- Email: [email protected]
Evaluation of in vitro cytotoxicity assays for contact lens multi-purpose solutions
Abstracts / Contact Lens & Anterior Eye 35S (2012) e1–e32
cells collected non-invasively via irrigation with the ocular surface cell collection apparatus (OSCCA). Method: Five healthy participants underwent contra-lateral instillation of 50 L of 0.01% BAK (Bausch&Lomb Moisture Eyes) or sterile saline on three separate days (n = 15). Corneal staining with fluorescein was graded on a 0–100 scale for the five corneal zones 15 minutes post-BAK instillation. The Global Staining Scores were calculated. Calcein AM Blue (live/metabolically active stain) and Ethidium Homodimer-1 (dead stain) were used to stain the cells collected from the eye wash. Using a fluorescent microscope, live, dead and sodium fluorescein stained cells were quantified. Results: Post BAK or saline instillation, the GSS were 42 ± 33 and 31 ± 24, respectively. There were 428 ± 153 cells collected from BAK treated eyes, compared to 434 ± 241 cells from the control. In the collected BAK cell population, 7 ± 5% of cells were metabolically active compared to 5 ± 5% in the control group. Similar numbers of fluorescein stained cells were observed in the BAK and control groups. Cells staining with sodium fluorescein stained exclusively with Calcein AM and not Ethidium homodimer-1, suggesting that sodium fluorescein enters metabolically active cells. For all the measured parameters, no significant difference was observed between the BAK and control groups (p > 0.5). Conclusions: Our results suggest that a single instillation of 0.01% BAK does not induce corneal staining or affect the shedding of collected human epithelial cells in an acute manner, as determined by ex vivo examinations. http://dx.doi.org/10.1016/j.clae.2012.08.071 69 Evaluation of in vitro cytotoxicity assays for contact lens multipurpose solutions Ling C. Huang ∗ , Mercedes Salvador-Silva, Charles H. Powell, Lisa Hoong, Rosanne M. Yetemian Abbott Medical Optics, Santa Ana, USA E-mail address: [email protected] (L.C. Huang)
∗
Purpose: Previously published in vitro cytotoxicity assessments of Contact Lens Multipurpose Solutions (MPS) lack correlation. This study correlates MPS effects on cell cytotoxicity, metabolic activity, membrane integrity, and biocompatibility. Method: Six MPS were used: MPS-1 [polyquaternium (PQ-1), alexidine], MPS-2 [PQ-1, 5 ppm myristamidopropyl dimethylamine (MAPD), nonanoyl-EDTA], MPS-3 [PQ-1, 5 ppm MAPD], MPS-4 [PQ-1, 6 ppm MAPD], MPS-5 [PQ-1, polyhexamethylene biguanide (PHMB)], and MPS-6 [PHMB, poloxamer 237]. Five soft contact lenses were used: balafilcon A, senofilcon A, galyfilcon A, lotrafilcon B, and comfilcon A. In vitro biocompatibility was assessed by placing MPDS-treated lenses (100 mL, 4 days, n = 3) onto confluent HCEC SV40 cells for 24 hrs [ISO 10993]. Cell reactivity was scored by USP Direct Contact Test. MPDS was evaluated at 100–25% diluted in culture medium. AlamarBlue assessed for cytotoxicity and metabolism. ZO-1 IHC assessed corneal epithelial barrier function. Lens preservative uptake-release was determined by HPLC. Results: MPS effects on HCEC depend on concentration, exposure time, and assay used. Lens uptake-release data support in vitro cytotoxicity of MPS and published clinical results. MPS-1, 5 and 6 biocompatibility by direct contact cytotoxicity score (0–2 vs. 2–4) and percent cell viability (>80% vs. <45%) outperform MPS 2–4. Corneal epithelial tight junction integrity under simulated in-use conditions [50% dilution, 5 min exposure] was not affected. Conclusions: MPS effects on in vitro cell cytotoxicity are best demonstrated through multiple assays: cell metabolic activity,
e23
membrane integrity, and biocompatibility. These results show MPS-1 as highly compatible with all soft hydrogel lenses examined [similar performance observed for MPS-5 and MPS-6] and better than MPS 2–4. http://dx.doi.org/10.1016/j.clae.2012.08.072 70 Comparison of total peroxide exposure values and biocidal efficacy of hydrogen peroxide disinfecting systems Jenilee Kilbury ∗ , Kimberly A. Millard, Suzanne F. Groemminger, Erning Xia Bausch & Lomb, Inc., Rochester, NY, USA E-mail address: [email protected] (J. Kilbury)
∗
