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.

Rose bengal and lissamine green inhibit detection of herpes simplex virus by PCR.

PURPOSE: To determine whether rose bengal and lissamine green affect polymerase chain reaction (PCR) detection of herpes simplex virus (HSV). DESIGN: Laboratory investigation. METHODS: Diagnostic corneal scrapings were evaluated for PCR inhibitory activity. Dacron swabs inoculated with rose bengal and lissamine green were processed as clinical samples, inoculated with control HSV, varicella zoster (VZV), cytomegalovirus (CMV), and toxoplasma DNA and prepared for PCR. The effects of calcium alginate and cotton swabs were also evaluated. RESULTS: Rose bengal, lissamine green, and calcium alginate not only inhibit PCR detection of HSV DNA, but also detection of VZV, CMV, and toxoplasma DNA. This inhibition could be overcome by serial dilution and by DNA purification of the sample before PCR. CONCLUSIONS: Rose bengal, lissamine green, and calcium alginate can inhibit PCR detection of HSV DNA. Clinical scrapings to be sent for PCR diagnostic testing should be taken before instillation of rose bengal or lissamine green.

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.

Staining characteristics and antiviral activity of sulforhodamine B and lissamine green B.

PURPOSE: Fluorescein and rose bengal are dyes used routinely in the examination of the ocular surface. As part of an ongoing search for a superior ophthalmic dye with optimal specificity and sensitivity and a lack of interference with subsequent viral cultures, and as part of studies that use chemical dyes to understand better the pathophysiology of ocular surface disorders, the staining characteristics and antiviral activity of sulforhodamine B and lissamine green B were investigated. METHODS: Staining of rabbit corneal epithelial cell cultures by sulforhodamine B and lissamine green B was compared to that of fluorescein and rose bengal. Diffusion of each dye through a collagen gel was measured. Uptake of lissamine green B by herpes simplex virus type 1 (HSV-1)-infected Vero cell cultures was compared at several times postinfection. The effect of sulforhodamine B and lissamine green B on HSV-1 plaque formation in Vero cells was determined. The cellular toxicity of sulforhodamine B and lissamine green B in vitro was examined by a quantitative 14C-amino acid uptake assay and by a qualitative cell viability assay. Finally, the effect of sulforhodamine B and lissamine green B on viral replication was compared in vivo with that of rose bengal in a rabbit model of herpetic epithelial keratitis. RESULTS: Rose bengal vividly stained cell monolayers of explant cultures of rabbit corneal epithelium. By light microscopy, sulforhodamine B and lissamine green B, like fluorescein, did not stain the epithelial cells, but did stain the corneal explant stroma. Pretreatment of epithelial cells with 0.25% trypsin for 5 minutes failed to induce dye uptake; however, pretreatment with 0.5% Triton X-100 for 5 minutes resulted in nuclear staining by lissamine green B, but not sulforhodamine B. When added to a collagen gel, the relative diffusion rate was fluorescein > lissamine green B > sulforhodamine B > rose bengal. By spectrophotometric analysis, HSV-1-infected and uninfected Vero cells bound equivalent amounts of lissamine green B until late in infection, when infected cells took up more dye (P < 0.001). A direct neutralization assay showed that 0.06% lissamine green B or 0.5% sulforhodamine B reduced HSV-1 plaque formation in Vero cells by greater than 50%, when present at the time of viral adsorption. By a quantitative 14C-amino acid uptake assay, lissamine green B was toxic to Vero cells in a dose-dependent manner, whereas sulforhodamine B was relatively nontoxic at the concentrations tested. By a cell viability assay, however, neither dye showed significant cellular toxicity. In a rabbit model of herpetic epithelial keratitis, rose bengal significantly reduced viral replication and recovery, whereas sulforhodamine B and lissamine green B had no effect. CONCLUSIONS: Neither sulforhodamine B nor lissamine green B stain healthy, normal cells. Lissamine green B stains membrane-damaged epithelial cells, but sulforhodamine B does not. Both sulforhodamine B and lissamine green B stain corneal stroma. Lissamine green B inhibits HSV-1 plaque formation at low concentrations of dye in vitro, which correlates with suppression of cellular metabolism as demonstrated by a 14C-amino acid uptake assay, but does not affect cell viability. Neither sulforhodamine B nor lissamine green B inhibit viral replication or recovery in vivo.

