O6-methylguanine levels and histopathological changes in the rat esophagus and liver following single and repeated administration of N-nitrosomethylbenzylamine.

In this study we investigated the time course of O6-methylguanine (O6-meGua) levels and concomitant histopathological effects in the rat esophagus and liver following single and repeated s.c. administration of the esophagus-specific carcinogen N-nitrosomethylbenzylamine (NMBA). The primary purpose of this study was to determine if differences in the induction and/or persistence of O6-meGua might account for differences in the tumorigenicity of NMBA observed with treatment regimens of 0.5 mg/kg/dose, 3 doses/week for 5 weeks (a proven tumorigenic regimen) and 1.67 mg/kg/dose, 3 doses/week for 2 weeks (an essentially non-tumorigenic regimen). Results of the single dose experiment indicated that enzymatic activation of NMBA in the rat esophagus was not dose limited, at least at doses up to and including 5.0 mg/kg. Results of the repeated dose experiment demonstrated that the non-tumorigenic NMBA regimen produced significantly higher levels of esophageal O6-meGua compared with the tumorigenic NMBA regimen. During the 2 week treatment period of the non-tumorigenic regimen esophageal O6-meGua levels decreased progressively, but remained significantly higher than in the tumorigenic regimen. In contrast, the relatively lower O6-meGua levels of the tumorigenic regimen remained essentially unchanged during the course of treatment. At 72 h following conclusion of dosing no O6-meGua was detected in the esophagi of rats treated with either regimen. Microscopic examinations revealed that the non-tumorigenic NMBA regimen produced a marked cytotoxic effect on the esophageal epithelium, while microscopic esophageal changes observed with the tumorigenic regimen were generally less severe. In the liver O6-meGua was detected in only a few rats and no remarkable microscopic pathology was observed in this organ. Together these findings indicate that: (i) abbreviated NMBA treatment induces tumors in the rat esophagus only at levels that induce DNA damage without causing extensive cytotoxicity; (ii) the lack of NMBA tumorigenicity in the rat liver may be due, at least in part, to the rapid and efficient repair of O6-meGua adducts, coupled with the lack of necrosis and compensatory cell division in this organ.

[A six-month intravenous repeated dose toxicity study of tazobactam/piperacillin and tazobactam in dogs].

Tazobactam (TAZ) is a newly developed beta-lactamase inhibitor. Tazobactam/Piperacillin (TAZ/PIPC) is a formulation consisting of TAZ and PIPC in a ratio of 1:4. A six-month intravenous repeated dose toxicity study of TAZ/PIPC and TAZ including a one-month recovery period were carried out using male and female dogs. The doses were 200, 400 and 800 mg/kg/day for TAZ/PIPC, and 40, 80 and 160 mg/kg/day for TAZ. The results were as follows. 1. No test article-related deaths occurred during the study period. No effects on clinical findings, body weight and food consumption were evident. 2. No test article-related changes were noted in hematological, serum biochemical and urinalysis evaluations, and opthalmological and electrocardiographic examinations. 3. There were no test article-related changes in macroscopic findings or organ weight. 4. The histopathological examination revealed deposition of marked PAS-positive aggregates in liver cells of dogs given 400 mg/kg/day or more of TAZ/PIPC and 80 mg/kg/day or more of TAZ. Electron micrographs of hepatocytes revealed glycogen granules to be accumulated in the cytoplasm, and an increase of smooth endoplasmic reticulum. 5. After a one-month recovery period, the histopathological changes had generally disappeared, suggesting that they were reversible. 6. From the histopathological changes of liver, the no-toxic dose levels for TAZ/PIPC and TAZ were 200 mg/kg/day and 40 mg/kg/day, respectively.

Two-week aerosol inhalation study in rats of ethylene oxide/propylene oxide copolymers.

