Thousands of chemicals have not been assessed for developmental neurotoxicity (DNT) despite potential human exposure, prompting efforts to screen chemicals for possible DNT hazard using in vitro approaches. Ninety-five chemicals were screened in in a battery including in vitro assays covering the key neurodevelopmental processes (KNDPs) of proliferation, apoptosis, cell migration, neuronal differentiation, neurite outgrowth, neuronal maturation and synaptogenesis, oligodendrocyte differentiation, and network formation using human and rat (primary cortical) cells. An in vivo assay of zebrafish neurobehavior was also included. This assay battery was sensitive enough to detect effects in the neurodevelopmental processes modeled. The chemicals comprised negative controls and several use classes: drugs, flame retardants, fungicides, herbicides, industrial chemicals (including PFAS), and insecticides. A data analysis pipeline was developed using a benchmark concentration approach and included multi-objective optimization (Pareto) and ToxPi analysis to integrate data from the individual assays to evaluate DNT hazard and prioritize chemicals, ranking them for further evaluation. Although the zebrafish assay was the most sensitive for detecting potency, selectivity varies significantly by assay and chemical class with neurite outgrowth assays and fungicides exhibiting the highest percentage of selective hits. Chemicals from the fungicide, drug, and insecticide use classes had higher priority in the Pareto and ToxPi analysis based on having the most potent and selective activity in the assays. These prioritization methods represent a novel and useful approach for comparative evaluation of potential developmental neurotoxicity hazard in large groups of chemicals.

Zusammenfassung Die Senatskommission zur gesundheitlichen Bewertung von Lebensmitteln (SKLM) der Deutschen Forschungsgemeinschaft (DFG) existiert seit den frühen 1950er‐Jahren und hat sich über Jahrzehnte kontinuierlich weiterentwickelt. Sie arbeitet in Themenauswahl und Prioritätensetzung in wissenschaftlicher Freiheit und ist im Bereich der Lebensmittelsicherheit in Deutschland eine verlässliche, mandats‐unabhängige wissenschaftsgetriebene Instanz. Das Ziel der Kommission ist es, offene wissenschaftliche Fragen zur Lebensmittelsicherheit systematisch zu analysieren, Bewertungskriterien weiterzuentwickeln und wissenschaftlich fundierte Empfehlungen für gesundheitliche Bewertungen abzuleiten. Dies schließt neue Lebensmittel, Zusatzstoffe, Prozesskontaminanten und Auswirkungen der Behandlungsverfahren auf Lebensmittel ein. Kontroverse Diskussionen über mögliche Risiken durch Lebensmittel begleiten die Arbeit der Lebensmittel‐ und Ernährungswissenschaften seit Jahrzehnten und kommen häufig dadurch zustande, dass für die Bewertung erforderliches Grundlagenwissen fehlt. Die Vorgehensweise der SKLM unterscheidet sich von jener offizieller öffentlicher Institutionen wie dem Bundesinstitut für Risikobewertung (BfR) oder der Europäischen Lebensmittelbehörde EFSA (European Food Safety Authority), da sie den Schwerpunkt auf das proaktive Schließen von Erkenntnislücken sowie die Entwicklung neuer Bewertungskonzepte und Sicherheitsstrategien legt. Ein Beispiel für den Mehrwert ist etwa die Diskussion der unterschiedlichen Richtwerte, die von Regulierungsbehörden zu Bisphenol A (BPA) herausgegeben wurden, und die Nutzung dieser Diskussion, um ein zukunftsorientiertes Konzept zur Verbesserung des Prozesses zu entwickeln. Die neu gegründete „International Commission on Food Safety“ (ICFS) wird solche Aufgaben künftig von der SKLM übernehmen.

