Developmental Toxicity of Ochratoxin A in Rat Embryo Midbrain Micromass Cultures

Embryonic midbrain micromass cultures were exposed for five days to ochratoxin A (OTA) at seven concentrations (ranging from 0.16 to 10 μg/mL). Cell viability was assessed in neutral red uptake test (NRU), and differentiation – by immunoenzymatic determination of structural proteins (βIII-tubulin, MAP2, GFAP) expression level as well as by computer image analysis. Dose dependent decrease in cell number and differentiation was observed. Concentration-response curves were analysed and the mean inhibition concentrations (μg/mL) for cytotoxicity (IC50) and differentiation (ID50) were calculated. There were no significant differences in the sensitivity of neurons in early and late stage of differentiation and astrocytes to the toxic activity of this compound. For all endpoints ID50 value was very low (< 10 μg/mL) so OTA was classified as a strong teratogen. IC50/ ID50 ratios <2 pointed out that with harmful action of OTA the basic cytotoxicity should be connected

Regulatory safety aspects of advanced therapy medicinal products (ATMPs)

Advanced therapy medicinal products (ATMPs) such as gene-, cell- and tissue engineered therapies offer tremendous potential for treating unmet medical needs. Due to the novelty, complexity and technical specificity of such products, specially tailored and harmonized rules are needed to evaluate their safety and efficacy. Despite rapid progress in research and the wide range of ATMPs undergoing pre-clinical and clinical development, the number of licenced products still remains low. In order to determine the main factors, which complicate the translation from ATMPs research into patient access, the feedback from main stakeholders i.e. representatives of product manufacturers, regulatory authorities and patients’ associations was collected in semi-structured interviews. The main focus was on pre-clinical safety testing and the relevant regulatory considerations. Numerous factors have been identified as limitations preventing more rapid market authorization for ATMPs. The key challenges in pre-clinical development considered availability of the right animal model for the specific disease, current approaches in immunogenicity testing and insertional site analysis as well as determining appropriate dosing strategy. Concerns regarding the lack of validated (bio)analytical methods and insufficient programs for environmental risk monitoring were raised. A specific identified challenge for the ATMPs sector was the difficulty to meet all the requirements to obtain market authorization from regulatory authorities, especially for products developed for European and non-European markets simultaneously, and a great heterogeneity in the legal requirements at the national level for products developed for non-routine use. To address these knowledge gaps and regulatory challenges, numerous international collaboration projects and initiatives have been already undertaken. It is likely that the introduction of ATMPs will follow an incremental pattern of technological and regulatory change, building on existing drug development and regulation heuristics and experiences. A necessary open dialog and continuous collaboration between stakeholders is considered as key to learn and grow together in this new field and to design solid guidelines for the safe implementation of advanced therapies as medicinal treatment

Biotransformation in vitro: an essential consideration in the quantitative in vitro-to-in vivo extrapolation (QIVIVE) of toxicity data

Early consideration of the multiplicity of factors that govern the biological fate of foreign compounds in living systems is a necessary prerequisite for the quantitative in vitro–in vivo extrapolation (QIVIVE) of toxicity data. Substantial technological advances in in vitro methodologies have facilitated the study of in vitro metabolism and the further use of such data for in vivo prediction. However, extrapolation to in vivo with a comfortable degree of confidence, requires continuous progress in the field to address challenges such as e.g., in vitro evaluation of chemical–chemical interactions, accounting for individual variability but also analytical challenges for ensuring sensitive measurement technologies. This paper discusses the current status of in vitro metabolism studies for QIVIVE extrapolation, serving today’s hazard and risk assessment needs. A short overview of the methodologies for in vitro metabolism studies is given. Furthermore, recommendations for priority research and other activities are provided to ensure further widespread uptake of in vitro metabolism methods in 21st century toxicology. The need for more streamlined and explicitly described integrated approaches to reflect the physiology and the related dynamic and kinetic processes of the human body is highlighted i.e., using in vitro data in combination with in silico approaches.JRC.F.3-Chemicals Safety and Alternative Method

