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.
The antimicrobial efficacy test (AET) is, at its heart, a suspension test for microbial kill (Figure 1). That is to say, a controlled inoculum of the challenge organism(s) is placed in suspension with the sample to be tested, and then the number of survivors determined at different time points. This simple design has some real strengths in terms of the opportunities to compare the biological activity of a wide array of preservative systems in a controlled manner, but some significant limitations as well. As just one example, the preservation of a finished product is a balance between the preservative system and the patient’s use of the product, a balance heavily affected by the container/closure properties of the packaging (1). This consideration of performance in the field is completely outside the scope of the AET and a task for which it is ill-suited (2).
Let’s take a brief look at the approaches used by the USP and the Pharm. Eur. to this question of the scope and purpose of the test.
For starters, let’s turn to the General Notices section of USP for guidance on the status of different chapters. Section 2 “Official Status and Legal Recognition” instructs us that
“…General chapters numbered from 1000 to 1999 are considered interpretive and are intended to provide information on, give definition to, or describe a particular subject. They contain no mandatory requirements applicable to any official article unless specifically referenced in these General Notices, a monograph, or a general chapter numbered below 1000.” In other words, those chapters numbered under 1000 are mandatory as they are referenced in monographs (and are designed to determine these monograph requirements) while those numbered over <1000> are for information only.”
Later, in section 3 “Conformance to Standards” we learn that “The standards in the relevant monograph, general chapter(s) and General Notices apply at any time in the life of the article from production to expiration.” In other words, these mandatory monograph requirements are also stability requirements.
More information on the specific purpose of chapter <51> can be found in the preface to the chapter:
“Antimicrobial preservatives are substances added to nonsterile dosage forms to protect them from microbiological growth or from microorganisms that are introduced inadvertently during or subsequent to the manufacturing process. In the case of sterile articles packaged in multiple-dose containers, antimicrobial preservatives are added to inhibit the growth of microorganisms that may be introduced from repeatedly withdrawing individual doses.
Antimicrobial preservatives should not be used as a substitute for good manufacturing practices or solely to reduce the viable microbial population of a nonsterile product or control the presterilization bioburden of multidose formulations during manufacturing…
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.
The concentration of an added antimicrobial preservative can be kept at minimum if 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 all injections packaged in multiple-dose containers or for other products containing antimicrobial preservatives. Antimicrobial effectiveness must be demonstrated for multiple-dose topical and oral dosage forms and for other dosage forms such as ophthalmic, otic, nasal, irrigation, and dialysis fluids (see Pharmaceutical Dosage Forms <1151>).
This chapter provides tests to demonstrate the effectiveness of antimicrobial protection. Added antimicrobial preservatives must be declared on the label. The tests and criteria for effectiveness apply to a product in the original, unopened container in which it was distributed by the manufacturer …”
So from this we can gather that AET is a requirement for multi-dose containers, that we want to keep the concentration of preservatives to a minimum, and that the test should be performed on material from the final product container. From the General Notices we take that this test is designed for demonstration of compliance with monograph requirements.
The non-mandatory chapter 5.1.3 “Efficacy of antimicrobial preservation” state:
“If a pharmaceutical preparation does not itself have adequate antimicrobial activity, antimicrobial preservatives may be added, particularly to aqueous preparations, to prevent proliferation or to limit microbial contamination which, during normal conditions of storage and use, particularly for multidose containers, could occur in a product and present a hazard to the patient from infection and spoilage of the preparation Antimicrobial preservatives must not be used as a substitute for good manufacturing practice.
The efficacy of an antimicrobial preservative may be enhanced or diminished by the active constituent of the preparation or by the formulation in which it is incorporated or by the container and closure used. The antimicrobial activity of the preparation in its final container is investigated over the period of validity to ensure that such activity has not been impaired by storage Such investigations may be carried out on samples removed from the final container immediately prior to testing.
During development of a pharmaceutical preparation, it shall be demonstrated that the antimicrobial activity of the preparation as such or, if necessary, with the addition of a suitable preservative or preservatives provides adequate protection from adverse effects that may arise from microbial contamination or proliferation during storage and use of the preparation.
The efficacy of the antimicrobial activity may be demonstrated by the test described below. The test is not intended to be used for routine control purposes.”
Thus we learn from the Pharm Eur that the test is viewed as a developmental test, and one that is not to be used on a routine basis to determine the finished product quality. While not to be performed on a particular product regularly, the product is expected to meet the requirements if tested (see reference 3 for review).
