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Terrestrial and aquatic ecosystems face various chemicals that might induce acute and/or long-term harm. To assess these impacts, ecotoxicological bioassays are essential. However, bioassays using animals, particularly mammals, are costly, time-consuming, and raise ethical concerns. In this context, terrestrial plants emerge as a viable alternative to conventional assays. Thus, the aim of this review was to address the history and evolution of plant bioassays, highlighting the main regulations, guidelines, and protocols governing the use of terrestrial plants in ecotoxicological tests. Initially, plant bioassays were employed to assess the cytogenotoxic effects of chemicals, gaining prominence with the GENE-TOX program in the 80s. Subsequently, plants were used in allelopathy bioassays and in studies aimed to examine the ecotoxicity of pesticides in soil. Currently, ecotoxicological bioassays with plants are regulated by specific standards, such as ASTM E1963–22, EPA 600/3–88/029, EPS 1/RM/45, ISO 11269-1, ISO 11269-2, ISO 17126, ISO 18763, ISO 29200, ISO 22030, OECD-208, OECD-227, OCSPP 850.4100, OCSPP 850.4230, OCSPP 850.4800 and OPPTS 850.4200. The existing protocols standardize bioassays in greenhouse and lab environments, and the duration of the tests varies from hours to months. The main ecotoxicological parameters to be analyzed after exposure include germination percentage, survival rate, root length, aerial part length, fresh mass of exposed plants, and phytotoxicity symptoms. In addition, the absorption rate of substances and genotoxic and mutagenic effects might also be assessed. Therefore, data in this review demonstrate that terrestrial plants represent an important tool in the analysis of environmental risks associated with chemicals and might serve as crucial allies in modern ecotoxicology. [ABSTRACT FROM AUTHOR]

The Food and Drug Administration's recent decision to eliminate mandatory animal testing for drug approval marks a significant shift to alternative methods. Similarly, the European Parliament is advocating for a faster transition, reflecting public preference for animal‐free research practices. In vitro tissue models are increasingly recognized as valuable tools for regulatory assessments before clinical trials, in line with the 3R principles (Replace, Reduce, Refine). Despite their potential, barriers such as the need for standardization, availability, and cost hinder their widespread adoption. To address these challenges, the Robotic Enabled Biological Automation (ReBiA) system is developed. This system uses a dual‐arm robot capable of standardizing laboratory processes within a closed automated environment, translating manual processes into automated ones. This reduces the need for process‐specific developments, making in vitro tissue models more consistent and cost‐effective. ReBiA's performance is demonstrated through producing human reconstructed epidermis, human airway epithelial models, and human intestinal organoids. Analyses confirm that these models match the morphology and protein expression of manually prepared and native tissues, with similar cell viability. These successes highlight ReBiA's potential to lower barriers to broader adoption of in vitro tissue models, supporting a shift toward more ethical and advanced research methods. [ABSTRACT FROM AUTHOR]

This document is a compilation of research articles in the field of immunology, focusing on advancements in immunology research through the use of new approach methods (NAMs) as alternatives to traditional animal models. NAMs aim to accurately replicate the complex and dynamic nature of the immune system, bridging the gap between animal studies and human clinical trials. The document highlights specific advancements in NAMs, such as using postmortem tissues for immunological studies and modeling vascular inflammation using a microfluidic platform. These developments contribute to a better understanding of immune responses and disease progression, offering a more ethical and potentially more accurate alternative to traditional animal-based research. [Extracted from the article]

