Q How can a preservative's efficacy be shown?

Efficacy of an antimicrobial preservation is commonly performed at two points in the product development process. The first point is during the formulation stage, where developers are looking at a range of the most promising preparations. The preservative is incorporated and the test carried out. This should narrow down the field of potentials, or confirm those that have already been chosen.

The second point is during stability storage, usually at the beginning and end of storage. A middle time point, however, can also be beneficial to catch failures earlier in the cycle. Testing during stability storage is perhaps the most important testing point because it highlights potential efficacy problems with the preservative. For example, it will show if the preservative degrades over time, which would require a higher starting level. Even if the preservative doesn't degrade, it may not remain biologically available. The test will show this. Different temperatures or humidity may cause the preservative to fall out of suspension or solution. The efficacy test will show any microbiological problems, the physical and chemical tests that are carried out concurrently can often then tell you why.

The Pharmacopoeias prescribe a set of test organisms that cover the basic types: Staphylococcus aureus for the Gram positives, Pseudomonas aeruginosa for the Gram negatives, Candida albicans for the yeasts and Aspergillus brasiliensis (formerly Aspergillus niger) for the moulds. The US Pharmacopeia includes Escherichia coli in its standard challenge organism list, while the European Pharmacopoeia reserves E.coli for use in oral preparations.

There is also the option of including organisms of interest, such as Zygosaccharomyces rouxii (anosmotolerant spoilage yeast) for high-sugar preparations, environmental isolates (either from a known contamination issue, or one that keeps appearing when you check the manufacturing area).

There is currently no requirement to test against antibiotic-resistant "super bugs". Although these organisms have evolved a method for protecting themselves from a variety of antibiotics, most preservative systems work along different pathways and so will have the same effect against both normal and 'super' varieties.

The Pharmacopoeias have a list of the minimum required efficacy based on the product type, be it oral, topical or parenteral. The requirement is expressed as a Log₁₀ reduction from the microbiological challenge level after a defined period of contact. Depending on the preparation type the fungi may only be required not to increase in numbers from the previous value. In addition, there are tests against a variety of organisms to ensure as wide an effect as possible. If the preparation doesn't meet these requirements, it's back to the formulation stage!

A common pitfall at this point is not realising that the Pharmacopoeias are not harmonised on efficacy testing. There may be different requirements and testing to one may not cover the others. A few things can be considered in the formulation stage that should help reduce the proportion of failures in the preservative efficacy test, and they all revolve around the premise of knowing your preservative:

• At what level is the preservative expected to be effective? For example, benzoic acid is effective against E.coli at 120 ppm, but requires 2000 ppm to be effective against A.brasiliensis.
• How soluble is the preservative in the product? The preservative may be efficacious at 1%, but if the solubility in the preparation is only 0.1% then it probably won't work.
• Is the preservative stable? Or is it likely to degrade with time, or temperature or by reaction with the preparation? All of these will reduce or negate its efficacy.

Some of above can be estimated in advance, but in the end the only way to know is to try.

Greg Snowdon is a microbiology scientist at RSSL.