2013
`
`USP 36
`
`THE UNITED STATES PHARMACOPEIA
`
`NF 31
`
`Volume 1
`
`THE NATIONAL FORMULARY
`
`By authority (if the United States Pimrrimcopgial Convent/om
`Prepared by the Council of Experts and its; Expert“ COrriiTliftem
`
`Official from May 7, 2073
`
`Tim «imiqriatiom or": the mver of thia publication, “USP NF 2013,” i5 er ease pf
`idemLii’iceaition MW. The publication coritaim two Separaie comi'mndia:
`(iii; Ui'izz'ieci
`3mm: F‘l’iam'i(iwpieiici, Thirtyvfiixth Reviraimn, and The National fO/T'HU/CII‘V, ThirtymFirsi
`Ed i ii (1m.
`
`THE UWTED STATES PHARMACOPEIAL CONVENWOM
`1260i ‘Twinbrmia Parkway, Roclwiile/ MD 20852
`
`Page 1
`
`CUBIST 2202
`AGILA V. CUBIST
`IPR2015-00143
`
`CUBIST 2202
`AGILA v. CUBIST
`IPR2015-00143
`
`

`

`ii
`
`USP 36
`
`SlX~lVlONTH IMPLEMENTATION GUIDELINE
`
`The United Rates Phormocopeia-Nntional Formulory and its supplements become official six months after being released to
`the public, The USP—NF, whit; 1
`is released on November 1 oi each year, becomes official on May 'I of the following year. This
`sixwmonth implementation timing gives users more time to bring their methods and procedures into compliance with new
`and revised USP—NF requirements.
`The table below describes the official dates of the USP—NF and its supplements. The 2011 USP 35—well: 30, and its supple
`merits, Interim Revision Announcements (IR/ls) and Revision Bulletins to that edition, will be official until May 1, 2013, at which
`time the USP 36-NF 37 becomes official.
`
`
`
`Official Date
` Release Date”
`_ __.Qifiti.al_ML“
`USP 36—-NF J]
`November 1, 2012
`May I, 201 s
`
`May 1, 2014 (except as superseded by supizilements, Ill/ls, and
`”Bet/sfooter/latirzs)
`_____________________________________________________
`August 1, 2013
`February 1, 2013s
`First Etrpplernenl to the
`May 1, 2014 (except as superseded by Second supplement, ill/ls,
`
`aindjievision Bulletins) ..
`USP 36 NF 31
`1
`June 1, 201%
`December 1, 201 '5
`Second Supplement to the
`May 1, 2014 (except as supers" :
` L
`USP “JG-«NF 3i
`‘
`USP 37~NF 3‘2
`May 1, 2014
`November 1, 20:1 3
`
`
`
`
`
`
`
` IRA__,,,,,,,, Pf Bestirmfla e___w____¢isw.cn_§11t_!2geflat£.m_“mLEA Postingjl
`
`
`
`The table below gives .he details of the IR/ls that will apply to USP 364% 37.
`
`
`
`titlLLJQ‘I 3
`39(1)
`lanuary L 2011s
`March 31, 2013
`,,,,§!y 31,2013
`
`
`.2013
`somber 1
`_________________
`March '1
`2012‘s_
`May 31 2013
`uly .26 2013 AAAAA_
`sea)
`
`
`timber 1 201 3%Wm.“........w
`i ,,_,May ‘1, 2013
`_
`lulv 31, 2013
`September 27 {91
`39g3)_‘
`
`39(4)
`lulv Z 2013
`‘_ W
`Beptember 30, 2013M_______
`ovember 29,.
`»
`anuaryj 20 'l 4 ___________
`
`l4
`secs)
`Eeptembe;4 2.913
`November 30 2013 W
`
`
`#320?) ,_
`l‘sloven‘rber is 2013
`lanuarv 31, 20H
`Revision Bulletins publis‘ ed on the USP website become official on the date specified in the Revision Bulletin.
`
`
`
`
`NOTICE AND WARNING
`
`Concerning U.5. Potent or Trademark Rights—The inclusion in The United States Phormncopeio or in the National Formulary of a
`monograph on any drug in respect to which patent or trademark rights may exist shall not be deemed and is not intended
`as, a grant of, or authority to exercise, any right or privilege protected by such patent or trademark. All, such rights and
`privileges are vested in the patent or trademark owner, and no other person may exercise the same without express
`permission, authority, or license secured from such patent or traden‘iark owner.
`H
`
`Concerning .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 Secretary otthe UBPC Board of
`rustees.
`
`Copyright to 2012 The United States Pl‘iarmacopeial Convention
`12601 Twinbroolr. Parkway, Rockville, MD 20852
`
`All rights reserved.
`ISSN: 01957996
`
`ISBN: 978-1936424424
`
`Printed in the United States by United Book Press, Inc, Baltimore, MD
`
`Page 2
`
`Page 2
`
`

