Unexpected tumour findings in lifetime rodent bioassay studies--what to do?

Currently, the majority of substances tested in lifetime bioassays in rodents are not mutagenic and, therefore, at the most weakly carcinogenic, generally by epigenetic mechanisms. It thus appears obvious that only marginal increases of tumour incidences can be expected in lifetime bioassays and that, therefore, every aspect of a potential carcinogenic effect must be thoroughly evaluated. This paper describes a series of key factors, which should be looked at in order to exclude that the lifetime bioassay in question is flawed for design, technical or qualification reasons. It also provides some hints whether there is indeed a real effect and not just a variation of the spontaneous tumour incidences. Tumour findings must be seen in the context of the animal model, the pharmcokinetics and pharmcodynamics of the test substance, as well as any other observation in the present or other studies with the test substance, including non-tumour findings and--in particular--potential precursor lesions and effects on feed intake and survival. The possibility that the observed carcinogenic effects may be species-specific and not relevant for man is discussed. It is also important to check what findings are reported with similar substances or substances with the same pharmacological effect. Data from additional investigations on material of the same study and/or mechanistic studies are often needed to support the final risk assessment.

A review of drug-induced Leydig cell hyperplasia and neoplasia in the rat and some comparisons with man.

This paper describes control of normal Leydig cell function and testosterone production. The macroscopic and histopathological appearances of spontaneous Leydig cell hyperplasias and tumors (LCT) in the rat are reviewed together with their incidence and hormonal status. Drugs which induce LCTs in chronic studies are discussed and include busereline, carbamazepine, cimetidine, finasteride, flutamide, gemfibrozil, histrelin, hydralazine, indomethacin, isradipine, lactitol, leuprolide, metronidazole, mesulergine, nafarelin, norprolac and vidarabine. The known mechanisms of LCT induction in the rat are reviewed together with other possible etiological factors. The incidence, clinical picture and etiological factors of LCTs in man are also surveyed. Hormone production in Leydig cells and LCTs in rats and man are compared. Differences between the two species are considered, particularly with regard to Leydig cell control mechanisms. The paper concludes that drug-induced LCTs in rats are most probably not predictive for man and their occurrence has little relevance in human safety assessment.

Proliferating cell nuclear antigen expression in Wistar rat livers. A retrospective immunohistochemical study of normal and neoplastic livers.

Formalin-fixed, paraffin-embedded liver specimens from 27 2-year-old Wistar rats, including 10 normal livers, 11 hepatocellular adenomas, 2 hepatocellular carcinomas, and 4 cystic cholangiomas, were immunostained using the streptavidin/peroxidase method and the PC10 monoclonal antibody (Mab), which recognizes an epitope on the proliferating cell nuclear antigen (PCNA). The following PCNA labeling index (LI) mean values were found for the above four groups of liver specimens: normal livers, 0.43 +/- 0.31%; hepatocellular adenomas, 1.51 +/- 0.59%; hepatocellular carcinomas, 24.80% +/- 10.28%; and cystic cholangiomas, 0.61 +/- 0.21%. Our findings indicate that PCNA LI clearly separates liver malignancies from other benign liver tumors, as well as from normal, non-neoplastic, liver tissues. Although the mean PCNA LI values seemed to reflect histological grading (i. e. normal, neoplastic benign, neoplastic malignant), overlapping between normal livers and hepatocellular adenomas was observed in five cases (i. e. in 2 normal livers and 3 hepatocellular adenomas, where the PCNA LI values varied between 0.74% and 0.96%). It thus appears that PCNA immunohistochemistry represents a promising tool for investigating liver cell proliferation in laboratory rats, and permits distinguishing between benign and malignant liver parenchymal tumors.

Hypertrophic osteopathy in rats following chronic administration of SDZ MNS 949, an isoquinoline.

