2000
`
`USP24
`
`THE UNITED STATES PHARMACOPEIA
`
`THE NATIONAL FORMULARY
`
`By authority of the United States Pharmacopeia!
`Convention, Inc., meeting at Washington, D.C.,
`March 9-12, 1995. Prepared by the Committee of
`Revision and published by the Board of Trustees
`
`Official from January 1, 2000
`
`UNITED STATES PHARMACOPEIAL CONVENTION, INC.
`12601 Twinbrook Parkway, Rockville, MD 20852
`
`LUPIN EX1051, Page 1
`
`

`
`NOTICE AND WARNING
`
`Concerning U.S. Patent or Trademark Rights
`
`The inclusion in the Pharmacopeia 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 trademark owner.
`
`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 of the
`USPC Board of Trustees.
`
`© 1999
`The United States Pharmacopeia! Convention, Inc.
`12601 Twinbrook Parkway, Rockville, MD 20852.
`All rights reserved
`ISSN 0195-7996
`ISBN 1-889788-03-1
`
`Printed by National Publishing, Philadelphia, PA
`
`LUPIN EX1051, Page 2
`
`

`
`Combined Index to USP 24 and NF 19
`
`Ampi-Bacit
`
`2543
`
`Ampicillin (continued)
`and sulbactam for injection, 140
`soluble powder, 138
`Amprolium, 141
`oral solution, 142
`soluble powder, 142
`Amrinone, 142
`injection, 143
`Amyl
`acetate, 217 4
`alcohol, 2174
`nitrite, 144
`nitrite inhalant, 145
`tert-Amy! alcohol, 2174
`a-Amylase, 2174
`Amylene hydrate, 2414
`Analysis
`automated methods of (16), 1801
`and design of biological assays (III), 1837
`phase-solubility (1171), 2122
`thermal (891), 1999
`Anethole, 2414
`Anileridine, 145
`hydrochloride, 146
`hydrochloride tablets, 146
`injection, 145
`Aniline, 2174
`blue, 2174
`Animal and vegetable substances, 13
`Anion-exchange
`resin, 50- to 100-mesh, styrene-divinylben(cid:173)
`zene, 2175
`resin, chloromethylated polystyrene-divinyl(cid:173)
`benzene, 217 4
`resin, strong, lightly cross-linked, in the chlo-
`ride form, 2175
`p-Anisidine, 2175
`Anisole, 2175
`Antazoline phosphate, 147
`Anthracene, 2175
`Anthralin, 147
`cream, 148
`ointment, 148
`Anthrone, 2175
`TS, 2233
`Antibiotics
`iodometric assay (425), 1872
`-microbial assays (81 ), 1823
`Anticoagulant
`citrate dextrose solution, 148
`citrate phosphate dextrose adenine solution,
`!50
`citrate phosphate dextrose solution, 149
`heparin solution, !51
`sodium citrate solution, 152
`Antihemophilic
`factor, 152
`factor, cryoprecipitated, 152
`Antinticrobial
`agents-<:ontent (341), 1864
`effectiveness testing (51), 1809
`Antimony
`pentachloride, 2175
`potassium tartrate, 153
`sodium tartrate, 153
`trichloride, 2175
`trichloride TS, 2233
`Antipyrine, 153
`and benzocaine otic solution, !54
`benzocaine, and phenylephrine hydrochloride
`otic solution, 154
`Antirabies serum, !55
`Anti-thrombin-III for anti-factor X, test, 2176
`Antivenin
`(crotalidae) polyvalent, !55
`(Iatrodectus mactans), 155
`(micrurus fulvius), !56
`Apomorphine
`hydrochloride, 156
`hydrochloride tablets, !56
`Apparatus, 6
`automated radiochemical synthesis (1015),
`2008
`cleaning glass (I 051), 2036
`for tests and assays, 1801
`
`volumetric (31), 1808
`volumetric, and prescription balances (1176),
`2124
`Approximate solubilities of USP and NF articles,
`2299
`Apraclonidine
`hydrochloride, 157
`ophthalntic solution, !57
`Aprobarbital, 2176
`L-Arabinitol, 2176
`Arginine, 158
`hydrochloride, 158
`hydrochloride injection, !59
`Aromatic elixir, 2415
`Arsanilic acid, 159
`Arsenic (211), 1856
`in reagents, 2167
`trioxide, 2176
`Articles
`admitted to USP 23 by supplement, !iii
`biotechnology-derived (1045), 2011
`included in USP 23 but not included in USP
`24, lix
`of incorporation, xxvi
`official, impurities in ( 1086), 2049
`Ascorbic
`acid, 160
