Article

How to Foster ‘New Approach Methodology’ Toxicologists

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

The need to reduce, refine and replace animal experimentation has led to a boom in the establishment of new approach methodologies (NAMs). This promising trend brings the hope that the replacement of animals by using NAMs will become increasingly accepted by regulators, included in legislation, and consequently more-often implemented by industry. The majority of NAMs, however, are still not very well understood, either due to the complexity of the applied approach or the data analysis workflow. A potential solution to this problem is the provision of better educational resources to scientists new to the area — showcasing the added value of NAMs and outlining various ways of overcoming issues associated with knowledge gaps. In this paper, the educational exchange between four institutions — namely, two universities and two SMEs — via a series of video training sessions, is described. The goal of this exchange was to showcase an exemplary event to help introduce scientists to non-animal approaches, and to actively support the development of resources enabling the use of alternatives to laboratory animals.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

ResearchGate has not been able to resolve any citations for this publication.
Full-text available
Article
The aim of this paper is to describe the essential points of Italian and European legislation governing the use of animals in biomedical experimentation. A close look will be taken at the principles of the 3Rs, which represent the mainstay of the legal architecture based on which a correct interpretation may be drawn of the legislative documents on animal experimentation. Furthermore, this paper will address the ways in which Directive 2010/63/EU is implemented in Italian legislation on the welfare of laboratory animals. In addition to an assessment of legal issues (such as the scope of jurisdiction of supervisory authorities tasked with issuing authorizations), it will include a discussion of cases of inadequate and insufficient implementation of the requirements laid down by Directive 2010/63/EU. Both the consistency of the interpretation of national legislation with the Directive and the direct effectiveness of the Directive in national law, in which animal testing has been and still is the subject of heated debate between supporters and opponents, will be examined.
Full-text available
Article
Since the Three Rs of replacement, reduction and refinement was proposed by Russel and Birch in 1959, researchers have a moral duty to minimize harm to animals. Even though animal experiments are performed by the Three Rs concept, animal researches which do not comply with international rules and standards are not accepted as well. As animal welfare has been important global issues, the methods to assess animal welfare compromise and distress have been proposed. Humanity is accepted as the goal of the Three Rs, however, another fourth R, ‘Refusal’ of fruitless protocol or ‘Responsibility’ for the experimental animal and social, scientific status of the animal experiments has been proposed. After establishing goals of animal research in a respective society, reliable knowledge can be obtained while improving laboratory animal welfare.
Full-text available
Article
The need to develop new tools and increase capacity to test pharmaceuticals and other chemicals for potential adverse impacts on human health and the environment is an active area of development. Much of this activity was sparked by two reports from the US National Research Council (NRC) of the National Academies of Sciences, Toxicity Testing in the Twenty-first Century: A Vision and a Strategy (2007) and Science and Decisions: Advancing Risk Assessment (2009), both of which advocated for "science-informed decision-making" in the field of human health risk assessment. The response to these challenges for a "paradigm shift" toward using new approach methodologies (NAMS) for safety assessment has resulted in an explosion of initiatives by numerous organizations, but, for the most part, these have been carried out independently and are not coordinated in any meaningful way. To help remedy this situation, a framework that presents a consistent set of criteria, universal across initiatives, to evaluate a NAM's fit-for-purpose was developed by a multi-stakeholder group of industry, academic, and regulatory experts. The goal of this framework is to support greater consistency across existing and future initiatives by providing a structure to collect relevant information to build confidence that will accelerate, facilitate and encourage development of new NAMs that can ultimately be used within the appropriate regulatory contexts. In addition, this framework provides a systematic approach to evaluate the currently-available NAMs and determine their suitability for potential regulatory application. This 3-step evaluation framework along with the demonstrated application with case studies, will help build confidence in the scientific understanding of these methods and their value for chemical assessment and regulatory decision-making.
Full-text available
Article
This paper summarizes current challenges, the potential use of novel scientific methodologies, and ways forward in the risk assessment and risk management of mixtures. Generally, methodologies to address mixtures have been agreed; however, there are still several data and methodological gaps to be addressed. New approach methodologies can support the filling of knowledge gaps on the toxicity and mode(s) of action of individual chemicals. (Bio)Monitoring, modeling, and better data sharing will support the derivation of more realistic co-exposure scenarios. As knowledge and data gaps often hamper an in-depth assessment of specific chemical mixtures, the option of taking account of possible mixture effects in single substance risk assessments is briefly discussed. To allow risk managers to take informed decisions, transparent documentation of assumptions and related uncertainties is recommended indicating the potential impact on the assessment. Considering the large number of possible combinations of chemicals in mixtures, prioritization is needed, so that actions first address mixtures of highest concern and chemicals that drive the mixture risk. As chemicals with different applications and regulated separately might lead to similar toxicological effects, it is important to consider chemical mixtures across legislative sectors.
