Carlton D. Jackson

2-year Toxicity Study of Doxylamine Succinate in the Fischer 344 Rat

Doxylamine succinate, a commonly used antihistamine, was administered as an admixture in the feed to groups of male and female Fischer 344 rats at dose levels of 0, 500, 1000, and 2000 parts per million (ppm) (based on free amine) for 65 weeks (9 per group) or two years (48 per group). There were no significant treatment-related differences in survival in either sex. Compared to controls, final body weights of rats in the 2000 ppm group were reduced 8.4% and 22.8% in males and females, respectively. Treatment-related, non-neoplastic lesions were found primarily in the liver and parotid salivary gland. Liver lesions consisted of fatty change and degeneration in both sexes; hyperplasia (resulting from degeneration due to leukemia) and atypical cells in males; and hypertrophy, chronic inflammation, and mixed cell foci in females. Both sexes exhibited a treatment-related increase in cytoplasmic alteration in the salivary glands. Liver neoplasms were found only in the highest dose group of male rats. The trend test was significant (p ≤ 0.05) for increased incidence of hepatocellular adenoma and carcinoma with increasing doses of doxylamine, but the increased incidence of either lesion in the high dose group was not significant compared to that in controls. However, when animals with carcinoma or adenoma were combined, the trend test remained significant (p ≤ 0.01), and the incidence of the highest dose group was significantly increased (p ≤ 0.05) over that in controls. No treatment-related increase in neoplasms was found in females, but doxylamine produced a marked dose-related decrease in mammary fibroadenomas. Although not statistically significant, a very rare pineal gland tumor was found in 1 male and 1 female rat in the 2000 ppm group.

ANALYSIS OF QUANTAL RESPONSE DATA FROM MIXTURE EXPERIMENTS

This paper presents a logistic regression procedure for the analysis of mixture data. The design of the mixture experiment is based on the simplex-lattice designs where the components represent the proportions of a mixture, i.e. each component is expressed as a fraction of the mixture with the sum equal to one. The Scheffe (1958) and Cox (1971) polynomial and Becker (1968) non-polynomial forms are used to model relationships between the expected response and individual components of the mixture. In a three components mixture it was shown that if one component is completely linear with the remaining two components, then the proper representation of such a system is the Becker non-polynomial model. An experimental data set designed to study the interrelating effects of calories from dietary fat, carbohydrate and fibre on tumour incidence is used to illustrate the analysis and the additive effect of the fibre component. Each mixture (diet) of fat, carbohydrate and fibre was administered to provide the same total caloric consumption.

Two-Year Toxicity Study of Doxylamine Succinate in B6C3F1 Mice:

Doxylamine succinate, a commonly used antihistamine, was administered as an admixture in the feed to groups of male and female B6C3F1 mice at dosage levels of 0,190,375, and 750 parts per million (ppm) (based on free amine) for 65 weeks (12 per group) or 2 years (48 per group). Survival to terminal sacrifice in the 2-year groups was 85–98% with no significant differences between groups of the same sex. Final body weights of the highest dose group were 3.4% and 8.7% less than controls in males and females, respectively. Doxylamine produced liver lesions in male mice including hepatocellular hypertrophy, atypical hepatocytes, clear cell and mixed cell foci, and necrosis. In females, doxylamine produced liver fatty change, hepatocellular hypertrophy, and necrosis. Doxylamine produced a significant increase in hepatocellular adenomas in the mid- and high-dosage groups of males and in the high-dosage group of females. Thyroid follicular cell hyperplasia and thyroid follicular cell adenomas also were increased in treated mice of both sexes. A treatment-related increase in cytoplasmic alteration of the parotid salivary gland in males and an increased incidence in hyperplasia of the pituitary gland in females were observed.

Dietary restriction modulated carcinogen-DNA adduct formation and the carcinogen-induced DNA strand breaks

