Abstract:
The antimicrobial preservative composition consisting essentially of (a) from 0.5 to 20 wt. % dihalogenated butane having terminal cyano groups and (b) from 80 to 99.5 wt. % of a heterocyclic poly(hydroxy lower alkylamide) in a weight ratio of between 0.5:2 and 0.5:25 which mixture exhibits a synergistic antimicrobial affect over each of components (a) or (b) taken individually.

Description:
BACKGROUND OF THE INVENTION 
     This invention relates to a water soluble antimicrobial agent suitable for incorporation into a commercial formulation for control of an extended spectrum of fungi, algae and bacterial microorganisms. 
     Diazolidinyl urea and dibromo dicyano butane have been used individually as antimicrobial agents in a wide range of applications, including cosmetic and pharmaceutical formulations, household and industrial cleaners, bactericides, fungicides and algaecides in paints, pools, paper pulp, animal dips and washes, latex emulsions and the like. The allowable concentration of dibromo dicyano butane in cosmetic and topical pharmaceutical applications is somewhat limited due to its skin irritating properties, water insolubility and color; the latter being particularly objectionable in cosmetics such as face creams and makeup. On the other hand, diazolidinyl urea is a bactericide less effective against fungi. 
     Thus, it is the aim of research to develop a colorless, odorless agent for control of a broader spectrum of biological activity which is not harmful to humans and which is effective at low dosage levels so as to minimize dilution and efficacy of other active components present in a formulation. 
     Accordingly, it is an object of this invention to accomplish all of the above objectives by providing a fast acting antimicrobial composition which is effective in small dosages. 
     Another object is to provide a long lasting bactericide effective against infectious microorganisms. 
     Still another object is to provide a non-irritating fungicidal composition which can be obtained by an economical and commercially feasible method of manufacture. 
     These and other objects and advantages will become apparent form the following description and disclosure. 
     SUMMARY OF THE INVENTION 
     In accordance with this invention there is provided a antimicrobial mixture of (a) from 0.5 to 20 wt. % of a dihalogenated butane having two terminal cyano groups and (b) from 80 to 99.5 wt. % of a heterocyclic poly(hydroxylated lower alkyl amide) in a weight ratio of between 0.5:2 and 0.5:25, preferably between about 0.5:3 and about 0.5:10. The halogenated component of the invention can be brominated, chlorinated or iodinated and is preferably a dibrominated, dichlorinated or bromochlorinated compound. The most preferred species is dibromo-1,4-dicyano butane. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The heterocyclic component (b) of the present mixture is defined by the formula: ##STR1## wherein each of R 1 , R 2 , R 3  and R 4  is independently lower alkyl, lower alkyl hydroxy or hydrogen with the proviso that at least one of R 1 , R 2 , R 3  and R 4  is lower alkyl hydroxy. The methyl group is preferred over other lower alkyl groups and diazolidinyl urea is the most preferred heterocyclic species of the invention. 
     Halogenated component (a) is defined by the formula: ##STR2## wherein two of R 5 , R 6 , R 7  and R 8  are halo substituents selected from the group of chlorine, bromine and iodine and the remainder of R 5 , R 6 , R 7  and R 8  are hydrogen, with 1,2-dibromo-1,4-dicyano butane being most preferred. 
     Within the above critical concentrations of components (a) and (b), a mixture of between 0.6 and about 12 wt. % dihalogenated dicyano butane and between about 99.4 and about 88 wt. % of the heterocyclic poly(hydroxyalkyl amide) is preferred. Below 0.5 wt. % of (a), fungicidal control is minimal and is substantially similar to the use of the heterocyclic component alone; whereas above 20 wt. % of (a) its water insolubility asserts its influence on the mixture and uniform distribution of the components in aqueous solutions and formulations is not achieved. Additionally, skin irritation occurs at concentrations above 20 wt. % (a). The ratio of (a) to (b) is also critical for obtaining synergism of the mixture. 
     The broad spectrum of pathogens controlled by the present antimicrobial mixture encompasses fungi, algae and microorganisms, including gram negative and gram positive bacteria, representative species of which are C. albicans (CAN. ATCC #10231); A. niger (AN, ATCC #16404); P. aeruginosa (PSA. ATCC #9027); E.coli (EC, ATCC #8739); S. aureus (SA. ATCC #6538); B. cepacia (BC, ATCC #25416); red algae, Rhodophyta; Clostridium botulinum, etc. 
     The concentration of the above mixture in a formulation can vary, depending in part on the degree of infection or infestation and the type of microbe to be controlled. Generally, the antimicrobial mixture can be incorporated in a commercial formulation at a concentration of between about 0.01 and about 3 wt. %, based on total composition. More specifically, for cosmetic or pharmaceutical formulations, the amount of the present mixture need not exceed 1.0 wt. %; however, for industrial or household cleaners, up to 5 wt. % can be employed when desired, depending on the pathogenic nature of the microorganism and the extent of infection. 
     The present mixture can be conveniently and economically prepared by merely mixing components (a) and (b) under ambient conditions of temperature and pressure until a homogeneous composition is obtained and then freeze dried to provide a free flowing powder. Alternatively, either or both of the components can be dissolved in a mutual solvent before or during mixing to provide a liquid solution or a stable emulsion, suspension or dispersion containing up to about 40% solids. Suitable solvents for the components include alkanols having from 1 to 6 carbon atoms, propylene glycol, butylene glycol, PEG 200-400, phenoxy ethanol and deionized water. Components (a) and/or (b) can be added to the solvent in the desired concentration or the components may be added directly, in a liquid or solid state, to a formulation which contains a suitable solvent or emulsifying agent or emulsifying mixture. 
     As stated above, the present mixture can be incorporated into a commercial formulation as a dry powder, aqueous dispersion, emulsion or suspension depending on the option of the consumer. When employed as a liquid in aqueous medium, the composition usually contains other excipients such as a wetting agent, emulsifier, anionic or cationic surfactant, etc. and additionally may contain a thickener when desired. 
     Some typical formulations for the present mixture when employed as an emulsion are the following. 
     