`acid injection, 160
`acid oral solution, 161
`Ascorbyl palmitate, 2415
`L-Asparagine, 2176
`Aspartame, 2415
`Aspirin, 161
`acetantinophen, and caffeine tablets, 21
`and acetaminophen tablets, 20
`aluntina, and magnesia tablets, 168
`aluntina, and magnesium oxide tablets, 169
`boluses, 162
`butalbital, and caffeine capsules, 266
`butalbital, and caffeine tablets, 267
`butalbital, caffeine, and codeine phosphate
`capsules, 268
`and butalbital tablets, 265
`caffeine, and dihydrocodeine bitartrate
`capsules, 170
`capsules, 163
`carisoprodol, and codeine phosphate tablets,
`316
`and carisoprodol tablets, 315
`codeine phosphate, alumina, and magnesia
`tablets, 172
`and codeine phosphate tablets, 171
`delayed-release capsules, 163
`delayed-release tablets, 166
`effervescent tablets for oral solution, 167
`extended-release tablets, 167
`and oxycodone tablets, 1238
`and pentazocine hydrochloride tablets, 1289
`propoxyphene hydrochloride, and caffeine
`capsules, 1422
`and propoxyphene napsylate tablets, 1427
`suppositories, 164
`tablets, 165
`tablets, buffered, 165
`Assay
`alginates (311 ), 1863
`alpha tocopherol (551), 1884
`amphetamine (3 11), 1864
`barbiturate (361 ), 1866
`calcium pantothenate (91 ), 1836
`cobalantin radiotracer (371), 1867
`dexpanthenol (115), 1847
`epinephrine (391 ), 1868
`folic acid (411), 1872
`iodometric, antibiotics (425), 1872
`niacin or niacinantide (441), 1873
`riboflavin (481), 1879
`single-steroid (511), 1880
`for steroids (351 ), 1866
`thiamine (531), 1881
`vitamin A (571), 1890
`vitamin B12 activity (171), 1851
`vitamin D (581), 1891
`Assays
`biological, design and analysis of (III), 1837
`insulin (121), 1848
`microbial, antibiotics (81), 1823
`
`tests and, 6
`and tests, apparatus for, 6, 180 I
`and tests, biological, 1823
`and tests, chentical, 1853
`Assemblies, transfusion and infusion (161), 1851
`Astentizole, 173
`tablets, 174
`Atenolol, 175
`and chlorthalidone tablets, 176
`injection, 175
`tablets, 176
`Atontic
`weights, 2305
`weights and chemical formulas, 3
`Atropine, 177
`sulfate, 178
`sulfate injection, 178
`sulfate ophthalmic ointment, 179
`sulfate ophthalmic solution, 179
`sulfate and diphenoxylate hydrochloride oral
`solution, 585
`sulfate tablets, 179
`sulfate and diphenoxylate hydrochloride tab(cid:173)
`lets, 586
`Attapulgite
`activated, 180
`colloidal activated, 180
`Aurothioglucose, 180
`injectable suspension, 181
`Automated
`methods of analysis (16), 180 I
`radiochentical synthesis apparatus (1015),
`2008
`A vobenzone, 181
`Azaperone, 182
`injection, 182
`Azatadine
`maleate, 183
`maleate tablets, 183
`Azathioprine, 184
`sodium for injection, 185
`tablets, 184
`Azithromycin, 185
`capsules, 186
`for oral suspension, 187
`Aztreonam, 187
`injection, 188
`for injection, 188
`
`B
`
`Bacampicillin
`hydrochloride, 189
`hydrochloride for oral suspension, 190
`hydrochloride tablets, 190
`Bacitracin, 190
`for injection, 191
`methylene disalicylate, soluble, 192
`methylene disalicylate soluble powder, 192
`neomycin and polymyxin B sulfates, and
`hydrocortisone acetate ointment, 1162
`neomycin and polymyxin B sulfates, and
`hydrocortisone ophthalmic ointment, 1163
`neomycin and polymyxin B sulfates, and
`lidocaine ointment, 1163
`and neomycin and polymyxin B sulfates
`ointment, 1162
`and neomycin and polymyxin B sulfates
`ophthalntic ointment, 1162
`and neomycin sulfate ointment, 1154
`ointment, 191
`ophthalmic ointment, 191
`and polymyxin B sulfate topical aerosol, 192
`zinc, 192
`zinc, neomycin and polymyxin B sulfates, and
`hydrocortisone ointment, 1164
`zinc, neomycin and polymyxin B sulfates, and
