Evaluation of the availability and applicability of computational approaches in the safety assessment of nanomaterials: Final report of the Nanocomput project

This is the final report of the Nanocomput project, the main aims of which were to review the current status of computational methods that are potentially useful for predicting the properties of engineered nanomaterials, and to assess their applicability in order to provide advice on the use of these approaches for the purposes of the REACH regulation. Since computational methods cover a broad range of models and tools, emphasis was placed on Quantitative Structure-Property Relationship (QSPR) and Quantitative Structure-Activity Relationship (QSAR) models, and their potential role in predicting NM properties. In addition, the status of a diverse array of compartment-based mathematical models was assessed. These models comprised toxicokinetic (TK), toxicodynamic (TD), in vitro and in vivo dosimetry, and environmental fate models. Finally, based on systematic reviews of the scientific literature, as well as the outputs of the EU-funded research projects, recommendations for further research and development were also made. The Nanocomput project was carried out by the European Commission’s Joint Research Centre (JRC) for the Directorate-General (DG) for Internal Market, Industry, Entrepreneurship and SMEs (DG GROW) under the terms of an Administrative Arrangement between JRC and DG GROW. The project lasted 39 months, from January 2014 to March 2017, and was supported by a steering group with representatives from DG GROW, DG Environment and the European Chemicals Agency (ECHA).JRC.F.3-Chemicals Safety and Alternative Method

The Adverse Outcome Pathway approach in nanotoxicology

An Adverse Outcome Pathway (AOP) is a conceptual construct that describes existing knowledge on the link between a molecular initiating event and an adverse outcome. A sequential chain of causally related events is portrayed at different levels of biological organisation. AOPs are considered to be useful mechanistic blueprints for the development of novel tools for human and environmental risk assessment. Following OECD guidance, an increasing number of AOPs for chemically-induced adverse effects in humans and environmental species are being proposed. Due to their unique properties, the toxicity of nanomaterials (NMs) and chemicals is often difficult to directly compare since their modes of actions usually differ. While there are still many knowledge gaps in our understanding of NM toxicity, an ever increasing number of mechanistic studies are shedding light on their toxicokinetic and toxicodynamic properties. In this paper, we argue that the differences between NM and chemically induced adversity are primarily related to differences in toxicokinetics and the nature of the initial key events in the AOP. Consequently, much of the mechanistic knowledge captured by AOPs that have been developed from consideration of chemical induced toxicity is also relevant to describe AOPs of NMs, at least in qualitative terms, and thus can be used to inform predictive modelling of NM-toxicity. In support of these claims, we illustrate how the AOP framework can be used to rationally combine mechanistic knowledge relating to both NM- and chemically-induced liver toxicity to fill information gaps and guide the development of toxicity testing strategies.JRC.F.3-Chemicals Safety and Alternative Method

Grouping of multi-walled carbon nanotubes to read across genotoxicity: a case study to evaluate the applicability of regulatory guidance

Multi-walled carbon nanotubes (MWCNTs) consist of multiple layers of graphene sheets in a tubular shape. Due to their specific material properties, such as electrical and thermal conductivity, strength, rigidity, and toughness they are useful in a wide variety of applications in electronics, optics and other fields of materials science. Depending on the synthesis and purification method, MWCNTs may differ in size, shape, rigidity and other properties. Previous research has shown that physicochemical properties can influence the translocation and toxicity of MWCNTs. This paper describes a case study following the “Recommendations for nanomaterials applicable to the Guidance on QSARs and Grouping”, developed by the European Chemicals Agency (ECHA). Based on the data availability genotoxicity was selected as the hazard endpoint to explore and illustrate read across. The grouping hypothesis was supported by the use of chemoinformatics techniques such as hierarchical clustering and principal components analysis. The uncertainties of the present case study were evaluated using the Read-Across Assessment Framework (RAAF) developed by ECHA. This study shows the practical application of the ECHA framework for grouping of nanomaterials (NMs) as well as use of the ECHA RAAF for NMs, and how this can be supported by chemoinformatics techniques. Some adaptations to the workflow are suggested for a more practical and straightforward narrative in the reporting.JRC.F.3-Chemicals Safety and Alternative Method

Practical use of the Virtual Cell Based Assay: Simulation of repeated exposure experiments in liver cell lines

