UUALIWCUNTRUL
`
`The Essentials of United States
`Pharmacopeia Chapter <51>
`Antimicrobial Effectiveness Testing
`
`
`
`ANALYTICAL METHODS
`
`AND ITS APPLICATION IN PHARMACEUTICAL COMPOUNDING
`
`NW“ V“ ‘W’
`Thomas C. Kupiec
`are affiliated with
`Analytical Research
`Laboratories, Inc,
`Oklahoma City, Okla-
`homa Kevin Nguyen
`
`iwP*wmDC«~d«‘-
`date and is afiiliated
`with the ojgzahoma
`Universityffealth
`
`Nicole Vu, PhD
`,
`Kevln Nguyen
`Thomas Kupiec, PhD
`
`INTRODUCTION
`Antimicrobial preservatives are excipi-
`ents added to multi-dose formulas ofboth
`
`
`
`S”'"'“ C‘”"’'‘-’'» Okl“'
`homa City, Oklahoma.
`
`
`sterile and nonsterile drug products for
` inhibition of microbial growth. Microbial
`contamination may occur during nonsterile
`processing or during the period of use due to
`the repeated withdrawal of individual doses
`from multi-dose containers.‘ Multi-dose
`pharmaceutical products containing preser-
`
`vatives ofier several advantages over single-
`
`
`dose packages. Multi-dose drugs minimize
`
`product wastage and allow flexibility for
`
`
`dosage adjustments; repeated doses may be
`
`
`obtained from the same container without
`
`concerns for microbial growth during use;
`
`and their packaging is reduced because mul-
`
`tiple doses are supplied in a single con-
`tainer? It is general knowledge that
`
`unit-dose packaging is the most optimal
`
`
`
`
`
`with respect to the maintenance of sterility,
`PHOTO SOURCE.
`but it is not efficient and cost effective as
`ANALYTICAL
`
`“5E‘R‘5"
`preserved multi-dose preparations.
`LABORATORIES
`.
`.
`.
`.
`‘ PICKENS
`Anumicrobial preservatives can be
`PHOTOGRAPHY
`rnicrocidal, microstatic, and sporicidal.
`
`
`
`
`www.IJ'PC.oom
`
`International Journal ofPhannuoeut.ical Compounding
`Vol. 18 No. 2 March 1 A ril \2o14 123
`SEN
`IT 21 4
`
`ABSTRACT Antimicrobial preservatives are excipients
`added to multi-dose containers of both sterile and non-
`
`sterile drug products. Antimicrobial preservatives are
`used primarily to inhibit growth of microbial contamina-
`tion occurring during the period of use. Demonstration of
`antimicrobialpreservative effectiveness is required for
`
`these functional exclplents. This article reviews key factors
`for consideration In the selection of preservatives, princi-
`ples of the preservative-effectiveness test, and the signifi-
`cance of requirements for preservative-effectiveness
`testing in the compounding practice.
`
`PAGE 1 OF 8
`
`LUPIN v SENJU
`
`IPR2015—0l 105
`
`

`
`Uualirv Uumrul
`
`They interfere with various mechanisms i.n microbial cells causing
`cellular damage or cell lysis. The mechanisms for antimicrobial
`effects are not always specific and can be difficult to elucidate.
`Some preservatives may act at the cell wall, others may target the
`cytoplasmic membrane or cytosolic components. Their activities
`may lead to irreversible cell membrane damage, precipitation of cel-
`lular proteins, or inhibition of critical pathways for signal induction
`and cellular transport. Preservatives may also act synergistically
`with other preservatives or with other components ofthe formula
`to enhance the total effects for microbial control. Due to the cyto-
`toxic effects they exert against microbial cells, these preservatives
`can not be regarded simply as inactive ingredients. Their inclusion
`in pharmaceutical preparations should be at a concentration that is
`effective but nontoxic to humans? An ideal preservative should be
`active against a broad spectrum ofmicroorganisms but nontoxic to
`human cells and should be tolerable by the intended patient groups;
`it must also be stable and compatible with the other components of
`the drug product to be effective. Activities of commonly used anti-
`microbial agents, which are relatively safe for use in pharmaceuti-
`cal compounding, will be discussed in the following sections. The
`principle of antimicrobial effectiveness testing and its require-
`ments in the compounding practice will also be discussed.