`hydrocortisone ophthalmic ointment, 1165
`zinc, neomycin an<l polymyxin B sulfates, and
`hydrocortisone acetate ophthalntic ointment,
`1165
`
`LUPIN EX1051, Page 3
`
`

`
`USP 24
`
`Microbiological Tests I (51) Antimicrobial-Effectiveness Testing
`
`1809
`
`the receiving vessel to drain the tips. Volume readings on burets
`should be estimated to the nearest 0.01 mL for 25- and 50-mL
`burets, and to the nearest 0.005 mL for 5- and 10-mL burets. Pipets
`calibrated "to contain" are called for in special cases, generally for
`------
`
`measuring viscous fluids like syrups; however, a volumetric flask
`may be substituted for a "to contain" pipet. In such cases, the
`pipet or flask should be washed clean, after draining, and the wash(cid:173)
`ings added to the measured portion.
`
`Designated volume, mL
`Limit of error, mL
`Limit of error, %
`
`10
`0.02
`0.20
`
`Designated volume, mL
`Limit of error, mL
`Limit of error, %
`
`0.006
`0.60
`
`Designated volume, mL
`Subdivisions, mL
`Limit of error, mL
`
`Volumetric Flasks
`50
`0.05
`0.10
`
`100
`0.08
`0.08
`
`Transfer Pipets
`5
`0.01
`0.20
`
`10
`0.02
`0.20
`
`25
`0.03
`0.12
`
`2
`0.006
`0.30
`
`Burets
`10 ("micro" type)
`0.02
`0.02
`
`250
`0.12
`0.05
`
`25
`0.03
`0.12
`
`25
`0.10
`0.03
`
`500
`0.15
`0.03
`
`50
`0.05
`0.10
`
`1000
`0.30
`0.03
`
`100
`0.08
`0.08
`
`50
`0.10
`0.05
`
`(41) WEIGHTS AND BALANCES
`The intent of this section is to bring the requirements for weights
`into conformity with American National Standard ANSI/ASTM
`E617, "Laboratory Weights and Precision Mass Standards." This
`standard is incorporated by reference and should be consulted for
`full descriptions and information on the tolerances and construction
`of weights. 1
`Pharmacopeia! tests and assays require balances that vary in ca(cid:173)
`pacity, sensitivity, and reproducibility. Unless otherwise specified,
`when substances are to be "accurately weighed" for Assay the
`weighing is to be performed with a weighing device whose mea(cid:173)
`surement uncertainty (random plus systematic error) does not ex(cid:173)
`ceed 0.1% of the reading. Measurement uncertainty is satisfactory
`if three times the standard deviation of not less than ten replicate
`weighings divided by the amount weighed, does not exceed 0.001.
`Unless otherwise specified, for titrimetric limits tests, the weighing
`shall be performed to provide the number of significant figures in
`the weight of the analyte that corresponds to the number of signif(cid:173)
`icant figures in the concentration of the titrant.
`The class designations below are
`in order of increasing
`tolerances.
`Class I. I weights are used for calibration of low-capacity, high(cid:173)
`sensitivity balances. They are available in various denominations
`from I to 500 mg. The tolerance for any denomination in this class
`is 5 f.Lg. They are recommended for calibration of balances using
`optical or electrical methods for accurately weighing quantities be(cid:173)
`low 20 mg .
`Class I weights are designated as high-precision standards for
`calibration. They may be used for weighing accurately quantities
`below 20 mg. (For weights of I 0 g or less, the requirements of
`class I are met by USP XXI class M.)
`Class 2 weights are used as working standards for calibration,
`built-in weights for analytical balances, and laboratory weights for
`routine analytical work. (The requirements of class 2 are met by
`USP XXI class S.)2
`1 Copies of ASTM Standard E 617-81 (Reapproved 1985) may
`be obtained from the American Society for Testing and Materials,
`1916 Race Street, Philadelphia, PA
`19103.
