EURL ECVAM strategy to avoid and reduce animal use in genotoxicity testing

The assessment of genotoxicity represents an important component of the safety assessment of all types of substances. Although several in vitro tests are available at different stages of development and acceptance, they cannot at present be considered to fully replace animal tests needed to evaluate the safety of substances. Based on an analysis of regulatory requirements for this endpoint within different pieces of EU legislation, EURL ECVAM proposes a pragmatic approach to improve the traditional genotoxicity testing paradigm that offers solutions in both the short- and medium-term and that draws on the considerable experience of 40 years of regulatory toxicology testing in this area. EURL ECVAM considers that efforts should be directed towards the overall improvement of the current testing strategy for better hazard and risk assessment approaches, which either avoids or minimises the use of animals, whilst satisfying regulatory information requirements, irrespective of regulatory context. Several opportunities for the improvement of the testing strategy have been identified which aim to i) enhance the performance of the in vitro testing battery so that fewer in vivo follow-up tests are necessary and ii) guide more intelligent in vivo follow-up testing to reduce unnecessary use of animals. The implementation of this strategic plan will rely on the cooperation of EURL ECVAM with other existing initiatives and the coordinated contribution from various stakeholders.JRC.I.5-Systems Toxicolog

EURL ECVAM Recommendation on the Cell Transformation Assay based on the Bhas 42 cell line

The carcinogenic potential of compounds is a crucial aspect in human hazard and risk assessment of substances. Among the various alternatives developed for carcinogenicity prediction, the cell transformation assays (CTAs) have been shown to closely model some key stages of the in vivo carcinogenesis process. Similar to previously validated in vitro CTAs, the CTA in Bhas 42 cells aims at predicting carcinogenic potential. Based on the results of a validation study coordinated by Hadano Research Institute (HRI) Food and Drug Safety Center (FDSC) and other published data, the Bhas 42 CTA protocol (including the 6-well and 96-well plate versions) was considered to be sufficiently standardised, transferable, reproducible between laboratories and relevant to support the identification of potential carcinogenicity of substances. Following independent scientific peer review by the EURL ECVAM’s Scientific Advisory Committee (ESAC) and having considered the input from regulators, stakeholders, international partners and the general public, EURL ECVAM concluded that the CTA in Bhas 42 cells shows promise for inclusion within weight of evidence or integrated testing strategy approaches to assess carcinogenic potential or to support chemical category formation and read-across. Thus EURL ECVAM recommends that an OECD test Guideline be developed. In addition, further investigations on the capability of the assay to detect tumour promoters would provide useful information on mode of action of carcinogens for risk assessment purposes.JRC.I.5-Systems Toxicolog

Moving Forward in Human Cancer Risk Assessment

The goal of human risk assessment is to decide whether a given exposure level to a particular chemical or substance is acceptable to human health, and to provide risk management measures based on an evaluation and prediction of the effects of that exposure on human health. Within this framework, the current safety paradigm for assessing possible carcinogenic properties of drugs, cosmetics, industrial chemicals and environmental exposures relies mainly on in vitro genotoxicity testing followed by 2-year bioassays in mice and rats. This testing paradigm was developed 40 to 50 years ago with the initial premise that ¿mutagens are also carcinogens¿ and that the carcinogenic risk to humans can be extrapolated from the tumor incidence after lifetime exposure to maximally tolerated doses of chemicals in rodents. Genotoxicity testing is used as a surrogate for carcinogenicity testing and is required for initiation of clinical trials (Jacobs and Jacobson-Kram 2004) and for most industrial chemicals safety assessment. Although the carcinogenicity-testing paradigm has effectively protected patients and consumers from introduction of harmful carcinogens as drugs and other products, the testing paradigm is clearly not sustainable in the future. The causal link between genetic damage and carcinogenicity is well documented; however, the limitations of genotoxicity/carcinogenicity testing assays, the presence of additional non-genotoxic mechanisms, issues of species-specific effects, and the lack of mechanistic insights provide an enormous scientific challenge. The 2-year rodent carcinogenicity bioassays are associated with technical complexity, high costs, high animal burden as well as the uncertainty associated with extrapolating from rodents to humans. Additional frustrations exist because of the limited predictability of the 2-year bioassay and, in particular, with regard to the problem of the prediction of false positives. For instance, in the Carcinogenic Potency Project DataBase (CPDB) which includes results from chronic, long-term animal cancer tests with mice, rats, hamsters amounting to a total of 6540 individual experiments with 1547 chemicals, 751 of those chemicals or 51% have positive findings in rodent studies. Similarly, when one considers all chronically used human pharmaceuticals, some 50% induce tumors in rodents. Yet only 20 human pharmaceutical compounds have been identified as carcinogens in epidemiological studies, despite the fact that quite a large number of epidemiological studies have been carried out on these compounds, e.g. NSAID¿s, benzodiazepines, phenobarbital. This high incidence of tumors in bioassays has lead to questions concerning the human relevance of tumors induced in rodents (Knight et al. 2006; Ward 2008). In summary, dependency on the rodent model as a golden standard of cancer risk assessment is neglecting the high number of false positives and clearly has serious limitations. Consequently, there is a growing appeal for a paradigm change after "50 years of rats and mice". For instance, the current demands for volume of carcinogenic testing together with limitations of animal usage as initially stipulated by REACH (Combes et al. 2006) will require revolutionary change in the testing paradigm. For the purpose of developing a road map for this needed paradigm change in carcinogenicity testing, a workshop was held in August 2009 in Venice, Italy entitled ¿Genomics in Cancer Risk Assessment.¿ This workshop brought together toxicologists from academia and industry with governmental regulators and risk assessors from the US and the EU, for discussing the state-of-the-art in developing alternative testing strategies for genotoxicity and carcinogenicity, thereby focusing on the contribution from the ¿omics technologies. What follows is a highlight of the major conclusions and suggestions from this workshop as a path forward.JRC.DG.I.3-In-vitro method

