1、May 2010 Translation by DIN-Sprachendienst.English price group 12No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 07
2、.100.30; 67.250!$bC 67.250 English Version Paper and board - Determination of the cytotoxicity of aqueous extracts Papier et carton - Dtermination de la cytotoxicit des extraits aqueux Papier und Pappe - Bestimmung der Zytotoxizitt von wssrigen Extrakten This European Standard was approved by CEN on
3、 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 m
4、ay be obtained on application to the CEN Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the
5、CEN Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta
6、, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2010 CEN All rights of exploi
7、tation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 15845:2010: EEN 15845:2010 (E) 2 Contents Page Foreword 31 Scope 42 Normative references 43 Terms and definitions .44 Principle 55 Reagents .56 Cell line .66.1 Generating the cell strain 66.2 Maintaining the
8、 cell strain .76.3 Storing the cell strain 77 Food simulants used for testing 78 Cleaning laboratory glassware .78.1 Cleaning fluids for laboratory glassware 78.2 Cleaning procedure for laboratory glassware 88.3 Alternative cleaning procedure 89 Equipment 89.1 Equipment for the migration test .89.2
9、Cell culture equipment 89.3 Equipment used for cytotoxicity testing .910 Preparation of specimens 1010.1 General . 1010.2 Paper and board intended for wet contact . 1011 Cytotoxicicity assessment . 1011.1 Principle . 1011.2 General . 1011.3 Preparation of samples 1111.4 Preparation of the reference
10、sample, control sample and the positive control sample . 1111.5 Incubation of cells obtained from monolayer culture . 1111.6 Preparation of the chromatography sheet . 1211.7 Kinetics of uridine incorporation in the cell RNA 1212 Expression of results . 1312.1 Graphic representation of the results (t
11、o be generated for each tube) 1312.2 Calculation of percentage RNA synthesis and the validity of the test 1313 Interpretation of the results . 1513.1 Results for the reference sample 1513.2 Results of the positive control sample . 1513.3 Results for the test sample 1514 Precision 1515 Test report .
12、16Annex A (informative) Pyrodistilled water . 17Bibliography . 18DIN EN 15845:2010-05 EN 15845:2010 (E) 3 Foreword This document (EN 15845:2010) has been prepared by Technical Committee CEN/TC 172 “Pulp, paper and board”, the secretariat of which is held by DIN. This European Standard shall be given
13、 the status of a national standard, either by publication of an identical text or by endorsement, at the latest by July 2010, and conflicting national standards shall be withdrawn at the latest by July 2010. Attention is drawn to the possibility that some of the elements of this document may be the
14、subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium
15、, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. DIN EN 15845
16、:2010-05 EN 15845:2010 (E) 4 1 Scope 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 doc
17、uments 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 Prepara
18、tion 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 a
19、s 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
20、(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 M/cm, Total organic carbon 3 ppb, no micro-organisms) or waters of grade 1 or 2 (see EN ISO 3696), ca
21、n 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 necess
22、ary 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 DIN EN 158
23、45:2010-05 EN 15845:2010 (E) 5 3.7 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
24、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
25、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 co
26、mpared 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
27、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
28、to guarantee optimal cell growth (see 12.2). They shall be stored in compliance with manufacturers 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
29、 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 Earles salts in the minimum essential medium. Glutamax I is an example of a suitable pr
30、oduct 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. DIN EN 1
31、5845:2010-05 EN 15845:2010 (E) 6 f) reference water 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) glutam
32、ine 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) Dulbeccos PBS (Phosphate buffered saline) solution, (10x)5)100 ml b) reference water added up to 1 000 ml 5.4 Cel
33、l 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 uridin
