Comparison of the effect of multipurpose contact lens solutions on the viability ofcultured corneal epithelial cells

Megan E. Cavet *, Karen L. Harrington, Karl R. VanDerMeid, Keith W. Ward, Jin-Zhong Zhang

Pharmaceutical R&D, Bausch & Lomb, 1400 North Goodman Street, Rochester, NY 14609, USA

Contact Lens & Anterior Eye 32 (2009) 171–175

A R T I C L E I N F O

Keywords:

Cellular ATP content

Resazurin reduction

LDH release

Cell culture

Ocular surface

A B S T R A C T

Purpose: To determine the effect of four marketed multipurpose contact lens solutions (MPSs) on corneal

epithelial cell viability.

Methods: Comparison of the effect of MPS A (Renu MultiPlus, Bausch & Lomb), MPS B (OPTI-FREE

Express, Alcon), MPS C (AQuify, CibaVision), and MPS D (OPTI-FREE RepleniSH, Alcon) on cell viability

was performed by quantifying cellular ATP content, resazurin reduction, and lactate dehydrogenase

(LDH) release in transformed human corneal epithelial cells (HCEpiC) and primary bovine corneal

epithelial cells (BCEpiC).

Results: Significant reductions in cellular ATP content were observed at 40% solution and above with

both MPS B and MPS D, compared to at 100% only for MPS A and MPS C, and similar results were obtained

in BCEpiC. Effects on resazurin reduction were also less in HCEpiC exposed to increasing doses of MPS A

and MPS C than in cells exposed to MPS B and MPS D. After 15 min, HCEpiC viability measured by both

resazurin reduction and cellular ATP levels was significantly lower for cells exposed to MPS B, MPS D, and

MPS C, while HCEpiC exposed to MPS A were not affected. MPS B and MPS D reduced cell viability more

than MPS A and MPS C over a 2-h time course in both HCEpiC and BCEpiC.

Conclusions: Both MPS B and MPS D can cause large decreases in the viability of cultured corneal

epithelial cells even with just a 2 h exposure at multiple doses. Significant reduction in cell viability is

evident at brief 15–30 min exposures. In contrast, MPS A and MPS C have significantly less effect on the

cell viability of corneal epithelial cells at multiple doses, after these short exposure times.

� 2009 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

Contents lists available at ScienceDirect

Contact Lens & Anterior Eye

journa l homepage: www.e lsev ier .com/ locate /c lae

1. Introduction

Multipurpose contact lens solutions (MPSs) are designed tostore, clean, disinfect, rinse and rewet contact lenses. However,since these solutions are routinely introduced directly into the eyevia insertion of the contact lens, they must both effectively reducemicroorganisms and have minimum effects at the ocular surface. In

vitro cell viability studies are often used to assess the effect ofophthalmic agents and provide a uniform, rapid, and sensitivescreening tool [1–5]. These studies have been used to evaluatemultipurpose contact lens solutions as well [6,7–11].

Multipurpose solutions generally consist of an antimicrobialagent for disinfectant and preservative qualities, a surfactant, achelator which may have antibiotic properties, wetting agents, anda buffering agent to maintain pH of the solution [12]. MPS B andMPS D contain the antimicrobial agent polyquaternium-1 (trade-name Polyquad) and myristamidopropyl dimethylamine (MAPDA,

* Corresponding author. Tel.: +1 585 338 6191; fax: +1 585 338 6223.

E-mail address: megan.cavet@bausch.com (M.E. Cavet).

1367-0484/$ – see front matter � 2009 British Contact Lens Association. Published by

doi:10.1016/j.clae.2009.05.002

trade name Aldox), while MPS A and MPS C are polyhexamethylenebiguanide (PHMB)-based. In addition, each of these solutions differin the surfactant and buffering system [13]. These differing agentsgive rise to the possibility of a variety of different biological effectsof different brands of solution.

