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NTP Toxicology and Carcinogenesis Studies of Oxazepam (CAS No. 604-75-1) in Swiss-Webster and B6C3F1 Mice (Feed Studies).

Abstract

Oxazepam is one of a number of benzodiazepines used therapeutically as a sedative-hypnotic and antianxiety agent. Toxicology and carcinogenesis studies were performed by administering oxazepam (greater than 99% pure) in feed to male and female Swiss-Webster and B6C3F1 mice for 14 weeks, 57 weeks (Swiss-Webster), or 2 years (B6C3F1). Neurobehavioral assessments were performed during the studies. Genetic toxicology studies were conducted in Salmonella typhimurium and cultured Chinese hamster ovary cells, and peripheral blood samples were analyzed for frequency of micronucleated normochromatic erythrocytes. Supplemental studies were performed to compare the metabolism and toxicokinetics of oxazepam in the two mouse strains, to evaluate the effect on liver cell replication rates, to perform clinical pathology assessments, and to examine the mutation spectrum and frequency of activated H-ras oncogenes in liver neoplasms from the 2-year study with B6C3F1 mice. 14-WEEK STUDY IN SWISS-WEBSTER MICE: Groups of 10 male and 10 female Swiss-Webster mice received oxazepam in feed at concentrations of 0, 625, 1,250, 5,000, 10,000 ppm for 14 weeks. One 625 ppm male and one 10,000 female were killed moribund before the end of the study, and the condition of the female mouse was attributed to oxazepam exposure. Mean body weight gains of exposed groups were similar to those of the controls. Exposed mice displayed chemical-related sedation and lethargy during the first study week, but appeared normal thereafter. In the neurobehavioral studies, reductions in grip strength were evident in both male and female mice at week 2 and persisted in males through week 11. An antianxiety effect was detected in exposed mice in measures of motor activity, startle response, and reactions to thermal stimulus. At necropsy, absolute and relative liver weights were increased in an exposure-related manner and were approximately two-fold greater in 10,000 ppm mice than in controls. Centrilobular hepatocellular hypertrophy was present only in exposed mice, and the severity increased with dose. 14-WEEK STUDY IN B6C3F1 MICE: Groups of 10 male and 10 female B6C3F1 mice received oxazepam in feed at concentrations of 0, Groups of 10 male and 10 female Swiss-Webster mice 625, 1,250, 2,500, 5,000, or 10,000 ppm for 14 weeks. received oxazepam in feed at concentrations of 0, There were no deaths that were clearly related to 625,1,250, 2,500, 5,000, or 10,000 ppm for 14 weeks. oxazepam exposure. Mean body weight gains of One 625 ppm male and one 10,000 ppm female were exposed groups were similar to those of the controls. Exposed mice displayed chemical-related sedation and lethargy during only the first study week. In neurobehavioral studies, reductions in grip strength were evident in males at week 2 but were no longer observed at week 12. An antianxiety effect was noted in exposed mice in measures of motor activity, startle response, and reactions to a thermal stimulus (females). At necropsy, absolute and relative liver weights were increased in an exposure-related manner and were approximately two-fold greater in 10,000 ppm mice than in controls. Centrilobular hepatocellular hypertrophy was present only in exposed mice, and the severity increased with dose.

CHRONIC STUDIES:

