Toward Nano-Specific In Silico NAMs: How to Adjust Nano-QSAR to the Recent Advancements of Nanotoxicology?

The rapid development of engineered nanomaterials (ENMs) causes humans to become increasingly exposed to them. Therefore, a better understanding of the health impact of ENMs is highly demanded. Considering the 3Rs (Replacement, Reduction, and Refinement) principle, in vitro and computational methods are excellent alternatives for testing on animals. Among computational methods, nano-quantitative structure-activity relationship (nano-QSAR), which links the physicochemical and structural properties of EMNs with biological activities, is one of the leading method. The nature of toxicological experiments has evolved over the last decades; currently, one experiment can provide thousands of measurements of the organism's functioning at the molecular level. At the same time, the capacity of the in vitro systems to mimic the human organism is also improving significantly. Hence, the authors would like to discuss whether the nano-QSAR approach follows modern toxicological studies and takes full advantage of the opportunities offered by modern toxicological platforms. Challenges and possibilities for improving data integration are underlined narratively, including the need for a consensus built between the in vitro and the QSAR domains.

The skin sensitization adverse outcome pathway: exploring the role of mechanistic understanding for higher tier risk assessment.

For over a decade, the skin sensitization Adverse Outcome Pathway (AOP) has served as a useful framework for development of novel in chemico and in vitro assays for use in skin sensitization hazard and risk assessment. Since its establishment, the AOP framework further fueled the existing efforts in new assay development and stimulated a plethora of activities with particular focus on validation, reproducibility and interpretation of individual assays and combination of assay outputs for use in hazard/risk assessment. In parallel, research efforts have also accelerated in pace, providing new molecular and dynamic insight into key events leading to sensitization. In light of novel hypotheses emerging from over a decade of focused research effort, mechanistic evidence relating to the key events in the skin sensitization AOP may complement the tools currently used in risk assessment. We reviewed the recent advances unraveling the complexity of molecular events in sensitization and signpost the most promising avenues for further exploration and development of useful assays.

Complex In Vitro Model Characterization for Context of Use in Toxicologic Pathology: Use Cases by Collaborative Teams of Biologists, Bioengineers, and Pathologists.

Complex in vitro models (CIVMs) offer the potential to increase the clinical relevance of preclinical efficacy and toxicity assessments and reduce the reliance on animals in drug development. The European Society of Toxicologic Pathology (ESTP) and Society for Toxicologic Pathology (STP) are collaborating to highlight the role of pathologists in the development and use of CIVM. Pathologists are trained in comparative animal medicine which enhances their understanding of mechanisms of human and animal diseases, thus allowing them to bridge between animal models and humans. This skill set is important for CIVM development, validation, and data interpretation. Ideally, diverse teams of scientists, including engineers, biologists, pathologists, and others, should collaboratively develop and characterize novel CIVM, and collectively assess their precise use cases (context of use). Implementing a morphological CIVM evaluation should be essential in this process. This requires robust histological technique workflows, image analysis techniques, and needs correlation with translational biomarkers. In this review, we demonstrate how such tissue technologies and analytics support the development and use of CIVM for drug efficacy and safety evaluations. We encourage the scientific community to explore similar options for their projects and to engage with health authorities on the use of CIVM in benefit-risk assessment.

Understanding the Impacts of Presystemic Metabolism on the Human Oral Bioavailability of Chemicals.

Animal-free new approach methods promote chemical assessments based on the comparison between in vitro bioactivity and human internal concentrations, which necessitates a dependable knowledge of human oral bioavailability, i.e., the fraction of an orally ingested chemical that escapes from presystemic ("first-pass") metabolic processes and eventually enters systemic circulation. Using a physiologically based toxicokinetic model, we show how human oral bioavailability is impacted by presystemic metabolism within the gut lumen, gut wall, and liver and how this impact differs among chemicals with various permeability and stability properties. Our results highlight the gut lumen as a primary site of presystemic metabolism of certain chemicals, such as di-2-ethylhexyl phthalate (DEHP), for which the gut lumen may even exceed the liver in importance of presystemic metabolism due to these metabolic processes occurring in sequence. For chemicals with low transmembrane permeability and low stability, metabolism within the gut lumen is the most remarkable of the three presystemic metabolic processes. Notably, for chemicals that undergo substantial metabolism within the gut lumen, where the metabolites have high permeability, there is a notable discrepancy between the "theoretical bioavailability" (bioavailability of the unchanged parent compound) and the "apparent bioavailability" in measurement practices (bioavailability inferred from measured metabolites). Our work highlights the importance of considering presystemic metabolism, notably within the gut lumen, in human exposure and toxicokinetic modeling.