`2 Note that the designations S and P no longer designate weight
`classes but rather weight grades, that is, design limitations such as
`range of density of materials, surface area, surface finish, corrosion
`resistance, and hardness.
`
`Class 3 and class 4 weights are used with moderate-precision
`laboratory balances. (Class 3 requirements are met by USP XXI
`class S-1; class 4 requirements are met by USP XXI class P.)2
`A weight class is chosen so that the tolerance of the weights used
`does not exceed 0.1% of the amount weighed. Generally, class 2
`may be used for quantities greater than 20 mg, class 3 for quantities
`of greater than 50 mg, and class 4 for quantities of greater than 100
`mg. Weights should be calibrated periodically, preferably against
`an absolute standard weight.
`
`Microbiological Tests
`
`(51) ANTIMICROBIAL
`EFFECTIVENESS TESTING
`Antimicrobial preservatives are substances added to nonsterile
`dosage forms to protect them from microbiological growth or from
`microorganisms that are introduced inadvertently during or subse(cid:173)
`quent to the manufacturing process. In the case of sterile articles
`packaged in multiple-dose containers, antimicrobial preservatives
`are added to inhibit the growth of microorganisms that may be
`introduced from repeatedly withdrawing individual doses.
`Antimicrobial preservatives should not be used as a substitute for
`good manufacturing practices or solely to reduce the viable micro(cid:173)
`bial population of a nonsterile product or control the presterilization
`bioburden of multidose formulations during manufacturing. Anti(cid:173)
`microbial preservatives in compendia! dosage forms meet the re(cid:173)
`quirements for Added Substances under Ingredients and Processes
`in the General Notices.
`All useful antimicrobial agents are toxic substances. For maxi(cid:173)
`mum protection of patients, the concentration of the preservative
`shown to be effective in the final packaged product should be below
`a level that may be toxic to human beings.
`
`LUPIN EX1051, Page 4
`
`

`
`1810
`
`(51) Antimicrobial-Effectiveness Testing I Microbiological Tests
`
`USP 24
`
`The concentration of an added antimicrobial preservative can be
`kept at a minimum if the active ingredients of the formulation pos(cid:173)
`sess an intrinsic antimicrobial activity . Antimicrobial effectiveness,
`whether inherent in the product or whether produced because of the
`addition of an antimicrobial preservative, must be demonstrated for
`all injections packaged in multiple-dose containers or for other
`products containing antimicrobial preservatives. Antimicrobial ef(cid:173)
`fectiveness must be demonstrated for multiple-dose topical and oral
`dosage forms and for other dosage forms such as ophthalmic, otic,
`nasal, irrigation, and dialysis fluids (see Pharmaceutical Dosage
`Forms (1151 )).
`This chapter provides tests to demonstrate the effectiveness of
`antimicrobial protection. Added antimicrobial preservatives must be
`declared on the label. The tests and criteria for effectiveness apply
`to a product in the original, unopened container in which it was
`distributed by the manufacturer.
`
`PRODUCT CATEGORIES
`For the purpose of testing, compendia! articles have been divided
`into two categories (see Table 1). Category I products are those
`made with aqueous bases or vehicles, and emulsions. The criteria
`of antimicrobial effectiveness for these products are a function of
`the route of administration. Category 2 products are all dosage
`forms made with nonaqueous (anhydrous) bases or vehicles that
`contain a preservative.
`
`Table 1. Compendia! Product Categories.
`Product Description
`
`Category
`Category I
`lA
`
`lB
`
`IC
`
`Category 2
`
`Injections, other parenterals including emul(cid:173)
`sions, otic, sterile nasal products, and oph(cid:173)
`thalmic products made with aqueous bases or
`vehicles.
`Topically used products made with aqueous
`bases or vehicles, nonsterile nasal products,
`and emulsions, including those applied to mu(cid:173)
`cous membranes.
`Oral products made with aqueous bases or
`vehicles.
`All preserved dosage forms listed under Cat(cid:173)
`egory I made with nonaqueous (anhydrous)
`bases or vehicles.
