2013
`
`U SP 36 THE UNITED STATES PHARMACOPEIA
`
`N F
`THE NATIONAL FORMULARY
`
`Voiu rne 1
`
`By authority of the United States Pharmacopeial Convent/on
`Prepared by the Council of Experts and its Expert Committees
`
`Official from May 7, 2073
`
`ubiication, ”USP NF 2013,” is for ease of
`The designation on the cover of this p
`eparate compendia: The United
`identification oniy. The publication contains two 5
`e Nationai Forrriu/ary, Thi.rty—First
`States Pi7cirmar.‘opeia, Thirty-Sixth Revision, and Th
`Edition.
`
`THE UNiTED STATES PHARMACOPEIAL CONVENTiON
`12601 Twinbrook Parkway, Rockvilie, MD 20852
`
`1
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`1
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`CUBIST 2002
`FRESENIUS-KABI v. CUBIST
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`IPR2015-01570
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`

`
`3;
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`USP 36
`
`SlX«MONTH IMPLEMENTATION GUIDELINE
`
`The United States Phormaco eia~Nationa/ Formtilciry and its supplements become official six months after being released to
`the public. The USP~NF, whici is released on November 1 oi each year, becomes official on May 1 of the following year. This
`si><—month implementation timing gives users more time to bring their methods and procedures into compliance with new
`and revised U$P—NF requirements.
`’
`The table below describes the official dates of the USP—NF and its supplements. The 2011 USP 35—NF 30, and its supple-
`ments, /nterim Revision Anriouncemerits (IRAs) and Revision Bulletins to that edition, will be official until May 1, 2013, at which
`time the USP 36—NF 37 becomes official.
`
`Publica_gi_9‘n,__‘_
`USP 36~NF 37
`
`_, Release Date
`November 1, 2012
`
`Official Date
`May 1, 2013
`
`M_~_ ___Q_‘r',fic_i_gl,_l,._|_§_i_ti|
`May 1, 2014 (except as superseded by supplements, IRAS, and
`Revision Bu/letiris)»
`
`First Supplement to the
`ugp 35...N; 31
`Fecond Supplement to the
`use 36—NF 37
`USP ?7—l\/F 32
`
`February 1, 2013
`
`lune 1, 2013
`November 1, 2013
`
`r’\U9USt 1, 2013
`l____
`December 1, 2013
`May 1, 7.014
`
`May 1, 2014 (except as superseded by Second Supplement, IR/is,
`and Revision BU//€il'r1Sl_H____
`May 1, 2014 (except as superseded by IRAs and Revision Bulletins)
`V
`,
`May 1, 2015 (except as superseded by supplements, ll?/‘is, and
`Revision Bulletins‘;
`
`L
`
`The table below gives .he details of the IR/is that will apply to USP 36—NF 37.
`
`
`
`32(5)
`_12L<’J_c____....._ ..
`
`. ___,_l_’__l_{§fg_>_§g§_§i31_D,at_g
`lanueyygL2O13
`
`March 1 2013
`
`_M_gy__1,__._7,013
`julv 2, 2013
`__§gptember 4_2013
`. N.<>..y.err>_.t>.:r....L..2...f>13
`
`_
`
`l Comment Due Date
`
`| March 31 2013
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`,
`
`l May 31 2013
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`July 31, 2013
`September 30L_Zj)13
`November 3i; 2013
`lanuarv 3141014
`
`IRA Posting Date
`l
`l M.ay..31,_2oi 3
`
`t My 25, 2013
`
`September 27 2013
`November 29_,__201 3
`l January 31,_g014
`l March 2.5‘ 2014
`
`IRA Date
`my 1 2013
`
`September 1, 2013
`
`November 1 2013
`lariuary l, 2()__14
`_ M;,uch_>_1>L_2o14
`Mg_y 1, 2014
`
`4
`
`Revision Bulletins published on the USP website become official on the date specified in the Revision Bulletin.
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`Concerning U.$. Potent or Trademark Rights_—5Ttie inclusion in The United States Pharmacopeia or in the Noiioncil Formulary of a
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`Cop right Co 2012 The United States Pharmacopeial Convention
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`

`
`90 (81) Antibiotics-—l\/licrobial Assa)/5 / Biological Tests
`
`Table A2-1. Test for outlier Measurements
`
`li sam les from a normal population, gaps equal to or larger than the following values of Gr, 6;, and 6; occur with a probability P
`dutliereneasurements can occur only at one end‘ or with P = 0.02. when their may OCCUF at elthei @nd-
`N
`_
`3
`4
`5
`,
`5,
`9,987
`0.889
`0.781
`..__........._.._.~»—-— --~--—-
`--~——-
`v«—-—--—~
`
`‘M.
