Abstract:
This invention relates to a reaction product of allantoin and formaldehyde made in a molar ratio of about 1:2.75-1:3.25, preferably 1:3, respectively, preferably under controlled pH (5.0 to 7.0) and temperature (40° to 85° C., preferably 50-60° C.) conditions, which product contains free formaldehyde (&lt;0.1%), and methylene diol of about 580-1385 ppm, preferably at a pH of 7.2, with advantageous long-lasting, anti-microbial properties, particularly against the organism  B. cepacia , and in combination products with parabens and iodopropynyl butyl carbamate (IPBC).

Description:
BACKGROUND OF THE INVENTION  
         [0001]    1. Field of the Invention  
           [0002]    This invention relates to reaction products of allantoin and formaldehyde and, more particularly, to such products which contain very low levels of free formaldehyde (&lt;0.1%), while retaining advantageous, long-lasting anti-microbial properties, particularly against the organism  B. cepacia.    
           [0003]    2. Description of the Prior Art  
           [0004]    Berke, P. A., in U.S. Pat. No. 3,248,285 described a reaction product of allantoin and formaldehyde (Germall® 115) from the reactants in a mole ratio of 1:1.5, respectively; and in U.S. Pat. No. 4,271,176 and Reissue 32,848, (Germall® II) in a mole ratio of 1:4. However, during recent evaluations using modern C 13  NMR techniques, these reaction products were found to contain 0.5% to 1.0% of free formaldehyde. This amount was considered in the past to be necessary to retain its anti-microbial activity; although now it is recognized that a considerable amount of free formaldehyde in the product is disadvantageous from a safety (irritation) and environmental standpoints.  
           [0005]    Accordingly, it is an object of this invention to prepare reaction products of allantoin and formaldehyde which have long-lasting anti-microbial properties with the presence therein of only very low levels of free formaldehyde (i.e. &lt;0.1%).  
           [0006]    Another object of the invention is to provide a method of making such effective reaction products.  
           [0007]    Still another object of this invention is to provide a synergistic combination of such products with other known fungicides to obtain broad spectrum activity against bacteria and fungi, yeast, molds, and the like.  
         SUMMARY OF THE INVENTION  
         [0008]    This invention relates to a reaction product of allantoin and formaldehyde,  
                         
 
           [0009]    made in a molar ratio of about 1:2.75-1:3.25, preferably 1:3, respectively, preferably under controlled pH (5.0 to 7.0) and temperature (40° to 60° C.) conditions, which contains substantially no free formaldehyde (&lt;0.1%), with advantageous long-lasting, broad spectrum anti-microbial properties, particularly against the organism  B. cepacia.    
           [0010]    What has been discovered herein is that reaction products of allantoin and formaldehyde result in free formaldehyde being present in equilibrium with methylene diol and N-methylol. Unexpectedly, it was discovered that N-methylol and methylene diol, work in synergy to act as long-lasting anti-microbial agents, i.e. both can slowly react to provide anti-microbial protection against a wide range of microbes, including the difficult to kill  B. cepacia , while free formaldehyde is present in very low concentrations (&lt;0.1%) in equilibrium with methylene diol.  
                         
 
           [0011]    Both equilibrium moieties can react with the amide moieties of allantoin to form several N-methylol of the allantoin compounds, which are stabilized by H-bonding; however they can slow hydrolyzed in water to give the methylene diol intermediate, which can react with amide to form N-methylols. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0012]    The following examples describe preparation of Comparative Reaction Products (CRP) for (A) Germall® 115 (1:1.5); at low caustic levels; and (B) at high caustic levels; and (C) Germall® II (1:4), low caustic and (D) high caustic; and Invention Reaction Products (IRP) Germall® III (1:3), (A) laboratory and (B) commercial runs, with only enough caustic to neutralize the formic acid present in the Formalin solution.  
       Comparative Reaction Products  
     A. GERMALL® 115 
       [0013]    [0013]                                                                 (1:1.5)                                        Allantoin   15.8   g (0.1 mole)           Formalin (37%)   12.2   g (0.15 mole)           Water   28.5   ml                        
         [0014]    The above mixture was refluxed for one hour to form a clear solution.  
