This editorial introduces the annual themed issue of the Journal of Pharmacological and Toxicological Methods (JPTM) dedicated to methodologies in safety pharmacology. The issue draws on content presented at the 2025 Safety Pharmacology Society Annual Meeting in Utrecht, The Netherlands, which featured 124 posters, many of which are reproduced as abstracts in this volume. The manuscripts primarily focus on advances in core battery safety evaluations and data analysis methodologies, while also highlighting emerging areas of investigation, including applications of artificial intelligence, digital monitoring technologies for enhanced safety assessment, in vitro assays recapitulating a range of organ systems and assessment of the hERG IC10 value as an alternative to the conventional IC50 value. The continued publication of these themed issues over the more than two decades underscores the importance of methodological innovation and evaluation, as well as the central role of safety pharmacology in driving these advancements.

5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) is a serotonin receptor agonist, in clinical development for the treatment of major depression and other psychiatric disorders. A critical safety concern for serotonergic compounds is their potential to induce seizures, as a severe manifestation of serotonin toxicity. The objective of this study was to evaluate the seizure liability of 5-MeO-DMT in a canine model. Beagle dogs (n = 8) were surgically instrumented for continuous telemetric recording of electroencephalography (EEG) and electromyography (EMG), coupled with behavioral video monitoring. Following a baseline period, animals were intranasally administered with control vehicle or escalating doses of 5-MeO-DMT (0.5, 1.0, and 1.5 mg/kg/day), once daily for nine consecutive days. Plasma samples for determination of pharmacokinetic parameters were collected from a satellite group (n = 3). Intranasal administration of 5-MeO-DMT resulted in centrally mediated and dose-dependent behavioral signs, such as head shaking, salivation, repetitive movements, dilated pupils, increased muscle tone, and tremors. A majority of these signs had a rapid onset and offset, which correlated with the peak plasma levels and resolved within 1 hour post-dose, and they were consistent with serotonergic agonism of 5-MeO-DMT. The continuous EEG recording revealed no evidence of seizures or epileptiform discharges at any dose levels throughout the study. In a canine model, considered sensitive to serotonergic drug-induced seizures, daily intranasal administration of escalating doses of 5-MeO-DMT did not induce seizures or epileptiform activity, even at doses causing significant physiological signs of serotonergic stimulation. These results provide robust critical safety information for a low seizure liability of 5-MeO-DMT.

Today, toxicologists have a diverse role in drug development and function in multidisciplinary teams with unique expertise. This article summarizes presentations from a continuing education (CE) course at the 2024 Annual Meeting of the American College of Toxicology (ACT), discussing different case studies with regulatory input. The six case studies from the CE course on regulatory toxicology in pharmaceutical drug development span small molecules, biologics, and gene therapy and illustrate key nonclinical challenges encountered across the development continuum, including formulation changes, impurities and degradants, route-specific toxicology, local tissue risk assessment, and interpretation of unexpected toxicity findings. Additional topics include evaluation of residual solvents, application of weight-of-evidence approaches, and integration of nonclinical and clinical data to support regulatory decision making. The case studies reflect real-world regulatory and scientific considerations across multiple stakeholder perspectives, including industry, contract research organizations, and regulatory agencies. Collectively, they demonstrate how toxicologists must integrate data from chemistry, manufacturing and controls (CMC), nonclinical studies, and clinical pharmacology to support risk assessment and clinical development strategies. Across the cases, common themes emerge, including increasing reliance on exposure-based assessments, application of integrated weight-of-evidence frameworks, adaptation of study designs to novel therapeutic modalities, and the importance of early and proactive regulatory engagement. These examples further highlight the need for toxicologists to manage uncertainty, interpret incomplete datasets, and provide scientifically justified recommendations within compressed development timelines. Overall, these case studies illustrate the evolving role of the toxicologist responsible for translating multidisciplinary data into regulatory strategies that enable safe and efficient drug development.

