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.

Automatic classification of experimental models in biomedical literature to support searching for alternative methods to animal experiments.

Current animal protection laws require replacement of animal experiments with alternative methods, whenever such methods are suitable to reach the intended scientific objective. However, searching for alternative methods in the scientific literature is a time-consuming task that requires careful screening of an enormously large number of experimental biomedical publications. The identification of potentially relevant methods, e.g. organ or cell culture models, or computer simulations, can be supported with text mining tools specifically built for this purpose. Such tools are trained (or fine tuned) on relevant data sets labeled by human experts. We developed the GoldHamster corpus, composed of 1,600 PubMed (Medline) articles (titles and abstracts), in which we manually identified the used experimental model according to a set of eight labels, namely: "in vivo", "organs", "primary cells", "immortal cell lines", "invertebrates", "humans", "in silico" and "other" (models). We recruited 13 annotators with expertise in the biomedical domain and assigned each article to two individuals. Four additional rounds of annotation aimed at improving the quality of the annotations with disagreements in the first round. Furthermore, we conducted various machine learning experiments based on supervised learning to evaluate the corpus for our classification task. We obtained more than 7,000 document-level annotations for the above labels. After the first round of annotation, the inter-annotator agreement (kappa coefficient) varied among labels, and ranged from 0.42 (for "others") to 0.82 (for "invertebrates"), with an overall score of 0.62. All disagreements were resolved in the subsequent rounds of annotation. The best-performing machine learning experiment used the PubMedBERT pre-trained model with fine-tuning to our corpus, which gained an overall f-score of 0.83. We obtained a corpus with high agreement for all labels, and our evaluation demonstrated that our corpus is suitable for training reliable predictive models for automatic classification of biomedical literature according to the used experimental models. Our SMAFIRA - "Smart feature-based interactive" - search tool ( https://smafira.bf3r.de ) will employ this classifier for supporting the retrieval of alternative methods to animal experiments. The corpus is available for download ( https://doi.org/10.5281/zenodo.7152295 ), as well as the source code ( https://github.com/mariananeves/goldhamster ) and the model ( https://huggingface.co/SMAFIRA/goldhamster ).

A microphysiological system for studying human bone biology under simultaneous control of oxygen tension and mechanical loading.

Throughout life, continuous remodelling is part of human bone biology and depends on the simultaneous action of physicochemical parameters such as oxygen tension and varying mechanical load. Thus, suitable model systems are needed, which allow concomitant modulation of these factors to recapitulate in vivo bone formation. Here, we report on the development of a first microphysiological system (MPS) that enables perfusion, environment-independent regulation of the oxygen tension as well as precise quantification and control of mechanical load. To demonstrate the use of the MPS for future studies on the (patho-)biology of bone, we built a simplified 3D model for early de novo bone formation. Primary human osteoblasts (OBs), which are the key players during this process, were seeded onto type I collagen scaffolds and cultured in the MPS. We could not only monitor cell viability and metabolism of OBs under varied physicochemical conditions, but also visualise the mineralisation of the extracellular matrix. In summary, we present a MPS that uniquely combines the independent control of physicochemical parameters and allows investigation of their influence on bone biology. We consider our MPS highly valuable to gain deeper insights into (patho-)physiological processes of bone formation in the future.

The fate of surplus laboratory animals: Minimizing the production of surplus animals has the greatest potential to reduce the number of laboratory animals: Minimizing the production of surplus animals has greatest potential to reduce the number of laboratory animals.

To meet regulatory requirements and the political pressure to minimize the number of animals used in research, it is critical to reduce the production of surplus animals.

In vitro models of human development and their potential application in developmental toxicity testing.

Recent publications describe the development of in vitro models of human development, for which applications in developmental toxicity testing can be envisaged. To date, these regulatory assessments have exclusively been performed in animal studies, the relevance of which to adverse reactions in humans may be questioned. Recently developed cell culture-based models of embryo-fetal development, however, do not yet exhibit sufficient levels of standardisation and reproducibility. Here, the advantages and shortcomings of both in vivo and in vitro developmental toxicity testing are addressed, as well as the possibility of integrated testing strategies as a viable option in the near future.