ECVAM Technical Report on the Status of Alternative Methods for Cosmetics Testing (2008-2009)

The ECVAM technical report presents the progress made in the development and validation of alternative methods for the human health effects relevant to the Cosmetics Directive. It provides an update on the activities described by ECVAM in 2005 , 2006 and 2007 . The report intends to present the latest scientific and technical developments in the field during 2008-2009. As required by Directive 2003/15/EC, the seventh amendment to Directive 76/768/EEC, developments in refinement and reduction methods are also described (EU, 2003). Most successes in the development of alternative methods are in acute local toxicity and short-term testing, such as e.g. skin and eye irritation/corrosion, phototoxicity and skin penetration The test methods consuming a high number of animals, however, are in long-term testing and systemic toxicity, such as e.g. reproductive toxicity and repeated dose toxicity. In these complex fields, several research initiatives are ongoing. However full replacement approaches are still lacking.JRC.DG.I.3-In-vitro method

Use of in vitro 3D tissue models in genotoxicity testing: Strategic fit, validation status and way forward. Report of the working group from the 7th International Workshop on Genotoxicity Testing (IWGT)

Use of three-dimensional (3D) tissue equivalents in toxicology has been increasing over the last decade as novel preclinical test systems and as alternatives to animal testing. In the area of genetic toxicology, progress has been made with establishing robust protocols for skin, airway (lung) and liver tissue equivalents. In light of these advancements, a “Use of 3D Tissues in Genotoxicity Testing” working group (WG) met at the 7th IWGT meeting in Tokyo in November 2017 to discuss progress with these models and how they may fit into a genotoxicity testing strategy. The workshop demonstrated that skin models have reached an advanced state of validation following over 10 years of development, while liver and airway model-based genotoxicity assays show promise but are at an early stage of development. Further effort in liver and airway model-based assays is needed to address the lack of coverage of the three main endpoints of genotoxicity (mutagenicity, clastogenicity and aneugenicity), and information on metabolic competence. The IWGT WG believes that the 3D skin comet and micronucleus assays are now sufficiently validated to undergo an independent peer review of the validation study, followed by development of individual OECD Test Guidelines

Screening methodology to identify potential endocrine disruptors according to different options in the context of an impact assessment

Several pieces of EU legislation regulate the marketing and use of chemical substances. While several regulations, including the regulations on Plant Protection Products (PPPR), Biocidal Products (BPR) and Chemicals (REACH), include provisions for endocrine disrupting substances (EDs), objective scientific criteria are lacking. In order to evaluate the potential health, socio-economic and environmental impacts of applying four different options for criteria defining EDs across these pieces of legislation, the Commission initiated an Impact Assessment (IA). This IA has been supported by two studies, focusing on (a) selection of substances for the IA and the screening of their potential for identification as EDs according to different options for defining criteria for identification of endocrine disruptors and (b) the potential impacts of various policy options on health, environment, trade, agriculture and socio-economy. This report describes a screening methodology that has been developed by the JRC to support the first study which has assessed all pesticide and biocide active ingredients and a selection of substances falling under REACH, the Cosmetic Products Regulation and the Water Framework Directive. This screening methodology is not intended to replace an in-depth risk assessment process, and the results obtained are not intended to pre-empt regulatory conclusions that may eventually be made under different pieces of EU legislation.JRC.I.5-Systems Toxicolog