       B. GERMALL® 115 (High Caustic)  
       [0015]    [0015]                                                                 (1:1.5)                                        Allantoin   600   g (3.8 mole)           Formalin (37%)   450   g (5.5 mole)           Sodium Hydroxide   123   g                        
         [0016]    Refluxed for one hour to form a clear solution. Concentrated acetic acid was added to adjust the Ph to 4.0. Removed water to give a white powder.  
       C. GERMALL® II  
       [0017]    [0017]                                                                 (1:4)                                        Allantoin   1053   g (6.66 mole)           Formalin (37%)   2160   g (26.64 mole)                        
         [0018]    The white suspension was heated to 85° C. and held for an additional hour; upon cooling a clear solution was obtained. Removed water under reduced pressure to give a white powder.  
       D. GERMALL® II  
       [0019]    [0019]                                                                 (1:4)                                        Allantoin   158.1   g (1.0 mole)           Formalin (37%)   324.2   g (3.99 mole)           Sodium Hydroxide 10%   32.0   g (0.08 mole)                        
         [0020]    Refluxed at 85° C. for one hour. The clear colorless solution obtained was dried under reduced pressure to give solid white powder residue.  
       Comparative Test Results  
       [0021]    Activity against yeast and mold:  
         [0022]    0.3% test solutions.  
                                                                 A. Niger                   C. Albican     ATCC 9642           TEST SOLUTIONS   ATTC1023   (for 3 Days)                           0.3% Germall ® 115   +   +           (Exs. A/B)           0.3% Germall ®     —       —             II(Exs. C/D)                                              
 
       Invention Reaction Product  
     A. (Laboratory Scale) (GERMALL® III)  
       [0023]    [0023]                                                                 (1:3)                                        Allantoin   1616   g (10.23 mole)           Formalin LM*(37%)   2488   g (30.68 mole)           Sodium Hydroxide 50%   24   g                        
         [0024]    Mixed and heated at 60° C. for 3 hours to give a clear solution. The Ph of the product was adjusted to 7.2 with the sodium hydroxide solution to neutralize formic acid in Formalin® and the solution was spray dried to give a free-flowing, white powder.  
         [0025]    LM=low methanol (&lt;0.5%) Borden Chemicals  
       Invention Reaction Product  
     B. (Commercial Scale) (GERMALL® III)  
       [0026]    [0026]                                                                               (1:3)                                        Allantoin.wet cake   2095   lbs (10.23 mole)           Formalin LM (37%)   2488   lbs (30.68 mole)           Sodium Hydroxide 50%   23.6   lbs                Ph   6.5-7.0           Reaction Temp   40-60° C.                        
         [0027]    The resultant mixture then was further reacted at 85° C. for 3 hours to give a clear solution at pH 7.2. The solution was spray dried to remove water and other volatile by-products to give a free-flowing, white powder.  
         [0028]    A study was conducted to determine the level of methylene diol in the reaction products versus the number of equivalents of formaldehyde added during formation. These results are based on quantitative 13C-NMR analysis and summarized in Table 1 below.  
                                         TABLE 1                                   Number of Equivalents               Formaldehyde/Allantoin   ppm Methylene Diol                                        4.00   3545           3.50   1916           3.25   1385           3.00   790           2.75   583           2.50   340           1.50   250                      
 
         [0029]    [0029]                                                                                                                 TABLE 2                           BIOACTIVITY OF GERMALL ® COMPOUNDS            INVENTION EXS.            PRESERVATIVE   ORGANISM   STATIC   CIDAL                    IRP - Germall ®     Staph aureus     300   ppm   1250   ppm       III (1:3)     E. coli     300   ppm   1250   ppm             P. aeruginosa     300   ppm   600   ppm             B. cepacia     150   ppm   300   ppm                  C. albicans     &gt;5000   ppm   —                  A. niger     2500   ppm   2500   ppm       CRP - Germall ®     Staph aureus     300   ppm   1250   ppm       II (1:4)     E. coli     600   ppm   1250   ppm             P. aeruginosa     600   ppm   1250   ppm             B. cepacia     150   ppm   600   ppm             C. albicans     5000   ppm   &gt;5000   ppm             A. niger     2500   ppm   2500   ppm       CRP - Germall ®     Staph aureus     1250   ppm   2500   ppm       115 (1:1.5)     E. coli     1250   ppm   2500   ppm             P. aeruginosa     1250   ppm   2500   ppm             B. cepacia     600   ppm   1250   ppm             C. albicans     &gt;5000   ppm             A. niger     5000   ppm   5000   ppm                    
       Protocol  
     Minimum Inhibitory Concentration (MIC) Test Method  
       [0030]    Scope  
         [0031]    The purpose of this test procedure is to screen experimental compounds for anti-microbial activity.  
