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

Alteration of liver N-glycome in patients with hepatocellular carcinoma

Purpose: Alteration of liver function during progression of hepatocellular carcinoma (HCC) and cirrhosis affects the serum glycoprotein pattern. In this study, the changes in the N-glycome in liver tissue from patients with hepatocellular carcinoma and cirrhosis caused by hepatitis B virus infection were investigated to find out the relationship between this maker and liver disease. Methods: Twenty patients, 11 with cirrhosis and 9 with hepatocellular carcinoma, and 15 healthy donors were involved in this study. Liver protein N-glycans were profiled using the DSA-FACE technique developed in our laboratory. To further analyze the fucosylation status of these liver glycans Western lectin blots of total liver proteins were performed using Aspergillus oryzae lectin (AOL) as probe, which is a carbohydrate-binding protein that recognizes specifically α-1,6-fucosylated glycans. Results: The N-glycome of liver proteins in patients with HBV related HCC and cirrhosis was analyzed. Compared with healthy donors, the N-glycome had significantly less (p < 0.05) high mannose (M8) in both groups of patients. The total core α-1,6-fucosylation in total liver glycopro-teins was dramatically increased during the progress of hepatocellular carcinoma and cirrhosis compared to the controls. Conclusion: These results show that fucosylation not only increases in serum proteins but also in liver tissue itself of patients with HBV related HCC and cirrhosis

Retrospective analysis of the Draize test for serious eye damage/eye irritation: importance of understanding the in vivo endpoints under UN GHS/EU CLP for the development and evaluation of in vitro test methods

For more than two decades, scientists have been trying to replace the regulatory in vivo Draize eye test by in vitro methods, but so far only partial replacement has been achieved. In order to better understand the reasons for this, historical in vivo rabbit data were analysed in detail and resampled with the purpose of (1) revealing which of the in vivo endpoints are most important in driving United Nations Globally Harmonized System/European Union Regulation on Classification, Labelling and Packaging (UN GHS/EU CLP) classification for serious eye damage/eye irritation and (2) evaluating the method’s within-test variability for proposing acceptable and justifiable target values of sensitivity and specificity for alternative methods and their combinations in testing strategies. Among the Cat 1 chemicals evaluated, 36–65 % (depending on the database) were classified based only on persistence of effects, with the remaining being classified mostly based on severe corneal effects. Iritis was found to rarely drive the classification (<4 % of both Cat 1 and Cat 2 chemicals). The two most important endpoints driving Cat 2 classification are conjunctiva redness (75–81 %) and corneal opacity (54–75 %). The resampling analyses demonstrated an overall probability of at least 11 % that chemicals classified as Cat 1 by the Draize eye test could be equally identified as Cat 2 and of about 12 % for Cat 2 chemicals to be equally identified as No Cat. On the other hand, the over-classification error for No Cat and Cat 2 was negligible (<1 %), which strongly suggests a high over-predictive power of the Draize eye test. Moreover, our analyses of the classification drivers suggest a critical revision of the UN GHS/EU CLP decision criteria for the classification of chemicals based on Draize eye test data, in particular Cat 1 based only on persistence of conjunctiva effects or corneal opacity scores of 4. In order to successfully replace the regulatory in vivo Draize eye test, it will be important to recognise these uncertainties and to have in vitro tools to address the most important in vivo endpoints identified in this paper.JRC.I.5-Systems Toxicolog

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

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

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

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 (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

The Neutral Red Release Assay. A Review.

Abstract not availableJRC.I-Institute for Health and Consumer Protection (Ispra

ECVAM's Research and Validation Actiuvities in the Fields of Topical Toxicity and Human Studies.

Abstract not availableJRC.I-Institute for Health and Consumer Protection (Ispra