Guidance for the identification of polymers in multilayer films used in food contact materials: User guide of selected practices to determine the nature of layers

This guidance describes how to characterize the composition of a multilayer plastic film for food packaging, with respect to the consecutive order of the layers and their identity. It provides necessary background information on the general composition of multilayer plastic packaging and it illustrates in detail the separation of layers for some examples. It also provides in annexes additional information related to the use of a microtome and of optical microscopy using one common instrument for illustrative purposes.JRC.I.1-Chemical Assessment and Testin

Report of an inter-laboratory comparison from the European Union Reference Laboratory for Food Contact Materials: ILC02 2015 – Specific migration from a multilayer in food simulant A

This report presents the results of an inter-laboratory comparison on the specific migration of caprolactam (CAP) and 2,4-di-tert butyl phenol (2,4-DTBP) from a plastic food contact material in food simulant A (ethanol 10 %, v/v) organised by the EURL-FCM, Ispra (Italy). Homogeneity and stability studies were conducted. Participants had to carry out a migration test by immersion with food simulant A for 10 days at 60oC, to quantify the migration of caprolactam (CAP) in food simulant A, and to provide details of the analytical and sample extraction procedure. In addition a by-product 2,4-di-tert butyl phenol (2,4-DTBP) was also present and had to be quantified. The assigned values of the migration of the migrant were calculated as the robust mean of the results reported by the participants by applying the Q/Hampel method robust statistics. The participation to the ILC was satisfactory with 28 out of 29 participating laboratories submitting results. The z-score values of the laboratories for each substance were calculated based on the assigned value. In the case of CAP and also for the more challenging non intentionally added substances 2,4-DTBP more than 82% of the results were fully satisfactory (|z-score|<2). This percentage rose to close to 90% for z-scores <3 in the compliance evaluation of the regulated substance CAP.JRC.F.7-Knowledge for Health and Consumer Safet

Inhibition of the Soluble Epoxide Hydrolase Promotes Albuminuria in Mice with Progressive Renal Disease

Epoxyeicotrienoic acids (EETs) are cytochrome P450-dependent anti-hypertensive and anti-inflammatory derivatives of arachidonic acid, which are highly abundant in the kidney and considered reno-protective. EETs are degraded by the enzyme soluble epoxide hydrolase (sEH) and sEH inhibitors are considered treatment for chronic renal failure (CRF). We determined whether sEH inhibition attenuates the progression of CRF in the 5/6-nephrectomy model (5/6-Nx) in mice. 5/6-Nx mice were treated with a placebo, an ACE-inhibitor (Ramipril, 40 mg/kg), the sEH-inhibitor cAUCB or the CYP-inhibitor fenbendazole for 8 weeks. 5/6-Nx induced hypertension, albuminuria, glomerulosclerosis and tubulo-interstitial damage and these effects were attenuated by Ramipril. In contrast, cAUCB failed to lower the blood pressure and albuminuria was more severe as compared to placebo. Plasma EET-levels were doubled in 5/6 Nx-mice as compared to sham mice receiving placebo. Renal sEH expression was attenuated in 5/6-Nx mice but cAUCB in these animals still further increased the EET-level. cAUCB also increased 5-HETE and 15-HETE, which derive from peroxidation or lipoxygenases. Similar to cAUCB, CYP450 inhibition increased HETEs and promoted albuminuria. Thus, sEH-inhibition failed to elicit protective effects in the 5/6-Nx model and showed a tendency to aggravate the disease. These effects might be consequence of a shift of arachidonic acid metabolism into the lipoxygenase pathway

Ansätze zur pharmakologischen Therapie abdominaler Aortenaneurismen am Beispiel von Diltiazem und LP105

