Electronic cigarettes: can dependable public health policies be developed and applied without relevant and reliable risk assessments?

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.

The replacement of animal tests.

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.

The use of in vivo, ex vivo, in vitro, computational models and volunteer studies in vision research and therapy, and their contribution to the Three Rs.

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).

A critical assessment of the scientific basis, and implementation, of regulations for the safety assessment and marketing of innovative tobacco-related products.

Our scientific, logistical, ethical and animal welfare-related concerns about the latest US Food and Drug Administration (FDA) regulations for existing and so-called 'new' tobacco products, aimed at reducing harmful exposures, are explained. Such claims for sales in the USA now have to be based on a wide range of information, a key part of which will increasingly be data on safety and risk. One of the pathways to achieve marketing authorisation is to demonstrate substantial equivalence (SE) with benchmark products, called predicates. However, the regulations are insufficiently transparent with regard to: a) a rationale for the cut-off date for 'old' and 'new' products, and for exempting the former from regulation; b) the scientific validity and operation of SE; c) options for product labelling to circumvent SE; d) the experimental data required to support, and criteria to judge, a claim; and e) a strategy for risk assessment/management. Scientific problems related to the traditional animal methods used in respiratory disease and inhalation toxicology, and the use of quantitative comparators of toxicity, such as the No Observed Adverse Effect Level, are discussed. We review the advantages of relevant in vitro, mechanism-based, target tissue-oriented technologies, which an advisory report of the Institute of Medicine of the US National Academy of Sciences largely overlooked. These benefits include: a) the availability, for every major site in the respiratory tract, of organotypic human cell-based tissue culture systems, many of which are already being used by the industry; b) the accurate determination of concentrations of test materials received by target cells; c) methods for exposure to particulate and vapour phases of smoke, separately or combined; d) the ability to study tissue-specific biotransformation; and e) the use of modern, human-focused methodologies, unaffected by species differences. How data extrapolation, for risk assessment, from tissue culture to the whole animal, could be addressed, is also discussed. A cost (to animal welfare)-benefit (to society, including industry and consumers) analysis was conducted, taking into account the above information; the potential for animal suffering; the extensive data already available; the existence of other, less hazardous forms of nicotine delivery; the fact that much data will be generated solely for benchmarking; and that many smokers (especially nicotine-dependents) ignore health warnings. It is concluded that, in common with policies of several tobacco companies and countries, the use of laboratory animals for tobacco testing is very difficult, if not impossible, to justify. Instead, we propose and argue for an integrated testing scheme, starting with extensive chemical analysis of the ingredients and by-products associated with the use of tobacco products and their toxicity, followed by use of in vitro systems and early clinical studies (involving specific biomarkers) with weight-of-evidence assessments at each stage. Appropriate adjustment factors could be developed to enable concentration-response data obtained in vitro, with the other information generated by the strategy, to enable the FDA to meet its objectives. It is hoped that our intentionally provocative ideas will stimulate further debate on this contentious area of regulatory testing and public safety.

The Three Rs--opportunities for improving animal welfare and the quality of scientific research.

In 2013, an undercover investigation by the BUAV raised serious concerns about the use, treatment and care of laboratory animals involved in regulated procedures at Imperial College, London. This led to an inquiry, set up by the college, which found deficiencies in the local ethical review process and a general lack of focus on the implementation of the Three Rs (Replacement, Refinement and Reduction). The Three Rs concept is the foundation of UK and EU legislation, but surveys of the published literature show that lack of its adoption is widespread. In spite of numerous guidelines, publications and publicity material extolling the benefits of the Three Rs to both animals and science, as well as substantial advances in the development, validation, and deployment of mechanistically-based non-animal methods, many scientists prefer to use traditional animal-based approaches. In addition, such scientists tend to pay less attention than they should to strategic planning, experimental design and the choice of appropriate statistical procedures. They are often unaware of the existence of replacement test methods to address all or some of their objectives, and are reluctant to develop and use new replacement methods. We explore some possible reasons for these shortcomings. We summarise the welfare and scientific effects of each of the Three Rs, and argue that: a) there is an urgent need for evidence to be made readily accessible to prospective licensees, which directly demonstrates the beneficial effects on animal welfare of the implementation of the Three Rs, separately and in combination, and the direct link this has with the quality of the scientific data obtained; b) a detailed systematic review of this evidence should be undertaken to augment the inadequate content of the prescribed Module 5 licensee training offered currently in the UK; c) such training (including that suggested in new EU-wide proposals) should be much more comprehensive, with stronger emphasis on the Three Rs, all parts of the syllabus should be fully examined, and there should be no exemptions from Module 5 training; and d) as the responsible Government department in the UK, the Home Office should take measures to tighten up its guidance for local ethical review, and its system of inspection of designated establishments, to obviate the justification for future undercover investigations.

