BS-6920-2.5-1996.pdf

| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS 6920 : Section 2.5 : 1996 ICS 13.060.20 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW Suitability of non-metallic products for use in contact with water intended for human consumption with regard to their effect on the quality of the water Part 2. Methods of test Section 2.5 The extraction of substances that may be of concern to public health Licensed Copy: sheffieldun sheffieldun, na, Tue Dec 05 01:30:49 GMT+00:00 2006, Uncontrolled Copy, (c) BSI This British Standard, having been prepared under the direction of the Health and Environment Sector Board, was published under the authority of the Standards Board and comes into effect on 15 December 1996 BSI 1996 First published May 1988 Second edition June 1990 Third edition December 1996 The following BSI references relate to the work on this standard: Committee reference EH/3/7 Draft for comment 95/521408 DC ISBN 0 580 26290 1 BS 6920 : Section 2.5 : 1996 Amendments issued since publication Amd. No.DateText affected Committees responsible for this British Standard The preparation of this British Standard was entrusted by Technical Committee EH/3, Water quality, to Subcommittee EH/3/7, Effects of materials on water quality, upon which the following bodies were represented: Association of Manufacturers of Domestic Electrical Appliances Automatic Vending Association of Britain British Adhesives and Sealants Association British Bathroom Council British Cement Association British Iron and Steel Producers' Association British Malleable Tube Fittings Association British Non-ferrous Metals Federation British Plastics Federation British Plumbing Fittings Manufacturers' Association British Rubber Manufacturers' Association Limited British Valve and Actuator Manufacturers' Association British Water Cadmium Association Department of the Environment (Drinking Water Inspectorate) Department of the Environment for Northern Ireland Department of Trade and Industry (Laboratory of the Government Chemist) Ductile Iron Producers' Association Galvanizers' Association Institute of Plumbing Lead Development Association Pipeline Industries Guild Pipeline Protection Association Scottish Association of Directors of Water and Sewerage Services UK Water Byelaws Scheme Water Research Centre Water Services Association of England and Wales Zinc Development Association Licensed Copy: sheffieldun sheffieldun, na, Tue Dec 05 01:30:49 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 6920 : Section 2.5 : 1996 BSI 1996i Contents Page Committees responsibleInside front cover Forewordii Method Introduction1 1Scope1 2References1 3Definitions1 4Principle2 5Test premises2 6Safety2 7Reagents2 8Apparatus3 9Samples3 10Test procedures3 11Expression of results4 12Test report5 List of referencesInside back cover Licensed Copy: sheffieldun sheffieldun, na, Tue Dec 05 01:30:49 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ii BSI 1996 BS 6920 : Section 2.5 : 1996 Foreword This Section of BS 6920 has been prepared by Subcommittee EH/3/7 under the direction of the Health and Environment Sector Board. This Section of BS 6920 supersedes BS 6920 : Section 2.5 : 1990, which is withdrawn. This edition introduces technical changes but it does not reflect a full review or revision of the standard, which will be undertaken in due course. The changes made include the deletion of subsection 3 of the Standard (this will be eventually replaced by a new part of BS 6920 in preparation), the clarification of method details and provision for the use of suitable alternative cell line and growth media ingredients, and the introduction of a positive/toxic validation test. BS 6920 is published in several Parts, namely Part 1 Specification, Part 2 Methods of test and Part 3 High temperature tests. Part 2 is further subdivided into a number of Sections and Subsections as follows: Section 2.1Samples for testing Section 2.2Taste of water Subsection 2.2.1General method of test Subsection 2.2.2Method of testing tastes imparted to water by hoses Subsection 2.2.3Method of testing tastes imparted to water by hoses for conveying water for food and drink preparation Section 2.3Appearance of water Section 2.4Growth of aquatic micro-organisms Section 2.5The extraction of substances that may be of concern to public health Section 2.6The extraction of metals Compliance with a British Standard does not of itself confer immunity from legal obligations. Licensed Copy: sheffieldun sheffieldun, na, Tue Dec 05 01:30:49 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BSI 19961 BS 6920 : Section 2.5 : 1996 Method Introduction WARNING. As well as observing safe working practices, take particular care in handling continuous cell lines since they may become infected with pathogenic viruses and bacteria during the course of their manipulation. The nutrient media used in this test are capable of