Transition of Historical Control Data for High Incidence Tumors in F344 Rats

Historical control data of tumor incidence were collected from the control groups (215 animals of each sex) in four recent carcinogenicity studies that were started between 2005 to 2009 (terminally sacrificed between 2007 and 2011) at BoZo Research Center Inc. (Gotemba, Shizuoka, Japan) using Fischer 344 rats (F344/DuCrlCrlj). These data were compared to the previous historical control data (from 1990 to 2004, previously reported) in the same facility. In the results, the incidence of C-cell adenoma in the thyroid tended to increase in both sexes in recent years (30.8% for males and 24.4% for females in 2005-2009) as compared with the previous data (17.4% and 20.1% for males and 11.5% and 11.8% for females in 1990–1999 and 2000–2004, respectively). In addition, the incidences of pancreatic islet cell adenoma in males and uterine adenocarcinoma tended to increase from around 2000 and remained high in recent years (incidences of islet cell adenoma in males of 10.5%, 17.1% and 20.5% in 1990–1999, 2000–2004 and 2005–2009; incidences of uterine adenocarcinoma of 3.3%, 12.0% and 13.5% in 1990–1999, 2000–2004 and 2005–2009, respectively). There was no apparent difference in the incidence of other tumors.     

Malignant Lymphoma with Severe Infiltrative Growth into Skeletal Muscles in WBN/Kob Rats

Although spontaneously occurring neoplasms have been reported repeatedly in F344, SD and Wistar rats, which are commonly used strains for routine toxicologic and carcinogenicity studies, there are only a few reports of malignant lymphoma or lymphatic leukemia except for large granular lymphocytic leukemia (LGL) in F344 rats. Malignant lymphoma (lymphosarcoma) is thought to be uncommon in F344 rats. The authors encountered malignant lymphomas of the non-LGL leukemia type with characteristic pathologic features in WBN/Kob rats. The mean age at onset of the disease in all 13 affected rats (8 males and 5 females) was about 60 weeks. Common and characteristic clinical signs were abnormal gait with hind limb paralysis. Macroscopically, the enlargement of the lymph nodes, spleen and liver was slight to moderate. Scattered multiple white-to-gray nodules encompassed the aorta and assumed a bead-like appearance near the thoracic and lumbar vertebrae. Histopathologically, neoplastic proliferative changes were predominant in the bone marrow tissue of the entire body, and many tumor cells infiltrated the spleen and several lymph nodes. The most striking histological features were constant and severe infiltration of tumor cells in the adipose tissue and skeletal muscle adjacent the thoracic and lumber vertebrae. Immunohistochemically, all tumor cells were positive for B-cell markers (PAX-5, CD79a and CD45) and negative for CD3. From the results of immunohistochemistry and morphological examination, these tumors were diagnosed as malignant B-cell lymphomas.     

Background data of 2-year-old male and female F344 gpt delta rats

Although gpt delta rats, as reporter gene-transgenic rats, were originally developed for in vivo mutation assays, they have also been used to evaluate chemical carcinogenesis and comprehensive toxicity. Therefore, it is necessary to accumulate background data on carcinogenicity and general toxicity in gpt delta rats. Here, we investigated the background data of 110-week-old male and female F344 gpt delta rats and wild-type rats. There was no effect of reporter gene transfection on animal survival rates and body weights during the experiment. The relative weight of male gpt delta rat adrenals was significantly higher than that of wild-type rats, possibly due to the higher incidence of pheochromocytoma. There were no intergenotype differences in the incidence of nonneoplastic lesions in both sexes, including chronic progressive nephropathy and focus of cellular alteration in the liver, which had a higher incidence in both genotypes. Additionally, the significantly higher incidence of adrenal pheochromocytoma in male gpt delta rats than that in wild-type rats was likely incidental because of the lack of differences in the incidences of preneoplastic (male and female) and neoplastic (female) adrenal lesions in both genotypes. Other neoplastic lesions in both sexes showed no intergenotype differences in incidence rates, although large granular lymphocytic leukemia in the spleen and Leydig cell tumors in the testes of males showed higher incidence rates. Overall, there were no effects of reporter gene transfection on the spectrum of spontaneous lesions in F344 gpt delta rats, thus supporting their applicability in evaluating chemical toxicity and carcinogenicity.     

