COMMENTARY
`
`Development of the Antimicrobial Effectiveness Test as USP Chapter <51>
`
`Scott V. W. Sutton*1 and David Porter2
`
`1Alcon Laboratories, Fort Worth, TX and 2U.S. Pharmacopeial Convention, Inc., Rockville, MD
`
`ABSTRACT: The antimicrobial effectiveness test first appeared as a USP General Chapter in the 18th revision, official
`September 1, 1970. This chapter, at the beginning, was designed to evaluate the performance of antimicrobials added
`to inhibit the growth of microorganisms that might be introduced during or subsequent to the manufacturing process.
`As Good Manufacturing Practices (GMPs) became a governing principal in pharmaceutical manufacturing, the purpose
`of the test was refined to focus on activity of the preservative system as a protection against inadvertent contamina-
`tion during storage and usage of the product. This article will review the history of the antimicrobial test; its function,
`technique, and the background discussions that resulted in the changes from the test that appeared in USP XVIII to that
`of the current USP 25.
`
`Introduction
`
`The antimicrobial effectiveness test (AET) is designed
`to provide a laboratory test that gauges the level of bio-
`logical activity possessed by the preservative system of a
`pharmaceutical product. It is not meant to be a simulation
`of a real-world situation, nor is it meant as a guarantor
`that a preservative system that meets its requirements
`will never allow a contaminant to grow in the product.
`It was originally designed, and remains to this day, an
`assay that a careful laboratory can reproducibly per-
`form and one that will yield comparable results among
`a variety of laboratories. The value of those results in
`estimating the performance of the preserved product in
`the field is a subject of significant debate. Before looking
`at this controversy, however, let’s look to the genesis of
`today’s AET.
`
`USP XVIII - The Original Test
`
`The first appearance of this chapter was in the 18th edi-
`tion of the USP in 1970 (1), and is closely related to the
`one suggested in 1967 to USP by the Biological Section
`of the Pharmaceutical Manufacturer’s Association (2).
`It is of interest to note that there were other potential
`preservative tests being used at this time.
`
`* Author to whom correspondence should be ad-
`dressed: Alcon Research Ltd., Mail Code R0-15,
`6201 South Freeway, Fort Worth, TX 76134. Email:
`sysop@microbiol.org
`
`The stated purpose of the chapter “Antimicrobial
`Agents–Effectiveness” was “to demonstrate, in paren-
`teral and ophthalmic products, the level of any added
`antimicrobial agent(s), the presence of which is declared
`on the label of the product concerned.” The introduction
`to the assay also cautions that the tests apply only to
`products in the original container and that if a specific
`inactivator of the preservative is available, a suitable
`amount should be added to the agar plating medium.
`
`Challenge Organisms
`
`The test organisms specified were to be tested sepa-
`rately. This method differed from the method supported
`by Squibb and Abbott Laboratories which used a test
`with a mixed population of 21 different organisms and
`assayed for survivors over a 10 week period (3). The
`USP method used the five species individually which
`was subsequently shown to be a better indicator of
`preservative effectiveness (4) than challenging with
`a mixed culture. Although the species are familiar to
`today’s practitioners, they are not the same strain in
`all cases:
`
`
`
`
`
`
`
`Candida albicans ATCC 10231
`Aspergillus niger ATCC 16404
`Escherichia coli ATCC 4352
`Pseudomonas aeruginosa ATCC 9027
`Staphylococcus aureus ATCC 6538
`
`These microorganisms were based on the recommenda-
`tions of a Committee of the Biological Section of the
`Pharmaceutical Manufacturer’s Association, which
`
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`mL.” Conditions are described in Table 1:
`
`Microorganism
`Bacterial Cultures
`C. albicans
`A. niger
`
`Table 1
`Preparation of Inocula per USP XVIII
`Table 1. Preparation of inocula per USP XVIII.
`Incubation
`Temperature
`37oC
`25oC
`25oC
`
`Incubation Time
`18 – 24 hours
`48 hours
`1 week
`
`Wash Solution
`Sterile Saline TS
`Sterile Saline TS
`Sterile Saline TS containing
`0.05% polysorbate 80
`
`The contemporary practitioner will note with interest that the original instructions were to
`prepared a draft proposal in 1967. Interestingly, the origi-
`bial recovery (7). Interestingly, the media composition
`nal list of candidates was much longer and consisted of
`was referenced to the Microbial Limits Tests chapter, a
`determine the number of CFU/mL in each solution, and then use this to determine the size of
`several groups:
`practice that continues to this day.
