Liver tumors induced in rats by oral administration of the antihistaminic methapyrilene hydrochloride.

The antihistaminic over-the-counter drug methapyrilene hydrochloride, mixed with food at a concentration of 0.1 percent, was administered to 50 male and 50 female Fischer rats. A second group of 50 male and 50 female rats was given the same treatment together with 0.2 percent of sodium nitrite added to the food. Almost all of the rats in both groups developed liver neoplasms, mainly hepatocellular carcinomas and cholangiocarcinomas. The first rat died with a liver neoplasm at the 43rd week. Over 50 percent of the rats in both groups had metastases from the carcinomas of the liver to distant organs. Control rats treated with nitrite only, or untreated, did not develop liver neoplasms. There was no discernible effect of nitrite on the carcinogenicity of methapyrilene hydrochloride.

Carcinogenicity of nitrosotrialkylureas in Fischer 344 rats.

Three nitrosotrialkylureas were administered to female F344 rats as approximately 1-mM solutions in drinking water. Nitrosotrimethylurea, given for 47 weeks, gave rise to astrocytomas of the brain and tumors of the forestomach; nitrosotriethylurea induced a high incidence of adenocarcinomas of the breast and uterus and tumors of the forestomach. Nitrosomethyldiethylurea induced almost exclusively a high incidence of both astrocytomas of the brain and tumors of the spinal cord.

Carcinogenicity of 3-chloronitrosopiperidine, 4-chloronitrosopiperidine, and 3,4-dichloronitrosopiperidine in Fischer rats.

Three chlorinated nitrosopiperidines, 3-chloro-, 4-chloro-, and 3,4-dichloronitrosopiperidine, were administered to groups of 20 male Fischer 344 rats at a concentration of 0.17 mM in drinking water. Treatment with the monochloro compounds lasted for 30 weeks, while treatment with the dichloro compound lasted for 21 weeks. Almost all of the animals died with esophageal tumors. There was also a significant incidence of tumors of the forestomach and tongue in the rats treated with the monochloro compounds. Using the rate of death of the animals with tumors as an index, the relative potency of the three compounds increases from 3-chloro- to 4-chloro- to 3,4-dichloronitrosopiperidine.

The effect of deuterium labeling on the carcinogenicity of nitroso-2,6-dimethylmorpholine in rats.

Nitroso-2,6-dimethylmorpholine (Me2NMOR) was labeled with deuterium in either the alpha or beta positions. Both the deuterium-labeled, and the unlabeled, compounds were administered to female Fischer 344 rats at equimolar concentrations in drinking water. The animals were then allowed to die naturally with tumors. The parent compound and the alpha-d4-labeled derivative were given at 50 mg/liter and 20 mg/liter, while, because of a shortage of the compound, the beta-d2-labeled derivative was given only at 20 mg/liter. Almost all of the animals died with basal cell carcinomas and papillomas of the esophagus; many animals fed the lower doses also had tumors of the nasal cavity and tongue. The rate of death from induced tumors was lower in the alpha-d4-treated group than in those treated with the unlabeled compound (at both dose levels), but was higher in the rats treated with the beta-d2 compound. It appears that deuterium in the alpha positions decreases carcinogenic potency, while deuterium in the beta positions increases it. This suggests that oxidation at the beta carbon atoms is less likely to be involved in esophageal carcinogenesis in the rat by Me2NMOR than is oxidation at the alpha carbon atoms.

Chronic toxicity/carcinogenicity studies of gentian violet in Fischer 344 rats: two-generation exposure.