There is a strong need for animal-free methods that properly predict drug adversity. Especially drug-induced liver injury (DILI) is a common adverse effect that is difficult to predict with current methodology. In this study, we developed a data-driven computational adversity model for predicting the relative number of apoptotic cells in vitro, based on stress pathway activity invoked by exposure of liver cells to nitrofurantoin, diclofenac, and ketoconazole. Next, we adapted this adversity model to represent an in vivo situation and developed physiologically-based pharmacokinetic (PBPK) models for all three drugs to simulate the concentration of the drugs in the liver of humans and rats. Coupling the PBPK, stress pathway and adversity models generated quantitative systems toxicology models for rats and humans at clinically-relevant doses. For all three drugs, low levels of toxicity were predicted in rats and humans, which is consistent with in vivo observations. A detailed analysis of modifying factors (sex, age) showed that the amount of apoptotic cells is expected to increase for females and elderly people compared to males. Coupling the different models generated a novel modelling pipeline that is an important step towards in silico DILI predictions.

Next generation risk assessment (NGRA) aims to enable transparent, reproducible chemical safety assessments based on human-relevant, animal-free new approach methodologies (NAMs). The Alternative Safety Profiling Algorithm (ASPA) was developed within the ASPIS cluster to provide an algorithmic workflow that structures problem formulation, evidence integration, and decision-making across three main pillars – hazard, ADME (toxicokinetics), and exposure. A stakeholder workshop was organized to refine ASPA. Four breakout groups systematically reviewed corresponding workflow sections, identifying strengths, conceptual gaps, and opportunities for harmonization. Across groups, participants endorsed ASPA’s modular, technology-neutral nature and its focus on standardizing processes rather than prescribing specific test batteries. The hazard pillar discussions emphasized a sensitive, hypothesis-generating Tier 1, complemented by a specific, mechanistic Tier 2, capable of deriving points of departure (PoDs). ADME experts supported a physiologically based kinetic (PBK) modelling strategy, advancing from generic towards more complex models, using mechanistic information and experimental data. The exposure group proposed refinements for transparent, tiered exposure modelling, with emphasis on realistic worst-case scenarios and explicit uncertainty communication. Cross-pillar discussions highlighted the importance of feedback loops among all pillars, and the documentation of decision points to achieve consistency and defensi­bility. The workshop outcomes informed three parallel developments: (i) algorithmic refinement and re-design toward the next ASPA version, (ii) the creation of detailed guidance for each building block, and (iii) the establishment of practical case studies to demonstrate workflow implementation. This report already contains a first case study (developmental neurotoxicity assessment of desnitro-imidacloprid). These advances increase the operability, transparency, and regulatory readiness of ASPA.

Little is known about how nicotinic signalling in human astrocytes may contribute to the functional neurotoxicity of compounds related to tobacco alkaloids and neonicotinoid pesticides. We generated a single-cell Ca2+-imaging assay in induced pluripotent stem cell (iPSC)-derived astrocytes, and profiled functional expressions of some neurotoxicologically relevant receptors. Responses to pharmacological tool compounds indicated the expression of nicotinic, muscarinic, purinergic, glutamatergic receptors and voltage-gated Na+/Ca2+ channels. Closer investigation of the nicotinic system, e.g., using the alpha7 nicotinic acetylcholine receptor (nAChR)-selective positive allosteric modulator PNU-120596 and alpha7-preferring agonist (AR-R17779) demonstrated that Ca2+ signals elicited by nicotine and neonicotinoids are dominated by alpha7 nAChRs and depend on the downstream activation of L-type Ca2+ channels and tetrodotoxin-sensitive Na+ channels. Crosstalk of nAChR activation/desensitization was not observed for the inflammatory response elicited by TNF or for activation of glutamatergic or purinergic signalling. However, pre-stimulation of nAChR by neonicotinoids significantly blunted the response to the neurotransmitter acetylcholine. Comparative experiments in the human neuronal cultures (LUHMES cells) revealed similar potency ranges and pharmacological fingerprints for several neonicotinoids and their human-relevant metabolites descyanothiacloprid and desnitroimidacloprid. The pesticide metabolites showed a high potency, compared with their respective parent compounds. After this basic system characterization, the hitherto data-poor pesticides cycloxaprid and flupyradifurone were comparatively profiled in astrocytic and neuronal test systems. They showed the typical features of alpha7 nAChR agonists. The disruption of cholinergic signalling in astrocytes suggests that neonicotinoids affect not only neurons in human brains. Therefore, future neurotoxicity screening approaches may need to consider astrocyte toxicity.

