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Tim Streit, Paul E. Miller, Ellison Bentley, Evan A. Thackaberry, Chris Schuetz, Peter Sonnentag, Cindy Farman, Brian J Christian, Vladimir Bantseev; Determination of a No Observable Effect Level (NOEL) for Endotoxin following a Single Intravitreal Administration to Male Dutch Belted Rabbits. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3368.
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Endotoxin level of intravitreal ophthalmic formulations is an important consideration in both manufacturing and safety testing. This 7-day study aimed to characterize the acute inflammatory response and determine a no observable effect level in rabbit eyes after administration of endotoxin solutions via intravitreal injection.
Male Dutch Belted rabbits were treated with a single intravitreal dose at concentrations ranging from 0.01 to 0.5 endotoxin units/eye (EU/eye; USP standard). An initial range‑finding study established a concentration range (0.05 to 0.5 EU/eye) for a larger definitive study with four treated groups (N=5 eyes per group). Animals were monitored for up to 1 week post treatment via clinical ophthalmic examination using the modified standardization of uveitis nomenclature (SUN) Working Group Grading Scheme to score aqueous cell and flare. Concurrent with most ophthalmic examinations, laser flare photometry was performed to quantitatively measure aqueous flare.
Intravitreal injection of endotoxin administration at concentrations ≥0.05 EU/eye resulted in a dose-responsive acute anterior segment inflammation characterized by an increase in aqueous flare and/or aqueous cell. Post treatment aqueous flare and aqueous cell peaked at 24 and 72 hours and resolved by 96 and 168 hours, respectively. While variable, quantitative measurements via laser flare photometry correlated with clinical grades of aqueous flare [Grade “0” correlated with a range of 2.8 to 142.6 (mean = 21.4, SD = 21.5), Grade “1” a range of 30.0 to 382.1 (mean = 137.8, SD = 110.3), Grade “2” a range of 303.2 to 387.4 (mean = 340.0, SD = 30.3)].
Endotoxin contamination in ophthalmic formulations intended to be administered via intravitreal injection may elicit an inflammatory response at concentrations as low as 0.05 EU/eye in rabbits. This concentration is approximately 4‑fold lower than a minimal effect extrapolated concentration for intracameral route of exposure (Sakimoto et al., 2009). The response to these relatively low doses of endotoxin are transient, resolving by 168 hours. These data highlight the importance of assessing endotoxin level in intravitreal formulations, as levels as low as 0.05 EU/eye may confound the safety evaluations of intravitreal therapeutics in rabbits.
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