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

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

Scientific methodologies for the assessment of combined effects of chemicals - a survey and literature review

Exposure of humans and wildlife to chemicals via food, consumer products, the environment etc. can imply exposure to an infinite number of different combinations of chemicals in mixtures. It is practically impossible to test all these possible mixtures experimentally and it is therefore needed to find smart strategies to assess the potential hazards using new tools that rely less on in vivo testing and incorporate instead alternative experimental and computational tools. In this report the current state of the art for the application of these alternative tools for assessing the hazard of chemical mixtures is briefly reviewed. The focus is hereby on the adverse outcome pathway (AOP) concept, in vitro methods, omics techniques, in silico approaches such as quantitative structure activity relationships (QSARs) and read-across, toxicokinetic and dynamic energy budget (DEB) modelling, and on integrated approaches to testing and assessment (IATA). Furthermore, an expert survey was performed to collect up to date information and experience on the current use of different approaches for assessing human and environmental health risks from exposure to chemical mixtures, with a view to informing the development of a consistent assessment approach. An online survey was performed among experts in the field of combined exposure assessment in the period of January to March 2014, addressing both, human health and environmental risk assessment. Fifty-eight experts from 21 countries, different stakeholder groups and sectors of legislation participated in the survey. The main sectors where most experience is already gained in assessing mixtures are in the area of plant protection products and chemicals. These were also rated highest regarding the priority for performing mixture assessments. Experts have experience with the whole mixture as well as the component-based approaches applying them to both, intentional and unintentional mixtures. Mostly concentration addition (CA) based methods are used for predicting mixture effects. Regarding the use of novel and alternative tools in the risk assessment of mixtures, expert opinions are split between those applying them (often more in a research context) and those that generally think these tools are valuable but their use is currently limited because of lack of guidance, lack of data, or lack of expertise. A general need for clear guidance for combined exposure assessments was identified. Overall, a high potential in applying novel tools and scientific methodologies for the assessment of chemical mixtures can be identified. They allow deriving meaningful information on individual mixture components or whole mixtures, enabling a better understanding of the underlying mechanisms of mixture effects. Their main strengths lie in their integrated use and smart combination to put different aspects regarding the hazard from combined exposure to multiple chemicals into context. In order to benefit from these tools in the hazard assessment of mixtures, more guidance on their use is needed to facilitate a more widespread application.JRC.I.5-Systems Toxicolog

Regulatory assessment of chemical mixtures: Requirements, current approaches and future perspectives

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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

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