21st Century Approaches for Evaluating Exposures, Biological Activity, and Risks of Complex Substances: Workshop highlights.

The June 2019 workshop 21st Century Approaches for Evaluating Exposures, Biological Activity, and Risks of Complex Substances, co-organised by the International Council of Chemical Association's Long-Range Research Initiative and the European Commission's Joint Research Centre, is summarised. Focus was the need for improved approaches to evaluate the safety of complex substances. Approximately 10% and 20% of substances registered under the EU chemicals legislation are 'multi-constituent substances' and 'substances of unknown or variable compositions, complex reaction products and biological substances' (UVCBs), respectively, and UVCBs comprise approximately 25% of the U.S. Toxic Substances Control Act Inventory. Workshop participants were asked to consider how the full promise of new approach methodologies (NAMs) could be brought to bear to evaluate complex substances. Sessions focused on using NAMs for screening, biological profiling, and in complex risk evaluations; improving read-across approaches employing new data streams; and methods to evaluate exposure and dosimetry. The workshop concluded with facilitated discussions to explore actionable steps forward. Given the diversity of complex substances, no single 'correct' approach was seen as workable. The path forward should focus on 'learning by doing' by developing and openly sharing NAM-based fit-for-purpose case examples for evaluating biological activity, exposures and risks of complex substances.

Applying non-animal strategies for assessing skin sensitisation report from an EPAA/cefic-LRI/IFRA Europe cross sector workshop, ECHA helsinki, February 7th and 8th 2019.

Four years on since the last cross sector workshop, experience of the practical application and interpretation of several non-animal assays that contribute to the predictive identification of skin sensitisers has begun to accumulate. Non-animal methods used for hazard assessments increasingly are contributing to the potency sub-categorisation for regulatory purposes. However, workshop participants generally supported the view that there remained a pressing need to build confidence in how information from multiple methods can be combined for classification, sub-categorisation and potency assessment. Furthermore, the practical experience gained over the last few years, highlighted the overall high potential value of using the newly validated methods and testing strategies, but also that limitations for certain substance/product classes may become evident with further use as had been the case with other new regulatory methods. As the available information increases, review of the data and collated experience could further determine strengths and limitations leading to more confidence in their use. Finally, the need for a substantial and universally accepted dataset of non-sensitisers and substances of different sensitising potencies, based on combined human and in vivo animal data for validation of methods and test strategies was re-emphasised.

Finding synergies for the 3Rs - Repeated Dose Toxicity testing: Report from an EPAA Partners' Forum.

The European Partnership for Alternative Approaches to Animal Testing (EPAA) convened a Partners' Forum on repeated dose toxicity (RDT) testing to identify synergies between industrial sectors and stakeholders along with opportunities to progress these in existing research frameworks. Although RTD testing is not performed across all industrial sectors, the OECD accepted tests can provide a rich source of information and play a pivotal role for safety decisions relating to the use of chemicals. Currently there are no validated alternatives to repeated dose testing and a direct one-to-one replacement is not appropriate. However, there are many projects and initiatives at the international level which aim to implement various aspects of replacement, reduction and refinement (the 3Rs) in RDT testing. Improved definition of use, through better problem formulation, aligned to harmonisation of regulations is a key area, as is the more rapid implementation of alternatives into the legislative framework. Existing test designs can be optimised to reduce animal use and increase information content. Greater use of exposure-led decisions and improvements in dose selection will be beneficial. In addition, EPAA facilitates sharing of case studies demonstrating the use of Next Generation Risk Assessment applying various New Approach Methodologies to assess RDT.

Finding synergies for 3Rs - Toxicokinetics and read-across: Report from an EPAA partners' Forum.

The European Partnership for Alternative Approaches to Animal Testing (EPAA) convened a Partners' Forum Toxicokinetics and Read-Across to provide an overview on research activities to develop in vitro toxicokinetics methods and physiologically-based kinetic (PBK) models and to find synergies to enhance use of toxicokinetic data to strengthen read-across. Currently, lacking toxicokinetic data often prevent the application of read-across. Preferably, toxicokinetic data should be generated using in vitro and in silico tools and anchored towards human relevance. In certain sectors, PBK modelling is being used for risk assessment, but less so in others. Specific activities were identified to facilitate the use of in vitro and in silico toxicokinetic data to support read-across: The collation of available tools indicating the parameters and applicability domains covered; endpoint-specific guidance on toxicokinetics parameters required for read-across; case studies exemplifying how toxicokinetic data help support read-across. Activities to enhance the scientific robustness of read-across include the further user-friendly combination of read-across tools and formal guidance by the authorities specifying the minimum information requirements to justify read-across for a given toxicity endpoint. The EPAA was invited to continue dissemination activities and to explore possibilities to collate a contemporaneous list of open toxicokinetics tools that assist risk assessment.

Applying 'omics technologies in chemicals risk assessment: Report of an ECETOC workshop.

Prevailing knowledge gaps in linking specific molecular changes to apical outcomes and methodological uncertainties in the generation, storage, processing, and interpretation of 'omics data limit the application of 'omics technologies in regulatory toxicology. Against this background, the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) convened a workshop Applying 'omics technologies in chemicals risk assessment that is reported herein. Ahead of the workshop, multi-expert teams drafted frameworks on best practices for (i) a Good-Laboratory Practice-like context for collecting, storing and curating 'omics data; (ii) the processing of 'omics data; and (iii) weight-of-evidence approaches for integrating 'omics data. The workshop participants confirmed the relevance of these Frameworks to facilitate the regulatory applicability and use of 'omics data, and the workshop discussions provided input for their further elaboration. Additionally, the key objective (iv) to establish approaches to connect 'omics perturbations to phenotypic alterations was addressed. Generally, it was considered promising to strive to link gene expression changes and pathway perturbations to the phenotype by mapping them to specific adverse outcome pathways. While further work is necessary before gene expression changes can be used to establish safe levels of substance exposure, the ECETOC workshop provided important incentives towards achieving this goal.

