Oral squamous cell carcinoma in ad libitum-fed and food-restricted Brown-Norway rats.

A high incidence of oral squamous cell carcinoma was present in male and female Brown-Norway rats fed ad libitum or food-restricted dietary formulations. One hundred eight-nine rats were examined from 4 dietary treatment groups: male ad libitum, male food-restricted, female ad libitum, and female food-restricted. The ad libitum treatment groups for both males and females had significantly more cases of oral squamous cell carcinoma than cohort food-restricted groups. In ad libitum rats, 10 of 47 (21%) males and 15 of 47 (32%) females had oral squamous cell carcinoma, whereas only 4 of 47 (9%) males and 5 of 48 (10%) females in the food-restricted groups were similarly affected. The food-restricted rats lived significantly longer than ad libitum cohorts, so the higher incidence of squamous cell carcinoma was not dependent on extended lifespans. In addition to the dietary influence, a significant difference in oral squamous cell carcinoma incidence occurred between various familial lines. Family lines having representatives in both ad libitum and food-restricted groups had lower oral squamous cell carcinoma incidences in the food-restricted group whether comparing affected litters or individuals. Results suggest that the incidence of oral squamous cell carcinoma in our colony of Brown-Norway rats can be influenced by both the dietary treatment group and genetic predilection within certain pedigrees.

Subchronic toxicity of triethylenetetramine dihydrochloride in B6C3F1 mice and F344 rats.

Triethylenetetramine dihydrochloride (trien-2HCl; CAS No. 38260-01-04), a chelating agent used to treat Wilson's disease patients who are intolerant of the drug of choice, was tested for subchronic toxicity in B6C3F1 mice and F344 rats. Mice and rats received trien-2HCl in the drinking water at concentrations of 0, 120, 600, or 3000 ppm for up to 92 days. Twenty mice and 18 rats of each sex were assigned to each dose group fed either a cereal-based (NIH-31) or a purified (AIN-76A) diet, both containing nutritionally adequate levels of copper. An additional control group of rats and mice received a Cu-deficient AIN-76A diet. This low copper diet resulted in Cu-deficiency symptoms, such as anemia, liver periportal cytomegaly, pancreatic atrophy and multifocal necrosis, spleen hematopoietic cell proliferation, and increased heart weight, together with undetectable levels of plasma copper in rats but not in mice. Trien-2HCl lowered plasma copper levels some-what (at 600 and 3000 ppm) in rats fed the AIN-76A diet, but did not induce the usual signs of copper deficiency. Trien-2HCl caused an increased frequency of uterine dilatation at 3000 ppm in rats fed AIN-76A diet that was not noted in females fed the Cu-deficient diet. Trien-2HCl toxicity occurred only in mice in the highest dose group fed an AIN-76A diet. Increased frequencies of inflammation of the lung interstitium and liver periportal fatty infiltration were seen in both sexes, and hematopoietic cell proliferation was seen in the spleen of males. Kidney and body weights were reduced in males as was the incidence of renal cytoplasmic vacuolization. There were no signs of copper deficiency in mice exposed to trien-2HCl. The only effect of trien-2HCl in animals fed the NIH-31 diet was a reduced liver copper level in both rat sexes, noted at 3000 ppm.

Triprolidine: 104-week feeding study in rats.

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 histopathological evaluation was performed on all other animals; survivors were killed at 2 years. Survival was significantly extended in triprolidine-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 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.

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.

Chronic feeding study of pyrilamine in Fischer 344 rats.

The antihistamine, pyrilamine maleate, was fed for up to 2 years to groups of 57 Fischer 344 (F344) rats of each sex at dietary levels of 0, 300, 1500, or 3000 ppm (free base). Eight or nine of these rats per sex and dose group were killed at 65 weeks to analyze hematology and clinical chemistry in all groups and histopathology of control and high-dose animals. Histopathology also was performed on all dead or moribund rats and on all that survived for 2 years. Average daily exposures were 11 to 150 mg/kg pyrilamine compared to human dosages up to 3 mg/kg. Pyrilamine treatment did not reduce survival. Final body weights were reduced relative to controls (mid-dose males, 93%, females, 82%: high-dose males, 82%, females, 70%). The incidences of inflammation of the nasolacrimal duct (chronic in females; suppurative in males), liver cytoplasmic vacuolization (males), and the combination of animals with either liver basophilic or clear cell foci (males) tended to significantly increase with dose. Adrenal pheochromocytomas, mammary gland fibroadenomas, and neoplasms of the clitoral gland, thyroid c-cell, and pituitary gland all tended to decrease with increasing dose in females. In males only preputial gland neoplasms exhibited a similar negative trend. While two ovarian granulosatheca cell benign tumors occurred in high-dose females, these were thought to be a random occurrence. There was no evidence for the carcinogenicity of pyrilamine in F344 rats in the current study.