Sister chromatid exchange and chromosome aberrations in mice after in vivo exposure of green S--a food colorant.

Sister chromatic exchanges (SCE) and chromosome aberrations (CA) in mice after in vivo exposure of Green S were carried out following single acute treatment. Except for the lowest dose (25 mg/kg body weight) a significant increase in the SCEs were observed in all the other doses (50, 100, and 200 mg/kg) tested. In CA study two higher doses (200 and 400 mg/kg) showed a significant increase in CA when compared with control. The minimum effective dose which induced SCE and CA was 50 and 200 mg/kg of body weight, respectively. The trend tests for the evidence of dose response effects were also significant for both SCE and CA. No significant differences were observed in cell replication kinetic (RI) analysis. A significant increase in the mitotic index (MI) was also observed in the highest dose (400 mg/kg) tested when compared with control. Thus the present study indicates that Green S can induce both SCE and CA in vivo in bone marrow cells of mice.

Fast Green FCF (Food Green 3) inhibits synaptic activity in rat hippocampal interneurons.

The effects of the certified food dye Fast Green FCF (Food Green 3) on miniature synaptic events in whole-cell voltage clamped hippocampal interneurons were examined. Fast Green FCF reduced the frequency, but did not affect the amplitude or kinetics, of miniature synaptic events in a dose-dependent manner. At 1 mg/ml (1.2 mM), the frequency was reduced to 21% of control. The half-maximum effective concentration was estimated to be 200 microg/ml (250 microM). In contrast, the dye Phenol Red neither affected frequency, amplitude nor kinetics of synaptic events. The results suggest that Fast Green FCF acts at a presynaptic locus, thereby inhibiting the release of neurotransmitter.

Evaluation of the effect of lissamine green and rose bengal on human corneal epithelial cells.

PURPOSE: To examine the effects of lissamine green and rose bengal on proliferating human corneal epithelial (HCE) cells in vitro. METHODS: HCE cells derived from explants of discarded corneoscleral rims were cultured by the standard technique. Experimental cells were exposed to 1, 0.5, or 0.1% of either lissamine green or rose bengal for 10 min while control cells were exposed to a phosphate buffer solution (PBS). RESULT: Cell viability was 92% greater for 1% lissamine green (p = 0.013) and 81.2% greater for 0.5% lissamine green (p = 0.006) compared to 1 and 0.5% rose bengal, respectively. The difference between the effect of 0.1% rose bengal and 0.1% lissamine green on cell viability was not statistically significant (p = 0.83). Rose bengal staining of HCE cells was immediate and readily detectable with unaided eyes at all three concentrations, whereas no observable staining of healthy HCE cells was noted with lissamine green. CONCLUSION: Rose bengal adversely affects HCE cell viability and stains normal proliferating HCE cells in contrast to lissamine green, which exhibited neither of these characteristics. Therefore, we recommend the use of lissamine green over rose bengal in evaluating ocular surface disorders.

Genotoxicity of industrial dyes under the inductive effect of ethanol on monooxygenase system in mice.

The genotoxic effects of triarylmethane (Acid Green 16, C.I.44025) and arylmonoazo (Basic Orange 28, developed by Boruta Pigment Plant, Poland, C.I. undisclosed) dyes, were evaluated in Balb/C mice. Animals were fed for 6 days nutritionally adequate Portagen liquid diet (1 kcal/ml) or isocaloric alcoholic diet containing 5% (w/v) ethanol (36% of total calories) in order to induce the cytochrome P-4502E1 monooxygenase. Dye compounds were administered intraperitoneally 30 h before the test at doses: 90 mg/kg of Acid Green 16 and 70 mg/kg of Basic Orange 28. Bone marrow micronucleus test was used for evaluation of genotoxicity of the dyes. Ethanol caused an increase of the level of cytochrome P-450 by 200% and activities of 7-ethoxycoumarin O-deethylase (ECOD) by 650%, 7-ethoxyresorufin O-deethylase (EROD) by 460% and glutathione (GSH)-S-transferase by 60% in the liver. Both dyes exerted genotoxic effect as inferred from a 3-fold increase of frequency of micronucleated polychromatic erythrocytes in bone marrow, and a further increase (2-fold) was caused by ethanol liquid diet combined with Acid Green 16 treatment. Basic Orange 28 genotoxicity remained unaffected by ethanol. It is concluded that: (1) enhancement of genotoxic effect of Acid Green 16 by ethanol is caused by induction of cytochrome P-4502E1 monooxygenases resulting in an increased bioactivation of the dye; (2) lack of enhancement of the genotoxic effect of Basic Orange 28 by ethanol probably results from the dye- and ethanol-mediated stimulation of GSH-S-transferase, bypassing the cytochrome P-4502E1 bioactivation step.

