Toxicologic Pathology

The design and execution of toxicology studies supporting vaccine development have some unique considerations relative to those supporting traditional small molecules and biologics. A working group of the Society of Toxicologic Pathology Scientific and Regulatory Policy Committee conducted a review of the scientific, technical, and regulatory considerations for veterinary pathologists and toxicologists related to the design and evaluation of regulatory toxicology studies supporting vaccine clinical trials. Much of the information in this document focuses on the development of prophylactic vaccines for infectious agents. Many of these considerations also apply to therapeutic vaccine development (such as vaccines directed against cancer epitopes); important differences will be identified in various sections as appropriate. The topics addressed in this Points to Consider article include regulatory guidelines for nonclinical vaccine studies, study design (including species selection), technical considerations in dosing and injection site collection, study end point evaluation, and data interpretation. The intent of this publication is to share learnings related to nonclinical studies to support vaccine development to help others as they move into this therapeutic area.

*This Points to Consider article is a product of a Society of Toxicologic Pathology (STP) Working Group commissioned by the Scientific and Regulatory Policy Committee (SRPC) of the STP. It has been reviewed and approved by the SRPC and Executive Committee of the STP but does not represent a formal best practice recommendation. The views expressed in this article are those of the authors and do not necessarily represent the policies, positions, or opinions of their respective agencies and organizations. Readers of Toxicologic Pathology are encouraged to send their thoughts on these articles or ideas for new topics to the editor.

Toxicologic pathology is transitioning from analog to digital methods. This transition seems inevitable due to a host of ongoing social and medical technological forces. Of these, artificial intelligence (AI) and in particular machine learning (ML) are globally disruptive, rapidly growing sectors of technology whose impact on the long-established field of histopathology is quickly being realized. The development of increasing numbers of algorithms, peering ever deeper into the histopathological space, has demonstrated to the scientific community that AI pathology platforms are now poised to truly impact the future of precision and personalized medicine. However, as with all great technological advances, there are implementation and adoption challenges. This review aims to define common and relevant AI and ML terminology, describe data generation and interpretation, outline current and potential future business cases, discuss validation and regulatory hurdles, and most importantly, propose how overcoming the challenges of this burgeoning technology may shape toxicologic pathology for years to come, enabling pathologists to contribute even more effectively to answering scientific questions and solving global health issues.

*This article is a product of a Special Interest Group of the Society of Toxicologic Pathology (STP). The views expressed in this article are those of the authors and do not necessarily represent the policies, positions, or opinions of the STP.

Educational activities and training opportunities in toxicologic pathology are major goals of 9 formally established Toxicologic Pathology Societies and the International Academy of Toxicologic Pathology. Some Toxicologic Pathology Societies have examination-based certification programs while others accept certification or registration by veterinary pathology organizations including the American College of Veterinary Pathologists, the European College of Veterinary Pathologists. We summarize here the membership numbers and current activities of formally established Toxicologic Pathology Socities.

Significant advances in immunotherapies have resulted in the increasing need of predictive preclinical models to improve immunotherapeutic drug development, treatment combination, and to prevent or minimize toxicity in clinical trials. Immunodeficient mice reconstituted with human immune system (HIS), termed humanized mice or HIS mice, permit detailed analysis of human immune biology, development, and function. Although this model constitutes a great translational model, some aspects need to be improved as the incomplete engraftment of immune cells, graft versus host disease and the lack of human cytokines and growth factors. In this review, we discuss current HIS platforms, their pathology, and recent advances in their development to improve the quality of human immune cell reconstitution. We also highlight new technologies that can be used to better understand these models and how improved characterization is needed for their application in immuno-oncology safety, efficacy, and new modalities therapy development.

Reticulum cell hyperplasia (RCH) was a term used for many years by the National Toxicology Program (NTP) to describe a certain non-neoplastic bone marrow lesion of rats. Retrospective microscopic evaluation of RCH lesions and immunohistochemistry analyses were performed to reassess and further characterize these lesions. The NTP database was searched to identify femoral bone marrow specimens diagnosed with RCH from 1981 to 2014 (n = 254). The diagnosis last occurred in 2003, after which the term “cellular infiltration” was used. Eighty-three RCH slides, spanning 22 years, representing 34 different chemicals, were selected for microscopic review, and a subset (23) was chosen for ionized calcium binding adapter molecule 1 (Iba1) immunohistochemical staining; initial investigations revealed Iba1 worked as a macrophage marker on decalcified tissue. The following diagnoses were made upon reevaluation: 36 were consistent with cellularity increased, macrophage, 22 with histiocytic sarcoma, 8 with increased myeloid cells, 4 with autolysis, and 13 were normal appearance. All 23 RCH lesions stained positive for Iba1. Fifty-eight of 83 bone marrows previously diagnosed with RCH are consistent morphologically and immunohistochemically with cells of histiocytic origin. These results will help with interpretation of historical data and demonstrates that Iba1 can be used in decalcified bone marrow sections.

