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The chronic toxicity and carcinogenicity of pyrilamine maleate were evaluated in B6C3F1 mice by feeding for up to 2 years to groups of 60 male or female mice at dietary levels of 0,150, 750, or 1,500 ppm (as the free base). Up to 12 mice per sex and dose group were killed after 65 weeks for clinical chemistry, hematology, and histopathologic evaluation of control and high-dose animals. Histopathology was performed on all animals that were dead or moribund or lived until the study was terminated at 2 years. Daily exposures to pyrilamine ranged from ∼16 to 176 mg/kg, compared with maximum human exposure of 3 mg/kg. Regarding longevity, pyrilamine-treated male mice tended to survive longer than controls, whereas no differences among treated and control female mice were noted. Body weights did not differ among treated and control male groups, but final body weight average of treated female mice was 6–10% lower than that for controls. Although no dose-response trends were noted in male mice for histopathologic end points, liver /body and liver/ brain weight ratios were significantly greater in high-dose male mice than in controls. Chronic liver inflammation exhibited a positive dose-response trend in female mice, but there was no evidence for a carcinogenic response to pyrilamine by either sex. [ABSTRACT FROM PUBLISHER]

The purpose of this study was to determine the effects of subchronic exposure to the antihistamine pyrilamine maleate and to establish dosage levels to use in a 2-year chronic study. A 14-day repeated-dose study in male and female B6C3F1 mice at dietary levels of pyrilamine (as the free base) of 0, 196, 392, 783, 1563, or 3122 ppm revealed no influence on body weight gain and resulted in no deaths, nor abnormal clinical or gross necropsy observations. Parotid gland cell necrosis was more apparent in high-dosage groups than in controls of either gender. Male and female B6C3F1 mice also were administered pyrilamine for 90 days at dietary concentrations of 0, 375, 750, 1500, 3000, or 6000 ppm. Weight gain was markedly suppressed in the 6000 ppm group, but less so at 1500 and 3000 ppm. Parotid gland cell necrosis was apparent in males receiving 750 ppm pyrilamine and above and in females at 1500 ppm and above. Cytomegaly was noted in the parotid glands of both sexes at midlevel dosages but not in control, low-dosage, or high-dosage animals. It was concluded that 1500 ppm pyrilamine would not be life-threatening to B6C3F1 mice in a chronic study. [ABSTRACT FROM PUBLISHER]

This study was designed to determine the subchronic effects of the antihistamine, pyrilamine maleate, and to establish dose levels for a 2-year chronic study. Six male and six female F344 rats received dietary pyrilamine concentrations of 0, 392, 783, 1563, 3122, or 6276 ppm (as the free base) for 14 days. Only weight gain suppression was noted, at 3122 and 6276 ppm, especially in males. When 12 male and 12 female F344 rats were fed pyrilamine for 90 days at 0, 750, 1500, 3000, 6000, or 12,000 ppm, weight gain was suppressed and organ/body weight ratios often were higher than in controls at 3000 ppm and above. Heart/brain and thymus/brain ratios were decreased at 3000 ppm and above in males and at 6000 or 12,000 ppm in females. All rats receiving 6000 or 12,000 ppm pyrilamine exhibited severe diffuse parenchymal cell cytomegaly in the parotid salivary glands with a dose-related decrease in severity and prevalence at lower concentrations. Hepatocellular vacuolization, present only in males, increased in severity with dose, starting at 750 ppm. The severity of liver fatty metamorphosis increased with dose at the three low doses but was less apparent and/or disappeared at 6000 and 12,000 ppm. It was concluded that 3000 ppm pyrilamine would not be life threatening to F344 rats in a chronic study. [ABSTRACT FROM PUBLISHER]