Dietary restriction (DR) alters the activities of hepatic drug metabolizing enzymes and modulates the formation of carcinogen-DNA adducts in carcinogen treated animals. Our previous results showed that a 40% restriction of diet (60% of ad libitum (AL) food consumption) reduced the hepatic metabolic activation of aflatoxin B1 (AFB1) but increased the activation of benzo[a]-pyrene (BaP) in both rats and mice. In this study, the focus was directed toward the levels of carcinogen-DNA adducts formation and the carcinogen-induced DNA strand breaks in mouse kidney and liver DNA. DR significantly inhibited both AFB1-DNA adduct formation and AFB1-induced DNA strand breaks in kidney DNA of mice that received a single dose of [3H]AFB1 (5 mg/kg). The levels of AFB1-DNA adduct formation in mouse kidney DNA correlated well with increased AFB1-induced DNA strand breaks. The correlation between the levels of AFB1-DNA-adducts formed and DNA strand breaks in kidney DNA of DR-mice was less linear than between its AL-counterpart suggesting that other factors, such as different rates of DNA repair, may be involved. In addition, DR enhanced hepatic BaP- and 6-nitrochrysene (6-NC)-DNA adduct formation in the mice treated with BaP and 6-NC, respectively. The formation of the specific BaP-adduct, 10-(N2-deoxyguanosinyl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP (N2-dG-BaP), in mouse liver was proportional to the dose, and was compatible to the BaP-induced DNA strand breaks affected by DR. The enhancement of the total 6-NC-DNA adduct formation in DR-mouse was also in correlation with the increased 6-NC-induced DNA strand breaks. The activity of mouse liver microsomal nitro-reductase increased by 2-fold in response to DR indicating that the nitroreduction may contribute to the increase of the metabolic activation of 6-NC. Our present results indicate that the effect of DR on the carcinogen activation is dependent upon the DR-modulated carcinogen metabolizing enzyme activities.

Subchronic Studies of Thenyldiamine in Fischer 344 Rats

Thenyldiamine, an antihistamine used extensively as a sleep aid, has not been adequately studied for subchronic or chronic toxicity. This report presents the results of subchronic studies of thenyldiamine in the rat. Fischer 344 rats were administered thenyldiamine hydrochloride in their feed at dose levels of 0, 250, 500, 1000, 2000, and 4000 ppm (calculated as free amine) for 14 days or at dose levels of 0, 125, 250, 500, 1000, and 2000 ppm for 90 days. There were no mortalities in either study. Except for decreased weight gain, no significant treatment-related toxicity was observed in the 14-day study. In the 90-day study, 500 ppm and 1000 ppm were the lowest doses responsible for causing a significant decrease in weight gain in females and males, respectively. Clinical signs, gross observations at necropsy, and organ weights in the 90-day study were not remarkable. Histologically, a treatment-related mild fatty change was found in the livers of male rats, but the effect was so mild as to make the significance questionable. Treatment-related changes were also observed in the parotid salivary glands, but the significance of these changes was also questionable in view of the variability of this gland and its recognized physiological responses to antihistamines.

Relative contribution of calories from dietary fat, carbohydrate, and fiber in the promotion of DMBA-induced mammary tumors in Sprague-Dawley rats

It is well known that caloric restriction inhibits, whereas excess calories promote, mammary tumorigenesis in rats. However, the relative contribution to carcinogenesis by calories derived from fat or from carbohydrate are not well established. To determine the relative effects of calories from fat or from carbohydrate, as well as any interaction of dietary fiber on the promotion of 7,12-dimethylbenz[a]anthracene-induced mammary tumors, we fed isocalorically nine diets containing different ratios of fat, carbohydrate, and fiber to female Sprague-Dawley rats treated with 7,12-dimethylbenz[a]anthracene (30/group). Under conditions of isocaloric consumption, at or near ad libitum feeding, calories from dietary fat had approximately twofold greater promoting effect on final body weight and tumor incidence than calories derived from dietary carbohydrate. Dietary fiber had an inhibitory effect on tumor development, but the effect was evident only in the high-fat groups. Logistic regression analysis of tumor incidence gave beta-coefficient estimates for the relative effects of fat, carbohydrate, and fiber of 0.866, 0.189, and -4.281, respectively. Time-to-tumor analysis by the Weibull model indicated beta-estimates of 3.016, 3.324, and 5.825 for dietary fat, carbohydrate, and fiber, respectively, indicating that fat shortens and fiber increases the length of time to tumor. The statistical model derived from these results also indicates a significant synergistic interaction of dietary fat and carbohydrate on final body weight and tumor incidence.

