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

THE U.S. EPA ENDOCRINE DISRUPTOR SCREENING PROGRAM 503dose. Uterine weights are evaluated by analysis of variance, with body weight at necropsy as acovariable.Although there are numerous issues that must be considered during the optimization andvalidation process for the uterotrophic assay, the OECD has developed a strategy and begun tomethodically move through the process. 90 Initially, three major variables that required considerationincluded species selection (e.g., mouse versus rat), age of the test animal (sexually immature versusadult OVX), and route of administration (oral gavage versus subcutaneous injection [s.c.]). Additionalfactors, such as animal strain, animal husbandry (influence of diet or vehicle), and length oftime between last dose and necropsy (6 versus 24 h), that could possibly confound the results alsorequired special consideration.Drawing upon the vast amount of historical data and published literature on the uterotrophicassay, the OECD has based its approach to the validation process on scientific rationale and practicalexperience. The group chose to standardize the protocol with the laboratory rat as opposed to themouse, as the former is the preferred rodent model in toxicology testing paradigms for regulatorypurposes. It was also decided that studies would be designed to address questions regarding dosingroute, strain variation, and animal husbandry during the multiple laboratory evaluation. Phase I ofthe OECD validation process was designed to test, refine, and standardize the sexually immatureand the adult OVX rat uterotrophic assays using a high-potency estrogen, ethinyl estradiol, and theestrogen receptor antagonist, ZM 189,154. This phase was also designed to provide an initialdetermination of intra- and interlaboratory variability. Phase 2 was designed to demonstrate thecapability of the standardized uterotrophic protocols to identify estrogenicity in a set of testchemicals composed of weak estrogens and a known negative, as well as to continue to documentintra- and interlaboratory variations. The potential effects of low levels of isoflavones in laboratoryanimal chow were also evaluated to determine if the animal chows containing higher concentrationsof phytoestrogens would hamper the detection of estrogenic activity during tests of weak agonists.The results from Phases 1 and 2 of the validation process for the uterotrophic assay have recentlybeen published. Phase I studies were conducted by 19 laboratories from Japan, Denmark, France,Germany, Korea, the Netherlands, the United Kingdom, and the United States. 90 The study evaluatedfour protocols: (1) sexually immature, female rat model, with oral dosing for 3 days, (2) sexuallyimmature, female rat model, with dosing by s.c. injection for 3 days, (3) adult, OVX rat model,with dosing by s.c. injection for 3 days, and (4) adult, OVX rat model with extended dosing bys.c. injection for 7 days. Animals were exposed to ethinyl estradiol (0.03 to 3.0 µg/kg/d), or ZM189,154 (0.1 and 1.0 µg/kg/d) coadministered with ethinyl estradiol (0.3 µg/kg/d). All animals werekilled 24 h after the last dose. Other experimental conditions, such as animal strain, vehicle, diet,and bedding, varied between laboratories. Data from these studies demonstrated that all laboratoriessuccessfully conducted each of the four protocols, and that each protocol detected a significantincrease in uterine growth following exposure to ethinyl estradiol. For each protocol there was goodagreement among laboratories with respect to the lowest effective dose (LOEL) of ethinyl estradiol.Although the shape of the dose-response curve for ethinyl estradiol did vary between laboratories, therewas general agreement in dose response when the 10% and 50% effective doses (ED 10 and ED 50 ) werecalculated. Given the differences in animal strain and husbandry, the robust nature of each of theprotocols for detecting increases in uterine weight was demonstrated. In addition, preliminary evidencethat the protocols are a feasible method for detecting antagonists was also provided. The recommendationsresulting from these studies included: (1) moving forward with the validation of both thesexually immature and adult, OVX animal models, (2) refining the protocols to address minor technicalissues, and (3) continuing to test the protocols, using several weak estrogen agonists.Studies conducted under Phase 2 of the OECD validation process are reported by Kanno etal. 91,92 and Owens et al. 93 One study evaluated the same four protocols as employed in Phase I,using ethinyl estradiol, several weak estrogens (genistein, methoxychlor, nonylphenol, bisphenolA, and o,p′-DDT), and a negative control, dibutylphthalate. Twenty-one laboratories from ninenations participated in the study, although not all conducted each protocol or tested all of the© 2006 by Taylor & Francis Group, LLC