supporting microbial growth, and the cell line is capable of being infected by human viruses. CAUTION. It is essential that this test procedure is carried out only by persons with experience of mammalian tissue culture techniques and cell line morphology. This Section of BS 6920 describes a simple cytotoxicity technique to test leachates from materials and articles, used in customer's premises in contact with water for human consumption, for biologically active compounds. Materials and chemicals used by water suppliers are subjected to a fuller assessment using an extraction procedure followed by sophisticated analytical methods. This method should be regarded as only an initial screening test for substances potentially hazardous to health. A satisfactory result indicates that the leachate probably does not contain significant amounts of acutely toxic substances, but it does not indicate the absence of small quantities of substances which may be harmful on prolonged exposure. 1 Scope This Section of BS 6920 specifies a screening procedure (simple cytotoxicity test) using a mammalian cell line and a leachate from a product. The results of this procedure will assist in the toxicological assessment of the product for use in contact with potable water. 2 References 2.1 Normative references This Section of BS 6920 incorporates, by dated or undated reference, provisions from other publications. These normative references are made at the appropriate places in the text and the cited publications are listed on the inside back cover. For dated references, only the edition cited applies; any subsequent amendments to or revisions of the cited publication apply to this Section of BS 6920 only when incorporated in the reference by amendment or revision. For undated references, the latest edition of the cited publication applies, together with any amendments. 2.2 Informative references This Section of BS 6920 refers to other publications that provide information or guidance. Editions of these publications current at the time of issue of this standard are listed on the inside back cover, but reference should be made to the latest editions. 3 Definitions For the purposes of this Section of BS 6920, the following definitions apply. 3.1 cytotoxic Poisonous (toxic) to cells under the conditions of the test. 3.2 monolayer Resulting cell layer, only one cell thick, formed when a cell culture grows in contact with a suitable solid surface, normally provided by the walls of the culture vessel. The cells are contact dependent and spread out onto the surface and multiply until adjacent cells are in contact whereupon the growth ceases. NOTE. Only certain types of cell culture (including the cell line used in this test) form monolayers. 3.3 tissue culture Technique whereby cells are grown and maintained as a monolayer sheet on the inner surface of a container in a nutrient (culture) medium. 3.4 cell line Cells capable of growth under laboratory conditions using a tissue culture technique; a cell line capable of unlimited growth in vitro is described as a continuous cell line. 3.5 biologically clean atmosphere Atmosphere in which the numbers of micro-organisms are sufficiently low to not have any adverse effect on the test. 3.6 morphology Microscopic study of physical form; when applied to a cell line it is the overall appearance of both the cell monolayer and the individual appearance of the constituent cells. 3.7 rounding off Abnormal cells which are spherical or oval in shape and exhibit no visible internal structures when using a low-power microscope. 3.8 cell suspension Suspension prepared from a monolayer by the application of digestive enzymes and/or chelating agents, thereby loosening the cells from the surface to which they are attached and dispersing them to give a suspension, which can be used to start fresh cultures or to perform tests. Licensed Copy: sheffieldun sheffieldun, na, Tue Dec 05 01:30:49 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 2 BSI 1996 BS 6920 : Section 2.5 : 1996 4 Principle The product is immersed in potable water for 24 h. This water is used subsequently in the preparation of a nutrient medium. The morphology of the mammalian cell line grown in this medium is observed. A blank extract is assessed in parallel with the material. 5 Test premises Only manipulate the cells and media in a laboratory with a biologically clean atmosphere that is dust free, and carry out the test in an environment which avoids infection of the cell line. NOTE. For example, the test can be carried out in a laminar flow cabinet conforming to BS 5726 : Part 3. 6 Safety Observe the warning given in the Introduction. 