Killing bacterial spores with glutaraldehyde sporicidal compositions
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a Continuation of U.S. Ser. No. 157,681 filed June 28, 1971 by the present inventors, now abandoned. 
    
    
     This invention relates to a new and improved sporicidal composition whose main sporicidal component is glutaraldehyde, the sporicidal kill activity of the composition being more rapid than previously possible and effective after prolonged periods of storage. Enhanced sporicidal performance is achieved by use of controlled amounts of certain detergents (surface active agents) which serve to potentiate the sporicidal activity of the composition, preferably in combination with controlling the pH of the composition within a specific range. 
     BACKGROUND OF THE INVENTION 
     The prior art is replete with a variety of compositions directed toward the effective killing of bacterial spores. Among the most important developments in this area are the compositions disclosed in U.S. Pat. No. 3,016,328 to Pepper et al and U.S. Pat. No. 3,282,775 to Stonehill. In the former patent saturated dialdehydes provide sporicidal activity and the pH of the composition is controlled so that it is maintained in excess of 7.4 by including an alkalinizing agent. The sporicidal composition disclosed in the latter patent to Stonehill is also characterized as containing a saturated dialdehyde, including cationic surface active agents. In the Stonehill patent; it is expressly stated that anionic and/or nonionic detergents do not increase the sporicidal activity of the compositions. 
     While the patent to Pepper et al limits the pH of the sporicidal composition to a minimum in excess of at least 7.4, the Stonehill patent discloses no such limitation as the compositions disclosed therein are stated to be effective over a wide pH range of 4.0-9.0. 
     Each of the sporicidal compositions disclosed in the above-identified patents claim very fast sporicidal action of about 3 hours or less. However, close examination of the conditions upon which such fast kill claims were determined reveals that the &#34;kills&#34; were obtained against readily susceptible bacterial spores and that neither patent discloses any sporicidal activity results for bacterial spores on silk sutures, which carrier is specified as being required by the AOAC test procedure. Futhermore, it is well known that resistance of bacterial spores to chemical sterilizing agents is lowest in free suspension, intermediate on porcelain rings and most resistant on silk sutures. Recently, the claims for sporicidal compositions marketed by the common assignee of the above-noted patents had to be revised in their registration with the USDA from a contact kill time of 3 hours upward to 10 hours. 
     Independent analyses of the sporicidal compositions disclosed in U.S. Pat. No. 3,016,328 to Pepper et al revealed that the 10 hour contact kill time was readily obtainable when using a fresh solution, but that the efficacy of the compositions markedly decreased upon standing for prolonged periods of up to about 2 weeks. Further, this reduction in effectiveness was found to be attributable to the diminution of glutaraldehyde, which lost a total of about 25% of its value by the end of a 2 week period. 
     It can be seen, therefore, that these two principal disclosures relating to sporicidal compositions, under the limited test conditions set forth therein may not, in reality, exhibit the effectiveness implied for them with respect to passing the complete AOAC test. Furthermore, the effectiveness of the disclosed compositions are based upon those bacterial spores and/or carriers which are known to favor relatively easy &#34;kills. 
    
    
     THE INVENTION 
     It has now been found that the shortcomings of the prior art can be overcome by use of the glutaraldehyde sporicidal compositions of the invention which, in general, can be obtained by providing a minimum amount of glutaraldehyde in a suitable sporicidal solvent and including an anionic, nonionic, or ampholytic detergent therein to obtain enhanced sporicidal activity. Further, by closely controlling the pH of the compositions, significantly improved shelf life is provided which is manifested by the sporicidal performance of the activated compositions, even after standing for prolonged periods of 2 to 3 weeks. 
     The amount of glutaraldehyde incorporated in the sporicidal solvent should be no less than about 0.5% by weight, since lesser amounts unduly prolong the kill times, while the maximum amount which can be used is essentially without limit. The term &#34;sporicidal solvent,&#34; as used throughout this application and in the claims, should be understood as referring to those solvents normally employed for sporicidal compositions and which include water and/or alcohols. For example, the U.S. Patents to Pepper et al and Stonehill et al discussed above, each disclose the use of alcohols as a sporicidal solvent. However, in this invention, water is the preferred sporicidal solvent to be used, although other sporicidal solvents can also be employed. 
