Utility of a next‐generation framework for assessment of genomic damage: A case study using the pharmaceutical drug candidate etoposide

We present a hypothetical case study to examine the use of a next-generation framework developed by the Genetic Toxicology Technical Committee of the Health and Environmental Sciences Institute for assessing the potential risk of genetic damage from a pharmaceutical perspective. We used etoposide, a genotoxic carcinogen, as a representative pharmaceutical for the purposes of this case study. Using the framework as guidance, we formulated a hypothetical scenario for the use of etoposide to illustrate the application of the framework to pharmaceuticals. We collected available data on etoposide considered relevant for assessment of genetic toxicity risk. From the data collected, we conducted a quantitative analysis to estimate margins of exposure (MOEs) to characterize the risk of genetic damage that could be used for decision-making regarding the predefined hypothetical use. We found the framework useful for guiding the selection of appropriate tests and selecting relevant endpoints that reflected the potential for genetic damage in patients. The risk characterization, presented as MOEs, allows decision makers to discern how much benefit is critical to balance any adverse effect(s) that may be induced by the pharmaceutical. Interestingly, pharmaceutical development already incorporates several aspects of the framework per regulations and health authority expectations. Moreover, we observed that quality dose response data can be obtained with carefully planned but routinely conducted genetic toxicity testing. This case study demonstrates the utility of the next-generation framework to quantitatively model human risk based on genetic damage, as applicable to pharmaceuticals

Analysis of negative historical control group data from the in vitro micronucleus assay using TK6 cells.

The recent revisions of the Organisation for Economic Co-operation and Development (OECD) genetic toxicology test guidelines emphasize the importance of historical negative controls both for data quality and interpretation. The goal of a HESI Genetic Toxicology Technical Committee (GTTC) workgroup was to collect data from participating laboratories and to conduct a statistical analysis to understand and publish the range of values that are normally seen in experienced laboratories using TK6 cells to conduct the in vitro micronucleus assay. Data from negative control samples from in vitro micronucleus assays using TK6 cells from 13 laboratories were collected using a standard collection form. Although in some cases statistically significant differences can be seen within laboratories for different test conditions, they were very small. The mean incidence of micronucleated cells/1000 cells ranged from 3.2/1000 to 13.8/1000. These almost four-fold differences in micronucleus levels cannot be explained by differences in scoring method, presence or absence of exogenous metabolic activation (S9), length of treatment, presence or absence of cytochalasin B or different solvents used as vehicles. The range of means from the four laboratories using flow cytometry methods (3.7-fold: 3.5-12.9 micronucleated cells/1000 cells) was similar to that from the nine laboratories using other scoring methods (4.3-fold: 3.2-13.8 micronucleated cells/1000 cells). No laboratory could be identified as an outlier or as showing unacceptably high variability. Quality Control (QC) methods applied to analyse the intra-laboratory variability showed that there was evidence of inter-experimental variability greater than would be expected by chance (i.e. over-dispersion). However, in general, this was low. This study demonstrates the value of QC methods in helping to analyse the reproducibility of results, building up a 'normal' range of values, and as an aid to identify variability within a laboratory in order to implement processes to maintain and improve uniformity

Compilation and Use of Genetic Toxicity Historical Control Data

The optimal use of historical control data for the interpretation of genotoxicity results was discussed at the 2009 International Workshop on Genotoxicity Testing (IWGT) in Basel, Switzerland. The historical control working group focused mainly on negative control data although positive control data were also considered to be important. Historical control data are typically used for comparison with the concurrent control data as part of the assay acceptance criteria. Historical control data are also important for providing evidence of the technical competence and familiarization of the assay at any given laboratory. Moreover, historical control data are increasingly being used to aid in the interpretation of genetic toxicity assay results. The objective of the working group was to provide generic advice for historical control data that could be applied to all assays rather than to give assay-specific recommendations. In brief, the recommendations include: 1. The experimental protocol should remain fixed throughout the period during which the historical control data relevant to the current experiment are being built up, unless it can be demonstrated that changes to the protocol have not affected the values. 2. All data (both individual and group mean values) should be accumulated. 3. No negative control values (i.e., vehicle /solvent controls and absolute/culture medium controls, when available) should be eliminated from the data set, even if considered unusual, unless there is a scientifically justified reason, such as when they were obtained by an identified technical error. However, experiments may need to be repeated if disqualified by historical control data. 4. A minimum set of data resulting from at least 10, preferably 20, independent experiments is recommended to create the historical data set, depending upon the complexity of the assay. 5. It is not appropriate to use the simple range (minimum and maximum value observed during the data accumulation period) of the accumulated historical, especially negative, control data for an assessment. Rather, the distribution of the data together with appropriate descriptive statistics should be considered (e.g., confidence intervals, 95-99% percentiles). 6. For an experiment, when statistically significant increases over the concurrent negative controls (i.e., vehicle/solvent controls) are comparable, i.e., within confidence intervals, with the negative historical data, the biological importance needs to be carefully considered. 7. Historical control data could potentially have an important role in the future to help interpret aspects of genotoxicity data, such as dose response relationships

