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This report gives an overview of the scientific literature on the migration of undesired substances from packages made from recycled paper & board to foodstuffs with a focus on mineral oils. The knowledge is placed into an independent scientific perspective with regard to analysis, technology, legislation and sources by Wageningen Food & Biobased Research (WFBR) and with regard to exposure, toxicology and risk assessment by TNO (organisation for applied scientific research). Dit rapport geeft een overzicht van de huidige wetenschappelijke kennis op het gebied van de migratie van ongewenste verbindingen uit verpakkingen gemaakt van gerecycleerd papier en karton naar levensmiddelen met een focus op minerale oliën. Deze kennis wordt in een onafhankelijk wetenschappelijk perspectief geplaatst ten aanzien van analyse, technologie, wetgeving en bronnen door Wageningen Food & Biobased Research (WFBR) en ten aanzien van blootstelling, toxicologie en risicobeoordeling door de organisatie voor Toegepast Natuurwetenschappelijk Onderzoek (TNO).

Consumer health risk assessment for feed additives is based on the estimated human exposure to the additive that may occur in livestock edible tissues compared to its hazard. We present an approach using alternative methods for consumer health risk assessment. The aim was to use the fewest possible number of animals to estimate its hazard and human exposure without jeopardizing the safety upon use. As an example we selected the feed flavoring substance piperine and applied in silico modeling for residue estimation, results from literature surveys, and Read-Across to assess metabolism in different species. Results were compared to experimental in vitro metabolism data in rat and chicken, and to quantitative analysis of residues' levels from the in vivo situation in livestock. In silico residue modeling showed to be a worst case: the modeled residual levels were considerably higher than the measured residual levels. The in vitro evaluation of livestock versus rodent metabolism revealed no major differences in metabolism between the species. We successfully performed a consumer health risk assessment without performing additional animal experiments. As shown, the use and combination of different alternative methods supports animal welfare consideration and provides future perspective to reducing the number of animals.

Recently, the European Food Safety Authority (EFSA) stated that the Threshold of Toxicological Concern (TTC) thresholds should not be used for substances that are known or predicted to accumulate. Bioaccumulation of substances is usually considered unfavourable but so far a relation with toxicity at low dose exposure is insufficiently investigated to draw conclusions on the relevance of bioaccumulation at low dose exposure. In this manuscript it is investigated which physical chemical properties are related to bioaccumulation in order to predict accumulating properties of a substance, and is evaluated if the toxicity of known bioaccumulating substances is higher than for non-accumulating substances. Based on the evaluation it is concluded that the current TTC thresholds are derived with a dataset in which bioaccumulating substances are present, whereas the toxicity of the bioaccumulating substances is already taken into account in the TTC thresholds. The authors demonstrated that there is no need to exclude potential bioaccumulating substances from the TTC concept.

To derive an acute TTC threshold, the correlation between Allowable Daily Intakes (ADIs, chronic values) and Acute Reference Doses (ARfDs) of pesticides evaluated in the EU was investigated and their distributions were compared. The correlation between ARfDs and ADIs was significant (p = 0.01), but weak (r(2) = 0.051). Consequently, using this approach to derive acute TTC values does not seem valid. Therefore, the distributions of ARfDs and ADIs were compared directly, in order to extrapolate from chronic to acute TTC values. This comparison made for the combined Cramer structural class II and III pesticides showed a ratio ARfD/ADI of approximately 3 at the fifth percentile of the distributions. Based on these results, it is justified to propose a TTC for acute effects for Cramer III substances by multiplying the Cramer class III TTC threshold of 90 μg/person/day with a factor 3. This leads to an acute TTC threshold based on the Munro dataset for Cramer class III substances of 270 μg/person/day.

There is growing concern regarding the presence of nonintentionally added substances (NIAS) in food contact materials. These substances cannot be excluded from the food contact materials and end-up in our food. Identification thereof is often difficult or in some cases even not possible, which makes it difficult to evaluate their safety upon exposure. This review provides an overview of the current European Regulatory requirements and the latest innovations in the safety assessment of NIAS. Main conclusion is that recently an innovative, pragmatic and scientifically justified approach is published which can be used for prioritization of NIAS, based on their exposure and hazard.