`
`• Group 1 – Vegetative bacteria or yeast from standard
`sources
` Candida albicans ATCC 10231
`
`Staphylococcus aureus ATCC 6538
`
`Escherichia coli ATCC 4352
`
`Pseudomonas aeruginosa ATCC 14502
`
`• Group 2 – Special organisms isolated from products
`or the manufacturing environment
`
`• Group 3 – Bacterial or mold spore-formers
`
`Bacillus subtilus ATCC 6633
`
`Aspergillus niger ATCC 16404
`
`This committee concluded that the types of test organ-
`isms should be those that were found to contaminate the
`product—either through use or introduced with the raw
`materials. This seems strange to us today, as the AET is
`now well established as a referee test and so must be suit-
`able for use with no prior knowledge of the product. At the
`time the test was first introduced however, there were no
`monographs that made explicit references to the chapter.
`A requirement for the testing contained in the chapter
`could be inferred from text in the “Added Substances of
`General Notices” requiring that an added substance such
`as a preservative not exceed the amount necessary to pro-
`vide its intended effect. It was not a mandatory test. In
`fact, it was not until publication of the First Supplement
`to USP XXII (official Jan 1, 1990) (5) that a monograph
`for a preserved product specifically stated that it must
`meet the requirements of “<51> Antimicrobial Preserva-
`tives–Effectiveness” (reviewed in 6).
`
`Media
`
`Preparation of Inoculum
`
`The practitioner was instructed to grow the inoculum
`on the surface of a suitable agar plate from a recently
`grown stock culture. The cells were harvested using
`the solutions shown below and suspended to result in a
`microbial count of “about 100 million microorganisms
`per mL.” Conditions are described in Table 1.
`
`The contemporary practitioner will note with interest
`that the original instructions were to determine the
`number of CFU/mL in each solution, and then use this
`to determine the size of the inoculum to use in the test
`(Table 1). Further, if the standardized solutions were not
`used promptly, the suspensions were to be stored under
`refrigeration (defined as not above –45oF).
`
`Procedure
`
`This original procedure stated that the product was to
`be transferred to five tubes of 20 mL each, and then
`inoculated with 0.1 mL of the appropriate microbial
`stock (inoculum at a concentration of approximately
`50 million CFU per mL) to yield a final suspension of
`between 125,000 and 500,000 organisms per mL. These
`tubes were to be held at 30o – 32oC during the test. The
`inoculated product was to be examined “at suitable times,
`making not less than two observations, 7 days apart, at
`any time not later than 28 days subsequent to adding the
`inoculum” The investigator was to record any changes
`observed in the appearance of the sample, and make a
`plate count of the number of viable microorganisms pres-
`ent. These counts were then converted to a percentage
`change from the inoculum.
`
`The user was instructed to use a suitable agar media
`for initial cultivation of the microorganisms. The only
`specific media mentioned was Soybean-Casein Digest
`media which had been shown to be effective in micro-
`
`Interpretation
`
`The preservative system was defined as effective if there
`was “no significant increase in the number of Candida
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`albicans or Aspergillus niger organisms, and if the
`number of viable vegetative microorganisms is reduced
`to not more than 0.1 percent of the initial number and
`remains below that level for a 7-day period within the
`28-day period.” These criteria are so confusing as to be
`almost unusable, and the next version includes many
`revisions to the text to make both the procedure and the
`criteria more comprehensible.
`
`It is interesting to read some of the early commentaries
`on this test (2, 4, 8). Practitioners were already concerned
`with questions of how to make the test more reliable,
`less variable, the physiological state of the challenge
`organisms, and the test’s predictive power. These con-
`cerns are continually being addressed as the revision
`process proceeds.
`
`(e.g., <621> Chromatography). General chap-
`ters that include general requirements for tests
`and assays are numbered from <1> to <999>,
`chapters that are informational are numbered
`from <1000> to <1999>, and chapters relating
`to nutritional supplements are numbered from
`<2000> to <2999>.”
