Patent Publication Number: US-5290512-A

Title: Method of disinfecting a contact lens and preserving a contact lens care solution

Description:
The present invention relates to an improved method for imparting antimicrobial activity to contact lens solutions. More particularly, the invention relates to the use of a mixture of 5-chloro-2-methyl-4-isothiazoline-3-one and 2-methyl-4-isothiazoline-3-one for the disinfection of contact lenses and the preservation of contact lens care solutions. 
     Soft contact lenses are characteristically prepared from hydrophilic polymers, such as polymers of hydroxyethyl methacrylate, crosslinked with a conventional crosslinking agent, such as ethylene glycol dimethacrylate (EDGMA), or more complex copolymer systems including copolymers of HEMA, EDGMA, methacrylic acid and/or poly-N-vinylpyrolidone, and the like. In general, such lenses exhibit marked hydrophilic properties and, when wet, absorb water and are soft and flexible. 
     An antimicrobial agent used in relation to a contact lens must possess a number of unique characteristics. On one hand, it must be effective against microoorganisms which may be dangerous to the eye. At the same time, it must be tolerated in the delicate ocular environment of the user, and also not damage the contact lens itself. A number of contact lens disinfecting and preserving solutions are known in the art. Typically such solutions employ either sorbic acid, thimerosal, chlorhexidine, a polyquaternary germicide, a synthetic antibiotic or a conventional quaternary germicide, such as benzalkonium chloride. However, these conventional antimicrobial agents have drawbacks that tend to restrict their use. For example, sorbic acid characteristically contains formaldehyde residues, thimerosal in some patients acts as an allergy sensitizer, and chlorhexidine is relatively toxic. Also, a problem exists in that soft contact lens materials have a tendency to bind and concentrate antimicrobial agents and other active ingredients commonly found in contact lens care solutions, in some cases to hazardous levels. For example, benzalkonium chloride is typically not used with soft contact lenses due to its tendency to be taken up into the lens matrix. In addition, many of the antimicrobial agents known to date are relatively ineffective against a number of fungi and yeasts which are problematic in the ocular environment. 
     Therefore, there exists a need for a method of disinfecting and/or preserving a contact lens or a contact lens care solution which is effective against fungi and yeasts, which is tolerable by the user, and which does not bind with or otherwise damage the contact lens. 
     SUMMARY OF THE INVENTION 
     Surprisingly and unexpectedly, it has been found that a mixture of 5-chloro-2-methyl-4-isothiazoline-3-one and 2-methyl-4-isothiazoline-3-one may be used in a method for disinfecting and/or preserving a contact lens or in a contact lens care solution with minimal or none of the above described problems. The mixture has been found to be effective against fungi and yeasts, while also exhibiting low in vitro toxicity and minimal lens uptake. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention relates to an improved method for disinfecting and/or preserving contact lenses and contact lens care solutions involving contacting the lens with a composition, typicially a solution, having an effective amount of certain substituted 3-isothiazolones comprising 5-chloro-2-methyl-4-isothiazoline-3-one (CAS Registry No. 26172-55-4) and 2-methyl-4-isothiazoline-3-one (CAS Registry No. 2682-20-4). Their structures are as follows: ##STR1## 
     3-isothiazolones are generally described in U.S. Pat. No. 3,761,488 to Lewis et al., which is incorporated herein by reference. The preferred agent used in the present method is composed of a mixture of about 1.15% of 5-chloro-2-methyl-4-isothiazolin-3-one and about 0.35% of 2-methyl-4-isothiazolin-3-one. This agent is available in a product sold under the tradename &#34;Kathon CG,&#34; which is available from Rohm and Haas Company, Philadelphia, Pa. The composition of Kathon CG also includes 23.0% Magnesium salts and 75.5% water. 