Histopathological and immunohistochemical features of proliferative lesions in the pituitary pars distalis of rats

Pituitary proliferative lesions derived from the endocrine cells of the pars distalis are frequently encountered and adenomas/carcinomas are a common cause of death in standard 2-year carcinogenicity studies using various rat strains, especially Sprague-Dawley. This report describes the immunohistochemical characteristics of pituitary tumors derived from the pars distalis in rats. Prolactin (PRL)-containing tumors are the most common, with PRL/growth hormone (GH) dual positive tumor masses (PRL/GH co-positive tumor masses) being more prevalent than only PRL-positive tumor masses (PRL single-positive tumor masses). GH-containing tumors are relatively numerous and many of these are also PRL/GH co-positive tumor masses. TSH-containing tumors are common in females. PRL-containing tumors have been shown to increase the incidence of hyperlactation in males and mammary adenomas/adenocarcinomas in females, suggesting that these masses are functional tumors.     

Spontaneous Harderian Gland Adenocarcinoma in a Female F344 Rat: A Case Report

Harderian gland tumors are extremely rare in female F344 rats. An expansive enlarging lesion of the Harderian gland with compression, distortion and invasion of the surrounding muscle was found in a 110-week-old female F344/DuCrj rat, which was diagnosed as a Harderian gland adenocarcinoma. Epithelial growth patterns such as glandular, lobular, papillary and duct forming patterns were exhibited in most areas of the tumor. The tumor cells were pleomorphic and atypical. In one part of the tumor, poorly differentiated areas were found. This case was observed in the middle dose group of a carcinogenicity study of diphenylamine, which was not carcinogenic, we determine to be this case was a spontaneous tumor.     

Natural occurrence of neoplastic lesions in young sprague-dawley rats

It is important for the assessment of toxicological effects of chemicals to know what kinds of neoplasms naturally occur in the early life of experimental animals. In the present study, we demonstrated spontaneous neoplasms in Sprague-Dawley rats used in 4-, 13- and 26-week toxicity studies conducted at Bozo Research Center in the last decade. The tumors, which were first observed in 19-week-old animals, included anterior adenoma of the pituitary, follicular cell adenocarcinoma and C cell adenoma of the thyroids, nephroblastoma of the kidneys, basal cell tumor of the skin and malignant lymphoma. Thereafter, hemangiosarcoma of the tongue, adenocarcinoma of the submandibular glands, histiocytic sarcoma of the spleen, oligodendroglioma of the brain and adenocarcinoma and fibroadenoma of the mammary glands were detected in 32-week-old animals. The incidences of mammary adenocarcinoma and pituitary anterior adenoma were higher than those of other tumors. The present results showed that the same tumors as reported in aged rats could also develop in younger rats.     

Orbital malignant schwannoma and embryonal rhabdomyosarcoma in rats caused by leakage of locally injected nickel subsulfide to the orbit

Nickel subsulfide (Ni₃S₂) is known to induce intraocular neoplasms when injected intravitreally into the eyes of rats. Here, we found two extraocular orbital neoplasms in two different rat strains, presumably due to the leakage of locally injected Ni₃S₂ to the extraocular orbital tissues. In the F344/DuCrlCrlj rat, an orbital mass arose at 30 weeks after injection, and invaded into the cranium. Histologically, the orbital mass was composed of areas arranged in parallel bundles formed by densely packed elongated or spindle-shaped cells with indistinct cytoplasmic borders, and of areas of hypocellular arrangement consisting of round cells in eosinophilic myxoid-like substances. Metastases were observed in the right submandibular and cervical lymph nodes. The neoplastic cells were immunopositive for S-100 protein and vimentin. Transmission electron microscopy revealed that the neoplastic cells had cellular processes and pericytoplasmic basal laminae. In the RccHan^TM:WIST rat, an orbital mass arose at 36 weeks after injection. Histologically, the mass consisted of rhabdoid-like large round cells with proliferation of small round-to-polygonal cells, and these neoplastic cells infiltrated into the extraocular muscles. Immunohistochemically, the neoplastic cells were positive for desmin and vimentin. Transmission electron microscopy detected immature myofibrils with Z-band structures in the cytoplasm of these neoplastic cells. Consequently, the tumors were diagnosed as an orbital malignant schwannoma in an F344/DuCrlCrlj rat and an orbital embryonal rhabdomyosarcoma in a RccHan^TM:WIST rat. The results of this case report suggest that leakage of Ni₃S₂ to the orbit caused the induction of orbital malignant schwannoma or rhabdomyosarcoma in rats.     