Abdominale Aortenaneurysmen sind in Industrienationen eine häufige Erkrankung der Personengruppe über 65 Jahre. Diese Dilatationen der abdominalen Aorta zeichnen sich durch eine lokale Inflammation aus, die mit der Infiltration von Immunzellen, dem Verlust von glatten vaskulären Muskelzellen und der Degeneration der extrazellulären Matrix einhergeht. Ursprünglich als Symptom einer Atherosklerose angesehen, sind die Ursachen dieser progressiv verlaufenden Erkrankung nach wie vor nicht vollständig verstanden; obwohl steigendes Alter, männliches Geschlecht, genetische Prädisposition, Rauchen und ein zuvor erlittener Myocardinfarkt als Risikofaktoren identifiziert werden konnten. Der lange Zeit asymptomatische Krankheitsverlauf, die Gefahr einer Ruptur mit häufig letalen Folgen und der Mangel einer effizienten pharmakologischen Therapie machen eine weitere Untersuchung dieser Erkrankung unabdingbar. Diltiazem ist ein Inhibitor spannungssensitiver L Typ-Calciumkanäle, der seit über 25 Jahren zur Behandlung von arterieller Hypertonie, verschiedener Arrhythmien und Angina pectoris verwendet wird. Im Rahmen der vorliegenden Arbeit sollte untersucht werden, ob Diltiazem auch einen antianeurysmatischen Effekt besitzt. Eine vierwöchige subcutane Infusion des blutdrucksteigernden Hormons Angiotensin II führte nach vier Wochen zur Bildung abdominaler Aortenaneurysmen, sowie zu atherosklerotischen Gefäßveränderungen der thorakalen Aorta Apolipoprotein E (ApoE)-defizienter Mäuse. Eine parallele Therapie mit Diltiazem über das Trinkwasser konnte diese Entwicklung unabhängig vom arteriellen Blutdruck und damit unabhängig von der antihypertensiven Wirkung verhindern. Im Aortenbogen Diltiazem-behandelter Tiere konnte im Rahmen dieses in vivo-Modells nach sechs Tagen eine deutlich geringere lokale Expression proinflammatorischer Cytokine, wie Tumornekrosefaktor-a, Interleukin-1ß (IL1B) und Interleukin-6 (IL6), Chemokine, wie CCL2, und degenerativer Proteasen, wie der Matrix-Metalloprotease 9 (MMP9), festgestellt werden. Dies war die Folge einer reduzierten Anzahl von Macrophagen in der Gefäßwand. Zirkulierende proinflammatorische Cytokine, wie CCL12, konnten im Serum teilweise ebenfalls vermindert nachgewiesen werden. Obwohl die antihypertensive Wirkung von Diltiazem in glatten vaskulären Muskelzellen vermittelt wird, war es nicht möglich, die Angiotensin II-induzierte Produktion von promigratorischem CCL2 und proinflammatorischem IL6 in isolierten Aortenringen ApoE-defizienter Mäuse oder in glatten vaskulären Muskelzellen der Ratte zu reduzieren. Diltiazem war zudem nicht in der Lage, die CCL2-induzierte Migration proinflammatorischer Ly6C+-Monocyten in vivo zu unterbinden. In isolierten peritonealen Macrophagen ApoE-defizienter Mäuse dagegen, konnte die IL6 induzierte Expression von IL1B- und CCL12-mRNA durch eine Inkubation mit Diltiazem verhindert werden. In der RAW264.7-Zelllinie, die morphologische und funktionelle Merkmale von Monocyten und Macrophagen aufweist, konnte die Dilitiazem-sensitive IL6-induzierte Expression von IL1B-mRNA in vitro ebenfalls nachgewiesen werden. Eine Stimulation mit IL6 war in diesen Zellen jedoch nicht ausreichend, um die Sekretion von IL1B-Protein auszulösen. Thorakales Aortengewebe wies im Vergleich mit RAW264.7-Zellen eine veränderte Ausstattung spannungssensitiver Calciumkanäle auf. In letzteren fanden sich keine muskelzellspezifischen L-Typ-Calciumkanäle (CACNA1C), aber eine relevante Expression neuronaler P/Q-Typ-Calciumkanäle (CACNA1A). Mittels fluorimetrischer Bestimmung mit Fura-2AM konnte jedoch festgestellt werden, dass die intrazelluläre Calciumkonzentration Diltiazem-behandelter RAW264.7-Zellen unverändert war und der antiinflammatorische Effekt somit calciumunabhängig vermittelt wurde. Diltiazem war nicht in der Lage, eine Lipopolysaccharid (LPS)-bedingte Inflammation in RAW264.7-Zellen zu unterbinden. Weder die LPS-induzierte Sekretion von IL1B Protein, noch die nucleäre Translokation des Transkriptionsfaktors