2013
`
`USP 36
`
`THE UNITED STATES PHARMACOPEIA
`
`NF 31
`
`Volume 1
`
`THE NATIONAL FORMULARY
`
`By Gut/'70/‘ity of the United Slates; Pimrriiacopeicil COI‘1V€‘I’1Z'/OM
`Prepared by the Council of Experts; and its; Expert“ C0rriI7”IifZ‘€e5
`
`Official from May 7, 2013
`
`"1"iw <f,i(Z’Si{.:]T"1£i1U(.‘)f""! on the mver of1;i"iia publication, ”USP NF 2013,” ifs for eeise of
`1£1€;?f1T,11Mi£Zi:“l'[‘i(‘JI"1 oiiiy. The publicjatiiori c0r11:airis two SEé§J<':‘1l"a1;E* CO!T1[f}F3i"1CiiaZ "!“i’ii.:*
`(/I"iI‘i'(%fI'
`Stcitm: /3‘/’i«:iri7'iciwpie?i'ci,
`'Tii1irty—;‘i3i><'th Revirsicm, and The Natioricil F50/mu/ary,
`'1"1"xii“ty»Firs't
`Ed i ti <1; ri .
`
`‘THE W-J1’1’E[“) ST/\TE.§ PHARMACOPEIAL CONVENTWM
`12601 ‘”fwinbrm1a Parkway, Roclizviile,
`1‘v1[") 2085.32
`
`Page 1
`
`CUBIST 2302
`AGILA V. CUBIST
`IPR2015-00141
`
`Page 1
`
`CUBIST 2302
`AGILA v. CUBIST
`IPR2015-00141
`
`

`
`ii
`
`USP 36
`
`SIX-lVl0l‘«lTl*l lMPl.El\/IENTATION GUIDELINE
`
`The United Ercites Pnarmcicopeici-Natiorial Forrriuiciry and its supplements become official six months after being released to
`the public. The USP—MF, whic i
`is r'e|‘eased on hlcivernher l oi eacli year, becomes official on May ‘I of the following year. This
`six—rrion‘th implemeritation timing gives users more time to bring their methods and procedures into compliance with new
`and revised UEP—/W3 requirements.
`The table below describes the official dates of the U5P——i\/F and its supplements. The 20ll USP 3.9---NF 30, and its supple»
`merits, /riterirri Revision Aririouncerrierirs (IRAs) and ltevisiori milletiris to that edition, will be official until May 1, 2013, at which
`time the USP 36~l\lF 37 becomes official.
`
`Publigajzjvgrlrww
`USP 36--NF Ji
`,
`First Euppierrienl to the
`USP 36 NF 3i
`Second Supp/ernent to the
`USP 35--NF 3 7
`_
`USP 37—i\iF 32
`
`Release Da’te__
`liioverriber 1, ZUIZ
`..
`February 1, 2013
`
`lime i, 201%
`
`.._,
`Noveiiiber l, 201 3
`__
`
`Official Date
`May I, 2t)‘l 5
`
`August 1, 2013
`
`L
`
`Decernber 1, 201'}
`
`May l, 2014
`
`__________a_M
`_ __,Q_f_flC_l__§l,l__ Until
`é3ll‘liIl
`IR/ls,
`May 1, 2014 (except as siipersedecl by supplemerits,
`....~.i?_cMfifr»L"i_A3.ii//e*tirz.s)
`____,,
`__________________________________________________________________‘_
`May l, 2014 (except as stiprzrsibclrrcl by Secorici Eu/J/J/einei‘ii, ill/ii,
`eintljievisiori Bu/leririsl,
`’
`May I, 2014 (except as supers" ‘
`
`......
`Ms_..__.......................
`L-<~—...............><l
`_
`May I, 2015 (except as siiperaeded by supp tincnts, IRAs, and
`i(e._\_./_i§iori _i3ii/ieriris)
`
`R'_
`
`The table below gives .he details of the IR/ls that will apply to USP 3r5~/VF 37.
`
`ABA........:.................,_......,. PF Ifestsir-_si..l2e §;i.,tg eesE::7 .._LI1.A.r:9_s1:r;g_I3:
`39(1)
`lariuary
`2011
`March 3'], 2013
`V __ay 3_jl,_,_20’l’3
`March ‘l
`20‘l‘:‘i_
`May si 2013
`u_ly 25 2oi3
`-
`,._,lV.l.€3.Y
`20”
`lulv 3'1. 2013
`Septerriber 27 Z91
`lulvz 2013 _ W
`Begtember 30, 20133“_____M ovember 29,_ ,
`N
`Septemi)e;4_gQ‘lT§
`Nor/erriber 30 20__l_'5__
`anuary 31, 20l__A_i
`l‘\lW.e,n‘i,bs‘,r. "1, Ni 3
`Ianuarv 3i, 201:4
`_____.
