Michael R. Elwell

Incidence of Nonneoplastic Lesions in Historical Control Male and Female Fischer-344 Rats from 90-Day Toxicity Studies

The incidence of all spontaneously occurring histologic lesions was determined for control Fischer-344 (F-344) rats from 90-day (13-wk) prechronic National Toxicology Program (NTP) toxicity studies. A total of 319 rats, represented by control groups of 10 males and 10 females each from dosed feed (n = 8), inhalation (n = 4), and gavage (n = 4) studies were included in the review. All protocol required tissues routinely collected for evaluation were reexamined for potential nonneoplastic and neoplastic lesions. Histopathologic findings in tissues included a spectrum of degenerative and inflammatory lesions. The most common lesions in male rats were nephropathy [145/160 (90.6%)] and cardiomyopathy [125/158 (79.1%)]. These changes were also present in the female rats, however, at much lower incidence rates [nephropathy = 30/157 (19.1%); cardiomyopathy = 36/158 (22.8%)]. Other less frequently occurring lesions included inflammation of the preputial [36/152 (23.7%)] and clitoral [34/155 (21.9%)] glands and inflammation of the liver consisting of either foci of mononuclear inflammatory cells [19/159 (11.9%) in males and 33/159 (20.8%) in females] or focal granulomatous inflammation [1/159 (0.6%) in males and 14/159 (8.8%) in females]. Pancreatic acinar cell atrophy occurred in both males [11/160 (6.9%)] and females [8/159 (5.0%)]. A variety of other less common nonneoplastic lesions were identified in both sexes of rats. Also recorded in this review are histologic changes generally considered to be components of the normal morphology of a particular tissue or organ for the F-344 rat (i.e., extramedullary hematopoiesis and hemosiderin deposition in the spleen, renal mineralization, uterine dilation, etc.). These findings were included and discussed due to potential treatment effects that may result in an increase or decrease in these changes compared to controls. Neoplasms were not observed in rats from the prechronic studies evaluated.

Influence of Dietary Protein Concentration on Severity of Nephropathy in Fischer-344 (F-344/N) Rats:

Nephropathy is an age-related spontaneous disease of most rat strains, and protein content of diet may affect the severity. The purpose of this study was to determine the effect of a 15% protein nonpurified diet on body weight and severity of nephropathy in comparison to a 23% protein NIH-07 diet. Groups of 25 male and 25 female Fischer-344 (F-344) rats, 6 wk of age, were fed the 23 or 15% protein diet ad libitum for 2 yr. Rats were weighed at 1–4-wk intervals, and mean body weights were determined. Water consumption measurements and urinalysis were done at approximately 3-mo intervals during the second year of the study. At the end of the 2-yr study, kidneys from all rats, including those that died or were euthanatized after the eightieth week of the study, were examined by light microscopy and graded for severity of nephropathy as grades 1–4 (minimal, mild, moderate, marked). Growth patterns and the maximum body weights attained by each sex fed the 23 or 15% protein diet were not significantly different. The severity of nephropathy in male rats was significantly higher when fed the 23% protein diet (2.8 moderate to marked) compared to the 15% protein diet (1.3 minimal to mild). The severity of nephropathy in female rats increased slightly when fed the 23% protein diet (1.5 minimal to mild) compared to the 15% protein diet (1.0 minimal). When the 23% protein diet group was compared to the 15% protein diet group, the 24-hr urine volume, proteinuria, and water consumption values were increased, especially during the last 3 mo of the study. Reduction of protein concentration in the diet from 23 to 15% decreased the protein consumption by30% and markedly decreased the severity of nephropathy, especially in male F-344 rats, without substantial changes in growth patterns and body weight gains.

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 concentration s 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 simultaneou s light and electron microscopic preparation by the pathologist of comparable specimens that can be stored long-term at 4°C in McDowells and Trumps 4F:1G fixative (4F:1G).

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.

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.