SDZ MNS 949, 6,7-dimethoxy-3-methyl-1-(3',5'-bis (methoxyethoxy) phenyl)-isoquinoline, a bronchodilating anti-inflammatory drug that inhibits phosphodiesterase, had been proposed for the treatment of bronchial asthma. Groups of 14 male and 14 female Wistar rats were administered doses of 12, 50, and 130 mg/kg/day in feed for 26 weeks. Periodic radiographic examinations were performed in addition to clinical observations, clinical chemistry measurements and urinalysis. At study termination full necropsy and histopathological examinations were performed on all animals. The principal clinical signs observed were unilateral edematous, red and painful swelling of the distal hindlimbs in 8 of 28 high dose animals, and abdominal swelling in 19 of 28 high dose animals. At radiographic examination periosteal new bone formation was predominantly along the tibia. Lesions at necropsy included dilated small and large intestines. Microscopically, enteritis was observed, and the periosteal new bone formation was confirmed. Hematological findings consisted of thrombocytosis and lymphocytosis, especially in high dose animals. The clinical, radiographical and histological findings in treated rats were consistent with the diagnosis of "hypertrophic osteopathy" or "Marie's Disease".

Causes of death in rodent toxicity and carcinogenicity studies.

Peto test procedures for the statistical evaluation of carcinogenicity studies require that each tumor in an animal that died intercurrently (or was sacrificed in extremis) be classified as either fatal, probably fatal, incidental, or probably incidental. There is considerable controversy as to whether or not the cause of death can be established with accuracy in rodent studies. In the present article, the causes of death or ill-being as found in 10 consecutive carcinogenicity studies--5 studies with 2400 OFA (Sprague-Dawley-derived) and Wistar rats and 5 studies with 2400 OF1 and NMRI mice--were re-examined. A cause of death or moribund state had been established in more than 80% of the cases in rats and in more than 70% in mice. These causes were, in rats, mainly pituitary tumors, chronic progressive nephropathy (males), mammary gland tumors (females), and subcutaneous tumors (males); in mice, mainly hemolymphoreticular tumors, lung tumors, liver tumors (males), and glomerulonephropathy. The criteria used for determining the tumorous or non-tumorous lesions as the cause of death were based on in-life and pathological findings. The validity of such procedures, the possibility of improving criteria in the future, and the usefulness of establishing causes of death in safety assessment are discussed.

Neoplastic lesions of questionable significance to humans.

Many compounds giving a positive result in animal carcinogenicity studies through mechanisms involving secondary carcinogenesis pose little or no risk to humans. This article provides an overview of current understanding, with particular reference to renal tumors in male rats with alpha 2mu-globulin nephropathy, urinary bladder neoplasia in rodents, mesovarian leiomyomas induced in rats by beta 2-receptor stimulants, carcinoid tumors in the rodent stomach induced by prolonged suppression of acid secretion, thyroid follicular cell tumors in rodents, canine mammary neoplasia due to administration of progestagens, rodent mammary neoplasia induced by estrogens, uterine endometrial carcinomas of rats induced by dopamine agonists, Leydig cell tumors in the testis of rats, and ovarian tubulostromal adenomas in mice. A positive result on a rodent carcinogenicity study should not automatically preclude further development of a compound; future progress in this field should increase the accuracy of the rodent carcinogenicity study as a tool in human safety assessment.

Glycoprotein hormone alpha-subunit-producing pituitary adenomas in rats treated for one year with calcitonin.

Calcitonin, a calcium-lowering hormone, has been associated with an increased incidence of nonfunctioning pituitary tumors in rats. In this study, rats were treated with calcitonin (80 IU/kg/d) for 52 weeks. After treatment with calcitonin, immunohistochemistry and in situ hybridization analyses demonstrated that most pituitary tumors expressed the glycoprotein hormone alpha-subunit. Expression of the alpha-subunit was identified rarely in hyperplastic lesions of control animals. Serum levels of GH, PRL, ACTH, LH, and FSH were unchanged in calcitonin-treated rats relative to controls. However, TSH levels were increased 2.1 fold after chronic treatment with calcitonin in both male and female rats (P less than 0.001). The level of glycoprotein hormone alpha-subunit was markedly increased (20-fold) in male rats with smaller elevations in female rats. Time course studies demonstrated that increases in serum alpha-subunit levels could be detected by 24 weeks of treatment and that elevations in alpha-subunit were present in the majority of animals by 40 weeks of treatment with calcitonin. The authors conclude that high doses of calcitonin, administered to rats for 6 months or longer, increases the incidence of alpha-subunit-producing pituitary tumors.

