The following abstract presents results of a study conducted by a contract laboratory for the National Toxicology Program. The findings were not evaluated in accordance with the levels of evidence for reproductive or developmental criteria established by NTP in March 2009. The findings and conclusions for this study should not be construed to represent the views of NTP or the U.S. Government.
Diazepam (DAP) is a benzodiazepine derivative drug widely used as a tranquilizer, a pre-anesthetic and as an anticonvulsant. Diazepam in feed was tested for its effects on fertility and reproduction in Sprague-Dawley rats according to the Continuous Breeding Protocol. An extended (28 days including a mating trial) dose range-finding study (Task l) was performed at 0.005, 0.015, 0.05, 0.15 and 0.3% w/v in feed. Male body weights were significantly decreased, but pup survival was not affected at the 0.3% dose level. Based on these results, the Maximum Tolerated Dose (MTD) was estimated to be greater than 0.3%, and 0.04, 0.13 and 0.38% levels were chosen for the continuous breeding phase (Task 2). Male and female rats (F0) were continuously exposed for the 7-day precohabitation and 112-day cohabitation period.
During Task 2 cohabitation, body weights were decreased by up to 13% in the animals in the two highest dose groups. Feed consumption for these groups was decreased by up to 10%. While fertility was not affected for F0 animals, some reproductive parameters were adversely affected. The proportion of pups born alive was decreased by 3-4% in all dose groups. At the 0.38% dose level, the number of live pups per litter and live pup weights (both absolute and adjusted for litter size) were decreased by 10, 6 and 7%, respectively. Pup survival on postnatal day 4 was decreased by ~10% in the 0.38% group. No clinical signs of sedation or toxicity were noted at any dose level.
Since these changes were judged to be of statistical significance but of little or no biological significance, a crossover mating trial was not performed. The F1 pups from the last litter in the control and highest dose groups were reared and weaned. Body weights at weaning for the 0.38% treated males were 13% lower than control values, but were similar thereafter. F1 female body weights in the treated group were lower than control values at weaning, mating, delivery and at necropsy by 15, 14, 18, and 10%, respectively.
As in the F0 generation, fertility was unaffected in the Task 4 mating, while the proportion of pups born alive and weight of live pups were decreased by 7% and 12% in the treated animals, respectively. At necropsy, liver weights, and liver- and kidney-to-body weight ratios were increased. For F1 males, prostate and absolute seminal vesicle weights were significantly decreased. Epididymal sperm parameters (motility, count and the incidence of abnormal sperm) were unaffected. For F1 females, organ-to-body weight ratios were significantly increased for liver, kidney, and right ovary. The average estrous cycle length was similar for treated and control F1 females.
In summary, diazepam exposure at up to 0.38% in the diet (producing estimated intakes of up to 300 mg/kg/day) produced some apparent developmental toxicity (fewer live pups, and smaller live pups in both generations) in the presence of systemic toxicity (decreased body weight and increased liver and kidney weights), in the absence of any adverse effects on "reproductive" indices (number of litters, time between litters, estrous cycle length, and sperm endpoints). This study confirms that doses approximately 1000-fold higher than human doses are required to produce observable effects on rodent pup parameters, in the absence of effects on fertility.