`

`90 (81) Antibioticawl‘viicrobiai Asaaya / Biological Team
`
`USP 36
`
`Table A241. Test for Outlier Meawrements
`
`in samples from a normal population, gaps equal to or larger than the following values oi Gr,
`(22, and C; occur with a probability P - 0.01, when
`
`outlier r‘neaturen‘ients can occur only at one end or with P} 0.92 when they ma
`occur__at either end.
`._
`-.
`________
`
`N
`3
`l
`4
`5
`6
`..
`_
`_, -2....._____-
`
`c,
`,,--
`09257
`i
`_
`0,889
`0781
`05998
`.
`0,5437
`_______
`
`
`it;
`a
`_ t '
`9
`.. -
`i 0
`.._.
`-
`-......
`- -
`
`i"
`g
`0.68]
`_
`0.634
`0,597
`-
`_..._-
`
`
`
`
`
`
`
`
`
`N
`g,
`
`’
`
`'
`
`7
`
`'1 'i
`0674
`
`i
`l_
`
`_
`12
`0.643 ,_
`
`-
`
`_
`........
`
`13
`0.617 _
`
`_
`
`-
`
`(85) BACTERiAL ENDOTOXWS
`TEST
`
`“Portions of this general chapter have been harn‘ionized _
`with the correaponding texts of the European Pharmacopoeia
`and/or the [oporiese Pharmacopoeia. ‘ihoae portions that are
`not harmonized are marked with symbols in) to specify this
`-.
`t.
`fagthwe Bacterial Endotoxina Test (BET) is a teat to detect or
`quantity endotoxina from Cram-rte" ative bacteria usinq
`amoebocyte lyaate from the horaes oe crab (limulus poly-
`phernus or ibcliyp/eus tridentotus‘).
`There are three techniques for this teat: the gelciot tech
`nique, which i5 based on
`ei ‘iorr‘nation; the turbidimetnc
`.
`technique, based on the tevelopment of turbidity after
`cleavage of an endogenout substrate; and the chromogenic
`technique, bated on the development of color after cleav~
`age of a aynthetic peptide~chromogen complex. Proceed by
`any of the three techniquet for the test.
`in the event of
`doubt or dispute, the final decision it made based upon the
`gei»ciot limit teat unieaa otherwise indicated in the mono— _
`graph for the product being tested.
`ihe‘teat iii carried out in
`a manner that avoids endotoxin contamination.
`
`APPARATUS
`
`Depyrogenate all glaaaware and other heaiettable materi—
`als in a hot air oven using a validated procetaflu A com~
`moniy used minimum time and temperature it 30 min at
`250% If employing plaatic apparatua, auch at microplatea .
`and pipet tips for automatic pipettera, use apparatua that it
`shown to be free oi detectable endotoxin and does not In-
`teriere in the teat. [NOTE-in this chapter, the term ”tube”
`inciudea any other receptacle SUCH as a microtiter welt]
`
`REAGENTS AND TEST EQLUHDNS
`
`Amoebocyte Lyaate‘A lyophilized product obtained
`from the Iyaate of amoebocytea (white biooci cella) from the
`horsesi‘ioe crab (Li/riulua polyphemua or Tochyp/eut
`tridentotut). Thia reagent. refers oni
`to a product manufac—
`tured in accordance with the regu ations of the competent
`authority. iN()Ttm—Amorabocyte Lyrote reacts to some [iglo—
`cans in addition to endotoxint. Amoebocyte L tote prepara-
`tions that do not react to qlucans are avaiiab e: they are
`prepared by removing the C3 factor reacting to glucant from
`Amoebocyte Lysate or by inhibiting the 6 factor reacting Sys—
`tern of Arnoebocyte Lysote and may be used for endotoxin
`teating in the presence of glucana.j
`Water for Bacterial Endotoxina Test (BEHMUse Water
`
`for injection or water produced by other proceduret that
`
