2.6. BIOLOGICAL TESTS - tailingood.com

1 EUROPEAN PHARMACOPOEIA BIOLOGICAL TESTS01/2005 STERILITYThe test is applied to substances, preparations or articleswhich, according to the Pharmacopoeia, are required to besterile. However, a satisfactory result only indicates thatno contaminating micro-organism has been found in thesample examined in the conditions of the test. Guidancefor using the test for sterility is given at the end of this AGAINST MICROBIAL CONTAMINATIONThe test for sterility is carried out under aseptic order to achieve such conditions, the test environmenthas to be adapted to the way in which the sterility test isperformed. The precautions taken to avoid contaminationare such that they do not affect any micro-organisms whichare to be revealed in the test. The working conditions inwhich the TESTS are performed are monitored regularly byappropriate sampling of the working area and by carryingout appropriate controls (such as those indicated in theappropriate European Community Directives and associatedguidance documents on GMP).

2 CULTURE MEDIA AND INCUBATION TEMPERATURESM edia for the test may be prepared as described below, orequivalent commercial media may be used provided thatthey comply with the growth promotion the test for sterility. Fluid thioglycollate medium isprimarily intended for the culture of anaerobic bacteria;however, it will also detect aerobic bacteria. Soya-beancasein digest medium is suitable for the culture of both fungiand aerobic media may be used provided that they pass the growthpromotion and the validation thioglycollate gAgar, granulated (moisture content not inexcess of 15 per cent) gSodium gGlucose gYeast extract (water-soluble) gPancreatic digest of gSodium thioglycollate gThioglycollic mlResazurin sodium solution (1 g/l ofresazurin sodium), freshly mlWater R1000 mlpH of the medium after sterilisation theL-cystine, agar, sodium chloride, glucose,water-soluble yeast extract and pancreatic digest of caseinwith thewater Rand heat until solution is effected.

3 Dissolvethe sodium thioglycollate or thioglycollic acid in the solutionand, if necessary, add1 M sodium hydroxideso that, aftersterilisation, the solution will have a pH of Iffiltration is necessary, heat the solution again without boilingand filter while hot through moistened filter paper. Add theresazurin sodium solution, mix and place the medium insuitable vessels which provide a ratio of surface to depthof medium such that not more than the upper half of themedium has undergone a colour change indicative of oxygenuptake at the end of the incubation period. Sterilise using avalidated process. If the medium is stored, store at 2-25 Cin a sterile, airtight container. If more than the upper thirdof the medium has acquired a pink colour, the medium maybe restored once by heating the containers in a water-bathor in free-flowing steam until the pink colour disappears andcooling quickly, taking care to prevent the introduction ofnon-sterile air into the container.

4 Do not use the medium fora longer storage period than has been thioglycollate medium is to be incubated at 30-35 casein digest mediumPancreatic digest of gPapaic digest of soya-bean gSodium gDipotassium hydrogen gGlucose gWater R1000 mlpH of the medium after sterilisation the solids inwater R,warmingslightlytoeffectsolution. Cool the solution to room temperature. Add1 Msodium hydroxide, if necessary, so that after sterilisationthe medium will have a pH of Filter, if necessary,to clarify, distribute into suitable vessels and sterilise usinga validated process. Store at 2-25 C in a sterile well-closedcontainer, unless it is intended for immediate use. Do notuse the medium for a longer storage period than has casein digest medium is to be incubated at20-25 media used comply with the following TESTS , carriedout before or in parallel with the test on the product to Incubate portions of the media for 14 days.

5 Nogrowth of micro-organisms promotion test of aerobes, anaerobes and each batch of ready-prepared medium and each batchof medium prepared either from dehydrated medium orfrom the ingredients. Suitable strains of micro-organismsare indicated in Table portions of fluid thioglycollate medium with asmall number (not more than 100 CFU) of the followingmicro-organisms, using a separate portion of medium foreach of the following species of micro-organism:Clostridiumsporogenes,Pse udomonas aeruginosa,Staphylococcusaureus. Inoculate portions of soya-bean casein digestmedium with a small number (not more than 100 CFU) ofthe following micro-organisms, using a separate portion ofmedium for each of the following species of micro-organism:Aspergillus niger,Bacillus subtilis,Candida for not more than 3 days in the case of bacteria lot culture maintenance techniques (seed-lot systems)are used so that the viable micro-organisms used forinoculation are not more than 5 passages removed from theoriginal master TESTC arry out a test as described below under Test for sterility ofthe product to be examined using exactly the same methodsexcept for the following (1)