It is well known that exposure of the corneal epithelia to contactlens solutions is brief. For instance, in dry eye subjects it has beenestimated that after 25 min, 1 h, and 2 h the concentration of adrop of fluid applied to the eye would be 50%, 20%, and 4%,respectively [2]. Ocular residence times measured using quanti-tative gamma scintigraphy gave estimates of between 39 and 321 sfor artificial tear solutions [14]. Using a high molecular weightfluorescent indicator, pre-corneal residence time was estimated asbetween 15 and 25 min for saline in human subjects [15]. For moreviscous solutions, using the same technique, residence time was36–41 min in dry eye subjects [16]. While uptake of contact lenssolution constituents including surfactants and antimicrobialagents into contact lenses could also increase exposure time tothe ocular surface, it is clear that studies involving multiple days ofincubation are not clinically relevant, and shorter-term studies arerequired [17–21].

Elsevier Ltd. All rights reserved.

Table 1Composition of MPSs studied.

Solutions Antimicrobial agent Buffers, electrolytes, cleaners and surfactant

MPS A, Renu MultiPlus

(Bausch & Lomb)

PHMB (0.00005%) Sodium borate/boric acid, sodium chloride, EDTA, tetronic 1107, HAP

MPS B, OPTI-FREE Express (Alcon) Polyquad (0.001%),

MAPDA (0.0005%)

Sodium citrate/boric acid, sodium chloride, sorbitol, disodium EDTA, AMP-95, tetronic 1304

MPS C, AQuify (Ciba Vision) PHMB (0.0001%) Sodium phosphate dihydrogen, sorbitol, EDTA, tromethamine, pluronic F127,

dexpanthenol

MPS D, OPTI-FREE RepleniSH (Alcon) Polyquad (0.001%),

MAPDA (0.0005%)

Sodium citrate/sodium borate, sodium chloride, C9-ED3A, tetronic 1304, propylene

glycol

AMP: 2 amino-2-methyl-1-propanol; C9-ED3A: nonanoyl ethylenediaminetriacetic acid; EDTA: ethylenediamine tetraacetic acid; HAP: hydroxyalkyl phosphonate; MAPDA:

myristamidopropyl dimethylamine; PAPB: polyaminopropyl biguanide; PHMB: polyhexamethylene biguanide; and Polyquad: polyquaternium-1.

M.E. Cavet et al. / Contact Lens & Anterior Eye 32 (2009) 171–175172

The present set of studies provides a consistent evaluationplatform using multiple assays and solution concentrations toinvestigate the effect of contact lens solutions at the ocularsurface. We studied the effect on corneal cell viability of a brief(0–2 h) time course of exposure to multiple contact lens solutionsand determined the effect of increasing doses of solution inmultiple biological assays in both human and bovine cornealepithelial cells. The results clearly demonstrate substantial inter-solution differences on the effects on cell viability, with MPS Band MPS D having significantly reduced cell viability. Part of thiswork was presented at the BCLA Annual Clinical Conference 2008[22].

2. Materials and methods

2.1. Materials

Contact lens solutions were obtained from commercial sourcesand used before their labeled expiration date. Tissue culture mediaand reagents were from Invitrogen (Carlsbad, CA). Dispase II wasfrom Roche (Indianapolis, IN).

2.2. Cell culture

Transformed human corneal epithelial cells (HCEpiC; 2.040pRSV-T) were obtained from ATCC (Manassas, VA) at passage 20.All testing was done between passages 21 and 31. HCEpiC weregrown in EpiLife medium supplemented with human cornealgrowth factors (HCGF), 50 IU/ml penicillin, and 50 mg/mlstreptomycin, containing 5.5 mM glucose in 5% CO2/95% airwith 95% humidity at 37 8C. Cells were seeded at 9 � 103 cells/well on a 96-well plate for the ATP assay and at 1 � 104 cells/wellon a 48-well plate for the resazurin assay. Cells were used when95% confluent, 4 days after seeding. Primary bovine cornealepithelial cells (BCEpiC) were isolated as described previously[23]. Briefly, freshly obtained bovine corneas from Joes MeatMarket (Ontario, New York) were incubated with 2 U/ml DispaseII in phosphate-buffered saline (PBS) at 37 8C for 90 min. Thecornea was placed into 0.05% trypsin and the epithelium wasstripped off with gentle scraping from the limbus to the center.After incubation at 37 8C for 15 min, the trypsin was deactivatedusing fetal bovine serum (FBS) and cells were centrifuged at100 � g for 5 min. Cells were re-suspended and cultured inDulbeccos Minimal Essential Medium (DMEM)/F12 mediacontaining 10% FBS, HCGF, 100 IU/ml penicillin, 100 mg/mlstreptomycin, and 50 ng/ml amphotericin B (fungizone) in 5%CO2/95% air with 95% humidity at 37 8C. All experiments wereperformed on passages 3 through 7. Cells were seeded at3 � 103 cells/well on a 96-well plate for the ATP and lactatedehydrogenase (LDH) assays and at 1 � 104 cells/well on a 48-well plate for the resazurin assay. Cells were used when 95%confluent, 5 days after seeding.