Quantitative high-throughput phenotypic screening for environmental estrogens using the E-Morph Screening Assay in combination with in silico predictions.

BACKGROUND: Exposure to environmental chemicals that interfere with normal estrogen function can lead to adverse health effects, including cancer. High-throughput screening (HTS) approaches facilitate the efficient identification and characterization of such substances. OBJECTIVES: We recently described the development of the E-Morph Assay, which measures changes at adherens junctions as a clinically-relevant phenotypic readout for estrogen receptor (ER) alpha signaling activity. Here, we describe its further development and application for automated robotic HTS. METHODS: Using the advanced E-Morph Screening Assay, we screened a substance library comprising 430 toxicologically-relevant industrial chemicals, biocides, and plant protection products to identify novel substances with estrogenic activities. Based on the primary screening data and the publicly available ToxCast dataset, we performed an insilico similarity search to identify further substances with potential estrogenic activity for follow-up hit expansion screening, and built seven insilico ER models using the conformal prediction (CP) framework to evaluate the HTS results. RESULTS: The primary and hit confirmation screens identified 27 'known' estrogenic substances with potencies correlating very well with the published ToxCast ER Agonist Score (r=+0.95). We additionally detected potential 'novel' estrogenic activities for 10 primary hit substances and for another nine out of 20 structurally similar substances from insilico predictions and follow-up hit expansion screening. The concordance of the E-Morph Screening Assay with the ToxCast ER reference data and the generated CP ER models was 71% and 73%, respectively, with a high predictivity for ER active substances of up to 87%, which is particularly important for regulatory purposes. DISCUSSION: These data provide a proof-of-concept for the combination of in vitro HTS approaches with insilico methods (similarity search, CP models) for efficient analysis of large substance libraries in order to prioritize substances with potential estrogenic activity for subsequent testing against higher tier human endpoints.

Declaration of common standards for the preregistration of animal research-speeding up the scientific progress.

Preregistration of studies is a recognized tool in clinical research to improve the quality and reporting of all gained results. In preclinical research, preregistration could boost the translation of published results into clinical breakthroughs. When studies rely on animal testing or form the basis of clinical trials, maximizing the validity and reliability of research outcomes becomes in addition an ethical obligation. Nevertheless, the implementation of preregistration in animal research is still slow. However, research institutions, funders, and publishers start valuing preregistration, and thereby level the way for its broader acceptance in the future. A total of 3 public registries, the OSF registry, preclinicaltrials.eu, and animalstudyregistry.org already encourage the preregistration of research involving animals. Here, they jointly declare common standards to make preregistration a valuable tool for better science. Registries should meet the following criteria: public accessibility, transparency in their financial sources, tracking of changes, and warranty and sustainability of data. Furthermore, registration templates should cover a minimum set of mandatory information and studies have to be uniquely identifiable. Finally, preregistered studies should be linked to any published outcome. To ensure that preregistration becomes a powerful instrument, publishers, funders, and institutions should refer to registries that fulfill these minimum standards.

How many animals are used for SARS-CoV-2 research?: An overview on animal experimentation in pre-clinical and basic research.

Non-technical summaries of research projects allow tracking the numbers and purpose of animal experiments related to SARS-CoV2 research so as to provide greater transparency on animal use.

The E-Morph Assay: Identification and characterization of environmental chemicals with estrogenic activity based on quantitative changes in cell-cell contact organization of breast cancer cells.

