In vivo inhalation methods for respiratory toxicity and disease in laboratory animals are time-consuming, technically demanding and of doubtful relevance for predicting effects in humans. They can also cause great suffering to the animals used. Fortunately, there has been increasing interest in developing in vitro alternatives. They can greatly simplify exposure and dosimetry, while using human cell lines and primary cells representative of all the cell types of the conducting airways. Maintained on membranes, fed basally and exposed apically at the air. liquid interface, the cells retain their in vivo-like properties long enough for even chronic exposure and response studies to be undertaken. The predictive performance of several models is showing encouraging signs of improvement, especially when undertaken in conjunction with the latest airway particle deposition and gaseous exchange algorithms. Additional improvements in test substance delivery, automation and miniaturisation continue to be made. However, there needs to be more guidance and harmonisation applied to: (a) exposure modelling; (b) the need for exogenous metabolic activation; and (c) the use of positive and negative controls. With levels of inhalation testing and pulmonary diseases increasing, the need to expedite validation is now urgent. There are signs that the necessary dialogue and cooperation between the principal stakeholders is starting to occur, but regulatory authorities need to take the initiative to ensure that 'actions speak louder than words'.

A short introduction to the principal technologies that have had the most influence on the development and use of alternatives in toxicology and safety testing is provided. Advances in analytical and diagnostic methods, and a greater understanding of cell biology and molecular mechanisms of toxicity, have enhanced the range and detection of toxicity endpoints and, therefore, the relevance of test systems and data interpretation, as well as enabling an increased role for human studies.

KEYWORDS: Alternative test methodsanimal testsdrug efficacydrug safetyhuman variationintegrated testinginterspecies differences

The challenge of the need for change was thrown down when Russell and Burch proposed the Three Rs in 1959, and, among others, Smyth responded by focusing on the concept of alternatives. The Fund for the Replacement of Animals in Medical Experiments (FRAME), formed in 1969 to seek the replacement of animal tests by better methods, played a major role, through its journal, ATLA (Alternatives to Laboratory Animals), its Toxicity Committee, its Research Programme, including the FRAME Alternatives Laboratory at the University of Nottingham, and its good relations with academia, government, industry and scientists in many parts of the world. FRAME played a pivotal role in the drafting and passage of the Animals (Scientific Procedures) Act 1986, which had a sound Three Rs basis, received the first government grant specifically for alternatives research, and also provided the first Head of the European Centre for the Validation of Alternative Methods (ECVAM). The National Centre for the Three Rs (NC3Rs), set up in 2004, rapidly established effective collaborations with science and industry, and its flagship initiative, CRACK IT, focuses on the development of new technologies to meet challenges put forward by its sponsors. Many other individuals and organisations have made major contributions to the promotion of the Three Rs, and, in particular, the Central Toxicology Laboratory, originally at Imperial Chemical Industries (ICI), and Unilever Research. Julia Fentem, now at Unilever, has had a notably unique career, including important periods at the University of Nottingham, FRAME and ECVAM.

A short introduction to the principal technologies that have had the most influence on the development and use of alternatives in toxicology and safety testing is provided. Advances in analytical and diagnostic methods, and a greater understanding of cell biology and molecular mechanisms of toxicity, have enhanced the range and detection of toxicity endpoints and, therefore, the relevance of test systems and data interpretation, as well as enabling an increased role for human studies.

The scientific rationale for undertaking first-in-human volunteer studies at earlier than the usual stages of pharmaceutical development involving efficacy and safety assessment, within the stipulations of the Declaration of Helsinki, is discussed. The advantages and disadvantages of various clinical techniques, including diagnostic imaging, biomonitoring to estimate levels of endogenous exposure from levels of biomarkers in body fluids, and the administration of microdose levels (< 100 mg/each administration) of drug candidates (phase 0 trials), coupled with ultra-sensitive analytical detection methods, are considered, with the use of examples. It is concluded that: (a) the above-mentioned techniques offer substantial scientific, financial and logistical benefits, while remaining largely non-invasive to volunteers; (b) recently developed procedures for real-time imaging, at the single cell level in humans require simplification; and (c) these advantages need to be more-widely publicised to increase the adoption of the methods into practice.

The development of alternative methods to living animals, originally in the area of toxicity testing for chemical safety assessment, and more recently in biomedical research, has a rich history going back to the 1950s. In the early days, the drivers for change were mostly based on animal welfare concerns, but the evidence has increasingly pointed to the scientific and practical limitations of reliance on traditional animal methods. As toxicology transitioned into the 21st century, the need to assess an ever increasing range of chemicals, products and exposure scenarios has been accompanied by a plethora of new tools and assessment approaches. This rapid pace of change, both in terms of societal expectations for safety assessment science and the need to evaluate and intelligently apply the emerging technologies, continues to present challenges, as well as opportunities. This chapter highlights a number of lessons learned from a historical review of the field, and reflects on how far we have come since the seminal work of Russell and Burch in 1959.

In the early 1980s, the Organisation for Economic Cooperation and Development (OECD) began to publish test guidelines (TGs) on how in vivo toxicity tests should be conducted in laboratory animals, although where and when such TGs should be applied was the responsibility of national and regional regulatory authorities. The first TGs for non-animal tests involved in vitro methods for geno-toxicity, but, by the mid-1990s, alternative tests for other types of toxicity began to be validated and proposed for regulatory acceptance. The OECD published guidance on the development of TGs and on the validation and acceptance of new or updated methods for hazard assessment, and by the mid-1990s, TGs for validated individual non-animal tests began to be published. Later, guidance was published on the development, validation and acceptance of Quantitative Structure-Activity Relationship models and integrated testing strategies.