Application of the Biological Toxicity Effect Ratio (BER) Method for Advancing Water Quality Criteria Derivation.

Water quality criteria (WQC) serve as a scientific foundation for pollutant risk assessment and control in aquatic ecosystems. The development of regionally differentiated WQC tailored to specific regional characteristics has become an emerging trend. However, the current WQC is constrained by a lack of regional species toxicity data. To address these limitations, this study proposes the biological toxicity effect ratio (BER) method, which indirectly reflects the toxicity sensitivity of the overall aquatic ecosystem through the toxicity information on a limited number of species, enabling rapid WQC prediction. Using the established WQC in China and the USA as a case study, we combined mathematical derivation and data validation to evaluate the BER method. Among various species-taxon groups of freshwater organisms, planktonic crustaceans demonstrated the highest predictive accuracy. Our analysis further revealed that species toxicity sensitivity and regional variability jointly influence the prediction accuracy. Regardless of the evaluation indexes, planktonic crustaceans emerged as the most suitable species-taxon group for the BER method. Additionally, the BER method is particularly applicable to pollutants with conserved mechanisms across species. This study systematically explores the feasibility of using the BER method and offers new insights for deriving regionally differentiated WQC.

Missing Genomic Resources for the Next Generation of Environmental Risk Assessment.

Environmental risk assessment traditionally relies on a wide range of in vivo testing to assess the potential hazards of chemicals in the environment. These tests are often time-consuming and costly and can cause test organisms' suffering. Recent developments of reliable low-cost alternatives, both in vivo- and in silico-based, opened the door to reconsider current toxicity assessment. However, many of these new approach methodologies (NAMs) rely on high-quality annotated genomes for surrogate species of regulatory risk assessment. Currently, a lack of genomic information slows the process of NAM development. Here, we present a phylogenetically resolved overview of missing genomic resources for surrogate species within a regulatory ecotoxicological risk assessment. We call for an organized and systematic effort within the (regulatory) ecotoxicological community to provide these missing genomic resources. Further, we discuss the potential of a standardized genomic surrogate species landscape to enable a robust and nonanimal-reliant ecotoxicological risk assessment in the systems ecotoxicology era.

Using a Novel Multiplexed Algal Cytological Imaging (MACI) Assay and Machine Learning as a Way to Characterize Complex Phenotypes in Plant-Type Organisms.

High-throughput phenotypic profiling assays, popular for their ability to characterize alternations in single-cell morphological feature data, have been useful in recent years for predicting cellular targets and mechanisms of action (MoAs) for different chemicals and novel drugs. However, this approach has not been extensively used in environmental toxicology due to the lack of studies and established methods for performing this kind of assay in environmentally relevant species. Here, we developed a multiplexed algal cytological imaging (MACI) assay, based on the subcellular structures of the unicellular microalgae, Raphidocelis subcapitata, a toxicology and ecological model species. Several different herbicides and antibiotics with unique MoAs were exposed to R. subcapitata cells, and MACI was used to characterize cellular impacts by measuring subtle changes in their morphological features, including metrics of area, shape, quantity, fluorescence intensity, and granularity of individual subcellular components. This study demonstrates that MACI offers a quick and effective framework for characterizing complex phenotypic responses to environmental chemicals that can be used for determining their MoAs and identifying their cellular targets in plant-type organisms.

A Novel Transgenic Model to Study Thyroid Axis Activity in Early Life Stage Medaka.

