1、BSI Standards PublicationBS EN 16418:2014Paper and board Determination of thecytotoxicity of aqueousextracts using a metabolicallycompetent hepatoma cell line(HepG2)BS EN 16418:2014 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 16418:2014.The UK participation
2、in its preparation was entrusted to TechnicalCommittee PAI/11, Methods of test for paper, board and pulps.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are
3、 responsible for its correctapplication. The British Standards Institution 2014. Published by BSI StandardsLimited 2014ISBN 978 0 580 78119 3ICS 85.060Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandar
4、ds Policy and Strategy Committee on 30 April 2014.Amendments issued since publicationDate Text affectedBS EN 16418:2014EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16418 April 2014 ICS 85.060 English Version Paper and board - Determination of the cytotoxicity of aqueous extracts using a meta
5、bolically competent hepatoma cell line (HepG2) Papier et carton - Dtermination de leffet cytotoxique dextraits aqueux en utilisant une ligne cellulaire dhpatome possdant des enzymes du mtabolisme (cellules HepG2) Papier und Pappe - Bestimmung der Zytotoxizitt von wssrigen Extrakten unter Verwendung
6、einer metabolisch kompetenten Hepatom-Zelllinie (HepG2) This European Standard was approved by CEN on 8 February 2014. 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
7、any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other lang
8、uage made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC 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, Denmar
9、k, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE F
10、OR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2014 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16418:2014 EBS EN 16418:2014EN 1
11、6418:2014 (E) 2 Contents Page Foreword 3 1 Scope 4 2 Normative references 4 3 Terms and definitions .4 4 Principle 5 5 Reagents .5 6 Cell line .7 6.1 Generating the cell strain 7 6.2 Maintaining the cell strain .7 6.3 Storing the cell strain 8 7 Food simulants used for testing 8 7.1 Reference water
12、(3.1) .8 8 Cleaning laboratory glassware .8 8.1 Cleaning liquids for laboratory glassware 8 8.2 Cleaning procedure for laboratory glassware 8 9 Equipment 9 9.1 Equipment for the migration test .9 9.2 Cell culture equipment 9 9.3 Equipment used for cytotoxicity testing .9 10 Preparation of specimens
13、10 10.1 General . 10 10.2 Paper and board intended for wet contact . 10 11 Cytotoxicity assessment 10 11.1 Principle . 10 11.2 General . 10 11.3 Cell seeding . 11 11.4 Preparation of samples 11 11.5 Cell culture treatment . 11 11.6 Preparation of the chromatography sheet . 12 11.7 Kinetics of uridin
14、e incorporation in the cell RNA 12 11.8 Measurements of the RNA synthesis . 12 12 Expression of the results . 13 12.1 Graphic representation of the results 13 12.2 Calculation of percentage RNA synthesis and the validity of the test 14 13 Interpretation of the results . 15 13.1 Results for the refer
15、ence sample 15 13.2 Results of the positive control sample . 15 13.3 Results for the test sample 15 14 Precision 15 15 Test report . 15 Annex A (informative) 96-well plates configuration 17 Annex B (informative) RNA Synthesis rate inhibition cytotoxicity test work flow 18 Annex C (informative) Valid
16、ation of the two methods (option A and B) . 19 Bibliography . 20 BS EN 16418:2014EN 16418:2014 (E) 3 Foreword This document (EN 16418:2014) 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 the
17、status of a national standard, either by publication of an identical text or by endorsement, at the latest by October 2014 and conflicting national standards shall be withdrawn at the latest by October 2014. Attention is drawn to the possibility that some of the elements of this document may be the
18、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
19、, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerla
20、nd, Turkey and the United Kingdom. BS EN 16418:2014EN 16418:2014 (E) 4 1 Scope This European Standard specifies a test method for the laboratory assessment of the potential cytotoxic effect of paper and board intended to come into contact with foodstuffs using specifically the HepG2 cell line. Compa
21、red to the EN 158451, HepG2 cells are more representative of a human oral exposure to xenobiotics, due to the presence in the cells of phase I, II and III enzymes of the metabolism. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and a
22、re indispensable for its application. 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 ex
23、tract 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 tap water which undergoes the following treatment sequence: pre-f
24、iltration, 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 1 to entry: Alternatively, any other purification reg
25、ime, which produces HPLC-quality water (resistance 18,0 M/cm, total organic carbon 3 ppb, no microorganism) or waters of grade 1 or 2 according to EN ISO 3696, can be used. 3.2 water extract reference water that has been exposed to contact with paper or board Note 1 to entry: See EN 645 or EN 647. 3
26、.3 negative control water reference water that has been treated according to the same conditions as the water extract but without being in contact to paper or board 3.4 positive control waters 1) fresh 2,5 mg/l solution of potassium dichromate (CAS 7778-50-9) prepared in reference water and steriliz
27、ed by filtration through 0,22 m filter and kept at room temperature in the dark and 2) a stock 2 mg/ml solution of benzo|apyrene (CAS 50-32-8) prepared in dimethylsulfoxide (DMSO) and kept at 4 C in the dark. Dilute this stock solution 1 000 times in reference water before use 3.5 reference sample c
28、ulture medium prepared with reference water as specified in 3.1 BS EN 16418:2014EN 16418:2014 (E) 5 3.6 test sample culture medium prepared with water extract as specified in 3.2 3.7 negative control sample culture medium prepared with negative control water (3.3) alone or with 0,1 % of DMSO (if ben