A chronic feeding study was carried out in the F1a generation of dosed Fischer 344 rats of both sexes with gentian violet (GV). The test substance was administered in the diet to 570 male and 570 female rats at dose levels of 0 (control), 100, 300 and 600 ppm for 24 months. Rats were killed and necropsied after 12, 18 and 24 months of continuous dosing. Measurements of body weights, food consumption (and dose rate) and mortality and the results of histopathological examination were analysed statistically. Male and female rats fed 600 ppm GV for 24 months showed a decrease in body weights. Average food consumption based on g food/kg average body weight was essentially equal in all groups. Mortality at the end of the study (24 months) was approximately 33% in the controls for both males and females and approximately 66% in females of the high-dose group and 48 and 39% in males of the mid- and high-dose groups, respectively. All dose-related neoplastic pathology was noted at the final necropsy. Following 24 months of dosing, there was a significant difference from the controls in the incidence of follicular cell adenocarcinoma of the thyroid gland for both males (600 ppm GV) and females (300 and 600 ppm GV). Although the incidences were very low, statistical analysis showed a significant difference from the controls for hepatocellular adenomas in the mid-dose group of the females and the mid- and high-dose groups of the males. A dose-time-related incidence of mononuclear cell leukaemia was also noted in the females. There was high background incidence of the leukaemia. Several non-neoplastic dose-related lesions were observed in both males and females, principally in the 18- and 24-month necropsies. Almost all of these lesions were focal changes in the liver, many of which were probably related to the mononuclear cell leukaemia.

Chronic toxicity/carcinogenicity studies of sulphamethazine in B6C3F1 mice.

A chronic feeding study was carried out in B6C3F1 mice with sulphamethazine (SMZ). The test substance was administered in the diet at dose levels of 0 (control), 300, 600, 1200, 2400 and 4800 ppm for 24 months. Mice were killed after 12, 18 and 24 months of continuous dosing. Body weights and food consumption were measured weekly, and mortality was recorded daily. All animals received a complete necropsy and histopathological examination, the results of which were analysed statistically. A slight decrease in body-weight gain was noted for mice of all dose groups with females showing the greater effect. Food consumption based on g food/g average body weight was relatively constant among the controls and various dose groups. The mortality rate for males and females of the control groups (8 and 8%, respectively) was higher than that for males and females of some of the higher dose groups. Neoplastic lesions associated with the ingestion of SMZ in the diet included follicular cell adenomas of the thyroid gland. At the 24-month necropsy, the incidence of this lesion for males and females of the 4800-ppm dose groups was 33 and 26%, respectively. Non-neoplastic dose-related lesions observed in both males and females included follicular cell hyperplasia (diffuse and focal) of the thyroid gland, haematopoietic cell proliferation of the spleen and pigmentation of the spleen. In females, pigmentation of the lymph nodes and hyperplasia of the mammary gland were also noted.

Subchronic studies of doxylamine in B6C3F1 mice.

Doxylamine succinate, a histamine (H1) antagonist (antihistamine), was administered as an admixture in the feed to male and female B6C3F1 mice for 14 or 90 days. Dose levels of 0, 100, 250, 500, 1000, and 2000 ppm doxylamine were administered to males and females in the 14-day study while dose levels of 0, 80, 162, 325, 750, and 1500 ppm were administered to both sexes in the 90-day study. Little toxicity was seen in the 14-day study. Final body weights in the highest dose group were reduced 4.0 and 7.3% in males and females, respectively. Treatment-related histopathological changes in the 14-day study were limited to a very low incidence of hepatic necrosis in both sexes. There was little toxicity observed in the 90-day study and no clear dose response relative to weight gain was observed. Histologically, the liver was the only organ affected by doxylamine administration. The liver lesions consisted of hepatic cell cytomegaly and/or karyomegaly which varied from mild to severe and a possible dose-related hepatic necrosis.

Subchronic studies of doxylamine in Fischer 344 rats.

Doxylamine succinate was administered as an admixture in the feed to male and female Fischer 344 rats for either 14 or 90 days. The 14-day study included dose levels of 0, 100, 250, 500, 1000, or 2000 ppm doxylamine. Except for a 7% decrease in final body weight in female rats in the 2000 ppm group, there were no significant clinical observations made in the 14-day study. Microscopic lesions judged to be treatment-related were limited to cytoplasmic vacuolization in the livers. The lesions were more numerous in the higher dose groups of males and present only in the 2000 ppm group of females. Dose levels of 0, 162, 405, 1012, 2530, and 6325 ppm doxylamine were administered in the 90-day study. There were no deaths during the study. Final body weights were decreased 13.3% in males of the 6325 ppm group and 5.2, 10.1, and 14.4% in females in the 1012, 2530, and 6325 ppm groups, respectively. Liver/brain weight ratios were increased in all treated male groups and in the two highest dose groups of females. Other organ weight changes were decreases and believed to result from general reduction in weight gain in those groups where the decreases occurred. Treatment-related histological changes were identified in the liver and parotid salivary gland. Cytoplasmic vacuolization or fatty change of the liver was found in all groups of males but was more severe in the higher dose groups. In females, these liver lesions were observed only in the two highest dose groups. A dose-related change in the parotid salivary gland, consisting of cytomegaly with basophilic and coarsely granular or vacuolated cytoplasm, was observed.