Abstract Much of nutrition research has been conventionally based on the use of simplistic in vitro systems or animal models, which have been extensively employed in an effort to better understand the relationships between diet and complex diseases as well as to evaluate food safety. Although these models have undeniably contributed to increase our mechanistic understanding of basic biological processes, they do not adequately model complex human physiopathological phenomena, creating concerns about the translatability to humans. During the last decade, extraordinary advancement in stem cell culturing, three‐dimensional cell cultures, sequencing technologies, and computer science has occurred, which has originated a wealth of novel human‐based and more physiologically relevant tools. These tools, also known as “new approach methodologies,” which comprise patient‐derived organoids, organs‐on‐chip, multi‐omics approach, along with computational models and analysis, represent innovative and exciting tools to forward nutrition research from a human‐biology‐oriented perspective. After considering some shortcomings of conventional in vitro and vivo approaches, here we describe the main novel available and emerging tools that are appropriate for designing a more human‐relevant nutrition research. Our aim is to encourage discussion on the opportunity to explore innovative paths in nutrition research and to promote a paradigm‐change toward a more human biology‐focused approach to better understand human nutritional pathophysiology, to evaluate novel food products, and to develop more effective targeted preventive or therapeutic strategies while helping in reducing the number and replacing animals employed in nutrition research.

Discogenic pain is associated with deep nerve ingrowth in annulus fibrosus tissue (AF) of intervertebral disc (IVD). To model AF nerve ingrowth, primary bovine dorsal root ganglion (DRG) micro‐scale tissue units are spatially organised around an AF explant by mild hydrodynamic forces within a collagen matrix. This results in a densely packed multicellular system mimicking the native DRG tissue morphology and a controlled AF‐neuron distance. Such a multicellular organisation is essential to evolve populational‐level cellular functions and in vivo‐like morphologies. Pro‐inflammatory cytokine‐primed AF demonstrates its neurotrophic and neurotropic effects on nociceptor axons. Both effects are dependent on the AF‐neuron distance underpinning the role of recapitulating inter‐tissue/organ anatomical proximity when investigating their crosstalk. This is the first in vitro model studying AF nerve ingrowth by engineering mature and large animal tissues in a morphologically and physiologically relevant environment. The new approach can be used to biofabricate multi‐tissue/organ models for untangling pathophysiological conditions and develop novel therapies. [ABSTRACT FROM AUTHOR]

Animals like mice and rats have long been used in medical research to help understand disease and test potential new treatments before human trials. However, while animal studies have contributed to important advances, too much reliance on animal models can also mislead drug development. This article explains for a general audience how animal research is used to develop new medicines, its benefits and limitations, and how more accurate and humane techniques—alternatives to animal testing—could improve this process.

Inhibition of angiogenesis is an important mode of action for the teratogenic effect of chemicals and drugs. There is a gap in the availability of simple, experimental screening models for the detection of angiogenesis inhibition. The zebrafish embryo represents an alternative test system which offers the complexity of developmental differentiation of an entire organism while allowing for small-scale and high-throughput screening. Here we present a novel automated imaging-based method to detect the inhibition of angiogenesis in early life stage zebrafish. Video subtraction was used to identify the location and number of functional intersegmental vessels according to the detection of moving blood cells. By exposing embryos to multiple tyrosine kinase inhibitors including SU4312, SU5416, Sorafenib, or PTK787, we confirmed that this method can detect concentration-dependent inhibition of angiogenesis. Parallel assessment of arterial and venal aorta ruled out a potential bias by impaired heart or blood cell development. In contrast, the histone deacetylase inhibitor valproic acid did not affect ISV formation supporting the specificity of the angiogenic effects. The new test method showed higher sensitivity, i.e. lower effect concentrations, relative to a fluorescent reporter gene strain (Tg(KDR:EGFP)) exposed to the same tyrosine kinase inhibitors indicating that functional effects due to altered tubulogenesis or blood transport can be detected before structural changes of the endothelium are visible by fluorescence imaging. Comparison of exposure windows indicated higher specificity for angiogenesis when exposure started at later embryonic stages (24 h post-fertilization). One of the test compounds was showing particularly high specificity for angiogenesis effects (SU4312) and was, therefore, suggested as a model compound for the identification of molecular markers of angiogenic disruption. Our findings establish video imaging in wild-type strains as viable, non-invasive, high-throughput method for the detection of chemical-induced angiogenic disruption in zebrafish embryos. [ABSTRACT FROM AUTHOR]