6 SterilityEUROPEAN PHARMACOPOEIA Strains of the test micro-organisms suitable for use in the Growth Promotion Test and the Validation TestAerobic bacteriaStaphylococcus aureusATCC 6538, CIP , NCTC 10788, NCIMB 9518 Bacillus subtilisATCC 6633, CIP , NCIMB 8054 Pseudomonas aeruginosaATCC 9027, NCIMB 8626, CIP bacteriumClostridium sporogenesATCC 19404, CIP , NCTC 532orATCC 11437 FungiCandida albicansATCC 10231, IP , NCPF 3179 Aspergillus nigerATCC 16404, IP , IMI 149007 Membrane filtration. After transferring the contents of thecontainerorcontainerstobetestedtothem embraneaddaninoculum of a small number of viable micro-organisms (notmore than 100 CFU) to the final portion of sterile diluentused to rinse the inoculation. After transferring the contents of thecontainer or containers to be tested (for catgut and othersurgical sutures for veterinary use: strands) to the culturemedium add an inoculum of a small number of viablemicro-organisms (not more than 100 CFU) to the both cases use the same micro-organisms as thosedescribed above under Growth promotion test of aerobes,anaerobes and fungi.

7 Perform a growth promotion test asa positive control. Incubate all the containers containingmedium for not more than 5 clearly visible growth of micro-organisms is obtainedafter the incubation, visually comparable to that in thecontrol vessel without product, either the product possessesno antimicrobial activity under the conditions of the testor such activity has been satisfactorily eliminated. Thetest for sterility may then be carried out without clearly visible growth is not obtained in the presence ofthe product to be tested, visually comparable to that inthe control vessels without product, the product possessesantimicrobial activity that has not been satisfactorilyeliminated under the conditions of the test. Modify theconditions in order to eliminate the antimicrobial activityand repeat the validation validation is performed:a) when the test for sterility has to be carried out on a newproduct,b) whenever there is a change in the experimental conditionsof the validation may be performed simultaneously with thetest for sterility of the product to be FOR STERILITY OF THE PRODUCT TO BEEXAMINEDThe test may be carried out using the technique of membranefiltration or by direct inoculation of the culture media withthe product to be examined.

8 Appropriate negative controlsare included. The technique of membrane filtration isused whenever the nature of the product permits, that is,for filterable aqueous preparations, for alcoholic or oilypreparations and for preparations miscible with or soluble inaqueousoroilysolventsprovidedthesesolv entsdonothavean antimicrobial effect in the conditions of the filtration. Use membrane filters having a nominalpore size not greater than m whose effectivenessto retain micro-organisms has been established. Cellulosenitrate filters, for example, are used for aqueous, oily andweakly alcoholic solutions and cellulose acetate filters, forexample, for strongly alcoholic solutions. Specially adaptedfilters may be needed for certain products, for technique described below assumes that membranesabout 50 mm in diameter will be used. If filters of a differentdiameter are used the volumes of the dilutions and thewashings should be adjusted accordingly.

9 The filtrationapparatus and membrane are sterilised by appropriatemeans. The apparatus is designed so that the solution tobe examined can be introduced and filtered under asepticconditions; it permits the aseptic removal of the membranefor transfer to the medium or it is suitable for carrying outthe incubation after adding the medium to the solutions. If appropriate, transfer a small quantityof a suitable, sterile diluent such as a 1 g/l neutral solutionof meat or casein peptone pH onto the membranein the apparatus and filter. The diluent may contain suitableneutralising substances and/or appropriate inactivatingsubstances for example in the case of the contents of the container or containers to betested to the membrane or membranes, if necessary afterdiluting to the volume used in the validation test with thechosen sterile diluent but in any case using not less thanthe quantities of the product to be examined prescribedin Table Filter immediately.

10 If the product hasantimicrobial properties, wash the membrane not less than3 times by filtering through it each time the volume of thechosen sterile diluent used in the validation test. Do notexceed a washing cycle of 5 times 200 ml, even if duringvalidation it has been demonstrated that such a cycle doesnot fully eliminate the antimicrobial activity. Transfer thewholemembranetotheculturemediumorcuti tasepticallyinto 2 equal parts and transfer one half to each of 2 suitablemedia. Use the same volume of each medium as in thevalidation test. Alternatively, transfer the medium onto themembrane in the apparatus. Incubate the media for not lessthan 14 solids. Use for each medium not less than thequantity prescribed in Table of the product dissolvedinasuitablesolventsuchasa1g/lne utralsolutionofmeator casein peptone and proceed with the test as describedabove for aqueous solutions using a membrane appropriateto the chosen and oilysolutions.