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.

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

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 minipig 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.

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

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) Project (www.toxpath.org/inhand.asp) is a joint initiative among the Societies of Toxicological 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 the endocrine organs (pituitary gland, pineal gland, thyroid gland, parathyroid glands, adrenal glands and pancreatic islets) of laboratory rats and mice, with color photomicrographs illustrating examples of the lesions. 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 and aging lesions as well as lesions induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for endocrine lesions in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Intranasal nanoemulsion-based inactivated respiratory syncytial virus vaccines protect against viral challenge in cotton rats.

Respiratory Syncytial Virus is a leading cause of bronchiolitis and pneumonia in infants, the elderly and individuals with compromised immune systems. Despite decades of research, there is currently no available vaccine for RSV. Our group has previously demonstrated that intranasal immunization of mice with RSV inactivated by and adjuvanted with W805EC nanoemulsion elicits robust humoral and cellular immune responses, resulting in protection against RSV infection. This protection was achieved without the induction of airway hyper-reactivity or a Th2-skewed immune response. The cotton rat Sigmodon hispidus has been used for years as an excellent small animal model of RSV disease. Thus, we extended these rodent studies to the more permissive cotton rat model. Intranasal immunization of the nanoemulsion-adjuvanted RSV vaccines induced high antibody titers and a robust Th1-skewed cellular response. Importantly, vaccination provided sterilizing cross-protective immunity against a heterologous RSV challenge and did not induce marked or severe histological effects or eosinophilia in the lung after viral challenge. Overall, these data demonstrate that nanoemulsion-formulated whole RSV vaccines are both safe and effective for immunization in multiple animal models.

Hemangiosarcoma in mice administered pregabalin: analysis of genotoxicity, tumor incidence, and tumor genetics.

Pregabalin, (S)-3-(aminomethyl)-5-methylhexanoic acid, binds with high affinity to the α(2)δ subunit of voltage-gated calcium channels and exerts analgesic, anxiolytic, and antiseizure activities. Two-year carcinogenicity studies were completed in B6C3F1 and CD-1 mice and two separate studies in Wistar rats. Doses in mice were 200, 1000, and 5000 mg/kg/day, with systemic exposures (AUC(0-24 h)) up to 31 times the mean exposure in humans, given the maximum recommended clinical dose. In rats, doses were 50, 150, and 450 mg/kg/day in males and 100, 300, and 900 mg/kg/day in females; systemic exposures up to 24 times were achieved in clinical trials. In both strains of mice, pregabalin treatment was associated with an increased incidence of hemangiosarcoma primarily in liver, spleen, and bone marrow. The incidence of hemangiosarcoma was higher in B6C3F1 mice than in CD-1 mice, consistent with its spontaneous incidence. Pregabalin did not increase the incidence of any other tumor type in rats and was not genotoxic, based on an extensive battery of in vivo and in vitro tests in bacterial and mammalian systems. Thus, pregabalin is a single-species, single tumor-type, nongenotoxic mouse carcinogen. Hemangiosarcomas occurring in mice treated with pregabalin were genotypically distinct from hemangiosarcomas induced by genotoxic carcinogens in humans with respect to ras and p53 mutation patterns and were similar to spontaneous tumors. Furthermore, there was a strong association between pregabalin treatment and bone marrow changes in these studies in mice, suggesting a possible link between the effects observed in bone marrow and the increase in tumor incidence in pregabalin-treated mice.

Mode of action associated with development of hemangiosarcoma in mice given pregabalin and assessment of human relevance.

Pregabalin increased the incidence of hemangiosarcomas in carcinogenicity studies of 2-year mice but was not tumorigenic in rats. Serum bicarbonate increased within 24 h of pregabalin administration in mice and rats. Rats compensated appropriately, but mice developed metabolic alkalosis and increased blood pH. Local tissue hypoxia and increased endothelial cell proliferation were also confirmed in mice alone. The combination of hypoxia and sustained increases in endothelial cell proliferation, angiogenic growth factors, dysregulated erythropoiesis, and macrophage activation is proposed as the key event in the mode of action (MOA) for hemangiosarcoma formation. Hemangiosarcomas occur spontaneously in untreated control mice but occur only rarely in humans. The International Programme on Chemical Safety and International Life Sciences Institute developed a Human Relevance Framework (HRF) analysis whereby presence or absence of key events can be used to assess human relevance. The HRF combines the MOA with an assessment of biologic plausibility in humans to assess human relevance. This manuscript compares the proposed MOA with Hill criteria, a component of the HRF, for strength, consistency, specificity, temporality, and dose response, with an assessment of key biomarkers in humans, species differences in response to disease conditions, and spontaneous incidence of hemangiosarcoma to evaluate human relevance. Lack of key biomarker events in the MOA in rats, monkeys, and humans supports a species-specific process and demonstrates that the tumor findings in mice are not relevant to humans at the clinical dose of pregabalin. Based on this collective dataset, clinical use of pregabalin would not pose an increased risk for hemangiosarcoma to humans.

Key components of the mode of action for hemangiosarcoma induction in pregabalin-treated mice: evidence of increased bicarbonate, dysregulated erythropoiesis, macrophage activation, and increased angiogenic growth factors in mice but not in rats.