Chronic study of triprolidine for oncogenicity in mice.

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 hepatocellular 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Ethynylestradiol protection against methyl insufficiency in castrated male Wistar/Furth rats fed a methionine-choline-deficient diet.

The interactive effects of dietary methyl insufficiency and the estrogenic compound ethynylestradiol (EE) on the levels of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) were examined in the liver, lungs and pancreas of rats. In addition, such effects on the hepatic content of 5-methyl-deoxycytidine (5-MC) in nuclear DNA were determined. Castrated male Wistar/Furth rats were fed various levels of EE in either: (i) a complete, amino acid-defined diet (diet 1); (ii) the same diet lacking in choline and methionine and supplemented with 0.9% of DL-homocystine (equimolar to methionine) (diet 2); or (iii) diet 2 but only with 0.3% DL-homocystine (diet 2M). Methyl deficiency and EE each independently produced decreased weight gains and increased relative liver weights (liver weight relative to total body weight) compared with control animals. Livers from rats fed diets 2 and 2M without EE had lower levels of SAM and lower SAM:SAH ratios than did the livers from diet 1-fed rats not treated with EE. Hepatic SAM:SAH ratios in diet 1-fed rats were not altered by EE treatment. However, EE treatment increased the hepatic contents of SAM and restored the SAM:SAH levels to normal in rats fed diet 2 or 2M. The levels of SAM + SAH in the livers of rats fed the low homocystine diet (diet 2M) were less than in those fed either diet 1 or diet 2. Thus, the addition of EE at 10 p.p.m. gave protection against reduced levels of SAM, and reduced SAM:SAH ratios in the liver, but had little effect when added to the methyl-adequate diet. No differences in hepatic 5-MC levels were observed in any of the groups as a result of either methyl deficiency or EE treatment. Methyl deprivation alone caused no discernible difference in pancreatic SAM levels but did result in a significant rise in SAH levels and thus in decreased SAM:SAH ratios. EE had no consistent effect on pancreatic SAM, SAH or SAM:SAH ratios in any of the diet groups examined. Similarly, the chronic feeding of diet 2, diet 2M or of EE had no significant effect on the SAM contents of lungs, compared with the corresponding levels in control rats. The protection conferred by EE against SAM insufficiency in the livers of rats fed a methionine- and choline-deficient diet is consistent with the relative insensitivity of female rats to the hepatotoxicity of dietary methyl insufficiency.

Bioassay for carcinogenicity of rotenone in female Wistar rats.

Rotenone, a pesticide extracted from the Derris 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.

Effects of diet type on incidence of spontaneous and 2-acetylaminofluorene-induced liver and bladder tumors in BALB/c mice fed AIN-76A diet versus NIH-07 diet.

Diet is a major influence on the responses of experimental animals to drugs, toxins, and carcinogens. Two diets used widely in toxicological and/or nutritional studies, and considered to be nutritionally adequate, were compared with respect to their influence on growth, body weight, lifespan, spontaneous neoplasia, and neoplastic responses to 2-acetylaminofluorene (2-AAF). Both sexes of weanling BALB/c mice were fed either a purified diet (AIN-76A) or a nonpurified, natural ingredient diet (NIH-07), with or without 2-AAF for up to 2 years. Dosages of 2-AAF were administered to males at 0, 20, 40, or 60 ppm in each diet and to females at 0, 100, 125, or 150 ppm. Each group consisted of 96 mice. In most instances, males and females fed purified diet (AIN-fed) gained weight more rapidly, attained higher maximum body weights, and died earlier than their non-purified diet (NIH-fed) counterparts. 2-AAF inhibited weight gain significantly only in AIN-fed females. Thus, females receiving 150 ppm 2-AAF gained little more than their NIH-fed counterparts. At the dosages used in males, 2-AAF did not induce liver neoplasia but the AIN diet was clearly associated with a higher spontaneous frequency of liver neoplasia than the NIH diet. Although 2-AAF induced liver tumors in females fed either diet at all dosages, a higher frequency and earlier appearance of liver tumors among AIN-fed females than their NIH-fed counterparts was apparent mainly at the lowest dosage. 2-AAF induced bladder neoplasia in both sexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased incidence of spontaneous and 2-acetylaminofluorene-induced liver and bladder tumors in B6C3F1 mice fed AIN-76A diet versus NIH-07 diet.