     The present invention may, in some cases, be used in conjunction with one or more microbicides to provide a wide range of antimicrobial efficacy. Moreover, surfactants and wetting agents may be provided into the solutions to offer cleaning and wetting features. 
     The present invention may be employed for preserving a contact lens care solution (such as, for example, a saline solution, a wetting solution, or any other solution coming into contact with the lens) by providing a concentration of approximately 1-300 ppm Kathon in the solution. The amount of the agent for preserving purposes may vary, although it has been found that about 3 ppm is preferred. 
     The preferred procedure for disinfecting a lens using any one of these solutions is to place the solution having a disinfecting concentration of the mixture into a cup and placing the lens into the solution for four hours. The amount of the agent for disinfecting purposes may vary, although it has been found that about 30 ppm is preferred. The following examples are illustrative of disinfecting solutions which may be used in accordance with the present invention: 
     EXAMPLE I 
     11.3 ppm Kathon CG solution 
     7.8 g sodium chloride 
     4.76 g disodium hydrogen phosphate 
     0.714 g sodium phosphate monobasic 
     0.0086 g 5-chloro-2-methyl-4-isothiazolin-3-one 
     0.0026 g 2-methyl-4-isothiazolin-3-one 
     distilled H 2  O q.s. 1 liter 
     EXAMPLE II 
     50 ppm Kathon CG solution 
     7.8 g sodium chloride 
     4.76 g disodium hydrogen phosphate 
     0.714 g sodium phosphate monobasic 
     0.0384 g 5-chloro-2-methyl-4-isothiazolin-3-one 
     0.0117 g 2-methyl-4-isothiazolin-3-one 
     distilled H 2  O q.s. 1 liter 
     EXAMPLE III 
     30 ppm Kathon CG solution 
     6.8 g sodium chloride 
     4.76 g disodium hydrogen phosphate 
     0.72 g sodium phosphate monobasic 
     1.0 g ethylenediamine tetraacid disodium salt 
     0.0230 g 5-chloro-2-methyl-4-isothiazolin-3-one 
     0.0070 g 2-methyl-4-isothiazolin-3-one 
     distilled H 2  O q.s 1 liter 
     EXAMPLE IV 
     300 ppm Kathon CG solution 
     6.8 g sodium chloride 
     4.76 g disodium hydrogen phosphate 
     0.72 g sodium phosphate monobasic 
     1.0 g ethylenediamine tetraacid disodium salt 
     5.0 hydroxyethylcellulose 
     0.230 g 5-chloro-2-methyl-4-isothiazolin-3-one 
     0.070 g 2-methyl-4-isothiazolin-3-one 
     distilled H 2  O q.s. 1 liter 
     EXAMPLE V 
     7.6 ppm Kathon CG solution 
     7.3 g sodium chloride 
     0.91 g disodium hydrogen phosphate 
     0.12 g potassium phosphate 
     1.6 g potassium chloride 
     0.0058 g 5-chloro-2-methyl-4-isothiazolin-3-one 
     0.0018 g 2-methyl-4-isothiazolin-3-one 
     distilled H 2  O q.s. 1 liter 
     EXAMPLE VI 
     15 ppm Kathon CG solution 
     7.3 g sodium chloride 
     0.91 g disodium hydrogen phosphate 
     0.12 g potassium phosphate 
     1.6 g potassium chloride 
     0.012 g 5-chloro-2-methyl-4-isothiazolin-3-one 
     0.0035 g 2-methyl-4-isothiazolin-3-one 
     distilled H 2  O q.s. 1 liter 
     UPTAKE STUDIES 
     A study of disinfectant/preservative uptake of solutions according to Examples I and II, as well as other known disinfectant/preservative solutions, was performed. A tetrafilcon A (43% H 2  O) contact lens and 5 ml of solution according to Example I was placed in a glass vial. The vial was then incubated at room temperature for 4 hours, after which the concentration of disinfectant/preservative in the solution was determined spectrophotometrically. The percentage of uptake was calculated according to the following: ##EQU1## wherein A c  =the absorption of the disinfectant/preservative solution without lens at 276 nm; and 
     A t  =the absorption of the disinfectant/preservative solution with the lens ay 276 nm. 
     The tetrafilcon A lens had less than 2.0% uptake of the Kathon CG (5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one). 
     The above procedure was repeated using a fresh Tetrafilcon A lens incubated for 7 days, as well as a number of different type lenses incubated for 4 hour and 7 day periods. Table I below details the results of the study, which clearly show that the method of the present invention results in significantly less lens uptake than benzalkonium chloride and chlorohexidine. 
     