`
`The type of information introduced into this chapter
`by the 1975 revision underscores the status of the test
`as a control test to be performed by the manufacturer.
`As mentioned above, it would not be until 1990 that
`a preserved product would be required to meet the
`criteria of this test. However, this text, or text very
`much like it, persisted in subsequent revisions to the
`present day.
`
`USP XIX - Clarification
`
`Test Organisms
`
`The response to the original chapter indicated a need
`for much more clarity in the procedure. This redefini-
`tion began with the title, which changed from “Anti-
`microbial Agents – Effectiveness” to “Antimicrobial
`Preservatives – Effectiveness” to prevent confusion
`about the chapter’s impact on antibiotic test methods.
`The introduction to the chapter also includes much
`more detail, describing antimicrobials as “substances
`added to dosage forms to protect them from microbial
`contamination…used primarily in multi-dose contain-
`ers to inhibit the growth of microorganisms that may
`be introduced inadvertently during or subsequent to the
`manufacturing process” (9). The USP goes on to caution
`that “antimicrobial agents should not be used solely
`to reduce the viable microbial count as a substitute
`for good manufacturing practice.” The chapter further
`notes “. . . all useful antimicrobial agents are toxic sub-
`stances. For maximum protection of the consumer, the
`concentration of the preservative shown to be effective
`in the final packaged product should be considerably
`below the concentration of the preservative that may
`be toxic to human beings.”
`
`This is far more information and guidance than what
`had originally appeared in this chapter and sets the
`stage for a fundamental conflict in the structure of this
`chapter. According to the USP General Notices in USP
`25 (para10, p4) there are three different categories of
`General Chapters:
`
`“Each general chapter is assigned a number that
`appears in brackets adjacent to the chapter name
`
`The test organisms specified in 1975 did not change from
`the original test, with the exception of E. coli ATCC
`4352, which upon examination turned out to be Klebsi-
`ella pneumoniae. The reference strain of E. coli for the
`AET became ATCC 8739. A new allowance was added
`to provide for the inclusion of other organisms that may
`be introduced during the use of the product. However, no
`information was provided on how the testing laboratory
`was to choose these challenge organisms.
`
`Media
`
`Instruction was provided on the media used for recovery
`of organisms from the test in the section “Preparation
`of Inoculum.” This recovery was to be performed on the
`same media used to grow the inoculum, and if a neutral-
`izer for the antimicrobial was known, then this neutral-
`izer was to be included in the solid agar media.
`
`Preparation of Inoculum
`
`Several significant changes occurred in this section.
`The incubation temperatures were changed from a
`specific temperature to a 5o range, and the concentration
`of CFU/mL in the inocula was significantly increased
`(see Table 2).
`
`These more detailed instructions stated that if the
`standardized solutions were not used promptly, the
`suspensions were to be monitored by the plate-count
`method and could be used until a drop-off in viability
`was observed (presumably several days after the test
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`
`USP PET Historical Review
`
`8
`
`Bacterial Cultures
`C. albicans
`A. niger
`
`
`
`
`
`Inoculum CFU/mL
`1970
`1975
`About 50
`About
`million
`100
`million
`23
`
`Table 2
`Preparation of Inocula per USP XVIII vs USP XIX
`Table 2. Preparation of inocula per USP XVIII vs. USP XIX.
`Microorganism
`Incubation Temperature
`1970
`1975
`37oC
`30o – 35oC
`25oC
`20o – 25oC
`25oC
`20o – 25oC
`Table 3
`Summary of USP Criteria Through Revisions*
`These more detailed instructions stated that if the standardized solutions were not used
`Table 3. Summary of USP criteria through revisions.*
`promptly, the suspensions were to be monitored by the plate-count method and could be used
`
`Inoculum
`Criteria
`
`(CFU)
`7 Day
`14 Day
`21 Day
`28 Day
`Comments
`until a drop-off in viability was observed (presumably several days after the test using those
`USP XVIII (1970)
`125,000-
`Take “. . .not less than two observations, not less than 7 days apart at
`This original test was
`500,000
`any time not later than 28 days subsequent to adding the inoculum. . . .
`fundamentally sound, but the
`inocula). The provision for refrigeration of the stock cultures was deleted from this revision.
