Bisphenol A (BPA, CAS 80-05-7) is frequently used in many types of consumer products, food packaging and thermal paper, which has recently been shown to be a high exposure source of BPA (FPF reported). Human exposure to BPA is widespread and raises concerns due to endocrine disrupting effects of the substance. In response, manufacturers started replacing BPA with other substances. On January 8, 2015 the peer-reviewed journal Regulatory Toxicology and Pharmacology published a study entitled “Endocrine activity of alternatives to BPA found in thermal paper in Switzerland”. The study investigates the occurrence of BPA and its alternatives in thermal papers in Switzerland and assesses the endocrine activity of these substances using in vitro and in silico tools. The target BPA alternatives included bisphenol S (BPS, CAS 80-09-1), bisphenol F (BPF, CAS 620-92-8), D-8 (also known as WinCon-8, CAS 95235-30-6) and Pergafast® 201 (CAS 232938-43-1). BPS and BPF can be found in food contact materials and have been previously detected in food (FPF reported).
Methods. A total of 124 thermal paper receipts were randomly sampled in Switzerland between September 2013 and January 2014. Samples were first analyzed for their BPA content. In a next step, all receipts without BPA (n = 23) and 14 randomly chosen BPA-containing receipts were screened for their content of possible alternative substances using liquid chromatography high resolution mass spectrometry (LC–HRMS). The screening method was designed to detect 17 out of the 19 BPA alternatives identified by the U.S. Environmental Protection Agency (EPA), BPA and 13 additional bisphenols. Those substances most frequently detected in the screening process (BPS, Pergafast® 201, D-8) were then quantified during a further analysis step using liquid chromatography tandem mass spectrometry (LC–MS/MS). BPS, Pergafast® 201, D-8, BPA and additionally another potential alternative BPF (not detected) were tested in vitro for their influence on the 17β-estradiol and free testosterone by a steroidogenesis assay using the human adrenocortical carcinoma cell line H295R. The steroidogenesis assay was performed following the OECD Test Guideline 456. VirtualToxLab™, an in silico tool, was then used to predict the endocrine and metabolic disruption potential of the substances. The VirtualToxLab™ calculates the toxic potential and the binding affinity of any molecule to 16 proteins (ten receptors, four members of the cytochrome P450 enzyme family, one transcription factor and one potassium ion channel).
Results and discussion. All receipts analyzed contained only a single developer substance. BPA was the most frequently found substance (n = 100, concentration range 5.6–30.4 mg/g). Thus BPA currently remains the most commonly used color developer in thermal paper in Switzerland. Only three alternative substances, namely BPS (n=4), Pergafast® 201 (n=11) and D-8 (n=9) were detected (concentration range of 3.3–13.2 mg/g). The authors point out that recently several Swiss retailers have announced that they would discontinue the use of BPA-containing thermal papers. Thus it is rather surprising to find only such a small number of thermal papers with BPA alternatives.
Cytotoxicity was not observed in tested concentrations, apart from the highest concentration (100 μM) that showed a significant decrease in viability for BPA, Pergafast® 201 and D-8. Overall, 17β-estradiol production was induced by BPA and BPF, which showed stronger response at the lowest observed effect concentration of 30 μM compared to BPA. Free testosterone production was inhibited by BPA and BPS. The authors highlight that the observed effects on steroidogenesis of BPA, BPS and BPF are in agreement with previous studies in the H295R assay (Rosenmai et al., 2014, Zhang et al., 2011). As specified in the OECD Test Guideline 456, the metabolic capability of the H295R cell line is unknown, but probably quite limited. Thus substances that require metabolic activation to show endocrine activity might be missed. Previous research shows that BPS might require metabolic activation to elicit estrogenic activity (Hashimoto et al., 2001). The current study gives no indication that Pergafast® 201 exhibits hormonal activity. Nevertheless, the authors emphasize that steroidogenesis could be affected by several mechanisms. Therefore, further tests would be necessary to make a final decision on this issue. Despite being structurally related to BPS, D-8 had effect neither on the concentration of 17β-estradiol nor free testosterone.
Except for Pergafast® 201, the toxic potential values determined by VirtualToxLab™ indicated a moderate risk of binding the receptors. The main target was the estrogen receptor β (ER β) (binding affinity range: 84.4 nM to 1.33 μM). Pergafast® 201 had a low risk of binding and the peroxisome proliferator-activated γ (PPARγ) receptor was its main target (binding affinity 22 μM). Overall, these findings support previous research showing that the affinity of BPA to the ERβ is stronger than for ERα (Kolsek et al., 2014) and that both BPF and BPS show ER-binding (Rosenmai et al., 2014). The binding affinity to the ERβ was strongest for BPA (0.084 μM), followed by BPF (0.161 μM) and BPS (0.742 μM). It was very weak for D-8 (1.33 μM). Further in vitro and/or in vivo analyses are nevertheless required for both non-bisphenols before drawing firm conclusions regarding their receptor binding activity, the authors stress.
Conclusions. In conclusion, the authors emphasize that substitution of BPA with BPF and BPS must be considered with caution, as they exhibit almost a similar endocrine activity as BPA. D-8 and Pergafast® 201 may be good BPA alternatives with regards to their in vitro effects on steroidogenesis. However, further research is needed to show that they do not elicit adverse effects on the hormonal system e.g. via metabolic activation.
Goldinger, D.M. et al. (2014). “Endocrine activity of alternatives to BPA found in thermal paper in Switzerland.” Regulatory Toxicology and Pharmacology (published online January 8, 2015).
Hashimoto, Y. et al. (2001). “Measurement of estrogenic activity of chemicals for the development of new dental polymers.” Toxicology in Vitro 15, 421–425.
Kolsek, K. et al. (2014). “Endocrine disruptome – an open source prediction tool for assessing endocrine disruption potential through nuclear receptor binding.” Journal of chemical information and modelling 54, 1254–1267.
OECD (July 28, 2011). “Test No. 456: H295R Steroidogenesis Assay.”
Rosenmai, A.K. et al. (2014). “Are structural analogues to bisphenol A safe alternatives?“ Toxicological Sciences 139, 35–47.
U.S. EPA (January 2014). “Bisphenol A alternatives in thermal paper.” (pdf)
Zhang, X. et al. (2011). “Bisphenol A disrupts steroidogenesis in human H295R cells.” Toxicological Sciences 121, 320–327