In vivo skin sensitization tests are required to evaluate the biological safety of medical devices in contact with living organisms to provide safe medical care to patients. Negative and positive reference materials have been developed for biological tests of cytotoxicity, implantation, hemolysis, and in vitro skin irritation. However, skin sensitization tests are lacking. In this study, polyurethane sheets containing 1 wt/wt % 2,4‐dinitrochlorobenzene (DNCB‐PU) were developed and evaluated as a positive reference material for skin sensitization tests. DNCB‐PU sheet extracts prepared with sesame oil elicited positive sensitization responses for in vivo sensitization potential in the guinea pig maximization test and the local lymph node assay. Furthermore, DNCB‐PU sheet extracts prepared with water and acetonitrile, 10% fetal bovine serum‐containing medium, or sesame oil elicited positive sensitization responses as alternatives to animal testing based on the amino acid derivative reactivity assay, human cell line activation test, and epidermal sensitization assay, respectively. These data suggest that the DNCB‐PU sheet is an effective extractable positive reference material for in vivo and in vitro skin sensitization testing in medical devices. The formulation of this reference material will lead to the development of safer medical devices that contribute to patient safety. [ABSTRACT FROM AUTHOR]

The pharmacokinetic (PK) and toxicokinetic profile of a drug from its preclinical evaluation helps the researcher determine whether the drug should be tested in humans based on its safety and toxicity. Preclinical studies require time and resources and are prone to error. Moreover, according to the United States Food and Drug Administration Modernisation Act 2, animal testing is no longer mandatory for new drug development, and an animal-free alternative, such as cell-based assay and computer models, can be used. Different physiologically based PK models were developed for an anaplastic lymphoma kinase inhibitor in rats and monkeys after intravenous and oral administration using its physicochemical properties and in vitro characterisation data. The developed model was validated against the in vivo data available in the literature, and the validation results were found within the acceptable limit. A parameter sensitivity analysis was performed to identify the properties of the compound influencing the PK profile. This work demonstrates the application of the physiologically based PK model to predict the PKs of a drug, which will eventually assist in reducing the number of animal studies and save time and cost of drug discovery and development. [ABSTRACT FROM AUTHOR]

There is growing recognition that animal methods bias, a preference for animal‐based methods where they are not necessary or where nonanimal‐based methods may already be suitable, can impact the likelihood or timeliness of a manuscript being accepted for publication. Following April 2022 workshop about animal methods bias in scientific publishing, a coalition of scientists and advocates formed a Coalition to Illuminate and Address Animal Methods Bias (COLAAB). The COLAAB has developed this guide to be used by authors who use nonanimal methods to avoid and respond to animal methods bias from manuscript reviewers. It contains information that researchers may use during 1) study design, including how to find and select appropriate nonanimal methods and preregister a research plan, 2) manuscript preparation and submission, including tips for discussing methods and choosing journals and reviewers that may be more receptive to nonanimal methods, and 3) the peer review process, providing suggested language and literature to aid authors in responding to biased reviews. The author's guide for addressing animal methods bias in publishing is a living resource also available online at animalmethodsbias.org, which aims to help ensure fair dissemination of research that uses nonanimal methods and prevent unnecessary experiments on animals. [ABSTRACT FROM AUTHOR]

Xenobiotics released in the environment can be taken up by aquatic and terrestrial organisms and can accumulate at higher concentrations through the trophic chain. Bioaccumulation is therefore one of the PBT properties that authorities require to assess for the evaluation of the risks that chemicals may pose to humans and the environment. The use of an integrated testing strategy (ITS) and the use of multiple sources of information are strongly encouraged by authorities in order to maximize the information available and reduce testing costs. Moreover, considering the increasing demand for development and the application of new approaches and alternatives to animal testing, the development of in silico cost-effective tools such as QSAR models becomes increasingly important. In this study, a large and curated literature database of fish laboratory-based values of dietary biomagnification factor (BMF) was used to create externally validated QSARs. The quality categories (high, medium, low) available in the database were used to extract reliable data to train and validate the models, and to further address the uncertainty in low-quality data. This procedure was useful for highlighting problematic compounds for which additional experimental effort would be required, such as siloxanes, highly brominated and chlorinated compounds. Two models were suggested as final outputs in this study, one based on good-quality data and the other developed on a larger dataset of consistent Log BMFL values, which included lower-quality data. The models had similar predictive ability; however, the second model had a larger applicability domain. These QSARs were based on simple MLR equations that could easily be applied for the predictions of dietary BMFL in fish, and support bioaccumulation assessment procedures at the regulatory level. To ease the application and dissemination of these QSARs, they were included with technical documentation (as QMRF Reports) in the QSAR-ME Profiler software for QSAR predictions available online. [ABSTRACT FROM AUTHOR]