Groups of 60 male and 60 female Swiss-Webster and B6C3F1 mice received oxazepam in feed at concentrations of 0, 2,500, or 5,000 ppm. Additional groups of 60 male and 60 female B6C3F1 mice received 125 ppm in feed to allow for study of a group with projected serum concentrations of oxazepam similar to those achieved in humans taking a therapeutic dose. Ten male and 10 female B6C3F1 mice per group were evaluated at 15 months. Average daily oxazepam consumption varied throughout the studies, and the overall daily average ranged from 10 to 29 mg/kg body weight for the 125 ppm groups, 234 to 512 mg/kg for the 2,500 ppm groups, and 444 to 1,085 mg/kg for the 5,000 ppm groups. Serum oxazepam concentrations determined at 57 weeks in Swiss-Webster mice and at the 15-month interim evaluation of B6C3F1 mice 1 mice were approximately 1 ug/mL in the 125 ppm groups, 4 to 7 μg/mL in the 2,500 ppm groups, and 7 to 10 μg/mL in the 5,000 ppm groups. Neurobehavioral assessments during the chronic studies of each strain of mice were confounded by the poor survival and deteriorating condition of mice with hepatic neoplasia. However, within the limitations of the studies, there were no notable changes in the types of behaviors observed compared to those observed in the 14-week studies, nor was there an enhancement in the degree to which they were exhibited. 57-Week Study in Swiss-Webster Mice: Survival, Body Weights, Feed and Compound Consumption, and Clinical Findings: At 57 weeks, survival of exposed mice was significantly lower than that of controls (males: O ppm, 45/60; 2,500 ppm, 19/60; 5,000 ppm, 10/60; females: 47/60, 28/59, 17/59), causing the study to be terminated. Mean body weights of exposed males were similar to controls until week 17; afterwards, mean body weights of exposed male groups were lower than those of controls. Final mean body weights of exposed males were 9% lower than that of the controls. The mean body weight of 2,500 ppm females was greater than that of the controls throughout the study. Females receiving 5,000 ppm had a mean body weight greater than that of the controls early in the study; after week 29, the mean body weight of this group was similar to that of the controls. Feed consumption by exposed males and females was slightly lower than that by the controls, and females in all groups, including controls, consumed slightly more feed than males throughout the study. Dietary levels of 2,500 and 5,000 ppm oxazepam resulted in average daily compound consumption levels of 270 and 570 mg/kg for males and 320 and 670 mg/kg for females. Hypoactivity and sedation were observed in exposed mice during the first week of the study. There were no other clinical findings associated with oxazepam exposure. Pathology Findings: Systemic amyloidosis was the principal cause of death in mice dying before the study was terminated. The lower survival of mice receiving oxazepam was attributed to an increase in the extent and severity of amyloid deposits in many organs, including the heart and kidney. Atrial thrombosis and pulmonary lesions consistent with chronic heart failure occurred at higher incidences and with greater severity in exposed mice. The incidence of hepatocellular adenomas (males: 1/60, 35/60, 50/60; females: 0/60, 22/59, 47/59) and carcinomas (males: 0/60, 5/60,19/60; females: 1/60, 1/59, 11/59) were increased in exposed mice. The incidences of eosinophilic foci were also increased in exposed mice (males: 0/60, 22/60, 22/60; females: 0/60, 20/59, 14/59), and there was evidence of increased centrilobular hepatocyte hypertrophy (males: 12/60, 46/60, 47/60; females: 3/60, 51/59, 53/59). 2-Year Study in B6C3F1 Mice: Survival, Body Weights, Feed and Compound Consumption, and Clinical Findings: Survival of mice receiving 2,500 and 5,000 ppm was significantly lower than that of controls (males: O ppm, 45/50; 125 ppm, 44/50; 2,500 ppm, 15/50; 5,000 ppm, 0/50; females: 39/50, 41/50, 2/50, 0/50). Mean body weight gains of exposed male and female mice were similar to controls until about week 15 when weight gains for mice exposed to 2,500 or 5,000 ppm slowed in relation to controls, resulting in weight gains approximately 30% to 40% lower than those of the controls throughout the remainder of the study. Mean body weight gain of male mice exposed to 125 ppm was similar to that of the controls, while that of female mice receiving 125 ppm was 10% to 15% lower than that of the controls after about week 45. Feed consumption by exposed males and females was similar to that by controls. Dietary levels of 125, 2,500, and 5,000 ppm resulted in average daily oxazepam consumption levels of 12, 310, and 690 mg/kg body weight for males and 15, 350, and 780 mg/kg for females. In the 5,000 ppm groups, lethargy and sedation were observed in a few mice during the first week of study. Pathology Findings: The early deaths of many of the B6C3F1 mice exposed to oxazepam were attributed to a marked increase in the incidences of hepatoblastoma (males: 0/49, 2/50, 21/50, 13/50; females: 0/50, 1/50, 8/50, 8/50), hepatocellular adenoma (males: 17/49,18/50, 34/50, 32/50; females: 25/50, 35/50, 35/50, 36/50), and hepatocellular carcinoma (males: 9/49, 5/50, 45/50, 50/50; females: 9/50, 5/50, 49/50, 44/50). Moderate hypertrophy of centrilobular hepatocytes occurred in mice receiving 2,500 and 5,000 ppm (males: 0/49, 2/50, 26/50, 43/50; females: 0/50, 2/50,11/50, 29/50). An increase in the incidence of follicular cell hyperplasia of the thyroid gland occurred in all exposed groups of mice (males: 4/49, 22/50, 49/50, 47/50; females: 16/50, 34/50, 49/50, 44/50), and thyroid gland follicular cell adenoma was increased in exposed females (0/50, 4/50, 5/50, 6/50). Testicular atrophy occurred in the 2,500 and 5,000 ppm groups (1/50, 0/50, 25/50, 38/50), and the incidence of epididymal Iymphocyte infiltration was increased in all exposed groups (2/50,14/50, 33/50, 21/50). The frequency of hepatocellular neoplasms with an activated H-ras oncogene in the B6C3F1 mice and the mutation spectrum of the H-ras gene were determined. The mutation spectrum of the H-ras genes in the relatively few neoplasms from exposed mice that did have an activated H-ras did not differ from the spectrum of mutations observed in neoplasms from controls, but the proportion of neoplasms with an activated H-ras gene decreased with increasing oxazepam dose. While 11 of 19 (58%) neoplasms from control mice had an activated H-ras gene, only 1 of 40 neoplasms from mice receiving 2,500 or 5,000 ppm oxazepam exhibited a similar molecular lesion. Thirteen of 37 (35%) neoplasms from mice in the 125 ppm group had an activated H-ras oncogene, suggesting that, although the incidence of all liver neoplasms was not statistically increased compared to controls, there was an increase in a similar subset of neoplasms (lacking an activated H-ras) that occurred with increased incidence at higher doses.

