The chemical universe to which humans and ecosystems are exposed is expanding at a rate that traditional toxicology cannot match. Estimates of the commercial global chemical inventory have moved from tens of thousands in the 1980s to roughly 350,000 distinct substances registered for commercial use in 2019, and exposures now arrive as mixtures, transformation products, and engineered materials whose number grows faster than any list can capture. Against this curve, con-ventional regulatory testing has at best advanced linearly, and in some jurisdictions it has stagnated outright. The mismatch is not a regulatory failure of will; it is a structural impossibility, because animal-based assays cannot be made exponentially cheaper, faster, or more numerous. The escape from the bottleneck has come from a different family of methods entirely. New approach methodologies (NAMs), and in particular high-throughput in vitro screening, computational toxicology, microphysiological systems, and most recently artificial intelligence, are themselves on exponential growth curves. This article argues that the only coherent response to exponential exposure is exponential testing, and that the field must now think and plan in exponential rather than linear terms. I examine why this is hard, drawing on cognitive science and on Hartmut Rosa's diagnosis of social acceleration, and propose how an exponential toxicology can be built without sacrificing scientific rigor.

Public health decisions are uniquely difficult, weighing population benefits against harms, equity, resource constraints, and feasibility, often under deep uncertainty. The GRADE Evidence-to- Decision (EtD) framework, rooted in evidence-based medicine, offers a transparent route from evidence to action through twelve explicit criteria, and has recently been adapted for environmental and occupational health. A Human Exposome Project would generate evidence of a volume and complexity that breaks the manual assumptions on which EtD was built. I argue that agentic artificial intelligence, autonomous agents orchestrating multi-step scientific workflows, can operationalize each EtD criterion and make exposome-scale decision-making feasible, but only if it inherits the rigor of the evidence-based disciplines it is asked to accelerate. Six families of agents (evidence extraction, risk-of-bias assessment, uncertainty quantification, causality reasoning, cost-outcome analysis, and post-deployment validation) map cleanly onto the EtD criteria. Five governance requirements (traceability, versioning, context-of-use benchmarking, honest uncertainty, and human accountability) separate an evidence engine from a confident hallucination machine. The exposome demands nothing less.

Assessment of the carcinogenic potential of chemicals is considered an important element of human health risk assessment. However, the approaches currently used for different regulatory sectors have some shortcomings. To overcome these, an alternative testing strategy, like an IATA (Integrated Approach to Testing and Assessment), for the detection of non-genotoxic carcinogens (NGTXCs) is in demand. Such an IATA should be mechanism-based and, wherever possible, consist of New Approach Methodologies (NAMs) to avoid testing in experimental animals. To explore which type of NAMs (in silico and in vitro) should be included in the first tier of an IATA for NGTXCs we performed a case study, in line with various international initiatives focusing on this need. The case study comprised a diverse set of 29 chemicals that together cover different modes of action relevant for non-genotoxic carcinogenesis. Different NAMs, including in silico tools (e.g., QSARS, ADME predictions) and high-throughput in vitro assays such as ToxCast and CALUX, were explored. The findings from the case study reveal the complementarity of the NAMs studied as well as the need for additional NAMs to be included, to ensure a broader coverage of MOAs relevant for carcinogenicity. As such, the case study nicely contributes to a more defined composition of a first tier for an IATA for NGTXC.

Male reproductive health has declined over recent decades, characterized by rising rates of reproductive birth defects, reduced semen quality, and an increased risk of testicular cancer. This global trend has been linked to exposure to endocrine-disrupting chemicals (EDCs), which can interfere with the endocrine system including disrupting steroidogenesis, the process by which steroid hormones are synthesized. Steroid hormones, not least testosterone, are essential for male sex differentiation, masculinization, and spermatogenesis. During human fetal development, Leydig and Sertoli cells of the testis together produce androgens, whereas in adults, Leydig cells can synthesize testosterone alone. EDCs can disrupt this process through various mechanisms. Advances in in vitro models of the steroidogenic system, particularly three-dimensional (3D) cell cultures, have provided a more accurate representation of the in vivo environment, enabling more realistic responses to external stimuli. These models offer a powerful tool for studying the effects of EDCs on steroidogenesis and will deepen our understanding of the mechanisms underlying endocrine disruption, knowledge that is important in developing new strategies for chemical safety assessment. This review provides a comprehensive analysis of the steroidogenic system, with a focus on key enzymes involved. The current regulatory test strategies to reduce the impact of EDCs on male fertility is discussed. Furthermore, 3D testis models are compared, highlighting their strengths and limitations for testing effects on testis steroidogenesis. Emphasis is placed on the urgent need for innovative, practical, and cost-effective alternatives to traditional in vivo testing to improve chemical risk assessments.

