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.

Mice as model organisms play a vital role in translational research for developmental biology by aiding our understanding of embryonic and placental evolution in health and various disease states. The close similarity between mouse and human placentas is a great asset in reproductive translational research, making the mouse one of the more prominent model organisms in this field. Mice are routinely used to investigate mechanisms of embryonic and placental development, often through experimental manipulations involving engineered or spontaneous genetic mutations and/or xenobiotic treatments. A crucial aspect of fully appreciating the mouse as a model organism for placental research is understanding the anatomy and physiology of the major embryonic (chorion, labyrinth, junctional zone, and yolk sac) and maternal (decidua basalis and metrial gland) placenta components, including knowledge of the specific cell types active during each gestational stage. This paper discusses histochemical and immunohistochemical (IHC) methods that highlight specific tissue layers and cell types present during normal development of the mouse placenta and maternal metrial gland. Altered numbers and/or locations of these common cell types have been demonstrated in embryonic lethal phenotypes, indicating that placental assessments must examine not only the tissue organization but also affected cell populations when determining the cause of early pregnancy loss.

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.