Mice exposed to the air pollutant ozone develop eosinophilic rhinitis that is mediated by group 2, GATA-3+, innate lymphoid cells (ILC2s). In the present study, we determined the influx, persistence, and recall of nasal ILC2s and eosinophils in ozone-exposed mice. C57BL/6 (T/B cell sufficient, ILC sufficient), Rag2^−/− (T/B cell deficient, ILC sufficient), and Rag2^−/−Il2rg^−/− (T/B cell deficient, ILC deficient) mice were exposed to 0 or 0.8 ppm ozone for 1 or 9 weekdays and killed 1 or 17 days postexposure. GATA-3+ lymphocytes were sparse in nasal tissue of air-exposed ILC-sufficient mice and absent in ILC-deficient mice. Nine-day, but not 1-day, ozone exposures induced nasal influxes of eosinophils and GATA-3+ lymphocytes in C57BL/6 and Rag2^−/− mice but not in Rag2^−/−Il2rg^−/− mice. Eosinophils waned 17 days postexposure in ILC-sufficient strains of mice. GATA-3+ lymphocytes in C57BL/6 mice also attenuated after exposure but not in ILC-sufficient Rag2^−/− mice. Eosinophils, but not GATA-3+ cells, increased rapidly with reexposure in ILC-sufficient mice. Type 2 immune-related messenger RNA expression correlated with cellular responses to ozone. These new findings in mice further elucidate the role of ILC2s in ozone-induced eosinophilic rhinitis and support epidemiologic associations between ozone exposure and eosinophilic inflammation in children.

Human exposure to pentabromodiphenyl ether (PBDE) mixture (DE-71) and its PBDE-47 congener can occur both in utero and during lactation. Here, we tested the hypothesis that PBDE-induced neonatal hepatic transcriptomic alterations in Wistar Han rat pups can inform on potential toxicity and carcinogenicity after longer term PBDE exposures. Wistar Han rat dams were exposed to either DE-71 or PBDE-47 daily from gestation day (GD 6) through postnatal day 4 (PND 4). Total plasma thyroxine (T4) was decreased in PND 4 pups. In liver, transcripts for CYPs and conjugation enzymes, Nrf2, and ABC transporters were upregulated. In general, the hepatic transcriptomic alterations after exposure to DE-71 or PBDE-47 were similar and provided early indicators of oxidative stress and metabolic alterations, key characteristics of toxicity processes. The transcriptional benchmark dose lower confidence limits of the most sensitive biological processes were lower for PBDE-47 than for the PBDE mixture. Neonatal rat liver transcriptomic data provide early indicators on molecular pathway alterations that may lead to toxicity and/or carcinogenicity if the exposures continue for longer durations. These early toxicogenomic indicators may be used to help prioritize chemicals for a more complete toxicity and cancer risk evaluation.

As ovarian toxicity is often a safety concern for cancer therapeutics, identification of ovarian pathology is important in early stages of preclinical drug development, particularly when the intended patient population include women of child-bearing potential. Microscopic evaluation by pathologists of hematoxylin and eosin (H&E)–stained tissues is the current gold standard for the assessment of organs in toxicity studies. However, digital pathology and advanced image analysis are being explored with greater frequency and broader applicability to tissue evaluations in toxicologic pathology. Our objective in this work was to develop an automated method that rapidly enumerates rat ovarian corpora lutea on standard H&E-stained slides with comparable accuracy to the gold standard assessment by a pathologist. Herein, we describe an algorithm generated by a deep learning network and tested on 5 rat toxicity studies, which included studies that both had and had not previously been diagnosed with effects on number of ovarian corpora lutea. Our algorithm could not only enumerate corpora lutea accurately in all studies but also revealed distinct trends for studies with and without reproductive toxicity. Our method could be a widely applied tool to aid analysis in general toxicity studies.

Daprodustat (GSK1278863) is a hypoxia-inducible factor (HIF)-prolyl hydroxylase (PHD) inhibitor in development for treatment of anemia of chronic kidney disease. Daprodustat’s biological activity simulates components of the natural response to hypoxia; inhibition of PHDs results in HIF stabilization and modulation of HIF-controlled gene products, including erythropoietin. The carcinogenic potential of daprodustat was evaluated in 2-year carcinogenicity studies in Sprague-Dawley rats and CD-1 mice, where once-daily doses were administered. The mouse study also included evaluation of daprodustat’s 3 major circulating human metabolites. There were no neoplastic findings that were considered treatment related in either study. Exaggerated pharmacology resulted in significantly increased red cell mass and subsequent multiorgan congestion and secondary non-neoplastic effects in both species, similar to those observed in chronic toxicity studies. In rats, these included aortic thrombosis and an exacerbation of spontaneous rodent cardiomyopathy, which contributed to a statistically significant decrease in survival in high-dose males (group terminated in week 94). Survival was not impacted in mice at any dose. Systemic exposures (area under the plasma concentration–time curve) to daprodustat at the high doses in rats and mice exceed predicted maximal human clinical exposure by ≥143-fold. These results suggest that daprodustat and metabolites do not pose a carcinogenic risk at clinical doses.

Pigs are used as a model of human obesity, both for metabolic characterization and for evaluation of pharmacological interventions. Over a period of 7 years, acute death or clinical signs requiring immediate euthanasia were observed in 12 obese Göttingen minipigs (GMs) included in different pharmacological studies. The GM were fed ad libitum on normal chow-diet and the unscheduled deaths occurred in animals treated with drug candidates as well as in untreated animals. The most prominent clinical signs requiring euthanasia included varying degrees of respiratory distress; and on histopathological examination, thickening of the alveolar septa due to vacuolation was observed throughout the lung in 10 of the 12 animals. Furthermore, vacuolation in glomeruli of the kidney was detected in 9 of the 10 animals. Oil red O staining of cryosections demonstrated that the vacuoles both in lung and kidney contained lipid, and immunohistochemistry with anti-von Willebrand factor and transmission electron microscopy revealed that the lipid was localized in the lumen of blood vessels establishing the occurrence of fatal pulmonary lipid embolism. Additionally, lipogranulomatous inflammation in the abdominal adipose tissue was observed in all the GMs with lipid emboli.