Biocompatibility Evaluation of Contact Lens Multipurpose Solutions Through Novel Genotoxicity Assays

Abstract

Introduction:

Genotoxicity testing is necessary to address potential mutagenic characteristics of pharmaceutical candidates and medical devices depending on their intended use. Adapting such tests to contact lens multipurpose solutions (MPS) may be required due to their antimicrobial nature or to fulfill specific regulatory requirements. We describe a novel methodology for genotoxicity and biocompatibility evaluation of MPS in the context of traditional testing.

Materials and Methods:

Blink RevitaLens^® Multipurpose Disinfecting Solution (MPDS) or contact lens (Acuvue^® Advance and Acuvue^® 2 brand) extracts were tested by Ames assay and Chromosome Aberration (ChrAb) Assays. A novel procedure for cycling contact lenses in MPDS was developed for testing. Before assays with the Blink RevitaLens MPDS disinfectant ingredient, Alexidine Dihydrochloride (ALX) was diluted for dose-range tests identifying TA100 or Chinese Hamster Ovary cytotoxicity levels to determine the amount of ALX appropriate for each assay.

Results:

MiniScreen Ames and Ames show Blink RevitaLens MPDS and the contact lens extracts did not increase the number of revertant colonies compared to saline, suggesting nonmutagenicity. The test articles also did not increase ChrAb, indicating they are nonclastogeneic. Below its cytotoxic limits, ALX did not increase the frequency of revertants in the Ames assay or demonstrate chromosome changes compared with controls.

Discussion/Conclusions:

Results from novel and standard genotoxicity assays demonstrated that Blink RevitaLens MPDS and ALX are not genotoxic or clastogenic. Novel cycling of contact lenses in MPDS is beneficial and accurate for biocompatibility testing on MPDS.

Introduction

Contact lens wear continues to be very common, with silicone hydrogel contact lenses being the most prescribed lens.^1,2 Despite lens popularity, contact lens wearers still encounter ocular surface discomfort and dryness.^3,4 The discomfort associated with contact lens wear is a leading cause for discontinuation and dissatisfaction.^5,6 While the mechanism and etiology of discomfort may vary, the contact lens care regimen that is used certainly impacts the user experience. Multipurpose solutions (MPS) are a single solution method of disinfecting, rinsing, and storing soft contact lenses.^7,8 MPS care systems have the ability to improve contact lens comfort and efficacy, potentially increasing compliance with contact lens wear.⁹ Given its multiple uses, a critical role of MPS is still to disinfect a wide range of microorganisms and remove contaminants from lenses that could result in irritation of the eye.¹⁰ Manufacturers of MPS care systems are committed to the safe use and efficacy of solutions, thus, most solutions have adequate antimicrobial activity.^11–13 During normal use, contact lenses soaked in MPS will release the MPS, including disinfectants, into the eye during wear.¹⁴ In fact, with some contact lens and solution combinations, corneal staining and ocular symptoms can be observed as early as 1–2 hours of wear in patients and in studies conducted in rabbits.^15–17 Thus, while MPS represent an opportunity to increase comfort and compliance while simultaneously disinfecting contact lenses, ensuring nontoxicity of the MPS is vital.

Effective disinfection ability of MPS is often contrasted with the requirement of nontoxicity in corneal cells to maintain ocular surface health and promote long-term contact lens use. Blink RevitaLens^® Multipurpose Disinfecting Solution (MPDS) is a buffered aqueous solution containing a dual disinfectant system consisting of polyquaternium-1 (PQ-1) and alexidine dihydrochloride (ALX) in addition to sodium chloride, boric acid, sodium borate, disodium edetate, Tetronic^® 904, and sodium citrate. Previous studies have shown that Blink RevitaLens MPDS demonstrates broad-spectrum disinfection and sustained antimicrobial efficacy.^11,18–20 Preclinical safety evaluations conducted according to ISO recommendations and additional in vitro studies have demonstrated that Blink RevitaLens MPDS is nonirritating, nonsensitizing, and noncytotoxic. Because lens MPS are antimicrobial in nature, special attention to assess genotoxicity is warranted during the development process. For certain regions such as Japan where MPS is classified as a quasidrug, genotoxicity testing would address potential mutagenic or clastogenic characteristics of pharmaceuticals and some medical devices and would identify any potentially problematic solutions or ingredients while taking into account the innate toxicity of disinfectants. Blink RevitaLens MPDS includes both PQ-1 and ALX for disinfection. While PQ-1 is present in other contact lens MPS, ALX has not previously been used in MPDS.

