EN 16418:2014

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Papier und Pappe - Bestimmung der Zytotoxizität von wässrigen Extrakten
unter Vewendung einer metabolisch kompetenten Hepatom-Zelllinie (HepG2)Papier et carton - Détermination de l'effet cytotoxique d'extraits aqueux en utilisant une lignée cellulaire d'hépatome possédant des enzymes du métabolisme (cellules HepG2)Paper and board - Determination of the cytotoxicity of aqueous extracts using a metabolically competent hepatoma cell line (HepG2)85.060Papir, karton in lepenkaPaper and boardICS:Ta slovenski standard je istoveten z:EN 16418:2014SIST EN 16418:2014en,fr,de01-september-2014SIST EN 16418:2014SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16418
April 2014 ICS 85.060 English Version
Paper and board - Determination of the cytotoxicity of aqueous extracts using a metabolically competent hepatoma cell line (HepG2)
Papier et carton - Détermination de l'effet cytotoxique d'extraits aqueux en utilisant une lignée cellulaire d'hépatome possédant des enzymes du métabolisme (cellules HepG2)
Papier und Pappe - Bestimmung der Zytotoxizität von wässrigen Extrakten unter Verwendung 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 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.
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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 (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 . 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 uridine 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 reference 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)
Validation of the two methods (option A and B) . 19 Bibliography . 20 SIST EN 16418:2014

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 medium for testing samples A 5,45-fold concentrated culture medium is prepared by successively mixing: a) minimum Essential Medium Eagle with Earl's 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)
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+. 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 Earle's salt with a minimum of the medium necessary. 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 16418:2014

[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 sterile 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 µCi/ml in culture medium (5.2.3) prepared with reference water (3.1). 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 the rate of RNA synthesis meets the conditions set out in 13.3. 6 Cell line 6.1 Generating the cell strain 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 cultured 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 at 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 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 that 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 volume of culture medium (5.2.2); SIST EN 16418:2014

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 Laboratory detergent: RBS 25® 4) at 5 % (v/V) or Aquet® at 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) 8.1.3.2 Water prepared by mixing 3,30 g of analytical-grade CaCl2 x 2H2O in 20 l of reference water (3.1). 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 RNA synthesis with negative control water (3.3). 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 rinsing using the rinsing water (8.1.3.1); — air-drying in a dust-free area away from toxic vapour;
4)
RBS and Aquet are examples of suitable products available commercially. This information is given for the convenience of the user of this European Standard and does not constitute an endorsement by CEN of these products SIST EN 16418:2014
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