Diet is a major influence on the responses of experimental animals to drugs, toxins, and carcinogens. Two diets used widely in toxicological and carcinogenic studies, and considered to be nutritionally adequate, were compared with respect to neoplastic responses to 2-acetylaminofluorene (2-AAF). Both sexes of weanling B6C3F1 mice were fed either AIN-76A (a purified diet) or NIH-07 (a natural ingredient diet), with or without 2-AAF, for up to 2 years. Dosages of 2-AAF were administered to males at 0, 40, 60, or 80 ppm in each diet and to females at 0, 150, 200, or 250 ppm. Each group consisted of 96 mice, except the groups of females dosed at 0 and 150 ppm, which consisted of 120 and 72 mice, respectively. The incidence of malignant liver tumors was significantly greater in all AIN-fed groups compared to corresponding NIH-fed groups, as was the total incidence of tumors (malignant + benign). Similarly, the incidence of malignant and total bladder tumors was greater in AIN-fed groups of mice administered the two high doses of 2-AAF for each sex compared to NIH-fed groups. No malignant bladder tumors were observed among any of the groups of mice receiving control diets. This was also true for the males on low dosages (40 ppm) of 2-AAF. The AIN-fed group of low dose females (150 ppm) developed 4% incidence of malignant bladder tumors. This study dramatically showed the importance of diet selection in chronic carcinogenic studies and suggests that results obtained with the use of a purified diet may differ both qualitatively and quantitatively from results obtained with a natural ingredient diet.

Diabetogenic response to streptozotocin varies among obese yellow and among lean agouti (BALB/c x VY)F1 hybrid mice.

To test the hypothesis that the elevated insulin levels in obese neoplasia-susceptible yellow Avy/- mice might be a major factor stimulating tumor formation, it is necessary to use normoinsulinemic yellow mice. Although our attempt to obtain normoinsulinemic, euglycemic mice by streptozotocin treatment was unsuccessful, we did observe significant differences in the responsiveness to this treatment among mice of identical genotype. These differences were observed among female yellow Avy/A and agouti A/a (BALB/c x VY)F1 hybrid mice in the responses of body weight gain, plasma glucose, and plasma insulin levels to a single intraperitoneal injection of either 150 or 200 mg/kg streptozotocin (STZ) at 4 weeks of age followed by a 22-week observation period. Among animals treated with the high streptozotocin dose, 80% of the yellow mice gained almost no weight and became grossly hyperglycemic and hypoinsulinemic; however, only 55% of the agouti mice exhibited such a strong response. In the low dose group, 25% of the yellow mice responded with reduced body weight gain, decreased insulin, and elevated glucose levels whereas none of the agouti mice exhibited such responses. More pancreatic islet tissue mass was present in the untreated yellow control mice than among the comparable agouti mice by the end of the study. In both streptozotocin dose groups and in both genotypes, islet tissue mass was reduced to a much greater extent in the more responsive mice than in the less responsive mice. There appeared to be no correlation between islet tissue mass and insulin level. The phenotypic variation in responsiveness to an exogenous agent among test animals of a single inbred or F1 hybrid genotype reported here is not unique to this F1 hybrid since it is seen in most chronic bioassays when relatively low levels of agent are used.

Estrogen-induced thyroid follicular cell adenomas in C57BL/6 mice.

Diethylstilbestrol (DES) was fed chronically to C57BL/6 mice at concentrations of 0, 5, 10, 20, 40, 160, 320, or 640 ppb in order to define the dose-response curve for neoplastic responses. The incidence of thyroid follicular cell adenomas was higher in control females than in males and was increased at mid-level doses of DES, especially in males. None were found in mice fed 640 ppb DES, probably because these mice died from other causes before follicular cell adenomas had developed. In both sexes, DES fed at 160 or 320 ppb significantly shortened time-to-onset of these tumors, and 40 ppb increased their probability late in life. It is concluded that DES has a causal relationship to thyroid neoplasia in C57BL/6 mice, and similarities between this and the human disease suggest that C57BL/6 mice may be an appropriate model for human thyroid neoplasia.

