Thirty healthy New Zealand White rabbits (15 female, 15 male) were assigned to one of three groups: Group 1 (6 female, 6 male) to receive 0.33% xCMHA-S; Group 2 (6 female, 6 male) to receive 0.75% xCMHA-S; Group 3 (3 female, 3 male) to receive PBS (control). One drop of xCMHA-S (0.33% or 0.75%) or PBS alone was administered six times a day (an exaggerated dosing regimen) via topical application to both eyes of the rabbits for 28 days. Each dose (40 μL) was pipetted with a calibrated pipette and sterile tip for each eye; to minimize runoff, each eye was gently closed for a few seconds after dosing. Blood and urine were collected prior to dosing and prior to necropsy for complete blood count, clinical chemistry, coagulation factor testing, and urinalysis. Gross ocular observations, which consisted of a visual appraisal for swelling, discharge, and/or irritation of the eye, were performed daily. Clinical ophthalmic examinations, using slit-lamp biomicroscopy and indirect ophthalmoscopy, were performed weekly for the study duration. Examinations used a modified McDonald-Shadduck scoring system, and were performed by board-certified veterinary ophthalmologists (R Merideth, E Moeller, Eye Care for Animals, San Diego, CA, USA). Intraocular pressure (IOP) measurements were taken prior to dosing and prior to euthanasia for all animals (Reichert Model 30 pneumotonometer, Depew, NY, USA). One male and one female from Group 3, and two males and two females from both Groups 1 and 2, were observed for an additional 14 days after the final dose was administered.
Following euthanasia, the globes and adnexa were collected, preserved in 10% formalin, and processed for histopathology. The tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The sections were examined under light microscopy, and specific areas that were evaluated included: conjunctiva, cornea, anterior chamber, iris, retina, lenses, choroid, vitreous, clear, and ocular adnexal tissue. For nonneoplastic findings, severity was graded on a five-point scale, where 1 = minimal, 2 = mild, 3 = moderate, 4 = marked, and 5 = severe. Corneal thickness was measured from the surface of the epithelium to the posterior surface of the endothelium in the central cornea of each eye in a masked manner. Three measurements were taken in the central and paracentral cornea using a micrometer eyepiece on the microscope. These measures were directly perpendicular to the surface of the cornea.
This study was approved by the Institutional Animal Care and Use Committee at Absorption Systems (San Diego, CA, USA) and conducted in accordance with the Good Laboratory Practice for Nonclinical Laboratory Studies (Code of Federal Regulations US FDA 21 CFR Part 58) and Authorized Service Interruption Standard Operating Procedures. All animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.