The workshop titled State of the Science on Assessing Developmental Neurotoxicity Using New Approach Methods was co-organized by University of Maryland’s Joint Institute for Food Safety and Applied Nutrition (JIFSAN) and the U.S. Food and Drug Administration’s (FDA) Center for Food Safety and Applied Nutrition (CFSAN; now called the Human Foods Program), and was hosted by FDA in College Park, MD on November 14-15, 2023. This event convened experts from inter­national organizations, governmental agencies, industry, and academia to explore the transition from traditional in vivo tests to innovative new approach methods (NAMs) in developmental neurotoxicity (DNT) testing. The discussions emphasized the heightened vulnerability of the developing human brain to toxic exposures and the potential of NAMs to provide more ethical, economical, and scientifically robust alternatives to traditional testing. Various NAMs for DNT were discussed, including in silico, in chemico, in vitro, non-mammalian whole organisms, and novel mammalian approaches. In addition to progress in the field, the workshop discussed ongoing chal­lenges such as expectations to perfectly replicate the complex biology of human neurodevelopment and integration of DNT NAMs into regulatory frameworks. Presentations and panel discussions pro­vided a comprehensive overview of the state of the science, assessed the capabilities and limitations of current DNT NAMs, and outlined critical next steps in advancing the field of DNT testing.

The bioconcentration factor (BCF) is one of the metrics used to evaluate the potential of a substance to bioaccumulate into aquatic organisms. In this work, linear and non-linear regression QSARs were developed for the prediction of log BCF using different computational approaches, and starting from a large and structurally heterogeneous dataset. The new MLR-OLS and ANN regression models have good fitting with R2 values of 0.62 and 0.70, respectively, and comparable external predictivity with R2ext 0.64 and 0.65 (RMSEext of 0.78 and 0.76), respectively. Furthermore, linear and non-linear classification models were developed using the regulatory threshold BCF >2000. A class balanced subset was used to develop classification models which were applied to chemicals not used to create the QSARs. These classification models are characterized by external and internal accuracy up to 84% and 90%, respectively, and sensitivity and specificity up to 90% and 80%, respectively. QSARs presented in this work are validated according to regulatory requirements and their quality is in line with other tools available for the same endpoint and dataset, with the advantage of low complexity and easy application through the software QSAR-ME Profiler. These QSARs can be used as alternatives for, or in combination with, existing models to support bioaccumulation assessment procedures. [ABSTRACT FROM AUTHOR]

Almost a decade after the stipulated deadline in the 7th amendment to the EU Cosmetics Directive, which bans the marketing of animal-tested cosmetics in the EU from 2013, animal experimentation for cosmetic-related purposes remains a topic of animated debate. Cosmetic industry continues to be scrutinised for the practice, despite its leading role in funding and adopting innovation in this field. This paper aims to provide a state-of-the-art review of the field on alternative testing methods, also known as New Approach Methodologies (NAMs), with the focus on assessing the safety of cosmetic ingredients and products. It starts with innovation drivers and global regulatory responses, followed by an extensive, endpoint-specific overview of accepted/prospective NAMs. The overview covers main developments in acute toxicity, skin corrosion/irritation, serious eye damage/irritation, skin sensitisation, repeated dose toxicity, reproductive toxicity/endocrine disruption, mutagenicity/genotoxicity, carcinogenicity, photo-induced toxicity, and toxicokinetics. Specific attention was paid to the emerging in silico methodology. This paper also provides a brief overview of the studies on public perception of animal testing in cosmetics. It concludes with a view that educating consumers and inviting them to take part in advocacy could be an effective tool to achieve policy changes, regulatory acceptance, and investment in innovation. [ABSTRACT FROM AUTHOR]

