Long-term toxicity and carcinogenicity study of cyclamate in nonhuman primates.

Twenty-one monkeys (cynomolgus, rhesus, African green) were fed cyclamate (100 mg/kg and 500 mg/kg) in the diet five times per week from a few days after birth and continuing for up to 24 years. Malignant tumors were diagnosed in three 24-year-old cyclamate monkeys; these were metastatic colon carcinoma (rhesus; 500 mg/kg), metastatic hepatocellular carcinoma (cynomolgus; 500 mg/kg), and a small, well differentiated adenocarcinoma of the prostate (cynomolgus; 100 mg/kg). Benign tumors were found at necropsy in three females; these were adenoma of the thyroid gland (rhesus; 100 mg/kg) and two cases of leiomyoma of the uterus (rhesus; 100 mg/kg and 500 mg/kg). No tumors were detected in an age-matched control group of 16 monkeys. Examination of the testes revealed complete testicular atrophy in one of the old cyclamate monkeys, and focal germ cell aplasia (Sertoli-only tubules) in two other cyclamate monkeys. Focal spermatogenic interruption (maturation arrest) at various germ cell levels mixed with normal spermatogenesis was observed in both the cyclamate-treated and the control monkeys, all of which were over 20 years old. Measurements of terminal cyclohexylamine concentrations showed that three of the males dosed with cyclamate at 500 mg/kg were high converters, with plasma concentrations comparable to the levels that produce testicular atrophy in rats. However, only one of the three high converters showed histologic evidence of irregular spermatogenesis. The overall conclusion is that the testicular abnormalities and the sporadic cases of different malignancies found after more than 20 years of dosing do not provide clear evidence of a toxic or carcinogenic effect of sodium cyclamate in monkeys.

Lack of carcinogenicity of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in cynomolgus monkeys.

The carcinogenic potential of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was evaluated in cynomolgus monkeys. The animals received MeIQx, beginning at the age of one year, at doses of 10 or 20 mg/kg body weight by gavage five times a week for 84 months and were autopsied 8 months thereafter. Although sporadic development of aberrant crypt foci in the colon and glutathione S-transferase pi-positive foci in the liver as well as hyperplastic changes of the lymphatic tissue in the lung and gastro-intestinal tract were observed in several monkeys, this was not treatment-related. No neoplastic or preneoplastic lesions were found in other organs. Serum chemistry data and organ weights were also within the normal ranges. From these data, it is concluded that MeIQx is not carcinogenic in the cynomolgus monkey under the conditions examined. This lack of carcinogenicity is probably related to the poor activation of MeIQx due to the lack of constitutive expression of CYP1A2 as well as an inability of other cytochrome P450s to catalyze N-hydroxylation of MeIQx in the cynomolgus monkey.

Effects of long-term oral administration of DDT on nonhuman primates.

Because of reports on tumorigenic activity in different animal species exposed to DDT a decision was made in 1969 to evaluate the long-term effects of DDT on 24 cynomolgus and rhesus monkeys. DDT (20 mg/kg) was given in the diet for 130 months, followed by an observation period that ended in 1994. The two cases of malignant tumor detected in the DDT group included a metastatic hepatocellular carcinoma in a 233-month-old male and a well-differentiated adenocarcinoma of the prostate in a 212-month-old monkey. Benign tumors detected in the DDT group included three cases of leiomyoma, two of which were uterine and one, esophageal. No tumor was detected in the control group of 17 monkeys. Fatty changes in the liver were observed in 52.9% of the DDT group and 29.4% of the control group. More specific signs of hepatotoxicity were documented microscopically in seven DDT monkeys. Severe tremors and histological evidence of CNS and spinal cord abnormalities were observed in six DDT monkeys. The present findings show clear evidence of hepatic and CNS toxicity following long-term DDT administration to cynomolgus and rhesus monkeys. However, the two cases involving malignant tumors of different types are inconclusive with respect to a carcinogenic effect of DDT in nonhuman primates.

Long-term feeding of sodium saccharin to nonhuman primates: implications for urinary tract cancer.