A main challenge in food safety research is to demonstrate that processing of foodstuffs does not lead to the formation of substances for which the safety upon consumption might be questioned. This is especially so since food is a complex matrix in which the analytical detection of substances, and consequent risk assessment thereof, is difficult to determine. Here, a pragmatic novel safety assessment strategy is applied to the production of non-selective extracts (NSEs), used for different purposes in food such as for colouring purposes, which are complex food mixtures prepared from reference juices. The Complex Mixture Safety Assessment Strategy (CoMSAS) is an exposure driven approach enabling to efficiently assess the safety of the NSE by focussing on newly formed substances or substances that may increase in exposure during the processing of the NSE. CoMSAS enables to distinguish toxicologically relevant from toxicologically less relevant substances, when related to their respective levels of exposure. This will reduce the amount of work needed for identification, characterisation and safety assessment of unknown substances detected at low concentration, without the need for toxicity testing using animal studies. In this paper, the CoMSAS approach has been applied for elderberry and pumpkin NSEs used for food colouring purposes.

One of the main challenges in food contact materials research is to prove that the presence of non-intentionally added substances (NIAS) is not a safety issue. Migration extracts may contain many unknown substances present at low concentrations. It is difficult and time-consuming to identify all these potential NIAS and concurrently to assess their health risk upon exposure, whereas the health relevance at low exposure levels might not even be an issue. This paper describes a scientifically based, but pragmatic safety assessment approach for unknown substances present at low exposure levels in food contact matrices. This complex mixture safety assessment strategy (CoMSAS) enables one to distinguish toxicologically relevant from toxicologically less relevant substances, when related to their respective levels of exposure, and allows one to focus on the substances of potential health concern. In particular, substances for which exposure will be below certain thresholds may be considered not of health relevance in case specific classes of substances are excluded. This can reduce the amount of work needed for identification, characterisation and evaluation of unknown substances at low concentration. The CoMSAS approach is presented in this paper using a safety assessment of unknown NIAS that may migrate from three carton samples. © 2014 Taylor & Francis. Chemicals/CAS: aflatoxin, 1402-68-2; aflatoxin B1, 1162-65-8; aflatoxin B2, 7220-81-7; aflatoxin G1, 1165-39-5; aflatoxin G2, 7241-98-7; aflatoxin M1, 6795-23-9; ergocornine, 564-36-3; ergocristine, 511-08-0; ergocryptine, 511-09-1; ergometrine, 60-79-7; ergotamine, 113-15-5, 52949-35-6; ether, 60-29-7; fumonisin B1, 116355-83-0; HT 2 toxin, 26934-87-2; nivalenol, 23282-20-4; ochratoxin, 303-47-9, 37203-43-3; T 2 toxin, 21259-20-1; vomitoxin, 51481-10-8; zearalenone, 17924-92-4

The threshold of toxicological concern (TTC) concept is a risk assessment tool for substances present at low oral exposure and lacking hazard data. In the past, several thresholds were elaborated by Munro et al. (1996) and Kroes et al. (2004). For these TTC thresholds, the Cramer class III threshold is based on a broad spectrum of substances, including organophosphates. For organophosphates a separate threshold was elaborated by Kroes et al. (2004), however without adjustment of the Cramer class III threshold. Moreover, reference was made by Munro et al. (2008) that for organohalogens a separate threshold also may apply whereas the EFSA (2012) considers that carbamate substances with anti-choline esterase activity can be included in the threshold for organophosphates. In this paper, a reevaluation of the Munro dataset (original TTC database) was performed, focused on the thresholds for organophosphates including carbamates, organohalogens and remaining Cramer class III substances. This way thresholds for each of these groups are elaborated. As a results of the current reevaluation of the Munro dataset, thresholds for life-time exposure are elaborated for the group of organophosphates including carbamates, the group of organohalogens and the remaining Cramer class III substances, being 0.30, 1.5 and 4.0. μg/kg bodyweight/day, respectively. © 2014 Elsevier Inc.