Every silver lining has a cloud: the scientific and animal welfare issues surrounding a new approach to the production of transgenic animals.

The scientific basis and advantages of using recently developed CRISPR/Cas-9 technology for transgenesis have been assessed with respect to other production methods, laboratory animal welfare, and the scientific relevance of transgenic models of human diseases in general. As the new technology is straightforward, causes targeted DNA double strand breaks and can result in homozygous changes in a single step, it is more accurate and more efficient than other production methods and speeds up transgenesis. CRISPR/Cas-9 also obviates the use of embryonic stem cells, and is being used to generate transgenic non-human primates (NHPs). While the use of this method reduces the level of animal wastage resulting from the production of each new strain, any long-term contribution to reduction will be offset by the overall increase in the numbers of transgenic animals likely to result from its widespread usage. Likewise, the contribution to refinement of using a more-precise technique, thereby minimising the occurrence of unwanted genetic effects, will be countered by a probable substantial increase in the production of transgenic strains of increasingly sentient species. For ethical and welfare reasons, we believe that the generation of transgenic NHPs should be allowed only in extremely exceptional circumstances. In addition, we present information, which, on both welfare and scientific grounds, leads us to question the current policy of generating ever-more new transgenic models in light of the general failure of many of them, after over two decades of ubiquitous use, to result in significant advances in the understanding and treatment of many key human diseases. Because this unsatisfactory situation is likely to be due to inherent, as well as possibly avoidable, limitations in the transgenic approach to studying disease, which are briefly reviewed, it is concluded that a thorough reappraisal of the rationale for using genetically-altered animals in fundamental research and by the pharmaceutical industry, and for its support by funding bodies, should be undertaken. In the meantime, the use of CRISPR/Cas-9 to generate new transgenic cells in culture is to be guardedly encouraged.

A critical review of anaesthetised animal models and alternatives for military research, testing and training, with a focus on blast damage, haemorrhage and resuscitation.

Military research, testing, and surgical and resuscitation training, are aimed at mitigating the consequences of warfare and terrorism to armed forces and civilians. Traumatisation and tissue damage due to explosions, and acute loss of blood due to haemorrhage, remain crucial, potentially preventable, causes of battlefield casualties and mortalities. There is also the additional threat from inhalation of chemical and aerosolised biological weapons. The use of anaesthetised animal models, and their respective replacement alternatives, for military purposes -- particularly for blast injury, haemorrhaging and resuscitation training -- is critically reviewed. Scientific problems with the animal models include the use of crude, uncontrolled and non-standardised methods for traumatisation, an inability to model all key trauma mechanisms, and complex modulating effects of general anaesthesia on target organ physiology. Such effects depend on the anaesthetic and influence the cardiovascular system, respiration, breathing, cerebral haemodynamics, neuroprotection, and the integrity of the blood-brain barrier. Some anaesthetics also bind to the NMDA brain receptor with possible differential consequences in control and anaesthetised animals. There is also some evidence for gender-specific effects. Despite the fact that these issues are widely known, there is little published information on their potential, at best, to complicate data interpretation and, at worst, to invalidate animal models. There is also a paucity of detail on the anaesthesiology used in studies, and this can hinder correct data evaluation. Welfare issues relate mainly to the possibility of acute pain as a side-effect of traumatisation in recovered animals. Moreover, there is the increased potential for animals to suffer when anaesthesia is temporary, and the procedures invasive. These dilemmas can be addressed, however, as a diverse range of replacement approaches exist, including computer and mathematical dynamic modelling of the human body, cadavers, interactive human patient simulators for training, in vitro techniques involving organotypic cultures of target organs, and epidemiological and clinical studies. While the first four of these have long proven useful for developing protective measures and predicting the consequences of trauma, and although many phenomena and their sequelae arising from different forms of trauma in vivo can be induced and reproduced in vitro, non-animal approaches require further development, and their validation and use need to be coordinated and harmonised. Recommendations to these ends are proposed, and the scientific and welfare problems associated with animal models are addressed, with the future focus being on the use of batteries of complementary replacement methods deployed in integrated strategies, and on greater transparency and scientific cooperation.

