Refinement Of Animal Use Research Articles

Fostering Animal Welfare and Advancing 3Rs Principles through the Establishment of a 3Rs Advisory Group.

Based on the current state of science, the use of animals remains essential in bringing safe and effective medicines to patients. Respect for laboratory animal welfare and the application of 3Rs principles (the replacement, reduction, and refinement of animal use in research) are a priority throughout the pharmaceutical industry. Given the rapid pace of development, technological progress, and the emergence of new-approach methodologies (NAMs) in the field of biomedical research, maintaining a leading position in scientific advancements with a focus on the principles of replace, reduce, and refine (3Rs) can be quite challenging. To effectively address these challenges and sustain a prominent position in the scientific community, organizations can derive significant advantages from establishing an internal 3Rs advisory group (3Rs AG). The primary objective of a 3Rs AG is to stay at the forefront of the knowledge of best practices related to the 3Rs principles in the industry. This group plays a crucial role in fostering innovation and facilitating the seamless integration and implementation of 3Rs principles into a company's policies and procedures. The thoughtful reduction in and replacement of animal studies and the refinement of study designs and practices, enabled by a 3Rs AG, can minimize animal use as well as guide resources and positively impact study and data quality. This article provides guidance on how to establish a successful and impactful 3Rs AG.

10 Validation of an Aerosol Exposure-Air Liquid Interface (AE-ALI) System to Facilitate Realistic Hazard Identification of Nano-Sized Aerosols

Abstract To facilitate hazard characterisation for inhaled engineered nanomaterials we have established an aerosol exposure air-liquid-interface (AE-ALI) system, incorporating a Cultex RFSTM cell exposure system. While offering potential benefits in terms of more realistic aerosol exposure mode over traditional submerged in vitro models and being in-line with the principles of the 3Rs, (replace, reduce and refine animal use), detailed system characterisation is required to produce reliable and reproducible results. As such, this work looked to fully characterise our AE-ALI system with a view to optimising cell viability of system controls and deposition quantity, uniformity, and reproducibility for engineered nanomaterials. Using a nano-CeO2 aerosol, the effects of exposure conditions and system parameters (e.g. temperature, electrostatic precipitator voltage and airflow) on deposition efficiency, pattern and reproducibility were investigated using ICP-MS. While results showed that appropriate choice of operating parameters can produce broadly uniform deposition, reproducibility was low, and dose monitoring during exposures is recommended. After initial optimisation of the system for deposition, the effect of exposure duration, well size and flowrate on cellular responses (e.g. cytotoxicity (LDH) and selected gene expression (IL-8, CXCL1, HMOX1, SPP1) of system controls (H2O aerosol) was investigated using human lung alveolar (A549) and primary small airway epithelial cells. Results indicate that even after optimisation for cell health, the system itself is “toxic” to cells in comparison to incubator controls, with increasing effects observed with exposure duration meaning exposure durations must be minimised. Results from this study highlight the need for detailed characterisation of AE-ALI systems prior to use.

Russell and Burch's 3Rs then and now: The case of Switzerland.

Since Russell and Burch introduced and defined the 3Rs, i.e., the replacement, reduction, and refinement of animal use in research, in 1959, different definitions have emerged and been implemented in guidelines and policies. Switzerland is known for having some of the most restrictive legislation regarding the use of animals, in which the 3Rs are also defined and implemented. To our knowledge, the purpose and definitions of the 3Rs used in the Swiss Animal Welfare Act, Animal Protection Ordinance and Animal Experimentation Ordinance have never been compared with Russell and Burch's original purpose and definitions. In this paper we make this comparison with two aims: to reveal ethically relevant departures from the original purpose and definitions, and to provide an ethical evaluation of the current Swiss law regarding the 3Rs. In doing so, we first expose the similarity of purposes. We then identify one risky departure from the original definition of replacement in Swiss law, which shows a problematic focus on species. Finally, we address Swiss law's failure to apply the 3Rs in the most effective way. With respect to this last point, we discuss the need for 3R conflict resolution, the timing of application of the 3Rs, problematic prioritizations and choices of convenience as well as a solution to apply the 3Rs more effectively using Russell and Burch's concept of total sum of distress.

