Inter-Rater and Intra-Rater Agreement in Scoring Severity of Rodent Cardiomyopathy and Relation to Artificial Intelligence-Based Scoring.

We previously developed a computer-assisted image analysis algorithm to detect and quantify the microscopic features of rodent progressive cardiomyopathy (PCM) in rat heart histologic sections and validated the results with a panel of five veterinary toxicologic pathologists using a multinomial logistic model. In this study, we assessed both the inter-rater and intra-rater agreement of the pathologists and compared pathologists' ratings to the artificial intelligence (AI)-predicted scores. Pathologists and the AI algorithm were presented with 500 slides of rodent heart. They quantified the amount of cardiomyopathy in each slide. A total of 200 of these slides were novel to this study, whereas 100 slides were intentionally selected for repetition from the previous study. After a washout period of more than six months, the repeated slides were examined to assess intra-rater agreement among pathologists. We found the intra-rater agreement to be substantial, with weighted Cohen's kappa values ranging from k = 0.64 to 0.80. Intra-rater variability is not a concern for the deterministic AI. The inter-rater agreement across pathologists was moderate (Cohen's kappa k = 0.56). These results demonstrate the utility of AI algorithms as a tool for pathologists to increase sensitivity and specificity for the histopathologic assessment of the heart in toxicology studies.

Scientific and Regulatory Policy Committee Points to Consider: Primary Digital Histopathology Evaluation and Peer Review for Good Laboratory Practice (GLP) Nonclinical Toxicology Studies.

The Society of Toxicologic Pathology's Scientific and Regulatory Policy Committee formed a working group to consider the present and future use of digital pathology in toxicologic pathology in general and specifically its use in primary evaluation and peer review in Good Laboratory Practice (GLP) environments. Digital histopathology systems can save costs by reducing travel, enhancing organizational flexibility, decreasing slide handling, improving collaboration, increasing access to historical images, and improving quality and efficiency through integration with laboratory information management systems. However, the resources to implement and operate a digital pathology system can be significant. Given the magnitude and risks involved in the decision to adopt digital histopathology, this working group used pertinent previously published survey results and its members' expertise to create a Points-to-Consider article to assist organizations with building and implementing digital pathology workflows. With the aim of providing a comprehensive perspective, the current publication summarizes aspects of digital whole-slide imaging relevant to nonclinical histopathology evaluations, and then presents points to consider applicable to both primary digital histopathology evaluation and digital peer review in GLP toxicology studies. The Supplemental Appendices provide additional tabulated resources.

Scientific and Regulatory Policy Committee Brief Communication: 2019 Survey on Use of Digital Histopathology Systems in Nonclinical Toxicology Studies.

Histopathologic evaluation and peer review using digital whole-slide images (WSIs) is a relatively new medium for assessing nonclinical toxicology studies in Good Laboratory Practice (GLP) environments. To better understand the present and future use of digital pathology in nonclinical toxicology studies, the Society of Toxicologic Pathology (STP) formed a working group to survey STP members with the goal of creating recommendations for implementation. The survey was administered in December 2019, immediately before the COVID-19 pandemic, and the results suggested that the use of digital histopathology for routine GLP histopathology assessment was not widespread. Subsequently, in follow-up correspondence during the pandemic, many responding institutions either began investigating or adopting digital WSI systems to reduce employee exposure to COVID-19. Therefore, the working group presents the survey results as a pre-pandemic baseline data set. Recommendations for use of WSI systems in GLP environments will be the subject of a separate publication.

Using Artificial Intelligence to Detect, Classify, and Objectively Score Severity of Rodent Cardiomyopathy.