BACKGROUND: It was observed in the early 1970s that saccharin produced bladder cancer in rats. However, it has been unclear whether sodium saccharin when consumed by humans poses a substantial carcinogenic hazard. Numerous epidemiologic studies have not shown any evidence of increased urothelial proliferation associated with ingestion of sodium saccharin. PURPOSE: Our purpose was to determine the effects of long-term feeding of sodium saccharin to three species of nonhuman primates. METHODS: Twenty monkeys of three species (six African green, seven rhesus, six cynomolgus, and one hybrid [of rhesus male and cynomolgus female parentage]) were treated with sodium saccharin (25 mg in the diet/kg body weight daily for 5 days a week) beginning within 24 hours after birth and continuing for up to 24 years. Sixteen monkeys (seven rhesus and nine cynomolgus) served as controls. During their last 2 years of life, urine was collected from selected treated and control animals and evaluated for various urinary chemistries and for the presence of calculi, microcrystalluria, and precipitate. Urinary bladders were examined by light microscopy and by scanning electron microscopy. RESULTS: Sodium saccharin treatment had no effect on the urine or urothelium in any of these monkeys. There was no evidence of increased urothelial cell proliferation, and there was no evidence of formation of solid material in the urine. CONCLUSION: Although the dose of sodium saccharin administered to these monkeys was only five to 10 times the allowable daily intake for humans, the results provide additional evidence that sodium saccharin is without a carcinogenic effect on the primate urinary tract.

Comparative subchronic and chronic dietary toxicity studies on 2,4-dichlorophenoxyacetic acid, amine, and ester in the dog.

Forms of 2,4-dichlorophenoxyacetic acid (2,4-D) are herbicides used in the control of a wide variety of broadleaf and woody plants. Subchronic toxicity studies in dogs were conducted on three forms of 2,4-D: the parent form, 2,4-D acid (ACID); 2,4-D dimethylamine salt (DMA); and 2,4-D 2-ethylhexyl ester (2-EHE). The three studies were designed to allow for comparison of the toxicity of the three forms. Doses in the subchronic studies, on an acid equivalent basis, were 0, 0.5 (ACID only), 1.0, 3.75, and 7.5 mg/kg/day. Treatment related findings in the three studies included reductions in body weight gain, and food consumption, and minor increases in blood urea nitrogen, creatinine, and alanine aminotransferase. The data from the three subchronic studies demonstrated the comparable toxicity of ACID, DMA, and 2-EHE and support a subchronic no observed adverse effect level (NOAEL) of 1.0 mg/kg/day for all three forms. Due to the similarity in toxicity of the three forms of 2,4-D, a 1-year chronic toxicity study was performed on the parent ACID to fully characterize the potential toxicity of 2,4-D in the dog. ACID was well tolerated at doses of 0, 1.0, 5.0, and 7.5 mg/kg/day. The clinical pathology alterations were similar to those seen in the subchronic studies and were not progressive. The histopathology alterations observed were not severe in nature and the no observed effect level in the chronic study was determined to be 1.0 mg/kg/day. There was no indication of any immunotoxic or oncogenic response in the studies. In conclusion, the findings of these studies indicate comparable toxicity among representative forms of 2,4-D and their generally low toxicity following subchronic and chronic dietary exposure in the dog.

Studies on the carcinogenic and myocardial effects of 2-amino-3-methylimidazo [4,5-f] quinoline (IQ) in nonhuman primates.

The heterocyclic aromatic amine 2-amino-3-methylimidazo [4,5-f] quinoline (IQ) is one of three heterocyclic amine mutagens currently being evaluated for carcinogenic activity in nonhuman primates, primarily cynomolgus monkeys. IQ was administered by gavage five times a week at doses of 10 or 20 mg/kg. Thus far IQ induced tumors in 50 percent of the monkeys at the 10 mg/kg dose and in 85 percent of the monkeys at the 20 mg/kg dose. Because H and E sections of the myocardium from IQ-treated animals demonstrated inflammatory infiltrate and studies of IQ-DNA adducts in monkeys, by the 32P-postlabeling method, showed high levels of adducts in the heart a systematic study of cardiac pathologic changes associated with chronic administration of IQ was undertaken. Both light and electron microscopic abnormalities were seen. The possibility exists that heterocyclic aromatic amines may be etiologic factors for human cancer and myocardial disease.

Laparoscopy as a means of monitoring liver tumor induction in nonhuman primates.

