Abstract-The U.S. Congress has passed legislation requiring the U.S. Environmental Protection Agency (U.S. EPA) to develop, validate, and implement screening tests for identifying potential endocrine-disrupting chemicals within 3 years. To aid in the identification of methods suitable for this purpose, the U.S. EPA, the Chemical Manufacturers Association, and the World Wildlife Fund sponsored several workshops, including the present one, which dealt with wildlife species. This workshop was convened with 30 international scientists representing multiple disciplines in March 1997 in Kansas City, Missouri, USA. Participants at the meeting identified methods in terms of their ability to indicate (anti-) estrogenic/androgenic effects, particularly in the context of developmental and reproductive processes. Data derived from structure-activity relationship models and in vitro test systems, although useful in certain contexts, cannot at present replace in vivo tests as the sole basis for screening. A consensus was reached that existing mammalian test methods (e.g., with rats or mice) generally are suitable as screens for assessing potential (anti-) estrogenic/ androgenic effects in mammalian wildlife. However, due to factors such as among-class variation in receptor structure and endocrine function, it is uncertain if these mammalian assays would be of broad utility as screens for other classes of vertebrate wildlife. Existing full and partial life-cycle tests with some avian and fish species could successfully identify chemicals causing endocrine disruption; however, these long-term tests are not suitable for routine screening. However, a number of short-term tests with species from these two classes exist that could serve as effective screening tools for chemicals inducing (anti-) estrogenic/androgenic effects. Existing methods suitable for identifying chemicals with these mechanisms of action in reptiles and amphibians are limited, but in the future, tests with species from these classes may prove highly effective as screens. In the case of invertebrate species, too little is known at present about the biological role of estrogens and androgens in reproduction and development to recommend specific assays.

In the female Sprague-Dawley rat uterus 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds exhibited a broad spectrum of antioestrogenic responses. For example 2,3,7,8-TCDD inhibited the 17 beta-oestradiol-induced uterine wet weight increase, peroxidase activity, oestrogen and progesterone receptor levels, epidermal growth factor (EGF) receptor binding, and EGF receptor and c-fos protooncogene mRNA levels. The aryl hydrocarbon (Ah) receptor was identified in the rat uterus and the antioestrogenic activities of TCDD and related compounds were structure-dependent. In parallel studies, the effects of TCDD as an antioestrogen in MCF-7 human breast cancer cells was also investigated. TCDD inhibited the 17 beta-oestradiol-induced proliferation of these cells and the secretion of the 34-, 52- and 160-kDa proteins. Treatment of MCF-7 cells with 1 nM [3H]-17 beta-oestradiol resulted in a rapid accumulation of nuclear oestrogen receptor (ER) complexes. Pretreatment of the cells with TCDD caused a rapid decrease in nuclear ER binding activity and immunoreactive protein; moreover, the structure-dependent potencies of TCDD and related compounds as antioestrogens were similar to their Ah receptor binding affinities. TCDD also caused a decrease in nuclear ER levels in wild-type Ah-responsive Hepa 1c1c7 cells but was inactive in Ah non-responsive mutant Hepa 1c1c7 cells. Moreover, in the wild-type cells, both actinomycin D and cycloheximide blocked the effects of TCDD. 6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) has previously been characterized as a TCDD antagonist in rodents and in transformed rodent cell lines. However, like TCDD, MCDF also exhibited a broad spectrum of antioestrogenic activities in both the female Sprague-Dawley rat uterus and MCF-7 cells. MCDF is relatively non-toxic compared to TCDD and is being investigated as a compound which may be clinically useful for the treatment of mammary cancer.

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Ecological risk assessments based on chemical residue analysis and species demographics tend to ignore the bioavailability and bioaccumulation of the chemicals of concern. This study describes the incorporation of mechanistically based biomarkers into an ecological risk assessment of a poly-cyclic aromatic hydrocarbon (PAH)-contaminated site. A combination of soil residue analysis, tissue residue analysis, biomarkers in one-site trapped animals and biomarkers in animals confined to enclosures was used. In particular, the use of captured deer mice (Peromyscus maniculatus) for these studies is compared to the use of laboratory-raised deer mice placed in enclosures. This study indicates that the higher degree of variability in the responses of wild deer mice make the use of enclosure studies advantageous. Positive control studies performed by dosing laboratory-raised deer mice with the same PAHs as found on the site were used to validate this approach. These studies indicate that immune suppression occurred at PAH concentrations an order of magnitude below those required for the induction of ethoxyresorufin-O-dealkylase activity.