Pathologic features of abdominal and thoracic paragangliomas in F344/N rats

Seventeen paragangliomas were identified in a retrospective review of 200 NTP/NCI carcinogenicity studies in F344/N rats that served either as control or treated animals. Most tumors were grossly visible and located in the retroperitoneum adjacent to the vertebrae and aorta near the kidneys. Three microscopically detected paragangliomas were found at the base of the heart. Microscopically, neoplastic cells were in nests separated by reticulin fibers and capillaries. Argyrophil granules were in the cytoplasm of the retroperitoneal and mediastinal paravertebral tumors. Dense granules were found in the one tumor examined ultrastructurally. Some tumors had areas of necrosis and tumor emboli were present in the lumen of the abdominal aorta and vena cava adjacent to the tumor with metastases present in pulmonary vessels. The incidence of retroperitoneal neoplasms was 3 times more frequent in male than in female F344/N rats.     

Transplantation of large granular lymphocyte leukemia in congenitally athymic rats.

Twenty-two congenitally athymic nude (rnu/rnu) rats were transplanted with large granular lymphocyte leukemia derived from F344 rats and then compared with ten similar rats inoculated with a suspension of normal F344 rat spleen cells. The normal spleen cells and tumor cells from a spontaneous, naturally occurring leukemia did not grow or cause clinical disease in any of the rats. All rats inoculated with a serially passaged leukemia cell inoculum had local growth at the inoculation site that spread widely and resulted in progressive tumor growth. Death occurred between 16 and 38 days after inoculation. The 22 rats that received passaged tumor cells developed leukemia and splenomegaly. Spleens were diffusely infiltrated by tumor cells and had severe depletion of lymphocytes in the white pulp. Leukemic rats were thrombocytopenic and had hemolytic anemia characterized by increased osmotic fragility, red cell width, and many nucleated erythrocytes. The disease syndrome appears similar to that of F344 rats transplanted with the same inoculum. Because the host rats lacked T cells, it is concluded that the hemolytic anemia and thrombocytopenia that develop in transplanted rats are independent of T cell function.     

Granular cell lesions in the distal female reproductive tract of aged Sprague-Dawley rats

During the review of a rat carcinogenicity study, a spectrum of granular cell lesions was recognized in the distal female reproductive tract. To verify the diagnoses, cell populations of nine granular cell alterations of the cervix or vagina were characterized immunohistochemically and four were evaluated ultrastructurally. Immunoreactivity was demonstrated in 8/9 cases with S100 protein, 6/9 cases with neuron-specific enolase, and 7/9 cases with Leu-7. Granular cells were negative for smooth muscle-specific actin and calretinin. The immunohistochemical profile of these lesions was similar to that previously reported in other species, including humans. Ultrastructurally, as expected many lysosomal bodies were present in the cytoplasm of granular cells in all specimens evaluated. Based on the detailed evaluation of a series of lesions, we adopted the following diagnostic criteria and nomenclature for the granular cell changes of the female reproductive tract of rats. Granular cell aggregates were non-space-occupying lesions composed of clusters of typical granular cells. Benign granular cell tumors were space occupying lesions that typically contained prominent interstitial collagen and were either discrete masses or were difficult to discern from the surrounding tissues. Some benign tumors also contained foci of spindle cells with decreased granularity. Malignant tumors exhibited pleomorphism and an increased nucleus: cytoplasm ratio morphologically but had the same biologic behavior as the benign tumors. We applied these diagnostic criteria during the review of controls from 9 carcinogenicity studies. Up to 23% of control females in those carcinogenicity studies had granular cell lesions that could be classified into one of the three categories. Granular cell lesions appear to be common in the cervix/vagina of the Sprague-Dawley rat, and tumors may develop from granular cell aggregates.     

