Toxicity Pathways – from concepts to application in chemical safety assessment

Few would deny that the NRC report (NRC, 2007), "Toxicity Testing in the 21st Century: A Vision and Strategy”, represented a re-orientation of thinking surrounding the risk assessment of environmental chemicals. The key take-home message was that by understanding Toxicity Pathways (TP) we could profile the potential hazard and assess risks to humans and the environment using intelligent combinations of computational and in vitro methods. In theory at least, shifting to this new paradigm promises more efficient, comprehensive and cost effective testing strategies for every chemical in commerce while minimising the use of animals. For those of us who embrace the vision and the strategy proposed to achieve it, attention has increasingly focused on how we can actually practice what we preach. For a start, 21st century concepts described in the report have to be carefully interpreted and then translated into processes that essentially define and operationalize a TP framework for chemical risk assessment. In September 2011 the European Commission's Joint Research Centre (JRC) and the Hamner Institutes for Health Sciences co-organised a "Toxicity Pathways" workshop. It was hosted by the JRC and took place in Ispra, Italy. There were 23 invited participants with more or less equal representation from Europe and North America. The purpose of the meeting was to address three key questions surrounding a TP based approach to chemical risk assessment, namely – What constitutes a TP? How can we use TPs to develop in vitro assays and testing strategies? And, How can the results from TP testing be used in human health risk assessments? The meeting ran over two days and comprised a series of thought-starter presentations, breakout sessions and plenty of group discussions. The outcome was captured by rapporteurs and compiled as a workshop report which is available for download (without charge) from the JRC website. Here we expand on selected deliberations of the workshop to illustrate how TP thinking is still evolving and to indicate what pieces of the puzzle still need to fall into place before TP based risk assessment can become a reality.JRC.I.5-Systems Toxicolog

EURL ECVAM Recommendation on the Direct Peptide Reactivity Assay (DPRA) for Skin Sensitisation Testing

Identification of the skin sensitisation hazard of chemicals has traditionally relied on the use of animals. Progress in the development of alternative methods has been prompted by the increasing knowledge of the key biological mechanisms underlying this human health effect, as documented by the OECD's recent report summarising the key biological events leading to skin sensitisation ("Adverse Outcome Pathway" (AOP) for skin sensitisation). The molecular initiating event defined within this AOP is the covalent binding of chemicals with skin proteins. Thus peptide reactivity assays may provide valuable information in the context of integrated approaches such as Weight of Evidence (WoE) or Integrated Testing Strategies (ITS) for skin sensitisation hazard and safety assessment. Based on these considerations, EURL ECVAM coordinated a validation study on the Direct Peptide Reactivity Assay (DPRA) addressing mainly the test method’s transferability and within- and between-laboratory reproducibility. Following independent scientific peer review by the EURL ECVAM’s Scientific Advisory Committee (ESAC) and having considered the input from regulators, stakeholders, international partners and the general public, EURL ECVAM concluded that the DPRA may prove a valuable component of a WoE or ITS for skin sensitisation hazard assessment. In addition to this, the DPRA may also be able to contribute to the assessment of sensitising potency, e.g. by supporting sub-categorisation of sensitisers according to UN GHS. However it is recognised that further efforts are required to explore how DPRA data may contribute to potency assessmentJRC.I.5-Systems Toxicolog

A Simple Phase-Shifting Lateral Shearing Interferometer

A phase-shifting electronic speckle pattern shearing interferometer with a very simple shearing device is proposed. Two partially reflective glass plates are used to introduce the shear in this new interferometer. The reflection coefficients of the coatings on the two plates are 0.3 and 0.7. The distance between the two glass plates controls the size of the shear. The proposed new interferometric system is simple, flexible, and low cost

Bridging Across Methods in the Biosciences

Cross-disciplinary research is essential if science is to properly address societal needs. In spite of several policy initiatives to foster such research across sectors, there is still a high level of compartmentalisation in the biosciences. The European Commission is preparing for a Missions based science and innovation strategy in which it will be important to consider how the goal of meaningful cross-disciplinarity can be achieved. This report aims to raise the question of cross-disciplinarity again, and to suggest specific actions to further the understanding, achievement and evaluation of cross-disciplinarity in the biosciences.JRC.F.3-Chemicals Safety and Alternative Method