Is phenylbutazone a genotoxic carcinogen? A weight-of-evidence assessment.

Published in silico, in vitro, in vivo laboratory animal and human data, together with information on biotransformation and data from structure-activity analyses with two decision-tree systems (ACToR and Toxtree), have been used in a weight-of-evidence (WoE) assessment to determine whether phenylbutazone (PBZ) is a genotoxic or a non-genotoxic carcinogen. This was undertaken to facilitate the risk assessment of human exposure to this veterinary drug via the consumption of horsemeat from treated animals. Despite problems with data interpretation at all tiers of the database, it was concluded that PBZ behaves like a genotoxic carcinogen with a threshold dose. This conclusion is based mainly on the results of a definitive rodent bioassay, and on the following observations: a) that PBZ has weak in vitro activity only at high concentrations in some genotoxicity assays, accompanied by high levels of cytotoxicity; b) that it (and a major metabolite) is able to cause sister chromatid exchanges in vivo in rodents; and c) that it can induce cytogenetic effects in vivo in humans. It also takes into account the known and predicted activities of the parent drug, some of its metabolites and two structural analogues, and, importantly, several of the drug's other biochemical effects that are unrelated to toxicity. However, this conclusion is not fully supported by all the evidence, and much of the information is based on old papers. Therefore, more studies are required to establish whether the concentration thresholds seen in vitro would translate to dose thresholds for carcinogenicity, such that a safe dose-level could be defined for the purposes of assessing risk. It was disappointing that a WoE approach to evaluating all of the available hazard data, as is increasingly being advocated to improve the hazard identification paradigm, was unable to provide definitive answers in this case, particularly in view of the large numbers of animals that had been used to provide much of the information.

Cell transformation assays: are we barking up the wrong tree?

There has been a current resurgence of interest in the use of cell transformation for predicting carcinogenicity, which is based mainly on rodent carcinogenicity data. In view of this renewed interest, this paper critically reviews the published literature concerning the ability of the available assays to detect IARC Group 1 agents (known human carcinogens) and Group 2A agents (probable human carcinogens). The predictivity of the available assays for human and rodent non-genotoxic carcinogens (NGCs), in comparison with standard and supplementary in vitro and in vivo genotoxicity tests, is also discussed. The principal finding is that a surprising number of human carcinogens have not been tested for cell transformation across the three main assays (SHE, Balb/c 3T3 and C3H10T1/2), confounding comparative assessment of these methods for detecting human carcinogens. This issue is not being addressed in the ongoing validation studies for the first two of these assays, despite the lack of any serious logistical issues associated with the use of most of these chemicals. In addition, there seem to be no plans for using exogenous bio-transformation systems for the metabolic activation of pro-carcinogens, as recommended in an ECVAM workshop held in 1999. To address these important issues, it is strongly recommended that consideration be given to the inclusion of more human carcinogens and an exogenous source of xenobiotic metabolism, such as an S9 fraction, in ongoing and future validation studies. While cell transformation systems detect a high level of NGCs, it is considered premature to rely only on this endpoint for screening for such chemicals, as recently suggested. This is particularly important, in view of the fact that there is still doubt as to the relevance of morphological transformation to tumorigenesis in vivo, and the wide diversity of potential mechanisms by which NGCs are known to act. Recent progress with regard to increasing the objectivity of scoring the transformed phenotype, and prospects for developing human cell-based transformation assays, are reviewed.

In silico methods for toxicity prediction.

