| Popis: |
Manufacturing intermediates and formulation components constitute the bulk of compounds evaluated in ocular irritation protocols. A cost-effective in vitro screen for predicting severe ocular reactions, which reduces the chance of exposing animals to caustic chemicals, was a logical first step in a tiered approach to the reduction, refinement, and eventual replacement of animals in ocular irritation test protocols. Our laboratory has validated the agar diffusion cytolysis method, which employs a monolayered SIRC cell culture as an initial biological ocular screen. Twenty-four hours after plating 100-mm dishes, the culture medium was replaced with a 0.01% neutral red/1% agar overlay and incubated for a minimum of 16 h. Solid or liquid compounds were applied atop the agar diffusion matrix in glass cylinders or on paper discs, respectively, at doses of 2, 5, 10, and 25 mg or μ1 and incubated at 37°C for 24 h. Clear zones resulting from the loss of the neutral red dye following compound-induced cytolysis were... |