Three-dimensional HepaRG model as an attractive tool for toxicity testing

The culture of HepaRG cells as 3D structures in the spinner-bioreactor may represent added value as a hepatic system for toxicological purposes. The use of a cost-effective commercially available bioreactor, which is compatible with high-throughput cell analysis, constitutes an attractive approach for routine use in the drug testing industry. In order to assess specific aspects of the biotransformation capacity of the bioreactor-based HepaRG system, the induction of CYP450 enzymes (i.e. CYP1A2, 2B6, 2C9 and 3A4) and the activity of the phase II enzyme, UGT, were tested. The long term functionality of the system was demonstrated by 7 weeks stable profiles of albumin secretion, CYP3A4 induction and UGT activities. Immunofluorescence-based staining showed formation of tissue-like arrangements including bile canaliculi-like structures and polar distribution of transporters. The use of in silico models to analyse the in vitro data related to hepatotoxic activity of acetaminophen (APAP) demonstrated the advantage of the integration of kinetic and dynamic aspects for a better understanding of the in vitro cell behaviour. The bioactivation of APAP and its related cytotoxicity was assessed in a system compatible to high-throughput screening. The approach also proved to be a good strategy to reduce the time necessary to obtain fully differentiated cell cultures. In conclusion, HepaRG cells cultured in a 3D spinner-bioreactors are an attractive tool for toxicological studies, showing a liver-like performance and demonstrating a practical applicability for toxicodynamic approaches.JRC.I.2-Public Health Policy Suppor

Validation of in vitro methods for human cytochrome P450 enzyme induction: outcome of a multi-laboratory study

CYP enzyme induction is a sensitive biomarker for phenotypic metabolic competence of in vitro test systems; it is a key event associated with thyroid disruption, and a biomarker for toxicologically relevant nuclear receptor-mediated pathways. This paper summarises the results of a multi-laboratory validation study of two in vitro methods that assess the potential of chemicals to induce cytochrome P450 (CYP) enzyme activity, in particular CYP1A2, CYP2B6, and CYP3A4. The methods are based on the use of cryopreserved primary human hepatocytes (PHH) and human HepaRG cells. The validation study was coordinated by the European Union Reference Laboratory for Alternatives to Animal Testing of the European Commission's Joint Research Centre and involved a ring trial among six laboratories. The reproducibility was assessed within and between laboratories using a validation set of 13 selected chemicals (known human inducers and non-inducers) tested under blind conditions. The ability of the two methods to predict human CYP induction potential was assessed. Chemical space analysis confirmed that the selected chemicals are broadly representative of a diverse range of chemicals. The two methods were found to be reliable and relevant in vitro tools for the assessment of human CYP induction, with the HepaRG method being better suited for routine testing. Recommendations for the practical application of the two methods are proposed.JRC.F.3-Chemicals Safety and Alternative Method

Modern science for better quality control of medicinal products “Towards global harmonization of 3Rs in biologicals”: The report of an EPAA Workshop

This article summarizes the outcome of an international workshop organized by the European Partnership for Alternative Approaches to Animal Testing (EPAA) on Modern science for better quality control of medicinal products: Towards global harmonization of 3Rs in biologicals. As regards the safety testing of biologicals, the workshop participants agreed to actively encourage the deletion of abnormal toxicity tests and target animal batch safety tests from all relevant legal requirements and guidance documents (country-specific guidelines, pharmacopoeia monographs, WHO recommendations). To facilitate the global regulatory acceptance of non-animal methods for the potency testing of, e.g., human diphtheria and tetanus vaccines and veterinary swine erysipelas vaccines, international convergence on the scientific principles of the use of appropriately validated in vitro assays for replacing in vivo methods was recognized as a further means to unify regulatory approaches in different jurisdictions. It was recommended to include key regulators and manufacturers early in the corresponding discussions. Manufacturers and responsible expert groups, e.g. at the European Directorate for the Quality of Medicines and Health Care of the Council of Europe or the European Medicines Agency, were invited to consider leadership for international collaboration.JRC.F.3-Chemicals Safety and Alternative Method

Considerations in the development of in vitro toxicity testing methods intended for regulatory use

It has been demonstrated that the performance of in vitro toxicity testing methods can be seriously affected by variability in the essential components of the method, including the experimental protocol, the test items (chemicals), the test system (the biological models), and the analytical or measurement technique(s) used. Therefore these aspects need careful consideration during the development of in vitro methods if they are to be used in regulatory applications where compliance with quality systems is typically a prerequisite. Concerning the test system, implementation of the basic concepts of Good Cell Culture Practices (GCCP) is essential in the identification and characterisation of the biological model (test system part of the in vitro method). An adequate quality system, preferably Good Laboratory Practice (GLP) together with GCCP leads to overall Good In vitro Method Practices (GIVIM). Additional guidance for test developers will help to ensure that in vitro methods destined for regulatory use will prove to be robust, reliable and ultimately useful for supporting regulatory decisions.JRC.I.5-Systems Toxicolog

Validation of in vitro methods for human cytochrome P450 enzyme induction:outcome of a multi-laboratory study