`
`._
`
`._...
`
`---~~-—
`
`-
`
`5
`0.698
`
`USP 36
`
`0.01, when
`_
`
`7
`0.637
`
`n‘
`
`N
`
`N
`G,
`
`A
`
`M
`
`'
`
`8
`0.681
`
`ii
`o_574
`
`‘__
`
`i
`
`9
`0.634
`
`i2
`0.543
`
`(85) BACTERIAL EN DOTOXl NS
`TEST
`
`‘Portions of this general chapter have been harmonized A
`with the corresponding texts of the European Pharmacopoeia
`and/or the japanese Pharmacopoeia. Those portions that are
`not harmonized are marked with symbols (”o) to specify tl'1lS
`fdCT[h‘e Bacterial Endotoxins Test (BET) is a test to detect or
`quantify endotoxins from Gram-rie ative bacteria using
`amoebocyte lysate from, the horses oe crab (Lima/us poly-
`pherrius or Tachyp/eus tr/dentatus).
`There are three techniques for this test: the gel-clot tech-
`nique, which is based on
`el formation; the turbidimetric
`.
`technique, based on the
`evelopmerit of turbidity after
`cleavage of an endogenous substrate; and the chromogenic
`technique, based on the development of color after cleav-
`age of a synthetic peptide-chromogen complex. Proceed by
`any of the three techniques for the test. in the event of
`doubt or dispute, the final decision is made based upon the
`gel-clot limit test unless otherwise indicated in the mono- ‘
`graph for the product being tested. Thetest is carried out in
`a manner that avoids endotoxin contamination.
`
`APPARATUS
`
`Depyrogenate all glassware and other heat-stable materi-
`als in a hot air oven using a validated process". A com-
`monly used minimum time and temperature is 30 min at
`250”. if employing plastic apparatus, such as microplates '
`and pipet tips for automatic pipetters, use apparatus that is
`shown to be free of detectable endotoxin and does not in-
`terfere in the test. [NoTE~—ln this chapter, the term "tube”
`includes any other receptacle such as a rriicrotlter well.]
`
`REAGENTS AND TEST SOLUTIONS
`
`Amoebocyte Lysate—/\ lyophilized product obtained
`from the lysate of amoebocytes (white blood cells) from the
`horseshoe crab (Limulus polyphemus or Tacnypleus
`tridentatus). This reagent refers onl
`to a product manufac-
`tured in accordance with the regu ations of the com eterit
`authority. [N0'rE~—Amoebocyte Lysate reacts to some -glu-
`cans in addition to endotoxins. Arrioebocyte L sate prepara-
`tions that do not react to giucaris are available: they are
`prepared by removing the 6 factor reacting to glucans from
`Amoebocyte Lysate or by inhibiting the (3 factor reacting sys-
`tem of Amoebocyte 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
` st of the procedure for inactivating encloto>_<ins, see _Dry~-
`Hen’ Sterilization under Sterilization and Sterility Assurance of Com endial Arti-
`.
`__
`,.
`.
`.
`rie; i/121 ]'i ure Lygc-:2 TS having it sensitivity of not less than 0, 5 Endotoxin
`Unit per rnL..
`
`i
`
`10
`0.597
`
`_
`
`-
`
`_
`
`M
`
`W
`
`i3
`_
`O.6l 7
`shows no reaction with the lysate employed, at the detec-
`tion limit of the reagent.
`Lysate TS—Dissolve Amoebocyte Lysate in Water for BET,
`or in a buffer recommended by the lysate manufacturer, by
`entle stirring. Store the reconstituted lysate, refrigerated or
`rozen, according to the specifications of the manufacturer.
`
`,
`
`..
`
`i
`
`PREPARATION OF SOLUTIONS
`
`Standard Endotoxin Stock Solution—A Standard Endo-
`toxin Stock Solution is prepared from a USP Eridotoxin Refer-
`ence Standard that has been calibrated to the current WHO
`international Standard for Endotoxin, Follow the specifica-
`tions in the package leaflet and on the label for preparation
`and storage of the Standard Endotoxin Stock Solution. Endo-
`toxin is expressed in Endotoxin Units (EU). [NOTE——One USP
`Endotoxin Unit (EU) is equal to one International Unit (IU)
`of endotoxin.]