The purpose of the current studies was to rule out any genotoxicity potential of both Blink RevitaLens MPDS and ALX, a novel ingredient to MPS. Established Ames and chromosome aberration (ChrAb) assays were used with test samples prepared using novel methodology to assess genotoxicity.

Materials and Methods

Contact lenses and MPS

Contact lenses used in these studies were galyfilcon A (silicone hydrogel, Acuvue^® Advance; Johnson & Johnson Vision) and etafilcon A (hydrogel material, Acuvue^® 2; Johnson & Johnson Vision).

Cell lines and tissues culture

Chinese Hamster Ovary (CHO) cells that served as a sensitive indicator of clastogenic damage were obtained from American Type Culture Collection (ATCC). CHO cells were cultured in Ham's F-12 containing 10 mM HEPES, 10% FBS, 1–2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. All cells were cultured at 37°C with 5% CO₂ and 95% humidity.

Bacteria strains

Histidine-dependent Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 were used to detect reverse mutations in the histidine gene that would allow for histidine independent growth. The tryptophan-dependent Escherichia coli strain WP2uvrA- was used to detect reverse mutations in the tryptophan gene that would allow for tryptophan independent growth. All bacterial strains were purchased from Moltox, Inc. (Boone, NC).

Metabolic activation

Both Ames and ChrAb assays were performed in the presence and absence of metabolic activation, because activation of the test system would aid in identifying the mutagenic potential of metabolites produced from the original test article. Metabolic activation was achieved through treatment by S9 activation system. For Ames assays involving metabolic activation, a solution comprised 10% S9 microsomal fraction from Sprague–Dawley rat livers induced with Aroclor^® 1254 (Moltox, Boone, NC) was made in 8 mM MgCl₂, 33 mM KCl, 5 mM Glucose-6-Phosphate, 4 mM NADP, and 0.1 M phosphate buffer (pH 7.4). S9 activation solution was used in place of phosphate buffer in preparation of the top agar. For ChrAb Assays involving metabolic activation, S9 was prepared at a concentration of 20 μL/mL in cell culture medium lacking FBS, supplemented with trisodium salt and NADP.

MiniScreen Ames

Undiluted Blink RevitaLens MPDS and contact lens extracts were tested in duplicate using TA98 and TA100 as previously described.²¹ In breif, six-well culture plates (Costar; Corning, Inc.) were filled with 5 mL of minimal agar. Top agar was supplemented with 0.5% agar, 0.5% sodium chloride, 0.5 mM L-histidine hydrochloride, and 0.5 mM D-biotin. All samples were tested at both naive and metabolically activated states. N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and 2-nitrofluorene (NF) were used as positive controls in naive assays, and 2-aminoanthracene (AA) was a positive control in metabolically activated assays. PBS was used as the vehicle control. To conduct the assay, TA98 and TA100 bacterial strains were exposed to test solutions and extracts with and without metabolic activation in top agar and then plated into the six-well culture dishes. Revertant colonies were counted on an automated colony counter after 48 hours of incubation at 37°C. Assuming a negative control value was within typical range, a test article was considered mutagenic if it produced a two-fold increase over the negative control that was also dose-dependent when dilutions were tested.