`
`TEST ORGANISMS
`Use cultures of the following microorganisms' : Candida albicans
`(ATCC No. 10231), Aspergillus niger (ATCC No. 16404), Esch(cid:173)
`erichia coli (ATCC No. 8739), Pseudomonas aeruginosa (ATCC
`No. 9027), and Staphylococcus aureus (ATCC No. 6538). The vi(cid:173)
`able microorganisms used in the test must not be more than five
`passages removed from the original ATCC culture. For purposes of
`the test, one passage is defined as the transfer of organisms from
`an established culture to fresh medium. All transfers are counted.
`In the case of organisms maintained by seed lot techniques, each
`cycle of freezing, thawing, and revival in fresh medium is taken as
`one transfer. A seed stock technique should be used for long-term
`storage of cultures. Cultures received from the ATCC should be
`resuscitated according to directions. If grown in broth, the cells are
`pelleted by centrifugation. Resuspend in !/20th the volume of fresh
`maintenance broth, and add an equal volume of 20% (v/v in water)
`sterile glycerol. Cells grown on agar may be scraped from the sur(cid:173)
`face into the 10% glycerol broth. Dispense small aliquots of the
`suspension into sterile vials. Store the vials in liquid nitrogen or in
`a mechanical freezer at no more than -50°. When a fresh seed stock
`vial is required, it may be removed and used to inoculate a series
`of working cultures. These working cultures may then be used pe(cid:173)
`riodically (each day in the case of bacteria and yeast) to start the
`inoculum culture.
`
`MEDIA
`All media used in the test must be tested for growth promotion.
`Use the microorganisms indicated above under Test Organisms.
`
`PREPARATION OF INOCULUM
`Preparatory to the test, inoculate the surface of a suitable volume
`of solid agar medium from a recently revived stock culture of each
`of the specified microorganisms. The culture conditions for the in(cid:173)
`oculum culture are described in Table 2 in which the suitable media
`are Soybean-Casein Digest or Sabouraud Dextrose Agar Medium
`(see Microbial Limits Testing (61)).
`
`1 Available from American Type Culture Collection, 12301 Park(cid:173)
`lawn Drive, Rockville, MD 20852.
`
`Table 2. Culture Conditions for Inoculum Preparation.
`
`Organism
`Escherichia coli
`(ATCC No. 8739)
`
`Pseudomonas aeruginosa
`(ATCC No. 9027)
`
`Staphylococcus aureus
`(ATCC No. 6538)
`
`Candida albicans
`(ATCC No. 10231)
`
`Aspergillus niger
`(ATCC No. 16404)
`
`Suitable Medium
`Soybean-Casein
`Digest Broth;
`Soybean-Casein
`Digest Agar
`Soybean-Casein
`Digest Broth;
`Soybean-Casein
`Digest Agar
`Soybean-Casein
`Digest Broth;
`Soybean-Casein
`Digest Agar
`Sabouraud Dextrose
`Agar; Sabouraud
`Dextrose Broth
`Sabouraud Dextrose
`Agar; Sabouraud
`Dextrose Broth
`
`Incubation Temperature
`32.5 ± 2.SO
`
`Inoculum
`Incubation Time
`18 to 24 hours
`
`Microbial Recovery
`Incubation Time
`3 to 5 days
`
`32.5 ± 2.SO
`
`I 8 to 24 hours
`
`3 to 5 days
`
`32.5 ± 2.SO
`
`18 to 24 hours
`
`3 to 5 days
`
`22.5 ± 2.SO
`
`44 to 52 hours
`
`3 to 5 days
`
`22.5 ± 2.5°
`
`6 to 10 days
`
`3 to 7 days
`
`LUPIN EX1051, Page 5
`
`

`
`USP 24
`
`Microbiological Tests I (55) Biological Indicators
`
`1811
`
`To harvest the bacterial and C. albicans cultures, use sterile saline
`TS, washing the surface growth, collecting it in a suitable vessel,
`and adding sufficient sterile saline TS to obtain a microbial count
`of about 1 X 108 colony-forming units (cfu) per mL. To harvest
`the cells of A. niger, use sterile saline TS containing 0.05% of
`polysorbate 80, and add sufficient sterile saline TS to obtain a count
`of about I X 1 08 cfu per mL.