                       TABLE I                                                     
______________________________________                                    
         Group I   Group II  Group III                                    
                                     Group IV                             
         (%)       (%)       (%)     (%)                                  
Solution 4 hr/7 day                                                       
                   4 hr/7 day                                             
                             4 hr/7 day                                   
                                     4 hr/7 day                           
______________________________________                                    
Example 1                                                                 
         &lt;1.0/&lt;1.0 &lt;1.0/&lt;1.0 &lt;2.0/&lt;3.0                                    
                                     &lt;1.0/&lt;2.0                            
Example 2                                                                 
         &lt;1.5/&lt;3.0 &lt;1.5/&lt;1.5 &lt;1.5/&lt;1.5                                    
                                     &lt;1.5/&lt;1.5                            
50 ppm BAK                                                                
         27/80     25/67     25/75   25/58                                
25 ppm chlo-                                                              
         35/82     27/45     34/82   30/73                                
rohexidine                                                                
50 ppm chlo-                                                              
         35/80     26/44     32/82   35/74                                
rohexidine                                                                
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     ANTIMICROBIAL EFFICACY STUDY 
     The results of a study described below shows that the present invention can provide disinfection level microbiological performance in a soft contact lens solution, particularly against key fungal microorganisms such as C. albicans and Aspergillus ssp. Experiments were conducted using a time course survival/reduction design involving various soaking periods with challenge organisms followed by standard plate count evaluation to characterize antimicrobial efficacy. In the experiments, disinfection panel bacteria and various fungi were used as challenge organisms (See Table II). Microbial survival was tested with exposure times of 4 hours, 24 hours and 7 days (fungi only). 
     
                       TABLE II                                                    
______________________________________                                    
Challenge Microorganisms                                                  
and Experimental Conditions                                               
                           Plating/                                       
Test Microbe    ATCC #     Incubation                                     
______________________________________                                    
Pseudomonas aeruginosa                                                    
                15442      TSA    35° C.                           
Serratia marcescens                                                       
                14041      TSA    35° C.                           
Staphylococcus epidermidis                                                
                17917      TSA    35° C.                           
Candida albicans                                                          
                10231      SDA    25° C.                           
Aspergillus fumigatus                                                     
                10894      SDA    25° C.                           
Aspergillus niger                                                         
                16404      SDA    25° C.                           
Fusarium oxysporum                                                        
                26225      SDA    25° C.                           
Cladosporium cladsporioides                                               
                26688      SDA    25° C.                           
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     Test solutions consisted of various concentrations of the antimicrobial active agent in a phosphate buffered saline--5, 7.5, 15, 30 and 50 ppm Kathon CG. 
     Phosphate buffered saline (PBS) was used as the control for determination of initial inoculums. Prior to testing, all solutions were filtered through a 0.2 um cellulose nitrate membrane to ensure sterility. Tables III through X show the results of the study. 
     
