Mona-Lise Binderup

Toxicity testing and chemical analyses of recycled fibre-based paper for food contact.

Food-contact materials, including paper, have to comply with a basic set of criteria concerning safety. This means that paper for food contact should not give rise to migration of components, which can endanger human health. The objectives of this pilot study were, first, to compare paper of different qualities as food-contact materials and to perform a preliminary evaluation of their suitability, from a safety point of view, and, second, to evaluate the use of different in vitro toxicity tests for screening of paper and board. Paper produced from three different categories of recycled fibres (B-D) and a raw material produced from virgin fibres (A) were obtained from industry, and extracts were examined by chemical analyses and diverse in vitro toxicity test systems. The products tested were either based on different raw materials or different treatments were applied. Paper category B was made from 40% virgin fibres, 40% unprinted cuttings from newspapers, and 20% de-inked newspapers and magazines. Paper categories C and D were based on newspapers and magazines. However, paper D was de-inked, whereas C was not. To identify constituents of the papers with a potential to migrate into foodstuff, samples of the paper products were extracted with either 99% ethanol or water. Potential migrants in the extracts were identified and semiquantified by GC-IR-MS or GC-HRMS. In parallel to the chemical analyses, a battery of four different in vitro toxicity tests with different endpoints were applied to the same extracts: (1) a cytotoxicity test using normal human skin fibroblasts. The test was based on measurements of the reduction of resazurin to resorufin by cellular redox processes and used as a screening test for acute or general toxicity; (2) a Salmonella/microsome assay (Ames test) as a screening test for mutagenic and potentially carcinogenic compounds; (3) a recombinant yeast cell bioassay as a screening test for compounds with oestrogenic activity; (4) an aryl hydrocarbon (Ah)-receptor assay (CALUX assay) as a screening test for compounds with dioxin-like activity. In addition, the papers were tested for microbial content and, in general, the microbiological load was quite low. The following microorganisms were counted and identified on both surface and homogenized pulp samples: the total number of aerobic bacteria, the number of aerobic and anaerobic spore formers, the number of Bacillus cereus/thuringiensis, and the number of yeast and moulds. The chemical analyses showed a significantly higher amount and different composition pattern of chemicals extracted with ethanol compared with water. Analyses of the ethanol extracts showed a distinctly smaller number and lower concentrations of chemicals in extracts prepared from sample A compared with extracts of samples B-D. The compounds identified in B-D were similar, but the amounts were lower in B compared with C and D. In accordance with the chemical analyses, the water extracts were less cytotoxic than the ethanol extracts. The extract prepared from virgin fibres was less cytotoxic than the extracts prepared from paper made from recycled fibres, and extracts prepared from C was the most cytotoxic. None of the extracts showed mutagenic activity. No conclusion about the oestrogenic activity could be made, because all extracts were cytotoxic to the test organism (yeast cells). Ethanol extracts of A and B showed a negligible positive response in the Ah-receptor assay at the highest nontoxic concentration, whereas C and D showed a more pronounced effect with C being the most potent. A comparable weak effect of water extracts of samples B-D was observed, too. However, the active compound(s) was not identified by chemical analyses.

Genotoxicity of unmodified and organo-modified montmorillonite

The natural clay mineral montmorillonite (Cloisite) Na+) and an organo-modified montmorillonite (Cloisite 30B) were investigated for genotoxic potential as crude suspensions and as suspensions filtrated through a 0.2-microm pore-size filter to remove particles above the nanometre range. Filtered and unfiltered water suspensions of both clays did not induce mutations in the Salmonella/microsome assay at concentrations up to 141microg/ml of the crude clay, using the tester strains TA98 and TA100. Filtered and unfiltered Cloisite) Na+ suspensions in culture medium did not induce DNA strand-breaks in Caco-2 cells after 24h of exposure, as tested in the alkaline comet assay. However, both the filtered and the unfiltered samples of Cloisite 30B induced DNA strand-breaks in a concentration-dependent manner and the two highest test concentrations produced statistically significantly different results from those seen with control samples (p<0.01 and p<0.001) and (p<0.05 and p<0.01), respectively. The unfiltered samples were tested up to concentrations of 170microg/ml and the filtered samples up to 216microg/ml before filtration. When tested in the same concentration range as used in the comet assay, none of the clays produced ROS in a cell-free test system (the DCFH-DA assay). Inductively coupled plasma mass-spectrometry (ICP-MS) was used to detect clay particles in the filtered samples using aluminium as a tracer element characteristic to clay. The results indicated that clay particles were absent in the filtered samples, which was independently confirmed by dynamic light-scattering measurements. Detection and identification of free quaternary ammonium modifier in the filtered sample was carried out by HPLC-Q-TOF/MS and revealed a total concentration of a mixture of quaternary ammonium analogues of 1.57microg/ml. These findings suggest that the genotoxicity of organo-modified montmorillonite was caused by the organo-modifier. The detected organo-modifier mixture was synthesized and comet-assay results showed that the genotoxic potency of this synthesized organo-modifier was in the same order of magnitude at equimolar concentrations of organo-modifier in filtrated Cloisite) 30B suspensions, and could therefore at least partly explain the genotoxic effect of Cloisite) 30B.