     The addition of activating agents to adjust the pH of sporicidal compositions is well known to those skilled in the art. Generally, glutaraldehyde compositions are stable almost indefinitely within the pH range of about 2.5-4.5 at which pH levels they are stored before use. Just prior to use, their pH levels are adjusted through the addition of activating agents. Hence, most sporicidal compositions are made commercially available as a two package system, one of which comprises the sporicide in a suitable solvent and the other of which contains the activating agent, either as a powder or in solution, which is to be added to the sporicidal composition to activate it and adjust its pH just prior to use. The addition of such agents has been noted above in discussing the patent to Pepper et al (U.S. Pat. No. 3,016,328) wherein this procedure is referred to as &#34;alkalinizing&#34; the composition. In the contest of this invention, the term &#34;activating&#34; is employed and should be understood to be equivalent to alkalinizing such compositions and adjusting their pH levels by adding well known buffering agents to them. Once activated in this manner, the sporicidal compositions of the prior art have been found to have limited shelf lives and are, therefore, generally utilized immediately, or within a very short time, after being activated. It is to this particular problem that one embodiment of this invention is directed for it has now been found that upon activating the sporicidal compositions of this invention so that their pH levels are maintained within a specified range, improved shelf life stability can be obtained. 
     The pH of the activated sporicidal compositions of the invention can be controlled by incorporating therein one or more of the suitable and well known buffering agents so that the pH of the composition is no greater than 7.4, preferably about 6.5 to 7.4, and optimumly at a pH of 7.0 ± 0.3. The selection of suitable buffering agents for controlling the pH level is not critical and such materials as phosphates, citrates, carbonates, bicarbonates and the like, can be readily employed, although the phosphates are particularly preferred due to their favorable dissociation constants. As is well known in the art, other ingredients such as anti-corrosion agents, dyes, and the like, can also be added to the compositions. 
     The detergents which can be employed in the composition serve to potentiate; that is, increase and enhance, the sporicidal activity of the compositions. The minimum amount of detergent which should be employed is about 0.01% by weight with a range of about 0.1 to 1.0% preferred. For the purposes of this invention the term &#34;detergent&#34; should be understood as referring to any nonionic, anionic or ampholytic detergent which, when added to water at a concentration of 0.1%, will depress the surface tension of water by at least 20 dynes per square centimeter. When exposed to some materials, such as metal instruments; for example, scalpels, anionic detergents may exhibit a corrosive effect, and for this reason the nonionic detergents are preferred. Exemplary of the nonionic detergents which can be employed are the alkylphenolethoxylates available under the Trademark &#34;Igepal.&#34; 
     In a further embodiment of the invention it has been found that the inclusion of one or more monoaldehydes results in a synergistic effect thereby further enhancing the efficacy of the composition. When such monoaldehydes are included, they should be present in amounts no less than about 0.5% with the upper amounts being limited only by their solubility in the sporicidal solvent being employed. Illustrative of the monoaldehydes which can be employed are formaldehyde, acetaldehyde, propionaldehyde, and butyraldehyde, formaldehyde being preferred. 
     The sporicidal compositions of the invention have been found to be effective in killing a wide range of bacterial spores such as Clostridium welchii (Cl. welchii), Clostridium tetani (Cl. tetani), Bacillus subtilis (B. subtilis), Bacillus pumilus (B. pumilus), Bacillus globigii (B. globigii), Clostridium sporogenes (Cl. sporogenes), and the like. Of these, the Cl. sporogenes and the B. subtilis are known to be among the most difficult bacterial spores to kill, and are the organisms specified in the AOAC test. 
     The invention will be more fully understood when considered in light of the following examples which are set forth as being merely illustrative of the invention and are not intended to be limitative thereof. Unless otherwise indicated, all parts and percentages are by weight. The sporicidal data presented in the examples was, in all instances, obtained according to the USDA approved sporicidal test method set forth on pages 64 and 65 of the A.O.A.C., 11th edition (1970). 
