Report of the First ONTOX Stakeholder Network Meeting: Digging Under the Surface of ONTOX Together With the Stakeholders.

The first Stakeholder Network Meeting of the EU Horizon 2020-funded ONTOX project was held on 13-14 March 2023, in Brussels, Belgium. The discussion centred around identifying specific challenges, barriers and drivers in relation to the implementation of non-animal new approach methodologies (NAMs) and probabilistic risk assessment (PRA), in order to help address the issues and rank them according to their associated level of difficulty. ONTOX aims to advance the assessment of chemical risk to humans, without the use of animal testing, by developing non-animal NAMs and PRA in line with 21st century toxicity testing principles. Stakeholder groups (regulatory authorities, companies, academia, non-governmental organisations) were identified and invited to participate in a meeting and a survey, by which their current position in relation to the implementation of NAMs and PRA was ascertained, as well as specific challenges and drivers highlighted. The survey analysis revealed areas of agreement and disagreement among stakeholders on topics such as capacity building, sustainability, regulatory acceptance, validation of adverse outcome pathways, acceptance of artificial intelligence (AI) in risk assessment, and guaranteeing consumer safety. The stakeholder network meeting resulted in the identification of barriers, drivers and specific challenges that need to be addressed. Breakout groups discussed topics such as hazard versus risk assessment, future reliance on AI and machine learning, regulatory requirements for industry and sustainability of the ONTOX Hub platform. The outputs from these discussions provided insights for overcoming barriers and leveraging drivers for implementing NAMs and PRA. It was concluded that there is a continued need for stakeholder engagement, including the organisation of a 'hackathon' to tackle challenges, to ensure the successful implementation of NAMs and PRA in chemical risk assessment.

Recommendations for robust and reproducible preclinical research in personalised medicine.

BACKGROUND: Personalised medicine is a medical model that aims to provide tailor-made prevention and treatment strategies for defined groups of individuals. The concept brings new challenges to the translational step, both in clinical relevance and validity of models. We have developed a set of recommendations aimed at improving the robustness of preclinical methods in translational research for personalised medicine. METHODS: These recommendations have been developed following four main steps: (1) a scoping review of the literature with a gap analysis, (2) working sessions with a wide range of experts in the field, (3) a consensus workshop, and (4) preparation of the final set of recommendations. RESULTS: Despite the progress in developing innovative and complex preclinical model systems, to date there are fundamental deficits in translational methods that prevent the further development of personalised medicine. The literature review highlighted five main gaps, relating to the relevance of experimental models, quality assessment practices, reporting, regulation, and a gap between preclinical and clinical research. We identified five points of focus for the recommendations, based on the consensus reached during the consultation meetings: (1) clinically relevant translational research, (2) robust model development, (3) transparency and education, (4) revised regulation, and (5) interaction with clinical research and patient engagement. Here, we present a set of 15 recommendations aimed at improving the robustness of preclinical methods in translational research for personalised medicine. CONCLUSIONS: Appropriate preclinical models should be an integral contributor to interventional clinical trial success rates, and predictive translational models are a fundamental requirement to realise the dream of personalised medicine. The implementation of these guidelines is ambitious, and it is only through the active involvement of all relevant stakeholders in this field that we will be able to make an impact and effectuate a change which will facilitate improved translation of personalised medicine in the future.

The 3Ranker: An AI-based Algorithm for Finding Non-animal Alternative Methods.

The search for existing non-animal alternative methods for use in experiments is currently challenging because of the lack of both comprehensive structured databases and balanced keyword-based search strategies to mine unstructured textual databases. In this paper we describe 3Ranker, which is a fast, keyword-independent algorithm for finding non-animal alternative methods for use in biomedical research. The 3Ranker algorithm was created by using a machine learning approach, consisting of a Random Forest model built on a dataset of 35 million abstracts and constructed with weak supervision, followed by iterative model improvement with expert curated data. We found a satisfactory trade-off between sensitivity and specificity, with Area Under the Curve (AUC) values ranging from 0.85-0.95. Trials showed that the AI-based classifier was able to identify articles that describe potential alternatives to animal use, among the thousands of articles returned by generic PubMed queries on dermatitis and Parkinson's disease. Application of the classification models on time series data showed the earlier implementation and acceptance of Three Rs principles in the area of cosmetics and skin research, as compared to the area of neurodegenerative disease research. The 3Ranker algorithm is freely available at www.open3r.org; the future goal is to expand this framework to cover multiple research domains and to enable its broad use by researchers, policymakers, funders and ethical review boards, in order to promote the replacement of animal use in research wherever possible.