Precision-cut liver slices are frequently used to study hepatic toxicity and metabolism of xenobiotics in vitro. Successful cryopreservation techniques will enhance an efficient and economic use of scarcely available (human) liver tissue. For primary hepatocytes, slow freezing has been accepted as the best approach towards successful cryopreservation. For slices, however, no agreement exists on the optimal way of cryopreservation and both slow and fast freezing techniques have been reported. The aim of the present study was to determine the applicability of a computer-controlled slow freezing technique for the cryopreservation of (rat) liver slices. Thus far, this technique has not been described in detail. Our studies confirmed that slow freezing was most successful in the cryopreservation of primary rat hepatocytes. Based on this observation, the slow freezing technique was applied to the cryopreservation of rat liver slices. Directly after thawing, slice viability was between 60 and 100% of fresh values, depending on the parameter determined. However, after additional culturing, slice viability was reduced. This decrease in slice viability was more pronounced in comparison to primary hepatocytes. In conclusion, the slow freezing technique was confirmed to be a successful approach for the cryopreservation of primary rat hepatocytes, and was found to be of limited use for the cryopreservation of rat liver slices. Copyright (C) 2000 Elsevier Science Ltd. Chemicals/CAS: Adenosine Triphosphate, 56-65-5; Dinitrochlorobenzene, 97-00-7; Formazans; Glutathione Transferase, EC 2.5.1.18; Glutathione, 70-18-8; Lactate Dehydrogenase, EC 1.1.1.27; MTT formazan, 23305-68-2; Proteins; Testosterone, 57-85-2; Tetrazolium Salts; Urea, 57-13-6

The susceptibility to nephrotoxins of freshly isolated porcine proximal tubular cells (PPTC) and cultured PPTC in suspension were compared, with a view to using PPTC as in vitro models for studying nephrotoxicity. In a previous paper, we described how, in freshly isolated PPTC, well-known nephrotoxins such as mercury (II) chloride, cisplatin, p-aminophenol and halogenated hydrocarbons caused a dose-dependent decrease in the viability and mitochondrial membrane potential of the PPTC. In this paper, we show that suspensions of cultured PPTC, harvested by trypsinisation, are less susceptible to nephrotoxins, possibly due to the synthesis of extracellular matrix components. PPTC in primary culture are suitable for relatively long-term nephrotoxicity studies. This was demonstrated by incubation with mercury (II) chloride for 24 hours, resulting in a dose-dependent loss of viability. Freshly isolated PPTC can be cryopreserved by computer-controlled freezing. The cryopreserved PPTC displayed an increased susceptibility to mercury (II) chloride and a decreased susceptibility to cisplatin and 1,1-dichloro-2,2-difluoroethylene-cysteine, the toxicity of the latter indicating that the renal enzyme β-lyase remains active during cryopreservation.

Publisher Summary This chapter focuses on the current nontargeted compliance-testing strategies of food contact material (FCM) migrants and presents an overview of the legislation involved in FCMs. The term “nontarget” is mostly a synonym for unknown. In nontarget compliance tests, all possible components that can migrate to the food are included. Compliance testing is currently being carried out for plastic FCMs that contain starting substances that are on the positive list. This includes verification of the composition of the packaging material to judge whether all starting substances—that is, monomers and additives—are on the positive list. A novel concept to investigate the presence of potential toxic compounds and to rate their health relevance is based on the threshold of toxicological concern (TTC) principle, which is discussed in the chapter.

Hepatotoxicity is a term used for many and distinct adverse effects that may occur in the liver after exposure to toxic substances. This article reviews the current methods and markers to assess hepatotoxicity, and discusses the introduction of new ‘functional genomics’ technologies in toxicology (toxicogenomics). Applications of toxicogenomics may facilitate the development of potential new markers of (hepato) toxicity.

The human risk assessment of feed contaminants has often been hampered by a lack of knowledge concerning their behaviour when consumed by livestock. To gain a better understanding of the transfer of contaminants from animal feed to animal products, a meta-analysis of public literature was made. Data concerning feed contaminant concentrations, feeding periods, residue levels in animal products, and other parameters were gathered and recorded. For each case a 'transfer factor', which was defined as the ratio of the concentration of a chemical in an animal product to the concentration of the chemical in animal feed, was calculated. Scientifically founded transfer factors were calculated and analysed for groups of chemicals based on their contaminant classes or physicochemical properties. These database-derived transfer factors enable a more accurate risk assessment in the case of a feed contamination, and enable rapid risk management decision-making and/or intervention.