Previous studies have shown that aerosols of an ethylene oxide/propylene oxide copolymer (UCON 50-HB-5100) produced an inflammatory response in lungs of rats in short-term repeated exposures at relatively low concentrations. This study was carried out on related polyalkylene glycols (EO/PO copolymers) to determine if similar effects would occur upon short-term repeated exposure. Rats were treated by whole body liquid droplet aerosol exposures of six hours per day, five days per week for two consecutive weeks to each of five EO/PO copolymers. The exposure level for the positive control (UCON 50-HB-5100) was 55 mg/m3, while the remaining 4 test copolymers were evaluated at 100 mg/m3. Each exposure group consisted of ten male albino rats. After three exposures, nine of ten rats exposed to UCON 50-HB-5100, and six of ten rats exposed to UCON 50-HB-2000 had died. At necropsy, congestion, consolidation and red discoloration of the lungs were noted. A moderate to severe alveolitis, characterized by intraalveolar edema, hemorrhage and fibrin deposition, was observed after five days of exposure. At necropsy, these rats exhibited elevated lung weights and similar macroscopic and microscopic lesions. Rats exposed to the other test materials (UCON 75-H-1400, Pluronic L17R1, Pluronic L31, and Pluronic L64) survived with essentially no signs of toxicity through the ten exposure days. Body weights, organ weights, hematological evaluation, pharmacotoxic signs, and macroscopic and microscopic evaluation after necropsy were similar between groups and when compared to the negative control group. Only a slight alveolitis was noted after two weeks of exposure which subsided by two-weeks post exposure.

Inhalation toxicity of chromium dioxide dust to rats after two years exposure.

Rats were exposed by inhalation to chromium dioxide (CrO2) dust at design concentrations of 0, 0.5 (stabilized and unstabilized, respectively) or 25 mg m-3 (stabilized) for 6 h day-1, 5 days week-1 for 2 years. No dust-exposure-related pathological changes were observed, other than lung lesions, in all exposed rats. There were no significant differences in pulmonary response between unstabilized and stabilized CrO2 at the 0.5 mg m-3 exposure level. The lungs showed minute dust deposition in the alveoli adjacent to the alveolar ducts, but maintained an intact general architecture. The pulmonary responses satisfied the biological criteria for a nuisance dust. At 25 mg m-3, dust deposition was sharply confined to the alveoli in the alveolar duct region. Alveolar walls enclosing dust-laden macrophage (dust cell) aggregates were thickened with hyperplastic Type II pneumocytes and slightly collagenized fibrosis. Alveoli adjacent to the terminal bronchioles were lined with bronchiolar epithelium (alveolar bronchiolarization). In addition, lungs showed foamy macrophage response, cholesterol granulomas, alveolar proteinosis, and minute fibrotic pleurisy. These pulmonary lesions occurred predominantly in female rats. Of 108 female rats, six developed keratin cysts and two had cystic keratinizing squamous cell carcinoma (CKSCC). None of 106 male rats had either a keratin cyst or a CKSCC. The lung tumors developed from metaplastic squamous cells in the areas of alveolar bronchiolarization in the alveolar duct region. The lung tumors were well differentiated and devoid of characteristics of true malignancy. The CKSCC is an experimentally-induced, unique tumor type and is different from the type of spontaneous lung tumor seen in man or animals. The relevance to man of ths type of lung tumor appears to be negligible.

Subchronic inhalation toxicity of tetramethoxysilane in rats.