`Merci‘! 28 Z91‘)....................
`
`mm
`
`39(4)
`39(5)
`42(6)
`
`V
`
`_
`
`l
`
`W N-:l£l:”i;:l:::‘ilLfl_i_¢lj&3I1:):z;__t_;e:_m::::'MMnn:::
`li,i_]y___l 2013
`_____
`
`§Opi.t3M_i_T_1lQfi§rl 20l3___m(((((,_
`_(:{_T"Il‘)I£‘i' i 201 ;i________‘_
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`4
`
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`
`_______________,_
`
`Revision i3‘ul/eriris publis' ed on the USP website become official on the date specified in the /ievi.sioi'i Biri/etin.
`
`NOTICE AND WARNING
`
`Concerning U.5. Pcitent or Trademark Ri'giits—The inclusion in The United States Plicirmricopeici or in the hltiZ'/O/’lCi/ Forrriuiary of
`monograph on any drug in respectto which patent or trademark rights may exist shall not be deemed, and is not iritenclecl
`as, a grant of, or authority to exercise, any right or privilege protected by such patent or tradernarlt. All such rights and
`privileges are vested in the patent or tradernarlt owner, and no other person may exercise the same without eiipress
`permission, authority, or license secured from such patent or trademarl< owner.
`
`Concernirig Use of USP or NF Text—«Attention is called to the fact that USP and NF text is fully copyrighted. Authors and
`others wishing to use portions of the text should request permission to do so from the Eiecretary Oll‘[l'iQ UBPC Board of
`Trustees.
`
`Copyright to 2012 The United States Pharrriacopeial Convention
`‘l 2601 Twinbroolr. Parkway, Rockville, MD 20852
`
`All rights reserved.
`ISSN: O"l95«7996
`
`ISBN: 978-L936424-i 22
`
`Printed in the United States by United Book Press, Inc., Baltimore, MD
`
`Page 2
`
`Page 2
`
`

`
`90 (81) Antibiotics-—ls/licrobial Assays / B/0/09/CCl/ T9515
`
`USP 36
`
`Table A2-ll. Test for Uutlier Measurements
`
`_
`
`..
`
`5
`o_._s9s
`
`W
`
`.,,
`
`.
`
`_,
`
`..,,_7
`0437
`
`______,
`
`
`
`i m gles lronn a normal population, gaps equal to or larger tiiaii the following values of Ci, ()2, and C; occur with a probability P 2 0.01, when“
`l
`oltitlilerlrneasurements can occur only at one end‘ or with P5 0.92 when they ma
`occur___at either end.
`__
`_M
`_________
`N
`M
`3
`l
`/i
`5
`C,
`may
`i
`oasa
`0.7231
`
`"E
`
`N
`
`N
`C,
`
`3
`ass:
`
`'1 l
`0.574
`
`A
`
`A
`
`7
`
`_ " '
`_
`0.634
`
`~_ _
`
`l
`l_
`
`‘H
`
`.
`12
`@643 ,_
`
`,.
`
`;_,
`
`.
`______..
`
`‘l0
`0.597
`
`____
`
`ll 3
`0.617 .
`
`W
`
`U
`
`W____,,
`___u_._
`
`, _,
`
`-
`
`._
`
`(85) BACTERlAL EhlDOTOXll‘slS
`TEST
`
`_
`“Portions of this general chapter have been l’lal’lTiOl"IlZ€‘Cl
`with the corresponding texts of the European Phorrricicripoeio
`and/or the /ciponese Pharmacopoeia. Those portions that are
`not harmonized are marlted with symbols (M) to specify this
`mgllhwe Bacterial Eridotoxins Test (BET) is a test to detect or
`quantity endotoxins from Cram-new ative bacteria using
`amoebocyte lysate frorri the horses oe crab (L/rriiilus poly-
`pherrius or Tochyp/eus ti‘/dentotus).
`There are three techniques for this test: the gel«clot tech
`nique, which is based on
`el formation; the turbidirnetric
`,
`technique, based on the tevelopmeni: of turbidity after
`cleavage of an endogenous substrate; and the chromogenic
`technique, based on the development of color after cleav-
`age of a synthetic peptideclirornogen 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 prodiict being tested.
