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

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 activities of identified substances in the YAS and AR 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 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

Antiandrogenic activity of samples spiked with 2.4/l DHT standard as measured by the YAS.

<p>Antiandrogenic activity of samples spiked with 2.4/l DHT standard as measured by the YAS.</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

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