Purpose: The total peroxide exposure of a hydrogen peroxide contact lens disinfecting system can be used to evaluate the antimicrobial effectiveness of the system. The total peroxide exposure is expressed as Area Under Curve (AUC) in units of (ppm hrs) and is calculated by integrating the neutralization profile curve of the hydrogen peroxide disinfecting system. This study compared AUC to biocidal efficacy of four hydrogen peroxide disinfecting systems. The solutions evaluated included high and low AUC models, and control products utilizing either a catalytic disk or a catalase tablet. Method: Hydrogen peroxide concentrations were measured at specified time points throughout a 6 hour neutralization period using standard redox titration methodology. Neutralization profiles were plotted as concentration (log ppm) versus time (hrs). The resulting curve was integrated to determine AUC (ppm hrs). The two model formulations were neutralized using contact lens cases containing catalytic disks. The control products were neutralized according to the manufacturers prescribed regimens. Biocidal efficacy was determined by ISO/FDA Stand Alone Procedure for Disinfecting Products. Results: The AUC values of the four formulations varied from 6000 to 16,000 ppm hr. For the most difficult organisms to kill, yeast and fungi, the total log kill directly correlated to the AUC for each of the systems which varied from <1 log for the lowest AUC model to >3 logs for the highest model. All systems demonstrated 99.99% kill after 6 hours for bacteria. Conclusions: The AUC value of a hydrogen peroxide contact lens disinfecting system is a quantitative assessment of the total peroxide exposure available to kill microorganisms. The AUC values can be used as an indicator of biocidal efficacy of the system, particularly against yeast and fungi. http://dx.doi.org/10.1016/j.clae.2012.08.073 71 Characterisation of bacteria from contact lens storage cases of corneal infiltrative event patients Simon Kilvington 1,∗ , Joseph Shovlin 2 , Marina Nikolic 1 , Anthony Lam 1 1 2 ∗
Abbott Medical Optics, Santa Ana, USA Northeastern Eye Institute, Scranton, USA E-mail address: [email protected] (S. Kilvington)
Purpose: Corneal infiltrative events (CIEs) are being reported with increasing frequency in lens wearers and may relate to specific multipurpose disinfecting solution (MPDS) or contact lens type usage. Here, the efficacy of MPDS’s against bacteria from contact lens storage cases (CLSC) of patients with CIEs was investigated.
cells collected non-invasively via irrigation with the ocular surface cell collection apparatus (OSCCA). Method: Five healthy participants underwent contra-lateral instillation of 50 L of 0.01% BAK (Bausch&Lomb Moisture Eyes) or sterile saline on three separate days (n = 15). Corneal staining with fluorescein was graded on a 0–100 scale for the five corneal zones 15 minutes post-BAK instillation. The Global Staining Scores were calculated. Calcein AM Blue (live/metabolically active stain) and Ethidium Homodimer-1 (dead stain) were used to stain the cells collected from the eye wash. Using a fluorescent microscope, live, dead and sodium fluorescein stained cells were quantified. Results: Post BAK or saline instillation, the GSS were 42 ± 33 and 31 ± 24, respectively. There were 428 ± 153 cells collected from BAK treated eyes, compared to 434 ± 241 cells from the control. In the collected BAK cell population, 7 ± 5% of cells were metabolically active compared to 5 ± 5% in the control group. Similar numbers of fluorescein stained cells were observed in the BAK and control groups. Cells staining with sodium fluorescein stained exclusively with Calcein AM and not Ethidium homodimer-1, suggesting that sodium fluorescein enters metabolically active cells. For all the measured parameters, no significant difference was observed between the BAK and control groups (p > 0.5). Conclusions: Our results suggest that a single instillation of 0.01% BAK does not induce corneal staining or affect the shedding of collected human epithelial cells in an acute manner, as determined by ex vivo examinations. http://dx.doi.org/10.1016/j.clae.2012.08.071 69 Evaluation of in vitro cytotoxicity assays for contact lens multipurpose solutions Ling C. Huang ∗ , Mercedes Salvador-Silva, Charles H. Powell, Lisa Hoong, Rosanne M. Yetemian Abbott Medical Optics, Santa Ana, USA E-mail address: [email protected] (L.C. Huang)
∗
