Guide for Combining Primary Tumors for Statistical Analysis in Rodent Carcinogenicity Studies.

The Tumor Combination Guide was created at the request of the U. S. Food and Drug Administration (FDA) by a Working Group of biopharmaceutical experts from international societies of toxicologic pathology, the Food and Drug Administration (FDA), and members of the Standard for Exchange of Nonclinical Data (SEND) initiative, to assist pharmacology/toxicology reviewers and biostatisticians in statistical analysis of nonclinical tumor data. The guide will also be useful to study and peer review pathologists in interpreting the tumor data. This guide provides a higher-level hierarchy of tumor types or categories correlating the tumor names from the International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) publications with those available in the NEOPLASM controlled terminology (CT) code list in SEND. The version of CT used in a study should be referenced in the nonclinical study data reviewer's guide (SDRG) (section 3.1) of electronic submissions to the FDA. The tumor combination guide instructions and examples are in a tabular format to make informed decisions for combining tumor data for statistical analysis. The strategy for combining tumor types for statistical analysis is based on scientific criteria gleaned from the current scientific literature; as SEND and INHAND terminology and information evolve, this guide will be updated.

Pancreatic Effects of a Bruton's Tyrosine Kinase Small-molecule Inhibitor in Rats Are Strain-dependent.

Inhibitors of Bruton's tyrosine kinase (BTK) are under development as potential therapies for various autoimmune diseases. In repeat-dose toxicity studies, small-molecule BTK inhibitors (BTKi) have been reported to cause a constellation of histologic effects at the pancreatic endocrine-exocrine interface in male rats; however, similar findings were not reported in other species. Since the BTKi-induced pancreatic effect is morphologically similar to well-documented spontaneous changes (predominantly characterized by insular/peri-insular hemorrhage, pigment deposition, chronic inflammation, and fibrosis) that are known to vary by rat strain, we investigated potential strain-dependent differences in the pancreatic effects of a small-molecule BTKi, LY3337641. Following 13 weeks of LY3337641 treatment, Crl:CD(SD) rats were most sensitive, Crl:WI(Han) rats were of intermediate sensitivity, and Hsd:SD rats were least sensitive. These strain differences appear to be related to differences in rate of weight gain across strains and sexes; however, a definitive mechanism was not determined. This study demonstrated that BTKi-induced pancreatic effects were highly dependent on rat strain and correlated with differences in the incidence and severity of the spontaneous background change. When considered with the lack of pancreas effects in nonrat species, these changes in rats are unlikely predictive of similar changes in humans administered a BTK inhibitor.

Scientific and Regulatory Policy Committee Points to Consider Review: Inclusion of Reproductive and Pathology End Points for Assessment of Reproductive and Developmental Toxicity in Pharmaceutical Drug Development.

Standard components of nonclinical toxicity testing for novel pharmaceuticals include clinical and anatomic pathology, as well as separate evaluation of effects on reproduction and development to inform clinical development and labeling. General study designs in regulatory guidances do not specifically mandate use of pathology or reproductive end points across all study types; thus, inclusion and use of these end points are variable. The Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathology (STP) formed a Working Group to assess the current guidelines and practices on the use of reproductive, anatomic pathology, and clinical pathology end points in general, reproductive, and developmental toxicology studies. The Working Group constructed a survey sent to pathologists and reproductive toxicologists, and responses from participating organizations were collected through the STP for evaluation by the Working Group. The regulatory context, relevant survey results, and collective experience of the Working Group are discussed and provide the basis of each assessment by study type. Overall, the current practice of including specific end points on a case-by-case basis is considered appropriate. Points to consider are summarized for inclusion of reproductive end points in general toxicity studies and for the informed use of pathology end points in reproductive and developmental toxicity studies.

Nonproliferative and Proliferative Lesions of the Gastrointestinal Tract, Pancreas and Salivary Glands of the Rat and Mouse.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) project 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 and diagnostic criteria for nonproliferative and proliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature and diagnostic criteria for classifying lesions in the digestive system including the salivary glands and the exocrine pancreas of laboratory rats and mice. Most lesions are illustrated by color photomicrographs. The standardized nomenclature, the diagnostic criteria, and the photomicrographs are 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 age related lesions as well as lesions induced by exposure to test items. Relevant infectious and parasitic lesions are included as well. A widely accepted and utilized international harmonization of nomenclature and diagnostic criteria for the digestive system will decrease misunderstandings 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.

Comparative pathophysiology, toxicology, and human cancer risk assessment of pharmaceutical-induced hibernoma.