2.3. Cell treatments

Four commercial contact lens solutions were tested: MPS A(Renu MultiPlus, Bausch & Lomb, Rochester, NY), MPS B (OPTI-FREE Express, Alcon, Fort Worth, TX), MPS C (AQuify, CibaVision,Duluth, GA), and MPS D (OPTI-FREE RepleniSH, Alcon) (Table 1).Cells were exposed to two sets of treatments: 100% contact lenssolution over a 2-h time frame; and a dose response of 20–100%solution (diluted in growth medium) for 2 h. The negative controlwas growth medium alone, and phosphate-buffered saline(Invitrogen; 1.1 mM potassium phosphate, 155.2 mM sodiumchloride and 3.0 mM sodium phosphate dibasic) was included todetermine the effect of a simple saline solution. After 2 h in 5% CO2

in the incubator, the pHs of the MPSs were within 0.36 pH units ofthe growth medium (pH 7.32). The pH of the 100% solutions(which had the greatest differences from growth medium) were asfollows: MPS A was 6.97, MPS B was 7.59, MPS C was 6.96 and MPSD was 7.1.

2.4. Cell viability assays

ATP quantitation: Cells were allowed to equilibrate to roomtemperature for the last 15 min of the incubation time with theMPS. After incubation in contact lens solutions, 100 ml ofCellTiter-Glo Reagent (Promega; Madison, WI) was added to each96-well of cells. The contents were mixed on an orbital shaker for15 min. A negative control containing culture medium andCellTiter-Glo reagent without cells was included. The lumines-cence was measured on a Victor3V plate reader (PerkinElmer,Waltham, MA).

Resazurin reduction assay which measured the reduction ofresazurin to resorufin as a measure of the number of viable andproliferating cells was performed using alamarBlue solution(Invitrogen; Carlsbad, CA) according to the manufacturer’sinstructions. After incubation with contact lens solutions, solutionwas removed and 10% alamarBlue in DMEM was added to eachwell. Cells were incubated for 3 h in a 5% CO2/95% O2 incubator at37 8C. A negative control containing culture medium andalamarBlue reagent without cells was included. Fluorescencemeasurements were made by excitation at 530–560 nm andmeasuring emission at 590 using the Victor3V fluorescence platereader.

Lactate dehydrogenase release into the culture medium wasmeasured using CytoTox 96 Non-Radioactive Cytotoxicity Assay(Promega; Madison, WI) according to the manufacturer’s instruc-tions. After incubation of cells in contact lens solutions, media werecentrifuged and 50 ml of media was mixed with 50 ml of CytoToxsubstrate. Samples were incubated for 30 min in the dark afterwhich 50 ml of stop solution was added. A negative controlcontaining culture medium and reagent without cells wasincluded. The absorbance was measured at 490 nm on a Gen5plate reader (BioTek, Winooski, VT).

Fig. 1. Effect of contact lens solutions on cell viability in HCEpiC. (A) Cellular ATP content was quantified after exposing cells to increasing doses of lens care solutions. (B)

Resaruzin reduction was used to assess viability after exposing cells to increasing doses of solutions. (C) Cellular ATP content was quantified after exposing cells to lens

solutions for 0–2 h. (D) Resazurin reduction was used to assess viability after exposing cells to lens solutions for 0–2 h. * = significantly different from media control, P < 0.05.

n = 3.

