Tobacco products in the European Union Common Entry Gate (EU-CEG): A tool for monitoring the EU tobacco products directive.

INTRODUCTION: Under the European Union (EU) Tobacco Products Directive (2014/40/EU) (TPD), manufacturers and importers of tobacco products are required to report information to the European Commission (EC) and Member States (MS) on products intended to be placed on the market. We describe the distribution of notifications to the EU Common Entry Gate (EU-CEG) and identify key fields for improvement on reporting cigarettes or roll-your-own (RYO) tobacco. METHODS: A cross-sectional analysis of secondary data reported in the EU-CEG was conducted for tobacco products notified within EU-CEG between June 2016 and October 2019 for 12 EU MS. Analysis of compliance to specific regulations for priority additives that refer to cigarettes and RYO was conducted for 10 EU countries. RESULTS: Overall, 39170 tobacco products were notified. This included 16762 (42.8%) notifications of cigars, followed by cigarettes 11242 (28.7 %), waterpipes 3291 (8.4%), cigarillos (n=1783), pipe (n=1715), RYO (n=1635), chewing tobacco (n=1021), novel tobacco products (n=839), herbal products for smoking (n=535), other (n=258), nasal (n=74) and oral tobacco (n=15). In cigarettes and RYO tobacco products, the proportion of ingredients notified in all countries that contained an unknown Chemical Abstract Services (CAS) number was 3.8% and 2.1%, respectively. The proportion of underreporting flagging of priority additives ranged from 15.9% in Malta to 41.3% in Lithuania, the mean proportion of underreporting of the variable 'priority additive' for the 10 countries together was 24.7%. CONCLUSIONS: In the EU-CEG data base, for the period of analysis, a significant number of product notifications took place while large variations in the number of types of tobacco products notified across EU countries was noted. The timely monitoring of these data is needed so that products non-compliant within the EU-CEG system are assessed.

Toxicological approaches for the quantitative inhalation risk assessment of toxic metals from tobacco smoke: application on the deterministic and probabilistic inhalation risk assessment of cadmium for Austrian smokers.

OBJECTIVE: The objective of this study was the assessment of risks from inhalation exposure of Austrian smokers to cadmium through established toxicological approaches with emphasis on the exposure assessment component, which is challenging regarding the actual amount of metal that is inhaled and the simulation of the smoking pattern. MATERIALS AND METHODS: Exposure assessment comprised an estimation of the proportion of cadmium inhaled through smoking and actual occurrence data in tobacco products and survey smoking habits, which were integrated in alternative scenarios through a deterministic and a probabilistic Monte Carlo simulation method. Risks were characterized through the comparison of the exposure with health-based guidance values, as well as through the assessment of the excess lifetime cancer risk (ELCR), the non-cancer hazard quotient (NCHQ), and the margin of exposure (MOE). The strengths, the uncertainties, and the limitations of the different methodologies were discussed. RESULTS AND DISCUSSION: Upper exposures are close or exceed the Permitted Daily Exposure. Respiratory ELCRs are unacceptable compared to the benchmark range of 1.0E-06 to 1.0E-04. Renal and respiratory NCHQs exceed the target value of 1.0 by 3- to 17-fold. MOEs are not protective enough for cancer and non-cancer effects. The amount of cadmium that reaches the lung is a key source of uncertainty. CONCLUSION: Probabilistic estimates provide a refined capture of the actual inhalation exposure. Risk estimates and gender and age profiles are alarming, especially for young smokers. Application of toxicological approaches, combined with realistic assessment of the inhalation exposure levels, can support risk communication and management.

A tiered approach to cumulative risk assessment for reproductive and developmental toxicity of food contaminants for the austrian population using the modified Reference Point Index (mRPI).

Through our daily diet, we are exposed to a variety of food contaminants. Yet, assessing the cumulative health risk of chemical mixtures remains a challenge. Using a recently developed method, the modified Reference Point Index (mRPI), the cumulative risks posed by contaminant mixtures were assessed for their effects on reproduction and development. Since these effects can be quite diverse, a tiered approach was adopted to elucidate the risks at a more detailed level based on specific toxicological endpoints. An additional analysis was performed using the modified Maximum Cumulative Ratio (mMCR), which provides the determination of risk-dominating substances in the mixture. Our method represents a novel useful tool to screen and prioritise contaminant mixtures regarding their potential health risks. We found, that in the majority of the calculated scenarios a single substance dominates the cumulative risks. Lead was found to be the primary factor for adverse effects on reproduction and neuronal development of children. Perchlorate was identified as the most prominent risk factor for child development in generalCumulative risks of trichothecenes were dominated by deoxynivalenol. Concerning the impact on pre- and neonatal development, the co-exposure of several substances resulted in increased risks, with none of the considered contaminants dominating substantially.

