An article published on May 29, 2017 in the peer-reviewed journal Food and Chemical Toxicology reported on an in vitro bioassays-based strategy for testing the toxicity of chemicals potentially migrating from paper and board food contact materials (FCMs).

Anna Rosenmai and colleagues from the Division of Diet, Disease Prevention, and Toxicology, National Food Institute, Technical University of Denmark tested 20 samples of paper and board FCMs in eight different in vitro bioassays. These assays included the Ames test for mutagenicity, as well as assays detecting agonists of estrogen receptor (ER), androgen receptor (AR), aryl hydrocarbon receptor (AhR), peroxisome proliferator-activated receptor gamma (PPARγ), and glucocorticoid receptor (GR), and assays detecting activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway (oxidative stress) and p53 pathway (cancer-related). With the AR assay, also antagonistic activity was assessed.

The samples were prepared by reflux in ethanol for four hours. This extraction procedure allows for a quick screening of most chemicals that could migrate from a given FCM sample; however, it likely overestimates the real migration in foods. Therefore, positive samples would need to be investigated further using migration-like procedures.

In the Ames test, two out of 20 FCM samples tested positive, namely the microwave pizza tray and the microwave popcorn bag. In the p53 assay, six out of 20 FCM samples tested positive; there were no samples which tested positive in both Ames test and p53 assay. In AhR assay, all 20 samples tested positive. The identity of the compounds activating the AhR is not yet known. In another study by the same group, an AhR-active sample of a corrugated pizza box made of recycled material was subjected to the effect-directed analysis (i.e., fractionation followed by re-testing to identify active fractions, detailed chemical analysis of active fractions, and testing of identified substances to confirm their activity and calculate their contribution to the overall activity observed in the sample). In that study, not all chemicals present could be identified and/or tested, and the observed AhR activity could not be fully explained by those chemicals that were indeed identified (FPF reported).

In the ER assay, nine out of 20 FCM samples tested positive. Effect-directed analysis carried out with one ER-positive sample, microwave pizza box, showed that this activity is likely due to the presence of bisphenol A (BPA, CAS 80-05-7) and several phthalates. Of note, BPA analogues were also shown to be estrogenic (FPF reported), and have been detected in paper and board FCMs (FPF reported). In the AR assay, six samples showed agonistic activity, and nine samples showed antagonistic activity. Of these, four samples (plain paper, baking paper, sandwich wrapper, and imported Chinese paper) showed both agonistic and antagonistic activities. An effect-directed analysis carried out with the sandwich wrapper demonstrated that the observed AR antagonism was likely caused by abietic acid (CAS 514-10-3) and dehydroabietic acid (CAS 1740-19-8). Other FCM-relevant (anti)androgenic compounds include, for example, phthalates, parabens, and phenols (FPF reported).

None of the samples tested positive in the GR assay. A similar observation has been reported previously in a study where a set of 65 chemicals shown to migrate from polycarbonate-alternative baby bottles has been tested (FPF reported). These findings suggest that interaction with GR does not appear to be of high importance for the safety assessment of these FCM types.

On the contrary, in the PPARγ assay, twelve out of 20 samples tested positive, demonstrating a high frequency of interactions with this receptor. In the above mentioned study on compounds migrating from baby bottles, ten out of 65 compounds showed PPARγ agonism, and 20 substances showed PPARγ antagonism. PPARs are known to be involved in the regulation of glucose and fat metabolism (FPF reported), and disruption of PPAR signaling has been implicated in obesity and diabetes (FPF reported). FCM-relevant compounds capable of interacting with PPARs include, for example, phthalates (FPF reported), and some of the per- and polyfluoroalkyl substances (PFASs), as was shown for example in a 2016 publication by the same group in the peer-reviewed journal Andrology.

Similarly of interest for FCMs testing were the results of Nrf2 assay, where 16 out of 20 paper and board FCM samples tested positive. This assay points to the oxidative stress. Further research is needed to explore the identity of the substances that cause this effect, as well as to better understand the relevance of these findings for human health.

In the same paper, the authors reported the results of an in silico analysis based on quantitative structure activity relationship (QSAR) models for genotoxic carcinogenicity, mutagenicity, developmental toxicity, and endocrine activity. This analysis was carried out on the set of 2,076 chemicals likely to be used in the manufacture of paper and board FCMs. Based on the availability of in-house structural information, only these 2,076 substances were analyzed from the initial set of 4,041 unique compounds, which included additives, monomers, solvents, photo-initiators, dyes, and pigments. Among these 2,076 chemicals, 599 compounds (29%) had positive predictions at least for one of the endpoints. 217 substances (10.5%) showed structural alerts for genotoxic carcinogenicity, 297 substances (14.7%) for in vivo mutagenicity, 184 substances (9.1%) for developmental toxicity, 73 substances (3.6%) for AR antagonism, 62 substances (3.1%) for ER binding, and 35 substances (1.7%) for ER activation.

References

Rosenmai, A., et al. (2017). “An effect-directed strategy for characterizing emerging chemicals in food contact materials made from paper and board.Food and Chemical Toxicology (published May 29, 2017).

Rosenmai, A., et al. (2016). “Fluorinated alkyl substances and technical mixtures used in food paper-packaging exhibit endocrine-related activity in vitro.Andrology 4:662-672.

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