The principles and uses of (Q)SAR models and expert systems for predicting toxicity and the biotransformation of foreign chemicals (xenobiotics) are described and illustrated for some key toxicity endpoints, with examples from the published literature. The advantages and disadvantages of the methods and issues concerned with their validation, acceptance and use by regulatory bodies are also discussed. In addition, consideration is given to the potential application of these techniques in regulatory toxicity testing, both individually and as part of a chemically-based read-across approach, particularly for the risk assessment of chemicals within intelligent, integrated decision-tree testing schemes. It is concluded that, while there has been great progress in recent years in the development and application of in silico approaches, there is still much that has to be achieved to enable them to fulfill their potential for regulatory toxicity testing. In particular, there is a need for the wider availability of appropriate biological data and international agreement on how the systems should be validated. In addition, it is important that correlations between activity and physicochemical properties are based on a mechanistic basis to maximize the predictivity of models for novel chemicals.

The use of integrated and intelligent testing strategies in the prediction of toxic hazard and in risk assessment.

There is increasing concern that insurmountable differences between humans and laboratory animals limit the relevance and reliability for hazard identification and risk assessment purposes of animal data produced by traditional toxicity test procedures. A way forward is offered by the emerging new technologies, which can be directly applied to human material or even to human beings themselves. This promises to revolutionise the evaluation of the safety of chemicals and chemical products of various kinds and, in particular, pharmaceuticals. The available and developing technologies are summarised and it is emphasised that they will need to be used selectively, in integrated and intelligent testing strategies, which, in addition to being scientifically sound, must be manageable and affordable. Examples are given of proposed testing strategies for general chemicals, cosmetic ingredients, candidate pharmaceuticals, inhaled substances, nanoparticles and neurotoxicity.

Integrated testing strategies for toxicity employing new and existing technologies.

We have developed individual, integrated testing strategies (ITS) for predicting the toxicity of general chemicals, cosmetics, pharmaceuticals, inhaled chemicals, and nanoparticles. These ITS are based on published schemes developed previously for the risk assessment of chemicals to fulfil the requirements of REACH, which have been updated to take account of the latest developments in advanced in chemico modelling and in vitro technologies. In addition, we propose an ITS for neurotoxicity, based on the same principles, for incorporation in the other ITS. The technologies are deployed in a step-wise manner, as a basis for decision-tree approaches, incorporating weight-of-evidence stages. This means that testing can be stopped at the point where a risk assessment and/or classification can be performed, with labelling in accordance with the requirements of the regulatory authority concerned, rather than following a checklist approach to hazard identification. In addition, the strategies are intelligent, in that they are based on the fundamental premise that there is no hazard in the absence of exposure - which is why pharmacokinetic modelling plays a key role in each ITS. The new technologies include the use of complex, three-dimensional human cell tissue culture systems with in vivo-like structural, physiological and biochemical features, as well as dosing conditions. In this way, problems of inter-species extrapolation and in vitro/in vivo extrapolation are minimised. This is reflected in the ITS placing more emphasis on the use of volunteers at the whole organism testing stage, rather than on existing animal testing, which is the current situation.

Challenges for computational structure-activity modelling for predicting chemical toxicity: future improvements?

INTRODUCTION: Structure-activity modelling for predicting toxicology as a discipline is now 50 years old, and great strides have been taken in developing methods for the physicochemical analysis of molecules and their toxicity evaluation, both essential stages in modelling. Computational toxicology also has huge potential for speeding up the screening and prioritisation of chemicals for further testing and for reducing the numbers of expensive and time-consuming conventional tests. Yet, the realisation of this potential has been largely stifled by many problems inherent in developing and validating new structure-activity models of toxicity. AREAS COVERED: Problems of computational toxicology discussed include i) the use of inappropriate molecular descriptors and tools that are not transparent; ii) the undetected existence of chemicals that cause large changes in toxicity with only small differences in molecular structure (causing 'activity cliffs' in the structure-activity landscape); iii) spurious correlations between structure and activity; iv) lack of quality control of toxicity data; v) difficulties in determining predictivity for novel chemicals; and vi) an over-reliance on complex mathematics and statistics. EXPERT OPINION: Greater emphasis needs to be placed on i) the selection of training and test sets of chemicals to enable both internal and external validation of models to be undertaken for more accurate assessment of model predictivity and ii) the use of recently developed techniques for characterising structure-activity landscapes.