The major difference between the AET of USP and that of Pharm Eur is in the acceptance criteria. Each test separates products into different categories based on criticality of the preservative system, and each has acceptance criteria for testing intervals lasting over 28 days. They differ in the extent of the required kill and in the time points for testing, with Pharm Eur having some very short testing intervals (relatively speaking). In addition, Pharm Eur provides a “target” acceptance level and a “mandatory” or “minimal” level. The acceptance criteria are reported as log10 unit reductions, and calculated as the log10 unit value of the inoculum from which is subtracted the log10 unit value of the survivors at the particular time point. An example of this difference is presented in Table 1 for the Category 1 products.
The reader will note that the criteria are expressed to two significant figures in the USP criteria.
|(cfu)||6 Hr||24 Hr||7 Day||14 Day||21 Day||28 Day|
The antimicrobial efficacy test is designed to be a test of the biocidal activity of a preserved formulation. As such, it is important to recover those viable cells remaining in suspension. Residual preservative in the recovery agar could artificially depress the recovery of viable cells, and so it is important to neutralize this residual activity to get accurate counts of survivors. Common methods for inhibition of residual biocide include dilution or chemical neutralization of the biocide. Dilution is useful for those biocides with a large concentration exponent and little propensity for binding to the cell (4-6). A variation on dilution is filtration of the suspension to remove the biocide (such as is done in the compendial Sterility Tests). This technique must be approached with caution, however, as the biocide may bind either to the membrane filter or to the cells, inhibiting recovery (7-12). Finally, one can inhibit residual biocide by chemical neutralization (see 13 and 14 for reviews). Several classes of biocides have well-established chemical neutralizers. Examples of these are noted in Table I (this type of table can also be found in USP chapters <1072> and <1227>). A potential difficulty of chemical neutralization of biocides is the toxicity displayed by several types of neutralizers. Examples of these are also provided in Table 2. Evaluation of a chemical neutralizer or a physical neutralization scheme must examine the potential toxicity of the neutralizer as well as its efficacy.
An excellent review by Russell (13) describes three criteria for an effective neutralizer. First, the neutralizer must effectively inhibit the action of the biocidal solution. Second, the neutralizer must not itself be unduly toxic to the challenge organisms. Finally, the neutralizer and active agent must not combine to form a toxic compound.
Three methods have been published describing methods of neutralizer evaluation. Dey and Engley (15) describe a procedure utilizing Staphylococcus aureus as the index organism that measures survival with time. The challenge organism is inoculated directly into the biocide and sampled with time. The relative efficacy of the neutralizer is measured by comparing the relative recovery of the challenge organism among different treatments. This protocol is useful in identifying neutralizers. However, it has several limitations. First, it does not distinguish between neutralization of the biocide versus recovery of organisms injured by sub-lethal exposure to the biocide. An apparent increase in recovery could result either from a decrease in the activity of the biocide upon treatment, or from improved recovery of crippled organisms. A second concern is that this method utilizes only one organism, S. aureus. The assumption is that a neutralizer acts upon the biocide, independent of the challenge organism.
Terleckyj and Axler (16) describe a control procedure to demonstrate neutralization for a fungicidal experiment. The basic design for this experiment was first described in 1972 by Bergan and Lystad (17). First, the biocide is exposed to the neutralizer for a specified time. The challenge organism (Candida albicans) is then added to a final concentration of approximately 106 CFU/mL. Survival is determined after an additional 15 minute incubation. While this design separates recovery from inactivation of the biocide, several concerns remain. This method assumes that a neutralizer proven effective for one organism will be equally effective for all. In addition, this method uses extensive dilution of the organism prior to plating. Dilution of the sample is required due to the high number of organisms in the challenge, but serves to dilute the biocide, which compromises the stringency of the procedure. Finally, this method does not allow the investigator to separate any toxic effect of the neutralization treatment from the effects of the biocide.
The final method was described for use in testing contact lens disinfectants (18). This method is similar in overall design to that described above (17). However, it addresses some of the concerns suggested by earlier protocols. This method employs a smaller inoculum concentration to avoid dilution of the sample. It also takes advantage of statistical analysis of the data to ensure that apparent differences among populations are supported. Finally, and perhaps most importantly, this method evaluates the potential neutralizer with all index organisms. This strategy is employed by the American Society for Testing and Materials (ASTM) in the evaluation of neutralizers (19). It is also the method recommended by USP for validation of microbial recovery (20).
|BIOCIDE CLASS||CHEMICAL NEUTRALIZER||POTENTIAL TOXICITY|
|Glutaraldehyde, Mercurials||Bisulphate||Non-sporing Bacteria|
|Phenolics, Alcohol, Glutaraldehyde||Dilution||- – -|
|Quaternary Ammonium Compounds (QACs), Parabens, Bis-biguanides||Lecithin||Bacteria|
|EDTA||Mg+2 or Ca+2 ions||- – -|
|QACs, Iodine, Parabens||Polysorbate||- – -|
|Mercurials||Thioglycollate||Staphylococci and Spores|
|Thiosulphate||Mercurials, Halogens, Glutaraldehyde||Staphylococci|
Regardless of the method used to evaluate a neutralizer, there must be a population of organisms included that serve as a growth control. This control population is not exposed to neither the potential neutralizer nor the biocide. We suggest two comparisons among three populations, providing evidence for fulfillment of the three criteria outlined by Russell. The first comparison is Neutralizer Efficacy (NE) which can be determined by evaluating survivors in the neutralizing broth in the presence and the absence of the biocide. The ability of the neutralizing broth alone to allow survival is a second important consideration in this analysis. The second comparison is Neutralizer Toxicity (NT). This aspect of neutralization is determined by comparing survivors in the neutralizing medium without the biocide with the viability (growth) control.