Biokinetic complexities (plastic sorption, protein binding, and cellular accumulation) may cause large discrepancies between nominal and biologically effective concentrations of test compounds assessed by new approach methods (NAMs). This case study was performed to explore a generally applicable workflow that addresses biokinetic complexities in the context of NAM-based hazard testing for next-generation risk assessment (NGRA). The pesticide tebufenpyrad (TEBU) is a challenging test compound, as it (i) is hydrophobic, (ii) has an intracellular target (mitochondrial respiration), and (iii) is acting at low concentrations (susceptible to biokinetic complexities). In the newly established NeuriTox-M neurotoxicity assay, based on human dopaminergic (LUHMES) neuron cultures, TEBU showed toxic effects at 20 nM. Mass spectrometric analyses of various experimental setups showed that a large fraction (75% to >90%) of TEBU was adsorbed to plastic. This effect was strongly attenuated by albumin in the medium. Cells, cultured on plastic, were considered unsuitable to assess cellular uptake. Therefore, alternatives were explored: when cells were used as suspension cultures (3% v/v) in albumin-containing medium, analysis worked best. Under such conditions, the concentration ratio (cells/medium) of TEBU was around 10. Data from an in vitro distribution (VIVD) model were in good agreement with the measurements. VIVD predicted the unbound medium TEBU concentration (Cu) to be 2-3 orders of magnitude below the nominal concentration and the total cellular concentration to be 10-100-fold above. Standard cell culture assays showed that the medium albumin content indeed altered the TEBU toxicity threshold. More such studies are needed to embed biokinetics information into NGRA.

While the genome codes for all proteins an organism can express, only certain sets of proteins are expressed in defined cell types. A cell's phenotype is influenced by the life-stage, exposure to signal molecules, as well as the exposome, i.e., external influences including physical stressors or chemicals from food, the environment or microorganisms. The interplay between genetics and these exposures is termed gene x environment interaction (GxE). Epigenetics contributes to GxE by modifying the accessibility of genes and thus their ability to be translated to proteins. Epigenetic mechanisms include DNA methylation, histone modifications, non-coding RNAs, and changes in local DNA packaging. As genetics and the exposome often jointly contribute to disease, understanding epigenetics may enable a better understanding of many human pathologies. Often, epigenetics will retain the memory of exposure, which changes an organism's susceptibility to subsequent, other exposures. This concept may allow new insights into mixture toxicity, especially when the exposures do not take place at the same time. Knowledge on epigenetic processes provides a basis for novel drugs that modify cell phenotypes (e.g., in cancer or neurodegenerative disease). Here, we provide an overview of the role of epigenetics in toxicology, and we call for a systematic assessment of epigenetic changes as part of investigative, and possibly regulatory, toxicity assessments. We propose tools and strategies for using human-relevant models, biomarkers, and AI to better predict who may be at risk. Ultimately, adding epigenetics to toxicology will help us create safer products and protect vulnerable individuals and future generations. Plain language summary Our genes are not the whole story when it comes to how our bodies respond to chemicals, medicines or environmental stress. A level of control-called epigenetics-determines which genes are turned on or off without changing the DNA itself. Detrimental epigenetic changes can be caused by external exposures like pollution, diet or drugs, and they may affect our health in long-lasting ways. This article explores how such "epigenetic toxicity" works, how it can lead to diseases like cancer or developmental disorders, and why current safety testing may miss it. We argue that epigenetic effects should be considered alongside traditional genetic damage in safety assessments of new drugs. We propose tools and strategies for using human-relevant models, biomarkers, and AI to better predict who may be at risk. Adding epigenetics to toxicology will help us create safer products and protectvulnerable individuals and future generations.