`*i For a validity teat oi the procedure for inactivating endotoains, s L‘ljlyrv
`i-Ieui Sterilization under Sterilization and Sterility Acaurunce of ternary/tow rim:
`(‘Ipr (121 ii Um tysoie T3 having a Semitivity of not less than 04 a Endotoxm
`Unit per mm
`
`Showt no reaction with the lysate employed, at the detec»
`tion limit of the reagent.
`Lyaate 'rfimDisaolve Amoebocyte Lytote in Water for BET,
`or in a butter recommended by the. lyaate manufacturer, by
`entle stirring. Store the reconttituted iysate, refrigerated or
`rozen, according to the Specifications of the manufacturer.
`
`PREPARATION OF SOLUTIONS
`
`Standard Endotoxin Stock SiolutionwA Standard Enclo—
`toxin Stock Solution i5 prepared from a USP Endotoxin Refer—
`ence Standard that lliléi been calibrated to the current WHO
`international Standard for Endotoxin. Follow the specificae
`tiona in the package ieailet and on the label ior preparation
`and Storage of the Standard Endoloxin Si‘ocli Solution. Endo«
`toxin it expreaaed in Endotoiiin Units (EU).
`[NOTE--------One USP
`Endotoxin Unit (EU) is equal to one International Unit (iii)
`of endotoxirti
`
`Standard Endotoxin Eolutiona-WAiter mixing the Eton”
`dorcl Endot’oxin Stock Solution vigorouaiy, prepare appropriate
`serial dilutiona of Standard Endotoxin Solution, usin Water
`for BET. Use dilutiona as aoon at possible to avoid on oi
`activity by adaorption.
`Sample Solutionawi’repare the Sample Solutions by dire
`solving or diluting druga uaing Water for BET. Some cubw
`stancea or preparations may be more appropriately dit~
`solved, or diluted in other aqueous aolutiona.
`if neceritary,
`adjuat the pH of the solution to be examined (or dilution
`thereof) 50 that the. pH of the mixture of the iyaate and
`Sample Solution tailt within the pH rance specified by the
`lysate manufacturer, utualiy oil-~80. Tie phi may be ad»
`justed b use of an acid, base, or suitable buffer as recom»
`mende
`by the lysate manuiacturer. Acida and baaea ma
`be prepared from concentrates or SOlldS with Water for BET
`in containers tree oi detectable endotoxin. Buttery mutt be
`validated to be free of detectable endotoxin and interfering
`factors.
`‘
`
`DETERMINATION OF MAXIMUM VALID
`DILUTIOM (MVD)
`
`The maximum valid dilution it the maximum allowable
`dilution of a specimen at which the endotuxin limit can be
`determined. Determine the MVD from the following
`equation:
`..
`
`MVD = (endotoxin limit >6. concentration of Sample Solution)/
`(1)
`
`Endotoxin Limit—The endotoxin limit for parenteral
`drugs, defined on the basis of dose, equaia lot/lira, where K
`'N K is 5 USl’nEU/kq oi body weight for any route oi administration other than
`intrathecai (for which it it. 0.2 USP~ilUlkq oi body W "
`t). For radiopharma-
`
`
`ceuiiral products not adminittered intrall’iecally, t‘
`.oxin limit i
`
`
`lated as.
`l 75 [EU/V, where i/ it. the maximum rectum
`oed dote in n
`..
`intrathecall
`administered radiopharn‘iamuticalt,
`the ondoroxin limit hi ob
`tamed by tie lorrmria 14 WW. For forn'iuiationr» (ritually ariticai‘iter products)
`administered on a per aquare meter of body ‘iuria
`_
`2, the iormula ii. K/M,
`
`where K _.-. itiil FU/m/ and M is the maximum (loan/ma.
`
`Page 3
`
`Page 3
`
`

