6508 (81) / Biological Tests usp 43 If Gy, Gy, oF Gy, as appropriate, exceeds the critical value In Table A2-1, forthe observed N, there isa statistical basis MTGE identiying the discordant measurement as an outlier and considering Is fernval-a Uiri.oe 305) ‘Table A2-1. Test for Outlier Measurements in samples rom anormal papain gapr equal too ger than the ellowing vals of Gy, Cand G, acc wha probably P= 0.0, when ouier meatruents eu csr ony ot a enor ith 30.03, when they may Sear ae en 3 4 5 é 7 @ aor on oreo 0698 0637 x @ 3 10 = S ae OARS T aay oe = = x a 12 3 = = G Ed oa Doe = = EXAMPLE Estimated potencies of sample in log scale = 1.561, 1.444, 1.517, 1.535. CCheck the lowest potency for outlier: Gy = (1.517 ~ 1.444)/(1.561 ~ 1.444) = 0.624 < 0.889 ‘Therefore, 1.444 is not an out. ‘Check the highest potency for outlier: G Therefore, 1.561 Is not an outer. utr potencies shouldbe marked as outlier values and excluded from the assay calculations, NMT 1 potency can be [561 ~ 1.535)/(1.561 ~ 1.444) = 0.222 < 0.889 (85) BACTERIAL ENDOTOXINS TEST ‘Portions ofthis general chapter have been harmonized with the corresponding texts ofthe European Phar 2a and/or the Japanese Pharmacopoeia, Those portions that are not harmonized are marked with symbols (°,) to specity this fact., The Bacterial Endotoxins Test (BEM) is atest to detect or quantify endotoxins from Gram-negative bacteria using amoebocyte lysate from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). There are three techniques fr this test: the gel-clot technique, which is based on gel formation; the turbidimetic technique, based on the development of turbidity after cleavage of an endogenous substrate: and the chromogenic technique, based on the development of color after cleavage ofa synthetic peptide-chromogen complex, Proceed by any of the three techniques {or the test. In the event of doubt or dispute, the final decision is made based upon the gel-cot limit test unless otherwise indicated inthe monograph forthe product being tested. The test scared out ina manner tat avlds endotoxin contamination, APPARATUS Depyrogenate all lassware and other heat-stable materials in ahot air oven using a validated process."” , A commonly used minimum time and temperature is 30 min at 250°. If employing plastic apparatus, such as microplates and pipet tps for automatic pipeters, use apparatus that ls shown tobe free of detectable endotoxin and does not interfere in the test. {Nore~In this chapter, the term “tube” includes any other receptacle such as a microtiter wel,} REAGENTS AND TEST SOLUTIONS Amoebocyte Lysate ‘A lyophilized product obtained from the lysate of amoebocytes (white bload cells) from the horseshoe crab (Limulus >polyphemus or Tachypleus tridentatus). This reagent refers only to a product manufactured in accordance with the regulations ofthe competent authority, [NovtAmoeboge Lysate eats o some glucans in addon to endotoxins. Armoeboyte Lysate preparations that do not react to glucans are availabe: they are prepared by removing the G factor reacting to glucans from Fer a valid txt ofthe procedure for inactivating endotoxins, see On-Heat Steiizain under Seri Assuonce (1211). Use iste TS having 2 sentvityof nat Iss than 615 Endotoxin Unt pe My, www.webofpharma.comUsp 43 Biological Tests / (85) 6509 ‘Amocbocyte Lysate or by inhibiting the G factor reacting system of Amocbocyte Lysate and may be used for endotoxin testing in the presence of glucans.] Water for Bacterial Endotoxins Test (BET) Use Water for Injection or water produced by other procedures that shows no reaction with the lysate employed, at the detection limit or the reagent. Lysate TS Dissolve Amoebocyt Lysate in Water for BET, or ina buffer recommended by the lysate manufacturer, by gentle stirring. Store the reconstituted iysate, refrigerated or frozen, according to the specifications of the manufacturer. PREPARATION OF SOLUTIONS Standard Endotoxin Stock Solution {A Standard Endotoxin tock Solution Is prepared from a USP Endotoxin Reference Standard that has been calibrated to the ‘current WHO International Standard for Endotoxin. Follow the specifications in the package leaflet and on the label for preparation and storage of the Standard Endotoxin Stock Solution, Endotoxin is expressed in Endotoxin Units (EU). [NoTe-One USP Endotoxin Unit (EU) is equal to one International Unit (1U) of endotoxin.] Standard Endotoxin Solutions ‘After mixing the Standard Endotoxin Stock Solution vigorously, prepare appropriate serial dilutions of Standard Endotoxin Solution, using Water