     In the examples, the preferred detergents employed are identified by letters and/or numerals and are described in the following tabulation according to their commercial Trademarks, where applicable, and their general chemical composition. However, as previously indicated, it should be understood that while the following list sets forth preferred detergents, they are, in a broad sense, only exemplary of the entire class of nonionic, anionic, and/or ampholytic detergents which can be employed. 
     
         ______________________________________                                    
Identification                                                            
           Trademark Type and Composition                                 
______________________________________                                    
IGP        &#34;Igepal   Nonionic--consisting                                 
            CO 710&#34;  essentially of nonyl                                 
                     phenol condensed with 10-11                          
                     mols of ethylene oxide.                              
P 65       &#34;Pluronic Nonionic--consisting essen-                          
            P 65&#34;    tially of hydrophilic poly-                          
                     oxyethylene groups and a                             
                     hydrophobic polyoxypropylene                         
                     group; Av. M.W.-3500; 50% EO.                        
P 123      &#34;Pluronic Nonionic--same general chem-                         
           P 123&#34;    ical composition as P 65;                            
                     Av. M.W.-5650; 30% EO.                               
T-XD       &#34;Tergitol Nonionic--a butoxy derivative                        
            XD&#34;      of a propylene oxide-ethylene                        
                     oxide block polymer.                                 
C.sub.12 A + 8 EO    Nonionic--C.sub.12 alkanol + 8                       
                     ethylene oxide groups.                               
C.sub.14 A + 10 EO   Nonionic--C.sub.14 alkanol + 10                      
                     ethylene oxide groups.                               
C.sub.12 A + 3 EO-S  Anionic--C.sub.12 alkanol + 3                        
                     sulfated ethylene oxide                              
                     groups.                                              
LAS                  Anionic--linear alkane                               
                     sulfonate.                                           
SLS                  Anionic--sodium lauryl sul-                          
                     fate.                                                
FC-128     &#34;FC-128&#34;  Anionic--fluorinated.                                
CAT                  Cationic--cetylpyridinium                            
                     chloride.                                            
AMPH       &#34;Deriphat Ampholytic--disodium N-lauryl                        
            160&#34;     B-imino-dipropionate.                                
______________________________________                                    
 
    
     EXAMPLE 1 
     In order to demonstrate the effect of detergents and pH on the sporicidal composition of the invention, various detergents at different levels were incorporated in the composition and the pH was adjusted at different levels by use of a phosphate salt as a buffering agent. Results were recorded at varying exposure times of the sporicidal composition tested against Cl. sporogenes on silk suture loops. These results are set forth in Table 1 below wherein each of the numbered samples of sporicidal compositions was obtained by dissolving 2% glutaraldehyde in water. 
     
                                           TABLE I                                 
__________________________________________________________________________
2% Glutaraldehyde Sporicidal Compositions at Different pH Levels          
and With Varying Amounts of Anionic and Nonionic Detergents               
Tested Against Cl. Sporogenes on Silk Suture Loops                        
        Det.        Positive (Failure) Tubes/Total                        
Sam-    Name        Tubes Tested                                          
ple     and         2   3   4   6   8                                     
No. pH  Type   Amt. Hrs.                                                  
                        Hrs.                                              
                            Hrs.                                          
                                Hrs.                                      
                                    Hrs.                                  