Modifications of azoxymethane-induced carcinogenesis and 90-day oral toxicities of 2-tetradecylcyclobutanone as a radiolytic product of stearic acid in F344 rats

A 90-day oral toxicity test in rats was performed to evaluate the toxicity of 2-tetradecylcyclobutanone (2-tDCB), a unique radiolytic product of stearic acid. Six-week-old male and female F344 rats (n=15/group) were given 2-tDCB at concentrations of 0, 12, 60 and 300 ppm in a powder diet for 13 weeks. Slight dose-dependent increases in serum total protein and albumin in male rats were found, but these changes were not considered to be a toxic effect. The fasting, but not non-fasting, blood glucose levels of the male rats in the 300 ppm group and female rats in the 60 and 300 ppm groups were lower than those of the controls. Gas chromatography-mass spectrometry analysis showed dose-dependent accumulation of 2-tDCB in adipose tissue, notably in males. Next, we performed an azoxymethane (AOM)-induced two-stage carcinogenesis study. After injection of 6-week-old male F344 rats (n=30/group) once a week for 3 weeks, the animals received 2-tDCB at concentrations of 0, 10, 50 and 250 ppm in a powder diet for 25 weeks. The incidences of colon tumors for the 2-tDCB dosages were 34%, 45%, 40% and 37%, respectively, and were not statistically significant. These data suggest that 2-tDCB shows no toxic or tumor-modifying effects under the present conditions, and that the no-observed-adverse-effect level for 2-tDCB is 300 ppm in both sexes, equivalent to 15.5 mg/kg b.w./day in males and 16.5 mg/kg b.w./day in females.     

Malignant Peritoneal Mesothelioma with a Sarcomatoid Growth Pattern and Signet-Ring-Like Structure in a Female F344 Rat

We report a biphasic malignant mesothelioma in an aged female F344/DuCrlCrlj rat. Macroscopically, multiple pale brown nodules were observed in the abdominal cavity with retention of bloody ascites. Histopathologically, the tumor cells spread over the peritoneum and formed masses on the surface and underlying adipose tissues. The tumor cells dominantly proliferated in a solid, nodular or nest-like pattern with modest amount of fibrillar connective tissues, which contained hyaluronan. The tumor consisted of ovoid, polygonal or spindle-shaped cells that possessed eosinophilic cytoplasms including glycogen; some tumor cells showed a signet-ring-like structure. Multinucleated cells and mitosis were found frequently, and direct invasion to intra-abdominal organs and intravascular metastasis to the liver were observed. Immunohistochemically, keratin and mesothelin were strongly positive in most of tumor cells, while vimentin was mainly positive in spindle-shaped cells. Podoplanin was also positive, particularly in the cell membrane of tumor cells. Electron microscopically, tumor cells showed an intercellular desmosome-like structure, basement membrane and microvillus. We diagnosed the case as a malignant peritoneal mesothelioma with a sarcomatoid growth pattern and signet-ring-like structure.     

Amelanotic melanocytic tumors of the pinna in six F344 rats.