Abstract CYP enzyme induction is a sensitive biomarker for phenotypic metabolic competence of in vitro test systems; it is a key event associated with thyroid disruption, and a biomarker for toxicologically relevant nuclear receptor-mediated pathways. This paper summarises the results of a multi-laboratory validation study of two in vitro methods that assess the potential of chemicals to induce cytochrome P450 (CYP) enzyme activity, in particular CYP1A2, CYP2B6, and CYP3A4. The methods are based on the use of cryopreserved primary human hepatocytes (PHH) and human HepaRG cells. The validation study was coordinated by the European Union Reference Laboratory for Alternatives to Animal Testing of the European Commission's Joint Research Centre and involved a ring trial among six laboratories. The reproducibility was assessed within and between laboratories using a validation set of 13 selected chemicals (known human inducers and non-inducers) tested under blind conditions. The ability of the two methods to predict human CYP induction potential was assessed. Chemical space analysis confirmed that the selected chemicals are broadly representative of a diverse range of chemicals. The two methods were found to be reliable and relevant in vitro tools for the assessment of human CYP induction, with the HepaRG method being better suited for routine testing. Recommendations for the practical application of the two methods are proposed

PBTK modelling platforms and parameter estimation tools to enable animal-free risk assessment Recommendations from a joint EPAA - EURL ECVAM ADME workshop

Information on toxicokinetics is critical for animal-free human risk assessment. Human external exposure must be translated into human tissue doses and compared with in vitro actual cell exposure associated to effects (in vitro-in vivo comparison). Data on absorption, distribution, metabolism and excretion in humans (ADME) could be generated using in vitro and QSAR tools. Physiologically-based toxicokinetic (PBTK) computer modelling could serve to integrate disparate in vitro and in silico findings. However, there are only few freely-available PBTK platforms currently available. And although some ADME parameters can be reasonably estimated in vitro or in silico, important gaps exist. Examples include unknown or limited applicability domains and lack of (high-throughput) tools to measure penetration of barriers, partitioning between blood and tissues and metabolic clearance. This paper is based on a joint EPAA - EURL ECVAM expert meeting. It provides a state-of-the-art overview of the availability of PBTK platforms as well as the in vitro and in silico methods to parameterise basic (Tier 1) PBTK models. Five high-priority issues are presented that provide the prerequisites for wider use of non-animal based PBTK modelling for animal-free chemical risk assessment

PBTK modelling platforms and parameter estimation tools to enable animal-free risk assessment: Recommendations from a joint EPAA – EURL ECVAM ADME workshop

Absorption, distribution, metabolism and excretion (ADME) determine target tissue doses upon human exposure to chemicals. In this respect, ADME is a critical piece of information in the framework of chemical risk assessment. This is especially true in an era where risk assessment must increasingly be based on in vitro toxicity experiments. Human external exposure must be translated into human tissue doses and compared with in vitro actual cell exposure associated to effects (in vitro - in vivo comparison). Much of the information needed can be generated at tissue/cell or sub-cellular level using in vitro and/or in silico (data-based such as QSAR - Quantitative Structure Activity Relationship) tools. Finally, (computer) modelling serves to integrate disparate in silico and in vitro findings in order to simulate whole body processes. However, there are few freely-available computational modelling platforms currently available in the public domain. Also, the absence of a complete toolbox of in silico and in vitro prediction tools to provide estimates for kinetic parameters is currently a problem. Although some ADME processes can be reasonably estimated in vitro or in silico, important gaps withhold physiologically-based toxicokinetic (PBTK) modelling from wider use for in vitro to in vivo dose extrapolation. Examples of gaps include unknown applicability domains and lack of high-throughput models to measure the following: (1) penetration of barriers, (2) partitioning between blood and tissues and (3) metabolic clearance. This paper is based on a joint EPAA - EURL ECVAM expert meeting and provides a summary of the state of art of in silico and in vitro ADME models used to deliver parameter values for basic (Tier 1) PBTK modelling. Furthermore insights and concrete recommendations are provided to in silico QSAR and in vitro test developers, toxicologists, safety assessors and regulators on priorities to further progress towards a new integrated in silico / in vitro risk assessment paradigm for systemic toxicity without or with significantly fewer animals. The paper, (1) describes a set of critical in vitro and in silico methods providing credible input data for Tier 1 PBTK modelling; (2) defines recommendations for public availability of PBTK modelling tools; (3) provides concrete suggestions where to address the scientific issues to foster progress with a new integrated risk assessment framework; (4) identifies gaps and lists a set of recommendations for initiating PBTK assessment in R&D and standardization and optimization efforts.JRC.I.5-Systems Toxicolog