EURL ECVAM Status Report on the Development, Validation and Regulatory Acceptance of Alternative Methods and Approaches (2013-April 2014)

The EURL ECVAM status report provides an update on the progress made in the development, validation and regulatory acceptance of alternative methods and approaches since the last report published in April 2013. It is informing on ongoing research and development activities, validation studies, peer reviews, recommendations, strategies and international acceptance of alternative methods and approaches. R&D activities are ongoing for the complex endpoints where the toxicological processes and the mechanistic understanding have not been sufficiently elucidated yet and for which 3Rs solutions are more difficult to find. On the other hand, good progress In the validation and regulatory acceptance is made in areas where non-animal alternative methods have been developed and validated and where the focus lies in an intelligent combination/ integration of the various non-animal approaches.JRC.I.5-Systems Toxicolog

Alternative methods for regulatory toxicology – a state-of-the-art review

This state-of-the art review is based on the final report of a project carried out by the European Commission’s Joint Research Centre (JRC) for the European Chemicals Agency (ECHA). The aim of the project was to review the state of the science of non-standard methods that are available for assessing the toxicological and ecotoxicological properties of chemicals. Non-standard methods refer to alternatives to animal experiments, such as in vitro tests and computational models, as well as animal methods that are not covered by current regulatory guidelines. This report therefore reviews the current scientific status of non-standard methods for a range of human health and ecotoxicological endpoints, and provides a commentary on the mechanistic basis and regulatory applicability of these methods. For completeness, and to provide context, currently accepted (standard) methods are also summarised. In particular, the following human health endpoints are covered: a) skin irritation and corrosion; b) serious eye damage and eye irritation; c) skin sensitisation; d) acute systemic toxicity; e) repeat dose toxicity; f) genotoxicity and mutagenicity; g) carcinogenicity; h) reproductive toxicity (including effects on development and fertility); i) endocrine disruption relevant to human health; and j) toxicokinetics. In relation to ecotoxicological endpoints, the report focuses on non-standard methods for acute and chronic fish toxicity. While specific reference is made to the information needs of REACH, the Biocidal Products Regulation and the Classification, Labelling and Packaging Regulation, this review is also expected to be informative in relation to the possible use of alternative and non-standard methods in other sectors, such as cosmetics and plant protection products.JRC.I.5-Systems Toxicolog

EURL ECVAM Status Report on the Development, Validation and Regulatory Acceptance of Alternative Methods and Approaches (2015)

The EURL ECVAM status report provides an update on the progress made in the development, validation and regulatory acceptance of alternative methods and approaches and their dissemination since the last report published in June 2014. It is informing on ongoing research and development activities, validation studies, peer reviews, recommendations, strategies and regulatory/international acceptance of alternative methods and approaches and dissemination activities. R&D activities within large European or International consortia continued in toxicity areas where 3Rs solutions are more difficult to find due to the underlying complexity of the area. On the other hand, toxicity areas where promising non-animal approaches have been developed, their validation and regulatory acceptance/international adoption could be progressed. Particular emphasis was given to the best and most intelligent combination and integration of these different non-animal approaches to ultimately obtain the required information without resorting to animal testing.JRC.I.5-Systems Toxicolog

Meeting Report: Validation of Toxicogenomics-Based Test Systems: ECVAM–ICCVAM/NICEATM Considerations for Regulatory Use

This is the report of the first workshop “Validation of Toxicogenomics-Based Test Systems” held 11–12 December 2003 in Ispra, Italy. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and organized jointly by ECVAM, the U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), and the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). The primary aim of the workshop was for participants to discuss and define principles applicable to the validation of toxicogenomics platforms as well as validation of specific toxicologic test methods that incorporate toxicogenomics technologies. The workshop was viewed as an opportunity for initiating a dialogue between technologic experts, regulators, and the principal validation bodies and for identifying those factors to which the validation process would be applicable. It was felt that to do so now, as the technology is evolving and associated challenges are identified, would be a basis for the future validation of the technology when it reaches the appropriate stage. Because of the complexity of the issue, different aspects of the validation of toxicogenomics-based test methods were covered. The three focus areas include a) biologic validation of toxicogenomics-based test methods for regulatory decision making, b) technical and bioinformatics aspects related to validation, and c) validation issues as they relate to regulatory acceptance and use of toxicogenomics-based test methods. In this report we summarize the discussions and describe in detail the recommendations for future direction and priorities