The OECD program to validate the rat uterotrophic bioassay. Phase 2: coded single-dose studies.

Abstract

The Organisation for Economic Co-operation and Development has completed phase 2 of an international validation program for the rodent uterotrophic bioassay. This portion of phase 2 assessed the reproducibility of the assay with a battery of positive and negative test substances. Positive agonists of the estrogen receptor included the potent reference estrogen 17α-ethinyl estradiol (EE), and the weak estrogen agonists bisphenol A, genistein, methoxychlor, nonylphenol, and o,p´-DDT. The negative test substance or nonagonist was n-dibutylphthalate. The test substances were coded, and prescribed doses of each test substance were administered in 16 laboratories. Two versions of the uterotrophic assay, the intact immature and the adult ovariectomized female rat, were tested and compared using four standardized protocols covering both sc and po administration. Assay reproducibility was compared using a) EE doses identical to those used in phase 1 and in parallel dose-response studies, b) single doses of the weak agonists identical to one of five doses from the dose-response studies, and c) a single dose of the negative test substance. The results were reproducible and in agreement both within individual laboratories and across the participating laboratories for the same test substance and protocol. The few exceptions are examined in detail. The reproducibility was achieved despite a variety of different experimental conditions (e.g., variations in animal strain, diet, housing protocol, bedding, vehicle, animal age). In conclusion, both versions of the uterotrophic bioassay and all protocols appear robust, reproducible, and transferable across laboratories and able to detect weak estrogen agonists. These results will be submitted along with other data for independent peer review to provide support for the validation of the uterotrophic bioassay.

Usefulness of immature golden hamster (Mesocricetus auratus) as a model for uterotrophic assay

“…The mean uterine relative weight was approximately 60% of EE2 control value and histopathological examination confirmed attenuated proliferative effect. Our results are largely consistent with the literature data obtained using immature rats, in which case significant uterotrophic effects of EE2 were observed at doses from 0.3 to 3 µg/kg b.w./d (9,12,13,14,16,24,28) and antagonistic effects of tamoxifen at the dose of 1 mg/kg b.w/d (7).…”

“…Although much attention has been focused on identifying protocol variables and reproducibility among laboratories (12,13,14,16,23,24), direct comparison of uterine responses among rodent species has been rarely addressed (26). However, it is expected that the risk to humans takes into account the results obtained in the most sensitive species (26,30).…”

The OECD program to validate the rat uterotrophic bioassay. Phase 2: dose-response studies.

“…The maximum relative ratio responses of EEtreated were 4 to 5 in protocol A, 4.5 to 6 in protocol B, 3.25 to 5 in protocol C, and approximately 4 in protocol D (Kanno et al 2003). The maximum uterine weights reached by the weak estrogen agonists in these phase 2 studies were route, protocol, and test substance dependent, as is apparent by comparing the data in Figures 1-5.…”

“…Four other laboratories, which either participated in phase 1 (Kanno et al 2001) or the coded single-dose studies in phase 2 (Kanno et al 2003), did Blair et al (2000) and Branham et al (2002). b The binding curves were generated in a single laboratory on the basis of a single protocol using closely interspersed concentrations and performed in triplicate.…”

Using a customized DNA microarray for expression profiling of the estrogen-responsive genes to evaluate estrogen activity among natural estrogens and industrial chemicals.

“…The chemicals used here have estrogen activity in reporter gene assays (Demirpence et al 1993;Gaido et al 1999;Inoue et al 2002a;Pons et al 1990) and cell proliferation/uterotrophic assays [reviewed by Kanno et al (2003)] and upregulate estrogen target genes in responsive cells (Nagel et al 2001;Vivacqua et al 2003). Dioxin does not have estrogen agonist activity (Astroff and Safe 1988;Spink et al 1990).…”