`
`51 ANTIMICROBIAL EFFECTIVENESS TESTING
`
` INTRODUCTION
`Antimicrobial preservatives are substances added to aqueous pharmaceutical products. Nonsterile dosage forms may
`have preservatives added to protect them from growth of microorganisms inadvertently introduced during or
`subsequent to the manufacturing process. In the case of sterile articles packaged in multipledose containers,
`antimicrobial preservatives are added to inhibit the growth of microorganisms that may be introduced from repeatedly
`withdrawing individual doses. One or more antimicrobial preservative(s) are expected in all sterile multidose units.
`Antimicrobial preservatives should not be used as a substitute for good manufacturing practices, solely to reduce the
`viable microbial population of a nonsterile product, or control the presterilization bioburden of a multidose formulation
`during manufacturing. Antimicrobial preservatives in compendial dosage forms meet the requirements for 5.20 Added
`Substances in General Notices and Requirements.
`All useful antimicrobial agents are toxic substances. For maximum 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 based on the recommended dosage of the medicinal product.
`The concentration of an added antimicrobial preservative can be kept to a minimum if the active ingredients of the
`formulation possess an intrinsic antimicrobial activity. Antimicrobial effectiveness, whether inherent in the product or
`produced because of the addition of an antimicrobial preservative, must be demonstrated for all injections packaged
`in multipledose containers or for other products containing antimicrobial preservatives. Antimicrobial effectiveness
`must be demonstrated for aqueousbased, multipledose topical and oral dosage forms and for other dosage forms
`such as ophthalmic, otic, nasal, irrigation, and dialysis fluids (see Pharmaceutical Dosage Forms 1151 ). For the
`purpose of the test, aqueous is defined as a water activity of more than 0.6 (see Application of Water Activity
`Determination to Nonsterile Pharmaceutical Products 1112 ).
`Challenge organisms are generally based on likely contaminants to a drug product while considering its physical
`attributes, formulation, and intended use. The standard battery of challenge organisms described in this test need not
`prevent the inclusion of other species of microorganisms if deemed useful to measure the biological activity of the
`preservative system for a specific product. These supplemental challenge organisms are not within the scope of this
`chapter, but may be added in addition to the described test organisms.
`
`GENERAL PROCEDURES
`This chapter provides procedures to demonstrate the effectiveness of added antimicrobial preservatives. Such
`antimicrobial preservatives must be declared on the label. The procedures and acceptance criteria for effectiveness
`apply to a product in the original, sealed container in which it was distributed by the manufacturer (see Table 1 for
`categories of products). The test need not be conducted in these containers, but care should be taken to avoid using
`materials that can interact with the preservative in the containers that are used for antimicrobial effectiveness testing.
`
`Growth Promotion Procedure and Suitability of the Recovery Method
`
`GENERAL CONSIDERATIONS
`The ability of the procedure to detect challenge microorganisms in the presence of a suitably neutralized product to be
`tested must be established. The suitability of the procedure must be reconfirmed if a change is made in materials or
`methods or if a change is made in the product or direct product contact materials that may affect the outcome of the
`test.
`The growthpromoting capabilities of media used in this procedure must be established.
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`PREPARATION OF TEST STRAINS
`Use standardized suspensions of test strains or prepare as stated below. Seedlot culture maintenance techniques
`(seedlot systems) are used so that the viable microorganisms used for inoculation are NMT five passages removed
`from the original master seed lot. Grow each of the bacterial and fungal test strains separately (see Table 2).
`Use cultures of the following microorganisms:1 Candida albicans (ATCC No. 10231), Aspergillus brasiliensis (ATCC No.
`16404), Escherichia coli (ATCC No. 8739), Pseudomonas aeruginosa (ATCC No. 9027), and Staphylococcus aureus
`(ATCC No. 6538). The viable microorganisms used in the procedure should be part of a freshly growing culture (e.g.,
`in logarithmic growth phase) with the exception of A. brasiliensis spores. The culture conditions for the inoculum
`culture are described (see Table 2) in which the suitable media are Soybean–Casein Digest or Sabouraud Dextrose
`Agar Medium.
`To harvest the bacterial and C. albicans cultures, use sterile saline TS to wash the surface growth, and collect it in a
`suitable vessel. To harvest the spores of A. brasiliensis, use sterile saline TS containing 0.05% of polysorbate 80.