Advancing the use of noncoding RNA in regulatory toxicology: Report of an ECETOC workshop.

The European Centre for the Ecotoxicology and Toxicology of Chemicals (ECETOC) organised a workshop to discuss the state-of-the-art research on noncoding RNAs (ncRNAs) as biomarkers in regulatory toxicology and as analytical and therapeutic agents. There was agreement that ncRNA expression profiling data requires careful evaluation to determine the utility of specific ncRNAs as biomarkers. To advance the use of ncRNA in regulatory toxicology, the following research priorities were identified: (1) Conduct comprehensive literature reviews to identify possibly suitable ncRNAs and areas of toxicology where ncRNA expression profiling could address prevailing scientific deficiencies. (2) Develop consensus on how to conduct ncRNA expression profiling in a toxicological context. (3) Conduct experimental projects, including, e.g., rat (90-day) oral toxicity studies, to evaluate the toxicological relevance of the expression profiles of selected ncRNAs. Thereby, physiological ncRNA expression profiles should be established, including the biological variability of healthy individuals. To substantiate the relevance of key ncRNAs for cell homeostasis or pathogenesis, molecular events should be dose-dependently linked with substance-induced apical effects. Applying a holistic approach, knowledge on ncRNAs, 'omics and epigenetics technologies should be integrated into adverse outcome pathways to improve the understanding of the functional roles of ncRNAs within a regulatory context.

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.

Workshop: use of "read-across" for chemical safety assessment under REACH.

Read-across has generated much attention, since it may be used as an alternative approach for addressing the information requirements under REACH. Experience in the application of "read-across" has undoubtedly been gained within the context of the 2010 registrations (>1000 tonnes/annum). Industry, European Chemicals Agency (ECHA) and EU Member States all conceptually accept read-across approaches but difficulties still remain in applying them consistently in practice. A workshop on the 'Use of Read-Across for Chemical Safety Assessment under REACH', organised by ECHA with the active support of Cefic LRI was held on the 3rd October 2012 to gain insight on how ECHA evaluates read-across justifications, to share Industry experiences with read-across approaches and to discuss practical strategies to help develop scientifically valid read-across for future submissions.

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.

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.

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.

Internationalization of read-across as a validated new approach method (NAM) for regulatory toxicology.

Read-across (RAx) translates available information from well-characterized chemicals to a substance for which there is a toxicological data gap. The OECD is working on case studies to probe general applicability of RAx, and several regulations (e.g., EU-REACH) already allow this procedure to be used to waive new in vivo tests. The decision to prepare a review on the state of the art of RAx as a tool for risk assessment for regulatory purposes was taken during a workshop with international experts in Ranco, Italy in July 2018. Three major issues were identified that need optimization to allow a higher regulatory acceptance rate of the RAx procedure: (i) the definition of similarity of source and target, (ii) the translation of biological/toxicological activity of source to target in the RAx procedure, and (iii) how to deal with issues of ADME that may differ between source and target. The use of new approach methodologies (NAM) was discussed as one of the most important innovations to improve the acceptability of RAx. At present, NAM data may be used to confirm chemical and toxicological similarity. In the future, the use of NAM may be broadened to fully characterize the hazard and toxicokinetic properties of RAx compounds. Concerning available guidance, documents on Good Read-Across Practice (GRAP) and on best practices to perform and evaluate the RAx process were identified. Here, in particular, the RAx guidance, being worked out by the European Commission's H2020 project EU-ToxRisk together with many external partners with regulatory experience, is given.

A mode-of-action ontology model for safety evaluation of chemicals: Outcome of a series of workshops on repeated dose toxicity.

Repeated dose toxicity evaluation aims at assessing the occurrence of adverse effects following chronic or repeated exposure to chemicals. Non-animal approaches have gained importance in the last decades because of ethical considerations as well as due to scientific reasons calling for more human-based strategies. A critical aspect of this challenge is linked to the capacity to cover a comprehensive set of interdependent mechanisms of action, link them to adverse effects and interpret their probability to be triggered in the light of the exposure at the (sub)cellular level. Inherent to its structured nature, an ontology addressing repeated dose toxicity could be a scientific and transparent way to achieve this goal. Additionally, repeated dose toxicity evaluation through the use of a harmonized ontology should be performed in a reproducible and consistent manner, while mimicking as accurately as possible human physiology and adaptivity. In this paper, the outcome of a series of workshops organized by Cosmetics Europe on this topic is reported. As such, this manuscript shows how experts set critical elements and ways of establishing a mode-of-action ontology model as a support to risk assessors aiming to perform animal-free safety evaluation of chemicals based on repeated dose toxicity data.

Integrated Testing Strategies (ITS) for safety assessment.

Integrated testing strategies (ITS), as opposed to single definitive tests or fixed batteries of tests, are expected to efficiently combine different information sources in a quantifiable fashion to satisfy an information need, in this case for regulatory safety assessments. With increasing awareness of the limitations of each individual tool and the development of highly targeted tests and predictions, the need for combining pieces of evidence increases. The discussions that took place during this workshop, which brought together a group of experts coming from different related areas, illustrate the current state of the art of ITS, as well as promising developments and identifiable challenges. The case of skin sensitization was taken as an example to understand how possible ITS can be constructed, optimized and validated. This will require embracing and developing new concepts such as adverse outcome pathways (AOP), advanced statistical learning algorithms and machine learning, mechanistic validation and "Good ITS Practices".