The rat mammary carcinogen, N-hydroxy-2-acetylamino-fluorene (N-hydroxy-2-AAF), has been proposed to be metabolically activated by mammary cytosolic N,O-acetyltransferase to a DNA binding species. To test this hypothesis, adult female Sprague-Dawley derived CD rats were treated, l.p., with 4.0 mg/kg [ring-H]N-hydroxy-2-AAF. After 4 h, 1, 3, 14, and 28 days, the animals were killed, the mammary epithelium DNA was isolated and the carcinogen-deoxyribonucleoside adducts present were analyzed by high pressure liquid chromatography. At each time, only one adduct was detected and it was chromatographically identical to N-(deoxyguanosin-8-yl)-2-aminofluorene. The level of the adduct was maximal at 4 h (1.5 adducts/10 nucleotides) and then decreased, following first order kinetics with a t of 14.2 days. The detection of a single non-acetylated aminofluorene adduct is consistent with N,O-acyltransferase being involved in the metabolic activation of N-hydroxy-2-AAF in the rat mammary gland. [ABSTRACT FROM PUBLISHER]

A single local injection of 2.5 μmol of N-hydroxy-N-formyl-2-aminofluorene (N-hydroxy-FAF), N-hydroxy-N-acetyl-2-aminofluorene (N-hydroxy-AAF), or N-hydroxy-N-propionyl-2-aminofluorene (N-hydroxy-PAF) to each of the six left mammary glands of female Sprague-Dawley derived CD rats gave a mammary tumor incidence, after 12 months, of 53% for the N-acetyl (42% adenocarcinoma, 11% fibroadenoma), 41% for the N-formyl (8% adenocarcinoma, 11% sarcoma, 22% fibroadenoma), and 33% for the N-propionyl (11% adenocarcinoma, 22% fibroadenoma) derivatives of N-hydroxy-N-2-aminofluorene. Latent periods for malignant tumor appearance (adenocarcinoma or sarcoma) was 210 days, 148 days, and 177 days, respectively, with no malignant tumors occurring in the vehicle-treated animals. In contrast, latent periods for benign tumor appearance (fibroadenoma) was 263 days for control animals, 289 days for the N-hydroxy-AAF, 324 days for the N-hydroxy-FAF, and 317 days for the N-hydroxy-PAF animals. When N-acetyl-2-aminofluorene (AAF) was applied as above there was only an 8% mammary tumor incidence (4% adenocarcinoma, 4% fibroadenoma) with a latent period of 207 days for malignant tumor (adenocarcinoma) and 221 days for benign tumor (fibroadenoma) appearance. Arylhydroxamic acid N,O-acyltransferase activity has been demonstrated in the mammary glands of male, and lactating and non-lactating female Sprague-Dawley derived CD rats by means of a nucleic acid binding assay. Mammary gland cytosol catalyzed tRNA adduct formation to a greater extent with N-hydroxy-FAF. AAF was not activated by this enzyme. Ammonium sulfate fractionation demonstrated the presence of two enzymes, one specific for N-hydroxy-FAF (70–80% fraction), the other specific for N-hydroxy-AAF and N-hydroxy-PAF (40–70% fraction). Moreover, gel filtration chromatography of mammary gland cytosol demonstrated the presence of two enzymes of differing acyl specificity. Mammary gland microsomes catalyzed the formation of tRNA adducts, but only with the N-hydroxy-FAF derivative. Assays that tested the mutagenic potential of the arylhydroxamic acids in TA-1538 with either mammary gland cytosol or microsomes demonstrated the order of mutagenicity to be N-hydroxy-FAF » N-hydroxy-AAF > N-hydroxy-PAF. A similar order of mutagenicity was demonstrated without an external metabolic activation system. These data demonstrate the presence of two distinct enzymes in the rat mammary gland that activate arylhydroxamic acids. [ABSTRACT FROM PUBLISHER]

