Feasibility Assessment of Micro-Electrode Chip Assay as a Method of Detecting Neurotoxicity in vitro

Detection and characterization of chemically induced toxic effects in the nervous system represent a challenge for the hazard assessment of chemicals. In vivo, neurotoxicological assessments exploit the fact that the activity of neurons in the central and peripheral nervous system has functional consequences. And so far, no in vitro method for evaluating the neurotoxic hazard has yet been validated and accepted for regulatory purpose. The micro-electrode array (MEA) assay consists of a culture chamber into which an integrated array of micro-electrodes is capable of measuring extracellular electrophysiology (spikes and bursts) from electro-active tissues. A wide variety of electrically excitable biological tissues may be placed onto the chips including primary cultures of nervous system tissue. Recordings from this type of in vitro cultured system are non-invasive, give label free evaluations and provide a higher throughput than conventional electrophysiological techniques. In this paper, 20 substances were tested in a blinded study for their toxicity and dose–response curves were obtained from fetal rat cortical neuronal networks coupled to MEAs. The experimental procedure consisted of evaluating the firing activity (spiking rate) and modification/reduction in response to chemical administration. Native/reference activity, 30 min of activity recording per dilution, plus the recovery points (after 24 h) were recorded. The preliminary data, using a set of chemicals with different mode-of-actions (13 known to be neurotoxic, 2 non-neuroactive and not toxic, and 5 non-neuroactive but toxic) show good predictivity (sensitivity: 0.77; specificity: 0.86; accuracy: 0.85). Thus, the MEA with a neuronal network has the potency to become an effective tool to evaluate the neurotoxicity of substances in vitro

Suitability of skin integrity tests for dermal absorption studies in vitro

AbstractSkin absorption testing in vitro is a regulatory accepted alternative method (OECD Guideline 428). Different tests can be applied to evaluate the integrity of the skin samples. Here, we compared the pre- or post-run integrity tests (transepidermal electrical resistance, TEER; transepidermal water loss, TEWL; absorption of the reference compounds water, TWF, or methylene blue, BLUE) and additionally focused on co-absorption of a 3H-labeled internal reference standard (ISTD) as integrity parameter. The results were correlated to absorption profiles of various test compounds. Limit values of 2kΩ, 10gm−2h−1 and 4.5∗10−3cmh−1 for the standard methods TEER, TEWL and TWF, respectively, allowed distinguishing between impaired and intact human skin samples in general. Single skin samples did, however, not, poorly and even inversely correlate with the test-compound absorption. In contrast, results with ISTD (e.g. 3H-testosterone) were highly correlated to the absorption of 14C-labeled test compounds. Importantly, ISTD did not influence analytics or absorption of test compounds. Therefore, ISTD, especially when adjusted to the physico-chemical properties of test compounds, is a promising concept to assess the integrity of skin samples during the whole course of absorption experiments. However, a historical control dataset is yet necessary for a potential routine application

Adverse outcome pathways:opportunities, limitations and open questions

Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event relationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field

Hazard Identification of Inhaled Nanomaterials: Making use of Short-term Inhalation Studies

A major health concern for nanomaterials is their potential toxic effect after inhalation of dusts. Correspondingly, the core element of tier 1 in the currently proposed Integrated Testing Strategy (ITS) is a short-term rat inhalation study (STIS) for this route of exposure. STIS comprises a comprehensive scheme of biological effects and marker determination in order to generate appropriate information on early key elements of pathogenesis, such as inflammatory reactions in the lung and indications of effects in other organs. Within the STIS information on the persistence, progression and/or regression of effects is obtained. The STIS also addresses organ burden in the lung and potential translocation to other tissues. Up to now STIS was performed in research projects and routine testing of nanomaterials. Meanwhile rat STIS results for more than 20 nanomaterials are available including the representative nanomaterials listed by the Organization for Economic Cooperation and Development (OECD) Working Party on Manufactured Nanomaterials (WPMN), which has endorsed a list of representative Manufactured Nanomaterials (MN) as well as a set of relevant endpoints to be addressed. Here, results of STIS carried out with different nanomaterials are discussed as case studies. The ranking of different nanomaterials potential to induce adverse effects and the ranking of the respective NOAEC is the same among the STIS and the corresponding sub-chronic and chronic studies. In another case study, a translocation of a coated silica nanomaterial was judged critical for its safety assessment. Thus, STIS enables application of the proposed ITS, as long as reliable and relevant in vitro methods for the tier 1 testing are still missing. Compared to traditional subacute and subchronic inhalation testing (according to OECD test guidelines (TG) 412 and 413), STIS uses less animals and resources and offers additional information on organ burden and pro-/regression of potential effects.JRC.D-Institute for Reference Materials and Measurements (Geel

Feasibility assessment of micro-electrode chip assay as a method of detecting neurotoxicity in vitro

Detection and characterization of chemically-induced toxic effects in the nervous system represent a major challenge for registration and assessment of chemicals. So far, no in vitro method for evaluating the neurotoxic hazard has yet been validated and accepted for regulatory purpose. In vivo, neurotoxicological assessments exploit the fact that the activity of neurons in the central and peripheral nervous system has functional consequences. The microelectrode array (MEA) assay consists of a culture chamber into which an integrated array of microelectrodes is capable of measuring extracellular electrophysiology (spikes and bursts) from electro-active tissues. A wide variety of electrically excitable biological tissues may be placed onto the chips including primary cultures of nervous system tissue. Recordings from this type of in vitro cultured system are non invasive, give label free evaluations and provide a higher throughput than conventional electrophysiological techniques. In this study 20 blinded substances were tested in a dose-response curve on embryonic rat cortical neuronal networks on a MEA for their toxicity. The experimental procedure consisted of evaluating the firing activity (spiking rate) and modification/reduction in response to chemical administration. Native/reference activity, 30 minutes of activity recording per dilution, plus the reversibility/recovery points (after 24 hours) were recorded. The IC50 ranges were indicated. The preliminary data, using a set of chemicals with different mode-of-actions (13 known to be neurotoxic, 2 non neuroactive and not toxic and 5 not neuroactive but toxic) show good predictivity (sensitivity: 0.69; specificity: 1.0; accuracy: 0.80). Thus, the MEA with a neuronal network has the potency to become a powerful tool to evaluate the neurotoxicity of substances in vitro.JRC.DG.I.6-Systems toxicolog

The challenge of the application of ‘omics technologies in chemicals risk assessment: background and outlook

This survey by the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) highlights that ‘omics technologies are generally not yet applied to meet standard information requirements during regulatory hazard assessment. While they are used within weight-of-evidence approaches to investigate substances’ modes-of-action, consistent approaches for the generation, processing and interpretation of ‘omics data are not applied. To date, no ‘omics technology has been standardised or validated. Best practices for performing ‘omics studies for regulatory purposes (e.g., microarrays for transcriptome profiling) remain to be established. Therefore, three frameworks for (i) establishing a Good-Laboratory Practice-like context for collecting, storing and curating ‘omics data; (ii) ‘omics data processing; and (iii) quantitative WoE approaches to interpret ‘omics data have been developed, that are presented in this journal issue. Application of the frameworks will enable between-study comparison of results, which will facilitate the regulatory applicability of ‘omics data. The frameworks do not constitute prescriptive protocols precluding any other data analysis method, but provide a baseline for analysis that can be applied to all data allowing ready cross-comparison. Data analysis that does not follow the frameworks can be justified and the resulting data can be compared with the Framework-based common analysis output.JRC.F.3-Chemicals Safety and Alternative Method

Food for Thought : 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