NF-?B oder die Aktivierung des NF-?B-Promotors konnten durch eine Inkubation der Zellen mit Diltiazem verhindert werden. Diltiazem reduzierte jedoch, die IL6-induzierte Aktivierung des AP 1-Promotors unabhängig von der MAPK1-Phosphorylierung oder der Phosphorylierung und nucleären Translokation des Transkriptionsfaktors STAT3 zu unterbinden. Eine Unterdrückung von c-Jun N-terminale Kinase JNK- oder p38 Proteinkinase-vermittelten Signalwegen ist damit wahrscheinlich. Das Pirinixinsäurederivat LP105 ist ein neuer Inhibitor der Arachidonat-5-Lipoxygenase (LOX5), der im Rahmen dieser Arbeit erstmals in vivo auf seine antianeurysmatischen Eigenschaften hin untersucht wurde. LOX5 katalysiert die Reaktion von Arachidonsäure zu Leukotrien A4 und kontrolliert damit einen wichtigen Schritt in der Synthese proinflammatorischer Leukotriene. LP105 war im Tiermodell nicht in der Lage die Angiotensin II-induzierte Bildung abdominaler Aortenaneurysmen in ApoE-defizienten Mäusen komplett zu unterbinden, führte aber über die Reduktion der vaskulären Inflammation zu einer deutlich verringerten Krankheitslast. LP105 selbst beeinflusste die mRNA-Expression verschiedener Enzyme des Arachidonsäuremetabolismus nicht, verstärkte jedoch durch die Blockade von LOX5 die Metabolisierung von Arachidonsäure über Arachidonat-15-Lipoxygenase und Cytochrom P450-Enzyme.Diltiazem (DIL), a benzothiazepine-type inhibitor of voltage gated L-type calcium channels, is known for its anti-hypertensive properties and used in clinics to treat angina, tachycardia and hypertension for more than 25 years. Based on its beneficial effects in the cardiovascular system, this thesis analyzes whether DIL is also effective in preventing abdominal aortic aneurysm (AAA) formation. AAA formation is a progressive disease, involving infiltration of inflammatory cells, loss of vascular smooth muscle cells as well as degradation of the extracellular matrix, leading to vessel rupture, severe hemorrhages and death of mainly male and elder patients. Angiotensin II (ATII) infusion by osmotic pumps for four weeks induced AAA formation in Apolipoprotein E-deficient (ApoE-/-) mice that was blocked by co-treatment with DIL via the drinking water. Additional treatment with phenylephrine to counteract the anti-hypertensive properties of DIL had no influence on its beneficial effect, demonstrating that this mechanism is blood pressure independent. DIL prevented the ATII-induced mRNA expression of pro-inflammatory cytokines in the aortic arch after six days of ATII infusion by reducing the amount of macrophages within the tissue. DIL did not reduce the ATII-induced IL6 and CCL2 mRNA expression in isolated aortic rings or in rat aortic smooth muscle cells. Although DIL was not able to prevent the migration of pro inflammatory Ly6C+ monocytes towards CCL2 in an in vivo monocyte recruitment assay, it diminished the IL6 induced IL1B and CCL12 mRNA expression in peritoneal macrophages isolated from ApoE-/- mice as well as in RAW264.7 macrophages. Despite of the different expression of L-type calcium channels in the aorta and in macrophages, DIL did not alter the intracellular calcium concentration assessed by fluorimetric measurements with Fura-2AM. The anti-inflammatory effect of DIL was mediated by a blockade of IL6-induced AP-1 promoter activity as observed by luciferase reporter gene assay. Howewer, IL6-induced phosphorylation and nuclear translocation of the transcription factor STAT3 and the mitogen activated protein kinase MAPK1 were not affected. The pirinixic acid derivative LP105 is a potent inhibitor of arachidonic-5-lipoxygenase (LOX5) and was tested in the model of ATII-induced AAA formation in ApoE-/- mice for the first time. LP105 attenuated the vascular inflammation and remodeling but did not prevent AAA development completely. The blockade of LOX5 did not alter the mRNA expression of the arachidonic acid metabolism but shifted it towards arachidonic-15-lipoxygenase and cytochrome P450 enzymes