The antihistamine, triprolidine hydrochloride, was fed at dietary concentrations of 0, 250, 1000, or 2000 ppm (as the free base) to groups of 60 Fischer 344 (F344) rats of each sex for up to 2 years to evaluate its potential carcinogenicity. Up to 12 per sex from each group were killed at 65 weeks, and hematology, clinical chemistry, and histopathology were evaluated. A complete histopatho-logical evaluation was performed on all other animals; survivors were killed at 2 years. Survival was significantly extended in tri-prolidine-treated males and females, particularly at the high dose. At the close of the study high-dose males and females had gained significantly less body weight than controls. Among rats killed at 65 weeks females in the mid- and high-dose groups weighed significantly less than controls, but weights of control and dosed males were not significantly different. The incidences of numerous lesions tended to decrease with increasing triprolidine dose. In females, clitoral gland adenomas, thyroid c-cell hyperplasia and neoplasia, mammary gland hyperplasia and fibroadenomas, and uterine stromal polyps, and in males, anterior pituitary gland adenomas, preputial gland neoplasia, thyroid c-cell hyperplasia, pancreatic islet neoplasia, mononuclear cell leukemia, and the combination of lymphocytic, histiocytic, and undifferentiated cell malignant lymphomas and mononuclear leukemia, all exhibited negative dose trends. Cytoplasmic alterations of the parotid gland and numerous liver lesions tended to be more frequent in treated than in control animals. Liver lesions that exhibited positive dose trends include chronic inflammation and centrilobular fatty change in both sexes, mixed cell foci, and the combination of mixed cell foci and eosinophilic foci in females, and in males, basophilic foci and eosinophilic foci. Triprolidine was not carcinogenic in F344 rats. [ABSTRACT FROM PUBLISHER]

Triprolidine hydrochloride was fed to groups of 60 B6C3F1 mice per sex at dietary levels of 0, 500, 2000, or 4000 ppm (as the free base) for up to 2 years. Up to 12 mice of each sex and dose group were terminated after 65 weeks for hematology and clinical chemistry. The control and high-dose groups were examined histologically. A complete histopathological examination was performed on the remaining 48 mice from each dose group when removed from study due to moribund condition, early death, or terminal euthanization at 105 weeks. Triprolidine did not significantly alter the survival of either sex. High-dose male and mid- and high-dose female body weights were significantly less than controls at the end of the study. Significant trends toward lower frequency with increasing dose were noted in females for fatty change in the liver and lymphomas (combination of lymphocytic, mixed, and histiocytic lymphomas). Similar negative trends in males were for lymphocytic cellular infiltration in multiple organs and lung alveolar/bronchiolar adenomas or the combination of alveolar/bronchiolar adenomas or carcinomas. Significant trends toward increased frequency with increasing dose were found in female mice for lymphocytic infiltration in multiple organs and cytoplasmic alterations of the acinar cells of the parotid gland. Similar positive trends were found in males for cytoplasmic alterations of the parotid gland and various he-patocellular changes (e.g., hypertrophy and altered foci). While there was a positive dose-response trend for hepatocellular adenomas in males the combination of these and hepatocellular carcinomas eliminated the significant trend, and it was concluded that there was no evidence of a carcinogenic response to triprolidine in B6C3F1 mice. No effects considered to be adverse were observed in either sex at 500 ppm. Body weight depression was noted in females and liver toxicity was indicated in males at 2000 and 4000 ppm. [ABSTRACT FROM PUBLISHER]

Rotenone, a pesticide extracted from the root, consistently was reported by a series of investigators to have induced mammary fibroadenomas in female Wistar rats when administered ip or by gavage in a sunflower (SF) oil or SF oil:chloroform vehicle. In contrast, no less than eight bioassays done in other laboratories with rotenone or rotenone-containing powders have given consistently negative carcinogenic results when different strains or species and different modes or vehicles of administration have been used. However, these studies were not designed to address the biological reproducibility of the positive data. Thus, the present study was designed to simulate conditions of the positive studies and to investigate a possible cocarcinogenic interaction between rotenone and chloroform. Each of eight treatment groups was assigned 72 weanling female Wistar rats. Groups were (1) untreated, (2) needle puncture, (3) SF oil: 10% chloroform (SF oil:chloroform), (4) 1.0 mg/kg rotenone in SF oil:chloroform, (5) 2.0 mg%kg rotenone in SF oil:chloroform, (6) SF oil, (7) 1.0 mg/kg rotenone in SF oil, and (8) 2.0 mg/kg rotenone in SF oil. Rats were injected ip 5 days a week for 8 weeks (42 injection days) and subsequently held for 16 months. The appearance of palpable tissue masses was recorded; over 50 tissues from each rat were histologically evaluated. There were no statistically significant differences in overall or individual tumor incidences among control and rotenone treated groups. Specifically, neither incidence nor time-to-palpation of mammary fibroadenoma significantly differed among control and rotenone-treated groups, regardless of the vehicle of administration. Thus, rotenone was not carcinogenic, and rotenone and chloroform did not interact to produce a carcinogenic effect in female Wistar rats in the current study. Thus, previous reports of carcinogenic activity were not reproducible under similar experimental conditions. [ABSTRACT FROM PUBLISHER]