Long-term toxicity study of Green S in mice.

Groups of 105 (control) or 65 (treated) female CD-1 mice were mated one to one with equal numbers of males after both sexes had received diets containing 0 (control), 0.033, 0.33 or 0.66% Green S for 9 wk. The number of animals pregnant, the number of young born and the number surviving were similar in all groups. One male and one female from each litter were used to provide groups of 85 (control) or 50 (treated) mice of each sex for the long-term study. Treatment with Green S continued throughout pregnancy and rearing. Body weight and condition were regularly monitored for each animal throughout the study. Blood was examined from groups of 20 mice from the control and highest treatment groups at wk 14, 28 and 51 and from all survivors at the end of the study. A post-mortem examination was carried out on all animals in the long-term study and a full range of tissues was preserved and examined by light microscopy. Organ weights were recorded at the autopsy of all mice reaching the end of the study. No effects that could be attributed to treatment were seen in any of the observations. The no-untoward-effect level of Green S fed to mice for 2 yr is concluded to be 0.66% of the diet, equivalent to intakes of approximately 530 and 660 mg/kg body weight/day in males and females, respectively.

Teratogenicity and embryotoxicity study of Green S in rats.

Daily oral doses of 0 (control), 250, 500 or 1000 mg Green S/kg body weight were given to groups of 30 pregnant rats on days 0-19 of pregnancy. This treatment did not adversely influence maternal body weight, the numbers of implantations, of pre- or post-implantation losses or of live foetuses, the sex ratio or the weight of the litters or foetuses. No definite abnormalities were seen and the only finding in the examination of stained skeletons was a slightly more advanced ossification of the forelimbs of the offspring from females given 500 or 1000 mg Green S/kg/day. More foetuses with mucus in the trachea were found in the treated groups than in the controls but this was not considered to be a teratogenic effect. Thus no embryotoxic or teratogenic effects were detected with doses of up to 1000 mg Green S/kg/day throughout pregnancy.

Short-term toxicity study of Green S in rats.

Groups of 15 rats of each sex were fed Green S at dietary concentrations to provide dose levels of 0 (control), 250, 500 or 1500 mg/kg body weight/day for 13 wk. Additional groups of five animals of each sex were given the same treatments for 2 or 6 wk. There was a marked excretion of green colour in the faeces and some green colouring of the urine, although the latter may have been due to contamination. The males showed increased water and food intakes associated, particularly at the highest dose, with a higher rate of body-weight gain. Haematological examination revealed a transitory mild anaemia at the highest dose level, whilst no findings indicative of a toxic effect were found in the renal concentration tests or the serum analyses. With a dose of 1500 mg Green S/kg a greater proportion of the rats showed higher urinary protein, protein casts, increased caecal weight, thyroid degeneration in female animals and enlargement of the lymph nodes in the intestine wall. The no-effect dose level for Green S in this study was considered to be 500 mg/kg.

Three-generation toxicity study of rats ingesting Green S in the diet.

