In April 2025, the US Food and Drug Administration (FDA) introduced a bold roadmap to phase out animal testing in drug development by promoting the integration of New Approach Methodologies (NAMs), beginning with monoclonal antibodies (mAbs). Similar roadmaps have also been created by European health authorities and chemical safety agencies around the globe. This opinion paper from the European Society of Toxicologic Pathology and US Society of Toxicologic Pathology complex in vitro model and pathology working groups provides a balanced and expert perspective on the promise and limitations of these initiatives. NAMs, including in vitro, in silico, and refined in vivo models, offer exciting opportunities to improve human relevance, efficiency, and ethical standards in nonclinical safety assessment. While these approaches are already making meaningful contributions, current scientific and technical limitations prevent them from fully replacing animal studies in all contexts. At this time, NAMs should be viewed as powerful complementary tools whose appropriate integration depends on the context of use, validation, and continued collaboration across disciplines. Pathologists and investigative molecular pathology leaders are uniquely positioned to support this transition by bridging traditional methods with emerging technologies to enhance the safety and effectiveness of future therapeutics.
Drug-induced taste dysfunctions are an underrecognized yet clinically significant class of adverse effects that can substantially impair patients’ quality of life and compromise adherence to therapeutic regimens. Despite the potential for many pharmacologic agents to alter taste perception, these effects are frequently overlooked in both clinical practice and drug development. Gustatory disorders are rarely assessed in toxicology studies, and current knowledge regarding the pathophysiology of dysgeusia, the relevant organ systems involved, and nonclinical evaluation methods remains limited. This review details the anatomy of the gustatory system including taste buds and papillae, the functional characteristics of taste cells, the molecular mechanisms of taste transduction, and the neural components (peripheral and central). We emphasize the multimodal nature of flavor perception encompassing gustatory, olfactory and somatosensory systems, including the role of saliva in modulating flavor. Furthermore, we propose a comprehensive framework for nonclinical histopathological assessment of the key organ systems involved in flavor perception, with relevant case examples. By advancing awareness and providing optimized approaches for gustatory system evaluation in nonclinical toxicology studies, we aim to enable earlier detection and mechanistic investigation of taste-related adverse effects and improve safety profiling during drug development.
An unsupervised, single-class anomaly detection approach based on a vision transformer architecture was developed to aid histopathology evaluation of rat liver from nonclinical toxicology studies. The approach was designed to detect any histopathology finding without requiring specific histopathology examples for training. The model was developed by training a vision transformer with approximately 135,000 whole slide images (WSIs) and then extracting high-dimensional feature representations from 750 unremarkable vehicle control WSIs, creating a rat liver histomorphology reference dataset representing typical biological variability. Each test WSI was compared against the reference dataset at the tile level, and the resulting anomaly detections were aggregated to derive an overall image-level anomaly score. Anomaly scores were higher in affected groups treated with established toxicants than in control groups, and based on comparing anomaly scores at the individual animal level with pathologist diagnoses, model sensitivity was 95.9% and specificity was 70.8%. Utility as a pathologist aiding tool was further studied by comparing tile-level anomaly annotations with diagnosis. For one treated group, histomorphology feature extraction was used to explore discordance between anomaly detection and diagnosis. Specificity was impacted by pre-analytical variables as well as detection of anomalies not considered noteworthy by pathologists.
Mononuclear cell infiltrates (MCIs) and inflammation in the central nervous system (CNS) can be challenging in nonclinical studies, particularly in nonrodent species. A retrospective study was performed to determine the incidences of spontaneous MCI and inflammation in the brain and spinal cord of rodent and nonrodent species considering age (rodents), administration routes and geographical origin (primates). Historical control data (HCD) were collected from rats, mice, beagle dogs, Göttingen minipigs, cynomolgus macaques, and New Zealand White (NZW) rabbits. Perivascular MCI in the brain (choroid plexus and meninges most frequently affected) had the highest incidence in cynomolgus macaques (8.25% males/7.38% females), followed by Göttingen minipigs (3.23% males/3.27% females), beagle dogs (3.18% males/3.14% females). CD-1 mice, Sprague-Dawley rats, Han Wistar rats and NZW rabbits were the species with the least commonly identified perivascular MCI in the brain. Brain inflammation was a rare event. Perivascular MCI in the spinal cord (meninges most frequently affected) had the highest incidence in cynomolgus macaques, Göttingen minipigs, and beagle dogs. This HCD review will help in the interpretation of inflammatory CNS findings in toxicological safety studies and should allow discrimination of spontaneous and induced lesions in nonclinical studies.