`l‘l'iE‘i;€:"S‘l':
`is carried out in
`a manner that avoids enclotoxiri contamination.
`
`APPARATUS
`
`Depyrogenate all glassware and other heat—stable materi-
`als in a hot air oven using a validated processfll. A coin
`monly used minimum time and temperature is 30 min at
`250“.
`If employing plastic apparatus, such as mici‘oplates .
`and pipet tips for automatic pipetters, use apparatus that is
`shown to be free oi detectable endotoxin and does not in-
`terfere in the test. [isiort-ln this chapter, the term ”tube”
`includes any other receptacle such as a microtiter well.j
`
`REAGENT5 AND TE"-.$T EQUJTIDNS
`
`Amoebocyte Lysatee/ii lyophilized product obtained
`from the lysate of amoebocytes (white blood cells) from the
`horseshoe crab (Liiriulus polypherrius or Tuchyp/eus‘
`trideritorus). This reagent refers oni
`to a product manufac—
`tured in accordance with the regu ations of the competent
`authority. [N(‘)TE~«-—Amoebotfyte Lysote reacts to some [3-glu-
`cans in addition to endotoxins. Arrioebocyte L sate prepara-
`tions that do not react to glucans are availab e: they are
`prepared by removing the C3 factor reacting to glucans from
`Arrioebocyte Lysote or by inhibiting the C factor reacting sys-
`tem of Arrioebocyte Lysqte and rnay be used for endotoxin
`testing in the presence of glucansj
`Water for Bacterial Endotoxins Test (BET)e-«Use Water
`for injection or water pr'oducer_l by other procedures that
`
`M pol; ti V/I-alidity test of the procedi.irr.: for iriactivaiing enclotmiiiis, s
`L‘/.‘3iy~
`i-lent S'rr~ri/izatiori under Siiarilizaiioii and sterility Assurance of (.017) Jendiol Art»
`rfp: ‘/1
`1 i um Lyscile T3 having a sensitivity of not less than 0." E Endotoxin
`Unit per mL..
`
`Page 3
`
`shows no reaction with the lysate employed, at the detec-»
`tion limit of the reagent.
`Lysate T5—~Dis.solve Arrioebocyte Lysoie in Water for BET,
`or in a butter recommended by the lysate mariuiacturer, by
`entle stirring. Store the reconstituted lysate, refrigerated or
`rozen, according to the specificatioris of the i"nanuia<:turer.
`
`PREPARATION OF SOLUTIGNS
`
`Standard Endotoxin Stock S2o|utiori-»A ;'itr.‘ir'ic/aid Eric/o«
`toxin Stock Solutiori is prepared from a USP Eiidotoxin Refer-
`ence Standard that has been calibrated to the current Wl-10
`international Standard for Endotoxin. Follow the specitica
`‘Lions in the package leaflet and on the label for preparation
`and storage of the Sioricr/ord EI"I(/Ol())tlIi Stocli .S"o/Litiori. El"it',lt')-
`toxin
`expressed in Endotoxin Units (EU).
`[l~J(_")"l"E------~~One USP
`Endotoxin Unit (EU) is equal to one International Unit (iii)
`of endotoxin]
`
`Standard Eridotoxin Eolutions--—~After mixiiig the fituri»
`dord EI’idOl'0XlI’i Stock Solution vigorously, prepare appropriate
`serial dilutions of Etaridord Eridotoxiri Solis/tion, usin Water’
`for BET. Use dilutions as soon as possible to avoid oss of
`activity by adsorption.
`Sample Eolutions—«~Prepare the Scirriple SO/l.Il’lt)I’lS 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 iieressary,
`adjust the pH of the solution to be examined (or dilution
`thereof) so that the pH of the mixture of the lysato and
`Sample Solution falls within the pH rance specified by the.
`lysate manufacturer, usually 6.0---8.0. Til? pi~l may be ad»
`justed b use of an acid, base, or suitable buffer as recom~
`mende
`by the lysate manufacturer. Acids and bases ma
`be prepared from concentrates or solids with Water for BET
`in containers free oi detectable eiidotoxln. Buffers must be
`validated to be free of detectable endotoxin and interfering
`factors.