Identifying endocrine disrupting chemicals in order to limit their usage is a priority and required according to the European Regulation. There are no Organization for Economic Co-operation and Development (OECD) test guidelines based on fish available for the detection of Thyroid axis Active Chemicals (TACs). This study aimed to fill this gap by developing an assay at eleuthero-embryonic life stages in a novel medaka (Oryzias latipes) transgenic line. This transgenic line expresses green fluorescent protein (GFP) in thyrocytes, under the control of the medaka thyroglobulin gene promoter. The fluorescence expressed in the thyrocytes is inversely proportional to the thyroid axis activity. When exposed for 72 h to activators (triiodothyronine (T3) and thyroxine (T4)) or inhibitors (6-N-propylthiouracil (PTU), Tetrabromobisphenol A (TBBPA)) of the thyroid axis, the thyrocytes can change their size and express lower or higher levels of fluorescence, respectively. This reflects the regulation of thyroglobulin by the negative feedback loop of the Hypothalamic-Pituitary-Thyroid axis. T3, T4, PTU, and TBBPA induced fluorescence changes with the lowest observable effect concentrations (LOECs) of 5 µg/L, 1 µg/L, 8 mg/L, and 5 mg/L, respectively. This promising tool could be used as a rapid screening assay and also to help decipher the mechanisms by which TACs can disrupt the thyroid axis in medaka.

Bioconcentration Assessment of Three Cationic Surfactants in Permanent Fish Cell Lines.

Cationic surfactants are used in many industrial processes and in consumer products with concurrent release into the aquatic environment, where they may accumulate in aquatic organisms to regulatoryly relevant thresholds. Here, we aimed to better understand the bioconcentration behavior of three selected cationic surfactants, namely N,N-dimethyldecylamine (T10), N-methyldodecylamine (S12), and N,N,N-trimethyltetradecylammonium cation (Q14), in the cells of fish liver (RTL-W1) and gill (RTgill-W1) cell lines. We conducted full mass balances for bioconcentration tests with the cell cultures, in which the medium, the cell surface, the cells themselves, and the plastic compartment were sampled and quantified for each surfactant by HPLC MS/MS. Accumulation in/to cells correlated with the surfactants' alkyl chain lengths and their membrane lipid-water partitioning coefficient, DMLW. Cell-derived bioconcentration factors (BCF) of T10 and S12 were within a factor of 3.5 to in vivo BCF obtained from the literature, while the cell-derived BCF values for Q14 were >100 times higher than the in vivo BCF. From our experiments, rainbow trout cell lines appear as a suitable conservative in vitro screening method for bioconcentration assessment of cationic surfactants and are promising for further testing.

Microbial pesticides - challenges and future perspectives for testing and safety assessment with respect to human health.

Plant protection measures are necessary to prevent pests and diseases from attacking and destroying crop plants and to meet consumer demands for agricultural produce. In the last decades the use of chemical pesticides has largely increased. Farmers are looking for alternatives. Biopesticides should be considered a sustainable solution. They may be less toxic than chemical pesticides, be very specific to the target pest, decompose quickly, and be less likely to cause resistance. On the other hand, lower efficacy and higher costs are two disadvantages of many biopesticides. Biopesticides include macroorganisms, natural compounds and microorganisms. Microbial pesticides are the most widely used and studied class of biopesticides. The greatest difference between microbial and chemical pesticides is the ability of the former to potentially multiply in the environment and on the crop plant after application. The data requirements for the European Union and the United States Environmental Protection Agency are highlighted, as these regulatory processes are the most followed in regions where local regulations for biopesticide products are not available or vague. New Approach Methods already proposed or harmonized for chemical pesticides are presented and discussed with respect to their use in evaluating microbial pesticide formulations. Evaluating the microbials themselves is not as simple as using the same validated New Approach Methods as for synthetic pesticides. Therefore, the authors suggest considering New Approach Method strategies specifically for microbials and global harmonization with acceptability with the advancements of such approaches. Further discussion is needed and greatly appreciated by the experts.

Recent advances and current challenges of new approach methodologies in developmental and adult neurotoxicity testing.

Adult neurotoxicity (ANT) and developmental neurotoxicity (DNT) assessments aim to understand the adverse effects and underlying mechanisms of toxicants on the human nervous system. In recent years, there has been an increasing focus on the so-called new approach methodologies (NAMs). The Organization for Economic Co-operation and Development (OECD), together with European and American regulatory agencies, promote the use of validated alternative test systems, but to date, guidelines for regulatory DNT and ANT assessment rely primarily on classical animal testing. Alternative methods include both non-animal approaches and test systems on non-vertebrates (e.g., nematodes) or non-mammals (e.g., fish). Therefore, this review summarizes the recent advances of NAMs focusing on ANT and DNT and highlights the potential and current critical issues for the full implementation of these methods in the future. The status of the DNT in vitro battery (DNT IVB) is also reviewed as a first step of NAMs for the assessment of neurotoxicity in the regulatory context. Critical issues such as (i) the need for test batteries and method integration (from in silico and in vitro to in vivo alternatives, e.g., zebrafish, C. elegans) requiring interdisciplinarity to manage complexity, (ii) interlaboratory transferability, and (iii) the urgent need for method validation are discussed.

