Beyond Anthropocentrism: The Moral and Strategic Philosophy behind Russell and Burch's 3Rs in Animal Experimentation.

The 3Rs framework in animal experimentation- "replace, reduce, refine" - has been alleged to be expressive of anthropocentrism, the view that only humans are directly morally relevant. After all, the 3Rs safeguard animal welfare only as far as given human research objectives permit, effectively prioritizing human use interests over animal interests. This article acknowledges this prioritization, but argues that the characterization as anthropocentric is inaccurate. In fact, the 3Rs prioritize research purposes even more strongly than an ethical anthropocentrist would. Drawing on the writings of Universities Federation for Animal Welfare (UFAW) founder Charles W. Hume, who employed Russell and Burch, it is argued that the 3Rs originally arose from an animal-centered ethic which was however restricted by an organizational strategy aiming at the voluntary cooperation of animal researchers. Research purposes thus had to be accepted as given. While this explains why the 3Rs focus narrowly on humane method selection, not on encouraging animal-free question selection in the first place, it suggests that governments should (also) focus on the latter if they recognize animals as deserving protection for their own sake.

Development of a monoclonal antibody sandwich ELISA for the quality control of human and animal tetanus vaccines.

Antigen identity, quantity and integrity are key factors to be evaluated as part of consistency testing of tetanus vaccines. Here we have developed a mAb sandwich ELISA to measure the relative amount and quality of tetanus toxoid (TTxd) in human and animal tetanus vaccines. The ELISA is highly specific, has good dilutional linearity and is suitable for detecting TTxd in a range of different products. We have demonstrated the ability of the assay to discriminate between batches of different content, using vaccine batches that had been prepared to contain differing amounts of TTxd, and of different quality, using samples of non-adjuvanted TTxd that had been exposed to sonication and final lot vaccines that had been exposed to heat or oxidative stress. We have also demonstrated successful transfer of the method to other laboratories and have shown that different tetanus antigen materials may be able to serve as a reference antigen for standardisation of the method. The results show this test has the potential to play a key role in a control strategy no longer including an in vivo potency test.

Tetanus vaccines help to protect against tetanus infection. Currently, animal tests are used to ensure the potency of such vaccines. Since these tests were first introduced, there have been improvements in non-animal technologies that can be used to ensure consistent production of potent vaccine batches. To demonstrate that a new batch of tetanus vaccine is consistent with a previous batch of known potency, the quality and amount of the component that stimulates the immune response upon vaccination must be assessed in comparison. We have developed an assay that can measure the quality of a range of different tetanus vaccine product types. The assay is very specific and reliable, and different laboratories obtained comparable results, showing that the assay is suited for routine use. Once validated by manufacturers and accepted by regulators, this assay will greatly reduce the number of animals needed for batch release of tetanus vaccines.

Experimental glaucoma microstent implantation in two animal models and human donor eyes-an ex vivo micro-computed tomography-based evaluation of applicability.

Background: Minimally invasive glaucoma surgery (MIGS) has become an important treatment approach for primary open angle glaucoma. Restoration of aqueous humour drainage by means of alloplastic implants represents a promising treatment option and is itself subject of methodological development. An adequate positioning in the targeted tissue regions is essential is important for the performance of our in-house developed Rostock glaucoma microstent (RGM). The aim of this study was to evaluate the applicability of two animal models and human donor eyes regarding RGM placement. Methods: Eyes were obtained from rabbits, pigs, and human body donations. After orbital exenterations, RGMs were placed in the anterior chamber draining in the subconjunctival space. X-ray contrast was increased by incubation in aqueous iodine solution for subsequent detailed micro-computed tomography (micro-CT)-based visualization and analysis. Results: In contrast to the human and porcine eyes, the stent extended far to the posterior pole with a more pronounced curvature along the globe in the rabbit eyes due to their smaller size. However, dysfunctional deformations were not depicted. Adequate positioning of the stent's inflow area in the anterior chamber and the outflow area in the Tenon space was achieved in both the animal models and the human eye. Conclusions: Micro-CT has proven to be a valuable tool for postoperative ex vivo evaluation of glaucoma drainage devices in its entire complexity. With regard to morphology, the porcine eye is the ideal animal model to test implantation procedures of the RGM. Nevertheless, rabbit eye morphology facilitates successful implantation results and provides all prerequisites for preclinical animal studies.