The Current Status and Work of Three Rs Centres and Platforms in Europe.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics.

The development of antimicrobials is an expensive process with increasingly low success rates, which makes further investment in antimicrobial discovery research less attractive. Antimicrobial drug discovery and subsequent commercialization can be made more lucrative if a fail-fast-and-fail-cheap approach can be implemented within the lead optimization stages where researchers have greater control over drug design and formulation. In this article, the setup of an ex vivo ovine wounded skin model infected with Staphylococcus aureusis described, which is simple, cost-effective, high throughput, and reproducible. The bacterial physiology in the model mimics that during infection as bacterial proliferation is dependent on the pathogen's ability to damage the tissue. The establishment of wound infection is verified by an increase in viable bacterial counts compared to the inoculum. This model can be used as a platform to test the efficacy of emerging antimicrobials in the lead optimization stage. It can be contended that the availability of this model will provide researchers developing antimicrobials with a fail-fast-and-fail-cheap model, which will help increase success rates in subsequent animal trials. The model will also facilitate the reduction and refinement of animal use for research and ultimately enable faster and more cost-effective translation of novel antimicrobials for skin and soft tissue infections to the clinic.

Transparency in Non-Technical Project Summaries to Promote the Three Rs in Respiratory Disease Research.

Non-Technical Project Summaries (NTS) are legal documents that were first introduced by the Directive 2010/63/EU to enhance transparency within scientific animal experimentation. Researchers intending to conduct biological research on animal models must fulfil the NTS requirements by outlining their proposed use of animals and how they plan to implement the Three Rs (replacement, reduction and refinement of animal use) in their experiments. This study outlines a novel systematic analysis approach that enables the assessment of NTS transparency based on the accuracy of reporting of certain Three Rs-specific information. This potentially customisable strategy could help toward the development of practical guidelines for use by Animal Welfare and Ethical Review Bodies (AWERBs) in establishments conducting animal research, in the process of scrutinising NTS during their pre-submission review of proposed licence applications. This could help to identify gaps in reporting of Three Rs-specific information relating to the planned animal experiments, which represents a remarkable step toward achieving greater openness in scientific communication. This study supports the concept that NTS transparency can promote the implementation of non-animal alternatives in fields where this is currently lacking, such as respiratory disease research. Although NTS were originally conceived as informative documents for a lay audience, we can conclude that data in NTS can be successfully used as a basis for systematic analysis. By reviewing the NTS, the experimental limitations of the currently available replacement strategies can also be highlighted, potentially pinpointing where there is a need for future method development.

Educators' Views on the Use of Dissection and Dissection Alternatives in American Biology Classrooms.

Animal dissection remains a common practice in American biology classrooms, despite the availability of dissection alternatives to study anatomy and physiology. Indeed, there is a growing body of evidence in the literature suggesting that the use of alternatives leads to the greater achievement of learning objectives, as compared to dissection. To better understand the current use of and attitudes toward dissection and alternatives, a nationwide survey of (mainly high-school) biology teachers (n = 2687) was conducted. Most educators believed that learning objectives related to biology subject content could be met through the use of alternatives, yet they preferred the hands-on experience of dissection. Most educators allow their students to use alternatives if requested, although few teachers ask students about their preference for using an animal specimen versus an alternative. Educators cited student engagement as the main factor driving their decision to choose between dissection specimens and alternatives, and felt that cost is the biggest barrier to implementing alternatives at their schools. Additional perspectives on dissection and alternatives were shared by survey participants. Since alternatives can be used to meet learning objectives associated with dissection, we recommend their use as replacements for traditional animal specimens, in line with the replacement, reduction and refinement of animal use in education, according to the Three Rs principles.

Application of Mitra® microsampling for pharmacokinetic bioanalysis of monoclonal antibodies in rats.