The evaluation of skin sensitization potential of the UVCB substance diisopentyl phthalate by in silico and in vitro methods.

Diisopentyl phthalate (DiPeP) is primarily used as a plasticizer or additive within the production of polyvinyl chloride (PVC), and has many additional industrial applications. Its metabolites were recently found in urinary samples of pregnant women; thus, this substance is of concern as relates to human exposure. Depending upon the nature of the alcohol used in its synthesis, DiPeP may exist either as a mixture consisting of several branched positional isomers, or as a single defined structure. This article investigates the skin sensitization potential and immunomodulatory effects of DiPeP CAS No. 84777-06-0, which is currently marketed and classified as a UVCB substance, by in silico and in vitro methods. Our findings showed an immunomodulatory effect for DiPeP in LPS-induced THP-1 activation assay (increased CD54 expression). In silico predictions using QSAR TOOLBOX 4.5, ToxTree, and VEGA did not identify DiPeP, in the form of a discrete compound, as a skin sensitizer. The keratinocyte activation (Key Event 2 (KE2) of the adverse outcome pathway (AOP) for skin sensitization) was evaluated by two different test methods (HaCaT assay and RHE assay), and results were discordant. While the HaCaT assay showed that DiPeP can activate keratinocytes (increased levels of IL-6, IL-8, IL-1α, and ILA gene expression), in the RHE assay, DiPeP slightly increased IL-6 release. Although inconclusive for KE2, the role of DiPeP in KE3 (dendritic cell activation) was demonstrated by the increased levels of CD54 and IL-8 and TNF-α in THP-1 cells (THP-1 activation assay). Altogether, findings were inconclusive regarding the skin sensitization potential of the UVCB DiPeP-disagreeing with the results of DiPeP in the form of discrete compound (skin sensitizer by the LLNA assay). Additional studies are needed to elucidate the differences between DiPeP isomer forms, and to better understand the applicability domains of non-animal methods in identifying skin sensitization hazards of UVCB substances.

Development of an adverse outcome pathway network for nephrotoxicity.

Adverse outcome pathways (AOPs) were introduced in modern toxicology to provide evidence-based representations of the events and processes involved in the progression of toxicological effects across varying levels of the biological organisation to better facilitate the safety assessment of chemicals. AOPs offer an opportunity to address knowledge gaps and help to identify novel therapeutic targets. They also aid in the selection and development of existing and new in vitro and in silico test methods for hazard identification and risk assessment of chemical compounds. However, many toxicological processes are too intricate to be captured in a single, linear AOP. As a result, AOP networks have been developed to aid in the comprehension and placement of associated events underlying the emergence of related forms of toxicity-where complex exposure scenarios and interactions may influence the ultimate adverse outcome. This study utilised established criteria to develop an AOP network that connects thirteen individual AOPs associated with nephrotoxicity (as sourced from the AOP-Wiki) to identify several key events (KEs) linked to various adverse outcomes, including kidney failure and chronic kidney disease. Analysis of the modelled AOP network and its topological features determined mitochondrial dysfunction, oxidative stress, and tubular necrosis to be the most connected and central KEs. These KEs can provide a logical foundation for guiding the selection and creation of in vitro assays and in silico tools to substitute for animal-based in vivo experiments in the prediction and assessment of chemical-induced nephrotoxicity in human health.

Utility of in vivo metabolomics to support read-across for UVCB substances under REACH.

