Validation of a Rapid Microbiological Method for Sterility Testing of Atropine Eye Drops

Context
Atropine eye drops are sterile ophthalmic preparations widely used in pediatrics to control the progression of myopia. To face increasing demand, batch production has been implemented. For sterility testing, an alternative method described in European Pharmacopeia (EP) (5.1.6) is implemented. This method relies on automated detection of microbial growth through CO₂ production during metabolism, inducing a pH change detected by a colorimetric sensor.
The aim of this study was to evaluate the performance of this rapid microbiological method and to compare it with the direct inoculation reference method (EP 2.6.1).

Materials/Methods
A batch of atropine 0.01% eye drops (N = 100) was prepared under aseptic conditions. Applicability assays were conducted in accordance with EP recommendations (5.1.6) by inoculating two levels (5 and 20 CFU) of the six microorganisms: S.aureus, P.aeruginosa, B. subtilis, C.sporogenes, C.albicans, and A. brasiliensis.
For both methods, as recommended in EP 2.6.1, half of the content (4.5 mL) of each container was inoculated for each microorganism.
For the alternative method, device specific bottles were inoculated for aerobic and anaerobic microorganisms. For sterility testing by direct inoculation, sterile liquid media (tryptone soya broth and thioglycolate medium) were used. Analyses were performed in triplicate.
For the alternative method, the bottles were incubated at 30 °C for 7 days, with automated readings every 10 minutes. For the direct method, the media were incubated for 14 days at 32.5 °C (±2.5 °C) for bacteria and at 22.5 °C (±2.5 °C) for fungi with daily visual inspection for turbidity.

Results/Discussion
For both inoculum levels, the average detection time for bacteria was approximately 37 hours with the direct method and 28 hours with the rapid microbiological method, demonstrating a significant reduction in time to detection. However, A.brasiliensis was not detected at 5 and 20 CFU using the rapid method, whereas growth was observed with the reference method. Detection with the rapid method was only achieved at higher inocula (50 and 100 CFU) and after increasing the incubation temperature to 35 °C. For fungi, detection times were approximately 76 hours with the reference method and 28 hours with the rapid method.
Negative controls remained sterile.

Conclusion
The rapid microbiological method is validated for sterility testing for atropine 0,01% eye drops. It allows reproducible detection of microorganisms and shows better performance than, the reference method described in the EP (2.6.1).
The rapid microbiological method provides faster results; however, it is applicable only to products without antimicrobial activity, for which membrane filtration sterility testing (14 days incubation) is the reference method. Both methods are qualitative, in case of a positive result, subculture on solid media is required for identification.