Volumetric absorptive microsampling (VAM)is being increasingly applied in nonclinical pharmacokinetic studies. Although there are published results for VAM use in small molecule pharmacokinetics (PK) studies, there is limited data on the utility of VAM for protein therapeutics. We describe the use of Mitra® microsampler for blood sampling, ELISA quantitation and PK analysis of two marketed therapeutic monoclonal antibodies administered to rat. Results generated for these monoclonal antibodies using Mitra® were compared with both serum and whole blood sampling methods in the same study. The low relative standard deviation among the three sets of PK data suggest that Mitra® microsampler could be useful in early nonclinical PK studies for protein therapeutics where reduction and refinement of animal use is desirable.

Exploring the use of alternatives to animals in undergraduate education in Australia.

The replacement, reduction and refinement of animal use in education is part of the regulatory legislation in Australia, and requires the use of alternatives to animals where appropriate. The aims of this study were: a) to explore the extent of the replacement of animals when teaching life sciences to Australian undergraduate students; b) to understand which alternative models were being used, and the learning objectives covered; and c) to gain some insight into the circumstances facilitating the use of alternatives to animals in education. An anonymous online survey, consisting of open and closed questions, was conducted among faculty members that used either animal or alternative models in their teaching. A total of 27 faculty members participated, from eight universities. Human anatomy and physiology had the highest number of survey participants who had replaced animals entirely with alternative models. These subjects also had the highest number of participants that were using animal models. According to the participants, most learning objectives were met effectively by both types of model. Participants who only used alternatives were influenced by ethical considerations significantly more than those who used animal models and alternatives. We concluded that, while some participants have replaced animals successfully, others in the same field are still employing them, and that there appears to be a range of barriers to the wider adoption of alternatives to animal use.

The Chicken Thigh Adductor Profundus Free Muscle Flap: A Novel Validated Non-Living Microsurgery Simulation Training Model.

BackgroundSimulation training is becoming an increasingly important component of skills acquisition in surgical specialties, including Plastic Surgery. Non-living simulation models have an established place in Plastic Surgical microsurgery training, and support the principles of replacement, reduction and refinement of animal use. A more sophisticated version of the basic chicken thigh microsurgery model has been developed to include dissection of a type 1-muscle flap and is described and validated here.MethodsA step-by-step dissection guide on how to perform the chicken thigh adductor profundus free muscle flap is demonstrated. Forty trainees performed the novel simulation muscle flap on the last day of a 5-day microsurgery course. Pre- and post-course microvascular anastomosis assessment, along with micro dissection and end product (anastomosis lapse index) assessment, demonstrated skills acquisition.ResultsThe average time to dissect the flap by novice trainees was 82±24 minutes, by core trainees 90±24 minutes, and by higher trainees 64±21 minutes (P=0.013). There was a statistically significant difference in the time to complete the anastomosis between the three levels of training (P=0.001) and there was a significant decrease in the time taken to perform the anastomosis following course completion (P<0.001). Anastomosis lapse index scores improved for all cohorts with post-test average anastomosis lapse index score of 3±1.4 (P<0.001).ConclusionsThe novel chicken thigh adductor profundus free muscle flap model demonstrates face and construct validity for the introduction of the principles of free tissue transfer. The low cost, constant, and reproducible anatomy makes this simulation model a recommended addition to any microsurgical training curriculum.

Microphysiological Systems (Tissue Chips) and their Utility for Rare Disease Research.

The scientific and technological development of microphysiological systems (MPS) modeling organs-on-chips, or "tissue chips" (TCs), has progressed rapidly over the past decade. Stem cell research and microfluidic concepts have combined to lead to the development of microphysiological platforms representing an ever-expanding list of different human organ systems. In the context of rare diseases, these bioengineered microfluidics platforms hold promise for modeling of disorders and could prove useful in the screening and efficacy testing of existing therapeutics. Additionally, they have the potential for replacing and refining animal use for new drugs and clinical treatments, or could even act as surrogate human systems for testing of new therapeutics in the future, which could be particularly useful in populations of rare disease sufferers. This chapter will discuss the current state of tissue chip research, and challenges facing the field. Additionally, we will discuss how these devices are being used to model basic cellular and molecular phenotypes of rare diseases, holding promise to provide new tools for understanding of disease pathologies and screening and efficacy testing of potential therapeutics for drug discovery.