The Virtual Cell Based Assay (VCBA) was applied to simulate the long-term (repeat dose) toxic effects of chemicals, including substances in cosmetics and personal care products. The presented model is an extension of the original VCBA for simulation of single exposure and is implemented in a KNIME workflow. This work illustrates the steps taken to simulate the repeated dose effects of two reference compounds, caffeine and amiodarone. Using caffeine, in vitro experimental viability data in single exposure from two human liver cell lines, HepG2 and HepaRG, were measured and used to optimize the VCBA, subsequently repeated exposure simulations were run. Amiodarone was then tested and simulations were performed under repeated exposure conditions in HepaRG. The results show that the VCBA can adequately predict repeated exposure experiments in liver cell lines. The refined VCBA model can be used not only to support the design of long term in vitro experiments but also practical applications in risk assessment. Our model is a step towards the development of in silico predictive approaches to replace, refine, and reduce the in vivo repeated dose systemic toxicity studies in the assessment of human safety.JRC.F.3-Chemicals Safety and Alternative Method

Investigating cell type specific mechanisms contributing to acute oral toxicity

The replacement of animals in acute systemic toxicity testing remains a considerable challenge. Only animal data are currently accepted by regulators, including data generated by reduction and refinement methods. The development of Integrated Approaches to Testing and Assessment (IATA) is hampered by an insufficient understanding of the numerous toxicity pathways that lead to acute systemic toxicity. Therefore, central to our work has been the collection and evaluation of the mechanistic information on eight organs identified as relevant for acute systemic toxicity (nervous system, cardiovascular system, liver, kidney, lung, blood, gastrointestinal system and immune system). While the nervous and cardiovascular systems are the most frequent targets, no clear relationship emerged between specific mechanisms of target organ toxicity and the level (category) of toxicity. From a list of 114 chemicals with acute oral in vivo and in vitro data, 97 were identified with target organ specific effects, of which 94% (91/97) were predicted as acutely toxic by the 3T3 neutral red uptake cytotoxicity assay and 6% (6/97) as non-toxic. Although specific target organ mechanisms of toxicity could in some cases explain the false negative prediction obtained with the cytotoxicity assay, in general it is difficult to explain in vitro misclassifications only on the basis of mechanistic information. This analysis will help to prioritise the development of adverse outcome pathways for acute oral toxicity, which will support the assessment of chemicals using mechanistically informed IATA.JRC.F.3-Chemicals Safety and Alternative Method

A high throughput imaging database of toxicological effects of nanomaterials tested on HepaRG cells

The large amount of existing nanomaterials demands rapid and reliable methods for testing their potential toxicological effect on human health, preferably by means of relevant in vitro techniques in order to reduce testing on animals. Combining high throughput workflows with automated high content imaging techniques allows deriving much more information from cell-based assays than the typical readouts (i.e. one measurement per well) with optical plate-readers. We present here a dataset including data based on a maximum of 14 different read outs (including viable cell count, cell membrane permeability, apoptotic cell death, mitochondrial membrane potential and steatosis) of the human hepatoma HepaRG cell line treated with a large set of nanomaterials, coatings and supernatants at different concentrations. The database, given its size, can be utilized in the development of in silico hazard assessment and prediction tools or can be combined with toxicity results from other in vitro test systems.JRC.F.3-Chemicals Safety and Alternative Method

Microscopy-based high-throughput assays enable multi-parametric analysis to assess adverse effects of nanomaterials in various cell lines

Manufactured nanomaterials (MNMs) selected from a library of over 120 different MNMs with varied compositions, sizes, and surface coatings were tested by four different laboratories for toxicity by high-throughput/-content (HT/C) techniques. The selected particles comprise 14 MNMs composed of CeO2, Ag, TiO2, ZnO and SiO2 with different coatings and surface characteristics at varying concentrations. The MNMs were tested in different mammalian cell lines at concentrations between 0.5 and 250 µg/mL to link physical-chemical properties to multiple adverse effects. The cell lines are derived from relevant organs such as liver, lung, colon and the immune system. Endpoints such as viable cell count, cell membrane permeability, apoptotic cell death, mitochondrial membrane potential, lysosomal acidification and steatosis have been studied. Soluble MNMs, Ag and ZnO, were the most toxic in all cell types. TiO2 and SiO2 MNMs also triggered toxicity in some, but not all, cell types and the cell-type specific effects were influenced by the specific coating. CeO2 MNMs were nearly ineffective in our test systems. Differentiated liver cells appear to be most sensitive to MNMs, in particular to TiO2 MNMs. Whereas most of the investigated MNMs showed no acute toxicity, it became clear that some show adverse effects dependent on the assay and cell line. Hence, it is advised that future nanosafety studies utilise a multi-parametric approach such as HT/C screening to avoid missing signs of toxicity. Furthermore, some of the cell type specific effects should be followed up in more detail and might also provide an incentive to address potential adverse effects in vivo in the relevant organ.JRC.F.3-Chemicals Safety and Alternative Method