Environmental toxicants are increasingly implicated in male reproductive disorders, highlighting the need for physiologically relevant in vitro testicular models for toxicological testing. We previously developed a testis organoid system that recapitulates key architectural and endocrine features of native tissue; however, its suitability for toxicological applications requires validation. In this study, cadmium, a well-characterized environmental heavy metal and testicular toxicant, was used as a positive control to evaluate model responsiveness. Testis organoids were exposed to increasing concentrations of cadmium (0.01, 1, 10, and 100 μM) to assess dose-dependent toxicological effects. Cadmium bioaccumulation within organoids was quantified, and multiple endpoints were evaluated, including testis cell assembly, cord formation, apoptosis/necrosis, germ cell number, DNA damage, cellular proliferation, and endocrine function. Cadmium accumulated in a concentration-dependent manner, with greater accumulation at 10 and 100 μM. At these concentrations, cadmium exposure reduced cord formation, germ cell numbers, and cellular proliferation, and decreased testosterone and estradiol production, while increasing apoptosis/necrosis and DNA damage. Disruption of testis cell assembly was observed only at 100 μM. Collectively, these findings demonstrate dose-dependent toxicological responses and support the use of this testis organoid system as a relevant in vitro model for reproductive toxicology and environmental toxicant evaluation.
RC-0315 is a chemotherapy-exposed mesenchymal stem cell-derived secretome developed as a potential advanced therapy for idiopathic pulmonary fibrosis (IPF). A Good Laboratory Practice (GLP)-compliant repeated-dose toxicity study was conducted to evaluate the safety of RC-0315 following intratracheal (ITR) instillation in immunocompetent ICR and SCID mice. Animals received two cycles of three repeated ITR instillations of RC-0315 performed within a 7-day period and separated by a two-week interval. Mice were assigned to saline, vehicle, low-dose, or high-dose groups and were monitored for 3 days (main phase) or 13 weeks (recovery phase) post last administration. No test article-related mortality or toxic clinical signs were observed in either strain. Clinical pathology parameters, body weight, food consumption, and ophthalmologic findings remained within normal limits, with no dose-dependent alterations. Histopathological examination revealed no RC-0315-related adverse findings in the lungs or other organs, and no local toxicity was evident at the site of administration. These findings support the favorable safety profile of RC-0315 when administered via the clinically intended route.
The impact of non-fasting on clinical pathology parameters in pharmacologic safety assessment has been incompletely characterized. The aim of this study was to characterize changes in hematology, coagulation, and clinical chemistry parameters between healthy fasted and fed rats and macaques. Adult Sprague-Dawley rats (41 males, 40 females) and cynomolgus macaques (10 per sex) were sampled after free access to food and after 8 hours of fasting. Fed rats demonstrated lower reticulocyte and neutrophil counts in males only. Higher alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase activities and higher glucose, triglyceride, cholesterol, and globulin concentrations were observed with lower urea nitrogen, albumin, and phosphorus concentrations in fed compared with fasted rats of both sexes. Fed macaques of both sexes demonstrated higher triglyceride concentrations and lower phosphorus and total bilirubin concentrations. All changes in fed compared with fasted rats and macaques were considered minor. Non-fasting did not impact coagulation parameters in rats and macaques or hematology parameters in macaques. This study supports that non-fasting has a minimal impact on standard clinical pathology parameters in Sprague-Dawley rats and cynomolgus macaques; however, fasting is still recommended when there are anticipated effects on glucose and lipid metabolism or on endocrine systems related to energy metabolism.
Timely tissue fixation is critical to prevent autolysis, which confounds histopathologic assessment. This study systematically mapped autolysis progression in ex vivo rat tissues under room temperature and refrigerated (4°C) storage conditions to establish an evidence-based, tissue-specific timeline for practical guidance. Thirty-six Sprague-Dawley rats were euthanized; major organs were rapidly collected and maintained in sterile saline at either room temperature or 4°C for intervals ranging from 0.5 to 16 hours before fixation in 10% neutral buffered formalin and H&E evaluation. The data provide a clear timeframe of autolytic change in rat tissues. At room temperature, most tissues exhibited minimal autolysis within the first 2 hours, and refrigeration at 4°C extended the time to autolysis. Immunohistochemistry for the endothelial marker CD31 suggested that CD31 antigenicity remained detectable in most tissues maintained at room temperature out to 16 hours with the exception of intestinal tissues demonstrating severe autolysis, indicating that advanced autolysis can compromise immunohistochemical interpretation. In conclusion, this work provides guidance for handling tissues that require temporary preservation in saline. The documented tissue-specific timelines serve as a valuable reference for pathologists.
