Neurotoxic effects in zebrafish embryos by valproic acid and nine of its analogues: the fish-mouse connection?

Since teratogenicity testing in mammals is a particular challenge from an animal welfare perspective, there is a great need for the development of alternative test systems. In this context, the zebrafish (Danio rerio) embryo has received increasing attention as a non-protected embryonic vertebrate in vivo model. The predictive power of zebrafish embryos for general vertebrate teratogenicity strongly depends on the correlation between fish and mammals with respect to both overall general toxicity and more specific endpoints indicative of certain modes-of-action. The present study was designed to analyze the correlation between (1) effects of valproic acid and nine of its analogues in zebrafish embryos and (2) their known neurodevelopmental effects in mice. To this end, zebrafish embryos exposed for 120 h in an extended version of the acute fish embryo toxicity test (FET; OECD TG 236) were analyzed with respect to an extended list of sublethal endpoints. Particular care was given to endpoints putatively related to neurodevelopmental toxicity, namely jitter/tremor, deformation of sensory organs (eyes) and craniofacial deformation, which might correlate to neural tube defects caused by valproic acid in mammals. A standard evaluation of lethal (LC according to OECD TG 236) and sublethal toxicity (EC) merely indicated that four out of ten compounds tested in zebrafish correlate with positive results in mouse in vivo studies. A detailed assessment of more specific effects, however, namely, jitter/tremor, small eyes and craniofacial deformation, resulted in a correspondence of 75% with in vivo mouse data. A refinement of endpoint analysis from an integration of all observations into one LCx or ECx data (as foreseen by current ecotoxicology-driven OECD guidelines) to a differential evaluation of endpoints specific of selected modes-of-action thus increases significantly the predictive power of the zebrafish embryo model for mammalian teratogenicity. However, for some of the endpoints observed, e.g., scoliosis, lordosis, pectoral fin deformation and lack of movement, further experiments are required for the identification of underlying modes-of-action and an unambiguous interpretation of their predictive power for mammalian toxicity.

Towards grouping concepts based on new approach methodologies in chemical hazard assessment: the read-across approach of the EU-ToxRisk project.

Read-across is one of the most frequently used alternative tools for hazard assessment, in particular for complex endpoints such as repeated dose or developmental and reproductive toxicity. Read-across extrapolates the outcome of a specific toxicological in vivo endpoint from tested (source) compounds to "similar" (target) compound(s). If appropriately applied, a read-across approach can be used instead of de novo animal testing. The read-across approach starts with structural/physicochemical similarity between target and source compounds, assuming that similar structural characteristics lead to similar human hazards. In addition, similarity also has to be shown for the toxicokinetic and toxicodynamic properties of the grouped compounds. To date, many read-across cases fail to demonstrate toxicokinetic and toxicodynamic similarities. New concepts, in vitro and in silico tools are needed to better characterise these properties, collectively called new approach methodologies (NAMs). This white paper outlines a general read-across assessment concept using NAMs to support hazard characterization of the grouped compounds by generating data on their dynamic and kinetic properties. Based on the overarching read-across hypothesis, the read-across workflow suggests targeted or untargeted NAM testing also outlining how mechanistic knowledge such as adverse outcome pathways (AOPs) can be utilized. Toxicokinetic models (biokinetic and PBPK), enriched by in vitro parameters such as plasma protein binding and hepatocellular clearance, are proposed to show (dis)similarity of target and source compound toxicokinetics. Furthermore, in vitro to in vivo extrapolation is proposed to predict a human equivalent dose, as potential point of departure for risk assessment. Finally, the generated NAM data are anchored to the existing in vivo data of source compounds to predict the hazard of the target compound in a qualitative and/or quantitative manner. To build this EU-ToxRisk read-across concept, case studies have been conducted and discussed with the regulatory community. These case studies are briefly outlined.

The OSIRIS Weight of Evidence approach: ITS for skin sensitisation.