29、zoapyrene is used as positive control) 3.8 positive control sample culture medium prepared with positive control water as specified in 3.4 3.9 test material a specified quantity of paper or board, that is randomly sampled from a batch 4 Principle The test method specified in this document is intende
30、d to evaluate the cytotoxic effect of water extracts from materials for wet foods intended for human consumption. The test evaluates the impact of the water extract on the rate of RNA (Ribonucleic Acid) synthesis by measuring the incorporation of a radioactive marker (tritiated uridine) in human cel
31、ls (HepG2). The food contact paper and board samples to be tested are extracted into the water as described in Clause 10. The extracts then undergo cytotoxicity assessment, and the results obtained are compared to the results of a non-cytotoxic control purified water (for which the rate of RNA synth
32、esis is considered optimal and arbitrarily set at 100 %). Potassium dichromate and/or benzoapyrene, prepared from stock solutions described in 3.4 and diluted as described in 11.4.4, are used as positive controls. 5 Reagents 5.1 Liquid scintillant, for tritium counts on dry filters 5.2 Culture media
33、 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
34、 (see 12.2). They shall be stored in compliance with manufacturers instructions, where given. Complete medium with additives and serum should be stored at 4 C for no longer than two weeks. 5.2.2 Medium for routine culture Composition: BS EN 16418:2014EN 16418:2014 (E) 6 a) minimum Essential Medium E
35、agle with Earles salts1), (10x)2)100 ml b) sodium bicarbonate solution1), 7,5 % (m/V) 30 ml c) glutamine solution, 200 mmol/l (or Glutamax I)1), (100x)2)10 ml d) non-essential amino acids solution1), (100x)2)10 ml e) foetal calf serum3)100 ml f) reference water 800 ml 5.2.3 Concentrated culture medi
36、um for testing samples A 5,45-fold concentrated culture medium is prepared by successively mixing: a) minimum Essential Medium Eagle with Earls salts1), (10x)2)100 ml b) sodium bicarbonate solution1), 7,5 % (m/V) 30 ml c) glutamine solution, 200 mmol/l (or Glutamax I)1), (100x)2)10 ml d) non-essenti
37、al amino acids solution1), (100x)2)10 ml e) foetal calf serum3)5 ml The serum concentration of reconstituted treatment medium is reduced to 0,5 % since serum proteins can mask the toxicity of the water extract. 5.3 Solution for rinsing cell lawns Phosphate buffered saline (PBS)1)without Ca2+and Mg2+
38、. 5.4 Cell dissociation reagent Cells are detached using a trypsin/EDTA solution1)(0,25 % m/V trypsin, 1mM EDTA/Na4). 1)Commercially available. Glutamax I is an example of a suitable product available commercially and based on Earles salt with a minimum of the medium necessary. This information is g
39、iven 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. BS EN 16418:2014EN 16418:2014 (E) 7 5.5 Trypan blue, at 0,4 %
40、 (w/V)1)5.6 Dimethyl sulfoxide (DMSO), analytical-grade 5.7 Sodium dodecyl sulphate (SDS), for analysis, at 3 % (m/V) 5.8 Trichloroacetic acid (TCA), for analysis 5.9 Ethanol, 95 % to 96 % (v/V) 5.10 5,6-3H uridine Use uridine (35 Ci/mmol to 50 ; 1 mCi/ml or 1,29 TBq/mmol to 1,85, 37MBq/ml): a steri
41、le and non-cytotoxic aqueous solution. On the day of kinetic measurement of uridine incorporation, the required volume of uridine is taken under sterile conditions and put into a sterile tube to prepare a uridine solution at 10 Ci/ml in culture medium (5.2.3) prepared with reference water (3.1). NOT
42、E 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 the rate of RNA synthesis meets the conditions set out in 13.3. 6 Cell line 6.1 Generating the cell str
43、ain The cell line used is HepG2, a human hepatoma cell line. This line shall be generated from recognised sources, such as the HB-8065 line of the American Type Culture Collection (ATCC) or European Collection of Cell Cultures (ECACC) No. 85011430. 6.2 Maintaining the cell strain Cells shall be cult
44、ured without antibiotics, and checks of absence of mycoplasma shall be run at frequent intervals: a) seed the HepG2 cells in the culture medium (5.2.2) in a flask (9.2.1) and incubate at (37 1) C until a 90 % confluent lawn of growth is formed; b) remove the culture medium, and rinse the cell lawn a
45、t least twice with about 10 ml of the rinse medium (5.3); c) cover the cell lawn with 2 ml of the dissociation solution (5.4) and swirl around gently to cover the entire bottom of the flask. Leave on for 1 min and remove excess trypsin/EDTA solution; d) incubate the flask at (37 1) C for 2 min to 5
46、min. Observe the cells detaching under the inverted microscope periodically tapping on the side of the flask to assist in detachment; e) once the cells are detached, add 10 ml of the culture medium (5.2.2) to stop the reaction. Pipette the medium up and down over the bottom of the flask ensuring tha
47、t all the cells are detached and resuspend in the medium; f) transfer the cell suspension to a sterilized polypropylene tube; g) homogenise cell suspension with a syringe and needle (9.2.3); h) redistribute the cell suspension obtained into the required number of flasks, and add the appropriate volu
48、me of culture medium (5.2.2); BS EN 16418:2014EN 16418:2014 (E) 8 i) place the flasks in an incubator at (37 1) C. When the cultures are approximately 90 % confluent, the cells can be subcultured or harvested for use in an experiment. 6.3 Storing the cell strain If a stock of the cell line culture i
49、s stored, then it shall be stored in liquid nitrogen, with the cells preserved in the culture medium with added dimethyl sulfoxide (5.6) (10 % v/V, final concentration). The cells should be propagated in monolayers through the minimum of three passages after thawing, before they can be used for testing. 7 Food simulants used for testing 7.1 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 liquids for laboratory glassware 8.1.1 Labor
