EN 15845:2010

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.VWUDNWRYPapier und Pappe - Bestimmung der Zytotoxizität in wässrigen ExtraktenPapier et carton - Détermination de la cytotoxicité des extraits aqueuxPaper and board - Determination of the cytotoxicity of aqueous extracts85.060Papir, karton in lepenkaPaper and board67.250Materiali in predmeti v stiku z živiliMaterials and articles in contact with foodstuffsICS:Ta slovenski standard je istoveten z:EN 15845:2010SIST EN 15845:2010en,fr,de01-marec-2010SIST EN 15845:2010SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 15845
January 2010 ICS 13.060.20; 67.250 English Version
Paper and board - Determination of the cytotoxicity of aqueous extracts
Papier et carton - Détermination de la cytotoxicité des extraits aqueux
Papier und Pappe - Bestimmung der Zytotoxizität von wässrigen Extrakten This European Standard was approved by CEN on 11 December 2009.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN Management Centre or to any CEN member.
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Pyrodistilled water . 17Bibliography . 18 SIST EN 15845:2010

This European Standard specifies a test method for the laboratory assessment of the potential cytotoxic effect of paper and board materials. This test method is intended to assess wet contact with food simulant. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN 645, Paper and board intended to come into contact with foodstuffs — Preparation of a cold water extract EN 647, Paper and board intended to come into contact with foodstuffs — Preparation of a hot water extract 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 reference water purified water or pyrodistilled water used as the reference in the cytotoxicity assay
3.2 purified water produced starting with tap water, which then undergoes the following treatment sequence: pre-filtration, reverse osmosis, filtering through activated carbon powder (adsorption) then through cartridges of mixed-bed ion exchange microresins (demineralisation), ultrafiltration (molecular weight cut-off at 10 kDa), and UV photo-oxidation
NOTE Alternatively, any other purification regime, which produces HPLC-quality water (resistance > 18,0 MΩ/cm, Total organic carbon < 3 ppb, no micro-organisms) or waters of grade 1 or 2 (see EN ISO 3696), can be used. 3.3
pyrodistilled water
water prepared as described in Annex A, that is used to maintain the cell line, and which can also be used for cleaning laboratory glassware 3.4 water extract reference water that has been exposed to contact with paper or board
NOTE See EN 645 or EN 647 with necessary modifications. 3.5 control water reference water that has been treated according to the same conditions as the water extract but without being exposed to contact with paper or board 3.6
positive control potassium dichromate (CAS 7778-50-9) 5 mM solution freshly made in reference water SIST EN 15845:2010

sample culture medium prepared with water extract as specified in 3.4 3.8 test material one or more components, or a specified quantity of paper or board, that is randomly sampled from a batch 4 Principle The test protocol specified in this document is intended to evaluate the cytotoxic effect of substances migrating from food contact paper and board into wet foods intended for human consumption. The food simulant used is water as described in Clauses 7 and 10. The test evaluates the impact of the water extract on the rate of RNA (Ribonucleic Acid) synthesis by measuring the incorporation of a radioactive tracer (tritiated uridine) in human cells (HeLa S3). The food contact paper and board samples to be tested are exposed to the extraction water as described in Clause 10. The extracts then undergo cytotoxicity assessment, and the results obtained are compared to the results for a non-cytotoxic control (a purified water for which the rate of RNA synthesis is considered optimal and is therefore arbitrarily set at 100 %). Potassium dichromate, as described in 11.4.3, is used as a positive control. 5 Reagents 5.1 Liquid scintillant for tritium counts on dry filters 5.2 Culture media The pH of all culture media used shall be 7,4 ± 0,1: pH to be adjusted using a sterile NaOH (or HCl) solution. 5.2.1 Culture media – quality and storage All culture media, foetal serum and solutions used for cell culture shall be sterile and of sufficiently high quality to guarantee optimal cell growth (see 12.2). They shall be stored in compliance with manufacturer's instructions, where given. 5.2.2 Medium for maintaining HeLa S3 cells in monolayer culture Composition: a) minimum Essential Medium Eagle1) (10x)2)
100 ml b) sodium bicarbonate solution1), 7,5 % (m/V)
30 ml c) glutamine solution, 200 mM (or Glutamax I®)1) (100x)2) 10 ml d) non-essential amino acids solution1) (100x)2)
10 ml e) foetal calf serum3)
50 ml
1) Commercially available, the composition is based on the used of Earle’s salts in the minimum essential medium. Glutamax I® is an example of a suitable product available commercially. This information is given for the convenience of this European Standard and does not constitute an endorsement by CEN of this product. 2) 10x or 100x imply tenfold or hundredfold concentrated media or solutions. 3) Heat inactivated (56 °C for 40 min) before use. SIST EN 15845:2010

800 ml 5.2.3 Concentrated culture medium for cytotoxicology testing A 5-fold concentrated culture medium is prepared by successively mixing: a) minimum Essential Medium Eagle4) (10x)5)
100 ml b) sodium bicarbonate solution, 7,5% (m/V)
30 ml c) glutamine solution, 200 mmol (or Glutamax I®)4) (100x)5) 10 ml d) non-essential amino acids solution4) (100x)5)
10 ml e) foetal calf serum6)
50 ml 5.3 Solution for rinsing cell lawns Composition: a) Dulbecco's PBS (Phosphate buffered saline) solution, (10x)5) 100 ml b) reference water added up to
1 000 ml 5.4 Cell dissociation reagent Cells are detached using a solution of Versene7) 1/5 000. 5.5 Analytical-grade dimethyl sulfoxide or glycerol 5.6 Sodium dodecyl sulphate (SDS) for analysis, at 3 % (m/v) 5.7 Trichloroacetic acid (TCA) for analysis, at 5 % (m/V) 5.8 Ethanol, 95 % to 98 % (v/v) 5.9 [5,6-3H] uridine (35Ci/mmol to 50 Ci/mmol; 1 m Ci/ml): a sterile and non-cytotoxic aqueous solution. NOTE The products and ma
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