                       TABLE III                                                   
______________________________________                                    
                              Exposure Time-                              
Test Solution       Initial   CFU/mL, [Log Drop]                          
[Kathon CG]                                                               
         Test Microbe                                                     
                    Inoculum  4 hour 24 hour                              
______________________________________                                    
5    ppm     P. aeruginosa                                                
                        1.2 × 10.sup.4                              
                                6.1 × 10.sup.4                      
                                       &lt;10                                
                                [1.3]  [TK]                               
7.5  ppm                        4.0 × 10.sup.4                      
                                       &lt;10                                
                                [1.5]  [TK]                               
15   ppm                        5.0 × 10.sup.4                      
                                       &lt;10                                
                                [1.4]  [TK]                               
30   ppm                        8.2 × 10.sup.4                      
                                       &lt;10                                
                                [1.2]  [TK]                               
50   ppm                        9.0 × 10.sup.5                      
                                       &lt;10                                
                                [1.1]  [TK]                               
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                       TABLE IV                                                    
______________________________________                                    
                              Exposure Time-                              
Test Solution       Initial   CFU/mL, [Log Drop]                          
[Kathon CG]                                                               
         Test Microbe                                                     
                    Inoculum  4 hour 24 hour                              
______________________________________                                    
5    ppm     S. epidermidis                                               
                        5.0 × 10.sup.5                              
                                4.1 × 10.sup.5                      
                                       7.4 × 10.sup.3               
                                [NK]   [1.8]                              
7.5  ppm                        3.7 × 10.sup.5                      
                                       9.4 × 10.sup.3               
                                [NK]   [1.7]                              
15   ppm                        3.8 × 10.sup.5                      
                                       1.0 × 10.sup.4               
                                [NK]   [1.7]                              
30   ppm                        ≧10.sup.5                          
                                       1.8 × 10.sup.4               
                                [NK]   [1.4]                              
50   ppm                        ≧10.sup.5                          
                                       1.7 × 10.sup.4               
                                [NK]   [1.5]                              
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                       TABLE V                                                     
______________________________________                                    
                              Exposure Time-                              
Test Solution       Initial   CFU/mL, [Log Drop]                          
[Kathon CG]                                                               
         Test Microbe                                                     
                    Inoculum  4 hour 24 hour                              
______________________________________                                    
5    ppm     S. marcescens                                                
                        1.2 × 10.sup.4                              
                                5.9 × 10.sup.5                      
                                       &lt;10                                
                                [NK]   [TK]                               
7.5  ppm                        5.8 × 10.sup.5                      
                                       1.0 × 10.sup.1               
                                [NK]   [5.1]                              
15   ppm                        5.8 × 10.sup.5                      
                                       1.5 × 10.sup.1               
                                [NK]   [4.9]                              
30   ppm                        ≧10.sup.5                          
                                       2.3 × 10.sup.1               
                                [NK]   [4.7]                              
50   ppm                        ≧10.sup.5                          
                                       8.7 × 10.sup.2               
(541-25-5)            [NK]     [3.1]                                      
______________________________________                                    
 
    
     
                                           TABLE VI                                
__________________________________________________________________________
                   Exposure Time-                                         
Test Solution Initial                                                     
                   CFU/mL, [Log Drop]                                     
[Kathon CG]                                                               
       Test Microbe                                                       
              Inoculum                                                    
                   4 hour                                                 
                         24 hour                                          
                               7 days                                     
__________________________________________________________________________
5  ppm C. albicans                                                        
              2.2 × 10.sup.6                                        
                   &lt;1 × 10.sup.1                                    
                         &lt;1 × 10.sup.1                              
                               &lt;1 × 10.sup.1                        
                   [TK]  [TK]  [TK]                                       
7.5                                                                       
   ppm             &lt;1 × 10.sup.1                                    
                         &lt;1 × 10.sup.1                              
                               &lt;1 × 10.sup.1                        
                   [TK]  [TK]  [TK]                                       
15 ppm             &lt;1 × 10.sup.1                                    
                         &lt;1 × 10.sup.1                              
                               &lt;1 × 10.sup.1                        
                   [TK]  [TK]  [TK]                                       
30 ppm             &lt;1 × 10.sup.1                                    
                         &lt;1 × 10.sup.1                              
                               &lt;1 × 10.sup.1                        
                   [TK]  [TK]  [TK]                                       
50 ppm             &lt; 1 × 10.sup.1                                   
                         &lt;1 × 10.sup.1                              
                               &lt;1 × 10.sup.1                        
                   [TK]  [TK]  [NK]                                       
__________________________________________________________________________
 