Spontaneous amelanotic melanocytic tumors of the pinna were found in six females of 960 male and 960 female albino (F344/DuCrj) rats which had been used in three different 24-month chronic toxicity studies. The age when the pinnal tumors were detected ranged from 37 to 59 weeks. The tumors were located unilaterally in the pinna and observed as subcutaneous spherical to irregular, solid white masses measuring 7 to 25 mm in diameter. The pinnal tumors were histologically classified into spindle cell and pleomorphic cell types. The spindle cell type was observed in four rats and composed of fusiform cells arranged in interlacing bundles. The pleomorphic cell type was observed in the remaining two rats and composed of pleomorphic large cells arranged in sheets. One tumor of the latter type metastasized to the submaxillary lymph node and lung. Melanin pigments were not demonstrated in any of the tumors. In immunohistochemistry, nuclei and cytoplasm of tumor cells in all the tumors were slightly positive for S-100 protein. Ultrastructurally, tumor cells contained a considerable number of premelanosomes in the cytoplasm. Desmosomes were occasionally observed between the cell membranes of the adjacent tumor cells. No distinct basal lamina was seen around tumor cells.     

Background Data on Developmental Parameters During the Gestation Period in Rats

Abstract: Background data during the gestation period were obtained from 128 Wistar Hannover GALAS rats and 26 Crl:CD(SD) pregnant rats in the control groups of our previous toxicity studies. The body weights of dams in the Wistar Hannover GALAS rats were significantly lower throughout the gestation period than those in the Crl:CD(SD) rats. In contrast, the time-dependent change in the body weight gain (%) of dams showed very similar trends in both strains. The mean number of live embryos/fetuses in the Wistar Hannover GALAS rats was 12.0, and was lower than that (14.5) in the Crl:CD(SD) rats. The placental weights gradually increased with pregnancy progression and reached a plateau on gestation day (GD) 19, although the embryo/fetal weights rapidly increased from GD 17 to GD 21. The embryo/fetal weights in the Wistar Hannover GALAS rats were significantly lower on only GD 21 than those in the Crl:CD(SD) rats. It is considered that this fetal weight difference between the strains develops during the fetal period, but not during the organogenesis period. In contrast, there were no differences in the placental weights between the two strains. Microscopically, the thickness of the labyrinth zone in the Wistar Hannover GALAS rats was thicker throughout the gestation period than that in the Crl:CD(SD) rats.     

Strain differences in morphometrical characteristics of rat kidneys

To clarify the strain differences in the morphology of the rat kidneys, we investigated the morphometrical characteristics of the kidneys of Slc:Wistar, Slc:SD, and F344/NSlc rats. The diameter of the renal corpuscles in female F344/N rats is smaller than that in female Wistar rats. Although sex differences (males>females) were shown in SD and F344/N rats, no effects of castration were detected in any of the groups. Strain-dependent differences in the percentage of renal corpuscles with a cuboidal parietal layer were found in both male and female groups. The highest percentage of them was noted in male Wistar rats. Effects of castration were observed in female Wistar and male F344/N rats, and the values after castration were significantly higher than those in the intact animals. As for the number of proximal convoluted tubular nuclei, no strain differences were detected in either the male or female groups. Although a sex difference was found in SD rats (female>male), no effects of castration were detected in any of the groups. In female F344/N rats, numerous numbers of PAS-positive granules, which were observed in the proximal convoluted and straight tubular epithelia, were noted. Orchiectomy induced an increase of these granules in male SD and F344/N rats, but ovariectomy showed no effects on them in any strains. This is the first study to clarify the strain differences in the morphological characteristics of the kidneys in ordinary rat strains.     

Spontaneous amelanotic melanomas of the uveal tract in F344 rats.

Five intraocular amelanotic melanomas were identified in the National Toxicology Program's database consisting of records from more than 60,000 female and 60,000 male F344 rats, which were used as control and treated animals in 2-year carcinogenicity studies. The five spontaneous melanomas were grossly observed as white or yellow, unilateral nodules, which originated in the region of the iris and ciliary body, often also involving the choroid. These amelanotic melanomas were composed predominantly of spindle cells arranged in a whorled pattern often with perivascular orientation. Mitotic figures were common in five tumors. The spindle cells had a positive immunoreactivity for S-100 protein but were negative for desmin. Electron microscopic studies provided clear evidence that these tumors originated from the uveal melanocytes. Ultrastructurally, the spindle cells contained numerous cytoplasmic premelanosomes (stage II melanosomes) that were not associated with melanin. Special histochemical studies showed that the spindle cells had a negative reaction for melanin. Although electron microscopic features are critical in the diagnosis of amelanotic melanomas of the uveal tract, the whorled pattern of spindle cells is a useful histologic criterion in differential diagnosis of this tumor in F344 rats.     