Full-text available
Article
In modern toxicology, substantial efforts are undertaken to develop alternative solutions for in vivo toxicity testing. The adverse outcome pathway (AOP) concept could facilitate knowledge-based safety assessment of chemicals that does not rely exclusively on in vivo toxicity testing. The construction of an AOP is based on understanding toxicological processes at different levels of biological organisation. Here, we present the developed AOP for liver fibrosis and demonstrate a linkage between hepatic injury caused by chemical protein alkylation and the formation of liver fibrosis, supported by coherent and consistent scientific data. This long-term process, in which inflammation, tissue destruction, and repair occur simultaneously, results from the complex interplay between various hepatic cell types, receptors, and signalling pathways. Due to the complexity of the process, an adequate liver fibrosis cell model for in vitro evaluation of a chemical's fibrogenic potential is not yet available. Liver fibrosis poses an important human health issue that is also relevant for regulatory purposes. An AOP described with enough mechanistic detail might support chemical risk assessment by indicating early markers for downstream events and thus facilitating the development of an in vitro testing strategy. With this work, we demonstrate how the AOP framework can support the assembly and coherent display of distributed mechanistic information from the literature to support the use of alternative approaches for prediction of toxicity. This AOP was developed according to the guidance document on developing and assessing AOPs and its supplement, the users' handbook, issued by the Organisation for Economic Co-operation and Development.
Full-text available
Article
Simple Summary This article reviews the use of non-human animals in biomedical research from a historical viewpoint, providing an insight into the most relevant social and moral issues on this topic across time, as well as to how the current paradigm for ethically and publically acceptable use of animals in biomedicine has been achieved. Abstract The use of non-human animals in biomedical research has given important contributions to the medical progress achieved in our day, but it has also been a cause of heated public, scientific and philosophical discussion for hundreds of years. This review, with a mainly European outlook, addresses the history of animal use in biomedical research, some of its main protagonists and antagonists, and its effect on society from Antiquity to the present day, while providing a historical context with which to understand how we have arrived at the current paradigm regarding the ethical treatment of animals in research.
Article
Exposure to environmental chemicals, particularly those with persistent and bioaccumulative properties have been linked to liver diseases. Induction of fibrotic pathways is considered as a pre-requirement of chemical induced liver fibrosis. Here, we applied 3D in vitro human liver microtissues (MTs) composed of HepaRG, THP-1 and hTERT-HSC that express relevant hepatic pathways (bile acid, sterol, and xenobiotic metabolism) and can recapitulate key events of liver fibrosis (e.g. extracellular matrix-deposition). The liver MTs were exposed to a known profibrotic chemical, thioacetamide (TAA) and three representative environmental chemicals (TCDD, benzo [a] pyrene (BaP) and PCB126). Both TAA and BaP triggered fibrotic pathway related events such as hepatocellular damage (cytotoxicity and decreased albumin release), hepatic stellate cell activation (transcriptional upregulation of α-SMA and Col1α1) and extracellular matrix remodelling. TCDD or PCB126 at measured concentrations did not elicit these responses in the 3D liver MTs system, though they caused cytotoxicity in HepaRG monoculture at high concentrations. Reduced human transcriptome (RHT) analysis captured molecular responses involved in liver fibrosis when MTs were treated with TAA and BaP. The results suggest that 3D, multicellular, human liver microtissues represent an alternative, human-relevant, in vitro liver model for assessing fibrotic pathways induced by environmental chemicals.
Chapter
This chapter reflects on the challenges and opportunities associated with establishing risk acceptance for risk assessment and management. Risk assessment and management have evolved from significant accidents associated with industrial activities—the same industrial activities that are created for our benefit to supply energy, food, and commodities. If risks cannot be eliminated, then how low is low enough in reducing the level of risk? The notation of acceptability immediately raises the question of acceptable to whom? At some point, someone needs to decide if risks are acceptable, but the general public has great difficulty with the concept of acceptable risk. Public perception plays a critical role in defining the risk versus development relationship. The public perception of risk is dependent on awareness and understanding of potential hazards and their likelihood of occurrence, and most importantly, effective communication of these along with the associated uncertainty. Public awareness can have a profound effect on the development of public policy, which in many cases is driven more by perception rather than by sound science. This chapter sheds light on this crucial issue. It investigates two commonly used concepts of policy and decision-making, the Precautionary Principle (PP) and As Low As Reasonably Practicable (ALARP). The paper provides a clearer understanding of both approaches and proposes a process to help readers understand where and when PP versus ALARP would be most applicable.
