Testing for Endocrine Disruption: How Much is Enough?

John F. Gierthy,1

New York State Department of Health, Wadsworth Center, Empire State Plaza, P.O. Box 509, Albany, NY 12201–0509

ABSTRACT

The article highlighted in this issue is "Comparison of the Developmental and Reproductive Toxicity of Diethylstilbestrol Administered to Rats in Utero, Lactationally, Preweaning or from Weaning" by J. Odum, P. A. Lefevre, H. Tinwell, J. P. Van Miller, R. L. Joiner, R. E. Chapin, N. T. Wallis and J. Ashby (pp. 147–163).

The inappropriate exposure to endocrine disruptors continues to be a significant, as well as controversial, health issue (Colborn et al., 1993Go, Safe, 2000Go). While early emphasis was often placed on examples of environmental exposure, more recent concerns have focused on endocrine active potential and potency of commercial products and formulations, both synthetic and natural. Typical examples include bisphenol A, nonyphenol, and genistein, whose presence may be ubiquitous in commonly used household products and foods, resulting in chronic human exposure to low levels of these weakly estrogenic compounds.

The 1996 Food Quality Protection Act and the 1996 amendment to the Safe Drinking Water Act have acknowledged public health relevance, as well as the more wide-ranging environmental impact, of such compounds. These laws direct the U.S. Environmental Protection Agency (EPA) to test approximately 87,000 compounds and formulations for their estrogen modulating capacity (Schmidt,1999Go). The initial emphasis on estrogen modulators has subsequently been expanded to include modulators of androgens and thyroid hormones, and also to consider the effects on wildlife as well as humans.

The response to this mandate was the initiation of the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC, 1998Go), whose purpose was to consider and propose to EPA the processes and procedures by which this assessment could be accomplished. A tiered approach was adapted, after much deliberation and public input from the various stakeholders. Taken into account were the various physiological markers of endocrine-based effects under the overarching principles of toxicological testing, as they were then known. This testing is currently proposed to include a prioritization stage that considers such aspects as potency and likelihood of exposure. Testing would then begin with tier 1 in vitro and in vivo short-term assessment to further prioritize the candidates for tier 2 testing, which would include multigenerational studies. The in vivo components of this testing program will utilize developmental and reproductive toxicology procedures that take into account the need for a complete physiology of the intact animal as well as offspring. This is considered essential due to the complexity of the interactions of the endocrine system.

The various testing methods under consideration include the comprehensive examination of various life stages, from in utero through multigenerational assays, to enable assessment of developmental and reproductive effects. This extensive examination is considered to be essential to determine the consequences of exposure to endocrine disruptors over the lifetime of the animal, beyond life stage-specific effects. Since alteration of hormone levels during in utero and early postnatal periods can result in sustained adverse effects, these particular windows of exposure are considered to be especially important (Bigsby et al., 1999Go). Because of this, in utero and early postnatal exposures are often used for evaluation of compounds for putative endocrine-disrupting activity.

The comprehensive nature of this proposed in vivo testing would clearly require significant resources. Concern has been expressed that such a comprehensive examination may not be required to evaluate the endocrine modulatory potential of the compound under examination.

Odum and colleagues, in the highlighted article, note that, while all the life stages have been examined in an individual fashion in various studies, there have been no reports for comparative life-stage evaluation of developmental and reproductive outcomes in a single study. The authors thus undertook a comprehensive study of the developmental and reproductive effects of a potent estrogen, diethystilbesterol (DES), at a concentration that causes a midrange increase in uterine weight (60 µg/l drinking water for a dose of 6.5 µg DES/kg body weight/day). They conducted studies involving various windows of exposure that included in utero, neonatal, and weaning, fertility of the F1 animals, and sexual development of the F2 offspring. Their conclusion is that all the relevant effects required for estrogenic evaluation in rats can be determined using developmental-landmark endpoints from weaning onwards. Essentially, these changes involved testes descent, prepuce separation, and vaginal opening. Other developmental landmarks of anogenital distance and caudal sperm counts were not affected. They also conclude that neither in utero exposure nor early neonatal exposure produces significant effects in this system, and that, in general, no additional information is generated by histopathological examination of tissues. No unique sensitivity was noted for exposure in utero or during the early neonatal period. The results of this study would thus seem to suggest that the costly and time-consuming in utero, neonatal, and multigenerational studies will provide no additional information, and therefore should not be required in an endocrine disrupter testing program.

