BS EN 16418:2014
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Paper and board. Determination of the cytotoxicity of aqueous extracts using a metabolically competent hepatoma cell line (HepG2)
| Published By | Publication Date | Number of Pages |
| BSI | 2014 | 24 |
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.
Compared to the EN 15845[1], 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.
PDF Catalog
| PDF Pages | PDF Title |
|---|---|
| 4 | Contents Page |
| 5 | Foreword |
| 6 | 1 Scope 2 Normative references 3 Terms and definitions |
| 7 | 4 Principle 5 Reagents 5.1 Liquid scintillant, for tritium counts on dry filters 5.2 Culture media 5.2.1 Culture media – quality and storage 5.2.2 Medium for routine culture |
| 8 | 5.2.3 Concentrated culture medium for testing samples 5.3 Solution for rinsing cell lawns 5.4 Cell dissociation reagent |
| 9 | 5.5 Trypan blue, at 0,4 % (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 6 Cell line 6.1 Generating the cell strain 6.2 Maintaining the cell strain |
| 10 | 6.3 Storing the cell strain 7 Food simulants used for testing 7.1 Reference water (3.1) 8 Cleaning laboratory glassware 8.1 Cleaning liquids for laboratory glassware 8.2 Cleaning procedure for laboratory glassware |
| 11 | 9 Equipment 9.1 Equipment for the migration test 9.2 Cell culture equipment 9.3 Equipment used for cytotoxicity testing |
| 12 | 10 Preparation of specimens 10.1 General 10.2 Paper and board intended for wet contact 11 Cytotoxicity assessment 11.1 Principle 11.2 General |
| 13 | 11.3 Cell seeding 11.4 Preparation of samples 11.5 Cell culture treatment |
| 14 | 11.6 Preparation of the chromatography sheet 11.7 Kinetics of uridine incorporation in the cell RNA 11.8 Measurements of the RNA synthesis 11.8.1 option A |
| 15 | 11.8.2 option B 12 Expression of the results 12.1 Graphic representation of the results |
| 16 | 12.2 Calculation of percentage RNA synthesis and the validity of the test 12.2.1 General 12.2.2 Reference sample 12.2.3 Negative control sample 12.2.4 Positive control sample |
| 17 | 12.2.5 Test sample 13 Interpretation of the results 13.1 Results for the reference sample 13.2 Results of the positive control sample 13.3 Results for the test sample 14 Precision Table 1 — Repeatability and reproducibility found in an interlaboratory test 15 Test report |
| 19 | Annex A (informative) 96-well plates configuration Table A.1 — 96-well plates configuration |
| 20 | Annex B (informative) RNA Synthesis rate inhibition cytotoxicity test work flow Table B.1 — RNA Synthesis rate inhibition cytotoxicity test work flow |
| 21 | Annex C (informative) Validation of the two methods (option A and B) Table C.1 — Validation of the two methods (option A and B) |
| 22 | Bibliography |
