New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals - a PARC (Partnership for the Assessment of Risk from Chemicals) project.

As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC).

Qualitative and quantitative concentration-response modelling of gene co-expression networks to unlock hepatotoxic mechanisms for next generation chemical safety assessment.

Next generation risk assessment of chemicals revolves around the use of mechanistic information without animal experimentation. In this regard, toxicogenomics has proven to be a useful tool to elucidate the underlying mechanisms of adverse effects of xenobiotics. In the present study, two widely used human in vitro hepatocyte culture systems, namely primary human hepatocytes (PHH) and human hepatoma HepaRG cells, were exposed to liver toxicants known to induce liver cholestasis, steatosis or necrosis. Benchmark concentration-response modelling was applied to transcriptomics gene co-expression networks (modules) to derive benchmark concentrations (BMCs) and to gain mechanistic insight into the hepatotoxic effects. BMCs derived by concentration-response modelling of gene co-expression modules recapitulated concentration-response modelling of individual genes. Although PHH and HepaRG cells showed overlap in deregulated genes and modules by the liver toxicants, PHH demonstrated a higher responsiveness, based on the lower BMCs of co-regulated gene modules. Such BMCs can be used as transcriptomics point of departure (tPOD) for assessing module-associated cellular (stress) pathways/processes. This approach identified clear tPODs of around maximum systemic concentration (Cmax) levels for the tested drugs, while for cosmetics ingredients the BMCs were 10-100-fold higher than the estimated plasma concentrations. This approach could serve next generation risk assessment practice to identify early responsive modules at low BMCs, that could be linked to key events in liver adverse outcome pathways. In turn, this can assist in delineating potential hazards of new test chemicals using in vitro systems and used in a risk assessment when BMCs are paired with chemical exposure assessment.

Risk assessment of chemicals has traditionally been focused on animal experiments. In contrast, next generation risk assessment uses biological information obtained from experiments in cell culture models without animals to identify potential hazards. Since the liver is the main target organ of toxicity, many liver cell (hepatocyte) models have been developed and applied for hazard assessment. In this study, two widely used human hepatocyte cell models, PHH and HepaRG, were exposed to liver toxic chemicals. Biological changes in gene expression were measured in a concentration range to identify the concentration at which a biological response was perturbed using concentration response modelling. Genes belonging to the same biological process were joined based on co-expression to derive an average concentration of this process. This animal-free approach could be applied for risk assessment when biological response concentrations were related to the expected human exposure to identify potential hazard of the test chemicals.

Adverse Outcome Pathways as Versatile Tools in Liver Toxicity Testing.

Adverse outcome pathways (AOPs) are tools to capture and visualize mechanisms driving toxicological effects. They share a common structure consisting of a molecular initiating event, a series of key events connected by key event relationships and an adverse outcome. Development and evaluation of AOPs ideally comply with guidelines issued by the Organization for Economic Cooperation and Development. AOPs have been introduced for major types of hepatotoxicity, which is not a surprise, as the liver is a frequent target for systemic adversity. Various applications for AOPs have been proposed in the areas of toxicology and chemical risk assessment, in particular in relation to the establishment of quantitative structure-activity relationships, the elaboration of prioritization strategies, and the development of novel in vitro toxicity screening tests and testing strategies.

An overview of current practices for regulatory risk assessment with lessons learnt from cosmetics in the European Union.

Risk assessments of various types of chemical compounds are carried out in the European Union (EU) foremost to comply with legislation and to support regulatory decision-making with respect to their safety. Historically, risk assessment has relied heavily on animal experiments. However, the EU is committed to reduce animal experimentation and has implemented several legislative changes, which have triggered a paradigm shift towards human-relevant animal-free testing in the field of toxicology, in particular for risk assessment. For some specific endpoints, such as skin corrosion and irritation, validated alternatives are available whilst for other endpoints, including repeated dose systemic toxicity, the use of animal data is still central to meet the information requirements stipulated in the different legislations. The present review aims to provide an overview of established and more recently introduced methods for hazard assessment and risk characterisation for human health, in particular in the context of the EU Cosmetics Regulation (EC No 1223/2009) as well as the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) Regulation (EC 1907/2006).

Derivation, characterisation and analysis of an adverse outcome pathway network for human hepatotoxicity.

Adverse outcome pathways (AOPs) and their networks are important tools for the development of mechanistically based non-animal testing approaches, such as in vitro and/or in silico assays, to assess toxicity induced by chemicals. In the present study, an AOP network connecting 14 linear AOPs related to human hepatotoxicity, currently available in the AOP-Wiki, was derived according to established criteria. The derived AOP network was characterised and analysed with regard to its structure and topological features. In-depth analysis of the AOP network showed that cell injury/death, oxidative stress, mitochondrial dysfunction and accumulation of fatty acids are the most highly connected and central key events. Consequently, these key events may be considered as the rational and mechanistically anchored basis for selecting, developing and/optimising in vitro and/or in silico assays to predict hepatotoxicity induced by chemicals in view of animal-free hazard identification.

In Vitro Liver Toxicity Testing of Chemicals: A Pragmatic Approach.

The liver is among the most frequently targeted organs by noxious chemicals of diverse nature. Liver toxicity testing using laboratory animals not only raises serious ethical questions, but is also rather poorly predictive of human safety towards chemicals. Increasing attention is, therefore, being paid to the development of non-animal and human-based testing schemes, which rely to a great extent on in vitro methodology. The present paper proposes a rationalized tiered in vitro testing strategy to detect liver toxicity triggered by chemicals, in which the first tier is focused on assessing general cytotoxicity, while the second tier is aimed at identifying liver-specific toxicity as such. A state-of-the-art overview is provided of the most commonly used in vitro assays that can be used in both tiers. Advantages and disadvantages of each assay as well as overall practical considerations are discussed.