`
`GENERAL CONSIDERATIONS IN THE
`
`SELECTION OF ANTIMICROBIAL
`
`PRESERVATIVES
`
`Most viable cells function optimally within a narrow pH range
`around neutrality, and growth is slow at pH beyond 6 or 8.4 This pH
`range may not always be optimal due to solubility and stability effor-
`mulation ingredients. Hence, the pH of a formula is often adjusted to
`enhance product quality. In terms of solubility, the optimum pH for
`formulation ingredients can be deduced from their dissociation con-
`
`stants (pKa) and their oil-water partition coefficients (LogP°/W).
`Both parameters are related to their aqueous solubility, where the
`antimicrobial effect is required, and their concentration in different
`phases ofa multiphasic system. However, the relationship between
`pH and antimicrobial activities is more complex. For example, the
`antifungal activity ofbenzoic acid is less susceptible to pH than its
`antibacterial activity. Similarly, sorbic acid has significant anti:fun-
`gal but little antibacterial activity at pH 6.0.4
`Antacid formulations and multiphase systems are more difficult
`to preserve than simple aqueous formulas. Such products require
`additional ingredients that have a high potential for interactions.
`Interactions ofpreservatives with formulation ingredients and con-
`tainers may compromise product stability and preservative effi-
`cacy. Interactions do not always lead to structural modification of
`the preservatives but may occur in the form of complex formation,
`precipitation, or adsorption to surfaces. Incompatibility among
`components occurs in the presence of strong oxidizing agents, or
`between a strong base and acidic preservatives. Cationic preserva-
`
`tives are incompatible with anionic surfactants, and non-ionic sur-
`factants (e.g., polysorbate 80) are incompatible with some alcohol
`phenolic preservatives. The parabens, benzoic acid, chlorobutanol,
`m-cresol, etc. are relatively volatfle and can be lost during process-
`ing and storage. Preservative precipitation in the presence ofpoly-
`valent cations was observed with sorbic acid, butylated
`hydroxyanisole, chlorhexidine, etc. Additionally, reconstitution of
`Activase, Proleukin, and Leukine with diluents containing preser-
`vatives may denature protein and peptide molecules}-5 Lab tech-
`niques such as size exclusion chromatography (SEC), dynamic light
`scattering (DLS), fourier transform infrared (FTIIR), electron
`microscopy, histologic analysis, and immunological assay have been
`used to characterize interactions in small-molecule drug products
`and in biopharmaceuticals?
`In addition to in vitro formulation issues, in viva adverse effects
`may further limit the availability of suitable agents for preserved
`products. As previously discussed, most preservatives are cytoxic
`to microbial cells, and their use may impart unintended side effects
`in patients. Notably, benzyl alcohol is not recommended in neona-
`tal parenteral products, as it has been linked to fatal toxic syn-
`drome in premature neonates. Irritants, such as parabens, were
`determined unsuitable for ophthalmic preparations, and benzalko-
`nium chloride may not be appropriate for soft contact lenses solu-
`tions. Concerns over neurotoxicity have lead to the declined usage
`of organomecuric compounds in parenteral products, and hexa-
`chlorophene in topical products.“
`The above discussion highlights formulation and external factors
`that must be considered in the preparation ofpreserved products.
`Optimization ofthe preservative system is often conducted during
`pre-formulation studies, which are not usually performed for com-
`pounded preparations, thus emphasizing the requirements for the
`demonstration ofantimicrobial efficacy in dispensing and beyond-
`use dating (BUD).
`
`COMMONLY UTILIZED ANTIMICROBIAL
`
`PRESERVATIVES
`
`In a review conducted by Meyer, et al,2 the authors observed that
`macromolecular biotech products such as peptides and proteins
`usually contain phenol and benzyl alcohol as preservatives.
`Vifhereas a combination ofparabens are found in small molecule
`parenterals, and phenoxyethanol is often found i.n vaccines. M-cre-
`sol and chlorobutanol are present in fewer products, and older prod-
`ucts may also contain thimerosal or phenylmercurric salts although
`they are no longer preferred agents in new formulas. Most intrace-
`cular and intrathecal products are preservative-free because of
`safety considerations."