Within the EU FP6 project OSIRIS approaches to Integrated Testing Strategies (ITSs) were developed, with the aim to facilitate the use of non-test and non-animal testing information in regulatory risk assessment of chemicals. This paper describes an analytical Weight-of-Evidence (WoE) approach to an ITS for the endpoint of skin sensitisation. It specifically addresses the European chemicals legislation REACH, but the concept is readily applicable to ITS and WoE procedures in other regulatory frameworks, and for other toxicological endpoints. Bayesian statistics are applied to estimate the reliability of a conclusion on the sensitisation potential of a chemical, combining evidence from different information sources such as QSAR model predictions, in vitro and in vivo test results. The methodology allows for adaptation of the weight of individual information sources to account for the different levels of reliability of the individual ITS components. The calculated reliability of the WoE conclusion gives an objective, transparent and reproducible measure to decide if the information requirements for data evaluation are satisfied. Furthermore, in case the WoE is not sufficient, it gives the possibility to evaluate a priori if and how it will be possible to fulfil the information requirements with additional tests and/or model predictions.

OSIRIS, a quest for proof of principle for integrated testing strategies of chemicals for four human health endpoints.

Chemical substances policies in Europe are aiming towards chemical safety and at the same time a reduction in animal testing. These goals are alleged to be reachable by mining as many relevant data as possible, evaluate these data with regard to validity, reliability and relevance, and use of these data in so-called Integrated Testing Strategies (ITS). This paper offers an overview of four human health endpoints that were part of the EU-funded OSIRIS project, aiming to develop ITS fit for the EU chemicals legislation REACH. The endpoints considered cover their categorical as well as continuous characteristics: skin sensitisation, repeated dose toxicity, mutagenicity and carcinogenicity. Detailed papers are published elsewhere in this volume. The stepwise ITS approach developed takes advantage of existing information, groups information about similar substances and integrates exposure considerations. The different and possibly contradictory information is weighted and the respective uncertainties taken into account in a weight of evidence (WoE) approach. In case of data gaps, the ITS proposes the most appropriate method to acquire the missing information. Each building block for the ITS, i.e. each in vivo test, in vitro test, (Q)SAR model or human evidence, is evaluated with regard to quality.

The OSIRIS Weight of Evidence approach: ITS mutagenicity and ITS carcinogenicity.

Risk assessment of chemicals usually implies data evaluation of in vivo tests in rodents to conclude on their hazards. The FP7 European project OSIRIS has developed integrated testing strategies (ITS) for relevant toxicological endpoints to avoid unnecessary animal testing and thus to reduce time and costs. This paper describes the implementation of ITS mutagenicity and carcinogenicity in the public OSIRIS webtool. The data requirements of REACH formed the basis for these ITS. The main goal was to implement procedures to reach a conclusion on the adequacy and validity of available data. For the mutagenicity ITS a quantitative Weight of Evidence approach based on Bayesian statistics was developed and implemented. The approach allows an overall quality assessment of all available data for the five types of mutagenicity data requirements: in vitro bacterial mutagenicity, in vitro and in vivo chromosome aberration, in vitro and in vivo mammalian mutagenicity. For the carcinogenicity ITS a tool was developed to evaluate the quality of studies not conforming (entirely) to guidelines. In a tiered approach three quality aspects are assessed: documentation (reliability), study design (adequacy) and scope of examination (validity). The quality assessment is based on expert and data driven quantitative Weight of Evidence.

The OSIRIS Weight of Evidence approach: ITS for the endpoints repeated-dose toxicity (RepDose ITS).

In the FP6 European project OSIRIS, Integrated Testing Strategies (ITSs) for relevant toxicological endpoints were developed to avoid new animal testing and thus to reduce time and costs. The present paper describes the development of an ITS for repeated-dose toxicity called RepDose ITS which evaluates the conditions under which in vivo non-guideline studies are reliable. In a tiered approach three aspects of these "non-guideline" studies are assessed: the documentation of the study (reliability), the quality of the study design (adequacy) and the scope of examination (validity). The reliability is addressed by the method "Knock-out criteria", which consists of four essential criteria for repeated-dose toxicity studies. A second tool, termed QUANTOS (Quality Assessment of Non-guideline Toxicity Studies), evaluates and weights the adequacy of the study by using intra-criterion and inter-criteria weighting. Finally, the Coverage approach calculates a probability that the detected Lowest-Observed-Effect-Level (LOEL) is similar to the LOEL of a guideline study dependent on the examined targets and organs of the non-guideline study. If the validity and adequacy of the non-guideline study are insufficient for risk assessment, the ITS proposes to apply category approach or the Threshold of Toxicological Concern (TTC) concept, and only as a last resort new animal-testing.

Stoffenmanager Nano version 1.0: a web-based tool for risk prioritization of airborne manufactured nano objects.

