Inaccurate Models for Mixtures: Kortenkamp's Response.

The aim of our paper (1) was to explore whether the combined estrogenic effects of mixtures of xenoestrogens could be predicted successfully on the basis of the potency of individual mixture components. Putzrath's criticism of our study focuses primarily on the responses we observed with high concentrations of xenoestrogen mixtures. At these concentrations marked reductions of estrogenic effects became noticable. This phenomenon is frequently observed in the YES (yeast estrogen screen) assay (2) and is a manifestation of toxic effects on the yeast cells, as was clearly pointed out on page 986 of our paper (1). It is definitely not due to competition for the estrogen receptor in yeast cells, as suggested by Putzrath.

During the assessment of estrogenic effects of chemicals in yeast cells, toxic effects introduce anomalies to the concentration-response curves for estrogen receptor activation, and these confound the assessment of estrogenic effects. Therefore, toxic effects must be carefully distinguished from estrogenic responses, and the assay should not be run with concentrations of test agents the yeast cells cannot tolerate. Furthermore, no dosimetric model is able to deal with estrogen receptor activation and toxic effects at the same time. For these reasons, the data points at high mixture concentrations could not be included in the regression analysis for estrogenic effects, which was also clearly stated in our paper. We have nevertheless chosen to present these observations because we (like Putzrath) were intrigued by the toxicity that occurred at high concentrations of all mixtures. However, toxicity was not--and could not be--the object of our analysis. We maintain that our data show decisively that the combined estrogenic effect of all four xenoestrogens is additive. There was good agreement between the various predictions made on the basis of the individual effects of each mixture component and the observed combination effects. Therefore, in emphasising the discrepancies between observed and predicted effects at high mixture concentrations Putzrath misses the point of our work entirely. Our models are not accurate at these concentrations, nor are they intended to be.

Putzrath also criticizes the spread of data points observed with the single agents and with some of the mixtures. Again, we disagree with his notion that this represents an unacceptably high variation. Our data were from different experiments, performed by different operators over a period of 3 months. Given the biological variation inherent in living organisms and the other possible sources of experimental error, we feel that the variation in our data is nothing out of the ordinary. The confidence limits shown in our figures are 95% confidence bands of the best estimate of the regression models, not the population means, and data points are bound to lie outside these limits.

Taken together, our studies have encouraged us to attempt the prediction and assessment of the effects of more than four xenoestrogens. It remains to be seen which of the two prediction models used in our paper can be applied productively to such mixtures.

REFERENCES AND NOTES

(1.) Payne J, Rajapakse N, Wilkins M, Kortenkamp A. Prediction and assessment of the effects of mixtures of four xenoestrogens. Environ Health Perspect 108:983-987 (2000).

(2.) Beresford N, Routledge EJ, Harris CA, Sumpter JP. Issues arising when interpreting results from an in vitro assay for estrogenic activity. Toxicol Appl Pharmacol 162:22-33 (2000).

Andreas Kortenkamp The School of Pharmacy Centre for Toxicology London, United Kingdom E-mail: [e-mail]