A critical appraisal of the sensitivity of in vivo genotoxicity assays in detecting human carcinogens.

Genotoxicity testing is an important part of standard safety testing strategies. Animal studies have always been a key component, either as a mandatory part of the regulatory test battery, or to follow-up questionable in vitro findings. The strengths and weaknesses of in vivo assays is a continuous matter of debate, including their capacity to predict (human) carcinogenicity. We have therefore analysed the sensitivity of five routinely used in vivo tests to determine, in addition to other aspects, which tests or combination of tests best identify 73 chemicals classified as IARC Group 1 and 2A carcinogens. The in vivo tests included the micronucleus (MN), unscheduled DNA synthesis (UDS), comet, Pig-a and transgenic rodent assays (TGR). The individual assays detect 74.2% (49/66, MN), 64.3% (9/14, UDS), 92.1% (35/38, comet), 82.4% (14/17, Pig-a) and 90.3% (28/31, TGR) of the probable and confirmed human carcinogens that were tested in these assays. Combining assays that cover different genotoxicity endpoints and multiple tissues, e.g. the bone marrow MN and the liver comet assays, increases the sensitivity further (to 94%). Correlations in terms of organ-specificity for these assays with human cancer target organs revealed only a limited correlation for the hematopoietic system but not for other organs. The data supports the use of the comet and TGR assays for detection of 'site-of-first-contact' genotoxicants, but these chemicals were generally also detected in assays that measure genotoxicity in tissues not directly exposed, e.g. liver and the hematopoietic system. In conclusion, our evaluation confirmed a high sensitivity of the five in vivo genotoxicity assays for prediction of human carcinogens, which can be further increased by combining assays. Moreover, the addition of the comet to the in vivo MN test would identify all DNA reactive human carcinogens. Importantly, integration of some of the study readouts into one experiment is an animal-saving alternative to performing separate experiments.

Moving forward in carcinogenicity assessment: Report of an EURL ECVAM/ESTIV workshop.

There is an increased need to develop novel alternative approaches to the two-year rodent bioassay for the carcinogenicity assessment of substances where the rodent bioassay is still a basic requirement, as well as for those substances where animal use is banned or limited or where information gaps are identified within legislation. The current progress in this area was addressed in a EURL ECVAM- ESTIV workshop held in October 2016, in Juan les Pins. A number of initiatives were presented and discussed, including data-driven, technology-driven and pathway-driven approaches. Despite a seemingly diverse range of strategic developments, commonalities are emerging. For example, providing insight into carcinogenicity mechanisms is becoming an increasingly appreciated aspect of hazard assessment and is suggested to be the best strategy to drive new developments. Thus, now more than ever, there is a need to combine and focus efforts towards the integration of available information between sectors. Such cross-sectorial harmonisation will aid in building confidence in new approach methods leading to increased implementation and thus a decreased necessity for the two-year rodent bioassay.

Regulatory Acceptance of Alternative Methods in the Development and Approval of Pharmaceuticals.

Animal studies may be carried out to support first administration of a new medicinal product to either humans or the target animal species, or before performing clinical trials in even larger populations, or before marketing authorisation, or to control quality during production. Ethical and animal welfare considerations require that animal use is limited as much as possible. Directive 2010/63/EU on the protection of animals used for scientific purposes unambiguously fosters the application of the principle of the 3Rs when considering the choice of methods to be used.As such, today, the 3Rs are embedded in the relevant regulatory guidance both at the European (European Medicines Agency (EMA)) and (Veterinary) International Conference on Harmonization ((V)ICH) levels. With respect to non-clinical testing requirements for human medicinal products, reduction and replacement of animal testing has been achieved by the regulatory acceptance of new in vitro methods, either as pivotal, supportive or exploratory mechanistic studies. Whilst replacement of animal studies remains the ultimate goal, approaches aimed at reducing or refining animal studies have also been routinely implemented in regulatory guidelines, where applicable. The chapter provides an overview of the implementation of 3Rs in the drafting of non-clinical testing guidelines for human medicinal products at the level of the ICH. In addition, the revision of the ICH S2 guideline on genotoxicity testing and data interpretation for pharmaceuticals intended for human use is discussed as a case study.In October 2010, the EMA established a Joint ad hoc Expert Group (JEG 3Rs) with the mandate to improve and foster the application of 3Rs principles to the regulatory testing of medicinal products throughout their lifecycle. As such, a Guideline on regulatory acceptance of 3R testing approaches was drafted that defines regulatory acceptance and provides guidance on the scientific and technical criteria for regulatory acceptance of 3R testing approaches, including a process for collection of real-life data (safe harbour). Pathways for regulatory acceptance of 3R testing approaches are depicted and a new procedure for submission and evaluation of a proposal for regulatory acceptance of 3R testing approaches is described.