`

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`Page 4
`
`Page 4
`
`

`

`92 (85) Bacterial Endotoxins Test / Biological Tests
`
`above usinc the preparation to be examined to which Stan-
`dard Endot'gixin has been added and which has then been
`submitted to the chosen treatment.
`
`Limit Test
`
`Procedurew-Prepare Solutions A, B, C, and D as shown in
`Table 2, and perform the test on these solutions followrn .
`the procedure above for Preporqtory Testing, Test for Con ir-
`motion of Lobe/ed tysote Sensitlvrty.
`
`
`
`
`
`Table 2. Preparation of Solutions for the Gel~Clot Limit Test
`Endotoxin Concentration/
`Solution to Which
`Number of
`
`Solution”
`Endotoxin Isr‘Agc‘Ied
`Replicates
`
`A
`N None/Diluted Sumo/e Solution
`2
`
`B
`ZK/Diluted Sample Solution
`2
`- L
`I
`ZA/ Woterfor BET
`_.
`2
`
`D 2 NonejWoter for BET
`
`* Prepare Solution A and the positive product control Solution it 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 13 and C contain the Standard Ericlotorrin Solution at a
`concentration corresponding to twice the labeled lysate sensitivity.
`The negative control Solution D consists of Water for BET.
`
`Interpretation—«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 under 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 is found for‘the other, repeat
`the test.
`in the repeat test, the preparation under test com-I
`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 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
`
`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 Lube/ed tysote Sensitivity.
`Calculation and interpretatioanhe 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 2A.
`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 by multiplying by the
`dilution factor.
`if none of the dilutions of the Sample Soluw
`tion is positive in a valid assay, report the endotoxln 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). It 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., initial dilution factor
`times eight times it in Table 3).
`The preparation under test meets the requirements of 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 kinetic-turbidimet
`
`
`
`
`
`
`
`
`
`Table 3. Preparation of Solutions for the GeI~Clot Assay
`
`Endotoxin Concentration/
`Solution to Which Endotoxin
`Dilution
`Endotoxin
`Number of
`
`Solution
`i“
`I5 Added
`Diluent
`m
`Factor
`Concentrgflgr!________ ______[itgpllcates,
`
`Au
`‘ “None/Sample Solution
`_
`M“ Water for: BET
`___Ml
`.
`_
`
`_
`r
`'2
`
`s
`..
`—-
`‘-
`~'
`4
`-
`
`
`Bl)
`ZMSornp/e Solution
`_
`_
`..
`.
`.
`L” m
`
`C5
`ZMWoter for BET
`_
`Writer for BF T
`l n
`w
`
`w .,
`as,
`_,
`,
`,2
`c
`M
`.4 m- m-.....m
`"8
`.—
`mm
`.._.,c
`M
`Dil _ _ None/Water for BET i ~--— _,
`
`
`
`
`
`
`A Solution A: Sorrip/e Solution under test at the dilution, not to exceed the MVD, with which the Test for interfering Factors was corripleteisl.
`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 i, ‘/1, ‘li, and Vi relative to the concentration used in the 72351 for Interfering Frictors, Other dilutions
`up to the MVD may be used as appropriate,
`A Solution ti: Solution A containing standard endotoxin at a concentration of 2')» (positive product control).
`a Solution C: Two replicates of four tubes of Water for BET containing the standard endotoxin at concentrations of 27c,
`respectively.
`it Solution D: Water for MT (negative control).
`
`’lt, 0.8x, and 0.23%,
`
`Page 5
`
`Page 5
`
`