As there were many compendial expectations (AET, bioburden, environmental monitoring, etc) that did not incorporate a specific method validation step, an informational chapter devoted to this topic was developed. <1227> describes a straight-forward design to determine the adequacy of a neutralization scheme:
In this scheme, multiple replicates (at least 3, preferably 5) of identical inocula are used in the three treatment groups.
The numbers of CFU arising on the recovery agar from each group is then compared. If the neutralizer is both effective and nontoxic, then all treatment groups will behave in identical manners. However, this is rarely so, and usually, some measure of neutralizer toxicity must be accepted to allow adequate neutralization. Neutralizer Toxicity can be estimated by the comparison between the viability group and the Peptone Control Group, Neutralizer Efficacy by comparison between the Peptone Control Group and the Test Group.
Modifications of this general design should be made for recovery by filtration to evaluate any loss of inocula by adherence to the filtration vessel. Similarly, USP <1227> describes modifications to this design for qualitative tests.
It should be stressed that even though this evaluation is not specifically required in the text of USP <51>, the results cannot be considered reliable without demonstration of an adequate recovery scheme.
The USP describes four product categories for multidose product products based on patient risk and route of administration. These categories have decreasing levels of antimicrobial effectiveness expected. By decreasing order:
1. Injections, otics, sterile nasals, ophthalmics with aqueous bases
2. Topicals, nonsterile nasals
3. Orals other than antacids (aqueous bases)
4. Antacids (aqueous base)
While the antimicrobial effectiveness test is not a batch release test, it is expected that all relevant products “if tested will comply” with its requirements. To this end, it is prudent to have some level of postmarket stability program in place. While the important time points for this stability program study would clearly be the initial and the AET at expiry, annual time point assays can provide useful information for many products.
Packaging components, especially dispensing closures, can be important considerations in preventing microbial contamination during consumer use. A study by Brannan and Dille (1) showed that, during consumer use, unpreserved shampoo with a flip-cap had the greatest degree of protection from contamination (0%). For an unpreserved skin lotion, a pump-top dispenser afforded the best protection from contamination (10%). Other types of closures tested included the standard screw-cap and slit-cap. The screw-cap closure provided the least amount of protection, while the slit-cap provided moderate protection from contamination. This study underscores the need for considering preservation as an attribute of the entire product, not just the active preservative.
We have examined the AET as a laboratory test, one that has value as a QC test but may not be appropriate for determining the behavior of the finished product preservative system in the field. There have been several recommendations for methods to determine this performance in the literature. One such example is the EMEA recommendation for in-use testing of preserved products (21).
The generation of this guidance prior to its final release was reviewed by Sutton et a (1998) from the perspective of the integrity of the biological and chemical integrity of the preservative system. In the authors opinion, in-use testing for chemical attributes makes sense if oxidation or evaporation of the preservative is a concern, especially in oxygen-permeable containers. The effects of repeated microbiological challenges is a reasonable consideration, and should be addressed in the lab by repeated low-level challenge studies, or in the field by simulated in-use studies.
Unfortunately the final EMEA guidance document is a rather shallow exercise for most product categories. The product is dispensed (under undetermined conditions) at a rate approximating normal use. Chemical integrity and product sterility is checked after each simulated. Little is learned by this study as the laboratory is sure to conduct it under highly aseptic conditions to prevent the contamination we would like to study.
The manufacturer would be well-advised to conduct a simulated in-use study as part of the product development, but this will have to be done using in-house resources as there are no useful regulatory guidance documents for pharmaceutical products in this area.
Investigations are a critical area of regulatory concern, as many are confused about expectations. The FDA has provided guidance on the topic, the “Guidance for Industry – Investigating Out of Specification (OOS) Test Results for Pharmaceutical Production” which was released in 2006. Interestingly, this guidance document only briefly touches upon microbiological data, stating that “the USP prefers the use of averages because of the innate variability of the biological test system.” In addition, this guidance document specifically excludes microbiology from its scope in footnote 3.