By applying "New Approach Methodologies (NAMs)" based on innovative technologies such as computer modeling, high throughput testing, omics, and sophisticated cell cultures, the use of experimental animals in the life sciences can be reduced or sometimes even completely avoided. Stimulating NAMs may benefit from a bottom-up approach, i.e., local initiatives mapping the available NAMs and promoting their use. An example of such an initiative in Belgium is the RE-Place project, which collects the available NAMs in one central database, and links this knowledge with the names of experts and research centers. To this extent, a template was created to collect the information of interest in a fast and consistent manner. Based on this template, a web-based application was developed to facilitate the entry of information, which was evaluated in a pilot study by experts in the field of NAMs. After integration of their feedback, a revised version of the RE-Place online tool was launched to the public. Aspects such as user-friendliness, quality of submitted information, protection of personal data and Intellectual Property (IP) rights were all considered in the development process. Hurdles like incentives for collaboration were also taken into account. Information submitted with the online tool is directly integrated in the RE-Place open access database. By consulting the database, scientists from various disciplines can easily identify the different types of NAMs and the experts using them in Belgium. As such, the RE-Place database contributes to building trust in the use of NAMs and stimulating their use and regulatory uptake. [ABSTRACT FROM AUTHOR]

By applying “New Approach Methodologies (NAMs)” based on innovative technologies such as computer modeling, high throughput testing, omics, and sophisticated cell cultures, the use of experimental animals in the life sciences can be reduced or sometimes even completely avoided. Stimulating NAMs may benefit from a bottom-up approach, i.e., local initiatives mapping the available NAMs and promoting their use. An example of such an initiative in Belgium is the RE-Place project, which collects the available NAMs in one central database, and links this knowledge with the names of experts and research centers. To this extent, a template was created to collect the information of interest in a fast and consistent manner. Based on this template, a web-based application was developed to facilitate the entry of information, which was evaluated in a pilot study by experts in the field of NAMs. After integration of their feedback, a revised version of the RE-Place online tool was launched to the public. Aspects such as user-friendliness, quality of submitted information, protection of personal data and Intellectual Property (IP) rights were all considered in the development process. Hurdles like incentives for collaboration were also taken into account. Information submitted with the online tool is directly integrated in the RE-Place open access database. By consulting the database, scientists from various disciplines can easily identify the different types of NAMs and the experts using them in Belgium. As such, the RE-Place database contributes to building trust in the use of NAMs and stimulating their use and regulatory uptake.

Skin sensitization testing is a regulatory requirement for safety evaluations of pesticides in multiple countries. Globally harmonized test guidelines that include in chemico and in vitro methods reduce animal use, but no single assay is recommended as a complete replacement for animal tests. Defined approaches (DAs) that integrate data from multiple non-animal methods are accepted; however, the methods that comprise them have been evaluated using monoconstituent substances rather than mixtures or formulations. To address this data gap, we tested 27 agrochemical formulations in the direct peptide reactivity assay (DPRA), the KeratinoSens™ assay, and the human cell line activation test (h-CLAT). These data were used as inputs to evaluate three DAs for hazard classification of skin sensitization potential and two DAs for potency categorization. When compared to historical animal results, balanced accuracy for the DAs for predicting in vivo skin sensitization hazard (i.e., sensitizer vs. nonsensitizer) ranged from 56 to 78%. The best performing DA was the “2 out of 3 (2o3)” DA, in which the hazard classification was based on two concordant results from the DPRA, KeratinoSens, or h-CLAT. The KE 3/1 sequential testing strategy (STS), which uses h-CLAT and DPRA results, and the integrated testing strategy (ITSv2), which uses h-CLAT, DPRA, and an in silico hazard prediction from OECD QSAR Toolbox, had balanced accuracies of 56–57% for hazard classification. Of the individual test methods, KeratinoSens had the best performance for predicting in vivo hazard outcomes. Its balanced accuracy of 81% was similar to that of the 2o3 DA (78%). For predicting potency categories defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), the correct classification rate of the STS was 52% and that of the ITSv2 was 43%. These results demonstrate that non-animal test methods have utility for evaluating the skin sensitization potential of agrochemical formulations as compared to animal reference data. While additional data generation is needed, testing strategies such as DAs anchored to human biology and mechanistic information provide a promising approach for agrochemical formulation testing.