`

`USP 36
`
`Biological Tests / (85) Bacterial Endotoxins Test 93
`
`ric assay. The endpointoturbidir‘netric assay is based on the
`c uantitative relationship between the concentration of en»
`clotoxins and the turbidity (absorbance or transmission) of
`the reaction mixture at the end of an incubation period.
`The lrineticvturbidimetric assay is a method to measure ei-1
`ther the time (onset time) needed to reach a predetermined
`absorbance or transn’rission of the reaction mixture, or the
`rate of turbidity development. The test is carried out at the
`ir‘icuhation temperature recommended by the lysate manu~
`facturer (which is usually 37 1:1“),
`
`Chromogenic Technique
`
`This technique is an assay to measure the chromophore
`released from a suitable cl‘irorriogeriic peptide b the reac~
`tion of endotoxins with | sate. On the basis of the particular
`assay principle en‘iployec, this technique may be classified as
`either an end oint-cl'irornogenic assay or a ltinetic»chromo~
`genic assay. ' he eridpointflcl‘iromogenic assay is based on
`the quantitative relationship between the concentration of
`endotoxins and the release of cl’rromophore at the end of
`an incubation period. The kinetic-chrornogenic 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 teiriperature recommended by the lysate
`manufacturer (which is usually 37 :I:
`'l ").
`
`Preparatory Testing
`
`To assure the precision or validity of the turbidimetric and
`ci‘irornogenic 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 concer‘itration according to the manufacturer’s instruc—
`tions for the lysate (volume ratios, incubation time, temper-
`ature,
`‘H, etc.) If the desired range is r reater than two logs
`in the tinetic methods, additional stan ards should be in-
`cluded to bracket each log increase in the rance of the stain“
`dard curve. The absolute value of the correlation coeffi—
`cient, r‘, must be greater than or equal to 0.980 for the
`range of endotoxrn concentrations set up.
`Test for Interferin Factors--8elect an endotoxin con
`centration at or near tie n‘tiddle of the endotoxin standard
`curve. Prepare Solutions A,
`ti, C, and D as shown in Too/e 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, concerninc volume of sample 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 adde
`endotoxiri by
`subtracting the mean endotoxin concentration in the solu-
`tion,
`if any (Solution A, Table 4), from that containing the
`added endotoxin (Solution B, 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 Earn/e Solution must be within
`50%....2000/0 of the known a ded 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 .Sorriple 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 fiolution or diluted Sump/e Solution not to ex-
`ceed the MVD may be eliminated by suitable validated
`treatment such as filtration, neutralization, dialysis, or heal;
`treatment. To establisi’r 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 Foc-
`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.
`‘l. The results of the control Solution C comply with the
`requirements for validation defined for Assurance of
`Enter/o for the Etondord Curve under Preparatory
`esring.
`2. The endotoxin recovery, calculated from the concen—
`tration found in solution B after subtracting the 90in
`centration of endotoxin found in Solution A,
`l9 W'll‘l”
`the range of soc/Moors,
`
`
`Table 4. We duration of Eolutionf or the Inhibition/Entire cement Test for Photometric Techniques
`Solution to Which
`“Endotogrjn Is Added
`
`Number of Reflicates
`
` 2 concentrations (lo
`
`ated }
`de '
`
`
`, NNune
`
`
`it may be diluted not to ext
`sump/e iii/u!
`“ .fiolullon ii: The preparation under test at the lidliilf‘. dilution as Solution A, cruitaining added eridotoxin at a concentration equal to or near the
`middle of the standard curve.
`. ”solution C: The staritlc-ird endutoxin at the concentrations used in the validation of the method described for Assurance of Criteria for the Standard
`(Tu/w: under l’nrpurnlru‘y lasting (positive controls).
`'1 solution 1,): Water for Biff (negative control).
`
`Not less than 2
`
` «jachwnot less than 2
`
`WNot less than 2
`
`
`
`,llljdlc’l’ for BC i'
`
`Page 6
`
`Page 6
`
`

`

`94 (85> Bacterial Endotoxins Test / Biological Tests
`
`3. The result of the negative control Solution D does not
`exceed the lirriit of the blank value required in the
`description of the iysate employed, or it is less than
`the endotoxin detection iirnit of the lysate reagent
`employed.
`
`interpretation
`
`in photometric: assays, the preparation under test com-
`plies with the test if the mean endotoxrn coiicentration of
`the replicates of Solution A, after correction for dilution and
`concentration, is less than the endotoxin limit for the
`product.
`
`(87) BlOLOGlCAL REACTlVlTY
`TESTS,
`lhl VlTRO
`
`The followinc tests are designed to determine the biologi~
`cal reactivity o' mammalian cell cultures following contact
`with the elastomeric plastics and other poiyrneric 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 elaston’ier 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 (let, 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 izsrocodures; contacts
`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 (1 Tim-«USP High-Density Poly“
`ethylene its. UEP Positive Bioreoction R5.
`Cell Culture Preparation—Prepare multiple cultures of L
`929 (ATCC cell line CCL i, NCTC clone 929) mammalian
`fibroblast cells in serum-sup.ilemented minimum essential
`medium having a seeding
`ensity of about 103 cells per mt.
`incubate the cultures at 37 :t: l” in a humidified incubator
`for not less than 24 hours in a 5 :r.
`'i% carbon dioxide at-
`mosphere until a monolayer, with greater than 30% conflum
`ence, is obtained. Examine the prepared cultures under a
`microscope to ensure uniform, riearconiluent monolayers.
`['l‘iloi‘E—The reproducibility of the In Vitro Biological Reactivity
`Tests depends upon obtaining uniform cell culture density]
`Extraction