To test for potential estrogenic activity of plant stanols and plant stanol esters, two short-term tests were performed. These were the E-screen test, which measures a substance's ability to induce proliferation of estrogen-responsive human breast adenocarcinoma (MCF-7) cells in culture, and an in vivo test, which measures uterotrophic activity in immature female rats fed the test substance. Four samples of vegetable oil-derived stanols (containing 88-99% stanols) were tested in the E-screen test, and one sample of wood-derived and one of vegetable oil-derived stanol fatty acid esters were tested in the in vivo test. In the E-screen test, the positive control substance, 17beta-estradiol, at 100 pM, produced a statistically significant, 11.6-fold increase in cell proliferation, as measured by sulforhodamine B staining. None of the stanol preparations produced any increase in cell proliferation when tested at 1, 10, and 100 microM. The highest dose of each stanol sample was associated with microscopic evidence of cytotoxicity and crystalline precipitation in the culture dishes. In the in vivo test, the positive control compound, diethylstilbestrol, produced a significant, dose-related increase in absolute and relative uterus weight in young female rats (17 days old at the start of treatment) fed the compound at 5, 10, and 20 ppb in the diet for 4 days. Neither of the two stanol ester preparations caused any significant change in absolute or relative uterus weight when fed at a concentration of 8.3% in the diet for 4 days. Thus, under the conditions of testing used, neither the free stanols nor the stanol fatty acid ester preparations showed evidence of estrogenic or uterotrophic activity.

Precision-cut liver slices are presently used for various research objects, e.g. to study metabolism, transport, and toxicity of xenobiotics. Various incubation systems are presently employed, but a systematic comparison between these incubation systems with respect to preservation of slice function has not been performed yet. Therefore, we started a comparative study to evaluate five of these systems: the shaken flask (an Erlenmeyer in a shaking water bath), the stirred-well (24-well culture plate equipped with grids and magnetic stirrers), rocker platform (6-well culture plate with Netwell insert rocked on a platform), the roller system (dynamic organ culture rolled on an insert in a glass vial), and the 6-well shaker (6-well culture plate in a shaking water bath). The liver slices were incubated in these incubation systems for 0.5, 1.5, and 24.5 h and subsequently subjected to viability and metabolic function tests. The viability of the incubated liver slices was evaluated by: potassium content, MTT assay, energy charge, histomorphology, and LDH leakage. Their metabolic functions were studied by determination of the metabolism of lidocaine, testosterone, and antipyrine. Up to 1.5 h of incubation all five incubation systems gave similar results with respect to viability and metabolic function of the liver slices. However, after 24 h, the shaken flask, the rocker platform, and the 6-well shaker incubation systems appeared to be superior to the stirred well and the roller incubation systems. Chemicals/CAS: L-Lactate Dehydrogenase, EC 1.1.1.27; Potassium, 7440-09-7; Xenobiotics

Precision-cut rat liver slices were used to develop a new dynamic incubation system in which histomorphology and measurement of the release of lactate dehydrogenase (LDH) and the conversion of MTT were applied to evaluate cytotoxicity. Liver slices, precision-cut using a Krumdieck tissue slicer, were cultured in a new system using 200-mum polyester mesh Netwell inserts in six-well cell-culture clusters on a rocker platform at 37 degrees C and 40% O(2). The major advantage of this new culture system is the easy way in which slices can be manipulated and the culture medium be sampled or changed. Rat liver slices were exposed for 4 hr to retinoic acid (RA), menadione or aflatoxin B(1) (AFB(1)). Directly after treatment and after an additional 20-hr recovery period, histomorphological observations of slices were made, and LDH release and MTT conversion were measured. Slices exposed to RA showed dose-related cytotoxicity in the MTT assay only. The cytotoxic response to AFB(1) was more pronounced in the assay of LDH release than in the MTT assay. Histomorphology, LDH release and the MTT assay revealed cytotoxic effects induced by menadione. We conclude that culturing liver slices using Netwell inserts is a good alternative to other culture systems for testing non-volatile compounds.

The present paper reports about an immunocytochemical inventory of the cell types involved in the metabolic activation of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to a DNA methylating metabolite. The formation and distribution of the methylated DNA bases O6-methylguanine (O6-meGua) and 7-methylguanine (7-MeGua) were studied in respiratory tissues, oesophagus, liver, kidneys, pancreas, small intestine, colon and prostate of rat, mouse and hamster 6 h after treatment with a single dose of 30 mg NNK/kg. The tissue- and cell-specific distribution of O6-meGua- and 7-meGua-specific nuclear staining showed the same patterns and were remarkably similar in rat, mouse and hamster in spite of the diverging spectra of NNK-induced tumours in these species. In nasal tissue, a target for NNK-induced tumourigenesis in rat and hamster, but not in mouse, adduct-specific nuclear staining was observed in all three species in sustentacular cells, Bowman glands, respiratory epithelial cells and serous glands. Both methylated DNA bases were also observed in basal cells of the olfactory epithelium of rat and (occasionally) hamster, but not in those of the mouse. In the trachea, a target for NNK-induced tumourigenesis in hamster only, substantial adduct-specific nuclear staining was found in basal epithelial and glandular cells of the hamster; in the same cells of rat and mouse only a weak nuclear staining was found. In the lung, a common target for NNK-induced tumourigenesis, the formation of O6-meGua and 7-meGua was restricted predominantly to bronchial and proximal bronchiolar epithelium. Nuclear staining in the rat was occasionally found in alveolar cells and was also observed in hepatocytes. In the three species investigated, O6-meGua- and 7-MeGua-specific nuclear staining was found in target and non-target tissues. Apparently, and in analogy with results obtained in other studies, the species-specific organotropy for tumour formation of NNK is not exclusively determined by DNA methylation. Expanding methylation data with literature data on factors considered to be involved in tumour formation, namely proliferation, toxicity and DNA repair among others, still did not lead to a satisfactory explanation for the species-specific organotropy observed. Additional factors (yet to be identified), need to be taken into account in order to explain (and predict) tumourigenic effects induced by monofunctional methylating agents.