Structure-based grouping of chemicals for targeted testing and read-across is an efficient way to reduce resources and animal usage. For substances of unknown or variable composition, complex reaction products, or biological materials (UVCBs), structure-based grouping is virtually impossible. Biology-based approaches such as metabolomics could provide a solution. Here, 15 steam-cracked distillates, registered in the EU through the Lower Olefins Aromatics Reach Consortium (LOA), as well as six of the major substance constituents, were tested in a 14-day rat oral gavage study, in line with the fundamental elements of the OECD 407 guideline, in combination with plasma metabolomics. Beyond signs of clinical toxicity, reduced body weight (gain), and food consumption, pathological investigations demonstrated the liver, thyroid, kidneys (males only), and hematological system to be the target organs. These targets were confirmed by metabolome pattern recognition, with no additional targets being identified. While classical toxicological parameters did not allow for a clear distinction between the substances, univariate and multivariate statistical analysis of the respective metabolomes allowed for the identification of several subclusters of biologically most similar substances. These groups were partly associated with the dominant (> 50%) constituents of these UVCBs, i.e., indene and dicyclopentadiene. Despite minor differences in clustering results based on the two statistical analyses, a proposal can be made for the grouping of these UVCBs. Both analyses correctly clustered the chemically most similar compounds, increasing the confidence that this biological approach may provide a solution for the grouping of UVCBs.

Evaluating the in vitro developmental toxicity potency of a series of petroleum substance extracts using new approach methodologies (NAMs).

The present study evaluates the in vitro developmental toxicity and the possible underlying mode of action of DMSO extracts of a series of highly complex petroleum substances in the mouse embryonic stem cell test (mEST), the zebrafish embryotoxicity test (ZET) and the aryl hydrocarbon receptor reporter gene assay (AhR CALUX assay). Results show that two out of sixteen samples tested, both being poorly refined products that may contain a substantial amount of 3- to 7-ring polycyclic aromatic compounds (PACs), induced sustained AhR activation in the AhR CALUX assay, and concentration-dependent developmental toxicity in both mEST and ZET. The other samples tested, representing highly refined petroleum substances and petroleum-derived waxes (containing typically a very low amount or no PACs at all), were negative in all assays applied, pointing to their inability to induce developmental toxicity in vitro. The refining processes applied during the production of highly refined petroleum products, such as solvent extraction and hydrotreatment which focus on the removal of undesired constituents, including 3- to 7-ring PACs, abolish the in vitro developmental toxicity. In conclusion, the obtained results support the hypothesis that 3- to 7-ring PACs are the primary inducers of the developmental toxicity induced by some (i.e., poorly refined) petroleum substances and that the observed effect is partially AhR-mediated.

Systematic evaluation of high-throughput PBK modelling strategies for the prediction of intravenous and oral pharmacokinetics in humans.

Physiologically based kinetic (PBK) modelling offers a mechanistic basis for predicting the pharmaco-/toxicokinetics of compounds and thereby provides critical information for integrating toxicity and exposure data to replace animal testing with in vitro or in silico methods. However, traditional PBK modelling depends on animal and human data, which limits its usefulness for non-animal methods. To address this limitation, high-throughput PBK modelling aims to rely exclusively on in vitro and in silico data for model generation. Here, we evaluate a variety of in silico tools and different strategies to parameterise PBK models with input values from various sources in a high-throughput manner. We gather 2000 + publicly available human in vivo concentration-time profiles of 200 + compounds (IV and oral administration), as well as in silico, in vitro and in vivo determined compound-specific parameters required for the PBK modelling of these compounds. Then, we systematically evaluate all possible PBK model parametrisation strategies in PK-Sim and quantify their prediction accuracy against the collected in vivo concentration-time profiles. Our results show that even simple, generic high-throughput PBK modelling can provide accurate predictions of the pharmacokinetics of most compounds (87% of Cmax and 84% of AUC within tenfold). Nevertheless, we also observe major differences in prediction accuracies between the different parameterisation strategies, as well as between different compounds. Finally, we outline a strategy for high-throughput PBK modelling that relies exclusively on freely available tools. Our findings contribute to a more robust understanding of the reliability of high-throughput PBK modelling, which is essential to establish the confidence necessary for its utilisation in Next-Generation Risk Assessment.

A roadmap towards a human-centric safety assessment of advanced therapy medicinal products.