Critical analysis of carcinogenicity study outcomes. Relationship with pharmacological properties.

Predicting the outcome of life-time carcinogenicity studies in rats based on chronic (6-month) toxicity studies in this species is possible in some instances. This should reduce the number of such studies and hence have a significant impact on the total number of animals used in safety assessment of new medicines. From a regulatory perspective, this should be sufficient to grant a waiver for a carcinogenicity study in those cases where there is confidence in the outcome of the prediction. Pharmacological properties are a frequent key factor for the carcinogenic mode of action of some pharmaceuticals, but data-analysis on a large dataset has never been formally conducted. We have conducted an analysis of a dataset based on the perspective of the pharmacology of 255 compounds from industrial and regulatory sources. It is proposed that a pharmacological, class-specific, model may consist of an overall causal relationship between the pharmacological class and the histopathology findings in rats after 6 months treatment, leading to carcinogenicity outcome after 2 years. Knowledge of the intended drug target and pathway pharmacology should enhance the prediction of either positive or negative outcomes of rat carcinogenicity studies. The goal of this analysis is to review the pharmacological properties of compounds together with the histopathology findings from the chronic toxicity study in rodents in order to introduce an integrated approach to estimate the risk of human carcinogenicity of pharmaceuticals. This approach would allow scientists to define conditions under which 2-year rat carcinogenicity studies will or will not add value to such an assessment. We have demonstrated the possibility of a regulatory waiver for a carcinogenicity study in rats, as currently discussed in the International Council for Harmonization (ICH) - formerly known as the International Conference on Harmonization (ICH), by applying the proposed prediction approach in a number of case studies.

OSWG Recommendations for Genotoxicity Testing of Novel Oligonucleotide-Based Therapeutics.

The Oligonucleotide Safety Working Group subcommittee on genotoxicity testing considers therapeutic oligonucleotides (ONs) unlikely to be genotoxic based on their properties and on the negative results for ONs tested to date. Nonetheless, the subcommittee believes that genotoxicity testing of new ONs is warranted because modified monomers could be liberated from a metabolized ON and incorporated into DNA and could hypothetically cause chain termination, miscoding, and/or faulty replication or repair. The standard test battery as described in Option 1 of International Conference on Harmonisation S2(R1) is generally adequate to assess such potential. However, for the in vitro assay for gene mutations, mammalian cells are considered more relevant than bacteria for most ONs due to their known responsiveness to nucleosides and their greater potential for ON uptake; on the other hand, bacterial assays may be more appropriate for ONs containing non-ON components. Testing is not recommended for ONs with only naturally occurring chemistries or for ONs with chemistries for which there is documented lack of genotoxicity in systems with demonstrated cellular uptake. Testing is recommended for ONs that contain non-natural chemical modifications and use of the complete drug product (including linkers, conjugates, and liposomes) is suggested to provide the most clinically relevant assessment. Documentation of uptake into cells comparable to those used for genotoxicity testing is proposed because intracellular exposure cannot be assumed for these large molecules. ONs could also hypothetically cause mutations through triple helix formation with genomic DNA and no tests are available for detection of such sequence-specific mutations across the entire genome. However, because the potential for triplex formation by therapeutic ONs is extremely low, this potential can be assessed adequately by sequence analysis.

