Mardas Daneshian

A roadmap for the development of alternative (non-animal) methods for systemic toxicity testing - t4 report

Systemic toxicity testing forms the cornerstone for the safety evaluation of substances. Pressures to move from traditional animal models to novel technologies arise from various concerns, including: the need to evaluate large numbers of previously untested chemicals and new products (such as nanoparticles or cell therapies), the limited predictivity of traditional tests for human health effects, duration and costs of current approaches, and animal welfare considerations. The latter holds especially true in the context of the scheduled 2013 marketing ban on cosmetic ingredients tested for systemic toxicity. Based on a major analysis of the status of alternative methods (Adler et al., 2011) and its independent review (Hartung et al., 2011), the present report proposes a roadmap for how to overcome the acknowledged scientific gaps for the full replacement of systemic toxicity testing using animals. Five whitepapers were commissioned addressing toxicokinetics, skin sensitization, repeated-dose toxicity, carcinogenicity, and reproductive toxicity testing. An expert workshop of 35 participants from Europe and the US discussed and refined these whitepapers, which were subsequently compiled to form the present report. By prioritizing the many options to move the field forward, the expert group hopes to advance regulatory science.

Metabolomics in toxicology and preclinical research.

Metabolomics, the comprehensive analysis of metabolites in a biological system, provides detailed information about the biochemical/physiological status of a biological system, and about the changes caused by chemicals. Metabolomics analysis is used in many fields, ranging from the analysis of the physiological status of genetically modified organisms in safety science to the evaluation of human health conditions. In toxicology, metabolomics is the -omics discipline that is most closely related to classical knowledge of disturbed biochemical pathways. It allows rapid identification of the potential targets of a hazardous compound. It can give information on target organs and often can help to improve our understanding regarding the mode-of-action of a given compound. Such insights aid the discovery of biomarkers that either indicate pathophysiological conditions or help the monitoring of the efficacy of drug therapies. The first toxicological applications of metabolomics were for mechanistic research, but different ways to use the technology in a regulatory context are being explored. Ideally, further progress in that direction will position the metabolomics approach to address the challenges of toxicology of the 21st century. To address these issues, scientists from academia, industry, and regulatory bodies came together in a workshop to discuss the current status of applied metabolomics and its potential in the safety assessment of compounds. We report here on the conclusions of three working groups addressing questions regarding 1) metabolomics for in vitro studies 2) the appropriate use of metabolomics in systems toxicology, and 3) use of metabolomics in a regulatory context.

Consensus Report on the Future of Animal-Free Systemic Toxicity Testing

Since March 2013, animal use for cosmetics testing for the European market has been banned. This requires a renewed view on risk assessment in this field. However, in other fields as well, traditional animal experimentation does not always satisfy requirements in safety testing, as the need for human-relevant information is ever increasing. A general strategy for animal-free test approaches was outlined by the US National Research Council`s vision document for Toxicity Testing in the 21st Century in 2007. It is now possible to provide a more defined roadmap on how to implement this vision for the four principal areas of systemic toxicity evaluation: repeat dose organ toxicity, carcinogenicity, reproductive toxicity and allergy induction (skin sensitization), as well as for the evaluation of toxicant metabolism (toxicokinetics) (Fig. 1). CAAT-Europe assembled experts from Europe, America and Asia to design a scientific roadmap for future risk assessment approaches and the outcome was then further discussed and refined in two consensus meetings with over 200 stakeholders. The key recommendations include: focusing on improving existing methods rather than favoring de novo design; combining hazard testing with toxicokinetics predictions; developing integrated test strategies; incorporating new high content endpoints to classical assays; evolving test validation procedures; promoting collaboration and data-sharing of different industrial sectors; integrating new disciplines, such as systems biology and high throughput screening; and involving regulators early on in the test development process. A focus on data quality, combined with increased attention to the scientific background of a test method, will be important drivers. Information from each test system should be mapped along adverse outcome pathways. Finally, quantitative information on all factors and key events will be fed into systems biology models that allow a probabilistic risk assessment with flexible adaptation to exposure scenarios and individual risk factors.

