The first session of the 2025 European Society of Toxicologic Pathology (ESTP) Congress reviewed routine and specialized methods for microscopic evaluation of neural tissues during nonclinical studies. Three longer presentations reviewed brain sampling approaches in safety assessments, including an example to accentuate topographical analysis and integration of toxicology data; specific brain and spinal cord sampling for molecular and protein analyses; and an overview of technical aspects of intraparenchymal drug delivery. Four shorter talks discussed the uses, advantages, disadvantages, and interpretation of several special neurohistological techniques (stains and immunohistochemical markers) for assessing test item-associated responses. Common special methods used (when warranted) for nonclinical studies include Fluoro-Jade or silver stains for detecting neuronal death, Luxol fast blue (LFB) for examining myelin, anti-glial fibrillary acidic protein (GFAP) to demonstrate reactive astrocytes, and anti-ionized calcium-binding adaptor molecule 1 (IBA1) to highlight reactive microglia and macrophages, though alternatives methods were described. The last presentation discussed artificial intelligence as an aid in detecting subtle toxicant-induced lesions during digital pathology analyses (using the Olney lesion [acute neuronal vacuolation and necrosis in the cerebral cortex] as an example). Taken together, talks in this session provided a cohesive overview of traditional and innovative approaches to facilitate microscopic evaluation for potential neurotoxicity in nonclinical studies.

The American College of Toxicology (ACT), the Safety Pharmacology Society (SPS) and the Society for Toxicological Pathology (STP) conducted an industry survey in 2024 to assess current industry practices as they relate to neurotoxicity and safety testing of therapeutics. This survey was developed as a follow-up to 2015 survey conducted by the Safety Pharmacology Society (SPS) to identify industry practices as they relate to central, peripheral and autonomic nervous system ('CNS') drug safety testing. In the current survey, there were one hundred thirty (130) respondents from Asia (5 %), Europe (32 %) and North America (64 %). Most respondents (54 %) were from pharmaceutical companies of over 1000 employees. Small molecules (89 %), large molecules (73 %), gene therapy (52 %), cell therapy (41 %) and vaccines (38 %) were the types of modalities developed by respondents. Oncology (72 %) and neurology/psychiatry (64 %) were the most frequent therapeutic indications pursued by companies followed by inflammation (56 %), cardiovascular (48 %), rare/orphan (44 %), metabolic (42 %), infectious (35 %) and respiratory (28 %) diseases. Tremors (81 %), emesis (75 %) and salivation (61 %) were more frequently reported than in the 2015 CNS survey while gait/coordination abnormalities (67 %), convulsion (65 %), and peripheral neuropathy (24 %) were unchanged or decreased when compared to the 2015 survey. Most respondents reported using a modified Irwin's test (90 %) added to toxicology studies (80 %) and/or as a standalone study (71 %), a major change from the 2015 survey where most respondents reported using a standalone study. Survey results reflect an industry shift towards the development of new therapies classified as biologics, cell and gene therapies.

Drug discovery and development is a complex, lengthy, and expensive process that takes on average 10-15 years and approximately $1-2 billion USD for approval of a new drug. While the studies needed to support clinical development are generally outlined in guidance documents, there is much less guidance on how to translate the nonclinical data into clinical designs. Nonclinical studies are performed to conduct the First-in-Human clinical trial, which is the first major milestone to advance new promising drug candidates, and are conducted primarily to determine the safe dose range for clinical development. Resolving how to move forward, and even when to move forward, requires significant cross-functional collaboration with pathologists, ADME scientists, biologists, and clinical staff. There are many reasons why drug candidates may fail; these could be as simple as insufficient understanding of the nature of the translational process, failure to effectively integrate the data from different pharmacologically relevant species, or erosion of the margin of safety during chronic toxicology studies. The case studies described here were designed to help participants in the 2024 American College of Toxicology (ACT) Continuing Education course "Translational Challenges from Nonclinical to Clinical Program: Case Study Examples" to improve their skills in managing translational challenges from nonclinical to clinical program encountered during drug development.