Adverse health effects that are caused by endocrine disrupting chemicals (EDCs) in the environment, food or consumer products are of high public concern. The identification and characterization of EDCs including substances with estrogenic activity still necessitates the use of animal testing as most of the approved alternative test methods only address single mechanistic events of endocrine activity. Therefore, novel human-relevant in vitro assays covering more complex functional endpoints of adversity, including hormone-related tumor formation and progression, are needed. This study describes the development and evaluation of a novel high-throughput screening-compatible assay called "E-Morph Assay". This image-based phenotypic screening assay facilitates robust predictions of the estrogenic potential of environmental chemicals using quantitative changes in the cell-cell contact morphology of human breast cancer cells as a novel functional endpoint. Based on a classification model, which was developed using six reference substances with known estrogenic activity, the E-Morph Assay correctly classified an additional set of 11 reference chemicals commonly used in OECD Test Guidelines and the U.S. EPA ToxCast program. For each of the tested substances, a relative ER bioactivity score was derived that allowed their grouping into four main categories of estrogenic activity, i.e. 'strong' (>0.9; four substances, i.e. natural hormones or pharmaceutical products), 'moderate' (0.9-0.6; six substances, i.e. phytoestrogens and Bisphenol AF), 'weak' (<0.6; three substances, i.e Bisphenol S, B, and A), and 'negative' (0.0; four substances). The E-Morph Assay considerably expands the portfolio of test methods providing the possibility to characterize the influence of environmental chemicals on estrogen-dependent tumor progression.

25th anniversary of the Berlin workshop on developmental toxicology: DevTox database update, challenges in risk assessment of developmental neurotoxicity and alternative methodologies in bone development and growth.

25 years after the first Berlin Workshop on Developmental Toxicity this 10th Berlin Workshop aimed to bring together international experts from authorities, academia and industry to consider scientific, methodologic and regulatory aspects in risk assessment of developmental toxicity and to debate alternative strategies in testing developmental effects in the future. Proposals for improvement of the categorization of developmental effects were discussed as well as the update of the DevTox database as valuable tool for harmonization. The development of adverse outcome pathways relevant to developmental neurotoxicity (DNT) was debated as a fundamental improvement to guide the screening and testing for DNT using alternatives to animal methods. A further focus was the implementation of an in vitro mechanism-based battery, which can support various regulatory applications associated with the assessment of chemicals and mixtures. More interdisciplinary and translation research should be initiated to accelerate the development of new technologies to test developmental toxicity. Technologies in the pipeline are (i) high throughput imaging techniques, (ii) models for DNT screening tests, (iii) use of computer tomography for assessment of thoracolumbar supernumerary ribs in animal models, and (iv) 3D biofabrication of bone development and regeneration tissue models. In addition, increased collaboration with the medical community was suggested to improve the relevance of test results to humans and identify more clinically relevant endpoints. Finally, the participants agreed that this conference facilitated better understanding innovative approaches that can be useful for the identification of developmental health risks due to exposure to chemical substances.

A quantitative medium-throughput assay to measure Caenorhabditis elegans development and reproduction.

Growth and offspring count are two commonly determined toxicological endpoints for chemical- or gene-induced developmental and reproductive effects in Caenorhabditis elegans. Here, we present a protocol for a 96 h, medium-throughput assay, assessing both endpoints quantitatively within an automated framework using open-source software. The assay utilizes whole 96-well fluorescence images taken with a high-content screening system. Alternatively, conventional fluorescence images can also be utilized with only a few adjustments. For complete details on the use and execution of this protocol, please refer to Wittkowski et al. (2019).

Estrogens Determine Adherens Junction Organization and E-Cadherin Clustering in Breast Cancer Cells via Amphiregulin.

Estrogens play an important role in the development and progression of human cancers, particularly in breast cancer. Breast cancer progression depends on the malignant destabilization of adherens junctions (AJs) and disruption of tissue integrity. We found that estrogen receptor alpha (ERα) inhibition led to a striking spatial reorganization of AJs and microclustering of E-Cadherin (E-Cad) in the cell membrane of breast cancer cells. This resulted in increased stability of AJs and cell stiffness and a reduction of cell motility. These effects were actomyosin-dependent and reversible by estrogens. Detailed investigations showed that the ERα target gene and epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) essentially regulates AJ reorganization and E-Cad microclustering. Our results not only describe a biological mechanism for the organization of AJs and the modulation of mechanical properties of cells but also provide a new perspective on how estrogens and anti-estrogens might influence the formation of breast tumors.