The Use of Biomarkers of Toxicity for Integrating In Vitro Hazard Estimates Into Risk Assessment for Humans

The role that in vitro systems can play in toxicological risk assessment is determined by the appropriateness of the chosen methods, with respect to the way in which in vitro data can be extrapolated to the in vivo situation. This report presents the results of a workshop aimed at better defining the use of in vitro-derived biomarkers of toxicity (BoT) and determining the place these data can have in human risk assessment. As a result, a conceptual framework is presented for the incorporation of in vitro-derived toxicity data into the risk assessment process. The selection of BoT takes into account that they need to distinguish adverse and adaptive changes in cells. The framework defines the place of in vitro systems in the context of data on exposure, structural and physico-chemical properties, and toxicodynamic and biokinetic modeling. It outlines the determination of a proper point-of-departure (PoD) for in vitro-in vivo extrapolation, allowing implementation in risk assessment procedures. A BoT will need to take into account both the dynamics and the kinetics of the compound in the in vitro systems. For the implementation of the proposed framework it will be necessary to collect and collate data from existing literature and new in vitro test systems, as well as to categorize biomarkers of toxicity and their relation to pathways-of-toxicity. Moreover, data selection and integration need to be driven by their usefulness in a quantitative in vitro-in vivo extrapolation (QIVIVE).

Biology-inspired microphysiological system approaches to solve the prediction dilemma of substance testing

The recent advent of microphysiological systems - microfluidic biomimetic devices that aspire to emulate the biology of human tissues, organs and circulation in vitro - is envisaged to enable a global paradigm shift in drug development. An extraordinary US governmental initiative and various dedicated research programs in Europe and Asia have led recently to the first cutting-edge achievements of human single-organ and multi-organ engineering based on microphysiological systems. The expectation is that test systems established on this basis would model various disease stages, and predict toxicity, immunogenicity, ADME profiles and treatment efficacy prior to clinical testing. Consequently, this technology could significantly affect the way drug substances are developed in the future. Furthermore, microphysiological system-based assays may revolutionize our current global programs of prioritization of hazard characterization for any new substances to be used, for example, in agriculture, food, ecosystems or cosmetics, thus, replacing laboratory animal models used currently. Thirty-six experts from academia, industry and regulatory bodies present here the results of an intensive workshop (held in June 2015, Berlin, Germany). They review the status quo of microphysiological systems available today against industry needs, and assess the broad variety of approaches with fit-for-purpose potential in the drug development cycle. Feasible technical solutions to reach the next levels of human biology in vitro are proposed. Furthermore, key organ-on-a-chip case studies, as well as various national and international programs are highlighted. Finally, a roadmap into the future is outlined, to allow for more predictive and regulatory-accepted substance testing on a global scale.

Validation and quality control of replacement alternatives – current status and future challenges

Alternatives to animal testing have been developed mainly in the fields of toxicology and vaccine testing. Typical examples are the evaluation of phototoxicity, eye irritation or skin corrosion/irritation of cosmetics and industrial chemicals. However, examples can also be found in other biomedical areas, such the control of the quality of drug preparations for pyrogens or for the control of the production process of biologics, such as botulinum neurotoxin. For regulatory purposes, the quality, transferability and predictivity of an alternative method needs to be evaluated. This procedure is called the “validation process” of a new method. It follows defined rules, and several governmental institutions have been established to perform, supervise or advise on this process. As this often results in a delay of method implementation, different alternatives for the evaluation of a methods suitability and quality are under discussion. We describe here the principles of model development and quality control. We also give an overview on methods that have undergone validation. Strengths and shortcomings of traditional approaches are discussed, and new developments and challenges are outlined.