The 21st ESTP's (European Society of Toxicology Pathology) Annual Congress (2024) included a 3-hour scientific session on developmental neurotoxicity (DNT) as applied to chemical safety assessments. Key concepts of this session were to provide an introduction to public concerns around this endpoint, a status update on practical aspects of DNT studies, insights into the use of DNT studies within a regulatory context, as well as some pointers on how to evaluate specific parameters. Understanding the biological and technical variability in performing neuropathology examinations (such as morphometric evaluation) is critical during the course of DNT evaluation. Using thyroid hormone disruption as an example, challenges and pitfalls impacting data interpretation were discussed. Results from the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) Thyroid Task Force regarding thyroid hormone-related neurodevelopmental toxicity in humans and rodents were presented. Histopathological findings in the brain and potential changes in the cochlea of pups associated with thyroid hormone imbalance in dams during pregnancy were shown. A case presentation from an Extended One-Generation Reproductive Toxicity Study (EOGRTS) showing histopathological findings in the absence of changes with morphometric endpoints was included. In conclusion, all session participants underscored the need for integrated data evaluation for DNT risk assessment.

Recent trends in toxicological pathology include implementation of digital platforms that have gained rapid momentum in the field. Are we ready to fully implement this new modality? This opinion piece provides some practical perspectives on digital pathology such as its cost limitations, relative time requirements, and a few technical issues, some of which are encountered for specific lesions, that warrant caution. Although the potential for digital pathology assessment with whole slide images has made great strides, we are of the opinion that it is not yet ready for complete replacement of glass slides in toxicologic pathology safety assessments.

The International Academy of Toxicologic Pathology (IATP) Satellite Symposium on "New Approach Methodologies (NAMs) for Neurotoxicity Assessment and Regulatory Perspectives," organized in Spain, addressed the growing need for improved assessment of neurotoxicity. Traditional neurotoxicity assessment using in vivo animal studies are impractical for testing the substantial number of environmental chemicals that currently lack data and in the early detection of neuro-related adverse reactions in drug discovery. The NAMs, including human in vitro assays and small model organisms, have been developed for faster and cost-effective assessment of neurotoxic potential. While NAMs offer improved practicality, utility, and valuable mechanistic insights, their integration into regulatory decision-making requires robust scientific validation and technical characterization. Confidence in and regulatory application of NAMs data can be supported by mapping cellular outcomes to neuropathological findings in mammals, including humans, through the Adverse Outcome Pathway (AOP) framework, and the Integrated Approach to Testing and Assessment (IATA). Case studies presented demonstrated the application of NAMs in chemical and drug safety evaluations, focusing on developmental neurotoxicity (DNT), Parkinson's disease, and drug-induced seizures. In conjunction with in vivo toxicology studies, NAMs represent a significant step toward advancing chemical and drug toxicity assessment via hazard identification and drug screening safety assessments.

Consistent and unambiguous diagnostic terminology describing morphological changes clearly impacts interpretation and risk assessment in routine nonclinical toxicology studies. This chapter addresses the importance of and the issues involved with the application of nomenclature in toxicologic pathology. Nomenclature comprises a hierarchical system of components for a diagnosis including topography (organ/tissue), morphology (pathological change), and modifiers (also known as qualifiers). The lexicon and components of nomenclature differ between neoplastic and nonneoplastic lesions. An overview of the challenges of assigning and managing nomenclature encountered during histopathological evaluation and recommended practices is detailed. Finally, harmonization efforts to develop standardized nomenclature are described, including the Standards for Exchange of Nonclinical Data, the International Harmonization of Toxicologic Pathology Nomenclature, the National Toxicology Program Nonneoplastic Lesion Atlas, and the global open Registry Nomenclature Information System.

Beagle dogs are a key nonrodent species in nonclinical safety evaluation of new biomedical products. The Society of Toxicologic Pathology (STP) has published "best practices" recommendations for nervous system sampling in nonrodents during general toxicity studies (Toxicol Pathol 41[7]: 1028-1048, 2013), but their adaptation to the Beagle dog has not been defined specifically. Here we provide 2 trimming schemes suitable for evaluating the unique neuroanatomic features of the dog brain in nonclinical toxicity studies. The first scheme is intended for general toxicity studies (Tier 1) to screen test articles with unknown or no anticipated neurotoxic potential; this plan using at least 7 coronal hemisections matches the STP "best practices" recommendations. The second trimming scheme for neurotoxicity studies (Tier 2) uses up to 14 coronal levels to investigate test articles where the brain is a suspected or known target organ. Collection of spinal cord, ganglia (somatic and autonomic), and nerves for dogs during nonclinical studies should follow published STP "best practices" recommendations for sampling the central (Toxicol Pathol 41[7]: 1028-1048, 2013) and peripheral (Toxicol Pathol 46[4]: 372-402, 2018) nervous systems. This technical guide also demonstrates the locations and approaches to collecting uncommonly sampled peripheral nervous system sites.