`‘
`
`DETERMINATION OF MAXIMUM VALID
`DILUTIOM (MVD)
`
`The maximum valid dilution is the rna>iirriurn allowable
`dilution of a specimen at which the erir_loti..i>v.iri limit can be
`determined. Determine the MVD from the following
`equation:
`"
`
`MVD = (endotoxin limit
`
`concentratiori of .’>7i.irrip/e .So/ui‘iori)/
`(iv)
`
`Endotoxin Limit-—The endotoxin limit for parenteral
`drugs, defined on the basis of dose, equals K/ll/1%,, where K
`f» K is 5 lJSl“’-~t.U/kg oi lziody wi:iql‘il. for any route of :idinli”iis‘tratlon otliui' than
`9. 3c.1~< E
`
`iiitratlwcal (tor whlcth it is 0.2 USP~l1l.J/liq
`.
`"
`t‘). For i'ei<.llopii;irim.
`ceuliral products not adiriinistoretl iriti‘a'll'iecully, t‘
`_oxin limit i
`;
`
`lal.er.:l as 'l 75 EU/l/, where l/ is the rnaximum iettoin
`oeci dose in n
`.
`ll‘ltt£iljli€tCfill
`admlnisleietl radiopharn‘ia(:eutir;a|s,
`the ondoioxin limit is ob»,
`tainsid by two lorrnula l4 EU/V. For forn'iulutioris (usiially 2‘irit'ii:ai‘icer products)
`
`aitlinimtered on a per square. rrieter of lziody iuria 2, the lormula is K/M
`where K ..-I ltiii Fl.)/nv and M is the inaxinium close:/n'i'..
`I
`
`Page 3
`
`

`
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`Page 4
`
`Page 4
`
`

`
`92 (85) Bacterial Endotoxins Test / Biological Tests
`
`tion to be examined to which Stan-
`th
`-'
`?lt:\?(;/eEil:llgl|(?’[C_g)Xlnehpari?¥lZ))?E‘i:n added and which has then been
`submitted to the chosen treatment.
`
`Limit Test
`
`Procedure-——Prepare Solutions‘ Al 3, C, arid D 35 5h0Wl"‘ in
`Table 2 and perform the test on these solutions iollowin
`the procedure above for Preparatory Testing, Test for Can ir~
`motion of Labeled Lysate Sensitivity.
`
`Table 2. Preparation of Solutions for the Gel-Clot Limit Test
`Endotoxin Concentration/
`Number of
`Solution to Which
`Replicates
`Endotoxin ls”A¢_lc_|ed
`Solution”
`2
`W None/Diluted Sonia/e Solution
`A
`2
`2K/Diluted Sample Solution
`3
`2
`2»./wiiiegior BET
`' C
`Z
`lslonejwater for BET
`[3
`* Prepare Solution A and the positive product control Solutlonli using
`a dilution not greater than the MVD and treatments as described for
`the Test for interfering Factors in Preparatory Testing. The positive cori-
`trol Solutions /3 and C Cf)i'lI'cill'i the Stanaora’ Enclotoirin Solution at «Li
`concentration corresponding to twice the labeled lysate sensitivity.
`The negative control Solution D consists of Water for BET.
`
`#
`
`'
`
`lnterpretation—-«The test is corisiderecl 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 under test
`complies with the test. When a positive result is found for
`both replicates of Solution A, the prepeiration 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 is found forgthe other, repeat
`the test.
`in the repeat test, the preparation under test com-A
`plies with the test it 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 ior_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
`
`USP 36
`
`be repeated using a greater dilution, not exceeding the
`MVD.
`
`Quantitative Test
`
`Procedure--The test quantifies bacterial (il’id0l;0%ll‘lS 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 Corifirrrioliori of lobelecl l..ysate Serisilivity.
`Calculation and lriterpretation»-The test is considered
`valid when the following three conditions are met: (1) lsotli
`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 2%.
`To determine the enclotoxin concentration of Solution A,
`calculate the endpoint concentration for each replicate by
`multiplying each endpoint: dilution factor by 1.. The erirlo-
`toxin concentration in the Sample Solution is the endpoint;
`concentration of the replicates. ii the test is conclucled with
`a diluted Sample Solution, calculate the concentration of en-
`dotoxin in the original Sample Solution by multiplying by the
`dilution factor.
`if none of the dilutions of the Sarnplc? Solu»
`tion is positive in a valid assay, report the endotoxlri concen-
`tration as less than it (if the diluted sample
`tested, re
`port as less than it times the lowest dilution factor of the
`sample). ll all dilutions are positive, the endotoxin concen-
`tration is reported as equal to or greater than the greatest
`dilution factor multiplied by it (e.g., iriitial dilution factor
`times eight times ‘it in Table 3).