Nonhuman primates are not commonly used in the study of carcinogenesis. For several years, the National Cancer Institute (NCI) has funded a large study to evaluate the relative susceptibility of primates to the effects of several known rodent carcinogens, various cancer chemotherapeutic agents, and several food additives/contaminants, such as cyclamate, saccharin, and aflatoxin. The current colony is comprised of over 300 macaque monkeys which have received a particular test material beginning shortly after birth and continuing for periods of at least 10 years or until tumor is detected. In 1985, a study of the potential carcinogenic effect of the heterocyclic aromatic amine, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), was initiated. Subsequently, studies on two other heterocyclic aromatic amines were initiated, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx) in 1988 and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in 1990. Tumors have been induced thus far only by IQ, and this data will be presented by Dr. Richard Adamson, while the detailed pathology of the neoplasms will be presented by Dr. Unnur Thorgeirsson. Palpation of the liver and monitoring of liver enzymes are not effective means for detecting early evidence of liver tumor. Laparoscopy, on the other hand, can be done easily and quickly and is an effective means of visualizing the liver and for obtaining samples of liver and suspected tumor tissue. Repeated laparoscopic examinations using videotape to document the size, location, and appearance of developing tumors allow more precise monitoring of the carcinogenic process.

Tumor incidence in a chemical carcinogenesis study of nonhuman primates.

This report covers a 32-year period of an ongoing chemical carcinogenesis study in nonhuman primates, which was initiated by the National Cancer Institute in 1961. Autopsy records of 373 breeders and normal controls showed very low incidence of spontaneous malignant tumors in cynomolgus (1.5%) and rhesus (2.8%) monkeys, but considerably higher incidence in African green monkeys (8%). A large number of substances including a variety of food additives, food components, environmental contaminants, N-nitroso compounds, "classical" rodent carcinogens, antineoplastic agents, and immunosuppressive agents have been evaluated for long-term carcinogenic activity. Food components tested which are probably most relevant to human exposure are the artificial sweeteners, cyclamate and saccharin. After 22 years of continuous dosing, neither cyclamate nor saccharin have shown any evidence of carcinogenic effects. Similarly, the tumorigenic potential of arsenic and DDT was negligible after dosing for 15-22 years. In contrast, the fungal food contaminants, aflatoxin B1 (AFB1) and sterigmatocystin (SMT), were found to be potent hepatocarcinogens. AFB1 also induced adenocarcinomas of the pancreas, osteosarcomas, and other tumors. Also, the aglycone of cycasin, MAM acetate, induced a variety of tumors, but primarily hepatocellular and renal cell carcinomas. The compounds most recently introduced into the colony include three heterocyclic amines present in cooked meat. One of these compounds, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) has proven to be one of the most potent hepatocarcinogens in the history of the monkey project, inducing malignant liver tumors in 65% of animals over a 7-year period of exposure. Of the classical rodent carcinogens studied, urethane was the only one which produced malignant tumors in the monkeys. Conversely, all except two of the N-nitroso compounds were carcinogenic. Diethylnitrosamine (DENA) was the most potent and predictable hepatocarcinogen in cynomolgus, rhesus, and African green monkeys. However, when administered intraperitoneally to galagos (a prosimian), DENA induced primarily mucoepidermoid carcinoma of the nasal cavity. N-Methyl-N-nitrosourea (MNU) was the only carcinogen persistently producing tumors in the digestive tract, mostly squamous cell carcinomas of the esophagus. Among the antineoplastic and immunosuppressive agents, procarbazine (MIH) was the only unequivocal carcinogen, with a 33% tumor incidence, causing acute nonlymphocytic leukemia in most of the cases.

Enzyme immunosorbent assay for Ebola virus antigens in tissues of infected primates.

A sandwich enzyme immunosorbent assay (EIA) using a mixture of mouse monoclonal antibodies for antigen capture and polyclonal hyperimmune rabbit anti-Ebola virus serum for antigen detection was developed and evaluated on the tissues of monkeys naturally or experimentally infected with strains of Ebola viruses. When compared with virus isolation, the antigen detection EIA was both sensitive and specific: 44 of 45 (97.7%) liver homogenates and 38 of 41 (92.7%) spleen homogenates that were culture positive and tested by both techniques were positive for viral antigen, while 85 of 87 (97.7%) culture-negative liver homogenates and 66 of 66 culture-negative spleen homogenates were found to be antigen negative. The assay, initially developed to detect antigens of prototype African strains of Ebola virus, reliably detected related strains of Ebola virus found during two recent outbreaks of Ebola virus infection among imported, quarantined Macaca fascicularis monkeys in the United States. The assay allows economical and rapid testing of large numbers of tissue specimens. Antigen was found in homogenates of spleen and liver and in serum.