Sprague-Dawley rats were exposed 6 hr/day, 5 days a week, for 28 days to tetramethoxysilane (TMOS) at concentrations of 0, 1, 5, and 10 ppm (Phase I study) and to 0, 15, 30, and 45 ppm (Phase II study). All of the rats exposed to 45 ppm TMOS died or were sacrificed in a moribund state during the 28-day study period. Statistically significant changes were observed in food consumption, body weights, and clinical chemistry parameters in the animals exposed to 30 ppm TMOS. Males exposed to 15 ppm TMOS showed a significant decrease in total protein. No effects were seen in rats exposed to 1, 5, and 10 ppm TMOS. Histopathological lesions related to TMOS exposure were observed in the respiratory tract tissues and eyes of rats exposed to 15, 30, and 45 ppm TMOS. The principal types of lesions observed were ulceration, inflammation, and necrosis of epithelium. At 45 ppm, changes at these sites were severe and present in all animals. Changes at 30 ppm, while occurring in all rats, were much less severe than those seen at 45 ppm. At 15 ppm, the changes were minimal and occurred only in three males and five females. The data of this study showed that TMOS has a steep dose-response curve with no observable effects at 10 ppm, very minimal effects at 15 ppm, moderate to severe effects at 30 ppm, and severe effects and lethality at 45 ppm.

Effects of inhaled chromium dioxide dust on rats exposed for two years.

Rats were exposed by inhalation to chromium dioxide (CrO2) dust at design concentrations of 0, 0.5 mg/m3 (stabilized and unstabilized, respectively), or 25 mg/m3 (stabilized) for 6 hr/day, 5 days/week for 2 years. No dust-exposure-related pathological changes other than lung lesions were observed in all exposed rats. There were no significant differences in pulmonary responses between unstabilized and stabilized CrO2 at the 0.5 mg/m3 exposure level. The lungs showed minute dust deposition in the alveoli adjacent to the alveolar ducts, but maintained an intact general architecture. The pulmonary responses satisfied the biological criteria for a nuisance dust. At 25 mg/m3, dust deposition was sharply confined to the alveoli in the alveolar duct region. Alveolar walls enclosing dust-laden macrophage (dust cell) aggregates were thickened with hyperplastic Type II pneumocytes and slightly collagenized fibrosis. Alveoli adjacent to the terminal bronchioles were lined with bronchiolar epithelium (alveolar bronchiolarization). In addition, lungs showed foamy macrophage response, cholesterol granulomas, alveolar proteinosis, and minute fibrotic pleurisy. These pulmonary lesions occurred predominantly in female rats. Of 108 female rats, 6 developed keratin cysts and 2 had cystic keratinizing squamous cell carcinoma (CKSCC). None of 106 male rats had either a keratin cyst or a CKSCC. The lung tumors developed from metaplastic squamous cells in the areas of alveolar bronchiolarization at the alveolar duct region. The lung tumors were well differentiated and devoid of characteristics of true malignancy. The CKSCC is an experimentally induced, unique tumor type and is different from the type of spontaneous lung tumor seen in man or animals. The relevance to man of this type of lung tumor appears to be negligible.

Evaluation of the dose response and in utero exposure to saccharin in the rat.

A two-generation bioassay on sodium saccharin (NaS), involving 2500 second-generation male rats, was designed to determine the dose response for urinary bladder tumours in male rats and to evaluate other changes possibly related to the occurrence of the tumours. Six treatment groups (125-700 rats/group) were fed dietary levels of NaS ranging from 1.0 to 7.5%. To evaluate the role of in utero exposure, two additional groups were exposed to NaS either only during gestation via dams fed diet containing 5.0% NaS or for a single generation beginning at birth. In the latter group, the nursing dams were placed on an NaS diet immediately after giving birth and their offspring were weaned onto diets containing 5.0% NaS. A third additional group, included to evaluate the specificity of NaS and the role of excess sodium in the occurrence of urinary bladder tumours, was fed diet containing sodium hippurate (NaH) for two generations--5.0% NaH to the first generation and to the second until 8 wk old, and subsequently 3.0% because of unexpected toxicity. A clear dose response for urinary bladder tumours was observed in the second-generation NaS-treated male rats. The steep slope of the dose-response curve indicated a rapid decline in tumour incidence with decreasing dose. The 1.0% dietary level (fed to 700 rats) was considered to be a no-effect level for bladder tumours. The only other treatment-related pathological changes were an increase in urinary bladder weight in rats fed greater than or equal to 3.0% and an increase in mineralization of the kidneys with greater than or equal to 1.0%. Several physiological effects were seen in the NaS-treated groups showing an increase in bladder tumours (i.e. those fed greater than or equal to 3.0%). Some changes, e.g. depressed growth and increased water consumption, were indicative of a general disturbance of these rats, but analysis of body-weight, food-consumption, compound-consumption and water-consumption data revealed no correlations within any dose group between these quantitative data and the occurrence of bladder tumours. Other changes indicative of the compromised situations of the rats fed high dietary levels of NaS were anaemia in weanling rats fed 5.0 or 7.5% and a reduction in litter size at dietary levels greater than or equal to 3.0%. Changes in urine volume and urine osmolality were highly correlated with the occurrence of the urinary bladder tumours.(ABSTRACT TRUNCATED AT 400 WORDS)