Xenobiotics released in the environment can be taken up by aquatic and terrestrial organisms and can accumulate at higher concentrations through the trophic chain. Bioaccumulation is therefore one of the PBT properties that authorities require to assess for the evaluation of the risks that chemicals may pose to humans and the environment. The use of an integrated testing strategy (ITS) and the use of multiple sources of information are strongly encouraged by authorities in order to maximize the information available and reduce testing costs. Moreover, considering the increasing demand for development and the application of new approaches and alternatives to animal testing, the development of in silico cost-effective tools such as QSAR models becomes increasingly important. In this study, a large and curated literature database of fish laboratory-based values of dietary biomagnification factor (BMF) was used to create externally validated QSARs. The quality categories (high, medium, low) available in the database were used to extract reliable data to train and validate the models, and to further address the uncertainty in low-quality data. This procedure was useful for highlighting problematic compounds for which additional experimental effort would be required, such as siloxanes, highly brominated and chlorinated compounds. Two models were suggested as final outputs in this study, one based on good-quality data and the other developed on a larger dataset of consistent Log BMFL values, which included lower-quality data. The models had similar predictive ability; however, the second model had a larger applicability domain. These QSARs were based on simple MLR equations that could easily be applied for the predictions of dietary BMFL in fish, and support bioaccumulation assessment procedures at the regulatory level. To ease the application and dissemination of these QSARs, they were included with technical documentation (as QMRF Reports) in the QSAR-ME Profiler software for QSAR predictions available online.

The bioconcentration factor (BCF) is one of the metrics used to evaluate the potential of a substance to bioaccumulate into aquatic organisms. In this work, linear and non-linear regression QSARs were developed for the prediction of log BCF using different computational approaches, and starting from a large and structurally heterogeneous dataset. The new MLR-OLS and ANN regression models have good fitting with R2 values of 0.62 and 0.70, respectively, and comparable external predictivity with R2ext 0.64 and 0.65 (RMSEext of 0.78 and 0.76), respectively. Furthermore, linear and non-linear classification models were developed using the regulatory threshold BCF >2000. A class balanced subset was used to develop classification models which were applied to chemicals not used to create the QSARs. These classification models are characterized by external and internal accuracy up to 84% and 90%, respectively, and sensitivity and specificity up to 90% and 80%, respectively. QSARs presented in this work are validated according to regulatory requirements and their quality is in line with other tools available for the same endpoint and dataset, with the advantage of low complexity and easy application through the software QSAR-ME Profiler. These QSARs can be used as alternatives for, or in combination with, existing models to support bioaccumulation assessment procedures.

Almost a decade after the stipulated deadline in the 7th amendment to the EU Cosmetics Directive, which bans the marketing of animal-tested cosmetics in the EU from 2013, animal experimentation for cosmetic-related purposes remains a topic of animated debate. Cosmetic industry continues to be scrutinised for the practice, despite its leading role in funding and adopting innovation in this field. This paper aims to provide a state-of-the-art review of the field on alternative testing methods, also known as New Approach Methodologies (NAMs), with the focus on assessing the safety of cosmetic ingredients and products. It starts with innovation drivers and global regulatory responses, followed by an extensive, endpoint-specific overview of accepted/prospective NAMs. The overview covers main developments in acute toxicity, skin corrosion/irritation, serious eye damage/irritation, skin sensitisation, repeated dose toxicity, reproductive toxicity/endocrine disruption, mutagenicity/genotoxicity, carcinogenicity, photo-induced toxicity, and toxicokinetics. Specific attention was paid to the emerging in silico methodology. This paper also provides a brief overview of the studies on public perception of animal testing in cosmetics. It concludes with a view that educating consumers and inviting them to take part in advocacy could be an effective tool to achieve policy changes, regulatory acceptance, and investment in innovation.