Green S was fed to rats of both sexes, over three generations, at dietary concentrations designed to provide daily intakes of 0, 50, 500 or 1000 mg Green S/kg body weight. At each generation, treated groups each consisted of 36 males and 36 females with 60 of each sex as controls. The F0 generation first received Green S as weanlings, but succeeding generations were exposed throughout life, including in utero, as treatment continued during gestation and lactation. There were no adverse effects of treatment on body-weight gain, food and water consumption or on the general condition of the animals. Green-coloured faeces were produced by all animals exposed to the colouring and pale green coloration of urine or the bladder was seen in a few animals at autopsy. The post-mortem examinations and organ weights of animals receiving up to 500 mg Green S/kg/day showed no adverse effects of treatment. At 1000 mg/kg/day findings related to treatment were increased spleen weight (both sexes) and increased kidney weight (male), but relevant histopathological changes were not seen in either of these organs. Caecal enlargement was the most consistent finding in animals receiving 500 or 1000 mg Green S/kg/day, but this was not considered to be a toxic effect. Reproductive performance and intra-uterine development were not affected by treatment despite green colouring being visible in the amniotic sacs of foetuses from dams given 500 or 1000 mg Green S/kg/day. Small differences in the degree of skeletal ossification of foetuses from F2 generation dams were not related to treatment. A slightly premature eruption of the incisors during postnatal development of treated animals was not considered to be an adverse effect. It is concluded that the no-untoward-effect level in this study is 500 mg Green S/kg body weight/day.

Cytochrome P-450 mediated genotoxicity of triarylmethane dye in mice fed ethanol liquid diet.

Genotoxic effect of synthetic triarylmethane dye (Acid Green 16) was evaluated in Balb C mice fed nutritionally adequate liquid diet (1 kcal/ml) or isocaloric alcoholic diet containing 5% (w/v) ethanol (36% of total calories) for 6 days. Dye compound was given intraperitoneally at dose 150 mg/kg body wt. 30 h before test. The micronucleus test was used for evaluation of genotoxicity of the dye. The level of cytochrome P-450 and the activity of 7-ethoxycoumarin O-deethylase (ECOD) and 7-ethoxyresorufin O-deethylase (EROD) in microsomes were determined to assess the metabolic efficiency of the microsomal system. Acid Green 16 dye provoked an increased frequency of micronucleated polychromatic erythrocytes in bone marrow and ethanol enhanced this genotoxic effect through induction of cytochrome P-4502E1 and stimulation of activities of microsomal monooxygenases (ECOD and EROD), presumably catalyziung bioactivation of the dye.

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.

[Substances for staining biological tissues: use of dyes in ophthalmology]. / Substanzen zur Anfärbung von biologischem Gewebe -- Farbstoffe in der Ophthalmologie.

The azo dye trypan blue and the cyanine dye indocyanine green have been used in ophthalmology since the 1980 s to dye the anterior capsule in cataract operations and to stain epiretinal membranes in order to remove the membrana limitans interna (ILM). By means of a standardized in vitro test in accordance with DIN EN ISO 10993, it has now been shown that both dyes and the theoretically possible alternatives - the anthrachinone alizarincyanine green and the trityl dyes fast green and lissamine green - are cytotoxic in the normal concentrations of maximum 1.5 g/l. On the other hand, the new product Blueron(R) with the trityl dye patent blue, which has been developed to dye the anterior capsule, is completely biocompatible, even at a high dosage of up to 2.8 g/l.

Genotoxic effect of triarylmethane dye--acid green 16 after chronic ethanol consumption in mice.

Genotoxic effect and hepatic microsomal monooxygenase activities were assessed in mice treated with Acid Green 16 (single i.p. injection at dose 75 mg/kg) superimposed on prolonged ethanol consumption (10% solution in drinking water for 2-4 months). Treatment of mice with Acid Green 16 led to an increased frequency of micronucleated erythrocytes in bone marrow. In animals pretreated with ethanol the frequency of micronucleated erythrocytes, produced by Acid Green 16, was significantly higher. The changes in frequency of micronucleated erythrocytes were accompanied by the enhanced activity of microsomal monooxygenases manifested by higher activity of 7-ethoxycoumarin o-deethylase (the level of cytochrome P-450 was not altered). The obtained results showed that ethanol tended to increase the genotoxic effect of Acid Green 16. However, the slight inductive effect of ethanol on microsomal monooxygenases did not provide clear evidence that the genotoxic effect of Acid Green 16 was associated with ethanol stimulation of the metabolic activation of the dye in the liver.