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Nonhuman primates (NHPs) are utilized in nonclinical safety testing due to their phylogenetic proximity to humans and similarity in physiology and anatomy. However, ethical considerations and the increased demand for NHPs, coupled with the current shortage in their supply, have increased the calls to minimize their use. In addition, the increased demand and supply shortage of NHPs have increased the use of animals sourced from different geographical origins, and animals of different ages, which can complicate the interpretation of study results. Coupled with the relative uniqueness of findings induced by novel therapeutic modalities, there is an increasing need for a deeper understanding of the systemic pathobiology of NHPs. Here we provide a brief preview of the two main themes discussed in this special issue, which include the influence of geographical origin, age, and sex on background pathology, clinical pathology reference values, other relevant toxicology endpoints, and organ system pathology.

To investigate the influence of geographical origin, age, and sex on toxicologically relevant spontaneous histopathology findings in cynomolgus macaques ( Macaca fascicularis), we performed a comparative analysis of historical control data (HCD) from 13 test sites that included 3351 animals (1645 females and 1706 males) sourced from Mauritius, China, Vietnam, and Cambodia, aged from 2 to 9.5 years, and from 446 toxicology studies evaluated between 2016 and 2021. The most common findings were mononuclear infiltrates in the kidney, liver, brain, and lung, which showed highest incidences in Mauritian macaques, and heart, salivary glands, and gastrointestinal tract (GIT), which showed highest incidences of mononuclear infiltrates in mainland Asian macaques. Developmental and degenerative findings were more common in Mauritian macaques, while lymphoid hyperplasia and lung pigment showed higher incidences in Asian macaques. Various sex and age-related differences were also present. Despite origin-related differences, the similarities in the nature and distribution of background lesions indicate that macaques from all geographical regions are suitable for toxicity testing and show comparable lesion spectrum. However, in a toxicity study, it is strongly recommended to use animals from a single geographical origin and to follow published guidelines when using HCD to evaluate and interpretate commonly diagnosed spontaneous lesions.

Two similar benign, nonneoplastic vascular lesions have been described in the lymph nodes of humans and animals: angiomyomatous hamartoma (AMH), which is characterized by the replacement of lymphoid tissue by blood vessels, smooth muscle, and fibrous tissue, and vascular transformation of sinuses (VTS), which is considered a reactive transformation of lymph node sinuses into capillary-like vascular channels. We hereby report a lesion with features common to both lesions in the mediastinal lymph nodes of a 1-year-old beagle dog in a 1-month toxicity study. Grossly, enlargement and red discoloration were observed, while microscopically, the lesion was characterized by effacement of the lymph node parenchyma with replacement by mature blood vessels, smooth muscle, and fibrous tissue, associated with lymphoid atrophy, which is consistent with AMH. However, multifocal areas of anastomosing or plexiform capillary-like channels lined by normal to slightly plump endothelium, similar to those described for VTS, were also present. Immunohistochemistry analysis revealed abundant positive staining for smooth muscle actin and endothelial cells (von Willebrand factor/factor VIII) and the absence of proliferation (Ki67). In conclusion, these lesions most likely represent a mixture of both AMH and VTS.

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. [Box: see text]

To further our understanding of the nonhuman primate kidney anatomy, histology, and incidences of spontaneous pathology, we retrospectively examined kidneys from a total of 505 control Cynomolgus monkeys ( Macaca fascicularis; 264 male and 241 females) aged 2 to 6 years, from toxicity studies. Kidney weights, urinalysis, and kidney-related clinical biochemistry parameters were also evaluated. Although the functional anatomy of the monkey kidney is relatively similar to that of other laboratory animals and humans, a few differences and species-specific peculiarities exist. Unlike humans, the macaque kidney is unipapillate, with a relatively underdeveloped papilla, scarce long loops of Henle, and a near-equivalent cortical to medullary ratio. The most common spontaneous microscopic findings were interstitial infiltrates or interstitial nephritis and other tubular lesions, but several forms of glomerulopathy that may be interpreted as drug-induced were occasionally observed. Common incidental findings of little pathological significance included: papillary mineralization, epithelial pigment, multinucleate cells, cuboidal metaplasia of the Bowman’s capsule, and urothelial inclusions. Kidney weights, and some clinical chemistry parameters, showed age- and sex-related variations. Taken together, these data will aid the toxicologic pathologist to better evaluate the nonhuman primate kidney and assess the species’ suitability as a model for identifying and characterizing drug-induced injury.

We previously reported that a single immunization with an adenovirus serotype 26 (Ad26) vector-based vaccine expressing an optimized SARS-CoV-2 spike (Ad26.COV2.S) protected rhesus macaques against SARS-CoV-2 challenge. To evaluate reduced doses of Ad26.COV2.S, 30 rhesus macaques were immunized once with 1x1011, 5x1010, 1.125x1010, or 2x109 vp Ad26.COV2.S or sham and were challenged with SARS-CoV-2. Vaccine doses as low as 2x109 vp provided robust protection in bronchoalveolar lavage, whereas doses of 1.125x1010 vp were required for protection in nasal swabs. Activated memory B cells and binding or neutralizing antibody titers following vaccination correlated with protective efficacy. At suboptimal vaccine doses, viral breakthrough was observed but did not show enhancement of disease. These data demonstrate that a single immunization with relatively low dose of Ad26.COV2.S effectively protected against SARS-CoV-2 challenge in rhesus macaques, although a higher vaccine dose may be required for protection in the upper respiratory tract., Evaluation of a reduced dosage of the single-shot Ad26.COV2.S reveals protection across different tissues protects against SARS-CoV-2 challenge and enhancement of disease. A higher dosage may be needed for protection in the upper respiratory tract.

The European Society of Toxicologic Pathology organized an expert workshop in May 2018 to address adversity considerations related to thyroid follicular cell hypertrophy and/or hyperplasia (FCHH), which is a common finding in nonclinical toxicity studies that can have important implications for risk assessment of pharmaceuticals, food additives, and environmental chemicals. The broad goal of the workshop was to facilitate better alignment in toxicologic pathology and regulatory sciences on how to determine adversity of FCHH. Key objectives were to describe common mechanisms leading to thyroid FCHH and potential functional consequences; provide working criteria to assess adversity of FCHH in context of associated findings; and describe additional methods and experimental data that may influence adversity determinations. The workshop panel was comprised of representatives from the European Union, Japan, and the United States. Participants shared case examples illustrating issues related to adversity assessments of thyroid changes. Provided here are summary discussions, key case presentations, and panel recommendations. This information should increase consistency in the interpretation of adverse changes in the thyroid based on pathology findings in nonclinical toxicity studies, help integrate new types of biomarker data into the review process, and facilitate a more systematic approach to communicating adversity determinations in toxicology reports.

This paper presents a review of the nature, range, and incidences of background pathology findings in the respiratory tract of cynomolgus monkeys and rats. Data were collected from 81 inhalation studies and 133 non-inhalation studies evaluated at 3 geographically distinct contract research organization facilities. The inhalation studies were comprised of 44 different small molecule pharmaceuticals or chemicals which were also analyzed in order to understand the patterns of induced changes within the respiratory tract. The lung was the most frequently affected organ in both species, with increased alveolar macrophages being the most common background and test article–related finding. In the upper respiratory tract (URT), inflammatory cell infiltrates were the most common background findings in the nasal cavity in monkeys. Induced URT findings were more frequent in rats than monkeys, with squamous metaplasia in the larynx, and goblet cell hyperplasia in the nasal cavity being the most common. Overall, the data revealed a limited pattern of response to inhaled molecules in the respiratory tract, with background and test article–related findings often occurring in the same regions. It is hoped that these data will assist in the interpretation of findings in the respiratory tract induced by novel inhaled small molecule entities.

To investigate the effects of common nanosuspension-stabilizing excipients on the nature and temporal evolution of histopathological changes at intramuscular (i.m.) administration sites, 5 groups of 39 male rats per group received a single injection of 1 of the 5 analogous crystalline drug nanosuspensions containing 200 mg/ml of an antiviral compound with particle sizes of ±200 nm and identical vehicle compositions, except for the type of nanosuspension stabilizer. The investigated stabilizers were poloxamer 338, poloxamer 407, d-α-tocopherol polyethylene glycol 1,000-succinate (TPGS), polysorbate 80, and polysorbate 80 combined with egg phosphatidylglycerol. Histopathology and immunohistochemistry revealed progressive inflammatory changes at the i.m. administration sites and the draining lymph nodes that differed according to the time point of sacrifice and the type of stabilizer. Although the overall time course of inflammatory changes was similar across the groups, differences in the nature, severity, and timing of the inflammatory response were observed between animals injected with poloxamer- or TPGS-containing nanosuspensions and those injected with formulations containing polysorbate 80. A more severe and prolonged active inflammatory phase, the presence of multinucleate giant cells, prolonged macrophage infiltration of the formulation depot, and more persistent histiocytic infiltrates in the lymph nodes were observed in the polysorbate 80–containing nanosuspension groups. Such vehicle-mediated effects could influence the overall tolerability profile of long-acting nanosuspensions.