Neurite degeneration (ND) precedes cell death in many neurodegenerative diseases. However, it remains unclear how this compartmentalized cell death process is orchestrated in the central nervous system (CNS). The establishment of a CNS axotomy model (using modified 3D LUHMES cultures) allowed us to study metabolic control of ND in human midbrain-derived neurons without the use of toxicants or other direct disturbance of cellular metabolism. Axotomy lead to a loss of the NAD+ synthesis enzyme NMNAT2 within 2 h and a depletion of NAD+ within 4-6 h. This process appeared specific, as isolated neurites maintained ATP levels and a coupled mitochondrial respiration for at least 6 h. In the peripheral nervous system (PNS) many studies observed that NAD+ metabolism, in particular by the NADase SARM1, plays a major role in the ND occurring after axotomy. Since neither ferroptosis nor necroptosis, nor caspase-dependent apoptosis seemed to be involved in neurite loss, we investigated SARM1 as potential executioner (or controller). Knock-down or expression of a dominant-negative isoform of SARM1 indeed drastically delayed ND. Various modifications of NAD+ metabolism known to modulate SARM1 activity showed the corresponding effects on ND. Moreover, supplementation with NAD+ attenuated ND. As a third approach to investigate the role of altered NAD+ metabolism, we made use of the WLD(s) protein, which has been found in a mutant mouse to inhibit Wallerian degeneration of axons. This protein, which has a stable NMNAT activity, and thus can buffer the loss of NMNAT2, protected the neurites by stabilizing neurite NAD+ levels. Thus CNS-type ND was tightly linked to neurite metabolism in multiple experimental setups. Based on this knowledge, several new strategies for treating neurodegenerative diseases can be envisaged.

The potent dihydroorotate dehydrogenase (DHODH) inhibitor brequinar has been investigated as an anticancer, immunosuppressive, and antiviral pharmaceutical agent. However, its toxicity is still poorly understood. We investigated the cellular responses of primary human hepatocytes (PHH) and telomerase-immortalised human renal proximal tubular epithelial cells (RPTEC/TERT1) after a single 24-h exposure up to 100 mu M brequinar. Additionally, RPTEC/TERT1 cells underwent repeated daily exposure for five consecutive days at 0.3, 3, and 20 mu M. Transcriptomic analysis revealed that PHH were less sensitive to brequinar treatment than RPTEC/TERT1 cells. Upregulation of various phase I and II drug-metabolising enzymes, particularly Cytochrome P450 (CYP) 1 A and 3 A enzymes, in PHH suggests potential detoxification. Furthermore, brequinar exposure led to a significant upregulation of several stress response pathways in PHH and RPTEC/TERT1 cells, including the unfolded protein response, Nrf2, p53, and inflammatory responses. RPTEC/TERT1 cells exhibited greater sensitivity to brequinar at 0.3 mu M with repeated exposure compared to a single exposure. Furthermore, brequinar could impair the mitochondrial respiration of RPTEC/TERT1 cells after 24 h. This study provides new insights into the differential responses of PHH and RPTEC/TERT1 cells in response to brequinar exposure and highlights the biological relevance of implementing repeated dosing regimens in in vitro studies.

The 5th International Conference on Developmental Neurotoxicity (DNT) Testing (DNT5) took place in April 2024 in Konstanz, Germany, organized by CAAT-Europe, the University of Konstanz, and scientists from the US EPA, SCAHT, and CAAT at Johns Hopkins University Bloomberg School of Public Health. The conference convened experts from regulatory agencies, industry, and academia to explore the latest advancements in DNT testing and the integration of animal-free new approach methodologies (NAMs) into next-generation risk assessment (NGRA). The key topic was the appli-cation and further development of the recently established DNT in vitro test battery (DNT-IVB). To support this, OECD held a satellite meeting to discuss necessary next steps for further implementation of the DNT-IVB in regulatory contexts. Validation of new DNT test methods and use of their data for in-vitro-to-in-vivo extrapolations in physiologically based kinetic models were also important themes of the main meeting. In this context, the question was raised when a comprehensive biological and chemical coverage by the DNT-IVB would be reached. A need for additional testing data was recognized. Context-specific validation approaches for the entire DNT-IVB and the potential for intelligent combinations of assays to enhance the predictive power of the test battery were also addressed. Many presentations demonstrated the field's embrace of novel developments, including the use of multi-endpoint embryonic zebrafish tests, the development of artificial intelligence-driven computational approaches, and the establishment of complex, electrically active brain organoids and other self-organizing structures. Through its highly interactive format, DNT5 promoted extensive collaborative efforts in advancing the field toward more human-relevant, scientifically reliable, and ethical toxicological assessments.