`A list ofcommon antimicrobial preservatives with proven perfor-
`mance characteristics in various dosage forms is provided in Table
`1. Although limited in content, the table contains historical data
`that may be useful as a quick reference in a busy pharmacy environ-
`
`International Journal ofPharmaceutical Gonnpounding
`124 Vol. 18 No. 2 | March | April | 2014
`
`PAGE 2 OF 8
`
`www.IJ'.PG.eom
`
`PAGE 2 OF 8
`
`

`
`Uualilvflunmil
`
`ment. TheHandbook ofPharmaceutical Excipients3 is a comprehen-
`sive source of data describing physicochernical properties and
`safety profiles ofavailable excipients, including antimicrobial pre-
`servatives. Interested readers are directed to this reference for
`additional information.
`
`level should occur in
`1 to 3 log reduction in bacteria from the
`one to two weeks, with no further increase in bacteria thereafier at
`28 days. For yeast and mold, no increase from the initial inoculum
`level is permitted at all sampling intervals.
`
`ANTIMICROBIAL EFFECTIVENESS TESTING
`
`(UNITED STATES PI-IARMA COPEIA
`
`CHAPTER <51>)
`SUMMARY OF TEST
`
`The USP Chapter <51> Antimicrobial Efiectiveness Test‘ is con-
`ducted by adding specified microorganisms individually to the test
`product at relatively high concentrations to simulate contarnina—
`tion. The product is held for 28 days, during which time the added
`microorganisms are enumerated at defined intervals to determine
`any change in microbial content. Inoculated microorganisms
`include Candida albicans, Aspergillus niger, Escherichia coli‘, Pseu-
`domonas aeruginosa, and Staphylococcus aureus. The acceptance
`criteria are specified for each drug product categories. In general, a
`
`PRODUCT CATEGORIES AND SPECIFICATIONS
`
`Pharmaceutical products are divided into four categories based
`on product risk.‘ As shown in Table 2,‘ shorter sampling intervals
`during a 28-day period, and more stringent criteria are associated
`with Category 1 products, which includes sterile parenteral in
`aqueous base or emulsions (e.g., injections, otic products, ophthal-
`mic products, nasal products). Adequate preservation is indicated
`by not less than 1 and 3 log reduction in bacterial count from the
`initial value at day 7 and day 14, respectively. Subsequently, bacte-
`rial counts at day 28 should not increase from counts at day 14. Less
`stringent criteria are applied to topical and oral products in catego-
`ries 2 and 3. For oral and topical products, at least 1 log (oral prod-
`ucts) and 2 log (topical products) reduction from
`bacterial
`count at day 14 should be observed, and no increase relative to
`day-14 counts at day-28 testing. Antacid products are qualified by
`
`EMDMILLIPOIE
`
`Do you need guidance on
`complying with USP <797>?
`Sterile compounding solutions
`from EMD Millipore.
`
`0 Sterility Testing
`
`0 Media Fill
`
`0 Environmental Monitoring
`
`0 Consulting Services
`
`www.emdmilfiporc.com/sterilecompounding
`
`EMD Mllllpore Is a division of Merck KGaA, Dannmdt, Gennany
`
`Contact us for a free consultation
`sterilecompounding@emdmi|lipore.com or 800-64543476
`
`www.lJPC.oom
`
`PAGE 3 OF 8
`
`International Journal ofPhnrmIeouticIl Compounding
`Vol. 18 No.2 | March|April |2o14 135
`
`

`
`Uualiivfluniml
`
`TABLE 1. Common Pharmaceutical Preservatives.
`
`PRESBRVATIVE
`4-Chlorocresol
`
`FORMIILATION
`Oral, Topical
`
`4-Chloroxylenol
`
`Topical
`
`Benzaltonlum
`
`Benzethonium
`chloride
`
`Benzoic acid
`
`Oral, Ophthaknlc,
`Topical
`
`0.01 to 0.02
`
`Topical Ophthahtic
`
`Up to 0.5
`
`Oral, Parenteral,
`Topical
`
`Benzyl alcohol
`
`Oral. Parenteral
`
`Boric acid
`
`Cetrlmide
`
`Ophthalnic. Topical
`
`Ophthalnlc, Topical
`
`<5.0
`
`3.5 to 4.1
`
`- Ophthalmic: 0.005
`- Topical: 0.1 to 1.0
`
`Neutral or
`slightly altalne
`
`Chlorhexidine
`
`Chlorobutanol
`Inidurea
`
`Parenteral
`Topical Ophthatnlc
`
`Up to 0.5
`0.03 to 0.5
`
`m-Cresol
`
`Parenteral
`
`0.15 to 0.3
`
`Methybaraben
`
`Oral. Parenteral
`
`Phenols 0.5%
`
`Parenteral
`
`0.0018
`
`0.01
`
`Phenoxycthanol
`
`Parenteral Topical
`
`Potassium sorbate
`
`Oral, Topical
`
`Proolonic acid
`Propybaraben
`
`Oral. Topical
`Oral, Parenteral
`
`Sodium benzoate
`
`Oral. Parenteral
`
`Sorbic acid
`
`Oral, Topical
`
`Ophthalnic.
