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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.

The new software QSARINS‐Chem standalone version is a multiplatform tool, freely downloadable, for the in silico profiling of multiple properties and activities of organic chemicals. This software, which is based on the concept of the QSARINS‐chem module embedded in the QSARINS software, has been fully redesigned and redeveloped in the Java™ language. In addition to a selection of models included in the old module, the new software predicts biotransformation rates and aquatic toxicities of pharmaceuticals and personal care products in multiple organisms, and offers a suite of tools for the analysis of predictions. Furthermore, a comprehensive and transparent database of molecular structures is provided. The new QSARINS‐Chem standalone version is an informative and solid tool, which is useful to support the assessment of the potential hazard and risks related to organic chemicals and is dedicated to users which are interested in the application of QSARs to generate reliable predictions. [ABSTRACT FROM AUTHOR]

The EU Directive 2010/63/EU on the protection of animals used for scientific purposes and other EU regulations, such as REACH and the Cosmetic Products Regulation advocate for a change in the way toxicity testing is conducted. Whilst the Cosmetic Products Regulation bans animal testing altogether, REACH aims for a progressive shift from in vivo testing towards quantitative in vitro and computational approaches. Several endpoints can already be addressed using non-animal approaches including skin corrosion and irritation, serious eye damage and irritation, skin sensitisation, and mutagenicity and genotoxicity. However, for systemic effects such as acute toxicity, repeated dose toxicity and reproductive and developmental toxicity, evaluation of chemicals under REACH still heavily relies on animal tests. Here we summarise current EU regulatory requirements for the human health assessment of chemicals under REACH and the Cosmetic Products Regulation, considering the more critical endpoints and identifying the main challenges in introducing alternative methods into regulatory testing practice. This supports a recent initiative taken by the International Cooperation on Alternative Test Methods (ICATM) to summarise current regulatory requirements specific for the assessment of chemicals and cosmetic products for several human health-related endpoints, with the aim of comparing different jurisdictions and coordinating the promotion and ultimately the implementation of non-animal approaches worldwide. Recent initiatives undertaken at European level to promote the 3Rs and the use of alternative methods in current regulatory practice are also discussed. [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.

In the field of skin tissue engineering, the development of physiologically relevant in vitro skin models comprising all skin layers, namely epidermis, dermis, and subcutis, is a great challenge. Increasing regulatory requirements and the ban on animal experiments for substance testing demand the development of reliable and in vivo-like test systems, which enable high-throughput screening of substances. However, the reproducibility and applicability of in vitro testing has so far been insufficient due to fibroblast-mediated contraction. To overcome this pitfall, an advanced 3-layered skin model was developed. While the epidermis of standard skin models showed an 80% contraction, the initial epidermal area of our advanced skin models was maintained. The improved barrier function of the advanced models was quantified by an indirect barrier function test and a permeability assay. Histochemical and immunofluorescence staining of the advanced model showed well-defined epidermal layers, a dermal part with distributed human dermal fibroblasts and a subcutis with round-shaped adipocytes. The successful response of these advanced 3-layered models for skin irritation testing demonstrated the suitability as an in vitro model for these clinical tests: only the advanced model classified irritative and non-irritative substances correctly. These results indicate that the advanced set up of the 3-layered in vitro skin model maintains skin barrier function and therefore makes them more suitable for irritation testing.

Seizures are life-threatening adverse drug reactions which are investigated late in drug development using rodent models. Consequently, if seizures are detected, a lot of time, money and animals have been used. Thus, there is a need for in vitro screening models using human cells to circumvent interspecies translation. We assessed the suitability of cocultures of human-induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes compared with rodent primary cortical cultures for in vitro seizure liability assessment using microelectrode arrays. hiPSC-derived and rodent primary cortical neuronal cocultures were exposed to 9 known (non)seizurogenic compounds (pentylenetetrazole, amoxapine, enoxacin, amoxicillin, linopirdine, pilocarpine, chlorpromazine, phenytoin, and acetaminophen) to assess effects on neuronal network activity using microelectrode array recordings. All compounds affect activity in hiPSC-derived cocultures. In rodent primary cultures all compounds, except amoxicillin changed activity. Changes in activity patterns for both cell models differ for different classes of compounds. Both models had a comparable sensitivity for exposure to amoxapine (lowest observed effect concentration [LOEC] 0.03 µM), linopirdine (LOEC 1 µM), and pilocarpine (LOEC 0.3 µM). However, hiPSC-derived cultures were about 3 times more sensitive for exposure to pentylenetetrazole (LOEC 30 µM) than rodent primary cortical cultures (LOEC 100 µM). Sensitivity of hiPSC-derived cultures for chlorpromazine, phenytoin, and enoxacin was 10-30 times higher (LOECs 0.1, 0.3, and 0.1 µM, respectively) than in rodent cultures (LOECs 10, 3, and 3 µM, respectively). Our data indicate that hiPSC-derived neuronal cocultures may outperform rodent primary cortical cultures with respect to detecting seizures, thereby paving the way towards animal-free seizure assessment. [ABSTRACT FROM AUTHOR]