One R or the other - an experimental bioethics approach to 3R dilemmas in animal research.

Sacrificial dilemmas such as the trolley problem play an important role in experimental philosophy (x-phi). But it is increasingly argued that, since we are not likely to encounter runaway trolleys in our daily life, the usefulness of such thought experiments for understanding moral judgments in more ecologically valid contexts may be limited. However, similar sacrificial dilemmas are experienced in real life by animal research decision makers. As part of their job, they must make decisions about the suffering, and often the death, of many non-human animals. For this reason, a context-specific investigation of so-called "3R dilemmas" (i.e., dilemmas where there is a conflict between the principles of replacement, reduction, and refinement of the use of animals in research) is essential to improve the situation of both non-human animals and human stakeholders. An approach well suited for such investigation is experimental philosophical bioethics ("bioxphi"), which draws on methods similar to x-phi to probe more realistic, practical scenarios with an eye to informing normative debates and ethical policy. In this article, we argue for a need to investigate 3R dilemmas among professional decision-makers using the tools of bioxphi. In a first step, we define 3R dilemmas and discuss previous investigations of professionals' attitudes in such cases. In a second step, we show how bioxphi is a promising method to investigate the whys and hows of professional decision-making in 3R dilemmas. In a last step, we provide a bioxphi template for 3R dilemmas, give recommendations on its use, explore the normative relevance of data collected by such means, and discuss important limitations.

Analysis of Meiotic Progression by Ex Vivo Culture of Mouse Embryonic Ovaries.

Oogenesis is the central process required to produce viable oocytes in female mammals. It is initiated during embryonic development, and it involves the specification of primordial germ cells (PGCs) and progresses through the activation of the meiotic program, reaching a crucial phase in prophase I before pausing at diplotene around the time of birth. The significance of meiosis, particularly the prophase I stage, cannot be overstated, as it plays a pivotal role in ensuring the formation of healthy gametes, a prerequisite for successful reproduction. While research has explored meiosis across various organisms, understanding how environmental factors, including radiation, drugs, endocrine disruptors, reproductive age, or diet, influence this complex developmental process remains incomplete. In this chapter, we describe an ex vivo culture method to investigate meiotic prophase I and beyond and the disruption of oogenesis by external factors. Using this methodology, it is possible to evaluate the effects of individual xenobiotics by administering chemicals at specific points during oogenesis. This culture technique was optimized to study the effects of two selected endocrine disruptors (vinclozolin and MEHP), demonstrating that vinclozolin exposure delayed meiotic differentiation and MEHP exposure reduced follicle size. This approach also opens avenues for future applications, involving the exploration of established or novel pharmaceutical substances and their influence on essential events during prophase I, such as homologous recombination and chromosome segregation. These processes collectively dictate the ultimate fitness of oocytes, with potential implications for factors relevant to the reproductive age and fertility.

The use of weight-of-evidence approaches to characterize developmental toxicity risk for therapeutic monoclonal antibodies in humans without in vivo studies.

Regulatory guidance for global drug development relies on animal studies to evaluate safety risks for humans, including risk of reproductive toxicity. Weight-of-evidence approaches (WoE) are increasingly becoming acceptable to evaluate risk. A WoE for developmental risk of monoclonal antibodies (mAbs) was evaluated for its ability to retrospectively characterize risk and to determine the need for further in vivo testing based on the remaining uncertainty. Reproductive toxicity studies of 65 mAbs were reviewed and compared to the WoE. Developmental toxicities were absent in 52/65 (80%) mAbs. Lack of toxicity was correctly predicted in 29/52 (56%) cases. False positive and equivocal predictions were made in 9/52 (17%) and 14/52 (27%) cases. For 3/65 (5%) mAbs, the findings were equivocal. Of mAbs with developmental toxicity findings (10/65, 15%), the WoE correctly anticipated pharmacology based reproductive toxicity without any false negative predictions in 9/10 (90%) cases, and in the remaining case (1/10, 10%) an in vivo study was recommended due to equivocal WoE outcome. Therefore, this WoE approach could characterize presence and absence of developmental risk without animal studies. The current WoE could have reduced the need for developmental toxicity studies by 42% without loss of important patient information in the label.