Report of an inter-laboratory comparison from the European Union Reference Laboratory for Food Contact Materials: ILC 02 2014 – Identifying the composition of multilayer plastic packaging films

This report presents the results of an inter-laboratory comparison on the identification of the composition of multilayer plastic packaging films organised by the EURL-FCM, Ispra (Italy). The exercise represents a follow-up to ILC002 2013 on the identification of plastic polymers. Participants had to determine the layer composition of three different multilayer plastic packaging films which were obtained from an Italian packaging producer. Although only few laboratories were able to fully identify the comprehensive set of compositions for complex multilayers ( i.e. inclusive of adhesive layers), the majority was able to identify most of the plastic layers and to characterise the respective polymer types correctly. All in all the laboratory performance improved compared to ILC002 2013 and was judged satisfactory considering the complexity of such materials.JRC.I.1-Chemical Assessment and Testin

Critical aspects in the determination of the surface in contact with foods for migration testing of kitchen utensils: Results from two investigations by interlaboratory comparisons organised by the European Reference Laboratory for food contact materials

This report presents the results of investigations by ILCs and follow-up questionnaires which focused on the determination of the food contact surface area of kitchen utensils. The study also included a voluntary exercise on the determination of the envelope volume which constitutes a different technique to estimate contact with foods. The objective of the study was to identify sources of error that appeared in a first ILC (ILC003 2013) previously reported (EUR 26477, 2013).A questionnaire was designed and all laboratories which had obtained a zU-score >2 or <-2 for any of the reported results in ILC003 2013 were kindly invited to reply. With the information gained from the questionnaire, some difficulties in the surface area measurement and the main issues in the determination of Hf and the envelope volume could be identified. Reasons why certain results reported during ILC003 2013 had been outside the tolerance limits could also be identified.JRC.I.1-Chemical Assessment and Testin

Report of an inter-laboratory comparison from the European Reference Laboratory for Food Contact Materials: ILC003 2013 – Food Contact Surface Area of Kitchen Utensils

This report presents the results of the ILC which focused on the determination of the food contact surface area of kitchen utensils. The general aim of the exercise was to assess the capability of official control laboratories to measure the food contact surface area of kitchen utensils and to compare the most commonly used approaches for the determination of the surface area in terms of reproducibility and trueness. the ILC01 2012 highlighted several issues in the determination of the surface area of kitchen utensils. The results for the food contact surface area of a melamine spoon sample ranged from 0.73-1.99 dm2. Iin order to figure out whether the approaches that were used for the determination of the surface area were unsuitable or the performance of the laboratories was unacceptable. The test materials were five different types of plastic kitchen utensils obtained from a global supplier. Homogeneity studies on width, depth, length and thickness of the samples were carried out by the EURL-FCM. They indicated sufficient sample homogeneity. Samples were dispatched to 67 participants (30 NRLs + 37 national official control laboratories from Belgium, Germany, Italy, Spain and the United Kingdom), 63 of them submitted results for the surface area and 53 for the envelope volume. Results showed a satisfactory laboratory performance.Difficulties were observed for the determination of the sample height that will be foreseeably in food contact (Hf). They would not affect migration results if the migration is constant over the entire sample surface, i.e. unless the tested articles are multi-material products or have a printing on the handle. For the determination of the surface area, the trueness and precision of the methods depended on the sample shape. With respect to the final migration result, the obtained reproducibility standard deviations for all four approaches were acceptable considering that the migration measurement itself can be affected by uncertainties of similar levels as those of the determination of the surface area.JRC.I.1-Chemical Assessment and Testin