Anna Kjerstine Rosenmai

Are Structural Analogues to Bisphenol A Safe Alternatives

BACKGROUND Bisphenol A (BPA) is a chemical with widespread human exposure suspected of causing low-dose effects. Thus, a need for developing alternatives to BPA exists. Structural analogues of BPA have already been detected in foods and humans. Due to the structural analogy of the alternatives, there is a risk of effects similar to BPA. OBJECTIVES The aim was to elucidate and compare the hazards of bisphenol B (BPB), bisphenol E (BPE), bisphenol F (BPF), bisphenol S (BPS) and 4-cumylphenol (HPP) to BPA. METHODS In vitro studies on steroidogenesis, receptor activity, and biomarkers of effect, as well as Quantitative Structure-Activity Relationship (QSAR) modeling. RESULTS All test compounds caused the same qualitative effects on estrogen receptor and androgen receptor activities, and most of the alternatives exhibited potencies within the same range as BPA. Hormone profiles for the compounds indicated a specific mechanism of action on steroidogenesis which generally lead to decreased androgen, and increased estrogen and progestagen levels. Differential effects on corticosteroid synthesis were observed suggesting a compound-specific mechanism. Overall, BPS was less estrogenic and antiandrogenic than BPA, but BPS showed the largest efficacy on 17α-hydroxyprogesterone (17α-OH progesterone). Finally, there were indications of DNA damage, carcinogenicity, oxidative stress, effects on metabolism, and skin sensitization of one or more of the test compounds. CONCLUSIONS Interference with the endocrine system was the predominant effect of the test compounds. A substitution of BPA with these structural analogues should be carried out with caution.

Fluorochemicals used in food packaging inhibit male sex hormone synthesis

Polyfluoroalkyl phosphate surfactants (PAPS) are widely used in food contact materials (FCMs) of paper and board and have recently been detected in 57% of investigated materials. Human exposure occurs as PAPS have been measured in blood; however knowledge is lacking on the toxicology of PAPS. The aim of this study was to elucidate the effects of six fluorochemicals on sex hormone synthesis and androgen receptor (AR) activation in vitro. Four PAPS and two metabolites, perfluorooctanoic acid (PFOA) and 8:2 fluorotelomer alcohol (8:2 FTOH) were tested. Hormone profiles, including eight steroid hormones, generally showed that 8:2 diPAPS, 8:2 monoPAPS and 8:2 FTOH led to decreases in androgens (testosterone, dehydroepiandrosterone, and androstenedione) in the H295R steroidogenesis assay. Decreases were observed for progesterone and 17-OH-progesterone as well. These observations indicated that a step prior to progestagen and androgen synthesis had been affected. Gene expression analysis of StAR, Bzrp, CYP11A, CYP17, CYP21 and CYP19 mRNA showed a decrease in Bzrp mRNA levels for 8:2 monoPAPS and 8:2 FTOH indicating interference with cholesterol transport to the inner mitochondria. Cortisol, estrone and 17β-estradiol levels were in several cases increased with exposure. In accordance with these data CYP19 gene expression increased with 8:2 diPAPS, 8:2 monoPAPS and 8:2 FTOH exposures indicating that this is a contributing factor to the decreased androgen and the increased estrogen levels. Overall, these results demonstrate that fluorochemicals present in food packaging materials and their metabolites can affect steroidogenesis through decreased Bzrp and increased CYP19 gene expression leading to lower androgen and higher estrogen levels.

Non-targeted screening for contaminants in paper and board food-contact materials using effect-directed analysis and accurate mass spectrometry

ABSTRACT Due to large knowledge gaps in chemical composition and toxicological data for substances involved, paper and board food-contact materials (P&B FCM) have been emerging as a FCM type of particular concern for consumer safety. This study describes the development of a step-by-step strategy, including extraction, high-performance liquid chromatography (HPLC) fractionation, tentative identification of relevant substances and in vitro testing of selected tentatively identified substances. As a case study, we used two fractions from a recycled pizza box sample which exhibited aryl hydrocarbon receptor (AhR) activity. These fractions were analysed by gas chromatography (GC) and ultra-HPLC (UHPLC) coupled to quadrupole time-of-flight mass spectrometers (QTOF MS) in order tentatively to identify substances. The elemental composition was determined for peaks above a threshold, and compared with entries in a commercial mass spectral library for GC-MS (GC-EI-QTOF MS) analysis and an in-house built library of accurate masses for substances known to be used in P&B packaging for UHPLC-QTOF analysis. Of 75 tentatively identified substances, 15 were initially selected for further testing in vitro; however, only seven were commercially available and subsequently tested in vitro and quantified. Of these seven, the identities of three pigments found in printing inks were confirmed by UHPLC tandem mass spectrometry (QqQ MS/MS). Two pigments had entries in the database, meaning that a material relevant accurate mass database can provide a fast tentative identification. Pure standards of the seven tentatively identified substances were tested in vitro but could not explain a significant proportion of the AhR-response in the extract. Targeted analyses of dioxins and PCBs, both well-known AhR agonists, was performed. However, the dioxins could explain approximately 3% of the activity observed in the pizza box extract indicating that some very AhR active substance(s) still remain to be identified in recycled low quality P&B.