Cycling of contact lenses

For novel cycling and testing procedure, all contact lenses were first rinsed with saline solution upon removal from the manufacturer's blister packets. Lenses were equilibrated once in approximately 4 mL of saline solution for 6 hours at room temperature. To expose contact lenses to 30 cycles of Blink RevitaLens MPDS approximately, three drops of Blink RevitaLens MPDS were placed on one side of the lens surface and digitally rubbed from the center to the edges 20–30 times. The lens was then turned over and procedure repeated on the opposite side of the lens. Each side of the lens was rinsed with fresh Blink RevitaLens MPDS and the lens placed in 3.5 mL of Blink RevitaLens MPDS in a lens case for a minimum of 4 hours with the cap closed. Before initiating the next cycle, lenses were soaked in saline in a lens case to simulate wearing for at least 4 hours. This procedure constitutes one cycle. To initiate the next cycle, approximately three drops of Blink RevitaLens MPDS were placed on the lens surface and digitally rubbed as described above. In the final cycle, the lenses were taken from the Blink RevitaLens MPDS test solution and extracted for further testing. Thirty cycles were completed before each contact lens extraction to simulate product use and enable evaluation of genotoxicity in extracts from cycled contact lenses.

Ames assay

Contact lenses were cycled in Blink RevitaLens MPDS for 30 cycles and extracted into saline by a 72-hour incubation at 37°C. For extraction, a total of six contact lenses of each brand were completely immersed and extracted in saline at a ratio of 3 cm² per 1 mL. Blink RevitaLens MPDS and saline were also incubated at 37°C for 72 hours for use as controls. Undiluted Blink RevitaLens MPDS and contact lens extracts were tested for their abilities to induce reverse mutations in TA98, TA100, TA1535, TA1537, and WP2uvrA- in comparison to positive controls and saline. Bacteria were prepared by isolating a single colony from a nutrient agar plate and inoculating into sterile nutrient broth for overnight growth. One hundred microliters of each bacterial strain grown overnight was combined with 100 μL of the test articles or controls, 0.5 mL of 0.2 M phosphate buffer (pH = 7.4) in 2 mL of top agar (supplemented with 0.5 mM histidine and 0.5 mM biotin or tryptophan), then added to minimal agar plates. For plates requiring metabolic activation, 0.5 mL of S9 activation solution was used in place of phosphate buffer. After 50 hours of incubation at 37°C, revertant colonies were counted and compared with spontaneous revertants in negative controls. The positive control for naive (nonactivated) strains of bacteria was 2-2-aminoanthracene. For S9-activated strains, positive controls were strain-specific (Sodium azide for TA100 and TA1535, 2-nitrofluorene for TA98, 9-aminoacridine for TA1537, and 4-nitrofuinoline 1-oxide for WP2uvrA-). Saline was used as a negative control. Revertant counts were considered mutagenic if they were twice as high as the negative control.

Chromosome aberration

Contact lenses were cycled in Blink RevitaLens MPDS for 30 cycles and extracted into Ham's F-12 medium by a 72-hour incubation at 37°C (3 cm² per 1 mL). Blink RevitaLens MPDS was also incubated at 37°C for 72 hours for use as a control. Blink RevitaLens MPDS diluted 1:1 with media and undiluted contact lens extracts were tested for the ability to induce chromosomal aberrations (structural or numerical) in CHO cells with and without S9 metabolic activation. Cells were seeded at 0.5 × 10⁶ cells per T25 flask for 24 hours before exposure. Cells were exposed to test articles for 3 hours, with or without activation by S9. The cells were then washed and complete medium replaced for 21 hours. Ham's F-12 served as a negative control. Cyclophosphamide and Mitomycin C were positive controls, for cells with and without activation, respectively. Cells were exposed to 0.1 μg/mL colcemid for 1 hour and 30 minutes before harvesting. To harvest, the cells were detached, centrifuged, and treated with 0.075 M potassium chloride at 37°C for 10 minutes. Following two rinses with 3:1 methanol:glacial acetic acid, cells were plated on slides with 5% Giemsa stain. Metaphase cells were analyzed for ChrAb. One hundred metaphase cells were counted for positive controls, while 200 metaphase cells were counted for each test article and negative control. A test article was considered mutagenic if it exhibited a statistically significant increase in chromosomal aberrations over the negative control using a chi-square test.