Morphologic and immunohistochemical characterization of Leydig cell tumor variants in Wistar rats.

During a routine long-term drug safety study, lasting approximately 2 1/2 yr, male Wistar rats, treated with a prolactin-inhibiting compound, developed an excess of Leydig cell tumors (LCTs). Most tumors were typical for the rat but a small number showed an unusual variation and some appeared malignant. The variation consisted of glandular and/or tubular structures within the tumor mass which occasionally anastomosed and contained an eosinophilic periodic-acid Schiff (PAS) positive material. In a few of these variants, malignant features such as cellular atypia, capsular, and lymphatic invasion and necrosis were seen. No metastases were detected. Detailed morphological and immunohistochemical investigations were conducted in order to establish the cell of origin of these variants. Glandular/tubular structures were found to stain with varying intensity for vimentin and cytokeratin, but were always negative for beta-tubulin. The results indicated that the cell of origin of these LCT variants was indeed the Leydig cell and that glandular and/or tubular structures within LCTs represented a form of Leydig cell metaplasia.

Chronic relapsing experimental allergic encephalomyelitis in the Lewis rat.

Chronic relapsing experimental allergic encephalomyelitis (CR-EAE) was induced in rats with an emulsion of guinea-pig spinal cord tissue (GPSC) in complete Freund's adjuvant (CFA) enriched with Mycobacterium tuberculosis H37RA (Tbc). 78% of the sensitized rats developed a CR-EAE showing 2 to 3 clinical relapses during the first 40 days. After 60-80 days, approximately half of the rats with CR-EAE had a further relapse which was followed by complete recovery in only 35% of the cases. The remaining 65% of these animals showed a progressive state of the disease, characterized by paralysis or severe motor deficit, eventually leading to death. CR-EAE in rats showed some similarities to multiple sclerosis in man (MS) and it may be a useful model for the study of this disease.

Long-term inhalation toxicity of hydrazine.

Year-long intermittent exposures of rats, mice, hamsters, and dogs to hydrazine were conducted using concentrations of 0.05, 0.25, 1.0, and 5.0 ppm. Rats were held 18 months postexposure; hamsters, 1 year postexposure; mice, 15 months postexposure; and dogs, 38 months postexposure. Male and female rats exhibited dose-dependent incidences of benign nasal adenomatous polyps and smaller numbers of malignant nasal epithelial tumors after 1 year of exposure to hydrazine and 18 months postexposure holding. Nasal tumors were often associated with chronic irritation and were most frequent in male rats, with an incidence of greater than 50% in the highest exposure group. Hamsters exposed to 0.25-ppm and higher concentrations showed pathologic changes characteristic of degenerative disease, including amyloidosis. After exposure to 0.5 ppm hydrazine, hamsters developed a 10% incidence of benign nasal polyps compared to 0.5% in controls. Small numbers of colon neoplasms and thyroid parafollicular cell adenomas were found in hamsters, but only in the highest concentrations tested. Lung adenomas appeared to be marginally increased in mice exposed to 1.0 ppm hydrazine, the highest concentration tested in this species. No consistent clinical or pathological effects were seen in dogs during or after exposure to hydrazine at any concentration. Using amyloidosis as a criterion, a no-effect level was not achieved in hamsters. In rats, there appeared to be a marginal production of nasal tumors at 0.05 ppm, while mice showed no effects at 0.25 ppm. This study has demonstrated that the nasal respiratory epithelia of rats and hamsters are the most sensitive tissues to the tumorigenic action of hydrazine following inhalation exposures. This is similar to the reaction of rats to formaldehyde, another highly reactive water-soluble compound.

