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

Background

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 introduces a challenge for industry, the regulatory agencies and the toxicological research community to develop and validate alternative high-throughput testing strategies and limit the testing with animals by implementing the refinement, reduction and replacement (the 3Rs) principle in ecotoxicological testing.

This applies in particular to the use of aquatic vertebrates such as fish, which is regulated under the animal research and welfare legislation.

Project

The Norwegian Institute for Water research (NIVA) is managing a Norwegian Research Council (NRC) funded project “Non-animal (alternative) testing methods for REACH (alterREACH)” that ran from 2010 to 2016.

Objectives

The aim of the project was to develop, evaluate and use non-animal (alternative) test methods for performing robust, reproducible and rapid screening of chemicals for their bioaccumulation and toxic potential without excessive use of experimental animals.

These objectives were achieved through use of in vitro experimental systems for studying the bioaccumulation and toxic potency in fish in combination with computational (in silico) assessment of chemical properties.

Work packages

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 proposed work was divided into three work packages (WPs) where data and experiences were shared between WPs (Fig 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, WP 1 has provided a list of reference compounds to be used in the assessment of alternative test methods for bioaccumulation and some of these chemicals have been assessed in different in vitro methods using fish enzymes and fish cells. The reference compounds were selected based on their broad coverage of physico-chemical properties, toxic mode of action (MOA) and being representative for high-production industrial compounds. 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.

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 tests). Work package 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 different effect and biomarker methods (both novel and already established assays) for testing a number of substances for potential identification of their MOA to provide links between their acute and chronic toxicity. 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 devices based on principles of passive dosing. The work has resulted in the publication of two articles in reputable journals. 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.

Comprehensive assessment of how various alternative methods can be used to reduce the use of fish in toxicological experiments is important to uncover future development needs and prioritize research. Work package 3 has 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 (www.niva.no/radb) to assist 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 a scientific journal and work is expected to be continue in the coming years.

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 popular press. Several scientific works are in the final phases of development and is expected to increase the dissemination activities from the project in the coming years.