Act on the Registration and Evaluation of Chemicals (K-REACH) and replacement, reduction or refinement best practices.

ObjectivesKorea’s Act on the Registration and Evaluation of Chemicals (K-REACH) was enacted for the protection of human health and the environment in 2015. Considering that about 2000 new substances are introduced annually across the globe, the extent of animal testing requirement could be overwhelming unless regulators and companies work proactively to institute and enforce global best practices to replace, reduce or refine animal use. In this review, the way to reduce the animal use for K-REACH is discussed.MethodsBackground of the enforcement of the K-REACH and its details was reviewed along with the papers and regulatory documents regarding the limitation of animal experiments and its alternatives in order to discuss the regulatory adoption of alternative tests.ResultsDepending on the tonnage of the chemical used, the data required ranges from acute and other short-term studies for a single exposure route to testing via multiple exposure routes and costly, longer-term studies such as a full two-generation reproducibility toxicity. The European Registration, Evaluation, Authorization and Restriction of Chemicals regulation provides for mandatory sharing of vertebrate test data to avoid unnecessary duplication of animal use and test costs, and obligation to revise data requirements and test guidelines “as soon as possible” after relevant, validated replacement, reduction or refinement (3R) methods become available. Furthermore, the Organization for Economic Cooperation and Development actively accepts alternative animal tests and 3R to chemical toxicity tests.ConclusionsAlternative tests which are more ethical and efficient than animal experiments should be widely used to assess the toxicity of chemicals for K-REACH registration. The relevant regulatory agencies will have to make efforts to actively adopt and uptake new alternative tests and 3R to K-REACH.

Automated Microsampling Technologies and Enhancements in the 3Rs.

Data collected in vivo is essential for advising decisions on drug screening and development and basic research, and animal models are used extensively for acquiring experimental measurements. Traditionally, collection of specimens has been invasive, stressful to animal subjects, labor intensive, time-consuming and costly, and required many animals when using small models with low fluid volumes, such as rats or mice. Utilizing automated microsampling (AMS) alone or in an integrative pharmacology approach to evaluate multiple physiological, pharmacokinetic, and pharmacodynamic endpoints simultaneously in the same animal accomplishes multiple experimental goals. Use of AMS robotics and associated instrumentation can assist in achieving significant reduction and refinement of animal use. Automated robotic instrumentation for specimen collection from living animal models can now be used to provide better quality pharmacokinetic and pharmacodynamic data, reduce time, provide more data with less variability, reduce animal use, and refine animal model use to reduce pain and stress. Instrumentation in common use for automated sampling and dosing is briefly discussed. In parallel with advances in automated instrumentation, recent advances in analytical detection methods complement the use of automated technology for data and specimen collection. Methods requiring much smaller volumes can now be utilized. Microsampling (small biological samples in volumes of 5-100 μL) can facilitate reduction in animal numbers while minimizing stresses associated with excessive fluid volume removal. Innovations in automation, microsampling techniques, and analytical methods have facilitated advances in data collection that allow for more robust and accurate data, reduction in animal use, and refinement in techniques that improve animal welfare. These innovations offer opportunities for wider application in nonclinical investigations by collection of multiple data streams simultaneously from individual animals. Many benefits are achievable through the use integrative pharmacology designs utilizing AMS, including decreased time for completion of composite data collection, decreased personnel resources, lower costs, improved safety, higher quality and multiple data-sets, and improvements in aspects of the 3Rs.

Opportunities to Apply the 3Rs in Safety Assessment Programs.