Table 2Cellular viability in HCEpiC exposed to 100% MPSs for 2 h.

Treatment Cellular ATP content (arbitrary

fluorescent units �105)

Resazurin reduction (arbitrary

chemiluminescent units �105)

Dose (Fig. 1A) Time (Fig. 1C) Dose (Fig. 1B) Time (Fig. 1D)

Control 13.19 � 1.00 14.39 � 0.40 30.13 � 1.19 21.58 � 0.65

PBS 11.86 � 0.47 10.76 � 0.12 22.74 � 0.45 17.05 � 0.66

MPS A 9.22 � 0.72 8.66 � 0.37 26.38 � 0.89 16.20 � 0.61

MPS B 3.53 � 0.16 3.35 � 0.05 0.77 � 0.10 0.27 � 0.084

MPS C 7.88 � 1.30 11.54 � 0.69 20.89 � 0.97 10.45 � 0.73

MPS D 2.71 � 0.26 3.50 � 0.030 2.24 � 0.41 0.57 � 0.19

Results are expressed as mean � SD; n = 3.

M.E. Cavet et al. / Contact Lens & Anterior Eye 32 (2009) 171–175 173

2.5. Data analysis and statistics

Experiments were performed with three repeats and forselected cellular ATP content and resazurin content experimentsresults were replicated in independent experiments with verysimilar results. All data were expressed as the ratio of the testsolution viability to the media control, and are reported asmean � SD. Statistical analysis comparing effects of treatmentacross groups was performed using a two-way ANOVA with a Tukey–Kramer post-hoc comparison test. Multiple comparisons were madeto media control and between contact lens treatment groups. For allassays, P � 0.05 was predetermined as the criterion of statisticalsignificance.

3. Results

3.1. HCEpiC

Results from both resazurin reduction and ATP quantitationassays demonstrated reduced viability in cells exposed to MPS Band MPS D as compared to the medium control. The ATP contentof cells exposed to MPS B and MPS D was also significantly lowerin the presence of 40%, 60%, 80%, and 100% concentration of thecontact lens solutions compared to the medium control andother tested solutions. The ATP content of cells exposed to MPS Aand MPS C only demonstrated a decrease when exposed to a100% solution concentration for 2 h (Fig. 1A and Table 2).Similarly, the resazurin reduction assay showed that cellviability of cells exposed to MPS D and MPS B in the presenceof 40%, 60%, 80%, and 100% concentration of contact lenssolutions was lower than MPS A and MPS C (Fig. 1B and Table 2).Therefore, based on both cellular viability assays, MPS A and MPSC had a minimal effect on cell viability in HCEpiC when compared

to the effects of both MPS D and MPS B on cell viability after 2 hexposure at 100% solution.

Next, time course studies were performed to evaluate the effectof exposure time on cell viability in the presence of varioussolutions. Exposure of HCEpiC to 100% contact lens solutionscaused a statistically significant reduction in cell viability ascompared to media control for MPS B and MPS D after 15 min ofexposure, while cells exposed to MPS A and MPS C were notsignificantly different from control as measured by ATP quantita-tion (Fig. 1C) and resazurin reduction (Fig. 1D) at 15 min. Table 2shows the ATP content at 100% solution after 2 h exposure. Thereduction in cell viability was statistically significantly greater forMPS B and MPS D than for MPS A and MPS C at all time points tested(Fig. 1 C and D, and Table 2).

3.2. BCEpiC

The effects of contact lens solutions on cell viability were alsodetermined in primary BCEpiC to verify that toxic effects were not

Fig. 2. Effect of contact lens solutions on cell viability in BCEpiC. (A) Cellular ATP content was quantified after exposing cells to increasing doses of lens solutions. (B) Cellular

ATP content was quantified after exposing cells to lens solutions for 0–2 h. (C) Resazurin reduction was used to assess viability after exposing cells to lens solutions for 0–2 h.

(D) LDH release was measured after exposing cells to lens solutions for 0–2 h. * = significantly different from media control, P < 0.05. n = 3.

Table 3Cellular viability in BCEpiC exposed to 100% MPSs for 2 h.