`An agent is adequate . . . if the number of viable vegetative
`criteria were very difficult to
`microorganisms is reduced to not more than 0.1 percent of the initial
`interpret.
`Instruction was provided on how to select the media used for recovery of organisms from the
`number and remains below that level for a 7-day period within the 28-day
`test period.”
`test. This recovery was to be performed on the same media used to grow the inoculum, and if a
`USP XIX (1975)
`100,000 –
`--
`0.1% Survival
`NI
`NI
`These criteria were introduced for
`1,000,000
`clarity. Although testing was
`required at Day 7 there was no
`neutralizer for the antimicrobial was known, then this neutralizer was to be included in the solid
`criterion at that time point.
`USP 24 (2000)
`
`The motive for all changes in
`agar media.
`1 x 105 -
`Category 1A
`
`criteria was the international
`harmonization effort. (see text)
`1 x 106
`--
`2.0
`--
`Category 1B
`
`Anhydrous medications included
`as “Category 2”
`--
`1.0
`--
`
`Category 1C
`
`
`Procedure
`NI
`NI
`NI
`Category 2
`
`
`Anhydrous medications deleted
`USP 25 (2002)
`Criteria same as categories 1A, 1B, and 1C, respectively
`to improve harmonization with
`
`Category 1-3
`This revision included a significant change in the procedure. Where the original
`Ph. Eur. Antacids were removed
`1 x 103 -
`
`Category 4
`NI
`NI
`NI
`NI
`as a class from Category 1C and
`1 x 104
`given a unique category based
`procedure clearly stated that the test solution should be transferred to test tubes prior to
`on market and regulatory input.
`
`1.0**
`
`3.0
`
`--
`
`NI
`
`NI
`NI
`NI
`
`inoculation, this version states a strong preference for conducting the test with the solution in
`* The USP test has required stasis for Aspergillus niger and Candida albicans since its inception. The criteria listed in this table are only for
`the bacterial challenge organisms.
`** All subsequent criteria are in terms of log10 unit reduction from the measured inoculum.
`the original container – even to the point of providing instruction on how to enter the container
`
`aseptically with a needle to inoculate and to sample the product. The inoculum volume was to
`using those inocula). The provision for refrigeration of
`on how to enter the container aseptically with a needle
`be equivalent to a ratio of 0.10 mL of inoculum (inoculum concentration of “about 100 million
`the stock cultures was deleted from this revision.
`to inoculate and to sample the product. The inoculum
`volume was to be equivalent to a ratio of 0.10 mL of
`CFU per mL”) to 20 mL of sample, so that the final concentration of microorganisms in the test
`Instruction was provided on how to select the media used
`inoculum (inoculum concentration of “about 100 mil-
`is between “100,000 and 1,000,000 microorganisms per mL” (see Table 3). The inoculated
`for recovery of organisms from the test. This recovery
`lion CFU per mL”) to 20 mL of sample, so that the final
`was to be performed on the same media used to grow
`concentration of microorganisms in the test is between
`samples were then stored at the storage temperature specified on the label or at 20o – 25oC if
`the inoculum, and if a neutralizer for the antimicrobial
`“100,000 and 1,000,000 microorganisms per mL” (see
`was known, then this neutralizer was to be included in
`Table 3). The inoculated samples were then stored at
`no storage temperature was specified. This point is worth exploring. The intent of stipulating
`the solid agar media.
`the storage temperature specified on the label or at
`20o–25oC if no storage temperature was specified. This
`point is worth exploring. The intent of stipulating the
`label storage temperature was to test the antimicrobial
`efficacy of the formulation under conditions similar to
`those of its intended storage conditions. This change in
`temperature (from USP XVIII to XIX) had the potential
`to dramatically affect the measured efficacy of the prod-
`ucts as a decrease in temperature usually has the affect
`of reducing the potency of a preservative (11). The test
`
`This revision included a significant change in the pro-
`cedure. Where the original procedure clearly stated that
`the test solution should be transferred to test tubes prior
`to inoculation, this version states a strong preference
`for conducting the test with the solution in the original
`container – even to the point of providing instruction
`
`Procedure
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`samples were examined at 7, 14, 21, and 28 days for
`surviving microorganisms. This section of the chapter
`most dramatically shows the push for additional clarity
`in the revision.