34、e (35Ci/mmol to 50 Ci/mmol; 1 m Ci/ml): a sterile and non-cytotoxic aqueous solution. NOTE The products and materials referred to in the present document are considered non-cytotoxic if they do not trigger a cytotoxic response, i.e. if the linear regression line generated by measuring rate of RNA sy
35、nthesis meets the conditions set out in 12.2. 6 Cell line 6.1 Generating the cell strain The cell line used is HeLa S3, a human cell line. 4) Commercially available, the composition is based on the used of Earles salts in the minimum essential medium. Glutamax I is an example of a suitable product a
36、vailable commercially. This information is given for the convenience of this European Standard and does not constitute an endorsement by CEN of this product. 5) 10x or 100x imply tenfold or hundredfold concentrated media or solutions. 6) Heat inactivated (56 C for 40 min) before use. 7) Or equivalen
37、t amount of aqueous solution of the tetrasodium salt of ethylenediaminetetraacetic acid (NA4EDTA). DIN EN 15845:2010-05 EN 15845:2010 (E) 7 This line shall be generated from recognized sources, such as the CCL2.2 HeLa S3 line of the American Type Culture Collection. 6.2 Maintaining the cell strain C
38、ells should be cultured without antibiotics, and checks should be run at frequent intervals to screen for mycoplasma: a) seed the HeLa S3 cells in the culture medium (5.2.2) in a flask as defined in 9.2.1 (75 cm3or 150 cm3) and incubate at (37 1) C, 5 % CO2and 95 % humidity until a confluent lawn of
39、 growth is formed; b) remove the culture medium, and rinse the cell lawn at least twice with around 10 ml of the rinse medium (5.3); c) cover the cell lawn in a film of the Versene solution or equivalent (5.4). Leave the flask at (37 1) C for a few minutes. Detach the cells by gently shaking: 1) add
40、 a few millilitres of the culture medium (5.2.2); 2) disperse the cells by repeat pipetting; d) redistribute the cell suspension obtained into the required number of flasks, and add the appropriate volume of culture medium. 6.3 Storing the cell strain If a stock of the cell line culture is stored, t
41、hen it shall be stored in liquid nitrogen, with the cells preserved in the culture medium with added dimethyl sulfoxide (10 % v/v, final concentration) or glycerol (10 % v/v, final concentration), as per 5.5. The cells should be propagated through the minimum of three passages after the storage, bef
42、ore they can be used for testing. 7 Food simulants used for testing 7.1 Test water: Reference water (3.1) that will be used directly for contact with paper and board intended for wet foodstuffs. 8 Cleaning laboratory glassware 8.1 Cleaning fluids for laboratory glassware 8.1.1 Laboratory detergent:
43、RBS 258)5 % (v/v) or Aquet8), 1 % (v/v) or any equivalent alkaline detergent prepared in reference water (3.1). 8.1.2 Nitric acid: in solution, at 5 % (v/v), prepared by diluting 65 % to 70 % analysis-grade nitric acid in the reference water (3.1). 8.1.3 Rinsing water 8.1.3.1 Reference water (3.1).
44、8.1.3.2 Water prepared by mixing 3,30 g of analytical-grade CaCl2x 2H2O in 20 l of reference water (3.1). 8) RBS 25 and Aquet are examples of suitable products available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsem
45、ent by CEN of these products. DIN EN 15845:2010-05 EN 15845:2010 (E) 8 8.2 Cleaning procedure for laboratory glassware Cleanliness of the laboratory glassware is a very important factor, since it affects the quality of the results. Glassware cleanliness is therefore checked by measuring the rate of
46、RNA synthesis with control water (3.5). The cleaning procedure consists of the following steps: soaking in the laboratory detergent (8.1.1) for at least 12 h; washing and copiously rinsing using the rinsing water (8.1.3.2); soaking in analytical grade nitric acid (8.1.2) for about 2 h; copiously rin
47、sing using the rinsing water (8.1.3.1); air-drying in a dust-free area away from toxic vapour; sterilisation by autoclaving (at 120 C for 30 min) of laboratory glassware intended for cell culturing. NOTE This procedure can be automated. Any traces of organic materials left on the incubation tubes (9
48、.2.2) after the last tube rinsing cycle should be eliminated by sterilising in an oven at 550 C for 2 h. 8.3 Alternative cleaning procedure Standard washing programme for laboratory glassware is applied, but the utensils intended for cell culture work are washed separated from other glassware; the g
49、lassware is subsequently dried at 100 C for 2 h; sterilisation by autoclaving (at 120 C for 30 min) of laboratory glassware intended for cell culturing; all cell culture glassware is subsequently heat sterilised in an oven at 550 C for 2 h. NOTE This alternative procedure can be applied, if it can be demonstrated that the cytotoxicity assay and its results as de
