Combination of Multidimensional Instrumental Analysis and the Ames Test for the Toxicological Evaluation of Mineral Oil Aromatic Hydrocarbons.

Mineral oil aromatic hydrocarbons (MOAHs) include mutagenic and carcinogenic substances and are considered a potential health risk. Current methods address the total MOAH content but cannot address the actual toxicological hazard of individual components. This work presents a combined methodology closing those gaps: high-performance liquid chromatography (HPLC) coupled to gas chromatography with flame ionization detection was used to determine the MOAH content. To characterize present substance classes, comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry was applied. Preparative HPLC separated MOAHs into subgroups, which were tested with a miniaturized Ames test evaluating DNA reactivity of isolated fractions. Combining these methods allowed a correlation between present subgroups and DNA reactivity. The developed approach was applied to a mineral oil and distinguished between not DNA-reactive mono- and diaromatics and DNA-reactive tri- and polyaromatics, providing a proof of concept. Hereinafter, it will be applied to diverse sample matrices including mineral oils, food, and food contact materials.

Potential endocrine disrupting properties of toys for babies and infants.

Plastic toys mouthed by children may be a source of exposure to endocrine active substances. The purpose of this study was to measure hormonal activity of substances leaching from toys and to identify potential endocrine disruptors causing that activity. For this purpose, migration experiments of toys were conducted in saliva simulants. The CALUX® assays were used to detect (anti-) estrogenic and (anti-) androgenic activity of 18 toys. Chemical trace analysis-namely, GC-MS and HPLC-MS- was used to identify which compounds may be responsible for endocrine activity in the sample migrates. Nine out of 18 tested toys showed significant estrogenic activity. For two samples, the detected estrogenic activity could be well explained by detecting the known endocrine active substance bisphenol A (BPA). For all identified substances, including BPA, a risk assessment for human health was performed by comparing the exposure dose, calculated based on the determined substance concentration, to toxicological reference values. Using worst-case scenarios, the exposure to BPA by mouthing of the two estrogen active, BPA-containing toys could be above the temporary TDI that EFSA has calculated. This demonstrates that some toys could significantly contribute to the total exposure to BPA of babies and infants. For seven out of nine estrogen active samples, the source of the estrogen activity could not be explained by analysis for 41 known or suspected endocrine active substances in plastic, indicating that the estrogen activities were caused by currently unknown endocrine active substances, or by endocrine active substances that would currently not be suspected in toys.

Characterization of estrogen and androgen activity of food contact materials by different in vitro bioassays (YES, YAS, ERα and AR CALUX) and chromatographic analysis (GC-MS, HPLC-MS).

Endocrine active substances (EAS) show structural similarities to natural hormones and are suspected to affect the human endocrine system by inducing hormone dependent effects. Recent studies with in vitro tests suggest that EAS can leach from packaging into food and may therefore pose a risk to human health. Sample migrates from food contact materials were tested for estrogen and androgen agonists and antagonists with different commonly used in vitro tests. Additionally, chemical trace analysis by GC-MS and HPLC-MS was used to identify potential hormone active substances in sample migrates. A GC-MS method to screen migrates for 29 known or potential endocrine active substances was established and validated. Samples were migrated according to EC 10/2011, concentrated by solid phase extraction and tested with estrogen and androgen responsive reporter gene assays based on yeast cells (YES and YAS) or human osteoblast cells (ERα and AR CALUX). A high level of agreement between the different bioassays could be observed by screening for estrogen agonists. Four out of 18 samples tested showed an estrogen activity in a similar range in both, YES and ERα CALUX. Two more samples tested positive in ERα CALUX due to the lower limits of detection in this assay. Androgen agonists could not be detected in any of the tested samples, neither with YAS nor with AR CALUX. When testing for antagonists, significant differences between yeast and human cell-based bioassays were noticed. Using YES and YAS many samples showed a strong antagonistic activity which was not observed using human cell-based CALUX assays. By GC-MS, some known or supposed EAS were identified in sample migrates that showed a biological activity in the in vitro tests. However, no firm conclusions about the sources of the observed hormone activity could be obtained from the chemical results.