Rodent progressive cardiomyopathy (PCM) encompasses a constellation of microscopic findings commonly seen as a spontaneous background change in rat and mouse hearts. Primary histologic features of PCM include varying degrees of cardiomyocyte degeneration/necrosis, mononuclear cell infiltration, and fibrosis. Mineralization can also occur. Cardiotoxicity may increase the incidence and severity of PCM, and toxicity-related morphologic changes can overlap with those of PCM. Consequently, sensitive and consistent detection and quantification of PCM features are needed to help differentiate spontaneous from test article-related findings. To address this, we developed a computer-assisted image analysis algorithm, facilitated by a fully convolutional network deep learning technique, to detect and quantify the microscopic features of PCM (degeneration/necrosis, fibrosis, mononuclear cell infiltration, mineralization) in rat heart histologic sections. The trained algorithm achieved high values for accuracy, intersection over union, and dice coefficient for each feature. Further, there was a strong positive correlation between the percentage area of the heart predicted to have PCM lesions by the algorithm and the median severity grade assigned by a panel of veterinary toxicologic pathologists following light microscopic evaluation. By providing objective and sensitive quantification of the microscopic features of PCM, deep learning algorithms could assist pathologists in discerning cardiotoxicity-associated changes.

Is It Adverse, Nonadverse, Adaptive, or Artifact?

One of the principal challenges facing a toxicologic pathologist is to determine and differentiate a true adverse effect from a nonadverse or an adaptive response. Recent publications from the Society of Toxicologic Pathology (STP) and the European STP provide guidance for determining and communicating adversity in nonclinical toxicology studies. In order to provide a forum to inform and engage in a discussion on this important topic, a continuing education (CE) course was held during the 2016 STP Annual meeting in San Diego, CA. The lectures at this course provided guidance on determining and communicating adversity using case studies involving both clinical pathology and anatomic pathology. In addition, one talk also focused on data quality, study design, and interpretation of artifacts that could hinder the determination of adversity. The CE course ended with a talk on understanding adversity in preclinical studies and engaging the regulatory agencies in the decision-making process. This manuscript is designed to provide brief summaries of all the talks in this well-received CE course.

Points to Consider in Designing and Conducting Juvenile Toxicology Studies.

In support of a clinical trial in the pediatric population, available nonclinical and clinical data provide input on the study design and safety monitoring considerations. When the existing data are lacking to support the safety of the planned pediatric clinical trial, a juvenile animal toxicity study is likely required. Usually a single relevant species, preferably a rodent, is chosen as the species of choice, while a nonrodent species can be appropriate when scientifically justified. Juvenile toxicology studies, in general, are complicated both conceptually and logistically. Development in young animals is a continuous process with different organs maturing at different rates and time. Structural and functional maturational differences have been shown to affect drug safety. Key points to consider in conducting a juvenile toxicology study include a comparative development of the organ systems, differences in the pharmacokinetics/absorption, distribution, metabolism, excretion (PK/ADME) profiles of the drug between young animal and child, and logistical requirement in the juvenile study design. The purpose of this publication is to note pertinent points to consider when designing and conducting juvenile toxicology studies and to aid in future modifications and enhancements of these studies to enable a superior predictability of safety of medicines in the pediatric population.

Juvenile Toxicology: Relevance and Challenges for Toxicologists and Pathologists.

The Society of Toxicologic Pathology (STP) Education Committee and the STP Reproductive Special Interest Group held a North Carolina regional meeting entitled, "Juvenile Toxicology: Relevance and Challenges for Toxicologists and Pathologists" on March 13, 2015, at the National Institute of Environmental Health Sciences/National Toxicology Program in Research Triangle Park, North Carolina. The purpose of this regional meeting was to familiarize attendees with the topic of juvenile toxicity testing and discuss its relevance to clinical pediatric medicine, regulatory perspectives, challenges of appropriate study design confronted by toxicologists, and challenges of histopathologic examination and interpretation of juvenile tissues faced by pathologists. The 1-day meeting was a success with over 60 attendees representing industry, government, research organizations, and academia.

(Q)SAR: A Tool for the Toxicologist.

A continuing education (CE) course at the 2014 American College of Toxicology annual meeting covered the topic of (Quantitative) Structure-Activity Relationships [(Q)SAR]. The (Q)SAR methodologies use predictive computer modeling based on predefined rules to describe the relationship between chemical structure and a chemical's associated biological activity or statistical tools to find correlations between biologic activity and the molecular structure or properties of a compound. The (Q)SAR has applications in risk assessment, drug discovery, and regulatory decision making. Pressure within industry to reduce the cost of drug development and societal pressure for government regulatory agencies to produce more accurate and timely risk assessment of drugs and chemicals have necessitated the use of (Q)SAR. Producing a high-quality (Q)SAR model depends on many factors including the choice of statistical methods and descriptors, but first and foremost the quality of the data input into the model. Understanding how a (Q)SAR model is developed and applied is critical to the successful use of such a tool. The CE session covered the basic principles of (Q)SAR, practical applications of these computational models in toxicology, how regulatory agencies use and interpret (Q)SAR models, and potential pitfalls of using them.

