Feasibility and potential gains of enhancing the subacute rat study protocol (OECD test guideline no. 407) by additional parameters selected to determine endocrine modulation. A pre-validation study to determine endocrine-mediated effects of the antiandrogenic drug flutamide

Groups of five male and five female Wistar rats were treated by gavage with 0, 1, 10, and 100 mg flutamide/kg body weight for at least 28 days to inve stigate whether proposed enhancements to the current subacute rodent OECD t est guideline no. 407 could be included into the testing routine, which of the current and/or additional parameters would detect endocrine-mediated ef fects of flutamide reliably and sensitively, and to provide information on intra-laboratory variability. Two identical studies were performed concurre ntly. The enhanced protocol requests the additional determination of the sp ecific hormones triiodothyronine, thyroxine, thyroid stimulating hormone, f ollicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol, pro lactin. testosterone, corticosterone; of oestrus cyclicity and necropsy of all females in the dioestrus stage; of the number of homogenization-resista nt testicular spermatids and the number: motility, viability, and morpholog y of cauda epididymal spermatozoa; of additional organ weights (pituitary, ovaries, uterus, thyroid, male accessory reproductive organs); and of the h istopathology of additional organs (pituitary, epididymides, coagulation gl ands, pancreas, vagina). From a technical standpoint, it was possible to co nduct a study according to the enhanced protocol, however, with substantial additional effort, an increase in costs by some 67%, and logistic problems . In line with the specific pharmacological effect of flutamide, treatment- related changes were mainly found in male rats, while females were hardly a ffected by 100 mg/kg. In male rats, 100 mg/kg was the maximal tolerated dos e resulting in reduced body weight gain, but no or little other effects on clinical, haematological, clinico-chemical, or behavioral parameters, and 1 mg/kg was the no-observed-adverse-effect level. Antagonism of peripheral a ndrogen receptors by flutamide resulted in decreased relative organ weights of male accessory reproductive organs, changes that were reliably detected in both studies at 100 mg/kg, but only in one of both studies at 10 mg/kg. Corresponding histopathological changes were also detected reliably at 100 mg/kg. Antagonism of central androgen receptors by flutamide increased LH and FSH levels. LH stimulation of testicular Leydig cells in turn increased testosterone and estradiol levels. Again, all these changes were detected reliably at 100 mg/kg, but only in one of both studies at 10 mg/kg. Corresp onding histopathological alterations (increase of LH- and FSH-secreting cel ls, Leydig cell hypertrophy) were detected reliably and sensitively at 10 m g/kg. Studies on liver enzymes performed outside the scope of the enhanced protocol showed that flutamide at 100 mg/kg generally induced hepatic enzym e activcities, but decreased the activity of the sex-specific testosterone- dependent liver enzyme CYP2C11 in male rats. The laboratory methods employe d yielded reliable results, i.e., 93.6% of the quantitative measurements ob tained in both studies were in agreement. Doubling the animal number from f ive to ten per ses and dose does not increase the sensitivity of detection of endocrine-mediated effects above the level already provided by histopath ological examination of groups of five animals. Some of the proposed enhanc ements evaluated (additional organgravimetry and histopathology) were helpf ul in detecting the endocrine-mediated effects of flutamide reliably, while others did not contribute towards this aim (spermatology resulted in doubt ful effects, female cyclicity was not affected, hormone determinations prov ided mechanistic information). Ongoing testing according to the revised ver sion of the enhanced OECD test guideline no. 407 protocol and using ten compounds interfering with the endocrine system by different mechanisms will result in the identification of the most appro priate enhancements.