Advanced therapy medicinal products (ATMPs) are among the most complex pharmaceuticals with high human specificity. Species differences severely limit the clinical relevance of in vivo data. We conducted interviews with stakeholders involved in ATMP development about their perspective on the use of in vivo studies, the perceived hurdles and associated potential solutions regarding non-clinical development of ATMPs. In total, 17 stakeholders from 9 different countries were interviewed. A workshop was held with key stakeholders to further discuss major topics identified from the interviews. Conducting in vivo studies remains the status quo for ATMPs development. The hurdles identified included determining the amount of information required before clinical entry and effective use of limited human samples to understand a treatment or for clinical monitoring. A number of key points defined the need for future in vivo studies as well as improved application and implementation of New Approach Methodology (NAM)-based approach for products within a well-known modality or technology platform. These included data transparency, understanding of the added value of in vivo studies, and continuous advancement, evaluation, and qualification of NAMs. Based on the outcome of the discussions, a roadmap with practical steps towards a human-centric safety assessment of ATMPs was established.

Hepatotoxicity of silver nanoparticles: Benchmark concentration modeling of an in vitro transcriptomics study in human iPSC-derived hepatocytes.

Despite two decades of research on silver nanoparticle (AgNP) toxicity, a safe threshold for exposure has not yet been established, albeit being critically needed for risk assessment and regulatory decision-making. Traditionally, a point-of-departure (PoD) value is derived from dose response of apical endpoints in animal studies using either the no-observed-adverse-effect level (NOAEL) approach, or benchmark dose (BMD) modeling. To develop new approach methodologies (NAMs) to inform human risk assessment of AgNPs, we conducted a concentration response modeling of the transcriptomic changes in hepatocytes derived from human induced pluripotent stem cells (iPSCs) after being exposed to a wide range concentration (0.01-25 µg/ml) of AgNPs for 24 h. A plausible transcriptomic PoD of 0.21 µg/ml was derived for a pathway related to the mode-of-action (MOA) of AgNPs, and a more conservative PoD of 0.10 µg/ml for a gene ontology (GO) term not apparently associated with the MOA of AgNPs. A reference dose (RfD) could be calculated from either of the PoDs as a safe threshold for AgNP exposure. The current study illustrates the usefulness of in vitro transcriptomic concentration response study using human cells as a NAM for toxicity study of chemicals that lack adequate toxicity data to inform human risk assessment.

Evaluating scientific confidence in the concordance of in vitro and in vivo protective points of departure.

To fulfil the promise of reducing reliance on mammalian in vivo laboratory animal studies, new approach methods (NAMs) need to provide a confident basis for regulatory decision-making. However, previous attempts to develop in vitro NAMs-based points of departure (PODs) have yielded mixed results, with PODs from U.S. EPA's ToxCast, for instance, appearing more conservative (protective) but poorly correlated with traditional in vivo studies. Here, we aimed to address this discordance by reducing the heterogeneity of in vivo PODs, accounting for species differences, and enhancing the biological relevance of in vitro PODs. However, we only found improved in vitro-to-in vivo concordance when combining the use of Bayesian model averaging-based benchmark dose modeling for in vivo PODs, allometric scaling for interspecies adjustments, and human-relevant in vitro assays with multiple induced pluripotent stem cell-derived models. Moreover, the available sample size was only 15 chemicals, and the resulting level of concordance was only fair, with correlation coefficients <0.5 and prediction intervals spanning several orders of magnitude. Overall, while this study suggests several ways to enhance concordance and thereby increase scientific confidence in vitro NAMs-based PODs, it also highlights challenges in their predictive accuracy and precision for use in regulatory decision making.

New Approach Methodologies (NAMs) for ad hoc human health risk assessment of food and non-food products - Proceedings of a workshop.

RIVM convened a workshop on the use of New Approach Methodologies (NAMs) for the ad hoc human health risk assessment of food and non-food products. Central to the workshop were two case studies of marketed products with a potential health concern: the botanical Tabernanthe iboga which is used to facilitate mental or spiritual insight or to (illegally) treat drug addiction and is associated with cardiotoxicity, and dermal creams containing female sex hormones, intended for use by perimenopausal women to reduce menopause symptoms without medical supervision. The workshop participants recognized that data from NAM approaches added valuable information for the ad hoc risk assessment of these products, although the available approaches were inadequate to derive health-based guidance values. Recommendations were provided on how to further enhance and implement NAM approaches in regulatory risk assessment, specifying both scientific and technical aspects as well as stakeholder engagement aspects.