The effect of deuterium on the carcinogenicity of nitroso-methyl-n-butylamine.

Nitrosomethyl-n-butylamine and its derivatives, labeled with deuterium in the methyl group or at the alpha position of the butyl group, were given to rats in drinking water at equimolar doses for approximately 20 weeks. Two concentrations were used, 16 mg/l and 6.25 mg/l. The unlabeled compound was not as toxic as its analog labeled in the methyl group, which caused the early death of some of the test animals. The methyl-labeled compound was a more effective carcinogen, and the butyl-labeled compound was a less effective carcinogen, than the unlabeled compound, as measured by the rate of death of animals with tumors of the esophagus. Almost all of the surviving animals died with esophageal tumors, basal cell papillomas and carcinomas.

Bladder and liver tumorigenesis induced by 2-acetylaminofluorene in different F1 mouse hybrids: variation within genotypes and effects of using more than one genotype on risk assessment.

Several F1 mouse hybrids were used in a chronic bioassay to determine whether such an experimental design would provide greater statistical power than using only the B6C3F1 hybrid. For this purpose, the dose response of formation of hepatocellular and bladder tumors after 30 mo of feeding 2-acetylaminofluorene (2-AAF) in the diet was assessed in 4 F1 mouse hybrids, including the B6C3F1 hybrid. No strain background-related differences in frequency of bladder neoplasms between any F1 hybrids were detected. Bladder tumors occurred only at the highest 2-AAF dose in female mice. In males the lowest dose was already sufficient to induce bladder neoplasms with incidences of 25-48% adjusted for different nontumor mortality patterns across doses. No marked strain-related differences in hepatocellular tumor rates were apparent in either sex. Higher frequencies of hepatocellular neoplasms were observed among the untreated control males of the B6C3, AY, and CVA F1 hybrids than among the comparable females. Among treated mice, the lowest 2-AAF dose increased liver tumor incidence, more so among the females than among the males. The different background genomes resulted in somewhat different risk assessments for liver tumor formation in males due to differences in the time-to-tumor curves. Except for the much higher background liver tumor rate in the CVY mice, the adjusted liver tumor incidences were similar across the four hybrids. Hence, the levels of statistical significance obtained for dose-response trends and comparisons of treated and control groups were similar using 48 animals per dose groups with B6C3 mice, or combinations of 24 animals per dose from 2 genotypes, or 12 animals per dose from the 4 hybrid genotypes. Estimates of carcinogenic potency for bladder tumors were similar, within a factor of two, across the four hybrids. However, estimates of liver tumor potency across genotypes varied by a factor of two and six for females and males, respectively. Thus, the mean of cancer potency estimates across these genotypes would be more representative for mice than results from any single genotype. As in chronic carcinogenesis studies with other test agents, neoplasms developed in only a certain proportion, rather than in all, of the genetically identical animals exposed to a given dose of the toxicant for the same length of time under the same controlled environmental conditions. This phenotypic variability in toxic responses may reflect differential regulation of gene expression among the genetically identical test animals.

Chronic toxicity and carcinogenicity studies of gentian violet in mice.

