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

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

Facilitating relaxation and stress reduction in healthy participants through a virtual reality intervention: study protocol for a non-inferiority randomized controlled trial

Finanziert im Rahmen der DEAL-Verträge durch die Universitätsbibliothek SiegenBackground Repeated or chronic stress is considered a major source of disease, in terms of both somatic and mental illnesses. The prevention of stress-related disease by interventions for relaxation has thus increased societal relevance. In this randomized controlled non-inferiority trial, we will compare a newly developed virtual reality (VR) environment for relaxation to an active control group applying a freely chosen relaxation method. To test if our VR environment supports relaxation in a situation of acute stress, a standardized stress induction protocol will precede the relaxation phase. Methods One hundred healthy participants will be recruited from the University of Siegen and randomly assigned to the VR or the active control group that will be free to choose their own relaxation strategy. The multi-sensory VR includes visual, acoustic, and haptic features to induce a strong feeling of presence. The laboratory testing will comprise a baseline measurement, a stress induction, a relaxation intervention, and a recovery measurement. The primary outcomes are self-reported stress and relaxation measured with a visual analog scale (VAS) at pre- and post-baseline, at the start, middle, and end of the stress induction, at pre- and post-relaxation, at pre- and post-recovery, and in the evening of testing. Secondary outcomes are the physiological parameters, namely heart rate and heart rate variability, tonic skin conductance level as well as the number of non-specific skin conductance responses, systolic and diastolic blood pressure and respiratory rate recorded during the four experimental phases as well as state mood, and state rumination assessed at four time points (pre- and post-stress, post-relaxation, and in the evening of testing). Finally, post-event processing will be assessed after relaxation and in the evening of testing. Repeated measures ANOVAs will be performed to test for statistical effects of group, time, and group × time interaction. Discussion The newly developed, multi-sensory VR offers an intervention for relaxation without prior training. Its immersive character might increase efficacy compared to other relaxation methods, especially in situations of acute stress. Future directions could be the development of a mobile version of the VR to enhance accessibility for users. To achieve a transfer of training effects to real life, VR components should successively be eliminated until relaxation is practiced without guidance by the VR

Identification of substances in migrates of samples which were hormone active in bioassay analysis.

a<p>… identification of the substance verified by comparison to a standard.</p>b<p>… identification of the substances by database comparison of mass spectra, not verified by comparison to a standard.</p><p>LOD…Limit of detection.</p><p>LOQ…Limit of quantification.</p><p>CAS#… Chemical Abstracts Service Number.</p

Androgen and antiandrogen activity of migrates from plastic samples.

a<p>… inhibition of response to DHT in undiluted samples.</p>b<p>… inhibition of yeast growth in undiluted samples.</p><p>−… no antiandrogen activity was detected.</p><p>+… antiandrogen activity in the range of the activity of 0.01 to 0,1 mg/l flutamide.</p><p>++… antiandrogen activity in the range of the activity of 0.1 to 1 mg/l flutamide.</p><p>+++… antiandrogen activity in the range of the activity of 1 to 10 mg/l flutamide.</p

Estrogen and antiestrogen activities of identified substances in the YES and ER CALUX.

<p>CAS#… Chemical Abstracts Service Number.</p>a<p>… inhibition of human U2-OS osteosarcoma cell growth at higher concentrations.</p>b<p>… inhibition of yeast growth at higher concentrations.</p

Validation parameters of GC-MS (SIM) analysis of 29 reference substances.

<p>Validation parameters of GC-MS (SIM) analysis of 29 reference substances.</p

Estrogen and antiestrogen activity of migrates from plastic samples.

a<p>… inhibition of response to 17β-estradiol in undiluted samples.</p>b<p>… inhibition of yeast growth in undiluted samples.</p><p>−… no antiestrogen activity was detected.</p><p>+… antiestrogen activity in the range of the activity of 0.01 to 0.1 mg/l 4-OHT.</p><p>++… antiestrogen activity in the range of the activity of 0.1 to 1 mg/l 4-OHT.</p><p>+++… antiestrogen activity in the range of the activity of 1 to 10 mg/l 4-OHT.</p

GC-MS-Analysis: Total Ion Chromatogram (TIC) of a migrate of sample CF 1.

<p>GC-MS-Analysis: Total Ion Chromatogram (TIC) of a migrate of sample CF 1.</p

Food simulants for the migration of packaging samples.

a<p>Sample was previously tested by Kirchnawy et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100952#pone.0100952-Kirchnawy1" target="_blank">[1]</a> using the YES.</p>b<p>Migration was done at 60°C for 10 days.</p