`Standard Endotoxin Solutions——After mixing the Stan-
`dard Endotoxin Stock Solution vigorously, prepare appropriate
`serial dilutions of Standard Endotoxin Solution, usin Water
`for BET. Use dilutions as soon as possible to avoid (fins of
`activity by adsorption.
`Sample Solutions—~Prepare the Sample Solutions by dis-
`solving or diluting drugs using Water for BET. Some sub
`stances or preparations may be more appropriately dis-
`solved, or diluted in other aqueous solutions.
`if necessary,
`adjust the pH of the solution to be examined (or dilution
`thereof) so that the pH of the mixture of the lysate and
`Sample Solution falls within the pH ran e specified by the
`lysate manufacturer, usually 6.0-8.0. T e pH may be ad-
`justed b use of an acid, base, or suitable buffer as recom-
`rnende
`by the lysate manufacturer. Acids and bases may
`be prepared from concentrates or solids with Water for BET
`in containers free of detectable eiidotoxin. Buffers must be
`validated to be free of detectable 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 >< concentration of Sample So/utiori)/
`0-)
`
`Endotoxin Limit-—The endotoxin limit for parenteral
`drugs, defined on the basis of dose, equals K/ll/i'??., where K
`"' K
`USP-Eifikg of body wei_ htgfor any route of admlnistraiion other than
`iritrathecal (for which K is 9.2 U P-LU/kg of body weight). For radiopharma-
`ceuiical products not administered iritrathecally, the endotoxiii limit is calcu-
`lated as i75 Eb/V, where V is the inaximum recomiriended dose in mt. For
`intrathecaliy administered radioonarmaceiiticals, the eridotoxiii limit is ob.
`tained by t e formula M EU/V. For formulations [iisually anticancer products)
`administered on a per square meter of body surface, the formula is K/M,
`where K = i00 EL)/mi and M is the maximum dose/mi.
`
`

`
`USP 36
`
`Biological Tests / (85) Bacterial Endotoxins Test 9l
`
`is a threshold pyrogenic dose of endoto><in per kg of body
`weight, and M IS equal to the maximum recommended bo-
`lus dose of product per kg of body wei ht. When the prod
`uct is to be injected at frequent interva s or infused continu-
`ously,
`Ivi is the maximum total dose administered in a single
`hour period. The endotoxin limit for parenteral drugs is
`specified in the individual monograph in units suchas EU/
`ml_., Ell/mg, EU/Unit of biological activity, etc.
`Concentration of Sample Solution——
`mg/mL: in the case of endotoxin limit specified by weight
`(EU/mg);
`Units/mL: in the case of endotoxin limit specified by unit
`of biological activity (EU/Unit);
`mL/mL: when the endotoxin limit is specified by volume
`(EU/ml_).
`)2: the labeled sensitivity in the (Lei-C/ot Techni. ue (EUlmL)
`or the lowest concentration used in the standarc curve for
`the lurr)i'd/metric Technique or Chromogenlc Teclinique.
`
`GEL—CLOT TECHNIQUE
`
`The gel-clot technique is used for detecting or quantifying
`cndoto>r.ins based on clotting of the lysatc: reagent in the
`presence of endotoxin. The minimum concentration of en-
`dotoxin required to cause the lysate to clot under standard
`conditions is the labeled sensitivity of the lysate reagent. To
`ensure both the Jrecision and validity of the test, perform
`the tests ior confirming the labeled lysate sensitivity and for
`interfering factors as described in Preparatory lestirg, imme-
`diately below,
`
`Preparatory Testing
`
`Test for Confirmation of Labeled Lysate Sensitivity-
`Confirm in four replicates the labeled sensitivity,
`ex-
`pressed in EU/mL of the lysate prior to use in the test. The
`tr.-st for confirmation of lysate sensitivity is to be carried out
`when a new batch of lysate is used or when there is any
`change in the test conditions that may aifc-ct the outcome
`of the test. Prepare standard solutions having at least four
`concentrations equivalent to 2).,
`l., 0.5/‘t-, and 0.25). by di-
`luting the USP Endotoxin RS with Water for BET.
