Drug/consumer product preservation: making sense of the USP 24 Chapter <51> Antimicrobial Effectiveness Test

By Robert R. Friedel
Perritt Laboratories

I recently had the opportunity to comment on the United States Pharmacopeia's Chapter <51> Antimicrobial Effectiveness Test during a recent query by a member of the Pharmaceutical Microbiology Forum's Internet discussion group. This represented an opportunity to provide real-world perspective as to what the in-vitro test actually provides in terms of product protection.

It is important to remember that the Antimicrobial Effectiveness Test (AET) is a laboratory assay, i.e., it is an artificial simulation of events, which could occur during the shelf life and subsequent consumer-use of a multiple-dose product. Because of this inherent limitation, the AET should not be used as an absolute predictor of a product's ability to resist microbial contamination when in storage or use. Furthermore, the AET will not predict what an environmental organism will do in terms of product spoilage.

The AET is essentially a tool, which measures "active" preservative and/or the antimicrobial effect, which certain materials can exert on a microbiological challenge. In terms of product development, the test's ultimate function is to differentiate (in the laboratory) poorly-preserved and marginally-preserved products, from well-preserved products. In the end, the goal is to present the latter to the consumer marketplace.

In this author's experience, too much emphasis has been placed on the test's ability to predict the product's resistance to microbial contamination in the "real world." Antacid product preservation is one example in which the experimental results can mislead the formulation scientist as to the relative merits of a particular preservative system and the microbiological stability it may provide.

There have always been issues regarding the results and interpretation of such laboratory evaluations. The following illustrates some of these criticisms:

The validity of the use of pure cultures from the American Type Culture Collection. These are the "wimps" of the world, having been in cryogenic storage for several decades. These organisms lack the "toughness" of environmental organisms that may have been conditioned (via environmental/chemical stresses) to resist the action of antimicrobial agents.

Organisms do not exist as pure cultures in nature. They are not single cell inocula (planktonic cells) from pure cultures as represented by the organisms cited in the AET. In the natural world, microorganisms live in dynamic ecological communities known as "biofilms." These microbial communities tend to persist on minimally available nutrients (oligotrophic) versus the nutrient rich media (copiotrophic) used in the AET. The reason behind the use of pure cultures is that they can be evaluated from both a qualitative (type) and quantitative (log reduction) perspective. Ironically, it is the "tough" environmental organisms which frequently die after subculturing onto artificial, laboratory media.

In the "real world," organisms do not stir themselves into the sample they are in the process of contaminating. This author is currently unaware of ANY microbe, which mixes itself homogeneously into the product when microbiological adulteration occurs.

Some organisms have the tendency to clump, providing protection to the innermost members of the aggregate and subsequently limiting/eliminating contact with the preservative system. If the clump happens to break apart during the laboratory recovery attempt and the contact time is short enough to allow the preservative-neutralizing diluents to cancel any residual antimicrobial action, the organism looks as if it "rose from the dead." This has been referred to as the "phoenix phenomenon." This author respectfully disagrees with this particular characterization. However, USP recognizes the fact that organism counts may inevitably spike higher, only to fall during subsequent recovery points. To address this, they have incorporated a corrective factor of 0.5 log10 when comparing counts to those established at previously determined time-points. In fact, the fungal spore inoculum preparation for A. niger (ATCC # 16404) incorporates the use of Polysorbate 80 (non-ionic surfactant) as a preventative measure to reduce clumping.

The amount of organism inoculated into the product is well beyond that experienced during consumer-use or for that matter, permitted in the controlled environment of a GMP manufacturing facility. A high inoculum value, e.g., 10⁵-10⁶ colony-forming-units (CFU) per 20 grams of product, is used solely to measure log reduction values. These will eventually be compared with the allowable levels for each route of administration as determined by the pharmacopeia.

The validity of the specifications themselves and the problems with the process of harmonization throughout the world. The process is on going but the major pharmacopeias have not yet harmonized their methodologies. What if the formula passes the USP but fails the EP criteria? Does that mean that the product will become contaminated if released to the public for use? Probably not, but in the universe of globalization, it makes sense to have one standard, thereby eliminating the need to sort product for various destinations around the world.

The usefulness of performing a subsequent challenge or "rechallenge." This is essentially a "capacity" test or how much of a microbial load a preservative system can take prior to failing the AET. There is no evidence that the "rechallenge" adds any more protection to the product than a normal 28-day assay. It may only indicate how the product will perform under gross contamination.

The errors inherent in quantifying living organisms. When one takes into account that plate counts can be in error as much as 100%, combined with variations in technique between individual analysts, the AET's predictive ability becomes less convincing. In addition, there is no scientific evidence to support the current USP practice of allowing a maximum of 5 transfers from the ATCC parent culture.

The effect of the various ways to grow and prepare the challenge inoculum (i.e., solid vs. liquid growth media OR fresh vs. frozen). Are there any discernable differences in the elimination or preservation of organisms when prepared by the various preparation/storage methods?

The utility of performing the test on non-aqueous, multiple-use dosage forms. In order to maintain homeostasis and reproduce, microorganisms require a certain level of "free" water. This can be determined experimentally by measuring the formulation's water activity (a_w). If the product is non-aqueous in nature, the dosage form is essentially self-preserving. In contrast, most of these formulation types contain additional preservation "just in case" (e.g., parabens).