Reproducibility of cell culture experiments between laboratories needs to be improved by ensuring more complete reporting of methodology in scientific papers. The minimum reporting standards sug-gested here cover various cell culture methods including monolayers, stem cells, organoids, and microphysiological systems (MPS). The standards build on existing guidance like Good Cell Culture Practice (GCCP 2.0) and OECD test reporting guidelines on how to quality-assure in vitro work, focusing on transparency and completeness of reporting. Key elements to be reported include full details of cell source and identity, cell quality control and characterization, materials and reagents used, culture conditions and protocols, experimental design, data analysis, data availability, and legal and ethical aspects. For complex models, additional details need to be provided such as cell ratios, microenvironment conditions, functional characterization, etc. The guidance for Good In Vitro Reporting Standards (GIVReSt) is part of a broader initiative of evidence-based toxicology encompassing the improvement of the quality of in vitro studies for safety assessments and regulatory decisions. In summary, GIVReSt addresses incomplete reporting as a major factor affecting repro-ducibility of cell culture experiments by providing clear standards around transparency and rigor in reporting. The integration of agentic artificial intelligence (AI) is envisioned to streamline compliance checking, providing real-time feedback and accelerating scientific discovery by making high-quality evidence more accessible. This should lead to more reliable cell culture research overall.

Although recent progress has been made, structure-based methods such as molecular docking are still underexplored in the context of toxicity prediction. These approaches offer added value, particularly in addressing challenges such as activity cliffs─i.e., caused by stereoisomerism─that are difficult to capture by conventional Quantitative Structure-Activity Relationship (QSAR) methods. In this study, we investigated the ability of docking scoring functions and protein-ligand interaction fingerprints to rank the potential hazard of compounds targeting the human mitochondrial complexes I and III (CI, NADH:ubiquinone oxidoreductase and CIII, cytochrome bc1 complex). We applied an induced fit docking protocol to account for binding site flexibility and performed a set of binding energy minimizations for rescoring of representative binding modes. Both individual scoring functions and consensus scoring approaches achieved acceptable rank correlation to experimentally derived data from CIII (Spearman r: 0.89 and 0.86). Moreover, consensus interaction fingerprints that combine molecular interactions from both docking outputs captured differences of inhibitor subtypes at CIII. Follow-up in vitro testing confirmed an isomerism-dependent activity cliff of E-/Z-Fenpyroximate at CI. These findings support the utility of using consensus docking and scoring as a screening-level tool for prioritizing compounds based on interpretable predicted relative binding affinities at CI and CIII.

Proteasome dysfunctions are observed in many human pathologies. To study their role and potential treatment strategies, models of proteasome inhibition are widely used in biomedical research. One frequently used tool is the proteasome inhibitor MG-132. It triggers the degeneration of human neurons, and several studies show protection from pathological events by glutathione or its precursors. It has therefore been concluded that glutathione protects cells from proteasome dysfunction. However, an alternative explanation is that MG-132, which is a peptide aldehyde, is chemically inactivated by thiols, and the apparent protection by glutathione from proteasome dysfunction is an artefact. To clarify this issue, we examined the chemical inactivation of MG-132 by thiols and the role of such reactions for neuroprotection. Using mass spectrometry and nuclear magnetic resonance spectroscopy, we found that MG-132 reacted with L-cysteine to form a stable end product and with glutathione to form an unstable intermediate. Using a cell-free proteasome inhibition assay, we found that high concentrations of L-cysteine can scavenge a substantial fraction of MG-132 and thus reduce proteasome inhibition. Glutathione (or N-acetyl-cysteine) did not alter proteasome inhibition (even at high concentrations). In a final step, we studied human neuronal cultures. We exposed them to MG-132, supplemented the culture medium with various thiols, and assessed intracellular L-cysteine concentrations. The transcriptome response pattern also indicated an inhibition of the proteasome by MG-132 in the presence of L-cysteine. We conclude that thiol concentrations that can be reached in cells do not inactivate MG-132 in pathological models. They rather act in a cytoprotective way as antioxidants.

