3Rs Principle Research Articles

Toward a common interpretation of the 3Rs principles in animal research.

Toward a common interpretation of the 3Rs principles in animal research.

A Novel Machine Learning Model and a Web Portal for Predicting the Human Skin Sensitization Effects of Chemical Agents

Skin sensitization is a significant concern for chemical safety assessments. Traditional animal assays often fail to predict human responses accurately, and ethical constraints limit the collection of human data, necessitating a need for reliable in silico models of skin sensitization prediction. This study introduces HuSSPred, an in silico tool based on the Human Predictive Patch Test (HPPT). HuSSPred aims to enhance the reliability of predicting human skin sensitization effects for chemical agents to support their regulatory assessment. We have curated an extensive HPPT database and performed chemical space analysis and grouping. Binary and multiclass QSAR models were developed with Bayesian hyperparameter optimization. Model performance was evaluated via five-fold cross-validation. We performed model validation with reference data from the Defined Approaches for Skin Sensitization (DASS) app. HuSSPred models demonstrated strong predictive performance with CCR ranging from 55 to 88%, sensitivity between 48 and 89%, and specificity between 37 and 92%. The positive predictive value (PPV) ranged from 84 to 97%, versus negative predictive value (NPV) from 22 to 65%, and coverage was between 75 and 93%. Our models exhibited comparable or improved performance compared to existing tools, and the external validation showed the high accuracy and sensitivity of the developed models. HuSSPred provides a reliable, open-access, and ethical alternative to traditional testing for skin sensitization. Its high accuracy and reasonable coverage make it a valuable resource for regulatory assessments, aligning with the 3Rs principles. The publicly accessible HuSSPred web tool offers a user-friendly interface for predicting skin sensitization based on chemical structure.

Are Repeat-Dose Toxicity Studies Informative for Safety Assessment of Vaccine Candidates? A Survey of Vaccine Developers.

A BioSafe-sponsored survey investigated how vaccine companies (n = 12) perceive the value of the repeat-dose toxicity studies for safety assessment of vaccine candidates. As all major vaccine developers were part of the survey, it was considered representative for the industry practices up to 2022. Vaccine developers indicated that they see scientific value in performing repeat-dose toxicity studies with vaccines, especially when novel components (e.g., adjuvant) or technology is being used. However, a few (3/12) also indicated that repeat-dose toxicity studies could be replaced by a pharmacology study with additional toxicity parameters. For the majority of companies (9/12), findings from the repeat-dose toxicity studies never prevented or postponed a first-in-human (FIH) trial. In the remaining 3 companies, a total of 4 occurrences of postponement or prevention of clinical development occurred and in only 2 of these cases was the finding considered related to the vaccine. A platform approach has been successfully implemented for influenza vaccines already in 2016, and an outline of the regulatory requirements for a platform approach has been recently documented in the latest infectious disease mRNA-LNP vaccine guideline, as well as in the guidance on the development and licensure of COVID-19 vaccines presented by the FDA. Vaccine developers are seeking to extend this platform approach to the development of new vaccines, building on established technologies and using well-defined manufacturing processes. This approach could support reduction of animal use (a principle of 3Rs) while still providing reassurance of the nonclinical safety of these products.

In vivo fertilization improved the cryotolerance and developmental ability of vitrified-warmed rat fertilized oocytes

The cryopreservation of rat embryos is useful for efficiently archiving rat resources in bioresource repositories. The cryopreserved fertilized oocytes can be quickly reanimated to rats with homozygous mutations using embryo transfer. In addition, cryopreserved rat fertilized oocytes are easier to transport than live animals. Before cryopreservation, fertilized oocytes are obtained by mating or in vitro fertilization. However, it is not clear which fertilized oocytes are most suited to cryopreservation. In this study, we performed a systematic comparison of the fertilizing ability, cryotolerance, and developmental ability of cryopreserved fertilized oocytes at the pronuclear stage produced either by mating (in vivo) or in vitro fertilization (in vitro) in SD and F344 rats. In vivo-fertilized oocytes had higher cryotolerance and developmental ability than in vitro-fertilized oocytes in SD and F344 rats. Furthermore, the fertilization ability, cryotolerance, and developmental ability of vitrified-warmed fertilized oocytes differed between SD and F344 rats. In conclusion, our study suggests that in vivo-fertilized rat oocytes were more suitable for cryopreservation. Our protocol provides an optimized system for the management of rat colonies using fertilized oocytes cryopreservation and contributes to the 3Rs principle by reducing the number of animals used for research.