Treatment Cellular ATP content (arbitrary

chemiluminescent units �105)

Resazurin reduction fluorescent units �105) LDH (arbitrary absorbance units)

Dose (Fig. 2A) Time (Fig. 2B) Time (Fig. 2C) Time (Fig. 2D)

Control 9.69 � 1.08 8.19 � 0.43 35.62 � 4.66 0.08 � 0.02

PBS 9.95 � 0.43 8.94 � 0.46 35.46 � 2.56 �0.03 � 0.02

MPS A 7.13 � 0.39 5.67 � 0.39 26.13 � 2.54 0.07 � 0.04

MPS B 4.75 � 0.60 4.08 � 0.22 0.90 � 0.015 1.72 � 0.07

MPS C 8.88 � 0.28 8.50 � 0.62 27.17 � 0.79 0.10 � 0.03

MPS D 4.25 � 0.21 3.21 � 0.10 10.69 � 4.79 0.43 � 0.09

Results are expressed as mean � SD; n = 3.

M.E. Cavet et al. / Contact Lens & Anterior Eye 32 (2009) 171–175174

limited to a transformed human cell line. Measurement of theeffect of increasing doses of contact lens solution for 2 h on cellularATP content showed that there was a significant reduction in cellviability at 40% and above for both MPS D and MPS B, whileviability of cells exposed to MPS A was only decreased at 100%solution (Fig. 2A and Table 3). A time course study measuring ATPcontent showed that MPS B and MPS D significantly reduced cellviability after 30 min of incubation (Fig. 2B) while MPS Asignificantly reduced cell viability after 2 h (Fig. 2B). Similarly,results from a resazurin reduction time course experimentdemonstrated that viability of MPS B was lower than MPS A andMPS C at 30 min and longer exposure, while viability of MPS D waslower than MPS A and MPS C at 90 and 120 min exposure (Fig. 2Cand Table 3). As further validation of these cytotoxicity results, athird viability assay, release of cytosolic LDH, was employed [24].Results from the LDH release assay, which is a measure ofmembrane integrity, gave similar data, with MPS B significantlyincreasing LDH release after 60 min and MPS D significantlyincreasing LDH release after 90 min, and no significant effect ofPBS, MPS A, or MPS C (Fig. 2D and Table 3).

4. Discussion

In vitro cell culture systems can be a rapid, convenient, andinformative way to gather safety and efficacy data on new productformulations, and such systems readily allow for facile manipula-tion of experimental conditions to probe dose- or time-responses.However, because of this ease of control of multiple experimentalconditions, substantial care must be taken in the interpretation ofresults from these studies. Specifically, it is very difficult to reliablycompare in vitro data from different laboratories on differentproducts, due to the sensitivity of relative outcomes to experi-mental conditions. In the present study, we provide a robust head-to-head analysis of the effect of four currently marketed multi-purpose contact lens solutions on cell viability using multiple assayformats and conditions. Using this test battery, it is apparent thatthere are substantial differences in the effects of these products oncorneal epithelial cell viability. While MPS B and MPS Dsignificantly reduced the cell viability compared to the mediumcontrol at 2 h and concentrations �40% solution, MPS A and MPS Ccaused only minimal changes in cell viability. MPS A and MPS C did

M.E. Cavet et al. / Contact Lens & Anterior Eye 32 (2009) 171–175 175

not decrease cell viability by more than 50% (a known benchmarkfor cytotoxicity) in either the ATP assay or the resazurin reductionassay. The outcomes of the present study agree with several otherstudies in the peer-reviewed literature in this regard, which pointto increased adverse effect on cell viability in cells exposed to MPSB compared to MPS A [6–8]. Two exceptions to this general rule inthe literature both employ unrealistically long exposure times –either 1–2 days or up to 4 days [9,10], which may account for thedifferences seen. Given the physiology of tear turnover rates andthe biology of the ocular surface, such lengthy exposure times arenot likely to mimic the effects of the brief exposure of the cornealepithelium to contact lens solutions in vivo. The largest inter-solution differences where obtained at the 2 h exposure time in thecurrent study, which is longer than published values for pre-corneal retention times [14,15]. However, smaller but significantdifferences in effect on cell viability between solutions were alsoobserved at 15–30 min of exposure, in the range of recentlypublished data regarding retention rates [15,16]. While exposure isstill greater than that seen in vivo in the current study, sincedilution of the MPS over time into the tear volume is not accountedfor, this study provides a more comprehensive assessment of cellviability under more physiologically relevant conditions thanthose previously which used much longer exposure times.

