A preservative claim has to be proven, and the proof is the antimicrobial effectiveness test in USP general chapter <51>. It is the reason 0.9% benzyl alcohol can be relied on in bacteriostatic water, and it is also the origin of the 28-day figure that laboratories use for opened vials.

How the test works

1. Separate portions of the product are inoculated with each of five standardized challenge organisms: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, the yeast Candida albicans and the mold Aspergillus brasiliensis.
2. The inoculum is heavy: between 100,000 and 1,000,000 viable cells per millilitre of product, far more than any realistic contamination event.
3. The inoculated containers are held at 20–25 °C, and the number of surviving organisms is counted at 7, 14 and 28 days.
4. The counts are compared with the starting count and expressed as log reductions: a 1-log reduction is a 90% kill, 3-log is 99.9%.

The acceptance criteria

Products are grouped into categories by route and dosage form; sterile aqueous products such as bacteriostatic water fall into Category 1, the strictest. For Category 1 the bacteria must show at least a 1.0-log reduction by day 7 and at least a 3.0-log reduction by day 14, with no increase from day 14 to day 28. The yeast and mold counts must show no increase from the starting count at days 7, 14 and 28. In other words the preservative must kill the bacteria quickly and hold the fungi in check for the full four weeks.

What the criteria reveal about benzyl alcohol

The asymmetry in the criteria matches what benzyl alcohol does: it is strongly active against bacteria and only required to prevent growth of yeasts and molds. That is why a vial that has been open for weeks is more at risk from fungal contamination than bacterial, and it is one reason the in-use period is capped at 28 days rather than extended to the vial being empty. See what does bacteriostatic mean? and benzyl alcohol vs. other preservatives.

Where the 28 days comes from

The test demonstrates preservative performance over a 28-day window after a single, deliberate, very large contamination. Laboratory practice borrows that window as the in-use limit for an opened vial, on the reasoning that the preservative has been shown to hold for 28 days under conditions harsher than repeated small entries. Beyond 28 days there is no test data to rely on. See shelf life after opening.

What the test does not cover

• It is done on the product at manufacture, not on an individual vial after it reaches a laboratory; storage abuse or dilution of the preservative can invalidate it. See can you mix bacteriostatic water with sterile water?
• It uses five organisms; unusual contaminants may respond differently.
• It is a preservation test, not a sterilization test: it proves the product resists contamination, not that it can be made sterile again. See five terms that are not interchangeable.

Related

What 0.9% benzyl alcohol means · Sterility testing (USP <71>) · How the preservative content is verified

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