7 Reagents 7.1 Waters 7.1.1 Test water, obtained from a tap connected directly to a service pipe at mains pressure. Before collection of the water, flush the tap until the temperature of the flowing water does not vary by more than 1 ÊC over a period of 1 min and does not exceed 25 ÊC. Alternatively, glass-distilled, demineralized or reverse osmosis treated water conforming to grade 3 of BS EN ISO 3696 shall be used. The test water shall be free from substances that are toxic to or inhibit the growth of the cell line. NOTE. For example, this may be verified by growing at least three successive generations of the cell line in a nutrient medium made with the test water and comparing the morphology of the cells with that of cells from the same generations grown in the same medium made with distilled water (see 10.2). 7.1.2 Distilled water (for the preparation of media), glass-distilled water, or water produced by reverse osmosis and conforming to grade 3 of BS EN ISO 3696. NOTE. Deionized water is not suitable. 7.2 Media 7.2.1 General. All reagents shall be of analytical quality. NOTE 1. Foetal calf serum may be used in place of newborn-calf serum. NOTE 2. Other established growth and maintenance media may be used in place of those specified provided that healthy growth of the test cell line can be maintained using them. The use of Gentamicin is to be preferred to other antibiotics to control possible bacterial growth/contamination from the non-sterile test samples and test water. NOTE 3. Commercially available sterile media constituents are available and should be used wherever possible. 7.2.2 Growth medium, prepared from the following. Sterile distilled water90 ml 199 concentrate (3 10) with Earle's salts but without sodium hydrogen carbonate buffer10 ml Newborn-calf serum7 ml Gentamicin solution (4000 i.u./ml)1 ml Sodium hydrogen carbonate buffer solution, 44 g/l, saturated with carbon dioxide (7.2.4)2 ml Use sterilized ingredients. Prepare the medium aseptically and store in the absence of light at (4±1) ÊC. NOTE. 199 concentrate (3 10) with Earle's salts but without glutamine and sodium hydrogen carbonate is available commercially. Before use, 0.34 ml of a 200 mmol/l solution of L-glutamine should be added to each 100 ml of concentrate. Newborn-calf serum, mycoplasma screened, is available commercially. 7.2.3 Maintenance medium, prepared from the following. Sterile distilled water90 ml 199 concentrate (3 10) with Earle's salts but without sodium hydrogen carbonate buffer10 ml Newborn-calf serum2 ml Gentamicin solution (4000 i.u./ml)1 ml Sodium hydrogen carbonate buffer solution, 44 g/l, saturated with carbon dioxide (7.3.4)3 ml Use sterilized ingredients. Prepare the medium aseptically and store in the absence of light at (4±1) ÊC. Licensed Copy: sheffieldun sheffieldun, na, Tue Dec 05 01:30:49 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BSI 19963 BS 6920 : Section 2.5 : 1996 7.2.4 Sodium hydrogen carbonate buffer solution, 44 g/l, saturated with carbon dioxide, prepared from the following. Sodium hydrogen carbonate8.8 g Phenol red (2 g/l aqueous solution)10 ml Distilled waterto 200 ml Mix the ingredients and saturate the solution with carbon dioxide either by bubbling the gas through it, or by the addition of pieces of solid carbon dioxide. Immediately after preparation, place the buffer into 10 ml glass bottles, filled to the rim, close tightly and sterilize at a temperature of 115 ÊC and at a gauge pressure of 69 kPa for 20 min in a small autoclave. Store at (4±1) ÊC. Discard bottles showing a bright magenta colouration. 7.2.5 Phosphate-buffered saline solution, prepared from the following. Sodium chloride8.0 g Potassium chloride0.2 g Disodium hydrogen orthophosphate1.15 g Potassium dihydrogen orthophosphate0.2 g Distilled waterto 1 l Mix the ingredients and dispense the solution into glass bottles in 20 ml amounts and sterilize at a temperature of 115 ÊC and at a gauge pressure of 69 kPa for 10 min. 7.2.6 Trypsin-EDTA solution, as commercially available trypsin-EDTA solution or equivalent. NOTE. This should be stored at a temperature lower than 218 ÊC. 7.2.7 Concentrated growth medium, prepared as described in 7.2.2, but omitting the distilled water; store at (4±1) ÊC in the absence of light. 7.3 Cell line Use the established VERO cell line of African green monkey kidney cells (ATCC number CCL 81). Establish that the cell line is healthy upon receipt (see 10.2). NOTE. The BGM monkey kidney cell line may be used in place of the VERO cell line for this test. 8 Apparatus 8.1 Tissue-culture ware, prepared as follows. Clean all glassware used for this test using an aqueous solution of a proprietary detergent specifically designed for use in tissue culture techniques. Rinse thoroughly after cleaning, and give two final rinses in distilled water (7.1.2). Sterilize glassware at a temperature of 121 ÊC and at a gauge pressure of 103 kPa for 15 min. Alternatively, use presterilized plastics containers supplied specifically for tissue culture work. 8.2 Haemacytometer counting chamber, conforming to BS 748. 8.3 Extraction containers, consisting of borosilicate glass beakers calibrated for a capa