`The spore suspension should be aseptically treated (e.g., filtration through sterile glass wool) to remove hyphae. All
`microbial suspensions should be prepared to ensure that there is no carry over of residual growth medium from the
`inoculum (e.g., centrifugation followed by resuspension in appropriate sterile suspending fluid.)
`Alternatively, the stock culture organisms may be grown in a suitable liquid medium (i.e., Soybean–Casein Digest Broth
`or Sabouraud Dextrose Broth) and the cells harvested by centrifugation, then washed and resuspended in appropriate
`sterile suspending fluid. The microbial suspensions used for inoculations should be adjusted to obtain a microbial
`count of about 1 × 108 cfu/mL. Use the bacterial and yeast suspensions within 2 h, or within 24 h if stored between 2
`and 8 . A stable spore suspension can be prepared and then may be maintained at 2 –8 for up to 7 days. [NOTE
`—The estimate of inoculum concentration may be obtained by turbidimetric procedures for the
`challenge microorganisms and later confirmed by plate count. ]
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`GROWTH PROMOTION OF THE MEDIA
`Media used in this procedure must be capable of supporting microbial growth. Test each batch of readyprepared
`medium and each batch of medium prepared either from dehydrated medium or from the ingredients described.
`For solid media, counts obtained must be at least 50% of the calculated value for a standardized inoculum. For a
`freshly prepared inoculum, growth of the microorganisms occurs comparable to that previously obtained with a
`previously tested and approved batch of medium.
`
`Suitability of the Counting Method in the Presence of Product
`
`Prepare a 10 1 dilution by adding 1 mL of product (by volume) to 9 mL of saline or other neutralizing diluent. Continue
`
`this dilution scheme to 10 2 and 10 3 dilution levels. Add an appropriate number of challenge organisms to each
`tube of diluted product, mix, and then plate a suitable volume from each dilution to yield less than 250 cfu/plate for
`bacteria and yeast (ideally between 25 and 250 cfu) or less than 80 cfu/plate for A. brasiliensis (ideally between 8 and
`80 cfu). This plating should be performed minimally in duplicate (although a greater number of replicates can be useful
`to minimize variability in the plate count estimate). A positive control for this procedure is to introduce the same
`inocula into saline, and transfer similar volumes of saline to agar plates. A suitable recovery scheme is the one that
`provides at least 50% of this saline control count (averaged).
`If the diluted product exhibits antimicrobial properties, specific neutralizers may need to be incorporated into the
`diluents or the recovery media. See Validation of Microbial Recovery from Pharmacopeial Articles 1227 for more
`information.
`The ability of the procedure to measure preservative efficacy may be compromised if the method suitability requires
`
`significant dilution (10 2 or 10 3) as this will affect the measured recovery (e.g., it may be difficult to measure a 3
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`log unit reduction for a 105–106 inoculum). If no suitable neutralizing agent or method is found and method suitability
`requires significant dilution, a higher level of inoculum (e.g., 107–108) may be used so that a 3 log unit reduction can
`be measured. Reported recovery cannot be less than 1 cfu/plate on average (or 100 cfu/mL if 1 mL is plated in
`
`duplicate at the 10 2 dilution).
`Membrane filtration may be used to filter larger volumes of dilutions to overcome this difficulty or to assist in the
`neutralization of antimicrobial properties.
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`Testing of Products
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`PRODUCT CATEGORIES
`For the purpose of testing, compendial articles have been divided into four categories (see Table 1). The criteria of
`antimicrobial effectiveness for these products are a function of the route of administration. It is expected that
`formulations containing preservatives will meet minimal efficacy standards, whether packaged as multidoses or unit
`doses.
