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Background The optimum test diet and rodent species/strain for evaluating endocrine-disrupting compounds (EDCs) are critical. Objectives We conducted studies to evaluate rodent species sensitivity and the effects of diets varying in phytoestrogen content on the time of vaginal opening (VO) in CD-1 mice, Fischer 344 (F344) rats, and CD Sprague-Dawley (S-D) rats. Methods Mice were weaned on postnatal day (PND) 15 and rats on PND19 and randomly assigned to control or test diets. Body weights, food consumption, and time of VO were recorded. Results The time of VO was significantly advanced in F344 rats fed diets containing daidzein and genistein, whereas these same diets did not advance VO in S-D rats. When animals were fed the AIN-76A diet spiked with genistein, time of VO was significantly advanced at all doses in CD-1 mice, at the two highest doses in F344 rats, and at the highest dose in S-D rats. The time of VO in F344 rats was more highly correlated with the phytoestrogen content than with the total metabolizable energy (ME) of 12 diets. Conclusions The S-D rat is less sensitive to dietary phytoestrogens compared with the F344 rat or the CD-1 mouse, suggesting that the S-D rat is not the ideal model for evaluating estrogenic activity of EDCs. The profound effects of dietary phytoestrogens on the time of VO, an estrogen-sensitive marker, indicate that a standardized open-formula phytoestrogen-free diet containing a low ME level should be used to optimize the sensitivity of estrogenic bioassays.

Selecting the optimum diet for endocrine disruptor (ED) research and testing studies in rodents is critical because the diet may determine the sensitivity to detect or properly evaluate an ED compound. Dietary estrogens can profoundly influence many molecular and cellular event actions on estrogen receptors and estrogen-sensitive genes. The source, concentration, relative potency, and significance of dietary estrogens in rodent diets are reviewed, including dietary factors that focus specifically on total metabolizable energy and phytoestrogen content, which potentially affect ED studies in rodents. Research efforts to determine dietary factors in commercially available rodent diets that affect uterotrophic assays and the time of vaginal opening in immature CD-1 mice are summarized. A checklist is provided of important factors to consider when selecting diets for ED research and testing studies in rodents. Specific metabolizable energy levels are recommended for particular bioassays. Discussions include the between-batch variation in content of the phytoestrogens daidzein and genistein, the effects of total metabolizable energy and phytoestrogens on the timing (i.e., acceleration) of vaginal opening, and increased uterine weight in immature CD-1 mice. It is concluded that rodent diets differ significantly in estrogenic activity primarily due to the large variations in phytoestrogen content; therefore animal diets used in all ED studies should ideally be free of endocrine-modulating compounds.

The purpose of the study reported here was to determine the effects of dietary phytoestrogens on the time of vaginal opening (VO) in immature CD-1 mice, and to correlate it with phytoestrogen and total metabolizable energy (ME) contents of the diet in an effort to determine the most appropriate diets(s) for comparing or evaluating the estrogenic or antiestrogenic activity of endocrine disruptor compounds (EDC). Mice were weaned at postnatal day (PND) 15 and fed the test diets from PND 15 to 30. Vaginal opening was recorded from PND 20 to 30. The phytoestrogen content of the diet was highly predictive (P0.0001) of the proportion of mice with VO at PND 24. Total ME content also was significantly (P0.01) correlated with time of VO, although this variable was somewhat less predictive than was phytoestrogen content. Time of VO in mice was significantly (P0.05) accelerated in mice fed diets high in phytoestrogens, compared with those containing low phytoestrogen content. It was concluded that: dietary daidzein and genistein can significantly (P0.01) accelerate the time of VO in CD-1 mice; the advancement in time of VO is more highly correlated with daidzein and genistein contents of the diets than with total ME content; advancement in the time of VO is a sensitive end point for evaluating the estrogenic activity of EDCs, and should be part of the standard protocol for evaluating EDCs. Phytoestrogen-free diet(s) containing the same amount of ME should be used in bioassays that compare the time of VO, or increases in uterine weight as end points for evaluating the estrogenic activity of an EDC.