Comparative in vitro safety analysis of dyes for chromovitrectomy: indocyanine green, brilliant blue green, bromophenol blue, and infracyanine green.

PURPOSE: Vital dyes such as infracyanine green (IfCG), brilliant blue green (BBG), and bromophenol blue (BPB) have been used as an alternative to indocyanine green (ICG) during chromovitrectomy. We compared the in vitro toxicity of IfCG, BBG, and BPB with ICG on the retinal pigment epithelial cells and retinal ganglion cells at various concentrations to optimize the safe dose and duration of exposure. METHODS: Cultured retinal ganglion cells (RGC-5) and human retinal pigment epithelial cells (ARPE-19) were exposed to 2 concentrations (0.25 and 0.5 mg/mL) of ICG, IfCG, BBG, and BPB at various time intervals (1, 5, 15, and 30 minutes). Cell viability was quantified with neutral red assay, and mode of cell death was evaluated with flow cytometry-based Annexin V and propidium iodide staining. RESULTS: Exposure to ICG resulted in 48%-74% reduction in neutral red uptake in both RGC-5 and ARPE-19 cells, after an exposure time of ≥5 minutes compared with control (P < 0.001). Infracyanine green, BBG, and BPB were significantly less toxic on the 2 cell lines at exposure times <15 minutes. (Reduction in cell viability ranged from 6.9% ± 3.3% to 29.3% ± 7.4% when compared with control, P > 0.5.) However, among the newer dyes, BBG caused necrosis in retinal pigment epithelial cells and retinal ganglion cells as the exposure time period increased beyond 5 minutes. CONCLUSION: Newer vital dyes, IfCG, BBG, and BPB, are significantly less toxic on retinal ganglion cells and retinal pigment epithelial cells' cell lines when compared with ICG. Infracyanine green was least toxic among the three newer dyes studied.

Comparative effects of six intraocular vital dyes on retinal pigment epithelial cells.

PURPOSE: To evaluate and compare the effects of the following dyes on human pigmented epithelial cells: indocyanine green (ICG), infracyanine green (IfCG), trypan blue (TB), bromophenol blue (BrB), patent blue (PB), and Brilliant Blue G (BBG). METHODS: ARPE-19 cells cultured in vitro were exposed to these dyes, and acute and chronic toxicity were evaluated. Cell viability was measured by colorimetry (MTT assay), morphology was observed by phase-contrast microscopy, membrane permeability (CMP) was evaluated by flow cytometry with propidium iodide (PI), and mitochondrial membrane potential (ΔΨm) was measured with 3,3'-dihexyloxacarbocyanine (DiOC(6)(3)). RESULTS: Each of the studied dyes exhibited toxicity after acute exposure at surgical doses. The presence of light often reduced cell viability, especially when measured 3 hours after incubation in the case of ICG, TB, BrB, and BBG. Morphologic changes were induced by ICG, IfCG, and BBG. Both CMP and ΔΨm were altered after exposure to surgical doses of ICG, TB, PB, and a fourfold surgical dose of BrB. Chronic exposure to residual amounts of some dyes was associated with reduced proliferation and even cell death. CONCLUSIONS: It appears to be prudent to use the lowest possible dose of each dye, to minimize the risk of toxic effects. This precaution may be particularly important in the case of BrB, which should not be used in excess of 0.5%. In addition, abundant irrigation of the vitreous cavity after surgery to completely remove traces of dye may be of crucial importance, particularly in the case of ICG, in minimizing chronic toxicity.

Administration of novel dyes for intraocular surgery: an in vivo toxicity animal study.

PURPOSE: To investigate the effect of intravitreal injections of new vital dyes on the retina, the retinal pigment epithelium (RPE) and the choroid in an in vivo rat model. METHODS: Rats were injected intravitreally with four dyes: light-green SF yellowish (LGSF), copper(II)phthalocyanine-tetrasulfonic acid (E68), bromphenol blue (BPB), and Chicago blue (CB) dissolved in physiologic saline solution (PSS) at concentrations of 0.5% and 0.02%. PSS served as the control. Additional animals were treated with single injections of 0.5%, 0.02%, 0.002%, and 0.0002% ICG or 0.002% E68 into one eye. Adverse effects on anterior and posterior segments were evaluated by slit lamp biomicroscopy and ophthalmoscopy. Retinal toxicity was assessed by histology and retinal ganglion cell (RGC) quantification 7 days after dye administration. RESULTS: Eyes treated with 0.5% E68, 0.5% ICG, or 0.5% CB showed discrete staining of both cornea and lens not seen at lower concentrations or with other dyes. Histology revealed dose-dependent reactions after E68 administration. ICG 0.5% induced significant thinning of inner retinal layers compared with PSS. ICG 0.02% caused focal degenerative changes of the outer retina in three of seven eyes, whereas 0.002% and 0.0002% ICG did not. CB led to heterogeneous morphologic alterations. BPB- or LGSF-treated eyes showed normal retinal morphology. ICG at all tested concentrations induced significant RGC loss, as did E68 at 0.5% but not at lower concentrations. CONCLUSIONS: BPB or LGSF produced no significantly detectable toxic effects on the retina in vivo. The safety of these new dyes must be established in other models and/or in preclinical studies before the clinical use of any of these dyes.