Investigation of the pulmonary bioactivity of double-walled carbon nanotubes.

Double-walled carbon nanotubes (DWCNT) are a rather new and unexplored variety of carbon nanotubes. Previously conducted studies established that exposure to a variety of carbon nanotubes produced lung inflammation and fibrosis in mice after pharyngeal aspiration. However, the bioactivity of double-walled carbon nanotubes (DWCNT) has not been determined. In this study, the hypothesis that DWCNT would induce pulmonary toxicity was explored by analyzing the pulmonary bioactivity of DWCNT. To test this hypothesis, C57Bl/6 mice were exposed to DWCNT by pharyngeal aspiration. Mice underwent whole-lung lavage (WLL) to assess pulmonary inflammation and injury, and lung tissue was examined histologically for development of pulmonary disease as a function of dose and time. The results showed that DWCNT exposure produced a dose-dependent increase in WLL polymorphonuclear leukocytes (PMN), indicating that DWCNT exposure initiated pulmonary inflammation. DWCNT exposure also produced a dose-dependent rise in lactate dehydrogenase (LDH) activity, as well as albumin levels, in WLL fluid, indicating that DWCNT exposure promoted cytotoxicity as well as decreases in the integrity of the blood-gas barrier in the lung, respectively. In addition, at 7 and 56 d postexposure, the presence of significant alveolitis and fibrosis was noted in mice exposed to 40 µg/mouse DWCNT. In conclusion, this study provides insight into previously uninvestigated pulmonary bioactivity of DWCNT exposure. Data indicate that DWCNT exposure promotes inflammation, injury, and fibrosis in the lung.

Susceptibility and lack of evidence for a viremic state of rabies in the night owl monkey, Aotus nancymaae.

BACKGROUND: Rabies causes an acute fatal encephalomyelitis in most mammals following infection with rhabdovirus of the genus Lyssavirus. Little is known about rabies virus infection in species of New World non-human Primates (NHP). To investigate the suitability of the owl monkey Aotus nancymaae asissue sections examined were unremarkable for inflammation or other histologic signs of rabies a viable animal model for rabies virus candidate vaccine testing, we used clinical presentation, serology, viral isolation, and PCR to evaluate the incubation period, immunity, and pathogenesis of infected animals. We tested the hypothesis that no viremic state exists for rabies virus. METHODS: Eight monkeys divided into two equal groups were inoculated intramuscularly either in the neck or footpad with 105 pfu of rabies virus (Pasteur/V-13R) and observed for >130 days. Oral and blood samples were collected and analyzed. RESULTS: Two monkeys inoculated in the neck displayed classic paralytic rabies. The mean incubation period was 11.5 days. The average maximum IgG response (antibody titer >0.200 O.D.) was achieved at day 10.0 and 62.3 in the clinical rabies and non-clinical rabies cases, respectively (p = 0.0429). No difference in IgM or IgG time to seroconversion or average maximum IgM level was observed between neck versus footpad inoculation groups. No viremia or viral shedding was detected by PCR or viral isolation during the observation period, including within the two symptomatic animals three days after disease onset. Tissue sections examined were unremarkable for inflammation or other histologic signs of rabies within the asymptomatic animal. Similarly none of the brain sections exhibited immunoreactivity for rabies virus antibody. DISCUSSION: This study demonstrates there is no difference in time to immune response between inoculation sites and distance to the brain; however, immune response tends to be more rapid in cases of clinically apparent disease and prolonged in cases infected at sites further from the brain. CONCLUSIONS: Our findings support the hypothesis that a viremic state for rabies does not exist in the New World Monkey, Aotus nancymaae, and it appears that this species may be refractory to infection. The species does provide a suitable model to assess post infection immune responses. Additional studies that address the limitations of sample size, length of observation, and lack of measurable infection should be conducted.

