Food contact materials: an effect-based evaluation of the presence of hazardous chemicals in paper and cardboard packaging

Figures & data

Table 1. Food packaging materials (FCMs) tested

FCM	Printing	Purchased at	No. of pooled samples
Baking moulds	Yes	Supermarket	1
Pizza slice trays	Yes	Store	1
Paper for baking and baking moulds	No, but contained bleached material	Manufacturer/bakery/supermarket	5
Boxes for cookies	Yes	Manufacturer	3
Popcorn boxes	Yes	Movie theatre/store	3
Cake/pastry boxes/mats	Yes	Bakery	5
Board samples	Yes	Manufacturer	2
Boxes for infant formula/skimmed milk	Yes	Manufacturer	3
Boxes for porridge and flour mixes	Yes	Manufacturer	6
Paper plate for warm food	No	Restaurant	1
Paper plate (coated)	Yes	Store	1
Boxes for cereals	Yes	Supermarkets	4
Boxes for cookies	Yes	Supermarket	3
Microwave popcorn bags	Yes	Supermarkets	6
Straws	Yes	Movie theatre	1
Pizza boxes	Yes	Restaurant	2
Papers for wraps	Yes	Restaurants	3
Hamburger/French fries’ papers	Yes	Restaurants	5
Boxes for fries’ and hamburgers	Yes	Restaurant	2
Paper for trays	Yes	Restaurant	1
Bag for cookies	Yes	Bakery	1
Coloured paper for baking moulds	Yes	Manufacturer	1
Packages for frozen food	Yes	Supermarket	2

Table 2. Summary of bioassays and respective endpoints

Endpoint	Cell line	Standard Concentration	Added treatment Concentration
Oxidative stress – Nrf2 activation (Lundqvist et al. 2019)	MCF7 AREc32	Butylhydroquinone (tBHQ) 0.8–25 µM	N/A
Genotoxicity – Micronucleus (MN) events (OECD 2016a)	TK6	N/A	N/A
Agonistic ER activity (OECD 2016b)	VM7Luc4E2	Oestradiol (E2) 0.4 to 367 ρM	N/A
Antiestrogenic activity (OECD 2016b)	VM7Luc4E2	Raloxifen (Ral) 0.1–24.5 nM	E2 0.1 nM
Agonistic AR activity (OECD 2016c)	AR-EcoScreen GR-KO M1	Dihydrotestosterone (DHT) 0.001–1000 ρM	N/A
Antiandrogenic activity (OECD 2016c)	AR-EcoScreen GR-KO M1	Hydroxyflutamide (OHF) 0.0001–10 µM	DHT 200 ρM
Xenobiotic metabolism – AhR activation (Rosenmai et al. 2018)	DR-EcoScreen	2,3,7,8-Tetrachlorodibenzodioxin (TCDD) 0.01–1000 ρM	N/A
Inflammatory response – NFκβ activation (Lundqvist et al. 2019)	HepG2-NFκβ	Tumour necrosis factor α (TNFα) 0.2–50 ng/mL	N/A
Cytotoxicity (MTS/ATPase*/%EMA**)	All mentioned above	N/A	N/A

* ATPase was only used for VM7Luc4E2 cells ** % EMA was only used for TK6 cells

Figure 1. Oxidative stress response in MCF7 AREc32 cells exposed to FCM extracts for 24 h (a, b). The graphs represent mean ± SD of quadruplicates (n = 4) from one representative experiment, and the dotted line is the induction ratio 1.7 fold change, which was defined as the cut-off for bioactivity. The hashed grey black bars represent concentrations that were cytotoxic

Figure 2. Micronuclei formation in TK6 cells after exposure to FCM extracts for 24 h (a) and cytotoxicity test (b) Mitomycin C (MMC) was used as a positive control at concentrations 100 and 200 nM (a,b). The graphs illustrate mean ± SD of quadruplicates (n = 4) from one representative experiment. The dotted line in graph B represent the cut-off limit determined by the manufacturer’s protocol. Data was analysed using one-way ANOVA, followed by Dunnett’s post-hoc test. Results that were statistical significant are indicated by asterisks (* p-value < 0.05, *** p-value < 0.001, **** p-value < 0.0001)

Figure 3. Antioestrogenic effects in VM7Luc4E2 cells after 24 h of exposure to FCM extracts (a,b) The graphs illustrate mean ± SD of quadruplicates (n = 4) from one representative experiment. Unspiked medium with MeOH/MQ water was used as a control for the assay (1%). The dotted line shows the cut-off limit of 0.7. Samples with an activity below the cut-off were defined as bioactive. The hashed grey black bars represent concentrations that were cytotoxic

Figure 4. Antiandrogenic response in AR-EcoScreen GR-KO M1 cells after 24 h of exposure to FCM extracts (a,b). Unspiked medium with MeOH/MQ water was used as the control for the assay (1%). The panel shows mean ± SD of quadruplicates (n = 4) from one representative experiment. The dotted line illustrates the cut-off of 0.7. Samples with an activity below the cut-off was defined as bioactive

Figure 5. AhR activity in DR-EcoScreen cells after 24 h of exposure to FCM extracts (a,b). The graphs illustrates mean ± SD of quadruplicates (n = 4) from one representative experiment. The dotted line shows the cut-off limit of 1.5. The hashed grey black bars represent concentrations that were cytotoxic

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