The rapidly evolving field of gene and nucleic acid therapy has presented exciting options for the treatment of many previously untreatable or poorly managed diseases and disorders. These therapies include several different families of molecular tools, such as antisense oligonucleotides (ASOs), designed to modulate gene and protein expression. In order to bring ASO-based therapies into a clinical setting, researchers must first assess their short- and long-term toxicity profiles in pre-clinical studies to establish parameters for safe clinical use. Here, a retrospective analysis of ASO-specific toxicology and safety pharmacology studies conducted across multiple test sites over a span of five (5) years are presented, with a focus on study designs and class effects. Emerging industry trends are highlighted with respect to dosing regimens, species selection, study duration, and study endpoints, and provide insights and recommendations on the design of future ASO therapy toxicology and safety pharmacology studies, including a commentary on biologically relevant dose ranges for these promising molecular tools. As well, ASO therapy -associated clinical signs, histopathological and clinical pathology findings are summarized to highlight class effects to consider during program design.

Central nervous system (CNS) toxicities remain a major cause of drug attrition and represent a persistent challenge in predicting neurological risk during drug development. Limitations in the predictive resolution and translational relevance of conventional nonclinical paradigms contribute to uncertainty in identifying and interpreting neurotoxicity signals. This manuscript examines key challenges in CNS safety assessment and highlights emerging strategies to improve early detection and prediction of neurological risk. Through a series of case studies, we demonstrate practical approaches for interpreting CNS safety signals and integrating emerging methodologies into nonclinical safety assessment. Examples include sensory and seizure-related endpoints in nonclinical studies and the use of electroencephalography (EEG) to improve detection and characterization of seizure liability. We also highlight the expanding role of advanced sensor technologies and artificial intelligence (AI) in enabling continuous, noninvasive monitoring of animal behavior. In addition, an Integrated Approach to Testing and Assessment (IATA) case study demonstrates how systematic integration of mechanistic data, traditional toxicology findings, and exposure modeling can support regulatory decision-making while aligning with the 3Rs principles (replace, reduce, refine animal testing). Finally, we present regulatory CNS case studies in drug development. Collectively, these approaches enable quantitative assessment of neurological function across circadian cycles, reduce reliance on episodic observer-dependent measurements, and illustrate how integrating refined in vivo methods with New Approach Methodologies (NAMs) and digital technologies can improve prediction of neurological risk and strengthen translation from nonclinical findings to human outcomes in CNS drug development.

Paroxysmal supraventricular tachycardia (PSVT) is a common cardiac arrhythmia associated with substantial health care burden. Etripamil, a fast-acting non-dihydropyridine calcium channel blocker developed for intranasal self-administration, is currently under investigation for acute treatment of PSVT episodes. A novel two-phase study design was used to test a series of five doses of intravenous etripamil (0, 0.025, 0.05, 0.15, and 0.3 mg/kg) in conscious cynomolgus monkeys. The cardiovascular effects (e.g., blood pressure and ECG recordings) were assessed during the first phase, and the pharmacokinetic profile was characterized during the second phase. Animals were dosed remotely to avoid the stress of intranasal dosing and minimize the impact of dose administration on measurements. Results were compared with findings from subsequent intranasal studies in cynomolgus monkeys and humans. Etripamil decreased systolic blood pressure and increased heart rate proportionately in a dose-dependent manner. Etripamil also induced dose-dependent increases in the PR interval. At a dose of 0.3 mg/kg (the highest dose), the mean highest PR prolongation from baseline during 20 minutes after dosing was 27.38%. Systemic exposure to etripamil increased in a dose-dependent manner. Mean area under the curve from administration to when drug was no longer present (AUC 0–∞ ) values ranged from 179 to 2364 ng • min/mL, peak plasma concentration ranged from 13.2 to 176 ng/mL, and mean half-life ranged from 12.3 to 20.8 minutes (Figure 1). Results were consistent with data from subsequent intranasal preclinical and clinical studies. Intravenous etripamil demonstrated the desired targeted pharmacokinetic and pharmacodynamic profiles in conscious cynomolgus monkeys.