`
`USP chapters carried numbers, and so the official title of
`the chapter changed from “Antimicrobial Preservatives
`– Effectiveness” to “<51> Antimicrobial Preservatives
`– Effectiveness” in USP XX.
`
`Interpretation
`
`This section was completely rewritten to improve
`the clarity, and account for the specific test intervals
`described in the procedure. The preservative system
`was defined as effective if “(a) the concentrations of
`viable bacteria are reduced to not more than 0.1% of
`the initial concentrations by the fourteenth day; (b) the
`concentrations of viable yeasts and molds remain at or
`below original levels during the first 14 days; and (c)
`the concentration of each test organism remains at or
`below these designated levels during the remainder of the
`test period.” These criteria, established in 1975, remain
`fundamentally unchanged to this day.
`
`USP XX, XXI & XXII – A Period of Calm
`
`There were several suggestions for change during these
`years in the published literature. Orth (16, 17, 18, 19)
`recommended the use of D-values to establish preserva-
`tive efficacy, despite the fact that many chemical systems
`do not yield linear kill slopes (20, 21). The FDA was
`also developing an antimicrobial efficacy test for use
`with contact lens solutions (22). In addition, there were
`suggestions that the container closure system may have
`much to do with an adequately preserved product (23).
`Finally, the problem of testing anhydrous ointments was
`receiving some attention (24).
`
`In summary, although there was little activity by USP on
`the topic of antimicrobial effectiveness, a good amount
`of thought was being directed at the topic. A good review
`of the contemporary thinking can be found in a 1989
`review article by Cooper (25). The main points are ques-
`tions of harmonization with the British Pharmacopeia,
`variability, validation of microbial recovery, testing of
`ointments, and the criteria for passage.
`
`The 15 years from 1975 through 1990 saw little change
`in the chapter. USP XX (1980 - 12), USP XXI (1985-13)
`and USP XXII (1990-14) were published with text nearly
`identical to that which first appeared in 1975. One change
`that did occur was to reverse the decision on incubating
`the test samples at the label condition. The reference to
`storage temperatures specified on labels was simplified
`Several proposals were made in the period of 1990
`to “incubate the inoculated containers or tubes at 20o to
`through the present with the goal of reducing the reputed
`25o[C]” (initially proposed in 1982 (15) and finalized in
`level of inter-laboratory variability in the test (summa-
`USP XXI (13)). The only other change occurred in USP
`
`
`24
`rized in Table 4). The use of the Phenol Coefficient as
`XXII where a provision was made for the inocula to be
`a method to determine the suitability of the challenge
`grown in liquid media rather than requiring growth on
`organisms was proposed in 1992 (26). This test was in-
`solid media. As an aside, 1980 was the first year that the
`tended to be used to qualify the stock cultures, provid-
`Table 4.
`Changes Proposed to Reduce Variability*
`Table 4. Changes proposed to reduce variability.*
`
`USP 23, 24, & 25 - Attempts to Reduce Variability
`
`Rationale
`Change
`Reduce variability in
`Phenol coefficient to validate stock
`inoculum
`cultures
`Biocide qualification of stock cultures Reduce variability in
`inoculum
`Reduce variability in
`inoculum
`Reduce variability in
`inoculum
`Reduce variability in
`inoculum
`
`Restrict number of passages to 5 from
`original ATCC
`Greater detail in media and incubation
`conditions for inoculum prep.
`Requirement that inoculum be
`prepared fresh
`
`Change in criteria from one significant
`figure to two significant figures
`* See text for details
`
`Reduce variability in
`interpretation of results
`
`Disposition
`Proposal Rejected
`
`Proposal Rejected
`
`Official
`
`Official
`
`24 hours was defined as “fresh” to
`allow different shifts in the same facility
`to use the same inoculum for testing
`Official
`
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`ing documentation that the resistance of the challenge
`organisms was not changing with time. Due to severe
`concerns over the adequacy and appropriateness of this
`method, the Subcommittee proposed several changes
`designed to qualify the stock cultures used in the assay
`(27, 28), the first of which was proposal for an Anti-
`microbial Resistance Suitability Test in 1995. This test
`was designed to address the shortcomings of the Phenol
`Coefficient. The challenge organisms would be qualified
`using several common preservative agents, rather than
`just the single agent phenol. This qualifying test was
`not well received either. On the basis of comments and
`recommendations made at the USP Microbiology Open
`Conference in 19961, the Microbiology Subcommittee
`(MCB) resubmitted the previously proposed revision of
`this general test chapter with substantive changes. The
`new proposals included the deletion of the Stock Culture
`Antimicrobial Resistance Suitability section, the require-
`ment for a 21-day sampling interval, and the requirement
`to use microorganisms that have been isolated from the
`environment. In addition, a new requirement was added
`to ensure that all stock cultures used were within five pas-
`sages from the original ATCC stock. This requirement,
`a component of the Sterility Test since USP XXI (13),
`was included in an attempt to establish control over the
`organisms used in the test.