Progress toward the acceptance and application of validated alternative test methods as replacements for animal tests, is being frustrated by the unsatisfactory procedures involved in approving new test guidelines and deleting existing ones.

Phase 0 approaches, including microdosing, involve the use of sub-therapeutic exposures to the tested drugs, thus enabling safer, more-relevant, quicker and cheaper first-in-human (FIH) testing. These approaches also have considerable potential to limit the use of animals in human drug development. Recent years have witnessed progress in applications, methodology, operations, and drug development culture. Advances in applications saw an expansion in therapeutic areas, developmental scenarios and scientific objectives, in, for example, protein drug development and paediatric drug development. In the operational area, the increased sensitivity of Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS), expansion of the utility of Positron Emission Tomography (PET) imaging, and the introduction of Cavity Ring-Down Spectroscopy (CRDS), have led to the increased accessibility and utility of Phase 0 approaches, while reducing costs and exposure to radioactivity. PET has extended the application of microdosing, from its use as a predominant tool to record pharmacokinetics, to a method for recording target expression and target engagement, as well as cellular and tissue responses. Advances in methodology include adaptive Phase 0/Phase 1 designs, cassette and cocktail microdosing, and Intra-Target Microdosing (ITM), as well as novel modelling opportunities and simulations. Importantly, these methodologies increase the predictive power of extrapolation from microdose to therapeutic level exposures. However, possibly the most challenging domain in which progress has been made, is the culture of drug development. One of the main potential values of Phase 0 approaches is the opportunity to terminate development early, thus not only applying the principle of 'kill-early-kill-cheap' to enhance the efficiency of drug development, but also obviating the need for the full package of animal testing required for therapeutic level Phase 1 studies. Finally, we list developmental scenarios that utilised Phase 0 approaches in novel drug development.

The use of electronic cigarettes is being encouraged as a way of escaping from the harm resulting from conventional tobacco smoking, while scant attention is being paid to the long-term risks of inhaling electronic cigarette vapour. More information is needed for an acceptable risk assessment, from integrated non-animal testing and sound clinical investigations

While the standard of animal experimentation in the UK is generally very high, the aim of the Home Office appears to be to maintain the status quo, rather than to achieve significant Replacement and Reduction, and ensure Refinement

The use of electronic cigarettes is being encouraged as a way of escaping from the harm resulting from conventional tobacco smoking, while scant attention is being paid to the long-term risks of inhaling electronic cigarette vapour. More information is needed for an acceptable risk assessment, from integrated non-animal testing and sound clinical investigations

Two hundred years after his seminal publication on the ‘shaking palsy’, what would James Parkinson now make of progress achieved in understanding and treating the disease that bears his name?

• As professional toxicologists interested in improving testing methods for assessing the safety to humans of chemicals, we starting collaborating with the tobacco industry, to help identify promising new methods, beginning with tobacco smoking harm reduction products and then e-cigarettes. • We soon became perplexed over the FDA’s tobacco deeming regulations and then became even more-concerned about the way in which the UK authorities were laying the foundations for using e-cigarettes in the fight against smoking-related disease. We are especially surprised by the lack of scholarship and scientific rigour that is being applied to the safety assessment of these products, and feel it important to exploit our independence by speaking out. • The current stipulations regarding the regulatory control and authorisation of electronic cigarettes (ECs) and vaping in the UK are scientifically flawed, as they are based on little more than conjecture and value judgment, backed only by poor science. • There has been over-reliance on chemical analysis, the use of incomplete data, and risk assessments confused with the perceived benefits of vaping versus smoking, all of which bear little resemblance to standard approaches in toxicological risk assessment. • The authorities, and other stakeholders, have systematically ignored, or erroneously dismissed, basic principles of pharmacology and toxicology, and inconvenient scientific observations, while promoting vaping as a way of ceasing smoking, instead of discouraging the use of nicotine in any form. • The research being overlooked includes evidence of the many pleiotropic adverse biological effects of nicotine, more of which continue to be revealed with increasing frequency, which are likely to be highly relevant to carcinogenicity and disease. • We discuss this very serious situation, and offer some suggestions for a better way forward, for the benefit of individual humans, now and in the future.

As a new consortium to promote animal experimentation is formed, the numbers of procedures reported to the UK Home Office continue to increase, and opportunities to replace them are not being taken

Much is known about mammalian vision, and considerable progress has been achieved in treating many vision disorders, especially those due to changes in the eye, by using various therapeutic methods, including stem cell and gene therapy. While cells and tissues from the main parts of the eye and the visual cortex (VC) can be maintained in culture, and many computer models exist, the current non-animal approaches are severely limiting in the study of visual perception and retinotopic imaging. Some of the early studies with cats and non-human primates (NHPs) are controversial for animal welfare reasons and are of questionable clinical relevance, particularly with respect to the treatment of amblyopia. More recently, the UK Home Office records have shown that attention is now more focused on rodents, especially the mouse. This is likely to be due to the perceived need for genetically-altered animals, rather than to knowledge of the similarities and differences of vision in cats, NHPs and rodents, and the fact that the same techniques can be used for all of the species. We discuss the advantages and limitations of animal and non-animal methods for vision research, and assess their relative contributions to basic knowledge and clinical practice, as well as outlining the opportunities they offer for implementing the principles of the Three Rs (Replacement, Reduction and Refinement).

Strategic policy decisions are being made about e-cigarettes, based on the plausibility of their greater safety, rather than on essential scientific evidence which would permit a proper risk assessment. If e-cigarettes are really ‘safer’, then their use should be recommended, but only after an intelligent analysis of their risk to human health, based on integrated in silico, in vitro and clinical studies for both scientific and logistical reasons