This is a well-done, comprehensive study and represents an initial example of comparative life-stage endocrine toxicology. However, a significant caveat is that it is limited to the responses to a single compound using, for the most part, a single dose, a single species, and a single strain. Variation of these parameters could well provide alternative outcomes. To this point, the recent National Toxicology Program (NTP) Workshop on Low-Dose Endocrine Effects Peer Review (Melnick et al., 2002Go) raises the issues of altered, dose-dependent mechanisms, as well as nonlinear dose response for endocrine disrupters. Perhaps the most significant outcome of this extensive peer review was the confirmation that some endocrine disrupters have nonlinear dose-response relationships. One of the consequences is that a low dose may thus be more potent than a higher dose for a given response to a particular compound. Most toxicological studies assume a linear dose response, which indeed appears to be valid for many adverse effects. However, in the case of perturbation of endocrine-associated effects, it is known that natural hormones at relatively high levels often have the ability to suppress an endocrine response that is apparent at lower levels. This results in a U-or inverted U-shaped dose-response curve. It is argued that xenoestrogens could have similar, nonlinear dose responses. The consequence of this, with regard to toxicity testing, is that the usual testing paradigm using a range of exposure doses, from high doses that cause obvious adverse effects to the highest low-level dose that lacks observable adverse effects, may miss potential low-dose effects outside the range of most testing. A further consideration is that this format often does not test the lower dose levels that are most relevant to human exposures. These lower exposures may have effects that are not apparent when higher dose exposures are used.

There is considerable controversy over the existence and/or relevancy of these low dose estrogen effects. The reported finding that low-dose bisphenol A exposure causes prostate enlargement in mice is an example (Nagel et al., 1997Go). Due to the potentially wide human exposure to this compound through its use in food and beverage containers, there is concern that exposure to this compound at low, environmentally relevant levels may enhance the risk for prostate cancer. While this study was shown to be credible by the NTP review, attempts to reproduce this low-dose effect have not been successful (Melnick et al., 2002Go). Some of the reasons being considered for this discrepancy include differences in animal strain, in dosing procedures, and in diet. Taken together, these disparate, but equally credible findings, suggest a low dose endocrine response of even greater complexity than originally thought.

The issue of a low-dose, nonlinear prostate enlargement response to DES, examined elsewhere (vom Saal et al., 1997Go), may be relevant to the highlighted study. Odum and colleagues suggest that histopathology may be of secondary importance to the developmental-landmarks effects of endocrine disruptors. It is of considerable interest that their study does show an apparently nonlinear dose response in postweaning DES-exposed animals. The ventral prostate gland was enlarged in animals exposed to the various DES levels from postnatal days 21 to 100. However, the only statistically significant increase was seen for the submaximum dose of 30 µg DES/l, rather than for the maximal dose of 60 µg DES/l. The authors considered this effect to be of "uncertain biological relevance." Considering the controversial nature of the endocrine disruptor issue, this histological result must be considered in light of the NTP Low-Dose Peer Review, which "focuses on biologic change rather than adverse effects because, in many cases, the long-term health consequences of altered endocrine functions during development have not been fully characterized" (Melnick et al., 2002Go). The actual health relevance of these newly emerging non-adverse, but significant, biological changes will continue to be debated. Therefore, caution should be used in considering the conclusions of the highlighted report. Nevertheless, by providing a limited model for how such studies should be performed, it exemplifies the extreme effort that will be required for an unequivocal assessment of endocrine-related effects, both adverse and benign.

NOTES

1 For correspondence via fax: (518) 486-1505. E-mail: gierthy{at}wadsworth.org. Back

REFERENCES

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vom Saal, F. S., Timms B. G., Montano, M. M., Palanza, P., Thayer, K. A., Nagel, S. C., Dhar, M. D., Ganjam, V. K., Parmigiani, S., and Welshons, W. V. (1997). Prostate enlargement in mice due to fetal exposure to low doses of estradiol or diethylstilbestrol and opposite effects at high doses. Proc. Natl. Acad. Sci. U.S.A. 94, 2056–2061.[Abstract/Free Full Text]





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