Modified Reference Point Index (mRPI) and a decision tree for deriving uncertainty factors: A practical approach to cumulative risk assessment of food contaminant mixtures.

Risk assessment of chemical mixtures remains a challenging task in all areas of food and consumer safety. So far, no general method has been developed that is best suited to several subject areas (e.g. food contaminants, additives and supplements, plant protection products). Especially for mixtures of food contaminants sophisticated methods are typically not applicable due to a general lack of complete toxicological data sets. We developed a new approach, the modified Reference Point Index (mRPI), that combines the advantages of the Hazard Index and the Reference Point Index. Furthermore, we developed a decision tree for the determination of specific uncertainty factors that makes the mRPI an easy to use method for cumulative risk assessment even in a data poor field such as food contaminants. To further characterise the estimated cumulative risks, the Maximum Cumulative Ratio (MCR) was adapted to be applied on the mRPI, and the modified Maximum Cumulative Ratio (mMCR) was established to identify whether the risks are dominated by a single substance. We present two case studies assessing the nephrotoxic and neurotoxic risks for the Austrian population originating from food contaminant mixtures. Calculations could not rule out potential cumulative risks, yet, they seemed to be dominated by single substances.

Risk assessment of dietary exposure to perchlorate for the Austrian population.

Perchlorate is frequently found as contaminant in a variety of food. Based on analytical data of perchlorate occurrence in food products from the Austrian market, this study calculated dietary perchlorate exposure of the Austrian population for the three age classes of adults, children and infants. Furthermore, a detailed risk assessment was conducted based on the tolerable daily intake (TDI) of 0.3 µg/kg body weight/day, established by the European Food Safety Authority in 2014. Calculations of a scenario of average food consumption did not indicate elevated health risks by dietary perchlorate uptake. Exposure estimates reached only 12%, 26% and 24% of the TDI for adults, children and infants, respectively. However, in a scenario of high consumption, the TDI was exceeded by all age classes with 132%, 161% and 156%. The major cause for this exceedance is the comparatively high perchlorate contamination of spinach, but also other leaf vegetables, legumes and pineapples, leading to elevated exposure of high consumers. Our calculations reveal that the current provisional intra-Union trade reference level for perchlorate in spinach of 0.2 mg/kg, advocated by the European Commission, is not sufficient to protect high consumers against possible health risks. In order to reduce health risks to a tolerable level for all consumers, lowering of the regulatory maximum perchlorate concentrations is indicated. Moreover, a generally diversified diet can also counteract excessive exposure to perchlorate as well as to other harmful food contaminants.

FGF5 is expressed in melanoma and enhances malignancy in vitro and in vivo.

Although FGF5 mRNA was previously found expressed in some melanoma cell lines in contrast to normal human melanocytes, neither its contribution to melanoma growth nor its expression in melanoma tissue has been investigated. Here we demonstrate that ectopic overexpression of FGF5 in human melanoma cells with low endogenous FGF5 expression increased clonogenicity and invasion but not short-term growth in vitro. Silencing of FGF5 in melanoma cells with high endogenous FGF5 expression had the opposite effect on clonogenicity. FGF overexpression led to increased signaling along the MAPK and NFAT axis but had no effect on STAT3 signaling. In an in vivo experiment in immunocompromised mice, human melanoma xenografts overexpressing FGF5 showed enhanced tumor growth, a higher Ki-67 proliferation index, decreased apoptosis and enhanced angiogenesis. Immunohistochemistry performed on a tissue microarray demonstrated FGF5 protein expression in more than 50% of samples of melanoma and benign nevi. These data suggest that FGF5 has oncogenic potential in melanoma cells and contributes to melanoma growth in a subset of patients. This highlights the importance of further evaluating FGF5 as potential biomarker and therapy target in melanoma.

Synergistic estrogenic effects of Fusarium and Alternaria mycotoxins in vitro.

Mycotoxins are toxic secondary metabolites formed by various fungal species that are found as natural contaminants in food. This very heterogeneous group of compounds triggers multiple toxic mechanisms, including endocrine disruptive potential. Current risk assessment of mycotoxins, as for most chemical substances, is based on the effects of single compounds. However, concern on a potential enhancement of risks by interactions of single substances in naturally occurring mixtures has greatly increased recently. In this study, the combinatory effects of three mycoestrogens were investigated in detail. This includes the endocrine disruptors zearalenone (ZEN) and α-zearalenol (α-ZEL) produced by Fusarium fungi and alternariol (AOH), a cytotoxic and estrogenic mycotoxin formed by Alternaria species. For evaluation of effects, estrogen-dependent activation of alkaline phosphatase (AlP) and cell proliferation were tested in the adenocarcinoma cell line Ishikawa. The estrogenic potential varied among the single substances. Half maximum effect concentrations (EC50) for AlP activation were evaluated for α-ZEL, ZEN and AOH as 37 pM, 562 pM and 995 nM, respectively. All three mycotoxins were found to act as partial agonists. The majority of binary combinations, even at very low concentrations in the case of α-ZEL, showed strong synergism in the AlP assay. These potentiating phenomena of mycotoxin mixtures highlight the urgent need to incorporate combinatory effects into future risk assessment, especially when endocrine disruptors are involved. To the best of our knowledge, this study presents the first investigation on synergistic effects of mycoestrogens.