With the increasing use of ketamine as an off-label treatment for depression and the recent FDA approval of (S)ketamine for treatment-resistant depression, there is an increased need to understand the long-term safety profile of chronic ketamine administration. Of particular concern is the neurotoxicity previously observed in rat models following acute exposure to high doses of ketamine, broadly referred to as 'Olney's lesions'. This type of toxicity presents as abnormal neuronal cellular vacuolization, followed by neuronal death and has been associated with ketamine's inhibition of the N-methyl-D-aspartate receptor (NMDAR). In this study, a pharmacological and neuropathological analysis of ketamine, the potent NMDAR antagonist MK-801, and the ketamine metabolite (2R,6R)-hydroxynorketamine [(2R,6R)-HNK)] in rats is described following both single dose and repeat dose drug exposures. Ketamine dosing was studied up to 20 mg/kg intravenously for the single-dose neuropathology study and up to 60 mg/kg intraperitoneally for the multiple-dose neuropathology study. MK-801 dosing was studied up to 0.8 mg/kg subcutaneously for both the single and multiple-dose neuropathology studies, while (2R,6R)-HNK dosing was studied up to 160 mg/kg intravenously in both studies. These studies confirm dosedependent induction of `Olney's lesions' following both single dose and repeat dosing of MK-801. Ketamine exposure, while showing common behavioral effects, did not induce wide-spread Olney's lesions. Treatment with (2R,6R)-HNK did not produce behavioral effects, toxicity or any evidence of Olney's lesion formation. Based on these results, future NMDAR-antagonist neurotoxicity studies should strongly consider taking pharmacokinetics more thoroughly into account.

The Society of Toxicologic Pathology's Annual Virtual Symposium (2021) included a session on "Regulatory Perspectives on Juvenile Animal Toxicologic Pathology." The following narrative summarizes the key concepts from the four talks included in this symposium session chaired by Drs Deepa Rao and Alan Hoberman. These encompass an overview of various global regulations impacting the conduct of juvenile animal studies in pharmaceutical drug development and chemical toxicity assessments in a talk by Dr Alan Hoberman. Given the numerous regulatory guidances and legal statutes that have covered the conduct of juvenile animal studies and the recent harmonization of these guidances for pharmaceuticals, Dr Paul Brown provided an update on the harmonization of these guidances for pharmaceuticals, in the recently finalized version of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use S11 guidance document, "Nonclinical Safety Testing in Support of Development of Pediatric Medicines." The first two talks on regulations were followed by two talks focused on an evaluation of the postnatal development of two major organ systems relevant in juvenile animals. Dr Aurore Varela covered study design and endpoints impacting the skeletal system (bone), while Dr Brad Bolon presented a talk on the study design and conduct of neuropathology evaluations for the developing nervous system.

Visual system toxicity may manifest anywhere in the visual system, from the eye proper to the visual brain. Therefore, effective screening for visual system toxicity must evaluate not only ocular structures (ie, eye and optic nerve) but also multiple key brain regions involved in vision (eg, optic tract, subcortical relay nuclei, and primary and secondary visual cortices). Despite a generally comparable pattern across species, the neuroanatomic organization and function of the visual brain in rodents and rabbits exhibit appreciable differences relative to nonrodents. Currently recognized sampling practices for general toxicity studies in animals, which are based on easily discerned external neuroanatomic landmarks and guided by extant stereotaxic brain atlases, typically will permit histopathologic evaluation of many brain centers involved in visual sensation (eg, optic chiasm, optic tract, dorsal lateral geniculate nucleus, primary and secondary visual cortices) and often some subcortical brain nuclei involved in light-modulated nonvisual activities needed for visual attention and orientation (eg, rostral colliculus in quadrupeds, termed the superior colliculus in bipeds; several cranial nerve nuclei). Pathologic findings induced by toxicants in the visual brain centers are similar to those that are produced in other brain regions.

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, 2013 1 ). 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 system 1 and peripheral nervous system ( Toxicol Pathol 46: 372-402, 2018 2 ).

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/).