for BET, Use dilutions as soon as possible to avoid loss of activity by adsorption. Sample Solutions Prepare the Sample Solutions by dissolving or diluting drugs using Water or BET. Some substances or preparations may be more appropriately dissolved, or diluted in other aqueous solutions f necessary, adjust the pH ofthe solution tobe examined {Cr dilution thereoh 50 thatthe pH of the mixture of the lysate and Sample Solution fas within the pH range specified by the Ite manuacturer, usualy 60-80 The pH maybe acute by use ofan ad base, of sulbl fra recommended by the lysate manufacturer. Acids and bases may be prepared from concentrates of solids with Water for BET in containers free of ddotectable endotoxin. Buffers must be validated to be free of datectable endotoxin and Interfering factors. DETERMINATION OF MAXIMUM VALID DILUTION (MVD) ‘The maximum valid dilution is the maximum allowable dilution of a specimen at which the endotoxin limit can be determined. Determine the MVD from the following equation: MVD = (endotoxin limit x concentration of Sample Solution)/Q) Endotoxin Limit ‘The andatoxin limit for parenteral drugs, defined an the hase of dose, equals KIM *? ., where Kis a threshold pyrogenic dose of endotoxin per kg of body weight, and M is equal to the maximum recommended bolus dose of product per kg of body ‘weight. When the product isto be injected at frequent intervals or infused continuously, M is the maximum total dose administered in a single hour period. The endotoxin limit for parenteral drugs is specified inthe individual monograp! such as EU/mt, EU/mg, EU/Unit of biological activity, etc. units Concentration of Sample Solution Poco pies tajml: in the case of endotoxin it specified by weight U/mg): Units inthe ease of endotoxin it specied by unt of biological actvy (EU/Unit) mlfm: when the endotoxin int spectied by volume (2m). Zr the labeled sent in the Gel-lt Technique (Ulm) or the lowest concentration used in the standard curve forthe Turidimetrc Technique or Chromogenic Technique. TFs § USP-EUg of body weight for any route of adminstaton other than intrathecal (or which K's 0.2 USP-EU/hg of body weight). For tglopharaceta proce ot nner aoc, he enti cated ay 175, where the an eormended Sfoseln me for ivathecaly administered rdloprarmaceutab, tre endotoxin hits obtained by the fra 14 EU/V: For formulations (usualy Sndcancer products) adinstered ona per square meter of body surface, the formulas K/M, where K= 100 EU and Mi the maximum dose, www.webofpharma.com6510 (85) / Biological Tests usp 43 GEL-CLOT TECHNIQUE The gel-lot technique i used for detecting or quoting endotoxins based on clting of thelysat eagen in the presence of endotoxin. The minimum concentration of endotoxin requled to case the iysate to clot under standard condton the labeled sens of the iysate reagent. To ensure both the precon and valdty ofthe tet, perform the tests for contrming Areal acts sensi an fr renga es esd in reporatery Tian, imelatey below Preparatory Testing ‘TEST FOR CONFIRMATION OF LABELED LYSATE SENSITIVITY Confirm in four replicates the labeled sensitivity, 3, expressed in EU/ml of the lysate prior to use in the test. The test for confirmation of lysate sensitivity is to be carried out when a new batch of isate Is used or when ther is any change in the test Conditions that may affect the outcome ofthe test. Prepare standard solutions having at least four concentrations equivalent to 23, i, 0.5, and 0.25% by diluting the USP Endotoxin RS with Water for BET. Mix a volume of the Lysate TS with an equal volume (such as 0. 1-mL aliquots) of one of the Standard Endotoxin Solutions in ach teste, When ingle tes vals or ampuls containing ophitzedlysat are sed, add solutions det othe valor amp Incubate the reaction mixture for a constant period according to the directions ofthe lysate manufacturer (usually at 37 #1" for 60:4 2min), avoiding vibration, To test the integrity of the gel, take each tube in tum directly from the incubator, and invert itthrough about 180° in one smooth motion. Ifa firm gel has formed that remains in place upon inversion, record the result as positive. A result s negative if an intact gels not formed. The testis considered valid when the lowest concentration of the standard solutions shows a negative result in all replicate tests. ‘The endpoint isthe smallest concentration in the Series of decreasing concentrations of standard endotoxin that cots the tsate, Determine the geometric mean endpoint by calcula the mean of he logartyns of he endpott concentrations o the