`Parenteral
`
`0.0002
`
`- Oral: 0.02 to 0.5
`- Parenteral: 0.5
`0.05 to 0.2
`
`0.001 to 0.01
`
`126
`
`International Journal ofPharmaceutical Compounding
`Vol. 18 No.2 | March |April | 2014
`
`PAGE 4 OF 8
`
`SPECTRUM
`
`- Bacteria. spores. molds. and yeasts
`- Active in acidic media
`Gram (+) bacteria
`Less active vs Gram (-) bacteria
`Synergistic with EDTA
`Gram (4) > Gram (-) bacteria
`Ineffective vs resistant P. aeruginosa strains
`Minimal activity vs bacterial endospores. acid-fast bacteria
`Bacteria. fungi. and molds
`Synergistic with ethanol
`Reduced efficacy by soaps and other anionic surfactants
`Moderate activity vs Gram (s) < Gram (-)
`Moderate activity vs fungal
`Moderate activity vs mold
`Moderate activity vs Gram (+) < Gram (-)
`Effective vs molds and yeasts
`Weak bacteriostatic
`Weak fungistatic
`Gram (+) > Gram (-) bacteria
`- Synergistic with alcohols
`Variable activity vs fungi
`Synergistic with EDTA vs resistant strains of P. aoruginosa,
`A. nioer. C. albicans
`Gram (+) > Gram (-)
`weak activity vs Proteus and Pseudomonas
`Inactive vs acid-fast bacilli
`weak activity vs molds. yeasts
`Activity Gram (+), Gram (-). and some fungi
`Broad-spectrum antibacteria
`Some antifungal properties
`Synergistic with parabens vs fungi
`Moderately Gram (+) > Gram (-)
`Weak activity vs yeasts and molds
`Broad spectrum antimicrobial activity
`Most effective vs yeasts and molds
`Moderate activity vs Gram (+) < Gram (-)
`Weak activity vs yeasts and molds
`Antibacterial vs P. aeruginosa < Proteus vulgaris
`Weak activity vs Gram (-)
`Frequently used in combination with other preservatives
`Predominantly antifungal
`Moderate antibacterial
`Bacteria. fungi. and molds
`Activity vs yeasts and molds > bacteria
`Gram (+) > Gram (-) bacteria
`Bacteriostatic
`Antifungal
`Primarily antifungal
`Weak antimicrobid
`Synergy with glycol
`Bactericidal at acidic pH
`Bacteriostatlc and funglstatic at akalne or neutral pH
`Ineffective vs spore-forming organisms
`
`www.IJPC.eorn
`
`

`
`Duality Uumml
`
`separate criteria in Category 4 due to the inherent issues with this
`product (e.g., high pH, interactions of preservatives with formula-
`tion ingredients). Effective antimicrobial activities in antacid prod-
`ucts are indicated by no increase in bacterial, yeast, and mold from
`initial counts when tested at day 14 and day 28. No increase or no
`change is equivalent to not more than a 0.5 log change from the
`initial inoculum level to account for variability ofthe test.
`
`organism are limited to no more than five passages removed from
`the original eed stock”
`The microbial enumeration test is performed to determine the
`number ofviable cells in each cell suspension. Bacteria are grown at
`30°C to 35°C on Soybean-Casein Digest Agar, while yeast and mold
`are grown at 20°C to 25°C on Sabouraud Dextrose Agar. Table 3
`describes the culture conditions for the preparation ofstandardized
`cell suspensions and microbial recovery study.