Gentian violet is a dye belonging to a chemical class known as the di- and triaminophenylmethanes. Although it has been used for many years for the control of fungal and intestinal parasites, for various uses in veterinary medicine, and as an additive to the feed of chickens to inhibit propagation of mold and fungus, very few long-term toxicity data are available. A life span dosing study of gentian violet in the diet of 720 males and 720 females of B6C3F1 mice (C57BL/6 X C3H) at dose levels of 0, 100, 300, and 600 ppm was done to determine its toxicity and carcinogenicity. Sacrifices were conducted after 12, 18, and 24 months of continuous dosing. There was no effect on food consumption or body weight gain; however, a dose effect was noted for mortality rates. Mortality (adjusted for sacrifices) in the controls of both sexes was less than 15% at 24 months, but was approximately 64% in the females and 23% in the males given the high dose. Females appeared to be more susceptible than males. A positive dose response for hepatocellular carcinoma was noted in males at 24 months and in females at 18 and 24 months. Statistical tests for dose-related trends with respect to mortality due to liver neoplasms, prevalence of liver neoplasms, and time to onset of liver neoplasms showed positive trends in both males and females. Other dose-related toxicological responses, particularly in the female mice, included erythropoiesis in the spleen, atrophy of the ovaries, adenoma of the Harderian gland, and the presence of type A reticulum cell sarcomas in the urinary bladder, uterus, ovaries, and vagina. The estimation of risk of 10(-6) over background for malignant liver neoplasms using linear extrapolations showed a lower bound on the virtually safe dose (VSD) to be 2 ppb for the female mice and 1 ppb for the male mice. For benign and malignant liver tumors together, the lower bound on the VSD was essentially the same as for malignant liver neoplasm alone. Under the conditions of the experiment described above, gentian violet appears to be a carcinogen in mice at several different organ sites.

Longevity, body weight, and neoplasia in ad libitum-fed and diet-restricted C57BL6 mice fed NIH-31 open formula diet.

Groups of C57BL6 mice of each sex were assigned to one of 2 dietary regimens, ad libitum (AL) or dietary restriction (DR), to study effects of food restriction on body weight, survival, and neoplasia. The AL and DR groups were subdivided into a scheduled sacrifice group for examination at 6-mo intervals, and a lifetime group to provide longevity data. Necropsies and microscopic examinations were conducted on 911 animals. In the lifetime group food consumption averaged 33.6 and 34.4 g per week by AL males and AL females, respectively; the DR counterparts were given 40% less. The diet contained 4.35 kcal/g. The average lifetime body weights were 34.8, 26.8, 22.6, and 21.6 g for AL males, AL females, DR males, and DR females, respectively, and their age at 50% survival was 27.5, 26.9, 31.7, and 33.5 mo. Maximal lifespan was increased 18% in DR males and females. Lifetime incidence of tumor-bearing mice was 89% and 86% for AL males and females, versus 64% for each sex of DR mice. Dramatic reduction occurred in female DR mice in lymphoma (9% vs 29%), pituitary neoplasms (1% vs 37%), and thyroid neoplasms (0.4% vs 8%). In males, hepatocellular tumors were reduced to 1% from 10% by DR. In contrast, the incidence of histiocytic sarcoma was increased in DR females and unaffected in DR males. Tumor onset was delayed in DR animals; 87% of all neoplasms in males and 95% in females had occurred in the AL mice by 24 mo, whereas the DR animals had only 52% and 39% of their lifetime incidence, respectively, by that age. This study provided comparative AL and DR data from C57BL6 mice examined randomly at 6-mo intervals (cross-sectional group) in parallel with data from animals in similar cohort that was unsampled and allowed to succumb naturally (longevity group). Dietary restriction reduced the lifetime percentage of tumor-bearing animals and the number of tumors per animal, and delayed the age at onset of most neoplasms.

Cause-of-death assignment at the National Center for Toxicological Research.

The system for assigning cause of death in animal studies of carcinogenicity at the National Center for Toxicological Research (NCTR) is described. An empirical study of the NCTR's experience with its current cause-of-death assignment system based on selected representative experiments is reported. Issues investigated include the degree of confidence associated with histologic cause-of-death assignment, potential age-, dose-, and sex-related differences in assigned grades of certainty of cause of death, and frequencies of identification of various organ-specific and systemic diagnoses as the cause of death. Implications for age-adjusted statistical tests of carcinogenicity that require cause-of-death data are discussed.

