Endocrine Disruption Research: Final Report. Exploring novel endpoints, exposure, low-dose and mixture effects in humans, aquatic wildlife and laboratory animals

Abstract

The EDEN project is an interdisciplinary effort conceived to address key issues that have hampered sound hazard and risk assessment for endocrine disrupting chemicals (EDCs) in the European Union. It has adopted an approach that fully integrates human, wildlife, exposures, mechanisms and low-dose/mixture evaluations. The project was structured into four interlocking themes focusing on EDC mixture exposures, mechanisms of action and novel endpoints, effects on male reproductive health and the evaluation of low-dose and mixture effects of EDCs.

For the first time, the simultaneous occurrence of nearly 150 different EDCs in human and wildlife tissues was measured. These studies revealed that numerous EDCs occur together in humans, yet differences in the levels of individual EDCs in specimens from boys with cryptorchidism and from women suffering from breast cancer and their respective controls did not become apparent. It appears that the likelihood of developing any of the above conditions cannot be attributed to any individual chemical at relevant exposure levels. However, there are signs that simultaneous exposure to many different EDCs may play a cumulative role in these disease processes. This means that efforts to develop biomarkers of cumulative EDC exposure should be re-doubled. In contrast, symptoms of endocrine disruption in fish could be explained largely in terms of exposure to estrogenic chemicals, but the possible role of antiandrogenic chemicals in disrupting sexual development in fish requires urgent attention. Fish caught in certain Dutch rivers exceeded the EU permissible levels for polychlorinated dioxins and furans.

Considerable progress has been made in establishing relevant modes of action of EDCs and in assessing new endpoints. The screening for EDCs, with its focus on steroid receptor interactions, has not kept up with the progress made in understanding rapid cellular signalling events that occur in the wake of receptor activation. In the interest of avoiding overlooking endocrine active agents it is necessary to expand screening tools to capture rapid signalling events. Substantial progress has been also achieved in characterising the role of the aromatase system in fish as a target of EDCs and the consequences of steroid synthesis disruption on sexual differentiation, reproduction as well as non-reproductive processes such as neurodifferentiation. Extensive investigations of the role of certain phthalates in disrupting male sexual development have revealed delays in germ cell differentiation and other molecular effects as key events underlying the induction of the testicular dysgenesis syndrome. The role of the InsL3 protein in promoting male sexual development proved to be more complex than thought previously. Although the development and validation of an assay for the measurement of InsL3 blood levels was successful, the differences in blood InsL3 levels in normal and cryptorchid boys were too small to exploit InsL3 as a biomarker indicative of disruption of testis descent. The hypothalamic pituitary unit proved to be exquisitely sensitive to the effects of several EDCs, and the effects may account for precocious sexual development observed after early EDC exposure. EDEN has advanced the study of endocrine disruption in fish with activities including the development of microarrays for assessing endocrine disruption in zebrafish and establishing sensitive screening tools for endocrine disruption in fish. The development of a transgenic fish model for the detection of EDC effects proved to be technically too demanding to be completed in time, but efforts continue to complete this project after conclusion of EDEN. An in vivo model in fish (the three-spined stickleback) for the detection of antiandrogenic EDC was developed as a complement to the Hershberger assay.

Male reproductive health in Denmark and Finland showed a worrying declining trend. For the first time, it could be established that the same is true for young men in Germany. It is of concern that semen quality among young Germans is similar to the values found in young Danes, a group previously thought to show the lowest semen quality in Europe. Foetal exposure to smoking has been identified as one reason for these effects. Observations of a declining total natural conception rate among the young Danish cohorts imply that the current poor semen quality has an impact on the population fertility in the future – a situation which will be difficult to reverse in the short term. The current and projected widespread use of assisted reproductive technologies may be a sign of such an emerging public health problem which also adds to the load of medical costs in young population. It is of vital importance to continue surveillance of semen quality and all efforts should be made to identify the factors that may cause harm in order to prevent further deterioration.

Extensive low-dose studies with EDCs have shown that the conventional estimation of no-observed-adverse-effect-levels (NOAEL), with their reliance on hypothesis testing methods is inadequate for capturing low-dose effects of EDCs. Whenever possible, regression-based approaches with benchmark dose limits should replace NOAEL as the basis for establishing acceptable human exposure levels. A framework was developed to combine the strengths of both methodologies by making considerations of statistical detection limits and statistical power the starting point of testing procedures. Implementation of this framework will require a significant change in toxicological testing practice.

Determinants of additivity for EDC mixtures have been characterised and are now well understood for combinations of similarly acting EDCs. Experimental studies have produced evidence that EDCs of relatively low potency and at low exposure levels can still work together to produce significant combination effects when they are present in sufficient numbers. The perceived low potency of many EDCs alone is uninformative in anticipating possible risks stemming from these chemicals. Where EDCs act in concert with endogenous hormones, significant additional effects may result under certain circumstances. Uncertainty still exists in relation to the likelihood of synergistic mixture effects, and concerted efforts should be made to fill this gap. Another source of uncertainty that will hamper sound EDC mixture risk assessment is incomplete knowledge about the identity of EDCs, their exposure levels and number. This issue can only be resolved through the development of dedicated exposure assessment strategies that take account of cumulative exposures. Despite these uncertainties, knowledge about determinants and factors that govern the joint action of similarly acting EDCs is now sufficiently advanced to come to pragmatic risk assessment approaches that take mixture effects into consideration. A modus operandi for EDC mixtures was developed which includes the use of dose addition (including the toxic equivalency factor approach) to arrive at a “mixture no-observed-adverse-effect-level” (MNOAEL) for endpoints relevant to endocrine disruption. These are then combined with a safety factor to arrive at estimates of tolerable human exposure. “Data-poor” situations may require estimation of a crude MNOAEL by dividing individual NOAEL of certain prototype chemicals by the anticipated number of relevant similarly acting chemicals.