`
`TEST ORGANISMS AND PREPARATION OF
`STANDARDIZED CELL SUSPENSIONS
`
`CHALLENGE TEST
`
`A panel of five challenge organisms are used in USP <51>, includ-
`ing Candida albicans (yeast), Aspergillus mlger (mold), Escherichia
`colt‘ (Gram-negative enterobacillus), Pseudomonas aeruginosa
`(Gram—ncgative bacillus), and Staphylococcus aureus (Gram—posi—
`tive coecus). Fresh cultures of each organism are harvested in
`sterile saline and standardized to about 10‘ colony forming units
`per mL (cfii/mL). Extensive propagating of microbial cells is dis-
`couraged because it could lead to changes in phenotypic expression
`and antimicrobial susceptibility. Therefore, seed-stock techniques
`are recommended for long-term storage, and stock cultures of each
`
`The standardized cell suspensions are added to the test product
`in five separate containers, one container for each challenge
`organism. The concentration of challenge organisms in product
`Categories 1 through 3 is between 105 and 10° efu/mL. The prod-
`ucts in Category 4 (antacids) contain between 103 to 10‘ efu/mL of
`each challenge organism. The inoculum volume should not exceed
`1% ofthe total volume of the product to be tested. Inoculated
`samples are incubated at 20°C to 25°C for 28 days. The microbial
`enumeration test is performed at days 7, 14, and 28 by the vali-
`dated method.
`
`USP<797> COMPLIANCE
`
`INFECTION CONTROL
`
`TERMINAL CLEANING
`
`SIMPLE SOLUTIONS FOR
`
`COMPLEX PROBLEMS
`
`Contec, Inc. manufacturer wipes, mops, disinlectantx, sterile IPA and other products for
`critical cleaning. Visit www.contecinc.com/product:/u:p-797-phannacy/
`_ E or contact us at +1-864-503-8333 to request an evaluation sample today!
`
`£5 coN'rEc°
`
`25 YEARS OF INNOVATION 1983-2013
`
`www.lJPC.oom
`
`PAGE 5 OF 8
`
`International Journal ofPharmaceutical Compounding
`Vol. 18 No.2 | March|April |2014 137
`
`

`
`Uualiivflunirnl
`
`METHOD SUITABILITY TEST
`
`The antimicrobial preservatives in the
`drug product must be neutralized to recover
`viable cells in the microbial enumeration
`test. This neutralization can be accom-
`
`plished by neutralizing agents, membrane
`filtration, dilution, or any combination of
`these methods. Neutralization conditions
`
`must be validated for efficiency and suit-
`ability by the counting method. All organ-
`isms used in the challenge test must be
`included in the validation of methods. The
`
`validation protocol should follow guidelines
`elaborated in USP General Chapters <61>
`and <1227>_ Briefly, the validation study
`must show that recovery of an inoculum
`containing S100 cfu ofthe challenge organ-
`ism is not inhibited by the test sample and
`by the neutralization method This is
`accomplished by comparing recovery
`results for three treatment groups:
`
`1 The test group: Neutralized product
`inoculated with 100 cfu of the challenge
`organism
`
`2. The peptone control youp: The same
`treatment as in the test group but peptone
`is used instead ofthe test product
`3. Inoculum control containing 100 cfu of
`the challenge organism, but no neutral-
`ization and no product present
`
`The validation study is conducted in
`three independent experiments. In each
`experiment, average recovery ofviable cells
`in the test group should be at least 70% rela-
`tive to the inoculum control.
`
`TABLE 2. Four Categories ofDrug Products and Specifications for
`Antimicrobial Eflicacy}
`PRODUCT
`CATEGORY DECRIPTION
`
`CRITERIA FORYEAST
`AND MOLD
`
`CRITERIA FOR
`BAGPERIA
`
`1
`
`- Parenterals (injections.
`emulsions)
`Otic, ophthalmic, and
`sterile nasal products
`in aqueous base
`
`Topical products in
`aqueous base
`Nonstenle nasal
`products
`Nonsterile emulsions
`Products for rnucosal
`applcation
`Oral products in aqueous
`base (exclidlng antacids)
`
`Antacids in aqueous base
`
`- 21.0 log reduction at day
`7 relative to initial count
`- 23.0 log reduction at day
`14 relative to initld count
`- No increase at day 28
`relative to day-14 count
`- 29.0 log reduction at day
`14 relative to initid count
`- No increase at day 28
`relative to day-I4 count
`
`No increase at days 7, I4. and 28
`relative to initial count
`
`No increase at days 7, I4, and 28
`relative to initial count
`
`No increase at days 7, la. and 28
`relative to initial count
`
`~ 21.0 log reduction at day
`14 relative to initial count
`- No increase at day 28
`relative to day-14 count
`No increase at days 14 and 28 relative to initial count
`
`COMPARISON AMONG
`COMPENDIA MICROBIAL-
`
`EFFICACY TESTS
`
`Procedures for antimicrobial efficacy
`determination are described in three
`
`major compendia: the USP (Chapter <51>
`Antimicrobial Effectiveness Testing), the
`European Pharmacopoeia (EP) (Chapter
`<5.1.3> Efficacy ofAntimicrobial Preser-
`vation), and the Japanese Pharmacopoeia
`(JP) (Chapter <19> Preservative Effective-
`ness Test). These chapters are essentially
`harmonized in principles, but minor dif-
`ferences exist with respect to the challenge
`organisms, test intervals, and acceptance
`criteria? In situations where compliance
`to three compendia are required, these dif-
`ferences should be incorporated into the
`test protocol.
