EURL ECVAM Strategy to replace, reduce and refine the use of fish in aquatic toxicity and bioaccumulation testing

The assessment of aquatic toxicity and bioaccumulation are important components of the environmental hazard and risk assessment of all types of chemicals, and are therefore included in several pieces of European Union and international legislation. In this document, the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) outlines approaches which will deliver an impact on the replacement, reduction and refinement (3Rs) of fish tests used for aquatic toxicity and bioaccumulation testing. The document is based on an assessment of the regulatory needs for these endpoints, the scientific state-of-the art and recent activities in these areas. It highlights ongoing efforts at research, validation, guideline development and regulatory level. The proposed strategy is also intended to provide a framework for the prioritisation of alternative test methods submitted to EURL ECVAM for validation. Implementation of the strategy will rely on the coordinated efforts of multiple stakeholders.JRC.I.5-Systems Toxicolog

EURL ECVAM Recommendation on Non-Animal-Derived Antibodies

Affinity reagents are binding molecules that have a high specificity for their unique target (antigen). They are crucial tools for research, diagnostics, therapeutic and regulatory applications. Based on their recognition properties and binding specificity, protein-based antibodies are currently still the most important tools for the specific detection of proteins or other molecules. The development and production of monoclonal and polyclonal antibodies as well as other affinity reagents is still involving animals despite the availability of technologies that do not entail the use of animals. In line with the legal requirements of EU Directive 2010/63/EU on the protection of animals used for scientific purposes, animals should not be used in procedures, where a non-animal alternative exists, which provide the same or higher level of information as obtained from animal procedures. For this reason, the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) mandated its Scientific Advisory Committee (ESAC) to review the available evidence and deliver an opinion on the scientific validity of antibodies and non-antibody affinity reagents produced using animal-free technologies. The review focused on non-animal-derived antibodies generated by phage-display technology since this is the most mature technology and already widely used. Taking into consideration the available evidence, the ESAC endorsed an opinion on the suitability of existing animal-free technologies to produce affinity reagents with equal or better quality (purity, activity, specificity, affinity, stability, reproducibility) than that offered by antibodies produced using the conventional animal-based methods. In addition, ESAC commented on the scientific benefits of using animal-free affinity reagents and assessed whether there are any production and/or application scenarios for which these are not fit-for-purpose and animal-derived antibodies are still indispensable. This science-for-policy report describes the EURL ECVAM recommendations on non-animal-derived antibodies developed on the basis of the ESAC Opinion (Annex 1) and its detailed Working Group Report (Annex 2).JRC.F.3-Chemicals Safety and Alternative Method

EURL ECVAM Recommendation on the Zebrafish Embryo Acute Toxicity Test Method (ZFET) for Acute Aquatic Toxicity Testing

Acute fish toxicity testing is an important component of the environmental hazard assessment of chemicals. Since many years, (zebra-)fish embryo-based methods have been proposed as alternatives to the acute fish toxicity test carried out with juvenile or adult fish. On behalf of the Organisation for Economic Cooperation and Development (OECD), the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) coordinated during 2008-2012 the validation of the zebrafish embryo acute toxicity test method (ZFET) to evaluate its reproducibility in support to the development of an OECD Test Guideline. In parallel to this study, Belanger and colleagues continued to collect acute fish embryo toxicity and acute fish toxicity data to assess the relevance, predictive capacity and applicability of the ZFET and submitted their report to EURL ECVAM in July 2012. Following independent scientific peer review by EURL ECVAM's Scientific Advisory Committee (ESAC) of both studies and having considered input from regulators, stakeholders, international partners and the general public, EURL ECVAM concluded that the ZFET - being available as OECD TG236 since 2013 – should be used for generating information on acute fish toxicity, where appropriate. Its use would result in an overall reduction of the numbers of juvenile and adult fish for aquatic toxicity testing. It is recognised that further guidance on the use of OECD TG236 across the various regulatory frameworks and regions should be developed addressing in particular the possible use of the ZFET to generate information on acute fish toxicity and its potential limitations.JRC.I.5-Systems Toxicolog

Vitamin K2 Needs an RDI Separate from Vitamin K1

Vitamin K and its essential role in coagulation (vitamin K [Koagulation]) have been well established and accepted the world over. Many countries have a Recommended Daily Intake (RDI) for vitamin K based on early research, and its necessary role in the activation of vitamin K-dependent coagulation proteins is known. In the past few decades, the role of vitamin K-dependent proteins in processes beyond coagulation has been discovered. Various isoforms of vitamin K have been identified, and vitamin K2 specifically has been highlighted for its long half-life and extrahepatic activity, whereas the dietary form vitamin K1 has a shorter half-life. In this review, we highlight the specific activity of vitamin K2 based upon proposed frameworks necessary for a bioactive substance to be recommended for an RDI. Vitamin K2 meets all these criteria and should be considered for a specific dietary recommendation intake

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

EURL ECVAM Status Report on the Development, Validation and Regulatory Acceptance of Alternative Methods and Approaches (2016)

Replacement, Reduction and Refinement of animal testing is anchored in EU legislation. Alternative non-animal approaches facilitate a shift away from animal testing. Cell-based methods and computational technologies are integrated to translate molecular mechanistic understanding of toxicity into safety testing strategies.JRC.F.3-Chemicals Safety and Alternative Method

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio