Incidental Gliosis in the Central Nervous System of Control Nonhuman Primates and Rats.

Gliosis, including microgliosis and astrocytosis, can be challenging to interpret in nonclinical studies. Incidences of glial foci in brains and spinal cords of control rats and nonhuman primates (NHPs) were reviewed in the historical control databases from two contract research organizations, including one specializing in neuropathology. In the brain, minimal to mild (grades 1-2) microgliosis was the most common diagnosis, especially in NHPs, although occasional moderate or marked microgliosis (grades 3 and 4) was encountered in both species. Microgliosis was more common in the cerebral cortex, cerebellum, and medulla oblongata in both species and was frequent in the white matter (brain), thalamus, and basal nuclei of NHPs. Gliosis ("not otherwise specified") of minimal severity was diagnosed in similar brain sub-sites for both species and was more common in NHPs compared with rats. Astrocytosis was most prominent in the cerebellum (molecular layer) of NHPs but was otherwise uncommon. In the spinal cord, microgliosis was most common in the lateral white matter tracts in rats and NHPs, and in the dorsal white matter tracts in NHPs. These data indicate that low-grade spontaneous glial responses occur with some frequency in control animals of two common nonclinical species.

Guide for Combining Primary Tumors for Statistical Analysis in Rodent Carcinogenicity Studies.

The Tumor Combination Guide was created at the request of the U. S. Food and Drug Administration (FDA) by a Working Group of biopharmaceutical experts from international societies of toxicologic pathology, the Food and Drug Administration (FDA), and members of the Standard for Exchange of Nonclinical Data (SEND) initiative, to assist pharmacology/toxicology reviewers and biostatisticians in statistical analysis of nonclinical tumor data. The guide will also be useful to study and peer review pathologists in interpreting the tumor data. This guide provides a higher-level hierarchy of tumor types or categories correlating the tumor names from the International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) publications with those available in the NEOPLASM controlled terminology (CT) code list in SEND. The version of CT used in a study should be referenced in the nonclinical study data reviewer's guide (SDRG) (section 3.1) of electronic submissions to the FDA. The tumor combination guide instructions and examples are in a tabular format to make informed decisions for combining tumor data for statistical analysis. The strategy for combining tumor types for statistical analysis is based on scientific criteria gleaned from the current scientific literature; as SEND and INHAND terminology and information evolve, this guide will be updated.

Toxicologic Pathology Forum: Opinion on Performing Good Laboratory Practice Histopathology Evaluation for Nonclinical Toxicity Studies in a Remote Location.

Toxicologic/veterinary pathologists are working remotely from Good Laboratory Practice (GLP) test facilities (TFs) in increasing numbers, most commonly in home-office settings. A study pathologist (SP) generating data on GLP-compliant nonclinical studies must be keenly aware of applicable national GLP regulations and comply with TF and protocol requirements. This Toxicological Pathology Forum Opinion Piece will summarize primary areas of emphasis for the SP generating GLP data using glass slides. Peer review and digital review of whole slide images are out of scope for this opinion piece. Key GLP considerations for primary pathology on glass slides are discussed with respect to SP location and employment status, including pathologist qualifications, specimen management, facilities, equipment, archive, and quality assurance. Notable differences between national GLP regulations of the United States, the United Kingdom, Germany, the Netherlands, France, Ireland, Switzerland, Italy, and Israel are presented. With the understanding that each combination of location and employment is unique, the authors provide a general overview of considerations for successful remote GLP work.

Idiopathic Aneurysms of the Ascending Aorta in the Mouse and Rat.

Aneurysms of the ascending aorta, unrelated to xenobiotic administration, are described in 5 rats and 2 mice in nonclinical safety studies conducted at Charles River Laboratories (CRL) sites over the past 10 years. The most prominent microscopic finding was focal dilation with disruption of the wall of the ascending aorta with chronic adventitial inflammation or fibroplasia. The pathogenesis of this finding is unknown. There were no associated macroscopic findings, clinical abnormalities, or vascular lesions elsewhere. The results of a search of historical control data from toxicology studies of 1 day to 72 weeks' duration performed at CRL for aortic findings from 5900 mice and 23,662 rats are also reported. Aortic lesions are uncommon in mice and rats used in nonclinical safety studies, but toxicologic pathologists should be aware that aneurysms of the ascending aorta with fibroplasia and inflammation in the aortic wall and adventitia may occur spontaneously or iatrogenically, as they have the potential to impact interpretation in toxicology studies.

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Nonproliferative and Proliferative Lesions of the Rabbit.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions Project (www.toxpath.org/inhand.asp) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally accepted nomenclature for proliferative and non-proliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in most tissues and organs from the laboratory rabbit used in nonclinical safety studies. Some of the lesions are illustrated by color photomicrographs. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions as well as lesions induced by exposure to test materials. Relevant infectious and parasitic lesions are included as well. A widely accepted and utilized international harmonization of nomenclature for lesions in laboratory animals will provide a common language among regulatory and scientific research organizations in different countries and increase and enrich international exchanges of information among toxicologists and pathologists.

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Non-proliferative and Proliferative Lesions of the Non-human Primate (M. fascicularis).

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions Project (www.toxpath.org/inhand.asp) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in most tissues and organs from the nonhuman primate used in nonclinical safety studies. Some of the lesions are illustrated by color photomicrographs. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions as well as lesions induced by exposure to test materials. Relevant infectious and parasitic lesions are included as well. A widely accepted and utilized international harmonization of nomenclature for lesions in laboratory animals will provide a common language among regulatory and scientific research organizations in different countries and increase and enrich international exchanges of information among toxicologists and pathologists.

Proof of Concept: The Use of Whole-Slide Images (WSI) for Peer Review of Tissues on Routine Regulatory Toxicology Studies.

This Proof of Concept (POC) study was to assess whether assessment of whole slide images (WSI) of the 2 target tissues for a contemporaneous peer review can elicit concordant results to the findings generated by the Study Pathologist from the glass slides. Well-focused WSI of liver and spleen from 4 groups of mice, that had previously been diagnosed to be the target tissues by an experienced veterinary toxicologic pathologist examining glass slides, were independently reviewed by 3 veterinary pathologists with varying experience in assessment of WSIs. Diagnostic discrepancies were then reviewed by an experienced adjudicating pathologist. Assessment of microscopic findings using WSI showed concordance with the glass slides, with only slight discrepancy in severity grades noted. None of the lesions recorded by the Study pathologist were "missed" and no lesions were added by the pathologists evaluating WSIs, thus demonstrating equivalence of the WSI to glass slides for this study.

Evaluation of Stem Cell-Derived Cellular Therapy Products.

Although there is an increase in the development of new cellular therapies, few guidelines have been published to assist in the design of preclinical studies. As no product and therapeutic intention is entirely alike regulators and Contract Research Organizations need to treat each project on a case-by-case basis. One of the most important considerations in study design is to retain all tissues from the study, thereby allowing for further analysis of tissues should unexpected effects be seen in clinical studies. Input from the pathologist at the earliest stages of study design regarding animal selection, cell markers, and phased tissue examination improves the scientific integrity of the study.

Nervous System Sampling for General Toxicity and Neurotoxicity Studies in Rabbits.

Although manuscripts for multiple species recommending nervous system sampling for histopathology evaluation in safety assessment have been published in the past 15 years, none have addressed the laboratory rabbit. Here, we describe 2 trimming schemes for evaluating the rabbit brain in nonclinical toxicity studies. In both schemes, the intact brain is cut in the coronal plane to permit bilateral assessment. The first scheme is recommended for general toxicity studies (tier 1) in screening agents where there is no anticipated neurotoxic potential; this 6-section approach is consistent with the Society of Toxicologic Pathology (STP) "best practice" recommendations for brain sampling in nonrodents (Toxicol Pathol 41: 1028-1048, 20131). The second trimming scheme is intended for dedicated neurotoxicity studies (tier 2) to characterize known or suspected neurotoxicants where the nervous system is a key target organ. This tier 2 strategy relies on coronal trimming of the whole brain into 3-mm-thick slices and then evaluating 12 sections. Collection of spinal cord, ganglia, and nerve specimens for rabbits during nonclinical studies should follow published STP "best practice" recommendations for sampling the central nervous system1 and peripheral nervous system (Toxicol Pathol 46: 372-402, 20182).

