Alternatives to Laboratory Animals

This year saw a Black Box Prize award, for definitive work on the Adverse Outcome Pathway for skin sensitisation

Efforts toward replacing the use of animals in toxicology testing have begun to make significant headway in the last several years, due to co-operative and pragmatic efforts on the part of many stakeholders, and the public pressure that non-governmental advocacy organisations represent. Science-focused advocacy organisations have a unique role to play in these efforts, as they often have flexibility to adapt quickly to keep a project going and forge connections among different kinds of stakeholders to help encourage buy-in. This year, meaningful progress has been made, especially in regulatory laws and policies, which will lead to the replacement of animals in toxicology testing. In order to keep this momentum, we need to measure progress — but this requires improved transparency and regular reporting of animal use. In addition, we should consider how strategies that have successfully reduced and replaced animal use in toxicology can be applied to basic biomedical research practices.

The Adverse Outcome Pathway (AOP) concept is expected to guide risk assessors in their work to use all existing information on the effects of chemicals on humans and wildlife, and to target the generation of additional information to the regulatory objective. AOPs will therefore be used in the Organisation for Economic Co-operation and Development (OECD) chemical safety programme, as underlying scientific rationales for the development of alternative methods for hazard assessment, such as read-across, in vitro test methods or the development of integrated testing strategies that have the potential to replace animal tests. As a proof-of-concept, the OECD has developed an AOP for skin sensitisation, and as a follow-up has: a) implemented the AOP into the OECD QSAR Toolbox, so that information related to the Key Events (KEs) in the AOP can be used to group chemicals that are expected to act by the same mechanism and hence have the same skin sensitisation potential; b) developed alternative test methods for the KEs, so that ultimately chemicals can be tested for skin sensitisation without the use of animal tests. The development of integrated testing strategies based on the AOP is ongoing. Building on this proof-of-concept, the OECD has launched an AOP development programme with a first batch of AOPs published in 2016. A number of IT tools, which together form an AOP Knowledge Base, are at various stages of development, and support the construction of AOPs and their use in the development of integrated approaches for testing and assessment. Following the publication of the first batch of AOPs, OECD member countries will decide on priorities for their use in supporting the development of tools for regulatory use.

For toxicologists who are in any way associated with skin sensitisation, the last two decades have seen a series of fundamental changes. We have migrated from old-style guinea-pig assays, via the refined and reduced Local Lymph Node Assay (LLNA), to witness the imminent dominance of in vitro and in silico methods. Yet, over the same period, the use of the output data for human safety assurance has evolved from ‘black box’ risk assessment, via the quantitative risk assessment enabled by the LLNA measurement of potency, to a new period of relative uncertainty. This short review will endeavour to address these topics, all the while keeping a focus on three essential principles: a) that skin sensitisation potential is intrinsic in the molecular structure of the chemical; b) that test methods should have a mechanistic foundation; and finally c) that the only reason for undertaking any skin sensitisation work has to be the protection of human health.

Over the past 20 years or more, investigators have been developing non-animal test methods for use in assessing the skin sensitisation potential of chemicals. In parallel with this effort, the key biological events of skin sensitisation have been well-characterised in an Adverse Outcome Pathway (AOP) proposed by the Organisation for Economic Co-operation and Development (OECD). The key molecular initiating event of this AOP is haptenation or covalent modification of epidermal proteins. In this review, the strengths and limitations of the Direct Peptide Reactivity Assay (DPRA) are described, and the more recently developed Peroxidase Peptide Reactivity Assay (PPRA). The DPRA has been formally validated and incorporated into an OECD Test Guideline (TG442C). The DPRA shows promise for assisting in hazard identification as well as for assessing skin sensitisation potency when used in an integrated testing strategy.

The 21st century paradigm for toxicology and the adverse outcome pathway concept envisage a future toxicology largely based on mechanistic in vitro assays and relying mainly on cellular models. In the skin sensitisation field, this concept was not intuitive at the beginning. Given the high structural diversity of skin sensitising molecules, classical receptor binding as the molecular initiating event in a cell-based assay could be excluded from the start, leaving the question of how cells could sense potential skin sensitising chemicals and be able to differentiate them from non-sensitisers. When we entered this field in 2006, we realised that, in another emerging field of toxicology, detailed work on the antioxidant/electrophile sensing pathway Keap1/Nrf2/ARE was being performed. We postulated that, based on their intrinsic electrophilicity, a large structural variety of skin sensitisers would activate this pathway. This was demonstrated in a preliminary pilot study with an existing, breast cancer-derived reporter cell line. Broader confirmation of this initial hypothesis then came from a multitude of genome-wide studies, in which sensitiser-induced changes to the transcriptome were investigated. The results showed that this regulatory pathway is indeed the most common regulatory pathway activated by sensitisers at the gene expression level, and the underlying event in keratinocytes has become formalised as a Key Event in the Organisation for Economic Co-operation and Development (OECD) Adverse Outcome Pathway for sensitisation. These studies led to the development of the KeratinoSens^® assay, which became the first cell-based in vitro test for skin sensitisation to be endorsed by a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) statement and an OECD Test Guideline. More recently, a number of studies have further developed this approach into 3-D skin models. Here, we review the underlying mechanism and the development of the KeratinoSens assay. We also present data on the stability of the assay over time, which is a key requirement for a cell-based biological assay to be endorsed in a regulatory context.