    
     
                                           TABLE VII                               
__________________________________________________________________________
                   Exposure Time-                                         
Test Solution Initial                                                     
                   CFU/mL, [Log Drop]                                     
[Kathon CG]                                                               
       Test Microbe                                                       
              Inoculum                                                    
                   4 hour                                                 
                         24 hour                                          
                               7 days                                     
__________________________________________________________________________
5  ppm A. fumigatus                                                       
              2.9 × 10.sup.4                                        
                   2.0 × 10.sup.4                                   
                         6.9 × 10.sup.3                             
                               6.9 × 10.sup.3                       
                   [2.2] [2.6] [2.6]                                      
7.5                                                                       
   ppm             9.5 × 10.sup.3                                   
                         2.3 × 10.sup.3                             
                               2.7 × 10.sup.3                       
                   [2.5] [3.1] [3.0]                                      
15 ppm             4.0 × 10.sup.3                                   
                         7.1 × 10.sup.2                             
                               &lt;1 × 10.sup.1                        
                   [2.9] [3.6] [TK]                                       
30 ppm             5.7 × 10.sup.3                                   
                         1.0 × 10.sup.1                             
                               &lt;1 × 10.sup.1                        
                   [2.7] [5.5] [TK]                                       
50 ppm             2.1 × 10.sup.4                                   
                         1.4 × 10.sup.6                             
                               4.3 × 10.sup.3                       
                   [NK]  [NK]  [0.5]                                      
__________________________________________________________________________
 
    
     
                                           TABLE VIII                              
__________________________________________________________________________
                   Exposure Time-                                         
Test Solution Initial                                                     
                   CFU/mL, [Log Drop]                                     
[Kathon CG]                                                               
       Test Microbe                                                       
              Inoculum                                                    
                   4 hour                                                 
                         24 hour                                          
                               7 days                                     
__________________________________________________________________________
5  ppm A. niger                                                           
              1.4 × 10.sup.6                                        
                   3.3 × 10.sup.4                                   
                         4.8 × 10.sup.3                             
                               4.1 × 10.sup.3                       
                   [1.6] [2.5] [2.5]                                      
7.5                                                                       
   ppm             1.4 × 10.sup.4                                   
                         4.0 × 10.sup.3                             
                               7.8 × 10.sup.1                       
                   [2]   [2.5] [4.2]                                      
15 ppm             6.5 × 10.sup.3                                   
                         8.2 × 10.sup.2                             
                               &lt;1 × 10.sup.1                        
                   [2.3] [3.2] [TK]                                       
30 ppm             1.0 × 10.sup.4                                   
                         &lt;1 × 10.sup.1                              
                               &lt;1 × 10.sup.1                        
                   [2.1] [TK]  [TK]                                       
50 ppm             1.3 × 10.sup.6                                   
                         8.2 × 10.sup.6                             
                               6.8 × 10.sup.5                       
                   [NK]  [NK]  [NK]                                       
__________________________________________________________________________
 
    
     