__________________________________________________________________________
1   9.0 none   --   10/10                                                 
                        --  8/10                                          
                                4/10                                      
                                    0/10                                  
2   8.0 none   --   10/10                                                 
                        --  7/10                                          
                                4/10                                      
                                    0/10                                  
3   7.0 none   --   10/10                                                 
                        --  8/10                                          
                                5/10                                      
                                    0/10                                  
4   8.0 IGP    0.01%                                                      
                    8/10                                                  
                        4/10                                              
                            1/10                                          
                                0/10                                      
                                    --                                    
5   8.0 IGP    0.1% 3/10                                                  
                        1/10                                              
                            0/10                                          
                                0/10                                      
                                    --                                    
6   8.0 IGP    1.0% 2/10                                                  
                        0/10                                              
                            0/10                                          
                                0/10                                      
                                    --                                    
7   8.0 IGP    10.0%                                                      
                    1/10                                                  
                        0/10                                              
                            0/10                                          
                                0/10                                      
                                    --                                    
8   7.0 IGP    1.0% 2/10                                                  
                        0/10                                              
                            0/10                                          
                                0/10                                      
                                    --                                    
9   8.0 P 65   0.1%         0/10                                          
10  7.0 P 123  1.0%         0/10                                          
11  8.0 A + 8 EO                                                          
               1.0%         0/10                                          
12  7.0 A + 10 EO                                                         
               1.0%         0/10                                          
13  8.0 T-XD   1.0%         0/10                                          
14  7.0 LAS    0.01%                                                      
                    5/10                                                  
                        3/10                                              
                            2/10                                          
                                0/10                                      
                                    0/10                                  
15  7.0 LAS    0.10%                                                      
                    2/10                                                  
                        1/10                                              
                            0/10                                          
                                0/10                                      
                                    --                                    
16  7.0 LAS    1.0% 2/10                                                  
                        0/10                                              
                            0/10                                          
                                0/10                                      
                                    --                                    
17  7.0 LAS    10.0%                                                      
                    1/10                                                  
                        0/10                                              
                            0/10                                          
                                --  --                                    
18  8.0 LAS    1.0%         0/10                                          
19  9.0 LAS    1.0%         0/10                                          
20  8.0 SLS    0.1%         0/10                                          
21  7.0 SLS    0.1%         0/10                                          
22  7.0 SLS    1.0%         0/10                                          
23  7.0 FC-128 1.0%         0/10                                          
24  7.0 C.sub.12 A +                                                      
         3 EO-S                                                           
               1.0%         0/10                                          
25  7.0 AMPH   1.0%         0/10                                          
__________________________________________________________________________
 
    
     The data set forth in Table 1 above reveals the improvement in sporicidal efficacy that is attained at various composition pH levels and utilizing a wide range of nonionic and anionic detergents at different concentrations. 
     EXAMPLE II 
     The effect of pH on sporicidal compositions over prolonged periods was determined by comparing three compositions at different pH levels. The sporicidal activity of the compositions were evaluated according to the above-identified AOAC test procedure against B. subtilis on silk suture loops beginning on the day of preparation and thereafter at 1 week intervals for a period of 4 weeks. Each of the compositions consisted of 2% aqueous glutaraldehyde which were stored in closed containers until tested. The results obtained are set forth in Table II below wherein Sample 26 was a commercially obtained product while Samples 27 and 28 were prepared by dissolving glutaraldehyde in water and adjusting their indicated initial pH levels with phosphate salts. 
     
                                           TABLE II                                
__________________________________________________________________________
Aged Samples of 2% Glutaraldehyde at Varying Initial pH Levels            
Tested Against B. Subtilis on Silk Suture Loops for 10 Hour               
Exposure Times                                                            
First      First  2nd    3rd    4th                                       
Day        Week   Week   Week   Week                                      
Sample Pos/   Pos/   Pos/   Pos/   Pos/                                   
No. pH Total                                                              
           pH Total                                                       
                  pH Total                                                
                         pH Total                                         
                                pH Total                                  
__________________________________________________________________________
26  8.4                                                                   
       0/10                                                               
           7.8                                                            
              0/10                                                        
                  7.6                                                     
                     1/10                                                 
                         7.5                                              
                            2/10                                          
                                7.5                                       
                                   3/10                                   
27  7.2                                                                   
       0/10                                                               
           7.3                                                            
              0/10                                                        
                  7.3                                                     
                     0/10                                                 
                         7.4                                              
                            0/10                                          
                                7.4                                       
                                   0/10                                   
28  6.9                                                                   
       0/10                                                               
           7.0                                                            
              0/10                                                        
                  7.1                                                     
                     0/10                                                 
                         7.2                                              
                            0/10                                          
                                7.2                                       
                                   1/10                                   
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     Contrary to the prior art teachings, the results in Table II above clearly show that good sporicidal performance can be obtained from sporicidal compositions whose pH is 7.4 or less. Furthermore, the efficacy of sporicidal compositions having initially low pH levels of about 6.9 to 7.4 was retained over prolonged storage periods of 2 weeks or more, compared with the pH range previously taught, a finding not suggested in the prior art. 