This article outlines the work of the Organisation for Economic Co-operation and Development (OECD) that led to being jointly awarded the 2015 Lush Black Box Prize. The award-winning work centred on the development of ‘The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins’. This Adverse Outcome Pathway (AOP) has provided the mechanistic basis for the integration of skin sensitisation-related information. Recent developments in integrated approaches to testing and assessment, based on the AOP, are summarised. The impact of the AOP on regulatory policy and on the Three Rs are discussed. An overview of the next generation of the skin sensitisation AOP module in the OECD QSAR Toolbox, based on more-recent work at the Laboratory of Mathematical Chemistry, is also presented.

The Lush Prize supports animal-free testing by rewarding the most effective projects and individuals who have been working toward the goal of replacing animals in product or ingredient safety testing. Prizes are awarded for developments in five strategic areas: Science; Lobbying; Training; Public Awareness; and Young Researchers. Should there be a major breakthrough in 21st century toxicology, a Black Box Prize equivalent to the entire annual fund of £250,000 is awarded. A Background Paper is prepared each year, prior to the judging process, to provide the panel with a brief overview of current developments in the field of Replacement alternatives, particularly those relevant to the concept of toxicity pathways. The Background Paper includes information on recent work by the relevant scientific institutions and projects in this area, including AXLR8, the OECD, The Hamner Institutes, the Human Toxome Project, EURL ECVAM, ICCVAM, the US Tox21 Programme, the ToxCast programme, and the Human Toxicology Project Consortium. Recent developments in toxicity pathway research are also assessed by reviewing the relevant literature (including conference proceedings), and the abstracts and papers receiving the highest score are presented to the judges for consideration.

Body-on-a-chip systems replicate the size relationships of organs, blood distribution and blood flow, in accordance with human physiology. When operated with tissues derived from human cell sources, these systems are capable of simulating human metabolism, including the conversion of a pro-drug to its effective metabolite, as well as its subsequent therapeutic actions and toxic side-effects. The system also permits the measurement of human tissue electrical and mechanical reactions, which provide a measure of functional response. Since these devices can be operated with human tissue samples or with in vitro tissues derived from induced pluripotent stem cells (iPS), they can play a significant role in determining the success of new pharmaceuticals, without resorting to the use of animals. By providing a platform for testing in the context of human metabolism, as opposed to animal models, the systems have the potential to eliminate the use of animals in preclinical trials. This article will review progress made and work achieved as a direct result of the 2015 Lush Science Prize in support of animal-free testing.

This paper highlights the work for which OncoTheis, a Swiss biotechnology company, engaged in the development of innovative bioengineered tissues and organoids for cancer research, was co-awarded the 2015 Lush Science Prize. Noting that the use of animal models failed to lead to the design of effective treatments for cancer, OncoTheis has opted to develop in vitro models based exclusively on human cells. The company currently focuses on lung cancer, which is the leading cause of cancer-related deaths worldwide, with more than one million deaths per year. To address this public health concern, we developed OncoCilAir^™, a new 3-D model that mimics in vitro the progression of the disease as it happens in patients. In this system, bronchial and lung tumour cells obtained from discarded surgical tissue are co-cultured in a Petri dish to reconstitute a fragment of the human lung. After appropriate differentiation, the culture closely reproduces malignant pulmonary nodules invading a small piece of functional airway tissue. As OncoCilAir includes both healthy and cancerous tissues, it can be used to test tumour-killing activity and the adverse effects of chemotherapies and other anti-cancer drugs. Moreover, a single culture can be maintained for up to three months, which permits studies of longer-term effects, including the assessment of drug resistance and tumour recurrence. OncoCilAir heralds a new generation of integrated in vitro models, which is expected to increase the quality of preclinical research while replacing animal testing.

In 2015, the PETA International Science Consortium Ltd. was awarded the Lush Training Prize for its broad approach to education and training on the effective use of human-relevant, non-animal research techniques. The prize was awarded for work that included hosting workshops and webinars, initiating in-person training sessions and developing educational resources. The Consortium works closely with industry and regulatory agencies to identify and overcome barriers to the validation and use of alternatives to animal testing, by using an approach that identifies, promotes and verifies the implementation of these methods. The Consortium's recent activities toward replacing tests on animals for nanomaterials, pesticides and medical devices, are described, as examples of projects with broad applicability aimed at large-scale regulatory change.