four replicate series and then taking the antiogarithm of the mean value, as indicated in the following formula: ‘geometric mean encipoint concentration = antilog (Ze/f) where Beis the sum of the log endpoint concentrations of the dilution series used, and f's the numberof replicate test tubes ‘The geometric mean endpoint concentration is the measured sensitivity ofthe lysate (in EU/ml). If this isnot less than 0.5h and not more than 2, the labeled sensitivity is confirmed and is used in tests performed with this lysate TEST FOR INTERFERING FACTORS Usually prepare solutions (A-D) as shown in Table 1, and perform the inhibition/enhancement test on the Sample Solutions ata diuton les than the MVD, not cantaining any detectable endotoxine, operating 3¢ decried fr Tet fr Contimation of Tabcied Lysate Sensitivity. The geometric mean endpoint concentrations of Solutions B and Care determined using the formula described inthe Test for Confirmation of Labeled lysote Sensi. The test fr interfering factors must be repeated when any Condition changes thats iely to Influence the result ofthe test ‘Table 1. Preparation of Solutions for the Inhibition/Enhancement Test for Gel-Clot Techniques: Solution tick Endotoxin ition Endotoxin Number of Solution eaaded ivent actor concentration Rephstes * ‘None/Sanple Sabon 7 = < * _BiSonple Soon ample aon 7 a a 4 os 4 @ ey * e Biot tr T Water rer 1 2 z z 7% a * ost a 3 02s z o eve ter or Ber = = = 2 & Soin A: A Somat ouon othe preparation under test hat see of detectable endotoxins 8 Soto Tet for interterence| «Sattar C: Cont or abe ate sn, 4 SotonD: Negative cond of Water for BT. The testis considered valid when all replicates of Solutions A and D show no reaction and the result of Solution C contirms_ the labeled sensitivity. www.webofpharma.comusp 43 Biological Tests / (85) 6511 If the sensitivity ofthe lysate determined in the presence of Solution B is nat les than 0.5; and not greater than 2), the ‘Sample Solution does not contain factors that interfere under the experimental conditions used. Otherwise, the Sample Solution to be examined interferes with the tet, Ifthe sample under test does not comply withthe testa a dilution less than the MVD, repeat the test using a greater dilution, not exceeding the MVD. The use of a more sensitive lysate permits a greater dilution of the sample to be examined, and this ‘may contribute to the elimination of interference. Interference may be overcome by suitable treatmant such as filtration, neutralization, dialysis, or heating. To establish that the chosen treatment effectively eliminates interference without loss of endotoxins, perform the assay described above using the reparation to be examined to which Standard Endotoxin hasbeen added and which has then Been submited to the chosen treatment, Limit Test PROCEDURE Prepare Solutions 4,8, C, and D as shown in Table 2, and perform the test on these solutions following the procedure above for Preparatory Testing, Test for Confirmation of Labeled Lysate Sensitivity. tok Lint Test ‘Table 2, Preparation of Solutions for the Gel Endotoxin Concentration) Solution to Which Soaton” Endotorn Ae Number of Repiats x ‘Nene/Diuted Sample Soliton 2 = 2UDIkted Sop Shain 2 € “iter or eT z D None Wateror BeT 2 “Teese enantio nea knoe eer ded a (So cy ip cate tensa mean i rr a ee te, INTERPRETATION The testis considered valid when both replicates of Solutions Band Care positive and those of Solution Dare negative, When a negative results found for both replicates of Solution A, the preparation under test complies with the test. When a postive result is found for both replicates of Solution 4, the proparation under test does not comply with the test ‘When a positive result is found for one replicate of Solution A and a negative result is found for the other, repeat the test. In the repeat test, the preparation under test complies with the test if negative result is found for both replicates of Solution A, The preparation does not comply with the test if a positive result is found for one or both replicates of Solution A. However, the preparation does nat comply with the test at a dilution less than the MVD, the test may be repeated using a greater dilution, not exceeding the MVD. Quantitative Test PROCEDURE ‘The test quantifies bacterial endotoxins in SompleSlutons by tization to an endpaint: Prepare Sluons A 8 Cand D as shown In Table 3, and test these solutions by following the procedure in Preparatory Testing, Fes for Confirmation of Labeled iysate Sensitivity ‘Table 3. Preparation of Solutions for the Gel-Clot Assay Endotoxin Concentration! Solution to Which Endotoxin Diuton Fae: Endotoxin Number of Solution tee iluent ‘fe canting ‘peste = ‘None Sonple Soon Water rer 1 = z z = z 4 = 2 = = 2 = Bisanpe Slaton = 1 = 2 © Dieter Water eT v a z 2 cy z a os 2 = 02s z www. webofpharma.com6512 (85) / Biological Tests use 43 Table 3. Preparation of Solutions for the Gel-Clot Assay (continued) Endotoxin Concentration! solution te Which Endotoxin Diation Fc Endotoxln umber of Soltlon tsadded Divent ca concentration Replies a None Water fo BET = = = z * Salton A: Sample San unde ast at he dion, ott exceed the MVD, with which the Tes or lntafeing Factors was competed Subsequent ston of tke Sa Stan mut nt eced re MVD. ee ole a tan fel ingesting te Sore seuon ander canes 1, Ye and crv othe concentaton used nthe Test rere Factor: Other sistons up tothe MV maybe used as appropte ® soon & Slain A canting standard endotoxin ts concentration of 2 (patie produ con). € Solan C Two eplats of four ibe of Water fr ET containing the standard endotoxin tconcantatons 2,2, 0.5, and 0.28, respectively. “Soliton O: Water for BE (negative conv), CALCULATION AND INTERPRETATION ‘The testis considered valid when the following three conditions are met: (1) Both replicates of negative control Solution D are negative; (2) Both replicates of postive product control Solution B are positive; and (3) The geometric mean endpoint Concentration of Solution Cs in the range of 0.5%. to 22. "To determine the endotoxin concentration of Solution A, calculate the endpoint concentration for each replicate by ‘multiplying each endpoint dilution factor by 2. The endotoxin concentration in the Sample Solution is the endpoint Concentration ofthe replicates. I the test is conducted with a dliuted Sample Solution, calculate the concentration of endotoxin in the original Sample Solution by multiplying by the dilution factor. I none of the dilutions of the Sample Solution is positive Ina valid assay, report the endotoxin concentration as less than 3. (if the luted sample was tested, report as less than 2. times ‘the lowest dilution factor of the sample). I all dilutions are postive, the endotoxin concentration is reported as equal to or {greater than the grestert dilution factor multiplied by 1 (e.g. intial cllution factor times aight times hin Table 3) “The preparation under test meets the requirements of the es if the concentration of endotoxin in both replicates isles than ‘that specified In the individual monograph, PHOTOMETRIC QUANTITATIVE TECHNIQUES Turbidimetric Technique ‘This technique isa photometric assay measuring increases in reactant turbidity. On the basi ofthe particular assay principle ‘employed, this technique may be classified as either an endpoint-turbidimetric assay or a kinetic-turbiimetic assay. The tendpoint-turbidimetie assay Is based on the quantitative relationship between the concentration of endotoxins and the furhidty Gahsoebanee or trancmiscion) of the reaction mixture at tha end af an incubation period. The kinetic.turbidimetric assay is a method to measure either the time (onset time) needed to reach a predetermined absorbance or transmission of the feaction mixture othe ate of turbidty development. The est cared out atthe Incubation temperature recommended by the lysate manufacturer (which is usually 37 + 1°. Chromogenic Technique ‘This technique isan assay to measure the chromophore release from a suitable chromogenic endotoxins with lysate. On the basis ofthe particular assay principle employed, this technique may be classtied as ether an éndpoin-chromagenie assay or a Kinetie-chromogenic asso. The endpolntchtomogenic assay is based on the quantitative felationship between the concentration of endotoxins and the release of chromophore atthe end ofan incubation period. The Kinetie-chromogenic asay isa method to measure ether the time (onset time) needed to reach a predetermined absorbance ofthe reaction mistute oF the rata of color davelopment. The test carted out at the incubation temperature recommended by the iysate manufacturer (wich i usally 37 #1 pti by the reaction of Preparatory Testing To assure the precision or validity of the turbidimetric and chromogenic techniques, preparatory tests are conducted to verify sea es ESAS aga Reamer any nace ela Sa ASSURANCE OF CRITERIA FOR THE STANDARD CURVE ‘The test must be carried out foreach lt of lsat reagent, Using the Standard Endotoxin Solution, prepare at least three ‘endotoxin concentrations vnthin the range indeated by the sate manufacturer