Proliferative and Nonproliferative Lesions of the Rat and Mouse Central and Peripheral Nervous Systems: New and Revised INHAND Terms.

Harmonization of diagnostic terminology used during the histopathologic analysis of rodent tissue sections from nonclinical toxicity studies will improve the consistency of data sets produced by laboratories located around the world. The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a cooperative enterprise of 4 major societies of toxicologic pathology to develop a globally accepted standard vocabulary for proliferative and nonproliferative lesions in rodents. A prior manuscript (Toxicol Pathol 2012;40[4 Suppl]:87S-157S) defined multiple diagnostic terms for toxicant-induced lesions, common spontaneous and age-related changes, and principal confounding artifacts in the rat and mouse central nervous system (CNS) and peripheral nervous system (PNS). The current article defines 9 new diagnostic terms and updates 2 previous terms for findings in the rodent CNS and PNS, the need for which has become evident in the years since the publication of the initial INHAND nomenclature for findings in rodent neural tissues. The nomenclature presented in this document is also available electronically on the Internet at the goRENI website (http://www.goreni.org/).

International Regulatory Guiding Documents and Best Practice Recommendations on Peripheral Nervous System (PNS) Histopathologic Evaluation in Good Laboratory Practice (GLP)-Compliant Animal Toxicity Studies.

Assessment of the peripheral nervous system (PNS) tissues during animal toxicity studies generally is included within guiding documents issued by regulatory agencies of individual nations (eg, US Environmental Protection Agency, US Food and Drug Administration) and multinational federations (eg, European Medicines Agency) as well as international cooperative efforts (eg, International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use, Organisation for Economic Co-operation and Development). The present list of major regulatory guiding documents categorizes recommendations from around the world for sampling and processing PNS tissues (nerves and ganglia) for general animal toxicity studies (ie, where neurotoxicity is not expected) and specialized neurotoxicity studies (ie, where neurotoxicity is anticipated or known to occur). In general, regulatory guidelines call for collection of one or more sensorimotor nerves (usually the sciatic trunk and its branches), though details vary among agencies. Regulatory guiding documents represent a "starting point," after which additional PNS samples and/or special methods may be implemented at the applicant's discretion. Best practice recommendations for PNS sampling and processing in animal toxicity studies endorsed by multiple global societies of toxicologic pathology encompass and expand on existing regulatory guidelines.

Nonproliferative and Proliferative Lesions of the Rat and Mouse Hematolymphoid System.

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative changes in rats and mice. The purpose of this publication is to provide a standardized nomenclature for classifying changes observed in the hematolymphoid organs, including the bone marrow, thymus, spleen, lymph nodes, mucosa-associated lymphoid tissues, and other lymphoid tissues (serosa-associated lymphoid clusters and tertiary lymphoid structures) with color photomicrographs illustrating examples of the lesions. Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions as well as lesions induced by exposure to test materials. The nomenclature for these organs is divided into 3 terminologies: descriptive, conventional, and enhanced. Three terms are listed for each diagnosis. The rationale for this approach and guidance for its application to toxicologic pathology are described in detail below.

Normal Anatomy, Histology, and Spontaneous Pathology of the Kidney, and Selected Renal Biomarker Reference Ranges in the Cynomolgus Monkey.

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.

Histopathological Findings in Cynomolgus Macaques ( Macaca fascicularis) Consistent with Secondary Immunological Reaction to Biotherapeutics with an Emphasis on the CNS and Eye.