Malignant mammary tumors in beagle dogs dosed with investigational oral contraceptive steroids.

Of 172 beagle dogs administered investigational oral contraceptive steroids for 2.4-5.2 years, 9 developed malignant mammary tumors. At necropsy their ages varied from 41 to 70 months, with a mean age of 4.9 years. The malignant tumors were observed in 1 dog that received ethynerone plus mestranol at 1.05 mg/kg/day and in 4 dogs that received chlorethynyl norgestrel plus mestranol at 1.05 mg/kg/day. Also, 4 dogs that received anagestone acetate plus mestranol at either 0.44 or 1.10 mg/kg/day developed malignant mammary tumors. Malignant tumors were not seen in 33 dogs administered mestranol at 0.02 and 0.05 mg/kg/day for 7 years or in 18 dogs given ethynerone without mestranol at 1.00 mg/kg/day for 5 years. No malignant tumors were observed in 18 control dogs maintained for 7 years without treatment. Three dogs had single malignant mammary nodules, 3 dogs had 2 malignant nodules, 2 dogs had 4-6 malignant nodules, and 1 dog in the treatment group given high dosages of ethynerone plus mestranol had 14 mammary nodules composed of fibrosarcoma. The malignant tumors were histologically classified as 5 anaplastic carcinomas, 2 solid carcinomas, 1 tubular adenocarcinoma, 1 squamous cell carcinoma, and 1 fibrosarcoma. Most dogs had only 1 histologic type of cancer (8/9 dogs); however, 1 dog had carcinomas of both solid and anaplastic types involving different glands. Metastases were present in 5 dogs and most often involved regional lymph nodes and lung.

Mammary nodules in beagle dogs administered investigational oral contraceptive steroids.

Of 172 beagle dogs administered oral contraceptive steroids for 5-7 years, 114 developed 1,156 nodules in the mammary gland region. Most of these nodules arose 2.5-3.5 years after initiation of treatment. Approximately 16% of the nodules were transient and disappeared spontaneously from the mammary gland during the study. A total of 925 nodules were present in 99 dogs at the time of death or necropsy. These nodules were classified as benign mammary dysplasias (7.0%), lobular or intraductal hyperplasias (31.4%), simple adenomas (20.8%), complex adenomas (25.4%), benign mixed tumors (5.3%), malignant tumors (3.6%), or nonmammary lesions (6.5%). Histologically, the mammary nodules were representative primarily of the hyperplasias and tumors that occur spontaneously in the mammary glands of the dog. The only major exception was the presence of 82 simple adenomas that had basaloid features. Most of the contraceptive-related mammary nodules developed in dogs receiving the combination of progestion and mestranol at 10 or 25 times the proposed human dosage. Control dogs and dogs receiving mestrenol alone had few mammary nodules. Combinations of anagestone acetate and mestranol and chloroethynyl norgestrel (WY-4355) and mestranol produced large numbers of nodules at 10 and 25 times the proposed human dosage, whereas ethynerone plus mestranol produced large numbers of nodules only at 25 times the proposed human dosage. Ethynerone, when given alone at 25 times the proposed human dosage, was associated with fewer mammary nodules. Malignant neoplasms were seen in dogs given 10 and 25 times the proposed human dosage of anagestone acetate plus mestranol and 25 times the proposed human dosage of WY-4355 plus mestranol and ethynerone plus mestranol. This study strongly associates certain combinations of progestin and mestranol with mammary neoplasia in dogs.

