Updated recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests

In 2008 we published recommendations on chemicals that would be appropriate to evaluate the sensitivity and specificity of new/modified mammalian cell genotoxicity tests, in particular to avoid misleading positive results. In light of new data it is appropriate to update these lists of chemicals. An expert panel was convened and has revised the recommended chemicals to fit the following different sets of characteristics:. •Group 1: chemicals that should be detected as positive in in vitro mammalian cell genotoxicity tests. Chemicals in this group are all in vivo genotoxins at one or more endpoints, either due to DNA-reactive or non DNA-reactive mechanisms. Many are known carcinogens with a mutagenic mode of action, but a sub-class of probable aneugens has been introduced.•Group 2: chemicals that should give negative results in in vitro mammalian cell genotoxicity tests. Chemicals in this group are usually negative in vivo and non-DNA-reactive. They are either non-carcinogenic or rodent carcinogens with a non-mutagenic mode of action.•Group 3: chemicals that should give negative results in in vitro mammalian cell genotoxicity tests, but have been reported to induce gene mutations in mouse lymphoma cells, chromosomal aberrations or micronuclei, often at high concentrations or at high levels of cytotoxicity. Chemicals in this group are generally negative in vivo and negative in the Ames test. They are either non-carcinogenic or rodent carcinogens with an accepted non-mutagenic mode of action. This group contains comments as to any conditions that can be identified under which misleading positive results are likely to occur.This paper, therefore, updates these three recommended lists of chemicals and describes how these should be used for any test evaluation program

Summary of major conclusions from the 6th International Workshop on Genotoxicity Testing (IWGT), Foz do Iguaçu, Brazil

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Bacterial Mutagenicity Screening in the pharmaceutical industry

Genetic toxicity testing is used as an early surrogate for carcinogenicity testing. Genetic toxicity testing is also required by regulatory agencies to be conducted prior to initiation of first in human clinical trials and subsequent marketing for most small molecule pharmaceutical compounds. To reduce the chances of advancing mutagenic pharmaceutical candidates through the drug discovery and development processes, companies have focused on developing testing strategies to maximize hazard identification while minimizing resource expenditure due to late stage attrition. With a large number of testing options, consensus has not been reached on the best mutagenicity platform to use or on the best time to use a specific test to aid in the selection of drug candidates for development. Most companies use a process in which compounds are initially screened for mutagenicity early in drug development using tests that require only a few milligrams of compound and then follow those studies up with a more robust mutagenicity test prior to selecting a compound for full development. This review summarizes the current applications of bacterial mutagenicity assays utilized by pharmaceutical companies in early and late discovery programs. The initial impetus for this review was derived from a workshop on bacterial mutagenicity screening in the pharmaceutical industry presented at the 40th Annual Environmental Mutagen Society Meeting held in St. Louis, MO in October, 2009. However, included in this review are succinct summaries of use and interpretation of genetic toxicity assays, several mutagenicity assays that were not presented at the meeting, and updates to testing strategies resulting in current state-of the art description of best practices. In addition, here we discuss the advantages and liabilities of many broadly used mutagenicity screening platforms and strategies used by pharmaceutical companies. The sensitivity and specificity of these early mutagenicity screening assays using proprietary compounds and their concordance (predictivity) with the regulatory bacterial mutation test are discussed

Evaluation of methyl methanesulfonate, 2,6-diaminotoluene and 5-fluorouracil: Part of the Japanese Center for the Validation of Alternative Methods (JaCVAM) international validation study of the in vivo rat alkaline Comet assay

As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline Comet assay, we examined methyl methanesulfonate, 2,6-diaminotoluene, and 5-fluorouracil under coded test conditions. Rats were treated orally with the maximum tolerated dose (MTD) and two additional descending doses of the respective compounds. In the MMS treated groups liver and stomach showed significantly elevated DNA damage at each dose level and a significant dose-response relationship. 2,6-diaminotoluene induced significantly elevated DNA damage in the liver at each dose and a statistically significant dose-response relationship whereas no DNA damage was obtained in the stomach. 5-fluorouracil did not induce DNA damage in either liver or stomach

Derisking drug-induced carcinogenicity for novel therapeutics

Assessing the carcinogenic potential of innovative drugs spanning diverse therapeutic modalities and target biology represents a major challenge during drug development. Novel modalities, such as cell and gene therapies that involve intrinsic genetic modification of the host genome, require distinct approaches for identification of cancer hazard. We emphasize the need for customized weight of evidence cancer risk assessments based on mode of action that balance multiple options for preclinical identification of cancer hazard with appropriate labeling of clinical products and risk management plans. We review how advances in molecular carcinogenesis can enhance mechanistic interpretation and preclinical indicators of neoplasia, and recommend that drug targets be systematically assessed for potential association with tumorigenic phenotypes via genetic models and cancer genome resources

Automatic Analysis of the Micronucleus Test in Primary Human Lymphocytes Using Image Analysis

The in vitro micronucleus test (MNT) is a well established test for early screening of new chemical entities in industrial toxicology. For assessing the clastogenic or aneugenic potential of a test compound, micronucleus induction in cells has been shown repeatedly to be a sensitive and specific parameter. Various automated systems to replace the tedious and time consuming manual slide analysis procedure have been described. Flow cytrometric approaches have been discussed elsewhere. The ROBIAS image analysis system for both automatic cytotoxicity assessment and highly sensitive micronucleus detection in primary human lymphocytes was developed at Novartis, where the assay is used as to confirm positive results obtained in the MNT in TK6 cells which serves as the primary screening system for genotoxicity profiling in early drug development. The comparison of manual with automatic analysis results showed a high degree of concordance for 27 independent experiments conducted for profiling of 12 compounds. For concentration series of Cyclophosphamide (CP) and Carbendazim (MBC), a very good correlation between automatic and manual analysis could be established, both for the relative division index used as cytotoxicity parameter, and for MN scoring in mono- and bi-nucleated cells. Generally, false positive micronucleus decisions could be controlled by fast and simple relocation of the automatically detected patterns. The possibility to analyze 24 slides within 65 hours by fully automatic analysis over the weekend and the high reproducibility of the results make automatic image processing a powerful tool for the micronucleus analysis in primary human lymphocytes. The automated slide analysis for the MNT in human lymphocytes complements the portfolio of image analysis applications on ROBIAS supporting various assays in genetic toxicology and other biomedical areas

Comprehensive review of genotoxicity data for diclofenac

Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) discovered decades ago, which has since been used by an estimated one billion patients, and demonstrated a well acceptable safety profile. In support of its marketing approval, a comprehensive set of genotoxicity studies had been conducted in vitro and in vivo. Despite the fact that these studies preceded both GLP requirements and ICH guidelines on genotoxicity testing, they were conducted using the best scientific principles and are considered appropriate by contemporary standards. In addition to bacterial mutagenicity and mammalian in vitro assays, repeat-dose, germ cell and dominant lethal assays had been conducted. These data are made available for the first time to offer researchers an opportunity to review the existing data set which unequivocally demonstrates that diclofenac is not genotoxic. The lack of a genotoxic potential is further substantiated by long-term bioassay data demonstrating that diclofenac has no carcinogenic potential in rodents. However, more recently, new studies were published showing a genotoxic potential for diclofenac in novel or modified in vitro test systems. These new data are discussed in the context of the existing comprehensive data package

Gamma-H2AX immunofluorescence for the detection of tissue-specific genotoxicity in vivo

The phosphorylation of histone H2AX in Serine 139 (gamma-H2AX) marks regions of DNA double strand breaks and contributes to the recruitment of DNA repair factors to the site of DNA damage. Gamma-H2AX is used widely as DNA damage marker in vitro, but its use for genotoxicity assessment in vivo has not been extensively investigated. Here, we developed an image analysis system for the precise quantification of the gamma-H2AX signal, which we used to monitor DNA damage in animals treated with known genotoxicants (EMS, ENU and doxorubicin). To compare this new assay to a validated standard procedure for DNA damage quantification, tissues from the same animals were also analyzed in the comet assay. An increase in the levels of gamma-H2AX was observed in most of the tissues from animals treated with doxorubicin and ENU. Interestingly, the lesions induced by doxorubicin were not easily detected by the standard comet assay, while they were clearly identified by gamma-H2AX staining. Conversely, EMS appeared strongly positive in the comet assay but only mildly in the gamma-H2AX immunofluorescence. These observations suggest that the two methods could complement each other for DNA damage analysis, where gamma-H2AX staining allows the detection of tissue-specific effects in situ. Moreover, since gamma-H2AX staining can be performed on formalin-fixed and paraffin-embedded tissue sections generated during repeated-dose toxicity studies, it does not require any further treatments or extra procedures during dissection, thus optimizing the use of resources and animals

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

Setting occupational exposure limits (OELs) for genotoxic substances in pharmaceutical industry

Pharmaceutical drug substances (DS) and intermediates (IM) with positive results in genotoxicity tests, indicatethat they induce directly or indirectly damage to the genetic material. The basic assumption for the shape of the dose-response relationship of genotoxic substances is that it is linear, meaning that there is no health based lower limit below which there is no risk, so that an occupational exposure limit (OEL) cannot be readily calculated. However, it has been accepted recently that threshold-like mechanisms may apply under certain conditions, indicating that in such cases an OEL can be calculated, instead of applying a limit associated with an acceptable excess cancer risk. We have investigated the possibility to determine the conditions that need to be encountered in order to define the threshold and therefore calculate an OEL. In addition we investigated the inhalatory toxicological threshold concentration (iTTC) which would provide acceptable level of protection for those substances for which the threshold cannot be determined such as mutagenic IM, in line with hypotheses applied in other areas of cancer risk assessment. The availability of thresholds for the substances must be determined on a case-by-case basis including detailed evaluation of the mode of action (MOA) based on convincing experimental evidence for possible threshold-like mechanisms. Several examples for threshold and non-threshold mechanism are presented and calculations of OEL for those substances, as well as an argument for iTTC of 1.6 ug/m3 sufficiently protective against the unacceptable risks