title: NTP Toxicology and Carcinogenesis Studies of Theophylline (CAS No. 58-55-9) in F344/N Rats and B6C3F1 Mice (Feed and Gavage Studies).
abstract: Theophylline is an alkaloid found in tea (Thea sinensis) and chocolate and is structurally related to caffeine and theobromine. Theophylline is used as a pharmaceutical agent. It stimulates the heart and central nervous system, relaxes the smooth muscles of the bronchi and blood vessels, and causes diuresis. The drug is used mainly as a bronchodilator in obstructive airway diseases, such as bronchial asthma, and for myocardial stimulation. Theophylline was nominated for toxicologic and carcinogenicity testing as a representative of the purine structural subclass, particularly because of its relationship to purines such as caffeine, 1-methyl-3-hydroxyguanine, and 3-hydroxy-1-methylxanthine, the latter two compounds having been shown to induce sarcomas in rats. Additional reasons for testing theophylline included its widespread use in humans as a pharmaceutical agent, its possible genotoxicity in vitro, and the lack of information on its potential toxicity and/or carcinogenicity under conditions of chronic oral usage. Based on reported teratogenicity and testicular toxicity, it was also recommended that reproductive studies be included in the evaluation of theophylline. The oral route of administration was selected because it is the primary route of human exposure, and the gavage route was selected because it mimics the pharmaceutical use of theophylline in humans. Male and female F344/N rats and B6C3F1 mice were given theophylline (greater than 99% pure) in feed or in corn oil by gavage for 16 days or 14 weeks or in corn oil by gavage for 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, mouse bone marrow, and mouse peripheral blood. 16-DAY FEED STUDY IN RATS: Groups of five male and five female F344/N rats were given 0, 500, 1,000, 2,000, 4,000, or 8,000 ppm theophylline in feed for 16 days, which resulted in approximate daily doses of 50, 100, 250, 450, or 1,000 mg theophylline/kg body weight to males and 75, 150, 250, 450, or 1,100 mg/kg to females. All rats survived until the end of the study. The final mean body weights and body weight gains of 8,000 ppm males and females were significantly less than those of the controls. The absolute and relative testis weights of 4,000 ppm males were significantly greater than those of the controls. Increased incidences of uterine hypoplasia were observed microscopically in exposed groups of females. 16-DAY GAVAGE STUDY IN RATS: Groups of five male and five female F344/N rats were given 0, 12.5 (twice daily), 25 (once daily), 50 (once daily), 50 (twice daily), 100 (once daily), 200 (once daily), 200 (twice daily), or 400 (once daily) mg theophylline/kg body weight in corn oil by gavage. All rats receiving 400 mg/kg once daily and all but one female receiving 200 mg/kg twice daily died during the study. In groups dosed once daily, final mean body weights and body weight gains of males receiving 100 or 200 mg/kg and mean body weight gains of females receiving 50, 100, or 200 mg/kg were less than those of controls. The final mean body weights and body weight gains of groups receiving theophylline twice daily were generally similar to those of groups receiving the same daily dosages once daily. Clinical findings included rapid or labored respiration, hunched posture, and squinting. In groups dosed once daily, absolute and relative uterus weights of females receiving 100 or 200 mg/kg once daily were significantly less than those of the controls, and the absolute and relative uterus weights of females receiving 100 mg/kg once daily were significantly less than those of females receiving 50 mg/kg twice daily. Uterine atrophy was observed in three females receiving 200 mg/kg twice daily. Periarteritis of the mesenteric arteries was observed in two males and two females receiving 400 mg/kg once daily. 16-DAY FEED STUDY IN MICE: Groups of five male and five female B6C3F1 mice were given 0, 500, 1,000, 2,000, 4,000, or 8,000 ppm theophylline in feed for 16 days, resulting in approximate daily doses of 250, 475, 950, 1,800, or800, or 2,000 mg theophylline/kg body weight to males and 300, 450, 1,225, 2,000, or 4,375 mg/kg to females. All mice survived until the end of the study. Final mean body weights of 4,000 and 8,000 ppm females and mean body weight gains of 2,000, 4,000, and 8,000 ppm females were significantly greater than those of the controls. Feed consumption by exposed groups was similar to that by the controls, except that by the 8,000 ppm males, which was approximately 40&percnt; the amount of feed consumed by the control group. Histopathologic examinations were not performed due to the absence of mortality and significant exposure-related lesions. 