Urinary crystal formation and urothelial effects of pyroxasulfone administered to male rats

Pyroxasulfone induced a low incidence of urinary bladder tumors in male rats in a 2-year bioassay at 1000 and 2000 ppm, with occasional urinary calculi. No increased incidence of tumors of any tissue occurred in female rats or in mice of either gender. We performed three short-term studies to evaluate early development of pyroxasulfone-induced urinary crystals and urothelial cytotoxicity with consequent regenerative proliferation. First, male rats were treated with dietary 50, 1000 or 2000 ppm pyroxasulfone for 1, 3 or 7 days. The urothelium was examined by light and scanning electron microscopy (LM, SEM) and bromodeoxyuridine labeling index (BrdU LI). In two other studies, male rats were treated with dietary 20 000 ppm pyroxasulfone for 1 week. Urine collected at various times of day was examined by SEM and energy dispersive spectroscopy (EDS) or by LM, SEM, EDS, and infrared spectroscopy (IFS). Urinary crystals were present at various time points. EDS and IFS showed some contained calcium; others contained organic matter. Cytotoxicity was detected by SEM as cellular swelling, craters, and necrosis and by LM as cellular hypertrophy. Increased cell proliferation was detected by LM (hyperplasia), SEM (piling up of round cells), and by increased BrdU LI. There was no evidence of increased apoptosis. These findings support a mode of action for pyroxasulfone-associated bladder tumors in male rats involving formation of urinary crystals leading to urothelial cytotoxicity and regenerative proliferation. This is a high dose phenomenon, therefore, pyroxasulfone is not likely to be carcinogenic to humans at exposure levels that do not cause crystals with subsequent calculi formation in the urinary tract.