Interactions of varying levels of dietary fat, carbohydrate, and fiber on food consumption and utilization, weight gain and fecal fat contents in female sprague-dawley rats

The purpose of this study was to better define a nutritional regime for the DMBA-induced rat mammary tumor model for studying the interdependent effects of fats, carbohydrates, and fiber on mammary tumor development. Female Sprague-Dawley rats were administered (ad libitum) a modified AIN-76A diet containing different ratios of dietary fat, carbohydrate, and fiber for 6 weeks. Food consumption was decreased in the higher fat groups, but the decrease did not compensate completely for the higher caloric density of the diets. Calorie consumption and body weight gain were greater in both the mid and high than in the low fat groups. Dietary fiber had only a marginally significant effect on food consumption but did decrease both calorie consumption and body weight gain in the high fat groups. There was no significant interaction between fat and fiber with respect to food or calorie consumption, but there was a marginally significant interaction between these two dietary components on body weight gain. Food utilization, with respect to weight gain, was increased by fat and decreased by fiber, with a significant antagonistic interaction between the two. Calorie utilization (g weight gain/100 kcal) was increased by increased fat but the effect of fiber was not significant. Dry fecal weight was increased by dietary fiber and decreased by dietary fat. The amount of fecal fat excreted was increased by dietary fiber but the effects of dietary fat on fecal fat excretion were inconsistent. The results indicate a complex interaction of dietary fat and fiber. Animals administered the low fat/high fiber diet consumed the least number of calories and thus would be the control group in a pair fed, isocaloric study.

Tissue binding of 2-acetylaminofluorene in BALB/c and C57B1/6 mice during chronic oral administration

Blood and tissue binding levels of [9-14C]2-acetylaminofluorene ([9-14C]2-AAF) administered in the diets of female BALB/c and C57Bl/6 mice, were determined. Blood and liver levels reached a plateau after 2--4 weeks, while levels in bladder tissue continued to increase. Binding levels two weeks administration were determined in liver, bladder, kidney, lung, spleen, heart and skeletal muscle. In every instance the binding was linear over a dose range of 0.5--500 ppm 2-AAF. Removal of the 2-AAF in the diet after 48 weeks resulted in a rapid loss of radioactivity from blood and liver, with little or no loss from bladder tissue. The linear dose-response relationship of tissue binding correlated well with the previously reported linear dose response curve for liver tumors but did not correlated with the reported non-linear dose response curve for bladder tumors. A reversed correlation was found in the ability of the tissue to remove bound fluorene residues and the observed rate of tumor occurrence following discontinued administration of the carcinogen.

Subchronic studies of tripelennamine in fischer 344 rats

The purpose of this study was to determine the subchronic toxicity of the antihistamine tripelennamine and to allow selection of appropriate doses for chronic studies. Male and female Fischer 344 rats were administered tripelennamine hydrochloride in the feed at dose levels of 0, 300, 600, 1200, 2400, and 6000 ppm (as the free amine) for 14 days or at dose levels of 0, 150, 300, 600, 1200, and 2400 ppm for 90 days. In the 14-day study, all of the animals in the 6000 ppm groups died. All others survived until killed. Final body weights of treated groups were reduced from 3% to 25%. Cytoplasmic vacuolization of the liver was observed. In the 90-day study, a dose-dependent reduction in body weight gain was produced by tripelennamine, with a 10% reduction in final body weight occurring between 300 and 600 ppm. Reductions in various organ weights were found to be correlated with reduced final body weights. Tripelennamine produced cytoplasmic vacuolization or fatty change in the liver, cytomegaly and granular basophilic cytoplasm in the parotid salivary gland, and vacuolar degeneration of the bronchial epithelium of the lung. Based on the results of this study, it was determined that 400 ppm tripelennamine administered ad libitum in the feed to both male and female Fischer 344 rats would be an appropriate highest dose for a 2-year carcinogenicity study.

Using Short-Term Tests to Predict Carcinogenic Activity in the Long-Term Bioassay

A method for classifying chemicals with respect to carcinogenic potential based on short-term test results is presented. The method utilizes the logistic regression model to translate results from short-term toxicity assays into predictions of the likelihood that a chemical will be carcinogenic if tested in a long-term bioassay. The proposed method differs from previous approaches in two ways. First, statistical confidence limits on probabilities of cancer rather than central estimates of those probabilities are used for classification. Second, the method does not classify all chemicals in a data base with respect to carcinogenic potential. Instead, it identifies chemicals with highest and lowest likelihood of testing positive for carcinogenicity in the bioassay. A subset of chemicals with intermediate likelihood of being positive remains unclassified, and will require further testing, perhaps in a long-term bioassay. Two data bases of binary short-term and long-term test results from the literature are u...