Endeavours made by trade associations, pharmaceutical companies and regulators in the replacement, reduction and refinement of animal experimentation in safety testing of pharmaceuticals.

Following the European Commission decision to develop a roadmap to phase out animal testing and the signing of the US Modernisation Act, there is additional pressure on regulators and the pharmaceutical industry to abandon animal experimentation in safety testing. Often, endeavours already made by governments, regulators, trade associations, and industry to replace, reduce and refine animal experimentation (3Rs) are unnoticed. Herein, we review such endeavours to promote wider application and acceptance of 3Rs. ICH guidelines have stated 3Rs objectives and have enjoyed many successes driven by global consensus. Initiatives driven by US and European regulators such as the removal of the Abnormal Toxicity Test are neutralised by reticent regional regulators. Stream-lined testing requirements have been proposed for new modalities, oncology, impurity management and animal pharmacokinetics/metabolism. Use of virtual controls, value of the second toxicity species, information sharing and expectations for life-threatening diseases, human specific or well-characterised targets are currently being scrutinised. Despite much effort, progress falls short of the ambitious intent of decisionmakers. From a clinical safety and litigation perspective pharmaceutical companies and regulators are reluctant to step away from current paradigms unless replacement approaches are validated and globally accepted. Such consensus has historically been best achieved through ICH initiatives.

Neural acetylcholinesterase and monoamine oxidase deregulation during streptozotocin-induced behavioral, metabolic and redox modification in Nauphoeta cinerea.

Genetic and environmental factors have been linked with neurodegeneration, especially in the elderly. Yet, efforts to impede neurodegenerative processes have at best addressed symptoms instead of underlying pathologies. The gap in the understanding of neuro-behavioral plasticity is consistent from insects to mammals, and cockroaches have been proven to be effective models for studying the toxicity mechanisms of various chemicals. We therefore used head injection of 74 and 740 nmol STZ in Nauphoeta cinerea to elucidate the mechanisms of chemical-induced neurotoxicity, as STZ is known to cross the blood-brain barrier. Neurolocomotor assessment was carried out in a new environment, while head homogenate was used to estimate metabolic, neurotransmitter and redox activities, followed by RT-qPCR validation of relevant cellular signaling. STZ treatment reduced the distance and maximum speed travelled by cockroaches, and increased glucose levels while reducing triglyceride levels in neural tissues. The activity of neurotransmitter regulators - AChE and MAO was exacerbated, with concurrent upregulation of glucose sensing and signaling, and increased mRNA levels of redox regulators and inflammation-related genes. Consequently, STZ neurotoxicity is conserved in insects, with possible implications for using N. cinerea to target the multi-faceted mechanisms of neurodegeneration and test potential anti-neurodegenerative agents.

Mutagenesis on a complex mouse genetic background by site-specific nucleases.

Mouse models with complex genetic backgrounds are increasingly used in preclinical research to accurately model human disease and to enable temporal and cell-specific evaluation of genetic manipulations. Backcrossing mice onto these complex genetic backgrounds takes time and leads to significant wastage of animals. In this study, we aimed to evaluate whether site-specific nucleases could be used to generate additional genetic mutations in a complex genetic background, using the REVERSA mouse model of atherosclerosis, a model harbouring four genetically altered alleles. The model is comprised of a functional null mutation in the Ldlr gene in combination with a ApoB100 allele, which, after high-fat diet, leads to the rapid development of atherosclerosis. The regression of the pathology is achieved by inducible knock-out of the Mttp gene. Here we report an investigation to establish if microinjection of site-specific nucleases directly into zygotes prepared from the REVERSA could be used to investigate the role of the ATP binding cassette transporter G1 (ABCG1) in atherosclerosis regression. We show that using this approach we could successfully generate two independent knockout lines on the REVERSA background, both of which exhibited the expected phenotype of a significant reduction in cholesterol efflux to HDL in bone marrow-derived macrophages. However, loss of Abcg1 did not impact atherosclerosis regression in either the aortic root or in aortic arch, demonstrating no important role for this transporter subtype. We have demonstrated that site-specific nucleases can be used to create genetic modifications directly onto complex disease backgrounds and can be used to explore gene function without the need for laborious backcrossing of independent strains, conveying a significant 3Rs advantage.