However, these preservatives favor the hydrocarbon phase of the formula. At this point one has to ask: "If the preservative is not available in the aqueous phase of the formulation (which is the one requiring protection), how is it going to act upon the microbiological challenge presented in the laboratory, not to mention the real world?" Furthermore, how are the results of such a challenge to be interpreted? Is the lack of log reduction due to the inability of the paraben(s) to redistribute into the aqueous from the hydrocarbon phase during the contact time outlined in the AET or is the log reduction due to lack of available water to sustain the organism's turgor pressure?

This is not by any means an all-inclusive list. Unfortunately, the AET is currently the best in-vitro, laboratory tool available for accessing the protective characteristics of a particular formulation and for determining a preservative's actual killing potential. Conversly, the chemical assay, which relies solely on the detection of preservative, will not recognize the inevitable preservative interactions and/or inactivation in the presence of a variety of pharmaceutical materials. Only the AET is capable of recognizing the possibility of such an event.

Over the years, the AET has reduced the number of microbiologically related drug/consumer product recalls and prevented countless clinical manifestations associated with the use of contaminated products. Despite its limitations, the AET has become an effective standard for those formulating safe, effective products for the drug/consumer product industry.

References

• Brannan, D.K. (1995) "Cosmetic Preservation," J. Soc. Cosmet. Chem., Vol. 46, July/August, pp. 199-220.
  
• Bloomfield, S.F. (1995) "Reproducibility and Predictivity of Disinfection and Biocide Tests," In: Microbiological Quality Assurance: A Guideline Towards Relevance and Reproducibility of Inocula, Brown, M.R.W. and P.Gilbert (eds.), Chapter 3.4, pp. 189- 220.
  
• Leitz, M. (1972) "Panel Discussion: Critique of Preservative Challenge Systems I. Critique of U.S.P. Microbiological Test," Bull. Parent. Drug Assoc., Vol. 26, No. 5, pp. 212-216.
  
• Davidson, A.L. et al. (1991) "The Validity of the Criteria of Pharmacopoeial Preservative Efficacy Tests: A Pilot Study," Pharm. J., Vol. 246, pp. 555-557.
  
• Leak, R.E. and R. Leech (1988) "Challenge Tests and Predictive Ability," In: Microbial Quality Assurance in Pharmaceuticals, Cosmetics and Toiletries, Bloomfield, S.F. et al. (eds.), Chapter 10, pp. 129-146.
  
• Davison, A.L. (1988) "Preservative Efficacy Testing of Non-Sterile Pharmaceuticals, Cosmetics and Toiletries and Its Limitations," In: Microbial Quality Assurance in Pharmaceuticals, Cosmetics and Toiletries, Bloomfield, S.F. et al. (eds.), Chapter 9, pp. 120-128.
  
• Spooner, D.F. and A.L. Davidson (1993) "The Validity of the Criteria for Pharmacopoeial Antimicrobial Preservative Efficacy Tests," Vol. 251, pp. 602-605.
  Cooper, M.S. (1994) "Some Issues Associated with the International Harmonization of the Preservative Effectiveness Test," Pharm. Forum, Vol. 20, No. 6, pp. 8589-8592.
  
• Barnes, M. and G.W. Denton (1969) "Capacity Tests for the Evaluation of Preservatives in Formulations," Soap Perfum. Cosmet., Vol. 42, October, pp. 729-733.
  
• Sabourin, J. (1990) "Evaluation of Preservatives for Cosmetic Products," Drug & Cosmet. Ind., December, pp. 24-65.
  
• Ramp, J.A. & R.J. Witkowski (1975) "Microbiological Parameters and Preservative Testing," Develop. Indus. Microbiol., Volume 16, p.48-56.
  
• Orth, D.S. et al. (1989) "Effect of Culture Conditions and Method of Inoculum Preparation on the Kinetics of Bacterial Death During Preservative Efficacy Testing," J. Soc. Cosmet. Chem., Vol. 40, pp. 193-204.
  
• Al-Hiti, M.M.A. and P. Gilbert (1983) "A Note on Inoculum Reproducibility: A Comparison Between Solid and Liquid Culture," J. Appl. Bacteriol., Vol. 55, pp. 173- 175.
  
• Al-Hiti. M.M.A. and P. Gilbert (1980) "Changes in Preservative Sensitivity for the USP Antimicrobial Agents Effectiveness Test Micro-organisms," J. Appl. Bacteriol., Vol. 49, pp. 119-126.
  
• Casey, W.M. and H. Muth (2000) "The Effects of Antimicrobial Preservatives on Organisms Derived from Fresh Versus Frozen Cultures," Pharmacopeial Forum, Vol. 26, No. 2, pp. 519-533.
  
• Bohannan, T.E. and Li Wen L.F. (1978) "Methods for Storage of Antimicrobial Effectiveness Test Inoculum Suspensions Below Freezing," J. Pharm. Sci., Vol. 67, No. 6, pp. 815-818.

For more information: Bob Friedel, Quality Assurance Manager, Perritt Laboratories, PO Box 147, 145 South Main St., Hightstown, NJ 08520. Tel: 609-443-4848. Fax: 609-443-5293. Email: rfriedel@perrittlab.com.