A History of Regulatory Animal Testing: What Can We Learn?

The contemporary pharmaceutical industry is voicing growing concerns about the translatability and reproducibility of animal models. In addition, the usefulness of certain of the required regulatory safety tests in animals is being increasingly questioned. It remains difficult, however, to make the move toward alternative testing methods, not least because of legislative demands. A historical analysis was performed, in order to study how the mandatory animal studies in legislative requirements came about. This article reflects on the role that specific public health disasters played in the creation of (more) regulatory requirements for animal testing. It will show how the regulatory changes prompted by the sulfanilamide elixir disaster in the 1930s and the thalidomide disaster in the early 1960s were based on the belief that extensive animal testing would prevent similar future human health tragedies. As scientists increasingly highlight issues with translatability between non-human animals and humans, the belief that current regulatory requirements ensure safety becomes more difficult to maintain. In addition, it means that some of the regulations now in place require animal tests that do not contribute to the safety of a drug, as shown in a third case study of the court case by Vanda industries against the FDA. We finally argue that regulations should be critically examined and altered where necessary, so that they are no longer a barrier in the transition toward animal-free testing and more human-relevant science.

Effects of sleep deprivation on maternal behaviour in animal models: A systematic review and meta-analysis.

Pregnancy is a period of numerous physical and emotional changes in women's lives, including alterations in sleep patterns and worsening of pre-existing sleep disturbances, which possibly lead to impaired postpartum maternal behaviour and mother-infant relationship. The effects of sleep deprivation during pregnancy in maternal behaviour have been evaluated in preclinical studies, but have provided inconsistent results. Thus, in the present study, we aimed to evaluate the effects of sleep deprivation during pregnancy on maternal behaviour of animals through a systematic review and meta-analyses. After a two-step selection process, six articles were included, all of them describing rat studies. The most frequently used method of sleep deprivation was rapid eye movement sleep restriction, using the multiple-platform method. Four meta-analyses were performed, none of them presenting significant impact of sleep deprivation on maternal behaviour, failing to reproduce the results observed in previous clinical studies. In conclusion, our results show a lack of translational applicability of animal models to evaluate the effects of sleep deprivation during pregnancy on maternal behaviour.

A health-based recommended occupational exposure limit for nitrous oxide using experimental animal data based on a systematic review and dose-response analysis.

BACKGROUND: Nitrous oxide (N2O) is a common inhalation anaesthetic used in medical, paramedical, and veterinary practice. Since the mid 1950's, concerns have been raised regarding occupational exposure to N2O, leading to many epidemiological and experimental animal studies. Previous evaluations resulted in the classification of N2O as a possible risk factor for adverse reproductive health outcomes based on animal data. Human data were deemed inadequate primarily because of simultaneous co-exposures to other risk factors for adverse reproductive and developmental outcomes, including other anaesthetic gases. Since previous evaluations, controversies regarding N2O use remained and new approaches for dose response modelling have been adopted, calling for an update and re-evaluation of the body of evidence. This review aims to assess available animal evidence on N2O reproductive and developmental outcomes to inform a health-based recommended occupational exposure limit (OEL) for N2O with a benchmark dose-response modelling (BMD) approach. METHODS: Comprehensive searches in PubMed, EMBASE, and Web of Science were performed to retrieve all relevant studies addressing reproductive and developmental outcomes related to inhalation of N2O in animals. The articles retrieved were screened based on title-abstract and full text by two independent reviewers. After data extraction, an overview of all studies was created for the different endpoints, namely foetal outcomes (e.g., resorption), female outcomes (e.g. implantations), and male outcomes (e.g. sperm count). A subset of studies reporting on exposure relevant to workplace settings and with a sufficient number of tested doses were included in dose-response modelling using the BMD approach. RESULTS: In total, 15.816 articles were retrieved, of which 47 articles were finally included while 4 of those were used for the quantitative data synthesis. The overall risk of bias was judged to be probably high (using OHAT risk of bias tool) and unclear (using SYRCLE's risk of bias tool). From eligible rat studies, three studies provided an acceptable result by fitting a Hill model to the dose-response data. The resulting benchmark dose lower bounds (BMDLs) from three studies converged to an average (±sd) exposure level of 925 ± 2 mg/m3 at an additional risk of one standard deviation of implantation losses above those observed in the control group (i.e. reduced number of live foetuses/mother). For extrapolation from rats to humans, an uncertainty factor of 10 was used and an additional factor of 5 was applied to account for interindividual variability within the population of workers. CONCLUSION: With this systematic review, all available evidence for reproductive toxicity and adverse developmental outcomes in animals resulting from inhalation exposure to N2O was used to derive a health-based OEL recommendation of 20 mg/m3 as 8-h time-weighted average.