Rethinking the Three Rs: Growing Momentum Toward a New Gold Standard in Australian Research.

Recent years have seen increasing recognition of the scientific, economic and ethical benefits of the use of non-animal models in advancing preclinical research, giving reason to rethink the application and framework of the Three Rs. However, to benefit from the economic advantages of shifting to such alternative methods, and to realise Australia's drug development potential, legislative reform is essential. Such reform should be responsive to international regulations that encourage the use of animal-free methods, and be coupled with a corresponding re-evaluation of current Three Rs frameworks and principles. If these supportive changes, and the recommendations from the 2023 Australian Commonwealth Scientific and Industrial Research Organisation (CSIRO) Futures Non-animal models report, are implemented concurrently - with government support paramount- then a new gold standard for scientific research in Australia could be created in which the use of non-animal models and animal-free methods is the default.

Collaborative study for the characterisation of the BINACLE Assay for in vitro detection of tetanus toxicity in toxoids - Part 2.

Tetanus vaccines for human and veterinary use are produced by formaldehyde-induced inactivation of tetanus neurotoxin (TeNT) purified from Clostridium tetani cultures. Due to the high morbidity caused by exposure to TeNT it is essential that the quality control of tetanus vaccines includes testing for absence of tetanus toxin as prescribed by European Pharmacopoeia monographs 0452 and 0697. Currently this test is carried out in guinea pigs for each bulk of tetanus toxoid. To test the applicability of the in vitro BINACLE ("binding and cleavage") assay as an alternative method for the quality control of tetanus vaccines, two collaborative studies were run by the European Directorate for the Quality of Medicines & HealthCare under the aegis of the Biological Standardisation Programme. The first collaborative study indicated that the method allows sensitive TeNT detection. However, a clear conclusion could not be drawn due to the high variability of the results. To address the variability, the protocol was optimised and further standardised for the second study. The study results demonstrated good assay precision, both with respect to repeatability and reproducibility. Importantly, the limit of detection was 0.11 ng/mL TeNT in five out of nine laboratories and 0.33 ng/mL in four out of nine laboratories, suggesting that the BINACLE assay can detect TeNT with similar sensitivity as in vivo toxicity tests and can thus be taken into consideration as an alternative method to the current compendial in vivo test.

Collaborative study for the characterisation of the BINACLE Assay for in vitro detection of tetanus toxicity in toxoids - Part 1.

For several decades the European Pharmacopoeia monographs Tetanus vaccine (adsorbed) (0452) and Tetanus vaccine for veterinary use (0697) required that Specific toxicity and Absence of toxin and irreversibility of the toxoidof each bulk of tetanus toxoids had to be tested by an in vivo toxicity test in guinea pigs before it could be included in vaccines for human or veterinary use. In line with the 3Rs concept of replacing, reducing and refining animal experiments, an in vitro method for the detection of active tetanus neurotoxin (TeNT) has been developed at the Paul-Ehrlich-Institut (PEI, Germany). This method, the so-called BINACLE (binding and cleavage) assay, uses the receptor-binding and proteolytic properties of TeNT for the specific detection of active toxin molecules. Successful in-house validation studies as well as a small-scale transferability study had demonstrated that this method may represent a suitable alternative to the compendial in vivo toxicity test. As a follow up, an international collaborative study aimed at verifying the suitability of the BINACLE assay as a potential alternative to the guinea pig toxicity test for tetanus toxoids was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) under the aegis of its Biological Standardisation Programme (BSP). Within the framework of this study, coded BSP136, a feasibility phase - also referred to as Phase 1 - was run to select and qualify critical study reagents and samples and to assess the performance of the BINACLE Standard Operating Procedure developed by the project leaders. Then the international collaborative study aimed at evaluating the BINACLE, referred to as BSP136 Phase 2, was started. A total of 19 international laboratories (comprising vaccine manufacturers as well as national control laboratories) were supplied with a detailed assay protocol, critical reagents required for the assay, three samples consisting of three different bulk tetanus toxoids donated by major European vaccine manufacturers and one international standard toxoid. Each of the participants was asked to perform three independent BINACLE assays following the provided protocol. The statistical analysis of the results showed that most of the participating laboratories were able to perform the BINACLE assay according to the provided protocol. However, the results obtained by the participants varied widely, and not all the laboratories were able to achieve a sensitive detection of active TeNT. Multiple factors may have contributed to the elevated variability of the BSP136 study results. From an analysis of these factors, strategies were developed to help increase the standardisation of the BINACLE assay and obtain more consistent results in a follow-up validation study, BSP 136 Phase 3 (Part 2), for which the experimental phase took place in 2023. The present manuscript summarises the outcome of Phases 1 and 2, which constitute Part 1 of the BSP136 project.