`
`Another change in inoculum handling dealt with the
`age of the inoculum suspension. Recall that in the
`original test the inoculum suspensions were to be used
`promptly, or held under refrigeration until use (1). The
`next revision (9) stated that if the suspensions were not
`used promptly, then the viability should be monitored.
`This 1996 proposal recommended changing the holding
`times to not more than 24 hours for bacteria and yeast,
`and not more than 7 days for fungal spores (28). It was
`in this proposal, made at the end of 1996, that media for
`growth of the challenge organisms was specified and
`finally stated bacteria were to be grown on Soy Casein
`Digest Media while the fungi were to be grown under
`different conditions on Sabouraud Dextrose Media.
`In addition, the text was changed to clarify that the
`inoculum suspensions were to be standardized using
`a spectrophotometer, and the numbers confirmed by
`plate count. This method had been shown, at least for
`yeast, to provide a reproducible concentration of cells in
`the inoculum (29). It must be noted, however, that this
`proposal remains controversial (30).
`
`Other changes in this revision did not deal expressly with
`reducing variability. These included renaming some of
`the product categories – Category “1D” for antacids ap-
`
`peared as Category 1C for oral products. After lengthy
`debate over the peculiar requirements of liquid antacids,
`it was decided that, if special requirements were indeed
`necessary for this product class, these requirements were
`to be included in the specific antacid monograph. The
`MCB Subcommittee planned to develop an informational
`chapter on the Antimicrobial Effectiveness Test, which
`would deal with a number of issues raised at the January
`1996 Open Conference.
`
`This proposal generated a great deal of discussion in the
`pharmaceutical community, and was the subject of more
`discussion at the 1996 Interpharmacopeial Conference2.
`An In-process Revision was published (31) clarifying
`the requirement that multi-dose products must fulfill the
`criteria in the chapter (thus finalizing the status of <51>
`as a referee test).
`
`The criteria for passage were modified as well. The
`criteria for passage had been expressed in percent sur-
`vival (for example, not more than 0.1% survivors after
`14 days), and then as log reduction (see discussion on
`harmonization below). There was confusion about the
`interpretation of this; however, as many practitioners
`looked to the General Notices discussion on significant
`figures and decided that a “3 log reduction” was satis-
`fied by data demonstrating at least a 2.5 log reduction.
`This was not the intent of the subcommittee and so the
`criteria were amended to two significant figures (i.e., “3.0
`log reduction”) to eliminate this source of variability in
`data interpretation.
`
`Final editorial changes were presented early in 1997
`(31). This version was approved by the United States
`Pharmacopeial Convention and published in the Eighth
`Supplement to USP 23 – NF 18 (p. 1681) effective May
`15, 1998 (32). At this point it seemed that the obvious
`steps had been taken on the part of the Pharmacopeia to
`clarify those factors that would reasonably be expected
`to contribute to variability in the test outcome.
`
`USP 23, 24 & 25 - Trying to Harmonize Internationally
`
`The desire to harmonize at least the European Phar-
`macopoeia (Ph. Eur.) and the USP versions of this
`test was well established by the early 1990s (33, 34).
`However, after the pair of meetings in Sanibel Harbor
`and in Barcelona on the topic, there was some confu-
`sion in the field about the status of the harmonization
`efforts for both the AET and the Sterility Test. A review
`of the status of this effort was published in 1997 (35)
`as a Stimuli to the Revision Process. At that time, the
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`test had reached a point where most of the contentious
`issues had been analyzed, discussed, and considered.
