Andreas Kortenkamp

Widely used pesticides with previously unknown endocrine activity revealed as in vitro antiandrogens.

Background Evidence suggests that there is widespread decline in male reproductive health and that antiandrogenic pollutants may play a significant role. There is also a clear disparity between pesticide exposure and data on endocrine disruption, with most of the published literature focused on pesticides that are no longer registered for use in developed countries. Objective We used estimated human exposure data to select pesticides to test for antiandrogenic activity, focusing on highest use pesticides. Methods We used European databases to select 134 candidate pesticides based on highest exposure, followed by a filtering step according to known or predicted receptor-mediated antiandrogenic potency, based on a previously published quantitative structure–activity relationship (QSAR) model. In total, 37 pesticides were tested for in vitro androgen receptor (AR) antagonism. Of these, 14 were previously reported to be AR antagonists (“active”), 4 were predicted AR antagonists using the QSAR, 6 were predicted to not be AR antagonists (“inactive”), and 13 had unknown activity, which were “out of domain” and therefore could not be classified with the QSAR (“unknown”). Results All 14 pesticides with previous evidence of AR antagonism were confirmed as antiandrogenic in our assay, and 9 previously untested pesticides were identified as antiandrogenic (dimethomorph, fenhexamid, quinoxyfen, cyprodinil, λ-cyhalothrin, pyrimethanil, fludioxonil, azinphos-methyl, pirimiphos-methyl). In addition, we classified 7 compounds as androgenic. Conclusions Due to estimated antiandrogenic potency, current use, estimated exposure, and lack of previous data, we strongly recommend that dimethomorph, fludioxonil, fenhexamid, imazalil, ortho-phenylphenol, and pirimiphos-methyl be tested for antiandrogenic effects in vivo. The lack of human biomonitoring data for environmentally relevant pesticides presents a barrier to current risk assessment of pesticides on humans.

Science and policy on endocrine disrupters must not be mixed: a reply to a “common sense” intervention by toxicology journal editors

The “common sense” intervention by toxicology journal editors regarding proposed European Union endocrine disrupter regulations ignores scientific evidence and well-established principles of chemical risk assessment. In this commentary, endocrine disrupter experts express their concerns about a recently published, and is in our considered opinion inaccurate and factually incorrect, editorial that has appeared in several journals in toxicology. Some of the shortcomings of the editorial are discussed in detail. We call for a better founded scientific debate which may help to overcome a polarisation of views detrimental to reaching a consensus about scientific foundations for endocrine disrupter regulation in the EU.

Seven benzimidazole pesticides combined at sub-threshold levels induce micronuclei in vitro

Benzimidazoles act by disrupting microtubule polymerisation and are capable of inducing the formation of micronuclei. Considering the similarities in their mechanisms of action (inhibition of microtubule assembly by binding to the colchicine-binding site on tubulin monomers), combination effects according to the principles of concentration addition might occur. If so, it is to be expected that several benzimidazoles contribute to micronucleus formation even when each single one is present at or below threshold levels. This would have profound implications for risk assessment, but the idea has never been tested rigorously. To fill this gap, we analysed micronucleus frequencies for seven benzimidazoles, including the fungicide benomyl, its metabolite carbendazim, the anthelmintics albendazole, albendazole oxide, flubendazole, mebendazole and oxibendazole. Thiabendazole was also tested but was inactive. We used the cytochalasin-blocked micronucleus assay with CHO-K1 cells according to OECD guidelines, and employed an automated micronucleus scoring system based on image analysis to establish quantitative concentration–response relationships for the seven active benzimidazoles. Based on this information, we predicted additive combination effects for a mixture of the seven benzimidazoles by using the concepts of concentration addition and independent action. The observed effects of the mixture agreed very well with those predicted by concentration addition. Independent action underestimated the observed combined effects by a large margin. With a mixture that combined all benzimidazoles at their estimated threshold concentrations for micronucleus induction, micronucleus frequencies of ~15.5% were observed, correctly anticipated by concentration addition. On the basis of independent action, this mixture was expected to produce no effects. Our data provide convincing evidence that concentration addition is applicable to combinations of benzimidazoles that form micronuclei by disrupting microtubule polymerisation. They present a rationale for grouping these chemicals together for the purpose of cumulative risk assessment.