In carcinogenicity studies, pregabalin increased hemangiosarcoma incidence in mice but not in rats. Investigative studies, ranging in length from 24 h to 12 months, were conducted in mice (1000 or 5000 mg/kg) and rats (900 mg/kg) to evaluate a potential mode-of-action scheme for tumor formation. Three areas were evaluated: (1) hematopoiesis (because endothelial and hematopoietic cells arise from the same precursor and hemangiosarcomas are primarily located in mouse hematopoietic tissues), (2) angiogenic growth factors (because increased angiogenic growth factors may stimulate vascular tumors), and (3) pulmonary/blood gas parameters (because hypoxia is a known driver for endothelial cell proliferation). In mice, pregabalin rapidly increased platelet and megakaryocyte counts, activated platelets and bone marrow erythrophages, decreased the myeloid-to-erythroid (M:E) ratio (49%), and produced bone marrow and splenic congestion and extramedullary hematopoiesis (EMH). Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor immunohistochemical staining were also increased in mouse bone marrow and spleen and vascular endothelial growth factor receptor 2 immunolabeling was increased in liver. Serum bicarbonate was increased within 24 h of pregabalin administration, persisted over time, and was accompanied by decreased respiratory rate (up to 34%) and increased partial pressure of carbon dioxide (pCO(2)), resulting in sustained metabolic alkalosis and elevated blood pH in mice. In contrast, in rats, pregabalin decreased overall bone marrow cellularity, including decreased number of megakaryocytes (24%) with no evidence of erythrophages, no change in M:E ratio, no EMH, and no increase in angiogenic growth factors or blood pH. Persistent alterations in serum bicarbonate, respiratory function, and blood gas parameters in mice, without adequate compensatory mechanisms, has the potential to create chronic tissue hypoxia, an accepted driver of endothelial cell proliferation.

Pregabalin induces hepatic hypoxia and increases endothelial cell proliferation in mice, a process inhibited by dietary vitamin E supplementation.

The preceding article identified key components of pregabalin's mode of action on nongenotoxic hemangiosarcoma formation in mice, including increased serum bicarbonate leading to decreased respiratory rate, increased blood pH, increased venous oxygen saturation, increased vascular endothelial growth factor and basic fibroblast growth factor expression, increased hepatic vascular endothelial growth factor receptor 2 expression, and increased iron-laden macrophages. Increased platelet count and platelet activation were early, species-specific biomarkers in mice. Dysregulated erythropoiesis, macrophage activation, and elevations of tissue growth factors were consistent with the unified mode of action for nongenotoxic hemangiosarcoma recently proposed at an international hemangiosarcoma workshop (Cohen, S. M., Storer, R. D., Criswell, K. A., Doerrer, N. G., Dellarco, V. L., Pegg, D. G., Wojcinski, Z. W., Malarkey, D. E., Jacobs, A. C., Klaunig, J. E., et al. (2009). Hemangiosarcoma in rodents: Mode-of-action evaluation and human relevance. Toxicol. Sci. 111, 4-18). In this article, we present evidence that pregabalin induces hypoxia and increases endothelial cell (EC) proliferation in a species-specific manner. Dietary administration of pregabalin produced a significant 35% increase in an immunohistochemical stain for hypoxia (Hypoxyprobe) in livers from pregabalin-treated mice. Increased Hypoxyprobe staining was not observed in the liver, bone marrow, or spleen of rats, supporting the hypothesis that pregabalin produces local tissue hypoxia in a species-specific manner. Transcriptional analysis supports that rats, unlike mice, adapt to pregabalin-induced hypoxia. Using a dual-label method, increased EC proliferation was observed as early as 2 weeks in mouse liver and 12 weeks in bone marrow following pregabalin administration. These same assays showed decreased EC proliferation in hepatic ECs of rats, further supporting species specificity. Dietary supplementation with vitamin E, which is known to have antioxidant and antiangiogenic activity, inhibited pregabalin-induced increases in mouse hepatic EC proliferation, providing confirmatory evidence for the proposed mode of action and its species-specific response.

Hemangiosarcoma in rodents: mode-of-action evaluation and human relevance.

Although rarely occurring in humans, hemangiosarcomas (HS) have become important in evaluating the potential human risk of several chemicals, including industrial, agricultural, and pharmaceutical agents. Spontaneous HS arise frequently in mice, less commonly in rats, and frequently in numerous breeds of dogs. This review explores knowledge gaps and uncertainties related to the mode of action (MOA) for the induction of HS in rodents, and evaluates the potential relevance for human risk. For genotoxic chemicals (vinyl chloride and thorotrast), significant information is available concerning the MOA. In contrast, numerous chemicals produce HS in rodents by nongenotoxic, proliferative mechanisms. An overall framework is presented, including direct and indirect actions on endothelial cells, paracrine effects in local tissues, activation of bone marrow endothelial precursor cells, and tissue hypoxia. Numerous obstacles are identified in investigations into the MOA for mouse HS and the relevance of the mouse tumors to humans, including lack of identifiable precursor lesions, usually late occurrence of the tumors, and complexities of endothelial biology. This review proposes a working MOA for HS induced by nongenotoxic compounds that can guide future research in this area. Importantly, a common MOA appears to exist for the nongenotoxic induction of HS, where there appears to be a convergence of multiple initiating events (e.g., hemolysis, decreased respiration, adipocyte growth) leading to either dysregulated angiogenesis and/or erythropoiesis that results from hypoxia and macrophage activation. These later events lead to the release of angiogenic growth factors and cytokines that stimulate endothelial cell proliferation, which, if sustained, provide the milieu that can lead to HS formation.