In vitro combinatory effects of the Alternaria mycotoxins alternariol and altertoxin II and potentially involved miRNAs.

Alternariol (AOH) and altertoxin II (ATX II) are mycotoxins formed by Alternaria spp. Since they are expected to co-occur in Alternaria-infested food and feed, we addressed the question of combinatory effects. In addition, potentially involved regulatory microRNAs were surveyed in an exploratory approach. Cytotoxicity measurements in constant ratio combinations of 1:10 or 1:1 (ATX II: AOH) mainly revealed additive effects in HepG2, HT29 and HCEC-1CT cells. Yet, in specific high doses antagonism was found. Microarray analysis of miRNA expression profiles in HepG2 cells indicated different patterns of miRNA regulation by AOH and ATX II, including several miRNA species for which no distinct functions are currently known. Among others, miR-4654, miR-4715_3p and miR-6720_3p were up-regulated by AOH and miR-5583_5p was down-regulated by ATX II. Additionally, miR-1323, involved in hindering DNA repair mechanisms, was decreased by ATX II. Digital droplet PCR (ddPCR) analysis of selected miRNAs indicated regulation of miR-29a by AOH, which might play a role in AOH-induced apoptosis. miR-192 and miR-224 regulation was associated with antagonistic cytotoxic effects of AOH and ATX II combinations. Our study represents the first evaluation on combinatory effects of AOH and ATX II.

Combinatory estrogenic effects between the isoflavone genistein and the mycotoxins zearalenone and alternariol in vitro.

SCOPE: The isoflavone genistein, present in soy-based food and dietary supplements, is known for its estrogenic potential. In addition to phytoestrogens, food may also contain mycotoxins with estrogenic properties like zearalenone or alternariol, raising the question on potential combinatory effects of these xenoestrogens. METHODS AND RESULTS: Combinatory estrogenic effects of genistein with zearalenone or alternariol were studied in the human endometrial adenocarcinoma cell line Ishikawa as expression of alkaline phosphatase (AlP) activity. Combinations of genistein with either zearalenone or alternariol, showed synergism and antagonism in the AlP assay, depending on the combination ratios and the concentration range. For combinations of zearalenone with genistein synergistic effects dominated. CONCLUSION: We demonstrate that mixture effects of phyto- and mycoestrogens potentially pose unexpected risks to consumers. Our study highlights the necessity of according considerations regarding combinatory effects in future risk assessment. The applied in vitro study design represents a cost-efficient screening method to discover interactive effects of estrogens as a basic decision tool for priority risk assessment.

Non-synergistic cytotoxic effects of Fusarium and Alternaria toxin combinations in Caco-2 cells.

Exposure of humans and animals to mycotoxins via food and feed generally involves a conglomeration of compounds contaminating the consumed products. Investigations on combinatory effects of mycotoxins are therefore of great importance. In this study, cytotoxic effects of binary mixtures of the Fusarium toxins enniatin B, aurofusarin, deoxynivalenol, nivalenol and zearalenone, and tenuazonic acid produced by Alternaria spp., were evaluated by the WST-1 assay in the colorectal carcinoma cell-line Caco-2 after 24h of incubation. The selection of these mycotoxins was based on typically occurring natural contamination patterns in grains. Aurofusarin, which can be found abundantly in contaminated foodstuff and has not been toxicologically characterized properly so far, showed pronounced cytotoxicity, decreasing the mitochondrial activity at 10µM to 51% compared to a solvent control. Combinations of other mycotoxins with aurofusarin showed additive effects. In contrast, binary mixtures of enniatin B, deoxynivalenol, nivalenol and zearalenone at cytotoxic concentrations, predominantly resulted in antagonistic effects. Binary combinations of these four Fusarium toxins with tenuazonic acid also revealed interacting effects leading to a decrease in cytotoxicity, compared to expected combinatory effects. Especially in combination with deoxynivalenol, tenuazonic acid was found to significantly reduce the cytotoxicity of this mycotoxin in Caco-2 cells. Synergistic effects were not observed for any toxin combination under the chosen conditions.