Short-term in vivo evaluation of novel vital dyes for intraocular surgery.

PURPOSE: To evaluate the staining characteristics and safety of potential new dyes for intraocular surgery in porcine eyes. METHODS: Four dyes in different solutions (light green SF yellowish [LGSF]: 2%; copper(II) phthalocyanine-tetrasulfonic acid [E68]: 2% and 0.5%; bromophenol blue [BPB]: 2%, 1%, and 0.2%; and Chicago blue [CB]: 2% and 0.5%) were included in this investigation. All dyes were dissolved and diluted using balanced salt solution (BSS plus; Alcon Laboratories, Inc., Fort Worth, TX). After triamcinolone-assisted vitrectomy on 10 porcine eyes in vivo, the dyes were first injected into the air-filled vitreous cavity. After 1 minute, the dye was removed by irrigation with BSS, and the staining effect was graded by two examiners. After vitrectomy, the same dyes and concentrations were injected in the air-filled anterior chamber to stain the lens capsule of the same eye. After surgery, the eyes were enucleated and underwent fixation for light and electron microscopy. The animals were killed by injection of pentobarbital (50 mg/kg). For controls, each BSS plus alone and indocyanine green 0.5% were applied in one eye. RESULTS: On the retinal surface, bright staining of the retinal surface was seen after application of BPB 2% and 1%. The staining effect was less pronounced but still very good using E68 2%, and CB 2% and weak using BPB 0.2%, E68 0.5% and CB 0.5% as well as indocyanine green 0.5%. No staining of the retinal surface but of the vitreous was seen after application of LGSF 2%. The lens capsule stained very well with E68 2%, CB 2% and 0.5%, and BPB 2%, 1%, and 0.2% but not with LGSF. No histologic abnormalities were seen after the application in any eye after dye injection. No dye-related complications occurred during surgery. CONCLUSION: In this study, we identified three dyes with satisfying staining characteristics in both anterior and posterior segments. Because BPB stained the retinal surface and lens capsule at a low concentration (0.2%) with no signs of toxicity, this dye seems to be the most promising candidate for application in humans.

An evaluation of novel vital dyes for intraocular surgery.

PURPOSE: To evaluate systematically the staining characteristics and safety of potential new dyes for intraocular surgery. METHODS: Six dyes were included in the investigation: light green SF (LGSF) yellowish, E68, bromophenol blue (BPB), Chicago blue (CB), rhodamine 6G, rhodulinblau-basic 3 (RDB-B3). All dyes were dissolved and diluted in a balanced saline saline solution. The light-absorbing properties of each dye were measured at a concentration of 0.05% between 200 and 1000 nm. Staining characteristics were examined by staining lens capsule tissue and epiretinal membranes (ERMs), removed intraoperatively, with dye concentrations of 1.0%, 0.5%, 0.2%, and 0.05%. Enucleated porcine eyes (postmortem time, 9 hours) were also stained. Dye-related toxicity was evaluated by a colorimetric test (MTT) measuring the inhibition of retinal pigment epithelium (RPE) cell proliferation (ARPE-19 and primary human RPE cells, passages 3-6). Cell viability was also quantified based on a two-color fluorescence cell-viability assay. Dyes were investigated in concentrations of 0.2% and 0.02%. RESULTS: All dyes investigated in this study stained human lens capsules, removed intraoperatively; ERMs, peeled during macular pucker surgery; and enucleated porcine eyes, depending on the concentration applied. The long-wavelength absorption maximum of the dyes was within the range of 527 to 655 nm at concentrations of 0.05%. Rhodamine G6 and RDB-B3 showed adverse effects on ARPE-19 cell proliferation at a concentration of 0.2% and were excluded from further investigation in primary RPE cells. The remaining four dyes showed no toxic effect on ARPE-19 and primary RPE cell proliferation at concentrations of 0.2% and 0.02%. Cell viability was affected by LGSF yellowish (0.2%) and CB (0.2% and 0.02%). Two dyes (E68 and BPB) showed no relevant toxicity in vitro. CONCLUSIONS: The systematic evaluation of dyes for intraocular use seems mandatory. In this study four dyes were identified with effective staining characteristics, with two of these dyes having no detectable toxic effect on RPE cells in vitro.

Mutagenicity studies on two triphenylmethane dyes, bromophenol blue and tetrabromophenol blue.

Bromophenol blue and tetrabromophenol blue are two triphenylmethane dyes. Triphenylmethane derivatives and their structurally related compounds, such as fluoresceins and xathenes, are widely used as industrial dyes for foods, drugs, cosmetics, textiles, printing inks or laboratory indicators. Since a number of these types of dyes have been reported to be genotoxic, safety concerns on these two dyes of interest have been raised. Consequently, a battery of genetic toxicology assays, including the Ames Salmonella/microsome assay, L5178Y TK+/- mouse lymphoma assay, mouse micronucleus test and mitotic recombination assay with yeast Saccharomyces cerevisiae strain D5, has been performed on each of the two dyes. The results of the evaluations indicate that both bromophenol blue and tetrabromophenol blue were not active and can be considered non-genotoxic for the three genetic endpoints assessed (gene mutation, chromosome aberrations and primary DNA damage). Genetic activities in some structurally related compounds of these dyes have been reported but may be attributed to the presence of mutagenic impurities rather than the compound itself.