2002
`
`THE UNITED STATES PHARMACOPEIA
`USP 25
`
`NF 20
`THE NATIONAL FORMULARY
`
`By authority of the United States Pharmacopeial
`Convention, Inc., meeting at Washington, Pee
`April 12-16, 2000. Prepared by the Council ofExperts
`and published by the Board of Trustees
`Official from January 1, 2002
`
`The designation on the cover of this publication, “USP NF
`2002,” is for ease of identification only. The publication
`contains two separate compendia: The Pharmacopeia of the
`United States Twenty-fifth Revision, and the National
`Formulary, Twentieth Edition.
`
`UNITED STATES PHARMACOPEIAL CONVENTION, INC.
`12601 Twinbrook Parkway, Rockville, MD 20852
`Se™~
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`NOTICE AND WARNING
`
`Concerning U.S. Patent or Trademark Rights
`Theinclusion in the Pharmacopeiaorin the National Formulary ofa monograph on any drugin respect
`to which patent or trademark rights may exist shall not be deemed,andis not intended as, a grant of, or
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`
`Concerning Use of USP or NF Text
`
`Use ofthe USP-NFis subject to the terms and conditions ofthe USP-NF License Agreement.Attention
`is called to the fact that the USP-NF is fully copyrighted. Authors and others wishing to use portions of
`the text or images in any mannernot expressly permitted by the License Agreement should request
`permission to do so from the Secretary of the USPC Board of Trustees.
`
`Copyright © 2001 The United States Pharmacopeial Convention,Inc.
`12601 Twinbrook Parkway, Rockville, MD 20852
`All rights reserved.
`ISSN 0195-7996
`ISBN 1-889788-10-4
`Printed in Canada by Webcom Limited, Toronto, Ontario
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`This material may be protected by Copyright law (Title 17 U.S. Code)
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`1870
`
`(51) Antimicrobial—Effectiveness Testing / Microbiological Tests
`
`USP 25
`
`MEDIA
`
`PREPARATION OF INOCULUM
`
`Testy and dialysis fluids (see Pharmaceutical Dosage Forms
`
`1151)).
`This chapter provides tests to demonstrate the effectiveness of anti-
`microbial protection. Added antimicrobial preservatives must be de-
`clared on the label. The tests and criteria for effectiveness apply to a
`productin the original, unopened container in which it was distribu-
`ted by the manufacturer.
`
`PRODUCT CATEGORIES
`
`All useful antimicrobial agents are toxic substances. For maximum
`protection ofpatients, the concentration of the preservative shown to
`be effective in the final packaged product should be belowalevel that All media usedin the test must be tested for growth promotion. Use
`
`maybe toxic to human beings.
`the microorganismsindicated above under Jest Organisms.
`The concentration of an added antimicrobial preservative can be
`kept at a minimumif the active ingredients of the formulation possess
`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
`Preparatory to the test, inoculate the surface of a suitable volume of
`all injections packaged in multiple-dose containers or for other pro-
`solid agar medium fromarecently revived stock culture ofeach ofthe
`ducts containing antimicrobial preservatives. Antimicrobialeffective-
`specified microorganisms. The culture conditions for the inoculum
`ness must be demonstrated for multiple-dose topical and oral dosage
`culture are described in Table 2 in which the suitable media are Soy-
`forms and for other dosage forms such as ophthalmic,otic, nasal, ir-
`bean-Casein Digest or Sabouraud Dextrose Agar Medium (see Mi-
`crobial Limits Testing (61)).
`To harvest the bacterial and C. albicans cultures,usesterile saline
`TS, washing the surface growth,collecting it in a suitable vessel, and
`addingsufficientsterile saline TS to obtain a microbial countof about
`1 x 10° colony-forming units (cfu) per mL. To harvest the cells of A.
`niger, use sterile saline TS containing 0.05% of polysorbate 80, and
`addsufficient sterile saline TS to obtain a count of about 1 x 10* cfu
`per mL.
`Changeto read:
`Alternatively, the stock culture organisms may be growninasuit-
`able liquid medium (i.e., Soybean-Casein Digest Broth or Sabouraud
`Dextrose Broth) and the cells harvested by centrifugation, then
`washed and resuspendedinsterile saline TS to obtain a microbial
`count of about | x 10* cfu per mL.
`For the purposeoftesting, compendial articles have been divided
`[NoTE—Theestimate of inoculum concentration may beper-
`into “four,usp2s two categories (see Table 1).~,usp2s The criteria of
`formedby turbidimetric measurements for the challenge microorgan-
`antimicrobial effectiveness for these products are a function of the
`isms. Refrigerate the suspension if it is not used within 2 hours.]
`route of administration.” , ysp2s
`Determine the numberof cfu per mL in each suspension,using the
`conditions of media and microbial recovery incubationtimeslisted in
`Table 2 to confirm theinitial cfu per mL estimate. This value serves to
`calibrate the size of inoculum usedinthetest. The bacterial and yeast
`suspensionsare to be used within 24 hours of harvest, but the fungal
`preparation may be stored underrefrigeration for up to’seven days.