`The preparation under test meets the r'eciuiremeiits oi the
`test it 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 turbidit
`. On the basis of the particular assay
`principle employe , tl is technique may be classified as ei»
`ther an endpoint-turbidimetric assay or a |<inetic—turbidimet~
`
`Table 3. Preparation of Solutions for the Gel~CIot Assay
`Endotoxin Concentration/
`Solution to Which Endotoxin
`
`Dilution
`
`Endotoxin
`
`Nurnher of
`
`Concentration_____"_ ,,,,__EtgpIlcate§_
`
`
`
`
`
`lt, 0.S'lt, and (3.2%,
`
`Solution
`Au
`
`_
`
`7“
`
`.
`
`Is Added
`_ __lf~lOfi€/§ClI7lpl(’ Solution
`
`Diluent
`M“ Water for_ BET
`
`_
`
`-
`
`m
`
`r
`-
`
`-.
`
`B0
`C5
`
`Zltfiiarnple Solution
`2’i.[Woter for BET
`
`__
`
`_
`
`_
`_
`_
`Wr,iiv2i' for BF T
`cc
`_,
`
`_..
`
`..
`
`_
`
`Pv
`
`._.
`
`-
`
`_
`
`._—.»._—4..
`
`Fac_:_tor_
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`_
`2
`4
`
`__
`
`..
`
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`l
`__
`__
`,2
`.4 M-
`"8
`4-
`
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`.....
`
`.._..c.
`._...,.
`
`g M _
`l
`lilone/Water for BET
`_
`D”
`5] Factors was corripleteisl.
`~ Solution A: Sample Solution under test at the dilution, not to exceedtlielmlx/lVU,Mvvitfi
`Subsequent dilution of the Sci/np/e Solution must not exceed the l\/l\/D. Use Water for BET to make a dilution series of four tubes containing lglie
`Sample Solution under test at concentrations of l, ‘/1, ‘/4, and ‘la. relative to the coriceritratiori used in the Tizsi for /ritei'fei'ii'i_i; Ff.lCl/.)l‘S, C‘)i;her dilutions
`up to the MVD may be used as appropriate,
`A Solution ti: Solution A containing standard endotoxin at ai concentration of 2')» (positive product control).
`4 Solution C: Two replicates of four tubes of Water for BET contairiirig the standard endotoxin at coricentrations of Zlt,
`respectively.
`ll Solution D: Water for BET (negative control).
`
`Page 5
`
`Page 5
`
`

`
`Ulzil’ 36
`
`Biological Tests / (85) Bacterial Endotoxins Test 93
`
`ric assay. The endpoint-turbidimetric assay is based on the
`c uantitative relationship between the concentration of en-
`clotoxins and the turbidity (absorbance or transmission) of
`the reaction mi>rture at the end of an incubation period.
`The lrinetic-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 1:1“),
`
`Chromogenir: Technique
`
`is an assay to measure the chromophore
`This lf_“‘,t‘Ll“Il‘ilCll.lf;‘
`released from a suitable chromogenic peptide b the reac-
`tion of endotoxins with I sate. On the basis of the particular
`assay principle employer, this technique may be classified as
`either an end oint-chrornogenic assay or a ltinetic»chrorno-
`genic assay. ’ he endpoint-chromogenic assay is based on
`the guarttitat.ive relationship between the concentration of
`enclotoxins and the release of chromophore at the end of
`an incubation period. The lrinetic-chrornogenic assay is a
`method to measure either the time (oriset time) needed to
`reach a predetermined absorbance of the reaction rni>o;ure,
`or the rate of color development. The test is carried out at
`the inculoation tenriperature rectornmended by the lysate
`lT‘li:1l'lUld(IllJl'FJl‘ (which is usually 37 :|: ‘l ").
`
`Preparatory Testing
`
`To assure the precision or validity of the turbidimetric and
`chrornogenic 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
`Storidard 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 mar‘itifacturer’s instruc-
`tions for the lysate (volume ratios,
`inc ubation time, temper-
`ature,
`‘H, etc.) If the desired range is 4 reater than two logs
`in the tinetic methods, additional stan ards should be in-
`cluded to braclret each log increase in the rance 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 COl‘lCE.'l”ill"E?ltl0l“\_'~; set up.
`Test for Interferin Factors---fielect an eridotoxin con-
`centration at or near tie middle of the endotoxin standard
`curve. l’repare Solutions A,
`ll, C, and D as shown in Table 4.