Before a potential new medicine can be administered to humans it is essential that its safety is adequately assessed. Safety assessment in animals forms an integral part of this process, from early drug discovery and initial candidate selection to the program of recommended regulatory tests in animals. The 3Rs (replacement, reduction, and refinement of animals in research) are integrated in the current regulatory requirements and expectations and, in the EU, provide a legal and ethical framework for in vivo research to ensure the scientific objectives are met whilst minimizing animal use and maintaining high animal welfare standards. Though the regulations are designed to uncover potential risks, they are intended to be flexible, so that the most appropriate approach can be taken for an individual product. This article outlines current and future opportunities to apply the 3Rs in safety assessment programs for pharmaceuticals, and the potential (scientific, financial, and ethical) benefits to the industry, across the drug discovery and development process. For example, improvements to, or the development of, novel, early screens (e.g., in vitro, in silico, or nonmammalian screens) designed to identify compounds with undesirable characteristics earlier in development have the potential to reduce late-stage attrition by improving the selection of compounds that require regulatory testing in animals. Opportunities also exist within the current regulatory framework to simultaneously reduce and/or refine animal use and improve scientific outcomes through improvements to technical procedures and/or adjustments to study designs. It is important that approaches to safety assessment are continuously reviewed and challenged to ensure they are science-driven and predictive of relevant effects in humans.

The use of thermographic imaging to evaluate therapeutic response in human tumour xenograft models.

Non-invasive methods to monitor tumour growth are an important goal in cancer drug development. Thermographic imaging systems offer potential in this area, since a change in temperature is known to be induced due to changes within the tumour microenvironment. This study demonstrates that this imaging modality can be applied to a broad range of tumour xenografts and also, for the first time, the methodology’s suitability to assess anti-cancer agent efficacy. Mice bearing subcutaneously implanted H460 lung cancer xenografts were treated with a novel vascular disrupting agent, ICT-2552, and the cytotoxin doxorubicin. The effects on tumour temperature were assessed using thermographic imaging over the first 6 hours post-administration and subsequently a further 7 days. For ICT-2552 a significant initial temperature drop was observed, whilst for both agents a significant temperature drop was seen compared to controls over the longer time period. Thus thermographic imaging can detect functional differences (manifesting as temperature reductions) in the tumour response to these anti-cancer agents compared to controls. Importantly, these effects can be detected in the first few hours following treatment and therefore the tumour is observable non-invasively. As discussed, this technique will have considerable 3Rs benefits in terms of reduction and refinement of animal use.

Cigarette smoke induced genotoxicity and respiratory tract pathology: evidence to support reduced exposure time and animal numbers in tobacco product testing

Many laboratories are working to develop in vitro models that will replace in vivo tests, but occasionally there remains a regulatory expectation of some in vivo testing. Historically, cigarettes have been tested in vivo for 90 days. Recently, methods to reduce and refine animal use have been explored. This study investigated the potential of reducing animal cigarette smoke (CS) exposure to 3 or 6 weeks, and the feasibility of separate lung lobes for histopathology or the Comet assay. Rats were exposed to sham air or CS (1 or 2 h) for 3 or 6 weeks. Respiratory tissues were processed for histopathological evaluation, and Alveolar type II cells (AEC II) isolated for the Comet assay. Blood was collected for Pig-a and micronucleus quantification. Histopathological analyses demonstrated exposure effects, which were generally dependent on CS dose (1 or 2 h, 5 days/week). Comet analysis identified that DNA damage increased in AEC II following 3 or 6 weeks CS exposure, and the level at 6 weeks was higher than 3 weeks. Pig-a mutation or micronucleus levels were not increased. In conclusion, this study showed that 3 weeks of CS exposure was sufficient to observe respiratory tract pathology and DNA damage in isolated AEC II. Differences between the 3 and 6 week data imply that DNA damage in the lung is cumulative. Reducing exposure time, plus analyzing separate lung lobes for DNA damage or histopathology, supports a strategy to reduce and refine animal use in tobacco product testing and is aligned to the 3Rs (replacement, reduction and refinement).

Empirical analysis of BMD metrics in genetic toxicology part I: in vitro analyses to provide robust potency rankings and support MOA determinations.