Early risk assessment of drug-induced liver injury (DILI) potential for drug candidates remains a major challenge for pharmaceutical development. We have previously developed a set of rat liver transcriptional biomarkers in short-term toxicity studies to inform the potential of drug candidates to generate a high burden of chemically reactive metabolites that presents higher risk for human DILI. Here, we describe translation of those NRF1-/NRF2-mediated liver tissue biomarkers to an in vitro assay using an advanced micropatterned coculture system (HEPATOPAC) with primary hepatocytes from male Wistar Han rats. A 9-day, resource-sparing and higher throughput approach designed to identify new chemical entities with lower reactive metabolite-forming potential was qualified for internal decision making using 93 DILI-positive and -negative drugs. This assay provides 81% sensitivity and 90% specificity in detecting hepatotoxicants when a positive test outcome is defined as the bioactivation signature score of a test drug exceeding the threshold value at an in vitro test concentration that falls within 3-fold of the estimated maximum drug concentration at the human liver inlet following highest recommended clinical dose administrations. Using paired examples of compounds from distinct chemical series and close structural analogs, we demonstrate that this assay can differentiate drugs with lower DILI risk. The utility of this in vitro transcriptomic approach was also examined using human HEPATOPAC from a single donor, yielding 68% sensitivity and 86% specificity when the aforementioned criteria are applied to the same 93-drug test set. Routine use of the rat model has been adopted with deployment of the human model as warranted on a case-by-case basis. This in vitro transcriptomic signature-based strategy can be used early in drug discovery to derisk DILI potential from chemically reactive metabolites by guiding structure-activity relationship hypotheses and candidate selection. [ABSTRACT FROM AUTHOR]

Animal models are 78% accurate in determining whether drugs will alter contractility of the human heart. To evaluate the suitability of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for predictive safety pharmacology, we quantified changes in contractility, voltage, and/or Ca2+ handling in 2D monolayers or 3D engineered heart tissues (EHTs). Protocols were unified via a drug training set, allowing subsequent blinded multicenter evaluation of drugs with known positive, negative, or neutral inotropic effects. Accuracy ranged from 44% to 85% across the platform-cell configurations, indicating the need to refine test conditions. This was achieved by adopting approaches to reduce signal-to-noise ratio, reduce spontaneous beat rate to ≤ 1 Hz or enable chronic testing, improving accuracy to 85% for monolayers and 93% for EHTs. Contraction amplitude was a good predictor of negative inotropes across all the platform-cell configurations and of positive inotropes in the 3D EHTs. Although contraction- and relaxation-time provided confirmatory readouts forpositive inotropes in 3D EHTs, these parameters typically served as the primary source of predictivity in 2D. The reliance of these "secondary" parameters to inotropy in the 2D systems was not automatically intuitive and may be a quirk of hiPSC-CMs, hence require adaptations in interpreting the data from this model system. Of the platform-cell configurations, responses in EHTs aligned most closely to the free therapeutic plasma concentration. This study adds to the notion that hiPSC-CMs could add value to drug safety evaluation. [ABSTRACT FROM AUTHOR]

Many substances for which consumer safety risk assessments need to be conducted are not associated with specific toxicity modes of action, but rather exhibit nonspecific toxicity leading to cell stress. In this work, a cellular stress panel is described, consisting of 36 biomarkers representing mitochondrial toxicity, cell stress, and cell health, measured predominantly using high content imaging. To evaluate the panel, data were generated for 13 substances at exposures consistent with typical use-case scenarios. These included some that have been shown to cause adverse effects in a proportion of exposed humans and have a toxicological mode-of-action associated with cellular stress (eg, doxorubicin, troglitazone, and diclofenac), and some that are not associated with adverse effects due to cellular stress at human-relevant exposures (eg, caffeine, niacinamide, and phenoxyethanol). For each substance, concentration response data were generated for each biomarker at 3 timepoints. A Bayesian model was then developed to quantify the evidence for a biological response, and if present, a credibility range for the estimated point of departure (PoD) was determined. PoDs were compared with the plasma C max associated with the typical substance exposures, and indicated a clear differentiation between "low" risk and "high" risk chemical exposure scenarios. Developing robust methods to characterize the in vitro bioactivity of xenobiotics is an important part of non-animal safety assessment. The results presented in this work show that the cellular stress panel can be used, together with other new approach methodologies, to identify chemical exposures that are protective of consumer health. [ABSTRACT FROM AUTHOR]