`Mix a volume of the lysate T5 with an equal volume
`(such as O.l ~ml_ aliquots) of one of the Standard Endotoxiri
`Solutions in each test tube. When single test vials or ampuls
`containinc lyophilizod lysate are used, add solutions directly
`to tho. viallor ampul. Incubate the reaction mixture for a
`constant period according to the directions of the lysate
`manufacturer (usually at 37 1 l" for 50 l. 2 min’), avoiding
`vibration. To test the integrity of the gel, take each tube in
`
`turn directly from the incubator, and invert it through about
`180'“ in one smooth motion.
`if a firm gel has formed that
`remains in place upon inversion, record the result as posi-
`tive. A resut is negative if an intact gel is not formed‘ The
`test is considered valid when the lowest concentration of
`the standard solutions shows a negative result in all replicate
`tests.
`
`The endpoint is the smallest concentration in the series of
`decreasing concentrations ol stanclard endotoxiri that clots
`fine lysate. Determine the geometric mean end oint by cal-
`culating the mean of the logarithms of the en point con-
`centrations of the four replicate series and then taking the
`antilogarithm of the mean value, as inciicatecl in the ollow-
`ing formula:
`
`geometric mean endpoint concentration = ant:log (_§.‘»3/l)
`
`where E8 is the sum of the log endpoint concentrations of
`the dilution series used, and fis the number of replicate test
`tubes The georrielrir. mean endpoint r.ori<'entration is the
`measured sensitivity of the lysate (tn EU/mL)_ If this is not
`less than 0.5?» and not more than 2’/_, the labeled sensitvity
`is confirmed and is used in tests performed vvith this lysate.
`Test for interfering Factors-—~-Usually prepare solutions
`(A---D) as shown in To'l'1le 7, anci psj:i’forii“i the inhibition/c-n-
`hancement test on the Sample Solutions at a dilution less
`than the MVD, not containing any detectable endotoxins,
`operating as described for Test for Coiflirniriiion of Labeled
`lystne Sensitivity. The geometric mean endpoint concentra-
`tions of Solutions 8 and C are deterrnined using the formula
`described in the Test for Corillrrnatlori of Labeled Lysote Serisi-
`tivity. The test for interfering factors must be repeated when
`any condition changes that is likely to influence the result of
`the test.
`The test, is considered valid when all replicates of Solu-
`tions A and D show no reaction and the result of Solution C
`confirms the labe-Eed sensitivity.
`if the sensitivity of the lysate determined in the presence
`of Solution 8 is not less than 0.5‘/». and not greater than 2/.,
`the Sample Soltrtion does rot contain factors that interfere
`under the experimental conditions used. Otherwise, the
`Sample Solution to be examined interferes with the test.
`If the sample under test does not comply with the tes: at
`a dilution less than the MVD, repeat the test using a greater
`dilution, not exceeding the ivlVD. The use of a more sensi-
`tive 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 treatment such
`as filtration, neutralization, dialysis, or heating. To establisii
`that the chosen treatment effectively c-liniinates intcriereace
`without oss of enclotoxins, perform the assay tzlescribed
`
`.__ _._.....,...__.___M...,..
`on/Enhancement Test for Gel—Clot Techniques
`
`W ____
`
`Table ‘I. Preparation of Solutions for the In ilvlti
`Endotoxln Concentration/
`Solution to Which Endotoxln
`..
`
`
`..P”.l-£8:!J3_.
`
`Dilution
`_Facto_r
`
`Endotoxi n
`Conc_e_Q_t_r_atioI’9..
`.74
`
`Number of
`,
`‘
`. _-,t.,._ILe.r>J.!ge:e2..l
`
`
`
`
` "1/J Vi/z1tgr,__fg_r Br
`
`
`
`‘DJix.‘
` /l’V0tt"r for ]=i}Ej_i:________,
`__
`oration under test tlmr.
` ' ‘>'ull/I/on A; A "
`5' §r_ilu.‘I-.n7 B‘
`lllltl for ll‘.lf‘rl(-‘lT.‘ll(l'."
`,’>o/i/(mu C; (_'or:ti‘ul ‘or ‘Lll)E‘l'l'3C: lystite Sl_‘l'l’;lliVlly.
`' Sr,/tiiioii I).
`i'-~lc-g.-iti~/t-- c.oriiro. of Wa(t'/ To/' 131.7’.
`
`
`
`
`
`
`l
`
`free» i
`
`l l
`
`ll
`
`‘InT
`
`

`
`USP 36
`
`be repeated using a greater dilution, not exceeding the
`MVD.