Carcinogenicity study of 3,3'-dimethylbenzidine dihydrochloride in BALB/c mice.

BALB/c mice (120/sex/dose) were given 0, 5, 9, 18, 35, 70 or 140 ppm of 3,3'-dimethylbenzidine dihydrochloride in their drinking-water and killed after 13, 26, 39, 52, 78 or 116 wk. Full histopathological evaluations were performed on all animals that were found dead or moribund, or that were killed on schedule. Fatal lung alveolar cell neoplasms began to appear in males receiving 140 ppm at 78 wk and there was a significant dose-related decrease in the time-to-death from this cause. There were no significant dose-related trends for this neoplasm in females, nor were there treatment-related effects on body weight, water consumption or other lesions in either sex. This is the first report of a neoplastic response to 3,3'-dimethylbenzidine dihydrochloride in mice.

Variability of selected nutrients and contaminants monitored in rodent diets: a 6-year study.

The results are given from monitoring a commercial closed-formula cereal-based rodent diet (Purina 5010), two open-formula cereal-based diets (NIH-31 and NIH-07), and one purified diet (AIN-76) for selected nutrients and contaminants. The observed concentrations of nutrients (protein, fat, vitamin A, and thiamine) approximated the manufacturer specifications for closed-formula cereal diet, while the average concentrations of nutrients found in the open-formula cereal diets were well above the nominal concentrations. Nominal concentrations for these open-formula diets tended to be close to the minimum values that were observed. Except for protein levels, greater variation in nutrient concentrations was found in the purified diet than in the cereal diets, but the variation of contaminants was about equal in the two types of diets. Open- and closed-formula cereal diets appear to be very similar to each other in the degree of variation of nutrients and contaminants. Cadmium, lead, and selenium are the constituents of greatest concern in assuring the quality of the rodent diets that were evaluated.

Tumorigenic responses to lindane in mice: potentiation by a dominant mutation.

Obese mottled yellow Avy/a, lean pseudoagouti Avy/a and lean black a/a (YS X VY) F-1 hybrid female mice were fed diet containing 160 p.p.m. lindane (gamma-hexachlorocyclohexane) for 6, 12, 18 or 24 months. Clara cell hyperplasia was present in a majority of the mice after six months of lindane ingestion; however, more yellow mice (77%) than pseudoagouti (50%) or black (56%) mice had developed this lesion. Continued ingestion of lindane increased the incidence of Clara cell hyperplasia and resulted in similar prevalences in the three phenotypes. Lung tumors associated with lindane ingestion for 24 months were found only in yellow (19%) and pseudoagouti (14%) mice but not in the black mice. Prevalences of hepatocellular adenomas and carcinomas were very low (less than 10%) in untreated pseudoagouti and black mice. Lindane ingestion for 24 months resulted in an hepatocellular adenoma prevalence of 12% in pseudoagouti mice and 3% in black mice; comparable hepatocellular carcinoma prevalences were 5% and 1%. Among yellow mice fed lindane diet for 24 months, adenoma prevalence was 35% (9% among untreated controls) but carcinoma prevalence was only 17% (13% among controls). The tumorigenic responses evoked by lindane feeding in the lean pseudoagouti Avy/a mice but not in the black a/a mice indicate, for the first time, that the Avy gene itself, in the absence of obesity, sensitizes cells to transformation. The greater prevalence of hepatocellular adenomas in obese yellow Avy/a than in lean pseudoagouti Avy/a mice implicates obesity-associated factors in tumor promotion. Similarly, the increased prevalence of hepatocellular carcinomas in untreated obese yellow Avy/a mice, as compared to lean pseudoagouti mice, implicates obesity-associated factor as favoring histiotypic progression of liver tumors. Thus, the Avy gene not only sensitizes cells to respond to tumorigenic stimuli but also, by the induction of obesity, enhances promotion and progression of transformed cells.

Influence of a purified diet and route of administration on the metabolism and disposition of estradiol in B6C3F1 mice.

