A Practical Approach, Second Edition=Ronald D. Ho.pdf

504 DEVELOPMENTAL REPRODUCTIVE TOXICOLOGY: A PRACTICAL APPROACH, SECOND EDITIONchemicals. Overall, the uterotrophic assay provided reproducible results within the same laboratoryand across the participating laboratories for all the test chemicals. 91 With the exception that thelowest effect levels (LOELs) differed for the test chemicals by dosing routes, no major differenceswere noted between the protocols. As a result, the sexually immature and adult, OVX animal modelswere judged to be qualitatively equivalent to one another, and both protocols will continue to moveforward through the validation process. There were some false positives for dibutylphthalate inwhich 5 of 36 experiments resulted in a statistical increase in uterine weight as compared with thecontrol (vehicle). Of these, three sets of data for the dibutylphthalate had uterine weights significantlyhigher than the vehicle control, yielding a false positive rate of 8% for this test chemical. 92These data reinforce the importance of using a weight-of-the-evidence approach for data interpretation,using all the assays within the TIS battery. Finally, a comparison of the phytoestrogen contentin the diets used in the participating laboratories showed that levels lower than 325 to 350 µg/gtotal genistein equivalents did not impair the responsiveness of the uterotrophic assay when testingthe weak estrogen-receptor agonists, nonylphenol and bisphenol A. 93 However, recommendationsto monitor dietary phytoestrogen content will most likely be included in the final OECD guidelinesfor the uterotrophic assay as well as recommendations to monitor control uterine weights to becertain they fall within a range consistent with historical data.The advantages of using the uterotrophic assay as one of the in vivo components of the TISbattery are exemplified by its extensive use as an indicator of an estrogen receptor–mediatedresponse. Historical data, as well as data recently collected during the OECD validation studies,have proven the assay to be both biologically relevant (e.g., it detected chemicals known to bindto the estrogen receptor) and reliable (in that results can be replicated within a laboratory andbetween laboratories). The robust nature of the assay has also been proven through the ability ofmultiple laboratories to replicate results, even with variations in experimental conditions, such asstrain, dosing route, animal husbandry, and animal model (e.g., sexually immature versus adult,OVX female rats). Within the TIS battery, the uterotrophic assay is one of the most technicallysimple. It requires less time to complete and fewer animals per treatment group than the femalepubertal protocol, and in some cases it appears to be more sensitive to weak environmentalestrogens. For example, bisphenol A is known to possess a weak binding affinity for the estrogenreceptor 89,94 and to increase uterine weight following oral or s.c. exposures. 89,95 However, thischemical did not alter the age of vaginal opening when administered by oral gavage from 21 to 35d of age at doses two to three times higher than the dose required to stimulate uterine growth. 89Ashby and Tinwell 95 also reported no change in the age of vaginal opening following a 3-d exposureto bisphenol A (600 and 800 mg/kg) by oral gavage, although uterine weights were significantlyincreased in the same animals. In the same study when bisphenol A was administered by s.c.injection, 4 of 7 (600 mg/kg) and 3 of 8 (800 mg/kg) animals displayed early vaginal opening.Thus, although uterotrophic responses were similar following oral or s.c. exposures to bisphenolA, only the s.c. route altered the age at vaginal opening.While the uterotrophic assay has many strengths, the method is limited in that it can only detectestrogenic or antiestrogenic activity. The female pubertal protocol, for example, is capable ofdetecting a broader range of mechanisms of action. In addition, there are some technical caveatsthat may avert false negatives: Guidelines for the dose selection for the uterotrophic assay must bedeveloped to ensure that test chemicals with short-term estrogenic activity will not be missed ifthe endpoints are measured 24 h after the last dose. Several reports have demonstrated that thelength of the uterotrophic response following exposure to some chemicals is much shorter than forothers. Thus, the dosing route and timing following exposure may dictate whether a significantchange in uterine weight may still be detected at necropsy. 79,85,96,97 A comparison of the uterotrophicresponses 6 and 24 h following oral exposures to 4-tert-octylphenol or bisphenol A showedsignificant increases in uterine weights at the 6 h but not at the 24 h time point. 89 Yamasaki et al. 98also reported a higher increase in uterine weight at 6 h as compared with the 24 h time point© 2006 by Taylor & Francis Group, LLC