Stoffenmanager Nano (version 1.0) is a risk-banding tool developed for employers and employees to prioritize health risks occurring as a result of exposure to manufactured nano objects (MNOs) for a broad range of worker scenarios and to assist implementation of control measures to reduce exposure levels. In order to prioritize the health risks, the Stoffenmanager Nano combines the available hazard information of a substance with a qualitative estimate of potential for inhalation exposure. The development of the Stoffenmanager Nano started with a review of the available literature on control banding. Input parameters for the hazard assessment of MNOs were selected based on the availability of these parameters in, for instance, Safety Data Sheets or product information sheets. The conceptual exposure model described by Schneider et al. (2011) was used as the starting point for exposure banding. During the development of the Stoffenmanager Nano tool, the precautionary principle was applied to deal with the uncertainty regarding hazard and exposure assessment of MNOs. Subsequently, the model was converted into an online tool (http://nano.stoffenmanager.nl), tested, and reviewed by a number of companies. In this paper, we describe the Stoffenmanager Nano. This tool offers a practical approach for risk prioritization in exposure situations where quantitative risk assessment is currently not possible. Updates of this first version are anticipated as more data become available in the future.

NAM-supported read-across: From case studies to regulatory guidance in safety assessment.

The use of new approach methodologies (NAMs) in support of read-across (RAx) approaches for regulatory purposes is a main goal of the EU-ToxRisk project. To bring this forward, EU-ToxRisk partners convened a workshop in close collaboration with regulatory representatives from key organizations including European regulatory agencies, such as the European Chemicals Agency (ECHA) and the European Food Safety Authority (EFSA), as well as the Scientific Committee on Consumer Safety (SCCS), national agencies from several European countries, Japan, Canada and the USA, as well as the Organisation for Economic Cooperation and Development (OECD). More than a hundred people actively participated in the discussions, bringing together diverse viewpoints across academia, regulators and industry. The discussion was organized starting from five practical cases of RAx applied to specific problems that offered the oppor-tunity to consider real examples. There was general consensus that NAMs can improve confidence in RAx, in particular in defining category boundaries as well as characterizing the similarities/dissimilarities between source and target substances. In addition to describing dynamics, NAMs can be helpful in terms of kinetics and metabolism that may play an important role in the demonstration of similarity or dissimilarity among the members of a category. NAMs were also noted as effective in providing quanti-tative data correlated with traditional no observed adverse effect levels (NOAELs) used in risk assessment, while reducing the uncertainty on the final conclusion. An interesting point of view was the advice on calibrating the number of new tests that should be carefully selected, avoiding the allure of "the more, the better". Unfortunately, yet unsurprisingly, there was no single approach befitting every case, requiring careful analysis delineating the optimal approach. Expert analysis and assessment of each specific case is still an important step in the process.

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.

Read-across approaches--misconceptions, promises and challenges ahead.

Read-across is a data gap filling technique used within category and analogue approaches. It has been utilized as an alternative approach to address information requirements under various past and present regulatory programs such as the OECD High Production Volume Programme as well as the EU's Registration, Evaluation, Authorisation and restriction of CHemicals (REACH) regulation. Although read-across raises a number of expectations, many misconceptions still remain around what it truly represents; how to address its associated justification in a robust and scientifically credible manner; what challenges/issues exist in terms of its application and acceptance; and what future efforts are needed to resolve them. In terms of future enhancements, read-across is likely to embrace more biologically-orientated approaches consistent with the Toxicity in the 21st Century vision (Tox-21c). This Food for Thought article, which is notably not a consensus report, aims to discuss a number of these aspects and, in doing so, to raise awareness of the ongoing efforts and activities to enhance read-across. It also intends to set the agenda for a CAAT read-across initiative in 2014-2015 to facilitate the proper use of this technique.

A category approach to predicting the developmental (neuro) toxicity of organotin compounds: the value of the zebrafish (Danio rerio) embryotoxicity test (ZET).

Zebrafish embryos were exposed to different organotin compounds during very early development (<100h post fertilization). Morphology, histopathology and swimming activity (in a motor activity test) were the endpoints analyzed. DBTC was, by far, the most embryotoxic compound at all time points and endpoints studied. In fact, we observed a clear concordance between the effects observed in our zebrafish embryo model, and those observed with these compounds in full rodent in vivo studies. All organotin compounds classified as developmental (neuro) toxicants in vivo, were correctly classified in the present assay. Together, our results support the ZET model as a valuable tool for providing biological verification for a grouping and a read-across approach to developmental (neuro) toxicity.