Genetic toxicity testing has traditionally been used for hazard identification, with dichotomous classification of test results serving to identify genotoxic agents. However, the utility of genotoxicity data can be augmented by employing dose-response analysis and point of departure determination. Via interpolation from a fitted dose-response model, the benchmark dose (BMD) approach estimates the dose that elicits a specified (small) effect size. BMD metrics and their confidence intervals can be used for compound potency ranking within an endpoint, as well as potency comparisons across other factors such as cell line or exposure duration. A recently developed computational method, the BMD covariate approach, permits combined analysis of multiple dose-response data sets that are differentiated by covariates such as compound, cell type or exposure regime. The approach provides increased BMD precision for effective potency rankings across compounds and other covariates that pertain to a hypothesised mode of action (MOA). To illustrate these applications, the covariate approach was applied to the analysis of published in vitro micronucleus frequency dose-response data for ionising radiations, a set of aneugens, two mutagenic azo compounds and a topoisomerase II inhibitor. The ionising radiation results show that the precision of BMD estimates can be improved by employing the covariate method. The aneugen analysis provided potency groupings based on the BMD confidence intervals, and analyses of azo compound data from cells lines with differing metabolic capacity confirmed the influence of endogenous metabolism on genotoxic potency. This work, which is the first of a two-part series, shows that BMD-derived potency rankings can be employed to support MOA evaluations as well as facilitate read across to expedite chemical evaluations and regulatory decision-making. The follow-up (Part II) employs the combined covariate approach to analyse in vivo genetic toxicity dose-response data focussing on how improvements in BMD precision can impact the reduction and refinement of animal use in toxicological research.

Basic research support for shared magnetic resonance imaging resources.

Procedures that enable the collection of longitudinal physiologic and anatomic information can contribute to the reduction and refinement of animal use. Scientists are increasingly turning to noninvasive magnetic resonance imaging (MRI) to obtain such information from animal research subjects. As they make this important investment, research support veterinarians are often tasked with ensuring the proper care and use of laboratory animal research subjects. A basic understanding of MRI equipment, personnel practices, safety, and monitoring of animals and their recoveries is key to implementing a centralized animal MRI facility.

In vitro models for peripheral nerve regeneration.

The study of peripheral nerve repair and regeneration is particularly relevant in the light of the high clinical incidence of nerve lesions. However, the clinical outcome after nerve lesions is often far from satisfactory and the functional recovery is almost never complete. Therefore, a number of therapeutic approaches are being investigated, ranging from local delivery of trophic factors and other molecules to bioactive biomaterials and complex nerve prostheses. Translation of the new therapeutic approaches to the patient always requires a final pre-clinical step using in vivo animal models. The need to limit as much as possible animal use in biomedical research, however, makes the preliminary use of in vitro models mandatory from an ethical point of view. In this article, the different types of in vitro models available today for the study of peripheral nerve regeneration have been ranked by adopting a three-step stair model based on their increasing ethical impact: (i) cell line-based models, which raise no ethical concern; (ii) primary cell-based models, which have low ethical impact as animal use, although necessary, is limited; and (iii) organotypic ex vivo-based models, which raise moderate ethical concerns as the use of laboratory animals is required although with much lower impact on animal wellbeing in comparison to in vivo models of peripheral nerve regeneration. This article aims to help researchers in selecting the best experimental approach for their scientific goals driven by the 'Three Rs' (3Rs) rules (Replacement, Reduction or Refinement of animal use in research) for scientific research.

Developing accurate models of the human airways.

Particle delivery to the airways is an attractive prospect for many potential therapeutics, including vaccines. Developing strategies for inhalation of particles provides a targeted, controlled and non-invasive delivery route but, as with all novel therapeutics, in vitro and in vivo testing are needed prior to clinical use. Whilst advanced vaccine testing demands the use of animal models to address safety issues, the production of robust in vitro cellular models would take account of the ethical framework known as the 3Rs (Replacement, Reduction and Refinement of animal use), by permitting initial screening of potential candidates prior to animal use. There is thus a need for relevant, realistic in vitro models of the human airways. Our laboratory has designed and characterised a multi-cellular model of human airways that takes account of the conditions in the airways and recapitulates many salient features, including the epithelial barrier and mucus secretion. Our human pulmonary models recreate many of the obstacles to successful pulmonary delivery of particles and therefore represent a valid test platform for screening compounds and delivery systems.