A unified approach to investigating 4 dpf zebrafish larval behaviour through a standardised light/dark assay.

Zebrafish are a dynamic research model in the domains of neuropsychopharmacology, biological psychiatry and behaviour. Working with larvae ≤4 days post-fertilisation (dpf) offers an avenue for high-throughput investigation whilst aligning with the 3Rs principles of animal research. The light/dark assay, which is the most widely used behavioural assay for larval neuropharmacology research, lacks experimental reliability and standardisation. This study aimed to formulate a robust, reproducible and standardised light/dark behavioural assay using 4 dpf zebrafish larvae. Considerable between-batch and inter-individual variability was found, which we rectified with a normalisation approach to ensure a reliable foundation for analysis. We then identified that 5-min light/dark transition periods are optimal for locomotor activity. We also found that a 30-min acclimation in the light was found to produce significantly increased dark phase larval locomotion. Next, we confirmed the pharmacological predictivity of the standardised assay using ethanol which, as predicted, caused hyperlocomotion at low concentrations and hypolocomotion at high concentrations. Finally, the assay was validated by assessing the behavioural phenotype of hyperactive transgenic (adgrl3.1-/-) larvae, which was rescued with psychostimulant medications. Our standardised assay not only provides a clear experimental and analytical framework to work with 4 dpf larvae, but also facilitates between-laboratory collaboration using our normalisation approach.

Neurobehavioral, neurotransmitter and redox modifications in Nauphoeta cinerea under mixed heavy metal (silver and mercury) exposure.

Heavy metals are encountered in nature, and are used in several human endeavors, including in dental fillings. It is well known that the safety of metals depends on their chemical form, as well as the dose and route through which biological systems are exposed to them. Here, we used the Nauphoeta cinerea model to examine the mechanism by which salts of the heavy metals used in dental fillings - silver and mercury - exert their neurotoxicity. Nymphs exposed to heavy metals presented with reduced motor and exploratory abilities as they spent more time immobile, especially in the periphery of a novel object, and covered less distance compared with control nymphs. Exposure to AgNO3 and HgCl2 also exacerbated levels of oxidative stress markers (MDA & ROS) and the neurotransmitter regulators - AChE and MAO, while reducing antioxidant activity markers, both in biochemical (thiol & GST) and RT-qPCR (TRX, GST, SOD, Catalase) examinations, in neural tissues of the cockroach. The observed disruptions in neurolocomotor control, synaptic transmission and redox balance explain how heavy metal salts may predispose organisms to neurological disorders.

Biochemical study of ZNF76 rs10947540 and SCUBE3 rs1888822 single nucleotide polymorphisms in the Egyptian patients with systemic lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a common autoimmune disease marked by the formation of apoptotic debris and the presence of autoantibodies that target nuclear components. At this moment, the actual cause of SLE is uncertain. Genetic variables have been well proven to have a significant role in the propensity of SLE. This study aimed to investigate the effect of (ZNF76) rs (10947540) and (SCUBE) rs (1888822) gene polymorphism in patients with systemic lupus erythematosus. A case control study has been carried out at Medical Biochemistry & Molecular biology and Rheumatology unit of Internal Medicine Departments, Faculty of Medicine, Menoufia University, Egypt, for 1-year duration between 1 June 2022 and 1 June 2023. Sixty patients were females (75%) and twenty patients were males (25%). Their ages ranged from 19 to 53 years. Their disease durations ranged from 7 months to 20 years. The findings indicated that the TC genotype of the ZNF76 rs10947540 gene increases the risk of SLE by 2.274-fold, while the dominant TC + CC increases the risk by 2.472-fold, and the C allele increases the risk by 2.115-fold. Additionally, the results showed that the TT genotype of the SCUBE3 rs1888822 gene increases the risk of SLE by 3.702-fold, the dominant GT + TT increases the risk by 2.304-fold, and the T allele increases the risk by 2.089-fold, while the GT genotype increases the risk by 1.918-fold. The study revealed significant associations between the genotypes of these polymorphisms and certain clinical parameters in SLE patients. These findings highlight the potential genetic contributions to SLE susceptibility and its clinical manifestations, providing valuable insights for future research and potential personalized approaches to the management of this complex autoimmune disease.

Feasibility of Using a Type I IFN-Based Non-Animal Approach to Predict Vaccine Efficacy and Safety Profiles.

Animal-based tests are used for the control of vaccine quality. However, because highly purified and safe vaccines are now available, alternative approaches that can replace or reduce animal use for the assessment of vaccine outcomes must be established. In vitro tests for vaccine quality control exist and have already been implemented. However, these tests are specifically designed for some next-generation vaccines, and this makes them not readily available for testing other vaccines. Therefore, universal non-animal tests are still needed. Specific signatures of the innate immune response could represent a promising approach to predict the outcome of vaccines by non-animal methods. Type I interferons (IFNs) have multiple immunomodulatory activities, which are exerted through effectors called interferon stimulated genes (ISGs), and are one of the most important immune signatures that might provide potential candidate molecular biomarkers for this purpose. This paper will mainly examine if this idea might be feasible by analyzing all relevant published studies that have provided type I IFN-related biomarkers for evaluating the safety and efficacy profiles of vaccines using an advanced transcriptomic approach as an alternative to the animal methods. Results revealed that such an approach could potentially provide biomarkers predictive of vaccine outcomes after addressing some limitations.

SMAFIRA: a literature-based web tool to assist researchers with retrieval of 3R-relevant information.

The search for 3R-relevant information is a prerequisite for any planned experimental approach considering animal use. Such a literature search includes all methods to replace, reduce and refine (3Rs) animal testing with the aim of improving animal welfare, and requires an intensive screening of literature databases reflecting the current state of knowledge in experimental biomedicine. We developed SMAFIRA, a freely available online tool to facilitate the screening of PubMed/MEDLINE for possible alternatives to animal testing. SMAFIRA employs state-of-the-art language models from the field of deep learning, and provides relevant literature citations in a ranked order, classified according to the experimental model used. By using this classification, the search for alternative methods in the biomedical literature will become much more efficient. The tool is available at https://smafira.bf3r.de.

Erratum: Editorial: Towards a new 3Rs era in experimental research.

[This corrects the article DOI: 10.3389/fnbeh.2024.1404294.].

Plexin B2 tissue expression and related gene polymorphisms in psoriasis and their relation to NB-UVB and Acitretin therapy.

Psoriasis is a chronic, immune-mediated, hyperproliferative skin disease. Etiopathogenesis of psoriasis is not well understood. Plexin B2 was found to have effects on CD100-mediated T-cell morphology and expressed in the immune system. It may play a role in the pathogenesis of psoriasis. To assess the tissue level of plexin-B2 and plexin B2 related gene polymorphism which is signal regulatory protein gamma (SIRPγ-rs71212732) in psoriatic patients before and after NB-UVB, acitretin therapy alone or in combination and to detect correlation between level of tissue plexin B2 and disease severity and improvement. This single blinded randomized controlled trial was carried on 50 psoriatic patients and 50 healthy controls. Psoriasis Area and Severity Index score (PASI) was used to evaluate the disease severity. Tissue plexin-b2 level was measured using ELISA and SIRPγ-rs71212732 (T\C) was assessed using TaqMan™ assays and real-time PCR. A significant lower tissue plexin-B2 level was observed in control group (2.9 ± 0.6 pg/g) than cases (25.8 ± 2.8, pg/g) (p < 0.001). Also, a significantly higher tissue plexin-B2 level was observed in sever psoriasis (32.7 ± 3.8 pg/ml) in than moderate psoriasis (13.6 ± 2.1 pg/ml, p = 0.001). Tissue plexin B2 was positively correlated with diseases severity. Significantly higher (TC& TT) genotypes and mutant (C) allele among patients compared to the controls, p < 0.001 for all. Tissue plexin-b2 level was high in psoriasis vulgaris with positive correlation with disease severity and decreased after treatment. This may indicate a role of plexin-b2 in psoriasis vulgaris pathogenesis.