Genotoxicity assessment of peptide/protein-related biotherapeutics: points to consider before testing.

The ICH S6(R1) recommendations on safety evaluation of biotherapeutics have led to uncertainty in determining what would constitute a cause for concern that would require genotoxicity testing. A Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee Workgroup was formed to review the current practice of genotoxicity assessment of peptide/protein-related biotherapeutics. There are a number of properties of peptide/protein-related biotherapeutics that distinguish such products from traditional 'small molecule' drugs and need to be taken into consideration when assessing whether genotoxicity testing may be warranted and if so, how to do it appropriately. Case examples were provided by participating companies and decision trees were elaborated to determine whether and when genotoxicity evaluation is needed for peptides containing natural amino acids, non-natural amino acids and other chemical entities and for unconjugated and conjugated proteins. From a scientific point of view, there is no reason for testing peptides containing exclusively natural amino acids irrespective of the manufacturing process. If non-natural amino acids, organic linkers and other non-linker chemical components have already been tested for genotoxicity, there is no need to re-evaluate them when used in different peptide/protein-related biotherapeutics. Unless the peptides have been modified to be able to enter the cells, it is generally more appropriate to evaluate the peptides containing the non-natural amino acids and other non-linker chemical moieties in vivo where the cleavage products can be formed. For linkers, it is important to determine if exposure to reactive forms are likely to occur and from which origin. When the linkers are anticipated to be potential mutagenic impurities they should be evaluated according to ICH M7. If linkers are expected to be catabolic products, it is recommended to test the entire conjugate in vivo, as this would ensure that the relevant 'free' linker forms stemming from in vivo catabolism are tested.

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.

Critical issues with the in vivo comet assay: A report of the comet assay working group in the 6th International Workshop on Genotoxicity Testing (IWGT).

As a part of the 6th IWGT, an expert working group on the comet assay evaluated critical topics related to the use of the in vivo comet assay in regulatory genotoxicity testing. The areas covered were: identification of the domain of applicability and regulatory acceptance, identification of critical parameters of the protocol and attempts to standardize the assay, experience with combination and integration with other in vivo studies, demonstration of laboratory proficiency, sensitivity and power of the protocol used, use of different tissues, freezing of samples, and choice of appropriate measures of cytotoxicity. The standard protocol detects various types of DNA lesions but it does not detect all types of DNA damage. Modifications of the standard protocol may be used to detect additional types of specific DNA damage (e.g., cross-links, bulky adducts, oxidized bases). In addition, the working group identified critical parameters that should be carefully controlled and described in detail in every published study protocol. In vivo comet assay results are more reliable if they were obtained in laboratories that have demonstrated proficiency. This includes demonstration of adequate response to vehicle controls and an adequate response to a positive control for each tissue being examined. There was a general agreement that freezing of samples is an option but more data are needed in order to establish generally accepted protocols. With regard to tissue toxicity, the working group concluded that cytotoxicity could be a confounder of comet results. It is recommended to look at multiple parameters such as histopathological observations, organ-specific clinical chemistry as well as indicators of tissue inflammation to decide whether compound-specific toxicity might influence the result. The expert working group concluded that the alkaline in vivo comet assay is a mature test for the evaluation of genotoxicity and can be recommended to regulatory agencies for use.

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 in rodents. This correlation is not perfect because 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, e.g., 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 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 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 associated with absence of carcinogenic or in vivo genotoxic activity despite a positive Ames test. 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 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.

The 3T3 neutral red uptake phototoxicity test: practical experience and implications for phototoxicity testing--the report of an ECVAM-EFPIA workshop.