Parkinson's disease (PD) is characterized by the pathologic aggregation of α-synuclein, which induces endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR), ultimately leading to the death of dopaminergic neurons. This study investigated whether drugs known to mitigate protein aggregation in prion disease models would attenuate the stress response and cell death in PD models. Flunarizine and ten of its structural analogues, previously identified through a drug repositioning approach to reduce the aggregation of PrPSc prion protein, were evaluated in a Chinese hamster ovary (CHO-K1) cell model. First, UPR was induced in CHO-K1 cells using tunicamycin, revealing that several of these drugs conferred protection and also reduced the expression of the UPR marker CHOP. Subsequently, we tested these anti-prion drugs in PD relevant cellular models. Neuronally-differentiated PC12 cells were employed, and all 11 drugs exhibited protective effect. Finally, human dopaminergic neurons (LUHMES cells) were exposed to 1-methyl-4-phenylpyridinium (MPP+), a compound commonly used to induce parkinsonian-like pathology. In this model, all 11 drugs demonstrated cytoprotective properties and attenuated UPR. Notably, several compounds - benzydamine, duloxetine, flunarizine, metixene and triflupromazine - protected LUHMES cells from MPP+ -induced ER stress at nanomolar concentrations. These findings provide the proof of concept that drugs selected for other indications, may be repurposed to ameliorate PD and related pathologies linked to ER stress.

Human microglia are central regulators and actors in brain infections and neuro-inflammatory pathologies. However, access to such cells is limited, and studies systematically mapping the spectrum of their inflammatory states are scarce. Here, we generated microglia-like cells (MGLCs) from human induced pluripotent stem cells and characterized them as a robust, accessible model system for studying inflammatory activation. We validated lineage identity through transcriptome profiling, revealing selective upregulation of microglial signature genes and enrichment of microglia/macrophage-related gene sets. MGLCs displayed distinct morphologies and produced stimulus- and time-dependent cytokine secretion profiles upon exposure to diverse inflammatory stimuli, including pro-inflammatory cytokines (TNFα, interferon-γ) and agonists of the Toll-like receptors TLR2 (FSL-1), TLR3 (Poly(I:C)), TLR4 (lipopolysaccharide, LPS), and TLR7 (imiquimod). Transcriptome profiling and bioinformatics analysis revealed distinct activation signatures. Functional assays demonstrated stimulus-specific engagement of NFκB and JAK-STAT signaling pathways. The shared NFκB nuclear translocation response of TLR ligands and TNFα was reflected in overlapping transcriptome profiles: they shared modules (e.g., oxidative stress response and TNFα-related signaling) identified by weighted gene co-expression network analysis. Finally, the potential consequences of microglia activation for neighboring cells were studied on the example of microglia-astrocyte crosstalk. The capacity of MGLC supernatants to stimulate astrocytes was measured by quantifying astrocytic NFκB translocation. MGLCs stimulated with FSL-1, LPS, or Poly(I:C) indirectly activated astrocytes via a strictly TNFα-dependent mechanism, highlighting the role of soluble mediators in the signal propagation. Altogether, this platform enables a dissection of microglia activation states and multi-parametric characterization of subsequent neuroinflammation.

