Pyrogens test
- 1. PYROGENSTEST: PYROGENS: pyrogen is fever producing substance,pyro means pyrexia and gen means producing. Pyrogens are the by-products of microorganisms mainly of bacteria, molds and viruses. Parenteral solutions are officially tested for the presence of pyrogens by a biological test in which “FEVER” response of rabbits is used as criteria. The presence of pyrogen in pharmaceutical product causes a febrile reaction in human beings. Other symptoms like chills, pain in back and legs, and malaise may occur. Pyrogen is rarely fatal. Chemical nature of pyrogen Chemicallythesepyrogensare lipidsubstancesassociatedwithcarrierusuallypolysaccharidesormaybe proteins. Sources of pyrogen contamination Solvent-possibly the most important source, the medicament, the apparatus, the method of storage between preparation and sterilization Depyrogenation: Depyrogenationisthe removalof pyrogen.Thisisachievedby, Inactivation - Applicationof veryhighdry heat (250 °C) for not less than 30 minutes is the desired method for rendering material pyrogen free. Types of Pyrogen test: Basically there are 2 tests performed to detect the presence of pyrogens and quantify them in sterile parenteral products, they are, 1) In Vivo pyrogen test (Rabbit Test) 2) In Vitro pyrogen test(Limulus Amebocyte Lysate Test) 1) In Vivo Pyrogen test (Rabbit Test): PRINCIPLE: Thistestconsistsof measuringthe rise inbodytemperatureevokedinrabbitsbythe injectionof asterile solution of the substance being examined. SELECTION & PROTOCOL: 1) SELECTION OF ANIMALS: Use healthy adult rabbits of any sex weighing not less than 1.5kg. Feed them a well-balanced diet not containinganyantibiotics.A rabbitshouldnotbe usedinthepyrogentestif:Ithasbeenusedinanegative pyrogen test in the preceding three days or it has been used in the precedingthree weeks in a pyrogen test in which the substance under examination fails to pass the test.
- 2. 2) MATERIALS NEEDED: (a) ANIMALS' QUARTERS: Keepthe rabbitsindividuallyina quietareawithauniformappropriatetemperature.Carryoutthe testin a quietroomwherethere isnoriskof disturbance andexcitingtheanimalsinwhichthe roomtemperature is within ±3 °C of that of the rabbits' living quarters. The rabbits have been kept for at least 18 h before the test. (b) THERMOMETERS: The thermometerorelectrical device whichindicatesthe temperature with aprecisionof 0.1 °C isused, and insert into the rectum of the rabbit to a depth of about 5 cm. (B.P specification) or 7.2cm (USP specification).The depthof insertionisconstant for any rabbitin everygroup. Whenan electrical device isused,itshouldbe insertedinthe rectumof the rabbit90 minutesbefore injectionof the solutiontobe examined and left in position throughout the test. (c) GLASSWARE, SYRINGES AND NEEDLES: All the glassware, syringes and needles must be thoroughly washed with water and heated in a hot air oven at 250°C for 30 minutes or at 200°C for an hour. (d) RETAINING BOXES: The retainingboxesforrabbitinwhichthe temperature isbeingmeasuredbyanelectrical device should be made in such a way that the animals are retained only by loosely fitting neck stocks, the rest of the body remains relatively free, so the rabbit may sit in a normal position. The animals must be put in box not less than one hour before the test and remain there throughout the test. PRELIMINARY TEST (SHAMTEST): One to three days before testing the product, inject pyrogen free isotonic NaCl solution (10ml/kg body weight warmed at 38.5°C intravenously) into animal, which has not been used during the two previous weeks. Recordthe temperature of animal,beginningatleast90 minutesbefore injectionandcontinuing for 3 hours after injection of solution. Any animal showing a temperature difference greater than 0.6°C must not be used in the main test. MAIN TEST: Carry out the test using a group of three rabbits. PREPARATION AND INJECTION OF SAMPLE: Warm the liquids to be examined to approximately 38.5°C before injection. The sample liquid to be injected may be diluted with a pyrogen free isotonic NaCl solution. Inject the solution slowly into the marginal veinof the ear of each rabbitover a periodof four minutes,unlessotherwise mentionedinthe monograph. DETERMINATION OF INITIAL AND MAXIMUMTEMPERATURE:
- 3. The initial temperature of eachrabbitisthe meanof twotemperature readings,recordedforthatrabbit at an interval of 30 minutes in the 40min immediately preceding the injection. While the maximum temperature is the highest temperature recorded for that rabbit three hours after the injection of the preparation being tested. Record the temperature of each animal at an interval of not more than 30 minutes,beginningatleast90minutesbefore the injectionof the producttobe examinedandcontinuing 3 h after the injection. The difference betweenthe initialtemperature andthe maximumtemperature of eachrabbitistakento be its response. When this difference is negative, the result is counted as zero response. TestInterpretationandContinuation: If no rabbit shows an individual rise in temperature of 0.5ºC or more above its respective control temperature, the product meets the requirements for the absence of pyrogens. If any rabbit shows an individual temperature rise of 0.5ºC or more, continue the test using five other rabbits. If not more than three of the eight rabbits show individual rises in temperature of 0.5ºC or more and if the sum of the eight individual maximum temperature rises does not exceed 3.3ºC, the material under examination meets the requirements for the absence of pyrogens. Note: If temperature of the rabbits decreases after the test than consider it as zero rise. No of rabbits Material passed if the sum of response does not exceed. Material failed if the sum of response exceeds. 3 RABBITS 1.15C 2.65C 6 RABBITS 2.80C 4.30C 9 RABBITS 4.45C 5.95C 12 RABBITS 6.10C 7.60C Advantages of Rabbit Test: The human and rabbits are equally responsive to threshold levels of the pyrogens. 2) In Vitro Pyrogen test OR(Limulus Amebocyte Lysate, LAL Test): The limulus amebocyte lysate test is also called as in-vitro pyrogen test (USP XXI Specified new test). Officially it is termedas bacterial endotoxin test(BET) used to detect or quantifyendotoxins from gram negative bacteria. Test principle: The test principle is based on the clotting of lysate of amebocyte (an enzyme obtained from the horse shoe crab) in the presence of pyrogens. The extract from the blood cells of horse shoe crab, Limulus
- 4. Polyphemus contains an enzyme system called "Limulus- Amebocyte Lysate" (LAL) which reacts with pyrogens so that an assay mixture increases in viscosity and opacity until an opaque gel is formed. Amebocyte + Pyrogen = Opaque gel The reaction accomplisheswithin 15-60 minutes, dependingon concentrationof pyrogensafter mixing. The concentrated pyrogens make the gel more turbid and thick. REQUIREMENTS: Limulus-Ambocyte Lysate is prepared by bleeding healthy mature specimens by heart puncture. The amebocytes are carefully concentrated, washed and lysed by osmotic effects. Prior to perform the LAL test,lysate assayis carriedout withpurifiedendotoxinsandare acceptedif it detects0.001ug/ml or less concentrationof the purifiedendotoxins.The glassware,suchasglasstesttubes(10 x 75mm) usedinthe test must be thoroughly cleaned, dry and heat sterilized. A buffer solution of potassium phosphate 2mEq/ml isusedto adjustthe pH of testsample at7. The alcoholiccontentinsample istobe removedas itcausesprecipitationof lysate.If the samplecontainsproteins,itproducesgelthusthe proteinsmustbe diluted to appropriate concentration before the test. Similarly, other interfering substances present in sample must also be removed before the test. PROCEDURE: The pH of test sample if specified is adjusted. The test solution and standardized LAL are separately mixed −in equal parts (0.05-0.2ml). The mixture is incubated immediately at 36̶̶̶̶ 38°C for 1 hour in assay tube. The assay tube must be remained undisturbed completely because agitation may irreversibly destroythe gel leadingtoa false negative result. The testtube isobservedafterthe specifiedtimeandis examinedforthe formationof opaque gel. Formationof gel representsapositivetestendpointreaction. The test is performed using a commercial LAL test kit. This kit contains a lyophilized LAL, and E. coli endotoxinandpure waterasstandardsand these latertwoare usedto check the sensitivity of the test. ADVANTAGE OF LAL TEST: 1. It is in-vitro and does not require animal handling, thus is more convenient. 2. It is 10 times more sensitive than that of the invivo rabbit test. 3. It is economical. 4. It consume less time, i.e., 1 vs 3 hours required by rabbit’s test. 5. It requires less laboratory facilities and minimum equipment’s. 6. It requires less test volume (as little as 0.1ml of test solution). 7. It is more accurate. TEST FOR STERILITY: It is a testingprocedure appliedtoproductsintendedtobe sterile before marketing,tocheckthat these productsare free fromall livingmicroorganisms. The testisappliedtosubstances,preprationsorarticles which,accordingtopharmacopiea,are requiredtobe sterile. However,asatisfactoryresultonlyindicates