`Changeto read:
`
` |s
`
`iaiaa
`
`Table 1. Compendial Product Categories.
`Category Product Description
`
`a
`Injections, other parenterals including
`1 AUSP25
`emulsions,otic, sterile nasal products, and
`ophthalmic products made with aqueous
`bases or vehicles.
`Topically used products made with aque-
`ous basesorvehicles, nonsterile nasal pro-
`ducts, and emulsions, including those
`applied to mucous membranes.
`Oral products
`“other than antacids, , ysp2s
`4AUSP2S
`the product container can be entered aseptically (i.e., needle and sy-
`made with aqueousbases or vehicles.
`i
`ringe throughan elastomeric rubberstopper), orin five sterile, capped
`
`4usps “Antacids made with an aqueousbase.,.
`bacteriological containers of suitable size into whichasufficient vol-
`USP25,
`umeof product has been transferred. Inoculate each container with
`oneofthe prepared andstandardized inoculum, and mix. The volume
`of the suspension inoculum used is between 0.5% and 1.0% ofthe
`volumeof the product. The concentration of test microorganismsthat
`is added to the product “(Categories 1, 2, and 3),usps are suchthat
`the final concentration of the test preparation after inoculation iis be-
`tween 1 x 10° and 1 x 10° cfu per mL ofthe product. “For Category
`4 products (antacids) the final concentration of the test preparation
`after inoculation is between 1 x 10° and 1 x 10* cfu per mL ofthe
`product. AUSP25
`The initial concentration of viable microorganismsin eachtest
`preparation is estimated based on the concentration of microorgan-
`isms in each of the standardized inoculum as determined by the
`plate-count method.
`Incubate the inoculated containers at 22.5 + 2.5°. Sample each
`containerat the appropriate intervals specified in Table 3. Record any
`changes observed in appearanceat these intervals. Determine by the
`plate-count procedure the numberofcfu present in each test prepara-
`tion for the applicable intervals (see Procedure under Microbial Limit
`Tests (61)). Incorporate an inactivator (neutralizer) of the specific
`antimicrobialin 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 microbialre-
`covery incubation timeslisted in Table 2. Using the calculated con-
`centrations of cfu per mL presentatthe start of the test, calculate the
`changein log,) values of the concentration of cfu per mL for each
`microorganism at the applicable test intervals, and express the
`changes in terms of log reductions.
`AUSP25
`
`A
`
`A
`
`2AUSP2S
`
`PROCEDURE
`
`ers if“tee volume ofproduct is available in each container and
`
`usp2s The test can be conductedeither in five original contain-
`
`TEST ORGANISMS
`
`Use cultures of the following microorganisms!: Candida albicans
`(ATCC No. 10231), Aspergillus niger (ATCC No. 16404), Escheri-
`chia coli (ATCC No. 8739), Pseudomonas aeruginosa (ATCC No.
`9027), and Staphylococcus aureus (ATCC No. 6538). The viable mi-
`croorganisms used in the test must not be more than five passages
`removed from the original ATCC culture. For purposesofthe test,
`one passage is defined as the transfer of organisms from an estab-
`lished culture to fresh medium.All transfers are counted. In the case
`of organisms maintained by seed lot techniques, each cycle of freez-
`ing, thawing,and revival in fresh medium is taken as onetransfer. 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.
`Resuspendin 1/20th the volumeof fresh maintenance broth, and add
`an equal volume of 20% (v/v in water) sterile glycerol. Cells grown
`on agar maybe scraped from the surface into the 10% glycerol broth.
`Dispense small aliquots of the suspensionintosterile vials. Store the
`vials in liquid nitrogen or in a mechanical freezer at no more than -
`50°. Whena fresh seed stock vial is required, it may be removed and
`usedto inoculate a series of working cultures. These working cultures
`maythen be used periodically (each day in the case of bacteria and
`yeast) to start the inoculum culture.
`
`Change to read:' Available from American Type Culture Collection,
`“10801 University Boulevard, Manassas, VA 20110-2209 (http://
`Www.atcc.Org). 4usp2s
`
`|| |
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`USP 25
`
`Microbiological Tests |
`
`(55) Biological Indicators—Resistance Performance Tests
`
`1871
`
`
`Table 2. Culture Conditions for Inoculum Preparation
`Inoculum
`Microbial Recovery
`Incubation Time
`Incubation Time
`Organism
`Suitable Medium
`Incubation Temperature
`
`Escherichia coli
`Soybean-Casein
`325:2207
`18 to 24 hours
`3 to 5 days
`(ATCC No. 8739)
`Digest Broth;
`Soybean-Casein
`Digest Agar
`Soybean-Casein
`Digest Broth;
`Soybean-Casein
`Digest Agar
`Soybean-Casein
`Digest Broth;
`Soybean-Casein
`Digest Agar
`Sabouraud Dextrose
`Candida albicans
`Agar; Sabouraud
`(ATCC No. 10231)
`Dextrose Broth
`3 to 7 days
`6 to 10 days
`22.5 = 2.85
`Sabouraud Dextrose
`Aspergillus niger
`Agar; Sabouraud
`(ATCC No. 16404)
`Dextrose BrothegEeeeeeeeee
`
`
`
`32.5 + 2:58
`.