On the international side, the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) has provided some guidance on what is meant by a “specification.” The guidance directs that
“Specifications are chosen to confirm the quality of the drug substance and drug product rather than to establish full characterization, and should focus on those characteristics found to be useful in ensuring the safety and efficacy of the drug substance and drug product.”
These guidance documents, although written for analytical chemistry tests, do provide some useful guidance to microbiology investigations. For example, the FDA guidance’s focus on investigating a potential laboratory component (with its respective responsibilities) prior to the full scale investigation is especially helpful. Great care should be taken, however, in over-applying these documents into areas for which they are not appropriate.
To separate true OOS investigations from microbiology lab investigations PDA has recommended the term Microbiological Data Deviations (MDD) which we will use in this discussion.
All investigations should happen in a timely fashion. This is also true for the laboratory investigation, and a method for this has been described by the author (22). The data deviation should be communicated immediately to the laboratory supervisor, who then informs management. The investigation should follow an established procedure (Standard Operating Procedure – SOP), a procedure that may be general or directed at a particular test type.
Prior to initiation of a formal investigation, it is prudent to check common data entry errors:
• Incorrect math
• Sample dilution series error
• Transcription error
• Clerical error
• Nonsensical microbial identification
The overall investigation will be coordinated though QA as per 21 CFR 211.160. The laboratory investigation should be accompanied by a review of the manufacturing record for that batch (or batches if deviation is in an environmental monitoring study). A proposed model for a general lab investigation is presented in Figure 3. If the data deviation was determined to be due to error in lab practice, then the test may potentially be declared invalid. An invalid test is, by its nature, not a valid test. Therefore, the subsequent test is not a retest but rather the first correct performance of the assay. This distinction is important as some assays may be, in fact, investigated by confirmatory testing due to the inherent variability of the assays, and the distinction between the two conditions must be kept clear in the investigation documentation.
Much of the investigation will require evaluation of data records and test samples. Physical constraints on space may prevent retention of all samples and test articles that might be of interest. However, it is prudent to retain as many of the critical samples as possible until the successful completion of the test to aid in the laboratory investigation of a potential issue.
The final phase in the laboratory investigation is closing it. The investigator should determine if the MDD was the result of:
1. Laboratory error (invalid test)
2. Assay variation
3. Failing product
4. Inconclusive causes
If the test is invalid, a valid test should be run immediately to determine compliance. In addition, a corrective action plan should be put in place in the laboratory to remedy the error and prevent its recurrence. This plan should include a measure of the efficacy of the corrective action.
If the MDD was due to assay variation, a determination should be made as to the appropriateness of confirmatory testing. This determination should be guided by SOP as assay variability is a known quantity in microbiological testing and procedures should be in place to resolve indeterminate results.
If the MDD was the result of failing product, the product should be handled as appropriate under management guidance.
If the MDD investigation is inconclusive, the investigation should be retained on record and considered in the batch or lot disposition decision. The results should not, however, be attributed to lab error.
In addition to the specific discussions in the tests, a general discussion of investigations can be found in USP chapter “<1117> Best Microbiological Laboratory Practices.” The final section in USP <1117> is entitled “Interpretation of Assay Results” and lists the difficulties associated with the practice:
The USP continues by reinforcing the point that these difficulties in interpretation are one reason that the supervisor of the lab should be one with academic training in microbiology or bacteriology. Microbiological data requires interpretation; it is not data that can be presented solely by test results. Investigations should be conducted from a broad perspective, not only looking at equipment, training, product and test, but also the underlying biology associated with the situation.
Identifying a problem is not enough – cGMP requires a solution to that problem and demonstration that the corrective action was effective. Adequate corrective action requires the correct identification of the root cause of the nonconformance.
Identifying a root cause for most MDD is difficult as microorganisms are ubiquitous, and humans are the primary source of contamination in most pharmaceutical manufacturing and testing environments. A second difficulty is that MDD usually occur as isolated events, with insufficient data to resolve a root cause. Not every investigation into a MDD will be successful in establishing a cause. Therefore, a common (and not unreasonable) corrective action for an MDD is training. This training should be coupled with proficiency testing and retrospective review of performance to establish the effectiveness of the training.
Note to reader – see also the monograph Investigation of Microbiological Data Deviations
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20. USP. <1227> Validation of Microbial Recovery From Pharmacopeial Articles. Tenth Supplement to The United States Pharmacopeia 23 / National Formulary 18. 1994. The United States Pharmacopeial Convention, Inc. Rockville, MD. p. 5063, effective May 15, 1999.
21. EMEA. Note for Guidance on In-Use Stability Testing of Human Medicinal Products. 2001.
22. Sutton, S. “Laboratory Investigations of Microbiological Data Deviations (MDD)” IN Laboratory Design: Establishing the Facility and Management Structure S. Sutton (ed) PDA/DHI Publishers pp. 81-100. 2010.
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