Purpose: Previously published in vitro cytotoxicity assessments of Contact Lens Multipurpose Solutions (MPS) lack correlation. This study correlates MPS effects on cell cytotoxicity, metabolic activity, membrane integrity, and biocompatibility. Method: Six MPS were used: MPS-1 [polyquaternium (PQ-1), alexidine], MPS-2 [PQ-1, 5 ppm myristamidopropyl dimethylamine (MAPD), nonanoyl-EDTA], MPS-3 [PQ-1, 5 ppm MAPD], MPS-4 [PQ-1, 6 ppm MAPD], MPS-5 [PQ-1, polyhexamethylene biguanide (PHMB)], and MPS-6 [PHMB, poloxamer 237]. Five soft contact lenses were used: balafilcon A, senofilcon A, galyfilcon A, lotrafilcon B, and comfilcon A. In vitro biocompatibility was assessed by placing MPDS-treated lenses (100 mL, 4 days, n = 3) onto confluent HCEC SV40 cells for 24 hrs [ISO 10993]. Cell reactivity was scored by USP Direct Contact Test. MPDS was evaluated at 100–25% diluted in culture medium. AlamarBlue assessed for cytotoxicity and metabolism. ZO-1 IHC assessed corneal epithelial barrier function. Lens preservative uptake-release was determined by HPLC. Results: MPS effects on HCEC depend on concentration, exposure time, and assay used. Lens uptake-release data support in vitro cytotoxicity of MPS and published clinical results. MPS-1, 5 and 6 biocompatibility by direct contact cytotoxicity score (0–2 vs. 2–4) and percent cell viability (>80% vs. <45%) outperform MPS 2–4. Corneal epithelial tight junction integrity under simulated in-use conditions [50% dilution, 5 min exposure] was not affected. Conclusions: MPS effects on in vitro cell cytotoxicity are best demonstrated through multiple assays: cell metabolic activity,
e23
membrane integrity, and biocompatibility. These results show MPS-1 as highly compatible with all soft hydrogel lenses examined [similar performance observed for MPS-5 and MPS-6] and better than MPS 2–4. http://dx.doi.org/10.1016/j.clae.2012.08.072 70 Comparison of total peroxide exposure values and biocidal efficacy of hydrogen peroxide disinfecting systems Jenilee Kilbury ∗ , Kimberly A. Millard, Suzanne F. Groemminger, Erning Xia Bausch & Lomb, Inc., Rochester, NY, USA E-mail address: [email protected] (J. Kilbury)
∗
Purpose: The total peroxide exposure of a hydrogen peroxide contact lens disinfecting system can be used to evaluate the antimicrobial effectiveness of the system. The total peroxide exposure is expressed as Area Under Curve (AUC) in units of (ppm hrs) and is calculated by integrating the neutralization profile curve of the hydrogen peroxide disinfecting system. This study compared AUC to biocidal efficacy of four hydrogen peroxide disinfecting systems. The solutions evaluated included high and low AUC models, and control products utilizing either a catalytic disk or a catalase tablet. Method: Hydrogen peroxide concentrations were measured at specified time points throughout a 6 hour neutralization period using standard redox titration methodology. Neutralization profiles were plotted as concentration (log ppm) versus time (hrs). The resulting curve was integrated to determine AUC (ppm hrs). The two model formulations were neutralized using contact lens cases containing catalytic disks. The control products were neutralized according to the manufacturers prescribed regimens. Biocidal efficacy was determined by ISO/FDA Stand Alone Procedure for Disinfecting Products. Results: The AUC values of the four formulations varied from 6000 to 16,000 ppm hr. For the most difficult organisms to kill, yeast and fungi, the total log kill directly correlated to the AUC for each of the systems which varied from <1 log for the lowest AUC model to >3 logs for the highest model. All systems demonstrated 99.99% kill after 6 hours for bacteria. Conclusions: The AUC value of a hydrogen peroxide contact lens disinfecting system is a quantitative assessment of the total peroxide exposure available to kill microorganisms. The AUC values can be used as an indicator of biocidal efficacy of the system, particularly against yeast and fungi. http://dx.doi.org/10.1016/j.clae.2012.08.073 71 Characterisation of bacteria from contact lens storage cases of corneal infiltrative event patients Simon Kilvington 1,∗ , Joseph Shovlin 2 , Marina Nikolic 1 , Anthony Lam 1 1 2 ∗
Abbott Medical Optics, Santa Ana, USA Northeastern Eye Institute, Scranton, USA E-mail address: [email protected] (S. Kilvington)
Purpose: Corneal infiltrative events (CIEs) are being reported with increasing frequency in lens wearers and may relate to specific multipurpose disinfecting solution (MPDS) or contact lens type usage. Here, the efficacy of MPDS’s against bacteria from contact lens storage cases (CLSC) of patients with CIEs was investigated.