Abstract We created earlier a large machine‐readable database of 10,000 chemicals and 800,000 associated studies by natural language processing of the public parts of Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) registrations until December 2014. This database was used to assess the reproducibility of the six most frequently used Organisation for Economic Co‐operation and Development (OECD) guideline tests. These tests consume 55% of all animals in safety testing in Europe, i.e. about 600,000 animals. With 350–750 chemicals with multiple results per test, reproducibility (balanced accuracy) was 81% and 69% of toxic substances were found again in a repeat experiment (sensitivity 69%). Inspired by the increasingly used read‐across approach, we created a new type of QSAR, which is based on similarity of chemicals and not on chemical descriptors. A landscape of the chemical universe using 10 million structures was calculated, when based on Tanimoto indices similar chemicals are close and dissimilar chemicals far from each other. This allows placing any chemical of interest into the map and evaluating the information available for surrounding chemicals. In a data fusion approach, in which 74 different properties were taken into consideration, machine learning (random forest) allowed a fivefold cross‐validation for 190,000 (non‐) hazard labels of chemicals for which nine hazards were predicted. The balanced accuracy of this approach was 87% with a sensitivity of 89%. Each prediction comes with a certainty measure based on the homogeneity of data and distance of neighbours. Ongoing developments and future opportunities are discussed.

No‐observed‐effect concentrations (NOECs) are used in environmental hazard classification and labeling of chemicals and their environmental risk assessment. They are typically obtained using standard tests such as the fish early‐life stage (FELS) toxicity test, the chronic Daphnia reproduction test, and the algae growth inhibition test. Given the demand to replace and reduce animal tests, we explored the impact of the FELS toxicity test on the determination of effect concentrations by comparing the FELS toxicity test and the Daphnia and algae acute or chronic toxicity tests. Lowest‐observed‐effect concentrations (LOECs) were used instead of NOECs for better comparison with median lethal or effect concentration data. A database of FELS toxicity data for 223 compounds was established. Corresponding Daphnia and algae toxicity tests were identified using established databases (US Environmental Protection Agency ECOTOX, Organisation for Economic Co‐operation and Development QSAR Toolbox, eChemPortal, EnviroTox, and OpenFoodTox). Approximately 9.5% of the investigated compounds showed a 10‐fold higher sensitivity with the FELS toxicity test in comparison with the lowest effect concentrations obtained with any of the other tests. Some of these compounds have been known or considered as endocrine disrupting, or are other non‐narcotic chemicals, indicating that the higher sensitivity in the FELS toxicity test is related to a specific mechanism of action. Targeting these mechanisms by alternative test systems or endpoints, using fish embryos for instance, may allow reduction or replacement of the FELS toxicity test or may allow us to prioritize compounds for conduction of the FELS toxicity test. Environ Toxicol Chem 2019;39:30–41. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC. [ABSTRACT FROM AUTHOR]

An important goal in toxicology is the development of new ways to increase the speed, accuracy, and applicability of chemical hazard and risk assessment approaches. A promising route is the integration of in vitro assays with biological pathway information. We examined how the adverse outcome pathway (AOP) framework can be used to develop pathway‐based quantitative models useful for regulatory chemical safety assessment. By using AOPs as initial conceptual models and the AOP knowledge base as a source of data on key event relationships, different methods can be applied to develop computational quantitative AOP models (qAOPs) relevant for decision making. A qAOP model may not necessarily have the same structure as the AOP it is based on. Useful AOP modeling methods range from statistical, Bayesian networks, regression, and ordinary differential equations to individual‐based models and should be chosen according to the questions being asked and the data available. We discuss the need for toxicokinetic models to provide linkages between exposure and qAOPs, to extrapolate from in vitro to in vivo, and to extrapolate across species. Finally, we identify best practices for modeling and model building and the necessity for transparent and comprehensive documentation to gain confidence in the use of qAOP models and ultimately their use in regulatory applications. Environ Toxicol Chem 2019;38:1850–1865. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC. [ABSTRACT FROM AUTHOR]

