*2.10. Antimicrobial <sup>E</sup>*ffi*cacy*

The antimicrobial efficacy of MM3 was determined by the agar diffusion test employing the cup plate technique and compared with control (commercial eye drops of moxifloxacin). The sterile products (MM3 or control) were poured (0.01 mL) into the cups of sterile nutrient agar previously inoculated with susceptible test organisms (*Pseudomonas aeruginosa* and *Staphylococcus aureus*) under horizontal laminar air flow hood. After allowing diffusion of the solutions for 2 h, the agar plates were incubated at 37 ◦C for 24 h. The zone of inhibition measured around each cup was compared with the control. Both positive (with test organisms) and negative controls (without test organisms) were maintained throughout the study.

## *2.11. Ocular Irritation*

Albino rabbits (2–3 kg) placed in an animal house under observation were given ad libitum access to water and diet for 24 h. The guidelines of the Institutional Animal Ethics Committee were strictly followed while performing experiments (IPS/PCEU/PHD10/002). In vivo ocular irritation studies were performed according to the Draize technique [19]. Single administration of 60 μL was instilled in the left eye of each rabbit while keeping the untreated eye as the control. The sterile MM3 was administered twice daily for a period of 21 days (1, 2, 3, 4, 7, 10, 15, 18 and 21 days). The rabbits were observed periodically for redness, swelling and watering of the eye.
