Declining semen quality and polybrominated diphenyl ethers (PBDEs): Review of the literature to support the derivation of a reference dose for a mixture risk assessment.

To support a mixture risk assessment for chemicals that interfere with male reproductive health, we reviewed the literature to identify studies of polybrominated diphenyl ethers (PBDEs) and poor semen quality. Several epidemiological studies have shown associations of PBDE exposures with declining semen quality, non-descending testes and penile malformations. In rodent studies, poor semen quality, changes in testosterone levels and reproductive tissues have been observed. In vitro studies with reporter gene constructs show PBDE congeners as androgen receptor antagonists, and mixture studies in these systems have demonstrated that PBDE congeners act together with other androgen receptor antagonists. These observations led us to attempt the estimation of reference doses for specific PBDE congeners that can be used in a future mixture risk assessment for deteriorations of semen quality. While epidemiological studies provide support for such associations, they were uninformative for derivations of reference doses, due to the incompatibility of dose metrics used in exposure assessments. We therefore based our estimates on animal studies. Using a rigorous confidence rating approach, we found robust evidence that BDE-47 produced reductions in semen quality. We identified only one high confidence study of BDE-99 and accordingly evaluated the strength of evidence as moderate. One high confidence, and several medium confidence experimental studies observed declines in semen quality after BDE-209 exposure. Using established risk assessment procedures, we estimated that BDE-47 exposures below 0.15 µg/kg/d are unlikely to lead to reductions in semen quality. The corresponding exposures for BDE-99 and BDE-209 are 0.003 µg/kg/d and 1000 µg/kg/d. It is planned to use these estimates as reference doses in a mixture risk assessment of deteriorations in semen quality, involving multiple other chemicals also contributing to poor semen quality.

The GOLIATH Project: Towards an Internationally Harmonised Approach for Testing Metabolism Disrupting Compounds.

The purpose of this project report is to introduce the European "GOLIATH" project, a new research project which addresses one of the most urgent regulatory needs in the testing of endocrine-disrupting chemicals (EDCs), namely the lack of methods for testing EDCs that disrupt metabolism and metabolic functions. These chemicals collectively referred to as "metabolism disrupting compounds" (MDCs) are natural and anthropogenic chemicals that can promote metabolic changes that can ultimately result in obesity, diabetes, and/or fatty liver in humans. This project report introduces the main approaches of the project and provides a focused review of the evidence of metabolic disruption for selected EDCs. GOLIATH will generate the world's first integrated approach to testing and assessment (IATA) specifically tailored to MDCs. GOLIATH will focus on the main cellular targets of metabolic disruption-hepatocytes, pancreatic endocrine cells, myocytes and adipocytes-and using an adverse outcome pathway (AOP) framework will provide key information on MDC-related mode of action by incorporating multi-omic analyses and translating results from in silico, in vitro, and in vivo models and assays to adverse metabolic health outcomes in humans at real-life exposures. Given the importance of international acceptance of the developed test methods for regulatory use, GOLIATH will link with ongoing initiatives of the Organisation for Economic Development (OECD) for test method (pre-)validation, IATA, and AOP development.

Genotoxic mixtures and dissimilar action: concepts for prediction and assessment.

Combinations of genotoxic agents have frequently been assessed without clear assumptions regarding their expected (additive) mixture effects, often leading to claims of synergisms that might in fact be compatible with additivity. We have shown earlier that the combined effects of chemicals, which induce micronuclei (MN) in the cytokinesis-block micronucleus assay in Chinese hamster ovary-K1 cells by a similar mechanism, were additive according to the concept of concentration addition (CA). Here, we extended these studies and investigated for the first time whether valid additivity expectations can be formulated for MN-inducing chemicals that operate through a variety of mechanisms, including aneugens and clastogens (DNA cross-linkers, topoisomerase II inhibitors, minor groove binders). We expected that their effects should follow the additivity principles of independent action (IA). With two mixtures, one composed of various aneugens (colchicine, flubendazole, vinblastine sulphate, griseofulvin, paclitaxel), and another composed of aneugens and clastogens (flubendazole, doxorubicin, etoposide, melphalan and mitomycin C), we observed mixture effects that fell between the additivity predictions derived from CA and IA. We achieved better agreement between observation and prediction by grouping the chemicals into common assessment groups and using hybrid CA/IA prediction models. The combined effects of four dissimilarly acting compounds (flubendazole, paclitaxel, doxorubicin and melphalan) also fell within CA and IA. Two binary mixtures (flubendazole/paclitaxel and flubendazole/doxorubicin) showed effects in reasonable agreement with IA additivity. Our studies provide a systematic basis for the investigation of mixtures that affect endpoints of relevance to genotoxicity and show that their effects are largely additive.