         [0032]    Principle  
         [0033]    The measurement of the lowest effective concentration of an anti-microbial or anti-microbial blend is important for recommending use concentrations. The MIC test is an in vitro tube dilution procedure used to identify effective concentrations of anti-microbials. In this test, the experimental compound is diluted by serial concentrations into nutrient culture media. Test organisms are then inoculated into the anti-microbial solutions.  
         [0034]    If the experimental compound is effective, there is no growth observed in the test dilution tubes and they are clear. If the experimental compound is not effective, the test dilution tubes are cloudy, indicating growth. This test will determine static as well as cidal activity concentrations.  
         [0035]    Materials  
         [0036]    1. Laminar flow hood (Baker Sterilgard SG 400)  
         [0037]    2. 18×150 mm culture tubes  
         [0038]    3. Stock antimicrobial solution  
         [0039]    4. Media: Trypticase soy broth (BBL 11043) and AOAC Letheen broth (BBL 10914)  
         [0040]    5. Test organisms:  Staphylococcus aureus  ATCC 6538,  Escherichia coli  ATCC 8739,  Pseudomonas aeruginosa  ATCC 9027,  Burkholderia cepacia  ATCC 25416,  Candida albicans  ATCC 10231, and  Aspergillus niger  ATCC 16404.  
         [0041]    6. Spectrophotometer (Spectronic 20D, Milton Roy)  
       Minimum Inhibitory Concentration Test  
       [0042]    Procedure  
         [0043]    1. Antimicrobial stock solutions are prepared at predetermined concentrations (i.e., 10% through 0.07%) depending on the test material. Serial doubling dilutions are made as follows. Each culture tube contains 5 mis of trypticase soy broth. Five mis of the stock solution are added to the first tube and vortexed. 5 mis are then removed and placed into the second tube, (and so on, until the last tube). At the final test concentration, 5 mis of the broth/antimicrobial mixture is decanted out.  
         [0044]    2. The test organisms are prepared as with any organism inoculum (MLM 100-3, MLM 100-4, and MLM 100-5). A saline suspension of each organism is prepared. The bacterial organisms and the yeast are a standardized at a concentration of 1×10 6  cfu/ml. The mold inoculum is approximately 1×10 5  cfu/ml.  
         [0045]    3. Inoculate each culture tube with 0.10 mls of organism inoculum and vortex.  
         [0046]    4. Incubate bacterial tubes for 24 hours at 35° C. Incubate yeast or mold tubes for 48 hours at 25° C. Read for growth; turbid tubes for bacteria and yeast; mold clearly visible tubes. This is the minimum inhibitory concentration (static activity).  
         [0047]    5. After the tubes are read, transfer all “clear” tubes and the first cloudy (growth) tube into Letheen broth containing neutralizers. Incubate the Letheen broth tubes for 48 hours at the bacterial or fungal incubation temperatures. Read for growth; turbid tubes for bacteria and yeast; mold clearly visible in mold tubes. This is the cidal activity concentration.  
         [0048]    Discussion  
         [0049]    The cidal activity of an anti-microbial can be rapidly screened by means of a MIC test before further evaluation tests, such as longer preservative efficacy tests, are performed. This test is a tube serial dilution procedure limited only by the water solubility of the material. Where anti-microbial materials are slightly insoluble, leaving the TSB broth turbid, a procedure modification can be made. Tubes are incubated for 24 hours (bacteria) or 48 hours (fungi) but instead of transfer to Letheen broth, the TSB tubes are streaked onto Letheen agar. The agar plates are then incubated appropriately and then read for absence or presence of growth. Depending on the degree of insolubility, a measure of cidal activity may be the only parameter measured.  