The metabolism and disposition of [6,7-3H]estradiol ([3H]E2) given by gavage (po) or intravenously (iv) were examined in female B6C3F1 mice fed either the purified AIN-76A (AIN) or cereal-based NIH-07 (NIH) diet for a period of 8 weeks prior to treatment with E2. Initially, 40.6 Ci of [3H]E2 was given iv to each mouse. Subsequently, 45.6 Ci of [3H]E2 was given po to the same mice. Samples of blood, urine, and feces were obtained during a 48-hr period after each dosing. Total radioactivity was determined for each sample. Urine and plasma samples were analyzed by HPLC, and the radiolabeled metabolites were tentatively identified and quantified. Statistical comparisons were made of the effects of diet, route of administration, and interactions among groups. Analysis revealed: 1) greater fecal than urinary excretion of radioactivity regardless of route of administration or diet fed, 2) more radioactivity excreted in the urine of AIN-fed than in NIH-fed mice, significantly different only after iv administration (p less than 0.02), and 3) a greater feces:urine ratio of excreted radioactivity following iv than po administration and from NIH-than AIN-fed mice. Metabolite profiles showed: 1) no differences in urine due to route of administration, 2) estriol conjugates dominated urinary metabolites, 3) accumulation of radioactive material in plasma that apparently was tritiated water, with more rapid accumulation of tritiated water after po than iv administration, 4) relatively more plasma estradiol-17-glucuronide, estriol-3-sulfate, and estriol in po- than in iv-treated mice, 5) estradiol-3, 17-sulfate in plasma of iv- but not po-treated mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Mammary tumorigenesis in C3H/HeN-MTV + mice treated with diethylstilboestrol for varying periods.

C3H/HeN-MTV+ female mice were fed diets containing 320 or 640 ppb diethylstilboestrol (DES). DES feeding was started at 3 wk of age and was either continued throughout life or discontinued after 4, 8 or 26 wk of administration. A control group consisted of mice fed the same diet without DES, for the duration of the experiment. Mice were killed when palpable body masses (presumed to be mammary adenocarcinomas) reached a diameter of 1 cm. Adenocarcinomas developed in 79% of control mice and 96% of the mice exposed to DES for 26 wk, irrespective of the dose. The frequency and rate of removal of tumour-bearing mice were not increased further with lifetime exposure at a given dose. The time at which the first tumour occurred was largely dependent on the duration of exposure, not dose. The rate of occurrence of subsequent tumours was dependent on dose and duration of exposure; the rate of removal of mice with mammary adenocarcinomas was significantly greater at 640 ppb than at 320 ppb DES. Tumour frequency was 83% in mice exposed to 320 ppb DES for 8 wk and in those exposed for 4 wk; however, tumours developed at a faster rate in mice exposed for 8 wk. Tumour frequency was 94-96% in mice exposed to 640 ppb DES for 4 wk and 8 wk, and tumours developed more rapidly in mice exposed for 8 wk than in those exposed for 4 wk. When data were plotted as log-dose v. log-t50 (time to a probability that half the mice would be removed with mammary tumours) linear extrapolation to the control log-t50 gave an estimate of the no-effect level of exposure to DES. This estimate was remarkably consistent for all data sets (40-93 ppb) and was independent of the duration of exposure.

Age-dependent responses to 2-acetylaminofluorene in BALB/c female mice.

In order to evaluate the influence of age on 2-acetylaminofluorene (2-AAF) carcinogenesis, female BALB/c mice were fed diets containing 500 ppm 2-AAF for 6 mo, starting at 1, 7, or 13 mo of age. Groups of mice were killed immediately, 3 or 6 mo after termination of treatment, or were allowed to complete their life span. Mice corresponding to the age of each sacrifice group of the 2-AAF-treated mice and a life-span group were also included as controls. The study was moved to a different animal room area after 25 wk into the study. Thus, certain treatment groups were replicated to evaluate the impact of the change in environment. Controls and the young 2-AAF-treatment group killed at 7 mo of age were replicated twice in the new animal room at 6-mo intervals. Similarly, controls and the mid-aged treatment group killed at 13 mo of age were repeated once in the new animal room. This resulted in mice of each age/treatment group being treated simultaneously in the same room and killed as soon as treatment was stopped. The main histopathologic responses to 2-AAF were observed in the urinary bladder, liver, and mammary glands. In all three replicates urinary bladder hyperplasia was less severe when young mice were treated than when mid-aged or old mice were treated. In contrast, there was no clear influence of age on bladder tumorigenesis, although replicate variation in this response was very great. Tentatively one also may conclude that (1) sensitivity to the induction of mammary tumorigenesis by 2-AAF increased with the age of mice at the time of treatment, and (2) old mice were sensitive to induction of liver karyomegaly and cytoplasmic and nuclear inclusions, while young and mid-aged mice were resistant to this influence. Perhaps the most important conclusion to be made from this study is that replicate variation, probably due to environmental factors, may result in false conclusions concerning age sensitivity to a carcinogen if differing age groups are not treated simultaneously and/or key parts of an experiment are not replicated to eliminate the possibility of an influence of environmental factors or nonrandom assignment of animals to age groups.