`Alternatively , the stock culture organisms may be grown in a
`suitable liquid medium (i.e., Soybean-Casein Digest Broth or Sa(cid:173)
`bouraud Dextrose Broth) and the cells harvested by centrifugation,
`then washed and resuspended in sterile saline TS to obtain a mi(cid:173)
`crobial count of about 1 X 108 cfu per mL. [NOTE-The estimate
`of inoculum concentration may be performed by turbidimetric
`measurements for the challenge microorganisms. Refrigerate the
`suspension if it is not used within 2 hours.]
`Determine the number of cfu per mL in each suspension, using
`the conditions of media and microbial recovery incubation times
`listed in Table 2 to confirm the initial cfu per mL estimate. This
`value serves to calibrate the size of inoculum used in the test. The
`bacterial and yeast suspensions are to be used within 24 hours of
`harvest, but the fungal preparation may be stored under refrigeration
`for up to seven days.
`
`PROCEDURE
`Category 1 Products--The test can be conducted either in five
`original containers if sufficient volume of product is available in
`each container and the product container can be entered aseptically
`(i.e., needle and syringe through an elastomeric rubber stopper), or
`in five steri1e, capped bacteriological containers of suitable size into
`which a sufficient volume of product has been transferred. In(cid:173)
`oculate each container with one of the prepared and standardized
`inoculum, and mix. The volume of the suspension inoculum used
`is between 0.5% and 1.0% of the volume of the product. The con(cid:173)
`centration of test microorganisms that is added to the product
`should be such that the final concentration of the test preparation
`after inoculation is between I X 105 and 1 X 106 cfu per mL of
`the product.
`The initial concentration of viable microorganisms in each test
`preparation is estimated based on the concentration of microor(cid:173)
`ganisms in each of the standardized inoculum as determined by the
`plate-count method.
`Incubate the inoculated containers at 22.5 ± 2.SO. Sample each
`container at the appropriate intervals specified in Table 3. Record
`any changes observed in appearance at these intervals. Determine
`by the plate-count procedure the number of cfu present in each test
`preparation for the applicable intervals (see Procedure under Mi(cid:173)
`crobial Limit Tests (61)). Incorporate an inactivator (neutralizer) of
`the specific antimicrobial in the plate count or in the appropriate
`dilution prepared for plating. These conditions are determined in
`the validation study for that sample based upon the conditions of
`media and microbial recovery incubation times listed in Table 2.
`Using the calculated concentrations of cfu per mL present at the
`start of the test, calculate the change in log 10 values of the concen(cid:173)
`tration of cfu per mL for each microorganism at the applicable test
`intervals, and express the changes in terms of log reductions.
`
`Bacteria:
`
`Table 3. Criteria for Tested Microorganisms.
`For Category IA Products
`Not less than 1.0 log reduction from the
`initial calculated count at 7 days, not less
`than 3.0 log reduction from the initial
`count at 14 days, and no increase from
`the 14 days' count at 28 days.
`No increase from the initial calculated
`count at 7, 14, and 28 days.
`
`Yeast and Molds:
`
`Bacteria:
`
`Yeast and Molds:
`
`For Category IB Products
`Not less than 2.0 log reduction from the
`initial count at 14 days. and no increase
`from the 14 days ' count at 28 days.
`No increase from the initial calculated
`count at 14 and 28 days.
`
`Bacteria:
`
`Table 3. Criteria for Tested Microorganisms. (continued)
`For Category JC Products
`Not less than 1.0 log reduction from the
`initial count at 14 days, and no increase
`from the 14 days' count at 28 days.
`No increase from the initial calculated
`count at 14 and 28 days.
`
`Yeast and· Molds:
`
`Bacteria, Yeast,
`and Molds:
`
`For Category 2 Products
`No increase from the initial calculated
`count at 14 and 28 days.
`
`Category 2 Products-Proceed as directed under Category 1
`Products to obtain a concentration of microorganisms in the inoc(cid:173)
`ulum between I X 105 and 1 X I 06 cfu per mL of product, except
`to treat the test preparations as follows . For products having solid
`ointment bases, heat the test preparations to a temperature at 47.5
`± 2.5°. Warm test preparations for oils to ambient temperature.
`Using a sterile glass rod or spatula, mix each inoculum of stan(cid:173)
`dardized microbial suspension with the test specimen for 1 minute
`or until homogeneity is achieved. A surfactant may be added to the
`inoculum to improve miscibility where the validation study dem(cid:173)
`onstrates that the addition of the surfactant does not affect the sur(cid:173)
`vival of the microorganism or potentiate the effectiveness of the
`preservative.