The effects of decreasing low-molecular weight hemoglobin components of hemoglobin-based oxygen carriers in swine with hemorrhagic shock.

BACKGROUND: Some hemoglobin-based oxygen carriers (HBOCs) improve outcome in animal models of hemorrhagic shock (HS) in comparison with standard asanguinous resuscitation fluids. Nevertheless, concern about intrinsic vasoactivity, linked in part to low-molecular weight (MW) hemoglobin (Hb), has slowed HBOC development. We assessed the impact of decreasing the low-MW Hb component of bovine HBOC on vasoactivity in severe HS. METHODS: Anesthetized invasively monitored swine were hemorrhaged 55% blood volume and resuscitated with bovine HBOC containing 31% (31 TD [HBOC-301]), 2% (2 TD [HBOC-201]), or 0.4% (0.4 TD) low-MW Hb. Pigs received four 10 mL/kg infusions over 60 minutes, hospital arrival was simulated at 75 minutes, organ blood flow (BF) was evaluated by microsphere injection, and monitoring was continued for 4 hours followed by complete necrotic evaluation. RESULTS: There were few differences between 2 TD and 0.4 TD. Thirty-one TD pigs had higher systemic and pulmonary blood pressure (BP), systemic vascular resistance index, and pulmonary artery wedge pressure, compared with 2 TD or 0.4 TD (p < 0.01); however, pigs in all groups had at least mildly elevated BP. Transcutaneous tissue oxygenation, base excess, and mixed venous oxygen saturation were similar across groups; lactate and methemoglobin were highest with 0.4 TD (p < 0.03). There were no group differences in BF. Over time, myocardial BF increased and hepatic BF decreased in all groups (for 31 TD, p < 0.05); renal BF was unchanged in all groups. There were no group differences in heart, lung, or liver histopathology, and survival. CONCLUSIONS: Although purification from 31% to 2% low-MW Hb content significantly decreased vasoactive responses, further purification to 0.4% had no additional clinically measurable effects in severe HS. If further diminution in HBOC vasoactivity is desired for use in HS, additional technical approaches may be required.

Karyomegaly and intranuclear inclusions in the renal tubules of sentinel ICR mice (mus musculus).

Among 585 sentinel ICR mice (Mus musculus), 8 (7 female, 1 male) had unusual microscopic lesions in the kidney. Light microscopy revealed occasional tubular epithelial cells with large, karyomegalic nuclei that contained intranuclear inclusions and marginated chromatin. These cells were randomly present in the cortex and medulla but were more prominent near the corticomedullary junc tion. Rare pyknotic cells and mild interstitial infiltrates of lymphocytes and plasma cells were associated with occasional foci of abnormal cells. Electron microscopy performed on 2 (1 female, 1 male) of the mice demonstrated intranuclear inclusions composed of abundant flocculent, electron-lucent material. No viral particles or other pathogens were identified. General health monitoring that included serology, microbiology, parasitology, necropsy, and histopathology was negative for pathogens. Polymerase chain reaction-based testing for polyomavirus and immunohistochemistry for adenovirus were performed on 5 of the 7 female mice; all were negative for both viruses. In light of microscopy findings and the lack of evidence for an infectious agent, the tubular lesions were considered degenerative changes, possibly due to a toxic insult. The cause and significance of the findings in these mice can not be explained fully.

Use of recombinant factor VIIa (rFVIIa) as pre-hospital treatment in a swine model of fluid percussion traumatic brain injury.