Can prospective systematic reviews of animal studies improve clinical translation?

Systematic reviews are powerful tools with the potential to generate high quality evidence. Their application to animal studies has been instrumental in exposing the poor quality of these studies, as well as a catalyst for improvements in study design, conduct and reporting. It has been suggested that prospective systematic reviews of animal studies (i.e. systematic reviews conducted prior to clinical trials) would allow scrutiny of the preclinical evidence, providing valuable information on safety and efficacy, and helping to determine whether clinical trials should proceed. However, while prospective systematic reviews allow valuable scrutiny of the preclinical animal data, they are not necessarily able to reliably predict the safety and efficacy of an intervention when trialled in humans. Consequently, they may not reliably safeguard humans participating in clinical trials and might potentially result in lost opportunities for beneficial clinical treatments. Furthermore, animal and human studies are often conducted concurrently, which not only makes prospective systematic reviews of animal studies impossible, but suggests that animal studies do not inform human studies in the manner presumed. We suggest that this points to a confused attitude regarding animal studies, whereby tradition demands that they precede human studies but practice indicates that their findings are often ignored. We argue that it is time to assess the relative contributions of animal and human research in order to better understand how clinical knowledge is actually produced.

The evidence for the physiological effects of lactate on the cerebral microcirculation: a systematic review.

Lactate's role in the brain is understood as a contributor to brain energy metabolism, but it may also regulate the cerebral microcirculation. The purpose of this systematic review was to evaluate evidence of lactate as a physiological effector within the normal cerebral microcirculation in reports ranging from in vitro experiments to in vivo studies in animals and humans. Following pre-registration of a review protocol, we systematically searched the PubMed, EMBASE, and Cochrane databases for literature covering themes of 'lactate', 'the brain', and 'microcirculation'. Abstracts were screened, and data extracted independently by two individuals. We excluded studies evaluating lactate in disease models. Twenty-eight papers were identified, 18 of which were in vivo animal experiments (65%), four on human studies (14%), and six on in vitro or ex vivo experiments (21%). Approximately half of the papers identified lactate as an augmenter of the hyperemic response to functional activation by a visual stimulus or as an instigator of hyperemia in a dose-dependent manner, without external stimulation. The mechanisms are likely to be coupled to NAD+ /NADH redox state influencing the production of nitric oxide. Unfortunately, only 38% of these studies demonstrated any control for bias, which makes reliable generalizations of the conclusions insecure. This systematic review identifies that lactate may act as a dose-dependent regulator of cerebral microcirculation by augmenting the hyperemic response to functional activation below 5 mmol/kg, and by initiating a hyperemic response above 5 mmol/kg. OPEN SCIENCE BADGES: This article has received a badge for *Pre-registration* because it made the data publicly available. The data can be accessed at www.radboudumc.nl/getmedia/53625326-d1df-432c-980f-27c7c80d1a90/THollyer_lactate_protocol.aspx. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Animal to human translation: a systematic scoping review of reported concordance rates.

BACKGROUND: Drug development is currently hampered by high attrition rates; many developed treatments fail during clinical testing. Part of the attrition may be due to low animal-to-human translational success rates; so-called "translational failure". As far as we know, no systematic overview of published translational success rates exists. SYSTEMATIC SCOPING REVIEW: The following research question was examined: "What is the observed range of the animal-to-human translational success (and failure) rates within the currently available empirical evidence?". We searched PubMed and Embase on 16 October 2017. We included reviews and all other types of "umbrella"-studies of meta-data quantitatively comparing the translational results of studies including at least two species with one being human. We supplemented our database searches with additional strategies. All abstracts and full-text papers were screened by two independent reviewers. Our scoping review comprises 121 references, with various units of measurement: compound or intervention (k = 104), study/experiment (k = 10), and symptom or event (k = 7). Diagnostic statistics corresponded with binary and continuous definitions of successful translation. Binary definitions comprise percentages below twofold error, percentages accurately predicted, and predictive values. Quantitative definitions comprise correlation/regression (r2) and meta-analyses (percentage overlap of 95% confidence intervals). Translational success rates ranged from 0 to 100%. CONCLUSION: The wide range of translational success rates observed in our study might indicate that translational success is unpredictable; i.e. it might be unclear upfront if the results of primary animal studies will contribute to translational knowledge. However, the risk of bias of the included studies was high, and much of the included evidence is old, while newer models have become available. Therefore, the reliability of the cumulative evidence from current papers on this topic is insufficient. Further in-depth "umbrella"-studies of translational success rates are still warranted. These are needed to evaluate the probabilistic evidence for predictivity of animal studies for the human situation more reliably, and to determine which factors affect this process.

Establishing a health-based recommended occupational exposure limit for nitrous oxide using experimental animal data - A systematic review protocol.

Nitrous oxide (N 2 O) is widely used as inhalation analgesic and anaesthetic in medical, paramedical, and veterinary practice. Previous evaluations resulted in classification of N 2 O as a possible risk factor for adverse reproductive health outcomes based on evidence from animal data. Available human data were considered inadequate, partly due to the possibility that other risk factors, such as co-exposures to other inhalation anaesthetics may have contributed to the adverse outcomes. As no substantial new human evidence has emerged since previous evaluations, this protocol describes a planned systematic review of the evidence obtained from animal studies. The aim is to assess the available evidence on the effects of N 2 O on reproductive and developmental outcomes in animals to inform a health-based recommended occupational exposure limit (OEL) for N 2 O. Comprehensive search strategies were designed to retrieve animal studies addressing N 2 O exposure from PubMed, EMBASE, and Web of Science. Screening of the studies retrieved will be performed by at least two independent reviewers, while discrepancies will be resolved by reaching consensus through repeated review and discussions. Articles will be included according to criteria specified in this protocol. Outcome data relevant for reproduction and development will be extracted and risk of bias will be assessed by two independent reviewers using the SYRCLE's risk of bias tool. Primary reproductive and developmental outcomes of interest will be the number of resorptions, malformations, and birth weight. We will focus on dose-response studies that allow to derive an OEL with the benchmark dose (BMD) approach. Adverse outcomes occurring at doses that are equivalent to the exposures occurring in human occupational settings will be particularly relevant for dose-response modelling. The proposed review has not been performed before. We will follow the procedures specified in this protocol. We will adhere to guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA), adapted for animal studies. Ethical approval will not be required, as the review will use existing data available in the public domain.

Is it possible to overcome issues of external validity in preclinical animal research? Why most animal models are bound to fail.

BACKGROUND: The pharmaceutical industry is in the midst of a productivity crisis and rates of translation from bench to bedside are dismal. Patients are being let down by the current system of drug discovery; of the several 1000 diseases that affect humans, only a minority have any approved treatments and many of these cause adverse reactions in humans. A predominant reason for the poor rate of translation from bench to bedside is generally held to be the failure of preclinical animal models to predict clinical efficacy and safety. Attempts to explain this failure have focused on problems of internal validity in preclinical animal studies (e.g. poor study design, lack of measures to control bias). However there has been less discussion of another key factor that influences translation, namely the external validity of preclinical animal models. REVIEW OF PROBLEMS OF EXTERNAL VALIDITY: External validity is the extent to which research findings derived in one setting, population or species can be reliably applied to other settings, populations and species. This paper argues that the reliable translation of findings from animals to humans will only occur if preclinical animal studies are both internally and externally valid. We review several key aspects that impact external validity in preclinical animal research, including unrepresentative animal samples, the inability of animal models to mimic the complexity of human conditions, the poor applicability of animal models to clinical settings and animal-human species differences. We suggest that while some problems of external validity can be overcome by improving animal models, the problem of species differences can never be overcome and will always undermine external validity and the reliable translation of preclinical findings to humans. CONCLUSION: We conclude that preclinical animal models can never be fully valid due to the uncertainties introduced by species differences. We suggest that even if the next several decades were spent improving the internal and external validity of animal models, the clinical relevance of those models would, in the end, only improve to some extent. This is because species differences would continue to make extrapolation from animals to humans unreliable. We suggest that to improve clinical translation and ultimately benefit patients, research should focus instead on human-relevant research methods and technologies.

Letter to the editor - round table unites to tackle culture change in an effort to improve animal research reporting.

A round table discussion was held during the LAVA-ESLAV-ECLAM conference on Reproducibility of Animal Studies on the 25th of September 2017 in Edinburgh. The aim of the round table was to discuss how to enhance the rate at which the quality of reporting animal research can be improved. This signed statement acknowledges the efforts that participant organizations have made towards improving the reporting of animal studies and confirms an ongoing commitment to drive further improvements, calling upon both academics and laboratory animal veterinarians to help make this cultural change.

SYRCLE's risk of bias tool for animal studies.

BACKGROUND: Systematic Reviews (SRs) of experimental animal studies are not yet common practice, but awareness of the merits of conducting such SRs is steadily increasing. As animal intervention studies differ from randomized clinical trials (RCT) in many aspects, the methodology for SRs of clinical trials needs to be adapted and optimized for animal intervention studies. The Cochrane Collaboration developed a Risk of Bias (RoB) tool to establish consistency and avoid discrepancies in assessing the methodological quality of RCTs. A similar initiative is warranted in the field of animal experimentation. METHODS: We provide an RoB tool for animal intervention studies (SYRCLE's RoB tool). This tool is based on the Cochrane RoB tool and has been adjusted for aspects of bias that play a specific role in animal intervention studies. To enhance transparency and applicability, we formulated signalling questions to facilitate judgment. RESULTS: The resulting RoB tool for animal studies contains 10 entries. These entries are related to selection bias, performance bias, detection bias, attrition bias, reporting bias and other biases. Half these items are in agreement with the items in the Cochrane RoB tool. Most of the variations between the two tools are due to differences in design between RCTs and animal studies. Shortcomings in, or unfamiliarity with, specific aspects of experimental design of animal studies compared to clinical studies also play a role. CONCLUSIONS: SYRCLE's RoB tool is an adapted version of the Cochrane RoB tool. Widespread adoption and implementation of this tool will facilitate and improve critical appraisal of evidence from animal studies. This may subsequently enhance the efficiency of translating animal research into clinical practice and increase awareness of the necessity of improving the methodological quality of animal studies.

Pollutant exposure and myocardial injury: Protocol and progress report for a toxicological systematic mapping review.

An increasing body of evidence identifies pollutant exposure as a risk factor for cardiovascular disease (CVD), while CVD incidence rises steadily with the aging population. Although numerous experimental studies are now available, the mechanisms through which lifetime exposure to environmental pollutants can result in CVD are not fully understood. To comprehensively describe and understand the pathways through which pollutant exposure leads to cardiotoxicity, a systematic mapping review of the available toxicological evidence is needed. This protocol outlines a step-by-step framework for conducting this review. Using the National Toxicology Program (NTP) Health Assessment and Translation (HAT) approach for conducting toxicological systematic reviews, we selected 362 out of 8111 in vitro (17%), in vivo (67%), and combined (16%) studies for 129 potential cardiotoxic environmental pollutants, including heavy metals (29%), air pollutants (16%), pesticides (27%), and other chemicals (28%). The internal validity of included studies is being assessed with HAT and SYRCLE Risk of Bias tools. Tabular templates are being used to extract key study elements regarding study setup, methodology, techniques, and (qualitative and quantitative) outcomes. Subsequent synthesis will consist of an explorative meta-analysis of possible pollutant-related cardiotoxicity. Evidence maps and interactive knowledge graphs will illustrate evidence streams, cardiotoxic effects and associated quality of evidence, helping researchers and regulators to efficiently identify pollutants of interest. The evidence will be integrated in novel Adverse Outcome Pathways to facilitate regulatory acceptance of non-animal methods for cardiotoxicity testing. The current article describes the progress of the steps made in the systematic mapping review process.