Can in vitro mammalian cell genotoxicity test results be used to complement positive results in the Ames test and help predict carcinogenic or in vivo genotoxic activity?: I. Reports of individual databases presented at an EURL ECVAM Workshop

Positive results in the Ames test correlate well with carcinogenic potential, at least in rodents. However, the correlation is not perfect because mutagenesis is not carcinogenesis, and mutations are only one of many stages in tumour development. Also, situations can be envisaged where the mutagenic response may be specific to the bacteria or the test protocol, for example bacterial-specific metabolism, exceeding a detoxification threshold or the induction of oxidative damage to which bacteria may be more sensitive than mammalian cells in vitro or tissues in vivo. Since most chemicals are also tested for genotoxicity in mammalian cells, the pattern of mammalian cell results (positive, negative) may help identify whether Ames-positive results predict carcinogenic or in vivo mutagenic activity. A workshop was therefore organised and sponsored by the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) to investigate this further. Participants presented data on results from other genotoxicity tests with Ames-positive compounds. Data came from published, regulatory agency and industry sources. The question was posed whether negative results in mammalian cell tests were often associated with absence of carcinogenic or in vivo genotoxic activity. In the limited time available, the presented data were combined and an initial analysis suggested that the association of negative in vitro mammalian cell test results with lack of in vivo genotoxic or carcinogenic activity could have some significance. Possible reasons why a positive Ames test may not be associated with in vivo activity and what additional investigations/tests might contribute to a more robust evaluation were discussed. Because a considerable overlap was identified among the different databases presented, it was recommended that a consolidated database should be built, with overlapping chemicals removed, so that a more robust analysis of the predictive capacity for potential carcinogenic and in vivo genotoxic activity could be derived from the patterns of mammalian cell test results obtained for Ames-positive compounds.JRC.I.5-Systems Toxicolog

Genetic Toxicology: Opportunities to Integrate New Approaches

The genetic toxicity tests currently used to identify and characterize potential human mutagens and carcinogens rely on measurements of primary DNA damage, gene mutation, and chromosome damage in vitro and in rodents. The International Life Sciences Institute/Health and Environmental Sciences Institute (ILSI/HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity Testing held a Workshop to consider the impact that new understanding of biology and new technologies might have on the identification and characterization of genotoxic substances, and to identify new approaches to inform more accurate human risk assessment for genetic and carcinogenic effects. Workshop organizers and invited speakers were from industry, academe, and government organizations. The biological effects and technologies that were selected as topics were those that would potentially yield the most useful information relevant to human hazard and risk assessment for genetic damage. Also addressed was the impact that the improved understanding of biology and the availability the new techniques might have on genetic toxicology practices. The primary topics addressed were Alternative Experimental Models to Improve Genetic Toxicity Testing; Biomarkers of Epigenetic Changes and their Applicability to Genetic Toxicology, and; New Technologies and Approaches. The ability of these new tests/technologies to be developed into tests to identify and characterize genotoxic agents, serve as a bridge between in vitro and in vivo rodent, or preferably human, data, or be used to provide dose response information for quantitative risk assessment, was also addressed

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

Workshop participants agreed that genotoxicity tests in mammalian cells in vitro produce a remarkably high and unacceptable occurrence of irrelevant positive results (e.g. when compared with rodent carcinogenicity). As reported in several recent reviews, the rate of irrelevant positives (i.e. low specificity) for some studies using in vitro methods (when compared to this "gold standard") means that an increased number of test articles are subjected to additional in vivo genotoxicity testing, in many cases before e.g. the efficacy (in the case of pharmaceuticals) of the compound has been evaluated. If in vitro tests were more predictive for in vivo genotoxicity and carcinogenicity (i.e. fewer false positives) then there would be a significant reduction in the number of animals used. Beyond animal (or human) carcinogenicity as the "gold standard", it is acknowledged that genotoxicity tests provide much information about cellular behaviour, cell division processes and cellular fate to a (geno)toxic insult. Since the disease impact of these effects is seldomly known, and a verification of relevant toxicity is normally also the subject of (sub)chronic animal studies, the prediction of in vivo relevant results from in vitro genotoxicity tests is also important for aspects that may not have a direct impact on carcinogenesis as the ultimate endpoint of concern. In order to address the high rate of in vitro false positive results, a 2-day workshop was held at the European Centre for the Validation of Alternative Methods (ECVAM), Ispra, Italy in April 2006. More than 20 genotoxicity experts from academia, government and industry were invited to review data from the currently available cell systems, to discuss whether there exist cells and test systems that have a reduced tendency to false positive results, to review potential modifications to existing protocols and cell systems that might result in improved specificity, and to review the performance of some new test systems that show promise of improved specificity without sacrificing sensitivity.JRC.I.2-Validation of biomedical testing method