The OECD program to validate the rat uterotrophic bioassay. Phase 2: dietary phytoestrogen analyses.

Abstract

Many commercial laboratory diets have detectable levels of isoflavones (e.g., phytoestrogens such as genistein [GN]) that have weak estrogenic activity both in vitro and in vivo. During validation studies of the uterotrophic bioassay, diet samples from 20 participating laboratories were collected and analyzed for three major phytoestrogens: GN, daidzein (DN), and coumestrol (CM). Soy phytoestrogens GN and DN were found at total phytoestrogen levels from 100 to 540 microg/g laboratory diet; a forage phytoestrogen, CM, ranged from nondetectable to 4 microg/g laboratory diet. The phytoestrogen levels were compared with both baseline uterine weights of the control groups and with the relative uterine weight increase of groups administered two weak estrogen agonists: bisphenol A (BPA) and nonylphenol (NP). The comparison uses a working assumption of additivity among the phytoestrogens, despite several significant qualifications to this assumption, to estimate total genistein equivalents (TGE). Some evidence was found that phytoestrogen levels in the diet > 325-350 microg/g TGE could diminish the responsiveness of the uterotrophic bioassay to weak agonists. This was especially true for the case of the intact, immature female version of the uterotrophic bioassay, where higher food consumption relative to body weight leads to higher intakes of dietary phytoestrogens versus ovariectomized adults. This dietary level is sufficient in the immature female to approach a biological lowest observable effect level for GN of 40-50 mg/kg/day. These same data, however, show that low to moderate levels of dietary phytoestrogens do not substantially affect the responsiveness of the assay with weak estrogen receptor agonists such as NP and BPA. Therefore, laboratories conducting the uterotrophic bioassay for either research or regulatory purposes may routinely use diets containing levels of phytoestrogens < 325-350 microg/g TGE without impairing the responsiveness of the bioassay.

Nutritional flavonoids impact on nuclear and extranuclear estrogen receptor activities

“…In 1997 the Organization for Economic Cooperation and Development (OECD) initiated a high priority activity to develop new and revised guidelines for screening and testing the estrogenicity of potential endocrine disrupters (OECD, 1998). One of the validated screening was the uterotrophic bioassay, which demonstrated the uterine growth of pre-pubertal or ovariectomized rats by following E2 or genistein treatment (i.e., gavage or intraperitoneal injection) (Owens et al, 2003;Naciff et al, 2004;Ashby and Odum, 2004).…”

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

“…First, DBP does not display binding affinity for the rat uterine estrogen receptor, i.e., there is no displacement of bound [ 3 H]17β-estradiol at concentrations up to 1 mM concentrations in vitro (Blair et al 2000). Second, in vivo toxicological studies, with some including gene activation profiles, indicate that DBP does not elicit responses indicative of an estrogen mode of action (Ema et al 2000;Ema and Miyawaki 2001;Mylchreest et al 1998Mylchreest et al , 1999Schulz et al 2001;Zacharewski et al 1998). A single data set that included data for all four standardized protocols was judged adequate for the negative chemical to conserve resources and animals.…”

Influence of dietary soy isoflavones on immature hamster uterotrophic and Hershberger assays

“…According to OECD post validation recommendations, laboratories conducting the uterotrophic bioassay for either research, or regulatory purposes may routinely use diets containing levels of phytooestrogens <325-350 µg/g of total genistein equivalents without impairing the responsiveness of the bioassay (24). However, this is contraindicated by current literature, which specifically states that diets containing less than this value still have the potential to alter the results of vaginal opening and uterotrophic assay (29).…”