POLYCYCLIC AROMATIC HYDROCARBONS (PAH) IN DANISH SMOKED FISH AND MEAT PRODUCTS

Twenty seven PAH were detected in 45 selected smoked food samples produced in Denmark, including mackerel, herring, trout, small sausages, salami, and bacon. The sum of PAH in smoked meat products ranged from 24 μg/kg for salami to 64 μg/kg in bacon, while those in fish products ranged from 22 μg/kg in smoked mackerel prepared in an electric oven to 1387 μg/kg in herring smoked by direct smoking. The concentration of benzo[a]pyrene for all sample types were below the maximum level of 5 μg/kg for smoked fish and meat set by the European Commission. Results from this survey confirm that the actual level of individual PAH in fish products is dependent on variables such as the type of wood used in the smoking process. Furthermore, the use of the benzo[a]pyrene approach for estimation of the carcinogenicity of PAH in food is confirmed. The Danish intake of benzo[a]pyrene from these smoked products is 2 to 4 ng/person/day.

Observations of the effect of atmospheric processes on the genotoxic potency of airborne particulate matter

Abstract In this study, the relationship between genotoxic potency and the occurrence of polycyclic aromatic hydrocarbons (PAH), including benzo(a)pyrene (BaP), and nitro-PAH in urban and semi-rural air masses has been investigated. The Salmonella/microsome assay has been used as a measure of genotoxic potency. We find that the ratios of BaP/mutagenicity and PAH/mutagenicity are highly variable. The processes responsible for the variation are formation and degradation of mutagens and transport of polluted air masses from heavily industrialized regions. Air masses from Central Europe are shown to be highly enriched in mutagens as well as in PAH and nitro-PAH. However, the mutagenic activity is much more elevated than the PAH levels when these air masses are mixed with local urban air. Part of the variation in the PAH/mutagenicity ratio can be explained by photochemical transformation. Since BaP has been used in the past as an indicator of the carcinogenic risk of airborne particles, it is suggested that the cancer risk of air pollution has to be re-evaluated.

In vitro cytotoxicity of fungi spoiling maize silage

Penicillium roqueforti, Penicillium paneum, Monascus ruber, Alternaria tenuissima, Fusarium graminearum, Fusarium avenaceum, Byssochlamys nivea and Aspergillus fumigatus have previously been identified as major fungal contaminants of Danish maize silage. In the present study their metabolite production and in vitro cytotoxicity have been determined for fungal agar and silage extracts. All 8 fungal species significantly affected Caco-2 cell viability in the resazurin assay, with large variations for each species and growth medium. The 50% inhibition concentrations (IC(50)) of the major P. roqueforti metabolites roquefortine C (48 μg/mL), andrastin A (>50 μg/mL), mycophenolic acid (>100 μg/mL) and 1-hydroxyeremophil-7(11),9(10)-dien-8-one (>280 μg/mL) were high. Fractionating of agar extracts identified PR-toxin as an important cytotoxic P. roqueforti metabolite, also detectable in maize silage. The strongly cytotoxic B. nivea and P. paneum agar extracts contained patulin above the IC(50) of 0.6 μg/mL, however inoculated onto maize silage B. nivea and P. paneum did not produce patulin (>371 μg/kg). Still B. nivea infected maize silage containing mycophenolic acid (∼50 mg/kg), byssochlamic acid and other metabolites, was cytotoxic. In contrast hot-spots of P. roqueforti, P. paneum, M. ruber and A. fumigatus were not more cytotoxic than uninoculated silage.

Effects of an onion by-product on bioactivity and safety markers in healthy rats

Onions are excellent sources of bioactive compounds including fructo-oligosaccharides (FOS) and polyphenols. An onion by-product was characterised in order to be developed as a potentially bioactive food ingredient. Our main aim was to investigate whether the potential health and safety effects of this onion by-product were shared by either of two derived fractions, an extract containing the onion FOS and polyphenols and a residue fraction containing mainly cell wall materials. We report here on the effects of feeding these products on markers of potential toxicity, protective enzymes and gut environment in healthy rats. Rats were fed during 4 weeks with a diet containing the products or a control feed balanced in carbohydrate. The onion by-product and the extract caused anaemia as expected in rodents for Allium products. No other toxicity was observed, including genotoxicity. Glutathione reductase (GR) and glutathione peroxidase (GPx1) activities in erythrocytes increased when rats were fed with the onion extract. Hepatic gene expression of Gr, Gpx1, catalase, 5-aminolevulinate synthase and NAD(P)H:quinone oxidoreductase was not altered in any group of the onion fed rats. By contrast, gamma-glutamate cysteine ligase catalytic subunit gene expression was upregulated but only in rats given the onion residue. The onion by-products as well as the soluble and insoluble fractions had prebiotic effects as evidenced by decreased pH, increased butyrate production and altered gut microbiota enzyme activities. In conclusion, the onion by-products have no in vivo genotoxicity, may support in vivo antioxidative defence and alter the functionality of the rat gut microbiota.