`
`Category
`
`1
`
`2
`3
`4
`
`Table 1. Compendial Product Categories
`Product Description
`Injections; other parenterals including emulsions, otic products, sterile nasal products,
`and ophthalmic 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 mucous membranes
`Oral products other than antacids, made with aqueous bases or vehicles
`Antacids made with an aqueous base
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`PROCEDURE
`The procedure can be conducted either in five original containers if a sufficient volume of product is available in each
`container and if the product container can be entered aseptically (i.e., needle and syringe through an elastomeric
`rubber stopper), or in five sterile, capped bacteriological containers [inert relative to the antimicrobial agent(s)] of
`suitable size into which a sufficient volume of product has been transferred. Inoculate each container with one of the
`prepared and standardized inocula, and mix. The volume of the suspension inoculum used is between 0.5% and 1.0%
`of the volume of the product to minimize potential effects on the product. The concentration of test microorganisms
`that is added to the product (Category 1, 2, or 3) is such that the final concentration of the test preparation after
`inoculation is between 1 × 105 and 1 × 106 cfu/mL of the product. For Category 4 products (antacids), the final
`concentration of the test preparation after inoculation is between 1 × 103 and 1 × 104 cfu/mL of the product.
`The initial concentration of viable microorganisms in each test preparation is estimated based on the concentration of
`microorganisms in each of the standardized inocula as determined by the platecount method. Incubate the inoculated
`containers at 22.5 ± 2.5 . Sample each container at the appropriate intervals (specified in Table 3). Record any
`changes observed in appearance at these intervals. Determine, by the platecount procedure, the number of cfu
`present in each test preparation for the applicable intervals (see General Procedures in Microbial Enumeration Tests
`61 ). Plate counts will be conducted using a minimum of duplicate plates, with the cfu averaged before
`determination of deduced cfu/mL. If membrane filtration is used, duplicate membrane filters will be used for each
`estimate. Using the calculated concentrations of cfu/mL present at the start of the test, calculate the change in log10
`values of the concentration of cfu/mL for each microorganism at the applicable test intervals, and express the
`changes in concentration in terms of log reductions. The log reduction is defined as the difference between the log10
`unit value of the starting concentration of cfu/mL in the suspension and the log10 unit value of cfu/mL of the survivors
`at that time point.
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`Table 2. Culture Conditions for Inoculum Preparation
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`Inoculum
`Incubation
`Time
`
`Microbial
`Recovery
`Incubation
`Time
`
`Incubation
`Temperature
`
`32.5 ± 2.5
`
`18–24 h
`
`3–5 days
`
`32.5 ± 2.5
`
`18–24 h
`
`3–5 days
`
`32.5 ± 2.5
`
`18–24 h
`
`3–5 days
`
`22.5 ± 2.5
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`44–52 h
`
`3–5 days
`
`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
`
`Organism
`Escherichia coli
`(ATCC No. 8739)
`Pseudomonas
`aeruginosa
`(ATCC No. 9027)
`Staphylococcus
`aureus
`(ATCC No. 6538)
`Candida albicans
`(ATCC No.
`10231)
`Aspergillus
`brasiliensis
`(ATCC No.
`16404)
`
`Sabouraud Dextrose Agar;
`Sabouraud Dextrose Broth
`
`22.5 ± 2.5
`
`6–10 days 3–7 days
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`Criteria for Antimicrobial Effectiveness
`The requirements for antimicrobial effectiveness are met if the criteria specified in Table 3 are met (see Test Results in
`General Notices). “No increase” in counts is defined as NMT 0.5 log10 unit more than the value to which it is
`compared.
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`Table 3. Criteria for Tested Microorganisms
`For Category 1 Products
`NLT 1.0 log reduction from the initial calculated count at 7 days, NLT 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
`For Category 2 Products
`NLT 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
`For Category 3 Products
`NLT 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
`For Category 4 Products
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`Bacteria
`Yeast and
`molds
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`Bacteria
`Yeast and
`molds
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`Bacteria
`Yeast and
`molds
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`Bacteria,
`yeast, and
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`molds
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`USP38
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`No increase from the initial calculated count at 14 and 28 days
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`1 Available from American Type Culture Collection, 10801 University Boulevard, Manassas, VA 201102209 (http://www.atcc.org).
`
`Auxiliary Information— Please check for your question in the FAQs before contacting USP.
`Topic/Question Contact
`Expert Committee
`General Chapter Radhakrishna S Tirumalai, Ph.D.
`(GCM2010) General Chapters Microbiology
`Principal Scientific Liaison
`(301) 8168339
`USP38–NF33 Page 100
`Pharmacopeial Forum: Volume No. 40(1)
`
`Innopharma EX1016, Page 5
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