                                           TABLE IX                                
__________________________________________________________________________
                     Exposure Time-                                       
Test Solution   Initial                                                   
                     CFU/mL, [Log Drop]                                   
[Kathon CG]                                                               
       Test Microbe                                                       
                Inoculum                                                  
                     4 hour                                               
                          24 hour                                         
                               7 days                                     
__________________________________________________________________________
5  ppm C. cladsporioides                                                  
                1.0 × 10.sup.6                                      
                     1.9 × 10.sup.5                                 
                          4.7 × 10.sup.2                            
                               &lt;1 × 10.sup.1                        
                     [0.7]                                                
                          [2.3]                                           
                               [TK]                                       
7.5                                                                       
   ppm               1.2 × 10.sup.5                                 
                          5.3 × 10.sup.2                            
                               &lt;1 × 10.sup.1                        
                     [0.9]                                                
                          [2.3]                                           
                               [TK]                                       
15 ppm               1.4 × 10.sup.5                                 
                          6.0 × 10.sup.2                            
                               &lt;1 × 10.sup.1                        
                     [0.9]                                                
                          [3.2]                                           
                               [TK]                                       
30 ppm               1.1 × 10.sup.4                                 
                          3.8 × 10.sup.2                            
                               &lt;1 × 10.sup.1                        
                     [2]  [3.4]                                           
                               [TK]                                       
50 ppm               7.0 × 10.sup.3                                 
                          2.5 × 10.sup. 2                           
                               &lt;1 × 10.sup.1                        
                     [2.2]                                                
                          [3.6]                                           
                               [TK]                                       
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                                           TABLE X                                 
__________________________________________________________________________
                     Exposure Time-                                       
Test Solution   Initial                                                   
                     CFU/mL, [Log Drop]                                   
[Kathon CG]                                                               
       Test Microbe                                                       
                Inoculum                                                  
                     4 hour                                               
                          24 hour                                         
                               7 days                                     
__________________________________________________________________________
5  ppm F. oxysporum                                                       
                7.1 × 10.sup.5                                      
                     2.5 × 10.sup.1                                 
                          &lt;1 × 10.sup.1                             
                               &lt;1 × 10.sup.1                        
                     [5.5]                                                
                          [TK] [TK]                                       
7.5                                                                       
   ppm               1.8 × 10.sup.2                                 
                          &lt;1 × 10.sup.1                             
                               &lt;1 × 10.sup.1                        
                     [4.6]                                                
                          [TK] [TK]                                       
15 ppm               9.8 × 10.sup.1                                 
                          &lt;1 × 10.sup.1                             
                               &lt;1 × 10.sup.1                        
                     [4.9]                                                
                          [TK] [TK]                                       
30 ppm               7.5 × 10.sup.0                                 
                          &lt;1 × 10.sup.1                             
                               &lt;1 × 10.sup.1                        
                     [TK] [TK] [TK]                                       
50 ppm               &lt;1 × 10.sup. 1                                 
                          &lt;1 × 10.sup.1                             
                               &lt;1 × 10.sup.1                        
                     [TK] [TK] [NK]                                       
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     CYTOTOXICITY STUDY 
     Cell biology experiments were conducted using L929 agar overlay diffusion and SIRC direct contact morphological toxicity assays in order to characterize in vitro cytotoxicity for Kathon CG at 5 to 50 ppm in PBS. L929 agar diffusion experiments showed no significant cytotoxicity for solutions up to 15 ppm; the 30 and 50 ppm solutions caused only mild cytotoxic responses. Evaluation of lenses following 72 hour exposure to the Kathon CG solutions showed no evidence of lens preservative uptake/cytotoxicity. Similarly, SIRC morphology studies demonstrate only mild to moderate cellular effects for Kathon CG under direct contact conditions with limited concentration dependence (e.g. For 30 minutes exposure, Reactivity=2 for 5 ppm and Reactivity=3 for 7.5 to 50 ppm). Overall results, summarized in Tables XI and XII below, indicate low in vitro cytotoxicity for Kathon CG at proposed usage concentrations. 
     According to the Agar Diffusion Test (as defined in the USP method), a culture shall be deemed to show a cytotoxic effect if microscopic examination reveals malformation, degeneration, sloughing, or lysis of cells within the affected zone, or a moderate to severe reduction in cell layer density. As set forth on the following scale, a Biological Reactivity Grade above 2 indicates significant cytotoxic response under experimental conditions. 
     