         [0050]    Anti-microbial neutralization is important in this screening test. Letheen broth or agar contains neutralizers but if these do not neutralize the anti-microbial adequately, others can be added. These are to be determined prior to testing.  
         [0051]    Aseptic technique is important in any microbiological procedure. All functional operations are performed under the laminar flow hood with use of sterile pipettes, tubes and media to eliminate cross-contamination. Surface sanitizers (i.e., alcohol) are used on the work surface before and after each operation. Ample time is allowed for recirculation of air within the sterile chamber of the hood.  
         [0052]    The bioactivity data show particular effectiveness against the organism cepacia B. (cidal=300 ppm vs. 600 ppm and 1250 ppm for Germall® II and Germall® 115, respectively). However, if desired, even broader spectrum antibacterial activity can be achieved by combination products with the invention composition whose formulations are given below.  
       Invention Combination Products  
       [0053]    [0053]                                                 COMBINATION BLENDS (BY WEIGHT)                                    (1)   Germall ® III   20-30%               MP - methyl paraben    8-12%               PP - propyl paraben   2-4%               PG - propylene glycol   q.s. 100           (2)   Germall ® III   40-45%               IPBC - iodopropynyl butyl carbamate   0.5-5%                 PG - propylene glycol   qs 100           (3)   Germall ® III   98.5-99.5%               IPBC - iodopropynyl butyl carbamate   0.5-1.5%               (Powder)                        
       Preservative Activity (Challenge Test)  
       [0054]    A typical cosmetic emulsion was prepared for microbiological challenge testing and predetermined admixtures of a methylol compound and IPBC were added at various use levels. The emulsion thus prepared had the following composition:  
                                                           NONIONIC EMULSION (UNPRESERVED CONTROL)            Phase   Ingredient   % wt.                    A   Water   69.80       A   Carbomer   10.00       B   Octyl Palmitate   5.00       B   Cetearyl alcohol and Ceteareth-20   2.00       B   Glyceryl Stearate and Laureth-23   2.50       B   Mineral Oil   5.00       C   Triethanolamine (99%)   0.20       D   Preservative   0.00       E   Hydrolyzed Collagen   0.50       E   Water   5.00           Total   100.00                  
 
         [0055]    Procedure:  
         [0056]    Heat Phase A to 75° C. Heat Phase B to 75° C.  
         [0057]    Add Phase B to Phase A. Mix until uniform.  
         [0058]    Add Phase C. Remove heat.  
         [0059]    Add Phase D at the appropriate temperature.  
         [0060]    Add Phase E at 40° C.  
         [0061]    Continue mixing to 30° C.  
                                                                                         STANDARD SCREENING EMULSIONS                % wt.                            Phase A                Stearic Acid   5.00           Mineral Oil   2.50           Cetyl Alcohol   1.00           Lareth-5 and Ceteth-5 and   0.50           Oleth-5 and Steareth-5           Glycerol Monostearate and   1.50           Polyoxyethylene Stearate                Phase B                Deionized Water   88.0           Triethanolamine 99%   1.00           Citric Acid 30% aqueous solution   0.60           Preservative Admixture   qs                      
 
         [0062]    To prepare the emulsion, Phases A and B were heated separately to 75°-80° C. Phase A then was added to Phase B with mixing. The mixture then was cooled to 55″-60° C. At this point the desired amount of the preservative admixture was added and the product was cooled to 50° C. while stirring. The citric acid solution then was added to adjust the pH and the mixture was stirred until a temperature of 30° C. was reached.  
         [0063]    The challenge tests were carried out using the following microorganisms: SA, ECOLI, PSA, PC, AN and CAN, in this manner. 50 g aliquots of the test emulsion containing various amounts of the preservative admixture were inoculated with approximately 10 7 -10 8  of the challenge organisms. The test samples then were stirred to disperse the challenge inoculum. The samples were incubated and assayed at 48 hours, 7, 14, 21 and 28 days. The assays were performed on 1 g of the test sample by serially diluting 10 1  to 10 6  of the original concentration. The plating medium for bacteria was Letheen agar and for fungi it was low pH Mycophil agar with Tween 20. Each plated sample was incubated for 48 hours at 37° C. for bacteria, 5 days at 25° C. for mold, and 3 days at 25° C. for fungi. After incubation, readings of the number of colonies per milliliter (cfu/ml) were made. At 21 days the test product was reinoculated with half of the original inoculum. The data is presented in Tables 3-11 below.  