Recently, the importance of inhalation toxicity assessment increased due to recent humidifier disinfectant-associated deaths in children. Benzalkonium chloride (BAC) is currently used as a cationic surfactant and germicide in food industry processing lines and as a hand sanitizer. Animal models are mainly used as a method of evaluating the inhalation toxicity of a hazardous substance, but that approach requires considerable amounts of time and cost. As a replacement for animal experiments, in vitro cell culture can be used to assess toxicity. However, such culture does not reflect the natural microenvironment of the lung, particularly its dynamic nature. In this study, we simulated normal breathing levels (tidal volume 10%, 0.2 Hz) through surface elongation of an elastic membrane in a dynamic culture system. The low-cost dynamic system provided easy control of breathing rate during lung cell culture. We assessed the toxicity using different concentrations of BAC (0, 2, 5, 10, 20, and 40 μg/mL) under static and dynamic culture conditions. Following 24 h of exposure to BAC, cellular metabolic activity, cell membrane integrity, interleukin-8 (IL-8) and reactive oxygen species (ROS) levels, and the total amount of protein in cells were analyzed. Our results showed that significant differences in cellular metabolic activity, as well as IL-8 and ROS profiles, between static and dynamic cell growth conditions, following BAC exposure. • Dynamic culture device used in this study more closely mimics lung conditions than that produced by a conventional monolayer culture method. • Screening toxic substances using a dynamic culture method can reduce the effects of cost and ethical issues on researchers. [ABSTRACT FROM AUTHOR]

Detection of developmental phenotypes in zebrafish embryos typically involves a visual assessment and scoring of morphological features by an individual researcher. Subjective scoring could impact results and be of particular concern when phenotypic effect patterns are also used as a diagnostic tool to classify compounds. Here we introduce a quantitative morphometric approach based on image analysis of zebrafish embryos. A software called FishInspector was developed to detect morphological features from images collected using an automated system to position zebrafish embryos. The analysis was verified and compared with visual assessments of 3 participating laboratories using 3 known developmental toxicants (methotrexate, dexamethasone, and topiramate) and 2 negative compounds (loratadine and glibenclamide). The quantitative approach exhibited higher sensitivity and made it possible to compare patterns of effects with the potential to establish a grouping and classification of developmental toxicants. Our approach improves the robustness of phenotype scoring and reliability of assay performance and, hence, is anticipated to improve the predictivity of developmental toxicity screening using the zebrafish embryo. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract 3D organ models have gained increasing attention as novel preclinical test systems and alternatives to animal testing. Over the years, many excellent in vitro tissue models have been developed. In parallel, microfluidic organ-on-a-chip tissue cultures have gained increasing interest for their ability to house several organ models on a single device and interlink these within a human-like environment. In contrast to these advancements, the development of human disease models is still in its infancy. Although major advances have recently been made, efforts still need to be intensified. Human disease models have proven valuable for their ability to closely mimic disease patterns in vitro, permitting the study of pathophysiological features and new treatment options. Although animal studies remain the gold standard for preclinical testing, they have major drawbacks such as high cost and ongoing controversy over their predictive value for several human conditions. Moreover, there is growing political and social pressure to develop alternatives to animal models, clearly promoting the search for valid, cost-efficient and easy-to-handle systems lacking interspecies-related differences. In this review, we discuss the current state of the art regarding 3D organ as well as the opportunities, limitations and future implications of their use. [ABSTRACT FROM AUTHOR]

Embryologic development involves cell differentiation and organization events that are unique to each tissue and organ and are susceptible to developmental toxicants. Animal models are the gold standard for identifying putative teratogens, but the limited throughput of developmental toxicological studies in animals coupled with the limited concordance between animal and human teratogenicity motivates a different approach. In vitro organoid models can mimic the three-dimensional (3D) morphogenesis of developing tissues and can thus be useful tools for studying developmental toxicology. Common themes during development like the involvement of epithelial-mesenchymal transition and tissue fusion present an opportunity to develop in vitro organoid models that capture key morphogenesis events that occur in the embryo. We previously described organoids composed of human stem and progenitor cells that recapitulated the cellular features of palate fusion, and here we further characterized the model by examining pharmacological inhibitors targeting known palatogenesis and epithelial morphogenesis pathways as well as 12 cleft palate teratogens identified from rodent models. Organoid survival was dependent on signaling through EGF, IGF, HGF, and FGF pathways, and organoid fusion was disrupted by inhibition of BMP signaling. We observed concordance between the effects of EGF, FGF, and BMP inhibitors on organoid fusion and epithelial cell migration in vitro, suggesting that organoid fusion is dependent on epithelial morphogenesis. Three of the 12 putative cleft palate teratogens studied here (theophylline, triamcinolone, and valproic acid) significantly disrupted in vitro organoid fusion, while tributyltin chloride and all-trans retinoic acid were cytotoxic to fusing organoids. The study herein demonstrates the utility of the in vitro fusion assay for identifying chemicals that disrupt human organoid morphogenesis in a scalable format amenable to toxicology screening. [ABSTRACT FROM AUTHOR]