CONTEXT: Recombinant factor VIIa (rFVIIa) has been used as an adjunctive therapy for acute post-traumatic hemorrhage and reversal of iatrogenic coagulopathy in trauma patients in the hospital setting. However, investigations regarding its potential use in pre-hospital management of traumatic brain injury (TBI) have not been conducted extensively. AIMS: In the present study, we investigated the physiology, hematology and histology effects of a single pre-hospital bolus injection of rFVIIa compared to current clinical practice of no pre-hospital intervention in a swine model of moderate fluid percussion TBI. MATERIALS AND METHODS: Animals were randomized to receive either a bolus of rFVIIa (90 µg/kg) or nothing 15 minutes (T15) post-injury. Hospital arrival was simulated at T60, and animals were euthanized at experimental endpoint (T360). RESULTS: Survival was 100% in both groups; baseline physiology parameters were similar, vital signs were comparable. Animals that received rFVIIa demonstrated less hemorrhage in subarachnoid space (P = 0.0037) and less neuronal degeneration in left hippocampus, pons, and cerebellum (P = 0.00009, P = 0.00008, and P = 0.251, respectively). Immunohistochemical staining of brain sections showed less overall loss of microtubule-associated protein 2 (MAP2) and less Flouro-Jade B positive cells in rFVIIa-treated animals. CONCLUSIONS: Early pre-hospital administration of rFVIIa in this swine TBI model reduced neuronal necrosis and intracranial hemorrhage (ICH). These results merit further investigation of this approach in pre-hospital trauma care.

Sex-associated effects on hematologic and serum chemistry analytes in sand rats (Psammomys obesus).

We sought to determine whether sex had a significant effect on the hematologic and serum chemistry analytes in adult sand rats (Psammomys obesus) maintained under normal laboratory conditions. According to the few data available for this species, we hypothesized that levels of hematologic and serum chemistry analytes would not differ significantly between clinically normal male and female sand rats. Data analysis revealed several significant differences in hematologic parameters between male and female sand rats but none for serum biochemistry analytes. The following hematologic parameters were greater in male than in female sand rats: RBC count, hemoglobin, hematocrit, red cell hemoglobin content, and percentage monocytes. Red cell distribution width, hemoglobin distribution width, mean platelet volume, and percentage lymphocytes were greater in female than in male sand rats. The sex of adult sand rats is a source of variation that must be considered in terms of clinical and research data. The data presented here likely will prove useful in the veterinary medical management of sand rat colonies and provide baseline hematologic and serum chemistry analyte information for researchers wishing to use this species.

Determination of threshold adverse effect doses of percutaneous VX exposure in African green monkeys.

Percutaneous exposure to the chemical warfare nerve agent VX was evaluated in African green monkeys (n=9). Doses of VX (7.5-100 µg/kg) were applied to the skin for 60 min and residual agent was quantified (before decontamination) to estimate the absorbed dose. Monkeys were evaluated for the presence or absence of clinical signs of toxicity and blood was sampled periodically (30 min--12 weeks) following exposure to measure the degree of circulating acetylcholinesterase (AChE) inhibition. Monkeys were also evaluated for behavioral changes from VX exposure using a serial probe recognition (SPR) task. The lowest observable adverse effect level (LOAEL) for the production of major clinical signs was determined to be 42.22 µg/kg (absorbed dose estimate=17.36 µg/kg) and the LOAEL for AChE inhibition was 13.33 µg/kg (absorbed dose estimate=6.53 µg/kg). Behavioral performance was unaffected at doses that, while producing substantial AChE inhibition, did not produce clinical signs. VX represents a substantial threat as a contact hazard and these results complement previous studies using the percutaneous route of exposure with VX and extend the findings to a non-human primate species.

Pharmacokinetic and pharmacodynamic evaluation of intramuscular artesunate in healthy beagle dogs.

Pharmacokinetic and pharmacodynamic responses were evaluated after intramuscular (i.m.) injection of artesunate (AS). Twelve dogs were injected with i.m. AS at 2.5, 5, or 10 mg/kg into the left gluteal muscle. A second injection of only diluent was given in the right gluteal muscle. At 24 hours post-injection, plasma creatine kinase (CK) concentrations were elevated above normal. Muscle biopsies showed myocyte necrosis and acute inflammation, which was worse on the treated side. At 7 days after injection, CK concentrations were normal. Muscle biopsies showed mineralization, fibrosis, and chronic inflammation with less difference between sides. Compared with intravenous administration, i.m. AS resulted in a prolonged half-life for both AS and DHA. Intramuscular AS also had a lower mean dose-adjusted C(max) and a higher mean dose-adjusted area under the curve; but produced similar concentrations of dihydroartemisinin. These findings suggest that adverse reactions to i.m. artesunate are minor and temporary which justify further study of this route in treating severe malaria.