       Challenge Tests  
       [0064]    [0064]                                                                                   TABLE 3                           COMPARISON OF ACTIVITY OF GERMALL III TO       GERMALL II AND 115 (SCREENING EMULSION)                    Organ-                           Preservative   Conc.   ism   48 hrs   7 days   14 days   21 days   28 days                    Germall II   1000 ppm   SA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   160,000   78,000   63,000   260,000   210,000               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germall III   1000 ppm   SA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   160,000   380,000   380,000   810,000   640,000               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germall 115   2000 ppm   SA   3000   &lt;10   &lt;10   &lt;10   &lt;10               EC   490   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   230,000   2,000,000   650,000   1,500,000   1,200,000               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germall II   2000 ppm   SA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   150   9,600   48,900   490,000   210,000               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germall III   2000 ppm   SA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   6000   195,000   460,000   690,000   1,070,000               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Unpreserved   0   SA   2,100,000   57,000   90   &lt;10   68,000               EC   37,000   96,000   96,000   43,000   790,000               PSA   70   4600   500   10,100   170,000               BC   2,100,000   860,000   1,520,000   3,520,000   &gt;10 E6               CAN   1,100,000   168,000   67,000   270,000   460,000               AN   700,000   56,000   44,000   190,000   320,000                    
         [0065]    [0065]                                                                                   TABLE 4                           COMPARISON OF ACTIVITY OF GERMALL III TO       GERMALL II AND 115 (NONIONIC EMULSION)                    Organ-                           Preservative   Conc.   ism   48 hrs   7 days   14 days   21 days   28 days                    Germall III   2000 ppm   SA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   820,000   1,680,000   1,350,000   700,000   &gt;1E6               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germall II   2000 ppm   SA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   320,000   720,000   650,000   730,000   &gt;1E6               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germall 115   2000 ppm   SA   1,500   &lt;10   &lt;10   &lt;10   &lt;10               EC   52,000   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   &gt;1E6   &gt;1E6   &gt;1E6   700,000   &gt;1E6               AN   &lt;10   20   390   370   &gt;1E4       Germall III   4000 ppm   SA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   24,000   &gt;1E6   &gt;1E6   730,000   &gt;1E6               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germall II   4000 ppm   SA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   50   10,000   620,000   460,000   &gt;1E6               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germall 115   4000 ppm   SA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               EC   1,500   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   1,060,000   1,000,000   &gt;1E6   &gt;1E6   &gt;1E6               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Unpreserved   0   SA   &gt;1E6   6,300   &gt;1E4   &lt;10   &gt;1E4               EC   &gt;1E6   900,000   &gt;1E4   &gt;1E4   &gt;1E4               PSA   20,000   &gt;1E6   &gt;1E4   &gt;1E4   &gt;1E4               BC   &gt;1E6   &gt;1E6   &gt;1E4   &gt;1E4   &gt;1E4               CAN   &gt;1E6   &gt;1E6   &gt;1E4   &gt;1E4   &gt;1E4               AN   500,000   510,000   &gt;1E4   &gt;1E4   &gt;1E4                    
