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The inhalation route is a relatively novel drug delivery route for biotherapeutics and, as a result, there is a paucity of published data and experience within the toxicology/pathology community. In recent years, findings arising in toxicology studies with inhaled biologics have provoked concern and regulatory challenges due, in part, to the lack of understanding of the expected pathology, mechanisms, and adversity induced by this mode of delivery. In this manuscript, the authors describe 12 case studies, comprising 18 toxicology studies, using a range of inhaled biotherapeutics (monoclonal antibodies, fragment antigen-binding antibodies, domain antibodies, therapeutic proteins/peptides, and an oligonucleotide) in rodents, nonhuman primates (NHPs), and the rabbit in subacute (1 week) to chronic (26 weeks) toxicology studies. Analysis of the data revealed that many of these molecules were associated with a characteristic pattern of toxicity with high levels of immunogenicity. Microscopic changes in the airways consisted of a predominantly lymphoid perivascular/peribronchiolar (PV/PB) mononuclear inflammatory cell (MIC) infiltrate, whereas changes in the terminal airways/alveoli were characterized by simple (“uncomplicated”) increases in macrophages or inflammatory cell infiltrates ranging from mixed inflammatory cell infiltration to inflammation. The PV/PB MIC changes were considered most likely secondary to immunogenicity, whereas simple increases in alveolar macrophages were most likely secondary to clearance mechanisms. Alveolar inflammatory cell infiltrates and inflammation were likely induced by immune modulation or stimulation through pharmacologic effects on target biology or type III hypersensitivity (immune complex disease). Finally, a group of experts provide introductory thoughts regarding the adversity of inhaled biotherapeutics and the basis for reasonable differences of opinion that might arise between toxicologists, pathologists, and regulators.

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 lesions observed in most tissues and organs from the dog 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, lesions induced by exposure to test materials, and relevant infectious and parasitic lesions. 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.

Afrezza delivers inhaled insulin using the Gen2 inhaler for the treatment of patients with type 1 and type 2 Diabetes. Afrezza was evaluated in long-term nonclinical pulmonary safety studies in 2 toxicology species. Chronic inhalation toxicology studies in rat (26 weeks) and dog (39 weeks) and an inhalation carcinogenicity study in rats were conducted with Technosphere insulin (Afrezza) and with Technosphere alone as a vehicle control. Respiratory tract tissues were evaluated by histopathology and cells expressing proliferating cell nuclear antigen (PCNA) were quantified in lungs of rats. Microscopic findings in rats exposed to Afrezza were attributed to the Technosphere particle component, were confined to nasal epithelia, and consisted of eosinophilic globules and nasal epithelial degeneration. There were no Afrezza-related changes in pulmonary PCNA labeling indices in alveoli, large bronchioles, or terminal bronchioles. Microscopic findings in rats exposed to Technosphere particles included eosinophilic globules, mucus cell hyperplasia, and epithelial degeneration in the nasal cavities. PCNA labeling indices were increased in large bronchioles and terminal bronchioles but not in alveoli. There were no Technosphere particle-related findings in the dog study. Afrezza did not exhibit carcinogenic potential in the 2-year study in rats. These nonclinical inhalation studies support the use of Afrezza in humans over extended periods.

The Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathology (STP) appointed a working group to address risk assessment for increases in alveolar macrophages following inhalation of pharmaceutical materials. This position paper provides recommendations for inhalation study–specific terminology and interpretation based on literature and information from marketed inhaled drugs. Based on a weight-of-the-evidence approach, and with appropriate consideration of the physical and pharmacological characteristics of the compound, uncomplicated increases in the size or number of alveolar macrophages in nonclinical species are interpreted as nonadverse.

Exposure to moderately selective p38α mitogen-activated protein kinase (MAPK) inhibitors in the Beagle dog results in an acute toxicity consisting of mild clinical signs (decreased activity, diarrhea, and fever), lymphoid necrosis and depletion in the gut-associated lymphoid tissue (GALT), mesenteric lymph nodes and spleen, and linear colonic and cecal mucosal hemorrhages. Lymphocyte apoptosis and necrosis in the GALT is the earliest and most prominent histopathologic change observed, followed temporally by neutrophilic infiltration and acute inflammation of the lymph nodes and spleen and multifocal mucosal epithelial necrosis and linear hemorrhages in the colon and cecum. These effects are not observed in the mouse, rat, or cynomolgus monkey. To further characterize the acute toxicity in the dog, a series of in vivo, in vitro, and immunohistochemical studies were conducted to determine the relationship between the lymphoid and gastrointestinal (GI) toxicity and p38 MAPK inhibition. Results of these studies demonstrate a direct correlation between p38α MAPK inhibition and the acute lymphoid and gastrointestinal toxicity in the dog. Similar effects were observed following exposure to inhibitors of MAPK-activated protein kinase-2 (MK2), further implicating the role of p38α MAPK signaling pathway inhibition in these effects. Based on these findings, the authors conclude that p38α MAPK inhibition results in acute lymphoid and GI toxicity in the dog and is unique among the species evaluated in these studies.

Necropsies conducted to support medical device development may be conducted in facilities that do not have the infrastructure in place to support Good Laboratory Practice for Nonclinical Laboratory Studies (GLP) studies and for a variety of reasons they may be conducted without a pathologist present at necropsy. However, when a novel medical device or one that is expected to significantly alter tissues is deployed, or when the surgical model confounds interpretation of device effects, it is the opinion of the authors that an experienced pathologist should be present at necropsy.