Multipurpose solutions consist of a buffered solution contain-ing antimicrobial agents and surfactants, however the excipientsdiffer widely depending on the individual solution [12]. A majordifference between the formulations tested is that MPS D and MPSB both use Polyquaterium-1 and MAPDA as preservatives whileMPS C and MPS A both use PHMB as a preservative. Reports in theliterature indicate that Polyquaterium-1 is not the major factorcausing the reduction in cell viability in MPS B and MPS D. A studyin rats has demonstrated that high concentrations of Polyquater-ium-1 (0.1% and 0.5%; 100 fold higher than used in contact lenssolutions) does not change tear production, slit lamp andfluorescein evaluation or histology as compared to control [25].In a human corneal cell based study Polyquaternium ammoniumchloride (0.001%) did not cause cytotoxicity [26]. It is likely that acombination of excipients in the lens care solutions (antimicrobialagents, buffering agents, surfactants, and chelating agents) inaddition to the ionic balance of the overall solution are thecausative factors in the adverse effects of MPS B and MPS D on theviability of corneal epithelial cells. Further studies in which thecellular effects of excipients from MPS B and MPS D alone and incombination are investigated may be of value in determining thespecific excipients responsible for the decreased viability.

In conclusion, this series of studies demonstrates in a consistentexperimental system that MPS B and MPS D significantly reducecell viability compared to the medium control after 2 h exposure inboth transformed and primary corneal epithelial cells as measuredby three different methods. This may be due to a combinationeffect on cellular metabolism by some of the active or inactiveingredients of the solutions. Important areas for future researchwill include further investigation into the biochemical mechan-ism(s) modulating the apparent effect on cell viability observed,and the development of more robust in vitro–in vivo correlations tofurther improve the ability to place these important in vitro

findings into a relevant clinical context.

Conflict of interest statement

Authors state no conflict of interest. MEC, KLH, KRV, KWW andJ-ZZ are all employees of Bausch & Lomb (Rochester, NY).

Acknowledgements

We thank Dr. Susan Burke (Bausch & Lomb) for providing somereagents used in this study and Sue Groemminger (Bausch & Lomb)for providing product formulation information.

References

[1] Saarinen-Savolainen P, Jarvinen T, Araki-Sasaki K, Watanabe H, Urtti A. Eva-luation of cytotoxicity of various ophthalmic drugs, eye drop excipients andcyclodextrins in an immortalized human corneal epithelial cell line. Pharm Res1998;15:1275.

[2] Geerling G, Daniels JT, Dart JK, Cree IA, Khaw PT. Toxicity of natural tearsubstitutes in a fully defined culture model of human corneal epithelial cells.Invest Ophthalmol Vis Sci 2001;42:948.

[3] Diebold Y, Herreras JM, Callejo S, Argueso P, Calonge M. Carbomer- versuscellulose-based artificial-tear formulations: morphologic and toxicologiceffects on a corneal cell line. Cornea 1998;17:433.

[4] North-Root H, Yackovich F, Demetrulias J, Gacula Jr M, Heinze JE. Evaluation ofan in vitro cell toxicity test using rabbit corneal cells to predict the eyeirritation potential of surfactants. Toxicol Lett 1982;14:207.

[5] Kahn CR, Young E, Lee IH, Rhim JS. Human corneal epithelial primary culturesand cell lines with extended life span: in vitro model for ocular studies. InvestOphthalmol Vis Sci 1993;34:3429.

[6] Mowrey-McKee M, Sills A, Wright A. Comparative cytotoxicity potential of softcontact lens care regimens. CLAO J 2002;28:160.