Novel ingredient assays

Dose-range assays for Alexidine dihydrochloride (ALX) tolerance were conducted before Ames or ChrAb assays by incubating ALX, with and without activation, with TA100 or CHO cells. In CHO cells, various concentrations of ALX diluted in media were incubated with the cells for 3 hours. ALX-media was then removed and normal complete media returned to the plate for 21 hours. Cytotoxicity was evaluated by analyzing the relative population doubling rate compared with negative controls. The three highest concentrations of ALX with less than 50% cytotoxicity were chosen for ChrAb assay. In TA100 cells, ALX was tested at various concentrations by diluting in water and incubating with TA100. Plates were seeded with 8 × 10⁷ CFU. Cytotoxicity was determined by identifying a statistically significant reduction in the number of revertant colonies in treated TA100 compared with untreated. Ames and ChrAb tests were performed as described above, with ALX at concentrations below cytotoxic limits.

Results

Initial screening assays utilizing MiniScreen Ames demonstrates Blink RevitaLens MPDS is nonmutagenic

A screening assay was conducted on a smaller scale than traditional Ames with two bacterial strains, TA98 and TA100.²² MiniScreen Ames assay allows for reliable screening of new solutions in a development stage because it is a modified test procedure conducted with limited bacterial strains and test material, while still producing highly consistent results with traditional Ames. TA98 is a frameshift mutation detection strain, while TA100 is a base-pair substitution mutation detection strain. Thus, the use of these two strains, instead of five, still allows reasonable coverage of potential mutations. This screening step allows for early identification of problematic solutions or ingredients because it can be conducted rapidly with a smaller amount of test material that may be available during early development. Acuvue 2 brand contact lenses were extracted into deionized water, saline, or Blink RevitaLens MPDS. S9-activated and naive TA98 and TA100 bacterial strains were exposed to the undiluted extracts and Blink RevitaLens MPDS, then compared with phosphate-buffered saline (PBS) treated bacteria as a control (Table 1). Based on these results, Blink RevitaLens MPDS and contact lens extracts are considered nonmutagenic by screening technology.

Table 1.

MiniScreen Ames Suggests Blink RevitaLens MPDS is Nonmutagenic

	TA98		TA100
	Naive	S9-activated	Naive	S9-activated
Positive control	149	461.0	325.0	402.0
Positive control	42.57 ^*	120.26 ^*	8.94 ^*	10.09 ^*
Negative control	3.5	3.8	36.3	39.8
Negative control	—	—	—	—
Blink RevitaLens^® MPDS	5.0	8.5	35.5	24.0
Blink RevitaLens^® MPDS	1.43 ^*	2.22 ^*	0.98 ^*	0.60 ^*
RL + Acuvue^® 2 Extract	2.0	2.0	35.5	30.0
RL + Acuvue^® 2 Extract	0.57 ^*	0.52 ^*	0.98 ^*	0.75 ^*

This screening assay was conducted with two bacterial strains and on a smaller scale than traditional Ames to allow for fast, reliable results. Blink RevitaLens MPDS and its undiluted contact lens extract demonstrated nonmutagenicity compared with the vehicle control (PBS). Positive controls were 2-aminoanthracene (activated) and MNNG/2-nitrofluorene (naive). The average number of revertant colonies from two wells is shown in bold; numbers designated with an asterisk are the factors of increase above negative control. Statistical analysis was not performed on this screening assay.

MPDS, Multipurpose Disinfecting Solution.