`
`CRITERIA FOR ANTIMICROBIAL
`EFFECTIVENESS
`The requirements for antimicrobial effectiveness are met if the
`criteria specified under Table 3 are met (see Significant Figures and
`Tolerances under General Notices). No increase is defined as not
`more than 0.5 log 10 unit higher than the previous value measured.
`
`(55) BIOLOGICAL INDICATORS(cid:173)
`RESISTANCE PERFORMANCE
`TESTS
`Total Viable Spore Count-Remove three specimens of the rele(cid:173)
`vant biological indicator from their original individual containers.
`Pulp the paper into component fibers by placing the test specimens
`in a sterile 250-mL cup of a suitable blender containing I 00 mL of
`chilled sterilized Purified Water and blending for 3 to 5 minutes to
`achieve a homogeneous suspension. Transfer a 10-mL aliquot of
`the suspension to a sterile, screw-capped 16- X 125-mm tube. For
`Biological Indicator for Steam Sterilization, Paper Strip, heat the
`tube containing the suspension in a water bath at 95• to 100• for
`15 minutes, starting the timing when the temperature reaches 95•.
`For Biological Indicator for Dry-Heat Sterilization, Paper Strip ,
`and for Biological Indicator for Ethylene Oxide Sterilization, Paper
`Strip, heat the tube containing the suspension in a water bath at so•
`to 85° for 10 minutes, starting the timing when the temperature
`reaches so•. Cool rapidly in an ice water bath at o• to 4•. Transfer
`two 1-mL aliquots to suitable tubes, and make appropriate serial
`dilutions in sterilized Purified Water, the dilutions being selected
`as calculated to yield preferably 30 to 300 colonies, but not less
`than 6, on each of a pair of plates when treated as described below.
`Where the biological indicator has a low spore concentration, it may
`be necessary to modify the dilution series and to use more plates
`at each dilution. Prepare a separate series of plates for each aliquot.
`Place 1.0 mL of each selected dilution in each of two 15- X I 00-
`mm petri dishes. Within 20 minutes, add to each plate 20 mL of
`Soybean-Casein Digest Agar Medium (see Microbial Limit Tests
`(61)) that has been melted and cooled to 45• to so•. Swirl to attain
`a homogeneous suspension. and allow to solidify . Incubate the
`plates in an inverted position at 55• to 60• for Biological Indicator
`for Steam Sterilization , Paper Strip, and at 30• to 35• for Biological
`Indicator for Ethylene Oxide Sterilization, Paper Strip, and for Bi(cid:173)
`ological Indicator for Dry-Heat Sterilization, Paper Strip, or at the
`optimal recovery temperature specified by the manufacturer, and
`
`LUPIN EX1051, Page 6
`
`

`
`1812
`
`(55) Biological Indicators I Microbiological Tests
`
`USP 24
`
`examine the plates after 24 and 48 hours, recording for each plate
`the number of colonies, and using the number of colonies after 48
`hours to calculate the results. Calculate the average number of
`spores per specimen from the results, using the appropriate dilution
`factor. The test is valid if the log number of spores per carrier at
`48 hours is equal to or greater than the log number after 48 hours
`in each case. For Biological Indicators for Steam Sterilization , Self(cid:173)
`Contained, aseptically remove the spore strip from the container,
`and proceed as directed for Biological Indicator for Steam Steril(cid:173)
`ization, Paper Strip .
`D Value Determination-For all tests described in this section,
`handle each test specimen with aseptic precautions, using sterilized
`equipment where applicable.
`Apparatus-For Biological Indicator for Dry-heat Sterilization,
`Paper Strip, use an apparatus of known thermodynamic character(cid:173)
`istics that has been validated for compliance with the requirements
`for safety' and performance,2 that consists of a sterilizing chamber
`equipped with a means of heating the contained air, preferably elec(cid:173)
`trically rather than gas fired, and that has adequate movement of
`the air through forced ventilation (by mechanical devices such as
`blowers), with sensing and control devices for temperature and tim(cid:173)
`ing capable of indicating with an accuracy of not more than OS
`and !-second intervals, respectively. The geometrical pattern of the
`heat source(s) is such as to enable the biological indicators under
`test to be uniformly heated under the specified conditions. The
`temperature profile in the chamber is known, and cold spots, hot
`spots, and slow heat zones identified. The chamber has the capa(cid:173)
`bility to work within a temperature range of 40° to 300°, with an
`accuracy at any particular setting of not less than ± 2°. The ap(cid:173)
`paratus is equipped with a suitable additional access door or port
`so as to enable the entry and insertion (or removal) of specimens
`within 6 seconds and to enable the temperature to return to the set
`temperature within 0.5 minute where the specified temperature is
`120° to 190° and within 1.0 minute where such temperature is 220°
`and above.