`
`PREPARATION OF CHALLENGE
`MICROORGANISMS
`The EP does not include E. coli in the
`panel ofchallenge microorganisms, but
`does allow supplementing the panel with
`additional species "that may represent
`likely contaminants,” and recommends the
`addition ofE. colt’ for all oral preparations.”
`USP <51> listed only the strains ofchallenge
`organisms sourced from American Type
`Culture Collection (ATCC), while both the
`EP and JP recognize additional source
`strains besides those listed in USP <51>.
`
`The incubation temperatures of subcul-
`tures are harmonized, but the incubation
`durations are slightly varied for yeast and
`mold. To comply with three compendia, G
`
`www.IJPC.eorn
`
`TABLE 3. Incubation Temperature and Incubation Time for Preparation
`of Standardized Cell Suspensions and Microbial Recovery Study.‘
`TIME
`(CELL
`SUSPENSION)
`18 to 24 hours
`
`ORGANISM
`E. coli
`
`P. aeruginosa
`
`$.aureus
`
`C albicans
`
`A. nlger
`
`CULTURE
`MEDIUM
`Soybean-Casein
`Digest (broth, agar)
`Soybean-Casein
`Digest (broth, agar)
`Soybean-Casein
`Digest (broth. agar)
`Sabouraud Dextrose
`(broth, agar)
`Sabouraud Dextrose
`(broth, agar)
`
`TIME
`(RECOVERY)
`3 to 5 days
`
`18 to 24 hours
`
`3 to 5 days
`
`18 to 24 hours
`
`3 to 5 days
`
`44 to 52 hours
`
`3 to 5 days
`
`6 to 10 days
`
`3 to 7 days
`
`International Journal ofPhnrmaeetrtical Compounding
`128 Vol. 18 No.2 | March |April | 2014
`
`PAGE 6 OF 8
`
`

`
`Dualnv Eumml
`
`TABLE 4. Comparison ofAcceptance Criteria for Parenteral Products
`byJP, USP, and EP.
`
`JP(% REDUCTION)
`FUNGAL
`AND MOLD
`
`TIREEBAGTERIA
`
`USP(LOG REDUCTION)
`FUNl.':‘zAI.-
`EA(}'l'EIIIA AND MOLD
`
`olbicons should be harvested at about 48
`
`hours. and A. niger should be harvested
`after 6 to 7 days “when good sporulaticn is
`obtained.” Standardized cell suspensions
`should be used within 8 hours, and stored at
`2°C to 8°C when not in use?
`
`| NT
`0.1% or less
`reduction
`s at day 14
`NI = no increase: NR = no recovery: NT = not tested
`50 = European Prrarrrraoqooeia-JP = Japanese u' U$P= United States Pharrrracooeia
`
`TEST INTERVALS AND
`ACCEPTANCE CRITERIA
`
`6 Hours
`24 Hours
`
`I NT
`NT
`
`7Days
`14 Days
`28 Days
`
`The acceptance criteria expressed in the
`EP are the most stringent compared to the
`USP and JP. The E? has two criteria (A
`and B) for products in Categories 1 (paren-
`teral intrauterine, intramammary prepara-
`tions) and 2 (car. nasal. inhalation,
`cutaneous preparations). The A criteria
`are "the recommended efficacy to be achieved,” and "In justified
`cases where the A criteria cannot be attained... the B criteria must
`be satisfied." The EP Category 1-A has approval criteria at 6 hours
`and 24 hours in addition to days ‘Ir’, 14, and 2.8. The JP has accep-
`tance criteria expressed as a percentage recovery for days 14 and
`28.9 To comply with three compendia requirements, sampling
`intervals should start at 6 hours for products in Category 1, and
`day 2 for products in Category 2. Table 4 shows sampling frequen-
`cies and acceptance criteria expressed by the EP, JP, and USP for
`sterile parenteral products.“-‘°
`
`SIGNIFICANCE OF ANTIMICROBIAL-
`
`EFFECTIVENESS TEST
`
`The purpose of USP <51> is to provide a guide to antimicrobial-
`effective testing. Preservatives are not meant to replace but to com-
`pliment current good manufacturing processes. USP <51> testing
`ensures the efficacy of pharmaceutical products containi.ng preser-
`vatives in original, unopened containers made a.nd distributed by
`the manufacturer. Measurement ofpreservation during in-use is
`outside the scope of the current protocol and requires different
`experimental designs (e.g., breaching study designs). In addition,
`the panel of five organisms employed in the challenge study does
`not represent resistant phenotypes that have acquired the ability to
`Withstand the activity ofthe preservative. The standard preserva-
`tive test may thenbe insufficient to demonstrate the survival capac-
`ity in pharmaceuticals of strains adapted to low-nutrient
`environment and low storage temperatures.”