Article
Disrupted regulation and accumulation of bile salts (BS) in the liver can contribute towards progressive liver damage and fibrosis. Here, we investigated the role of BS in the progression of cholestatic injury and liver fibrosis using 3D scaffold-free multicellular human liver microtissues (MTs) comprising the cell lines HepaRG, THP-1 and hTERT-HSCs. This in vitro model has been shown to recapitulate cellular events leading to fibrosis including hepatocellular injury, inflammation and activation of HSCs, ultimately leading to increased deposition of extracellular matrix (ECM). In order to better differentiate the contribution of individual cells during cholestasis, the effects of BS were evaluated either on each of the three cell types individually or on the multicellular MTs. Our data corroborate the toxic effects of BS on HepaRG cells and indicate that BS exposure elicited a slight increase in cytokines without causing stellate cell activation. Contrarily, using the MTs, we could demonstrate that low concentrations of BS led to cellular damage and triggered a fibrotic response. This indicates that cellular interplay is required to achieve BS-triggered activation of HSC. Moreover, BS were capable of down-regulating CYP7A1 expression in MTs and elicited abnormal lipid production (accumulation) concordant with clinical cases where chronic cholestasis results in hypercholesterolemia.
Chapter
Drug-induced liver injury (DILI) is a major clinical and regulatory challenge. As a result, interest in DILI biomarkers is growing. So far, considerable progress has been made in identification of biomarkers for diagnosis (acetaminophen-cysteine protein adducts), prediction (genetic biomarkers), and prognosis (microRNA-122, high mobility group box 1 protein, keratin-18, glutamate dehydrogenase, mitochondrial DNA). Many of those biomarkers also provide mechanistic insight. The purpose of this chapter is to review major advances in DILI biomarker research over the last decade, and to highlight some of the challenges involved in implementation. Although much work has been done, more liver-specific biomarkers, more DILI-specific biomarkers, and better prognostic biomarkers for survival are all still needed. Furthermore, more work is needed to define reference intervals and medical decision limits.
Since the 1990 s, science based ecological risk assessments constitute an essential tool for supporting decision making in the regulatory context. Using the European REACH Regulation as example, this paper presents the challenges and opportunities for new scientific developments within the area of chemical control and environmental protection. These challenges can be sorted out in three main related topics. In the short term, the challenges are directly associated with the regulatory requirements, required for facilitating a scientifically sound implementation of the different obligations for industry and authorities. It is important to mention that although the actual tools are different due to the regulatory requirements, the basic needs are still the same than those addressed in the early 1990 s: understanding the ecological relevance of the predicted effects, including the uncertainty, and facilitating the link with the socio-economic assessment. The second set covers the opportunities for getting an added value from the regulatory efforts. The information compiled through REACH registration and notification processes is analyzed as source for new integrative developments for assessing the combined chemical risk at the regional level. Finally, the paper discusses the challenge of inverting the process and developing risk assessment methods focusing on the receptor, the individual or ecosystem, instead of on the stressor or source. These approaches were limited in the past due to the lack of information, but the identification and dissemination of standard information, including uses, manufacturing sites, physical-chemical, environmental, ecotoxicological and toxicological properties as well as operational conditions and risk management measures for thousands of chemicals, combined by the knowledge gathered through large scale monitoring programs and spatial information systems is generating new opportunities. The challenge is liking predictions and measured data in an integral "-omic type" approach considering collectively data from different sources, and offering a complete assessment of the chemical risk of individuals and ecosystems, with new conceptual approaches that could be defined as "risk-omics based" paradigms and models. Integr Environ Assess Manag © 2013 SETAC.
Article
Although they have several important limitations primary human hepatocytes still represent the in vitro gold standard model for xenobiotic metabolism and toxicity studies. The large use of human liver cell lines either from tumoral origin or obtained by oncogenic immortalisation is prevented by the loss of various liver-specific functions, especially many cytochrome P450 (CYP)-related enzyme activities. We review here recent results obtained with a new human hepatoma cell line, named HepaRG, derived from a human hepatocellular carcinoma. These cells exhibit unique features: when seeded at low density they acquire an elongated undifferentiated morphology, actively divided and after having reached confluency formed typical hepatocyte-like colonies surrounded by biliary epithelial-like cells. Moreover contrary to other human hepatoma cell lines including HepG2 cells, HepaRG cells express various CYPs (CYP1A2, 2B6, 2C9, 2E1, 3A4) and the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) at levels comparable to those found in cultured primary human hepatocytes. They also express various other functions such phase 2 enzymes, apical and canalicular ABC transporters and basolateral solute carrier transporters, albumin, haptoglobin as well as aldolase B that is a specific marker of adult hepatocytes. HepaRG cells could represent a surrogate to primary human hepatocytes for xenobiotic metabolism and toxicity studies and even more, a unique model system for analysing genotoxic compounds.