Ames assays showed that Blink RevitaLens MPDS is nonmutagenic alone or in combination with contact lenses

To rule out the mutagenic potential of Blink RevitaLens MPDS and contact lens extracts, Ames assays were conducted on four strains of S. typhimurium and one strain E. coli with the presence and absence of metabolic activation. To simulate use, Acuvue Advance and Acuvue 2 contact lenses were cycled through one change of Blink RevitaLens MPDS per day for 30 days before extraction in saline and compared with Blink RevitaLens MPDS in this assay. The extracts, Blink RevitaLens MPDS, and controls were evaluated for their ability to induce histidine (S. typhimurium) or tryptophan (E. coli) independent growth. The process of cycling contact lenses through MPDS before extraction was a novel procedure that is not defined in current standards or guidelines for testing of MPS solutions and was included to better represent actual usage. Thus instead of simply testing Blink RevitaLens MPDS, the solution itself was tested as well as extracts from contact lenses that were soaked in the solution cycling protocol. Results demonstrate that contact lens extracts and Blink RevitaLens MPDS did not induce a statistically significant increase in revertant colonies when compared with negative controls for both naive and metabolically activated conditions (Table 2). In both activated and naive metabolic conditions, all positive controls exhibited a statistically significant increase in revertant colonies compared to saline. These results suggest Blink RevitaLens MPDS is nonmutagenic either alone or in combination with silicone hydrogel contact lenses as determined by Ames.

Table 2.

Summary of Ames Assay

	TA98		TA100		TA1535		TA1537		Wp2uvrA-
	Naive	Activated	Naive	Activated	Naive	Activated	Naive	Activated	Naive	Activated
Positive control	291.7	282.3	915.3	773.7	226.7	219.0	215.7	204.0	365.3	421.3
Positive control	21.91	38.13	6.18	33.88	15.43	7.48	7.32	7.87	38.42	6.94
Negative control	17.3	17.7	72.0	76.3	8.0	9.0	3.3	7.3	42.0	42.7
Negative control	0.47	0.47	2.16	3.86	0.82	2.16	1.25	1.25	4.55	5.44
Blink RevitaLens MPDS	17.0	16.7	70.3	71.3	7.0	7.7	2.3	5.0	40.0	45.7
Blink RevitaLens MPDS	0.82	0.47	0.47	1.25	0.82	1.25	0.47	0.82	4.55	6.18
RL + Acuvue^® Adv extract	16.3	16.3	70.7	71.3	7.3	7.0	3.3	4.3	45.7	47.3
RL + Acuvue^® Adv extract	1.70	0.47	0.94	1.25	0.47	0.82	1.89	0.94	4.78	4.64
RL + Acuvue 2 extract	16.3	17.3	73.3	73.0	7.3	7.3	4.3	4.7	49.0	37.7
RL + Acuvue 2 extract	0.47	0.47	2.49	0.82	1.25	0.47	1.25	1.70	6.16	1.70

Four strains of salmonella and 1 strain of Escherichia coli were tested at both naive and metabolically activated states with Blink RevitaLens MPDS, and Acuvue Advance and Acuvue 2 contact lens extracts. The average of 3 values is shown in bold; standard deviations are in italics below. For each test article and bacteria strain, the number of revertant colonies by Blink RevitaLens MPDS and Acuvue Advance and Acuvue 2 contact lens extracts were not statistically different from the negative control. All were considered dramatically lower than the positive controls (p < 0.0001 by 1-way ANOVA with Dunnett's multiple comparisons test). RL–Blink RevitaLens MPDS

Chromosomal aberration assays showed that Blink RevitaLens MPDS alone and in combination with contact lenses is nonclastogenic