Synchronized oscillatory fluctuations in intracellular calcium concentration across extended neuronal networks represent a functional indicator of connectivity and signal coordination. In this study, a model of human immature neurons (differentiated from LUHMES precursors) has been used to establish a robust protocol for generating reproducible intracellular Ca2+ oscillations in both two-dimensional monolayers and three-dimensional spheroids. Oscillatory activity was induced by defined ionic conditions in combination with potassium channel blockade. It was characterized by stable frequencies of approximately 0.2 Hz and high synchronization indices across millimeter-scale cultures. These properties were consistently reproduced in independent experiments and across laboratories. Single-cell imaging confirmed that oscillations were coordinated throughout large cell populations. Pharmacological interventions demonstrated that neither excitatory nor inhibitory chemical synaptic transmission influenced oscillatory dynamics. Gap junction blockers completely disrupted synchronization, while leaving individual cell activity unaffected. Functional dye-transfer assays provided additional evidence for electrical coupling. This was further supported by connexin-43 expression profiles and immunostaining. Collectively, these findings indicate that synchronized Ca2+ oscillations in LUHMES cultures are mediated by gap junctional communication rather than by conventional synaptic mechanisms. This system offers a practical platform for studying fundamental principles of network coordination and for evaluating pharmacological or toxicological modulators of intercellular coupling. Moreover, it may provide a relevant human-based model to explore aspects of neuronal maturation and to assess compounds with potential neurodevelopmental toxicity.

Human cell-based assays for neurotoxicity (NT) and developmental neurotoxicity (DNT) have reached a high level of readiness, but some tests require improvements in the specificity and sensitivity at which mitochondrial toxicants are detected. This study aimed to optimize the PeriTox assay, which uses peripheral neurons (PNs) and predicts the potential of chemicals to trigger peripheral neuropathies. By introducing a glucose-to-galactose switch in the medium composition, cells were forced to rely on mitochondrial respiration. Using pre-differentiated PNs cultured in either glucose (Glc) or galactose (Gal), we observed no major differences in baseline phenotype, gene expression, neurite outgrowth, or total ATP content. However, a marked metabolic shift was confirmed by the increased oxygen consumption in Gal conditions. Based on measurements of neurite growth and ATP levels, Gal-adapted neurons showed a heightened sensitivity, up to 7500-fold, to a range of mitochondrial respiratory chain (MRC) inhibitors. The sensitivity shift was high for inhibitors of MRC complexes I and III and modest or absent for unrelated compounds such as proteasome inhibitors or cytoskeletal poisons. For complex I-III inhibitors, the enhanced detection of mitochondrial neurotoxicants was coupled with a more accurate distinction between cytotoxic and neurite-specific effects, i.e., an improved assay specificity. In conclusion, our study on 39 compounds suggests that running the PeriTox assay in galactose increases its sensitivity and specificity for several mitochondrial toxicants, while no general disadvantages or shortcomings were observed. The modified version (PeriTox-M) may increase the performance of in vitro test batteries for scientific and regulatory applications.

While the genome codes for all proteins an organism can express, only certain sets of proteins are expressed in defined cell types. A cell's phenotype is influenced by the life-stage, exposure to signal molecules, as well as the exposome, i.e., external influences including physical stressors or chemicals from food, the environment or microorganisms. The interplay between genetics and these exposures is termed gene x environment interaction (GxE). Epigenetics contributes to GxE by modifying the accessibility of genes and thus their ability to be translated to proteins. Epigenetic mechanisms include DNA methylation, histone modifications, non-coding RNAs, and changes in local DNA packaging. As genetics and the exposome often jointly contribute to disease, understanding epigenetics may enable a better understanding of many human pathologies. Often, epigenetics will retain the memory of exposure, which changes an organism's susceptibility to subsequent, other exposures. This concept may allow new insights into mixture toxicity, especially when the exposures do not take place at the same time. Knowledge on epigenetic processes provides a basis for novel drugs that modify cell phenotypes (e.g., in cancer or neurodegenerative disease). Here, we provide an overview of the role of epigenetics in toxicology, and we call for a systematic assessment of epigenetic changes as part of investigative, and possibly regulatory, toxicity assessments. We propose tools and strategies for using human-relevant models, biomarkers, and AI to better predict who may be at risk. Ultimately, adding epigenetics to toxicology will help us create safer products and protect vulnerable individuals and future generations.