- 5. that no contaminating microorganismshas been found in the sample examinedin the conditionsof the test. PRECAUTIONS: The testfor sterilityiscarried outunderasepticconditions. The workingconditionsinwhichthe testsare performed are regularly monitored by appropriate sampling of working area. Culture media & incubation temperature: The following culture media have been found to be suitable for test of sterility. Fluid thioglcollate medium: It is primarily intended for culture of anaerobic bacteria, but can also detect aerobic bacteria. Soya bean casein digest medium: It is suitable for culture of both fungi and aerobic bacteria. Sterility of medium: The sterility behavior of each culture medium is confirmed by incubating a portion of the media at the specified incubationtemperature for 14 days. Incubate portions of media for 14 days. No growth of microorganisms occurs. Growth promotion test of aerobes, anaerobes and fungi: Perform to check the suitability of medium for sterility test. Test each batch of medium. Inoculate portionsof thioglycolate mediumwithasmall no, (not more than 100CFU) of followingmicroorganisms; Clostridium sporogenes Pseudomonas aeruginnosa Staphylococcus aureus Use separate portion of media for each species of microorganisms. Inoculate portions of soya bean casein digest medium with a small no, (not more than 100CFU) of following microorganisms; Aspergillus brasiliensis Bacillus subtilis Candida albicans Use separate portion of media for each specie of microorganisms. Incubate for not more than 3 days in the case of bacteria. Not more than 5 days in the case of fungi. RESULT: The media are suitable if a clearly visible growth of microorganisms occur. BACTERIOSTASIS & FUNGISTASIS:
- 6. In additiontothe foregoingmediumtests,priortoconductinga sterilitytestona product,determine its level of bacteriostatic and fungistatic activity by the following procedure, To each of several vessels containing the specified quantity (15, 40, 80ml) of appropriate test medium, add the specified quantity of product. Inoculate these vessels of product-medium mixtures and the control vesselsof mediumwithdilute cultureof bacteriaandfungi thatare sensitivetothe productbeing tested,includingsporesof aerobicandanaerobicbacilli. Incubate all vesselsatan appropriate temp.for not less than 7 days. If growthof the test organismsiscomparable incontrol vesselsandinproduct-mediummixture vessels, the product is not bacteriostatic or fungistatic. If the product is fungstatic or bacteriostatic, either use a suitable sterile inactivating agent or diluting the product with sufficient quantity of culture medium. Test procedure for sterility of the product: The test may be carried out using the technique of membrane filtration or by direct inoculation of the culture media with the product to be examined. OPENING CONTAINERS: Cleanthe exteriorsurfaceof ampulesandclosuresof vialsandbottleswithantimicrobialagentsandmake access to the contents in a suitable manner. SAMPLING: For each unit, use not less than the volume of product and medium specified in BP. 1. Direct inoculation of culture medium: Transferthe specifiedquantityof thepreparationtobe examineddirectlyintoculture mediumsothatthe volume of the productis notmore than 10% of the volume of the medium. If the product has antimicrobial activity, carry out test after neutralizing this with a suitable neutralizing substance orbydilutinginasufficientquantityof culture medium. Whenitisnecessarytouse alarge volume of the product it may be preferable to use a concentrated medium. 2. Membrane Filtration: The techniqueof membranefiltrationisusedwheneverthe natureof the productpermits, that is,forfilterableaqueouspreparationforalcoholicoroilypreparations.Forpreparationsmiscible with orsoluble inaqueousoroilysolventsprovidedthese solventsdonothave antimicrobial activity. Use membrane filters having a nominal pore size of not greater than 0.45um with effectiveness to retain microorganisms. For example: Cellulose nitrate filters are used for For example: Cellulosenitrate filtersare usedforaqueous,oilyandweaklyalcoholicsolutions. Cellulose acetate filters are usedforstronglyalcoholicsolutions. Specialfiltersmaybe neededforcertainproducts .e.g.Antibiotics. The diameterof membrane isabout50mm. The filtrationapparatusandmembrane are sterilized under aseptic conditions. If appropriate, transfer a small quantityof a suitable,sterile diluent such as a 1 g/L neutral soln. Of meat or casein peptone on to membrane in the apparatus and filter. Transf
- 7. has antimicrobial activity,washthe membrane notlessthan3 timesbyfilteringthroughitthe volume of chosensterilediluent. Transferthe wholemembranetothe culturemediumorcutitasepticallyintoequal parts and transfer one half to each of suitable media. Alternately transfer the medium on to the membrane in the apparatus. Incubate the medium for not less than 14 days.