`For Biological Indicator for Ethylene Oxide Sterilization, Paper
`Strip, use an apparatus that consists of a test chamber with a means
`of ensuring adequate mixing of the sterilant gas and a means of
`heating the sterilant gas to not higher than the preselected operating
`temperature so that no liquid enters the test chamber, equipped with
`temperature control and monitoring, pressure control, humidifica(cid:173)
`tion, and gas concentration monitoring devices. Detailed specifi(cid:173)
`cations and operational parameters for suitable apparatus are those
`published in Standard for a Biological Indicator-Evaluator Resis(cid:173)
`tometer for Ethylene Oxide Gas Vessels (BIER/EO) Gas Vessels .3·
`For Biological Indicator for Steam Sterilization, Paper Strip, and
`for Biological Indicator for Steam Sterilization , Self-Contained, use
`an apparatus that consists of a chamber equipped with heating , tem(cid:173)
`perature, and steam control and monitoring devices. Detailed spec(cid:173)
`ifications and operational parameters for suitable apparatus are those
`published in Standard for a Biological Indicator-Evaluator Re(cid:173)
`sistometer for Saturated Steam (BIER/Steam Vessels)•
`Procedure--Carry out the tests forD value at each of the appli(cid:173)
`cable sets of sterilization conditions for which the packaged bio(cid:173)
`logical indicator under test is labeled for use. Take a sufficient
`number of groups of specimens of biological indicators in their
`
`1 Safety includes design to prevent electric shock or gas exposi(cid:173)
`tion and burns, where operators can wear protective clothing and
`gloves against burns_ from touching hot surfaces.
`2 Descriptions of different types of dry-heat sterilizing equipment
`and detailed guidelines for determining, monitoring , and controlling
`the operating parameters have been published by the Health Indus(cid:173)
`try Manufacturers Association (HIM A) in Report No. 78-1.7 , Op(cid:173)
`erator Training for Dry Heat Sterilizing Equipment , and by the
`Parenteral Drug Association , Inc. , (PDA) in Technical Report No.
`3, Validation of Dry Heat Processes used for Sterilization and
`Depyrogenation.
`3 Standard for BIER/EO Gas Vessels, 27 March 1992, Associa(cid:173)
`tion for the Advancement of Medical Instrumentation (AAMI),
`3330 Washington Boulevard, Suite 440 Arlington VA 22201-
`4598.
`.
`'
`4 Standard for BIER/Steam Vessels, 27 March 1981 , Association
`for th_e Advancement of Medical Instrumentation (AAMI) 3330
`Washtngton Boulevard, Suite 400, Arlington, v A 22201-4598.
`
`original individual containers, each group cons1stmg of 5 to 10
`specimens. The number of groups provides a range of observations
`from not less than one labeled D value below the labeled survival
`time through not less than one labeled D value above the labeled
`kill time. Place each group on a separate suitable specimen holder
`that permits each specimen to be exposed to the prescribed steril(cid:173)
`izing condition at a specific location in the sterilizing chamber.
`Check the apparatus for operating parameters using specimen hold(cid:173)
`ers without specimens. Select a series of sterilizing times in incre(cid:173)
`ments from the shortest time for the specimens to be tested. The
`differences in sterilizing times over the series are as constant as
`feasible, and the difference between adjacent times is no greater
`than 75% of the labeled D value.
`For Biological Indicator for Dry-Heat Sterilization, Paper Strip,
`preheat the sterilizing chamber for 30 minutes. Open the access
`door or port, place one of the holders with a group of specimens
`in the sterilizing chamber, close the access door or port, and con(cid:173)
`tinue to operate the apparatus. Commence timing the heat exposure
`when the chamber temperature returns to 2° below the specified
`temperature. After the contents have been subjected to the steril(c