Bruno Hubesch

A European perspective on alternatives to animal testing for environmental hazard identification and risk assessment

Tests with vertebrates are an integral part of environmental hazard identification and risk assessment of chemicals, plant protection products, pharmaceuticals, biocides, feed additives and effluents. These tests raise ethical and economic concerns and are considered as inappropriate for assessing all of the substances and effluents that require regulatory testing. Hence, there is a strong demand for replacement, reduction and refinement strategies and methods. However, until now alternative approaches have only rarely been used in regulatory settings. This review provides an overview on current regulations of chemicals and the requirements for animal tests in environmental hazard and risk assessment. It aims to highlight the potential areas for alternative approaches in environmental hazard identification and risk assessment. Perspectives and limitations of alternative approaches to animal tests using vertebrates in environmental toxicology, i.e. mainly fish and amphibians, are discussed. Free access to existing (proprietary) animal test data, availability of validated alternative methods and a practical implementation of conceptual approaches such as the Adverse Outcome Pathways and Integrated Testing Strategies were identified as major requirements towards the successful development and implementation of alternative approaches. Although this article focusses on European regulations, its considerations and conclusions are of global relevance.

Skin sensitisation – Moving forward with non-animal testing strategies for regulatory purposes in the EU

In a previous EPAA-Cefic LRI workshop in 2011, issues surrounding the use and interpretation of results from the local lymph node assay were addressed. At the beginning of 2013 a second joint workshop focused greater attention on the opportunities to make use of non-animal test data, not least since a number of in vitro assays have progressed to an advanced position in terms of their formal validation. It is already recognised that information produced from non-animal assays can be used in regulatory decision-making, notably in terms of classifying a substance as a skin sensitiser. The evolution into a full replacement for hazard identification, where the decision is not to classify, requires the generation of confidence in the in vitro alternative, e.g. via formal validation, the existence of peer reviewed publications and the knowledge that the assay(s) are founded on key elements of the Adverse Outcome Pathway for skin sensitisation. It is foreseen that the validated in vitro assays and relevant QSAR models can be organised into formal testing strategies to be applied for regulatory purposes by the industry. To facilitate progress, the European Partnership for Alternative Approaches to animal testing (EPAA) provided the platform for cross-industry and regulatory dialogue, enabling an essential and open debate on the acceptability of an in vitro based integrated strategy. Based on these considerations, a follow up activity was agreed upon to explore an example of an Integrated Testing Strategy for skin sensitisation hazard identification purposes in the context of REACH submissions.

Alternatives for skin sensitisation: Hazard identification and potency categorisation: Report from an EPAA/CEFIC LRI/Cosmetics Europe cross sector workshop, ECHA Helsinki, April 23rd and 24th 2015.

In the two years since the last workshop report, the environment surrounding the prediction of skin sensitisation hazards has experienced major change. Validated non-animal tests are now OECD Test Guidelines. Accordingly, the recent cross sector workshop focused on how to use in vitro data for regulatory decision-making. After a review of general approaches and six case studies, there was broad consensus that a simple, transparent stepwise process involving non-animal methods was an opportunity waiting to be seized. There was also strong feeling the approach should not be so rigidly defined that assay variations/additional tests are locked out. Neither should it preclude more complex integrated approaches being used for other purposes, e.g. potency estimation. All agreed the ultimate goal is a high level of protection of human health. Thus, experience in the population will be the final arbiter of whether toxicological predictions are fit for purpose. Central to this is the reflection that none of the existing animal assays is perfect; the non-animal methods should not be expected to be so either, but by integrated use of methods and all other relevant information, including clinical feedback, we have the opportunity to continue to improve toxicology whilst avoiding animal use.

Optimised testing strategies for skin sensitization--the LLNA and beyond.