This is the report from the "ECVAM-EFPIA workshop on 3T3 NRU Phototoxicity Test: Practical Experience and Implications for Phototoxicity Testing", jointly organized by ECVAM and EFPIA and held on the 25-27 October 2010 in Somma Lombardo, Italy. The European Centre for the Validation of Alternative Methods (ECVAM) was established in 1991 within the European Commission Joint Research, based on a Communication from the European Commission (1991). The main objective of ECVAM is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine and replace the use of laboratory animals. The European Federation of Pharmaceuticals Industries and Association (EFPIA) represent the pharmaceutical industry operating in Europe. Through its direct membership of 31 national associations and 40 leading pharmaceutical companies, EFPIA is the voice on the EU scene of 2200 companies committed to researching, developing and bringing to patients new medicines that improve health and the quality of life around the world. The workshop, co-chaired by Joachim Kreysa (ECVAM) and Phil Wilcox (GSK, EFPIA) involved thirty-five experts from academia, regulatory authorities and industry, invited to contribute with their experiences in the field of phototoxicology. The main objectives of the workshop were: -to present 'in use' experience of the pharmaceutical industry with the 3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU-PT), -to discuss why it differs from the results in the original validation exercise, -to discuss technical issues and consider ways to improve the usability of the 3T3 NRU-PT for (non-topical) pharmaceuticals, e.g., by modifying the threshold of chemical light absorption to trigger photo-toxicological testing, and by modifying technical aspects of the assay, or adjusting the criteria used to classify a positive response. During the workshop, the assay methodology was reviewed by comparing the OECD Test Guideline (TG 432) with the protocols used in testing laboratories, data from EFPIA and JPMA 'surveys' were presented and possible reasons for the outcomes were discussed. Experts from cosmetics and pharmaceutical industries reported on their experience with the 3T3 NRU-PT and evidence was presented for phototoxic clinical symptoms that could be linked to certain relevant molecules. Brainstorming sessions discussed if the 3T3 NRU-PT needed to be improved and whether alternatives to the 3T3 NRU-PT exist. Finally, the viewpoint from EU and US regulators was presented. In the final session, the conclusions of the meeting were summarized, with action points. It was concluded that the 3T3 NRU-PT identifies phototoxicological hazards with a 100% sensitivity, and thus is accepted as the tier one test that correctly identifies the absence of phototoxic potential. Consequently, positive results in the 3T3 NRU-PT often do not translate into a clinical phototoxicity risk. Possible ways to improve the practical use of this assay include: (i) adaptation of changed UV/vis-absorption criteria as a means to reduce the number of materials tested, (ii) reduction of the highest concentration to be tested, and (iii) consideration of modifying the threshold criteria for the prediction of a positive call in the test.

Considerations on photochemical genotoxicity. II: report of the 2009 International Workshop on Genotoxicity Testing Working Group.

A workshop to reappraise the previous IWGT recommendations for photogenotoxicity testing [E. Gocke, L. Muller, P.J. Guzzie, S. Brendler-Schwaab, S. Bulera, C.F. Chignell, L.M. Henderson, A. Jacobs, H. Murli, R.D. Snyder, N. Tanaka, Considerations on photochemical genotoxicity: report of the International Workshop on Genotoxicity Test Procedures working group, Environ. Mol. Mutagen., 35 (2000) 173-184] was recently held as part of the 5th International Workshop on Genotoxicity Testing (IWGT) meeting in Basel, Switzerland (August 17-19, 2009). An Expert Panel was convened from regulatory, academic and industrial scientists (with several members serving on the original panel) and chaired by Dr Peter Kasper (BfArM, Germany). The aim of the workshop was to review progress made in photo(geno)toxicity testing over the past decade; a period which saw the introduction of several regulatory photosafety guidances in particular in Europe and the USA. Based on current regulatory guidelines a substantial proportion of compounds trigger the requirements for photosafety testing. Moreover, there has been growing concern within industry about the performance of the in vitro photosafety tests in the "real world" of compound development. Therefore, the expert group reviewed the status of the current regulatory guidance's and the impact these have had on compound development in the context of the various triggers for photosafety testing. In addition, the performance of photogenotoxicity assays (old and new) was discussed, particularly in view of reports of pseudophotoclastogencity. The Expert Panel finished with an assessment of the positioning of photogenotoxicity testing within a photosafety testing strategy. The most significant conclusion made by the Expert Panel was that photogenotoxicity testing should no longer be recommended as part of the standard photosafety testing strategy. In addition, progress was made on the refinement of triggers for photosafety testing. For example, there was support for harmonisation of methods to determine the Molar Extinction Coefficient (MEC) and a consensus agreement that there should be no requirement for testing of compounds with a MEC<1000Lmol(-1)cm(-1).