Fluorinated alkyl substances and technical mixtures used in food paper-packaging exhibit endocrine-related activity in vitro

Migration of chemicals from packaging materials to foods may lead to human exposure. Polyfluoroalkyl substances (PFAS) can be used in technical mixtures (TMs) for use in food packaging of paper and board, and PFAS have been detected in human serum and umbilical cord blood. The specific structures of the PFAS in TMs are often unknown, but polyfluorinated alkyl phosphate esters (PAPs) have been characterized in TMs, food packaging, and in food. PAPs can be metabolized into fluorotelomer alcohols (FTOHs) and perfluoroalkyl carboxylic acids (PFCAs). Some PFAS have endocrine activities, highlighting the need to investigate these effects. Herein, we studied the endocrine activity of less characterized PFAS, including short‐chain PFCAs and FTOHs, PAPs, and TMs of unknown chemical composition. Long‐chain PFCAs were also included. We applied seven assays covering effects on estrogen, glucocorticoid, androgen, and peroxisome proliferator‐activated receptor (PPAR) activity, as well as steroidogenesis in vitro and ex vivo. In general, PAPs, FTOHs, TMs, and long‐chain PFCAs showed estrogenic activity through receptor activation and/or increasing 17β‐estradiol levels. Furthermore, short‐ and long‐chain PFCAs activated PPARα and PPARγ. Collectively, this means that (i) PAPs, FTOHs, and PFCAs exhibit endocrine activity through distinct and sometimes different mechanisms, (ii) two out of three tested TMs exhibited estrogenic activity, and (iii) short‐chain FTOHs showed estrogenic activity and short‐chain PFCAs generally activate both PPARα and PPARγ with similar potency and efficacy as long‐chain PFCAs. In conclusion, several new and divergent toxicological targets were identified for different groups of PFAS.

Polyfluorinated Alkyl Phosphate Ester Surfactants – Current Knowledge and Knowledge Gaps

Fluorochemicals are a diverse group of synthetically produced compounds with the unique ability to repel water as well as oil. This property makes them ideal for multiple purposes in a variety of consumer and industrial products. Fluorochemicals have been detected in the environment, as well as in human blood, urine and milk. Due to their long half-life in human beings, there is an increased risk that exposure to these compounds can cause adverse effects. However, except for perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), there is a large data gap regarding toxicological information on fluorochemicals. Polyfluorinated alkyl phosphate ester surfactants (PAPs) belong to the group of polyfluorinated alkyl surfactants. They have been detected in indoor dust and are widely used in food-contact materials, from which they have the ability to migrate into food. Toxicological data on PAPs are very limited, but current studies indicate that some PAPs have the potential to interfere with sex hormone synthesis in vitro. Disturbance of the sex hormone balance in foetal life has been suggested to be an important mechanism involved in adverse effects on, for example, male reproductive health and development. The current lack of toxicological data on PAPs impairs the risk assessment of this group of compounds. However, until more toxicological data on PAPs are available, the limited data currently accessible give reason to believe that these compounds might have the ability to cause potentially adverse effects, as seen for other perfluorinated chemicals, including some metabolic products of PAPs.

An effect-directed strategy for characterizing emerging chemicals in food contact materials made from paper and board

Food contact materials (FCM) are any type of item intended to come into contact with foods and thus represent a potential source for human exposure to chemicals. Regarding FCMs made of paper and board, information pertaining to their chemical constituents and the potential impacts on human health remains scarce, which hampers safety evaluation. We describe an effect-directed strategy to identify and characterize emerging chemicals in paper and board FCMs. Twenty FCMs were tested in eight reporter gene assays, including assays for the AR, ER, AhR, PPARγ, Nrf2 and p53, as well as mutagenicity. All FCMs exhibited activities in at least one assay. As proof-of-principle, FCM samples obtained from a sandwich wrapper and a pizza box were carried through a complete step-by-step multi-tiered approach. The pizza box exhibited ER activity, likely caused by the presence of bisphenol A, dibutyl phthalate, and benzylbutyl phthalate. The sandwich wrapper exhibited AR antagonism, likely caused by abietic acid and dehydroabietic acid. Migration studies confirmed that the active chemicals can transfer from FCMs to food simulants. In conclusion, we report an effect-directed strategy that can identify hazards posed by FCMs made from paper and board, including the identification of the chemical(s) responsible for the observed activity.

Fractionation of extracts from paper and board food contact materials for in vitro screening of toxicity

Paper and board used as food contact materials (FCMs) are chemically complex matrices, partly due to the naturally occurring substances in paper and board, but also due to the chemical treatment of the paper used to make it suitable for food contact. In order to assure the safety of packaging materials, information on the exposure as well as on the toxicity of substances in the packaging must be obtained. This study describes a comprehensive method for the extraction and fractionation of substances present in paper and board FCMs for further investigation by in vitro testing and chemical analysis. The extraction efficiency and the fractionation process were validated by determining recoveries in extracts from paper and board fortified with five surrogates of known concentration. The recoveries for the five surrogates were between 20% and 104% in the raw extract and between 21% and 109% after extraction and fractionation. The fractionation both reduces the number of compounds to be identified and works as a sample clean-up by reducing matrix effects. Raw extracts and fractions from two paper and board FCMs were furthermore tested in the aryl hydrocarbon receptor (AhR) reporter gene assay. Both raw extracts and two of the fractions of the raw extracts gave a positive response in the AhR assay. The strategy of extraction followed by fractionation offers a powerful tool in order to make the workflow for screening FCMs for potentially adverse effects more efficient.

PFAS in Paper and Board for Food Contact

Poly- and perfluorinated alkyl substances (PFAS) are used in paper and board food contact materials (FCMs) and they have been found to be highly persistent, bioaccumulative and toxic. The purpose of the Nordic workshop and of this report is to:* create an overview of the use of PFAS in FCMs of paper and board and of the toxicity and migration into food of the various substances* provide an overview of whether appropriate risk assessments for fluorinated substances exist as a basis for specific regulations or recommendations* provide an overview of whether analytical methods suitable for analysing and regulating the substances are available* discuss the possibility and structure of national regulations or Nordic recommendations for PFAS in FCMs of paper and board. Risk management to reduce the total content of organically bound fluorine in paper and board FCMs is supported.