`
`Quantitative Test
`
`Procedure——The test quantifies bacterial endotoxins in
`Sample Solutions by titration to an endpoint. Prepare Solu-
`tions A, B, C, and D as shown in Table 3, and test these
`solutions by following the procedure in Preparatory Testing,
`Test for Confirmation of Labeled Lysate Sensitivity.
`Calculation and lnterpretation——The test is considered
`valid when the following three conditions are met: (1) Both
`replicates of negative control Solution D are negative; (2)
`Both replicates of positive product control Solution 8 are
`positive; and (3) The geometric mean endpoint concentra-
`tion of Solution C is in the range of 0.5?» to 27».
`To determine the endotoxin concentration of Solution A,
`calculate the endpoint concentration for each replicate by
`multiplying each endpoint dilution factor by it. The endo-
`toxin concentration in the Sample Solution is the endpoint
`concentration of the replicates. if the test is conducted with
`a diluted Sample Solution, calculate the concentration of en-
`dotoxin in the original Sample Solution b multiplying by the
`dilution factor. if none of the dilutions 0 the Sample Solu-
`tion is positive in a valid assay, report the endotoxin concen-
`tration as less than it (if the diluted sample was tested, re-
`port as less than 7» times the lowest dilution factor of the
`sample). if all dilutions are positive, the endotoxin concen-
`tration is reported as equal to or greater than the greatest
`dilution factor multiplied by 7» (e.g., initial dilution factor
`times eight times it in Table 3).
`The preparation under test meets the requirements of the
`test if the concentration of endotoxin in both replicates is
`less than that specified in the individual monograph.
`
`PHOTOMETRIC QUANTITATIVE TECHNIQUES
`
`Turbidimetric Technique
`
`_ This technique is a photometric assay measuring increases
`in reactant turbidrcty On the basis of the particular assay
`principle employe , this technique may bevclassified as ei—
`ther an endpoint-turbidimetric assay or a kinetic-turbidimet—
`
`92 (85) Bacterial Endotoxins Test / Biological Tests
`
`t’ n to be examined to which Stan-
`h
`-'
`igltagd/eEi‘i1:i|o‘g)>Einehparfgignmadded and which has then been
`submitted to the chosen treatment.
`
`Limit Test
`
`Procedure——Prepare Solutions A, 3, C, and D 35 5l'l0‘{*’” ll‘
`Table 2 and perform the test on these solutions followin
`the procedure above for Preparatory Testing, Test for Can ir-
`mation of Labeled Lysate Sensitivity.
`
`Solution’
`
`Table 2. Preparation of Solutions for the Gel-Clot Limit Test
`Endotoxin Concentration/
`Number of
`Solution to Which
`Repllcates
`Endotoxin ls__Ad_¢jed
`2
`>__MNone/Diluted Sample Solution
`2
`Zit Diluted Sample Solution
`2
`l
`2)»! Wciterjor BET
`C
`2
`_l
`None/Water for BET
`J
`D
`* Prepare Solution A and the positive product control So/ution_8 using
`a dilution not greater than the MVD and treatments as described for
`the Test for Interfering Factors in Preparatory Testing. The positive con-
`trol Solutions 8 and C contain the Standard Endotoxin Solution at a
`concentration corresponding to twice the labeled lysate sensitivity.
`The negative control Solution D consists of Water for BET.
`
`F
`
`L L
`
`
`
`lnterpretatlon———The test is considered valid when both
`replicates of Solutions 3 and C are positive and those of Solu-
`tion D are negative. When a negative result is found for
`both replicates of Solution A, the preparation u_nder test
`complies with the test. ‘When a positive result is found for
`both replicates of Solution A, the preparation under test
`does not comply with the test.
`_
`When a positive result is found for one replicate of Solu-
`tion A and a negative result lS found for_the other, repeat
`the test. in the repeat test, the preparation under test com-I
`plies with the test if a negative result is found for both repli-
`cates 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, if the preparation does not comply
`with the test at a dilution less than the MVD, the test may
`
`1
`
`Solution 4
`A.
`A
`
`Diluent
`Water for BET
`
`_l
`
`Table 3. Preparation of Solutions for the Gel-Clot Assay
`EndotolitihilConcentration/
`Solution to Which Endotoxin
`Is Added
`m____,None/Sample Solution
`
`B”
`C'-
`
`l
`
`_.,
`....-_.._..........,....__
`27tLSampe otition
`Zit/Water for 8ET
`
`..
