expert computer systems can be used to identify structural alerts. In cases where structural alerts are identified, mutagenicity testing limited to bacteria can be conducted. The sequence of computer-aided analysis and limited testing using bacteria allows for screening a comparatively large number of drug candidates. In contrast, considerably more resources, in terms of supplies, technical time, and the amount of a test substance needed, are required when screening for clastogenic activity in mammalian cells. In addition, the relatively large percentage of false positive results for rodent carcinogenicity associated with clastogenicity assays is of considerable concern. As a consequence, mammalian cell–based alternatives to clastogenicity assays are needed for early screening of mammalian genotoxicity. The comet assay is a relatively fast, simple, and sensitive technique for the analysis of DNA damage in mammalian cells. This assay seems especially useful for screening purposes because false positives associated with excessive toxicity appear to occur less frequently, only relatively small amounts of a test compound are needed, and certain steps of the test procedure can be automated. Therefore, the in vitro comet assay is proposed as an alternative to cytogenetic assays in early genotoxicity/photogenotoxicity screening of drug candidates.
June 2007; Volume 97, Number 2
Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation Part IV:Recommendation of a Working Group of the Gesellschaft fuer Umwelt-Mutations for schung (GUM) for a Simple and Straight forward Approach to Genotoxicity Testing
Based on new scientific developments and experience of the regulation of chemical compounds, a working group of the Gesellschaft fuer Umweltmutationsforschung (GUM), a German-speaking section of the European Environmental Mutagen Society, proposes a simple and straightforward approach to genotoxicity testing. This strategy is divided into basic testing (stage I) and follow-up testing (stage II). Stage I consists of a bacterial gene mutation test plus an in vitro micronucleus test, therewith covering all mutagenicity endpoints. Stage II testing is in general required only if relevant positive results occur in stage I testing and will usually be in vivo. However, an isolated positive bacterial gene mutation test in stage I can be followed up with a gene mutation assay in mammalian cells. If this assay turns out negative and there are no compound-specific reasons for concern, in vivo follow-up testing may not be required. In those cases where in vivo testing is indicated, a single study combining the analysis of micronuclei in bone marrow with the comet assay in appropriately selected tissues is suggested. Negative results for both end points in relevant tissues will generally provide sufficient evidence to conclude that the test compound is nongenotoxic in vivo. Compounds which were recognized as in vivo somatic cell mutagens/genotoxicants in this hazard identification step will need further testing. In the absence of additional data, such compounds will have to be assumed to be potential genotoxic carcinogens and potential germ cell mutagens.
July 2007; Volume 98, Number 1
Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation Part IV: A Strategy in Genotoxicity Testing in Drug Development: Some Examples
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.
August 2007; Volume 98, Number 2
Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation Part VI: When Salt and Sugar and Vegetables Are Positive, How Can Genotoxicity Data Serve to Inform Risk Assessment?
This opinion piece examines the current approaches in the design and evaluation of genotoxicity data and recommends an alternative that would provide information that could be more useful to human risk assessment. It is suggested that genotoxicity studies, both in vitro and in vivo, be designed similar to other traditional toxicology studies, such that a dose-response relationship is characterized, including identification of a "no-observed-adverse-effect-level" dose. It is further suggested that genotoxicity tests should no longer be designed or interpreted in isolation but should be examined in the context of other available data including toxicokinetics, mechanism of genotoxicity, and relevant exposure information. The answer to improving genetic toxicology testing does not lie in coming up with better, "more sensitive" genotoxicity test systems but rather in the incorporation of contextual improvements in both the experimental design and the interpretation of data collected using the current models. Such a strategy will better position the toxicology and risk assessment communities to cope with the current intellectually uncomfortable dichotomy that directs disproportionate scientific resource to addressing genetic toxicity findings of anthropogenic substances, regardless of dose-exposure context, while at the same time ignoring the plethora and comparatively large amounts of genotoxic and toxic substances that are inescapably present in what are otherwise regarded as healthy foods (salt, sugar, and vegetables).