The second session of the 2nd Joint BSTP/ESTP Toxicologic Pathology Congress, Manchester, UK, September 23-26, 2025, entitled “New Approach Methodologies for Carcinogenicity Evaluation,” was dedicated to innovative strategies for assessing carcinogenic risks in various substances, particularly in drug development and agrochemicals. The two-year rodent cancer bioassay in two species (generally rats and mice) is currently the standard method for assessing the carcinogenic potential of agrochemicals for humans. However, this method has some weaknesses and is subject to ethical and scientific debate. Attempts to waive those studies have been proposed, but more relevant methods using less or preferably no animals are still being sought. The session featured five key presentations that explored cutting-edge methodologies aimed at improving the accuracy and reliability of carcinogenicity predictions. Below is a summary of each talk presented.
The International Academy of Toxicologic Pathology (IATP) Satellite Symposium on Pathology Working Groups (PWGs) in Toxicologic Pathology, organized in the United Kingdom, addressed the value and importance of PWGs and expert working groups in assuring the accurate evaluation of chronic/carcinogenicity studies. These specialized panels of expert pathologists play a crucial role in confirming data quality and accurate interpretation in support of human safety and risk decisions of xenobiotics. While not every carcinogenicity bioassay may require a PWG to evaluate the quality and accuracy of study interpretation, it was the consensus of the workshop that it can be a highly valuable addition to traditional peer review. Performing a PWG should be given serious consideration, especially when it is anticipated that the chronic/carcinogenicity study will be used for making significant regulatory or business decisions.
Emerging modalities in drug development including adeno-associated virus (AAV) and other viral-mediated gene therapy, targeted protein degraders, oligonucleotide, and chimeric antigen receptor-T cell (CAR-T) therapies require updated approaches in strategy and context for meaningful human carcinogenicity risk assessment. This may involve, but is not be limited to, traditional genetic toxicology and rodent bioassays. Speakers provided unique perspectives on these topics as well as an applicable and real-world case study that interrogated whether rat mammary gland cell proliferation was a useful tool with which to evaluate the carcinogenic potential of exogenous insulin.
Urethane has been used as a positive control material for carcinogenicity studies in the TgrasH2 mouse that is a valuable model in pre-clinical safety investigations. This article describes the incidence of hemangiosarcomas (HSAs) in the intercostal sternal brown adipose tissue that is routinely collected along with the sternum following the administration of 1000 mg/kg urethane to a total of 230 TgrasH2 mice (115 mice/sex). We report that HSAs were observed in 27/115 (23.48%) males and 17/115 (14.78%) females in the intercostal brown adipose tissue of the sternum, which was the third most common tissue in which these findings were observed, following the spleen (100/115 [86.96%]) in males and (90/115 [78.26%]) in females and lungs (30/115 [26.09%]) in males and (23/115 [20.00%]) in females. The rare investigations of HSAs in brown adipose tissue highlight unusual locations where the elevated incidence of HSA is observed, which is important when performing histopathological evaluation in the context of a pre-clinical carcinogenicity study.
Adeno-associated virus (AAV) gene therapy has received approvals for the treatment of a number of rare monogenic disorders. There are questions surrounding the natural history of recombinant AAV (rAAV) vectors, which could provide insights into durability and long-term safety. Wild-type AAV (wtAAV) persists in extrachromosomal episomes and is also able to actively or passively integrate into the host genome. Although clonal wtAAV integration has been described in a small number of hepatocellular carcinoma cases, this at most represents a minor risk factor. Differences in the structure of wtAAV and rAAV limit translation of these studies. Recombinant AAV also persists in episomal forms with a small proportion becoming integrated. Some early murine studies demonstrated insertional mutagenesis after treatment with rAAV. These findings have not consistently been seen and are likely context-dependent (high-dose or neonatal treatment). There have been no reports of rAAV insertional mutagenesis in large animal models or human biopsy samples.