`International face-to-face meetings of the pharmacopeial
`experts along with Open Conferences have resulted in
`advances in harmonization. However, the criteria for
`antimicrobial effectiveness were outstanding among the
`issues that were not harmonized.
`
`marized in Table 5). There are three main areas where
`USP has attempted to improve harmonization with the
`Ph. Eur. – criteria, inoculum, and product categories.
`The first is a change in the manner that the criteria are
`expressed. Prior to the 8th Supplement, the criteria for
`bacteria were expressed as “per cent reduction.” The Ph.
`Eur. suggested that this did not accurately convey the
`level of precision available to the microbiology labora-
`tory, and so these reductions should be expressed in
`terms of their log10 values. The Ph. Eur. also insisted
`that 14 days was too long to wait for the first evidence
`of activity, urging 6 hour, 24 hour, and 7 day criteria
`(see 37 for rationale). USP added a 7 day time point but
`could not add criteria at 6 and 24 hour as no informa-
`tion existed as to the performance of currently marketed
`products at these time points. Finally, Ph. Eur. suggested
`that the term “No Increase” was too stringent, and that an
`increase of 0.5 log10 units should be allowed to account
`for variability (38), a position supported by independent
`research (39). USP had previously suggested a factor
`There have been several substantive changes in the of-
`
`USP PET Historical Review
`25
`ficial USP AET test from 1995 to the present that have
`of 150% to address this issue (26), but accepted the
`been directly linked to the harmonization effort (sum-
`Ph. Eur. recommendation. Ph. Eur. later changed this
`
`Several new concerns were raised at the 1998 USP
`Open Conference on Microbiology3. Among these was
`the need to delete the requirements for antimicrobial ef-
`fectiveness testing of products with a nonaqueous base or
`vehicle. The deletion of this requirement would improve
`harmonization with the European and Japanese Pharma-
`copoeias. Therefore, a proposed revision was published
`in 1999 (36) with this change. This became official with
`the publication of USP 25, in January of 2002 (10) (this
`volume is alternately referred to as USP 2002).
`
`Table 5
`Changes in USP to Promote Harmonization with Pharm. Eur.*
`Table 5. Changes in USP to promote harmonization with Ph. Eur.*
`Change
`Rationale
`Change in criteria from %
`More accurately expresses
`reduction to log reduction
`level of precision in results
`Requirement that inoculum
`Reduce variability in
`be prepared fresh
`inoculum
`
`* See text for details
`
`306
`
`PDA Journal of Pharmaceutical Science and Technology
`
`Addition of 7 day criterion
`
`Ph. Eur. insistence on
`need for short time points
`
`Inoculum in Ph. Eur. is 1%,
`0.5% in USP
`Product Categories
`
`Non-sterile Otic and Nasal
`products should not be in
`Parenteral category
`Non-aqueous category is
`unnecessary
`
`Variability in counting
`should allow 0.5 log units as
`“no increase”
`
`Compromise
`
`Different routes of
`administration have
`different risks
`Sterility not required
`
`Low water activity prevents
`growth of microorganisms,
`therefore no need to test
`This recommendation by
`Ph. Eur. was later changed
`on part of Ph. Eur. to 0.3
`log
`
`Disposition
`USP Adopted Ph. Eur. suggestion
`
`USP settled on 24 hours to take
`shifts into account, Ph. Eur. at 8
`hours
`Concern over products on market
`prevented 6 hr and 24 hour time-
`points
`USP widened inoculum range to
`include the Ph. Eur. preference
`USP adopted product categories
`
`USP changed categories to reflect
`non-sterile attributes of products
`
`USP removed this product category
`from testing requirements at Ph.
`Eur. recommendation
`USP rejected own suggestion of
`150% and adopted original
`European suggestion of 0.5 log.
`Unlikely to change again to 0.3
`log10 unit definition of variability
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`recommendation to 0.3 log10 units without explanation
`(40), and this difference is now a point of disagreement
`between the pharmacopeia.
`
`A second point bears some discussion. The Ph. Eur.
`AET contains a “zero” time point. The intent of this
`time point is to validate the test for its ability to recover
`organisms in the presence of the preserved product (41).
`However, in practice it is found that strongly preserved
`formulations immediately reduce the viable microor-
`ganisms recoverable from the sus