Combined simian hemorrhagic fever and Ebola virus infection in cynomolgus monkeys.

Simian hemorrhagic fever (SHF) virus and a new strain of Ebola virus were isolated concurrently in recently imported cynomolgus monkeys (Macaca fascicularis) being maintained in a quarantine facility. Ebola virus had never been isolated in the U.S. previously and was presumed to be highly pathogenic for humans. A chronology of events including measures taken to address the public health concerns is presented. The clinicopathologic features of the disease were abrupt anorexia, splenomegaly, marked elevations of lactate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase, with less prominent elevations of blood urea nitrogen, creatinine, and other serum chemistry parameters. Histologically, fibrin deposition, hemorrhage, and necrosis of lymphoid cells and reticular mononuclear phagocytes were present in the spleens of SHF and of Ebola virus-infected animals. Intravascular fibrin thrombi and hemorrhage were also present in the renal medulla and multifocally in the gastrointestinal tract. Necrosis of lymphoid and epithelial cells was occasionally noted in the gastrointestinal tract. The histopathologic findings considered specific for Ebola virus infection include hepatocellular necrosis, necrosis of the zona glomerulosa of the adrenal cortex, and interstitial pneumonia, all of which were generally associated with the presence of 1 to 4 mu intracytoplasmic amphophilic inclusion bodies. The disease spread within rooms despite discontinuation of all direct contact with animals, and droplet or aerosol transmission was suspected. Antibody to Ebola virus developed in animal handlers but no clinical disease was noted, suggesting a less virulent strain of virus.(ABSTRACT TRUNCATED AT 250 WORDS)

Preliminary report: isolation of Ebola virus from monkeys imported to USA.

An epizootic caused by an Ebola-related filovirus and by simian haemorrhagic fever virus began among cynomolgus monkeys in a US quarantine facility after introduction of monkeys from the Philippines. This incident, the first in which a filovirus has been isolated from non-human primates without deliberate infection, raises the possibility that cynomolgus monkeys could be a reservoir of Ebola virus infection.

Carcinogenicity of 2-amino-3-methylimidazo[4,5-f]quinoline in nonhuman primates: induction of tumors in three macaques.

The carcinogenic potential of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was evaluated in cynomolgus monkeys. Monkeys received IQ, beginning at the age of one year, at doses of 10 or 20 mg/kg by gavage. Thus far, IQ has induced hepatocellular carcinoma in three monkeys with a latent period of 27 to 37 months. Metastases to the lung occurred in two of the three monkeys. Microscopically, the hepatocellular carcinoma in all three cases demonstrated a trabecular pattern. These data demonstrate that IQ is a potent carcinogen in nonhuman primates and support the idea that it is a potential carcinogen for humans.

Carcinogenicity and hepatotoxicity of cycasin and its aglycone methylazoxymethanol acetate in nonhuman primates.

The carcinogenic potential of cycasin and methylazoxymethanol (MAM) acetate was investigated in nonhuman primates. Old-world monkeys (rhesus, cynomolgus, and African green monkeys) received cycasin and/or MAM acetate by oral or ip routes up to 11 years. Eighteen monkeys survived longer than 2 months after initiation of treatment with cycasin (50-75 mg/kg) or MAM acetate (1.5-3.0 mg/kg) given orally 5 days/week; 9 of the animals were necropsied. Histopathologic examination of a liver tumor from 1 of these monkeys revealed well-differentiated hepatocellular carcinoma. A second monkey had multiple tumors, including hepatocellular carcinoma, intrahepatic bile duct adenocarcinoma, renal carcinoma and adenomas, and adenomatous polyps of the colon. Although liver tumors were not observed in the other monkeys, all but 1 monkey had hepatic lesions such as toxic hepatitis and cirrhosis. These monkeys had received cycasin and/or MAM acetate for an average of 57 months (range, 2-133 mo). A group of 10 monkeys received MAM acetate by weekly ip injections (3-10 mg/kg). Six of these animals developed tumors after receiving an average of 6.14 g (range, 3.58-9.66 g) of MAM acetate for an average of 75 months (range, 50-89 mo). Four of the monkeys developed hepatocellular carcinomas, and 2 had multiple primary tumors including hepatocellular carcinomas, renal carcinomas, squamous cell carcinomas of the esophagus, and adenocarcinomas of the small intestine. Our results showed that long-term administration of cycasin and/or MAM acetate by oral and ip routes was hepatotoxic and carcinogenic in old-world monkeys.