`
`WHEN ANTIMICROBIAL-EFFECTIVENESS
`
`TEST IS PERFORMED
`
`The antimicrobial-effectiveness test is ofien performed during
`drug development for optimization of formulation ingredients. The
`International Conference on Harmonization (ICH) requires“:
`
`NT
`NT
`NT
`S at day 14
`5 at day ‘I4
`
`.EP(I.nOG REDUUHON)
`FUNGAL
`BACTERIA AND MOLD
`A
`‘B
`A
`3
`2
`NT
`NT
`NT
`3
`I
`NT
`NT
`NT
`3
`2
`NT
`NT
`NT
`NR
`NI
`Ni
`
`Antimicrobial preservative effectiveness should be demonstrated
`during development, during scale—up, and throughout the shelf-
`life.... although chemical testing for preservative content is the
`attribute normally included in the specification.
`
`HARDY oyacuosncs
`
`ultedlnidans and;-I
`
`International Journal ofPI|a:-maeenfioal Compounding
`Vol.1!-] NCL2 | Ma.rch.Apri] 2014
`
`PAGE 7 OF 8
`
`

`
`Uualirv Uumrul
`
`The ICH Q,6A further specifies”:
`
`CONCLUSION
`
`The testing for antimicrobial preserva-
`tive content should normally be per-
`formed at release. Acceptance criteria for
`preservative content should be based
`upon the levels of antimicrobial preser-
`vative necessary to maintain microbio-
`logical quality ofthe product at all stages
`throughout its proposed usage and shelf
`life. The lowest specified concentration
`of antimicrobial preservative should be
`demonstrated to be effective in control-
`ling microorganisms by using a pharma-
`copeial antimicrobial preservative
`effectiveness test.
`
`The ICH further states”:
`
`A single primary stability batch ofdrug
`product should be tested for antimicrobial
`preservative effectiveness (in addition to
`preservative content) at the proposed
`shelflife for verification purpose.
`
`In pharmaceutical compounding, USP
`Chapter <51> forms a part ofthe product
`quality test for preserved preparations due
`to limited pre-formulation data. Preserva-
`tive content and effectiveness testing should
`be a part of a stability program for BUD of all
`preserved preparations, “when such a test is
`performed, the results shall support the
`BUD assigned to the compounded prepara-
`tions."1 One strategy is to prepare formulas
`with 100% and 70% ofthe label concentra-
`tion for the preservative (limit for assay t
`20% ofpreservative label content). Preser-
`vative effectiveness and content are estab-
`lished for these samples at the initial time
`point, and then content testing will be con-
`ducted for the remainder ofthe stability
`time intervals. It is also prudent to confirm
`the preservative effectiveness at the BUD
`accordingto USP Chapter <51>. Based on
`the stability results, only the content test
`will need to be conducted for future batches
`
`unless fundamental changes occur in the
`formulation or compounding procedure.
`The above discussion pertains to antimicro-
`bial-preservative testing only and does not
`address other testing requirements for com-
`pounded preparations.
`
`The USP Chapter <51> Antimicrobial
`Effectiveness Testing is a culture -based
`method and accuracy of results is depen-
`dent upon adequate neutralization of anti-
`microbial activities in test samples for
`enumeration testing. The efficiency ofthe
`neutralization method employed must be
`validated for all five challenge organisms.
`Alternative methods can be substituted if
`
`proven to be equivalent to compendia testing.