In humans, hibernoma is a very rare, benign neoplasm of brown adipose tissue (BAT) that typically occurs at subcutaneous locations and is successfully treated by surgical excision. No single cause has been accepted to explain these very rare human tumors. In contrast, spontaneous hibernoma in rats is rare, often malignant, usually occurs in the thoracic or abdominal cavity, and metastases are common. In recent years, there has been an increased incidence of spontaneous hibernomas in rat carcinogenicity studies, but overall the occurrence remains relatively low and highly variable across studies. There have only been four reported examples of pharmaceutical-induced hibernoma in rat carcinogenicity studies. These include phentolamine, an alpha-adrenergic antagonist; varenicline, a nicotine partial agonist; tofacitinib, a Janus kinase (JAK) inhibitor; and hydromorphone, an opiod analgesic. Potential non-genotoxic mechanisms that may contribute to the pathogenesis of BAT activation/proliferation and/or subsequent hibernoma development in rats include: (1) physiological stimuli, (2) sympathetic stimulation, (3) peroxisome proliferator-activated receptor (PPAR) agonism, and/or (4) interference or inhibition of JAK/Signal Transducer and Activator of Transcription (JAK/STAT) signaling. The evaluation of an apparent increase of hibernoma in rats from 2-year carcinogenicity studies of novel pharmaceutical therapeutics and its relevance to human safety risk assessment is complex. One should consider: the genotoxicity of the test article, dose/exposure and safety margins, and pathophysiologic and morphologic differences and similarities of hibernoma between rats and humans. Hibernomas observed to date in carcinogenicity studies of pharmaceutical agents do not appear to be relevant for human risk at therapeutic dosages.

Pharmacological effects of nicotine on norepinephrine metabolism in rat brown adipose tissue: relevance to nicotinic therapies for smoking cessation.

In a two-year carcinogenicity study with administration of high doses of the partial nicotinic agonist varenicline (recently approved for smoking cessation), mediastinal hibernomas occurred in three male rats. To investigate potential mechanisms for partial and full nicotinic agonists to contribute to development of hibernomas, the effects of nicotine on rat brown adipose tissue (BAT) were studied. Male and female rats were administered nicotine at doses of 0, 0.3, and 1 mg/kg subcutaneously for fourteen days. Intrathoracic (mediastinal periaortic and mediastinal perithymic) BAT and interscapular BAT were examined microscopically, and determinations of uncoupling protein-1 (UCP-1) expression and norepinephrine (NE) content were made. Additionally, NE turnover was measured in mediastinal periaortic and perithymic BAT. Nicotine (1 mg/kg) administration resulted in decreased vacuolation only in mediastinal periaortic and mediastinal perithymic BAT of males and elevated UCP-1 in mediastinal periaortic BAT of males and females. Increased NE content occurred only in mediastinal periaortic BAT of males given 0.3 and 1 mg/kg doses, whereas NE turnover was decreased in both males and females given 1 mg/kg. Together, these data demonstrate that nicotine primarily affects mediastinal BAT in male rats, consistent with the gender and location of the hibernomas observed in the two-year carcinogenicity study.

Revised guides for organ sampling and trimming in rats and mice--Part 2. A joint publication of the RITA and NACAD groups.

This is the second part of a series of three articles on trimming instructions of rat and mouse protocol organs and tissues in regulatory type toxicity studies, covering the respiratory, male and female genital, and the endocrine systems. The article is based on the experience of the European RITA and American NACAD working groups and is an extended revision of trimming guides published in 1995 (Bahnemann et al.). The optimum localization for tissue preparation, the sample size, the direction of sectioning and the number of sections to be prepared is described organ by organ. These descriptions are illustrated for each organ by a schematic drawing and/or a macro-photograph showing the plane of section as well as a low magnification of the H&E stained slide demonstrating the optimum "end-product". The objectives of this work, as addressed in detail in the first part (Ruehl-Fehlert et al. 2003), are to standardize tissue sampling and trimming, to improve the comparability of historical data obtained from different studies and different laboratories, ensure the presence of all relevant target sites for histopathological evaluation and provide technical advice for preparatory techniques during necropsy, fixation and trimming. dardize tissue sampling and trimming, to improve the comparability of historical data obtained from different studies and different laboratories, ensure the presence of all relevant target sites for histopathological evaluation and provide technical advice for preparatory techniques during necropsy, fixation and trimming.

Histopathology of hemangiosarcomas in mice and hamsters and liposarcomas/fibrosarcomas in rats associated with PPAR agonists.

Peroxisome proliferator-activated receptors (PPAR) are involved in the pathogenesis of insulin resistance, diabetes, and related complications. Consequently, the identification of PPAR subtypes and the potential for their activation provides promising therapeutic targets for the management of type 2 diabetes mellitus. Available data from rodent carcinogenicity studies, however, demonstrate that PPAR agonists can be tumorigenic in one or more species of rodents at multiple sites. In 2005, the Health and Environmental Sciences Institute (HESI) PPAR Agonist Project Committee was established by a group of pharmaceutical companies to advance research on and to understand the modes of action and human relevance of this emerging rodent tumor data for PPAR agonists. Since the most commonly observed tumor types reported in rodents are hemangiosarcomas, fibrosarcomas and liposarcomas, the PPAR Agonist Project Committee approved a Pathology Working Group (PWG) to develop consensus of morphologic criteria for tumor diagnoses and consistency of diagnoses across multiple studies for hemangiosarcomas in mice and hamsters and liposarcomas/fibrosarcomas in rats. Therefore, the focus of the PWG review was to establish consistent tumor diagnostic criteria, to assess evidence of potentially preneoplastic changes and to identify distinguishing morphologic differences which may exist between spontaneous changes present in control animals with similar changes from treated animals. Specific diagnostic criteria and nomenclature are recommended for the classification of proliferative vascular lesions which may be present in mice or hamsters and for proliferative mesenchymal changes in rats in studies that are conducted with PPAR agonists.

