Boobis (2019) Value and limitation of invitro bioassays to support the application of the threshold of toxicological concern to prioritise unidentified chemicals in food contact materials, Food Additives & Contaminants:

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.

Migrates from plastic food packaging were tested for oestrogen activity by yeast oestrogen screen and subsequently analysed by gas chromatography-mass spectrometry and high-performance liquid chromatography -mass spectrometry to identify oestrogen-active chemicals. Plastic samples were migrated according to EC 10/2011 with worst case scenarios being used. Food simulants consisted of either 10-95% ethanol or ultrapure water. Migrates were concentrated afterwards by solid phase extraction. Food contact material tested included polyethylene terephthalate, polypropylene, polyethylene, polystyrene and composite films. Oestrogenic activity ranging from 0.7 to 59 ng/l 17ß-estradiol equivalents was detected in seven out of 42 samples tested. The highest activity was found in a composite film sample. All 11 polyethylene terephthalate samples tested negative for oestrogen activity. A number of chemicals with known or supposed oestrogen activity were identified in migrates of oestrogen-active samples. These include butylated hydroxytoluene, 1,3-diphenylpropane, 1,2-diphenylcyclobutane and dibutyl phthalate. packaging surface during processing, e.g. set off effects. Most of the detected activities were low compared with the high oestrogen burden that was detected in bottled mineral water in recent studies. [13][14][15] Only one out of 42 tested samples showed an oestrogen activity of more than 18 ng EEQ/l, which was the average activity that Wagner and Oehlmann detected in bottled mineral water. 15 No PET samples tested (including recycling flakes) showed oestrogen activity, suggesting that PET bottles are not the source of the high oestrogen activities detected in mineral water. However, oestrogen activity in the range of 1.9 to 8.0 pg/l, as detected in mineral water samples by Wagner and Oehlmann 16 and by the Swiss Federal Office of Public Health 17 using more sensitive human cell-based assays are below the limit of detection of 0.1, respectively 0.2 ng EEQ/l. 478 C. KIRCHNAWY ET AL.

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