Analysis of intraepidermal nerve fibers (IENFs) in skin biopsy samples has become a standard clinical tool for diagnosing peripheral neuropathies in human patients. Compared to sural nerve biopsy, skin biopsy is safer, less invasive, and can be performed repeatedly to facilitate longitudinal assessment. Intraepidermal nerve fiber analysis is also more sensitive than conventional nerve histology or electrophysiological tests for detecting damage to small-diameter sensory nerve fibers. The techniques used for IENF analysis in humans have been adapted for large and small animal models and successfully used in studies of diabetic neuropathy, chemotherapy-induced peripheral neuropathy, HIV-associated sensory neuropathy, among others. Although IENF analysis has yet to become a routine end point in nonclinical safety testing, it has the potential to serve as a highly relevant indicator of sensory nerve fiber status in neurotoxicity studies, as well as development of neuroprotective and neuroregenerative therapies. Recently, there is also interest in the evaluation of IENF via skin biopsy as a biomarker of small fiber neuropathy in the regulatory setting. This article provides an overview of the anatomic and pathophysiologic principles behind IENF analysis, its use as a diagnostic tool in humans, and applications in animal models with focus on comparative methodology and considerations for study design.

Neuropathology of the peripheral nervous system (PNS) is an underappreciated area in toxicologic pathology. Toxicity to nerves and ganglia can result from toxic insults following exposure to environmental, occupational, and industrial chemicals; drugs and biologics; cosmetics and food additives; and even physical agents such as noise. The following introduction provides an overview of this special issue of Toxicologic Pathology on toxicologic neuropathology of the PNS and highlights the range of key topics in this field that are reviewed in this compilation.

Manganese (Mn), an essential metal and nutrient, is toxic in excess. Toxicity classically results from inhalational exposures in individuals who work in industrial settings. The first known disease of inherited Mn excess, identified in 2012, is caused by mutations in the metal exporter SLC30A10 and is characterized by Mn excess, dystonia, cirrhosis, and polycythemia. To investigate the role of SLC30A10 in Mn homeostasis, we first generated whole-body Slc30a10-deficient mice, which developed severe Mn excess and impaired systemic and biliary Mn excretion. Slc30a10 localized to canalicular membranes of hepatocytes, but mice with liver Slc30a10 deficiency developed minimal Mn excess despite impaired biliary Mn excretion. Slc30a10 also localized to the apical membrane of enterocytes, but mice with Slc30a10 deficiency in small intestines developed minimal Mn excess despite impaired Mn export into the lumen of the small intestines. Finally, mice with Slc30a10 deficiency in liver and small intestines developed Mn excess that was less severe than that observed in mice with whole-body Slc30a10 deficiency, suggesting that additional sites of Slc30a10 expression contribute to Mn homeostasis. Overall, these results indicated that Slc30a10 is essential for Mn excretion by hepatocytes and enterocytes and could be an effective target for pharmacological intervention to treat Mn toxicity.

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.

Peripheral nervous system (PNS) toxicity is surveyed inconsistently in nonclinical general toxicity studies. These Society of Toxicologic Pathology "best practice" recommendations are designed to ensure consistent, efficient, and effective sampling, processing, and evaluation of PNS tissues for four different situations encountered during nonclinical general toxicity (screening) and dedicated neurotoxicity studies. For toxicity studies where neurotoxicity is unknown or not anticipated (situation 1), PNS evaluation may be limited to one sensorimotor spinal nerve. If somatic PNS neurotoxicity is suspected (situation 2), analysis minimally should include three spinal nerves, multiple dorsal root ganglia, and a trigeminal ganglion. If autonomic PNS neuropathy is suspected (situation 3), parasympathetic and sympathetic ganglia should be assessed. For dedicated neurotoxicity studies where a neurotoxic effect is expected (situation 4), PNS sampling follows the strategy for situations 2 and/or 3, as dictated by functional or other compound/target-specific data. For all situations, bilateral sampling with unilateral processing is acceptable. For situations 1-3, PNS is processed conventionally (immersion in buffered formalin, paraffin embedding, and hematoxylin and eosin staining). For situation 4 (and situations 2 and 3 if resources and timing permit), perfusion fixation with methanol-free fixative is recommended. Where PNS neurotoxicity is suspected or likely, at least one (situations 2 and 3) or two (situation 4) nerve cross sections should be postfixed with glutaraldehyde and osmium before hard plastic resin embedding; soft plastic embedding is not a suitable substitute for hard plastic. Special methods may be used if warranted to further characterize PNS findings. Initial PNS analysis should be informed, not masked ("blinded"). Institutions may adapt these recommendations to fit their specific programmatic requirements but may need to explain in project documentation the rationale for their chosen PNS sampling, processing, and evaluation strategy.