Carcinogenicity of the isomeric, N-nitroso-delta3-and N-nitroso-delta2-piperidines in rats and the in vivo isomerization of the delta3-to the delta2-isomer.

N-Nitroso-1,2,3,6-tetrahydropyridine (N-nitroso-delta3-piperidine), N-nitroso-1,2,3,4-tetrahydropyridine (N-nitroso-delta2-piperidine) and N-nitroso-3,4-epoxypiperidine were tested for carcinogenicity in Fischer 344 rats. The unsaturated nitrosamines were administered in drinking water (100 mg/l). The epoxide was administered by gavage in corn oil (11.5 mg/ml, 0.2 ml twice a week). Both of the unsaturated nitrosamines were potent carcinogens (most of the animals died by the 35th week), and both produced many esophageal tumors, a property which they have in common with the parent compound, N-nitrosopiperidine. The spectrum of the other tumors formed, however, was different. The delta3-isomer produced hemangioendothelial sarcomas in the liver, which were absent in the tumor spectrum of the delta2-isomer and N-nitrosopiperidine. The delta2-isomer, on the other hand, produced tumors of the forestomach and the oropharynx, which were essentially absent in the rats treated with the delta3-isomer. N-nitroso-3,4-epoxypiperidine was a toxic compound (8 deaths in the first 5 weeks), but most of the remaining animals survived to 40 weeks. Of these, 8 animals died of induced tumors (esophagus and liver). The delta2- and the delta3-isomers were administered by gavage to groups of rats and the blood of these animals was withdrawn at timed intervals. Analysis of the serum revealed that both of the nitrosamines were cleared rapidly from circulation but that at the same time the delta3-isomer was being isomerized to the delta2. The reverse transformation did not occur in vivo.

Bile duct-specific lectins, Dolichos biflorus agglutinin and peanut agglutinin, as probes in mouse hepatocarcinogenesis.

BACKGROUND: It is well established that alterations in the expression of cell surface glycoproteins occur during the course of tumorigenesis and can be detected immunohistochemically. However, no consistent markers of malignancy in mouse hepatocellular tumors have yet been identified. EXPERIMENTAL DESIGN: Lectin histochemistry, using three bile duct-specific lectins, Dolichos biflorus agglutinin (DBA), peanut agglutinin (PNA) and soybean agglutinin (SBA), and anti-epidermal keratin immunohistochemistry, was conducted on formalin-fixed, paraffin-embedded tissues of a spectrum of benign and malignant hepatocellular proliferative lesions of mice, including hepatocholangiocarcinomas. DBA- and PNA-binding glycoproteins in normal livers and in bile and liver tumors of mice were verified by SDS-PAGE and Western blot analysis. RESULTS: Normal bile duct cells stained strongly with DBA but minimally to moderately with PNA and SBA. DBA-positive tumor cells were present in 96% of hepatocholangiocarcinomas, 89% of hepatocellular carcinomas, and 35% of hepatocellular adenomas. In comparison, 43% of hepatocholangiocarcinomas, 37% of hepatocellular carcinomas, and 24% of hepatocellular adenomas exhibited PNA staining. SBA did not specifically stain tumor cells. Normal hepatocytes and those in altered foci were consistently negative for these three lectins. Keratin-positive staining was found only in normal bile ductular cells and ductal elements in 70% of hepatocholangiocarcinomas. Electrophoresis and Western blot analysis demonstrated that, in normal livers, DBA and PNA bound to the 13- to 16-kDa and 27- to 30-kDa glycoproteins believed to be of bile duct cell origin and commonly present in hepatocellular adenomas, hepatocellular carcinomas, and hepatocholangiocarcinomas, with strongest expression in the last. In addition, hepatocholangiocarcinomas had the same high molecular mass glycoprotein (> 200 kDa) labeled with DBA as detected in bile. CONCLUSIONS: Our results suggest that some malignant hepatocytes, especially in mouse hepatocholangiocarcinomas, have the potential of biliary differentiation. DBA is a sensitive marker for malignant hepatocytes in mice.