FDA studies of estrogen, progestogens, and estrogen/progestogen combinations in the dog and monkey.

PIP: A study begun by a drug company and taken over by the FDA (Food and Drug Administration) in 1970 attempted to assess the role of oral contraceptives in tumorigenesis and clotting abnormalities in animals. The study used ethynerone (MK 665) + mestranol; chloroethynyl norgestrel (Wy-4355) + mestranol; anagestone acetate + mestranol; ethynerone, and; mestranol administered at levels up to 25 times the human use level to female beagle dogs and 50 times the human use level to female rhesus monkeys. No behavioral changes related to compound or dose were observed in either species. Both species exhibited pharmacologic effects of hormone administration. Inhibition of the estrous cycle and vulvar enlargement were seen in all dosed dogs. Both species exhibited a dose-dependent, nonprogressive decrease in hemoglobin and hematocrits, with the anagestone acetate-mestranol combination showing the greatest effect. More nodules developed in the mammary glands of dogs who received the progestogen-mestranol combinations and who received ethynerone alone than control dogs. The 3 progestogen-mestranol combination showed the greatest tumorigenic effect as expressed by the number of dogs affected and by numbers of mammary nodules. This effect was dose-dependent for the ethynerone-mestranol and chloroethynyl norgestrel-mestranol combinations, but for the anagestone acetate-mestranol combination was maximal at the lower dose. A small number of dogs that received each progestogen-mestranol combination developed clinically malignant tumors; control dogs or dogs that received only mestranol or ethynerone were unaffected. In contrast, none of the drugs was associated with an increased incidence of mammary nodules in the monkeys. Some monkeys that received each drug showed ductal epithelial hyperplasia in mammary gland biopsies. Diabetes mellitus occurred in 10 dogs from the chloroethynyl norgestrel-mestranol and anagestone acetate-mestranol groups and in 3 monkeys from the ethynerone-mestranol high dose and anagestone acetate-mestranol high dose groups. Generalized cystic hyperplasia of the gallbladder mucosa was seen in a small number of dogs from the anagestone acetate-mestranol group. The suitability of the dog as test species for the tumorigenic and carcinogenic study of oral contraceptives is indicated.^ieng

Teratology studies of a mixture of tallow alkyl ethoxylate and linear alkylbenzene sulfonate in rats and rabbits.

A mixture of 55% tallow alkyl ethoxylate sulfate (TAE3S) and 45% of linear alkylbenzene sulfonate (LAS) was fed to two generations of rats at dietary levels of 0.1, 0.5 or 1.0%. The rats were fed the surfactant mixture either continuously or during the organogenesis (days 6-15) period of six pregnancies. In addition, pregnant New Zealand rabbits were given 50, 100 or 300 mg/kg doses of the surfactant mixture by intubation on days 2-16 of gestation during a single pregnancy. No adverse effects were noted on conception, fetal viability or post-natal survival in either generation of rats. There were no statistical differences among the groups of rat fetuses taken by Caesarian section and examined for birth defects. Of 1210 rat fetuses, the overall incidence of abnormal young was 9.0%. Similarly, no adverse effects were seen in rabbits treated with the surfactant mixture. Of 855 rabbit fetuses, 5.7% were abnormal, but the incidences of defective fetuses in the test groups were not significantly different from those in controls. Thus, no test related effects were seen on reproduction or embryonic development in either animal species.