16-DAY GAVAGE STUDY IN MICE: Groups of five male and five female B6C3F1 mice were given 0, 12.5 (twice daily), 25 (once daily), 50 (once daily), 50 (twice daily), 100 (once daily), 200 (once daily), 200 (twice daily), or 400 (once daily) mg theophylline/kg body weight in corn oil by gavage. Three males and all females receiving 400 mg/kg once daily died on day 1. There were no significant differences in final mean body weights or body weight gains. There were no histopathologic findings attributed directly to theophylline. 14-WEEK FEED STUDY IN RATS: Groups of 10 male and 10 female F344/N rats were given 0, 1,000, 2,000, or 4,000 ppm theophylline in feed for 14 weeks, which resulted in approximate daily doses of 75, 125, or 250 mg theophylline/kg body weight to males and 75, 125, or 275 mg/kg to females. The final mean body weight of 1,000 ppm females was significantly greater than that of the control group. Feed consumption by exposed groups was similar to that by the controls. Mean cell volume and mean cell hemoglobin were significantly greater in males exposed to 2,000 or 4,000 ppm than those in the control group. Segmented neutrophil counts of all groups of exposed females were significantly greater than that of the control group. The absolute and relative kidney weights of 4,000 ppm males were significantly greater than those of the controls, and there was an exposure-related increase in the severity of nephropathy in males. Exposure-related increases in the incidences of mesenteric and/or pancreatic periarteritis were observed in males and females. 14-WEEK GAVAGE STUDY IN RATS: Groups of 10 male and 10 female F344/N rats were given 0, 37.5, 75, or 150 mg theophylline/kg body weight in corn oil by gavage for 14 weeks. One male and one female receiving 150 mg/kg died before the end of the study. The mean body weight gain of 150 mg/kg females was significantly greater than that of the controls. Mean cell volume of 150 mg/kg males and mean cell hemoglobin of all groups of dosed males were significantly greater than those of the control group. There were slight dose-dependent increases in the incidences of mesenteric periarteritis in dosed males and females. 14-WEEK FEED STUDY IN MICE: Groups of 10 male and 10 female B6C3F1 mice were given 0, 1,000, 2,000, or 4,000 ppm theophylline in feed for 14 weeks, resulting in approximate daily doses of 175, 400, or 800 mg theophylline/kg body weight to males and 225, 425, or 850 mg/kg to females. All mice survived until the end of the study. The final mean body weights and body weight gains of all exposed groups of males and females were significantly less than those of the controls. Feed consumption by exposed groups was similar to that by the controls. Leukocyte, segmented neutrophil, and lymphocyte counts of 4,000 ppm males were significantly greater than those of the controls. Leukocyte and segmented neutrophil counts of 2,000 or 4,000 ppm females were significantly greater than those of the controls. There were no histopathologic findings attributed directly to theophylline exposure. 14-WEEK GAVAGE STUDY IN MICE: Groups of 10 male and 10 female B6C3F1 mice were given 0, 75, 150, or 300 mg theophylline/kg body weight in corn oil by gavage for 14 weeks. Three males and all females receiving 300 mg/kg, one 75 mg/kg male, and one control female died before the end of the study. Final mean body weights and body weight gains of 150 and 300 mg/kg males were significantly less than those of the controls. Mean cell volume and mean cell hemoglobin of 300 mg/kg males were significantly greater than those of the controls. There were no histopathologic findings attributed directly to theophylline treatment. 2-YEAR GAVAGE STUDY IN RATS: Groups of 50 male and 50 female rats were given 7.5, 25, or 75 mg theophylline/kg body weight in corn oil by gavage for 2 years. Survival and Body Weights: There were no significant differences in survival between dosed and control groups. Final mean body weights of all groups of dosed males and females were significantly less than those of the controls. Pathology Findings: There were no significantly increased incidences of neoplasms in dosed rats. The incidence of chronic inflammation of the mesenteric arteries was significantly increased in males receiving 75 mg/kg compared to the controls. There were doserelated negative trends in the incidences of mammary gland fibroadenoma and fibroadenoma or carcinoma (combined) in females; these differences correlated with decreased body weights. 