Ten years of mixing cocktails: a review of combination effects of endocrine-disrupting chemicals.

In the last 10 years, good evidence has become available to show that the combined effects of endocrine disruptors (EDs) belonging to the same category (e.g., estrogenic, antiandrogenic, or thyroid-disrupting agents) can be predicted by using dose addition. This is true for a variety of end points representing a wide range of organizational levels and biological complexity. Combinations of EDs are able to produce significant effect, even when each chemical is present at low doses that individually do not induce observable effects. However, comparatively little is known about mixtures composed of chemicals from different classes of EDs. Nevertheless, I argue that the accumulated evidence seriously undermines continuation with the customary chemical-by-chemical approach to risk assessment for EDs. Instead, we should seriously consider group-wise regulation of classes of EDs. Great care should be taken to define such classes by using suitable similarity criteria. Criteria should focus on common effects, rather than common mechanisms. In this review I also highlight research needs and identify the lack of information about exposure scenarios as a knowledge gap that seriously hampers progress with ED risk assessment. Future research should focus on investigating the effects of combinations of EDs from different categories, with considerable emphasis on elucidating mechanisms. This strategy may lead to better-defined criteria for grouping EDs for regulatory purposes. Also, steps should be taken to develop dedicated mixtures exposure assessment for EDs.

Sediments are major sinks of steroidal estrogens in two United Kingdom rivers

The occurrence of intersex fish in a number of European rivers has been attributed to exposure to estrogenic chemicals present in sewage treatment work (STW) effluents. To further understand the environmental fate of these contaminants, the estrogenic activity of effluents, water, and sediments were investigated both upstream and downstream of the major STW discharge in two United Kingdom rivers. Estrogenic activity, determined using the yeast estrogen-receptor transcription screen, of the major STW effluents on both rivers was similar, ranging from 1.4 to 2.9 ng 17beta-estradiol equivalents (EEQ)/L. Estrogenic activities of surface waters 1 km upstream and downstream of both STW inputs were less than the limits of detection (0.04 ng/L); however, levels of estrogenic activity in sediments were between 21.3 and 29.9 ng EEQ/kg and were similar at both upstream and downstream sites. Effluent and sediment extracts were fractionated by reverse phase-high-performance liquid chromatography, and estrogenic active fractions were further analyzed by gas chromatography-mass spectrometry. The major active chemicals in the two effluents and in the sediments were estrone with lesser amounts of 17beta-estradiol; however, at one site, a number of other unidentified estrogenic fractions were detected in the sediments. These results suggest that riverine sediments are a major sink and a potential source of persistent estrogenic contaminants.

Low dose mixture effects of endocrine disrupters: implications for risk assessment and epidemiology

During the last years, a series of studies on combinations of oestrogenic, thyroid-disrupting and anti-androgenic chemicals at low doses have been published. The available experimental evidence shows that combination effects may result from endocrine disrupters that each produces very small effects, if they are present in sufficiently large numbers. This review examines the implications of these findings for chemicals risk assessment and epidemiology. It is concluded that a lack of knowledge about relevant exposure scenarios presents serious obstacles for better human risk assessment. Epidemiology needs to abandon its focus on single endocrine disrupters and has to embrace the reality of endocrine disrupter mixture effects by developing biomarkers that capture cumulative exposure to endocrine disrupters.