Effects of purified (AIN-76A) and natural-ingredient (NIH-07) diets on responses of BALB/c and B6C3F1 female mice to estradiol.

Female BALB/c and B6C3F1 mice were examined after a 3-wk exposure to dietary estradiol (0, 400, 800, 1600, and 3200 ppb) in a purified (AIN-76A) or a natural-ingredient (NIH-07) diet. The use of AIN-76A was associated with a 9-13% greater (p less than 0.001) body weight and a 36-43% higher (p less than 0.001) serum cholesterol in both mouse genotypes when compared to mice fed NIH-07. Conversely, when fed NIH-07, both mouse genotypes had a 20-22% higher (p less than 0.003) serum urea nitogren and 2-3.5% higher erythrocyte count (p less than 0.001) and hemoglobin concentration (p less than 0.04) than when fed AIN-76A. Reduced erythrocyte parameters suggest that chronic feeding of the purified diet might result in anemia. No significant compound or diet-related differences were noted for serum creatinine, alanine aminotransferase, aspartate aminotransferase, or gamma-glutamyl transferase. Although there was no diet effect on absolute or differential white blood cell count, estradiol caused a decrease in the total white blood cell count (p less than 0.014) and an increase in the percentage of polymorphonuclear leukocytes (p less than 0.014) in BALB/c and decreased the percentage of lymphocytes (p less than 0.005) in B6C3F1 females. In addition, estradiol increased uterine weight and inhibited thymic and splenic weights in one or both genotypes. Spleen and thymus weight responses to estradiol were not significantly influenced by diet. However, the uterine weight responses to estradiol were apparently influenced by diet in both genotypes. In B6C3F1 mice, the uterus weighed more at each level of estradiol when mice were fed AIN-76A compared to NIH-07 diet. In BALB/c mice, this was true only at the two lower dietary concentrations of estradiol. In conclusion, mice fed the purified diet, AIN-76A, differed from those fed the cereal-based diet, NIH-07, in hematology, clinical chemistry, and uterine weight response to estradiol.

Sex specificity of myocardial damage in mice fed a purified diet.

Both sexes of BALB/c and B6C3F1 mice were divided into test groups and fed either a purified diet (AIN-76A) or a natural ingredient diet (NIH-07) containing graded levels of 2-acetylaminofluorine (2-AAF) for 90 days. A large number of dead or moribund B6C3F1 males fed the AIN diet were removed from the study prematurely. AIN-fed B6C3F1 mice removed early as well as some sacrificed at the end of the study showed myocardial damage with hemorrhage. A much smaller number of BALB/c males fed the AIN diet also exhibited these signs while none of the females from either stock were affected. Mice having these lesions were confined largely to 2 of 5 treatment groups. Increased levels of serum aspartate aminotransferase (GOT) (P less than .01) occurred in the AIN-fed B6C3F1 male mice that were sacrificed, supporting the histopathological observation of myocardial damage. There was no other significant difference in the GOT between diets or 2-AAF doses. No environmental factors could be associated with the problem and microbiological and chemical analyses of the diets showed no convincing evidence of specific pathogenic organisms or nutritional deficiencies that might have caused these lesions. Extended storage (up to 4 months) and one batch of feed in particular seemed to be associated with mice having myocardial damage. These associations were highly strain and sex dependent and suggest that great care must be taken in the manufacture and handling of the diet. Furthermore, it seems likely that the diet may be marginally adequate for some strains of mice and may require modification before it will become generally useful.