Inhalation of beryllium (Be) has been associated with 2 syndromes: an acute chemical pneumonitis and a granulomatous lung disease known as chronic beryllium disease (CBD). Key to the pathogenesis of CBD is a delayed-type hypersensitivity reaction, in which Be most likely functions as a hapten and acts as a Class II-restricted antigen, stimulating local proliferation and accumulation in the lung of Be-specific CD4+ T cells. The purpose of this study was to establish a mouse model of CBD using the inhalation route of exposure. A/J (H-2a haplotype) and C3H/HeJ (H-2k) mice were exposed once for 90 min in nose-only exposure tubes to aerosols of Be metal. Six mo later, lung histopathologic responses were assessed. Further analyses defined the phenotypic profile of lymphocytes in pulmonary lesions and evaluated proliferation of lymphocytes in situ and in response to Be in vitro. Responses were similar in both strains of mice. The lungs of all Be-exposed mice had interstitial compact aggregates of lymphocytes, and granulomatous pneumonia characterized by vacuolated macrophages and giant cells in alveoli, neutrophils in alveoli and alveolar septa, multifocal interstitial granulomas, and interstitial infiltrates of lymphocytes, plasma cells, monocytes, and macrophages. Most Be-exposed mice had minimal to mild interstitial fibrosis. The majority of lymphocytes in interstitial infiltrates and in microgranulomas were CD4+ T cells. Interstitial compact aggregates of lymphocytes contained B cells centrally and CD4+ cells peripherally. Lymphocyte labeling indices, used to assess proliferation in situ, were significantly greater within microgranulomas compared to compact lymphocytic aggregates. Lymphocyte stimulation indices in response to BeSO4 in vitro were not positive in blood, spleen, or tracheobronchial lymph node samples. Be-specific immune responses and nonspecific inflammatory responses to toxic and foreign-body properties of Be may have contributed to the histopathology in both strains of mice. The interstitial mononuclear cell infiltrates, presence of microgranulomas, multinucleated foreign-body and Langhans' giant cells, interstitial fibrosis, and CD4+ T-cell predominance with local proliferation are features similar to CBD in humans. The chronic lung disease induced in these mice by inhaled Be can be used to investigate the importance of variables such as dose, exposure pattern, and physicochemical form of Be in producing this disease.

Most government regulatory guidelines do not provide speciŽ c details regarding the method of Ž xation of respiratory tract tissues for toxicity studies. Differences of opinion exist among regulatory agency personnel regarding the optimal methods for routine formalin Ž xation of lungs from rodent toxicology studies. Although there is a consensus that intratracheal instillation of Ž xative provides the best preservation of airways and alveoli, concern was expressed by toxicologists responsible for evaluation of toxicity study data for regulatory purposes. The main concerns were that intratracheal infusion of Ž xative could signiŽ cantly mask subtle in ammatory changes by washing out lung exudates and in ammatory cells, may produce artifactual peribronchiolar and perivascular cuffs resulting from diffusion of Ž xative into these areas, and may rupture alveolar walls, inducing artifacts that could be interpreted as emphysema. These concerns stimulated a request from the US. Environmental Protection Agency to the Society of Toxicologic Pathology to address this issue. A subcommittee consisting of the authors of this document was formed to review the various methods of lung Ž xation with formalin for use in rodent toxicology studies and the advantages and disadvantages of each method, and to recommend the most suitable method for rodent toxicology studies.

The transgenic heterozygous p53+/- knockout mouse has been a model for assessing the tumorigenicity of selected carcinogens administered by noninhalation routes of exposure. The sensitivity of the model for predicting cancer by inhaled chemicals has not been examined. This study addresses this issue by acutely exposing p53+/- mice of both sexes by nose-only inhalation to either air (controls), or to 1 of 2 levels of 239PuO2 (500 or 100 Bq 239Pu) or beryllium (Be) metal (60 or 15 μg). Additional wild-type p53+/- mice were exposed by inhalation to either 500 Bq of 239PuO2 or 60 μg of Be metal. These carcinogens were selected because they operate by differing mechanisms and because of their use in other pulmonary carcinogenesis studies in our laboratory. Four or 5 of the 15 mice per sex from each group were sacrificed 6 mo after exposure, and only 2 pulmonary neoplasms were observed. The remainder of the mice were held for life-span observation and euthanasia as they became moribund. Survival of the p53+/- knockout mice was reduced compared to the p53+/+ wild-type mice. No lung neoplasms were observed in p53+/- mice exposed to air alone. Eleven of the p53+/- mice inhaling 239PuO2 developed pulmonary neoplasms. Seven p53+/+ mice exposed to 239PuO2 also developed pulmonary neoplasms, but the latency period for pulmonary neoplasia was significantly shorter in the p53+/- mice. Four pulmonary neoplasms were observed in p53+/- mice exposed to the higher dose of Be, whereas none were observed in the wild-type mice or in the heterozygous mice exposed to the lower dose of Be. Thus, both p53+/- and p53+/+ mice were susceptible to 239Pu-induced carcinogenesis, whereas the 53+/- but not the p53+/+ mice were susceptible to Be-induced carcinogenesis. However, only 2 pulmonary neoplasms (1 in each of the 239PuO2 exposure groups) were observed in the 59 p53+/- mice that were sacrificed or euthanatized within 9 mo after exposure, indicating that the p53+/- knockout mouse might not be appropriate for a 6-mo model of carcinogenesis for these inhaled carcinogens.