     Similar improved results were also obtained when anionic and nonionic detergents were included in amounts of 0.01% to 10% in the sample compositions of Table II. Again, it should be noted that improved sporicidal performance was obtained utilizing anionic and nonionic detergents which is also contrary to the teachings of the prior art. 
     The glutaraldehyde content of Samples 26, 27 and 28 above was determined by conventional chemical analysis and it was found that the higher the initial pH level of the composition, such as in the range of about pH 6 to 10, the more rapid was the decomposition of the glutaraldehyde. The rate of sporicidal effectiveness of glutaraldehyde was found to diminish in those compositions having an initial pH of 6.5 and less, but in those compositions having an initial pH of 7.0 ± 0.3, good initial sporicidal performance was obtained and these compositions also maintained acceptable sporicidal performance for the greatest length of time. Therefore, an initial pH of 7.0 ± 0.3 is optimum for the sporicidal compositions of the invention. 
     EXAMPLE III 
     It has also been found that a synergistic effect can be obtained in the sporicidal composition of the invention when one or more monoaldehydes are added. To illustrate this synergistic effect, samples of sporicidal compositions were prepared in which the pH level was maintained constant at pH 7.0 and in which different detergents were either included in the amounts shown or were not included at all. The glutaraldehyde content of the compositions was varied from 2% to 4% and the amount of monoaldehyde added was also varied over a range of 0% to 6%. Since formaldehyde is the most important sporicidal monoaldehyde, it was selected to demonstrate the synergistic effect obtained. Furthermore, it is known that in order to obtain a sporicidal activity about equivalent to that when using 2% glutaraldehyde, about 10% formaldehyde would be needed. Hence, a good comparative basis was provided between the use of only glutaraldehyde and the use of only formaldehyde, although it was also found that the other higher monoaldehydes such as acetaldehyde, propionaldehyde, and butyraldehyde yielded similar results. The prepared sporicidal compositions were subjected to the above-identified AOAC test against B. subtilis on silk suture loops over a period of 10 hours and the results obtained are set forth below in Table III wherein glutaraldehyde is identified by the term &#34;BLU&#34; and formaldehyde is identified by its chemical abbreviation &#34;HCHO.&#34; In Table III, the results are shown on a &#34;pass&#34; or &#34;fail&#34; basis respectively indicated by the letter &#34;P&#34;, which denotes no growth in any of 10 tubes, and the letter &#34;F&#34;, which denotes one to 10 tubes having bacterial growth in a set of 10  tubes. 
     
                                           TABLE III                               
__________________________________________________________________________
Synergistic Effect of Formaldehyde with Glutaraldehyde                    
Sam-      Det.   Elapsed Time Results                                     
ple                                                                       
   %  %       Amt.                                                        
                 2  3  4  6  8  9  10                                     
No.                                                                       
   GLU                                                                    
      HCHO                                                                
          Type                                                            
              (%)                                                         
                 hrs.                                                     
                    hrs.                                                  
                       hrs.                                               
                          hrs.                                            
                             hrs.                                         
                                hrs.                                      
                                   hrs.                                   