Imatinib, a tyrosine kinase inhibitor that targets BCR-ABL, platelet-derived growth factor receptors (PDGFRs), and c-KIT, is known to cause rare ovarian hemorrhagic events. This study investigated the mechanisms underlying imatinib-induced hemorrhage in the corpus luteum by comparing normal, pseudopregnant, and hypophysectomized rats. While normal rats treated with imatinib exhibited significant luteal cyst with hemorrhage, this effect was markedly attenuated in pseudopregnant and hypophysectomized rats, whose estrous cycles were halted. Immunohistochemical analysis revealed that imatinib reduced the ratio of α-SMA-positive pericytes to CD31-positive endothelial cells in the corpus luteum of normal rats, but not in the other groups. These findings suggest that the susceptibility of rats to imatinib-induced hemorrhage is linked to active angiogenesis in the early luteal phase. In contrast, resistance is conferred by the stability of luteal capillaries in pseudopregnant and hypophysectomized rats with stable corpus luteum following arrest of the estrous cycle. This study provides new insights into the risk and mechanism of imatinib-induced luteal hemorrhage and suggests that the suppression of ovulation may mitigate this adverse effect in clinical use.
Exposure to certain per- and polyfluoroalkyl substances (PFAS) is associated with adverse pregnancy outcomes in mice and humans. These adverse outcomes are suspected to be due to exposure-related toxic effects on the placenta. This study was designed to more explicitly define the effects of selected PFAS on all regions of the placenta of pregnant CD-1 mice. Pregnant CD-1 mice were gavaged with perfluorooctanoic acid (PFOA; 1 or 5 mg/kg-d) or hexafluoropropylene oxide dimer acid (GenX; 2 or 10 mg/kg/day) from embryonic day (E) 1.5 to E17.5. Exposure to either PFOA or GenX resulted in higher placenta weights and lower placental efficiency (embryo:placenta weight ratio). Light microscopic evaluation of the placentae revealed treatment-associated lesions in the decidua, junctional zone, and labyrinth. The reduced placental efficiency and microscopic findings were consistent with maternal vascular malperfusion. With few exceptions, there were no differences between the effects of PFOA when compared with GenX, except for increased necrosis of spongiotrophoblasts in the high-dose PFOA-treated group. Taken together, these findings suggest both PFOA and GenX cause multiple pathological changes at the microscopic and tissue level of the placenta consistent with adverse maternal-fetal health outcomes.
Science is a lodestar in the search for knowledge to successfully steer societies through the stormy seas of a changeable world. Scientists are the navigators, using experiments and reason to find answers that expand basic knowledge and influence applications (e.g., public policymaking and risk assessment). An understanding of the scientific method generally and in-depth comprehension of a scientific field specifically are limited in many adults today, both among the public and in cultural and political leaders. Accordingly, scientists must vigorously defend science by upholding two core principles: (1) scientists must be free to ask and answer questions without concern for subjective cultural, ideological, and political constraints, and (2) the diverse perspectives of scientists based on their unique experiences are essential drivers of scientific innovation and attainment. Toxicologic pathologists have a responsibility to uphold these principles, whether their actions are directed to their institutions, the entire toxicologic pathology profession, or the scientific community and society. Concrete actions available to individual pathologists include communicating support for intellectual freedom of thought and association (orally or in writing) among colleagues or to the public as well as providing services (professional society governance, public political office, scientific advocacy, teaching, etc.) that maintain these principles.
Targeted protein degraders (TPDs) have recently emerged as a novel drug class. Targeted protein degraders engage E3 ubiquitin ligase complexes to degrade therapeutic proteins of interest via cereblon and other adapter proteins, acting as either molecular glue degraders (MGDs) or proteolysis-targeting chimeras (PROTACs). Several cereblon-based MGDs and PROTACs are in late-stage clinical development for oncology indications. However, as TPD drug discovery includes non-life-threatening indications, carcinogenicity risk assessment will be required. Although there is no regulatory requirement to treat TPDs differently from conventional small molecules in carcinogenesis risk assessment, several properties of TPDs could influence weight of evidence (WoE) assessments and carcinogenicity study design. A series of case studies is presented to provide examples for evolved, modified WoE approaches for carcinogenicity assessment that may be acceptable to health authorities and regulatory agencies. These examples also highlight that the biological assessment of E3 ligase is as critically important to the carcinogenicity and toxicology assessment as is the assessment of the primary target. Finally, drug developers must contend with limitations for early-generation cereblon MGDs related to the translatability of findings that may challenge traditional interpretation paradigms.