The borehole deformation type of rock stress measuring instrument, which is the subject of this paper measures radial displacements in the direction of one or more diameters of the borehole in which it is installed for the purpose of determining the stresses in the rock in which the borehole is drilled. The stresses are calculated from the changes in borehole diameter using formular derived by means of the theory of elasticity. Not included in the considerations of this paper is the so-called borehole inclusion type of rock stress measuring instrument. In principle, it also measures radial displacements in boreholes, but it is a ‘rigid’ or ‘stiff’ type while the one dealt with in this article is a ‘soft’ type of displacement measuring device. The former offers considerable resistance to the radial displacements of the borehole, the latter, a negligible resistance. A full discussion of the influence of the ‘rigidity’ of rock stress measuring instruments upon the scope of their usefulness was recently published in this journal by Hult et al.[1] Up to the present, many instruments of the soft borehole deformation type have been developed. In this paper an analysis is given of their operating principles in the hope that a clearer picture of their potentialities on the one hand and their limitations on the other may be obtained.

Male Wistar rats were exposed for 4 weeks, 5 days a week, to 0 (controls), 5 or 10 ppm formaldehyde continuously (8 hours a day), or to 10 or 20 ppm formaldehyde interruptedly (eight 30 min exposure periods separated by 30 min non-exposure periods). Histopathology and cell proliferation studies indicated that under the conditions of exposure used, concentration rather than the total dose of formaldehyde determined the severity of the cytotoxic effects on the nasal epithelium.

Male Wistar rats were exposed for 13 weeks, 5 days a week to 0 (controls), 1 or 2 ppm formaldehyde continuously (8 h a day), or to 2 or 4 ppm formaldehyde interruptedly (eight 30-min exposure periods separated by 30-min non-exposure periods a day). Histopathological changes were only found in the nose of animals (interruptedly) exposed to 4 ppm formaldehyde and comprised an increased degree and incidence of disarrangement and squamous metaplasia accompanied by basal cell hyperplasia and occasionally by keratinization of the respiratory epithelium. Two ppm formaldehyde was the non-toxic effect level. Cell proliferation studies demonstrated a slightly higher cell turnover of the nasal respiratory epithelium exposed (interruptedly) to 4 ppm formaldehyde than in controls. It was concluded that under the conditions of repeated exposure to marginally cytotoxic concentrations during a period of 13 weeks the exposure concentration rather than the total 'dose' (= concentration x exposure time) determined the severity of the cytotoxic effects of formaldehyde on the nasal epithelium.

Cyanoketone administered via the food (0.1, 0.2 and 2 mg/g) for 8 weeks from the first feeding (day 46 after fertilization) or via the aquarium water (3 and 30 mg/100 l) for 4 weeks from day 41 does not influence the activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in the interstitial cells of the gonads or interrenal cells of juvenile trout in vivo. However, the number of 3 beta-HSD-positive interrenal cells was strongly increased by administration of the highest dose of cyanoketone via both routes. These high doses furthermore affect the sex ratio in favor of males. It is concluded that interrenal tissue is responsible for the masculinizing effect of cyanoketone via increased production of androgens and/or corticosteroids. Cyanoketone at concentrations of 0.01 to 100 micrograms/ml causes a dose-response inhibition of 3 beta-HSD activity in the interrenal cells, when the substance is administered to an incubation medium for demonstration of this enzyme in tissue sections. The controversial in-vivo and in-vitro effects of cyanoketone on 3 beta-HSD activity are discussed.