Characterisation of a preservative system in an oily preparation

The influence of three variables, i.e. the concentrations of benzyl alcohol (BA), butylated hydroxytoluene (BHT) and tert-butyl-4-hydroxyanisol (BHA), on the preservative efficacy and antioxidant activity of an oily veterinary formulation was investigated using quantitative experimental designs and applying pharmacopoeial methods as part of the robustness-evaluation. Preservative Efficacy Tests (PETs) were performed using the validated European Pharmacpoeia (EP) methodology with 7 test-organisms over one month on lab-scale test-formulations. These were independently prepared according to a Box-Behnken experimental design with a triplicate central point at 0.75% m/V BA, 0.05% m/V BHT and 0.05% m/V BHA, and with an additional control-point outside the Box-Behnken cube containing no preservative ingredient. The preservative efficacies were evaluated against the USP and EP criteria for formulations for oral use, as well as by the statistical comparison of the slopes obtained by linear regression of the log of CFU/g versus time. The peroxide values were determined after two months storage at 50 °C, using the EP titrimetric method. No interactions between the preservatives were observed for any of the seven tested micro-organisms in the PETs. BA had a very significant preservative effect against several of the tested microorganisms, while no antimicrobial effect for BHT and BHA was observed. Aspergillus niger was the most preservative-resistant micro-organism, while Staphylococcus aureus was the most sensitive test-germ. Compliance with USP-PET criteria was found for all formulations tested, even those without preservatives, while the EP-PET criteria showed compliance for those formulations with the highest BA concentration only. Stored in glass vials, a statistically significant antioxidant effect was demonstrated for BA only, although all tested formulations showed acceptable anti-oxidative properties. No significant antioxidant effects were shown for BHT or BHA.