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.
Despite ethical concerns and scientific drawbacks, fetal bovine serum (FBS) remains a common supplement of culture media for continuous cancer cell lines. Although FBS alternatives like human platelet lysate (hPL) and animal component-free, chemically defined media (CDM) have been commercially available for many years, acceptance of alternative media is limited, as data verifying the stability of the phenotype and function of a cell line cultured using alternative media are often lacking. Here, we adapted four widely used human cancer cell lines (HELA, HL-60, JIMT-1, K-562) to hPL-supplemented media and different CDM. To evaluate the FBS-free replacements in comparison to FBS-containing media, we systematically analyzed the cultures with respect to recovery after cryopreservation, short tandem repeat (STR) profile stability, proliferation, morphology, and transcriptomic alterations. Neither changes in STR profiles nor difficulties after cryopreservation were found. Except for K-562, FBS-free cultures showed a reduced proliferation rate and, in some conditions, slight morphological alterations in comparison to cultures with FBS. In all cell lines, gene set enrichment analyses revealed that culture media mainly affected expression of cholesterol homeostasis genes. In HELA and JIMT-1, media also influenced genes of epithelial-mesenchymal transition; nevertheless, their overall phenotypic hallmarks remained stable. Only a few differentiation markers were among the differentially expressed genes of HL-60 and K-562 cultures, while their main phenotypes remained unchanged. This was further confirmed by successful induction of differentiation of HL-60 in FBS-free media. In conclusion, our multiparametric approach provides strong evidence supporting the transition to FBS-free media for long-established cancer cell lines.
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.
Since 2023 the European Commission has been working on a roadmap for phasing out animal testing in chemical safety assessment, and in 2025 the US FDA published a roadmap to phase out animal testing for the pharmaceutical industry. We describe our Merck KGaA strategy across Life Science, Healthcare, and Electronics, introduced in 2021, to reduce animal testing by 50% by 2032 and by 75% by 2040 through our 4R program (Replace, Reduce, Refine, Responsibility). The approach focuses on what we can achieve today and avoids obstructive discussions about unresolved issues. We have categorized all animal tests currently required for our products into three "baskets" (3B). Basket 1 (Adoption) includes animal tests for which alternatives are available and accepted, including those still required in certain regions. Basket 2 (Adaptation) contains tests for which alternatives are proposed or being developed but that cannot yet be replaced. Basket 3 (Assessment) contains tests for which innovative replacement strategies still need to be developed. This strategy guides effective short-term replacement and directs investments into areas where innovation can replace animal testing in the future. It coordinates the way forward for all stakeholders and creates actionable milestones toward a genuine replacement of animal testing. The collaborative agreement in 2024 among the members of the European Federation of Pharmaceutical Industries and Associations to adopt the 3B approach, recognized by the European Commission and the European Medicines Agency, highlights its significance as a valuable tool for fostering a more ethical and sustainable science environment across Europe.
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.
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.
The whole blood pyrogen test was first described in this journal exactly thirty years ago. Its variant based on cryopreserved blood followed one year later. Together with other monocyte activation tests (MATs), it has fundamentally changed the landscape of pyrogen testing. In the five years since the 25th anniversary article in this series, progress has been remarkable: The European Pharmaco-poeia deleted the rabbit pyrogen test (RPT) effective January 2026, ending a 55-year era; ISO 10993-1:2025 removed material-mediated pyrogenicity from mandatory evaluation endpoints for medical devices; the U.S. FDA updated its guidance on pyrogen testing; and the MAT market grew to over $600 million. New validation studies have demonstrated MAT equivalence to the RPT for both endotoxin and non-endotoxin pyrogens, and the first product-specific MAT validations have been accepted in regulatory filings in Europe and the United States. Reporter cell lines and tran-scriptomic approaches are opening next-generation detection capabilities. Yet implementation gaps persist: The MAT is still underutilized for blood transfusions, cell therapies, and airborne pyrogens. Recombinant alternatives to the Limulus amebocyte lysate assay (LAL) have finally achieved phar-macopeial recognition, addressing horseshoe crab conservation concerns. This article reviews the developments of the last five years, updates the lessons learned, and reflects on three decades of bringing a human cell-based test from the laboratory bench to global regulatory acceptance.