The background incidence of common neurological clinical signs was evaluated in commonly used laboratory animal species. Salivation, tremors and ataxia can be considered as premonitory clinical signs to seizures but can also be observed in normal healthy animals in the absence of seizures. We characterized and compared the incidence of spontaneous convulsions, tremors, salivation and uncoordination/ataxia in control non-human primates, dogs, minipigs, rabbits, rats and mice from drug safety testing studies. A retrospective analysis was conducted with data from GLP facilities in North America and Europe including non-human primates (n = 8805), dogs (n = 24,553), minipigs (n = 2359), rabbits (n = 21,476), rats (n = 312,261) and mice (n = 131,272). Data from telemetered and jacketed animals were included. For rats and mice, the incidence of spontaneous convulsions was lowest at less than 6 weeks of age, was stable from 6 to 26 weeks of age and then increased progressively for older animals reaching 0.66 % in rats and 0.60 % in mice above 38 weeks. When comparing species, the incidence of spontaneous convulsion was lowest in minipigs (0 %) followed by mice (0.03 %), rats (0.06 %), rabbits (0.07 %), dogs (0.11 %) and non-human primates (0.17 %). Uncoordination/ataxia was observed in all species with increasing incidence in mice (0.04 %), minipigs (0.21 %), rats (0.23 %), rabbits (0.32), non-human primates (0.79 %) and dogs (1.00 %). As expected, dogs presented the highest incidence of salivation followed by non-human primates and minipigs. Spontaneous salivation was minimal in mice, rats and rabbits. Spontaneous tremors were observed in 1.6 % of dogs and non-human primates and reached 1 % in minipigs. The incidence of tremors in mice, rats and rabbits was negligible. The data summarized provides a robust characterization of spontaneous neurological clinical signs across multiple research facilities which can help during interpretation of safety pharmacology studies involving neurological assessments.

Seizures are complex electrophysiological disturbances affecting one or more populations of brain neurons. Seizures following test article (TA) exposure pose significant challenges in drug development. This paper considers the diverse neurological manifestations, mechanisms, and functional and structural assessments needed to investigate TA-related seizure liabilities, with a particular focus on nonclinical species. Accurate discrimination of seizures from convulsions (irregular involuntary body and/or limb movements) and the nuanced presentation of different seizure types (partial vs. general) and phases (prodromal, ictal, and postictal) are essential for discerning their clinical implications. In nonclinical safety testing, the most direct evaluation method to confirm existence of seizures is electroencephalography (EEG) while clinical endpoints (e.g., functional observational batteries [FOB], comprehensive neurological examinations) and neuropathological findings (e.g., neuronal necrosis in tissue sections, raised biomarker levels in cerebrospinal fluid or serum) can indicate a seizure liability and provide additional guidance to identify the origin, frequency, and severity of seizures needed to align nonclinical effects with clinical relevance. In general, the regulatory perspective is that seizures identified in nonclinical species as well as potential risk management strategies (e.g., safety margin considerations, dosing paradigms, and clinical monitoring) translate effectively for purposes of clinical risk assessment.

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.

QTc is an established biomarker for drug-induced Torsade de Pointes (TdP) but with concerns for a false positive signal. Clinically, JTpc and TpTec have emerged as ECG sub-intervals to differentiate predominant hERG vs. mixed ion channel blocking drugs that prolong QTc. Drugs that prolong QTc with increased TpTe, but without JTpc effects, are likely mixed ion channel blockers with low TdP risk. Drug effects on QTc, JTpc and TpTec were characterized with cynomolgus monkeys using telemetry in a Lead II configuration. Drugs and vehicle were administered orally to group sizes of 4 to 8 animals, in 4 laboratories. In monkeys, dofetilide (0.03–0.3 mg/kg, PO) was associated with exposure dependent QTc and JTpc increases but no significant TpTec effect. Quinidine (2 to 50 mg/kg, PO) increased QTc and JTpc but did not change TpTec. Mexiletine (1–15 mg/kg, PO) and verapamil (50 mg/kg, PO) did not induce any significant effect on QTc, JTpc or TpTec. Clinically, predominant hERG blockers (dofetilide and quinidine) prolong QTc, JTpc and TpTec and are associated with an increased risk for TdP. Results from the current study demonstrate that ECG changes after dofetilide and quinidine administration to telemetered monkeys differ from the clinical response, lacking the expected effects on TpTec. Potential explanations for the lack of translation include physio-pharmacology species differences or ECG recording and analysis methodology variations. Mixed ion channel blockers verapamil and mexiletine administered to monkeys showed no significant QTc, JTpc or TpTec prolongation as expected based on the similar clinical response for these agents.