Mononuclear cell leukemia in aged Sprague-Dawley rats.

A mononuclear cell leukemia in Sprague-Dawley rats is described in which liver, spleen, and lung involvement was a constant feature. The cell was 16 to 25 microns in diameter with round, oval or indented nucleus, and the cytoplasm contained bright red granules. This is the first report of such a leukemia in Sprague-Dawley rats and a comparison with mononuclear cell leukemia in other strains of laboratory rats is made.

The toxicology of a ganglioside extract (Cronassial).

The accepted animal toxicity studies indicate that the ganglioside mixture extracted and purified from the bovine brain cortex (Cronassial) is without detectable toxicity. It did not induce any adverse effects on any of the characteristics of reproduction and it is not antigenic.

Influence of fenofibrate on cellular and subcellular liver structure in hyperlipidemic patients.

The administration of lipid-lowering drugs to rodents, notably those related to clofibrate, rapidly provokes a hepatic response characterized by hepatomegaly, proliferation of smooth endoplasmic reticulum and proliferation of peroxisomes in hepatocytes. In some studies hepatocellular carcinoma has been found in rats or mice exposed for their entire life-span to high dose levels of various fibrates. In the present study liver biopsy samples were obtained from 38 hyperlipidemic patients, 28 of whom had been receiving fenofibrate for between 2 months and approximately 3 years (mean values: males 1.79, females 1.98 years). The remaining 10 patients had never been treated with a lipid-lowering drug. Examination of the biopsy samples by a variety of optical techniques and by electron microscopy failed to reveal any difference between the groups. Peroxisomes were relatively rare, there being no evidence of the clear proliferation seen in rodent studies. Other microscopic features of interest were some variation of nuclear size, mitochondria containing paracrystalline inclusions, dilated endoplasmic reticulum associated with reduced amounts of rough endoplasmic reticulum, and the presence of lipid droplets in the liver cells. However, these variations from normal were in general not much more apparent in samples from the fenofibrate-treated patients than in the untreated group. Light- and electron-microscopic observations did not suggest liver intoxication or a carcinogenic pattern.

The effect of dose and vehicle on early tissue damage and regenerative activity after chloroform administration to mice.

The relationship between the acute toxicity of orally-administered chloroform and its long-term tumorigenic potential was studied in male mice of the CFLP outbred Swiss albino mouse strain. A single dose of approximately 18 mg CHCl3/kg had no detectable acute toxic effect on the liver or kidneys and did not stimulate regenerative activity, whereas both toxicity and subsequent tissue regeneration were observed with single doses of about 60 mg/kg or higher. The severity of the toxic effects and regenerative changes was greater when corn oil was used as a vehicle for chloroform than when the vehicle was a toothpaste base. In earlier long-term studies in mice of the same strain, kidney tumours occurred in males given 60 mg/kg/day throughout life but not in mice given 17 mg/kg/day. The tumour response was greater when the 60-mg/kg/day dose was given in an oily vehicle than when it was given in toothpaste. The findings are consistent with the hypothesis that early acute toxic change and subsequent repair are a sine qua non for tumorigenesis in the kidney and liver in response to chloroform.

Sub-acute toxicity studies on a new piperidine derivative (HSR-902) in dogs.

The administration of a new piperidine antispasmodic agent (HSR-902) to the dog by the oral and intravenous routes, induced clinical signs attributable to parasympathetic blockage. The only significant toxicological finding was rarefied appearance and enlargement of the hepatocytes at the high dose level (50 mg/kg/day); this was shown to be reversible.

The intravenous toxicity of lentinan to the beagle dog.

The i.v administration of lentinan to the Beagle dog induced changes in the cytoplasm of macrophagic cells in the liver, spleen, kidney, lungs, lymph nodes, small intestine. Electron-lucent or filamentous inclusions were demonstrated in the liver, kidney and spleen. A dose level of 0.5 mg/kg/day was without adverse effect.