         [0066]    [0066]                                                                                   TABLE 5                           COMPARISON OF ACTIVITY OF GERMALL III TO       GERMALL II AND 115 (SCREENING EMULSION)                    Organ-                           Preservative   Conc.   ism   48 hrs   7 days   14 days   21 days   28 days                    Germall III   250 ppm   SA   69,000   &lt;10   &lt;10   &lt;10   &lt;10               EC   11,000   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   200   &lt;10   &lt;10   &lt;10   &lt;10               CAN   430,000   120,000   70,000   150,000   850,000               AN   100,000   200   70   40   1,300       Germall II   250 ppm   SA   55,000   &lt;10   &lt;10   &lt;10   &lt;10               EC   5500   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   290,000   170,000   71,000   46,000   680,000               AN   50,000   &lt;10   &lt;10   &lt;10   100       Germall 115   250 ppm   SA   117,000   30   &lt;10   &lt;10   1,500               EC   40,000   20   &lt;10   &lt;10   3600               PSA   &lt;10   320   &gt;1E4   &gt;1E6   &gt;1E6               BC   11,000   &gt;1E6   &gt;1E6   &gt;1E6   &gt;1E6               CAN   1,090,000   270,000   1,120,000   770,000   &gt;1E6               AN   90,000   20,000   20,000   29,000   300,000       Germall III   500 ppm   SA   38,000   &lt;10   &lt;10   &lt;10   &lt;10               EC   18,000   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   100,000   210,000   310,000   270,000   &gt;1E6               AN   9000   &lt;10   &lt;10   &lt;10   &lt;10       Germall II   500 ppm   SA   17,000   &lt;10   &lt;10   &lt;10   &lt;10               EC   610   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   170,000   100,000   90,000   320,000   930,000               AN   40   &lt;10   &lt;10   &lt;10   &lt;10       Germall 115   500 ppm   SA   140,000   &lt;10   &lt;10   &lt;10   &lt;10               EC   24,000   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   290,000   760,000   790,000   1,210,000   &gt;1E6               AN   130,000   1,000   40   290   80,000       Unpreserved   0   SA   &gt;1E6   34,000   6,800   20   &gt;1E4               EC   18,000   4,900   &gt;1E4   &gt;1E4   &gt;1E4               PSA   50   &gt;1E4   &gt;1E4   &gt;1E4   &gt;1E4               BC   &gt;1E6   &gt;1E6   &gt;1E4   &gt;1E4   &gt;1E4               CAN   970,000   270,000   &gt;1E4   &gt;1E4   &gt;1E4               AN   150,000   280,000   &gt;1E4   &gt;1E4   &gt;1E4                    
         [0067]    [0067]                                                                                   TABLE 6                           COMPARISON OF ACTIVITY OF GERMALL PLUS AND       GERMALL III/ IPBC (SCREENING EMULSION)                    Organ-                           Preservative   Conc.   ism   48 hrs   7 days   14 days   21 days   28 days                    Germall Plus       SA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germall II   1980   EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       IPBC   20   PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germall III   1980   SA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       IPBC   20   EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               AN   &lt;10   &lt;10   &lt;10   &lt;10   100       Germall III   1960   SA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       IPBC   40   EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Unpreserved       SA   580,000   3200   180   &lt;10   &gt;1E4               EC   5,200   70,000   &gt;1E4   &gt;1E4   &gt;1E4               PSA   18,000   40,000   &gt;1E4   &gt;1E4   &gt;1E4               BC   &gt;1E6   &gt;1E6   &gt;1E4   &gt;1E4   &gt;1E4               CAN   &gt;1E6   200,000   &gt;1E4   &gt;1E4   &gt;1E4               AN   210,000   270,000   &gt;1E4   &gt;1E4   &gt;1E4                    
         [0068]    [0068]                                                                                   TABLE 7                           COMPARISON OF ACTIVITY OF LIQUID GERMALL PLUS AND       GERMALL III / IPBC -LIQ (SCREENING EMULSION)                    Organ-                           Preservative   Conc.   ism   48 hrs   7 days   14 days   21 days   28 days                    LiqGermPlus       SA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germall II   790   EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       IPBC   10   PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   8,000   &lt;10   &lt;10   &lt;10   &lt;10               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       LiqGermPlus       SA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germall II   1580   EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       IPBC   20   PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               AN   &lt;10   &lt;10   &lt;10   &lt;10   100       Germall III   790   SA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       IPBC   10   EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Liquid       PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   26,000   &lt;10   &lt;10   &lt;10   &lt;10               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germall III   1580   SA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       IPBC   20   EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Liquid       PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Unpreserved   0   SA   580,000   3200   180   &lt;10   &gt;1E4               EC   5200   70,000   &gt;1E4   &gt;1E4   &gt;1E4               PSA   18,000   40,000   &gt;1E4   &gt;1E4   &gt;1E4               BC   &gt;1E6   &gt;1E6   &gt;1E4   &gt;1E4   &gt;1E4               CAN   &gt;1E6   200,000   &gt;1E4   &gt;1E4   &gt;1E4               AN   210,000   270,000   &gt;1E4   &gt;1E4   &gt;1E4                    