         ______________________________________                                    
Grade  Reactivity                                                         
                 Description of Reactivity Zone                           
______________________________________                                    
0      None      No detectable zone around or under                       
                 sample                                                   
1      Slight    Zone limited to area under the sample                    
2      Mild      Zone extends &lt;0.5 cm beyond sample                       
3      Moderate  Zone extends 0.5 to 1.0 cm beyond                        
                 sample                                                   
4      Severe    Zone extends &gt;1.0 cm beyond the                          
                 sample                                                   
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                       TABLE XI                                                    
______________________________________                                    
Evaluation of Kathon CG Solutions                                         
using the L929 Agar Overlay Diffusion Assay                               
Test Sample  Zone of Lysis   Reactivity                                   
______________________________________                                    
[Kathon CG]                                                               
5      ppm       0.0     0.0 0.0   0.0 0                                  
7.5    ppm       0.0     0.0 0.0   0.0 0                                  
15     ppm       0.0     0.0 0.0   0.0 0                                  
30     ppm       0.2     0.2 0.1   0.2 2                                  
50     ppm       0.3     0.2 0.3   0.2 2                                  
Controls                                                                  
100    ppm BAK   0.7     0.7 0.8       3                                  
PBS              0.0     0.0 0.0       0                                  
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                       TABLE XII                                                   
______________________________________                                    
Evaluation of Lenses Following 72 Hour Exposure to Kathon                 
CG Solutions using the L929 Agar Overlay Diffusion Assay                  
Test Sample  Zone of Lysis   Reactivity                                   
______________________________________                                    
[Kathon CG]                                                               
5      ppm       0.0     0.0 0.0   0.0 0                                  
7.5    ppm       0.0     0.0 0.0   0.0 0                                  
15     ppm       0.0     0.0 0.0   0.0 0                                  
30     ppm       0.0     0.0 0.0   0.0 0                                  
50     ppm       0.1     0.0 0.0   0.1 0                                  
Controls                                                                  
100    ppm BAK   0.7     0.9           3                                  
PBS              0.0     0.0           0                                  
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     Direct Contact SIRC Morphological Toxicity was studied based on Direct Contact USP method. Cytotoxic response is qualitatively measured using a 1-5 grading scale. The following morphological grading system allows estimation of the percentage of cells adversely affected by a given test solution and comparative evaluation of solution cytotoxicity. 
     
         ______________________________________                                    
MORPHOLOGICAL TOXICITY GRADING SYSTEM                                     
Grade     Membrane Damage/Cytotoxicity                                    
______________________________________                                    
1         &lt;10%                                                            
2         &lt;30%                                                            
3         &lt;60%                                                            
4         &lt;80%                                                            
5          &lt;100%                                                          
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     A culture is judged to show a cytotoxic effect if microscopic examination reveals malformation, degeneration, sloughing, or lysis or cells. A morphological toxicity grade above 2 indicates significant adverse cellular response under direct exposure conditions. The results are set forth as follows: 
     
                       TABLE XIII                                                  
______________________________________                                    
Evaluation of Kathon CG Solutions using                                   
the Direct Contact SIRC Morphological Toxicity                            
Assay        Toxicity Grades                                              
Test Sample  10 Min      30 Min  90 Min                                   
______________________________________                                    
[Kathon CG]                                                               
5      ppm       1 1 1 1     2 2 2 2                                      
                                   3 3 3 3                                
7.5    ppm       1 1 2 1     3 3 3 2                                      
                                   4 4 4 4                                
15     ppm       2 2 2 2     3 3 3 3                                      
                                   4 4 4 5                                
30     ppm       2 2 2 2     3 3 3 3                                      
                                   4 4 4 4                                
50     ppm       2 2 2 2     3 3 3 3                                      
                                   4 4 4 5                                
Controls                                                                  
100    ppm BAK   5 5 5 5     5 5 5 5                                      
                                   5 5 5 5                                
10     ppm BAK   2 2 2 2     3 3 3 3                                      
                                   4 5 5 5                                
PBS              1 1 1 1     1 1 1 1                                      
                                   1 1 1 1                                
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