`Perfrirrii the test on 3‘olutiori.r /l,
`[3, C, and D at least in dupli-
`
`cate, according to the instructions for the lysate employed,
`for example, concerninc volume of Earn/ale Solution and Ly-
`sate TS, volume ratio 0 Sample Solution to Lysote T5, incu-
`bation time, etc.
`The test is considered valid when the following conditions
`are met.
`‘l. 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 reac ent employed.
`Calculate the mean recovery of the adcle
`endotoxin by
`subtracting the mean endotoxin concentration in the solu-
`tion,
`if any l_.S7olut‘ion A, Table 4), from that containing the
`added endotoxin (Solution [3, 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 §urn‘le Solution must be within
`50%---200% of the ltnown a ded endotoxin concentration
`after subtraction of any €;‘l'lCl(')tOXll'"l detected in the solution
`without acliiiled enclotoxiri.
`When the endoto>rin recovery is out of the specified
`range, the Sornple Solution under test is considered to con-
`tain interfering factors. Then, repeat the test using a greater
`dilution, not exceeding the it/IVD. Furthermore, interference
`of the Sample fiolution or diluted Surnple Solution not to ex-
`ceed the lvl‘i/D 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 endotoxins, 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 Interfering fac-
`tors under l-lreporotory 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.
`‘l. The results of the control Solution C comply with the
`i‘eqtiiren"iei'ils for validation defined for Assurance of
`gr/terio for the standard Curve under Preparatory
`esring.
`2. The enclotoxin recovery, calculated from the concen-
`tration found in Solution 8 after subtractiiig the’ C0”:
`centration of eridotoxin found in Solution A, 59 W”:l””
`the range of 500/0-2000/o.
`
`_,
`
`Number of Regllciltei
`________N
`|‘\.l,c_'it less irigi;i_,2,,, MM
`Not less than 2
`
`
`
`Table 4. We aratlon of 5oIq_t_l_grLs or the Inhibition/Enhai cement Test for Photometric Techniques
`Solution to which
`__Endoto_>_r_i_n Is Added
`....................,_§er1:ii2l.c:_f12ti_it!92L.
`Sninple Solution
`
`
`
`:» (IOl‘lC;(3l”lll‘i‘lill')l)ti (lo
` de
`ated }
`'
`
`
`
` lu
`n A:
`iurriplizr 5o/ul n may be diluted not to exc
`N iiolullon ll; lhe pi'oparv:itioi‘i imder test at the ziairie dilution as Solution A, contairiing adclr.-d enclotoxin at a r:oni:eritration equal to or near “CW-‘
`inid<,lle of lltl-i <il,rindnrtl curve,
`‘ ‘solution C: The l5l£Ll{'ltllC'3l“iL]l endoto>v.in at lghe corir:oriti'ations used in the validation of the method described for /lssurunce ol Criteria for the 5lUI‘IC/Uftl
`(furvii ul"lf;ll”:F l’i'ir/>tii'r1loI‘y liaslirir] (positive controls).
`'1 to/ullori U: Writer‘ for Blil (negative control).
`
`“ii coriizeritration is
`
`.......................tt(tI1r’L_l0I' W
`Al,/l_/olcir lor BC I’
`
`Page 6
`
`wgEacl‘i___,not less t_ltEll“I 2
`,/____l\lot less than 2
`
`
`
`Page 6
`
`

`
`94 (85) Bacterial Endotoxiris 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 endotoitin detection limit of the lysate reagent
`employed.
`
`interpretation
`
`in photometric assays, the preparation under test com-
`plies with the test if the mean eridotoxiii coriceritratiori of
`the replicates of Solution A, after correction for dilution and
`concentration, is less than the endotomn limit for the
`product.
`
`(87) BIOLOCJCAL RE/\CTlVlTY
`TESTS,
`lhl VlTRO
`
`The follovviri1g tests are designed to determine the bio|ogi~
`cal reactivity o’ mammalian cell cultures following contact
`with the elastomeric plastics and other polymeric materials
`with direct or indirect patient contaci: or of specific extracts
`prepared from the rriateriais under test.
`it is essential that
`the tests be performed on the specified surface area. When
`the surface area of the specimen cannot be determineci, use
`0.1 g of elasi:on'ier or 0.2 g of plastic or other material for
`every rriL of extraction fluid. Exercise care in the preparation
`of the rriaterials to prevent contamination with rriicroorgan
`isms and other foreign matter.
`Three tests are described (ie, the Agar Difitisiori Test, the
`Direct Contact Test