The transition toward non-animal safety assessment has driven the development of in vitro models for skin sensitization, particularly those targeting key event 2 (KE2) within the adverse outcome pathway (AOP). While conventional KE2 assays often rely on randomly integrated reporter systems, the EndoSens model utilizes CRISPR/Cas9-mediated knock-in to precisely insert a luciferase reporter into the endogenous HMOX1 locus in HaCaT keratinocytes, providing a genomically anchored and physiologically relevant system. To enable reproducible adoption across laboratories, we established a standardized protocol with defined quality control (QC) criteria, incorporating cinnamyl alcohol as a quantitative positive control and a cytotoxicity pre-screen to identify non-cytotoxic test concentrations. Evaluation using ten OECD TG 442D reference chemicals and ten additional compounds demonstrated the assay's robust performance in discriminating skin sensitizers with high inter-laboratory consistency, supporting its integration as a reliable in vitro component within defined approaches (DA) for animal-free skin sensitization assessment.

Endocrine disruptors, which pose a risk to organisms and entire ecosystems even at low concentra­tions, can be detected by standardized in vitro methods according to OECD test guidelines 455 and 458. However, these methods require the undefined animal-derived supplement fetal bovine serum (FBS), which is associated with ethical concerns and may lead to variable results, and they lack representation of metabolic processes, which can result in the over- or underestimation of a chemical’s endocrine activity. We addressed both aspects by developing a simple, cost-effective FBS-free medium for long-term culture of the cell lines ERα-CALUX® and AR-CALUX® and by inte­grating phase I mammalian metabolism by including S9-homogenates (rat-S9, human S9) or their animal-free alternatives (ewoS9R and ewoS9H) into test procedures. Both OECD test procedures could be adapted to FBS-free conditions, yielding similar results to the FBS-containing assays, though some of the assessed test compounds were only metabolized in FBS-free or FBS-containing medium. Rat- and human S9 metabolized most of the compounds assessed, whereas ewoS9R and ewoS9H transformed fewer substances and thus do not yet represent equivalent alternatives. The maximum changes in the biologically equivalent concentrations of 296-fold bioactivation and 1,540-fold detoxification indicate that including metabolism can make a substantial difference for risk assessment. Overall, our study demonstrates that OECD TG 455 and 458 can be performed under FBS-free conditions and with S9-homogenates of different origins, enabling endocrine activity testing using fewer or no animal components while improving the prediction of the in vivo activity.

Grouping and read-across are applied to human health toxicity classification of metal substances under EU REACH to reduce animal testing. Metal ion release is responsible for the systemic (and often local) toxicity of metal-containing materials. Metal release in simulated gastric fluid is relevant to the oral route of exposure. In 2010, the metals industry initiated the development of a method to assess relative metal release in HCl pH 1.5 from different substances of the same metal, including metal-containing materials such as massive and powder forms of alloys, metals, inorganic metal compounds, and other inorganic complex metal-containing materials. These data could also identify matrix effects in complex materials that increase or decrease metal release compared to what could be predicted from the content of the classified ingredients. The method underwent a round robin and multiple reviews over the past 15 years (e.g., by EURL ECVAM, ESAC and OECD), leading to revi­sions of the protocol. This paper provides a history of the protocol’s evolution and details supporting studies and revisions made to address the comments and concerns of its reviewers. The 2025 version of the protocol (publicly available via TSAR) significantly improved the clarity and robustness of the method without changing the way in which data are generated. The method is relevant to the oral route and is suitable for supporting grouping and read-across of metal-containing materials and refining the classification of metal materials showing a matrix effect, which might contribute to reducing animal testing.

The OECD’s test guidelines programme provides internationally recognized standard methods for non-clinical health and environmental safety testing of chemicals. They act as a harmonization mechanism and can support the replacement, reduction and refinement of regulatory tests using animals. We reviewed the reports of 300 repeated dose tests using rats conducted for chemical safety purposes to determine the extent to which current reduction- and refinement-related param­eters in TG 407 and 408 had been taken up. We found strong adherence to minimum group size and number of dose groups and strong adherence to the maximum dose level and dose volume. There was, however, very low uptake of the use of a limit test as a reduction method in both TGs, and some evidence of continued use of individual housing and larger than the minimum group sizes in the 28-day studies (TG 407). There are other opportunities to improve the TGs with respect to reduction and refinement, and we recommend that there is a systematic review by the OECD test guidelines programme to ensure their guidelines comply with the aims of the 3Rs.

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.