To confirm that Blink RevitaLens MPDS and/or its contact lens extracts do not induce genotoxic effects such as chromosomal changes, CHO cells were used in a Chromosomal Aberrations Assay according to ISO 10993-3 Biological Evaluation of Medical Devices–Part 3: Tests for Genotoxicity, Carcinogenicity, and Reproductive Toxicity, as well as OECD 473 Guidelines for Testing of Chemicals, “In Vitro Mammalian Chromosomal Aberration Test.”^23,24 Acuvue Advance and Acuvue 2 contact lenses were cycled through one change of Blink RevitaLens MPDS per day for 30 days before extraction in Ham's F-12 Medium and compared to Blink RevitaLens in this assay. Again, this novel procedure of cycling lenses through MPDS was included to better simulate actual usage. Results for both S9-activated and naive CHO cells indicate that contact lens soaked in Blink RevitaLens MPDS and then extracted did not cause a statistically significant increase in the number of ChrAb (Table 3). Blink RevitaLens MPDS diluted by 50% also did not demonstrate an increase in ChrAb. In fact, test articles exhibit very similar results to negative controls. These data suggest that Blink RevitaLens MPDS alone or in combination with contact lenses is nonclastogenic, using the ChrAb method.

Table 3.

Chromosome Aberrations Assay in CHO Demonstrates Blink RevitaLens MPDS is Nonclastogenic

	Naive		S9-activated
	Total # aberrations	% Cells with aberrations	Total # aberrations	% Cells with aberrations
Positive Control	46	18	41	16
Negative Control	4	2	3	1.5
Blink RevitaLens MPDS	3	1.5	2	1
RL + Acuvue Adv. Extract	2	1	2	1
RL + Acuvue 2 Extract	3	1.5	2	1

Neither Blink RevitaLens MPDS nor the contact lens extracts induced a statistically significant (chi-square Test) increase in the number of structural or numerical chromosomal aberrations compared to Ham's F-12 negative control. All test articles were considered nonclastogenic. RL- Blink RevitaLens MPDS

CHO, Chinese Hamster Ovary.

Alexidine Cytotoxicity Assays determine relevant concentrations for genotoxicity assays

Blink RevitaLens MPDS comprised two antimicrobials, including PQ-1 and ALX. Although both these antimicrobials are approved for human use and can be found in other commercially available products, to satisfy safety regulations globally, further investigation of ALX was warranted. Because antimicrobials are by nature cytotoxic, use of ALX at high concentrations in Ames or ChrAb assays would be limited. Instead, ALX was diluted to first identify a dose curve of cytotoxicity in both CHO cells and representative bacterial strains for ChrAb and Ames assays, respectively. Both S9 metabolically activated and naive CHO cells were exposed to 20, 6.3, 2.0, 0.63, 0.20, 0.06, 0.02, 0.006, 0.002, and 0.0006 μg/mL of ALX diluted in culture media to assess cytotoxicity. Cytotoxicity was evaluated on the basis of relative population doubling compared with the negative control, performing a dose curve allowed for confidence in the results. ALX concentrations at and above 2 μg/mL demonstrated cytotoxicity in naive CHO cells and at 20 μg/mL in activated CHO cells. Based on these results, the three highest concentrations of ALX without significant toxicity were used for each assay. To find an acceptable dose range for Ames assays, various concentrations of ALX were applied to TA100 cells, that would serve as a representative of all 5 bacterial strains used in Ames. S9 metabolically activated and naive TA100 cells were exposed to 200, 100, 50, 25, 12.5, and 6.25 μg/mL of ALX. ALX concentrations above 12 μg/mL demonstrated cytotoxicity in naive TA100 cells and above 100 μg/mL in activated CHO cells.