Why choose the rabbit to work in reproductive technology?

Rabbits have played a significant role in both livestock production and the advancement of reproductive scientific research. Their unique biological traits, including induced ovulation and a reproductive process that closely mirrors that of humans, have been pivotal in their use as a model. Moreover, their body size is perfectly aligned with the 3Rs principles: Replacement, Reduction, and Refinement. Consequently, techniques for gamete collection and embryo recovery, followed by their use in artificial insemination or embryo transfer, are characterized by being minimally invasive. However, refining invitro fertilization and embryo culture techniques continues to present challenges. The incorporation of cutting-edge genomic editing tools, such as CRISPR/Cas9, has reestablished rabbits as essential models in genetic and biomedical research, driving scientific progress. This review aims to describe the most effective reproductive biotechnologies for both male and female rabbits and how these methodologies are in line with the 3Rs principles-Replacement, Reduction, and Refinement-highlighting their significance in conducting ethical research.

Statistical applications of virtual control groups to nonrodent animal toxicity studies: An initial evaluation

Utilization of data from historical control animals to form virtual control groups (VCGs) is an innovative approach to embody the 3Rs (reduce, refine, and replace use of control animals) principle in research. However, there is no available systematic comparison of statistical performance between concurrent control groups (CCGs) and VCGs in nonrodent safety assessment. The optimal selection criteria and combination of VCGs and CCGs also remain unclear. This study retrospectively evaluated VCGs’ statistical performance to detect test article effects on body weight and clinical pathology endpoints in dog and nonhuman primate (NHP) systemic toxicity studies. Body weight and six clinical pathology endpoints were analyzed against the reported study findings from a cohort of 22 previously reported nonrodent 1-month oral gavage toxicity using three different methods of generating VCGs. When the fold change from baseline was used, VCGs yielded a similar or higher statistical sensitivity to detect test article relatedness than CCGs. Compared to simple random sampling or using fixed criteria, the propensity score matching by BW, age, and year of study initiation yielded higher sensitivities. Our analysis supports the hypothesis that VCGs can be a viable instrument in nonrodent toxicity studies.

A novel integrated testing strategy (ITS) for evaluating acute fish toxicity with new approach methodologies (NAMs)

Acute fish toxicity (AFT) tests are performed in aquatic risk assessments of chemical compounds globally. However, the specific endpoint of in vivo AFT is based on the lethal concentration 50 (LC50), which is a serious challenge in terms of animal welfare. To support the 3Rs principle of replacing, reducing, and refining use of animals, integrated testing strategies (ITS) have recently been developed for environmental risk assessment. ITS efficiently integrates multiple types of information, especially new approach methodologies (NAMs), and further supports regulatory decision-making. Currently, an effective ITS framework for evaluating aquatic toxicity is lacking. Therefore, we aimed to develop a promising ITS for AFT using in silico, in vitro, and in vivo data. We established the ITS via in silico (OECD QSAR Toolbox 4.6), fish cell line acute toxicity (FCT), and fish embryo acute toxicity (FET) tests and then validated the NAMs with AFT testing. The NAM data were derived from the European Chemicals Agency (ECHA) dossier, toxicology databases, peer-reviewed research articles, and this study. For the first step in the ITS process, we aimed to design a high-throughput screening tool to identify non-toxic and toxic chemicals. We found that results of in silico, FCT, and FET tests alone were strongly correlated with AFT. Among the models, the in silico model was most suitable for identifying toxicants due to its high sensitivity and minimal animal use. Next, considering regulatory purposes and flexibility, we determined the predictive LC50 of toxic chemicals by pursuing a preference-dependent strategy, sequential testing strategy, and sensitivity-dependent strategy. All the strategies demonstrated a predictive power equal to or greater than 73%. In addition, to meet user preferences, our ITS approach has high flexibility and supports animal welfare and environmental protection. We have therefore developed multiple powerful, flexible, and more humane ITS methods for acute fish toxicity assessment by integrating NAMs.