Cardiotoxic and possible leukemogenic effects of adriamycin in nonhuman primates.

10 monkeys (macaques) received adriamycin by monthly intravenous injections at 12 mg/m2 (1 mg/kg). 8 of the 10 monkeys developed congestive heart failure at an average cumulative adriamycin dose (310 mg/m2) well below that considered the safe upper limit (550 mg/m2) in man. Histologically, the myocardial lesions resembled those found in human anthracycline-induced cardiomyopathy. 1 of the 10 monkeys developed acute myeloblastic leukemia after receiving 324 mg/m2 of adriamycin; the 10th monkey is alive and well 26 months after the last dose of drug. Our results suggest that adriamycin is a more potent cardiotoxin in monkeys than in man, and that leukemia may be a consequence of prolonged treatment with this drug.

Lactose malabsorption in two sibling rhesus monkeys (Macaca mulatta).

Observation of two sibling rhesus monkeys, Macaca mulatta, indicated that they were intolerant to lactose. While on a lactose formula the monkeys had severe diarrhea and weight loss but were otherwise healthy. When a lactose-free formula was instituted, the diarrhea abated and there was a steady weight gain.

Induction of osteogenic sarcomas and tumors of the hepatobiliary system in nonhuman primates with aflatoxin B1.

The carcinogenicity of aflatoxin B1 (AFB1) has been under evaluation in nonhuman primates for the past 13 years. A total of 47 Old World monkeys, chiefly rhesus and cynomolgus, have received AFB1 i.p. (0.125 to 0.25 mg/kg) and/or p.o. (0.1 to 0.8 mg/kg) for 2 months or longer, and 12 are currently alive and without evidence of tumor. Thirteen of the 35 monkeys necropsied to date (37%) developed one or more malignant neoplasms, yielding an overall tumor incidence of 28%. Five of the neoplasms were primary liver tumors (2 hepatocellular carcinomas and 3 hemangioendothelial sarcomas), and 2 cases of osteogenic sarcoma were found. Other tumors diagnosed were 6 carcinomas of the gall bladder or bile duct, 3 tumors of the pancreas or its ducts, and one papillary Grade I carcinoma of the urinary bladder. The tumors developed in animals receiving an average total AFB1 dose of 709 mg (range, 99 to 1354 mg) for an average of 114 months (range, 47 to 147 months). Fifteen of the 22 necropsied monkeys (68%) without tumor showed histological evidence of liver damage, including toxic hepatitis, cirrhosis, and hyperplastic liver nodules. These animals had received an average total AFB1 dose of 363 mg (range, 0.35 to 1368 mg) for an average of 55 months (range, 2 to 141 months). Our results indicate that AFB1 is a potent hepatotoxin and carcinogen in nonhuman primates and further support the hypothesis that humans exposed to this substance may be at risk of developing cancer.

Surgical technique for a permanent tracheostomy in Beagle dogs.

A surgical procedure to produce a permanent tracheostomy in dogs was developed. The procedure consisted of dissecting portions of the cartilaginous rings free from the underlying tracheal mucosa, cutting through the mucosa and suturing the mucosa to the skin. The procedure was performed in approximately 30 minutes and resulted in a permanent, maintenance-free, mucocutaneous stoma that also permitted breathing through the upper respiratory airways.

Carcinogenic and other adverse effects of procarbazine in nonhuman primates.

The carcinogenic potential of procarbazine is under investigation in three species (Macaca mulatta, Macaca fascicularis, and Cercopithecus aethiops) of nonhuman primates. A total of 55 monkeys have received procarbazine s.c., p.o., or i.p. for periods of up to 12 years. Eleven of the 42 monkeys (26%) necropsied thus far have had malignant neoplasms, 6 of which were acute leukemia. Two monkeys developed osteogenic sarcomas, two monkeys developed hemangiosarcomas, and a single case of lymphocytic lymphoma was found. The average total dose of procarbazine received by the monkeys developing cancers was 36.0 g; the average duration of procarbazine treatment was 75 months. A number of the toxic effects of procarbazine seen in humans were also noted in this series of monkeys, including vomiting and myelosuppression. Another striking toxic effect was on the reproductive system of the males. The majority of the adult males necropsied to date have had testicular atrophy with complete aplasia of the germinal epithelium.