Replacement, Reduction and Refinement of Animal Testing in the Quality Control of Human Vaccines

Vaccines are recognised as a highly cost effective tool for preventing infectious diseases. They are derived from biological sources and due to the complexity of composition and heterogeneity of products, vaccine lots undergo legally required quality control before they are released. Traditionally, laboratory animals have played an important role in quality control of vaccines and still, many laboratory animals are used in Europe for this purpose. Over the last decades, Replacement, Reduction and Refinement (3Rs) methods to classical animal tests have been developed by control authorities, academia and vaccine manufacturers. The purpose of this report is to inform the EURL ECVAM stakeholders on ongoing activities in development and validation of 3Rs methods for the quality control of vaccines for human use. The focus of the report is on methods for lot release testing (e.g. safety, pyrogenicity, potency) and projects related to the implementation of the consistency approach to established vaccines such as diphtheria, tetanus, pertussis and rabies vaccines.JRC.I.5-Systems Toxicolog

Scientific options for avoiding chronic fish testing on the basis of existing data and extrapolation approaches

The assessment of aquatic toxicity is an important component of the environmental hazard and risk assessment of all types of chemicals, and is therefore included in several pieces of EU chemicals legislation. Aquatic toxicity refers to the effects of chemicals on organisms living in the water and is usually determined by testing on organisms representing three trophic levels, i.e. plants (or algae), invertebrates (crustaceans such as Daphnia spp.) and vertebrates (fish). Whereas acute aquatic toxicity testing is a basic requirement in most pieces of EU chemicals legislation, chronic aquatic toxicity testing may be required when the outcome of the acute testing indicates a risk, or in the case that long-term exposure is expected. EU chemicals legislation encourages the use of all available information for hazard and risk assessment before new tests on vertebrates are proposed or conducted. In this context, scientific options for avoiding chronic fish testing on the basis of existing data and extrapolation approaches have been explored. For the purposes of this work, data on acute and chronic aquatic toxicity (Daphnia and fish) from several databases (US EPA Ecotox database, Aquatic ECETOC, Aquatic OASIS, Aquatic Japan MoE databases and ECHA database as implemented in the OECD QSAR Toolbox Version 2.3) were collated and analysed. Simple linear relationships and interspecies sensitivity ratios were calculated using either acute Daphnia data (48h LC50) or chronic Daphnia data (14 days NOEC) and chronic fish data (>21 days NOEC). Acute to chronic relationships and acute to chronic ratios (ACR) were also calculated based on acute fish data (96h LC50) and chronic fish data. These analyses were carried out on the whole set of chemicals and on subgroups of chemicals classified according to the Verhaar mode of action (MOA) scheme, which attribute general mode of acute aquatic toxic action based on the chemical structure of the molecule. Outliers were identified applying the Robust regression and Outlier removal (ROUT) method. Our results show that the best fitted relationships for the prediction of chronic fish toxicity are obtained based on acute fish data (r2=0.87) and acute Daphnia data (r2=0.64) when dealing with the whole set of chemicals regardless of the MOA. The quality of the relationships was increased by using the geometric mean (calculated across all the values extracted for a given chemical and a given endpoint) instead of the lowest value for a given endpoint. When considering the MOA, MOA 3 and MOA 1 chemicals give the strongest acute Daphnia to chronic fish relationship and chronic Daphnia to chronic fish relationship; however the relationships obtained with acute Daphnia data are better (r2= 0.83 and 0.69 for MOA 3 and MOA 1 respectively) than the one obtained with chronic Daphnia data (r2= 0.66 and 0.65 for MOA 1 and 3 respectively). When considering acute fish data, all the MOA classes give strong relationships (r2=0.88 for MOA 3 and MOA 5 chemicals, 0.85 for MOA 4 chemicals and 0.83 for MOA 1 and MOA 2 chemicals). Therefore when acute toxicity data on fish are available, they might give a reliable basis to extrapolate the chronic toxicity on fish as a first tier assessment or within a weight of evidence approach. There is a correlation between chemicals with high ACR values or interspecies sensitivity ratios and the outliers identified in the above-mentioned relationships. When considering chemicals with a high interspecies sensitivity ratio, Daphnia being more sensitive than fish, several aniline derivatives and pesticides acting through cholinesterase inhibition were identified. When considering high interspecies sensitivity ratio chemicals for which Daphnia is less sensitive than fish, we found pesticides and known endocrine disruptors such as ethynil oestradiol and 17ß-oestradiol. Extreme (i.e. 100) interspecies sensitivity ratios were mainly evident for MOA 2, 4 and 5 chemicals. Regarding ACR for fish, around 50% of the chemicals in each MOA class have an ACR within a factor of 10; whereas 100% of MOA 3, 90.9% of MOA 2, 88.3% of MOA 4 and 85.5% of MOA 1 chemicals have an ACR within a factor of 100. Therefore, the safety factor of 100 commonly applied in environmental risk assessment does not seem to be equally protective for every MOA.JRC.I.5-Systems Toxicolog

Expert survey on identification of gaps in available test methods for evaluation of endocrine disruptors

According to the 2012 WHO/UNEP publication 'State of the Science of Endocrine Disrupting Chemicals' research into endocrine disrupting chemicals over the last decade has indicated that, despite the progress achieved in development and validation of test methods for evaluation of endocrine disruptors, there are still several gaps that need to be addressed. Considering the expected significant amount of work needed to fill the gaps and the limited resources available, it will be important to set priorities for the upcoming period (next 5-10 years) for the development and validation of test methods. Thus there is a need to focus the European input to the OECD test guideline programme to effectively enhance the identification of chemical substances with endocrine disrupting properties whilst making best use of existing resources. With this objective in mind, DG Environment, supported by JRC, is organising a European expert workshop on setting priorities for further development and validation of test methods for evaluating endocrine disruption. The workshop will take place on 30 May - 01 June 2017 in Brussels. The deliberations will focus on what is necessary and achievable in the context of resources, timescales and animal welfare considerations. In preparation for the workshop, JRC has drawn up a questionnaire to gather input from experts in the field on key issues to be used as a basis for the further discussions at the workshop. An online survey with the title "Identifying gaps in available test methods for evaluation of endocrine disruptors" was performed on the EU Survey platform and open for commenting from 19/05/2015 until 15/06/2015. A selected group of experts (EFSA Scientific Committee and WG on EDs, ECHA ED WG and RAC, WNT (European members from OECD webpage), Experts identified in Annex 3 of the "Roadmap for setting priorities for further development and validation of test methods and testing approaches for evaluating endocrine disruptors") was invited to participate in the survey. Experts were asked to rank endocrine related diseases/disorders regarding the possibility to predict them with existing test methods (TMs). They were further asked to rank diseases/disorders regarding the need to develop new test methods to better cover those. Experts were then requested to provide their views on including further tests based on those discussed in the OECD (2012) "Detailed Review Paper on the state of the science on novel in vitro and in vivo screening and testing methods and endpoints for evaluating endocrine disruptors" and their views on the current OECD Conceptual Framework and proposals for improvements. Forty experts representing 15 countries and different stakeholder groups (authorities; academia; civil society organisation; industry) replied. The purpose of this report is to present the detailed survey results. Multiple choice questions were evaluated and where possible quantitative rankings were performed. In addition, the survey respondents provided a lot of valuable information in numerous free text comments. Those are included in the report in tables as they were received, without editing them, unless personal information had to be removed. Brief summaries of the main points raised are added after each section.JRC.F.3-Chemicals Safety and Alternative Method

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

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

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 (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