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:

Towards consensus practices to qualify safety biomarkers for use in early drug development.

Application of any new biomarker to support safety-related decisions during regulated phases of drug development requires provision of a substantial data set that critically assesses analytical and biological performance of that biomarker. Such an approach enables stakeholders from industry and regulatory bodies to objectively evaluate whether superior standards of performance have been met and whether specific claims of fit-for-purpose use are supported. It is therefore important during the biomarker evaluation process that stakeholders seek agreement on which critical experiments are needed to test that a biomarker meets specific performance claims, how new biomarker and traditional comparators will be measured and how the resulting data will be merged, analyzed and interpreted.

Renal biomarker qualification submission: a dialog between the FDA-EMEA and Predictive Safety Testing Consortium.

The first formal qualification of safety biomarkers for regulatory decision making marks a milestone in the application of biomarkers to drug development. Following submission of drug toxicity studies and analyses of biomarker performance to the Food and Drug Administration (FDA) and European Medicines Agency (EMEA) by the Predictive Safety Testing Consortium's (PSTC) Nephrotoxicity Working Group, seven renal safety biomarkers have been qualified for limited use in nonclinical and clinical drug development to help guide safety assessments. This was a pilot process, and the experience gained will both facilitate better understanding of how the qualification process will probably evolve and clarify the minimal requirements necessary to evaluate the performance of biomarkers of organ injury within specific contexts.

Reduction of use of animals in regulatory genotoxicity testing: Identification and implementation opportunities-Report from an ECVAM workshop.

In vivo genetic toxicology tests measure direct DNA damage or the formation of gene or chromosomal mutations, and are used to predict the mutagenic and carcinogenic potential of compounds for regulatory purposes and/or to follow-up positive results from in vitro testing. These tests are widely used and consume large numbers of animals, with a foreseeable marked increase as a result of the EU chemicals legislation (REACH), which may require follow-up of any positive outcome in the in vitro standard battery with appropriate in vivo tests, regardless of the tonnage level of the chemical. A 2-day workshop with genotoxicity experts from academia, regulatory agencies and industry was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) in Ranco, Italy from 24 to 25 June 2008. The objectives of the workshop were to discuss how to reduce the number of animals in standard genotoxicity tests, whether the application of smarter test strategies can lead to lower animal numbers, and how the possibilities for reduction can be promoted and implemented. The workshop agreed that there are many reduction options available that are scientifically credible and therefore ready for use. Most of these are compliant with regulatory guidelines, i.e. the use of one sex only, one administration and two sampling times versus two or three administrations and one sampling time for micronucleus (MN), chromosomal aberration (CA) and Comet assays; and the integration of the MN endpoint into repeat-dose toxicity studies. The omission of a concurrent positive control in routine CA and MN tests has been proven to be scientifically acceptable, although the OECD guidelines still require this; also the combination of acute MN and Comet assay studies are compliant with guidelines, except for sampling times. Based on the data presented at the workshop, the participants concluded that these options have not been sufficiently utilized to date. Key factors for this seem to be the uncertainty regarding regulatory compliance/acceptance, lack of awareness, and an in many cases unjustified uncertainty regarding the scientific acceptance of reduction options. The workshop therefore encourages the use and promotion of these options as well as the dissemination of data related to reduction opportunities by the scientific community in order to boost the acceptance level of these approaches. Furthermore, experimental proof is needed and under way to demonstrate the credibility of additional options for reduction of the number of animals, such as the integration of the Comet assay into repeat-dose toxicity studies.

Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: a follow-up to an ECVAM workshop.

At a recent ECVAM workshop considering ways to reduce the frequency of irrelevant positive results in mammalian cell genotoxicity tests [D. Kirkland, S. Pfuhler, D. Tweats, M. Aardema, R. Corvi, F. Darroudi, A. Elhajouji, H.-R. Glatt, P. Hastwell, M. Hayashi, P. Kasper, S. Kirchner, A. Lynch, D. Marzin, D. Maurici, J.-R. Meunier, L. Müller, G. Nohynek, J. Parry, E. Parry, V. Thybaud, R. Tice, J. van Benthem, P. Vanparys, P. White, How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary followup animal tests: Report of an ECVAM Workshop, Mutat. Res. 628 (2007) 31-55], recommendations for improvements/modifications to existing tests, and suggestions for new assays were made. Following on from this, it was important to identify chemicals that could be used in the evaluation of modified or new assays. An expert panel was therefore convened and recommendations made for chemicals to fit three different sets of characteristics, namely: This paper therefore contains these three recommended lists of chemicals and describes how these should be used for any test-evaluation programme.

Interlaboratory evaluation of genomic signatures for predicting carcinogenicity in the rat.

The Critical Path Institute recently established the Predictive Safety Testing Consortium, a collaboration between several companies and the U.S. Food and Drug Administration, aimed at evaluating and qualifying biomarkers for a variety of toxicological endpoints. The Carcinogenicity Working Group of the Predictive Safety Testing Consortium has concentrated on sharing data to test the predictivity of two published hepatic gene expression signatures, including the signature by Fielden et al. (2007, Toxicol. Sci. 99, 90-100) for predicting nongenotoxic hepatocarcinogens, and the signature by Nie et al. (2006, Mol. Carcinog. 45, 914-933) for predicting nongenotoxic carcinogens. Although not a rigorous prospective validation exercise, the consortium approach created an opportunity to perform a meta-analysis to evaluate microarray data from short-term rat studies on over 150 compounds. Despite significant differences in study designs and microarray platforms between laboratories, the signatures proved to be relatively robust and more accurate than expected by chance. The accuracy of the Fielden et al. signature was between 63 and 69%, whereas the accuracy of the Nie et al. signature was between 55 and 64%. As expected, the predictivity was reduced relative to internal validation estimates reported under identical test conditions. Although the signatures were not deemed suitable for use in regulatory decision making, they were deemed worthwhile in the early assessment of drugs to aid decision making in drug development. These results have prompted additional efforts to rederive and evaluate a QPCR-based signature using these samples. When combined with a standardized test procedure and prospective interlaboratory validation, the accuracy and potential utility in preclinical applications can be ascertained.

Relevance and follow-up of positive results in in vitro genetic toxicity assays: an ILSI-HESI initiative.

In vitro genotoxicity assays are often used to screen and predict whether chemicals might represent mutagenic and carcinogenic risks for humans. Recent discussions have focused on the high rate of positive results in in vitro tests, especially in those assays performed in mammalian cells that are not confirmed in vivo. Currently, there is no general consensus in the scientific community on the interpretation of the significance of positive results from the in vitro genotoxicity assays. To address this issue, the Health and Environmental Sciences Institute (HESI), held an international workshop in June 2006 to discuss the relevance and follow-up of positive results in in vitro genetic toxicity assays. The goals of the meeting were to examine ways to advance the scientific basis for the interpretation of positive findings in in vitro assays, to facilitate the development of follow-up testing strategies and to define criteria for determining the relevance to human health. The workshop identified specific needs in two general categories, i.e., improved testing and improved data interpretation and risk assessment. Recommendations to improve testing included: (1) re-examine the maximum level of cytotoxicity currently required for in vitro tests; (2) re-examine the upper limit concentration for in vitro mammalian studies; (3) develop improved testing strategies using current in vitro assays; (4) define criteria to guide selection of the appropriate follow-up in vivo studies; (5) develop new and more predictive in vitro and in vivo tests. Recommendations for improving interpretation and assessment included: (1) examine the suitability of applying the threshold of toxicological concern concepts to genotoxicity data; (2) develop a structured weight of evidence approach for assessing genotoxic/carcinogenic hazard; and (3) re-examine in vitro and in vivo correlations qualitatively and quantitatively. Conclusions from the workshop highlighted a willingness of scientists from various sectors to change and improve the current paradigm and move from a hazard identification approach to a "realistic" risk-based approach that incorporates information on mechanism of action, kinetics, and human exposure..