Toxicity testing in the 21st century beyond environmental chemicals

Summary After the publication of the report titled Toxicity Testing in the 21st Century – A Vision and a Strategy, many initiatives started to foster a major paradigm shift for toxicity testing – from apical endpoints in animal-based tests to mechanistic endpoints through delineation of pathways of toxicity (PoT) in human cell based systems. The US EPA has funded an important project to develop new high throughput technologies based on human cell based in vitro technologies. These methods are currently being incorporated into the chemical risk assessment process. In the pharmaceutical industry, the efficacy and toxicity of new drugs are evaluated during preclinical investigations that include drug metabolism, pharmacokinetics, pharmacodynamics and safety toxicology studies. The results of these studies are analyzed and extrapolated to predict efficacy and potential adverse effects in humans. However, due to the high failure rate of drugs during the clinical phases, a new approach for a more predictive assessment of drugs both in terms of efficacy and adverse effects is getting urgent. The food industry faces the challenge of assessing novel foods and food ingredients for the general population, while using animal safety testing for extrapolation purposes is often of limited relevance. The question is whether the latest paradigm shift proposed by the Tox21c report for chemicals may provide a useful tool to improve the risk assessment approach also for drugs and food ingredients.

Evidence for the detection of non-endotoxin pyrogens by the whole blood monocyte activation test.

Threats of pyrogenicity were discovered more than a century ago. Measures to determine the safety of parenterals and, more recently, medical devices and cell therapies for human use have been in place for 70 years. Currently, there are three testing possibilities available: the Rabbit Pyrogen Test, the Limulus Amebocyte Lysate test (Bacterial Endotoxin Test), and test systems using human whole blood or human monocytes, called Monocyte Activation Test (MAT). The MAT is based on the human fever reaction and thus most closely reflects the human situation. Unfortunately, regulations and testing guidelines are not fully harmonized, despite formal international validation. Furthermore, data showing that the MAT is capable of covering the totality of possible pyrogens relevant to humans were not included in the MAT validations of the last decade. For this review we collate evidence from published literature, unpublished data of our own, and results from the international validation study to show that there is overwhelming scientific evidence to conclude that the whole blood MAT reliably detects non-endotoxin pyrogens. Therefore, further validation exercises do not seem warranted.

Assessment of pyrogenic contaminations with validated human whole-blood assay

We present an internationally validated protocol for the evaluation of pyrogenic contaminations using human whole blood. In the in vitro pyrogen test (IPT) the sample is incubated with fresh or cryopreserved human whole blood, and the proinflammatory cytokine interleukin-1β (IL-1β) is detected by enzyme-linked immunosorbent assay (ELISA). In addition to detecting pyrogenic contaminations in aqueous samples, e.g., parenteral drugs; adaptations allow the assessment of lipidic, toxic or immunomodulatory substances; detection of low-grade contaminations in large-volume parenterals, e.g., dialysis water and fluids; pyrogenicity assessment of solid materials, e.g., medical devices; and evaluation of airborne pyrogenic burden. In contrast to the rabbit pyrogen test and the limulus amoebocyte lysate (LAL) test, it requires no components of animal origin. In comparison with the LAL, it also detects nonlipopolysaccharide pyrogens. In comparison with other monocyte activation tests it requires no cell preparation steps or cell culture facilities. The procedure takes 21–35 h to complete.

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

Models of the outer epithelia of the human body - namely the skin, the intestine and the lung - have found valid applications in both research and industrial settings as attractive alternatives to animal testing. A variety of approaches to model these barriers are currently employed in such fields, ranging from the utilization of ex vivo tissue to reconstructed in vitro models, and further to chip-based technologies, synthetic membrane systems and, of increasing current interest, in silico modeling approaches. An international group of experts in the field of epithelial barriers was convened from academia, industry and regulatory bodies to present both the current state of the art of non-animal models of the skin, intestinal and pulmonary barriers in their various fields of application, and to discuss research-based, industry-driven and regulatory-relevant future directions for both the development of new models and the refinement of existing test methods. Issues of model relevance and preference, validation and standardization, acceptance, and the need for simplicity versus complexity were focal themes of the discussions. The outcomes of workshop presentations and discussions, in relation to both current status and future directions in the utilization and development of epithelial barrier models, are presented by the attending experts in the current report.