As toxicology in the 21st century progresses towards a future which aims at avoiding the use of in vivo testing, the endpoint of skin sensitisation can now be found in the front line. Accordingly, it was appropriate for several industry sectors to meet and review what has been learned from the currently most widely used in vivo method, the local lymph node assay (LLNA), and to consider the status of progress as we attempt to move beyond that test. No toxicology test is perfect, an experience brought into focus by issues of false positives and, to a lesser extent, false negatives in the LLNA. Use of weight of evidence arguments for classification and labelling, as well as for risk assessment was emphasised and it was also noted that a sufficient body of evidence now exists for conduct of methods other than the LLNA for carefully defined chemical classes. In terms of in vitro alternatives, progress towards methods which will deliver mainly hazard identification is being made, with some entering the final stages of validation, whereby (Q)SAR tools still need improvement to be used on a large scale in practise. As various other challenges also remain, e.g. testing lipophilic substances, as well as the development of non-animal methods which deliver reliable information on potency for risk assessment, these will remain a topic for continuing research and development.

Two Good Read-across Practice workshops. Making it work for you!

324 neous physical-chemical properties that may change with a regular trend within the category, but it is not sufficient for a proper justification. For some simple homologue substances similarity may be straightforward, but with more complex molecules or isomers this is not trivial. In fact, one of the main arguments against read-across is the presence of activity cliffs: Many chemicals that are identical in terms of 2but not 3-dimensional structure – thalidomide being a well-known example – markedly differ in terms of bioactivity. Therefore, the grouping of similar molecules has to be based on more than just chemically similar molecules. Biological similarity approaches can take many forms, i.e., using several bioassays to identify common molecular targets of toxicity, narrowing the applicability domain to identify areas of “local validity,” or identifying Adverse Outcome Pathways (AOP) or Pathways of Toxicity (PoT). The AOP is a mechanistically-based approach that may explain the similarity in the biological behavior of two or more substances with the complement of pharmacokinetics considerations that should consider metabolism, distribution in the organism and kinetics of the excretion. Information on substance metabolism, in particular, is central to supporting similarity between two or more substances for read-across purposes as metabolism may determine a biological difference between two substances that look similar from a chemical point of view. However, this information is not often available or the biological data often lacks standardization, preventing good automatic comparison. The definition of the principle and the format for justifying and presenting the data in read-across is not enough and users need suitable tools to exploit the read-across opportunity on a strong scientific basis, including clear applicability domain, robust statistical evaluation plus transparent and objective outcome. The availability of large quantities of data and test results acquired for chemicals whose structure and physical chemical properties are well defined is a fundamental basis for a robust statistical evaluation and feed the read-across approach. In this sense, the public access of the REACH registration dossiers on the ECHA website represents a tremendous opportunity with 14,000 registration dossiers that contain relevant chemicals assessment data (http://www.echa.europa. Read-across is an innovative approach that can be considered an alternative to animal testing – and at the moment it is probably the most effective method of reducing the use of lab animals. In fact, the latest ECHA report on the use of alternative methods revealed that 85% of REACH registration dossiers waived in vivo test requirements by using the readacross option (ECHA, 2014). However, the applicability of the read-across principle goes far beyond REACH, and experience gained in this field will help to clarify what “Good Read-across Practice” involves (Ball et al., 2016; Zhu et al., 2016). Following recent publications on read-across (Hartung, 2016; Luechtefeld et al., 2016a-d), CAAT-Europe in collaboration with the integrated project EU-ToxRisk (www. eu-toxrisk.eu) and CEFIC-LRI (European Chemical Industry Council – Long Range Research Initiative, http://www. cefic-lri.org) convened a workshop to learn the opinions of main stakeholders in the field. This workshop took place in Brussels on February 26, 2016. About 100 individuals, representing toxicologists, industry, regulators, academia and other associations, took part. Full details and copies of the presentations are available at http://bit.ly/25NqtFf. A further workshop was held in March 2016 at the U.S. Food and Drug Administration in College Park, MD, also well attended by representatives from academia, regulators and industry, and webcasted to many more. In 2015, the European Chemicals Agency (ECHA) published a document showing how to present a read-across strategy (RAAF, Read Across Assessment Framework; ECHA, 2015). While this document does not claim to demonstrate the scientific basis of the read-across principle, it does explain how to present data to regulators through a robust scientific justification. It considers two cases: i) different substances that give rise to (the same) common compounds to which the organism is exposed (biotic or abiotic transformation to common compounds) or ii) different compounds that cause the same type of effects on the organism as a result of structural similarity. This means that the justification for a read-across or category approach is the result of a complex assessment of both the chemical structure and biological behavior of the substances; chemical similarity is the basis, with homogeWorkshop report