Ames assay demonstrates that Alexidine is nonmutagenic

To exclude the potential for mutagenesis by ALX, six dilutions of ALX were assayed on five different bacterial strains in an Ames assay. The concentrations of ALX used were determined by dose-range finding cytotoxicity assays described above. Nonactivated bacteria were treated with 12.5, 3.95, 1.25, 0.395, 0.125, and 0.0395 μg/mL ALX, while metabolically activated bacteria were treated with 200, 63.3, 20, 6.33, 2.0, and 0.633 μg/mL ALX. Results of Ames assays with ALX demonstrate that all dilutions of ALX studied did not induce a statistically significant increase in revertant colonies when compared with negative controls for both naive and metabolically activated conditions (Table 4). Performing the assay with a dose range, in triplicate, with five different strains of bacteria, provides confidence for the results. Because the concentration of ALX in Blink RevitaLens MPDS is 1.6 ppm, or 1.6 μg/mL, Ames assays were conducted with levels of ALX at or significantly above what is found in the Blink RevitaLens MPDS formulation. These results suggest that ALX is nonmutagenic, even at concentrations higher than that used in Blink RevitaLens MPDS.

Table 4.

Summary of Ames Assay with Alexidine

	TA98	TA100	TA1535	TA1537	Wp2uvrA-
(A) Naive bacterial strains
Positive control	59.0	1118.7	127.3	289.3	104.0
Positive control	1.0	155.7	3.8	16.1	10.4
Negative control	11.3	48.0	3.7	1.7	40.0
Negative control	5.7	8.5	1.5	0.6	2.6
ALX 12.5	4.7	34.7	1.3	1.3	45.7
ALX 12.5	2.3	9.6	0.6	0.6	5.0
ALX 3.95	11.7	57.7	3.3	1.7	50.3
ALX 3.95	0.6	8.6	2.5	0.6	10.4
ALX 1.25	6.7	61.3	4.3	2	42.7
ALX 1.25	2.3	11.1	0.6	1.0	8.6
ALX 0.395	10	73.7	4.7	1.3	46
ALX 0.395	1.0	11.5	1.2	0.6	6.1
ALX 0.125	11.3	71	3.7	1.7	42.7
ALX 0.125	5.0	11.1	2.1	1.2	3.1
ALX 0.0395	10.7	58	3.7	1.7	45.7
ALX 0.0395	1.5	7.5	1.5	1.2	10.1
(B) Metabolically Activated bacterial strains
Positive control	65.7	212.0	46.3	38.7	245.3
Positive control	3.1	22.0	6.0	10.0	36.5
Negative control	11	72.3	4.7	3.7	47.7
Negative control	5.0	5.0	1.2	2.5	17.2
ALX 200	13	29.3	1.7	5.3	51
ALX 200	5.6	5.1	0.6	2.5	6.1
ALX 63.3	16.3	70	2.7	3.7	55.7
ALX 63.3	4.0	13.7	0.6	1.5	8.1
ALX 20	14.7	94.3	5.3	4	45
ALX 20	3.1	14.0	2.9	2.0	3.5
ALX 6.33	14.3	103.3	4	4.3	54.3
ALX 6.33	2.9	18.4	1.0	2.1	9.5
ALX 2.0	12.7	95.7	6	4.3	52.7
ALX 2.0	1.5	30.6	1.7	1.5	6.4
ALX 0.633	18	93.3	3.7	3.7	61.3
ALX 0.633	8.9	10.8	2.3	2.3	15.0

Four strains of salmonella and 1 strain of E. coli were tested at both naive (A) and metabolically activated (B) states with a serial dilution of Alexidine (ALX). Bold numbers are averages of three values; standard deviations are shown in italics below. For each test article and bacteria strain, the number of revertant colonies induced by ALX (any dilution) was not statistically different from the negative control. All were considered dramatically lower than the positive controls (p < 0.0001 by 1-way ANOVA with Dunnett's multiple comparison test).