The evaluation of inhalation studies in monkeys is often hampered by the scarcity of published information on the relevant nasal anatomy and pathology. We examined nasal cavities of 114 control cynomolgus monkeys from 11 inhalation studies evaluated 2008 to 2013, in order to characterize and document the anatomic features and spontaneous pathology. Compared to other laboratory animals, the cynomolgus monkey has a relatively simple nose with 2 unbranched, dorsoventrally stacked turbinates, large maxillary sinuses, and a nasal septum that continues into the nasopharynx. The vomeronasal organ is absent, but nasopalatine ducts are present. Microscopically, the nasal epithelium is thicker than that in rodents, and the respiratory (RE) and transitional epithelium (TE) rest on a thick basal lamina. Generally, squamous epithelia and TE line the vestibule, RE, the main chamber and nasopharynx, olfactory epithelium, a small caudodorsal region, while TE is observed intermittently along the passages. Relatively high incidences of spontaneous pathology findings, some resembling induced lesions, were observed and included inflammation, luminal exudate, scabs, squamous and respiratory metaplasia or hyperplasia, mucous cell hyperplasia/metaplasia, and olfactory degeneration. Regions of epithelial transition were the most affected. This information is considered helpful in the histopathology evaluation and interpretation of inhalation studies in monkeys.

This retrospective study was performed to determine the range, occurrence and incidence of spontaneously arising histopathological findings of the cardiovascular system in purpose-bred laboratory nonhuman primates. Data were collected from 84 controlled toxicological studies with equal numbers of male and female animals and full tissue lists. Attempts were also made to standardize pathological terms used by various original pathologists. Tissue sections from 2464 animals, which included 2050 cynomolgus monkeys ( Macaca fascicularis), 284 common marmosets ( Callithrix jacchus) and 130 rhesus monkeys ( Macaca mulatta) were examined. The most common cardiac finding was focal myocardial inflammation, subcategorized as either “inflammatory cell infiltration” (339) or “focal myocarditis” (131). Other cardiac findings included mineralization (29), endocarditis (16), pericarditis (10), squamous cysts (6) and ectopic thyroid tissue (5). Perivasculitis/vasculitis in the kidney, lung, meninges, sciatic nerve, and other tissues (206) was the most common vascular lesion. Focal myocarditis was more common in male (60%) than female (40%) animals. Cardiac mineralization and extramedullary hematopoiesis were more common in marmosets than other species while ectopic thyroid tissue was present in marmosets and cynomolgus monkeys. To our knowledge, this is the first study to demonstrate the range and incidence of spontaneous cardiovascular lesions in laboratory nonhuman primates.

The authors performed a retrospective study to determine the incidences and range of spontaneous pathology findings in control cynomolgus monkeys. Data were collected from 570 monkeys (285 animals per sex), aged twelve to thirty-six months, from sixty regulatory studies evaluated at our laboratory between 2003 and 2009. The most common finding overall was lymphoplasmacytic infiltrates observed in the following incidence: liver (60.7%), kidneys (28.8%), heart (25.8%), salivary glands (21.2%), and stomach (12.1%). Inflammation also commonly occurred in the heart, kidneys, lungs, and stomach. The most common degenerative changes were localized fatty change in the liver, myocardial degeneration, and mineralization and pigment deposits in various tissues. Parathyroid, thyroid, and pituitary cysts; ectopic thymus in the parathyroid or thyroid gland; accessory spleen within the pancreas; and adrenohepatic fusion were among the most common congenital findings. Some incidental findings bearing similarities to drug-induced lesions were also encountered in various organs. It is hoped that the results presented here and elsewhere could form the groundwork for the creation of a reliable database of incidental pathology findings in laboratory nonhuman primates.