Repeatability analysis improves the reliability of behavioral data.

Reliability of data has become a major concern in the course of the reproducibility crisis. Especially when studying animal behavior, confounding factors such as novelty of the test apparatus can lead to a wide variability of data which may mask treatment effects and consequently lead to misinterpretation. Habituation to the test situation is a common practice to circumvent novelty induced increases in variance and to improve the reliability of the respective measurements. However, there is a lack of published empirical knowledge regarding reasonable habituation procedures and a method validation seems to be overdue. This study aimed at setting up a simple strategy to increase reliability of behavioral data measured in a familiar test apparatus. Therefore, exemplary data from mice tested in an Open Field (OF) arena were used to elucidate the potential of habituation and how reliability of measures can be confirmed by means of a repeatability analysis using the software R. On seven consecutive days, male C57BL/6J, BALB/cJ and 129S1/SvImJ mice were tested in an OF arena once daily and individual mouse behavior was recorded. A repeatability analysis was conducted with regard to repeated trials of habituation. Our data analysis revealed that monitoring animal behavior during habituation is important to determine when individual differences of the measurements are stable. Repeatability values from distance travelled and average activity increased over the habituation period, revealing that around 60% of the variance of the data can be explained by individual differences between mice. The first day of habituation was significantly different from the following 6 days. A three-day habituation period appeared to be sufficient in this study. Overall, these results emphasize the importance of habituation and in depth analysis of habituation data to define the correct starting point of the experiment for improving the reliability and reproducibility of experimental data.

Analgesic treatment with buprenorphine should be adapted to the mouse strain.

Buprenorphine is a commonly used opioid to treat moderate to severe pain in mice. Although strain differences regarding basal pain sensitivity and the analgesic effect of other opioids have been described for mice, the data for buprenorphine is incomplete. Hence, we investigated basal pain sensitivity and the analgesic effect of buprenorphine (0.42, 4.0 mg·kg-1) in male C57BL/6J, Balb/cJ and 129S1/SvImJ mice using the incremental hot plate. Additionally, we verified single nucleotide polymorphisms in Cytochrome P450 3a (Cyp3a) genes, which encode for enzymes that are relevant for buprenorphine metabolism, and analyzed serum and brain concentrations of buprenorphine and its metabolites. Finally, in a pilot survey we determined µ-opioid receptor (MOR) protein expression in whole brain lysates. Basal pain sensitivity differed significantly between the mouse strains (Balb/cJ > C57BL/6J > 129S1/SvImJ). Additionally, buprenorphine showed a dose- and strain-dependent effect: at a higher dose it led to increased antinociception in C57BL/6J and Balb/cJ mice, whereas in 129S1/SvImJ mice this effect was diminished. Serum and brain concentrations of buprenorphine and its metabolites dose-dependently increased and differed slightly between the strains at the high dose. However, these slight strain differences did not correlate with pain behavior. Furthermore, serum buprenorphine metabolic ratio and distribution of buprenorphine and its metabolites between brain and blood showed no dose- and only some strain-dependent differences independent from nociceptive behavior. Western blot analysis revealed no strain difference in the basal MOR protein expression in brain lysates. Our results indicate that buprenorphine dosing should be determined in a pilot study for the respective mouse strain to optimize pain treatment and to avoid unwanted side effects. The present pharmacokinetic data and the coarse determination of MOR expression do not explain the strain differences in the analgesic effect of buprenorphine. However, follow-up studies focusing on more specific pharmacodynamic factors could further elucidate the reasons.

Rethinking the incentive system in science: animal study registries: Preregistering experiments using animals could greatly improve transparency and reliability of biomedical studies and improve animal welfare.

The Animal Study Registry offers scientists a range of benefits by preregistering their studies. Wider adoption could address the reproducibility problem in biomedical research and enhance animal welfare.

Restoring circadian synchrony in vitro facilitates physiological responses to environmental chemicals.