Highly invasive intracranial malignant schwannoma in a rat

A highly invasive intracranial malignant schwannoma containing several masses was detected in a 28-week-old male Crl:CD(SD) rat. Macroscopically, 3 masses were noted in the cranial cavity; one was present at the bottom of the cranial cavity and involved the trigeminal nerve, and the other two were in the parietal bone. Histologically, each mass consisted of fusiform cells with interlacing fascicular, wavy and nuclear pseudopalisading arrangements and round cells with cystic lesions. The tumor cells invaded not only the brain but also the parietal bone. In the brain, the tumor cells infiltrated diffusely into the leptomeningeal and perivascular spaces and parenchyma, in which the tumor cell morphology and invasive pattern closely resembled those of malignant astrocytoma and malignant reticulosis. Immunohistochemically, the tumor cells in the masses showed positive reactions for both S-100 protein and GFAP, while those in the cerebral invasion sites were negative for GFAP and less positive for S-100 protein. Electron microscopically, a single basal lamina layer and short intricate cell processes were confirmed in the tumor cells. From these results, the present tumor was diagnosed as a malignant schwannoma arising in the cranial cavity, probably originating from the trigeminal nerve. The present tumor is considered to be a relatively unique malignant schwannoma based on its growth and invasion patterns.     

Comparison of the effectiveness of high and low LET radiations for the proportion of survivals with liver tumors at every age in (C57BL/6N x C3H/HeN) F1 mice

To investigate the late effects of neutrons at the energy below 1 MeV on the liver carcinogenesis as a function of age, one-week old mice were exposed to 1.0 Gy monoenergetic neutrons (0.317, 0.525 and 1.026 MeV) or 137Cs gamma rays. Survival and carcinogenesis were examined by 18 months of age. Following radiation, tumor incidences in liver, Harderian gland, lung, ovary and pituitary gland were compared. The proportion of the lifespan with liver tumors exposed to neutrons to that exposed to gamma rays was calculated as a function of age. Survival rates among the three groups exposed to neutrons of different energies were not significantly different from one another but shorter than those treated with gamma rays for both sexes. With regard to liver tumor incidence evaluated at 18 months of age, the effectiveness of neutrons to gamma rays was 2.54 for females, and 2.08 for males by the factor. Levels of estrogen in the serum were similar between mice bearing liver tumors and those devoid of tumors. In conclusion, all three energies of neutrons induced similar effectiveness with respect to liver carcinogenicity. Proportions of the lifespan with liver tumors of neutron-exposed to gamma-exposed were shorter in females than males along with ages over 12 months. To obtain this factor at every age contributed for the evaluation of the biological effectiveness of radiations with the parameter of tumor incidence and latency simultaneously.     

Age-related Histological Findings in the Pineal Gland of Crl:CD(SD) Rats

To provide background data as the pathologic basis, the pineal glands of 190 male and 193 female Crl:CD(SD) rats at ages of 0–7, 51–58, 70–85 and 111 weeks were examined histologically in this study. Mineralization and fibrosis were common findings in the aged rats, whereas they were rarely found in the young ones; mineralization was present in 7, 44, 67 and 79% of males and in 0, 32, 67 and 79% in females, and fibrosis was present in 0, 29, 48 and 44% of males and 0, 18, 40 and 35% of females at ages of 0–7, 51–58, 70–85 and 111 weeks, respectively. Striated muscle fiber appeared regularly in the fibrosis region from 51–58 weeks of age when fibrosis increased, while the origin of this fiber remained unclear. Vacuolation of pineal cells also increased with age in both sexes, though the total incidence was low. There was a low incidence of lymphocytic infiltration in both sexes, but this was not related to age.