Examines the impact of body weight on spontaneous diseases in chronic biological assays. Recommended use of nutritionally adequate dietary manipulation in chronic studies; Importance of chronic toxicity evaluations in the estimation of risks associated with regulated products to humans.

In B6C3F1 mice, the rate of body growth influences susceptibility to liver neoplasia and large variations in body weight can complicate the interpretation of bioassay data. The relationship between body weight and liver tumor incidence was calculated for historical control populations of male and female ad libitum-fed mice (approx. 2750 and 2300 animals, respectively) and in populations of male and female mice which had been subjected to forced body weight reduction due to either dietary restriction or exposure to noncarcinogenic chemicals (approx. 1600 and 1700, respectively). Resulting tumor risk data were then used to construct idealized weight curves for male and female B6C3F1 mice; these curves predict a terminal background liver tumor incidence of 15–20%. Use of dietary control to manipulate body growth of male B6C3F1 mice to fit the idealized weight curve was evaluated in a 2-year bioassay of chloral hydrate. Cohorts of mice were successfully maintained at weights approximating their idealized target weights throughout the study. These mice exhibited less body weight variation than their ad libitum-fed counterparts (e.g., standard deviations of body weight were 1.4 and 3.4 g for respective control groups at 36 weeks). Historical control body weight and tumor risk data from the two male mouse populations were utilized to predict background liver tumor rates for each experimental group of the chloral hydrate study. The predicted background tumor rates closely matched the observed rates for both the dietary controlled and ad libitum-fed chloral hydrate control groups when each mouse was evaluated according to either its weekly food consumption or its weekly change in body weight. [Copyright &y& Elsevier]

Abstract: Usnic acid is a lichen metabolite used as a weight-loss dietary supplement due to its uncoupling action on mitochondria. However, its use has been associated with severe liver disorders in some individuals. Animal studies conducted thus far evaluated the effects of usnic acid on mitochondria primarily by measuring the rate of oxygen consumption and/or ATP generation. To obtain further insight into usnic acid-mediated effects on mitochondria, we examined the expression levels of 542 genes associated with mitochondrial structure and functions in liver of B6C3F1 female mice using a mitochondria-specific microarray. Beginning at 8weeks of age, mice received usnic acid at 0, 60, 180, and 600ppm in ground, irradiated 5LG6 diet for 14days. Microarray analysis showed a significant effect of usnic acid on the expression of several genes only at the highest dose of 600ppm. A prominent finding of the study was a significant induction of genes associated with complexes I through IV of the electron transport chain. Moreover, several genes involved in fatty acid oxidation, the Krebs cycle, apoptosis, and membrane transporters were over-expressed. Usnic acid is a lipophilic weak acid that can diffuse through mitochondrial membranes and cause a proton leak (uncoupling). The up-regulation of complexes I–IV may be a compensatory mechanism to maintain the proton gradient across the mitochondrial inner membrane. In addition, induction of fatty acid oxidation and the Krebs cycle may be an adaptive response to uncoupling of mitochondria. [Copyright &y& Elsevier]