Synergistic disruption of external male sex organ development by a mixture of four antiandrogens

Background By disrupting the action of androgens during gestation, certain chemicals present in food, consumer products, and the environment can induce irreversible demasculinization and malformations of sex organs among male offspring. However, the consequences of simultaneous exposure to such chemicals are not well described, especially when they exert their actions by differing molecular mechanisms. Objectives To fill this gap, we investigated the effects of mixtures of a widely used plasticizer, di(2-ethylhexyl) phthalate (DEHP); two fungicides present in food, vinclozolin and prochloraz; and a pharmaceutical, finasteride, on landmarks of male sexual development in the rat, including changes in anogenital distance (AGD), retained nipples, sex organ weights, and malformations of genitalia. These chemicals were chosen because they disrupt androgen action with differing mechanisms of action. Results Strikingly, the effect of combined exposure to the selected chemicals on malformations of external sex organs was synergistic, and the observed responses were greater than would be predicted from the toxicities of the individual chemicals. In relation to other hallmarks of disrupted male sexual development, including changes in AGD, retained nipples, and sex organ weights, the combined effects were dose additive. When the four chemicals were combined at doses equal to no observed adverse effect levels estimated for nipple retention, significant reductions in AGD were observed in male offspring. Conclusions Because unhindered androgen action is essential for human male development in fetal life, these findings are highly relevant to human risk assessment. Evaluations that ignore the possibility of combination effects may lead to considerable underestimations of risks associated with exposures to chemicals that disrupt male sexual differentiation.

Combined Exposure to Anti-Androgens Exacerbates Disruption of Sexual Differentiation in the Rat

Objective The aim of this study was to assess whether the joint effects of three androgen receptor antagonists (vinclozolin, flutamide, procymidone) on male sexual differentiation after in utero and postnatal exposures can be predicted based on dose–response data of the individual chemicals. Methods Test chemicals and mixtures were administered by gavage to time-mated nulliparous, young adult Wistar rats from gestational day 7 to the day before expected birth, and from postnatal days 1–16. Changes in anogenital distance (AGD) and nipple retention (NR) in male offspring rats were chosen as end points for extensive dose–response studies. Vinclozolin, flutamide, and procymidone were combined at a mixture ratio proportional to their individual potencies for causing retention of six nipples in male offspring. Results With AGD as the end point, the joint effects of the three anti-androgens were essentially dose additive. The observed responses for NR were slightly higher than those expected on the basis of dose addition. A combination of doses of each chemical, which on its own did not produce statistically significant AGD alterations, induced half-maximal mixture effects. At individual doses associated with only modest effects on NR, the mixture induced NR approaching female values in the males. Conclusions Effects of a mixture of similarly acting anti-androgens can be predicted fairly accurately on the basis of the potency of the individual mixture components by using the dose addition concept. Exposure to anti-androgens, which individually appears to exert only small effects, may induce marked responses in concert with, possibly unrecognized, similarly acting chemicals.

Low-Level Exposure to Multiple Chemicals: Reason for Human Health Concerns?

Background A key question in the risk assessment of exposures to multiple chemicals is whether mixture effects may occur when chemicals are combined at low doses which individually do not induce observable effects. However, a systematic evaluation of experimental studies addressing this issue is missing. Objectives With this contribution, we wish to bridge this gap by providing a systematic assessment of published studies against well-defined quality criteria. Results On reviewing the low-dose mixture literature, we found good evidence demonstrating significant mixture effects with combinations of chemicals well below their individual no observable adverse effect levels (NOAELs), both with mixtures composed of similarly and dissimilarly acting agents. Conclusions The widely held view that mixtures of dissimilarly acting chemicals are “safe” at levels below NOAELs is not supported by empirical evidence. We show that this view is also based on the erroneous assumption that NOAELs can be equated with zero-effect levels. Thus, on the basis of published evidence, it is difficult to rule out the possibility of mixture effects from low-dose multiple exposures.

Synergisms with mixtures of xenoestrogens: a reevaluation using the method of isoboles.

There are concerns about possible combination effects of environmental chemicals with oestrogenic activity and their implications for human health. Such chemicals are present in complex mixtures in our environment. A number of studies searching for possible synergistic interactions between xenoestrogens have appeared in the literature. However, in these studies no account was taken of established concepts and methods for analysing combination effects. In the present review, we highlight conceptual issues which may be useful for a sound analysis of the effects of mixtures of xenoestrogens. We find that much published work suffers from an undue focus on measuring effects of mixtures at only one dose level. Assessments of combination effects are frequently complicated by a lack of information on dose-response relationships. Some studies which purportedly show absence of synergy have in fact overlooked synergisms.