EURL ECVAM strategy to avoid and reduce animal use in genotoxicity testing

The assessment of genotoxicity represents an important component of the safety assessment of all types of substances. Although several in vitro tests are available at different stages of development and acceptance, they cannot at present be considered to fully replace animal tests needed to evaluate the safety of substances. Based on an analysis of regulatory requirements for this endpoint within different pieces of EU legislation, EURL ECVAM proposes a pragmatic approach to improve the traditional genotoxicity testing paradigm that offers solutions in both the short- and medium-term and that draws on the considerable experience of 40 years of regulatory toxicology testing in this area. EURL ECVAM considers that efforts should be directed towards the overall improvement of the current testing strategy for better hazard and risk assessment approaches, which either avoids or minimises the use of animals, whilst satisfying regulatory information requirements, irrespective of regulatory context. Several opportunities for the improvement of the testing strategy have been identified which aim to i) enhance the performance of the in vitro testing battery so that fewer in vivo follow-up tests are necessary and ii) guide more intelligent in vivo follow-up testing to reduce unnecessary use of animals. The implementation of this strategic plan will rely on the cooperation of EURL ECVAM with other existing initiatives and the coordinated contribution from various stakeholders.JRC.I.5-Systems Toxicolog

An investigation into the use of computational and in vitro methods for acute systemic toxicity prediction

We have assessed the abilities of five alternative (non-animal) approaches to predict acute oral toxicity, a toxicological endpoint relevant to multiple pieces of legislation on chemicals and consumer products. In particular, we have investigated four QSAR models (ToxSuite, TOPKAT, TEST and ADMET Predictor) and one in vitro method (3T3 NRU). Based on a test set of in vitro and in vivo data for 180 compounds, we have characterized the predictive performance of each method when used alone (both for LD50 prediction and acute toxicity classification into three categories), as well as multiple test combinations (batteries) and stepwise testing strategies (for acute toxicity classification into three categories). When used individually, the alternative methods showed an ability to predict LD50 with correlation coefficients in the range from 49% to 84%, and to classify into three toxicity groups with accuracies in the range from 41% to 72%. When the alternative methods were combined into batteries or testing strategies, the overall accuracy of prediction could reach 76%. We also illustrate how different combinations of methods can be used to optimize sensitivity or specificity.JRC.I.5-Systems Toxicolog

Enrichment of hepatocytes in a HepaRG culture using spatially selective photodynamic treatment

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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

Description of Prototype Modes-of-Action Related to Repeated Dose Toxicity

This report presents the definition and detailed documentation of chosen toxicological MoAs associated with repeated dose target organ toxicity as a first step in building a "prototype" safety assessment framework. In addition to providing a detailed description of the two chosen MoAs related to chronic liver toxicity, namely "MoA from Protein Alkylation to Liver Fibrosis" and "MoA from Liver X Receptor Activation to Liver Steatosis", the report also describes the working process leading to this result including the problems that have been encountered. The exercise followed as far as possible relevant WHO-IPCS and OECD guidance. The report represents the first deliverable of a contract of work between Cosmetics Europe and the European Commission's Joint Research Centre for supplementing the work of the SEURAT-1 research cluster.JRC.I.5-Systems Toxicolog

EURL ECVAM Recommendation on the Cell Transformation Assay based on the Bhas 42 cell line

The carcinogenic potential of compounds is a crucial aspect in human hazard and risk assessment of substances. Among the various alternatives developed for carcinogenicity prediction, the cell transformation assays (CTAs) have been shown to closely model some key stages of the in vivo carcinogenesis process. Similar to previously validated in vitro CTAs, the CTA in Bhas 42 cells aims at predicting carcinogenic potential. Based on the results of a validation study coordinated by Hadano Research Institute (HRI) Food and Drug Safety Center (FDSC) and other published data, the Bhas 42 CTA protocol (including the 6-well and 96-well plate versions) was considered to be sufficiently standardised, transferable, reproducible between laboratories and relevant to support the identification of potential carcinogenicity of substances. Following independent scientific peer review by the EURL ECVAM’s Scientific Advisory Committee (ESAC) and having considered the input from regulators, stakeholders, international partners and the general public, EURL ECVAM concluded that the CTA in Bhas 42 cells shows promise for inclusion within weight of evidence or integrated testing strategy approaches to assess carcinogenic potential or to support chemical category formation and read-across. Thus EURL ECVAM recommends that an OECD test Guideline be developed. In addition, further investigations on the capability of the assay to detect tumour promoters would provide useful information on mode of action of carcinogens for risk assessment purposes.JRC.I.5-Systems Toxicolog