Biotherapeutics are pharmaceutical products derived from or synthesized by biological systems. Such molecules carry the potential for immunogenicity which may lead to adverse immune responses. The cynomolgus macaque ( Macaca fascicularis) is the species of choice in nonclinical safety assessment of biotherapeutics. The main aim of this study was to confirm whether mononuclear cell infiltrates at specific locations represent a generic effect of biotherapeutics, and therefore the result of their immunogenicity. Following a review of microscopic findings in studies conducted over a 10-year period at one test facility, 15% of biotherapeutics were reported to have such findings. The most commonly affected site was the choroid plexus and less frequently the meninges and ciliary body. The reporting of such findings as test article-related becomes more subjective as the severity and incidence decreases. To assess the accuracy of such associations, a mathematical approach was employed to determine the probability of obtaining the observed results by chance. There was good agreement between this approach and the original findings. In addition to an increased number and size of mononuclear cell infiltrates in the brain, biotherapeutic administration was strongly associated with the presence of plasma cells and eosinophils.

Toxicologic Pathology Forum*: Opinion on Considerations for the Use of Whole Slide Images in GLP Pathology Peer Review.

Whole slide imaging (WSI) technology has advanced to a point where it has replaced the glass slide as the primary means of pathology evaluation within many areas of medical pathology. The deployment of WSI in the field of toxicologic pathology has been delayed by a lack of clarity around the degree of validation required for its use on Good Laboratory Practice (GLP) studies. The current opinion piece attempts to provide a high-level overview of WSI technology to include basic methodology, advantages and disadvantages over a conventional microscope, validation status of WSI scanners, and perceived concerns over regulatory acceptance for the use of WSI for (GLP) peer review in the field of toxicologic pathology. Observations are based on the extensive use by AstraZeneca of WSI for the peer review of non-GLP studies conducted at Charles River facilities and represent the experiences of the authors. Note: This is an opinion article submitted to the Toxicologic Pathology Forum. It represents the views of the author(s). It does not constitute an official position of the Society of Toxicologic Pathology, British Society of Toxicological Pathology, or European Society of Toxicologic Pathology, and the views expressed might not reflect the best practices recommended by these Societies. This article should not be construed to represent the policies, positions, or opinions of their respective organizations, employers, or regulatory agencies.

Historical control background incidence of spontaneous pituitary gland lesions of Han-Wistar and Sprague-Dawley rats and CD-1 mice used in 104-week carcinogenicity studies.

The aim of this study was to determine the range and incidences of spontaneous microscopic lesions of the pituitary gland in control Han-Wistar and Sprague-Dawley rats and CD-1 mice from 104-week carcinogenicity studies carried out between 1998 and 2010 at Charles River Edinburgh. In both strains of rats and in CD-1 mice, non-proliferative lesions of the pituitary gland were generally uncommon, excluding cysts/pseudocysts (6.42% in Han-Wistar rats, 5.85% in Sprague-Dawley rats, and 2.08% in CD-1 mice). Primary proliferative lesions were most frequently found in the pars distalis of the pituitary gland. Adenomas and carcinomas of the pars distalis were more common in Sprague-Dawley rats (49.33% and 2.85%, respectively) than in Han-Wistar rats (27.29% and 0.21%, respectively), and adenomas in both strains of rats and CD-1 mice exhibited a marked sex predisposition, with females more commonly affected.

Evaluation of the PhysioTel™ Digital M11 cardiovascular telemetry implant in socially housed cynomolgus monkeys up to 16weeks after surgery.