         [0069]    [0069]                                                                                   TABLE 8                           COMPARISON OF ACTIVITY OF GERMALL PLUS AND       GERMALL III/ IPBC (SCREENING EMULSION)                    Organ-                           Preservative   Conc.   ism   48 hrs   7 days   14 days   21 days   28 days                    Germall Plus       SA   42,000   &lt;10   &lt;10   &lt;10   &lt;10       Germall II   495   EC   40   &lt;10   &lt;10   &lt;10   &lt;10       IPBC   5   PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   48,000   &lt;10   &lt;10   &lt;10   &lt;10               AN   100   &lt;10   &lt;10   &lt;10   &lt;10       Germall Plus       SA   300   &lt;10   &lt;10   &lt;10   &lt;10       Germall II   990   EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       IPBC   10   PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germall III   495   SA   46,000   &lt;10   &lt;10   &lt;10   &lt;10       IPBC   5   EC   25,000   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   11,000   &lt;10   &lt;10   &lt;10   &lt;10               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germall III   990   SA   24,000   &lt;10   &lt;10   &lt;10   &lt;10       IPBC   10   EC   1,000   &lt;10   &lt;10   &lt;10   &lt;10               PSA   19,000   &lt;10   &lt;10   &lt;10   &lt;10               BC   &gt;1E6   &lt;10   &lt;10   &lt;10   &lt;10               CAN   2,500   &lt;10   &lt;10   &lt;10   &lt;10               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germall III   490   SA   23,000   &lt;10   &lt;10   &lt;10   &lt;10       IPBC   10   EC   900   &lt;10   &lt;10   &lt;10   &lt;10               PSA   18,000   &lt;10   &lt;10   &lt;10   &lt;10               BC   &gt;1E6   &lt;10   &lt;10   &lt;10   &lt;10               CAN   2800   &lt;10   &lt;10   &lt;10   &lt;10               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germall III   980   SA   2,700   &lt;10   &lt;10   &lt;10   &lt;10       IPBC   20   EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Unpreserved   0   SA   &gt;1E6   54,000   4,400   20   &gt;1E4               EC   80,000   67,000   &gt;1E4   &gt;1E4   &gt;1E4               PSA   2,000   4200   &gt;1E4   &gt;1E4   &gt;1E4               BC   &gt;1E6   &gt;1E6   &gt;1E4   &gt;1E4   &gt;1E4               CAN   990,000   320,000   &gt;1E4   &gt;1E4   &gt;1E4               AN   380,000   170,000   &gt;1E4   &gt;1E4   &gt;1E4                    
         [0070]    [0070]                                                                                   TABLE 9                           COMPARISON OF ACTIVITY OF LIQUID GERMALL PLUS AND GERMALL       111/ 0.5% OR 0.8% IPBC (SCREENING EMULSION)                    Organ-                           Preservative   Conc.   ism   48 hrs   7 days   14 days   21 days   28 days                    LiqGermPlus       SA   110,000   &lt;10   &lt;10   &lt;10   &lt;10       Germall II   195   EC   2,600   &lt;10   &lt;10   &lt;10   &lt;10       IPBC   2.5   PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   240,000   120   &lt;10   &lt;10   &gt;1E4               AN   230   &lt;10   &lt;10   &lt;10   &lt;10       LiqGermPlus       SA   2,800   &lt;10   &lt;10   &lt;10   &lt;10       Germall II   390   EC   1100   &lt;10   &lt;10   &lt;10   &lt;10       IPBC   5   PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   11,000   &lt;10   &lt;10   &lt;10   20               AN   &lt;10   &lt;10   &lt;10   &lt;10   100       Germall III   195   SA   260,000   &lt;10   &lt;10   &lt;10   &lt;10       IPBC   2.5   EC   4,300   &lt;10   &lt;10   &lt;10   &lt;10       Liquid       PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   150,000   &lt;10   &lt;10   &lt;10   &gt;1E4               AN   200   &lt;10   &lt;10   &lt;10   &lt;10       Germall III   390   SA   170,000   &lt;10   &lt;10   &lt;10   &lt;10       IPBC   5   EC   2,500   &lt;10   &lt;10   &lt;10   &lt;10       Liquid       PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   50,000   &lt;10   &lt;10   &lt;10   &gt;1E4               