BACKGROUND: The growing requirement of hazard and risk assessment of environmental chemicals and the efforts to minimize animal testing, increases the demand for innovative and predictive in vitro test systems in toxicology, reflecting the physiological conditions of human nature. Here, an elemental factor regulating a variety of physiological processes is the day-night rhythm. This circadian rhythm, describing a biological oscillation with a 24-h period is hardly acknowledged in toxicology and test method development. Whilst, in animals or humans the entire organism exhibits a rigorous cellular circadian synchrony, in conventional in vitro systems each cell follows its own rhythm, due to the absence of appropriate synchronizing signals. OBJECTIVE: Here we investigated whether circadian synchronization of human cells in an in vitro system improves the cellular response and, thus, increases the sensitivity of the test system. Since the circadian regulation of metabolism is particularly well understood, and dioxin and dioxin-like compounds are of major concern for environmental health we focused on the ubiquitous drug metabolizing detoxification system mediated by the aryl hydrocarbon receptor (AHR). METHODS: To this end, we applied various prototypical AHR activators onto different human cell lines under non-synchronized or circadian synchronized conditions and determined the dose response on representative endogenous target genes. RESULTS: Remarkably, the cellular response dynamic upon chemical treatment was substantially enhanced in circadian synchronized cells and followed a rhythmic expression pattern. This broader dynamic range was associated with a strikingly higher induction of AHR target genes and the corresponding enzymatic activity, thereby rather mimicking the in vivo situation. CONCLUSION: Our findings indicate that a synchronized circadian rhythm in a cell culture based test system can improve the physiological relevance of an appropriate in vitro method by reflecting the biological in vivo situation more closely. Accordingly, it is a promising tool to facilitate the wide acceptance of in vitro methods in the field of regulatory toxicology and to further optimize the toxicological assessment of environmental chemicals.

Refining animal research: The Animal Study Registry.

The Animal Study Registry (ASR; www.animalstudyregistry.org) was launched in January 2019 for preregistration of animal studies in order to increase transparency and reproducibility of bioscience research and to promote animal welfare. The registry is free of charge and is designed for exploratory and confirmatory studies within applied science as well as basic and preclinical research. The registration form helps scientists plan their study thoroughly by asking detailed questions concerning study design, methods, and statistics. With registration, the study automatically receives a digital object identifier (DOI) that marks it as intellectual property of the researcher. To accommodate the researchers concerns about theft of ideas, users can restrict the visibility of their registered studies for up to 5 years. The full content of the study becomes publicly accessible at the end of the embargo period. Because the platform is embedded in the infrastructure of the German Federal Government, continuity and data security are provided. By registering a study in the ASR, researchers can show their commitment to transparency and data quality to reviewers and editors, to third-party donors, and to the general public.

Caenorhabditis elegans As a Promising Alternative Model for Environmental Chemical Mixture Effect Assessment-A Comparative Study.

A key challenge of mixture toxicity testing is that a multitude of substances with even more combinations need to be tested in a broad dose range. Consequently testing in rodent bioassays, the current gold standard of toxicity testing, is hardly feasible. High-throughput compatible cell culture systems, however, suffer from limitations with respect to toxicokinetics, tissue interactions, and compensatory mechanisms. Therefore, simple organisms like the nematode Caenorhabditis elegans, combining relevant advantages of complex in vivo and fast in vitro assays might prove highly valuable within a testing strategy for mixtures. To investigate the comparability between results obtained with C. elegans and traditional rodent assays, we used five azole fungicides as well investigated model substances. Our findings suggest that azoles act additively in C. elegans which is in line with previous results in rats. Additionally, we show that toxicokinetics are one important factor for the differences in the relative toxicity of the azoles in both species. Importantly, we also demonstrate that in contrast to most rodent in vivo studies, C. elegans assays provide well-defined concentration-response relationships which are a very good basis for the prediction of mixture effects. We conclude that C. elegans may be an appropriate model for mixture toxicity testing at least within a first step to identify and prioritize relevant mixtures for further testing.