SUPPLEMENTAL STUDIES:

Because exposure to oxazepam caused increased incidences of liver neoplasms, supplemental short-term studies were performed. Oxazepam given in feed to male B6C3F1 mice at 25, 125, 2,500, or 5,000 ppm for up to 13 weeks was found to cause a dose-related increase in nuclear labeling index in studies measuring the incorporation of bromodeoxyuridine into replicating liver cells. This increase was statistically significant at all but the 25 ppm exposure level and was limited to mice evaluated at 15 days. Cell replication rates in most groups evaluated at 30 days and after were similar to control rates. There was minimal evidence suggestive of hepatocyte necrosis either by light microscopy or in clinical chemistry measures. There was, however, evidence of cholestasis, likely due to physical obstruction of bile canaliculi by swollen hepatocytes. The metabolic fate and toxicokinetics of oxazepam were evaluated in each strain of mice and were compared to published data from human studies. Both mice and humans form glucuronides of oxazepam and form 3- and 4-hydroxy and methoxy derivatives of the phenyl group. Oxidative metabolism of the phenyl group appears to be more prevalent in mice than is reported for humans. Elimination half-lives of parent compound do not differ between Swiss-Webster and B6C3F1 mice and are similar to values reported for humans.

GENETIC TOXICOLOGY:

Oxazepam was not mutagenic in any of several strains of Salmonella typhimurium, nor did it induce sister chromatid exchanges or chromosomal aberrations in cultured Chinese hamster ovary cells. These in vitro tests were performed with and without S9 metabolic activation. Results from an in vivo mouse peripheral blood micronucleus test performed on the B6C3F1 mice used in the 14-week study were also negative.

CONCLUSIONS:

Under the conditions of these feed studies, there was clear evidence of carcinogenic activity of oxazepam in male and female Swiss-Webster mice based on increased incidences of hepatocellular adenoma and carcinoma. There was clear evidence of carcinogenic activity of oxazepam in male and female B6C3F1 mice based on increased incidences of hepatoblastoma and hepatocellular adenoma and carcinoma. Increased incidences of hyperplasia of thyroid gland follicular cells in male and female B6C3F1 mice and of follicular cell adenomas in female B6C3F1 mice were also related to oxazepam exposure. Administration of oxazepam to Swiss-Webster mice resulted in centrilobular hepatocellular hypertrophy and increased incidences and severity of systemic amyloidosis. Administration of oxazepam to B6C3F1 mice also resulted in centrilobular hepatocellular hypertrophy. Synonyms: 7-Chloro-1,3-dihydro-3-hydroxy-5-phenyl-2 H - 1,4-benzodiazepin-2-one Trade Names: Tazepam, Wy-3498, Serax

PMID:
12595920
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