[7] Horwath-Winter J, Simon M, Kolli H, Trummer G, Schmut O. Cytotoxicityevaluation of soft contact lens care solutions on human conjunctival fibro-blasts. Ophthalmologica 2004;218:385.

[8] McCanna DJ, Harrington KL, Driot JY, Ward KW, Tchao R. Use of a humancorneal epithelial cell line for screening the safety of contact lens care solutionsin vitro. Eye Contact Lens 2008;34:6.

[9] Wright A, Mowrey-McKee M. Comparative cytotoxicity potential of soft con-tact lens care products. Cutan Ocul Toxicol 2005;24:53.

[10] Pham XT, Huff JW. Cytotoxicity evaluation of multipurpose contact lenssolutions using an in vitro test battery. CLAO J 1999;25:28.

[11] Chuang EY, Li DQ, Bian F, Zheng X, Pflugfelder SC. Effects of contact lensmultipurpose solutions on human corneal epithelial survival and barrierfunction. Eye Contact Lens 2008;34:281.

[12] Christie CL, Meyler JG. Contemporary contact lens care products. Contact LensAnterior Eye 1997;20(Suppl. 1):S11.

[13] Jones L, Senchyna M. Soft contact lens solutions. Part 1: Components ofmodern care regimens. Optom Pract 2007;8:45.

[14] Snibson GR, Greaves JL, Soper ND, Tiffany JM, Wilson CG, Bron AJ. Ocularsurface residence times of artificial tear solutions. Cornea 1992;11:288.

[15] Meadows DL, Paugh JR, Joshi A, Mordaunt J. A novel method to evaluateresidence time in humans using a nonpenetrating fluorescent tracer. InvestOphthalmol Vis Sci 2002;43:1032.

[16] Paugh JR, Nguyen AL, Ketelson HA, Christensen MT, Meadows DL. Precornealresidence time of artificial tears measured in dry eye subjects. Optom Vis Sci2008;85:725.

[17] Chapman JM, Cheeks L, Green K. Interactions of benzalkonium chloride withsoft and hard contact lenses. Arch Ophthalmol 1990;108:244.

[18] Christensen MT, Barry JR, Turner FD. Five-minute removal of soft lensesprevents most absorption of a topical ophthalmic solution. CLAO J1998;24:227.

[19] Jones L, MacDougall N, Sorbara LG. Asymptomatic corneal staining associatedwith the use of balafilcon silicone-hydrogel contact lenses disinfected with apolyaminopropyl biguanide-preserved care regimen. Optom Vis Sci2002;79:753.

[20] Dassanayake NL, Garofalo R, Carey C, David R, Meadows DL, Stone RP. Corre-lating biocide uptake and release profiles with corneal staining and subjectivesymptoms. Invest Ophthalmol Vis Sci 2005;2005:46E.

[21] Ketelson HA, Meadows DL, Stone RP. Dynamic wettability properties of a softcontact lens hydrogel. Colloids Surf B Biointerfaces 2005;40:1.

[22] Zhang J, Vandermeid K, Cavet M, Ward K. The effect of multipurpose contactlens solutions on cell viability in human corneal epithelial cells. Contact LensAnterior Eye 2008;31:252.

[23] Guo Y, Satpathy M, Wilson G, Srinivas SP. Benzalkonium chloride inducesdephosphorylation of Myosin light chain in cultured corneal epithelial cells.Invest Ophthalmol Vis Sci 2007;48:2001.

[24] Babich H, Zuckerbraun HL, Wurzburger BJ, Rubin YL, Borenfreund E, Blau L.Benzoyl peroxide cytotoxicity evaluated in vitro with the human keratinocytecell line RHEK-1. Toxicology 1996;106:187.

[25] Labbe A, Pauly A, Liang H, Brignole-Baudouin F, Martin C, Warnet JM, et al.Comparison of toxicological profiles of benzalkonium chloride and polyqua-ternium-1: an experimental study. J Ocul Pharmacol Ther 2006;22:267.

[26] Tripathi BJ, Tripathi RC, Kolli SP. Cytotoxicity of ophthalmic preservatives onhuman corneal epithelium. Lens Eye Toxic Res 1992;9:361.