Although risk assessments are typically conducted on a chemical-by-chemical basis, regulatory agencies and the scientific community are actively involved in discussions of how to conduct cumulative risk assessments for mixtures of chemicals. At present, there is no consistent framework for conducting cumulative assessments and experimental data are needed to facilitate the development of such a framework. Concern about mixtures of chemicals has arisen because studies show that humans [1
], fish [2
] and wildlife [6
] are exposed to multiple chemicals. As a result, the study of the effects of mixtures of chemicals is an emerging area of increasing scientific and regulatory focus.
The US EPA began considering the cumulative risk of some of the chemicals that act via a common mechanism of toxicity under the 1996 Food Quality Protection Act (FQPA, Public Law 104–170, formerly known as H.R. 1627 at http://www.epa.gov/pesticides/regulating/laws/fqpa/gpogate.pdf
). The US EPA’s Offices of Water and Research and Development and the EPA Superfund, Solid Waste and Air Programs have ongoing programs in this area. In this regard, the objective of our research is to provide data that facilitate the development of a guidance framework for assessing cumulative risk to reproduction and development from exposures that occur during pregnancy. Our working hypothesis is that chemicals that disrupt a common system or tissue during development, whether or not they have the same mechanism of toxicity, contribute to cumulative toxicity and should, therefore, be included in cumulative risk assessments. Several investigators have shown that mixtures of chemicals with diverse mechanisms of toxicity produce effects that far exceed those predicted by response addition models, which assume independent action for chemicals with different mechanisms of toxicity [7
]. Our mixture research has included different combinations of pesticides, phthalates and 2,3,7,8-tetrachlorodibenzodioxin (TCDD) [9
]. These chemicals disrupt sexual differentiation by acting as androgen receptor (AR) antagonists, inhibitors of fetal testosterone synthesis or as an aryl hydrocarbon receptor (AhR) agonist, respectively. Our results are in agreement with the conclusions of the National Academy of Science (NAS) Report on Phthalates [16
] which reviewed the current research on mixtures for the US EPA. This approach represents a fundamental shift from the current cumulative risk assessment frameworks that only include chemicals sharing a narrowly defined mechanism of action in a cumulative assessment.
In the current study, we conducted two mixture experiments to determine how two chemicals with very distinct mechanisms of toxicity interact in a mixture. In these studies, pregnant rats were dosed during sexual differentiation on gestational days (GD) 14–18 with either the individual compounds or a mixture of them, and the postnatal development of the male offspring was monitored through adulthood.
The two chemicals selected for this investigation were chosen because their modes of action have been well characterized and they disrupt fetal male rat reproductive development via different cellular and molecular mechanisms of toxicity. They do not share a “common mechanism of toxicity”. procymidone (PRO) is a fungicide with androgen receptor (AR) antagonist activity, displayed both in vitro and in vivo [17
]. PRO competitively inhibits the binding of androgens to the AR which leads to an inhibition of androgen-dependent gene expression in vitro and in vivo. In contrast, di-n-butyl phthalate (DBP) does not bind the AR, but rather, it alters male rat sexual differentiation by, in part, disrupting Leydig cell migration, differentiation and function [21
]. This in turn results in reductions in (1) fetal testis testosterone production, (2) mRNA levels for key proteins in the steroidogenic pathway including StAR and CYP11, and (3) reductions in insl-3 mRNA: insl3 is a peptide that is critical for gubernacular development and normal testis descent [25
The first experiment was a 2 × 2 factorial design with a control group, a PRO group, a DBP group and one mixture group, which combined the pesticide with the phthalate (this experiment has been briefly reviewed in [9
] but the detailed results of this experiment have not been presented). We combined 500 mg/kg-d of the fetal testosterone synthesis inhibitor DBP with 50 mg/kg-d of the AR antagonist PRO. These dosage levels were based upon previous dose–response studies on each chemical and the mixture was expected to induce malformations of the external genitalia (hypospadias) in approximately half the male offspring if it behaved in a dose additive fashion.
In the second DBP and PRO mixture experiment we increased the number of dose groups and expanded the dose range with the mixture. This experiment used a fixed ratio design, with a control group and eight dilutions of the mixture of PRO and DBP and was designed to examine the interaction in utero of these two chemicals over a broad dose range. To make predictions the ED50s and Hillslope values from individual chemical dose–response experiments for each chemical were calculated using logistic regression analyses for several androgen-dependent endpoints (). These values were used in dose addition (DA) and response addition (RA) models to predict the effects of the DBP plus PRO mixture in the second mixture experiment. Then, the ED50s and Hillslope values from this second mixture experiment were calculated using logistic regression analyses and the results were compared to the predictions from the DA and RA models.
Table 1 A comparison of the effects of the individual chemical dose–response studies with procymidone (PRO) and di-n-butyl phthalate (DBP) on male rat reproductive tract and sex accessory tissues. The table shows the ED50 values, the relative potencies, (more ...)
Even though each of these modes of disruption of the androgen signaling pathway results in different phenotypes in male rat offspring, our studies indicate that they both affect some of the same tissues. However, the relative potency factors (RPF) for PRO and DBP vary greatly from tissue to tissue ( and ) [9
]. Therefore, the contribution of each chemical to the overall mixture response depends upon the specific tissue and is not uniform across the reproductive tract. With this in mind, induction of hypospadias was chosen as the focal endpoint and the ratio of PRO and DBP in the mixture was selected such that each chemical would contribute equally to the induction of hypospadias () if they behaved in a dose additive manner. Since this endpoint typically displays a steep threshold, DA and RA models should predict very different values. In contrast, DA and RA predictions for anogenital distance (AGD) and retained nipples in infant male rats are quite similar because the dose–response curves are more linear and, as a consequence, one cannot determine for this endpoint “which is the best model”: DA or RA. In summary, our hypothesis was that DA predictions would provide a more accurate fit than the RA model to the effects of the mixture of PRO and DBP on the induction of hypospadias, and, in addition, that DA models would provide predictions that were as good, or better, than RA models for the other androgen-dependent endpoints.
Fig. 1 Individual chemical dose–response data for procymidone and di-n-butyl phthalate (DBP) were analyzed using a four parameter logistic regression model with GraphPad Prism 5.0 software. The ED50 and Hillslope values from the logistic regression analyses (more ...)