Neurotoxicity testing still relies on ethically debated, expensive and time consuming in vivo experiments, which are unsuitable for high-throughput toxicity screening. There is thus a clear need for a rapid in vitro screening strategy that is preferably based on human-derived neurons to circumvent interspecies translation. Recent availability of commercially obtainable human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes holds great promise in assisting the transition from the current standard of rat primary cortical cultures to an animal-free alternative. We therefore composed several hiPSC-derived neuronal models with different ratios of excitatory and inhibitory neurons in the presence or absence of astrocytes. Using immunofluorescent stainings and multi-well micro-electrode array (mwMEA) recordings we demonstrate that these models form functional neuronal networks that become spontaneously active. The differences in development of spontaneous neuronal activity and bursting behavior as well as spiking patterns between our models confirm the importance of the presence of astrocytes. Preliminary neurotoxicity assessment demonstrates that these cultures can be modulated with known seizurogenic compounds, such as picrotoxin (PTX) and endosulfan, and the neurotoxicant methylmercury (MeHg). However, the chemical-induced effects on different parameters for neuronal activity, such as mean spike rate (MSR) and mean burst rate (MBR), may depend on the ratio of inhibitory and excitatory neurons. Our results thus indicate that hiPSC-derived neuronal models must be carefully designed and characterized prior to large-scale use in neurotoxicity screening. [ABSTRACT FROM AUTHOR]

Abstract: Fish early life stage (ELS) tests (Organisation for Economic Co‐operation and Development test guideline 210) are widely conducted to estimate chronic fish toxicity. In these tests, fish are exposed from the embryonic to the juvenile life stages. To analyze whether certain modes of action are related to high toxic ratios (i.e., ratios between baseline toxicity and experimental effect) and/or acute‐to‐chronic ratios (ACRs) in the fish ELS test, effect concentrations (ECs) for 183 compounds were extracted from the US Environmental Protection Agency's ecotoxicity database. Analysis of ECs of narcotic compounds indicated that baseline toxicity could be observed in the fish ELS test at similar concentrations as in the acute fish toxicity test. All nonnarcotic modes of action were associated with higher toxic ratios, with median values ranging from 4 to 9.3 × 104 (uncoupling < reactivity < neuromuscular toxicity < methemoglobin formation < endocrine disruption < extracellular matrix formation inhibition). Four modes of action were also found to be associated with high ACRs: 1) lysyl oxidase inhibition leading to notochord distortion, 2) putative methemoglobin formation or hemolytic anemia, 3) endocrine disruption, and 4) compounds with neuromuscular toxicity. For the prediction of ECs in the fish ELS test with alternative test systems, endpoints targeted to the modes of action of compounds with enhanced toxic ratios or ACRs could be used to trigger fish ELS tests or even replace these tests. Environ Toxicol Chem 2018;37:955–969. © 2018 SETAC [ABSTRACT FROM AUTHOR]

Increasing amounts of systems toxicology data, including omics results, are becoming publically available and accessible in databases. Data-driven and informatics-tool supported pipeline schemas for fitting such data into Adverse Outcome Pathway (AOP) descriptions could potentially aid the development of nonanimal-based hazard and risk assessment methods.We devised a 6-step workflow that integrated diverse types of toxicology data into a novel AOP scheme for pulmonary fibrosis. Mining of literature references and diverse data sources covering previous pathway descriptions and molecular results were coupled in a stepwise manner with informatics tools applications that enabled gene linkage and pathway identification in molecular interaction maps. Ultimately, a network of functional elements coupled 64 pulmonary fibrosis-associated genes into a novel, open-source AOP-linked molecular pathway, now available for commenting and improvements in WikiPathways (WP3624). Applying in silico-based knowledge extraction and modeling, the pipeline enabled screening and fusion of many different complex data types, including the integration of omics results. Overall, the taken, stepwise approach should be generally useful to construct novel AOP descriptions as well as to enrich developing AOP descriptions in progress. [ABSTRACT FROM AUTHOR]