Heart disease is a leading global cause of death. Recent research indicates that certain environmental chemicals can worsen heart problems. We're conducting a rigorous review of scientific studies to understand how these chemicals affect the heart. This will inform policymakers and promote non-animal testing methods for cardiotoxicity by providing a clear overview of the toxicological evidence. We have reviewed over 8,000 articles and focused on 362 studies about 129 chemicals, including heavy metals, air pollutants and pesticides, and their effects on the heart. The current manuscript describes the used methods and steps made in this process. The outcome of our systematic review of these 362 articles will be a comprehensive database that will aid the development of alternative testing methods for cardiotoxicity.

Methodological quality assessment should move beyond design specificity.

OBJECTIVE: This study aimed to assess the utility of a unified tool (MASTER) for bias assessment against design-specific tools in terms of content and coverage. METHODS: Each of the safeguards in the design-specific tools was compared and matched to safeguards in the unified MASTER scale. The design-specific tools were the JBI, Scottish Intercollegiate Guidelines Network (SIGN), and the Newcastle-Ottawa Scale (NOS) tools for analytic study designs. Duplicates, safeguards that could not be mapped to the MASTER scale, and items not applicable as safeguards against bias were flagged and described. RESULTS: Many safeguards across the JBI, SIGN, and NOS tools were common, with a minimum of 10 to a maximum of 23 unique safeguards across various tools. These 3 design-specific toolsets were missing 14 to 26 safeguards from the MASTER scale. The MASTER scale had complete coverage of safeguards within the 3 toolsets for analytic designs. CONCLUSIONS: The MASTER scale provides a unified framework for bias assessment of analytic study designs, has good coverage, avoids duplication, has less redundancy, and is more convenient when used for methodological quality assessment in evidence synthesis. It also allows assessment across designs that cannot be done using a design-specific tool.

Comparing translational success rates across medical research fields - A combined analysis of literature and clinical trial data.

Many interventions that show promising results in preclinical development do not pass clinical tests. Part of this may be explained by poor animal-to-human translation. Using animal models with low predictability for humans is neither ethical nor efficient. If translational success shows variation between medical research fields, analyses of common practices in these fields could identify factors contributing to successful translation. We assessed translational success rates in medical research fields using two approaches: through literature and clinical trial registers. Literature: We comprehensively searched PubMed for pharmacology, neuroscience, cancer research, animal models, clinical trials, and translation. After screening, 117 review papers were included in this scoping review. Translational success rates were not different within pharmacology (72%), neuroscience (62%), and cancer research (69%). Clinical trials: The fraction of phase-2 clinical trials with a positive outcome was used as a proxy (i.e., an indirect resemblance measure) for translational success. Trials were retrieved from the WHO trial register and categorized into medical research fields following the international classification of disease (ICD-10). Of the phase-2 trials analyzed, 65.2% were successful. Fields with the highest success rates were disorders of lipoprotein metabolism (86.0%) and epilepsy (85.0%). Fields with the lowest success rates were schizophrenia (45.4%) and pancreatic cancer (46.0%). Our combined analyses suggest relevant differences in success rates between medical research fields. Based on the clinical trials, comparisons of practice, e.g., between epilepsy and schizophrenia, might identify factors that influence translational success.

Reporting of 3Rs Approaches in Preclinical Animal Experimental Studies-A Nationwide Study.

The 3Rs aim to refine animal welfare, reduce animal numbers, and replace animal experiments. Investigations disclose that researchers are positive towards 3Rs recommendations from peers. Communication of 3Rs approaches via primary preclinical animal experimental literature may become a fast-forward extension to learn relevant 3Rs approaches if such are reported. This study investigates 3Rs-reporting in peer-reviewed preclinical animal research with at least one author affiliated to a Danish university. Using a systematic search and random sampling, we included 500 studies from 2009 and 2018. Reporting was low and improvement over time limited. A word search for 3R retrieved zero results in 2009 and 3.2% in 2018. Reporting on 3Rs-related sentences increased from 6.4% in 2009 to 18.4% in 2018, "reduction" increased from 2.4% to 8.0%, and "refinement" from 5.2% to 14.4%. Replacement was not reported. Reporting of the methodology was missing. For "reduction", methodology was mentioned in one study in 2009 and 11 studies in 2018, and for "refinement" in 9 and 21, respectively. Twenty-one studies stated compliance with ARRIVE-guidelines or similar without disclosure of details. Reporting of 3Rs approaches in preclinical publications is currently insufficient to guide researchers. Other strategies, e.g., education, interdisciplinary collaboration, and 3Rs funding initiatives, are needed.