`
`r
`
`.
`
`.,
`
`.
`—
`Water for BET
`
`i.
`
`Dilution
`Factor
`l
`2
`4
`8
`1
`1
`
`V
`
`“
`
`__
`
`_
`
`l
`
`_.
`
`r_
`
`L_
`
`
`
`T
`y D T
`Number of
`Endotoxin
`__ Bgpllcates
`Concentration
`__M_ 2m _
`__
`——
`2
`—
`2 "
`‘l
`—
`2 ___M__
`*‘*_,,W,,__ ,,,,,,,,__
`27;
`i
`_ 2
`7'1
`_2
`
`,-
`
`0.5‘).
`4
`_
`rt
`O.25lt
`8
`_,,_,___—___
`-—
`__ :,-___ .
`F
`Noneljzqter for BET
`_
`D“
`-‘ Solution A: Sample Solution under test at the dilution, not to exceed the MVD, with which the Test lorlnterfering Factors was completed.
`Subsequent dilution of the Sample Solution must not exceed the MVD. Use Water for BET to make a dilution series of four tubes containing the
`Sample Solution under test at concentrations of 1, ‘/2,
`‘/.i, and ‘/ii relative to the concentration used in the Test for Interfering Factors. Other dilutions
`up to the MVD may be used as appropriate.
`0 Solution 8: Solution A containing standard endotoxin at a concentration of 2‘li (positive product control).
`'» Solution C: Two replicates of four tubes of Water for BET containing the staiidard endotoxin at concentrations of 22., it, 0.5lt, and 0.251,
`respectively.
`0 Solution D: Water for BET (negative control).
`
`i
`
`L
`
`j
`
`2 __
`2
`2
`
`i
`‘
`
`

`
`USP 36
`
`Biological Tests / (85) Bacterial Endotoxins Test 93
`
`ric assay. The endpoint-turbidimetric assay is based on the
`uantitative relationship between the concentration of en-
`otoxins and the turbidity (absorbance or transmission) of
`the reaction mixture at the end of an incubation period.
`The kinetic-turbidimetric assay is a method to measure ei-
`ther the time (onset time) needed to reach a predetermined
`absorbance or transmission of the reaction mixture, or the
`rate of turbidity development. The test is carried out at the
`incubation temperature recommended by the lysate manu-
`facturer (which is usually 37 -:1“).
`
`Chromogenic Technique
`
`This technique is an assay to measure the chromophore
`released from a suitable chrornogenic peptide b‘
`the reac-
`tion of endotoxins with I sate, On the basis of the particular
`assay principle employe , this technique may be classified as
`either an end oint-chromogenic assay or a kinetic-chromo-
`genic assay. he endpoint-chromogenic assay is based on
`the quantitative relationship between the concentration of
`endotoxins and the release of chromophore at the end of
`an incubation period. The kinetic-chromogeniri assay is a
`method to measure either the time (onset time) needed to
`reach a predetermined absorbance of the reaction mixture,
`or the rate of color development. The test is carried out at
`the incubation temperature recommended by the lysate
`rnanufar.:turer (which is usually 37 i l”).
`
`Preparatory Testing
`
`To assure the precision or validity of the turbidimetric and
`chromogenic techniques, preparatory tests are conducted to
`verify that the criteria for the standard curve are valid and
`that the sample solution does not interfere with the test.
`Validation for the test method is required when conditions
`that are likely to influence the test result change.
`Assurance of Criteria for the Standard Curve——The test
`must be carried out for each lot of lysate reagent. Using the
`Standard Endotoxin Solution, prepare at least three endotoxin
`concentrations within the range indicated by the lysate
`manufacturer to generate the standard curve. Perform the
`assay using at least three replicates of each standard endo-
`toxin concentration according to the manufacturer's instruc-
`tions for the lysate (volume ratios, incubation time, temper-
`ature, H, etc.) if the desired range is
`reater than two logs
`in the liinetic methods, additional stan ards should be in-
`cluded to bracket each log increase in the ran e of the stan-
`dard curve. The absolute value of the correlation coeffi-
`cient, r, must be greater than or equal to 0.980 for the
`range of endotoxin concentrations set up.