__________________________________________________________________________
29 2  0   --  0  F  F  F  F  F  F  P                                      
30 2  1   --  0  F  F  F  F  P  P  P                                      
31 2  3   --  0  F  F  P  P  P  P  P                                      
32 4  0   --  0  F  F  F  F  P  P  P                                      
33 4  3   --  0  F  P  P  P  P  P  P                                      
34 2  0   IGP 1  F  F  F  F  F  P  P                                      
35 2  3   &#34;   1  F  F  P  P  P  P  P                                      
36 2  6   &#34;   1  F  P  P  P  P  P  P                                      
37 4  0   &#34;   1  F  F  F  P  P  P  P                                      
38 4  3   &#34;   1  F  P  P  P  P  P  P                                      
39 2  1   LAS 5  F  F  F  P  P  P  P                                      
40 4  3   LAS 1  F  P  P  P  P  P  P                                      
41 2  1   CAT 1  F  F  F  P  P  P  P                                      
42 2  6   CAT 0.1                                                         
                 F  F  P  P  P  P  P                                      
43 4  3   CAT 5  F  P  P  P  P  P  P                                      
44 2  3   AMPH                                                            
              1  F  F  P  P  P  P  P                                      
__________________________________________________________________________
 
    
     As can be seen from the above results, increasing the glutaraldehyde concentration from 2% to 4% decreases the sporicidal performance time of the composition from 10 hours to about 8 hours. However, when only 3% formaldehyde was added to the 2% glutaraldehyde, its sporicidal performance time was decreased from 10 to 4 hours. In this regard, it is significant to note that essentially the same results are obtained regardless of the type of detergent used; that is, nonionic, anionic, cationic or ampholytic detergent. 
     In other, similar tests conducted over a pH range of about 1 to 9, the synergistic effect illustrated in Example III was found to function at each pH level over this entire pH range. Similar synergistic results were also obtained when fomaldehyde at concentrations of about 0.5 to 10% were combined with glutaraldehyde at concentrations of about 0.5 to 6%. However, the most effective synergism was found to be obtained with formaldehyde concentrations of about 1 to 6% in combination with glutaraldehyde concentrations of about 2 to 4% and these concentration ranges are preferred. 
     EXAMPLE IV 
     In order to further demonstrate the improved sporicidal performance of the compositions of the invention, a sporicidal composition was provided from 1 gallon of stock solution containing 4% glutaraldehyde, 3% formaldehyde and 1% of a nonionic detergent (IGP). The pH of the stock solution was adjusted to pH 4 by adding a few drops of phosphoric acid. Thereafter, this stock solution was activated by adding 16 grams of a mixture of di- and trisodium phosphate and sodium carbonate to provide a pH of 7.1. Following the normal practice in the art, incidental amounts of sodium nitrite as a corrosion inhibitor and incidental amounts of D and C Green No. 8 as a dye were also included in the activating salt mixture. 
     The activated solution passed the A.O.A.C. Sporicidal Test against B. subtilis and Cl. sporogenes on suture carriers within 3 hours and 2 hours, respectively, and passed the same test against both of these organisms on porcelain cylinders within even shorter time periods. 
     After storage for 3 weeks at room temperature (20° C. - 25° C.), the same activated solution passed the same A.O.A.C. test against the same two spores types and test carriers (a total of four test conditions) within a maximum interval of 5 hours. 
     Upon further testing, this activated solution was found to also successfully sterilize bronchoscopes, cystoscopes, rubber tubing and scalpels upon immersion of these materials for a period of 5 hours. 
     It can be seen from the above that the present invention is, in part, an improvement over the references to Pepper et al and Stonehill et al discussed earlier. For example, it has been demonstrated in Examples I and II that faster kills can be obtained and that the sporicidal composition of the invention exhibits sporicidal activity over prolonged periods, and that these results were obtained at lower pH levels than those indicated as being critical in the patent to Pepper et al (U.S. Pat. No. 3,016,328). Furthermore, Example III illustrates that the detergents which can be employed need not be limited to the cationic group as disclosed in the patent to Stonehill et al (U.S. Pat. No. 3,282,775), but can be any detergent selected from the nonionic, anionic or ampholytic groups, provided, however, that the surface tension of the detergent selected meets the criteria set forth hereinabove. In addition, none of the prior art suggests that a monoaldehyde can be combined with glutaraldehyde to obtain a synergistic effect, and this is also demonstrated in Example III.