INTRODUCTION: The novel PhysioTel™ Digital M11 telemetry implant was evaluated in socially housed monkeys with respect to both safety pharmacological cardiovascular (arterial blood pressure (BP), heart rate (HR) and electrocardiogram (ECG)) and toxicological (clinical pathology and histopathology) endpoints. METHODS: Telemetry and clinical pathology data were obtained repeatedly up to 16weeks after surgery in four female cynomolgus monkeys, followed by necropsy. Due to postsurgical complications, one spare animal was included and only toxicological endpoints from the affected (fifth animal) were reported. Continuous telemetry recordings were conducted at periods without dosing and after ascending doses of moxifloxacin (0, 10, 30, 100mg/kg) and L-NAME (0, 0.1, 1, 10mg/kg). Additionally, a retrospective power analysis was conducted based on baseline M11 implant data from 32 other animals. RESULTS: During periods without dosing, the cardiovascular endpoints were stable over time and within normal ranges. Moxifloxacin and L-NAME elicited the expected pharmacological responses with dose-dependent increase in QTca (8, 17, 22ms) and BP (mean BP: 12, 21, 34mmHg), respectively. Expected intravascular and tissue reactions were observed at the sites of the BP catheter and the transmitter. Signs of infection (localised to the transmitter implantation site with associated systemic effects) was noted in the fifth animal. No systemic pathologies were seen in any animals. Power analysis (80% power) indicated that the minimal differences which can be detected in a parallel group design (n=6) are 7mmHg (mean BP), 16bpm (HR), 12ms (QTca). DISCUSSION: The M11 implant provided stable, high quality ECG and BP data for a duration covering the length of sub-chronic repeated dose toxicity studies without important impact on toxicological endpoints. Adequate power in order to elucidate major treatment-related cardiovascular effects was demonstrated. However to avoid post-surgical complications the implantation procedures should be carefully considered before using the method.

Historical control background incidence of spontaneous thyroid and parathyroid glands lesions of rats and CD-1 mice used in 104-week carcinogenicity studies.

The incidence and range of spontaneous thyroid and parathyroid glands findings were determined in control Han-Wistar and Sprague-Dawley rats, and CD-1 mice from 104-week carcinogenicity studies carried out between 1998 and 2010 at Charles River Edinburgh. In both strains of rats and in CD-1 mice, non-proliferative lesions of the thyroid or parathyroid glands were generally uncommon apart from some findings in CD-1 mice such as ultimobranchial duct/cyst (5.72%), follicular distension/dilatation (3.84%), and cystic follicles (3.53%). In Han-Wistar rats, thyroid proliferative lesions were slightly more frequent in males than in females, but in Sprague-Dawley rats, they were of similar incidence in both sexes. The most common findings overall in Han-Wistar and Sprague-Dawley rats were C-cell hyperplasia (48.11% and 36.56%, respectively) and adenoma (10.87% and 9.52%, respectively), follicular cell hyperplasia (4.21% and 0.91%, respectively) and adenoma (4.32% and 1.36%, respectively). Secondary neoplastic lesions either in thyroid or parathyroid gland were poorly represented.

Spontaneous and Dosing Route-related Lung Lesions in Beagle Dogs from Oral Gavage and Inhalation Toxicity Studies: Differentiation from Compound-induced Lesions.

This study was conducted to characterize lung microscopic lesions in control beagle dogs from inhalation and oral gavage toxicity studies, to determine differences associated with the route of administration, and to discuss distinguishing features from compound-induced lung lesions. Samples from 138 control dogs from oral gavage studies and 124 control dogs from inhalation (vehicle control) studies were evaluated microscopically. There was no significant sex-related difference in the incidence of all lesions. Perivascular mononuclear cell infiltration, centriacinar mixed cell infiltration, bronchopneumonia, subpleural septal fibrosis, and alveolar macrophage accumulation were the most common lesions. Aspiration pneumonia was more common in dogs from gavage studies, suggesting reflux after gavage dosing or accidental administration of test formulation as possible causes. Centriacinar mixed cell infiltration was more common in dogs from inhalation studies, suggesting mild irritation by the vehicles used. Vascular lesions, which included pulmonary arteriopathy and smooth muscle mineralization, were observed in a few animals. Some of the spontaneous lesions are similar to lesions induced by test compounds. Compared to spontaneous lesions, compound-induced lesions tend to be multifocal or diffuse, follow a pattern of distribution (e.g., centriacinar, perivascular, and interstitial), show a dose response in the incidence and severity, and may show cell-specific toxicity.