`Test for lnterferin Factorsw-Select an endotoxin con-
`centration at or near t e middle of the endotoxin standard
`curve. Prepare Solutions A, B, C, and D as shown in Table 4.
`Perform the test on Solutions A, B, C, and D at least in dupli-
`
`cate, according to the instructions for the lysate employed,
`for example, concernin volume of Sample Solution and Ly-
`sate TS, volume ratio 0 Sample Solution to Lysote TS, incu-
`bation time, etc.
`The test is considered valid when the following conditions
`are met.
`1. The absolute value of the correlation coefficient of the
`standard curve generated using Solution C is greater
`than or equal to 0.980.
`2. The result with Solution D does not exceed the limit
`of the blank value required in the description of the
`lysate reagent employed, or it is less than the endo«
`toxin detection limit of the lysate rea ent employed.
`Calculate the mean recovery of the addedgendotoxiri by
`subtracting the mean endotoxin concentration in the solu-
`tion, if any (Solution A, Table 4), from that containing the
`added endotoxin (Solution 8, Table 4').
`in order to be consid-
`ered free of factors that interfere with the assay under the
`conditions of the test, the measured concentration of the
`endotoxin added to the Sam le Solution must be within
`50%-200% of the known a cled endotoxin concentration
`after subtraction of any endotoxin detected in the solution
`without added endotoxin.
`When the endotoxin recovery is out of the specified
`range, the Sample Solution under test is considered to con-
`tain interfering factors. Then, repeat the test using a greater
`dilution, not exceeding the MVD. Furthermore, interference
`of the Sample Solution or diluted Sample Solution not to ex-
`ceed the MVD may be eliminated by suitable validated
`treatment such as filtration, neutralization, dialysis, or heat
`treatment. To establish that the chosen treatment effectively
`eliminates interference without loss of endotoxiris, perform
`the assay described above, using the preparation to be ex-
`amined to which Standard Endotoxin has been added and
`which has then been submitted to the chosen treatment.
`
`Test Procedure
`
`Follow the procedure described for Test for lriterlering Fac-
`tors under Preparatory Testing, immediately above.
`
`Calculation
`
`Calculate the endotoxin concentration of each of the rep-
`licates of Solution A, using the standard curve generated by
`the positive control Solution C. The test is considered valid
`when the following three requirements are met.
`1. The results of the control Solution C comply with the
`requirements for validation defined for Assurance of
`Criteria for the Standard Curve under Preparatory
`Testing.
`2. The endotoxin recovery, calculated from the concen-
`tration found in Solution 8 after subtracting the con-
`centration of endotoxin found in Solution A.
`l5 Wll5l"”
`the range of 50°/o—200%,
`
`,____
`
`Table 4. Prepfratlon of Solutions for the Inhibition/Enhancement Test for Photometric Techniques
`Solution to which
`Endotoxln Is Added
`
`.
`50'Ili2l_€i Solut/0.17.
`_._
`Stiiiple Solution
`
`Number of Repllcates
`__
`N__ot less than 2
`Not less than 2
`
`
`
`
`Endojgxln C_oncentration__
`_ NEW‘
`.
`...... ._
`Middle concentration of the stqn_<,l__;a__rd curve
`At least three concentrations (lowest concentration is
`_ (lET>l(_]__l_l_§llB(.Il K)
`_
`__
`_,N<>.n.<§.-.._
`—' Solution ‘Solution may be diluted not to exceed MVD.
`‘i Solution B: The preparation under test at the same dilution as Solution A, containing added endotoxin at a concentration equal to or near the
`middle of the standard curve.
`' Solution C: The standard endotoxin at the concentrations used in the validation of the method described for Assurance of Criteria for ill? 5lU”°'0’d
`Curve under Pi‘epc1ralory Testing (positive controls).
`'1 Solution D: Writer for BET (negative control).
`
`_
`
`L
`
`Water for BF T
`Wate_r_Lor BE T
`
`Each not less t_h_an 2
`‘___,Not less than 2
`
`l
`
`

`
`94 (85) Bacterial Endotoxins Test / Biological Tests
`
`3. The result of the negative control Solution D does not
`exceed the limit of the blank value required in the
`description of the lysate employed, or it is less than
`the endotoxin detection limit of the lysate reagent
`employed.
`
`Interpretation
`
`In photometric assays, the preparation under test com-
`plies with the test if the mean endotoxin concentration of
`the replicates of Solution A, after correction for dilution and
`concentration, is less than the endotoxin limit for the
`product.