`
`18 to 24 hours
`
`3 to 5 days
`
`32.5 2245
`
`18 to 24 hours
`
`3 to 5 days
`
`2215 + 25%
`
`44 to 52 hours
`
`3 to 5 days
`
`Pseudomonas aeruginosa
`(ATCC No. 9027)
`
`Staphylococcus aureus
`(ATCC No. 6538)
`
`CRITERIA FOR ANTIMICROBIAL
`EFFECTIVENESS
`
`The requirements for antimicrobial effectiveness are metif the cri-
`teria specified under Table 3 are met (see Significant Figures and Tol-
`erances under General Notices). No increase is defined as not more
`than 0.5 log, unit higher than the previous value measured.
`
`Biological Indicator for Steam Sterilization, Paper Carrier, 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 Indicatorfor Dry-HeatSterilization, Paper Car-
`rier, and for Biological Indicator for Ethylene Oxide Sterilization,
`PaperCarrier, heat the tube containing the suspension in a water bath
`at 80° to 85° for 10 minutes, starting the timing when the temperature
`reaches 80°. Coolrapidly in an ice water bath at 0° to 4°. Transfer two
`1-mL aliquots to suitable tubes, and make appropriatescrial dilutions
`in sterilized Purified Water, the dilutions being selected as calculated
`to yield preferably 30 to 300 colonies, butnotless than 6, on each of a
`pair of plates whentreated as described below. Wherethe biological
`indicator has a low spore concentration, it may be necessary to mod-
`ify the dilution series and to use moreplates at each dilution. Prepare
`a separate series of plates for each aliquot. Place 1.0 mL ofcach se-
`lected dilution in each of two 15- x 100-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 50°. 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
`Carrier, and at 30° to 35° for Biological Indicatorfor Ethylene Oxide
`Sterilization, Paper Carrier, and for Biological Indicator for Dry-
`Heat Sterilization, Paper Carrier, or at the optimal recovery tempera-
`ture specified by the manufacturer, and examinethe plates after 24
`and 48 hours, recording for each plate the numberof colonies, and
`using the numberofcolonies after 48 hours to calculate the results.
`Calculate the average number of spores per specimen from the re-
`sults, using the appropriate dilution factor. Thetest is valid if the
`log numberofspores per Carrier at 48 hours is equal to or greater
`than the log numberafter 24 hours in each case. For BiologicalIndi-
`catorsfor Steam Sterilization, Self-Contained, aseptically remove the
`sporestrip from the container, and proceed as directed for Biological
`Indicatorfor Steam Sterilization, Paper Carrier.
`D Value Determination—Forall tests described in this section,
`handle each test specimen with aseptic precautions, using sterilized
`equipment where applicable.
`Apparatus—ForBiological Indicator for Dry-HeatSterilization,
`Paper Carrier, use an apparatus of known thermodynamic character-
`istics that has been validated for compliance with the requirements for
`safety’ and performance,” that consists of a sterilizing chamber
`equipped with a meansof heating the contained air, preferably clec-
`trically rather than gas fired, and that has adequate movementofthe
`' Safety includes design to preventelectric shock or gas exposition and burns,
`whereoperators can wearprotective clothing and gloves against burns from
`touching hot surfaces.
`Total Viable Spore Count—Removethree specimensofthe rele-
`2 Descriptionsofdifferent types ofdry-heatsterilizing equipmentanddetailed
`vant biological indicator from their original individual containers.
`guidelines for determining, monitoring, and controlling the operating para-
`Pulp the paper into component fibers by placing the test specimens
`meters have been published by the Health Industry Manufacturers Association
`in a sterile 250-mL cup ofa suitable blender containing 100 mL of
`(HIMA)in Report No. 78-1.7, Operator Training for Dry HeatSterilizing
`chilled sterilized Purified Water and blending for 3 to 5 minutes to
`Equipment, and by the Parenteral Drug Association, Inc., (PDA) in Technical
`achieve a homogeneous suspension. Transfer a 10-mL aliquot of
`Report No. 3, Validation ofDry Heat Processes usedforSterilization and De-
`the suspension toasterile, screw-capped 16- x 125-mm tube. For
`pyrogenation.
`
`Bacteria:
`
`Bacteria:
`
`Table 3. Criteria for Tested Microorganisms
`For Category rail ausp2s Products
`Notless than 1.0 log reduction from the
`initial calculated countat 7 days, notless
`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
`Yeast and Molds:
`
`count at 7, 14, and 28 days.
`For Category 49 spos Products
`Notless 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 theinitial calculated
`Yeast and Molds:
`
`count at 14 and 28 days.
`
`For Category £3 4 usp2s Products
`Bacteria:
`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.
`Yeast and Molds:
`Noincrease from theinitial calculated
`
`count at 14 and 28 days.
`
`For Category “4 4 UsP25 Products
`Noincrease from the initial calculated
`Bacteria, Yeast,
`and Molds:
`count at 14 and 28 days.
`
`(55) BIOLOGICAL INDICATORS—
`RESISTANCE PERFORMANCE
`TESTS
`
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