to generate the sandra curve, Perform the Sssay using at leas three replicates ofeach standard endotoxin concentration according tothe marfacturer’ instructions for the hsate (volume ratios, incubation time, temperature, pH et) I the desired range fs greater than two logs in the kinetic ‘methods, adctona standards shouldbe included to bracket each ig increase in the range ofthe standard curve. The absolute ‘ale ofthe correlation coefficient, , must be greater than or equal t0 0.980 for the range of endotoxin concentrations set Up. www.webofpharma.comusp 43 Biological Tests / (85) 6513, TEST FOR INTERFERING FACTORS Select an endotoxin concentration at or near the middle ofthe endotoxin standard curve. Prepare Solutions 4, 8, C, and D as shown in Table 4, Perform the test on Solutions A,B,C, and D at least in duplicate, according to the instructions forthe lysate employed, er ample, cancening volume of Sample olution and Lysate, volume rato of Sample Sltion to Lysote 7, incubation time, ete, ‘Table 4 Preparation of Solutions for the nhibition/Enhancement Test for Photometric Techniques Solution Endotoxin Concentration ndotoni'e aes Number of Replates ® None Sample Stion Noble than = Tie cncanonton ot he Nando ne Sap tion Totienihon cP tiecenenaon re rent aa veauaraer cvnoanans oF None Water rae Notes than? ° Satin Te Sample Soliton maybe dButed ott exceed MVD, ® Stren 8 The preparation uncer et at the sore ton Slit A, cotasng added endotoxin at concentration equal oor ner the mie the standard Slitan C:The andar endotoxin atthe concentatons used Inthe valdaton ofthe method described for Assurance of Crea othe Standard Curve under frepartay Ting (postive contol. Scan b Water or BE (neg conto). “The testi cansideredl vale when the following conditions ae met. 1. The absolute value of the corelation coefficient ofthe standard curve generated usin Solution Cis greater than or equal 100.980, 2, The result with Solution D does not exceed the lint of the blank value required inthe description ofthe lysate reagent ‘employed, or its less than the endotoxin detection limit ofthe sate reagent employed. Calculate the mean recovery of the added endotoxin by subtracting the mean endotoxin concentration in the solution, it any (Sohtion Ay Table), rom that contalring the added éadotonin Galton 8, Yoble +) In order to be considered free ot factor that interfere with the assay under the conditions ofthe tex, the measured concentation ofthe endotoxin added to the Sample Solution must be within 50%-200%6of the known added endotoxin concentration after subtraction ofan endotoxin detected In the solution without added endotoxin ‘When the endotoxin recovery is out of the spectied range, the Somple Solution under tests considered to contain interfering factors. Then repeat the test using a greater ction, not exceeding the MVD. Furthermore, interference ofthe Sample Salton or dues Semple Solution ot to exceed the MVD may be elinated by sltablovaldated treatment such arfitration, ‘eutraizaton, alysis or heat treatment. To establish thatthe chosen treatment effectively eliminates interference without los ‘of endotoxins, perforin the assay described above, using the preparation to be examined to which Standard Endotoxin has been added and which has then been submitted fo the chosen treatment. Test Procedure Follow the procedure described for Test for interfering Factors under Preparatory Testing, immediately above. Calculation Calculate the endotoxin concentration of each of the replicates of Solution A, using the standard curve generated by the positive contrat Solution C. the tests considered valid when the following three requtrements are met, 1. The results of the control Solution C comply with the requirements for validation defined for Assurance of Criteria forthe Standard Curve under Preparatory Testing. 2. The endotoxin recovery, calculated from the concentration found in Solution after subtracting the concentration of ‘endotoxin found in Solution A, is within the range of 50%-200%. 3. The result ofthe negative control Solution D does not exceed the limit ofthe blank value required in the description of the lysate employed, o tis less than the endotoxin detection limit of the lysate reagent employed. Interpretation In photometric assays, the preparation under test complies with the testi the mean endotoxin concentration ofthe replicates of Solution A, after correction for dilution and concentration, i less than the endotoxin limit for the product, USP Reference Standards (11) USP Endotoxin RS www. webofpharma.com