DNA damage detected by the alkaline comet assay in the liver of mice after oral administration of tetrachloroethylene

Induction of DNA damage in the liver and kidney of male CD1 mice was studied by means of the alkaline Comet assay after oral administration of tetrachloroethylene at the doses of 1000 and 2000 mg/kg/day. A statistically significant dose-related increase in tail intensity was established in hepatocytes, indicating that tetrachloroethylene induced DNA damage in the liver. No effect on DNA damage was observed in the kidney. The results are in agreement with carcinogenicity data in mice, in which tetrachloroethylene induced tumours in the liver but not in the kidney, and support that a genotoxic mode of action might be involved in liver carcinogenicity in mice. An alternative interpretation of the results conveyed by the Study director at the test facility, involving that tetrachloroethylene did not induce DNA damage in the liver and kidney of mice, is also presented and discussed.

The Effect of Apple Feeding on Markers of Colon Carcinogenesis

Regular consumption of fruits and vegetables is associated with reduced risks of certain cancers and other diseases in observational studies and animal models of human diseases. The aim of the present study was to investigate whether feeding of rats with whole raw apple has potentially chemopreventive properties by affecting markers of colon cancer. The end-point was preneoplastic changes in the colon known as aberrant crypt foci (ACF). Rats initiated with the colon carcinogen 1,2-dimethylhydrazine dihydrochloride (DMH) were given 0, 5, or 10 g apple/day for 13 wk. The group fed 5 g apple but not 10 g had a significantly lower number of ACF (P = 0.009) compared to the initiated control. DNA damage evaluated by the comet assay was significantly increased in leucocytes of DMH-treated animals (P = 0.021). No antigenotoxic effect of apple feeding was apparent in the colon. Apple feeding tended to lower DNA damage in the liver (P = 0.136 in DMH-initiated and P = 0.284 in noninitiated rats). Liver alanine aminotransferase was significantly increased in rats fed apples (P = 0.008 in DMH-initiated and P = 0.019 in noninitiated rats). In conclusion, feeding whole fresh apple may affect the occurrence of preneoplastic changes in the rat colon, but the effect was not gradual.

Primary aromatic amines (PAAs) in black nylon and other food-contact materials, 2004–2009

Primary aromatic amines (PAAs) were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in migrates from 234 samples of food-contact materials, including black nylon (polyamide) kitchen utensils (n = 136), coloured plastics (28), and clear/printed multilayer film/laminates (41), from retailers, importers, and food producers. A further 29 utensils in use were obtained from colleagues. Very high PAA migration was found from black nylon kitchen utensils to the food simulant 3% acetic acid: the ‘non-detectable’ limit (20 µg aniline equivalents kg−1 food) was exceeded by up to 2100 times. All the other materials were compliant. The majority of the non-compliant utensils came from China. The predominant PAAs were aniline and 4,4′-methylenedianiline (4,4′-MDA). The frequency of violations decreased from the year 2004 (55%) to the autumn of 2005 (13%), possibly due to increased demands for in-house documentation, but they remained almost constant from 2005 to 2009. The validity of the results was shown by recovery studies, participation in proficiency testing, and comparative testing of utensils by two laboratories. Migration modelling was used to compare how various compliance migration test conditions influenced the final test results. Long-term release of PAAs was fitted by diffusion modelling experiments and long-term release was also seen as expected from used utensils. Toxicologists consider these migration levels of the suspected carcinogenic PAAs as a problem of major concern.

In-vivo study of genotoxic and inflammatory effects of the organo-modified Montmorillonite Cloisite ® 30B

Because of the increasing use of clays and organoclays in industrial applications it is of importance to consider the toxicity of these materials. Recently it was reported that the commercially available Montmorillonite clay, Cloisite(®) 30B, which is surface-modified by organic quaternary ammonium compounds, was genotoxic in vitro. In the present study the in-vivo genotoxic and inflammatory potential of Cloisite(®) 30B was investigated as a follow-up of the in-vitro studies. Wistar rats were exposed to Cloisite(®) 30B twice 24h apart by oral gavage, at doses ranging from 250 to 1000 mg/kg body weight [indicate duration of treatment; Ed.]. There was no induction of DNA strand-breaks in colon, liver and kidney cells and there was no increase in inflammatory cytokine markers in blood-plasma samples. In order to verify the possible absorption of Cloisite(®) 30B from the gastrointestinal tract, inductively coupled plasma mass-spectrometry (ICP-MS) analysis was performed on samples of liver, kidney and faeces, with aluminium as a tracer element characteristic to clay. The results showed that aluminium could be detected in faeces, but not in the liver or kidneys. This indicated that there was no systemic exposure to clay particles from Cloisite(®) 30B. Detection and identification of free quaternary ammonium modifier in the highest dose of Cloisite(®) 30B was carried out by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS). This analysis revealed a mixture of three quaternary ammonium analogues. The detected concentration of the organomodifier corresponded to an exposure of rats to about 5mg quaternary ammonium analogues/kg body weight.