Chloral hydrate is widely used as a sedative in pediatric medicine and is a by-product of water chlorination and a metabolic intermediate in the biotransformation of trichloroethylene. Chloral hydrate and its major metabolite, trichloroacetic acid, induce liver tumors in B6C3F1 mice, a strain that can exhibit high rates of background liver tumor incidence, which is associated with increased body weight. This report describes the influence of diet and body weight on the acute toxicity, hepatic enzyme response, and toxickinetics of chloral hydrate as part of a larger study investigating the carcinogenicity of chloral hydrate in ad libitum-fed and dietary controlled mice. Dietary control involves moderate food restriction to maintain the test animals at an idealized body weight. Mice were dosed with chloral hydrate at 0, 50, 100, 250, 500, and 1000 mg/kg daily, 5 days/week, by aqueous gavage for 2 weekly dosing cycles. Three diet groups were used: ad libitum, dietary control, and 40% caloric restriction. Both dietary control and caloric restriction slightly reduced acute toxicity of high doses of chloral hydrate and potentiated the induction of hepatic enzymes associated with peroxisome proliferation. Chloral hydrate toxicokinetics were investigated using blood samples obtained by sequential tail clipping and a microscale gas chromatography technique. It was rapidly cleared from serum within 3 h of dosing. Trichloroacetate was the major metabolite in serum in all three diet groups. Although the area under the curve values for serum trichloroacetate were slightly greater in the dietary controlled and calorically restricted groups than in the ad libitum-fed groups, this increase did not appear to completely account for the potentiation of hepatic enzyme induction by dietary restriction. [Copyright &y& Elsevier]

Chloral hydrate, which is used as a sedative in pediatric medicine and is a by-product of water chlorination, is hepatocarcinogenic in B6C3F1 mice, a strain that can exhibit high rates of background liver tumor incidence, which are associated with increased body weight. In this study, dietary control was used to manipulate body growth in male B6C3F1 mice in a 2-year bioassay of chloral hydrate. Male B6C3F1 mice were treated with water or 25, 50, or 100 mg/kg chloral hydrate by gavage. The study compared ad libitum-fed mice with dietary controlled mice. The latter received variably restricted feed allocations to maintain their body weights on a predetermined “idealized” weight curve predictive of a terminal background liver tumor incidence of 15–20%. These mice exhibited less individual body weight variation than did their ad libitum-fed counterparts. This was associated with a decreased variation in liver to body weight ratios, which allowed the demonstration of a statistically significant dose response to chloral hydrate in the dietary controlled, but not the ad libitum-fed, test groups. Chloral hydrate increased terminally adjusted liver tumor incidence in both dietary controlled (23.4, 23.9, 29.7, and 38.6% for the four dose groups, respectively) and ad libitum-fed mice (33.4, 52.6, 50.6, and 46.2%), but a statistically significant dose response was observed only in the dietary controlled mice. This dose response positively correlated with markers of peroxisomal proliferation in the dietary controlled mice only. The study suggests that dietary control not only improves terminal survival and decreases interassay variation, but also can increase assay sensitivity by decreasing intra-assay variation. [Copyright &y& Elsevier]

Genistein is a plant-derived estrogenic isoflavone commonly found in soy-based products such as soymilk and soy-based dietary supplements for treating menopausal symptoms, for example. Vasopressin is a neurosecretory nonapeptide synthesized primarily in neurons of the hypothalamus and secreted into the bloodstream from the posterior lobe of the pituitary. The endogenous opiate peptide beta-endorphin is synthesized both in neurons of the hypothalamus and in pituitary cells, primarily of the neurointermediate lobe. It has been reported that exposure to 17beta-estradiol or diethylstilbesterol increased the vasopressin content of the hypothalamus, and that estradiol valerate selectively damages hypothalamic beta-endorphin-containing neurons. Since little was known of the potential effects of estrogenic endocrine-disruptor compounds on hypothalamic neuropeptides, we fed Sprague-Dawley fetuses from day 7 in utero until sacrifice at postnatal day 77, with either a control diet (<1 ppm) or an experimental diet containing 25, 250, or 1250 ppm of genistein. We then conducted ELISA assays for hypothalamic content of both beta-endorphin and vasopressin immunoreactivity. Whereas there were no statistically reliable effects of dietary genistein on hypothalamic beta-endorphin content, vasopressin levels were significantly elevated in the 1250-ppm genistein group (p < 0.05). Elevated vasopressin levels may be associated with fluid balance, altered blood pressure, and cardiovascular effects. These data are consistent with the known actions of estradiol and may serve to explain our finding in a previous study that estrogenic endocrine-disruptors such as genistein increased sodium preference in rats exposed through their diet.