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 seldom 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. It was concluded that better guidance on the likely mechanisms resulting in positive results that are not biologically relevant for human health, and how to obtain evidence for those mechanisms, is needed both for practitioners and regulatory reviewers. Participants discussed the fact that cell lines commonly used for genotoxicity testing have a number of deficiencies that may contribute to the high false positive rate. These include, amongst others, lack of normal metabolism leading to reliance on exogenous metabolic activation systems (e.g. Aroclor-induced rat S9), impaired p53 function and altered DNA repair capability. The high concentrations of test chemicals (i.e. 10 mM or 5000 microg/ml, unless precluded by solubility or excessive toxicity) and the high levels of cytotoxicity currently required in mammalian cell genotoxicity tests were discussed as further potential sources of false positive results. Even if the goal is to detect carcinogens with short in vitro tests under more or less acute conditions, it does not seem logical to exceed the capabilities of cellular metabolic turnover, activation and defence processes. The concept of "promiscuous activation" was discussed. For numerous mutagens, the decisive in vivo enzymes are missing in vitro. However, if the substrate concentration is increased sufficiently, some other enzymes (that are unimportant in vivo) may take over the activation-leading to the same or a different active metabolite. Since we often do not use the right enzyme systems for positive controls in vitro, we have to rely on their promiscuous activation, i.e. to use excessive concentrations to get an empirical correlation between genotoxicity and carcinogenicity. A thorough review of published and industry data is urgently needed to determine whether the currently required limit concentration of 10mM or 5000 microg/ml, and high levels of cytotoxicity, are necessary for the detection of in vivo genotoxins and DNA-reactive, mutagenic carcinogens. In addition, various measures of cytotoxicity are currently allowable under OECD test guidelines, but there are few comparative data on whether different measures would result in different maximum concentrations for testing. A detailed comparison of cytotoxicity assessment strategies is needed. An assessment of whether test endpoints can be selected that are not intrinsically associated with cytotoxicity, and therefore are less susceptible to artefacts produced by cytotoxicity, should also be undertaken. There was agreement amongst the workshop participants that cell systems which are p53 and DNA-repair proficient, and have defined Phase 1 and Phase 2 metabolism, covering a broad set of enzyme forms, and used within the context of appropriately set limits of concentration and cytotoxicity, offer the best hope for reduced false positives. Whilst there is some evidence that human lymphocytes are less susceptible to false positives than the current rodent cell lines, other cell systems based on HepG2, TK6 and MCL-5 cells, as well as 3D skin models based on primary human keratinocytes also show some promise. Other human cell lines such as HepaRG, and human stem cells (the target for carcinogenicity) have not been used for genotoxicity investigations and should be considered for evaluation. Genetic engineering is also a valuable tool to incorporate missing enzyme systems into target cells. A collaborative research programme is needed to identify, further develop and evaluate new cell systems with appropriate sensitivity but improved specificity. In order to review current data for selection of appropriate top concentrations, measures and levels of cytotoxicity, metabolism, and to be able to improve existing or validate new assay systems, the participants called for the establishment of an expert group to identify the in vivo genotoxins and DNA-reactive, mutagenic carcinogens that we expect our in vitro genotoxicity assays to detect as well as the non-genotoxins and non-carcinogens we expect them not to detect.