The field of experimental toxicology is rapidly advancing by incorporating novel techniques and methods that provide a much more granular view into the mechanisms of potential adverse effects of chemical exposures on human health. The data from various in vitro assays and computational models are useful not only for increasing confidence in hazard and risk decisions, but also are enabling better, faster and cheaper assessment of a greater number of compounds, mixtures, and complex products. This is of special value to the field of green chemistry where design of new materials or alternative uses of existing ones is driven, at least in part, by considerations of safety. This article reviews the state of the science and decision-making in scenarios when little to no data may be available to draw conclusions about which choice in green chemistry is "safer." It is clear that there is no "one size fits all" solution and multiple data streams need to be weighed in making a decision. Moreover, the overall level of familiarity of the decision-makers and scientists alike with new assessment methodologies, their validity, value and limitations is evolving. Thus, while the "impact" of the new developments in toxicology on the field of green chemistry is great already, it is premature to conclude that the data from new assessment methodologies have been widely accepted yet. [ABSTRACT FROM AUTHOR]

Inclusion of alternative methods that replace, reduce, or refine (3R) animal testing within regulatory safety evaluations of chemicals generally faces many hurdles. The goal of the current work is to i) collect responses from key stakeholders involved in food safety evaluations on what they consider the most relevant factors that influence the acceptance and use of 3R methods and to ii) use these responses to formulate activities needed to increase the acceptance and use of 3R methods, particularly for kinetics. The stakeholders were contacted by e-mail for their opinions, asking the respondents to write down three barriers and/or drivers and scoring these by distributing 5 points over the three factors. The main barriers that obtained the highest aggregated scores were i) uncertain predictability 3R methods/lack of validation, ii) insufficient guidance regulators/industry and iii) insufficient harmonization of legislation. The major driver identified was the possibility of 3R methods to provide more mechanistic information. Based on the results, recommendations are given to enhance the acceptance and application of 3R toxicokinetic methods in food safety evaluations. These include steering of regulatory data requirements as well as creating (funding) opportunities for development and validation of alternative methods for kinetics and development of guidances. [ABSTRACT FROM AUTHOR]

Evaluating the safety of newly expressed proteins in genetically modified (GM) crops is conducted prior to commercialization to determine whether they could present a hazard upon consumption. A multicomponent, weight of evidence approach has been applied to individual proteins that has often included acute oral toxicology studies. Based on resources required to produce and purify the proteins, the number of animals necessary for these studies and the fact that no evidence of hazard has been observed for any of the proteins tested to date, it is questionable whether acute toxicology studies should be conducted for all proteins. This article reviews the chronology of the acute toxicology study from its origins into application for hazard assessment and classification of individual substances including proteins expressed in GM crops. It further proposes that a physiologic approach using cultured intestinal epithelial cell (IEC) line monolayers as an in vitro model of the gastrointestinal system provides results relevant to the hazard characterization of proteins when necessary. Benefits of this approach would include reduced quantities of proteins for testing and minimization or elimination of animal studies while maintaining confidence in the safety assessment process. [ABSTRACT FROM AUTHOR]

The objective of the present work was to review the availability and predictive value of non-animal toxicokinetic approaches and to evaluate their current use in European risk evaluations of food contaminants, additives and food contact materials, as well as pesticides and medicines. Results revealed little use of quantitative animal or human kinetic data in risk evaluations of food chemicals, compared with pesticides and medicines. Risk evaluations of medicines provided sufficient in vivo kinetic data from different species to evaluate the predictive value of animal kinetic data for humans. These data showed a relatively poor correlation between the in vivo bioavailability in rats and dogs versus that in humans. In contrast, in vitro (human) kinetic data have been demonstrated to provide adequate predictions of the fate of compounds in humans, using appropriate in vitro-in vivo scalers and by integration of in vitro kinetic data with in silico kinetic modelling. Even though in vitro kinetic data were found to be occasionally included within risk evaluations of food chemicals, particularly results from Caco-2 absorption experiments and in vitro data on gut-microbial conversions, only minor use of in vitro methods for metabolism and quantitative in vitro-in vivo extrapolation methods was identified. Yet, such quantitative predictions are essential in the development of alternatives to animal testing as well as to increase human relevance of toxicological risk evaluations. Future research should aim at further improving and validating quantitative alternative methods for kinetics, thereby increasing regulatory acceptance of non-animal kinetic data. [ABSTRACT FROM AUTHOR]