`USP <51> does not address contamination
`by end users. Evaluating the efficacy ofthe
`preservative system for these in-use condi-
`tions requires different experimental
`designs that simulate in-use. The three
`major compendia (EP, JP, USP) are harmo-
`nized in principles but different in some
`aspects, which must be incorporated into
`the test protocol for compliance.
`
`REFERENCES
`1. United States Pharmacopeial Convention,
`Inc. United States Pharmacopeia 36-
`NationalFormulary 31. Rockville, MD:
`U.S. Pharmacopeial Convention, Inc.;
`2012: 54-55, 885- 889.
`Meyer BK, Ni A, Hu B et al. Antimicrobial
`preservative use in parenteral products:
`Past and present. JPharm Sci 2007;
`96(12): 3155-3167.
`Rowe RC, Sheskey PJ, Quinn ME, eds.
`Handbook ofPharmaceutical Excipients.
`6th ed. Washington, DC: American Phar-
`maceutical Association; 2009.
`Elder DP, Crowley PJ. AntimicrobialPre-
`servatives Part Two: Choosing a Preserva-
`tive. [American Pharmaceutical Review
`Website.] January 1, 2012. Available at:
`wwwamericanpharmaceuticalreview.
`com/Featured-Arl:icles/38885-Anti.micro-
`bial-Preservatives-Part-Two-Choosing-a-
`Preservative. Accessed January 16, 2014.
`Akers, MJ. Excipient-drug interactions in
`parenteral formulations. JPharm Sci
`2002; 91(11): 2283.
`Elder DP, Crowley PJ. AntimicrobialPre-
`servatives Part Three: Challenges Facing
`Preservatioe Systems. [American Pharma-
`ceutical Review Website.] January 1, 2012.
`Available at: www.americanpharmaceuti-
`calreview.com/Featured-Articles/38874-
`Antimicrobial-Preservatives-Part-Three
`
`Challenges-Facing-Preservative-Systems.
`Accessed January 16, 2014.
`Elder DP, Crowley PJ. AntimicrobialPre-
`servativesPart One: Choosing a Preserva-
`tive System [American Pharmaceutical
`Review Website.] January 1, 2012. Avail-
`able at: www.americanpharmaceuticalre-
`view.com/Featured-Articles/38886-
`Antimicrobial-Preservatives-Part-One-
`Choosing-a-Preservative-System.
`Accessed January 16, 2014.
`Sutton S. GMP and Compounding Phar-
`macies. [American Pharmaceutical
`Review Website.] April 30, 2013. Available
`at: www.arnerica.npharmaceuticalreview.
`com/Featured-Articles/135985-GMP-
`and-Compounding-Pharmacies. Accessed
`January 16, 2014.
`Moser CL, Meyer BK. Comparison of com-
`pendia] antimicrobial effectiveness tests:
`A review. AAPSPharmSciTech 2011; 12(1):
`222-226.
`. European Pharmacopoeia. EP 7.0-
`01/2011:50103 <5.1.3> Efficacy of antirni-
`crobial preservatives.
`. Chamock C, Otterholt E. Evaluation ofpre-
`servative efficacy in pharmaceutical prod-
`ucts: The use ofpsychrotolerant,
`low-nutrient preferring microbes in chal-
`lenge tests. J ClinPharm Ther 2012; 37(5):
`558-564.
`. International Conference on Harmonisa-
`tion ofTechnical Requirements for Regis-
`tration ofPharmaceuticals for Human
`Use. ICHHarmonised Tripartite Guide-
`line. Specifications: Testprocedures and
`acceptance criteriafor new drug sub-
`stances and new drugproducts: chemical
`substances QGA (4). October 1999.
`. International Conference on Harmonisa-
`tion ofTechnical Requirements for Regis-
`tration ofPharmaceuticals for Human Use.
`ICHHarmonised Tripartite Guideline. Sta-
`bility testing ofnew drug substances and
`products QIA (R2). February 2003.
`
`Address correspondence to Nicole Vu, PhD,
`Analytical Research Laboratories, Inc., 840
`Research Parkway, Suite 546, Oklahoma
`City, or: 73104. E-mail: nvu@arlok.com 1/
`
`International Journal ofPharmaceutical Compounding
`130 Vo1.18 No.2 | March |April | 2014
`
`PAGE 8 OF 8
`
`www.IJ'.PG.eon1
`
`PAGE 8 OF 8