Pathology and Incidence of Amelanotic Melanomas of the Skin in F-344/N Rats

A total of 121 spontaneous amelanotic melanomas of the skin were identified in 70 of 11,171 male and 51 of 10,927 female Fischer-344/N rats in 63 2-yr carcinogenicity studies conducted by the National Cancer Institutes Carcinogenicity Testing Program/National Toxicology Program. Amelanotic melanomas had characteristic anatomical locations and histologic features distinguishable from Schwann cell tumors. Of the 121 tumors, 84, 19, 10, and 8 cases occurred in the pinna, eyelid, scrotum, and perianal region (anus and tail), respectively. Amelanotic melanomas originated from the dermis and consisted of spindle cells arranged in an interlacing fascicular pattern often with a perivascular orientation; epithelioid cells were rarely seen. Only the tumors arising in the pinna metastasized to the lung and/or mandibular lymph nodes. The metastatic rate was 19% (16/84) of the tumors and was clearly increased with an increase in tumor size. Most metastasizing tumors had focal areas consisting of anaplastic spindle cells with an increased number of mitosis. The tumor cells stained positive for S-100 protein but negative for melanin. Ultrastructurally, the tumors were diagnosed as amelanotic melanomas based on the identification of numerous, intracytoplasmic pre-melanosomes without melanin formation in the tumor cells which were not enveloped by pericytoplasmic basal laminae. One localized amelanotic melanoma of the pinna was successfully transplanted to the subcutaneous tissue in the flank of 3 Fischer-344/N rats.

Demonstration of a Temporal Relationship Between Ethyl Acrylate-Induced Forestomach Cell Proliferation and Carcinogenicity

Ethyl acrylate (EA) is a known forestomach carcinogen in both rats and mice. Recent work in this laboratory indicated that carcinogenic doses of EA administered by gavage for 13 wk resulted in a sustained increase in forestomach epithelial hyperplasia as long as exposure to EA continued. However, hyperplasia regressed and no forestomach neoplasms were seen after a 19-mo recovery period. Current studies were designed to further investigate the time required for sustained hyperplasia to lead to neoplasia as well as the organ specificity of EA-induced cell proliferation/hyperplasia vs carcinogenicity. EA was administered at 200 mg/ kg (po) to male Fischer-344 rats, 5 days/wk. Squamous cell proliferation/hyperplasia was observed in the forestomach of all rats that received EA for 6 or 12 mo. Treatment of rats with EA for 12 mo followed by 2 mo of recovery resulted in the development of forestomach papillomas in 2 of 5 treated rats. Furthermore, animals treated for 12 mo and allowed 9 mo of recovery exhibited an increase in forestomach squamous cell carcinomas and papillomas at a combined incidence of 4 in 13. In contrast, animals treated with EA for 6 mo and allowed 2 or 15 mo of recovery exhibited a time-dependent regression of cell proliferation and did not develop forestomach neoplasms. No significant elevation in liver cell proliferation or neoplasia was seen at any time in any of the rats included in the present study, further confirming the organ specificity in the relationship between EA-induced cell proliferation and carcinogenicity. In conclusion, EA resulted in increased cell proliferation in the target organ of carcinogenicity (forestomach) but not in nontarget organs such as the liver. This work indicates that cell proliferation, sustained for a sufficient period of time, results in the development of neoplasia despite cessation of chemical administration. Furthermore, a temporal relationship exists between EA-induced epithelial cell proliferation and forestomach carcinogenicity.

Necropsy Techniques with Standard Collection and Trimming of Tissues

Publisher Summary This chapter provided details related to performance of necropsy examination, tissue collection and fixation. Instructions for standard trimming and blocking of the common protocol-required tissues for microscopic evaluation are also provided. The necropsy examination, including tissue collection and preservation, represents one of the most critical phases for the histopathological evaluation of animals from toxicity and carcinogenicity studies. At the time of necropsy, fluids such as urine can frequently be obtained by using a tuberculin syringe and needle to aspirate the contents of the urinary bladder. There are various standard methods and sites for blood collection from rats during the in-life phase. They all require appropriate anesthesia and have certain limitations, depending on volume requirements and type of analyses to be performed on the sample. In exsanguination, the gauze is used primarily to prevent excessive blood staining and discoloration of surrounding tissues that might otherwise adversely affect the subsequent gross examination and tissue collection. Further, discussion addresses the most common fixatives and some of the basic rules governing good fixation. Most routine fixation is done by immersion. That is to say, the tissues are placed whole or in part into the fixative solution and fixed from the outside in. Perfusion is a much more effective method for rapid fixation. Perfusion may be done for selected organs of interest, or the whole body can be perfused. Normally, whole-body perfusion is done for neurotoxicity studies.

Seminoma, Testis, Rat

A seminoma reported in a rat was described as a soft, tan mass 1 cm in diameter that replaced most of the testicle (Kim et al. 1985).

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.