`
`(87) BIOLOGICAL REACTIVITY
`TESTS,
`IN VITRO
`
`The following tests are designed to determine the biologi-
`cal reactivity o mammalian cell cultures following Contact
`with the elastomeric plastics and other polymeric materials
`with direct or indirect patient contact or of specific extracts
`prepared from the materials under test. it is essential that
`the tests be performed on the specified surface area. When
`the surface area of the specimen cannot be determined, use
`0.1 g of elastomer or 0.2 g of plastic or other material for
`every mL of extraction fluid. Exercise care in the preparation
`of the materials to prevent contamination with microorgan-
`isms and other foreign matter.
`Three tests are described (i.e., the Agar Diffusion Test, the
`Direct Contact Test, and the Elation Test)?’ The decision as to
`which type of test or the number of tests to be performed
`to assess the potential biological response of a specific sam-
`ple or extract depends upon the material, the final product,
`and its intended use. Other factors that may also affect the
`suitability of sample for a specific use are the polymeric
`composition; processing and cleaning procedures; contact-
`ing media; inks; adhesives; absorption, adsorption, and per-
`meability of preservatives; and conditions of storage. Evalua-
`tion of such factors should be made by appropriate
`additional specific tests before determining that a product
`made from a specific material is suitable for its intended use.
`USP Reference Standards ./_‘l‘l‘;—~USP Higli-Density Poly»-
`etfiylene RS. USP Positive Bioreactiori R5.
`Cell Culture Preparation~Prepare multiple cultures of L-
`929 (ATCC cell line CCL T, NCTC clone 929) mammalian
`fibroblast cells in serum-sup lemented rninirnum essential
`medium having a seeding ensity of about i05 cells per mL..
`Incubate the cultures at 37 :t I’-’ in a humidified incubator
`for not less than 24 hours in a 5 Jr. 1% carbon dioxide at-
`mosphere until a monolayer, with greater than 80% conflu-
`ence, is obtained. Examine the prepared cultures under a
`microscope to ensure uniform, near-confluent monolayers.
`[Non-:—The reproducibility of the in Vitro Biological Reactivity
`Tests depends upon obtaining uniform cell culture density.]
`Extraction So|vents——-Sodium Chloride Injection (see mon-
`ogra h-—use Sodium Chloride Injection containing 0.9% of
`l\laC ). Alternatively, serum—free mammalian cell culture me-
`dia or serum-supp emented mammalian cell culture media
`may be used. Serum supplementation is used when extrac-
`tion is done at 37" for 24 hours.
`’ Further details are given in the following ptiblicalions of the American Soci-
`ety forTesting and lvlaterials, l9l6 Race SL, Philadelphia, PA l9lO3: "Stan-
`dard Test lvletnod for Agar Diffusion Cell Culture Scieeriirig for (.ytotox1cily.”
`ASIM Desigriation F 895-84; "Staridard Practice for DETECI Contact Cell Cul-
`ture Evaluation of Materials for Medical Devices," ASTIVI D€3'9”'<m0“
`F 813-83.
`
`USP 36
`
`Apparatus-
`Autoclcive-—Emp|oy an autoclave capable of maintaining a
`temperature of 121 : 2", equipped with a thermometer, a
`pressure gauge, a vent cock, a rack adequate to accommo-
`date the test containers above the water level, and a water
`cooling system that will allow for cooling of the test con-
`tainers to about 20°, but not below 20", immediately fol-
`lowing the heating cycle.
`Oven-—---Use an oven, preferably a mechanical convection
`model, that will maintain operating temperatures in the
`range of 50° to 70’ within i 2".
`Incubator-——Use an incubator capable of maintaining a
`terriperature of 37ii'* and a humidified atmosphere of
`5 -t 1% carbon dioxide in air.
`
`Extraction Contairiers——Use only containers, such as am-
`puls or screw—cap culture test tubes, or their equivalent, of
`Type I glass.
`If used, culture test tubes, or their equivalent,
`are closed with a screw cap having a suitable elastomeric
`liner. The exposed surface of the elastomeric liner is com-
`pletely protected with an inert solid disk 50 to 75 pm in
`thickriess. A suitable disk can be fabricatecl frorri polytef.
`Preparation of Apparatus——Cleanse all glassware thor-
`oughly with chromic acid cleansinc mixture and, if neces-
`sary, wi