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1.
Toxicol In Vitro ; 60: 212-228, 2019 Oct.
Article in English | MEDLINE | ID: mdl-31158489

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.


Subject(s)
Cytochrome P-450 Enzyme Inducers/pharmacology , Cytochrome P-450 Enzyme System/biosynthesis , Animal Testing Alternatives , Cell Survival/drug effects , Cells, Cultured , Cytochrome P-450 Enzyme Inducers/chemistry , Enzyme Induction , Hepatocytes/drug effects , Humans , Laboratories , Reproducibility of Results , Solubility
2.
Biologicals ; 48: 55-65, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28596049

ABSTRACT

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 identified as an overarching goal. The establishment of scientific requirements for new assays 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.


Subject(s)
Drug Industry/standards , Pharmaceutical Preparations/standards , Quality Control , Animals , Congresses as Topic , Drug Evaluation, Preclinical/methods , Drug Evaluation, Preclinical/standards , Humans
3.
Adv Exp Med Biol ; 856: 133-163, 2016.
Article in English | MEDLINE | ID: mdl-27671721

ABSTRACT

This chapter focuses on practical aspects of conducting prospective in vitro validation studies, and in particular, by laboratories that are members of the European Union Network of Laboratories for the Validation of Alternative Methods (EU-NETVAL) that is coordinated by the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). Prospective validation studies involving EU-NETVAL, comprising a multi-study trial involving several laboratories or "test facilities", typically consist of two main steps: (1) the design of the validation study by EURL ECVAM and (2) the execution of the multi-study trial by a number of qualified laboratories within EU-NETVAL, coordinated and supported by EURL ECVAM. The approach adopted in the conduct of these validation studies adheres to the principles described in the OECD Guidance Document on the Validation and International Acceptance of new or updated test methods for Hazard Assessment No. 34 (OECD 2005). The context and scope of conducting prospective in vitro validation studies is dealt with in Chap. 4 . Here we focus mainly on the processes followed to carry out a prospective validation of in vitro methods involving different laboratories with the ultimate aim of generating a dataset that can support a decision in relation to the possible development of an international test guideline (e.g. by the OECD) or the establishment of performance standards.


Subject(s)
Animal Testing Alternatives/methods , Research Design , Toxicity Tests/methods , Validation Studies as Topic , Animals , European Union
4.
Toxicology ; 332: 8-19, 2015 Jun 05.
Article in English | MEDLINE | ID: mdl-25456264

ABSTRACT

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.


Subject(s)
Biotransformation , In Vitro Techniques , Models, Biological , Toxicity Tests/methods , Toxicology/methods , Animal Testing Alternatives , Animals , Computer Simulation , Humans , Risk Assessment , Risk Factors , Species Specificity , Systems Biology
5.
Regul Toxicol Pharmacol ; 68(1): 119-39, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24287156

ABSTRACT

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.


Subject(s)
Environmental Pollutants/pharmacokinetics , Environmental Pollutants/toxicity , Models, Biological , Animal Testing Alternatives , Drug-Related Side Effects and Adverse Reactions , Environmental Exposure/adverse effects , Humans , Pharmacokinetics , Risk Assessment
6.
Toxicol Sci ; 130(1): 106-16, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22843569

ABSTRACT

The culture of HepaRG cells as three dimensional (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, uridine diphosphate glucuronoltransferase (UGT), were tested. The long-term functionality of the system was demonstrated by 7-week 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 analyze 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 behavior. 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 3D spinner-bioreactors are an attractive tool for toxicological studies, showing a liver-like performance and demonstrating a practical applicability for toxicodynamic approaches.


Subject(s)
Acetaminophen/toxicity , Analgesics, Non-Narcotic/toxicity , Animal Testing Alternatives/methods , Bioreactors , Hepatocytes/drug effects , Toxicity Tests , Acetaminophen/chemistry , Acetaminophen/metabolism , Albumins/metabolism , Analgesics, Non-Narcotic/chemistry , Analgesics, Non-Narcotic/metabolism , Cell Culture Techniques , Cell Survival/drug effects , Computer Simulation , Cytochrome P-450 CYP3A/biosynthesis , Enzyme Induction/drug effects , Glucuronosyltransferase/metabolism , Hep G2 Cells , Hepatocytes/metabolism , Hepatocytes/pathology , Humans
7.
Int J Mol Sci ; 10(1): 37-49, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19333434

ABSTRACT

Embryonic midbrain micromass cultures were exposed for five days to ochratoxin A (OTA) at seven concentrations (ranging from 0.16 to 10 microg/mL). Cell viability was assessed in neutral red uptake test (NRU), and differentiation - by immunoenzymatic determination of structural proteins (beta(III)-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 (microg/mL) for cytotoxicity (IC(50)) and differentiation (ID(50)) 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 ID(50) value was very low (< 10 microg/mL) so OTA was classified as a strong teratogen. IC(50)/ ID(50) ratios <2 pointed out that with harmful action of OTA the basic cytotoxicity should be connected.


Subject(s)
Astrocytes/drug effects , Carcinogens/toxicity , Mesencephalon/drug effects , Neurons/drug effects , Ochratoxins/toxicity , Animals , Cell Differentiation/drug effects , Cell Survival/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Mesencephalon/cytology , Mesencephalon/embryology , Rats , Rats, Wistar
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