Bisphenol A is a high production volume chemical used extensively in the manufacture of polycarbonate plastics, epoxy resins, and thermal printer paper with a high potential for occupational and post-production dermal exposure. Bisphenol A-containing plastics were commonly used in food packaging, resulting in significant public exposure through leaching into foodstuff. The public is also at risk of dermal exposure due to environmental contamination. Due to public health concerns regarding the potential for endocrine disrupting effects, efforts have been applied to replace bisphenol A with safer alternatives. Bisphenol A has been shown to cause skin sensitization in humans; however, there is a paucity of information available on the sensitizing potential of the structural analogues which are increasingly being employed as substitutes. We utilized new approach methodologies (NAMs) addressing key events 1-3 of the adverse outcome pathway for skin sensitization to assess the potential of bisphenol A substitutes to induce dermal sensitization. Defined approaches (DA) were applied to further classify and categorize potency according to OECD TG 497. The NAMs and DAs confirmed that bisphenol A was a skin sensitizer in potency category UN GHS 1B. Bisphenol B, AP, and E were also classified as UN GHS 1B sensitizers, and bisphenol AF as UN GHS 1A/1B depending on the DA, while 2,4-bisphenol S and F were borderline sensitizers, and bisphenol S was classified as a non-sensitizer. These data provide evidence of skin sensitization hazard for the bisphenol structural analogues tested, except for bisphenol S, suggesting that they present risks for dermal allergy.
According to ISO 10993-23 (Biological evaluation of medical devices – Part 23: Tests for irritation, Annex D2), no specific alternative test method has yet been established to identify substances that may cause eye irritation or serious eye damage in medical devices. Therefore, the in vivo animal test OECD TG 405 continues to be used. This study adapted the in vitro eye irritation test (EIT) using the MCTT HCE™ Reconstructed Human Corneal Epithelial (RHCE) model, which was originally developed for testing chemicals, to the testing of ophthalmic medical devices such as contact lenses and intraocular lenses. A validation study, carried out in accordance with OECD GD 34, aimed to confirm transferability (2 produced medical devices and 2 spiked extract vehicles), proficiency (4 produced medical devices and 3 spiked extract vehicles), and reproducibility (8 produced medical devices and 9 spiked extract vehicles). The results demonstrated a combined predictive capacity of 89.6% sensitivity, 87.3% specificity, and 88.4% accuracy based on 285 runs. Specifically, the test showed 85.7% sensitivity, 100% specificity, and 92.9% accuracy for classifying produced medical devices (140 runs) and 93.8% sensitivity, 76.3% specificity, and 84.1% accuracy for classifying solutions spiked with chemicals before extraction (145 runs). These findings confirm the reliability of the “MCTT HCE™ EIT for medical devices” in assessing the irritation potential of medical device extracts, including the detection of low concentrations of potent irritants.
The endothelial cells of the capillary network in the brain, in conjunction with pericytes and astrocyte end feet, form the blood-brain barrier (BBB). This highly selective cellular barrier helps to protect and maintain brain homeostasis. This barrier function must be circumvented to deliver therapeutics to the brain, a growing need owing to the increasing incidence in neurological disorders in progressively aging populations. Animal models may exhibit species-specific differences in response, ultimately leading to problems with predicting efficacy of treatments in humans, and conventional in vitro models of the BBB face challenges of reproducing cell-cell interactions, the human brain microenvironment, and transporter abundance and type. Microphysiological systems (MPS) and other complex in vitro models aim to address these challenges by combining engineered shear forces coupled with cell biology advancements to create models with greater predictive power, thereby contributing towards the replacement, reduction and/or refinement of animal use (3Rs). Here, we provide an industry perspective on the application of MPS to test drug delivery to the brain, including on newer modalities such as targeted protein degraders, biologics, nanoparticles, and viruses. We also highlight important considerations for MPS model qualification centered on cell sourcing, technological platform, and qualification of functionality. This commentary from members of the Innovation & Quality Microphysiological Systems Affiliate also aims to highlight areas where developers and suppliers can help address gaps in model development.
OECD Guideline 497 includes defined approaches (DAs) that combine new approach methodologies (NAMs) to predict skin sensitization hazard and categorize potency according to the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS). To increase flexibility, the OECD allows the substitution in the DAs of NAMs addressing the same key events (KEs) in the adverse outcome pathway (AOP). This study evaluated the implementation of the U-SENS™ assay for dendritic cell activation (KE3) within two integrated testing strategy (ITS) DAs. First, EC150 threshold values were determined via computational modeling to convert CD86 stimulation index data into ITS DA scores. The performance of the modified ITS DAs was then evaluated against available Local Lymph Node Assay (LLNA) and human reference data. For hazard identification, the ITS DAs achieved 79% balanced accuracy compared with LLNA data, and 73-74% compared with human data. For potency categorization, correct classification rates were 67-68% against LLNA and 65-67% against human data. In comparison, the LLNA showed lower performance, with 58% balanced accuracy for hazard identification and 60% for potency categorization against human data. Selected case studies illustrate the practical application of these DAs following the Guideline 497 decision flowchart. Overall, findings underscore that incorporating U-SENS™ into ITS DAs enhances flexibility without compromising predictive capacity and outperforms traditional animal testing in providing reliable skin sensitization classification outcomes.