This editorial prefaces the annual themed issue on those methods with application to safety pharmacology (SP) in the Journal of Pharmacological and Toxicological Methods (JPTM). Highlighted content is derived from the 2024 Safety Pharmacology Society (SPS) meeting held in San Diego, CA, USA. The meeting showcased 122 posters, many of which are reproduced as abstracts published in JPTM. The manuscripts predominantly reflect updates to core battery safety evaluation and data analysis methods and include areas of novel investigation within SP. The results from several surveys including an updated salary survey by the SPS, current industry practices on neurotoxicity by the ACT, SPS and STP and results on the need to revisit the ICH S7A guidance on safety and secondary pharmacology by EFPIA, JPMA, and PhRMA provide timely updates. Other manuscripts include in vitro assessment methods for sodium channel block using MEA arrays, and a comparison of the in vitro effects of positive control drugs on the hERG channel current using different testing procedures. In vivo cardiovascular manuscripts include an overview of surgical telemetry implantation methods, utility of automated blood sampling methods in SP studies, evaluation of the performance characteristics for common QTc data collection methods, and an evaluation of a clinically used wearable ECG device for use in SP studies. There is also an overview of the impact of respiratory SP and a comprehensive review and comparison of current practices regarding methodological approaches used to acquire CV data in repeat-dose non-rodent toxicology studies. The 21 years of consecutive themed issues on SP methods attends to the importance of methods evaluation and the contribution of SP to the process of methods evaluation.

In the event of a large-scale radiological emergency, delivering timely medical aid to individuals receiving potentially lethal doses of radiation will result in improved survival and decreased severity of injuries. While it may be possible to reconstruct a dose estimate based on a location during the event and/or early symptoms presenting after the event, limitations with readily available information and inaccuracy of that estimate may not provide enough certainty for successful medical triage. Thus, individual biodosimetry assessments would assist medical professionals in providing prompt care to those who would benefit the most. In this study, a variety of accessible biospecimens (blood, plasma, serum, feces, saliva, and urine) from eight rhesus macaques irradiated with a single total body sublethal dose of 4 Gy of 60Co γ rays were collected before and up to 60 days after exposure for distribution to 10 different investigators' work sites for site-specific analyses. Results showing statistically significant changes in hematology parameters as well as gene, protein, and metabolite expression have since been published. Here, these results are combined and integrated with new data from microRNA (miRNA) expression in plasma samples as well as 16S rRNA sequencing and metabolomics data from fecal samples. A total of 40 unique miRNAs were significantly expressed on days 3, 6, 30, or 60. Metabolomic analysis of fecal samples found changes in multiple pathways, including steroid hormones, C18 (sex) hormones, and bile acid synthesis. Temporal changes were found in the gut microbiome for microbial abundance and richness. Finally, a retrospective view of the collective results demonstrated common overlapping pathways that were enriched from significantly altered biomarkers. This large, collaborative study from a single irradiated cohort demonstrates the utility of multiple timepoints, biospecimen types, and omics technologies that collectively identified 61 common biomarkers across 4 omics platforms that were enriched for pathways relevant to an acute radiation injury to the hematopoietic system that may aid future radiation biodosimetry efforts.

The second salary survey of safety pharmacology professionals was conducted in late 2023. This electronic survey was distributed to all members of the Safety Pharmacology Society (SPS). Survey questions assessed demographic variables, professional background, employment type, and annual incomes. Of 463 active SPS members, 120 responses were received. A majority of the responses (56 %) were from North America, followed by Europe (35 %), then Asia-Pacific (9 %). The results of the 2023 salary survey highlight the continued growth in salaries in the field of safety pharmacology and may serve as a valuable resource for current and future safety pharmacologists.