Alexidine Dihydrochloride

ChrAb assay determined that Alexidine is nonclastogenic

Based on cytotoxicity range-finding assays, naive CHO cells were exposed to 0.63, 0.20, and 0.06 μg/mL of ALX and metabolically activated cells were exposed to 6.3, 2.0, and 0.63 μg/mL of ALX for ChrAb analysis. Results from both the activated and naive CHO cells show a similar levels of ChrAb between ALX-treated cells and the negative control water (Table 5). The positive controls mitomycin-C and cyclophosphamide demonstrated statistically significant increases in ChrAb, validating the assay results. On average, in positive controls, 43% of nonactivated CHO cells had ChrAb; while negative controls and ALX-treated (all doses) had 2.5% and 2.5%–4.0% aberrations, respectively. For activated cells, 56% of positive control-treated CHO cells had aberrations, while 3.0% of control cells and between 1.5% and 2.0% of ALX-treated cells did. These data suggest that ALX is nonclastogenic even at doses higher than what is found in Blink RevitaLens MPDS.

Table 5.

Chromosome Aberration Assays with ALX Dilutions: Percentage of Cells with Chromosome Aberrations

	Naïve		S9-activated
Positive control	43 ± 4.2		56 ± 8.5
Negative control	2.5 ± 0.7		3.0 ± 1.4
ALX (dilution \| mean ± SD)	0.06 μg/mL	2.5 ± 0.7	0.63 μg/mL	1.5 ± 0.7
ALX (dilution \| mean ± SD)	0.2 μg/mL	4.0 ± 0.0	2.0 μg/mL	2.0 ± 0.0
ALX (dilution \| mean ± SD)	0.63 μg/mL	3.0 ± 1.4	6.3 μg/mL	2.0 ± 1.4

ALX was tested at three dilutions that did not induce cytotoxicity in CHO cells. Based on the results, ALX is nonclastogenic at all dilutions tested. The aberration percentage for each test article and negative control was compared with a chi-square statistical test. In each case, the chi-square p-value was greater than 0.05, thus indicating nonsignificance. (n = 3; mean results reported ± standard deviation).

Discussion/Conclusion

These studies were designed to exclude the genotoxicity potential of Blink RevitaLens MPDS and one of its active disinfecting ingredients, alexidine dihydrochloride. Regulatory agencies would typically require testing to demonstrate Blink RevitaLens MPDS is nontoxic, nonirritating, and nonsensitizing based on ISO 10993-1 Biological evaluation of medical devices–Evaluation and testing within a risk management process.²⁵ Because Blink RevitaLens MPDS is designed for disinfection, rinsing, and storing of soft contact lenses,¹¹ some regulatory agencies worldwide where MPDS is classified as a prescription or quasi-drug may prefer further genotoxicity testing in addition to testing described in ISO 10993-1. While genotoxicity is not a requirement for MPDS, due to the nature of disinfectants, it is an important step to consider during the development process to ensure product safety.

After rigorous biocompatibility testing according to ISO 10993 standards, genotoxicity of Blink RevitaLens MPDS and its disinfectant ALX was also evaluated. While ALX had been used in other medical devices with direct human contact (oral hygiene products), very little toxicity information is available for this disinfectant. Blink RevitaLens MPDS was initially evaluated with a MiniScreen Ames assay to provide rapid results before lengthy and comprehensive genotoxicity testing in mammalian and bacterial cells. Due to the nature of disinfectants, ALX could not be tested undiluted in any mammalian or bacterial test system. To circumvent this challenge, range-finding assays were first used to determine dosages of ALX that would not result in toxicity of CHO cells or bacterial cells used in ChrAb or Ames assays. The dosages that were tested varied between the test system and activation status, ranging from as low as 0.0395–200 μg/mL. Blink RevitaLens MPDS contains 1.6 μg/mL of ALX in solution. Following soaking of contact lens in Blink RevitaLens MPDS, the amount of ALX that potentially releases into the eye of the contact lens wearer would be considerably lower. Taken together, these data demonstrate the safe use of Blink RevitaLens MPDS, as demonstrated through cytotoxicity testing and genotoxicity testing.

Footnotes

Author Disclosure Statement

This study is sponsored by Abbott Medical Optics Inc., part of the Johnson & Johnson Vision family of companies. The authors, Ling C. Huang and Ronika S. Leang, are employees of Abbott Medical Optics, Inc.

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