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germall III   195   SA   70,000   &lt;10   &lt;10   &lt;10   &lt;10       IPBC   4   EC   1400   &lt;10   &lt;10   &lt;10   &lt;10       Liquid       PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   41,000   &lt;10   &lt;10   &lt;10   40               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germall III   390   SA   76,000   &lt;10   &lt;10   &lt;10   &lt;10       IPBC   8   EC   3,400   &lt;10   &lt;10   &lt;10   &lt;10       Liquid       PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   14,000   &lt;10   &lt;10   &lt;10   &lt;10               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Unpreserved   0   SA   &gt;1E6   54,000   4,400   20   &gt;1E4               EC   80,000   67,000   &gt;1E4   &gt;1E4   &gt;1E4               PSA   2,000   4200   &gt;1E4   &gt;1E4   &gt;1E4               BC   &gt;1E6   &gt;1E6   &gt;1E4   &gt;1E4   &gt;1E4               CAN   990,000   320,000   &gt;1E4   &gt;1E4   &gt;1E4               AN   380,000   170,000   &gt;1E4   &gt;1E4   &gt;1E4                    
         [0071]    [0071]                                                                                   TABLE 10                           COMPARISON OF ACTIVITY OF GERMABEN II       AND GERMABEN III (SCREENING EMULSION)                Use   Organ-                           Preservative   Level   ism   8 hrs   7 days   14 days   21 days   28 days                    Germaben II   0.30%   SA   480   &lt;10   &lt;10   &lt;10   &lt;10               EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   20,000   100   2,600   380,000   380,000               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germaben II   0.75%   SA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               AN   &lt;10   &lt;10   &lt;10   &lt;10   100       Germaben III   0.30%   SA   7,000   &lt;10   &lt;10   &lt;10   &lt;10               EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   14,000   120   &gt;1E4   470,000   190,000               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germaben III   0.75%   SA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10           5   EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Unpreserved   0   SA   &gt;1E6   46,000   &gt;1E4   60   &gt;1E4               EC   &gt;1E6   170,000   &gt;1E4   &gt;1E4   &gt;1E4               PSA   690   24000   &gt;1E4   &gt;1E4   &gt;1E4               BC   &gt;1E6   &gt;1E6   &gt;1E4   &gt;1E4   &gt;1E4               CAN   440,000   &gt;1E4   &gt;1E4   &gt;1E4   &gt;1E4               AN   87,000   &gt;1E4   &gt;1E4   &gt;1E4   &gt;1E4                    
         [0072]    [0072]                                                                                   TABLE 11                           COMPARISON OF ACTIVITY OF GERMABEN IIE       AND GERMABEN IIIE (SCREENING EMULSION)                Use   Organ-                           Preservative   Level   ism   48 hrs   7 days   14 days   21 days   28 days                    Germaben IIE   0.30%   SA   580   &lt;10   &lt;10   &lt;10   &lt;10               EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   1,600   &lt;10   &lt;10   &lt;10   &lt;10               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germaben IIE   0.75%   SA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germaben IIIE   0.30%   SA   270   &lt;10   &lt;10   &lt;10   &lt;10               EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   4,000   &lt;10   &lt;10   &lt;10   90               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10       Germaben IIIE   0.75%   SA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               EC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               PSA   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               BC   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               CAN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10               AN   &lt;10   &lt;10   &lt;10   &lt;10   &lt;10                    
         [0073]    Discussion of Challenge Testing Results  
         [0074]    The 28 day challenge results reported in Tables 3-11 above demonstrate the effectiveness of the preservative composition of the invention in a use emulsion composition against a wide range of bacteria and fungi organisms.  
         [0075]    While the invention has been described with particular reference to certain embodiments thereof, it will be understood that changes and modifications may be made which are within the skill of the art. Accordingly, it is intended to be bound only by the following claims, in which: