The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.

 

Johns Hopkins School of Public Health

Abstract for TestSmart -- Endocrine Disruptors

An Alternative to the EDSTAC Screening Battery for Detecting Endocrine-Active Compounds

John C. O'Conner
DuPont-Haskell Laboratory, Newark, DE

It has been argued that agricultural products, industrial chemicals, and some environmental pollutants may be exerting adverse effects by disrupting hormonal homeostasis in humans and in wildlife. Several examples of EACs producing adverse effects in humans (e.g., diethylstilbestrol) and wildlife (e.g., DDT) have been documented, and are scientifically substantiated. In most cases, however, the link between EACs and adverse effects on humans, fish, and wildlife is more tenuous. Responding to concerns that EACs may impact endocrine function in humans and wildlife, Congress passed amendments to the Food Quality Protection Act of 1996 and the Safe Drinking Water Act of 1996 requiring the U.S. Environmental Protection Agency (EPA) to implement screening/testing strategies for EACs before August 1999. In response to the 1996 legislation, EPA convened the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) to advise the agency on a strategy to screen and test xenobiotics for endocrine disruption. EDSTAC completed their charter in 1998 by recommending a tiered screening and testing scheme to evaluate compounds for their potential to act as agonists or antagonists to the estrogen (ER) or androgen (AR) receptors, steroid biosynthesis inhibitors, or their ability to alter thyroid function. EDSTAC outlined three potential screening batteries. All three EDSTAC batteries incorporate an array of 6-8 in vitro, in vivo mammalian, and environmental assays for examining effects of EACs. The in vitro receptor binding/transactivation assays, uterotrophic assay, and environmental assays are consistent between the three potential batteries. The EDSTAC-recommended battery also incorporates a Hershberger assay, pubertal female assay, and an in vitro steroidogenesis assay. For the alternate screening batteries, these three tests are replaced by either a 15-day intact male assay (alternate 1) or a pubertal male assay (alternate 2).

Over the last eleven years, DuPont has used short-duration in vivo testing models to identify endocrine modes of action for many different compounds. For instance, ammonium perfluorooctanoate (C8), a peroxisome proliferator, was shown to induce aromatase and increase serum estradiol levels in a 2-week intact male model (Cook et al. 1992). Similarly, the herbicide linuron was identified as being a weak androgen receptor antagonist (Cook et al. 1993), while 1-methyl-3-propylimidazole-2-thione (PTI) was shown to alter thyroid function by directly inhibiting thyroid hormone synthesis and by enhancing thyroid hormone excretion via UDP-glucuronyltransferase induction (Biegel et al. 1995). In addition, the modes of action for five other proprietary compounds were identified using this 2-week study design, all of which produced either Leydig cell or thyroid tumors in two-year rat bioassays. These experiences led to the development of a 15-day screening battery using intact male rats for identifying EACs (Cook et al. 1997).

The 15-day intact male battery, which is designed to be run in parallel with the uterotrophic assay and the in vitro receptor binding assays, identifies a broader spectrum of EACs than proposed by EDSTAC. It is designed to identify compounds that have the potential to act as agonists or antagonists to the ER, AR, or PR, dopamine modulators, steroid biosynthesis inhibitors (aromatase, 5a-reductase, and testosterone biosynthesis), or compounds that alter thyroid function. The 15-day intact male battery combines organ weight measurements, a comprehensive hormonal battery, and limited histopathology to achieve these goals. It provides specific information on the mode of action of a compound by using a "fingerprint" of changes in the endpoints to identify EACs. Male rats are dosed daily for 15 days with the test compound and euthanized on the morning of test day 15, approximately 2 hours after the last administered dose. At the terminal euthanization, the liver, thyroid gland, and reproductive organs [testes, epididymides, prostate, seminal vesicles with fluid, accessory sex gland unit (ASG; composed of the prostate, seminal vesicles with fluid, and coagulating glands)] are weighed, and the testes, epididymides, and thyroid gland are saved for histopathological evaluation. Blood is collected and serum is prepared for hormonal evaluation (testosterone, estradiol, DHT, LH, FSH, prolactin, T3, T4, TSH).

O'Connor and co-workers recently completed a pre-validation exercise for an integrated Tier I testing strategy using 20 compounds with well-characterized endocrine activities (O'Connor et al. 1996, 1998a,b, 1999a,b, 2000a,b). The two primary goals of the pre-validation exercise were to test the hypothesis that distinct "fingerprints" could be identified for each type of endocrine activity, and to determine which of the endpoints evaluated in the pre-validation exercise should be included in a final screen. By developing a "fingerprint" for each type of endocrine activity, the pattern of the responses for compounds with unknown endocrine activity can be compared to those from the positive controls. To accomplish these goals, each positive control was examined in an integrated Tier I screening battery consisting of the uterotrophic assay, 15-day intact male assay, and in vitro yeast transactivation assay (YTS). Each endpoint was evaluated for the variability, stability over time, predictability, and dose-dependency for each of the positive endocrine controls.

Based on the current data, the Tier I screening battery recommended by industry is composed of three components; the in vitro receptor binding assays, uterotrophic assay, and the 15-day intact male assay. The advantage of an integrated holistic approach is that it identifies more endocrine activities while using less animals and costing less than the EDSTAC-recommended approach. The ability to identify several different classes of EACs provides a more comprehensive approach than that proposed by EDSTAC, which attempts to develop stand-alone tests for each type of endocrine activity. Because the three components of the alternate Tier I battery are run in parallel and utilize a weight-of-evidence approach for identifying potential EACs rather than alterations in apical end points, this Tier I screening battery decreases the probability of false-positive and false-negative responses. Since it integrates (i.e., utilizes many endpoints from a few in vivo tests), it is cost-effective and comprehensive compared to the approaches recommended by EDSTAC, and the most efficient in terms of animal usage. By identifying the potential mode of action, the Tier I screening battery also focuses the direction of future testing (i.e., Tier II tests). Since the 15-day male battery utilizes male rats with a physiologically intact endocrine system, it is a more flexible assay that can be adapted to evaluate a wider range of endpoints and endocrine activities. By identifying the potential mode of action, this Tier I battery focuses the direction of future testing by identifying critical endpoints to be included in additional studies that will help define the dose-response curves and NOAELs/NOELs for the compound. As we gain more understanding of the effects EACs have on the environment and humans, mode of action data will become a critical component in decisions to reduce the associated risks.

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