A replacement approach to rodent model for the toxicity evaluation of gram-positive autogenous vaccine

The Abnormal Toxicity Test (ATT) is an in vivo assay traditionally used in the autogenous vaccine release. That test, commonly applied to verify vaccine quality, is subject to ethical and practical limitations and should be removed as reference methods.This approach is necessary to update the regulatory framework and to apply the 3Rs principle in the pharmaceutical field.In this study, two in vitro cell viability assays were used to evaluate the potential toxicity of autogenous vaccines. Cells were treated with base-2 dilutions of autogenous vaccines, and viability was detected by MTT and ATP assays.Differences and similarities between these tests were analysed. The ATP test showed good cell viability at 1:32 or 1:64 vaccine dilutions. The MTT assay gave discordant results and vaccines were not cytotoxic from the first dilutions. For this reason, a preference has been highlighted for the ATP method, a more sensitive test, capable of providing more accurate results.

An alternative method for comparing hemorrhagic activity of snake venoms following one of the 3R's principles

Venom-induced hemorrhage analysis usually is performed by Minimum Hemorrhagic Dose (MHD), however a similar method can be used to compare venoms with fewer laboratory animals. Our work compared the MHD of five different venoms, with the size of hemorrhagic spot, finding good correlations in the results. Considering the 3Rs principle, we propose the use of the hemorrhagic spot method to compare hemorrhagic activity of snake venoms, rather than using the MHD method, since the first one needs 5 times less animals than the other.

Artificial intelligence-based analysis of behavior and brain images in cocaine-self-administered marmosets

BackgroundThe sophisticated behavioral and cognitive repertoires of non-human primates (NHPs) make them suitable subjects for studies involving cocaine self-administration (SA) schedules. However, ethical considerations, adherence to the 3Rs principle (replacement, reduction and refinement), and other factors make it challenging to obtain NHPs individuals for research. Consequently, there is a need for methods that can comprehensively analyze small datasets using artificial intelligence (AI). New methodsWe employed AI to identify cocaine dependence patterns from collected data. First, we collected behavioral data from cocaine SA marmosets (Callithrix jacchus) to develop a dependence prediction model. SHapley Additive exPlanations (SHAP) values were used to demonstrate the importance of various variables. Additionally, we collected positron emission tomographic (PET) images showing dopamine transporter (DAT) binding potential and developed an algorithm for PET image segmentation. ResultsThe prediction model indicated that the Random Forest (RF) algorithm performed best, with an area under the curve (AUC) of 0.92. The top five variables influencing the model were identified using SHAP values. The PET image segmentation model achieved an accuracy of 0.97, a mean squared error of 0.02, an intersection over union (IoU) of 0.845, and a Dice coefficient of 0.913. Comparison with existing methods and conclusionUtilizing data from the marmoset SA experiment, we developed an ML-based dependence prediction model and analyzed variable importance rankings using SHAP. AI-based imaging segmentation methods offer a valuable tool for evaluating DAT availability in NHPs following chronic cocaine administration.

Culture of care: the question of animal agency in laboratory animal science.

A majority of the current debates in experimental animal science research focus to a large extent on the significance and implementation of the 3Rs principle according to Russell and Burch. In this context, not least due to the EU Directive 2010/63/EU, the concept of a culture of care has become more prevalent. Although animals are essential actors in the field of laboratory science, the discussion around animal agency, as well as the resulting consequences for laboratory animal science, is currently unconsidered. The purpose of this qualitative survey was to identify the perception and understanding of professional workers in laboratory animal science regarding the culture of care in general and aspects of animal agency in particular. Using a non-standardized qualitative survey method (topic-oriented, guideline-based expert interviews), persons involved in animal experimentation in different groups (management, science, regulation, and care) were interviewed. Overall, the results of the qualitative survey showed that animal agency plays a subordinate role in the question of a culture of care in animal research. Although not all groups explicitly applied the construct of animal agency or comparable terminology for this, there were links to the theoretical construct. Overall, the interviews showed a recognized network between humans and animals and that animals can interact dialogically in research. This is justified, for example, by the transfer of emotions from carers or scientists to animals. Nevertheless, a differentiated reflection of an animal's agency remains disregarded. The present qualitative survey approached the understanding of a culture of care among experts in the field of animal research. Animal agency does occur in the theoretical reception of the culture of care model. However, it is not conclusively established in everyday practice. Rather, the results lead to the assumption that strategies are being implemented to largely fade out animal agency.

Transcriptomic Point of Departure (tPOD) of androstenedione in zebrafish embryos as a potential surrogate for chronic endpoints

The transcriptomic Point of Departure (tPOD) is increasingly used in ecotoxicology to derive quantitative endpoints from RNA sequencing studies. Utilizing transcriptomic data in zebrafish embryos as a New Approach Methodology (NAM) is beneficial due to its acknowledgment as an alternative to animal testing under EU Directive 2010/63/EU. Transcriptomic profiles are available in zebrafish for various modes of action (MoA). The limited literature available suggest that tPOD values from Fish Embryo Toxicity (FET) tests align with, but are generally lower than, No Observed Effect Concentrations (NOEC) from long-term chronic fish toxicity tests.In studies with the androgenic hormone androstenedione in a Fish Sexual Development Test (FSDT), a significant shift in the sex ratio towards males was noted at all test concentrations, making it impossible to determine a NOEC (NOEC <4.34 μg/L). To avoid additional animal testing in a repetition of the FSDT and adhere to the 3Rs principle (replacement, reduction, and refinement), a modified zebrafish FET (zFET) was conducted aiming to determine a regulatory acceptable effect threshold. This involved lower concentration ranges (20 to 6105 ng/L), overlapping with the masculinization-observed concentrations in the FSDT. The tPOD analysis in zFET showed consistent results with previous FSDT findings, observing strong expression changes in androgen-dependent genes at higher concentrations but not at lower ones, demonstrating a concentration-response relationship.The tPOD values for androstenedione were determined as 24 ng/L (10th percentile), 60 ng/L (20th gene), and 69 ng/L (1st peak). The 10th percentile tPOD value in zFET was 200 times lower than the lowest concentration in the FSDT. Comparing the tPOD values to literature suggests their potential to inform on the NOEC range in FSDT tests.

Māori Concepts in Animal Ethics: Implications for the Three Rs

ABSTRACT Our aim is to explore how Māori concepts can refresh thinking about animal ethics. As sources of data for this exploration, we draw on interviews with Māori expert informants, as well as information gathered from relevant literature. First, we overview the two main theoretical traditions of animal ethics, unifying and relational, followed by an account of the development and current status of the Three Rs (Replace, Reduce, and Refine) for animal ethics in practice. We then introduce central Māori ethical concepts and how they interconnect within the traditional Māori cosmos. Two more “Rs” fall out of the Māori concepts: Relate and Respect. Māori views of animals center on whakapapa, a unique Indigenous Māori concept, roughly equated to genealogy, which encapsulates in Māori terms how animals are related, both to humans and to primordial forces, thus commanding respect from humans – as kin, and as ethically significant beings in their own right. This Māori argument illuminates the logic of respect that underpins the Three Rs principles for reducing harm to animals in research, testing, and teaching.

Round Robin Study for Evaluation an in vitro skin irritation test for medical device extracts using KeraSkinTM in Korea

With the growing importance of alternative test methods that implement the 3Rs principles (Reduction, Refinement and Replacement) and the global importance of biological safety assessment data for medical devices is increasing. We have developed and optimized the ‘KeraSkin™ Skin Irritation Test (KeraSkin™ SIT) for medical device’ for regulatory application in biological evaluation according to ISO 10993-23. We conducted a round robin study to optimize and evaluate the performance of KeraSkin™ SIT for medical devices using KeraSkin™ Reconstructed Human Epidermis (RhE), which is developed and manufactured in Korea. This round robin study was performed to assess the transferability, reproducibility (within and between laboratories) and predictive capacity in 1 lead laboratory and 3 participating laboratories based on OECD Guidance Document 34. The predictive capacity, the results showed 83.3 % of sensitivity, 100 % of specificity and 91.6 % of accuracy. In conclusion, the results demonstrate that ‘KeraSkin™ SIT for medical device’ provides a robust test method for detecting irritant activity of medical device extracts and can be utilized for identifying low levels of potent irritants in medical device extracts. Therefore, it fulfills the requirements to be included as a ‘me-too’ test method to EpiDerm™ and SkinEthic™ skin irritation test in ISO 10993-23.

Linking One Health to 3R. Culture of Care as a bridge towards Russell and Burch's highest goal: Replacement

Animal testing has long been ethically controversial, initially due to religious and cultural beliefs and increasingly because of the obvious suffering inflicted. The 3Rs principles are now a central part of laboratory practice and have prompted discussions about adding further Rs to take account of societal values. However, this expansion can lead to contradictions, such as the emphasis on reproducibility, which implies the continued use of animals and thus conflicts with the original highest goal of the 3Rs, replacement. At the same time, the Culture of Care approach associated with the 3Rs promotes a change in the human-animal relationship but has not significantly challenged the use of animals in research. The One Health approach, which recognizes the connection between human, animal and environmental health, is also often criticized for prioritizing human health over animal welfare. This article aims to show how the One Health approach can be consistently extended to interdisciplinarity and intersectionality to include animals as stakeholders in the debate. It will also discuss the potential of the Culture of Care approach to transfer the implications of a consistently conceived One Health approach to the field of animal experimentation, in order to bring the complete renunciation of the use of sentient beings back into the focus of 3R research

Development of LC-MS/MS method for quantification of Lurasidone using volumetric absorptive microsampling (VAMS); a comparative study between dried blood and plasma samples

The ecological impact of biological, chemical, and analytical research practices, including toxic reagents and biohazardous waste, has led to the development of alternative sampling and extraction techniques for bioanalysis. Microsampling (sample volume < 50 µL) aligns with the 3Rs principle, allowing multiple sampling points from the same animal at different time points and improving animal welfare. A bioanalytical method was developed to investigate factors related to bioanalytical challenges and the implementation of microsampling techniques. An LC-MS/MS method for Volumetric Absorptive Microsampling (VAMS), 20 µL, was developed for quantifying Lurasidone using a liquid–liquid extraction technique. The method uses a C18, Phenomenex column for chromatographic separation and a mobile phase composition of Methanol, Acetonitrile, and Water with 0.1 % HFBA. The method was validated over a concentration range of 5.0 to 1200.0 ng/mL and achieved acceptable precision and accuracy. The recovery for analyte from VAMS was approximately 40% at four different concentrations and is consistent (%CV < 15), with no significant differences among HCT levels. The matrix factor ranged between 85.00 and 115.00 %, showing no substantial issues with reduced or enhanced signal. The stability data showed no significant degradation of LUR in VAMS samples when stored at room temperature for 15 days. The newly established method for Lurasidone confirmed the use of VAMS sampling method and its analysis on LC-MS/MS. Further, the data obtained from microsampling techniques was compared with conventional (plasma) technique, as proof-of-concept, and it confirms the agreement between the two methods. The study supports the advantages of microsampling in protecting the environment and animals while maintaining scientific judgement.

An anesthetized rat assay for evaluating the effects of organophosphate-based compounds and countermeasures on intracranial EEG and respiratory rate

The development of medical countermeasures (MCMs) against organophosphate (OP) induced poisoning is of substantial importance. Use of conventional therapeutics is complicated by off-target effects and restricted penetration of the blood-brain barrier (BBB). Therefore, a concerted effort is underway to discover improved acetylcholinesterase (AChE) reactivators, muscarinic acetylcholine receptor (mAChR) antagonists, and other countermeasures with broader spectrum activity and enhanced CNS efficacy. We recently developed a rat brain slice assay to assess the efficacy of AChE reactivators and mAChR antagonists against the acute effects of the organophosphorus AChE inhibitor 4-nitrophenyl isopropyl methylphosphonate (NIMP) in the basolateral amygdala (BLA). Here we introduce a complimentary anesthetized animal model to evaluate the same compounds in vivo with concurrent monitoring of EEG and respiratory rate. We find that intravenous delivery of 0.5 mg/kg NIMP reliably produces seizure like activity in the BLA, with concurrent respiratory depression and eventual respiratory failure. The central effects of AChE reactivators and mAChR antagonists delivered intravenously are consistent with their expected ability to cross the BBB. Combining our previously described in vitro assay with the methods described here provides a relatively comprehensive set of preclinical tools for evaluating the efficacy of novel MCMs. Notably, using these methods is expected to reduce the need to subject conscious animals to cholinergic crises, which aligns with the AAALAC’s 3Rs principle of refinement.

Transforming Culture: a Culture of Care in Animal Research

Abstract The EU adopted Directive 2010/63/EU, thereby creating an instrument for legally implementing effective measures to regulate animal experimentation. In this framework, the 3Rs principle for replacement, reduction, and refinement of animal experiments proposed by Russell and Burch (1959) is of paramount importance. The highest goal of the 3Rs can be understood as a Culture of Care (CoC), entailing complete renunciation of sentient beings as test subjects. The implementation of a CoC is explicitly mentioned in Recital 31 of the EU-Directive. This article elaborates on historical underpinnings of the CoC concept and uses qualitative social research to trace its characteristics. Data were based on non-standardized survey procedures (topic-oriented, guideline-based expert interviews) and were collected at the management, scientific, supervisory and care levels in relation to animal experimentation. The goal of the qualitative approach is to understand contexts, conditions, strategies, and consequences of the CoC and depict them as a theoretical model.

Glial Response and Neuronal Modulation Induced by Epidural Electrode Implant in the Pilocarpine Mouse Model of Epilepsy.

In animal models of epilepsy, cranial surgery is often required to implant electrodes for electroencephalography (EEG) recording. However, electrode implants can lead to the activation of glial cells and interfere with physiological neuronal activity. In this study, we evaluated the impact of epidural electrode implants in the pilocarpine mouse model of temporal lobe epilepsy. Brain neuroinflammation was assessed 1 and 3 weeks after surgery by cytokines quantification, immunohistochemistry, and western blotting. Moreover, we investigated the effect of pilocarpine, administered two weeks after surgery, on mice mortality rate. The reported results indicate that implanted mice suffer from neuroinflammation, characterized by an early release of pro-inflammatory cytokines, microglia activation, and subsequent astrogliosis, which persists after three weeks. Notably, mice subjected to electrode implants displayed a higher mortality rate following pilocarpine injection 2 weeks after the surgery. Moreover, the analysis of EEGs recorded from implanted mice revealed a high number of single spikes, indicating a possible increased susceptibility to seizures. In conclusion, epidural electrode implant in mice promotes neuroinflammation that could lower the seizure thresholds to pilocarpine and increase the death rate. An improved protocol considering the persistent neuroinflammation induced by electrode implants will address refinement and reduction, two of the 3Rs principles for the ethical use of animals in scientific research.

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.