Suggested Standard Operating Procedures (SOPs) for the preparation of electron microscopy samples for toxicology/pathology studies in a GLP environment.

We provide a set of Standard Operating Procedures (SOPs) for preparing samples for electron microscopic evaluation that allow storage of samples in the primary fixative for at least 17 years without noticeable degradation, do not compromise the ability to prepare the same samples for standard light microscopic evaluation, and provide tips for orientation of samples that may be necessary for evaluation. Guidelines for proper sample size, buffer composition, and fluid concentrations during processing are given. The impact of these procedures on specimen quality, ability to produce truly comparable samples for drug development studies, and ways to minimize time spent by technicians preparing these samples during necropsies is evaluated. Although many laboratories routinely employ most of these techniques, this compilation will facilitate the simultaneous light and electron microscopic preparation by the pathologist of comparable specimens that can be stored long-term at 4 degrees C in McDowell's and Trump's 4F:1G fixative (4F:1G).

Morphogenesis of postmortem hepatocyte vacuolation and liver weight increases in Sprague-Dawley rats.

Accurate interpretation of microscopic changes in tissues is critical in hazard identification and risk assessment. To address a possible confounder, the effects of postmortem interval on hepatocyte vacuolation and liver weight were studied in fasted and nonfasted Sprague-Dawley rats. Male and female rats (5/sex/interval) were euthanized with CO2, weighed, and necropsied either immediately or after remaining in the closed CO2 chamber for 5, 10, or 25 minutes after respirations ceased. The liver was removed, weighed, and fixed for light microscopy, immunohistochemistry, and electron microscopy. The liver weight and liver to body weight ratio increased significantly in both male and female rats. Postmortem hepatocellular vacuolation was more prominent in males than in females. Both fasted and nonfasted males were similarly affected, however, fasted females were affected more than nonfasted females at the 25-minute interval. Ultrastructurally, intracytoplasmic vacuoles in hepatocytes and/or endothelial cells contained electron-lucent material that was morphologically similar to plasma in sinusoidal spaces. Results of our study suggest that hepatocyte vacuoles were formed in a postmortem time-dependent manner as a result of plasma influx into the cytoplasm. This change was associated with hepatic sinusoidal congestion and increases in liver weight. Males were more sensitive than females to postmortem hepatocyte vacuolation.

Inhalation toxicology of octamethylcyclotetrasiloxane (D4) following a 3-month nose-only exposure in Fischer 344 rats.

Octamethylcyclotetrasiloxane (D4) is a low-molecular-weight cyclic siloxane used primarily in the synthesis of silicone polymers. The objective of the present study was to evaluate the subchronic toxicity of D4 following a 3-month nose-only inhalation exposure. Male and female Fischer 344 rats (20/sex/group) were exposed 6 h/day, 5 days/week for 3 months to vapor concentrations of 0, 35, 122, 488, and 898 ppm D4. Also, an additional 10 per sex in the control and high-exposure groups were allowed a 4-week recovery period to observe reversibility, persistence, or delayed occurrence of any potential adverse effects. Body weights and food consumption were monitored at least twice weekly over the course of exposures. Approximately 18 hours preceding euthanasia, animals were transferred into metabolism cages for urine collection, and were fasted. At necropsy, rats were anesthetized with pentobarbital and euthanized by exsanguination. Blood was collected for hematological and clinical biochemical analyses. Selected organ weights were measured and a complete set of tissues was taken for histopathological examination. A concentration-dependent increase in absolute and relative liver weight (488 to 898 ppm) and a significant decrease in ovarian weight (898 ppm) were observed in female rats. Exposure to D4 via nose-only inhalation (35 to 898 ppm) produced minor alterations in hematological and serum chemistry parameters that were considered either incidental and of little toxicological significance (hematology) or suggestive of metabolic adaptation/alteration (serum chemistry) in response to exposure-related hepatomegaly. There were no histopathological findings noted in the liver. Histopathological evidence indicated the primary target organs following D4 inhalation exposure to be components of the female reproductive tract. Reversible histopathological changes were observed in the ovary (hypoactivity) and vagina (mucification) of female rats in the high-dose group only (898 ppm). Although an increase in the incidence and severity of both macrophage accumulation, interstitial inflammation, and eosinophil infiltration was observed in the lungs of male and female rats exposed to D4, the toxicological significance is uncertain as other inhalation studies at similar concentrations failed to show these effects. In summary, nose-only inhalation of a high concentration of D4 resulted in reversible histopathological changes in the female rat reproductive tract. Lower concentrations did not elicit these same effects.