FORURENS: Non-animal (alternative) testing methods for REACH

The project alterREACH has in the period 2010-2016 conducted studies to develop and evaluate alternatives to animal studies. This work has led to the development and use of computer-assisted predictive models and experimental methods with fish cells as alternatives to fish studies in order to identify toxic substances, assess their bioaccumulation potential, and perform extrapolation of in vitro responses to in vivo effects in fish. The Project has fully financed one PhD student and partly funded another PhD student. Both candidates have graduated and their scientific achievements are published in reputable international scientific journals. The project was divided into three different work packages described in detail below. Work package 1: biological tests to quantify the potential for bioaccumulation of organic compounds from water to fish is often conducted with intact fish. As contribution to the 3Rs (improvement, reduction or replacement), work package 1 has provided a list of reference compounds to be used in the assessment of alternative test methods and some of these chemicals have bene tested in different in vitro methods using fish enzymes and fish cells. The work is a key part of the PhD scholar Neus Rodriguez Sanchez work at Liverpool John Moores University (United Kingdom), which was completed and led to a PhD in December 2015. Work package 2: Testing the toxicity potential of various chemical substances involves the use of fish in short-term studies (acute tests) and in long-term studies (chronic test). Project 2 (WP2) has developed and applied alternative test methods to measure the acute toxicity, dioxin-like toxicity and endocrine disruption in two different cell cultures from salmonids. This work has resulted in optimization, evaluation and use of the methods using different effect and biomarker methods for testing a number of substances for potential identification of the mode of action (MOA) of these. The work has revealed that the methods appear satisfactory for various toxic endpoints, but the dosage of chemicals with low water solubility is challenging in such test systems, and thus led to the establishment of specialized dosing device based on principles of passive dosing. The work has resulted in the publication of two articles in reputable journals, submission of two articles for publication, and the preparation of 3 manuscripts for publication in 2017 as a collaborative effort with other initiatives. The work was a key part of the PhD thesis of Maria Hultman at the Norwegian University of Life Sciences (NMBU) and she was awarded a PhD degree in July 2016. Work package 3: comprehensive assessment of how various alternative methods can be used to reduce the use of fish in experiments is important to uncover future development needs and prioritize research. Subproject 3 have contributed to this process by evaluating existing methods, contributed to the practical evaluation of standardized in vitro methods for the testing of acute toxicity and bioaccumulation of organic pollutants. The work package has also helped to develop a database to organize various effect data from in vitro and in vivo methods in order to carry out in vitro in vivo extrapolations, support comparison across substances and methods (read-across) and to help develop predictive methods for acute and chronic toxicity. The work has resulted in the publication of one article in scientific journals and is expected to continue in other/new projects in the coming years. An update in alternative test methods and establishment of new approaches is expected developed after completion of the project. The project has resulted in the 28 presentations (oral and posters) at scientific meetings, publication of 7 articles in reputable scientific journals, one honorary award for work on the development of alternative test development in regulatory research and 2 popular scientific contributions in the press (see www.niva.no/alterREACH for details). Several scientific works are in the final phase and is expected to increase the dissemination activities from the project in the coming years.

The Registration, Evaluation, Authorisation and Restriction of Chemical substance (REACH) is the new European Community Regulation on chemicals and their safe use. Estimates indicate that as much as 30,000 single chemicals may be required to be registered with a potentially risk assessment requirement based on substance-specific data for the persistence, bioaccumulation and toxic (PBT) properties for several thousand chemicals. This introduce a challenge for industry, the regulatory agencies and the toxic ological research community to develop and validate alternative high-throughput testing strategies and limit the testing with animals by implementing the reduction, refinement and replacement (the 3Rs) principle in ecotoxicological testing. This applies i n particular to the use of aquatic vertebrates such as fish, which under the legislation . This proposal seeks to provide theoretical and experimental data to develop and evaluate alternative ecotoxicological test methods for fish in risk assessment and to provide input to risk regulators, industry and academia on the applicability of the different approaches to assess the PBT properties of chemicals. The proposed work will be performed through use of a combination between computational (in silico) and experimental (in vitro, in vivo screening) investigations with comparison with existing in vivo testing data. The project is multi-disciplinary and highly integrated with partners from key environmental institutes in Germany, United Kingdom, Switzerlan d and Norway will and the project will be lead by the Norwegian Institute for Water Research (NIVA). The proposed project will support both research by established research groups as well as education of one PhD and 3 Ms. Sc. student to be working in the different collaborating laboratories.