2-YEAR GAVAGE STUDY IN MICE: Groups of 50 male B6C3F1 mice were given 0, 15, 50, or 150 mg theophylline/kg body weight and groups of 50 female B6C3F1 mice were given 0, 7.5, 25, or 75 mg/kg in corn oil by gavage for 2 years. Survival and Body Weights: Survival of 150 mg/kg males was significantly less than that of the controls. The final mean body weights of 150 mg/kg males, 25 mg/kg females, and 75 mg/kg females were significantly less than those of the control groups. Pathology Findings: There were no treatment-related increases in incidences of nonneoplastic lesions or neoplasms. In males and females, there were decreased incidences of hepatocellular adenoma and of the combined incidences of hepatocellular adenoma or carcinoma compared to the controls. Male mice had a pattern of nonneoplastic liver lesions along with silver-staining helical organisms in the liver consistent with Helicobacter hepaticus infection. The incidences of these liver lesions in 150 mg/kg males were significantly lower than those in control males. Increases in the incidences of hepatocellular neoplasms in male mice have been shown to be associated with H. hepaticus infection when hepatitis is also present. Because of this association, interpretation of the decreased incidence of liver neoplasms in male mice was more difficult. Incidences of lesions at other sites in this study were not considered to have been significantly impacted by H. hepaticus infection or its associated hepatitis. GENETIC TOXICOLOGY: Theophylline was not mutagenic in Salmonella typhimurium, with or without metabolic activation (S9). It induced sister chromatid exchanges but not chromosomal aberrations in cultured Chinese hamster ovary cells. The positive sister chromatid exchange response was noted only in the absence of S9. In vivo, a mouse bone marrow sister chromatid exchange test showed positive results at a standard 23-hour harvest time; however, this test was not repeated and the response is unconfirmed. An in vivo mouse bone marrow chromosomal aberrations test, that employed both standard and extended exposure protocols, gave negative results. The frequency of micronucleated erythrocytes was determined in peripheral blood of male and female mice exposed to theophylline in dosed feed or in corn oil by gavage for 14 weeks. No significant increases in the frequencies of micronucleated cells were seen in male or female mice in either of the studies. CONCLUSIONS: Under the conditions of these 2-year gavage studies, there was no evidence of carcinogenic activity of theophylline in male or female F344/N rats administered 7.5, 25, or 75 mg/kg. There was no evidence of carcinogenic activity of theophylline in male B6C3F1 mice administered 15, 50, or 150 mg/kg or female B6C3F1 mice administered 7.5, 25, or 75 mg/kg. Gavage administration of theophylline caused chronic inflammation of the mesenteric arteries in dosed male rats. Decreased incidences of mammary neoplasms in female rats were likely associated with lower body weights. There were dose-related decreases in the incidences of hepatocellular adenoma and hepatocellular carcinoma in male and female mice. Synonyms: 3,7-dihydro-1,3-dimethyl-1H-purine-2,6-dione; 1,3-dimethylxanthine; 1H-purine-2,6-dione; NSC 2066; pseusdotheophylline; theocin; theophyllin; theophylline, anhydrous Trade names: Accurbron; Aerobin; Aerolate III; Afonilum; Aminophylline; Aquaphyllin; Armophylline; Asmalix; Bilordyl; Bronchoretard; Bronkodyl; Cetraphylline; Constant-T; Diffumal; Duraphyl; Duraphyllin; Elixicon; Elixophyllin; Euphylline L.A.; Euphylong; LaBID; Labophylline; Lanophyllin; Lasma; Liquophylline; Optiphyllin; Parkophyllin; Phylocontin; Physpan; Pro-Vent; PulmiDur; Pulmo-Timelets; Quibron; Respbid; Rona-Phyllin; Sabidal; Slo-bid; Slo-Phyllin; Solosin; Sustaire; Tefamin; Teobid; Teofyllamin; Tesona; Theal tablets; Theo-24; Theobid; Theocap; Theochron; Theoclear; Theocontin; Theo-Dur; Theofol; Theograd; Theolair; Theolan; Theolix; Theophyl; Theoplus; Theo-Sav; Theosol; Theospan; Theostat; Theovent; TheoX; T-Phyl; Truphylline; Uni-Dur; Unifyl; Uniphyl; Uniphyllin; Xanthium