         ______________________________________STANDARD EMULSIONPhase     Ingredient       Wt. %______________________________________A         Stearic Acid     5.0A         Mineral Oil      2.5A         Cetyl Alcohol    1.0A         Laneth-5 + Ceteth-5 +                      0.5     oleth-5 + Steareth-5*A         Glycerol Monostearate +                      1.5     Polyoxyethylene StearateB         Triethanol amine (99%)                      1.0D         Citric Acid (30%)                      0.6C         Present Antimicrobial mixture                      0.1-3B         Distilled Water  qs     Total            100.0______________________________________ 
    
     The above emulsion is prepared by mixing the ingredients of Phase A and heating the mixture to 75° C. Separately mixing and heating the ingredients of Phase B to 75° C. and then adding Phase A to Phase B under constant agitation. Cooling the resulting mixture to about 50° C. before adding C during agitation and then cooling to 40° C. and adding D with continued stirring; after which the mixture is allowed to cool to room temperature. The pH of the emulsion should be about 7. 
     
         ______________________________________ANIONIC EMULSIONPhase    Ingredient         Wt. %______________________________________A        Distilled water    59.1A        Carbomer (Crosslinked polymer                       10.0    of acrylic acid)B        Octyl palmitate    5.0B        Stearic acid       3.0B        Caprylic, caproic triglyceride                       5.0B        Ethyl, hexyl p-methoxy cinnamate                       5.0B        Cetearyl alcohol ceteareth-20                       1.5B        Cetearyl alcohol   0.5C        Triethanolamine (99%)                       1.3D        Propylene glycol   4.0D        Present antimicrobial mixture                       1.6E        Hydrolyzed Collagen                       0.5E        Aloe powder 200X   0.1E        Distilled water    qs    Total              100.0%______________________________________ 
    
     The above emulsion was prepared by separately mixing and heating phases A and B to 75° C. and adding phase B to A with continued stirring to obtain a uniform mixture. Phase C is then added followed by Phase D during cooling of the mixture. Finally Phase E is added at 35-40° C. and mixing continued while cooling to room temperature. 
     
         ______________________________________NONIONIC EMULSIONPhase   Ingredient           Wt. %______________________________________A       Distilled water      69.8A       Carbomer             10.0B       Octyl palmitate      5.0B       Cetearyl alcohol Ceteareth-20                        2.0B       Glyceryl stearate, Laureth-23                        2.5B       Mineral oil          5.0C       Triethanolamine (99%)                        0.2D       Antimicrobial mixture of this invention                        0.5E       Hydrolyzed Collagen  0.5E       Distilled water      qs   Total                100.0%______________________________________ 
    
     The non-ionic emulsion is prepared by separately mixing and heating Phases A and B to 75° C. and adding Phase B to Phase A with constant stirring to obtain a uniform mixture. Phase C, followed by Phase D are the added and mixed while allowing the mixture to cool. Finally Phase E is added and mixed while the mixture is cooled to room temperature. 
     A few representative formulations in which the present mixture is employed as the antimicrobial agent include the following. 
     
         ______________________________________SHAMPOOIngredient           wt. %______________________________________Distilled water      qsBlend                30.00Sodium laureth sulfateSodium lauryl sulfateLauramide DEACocamide DEACocamidopropyl betaineSodium Cumene sulfonatePropylene glycolCitric acidBlend                6.00Sodium laureth sulfateGlycol distearateLaureth alcoholCocamide MEAPresent antimicrobial mixture                1.00Sodium chloride (20% aqueous)                2.25Citric acid (20% aqueous)                0.10Total                100.00%______________________________________ 
    
     This formulation is prepared by heating water to 40° C. and adding the remaining ingredients sequentially. The following formulations are similarly prepared. 
     
         ______________________________________Ingredients             wt. %______________________________________CONTACT LENS CLEANERPolyhexamethylene biguanide (20% active)                   5.0HCl saltMicrobicidal mixture of this invention                   0.4Distilled water         qsTotal                   100.0%DENTURE ADHESIVELight mineral oil       19.0White petroleum         19.0Na/Ca salts of MVE/MA copolymer*                   18.5Natural non-crosslinked guar                   42.5Microbicidal mixture of this invention                   1.0Total                   100.0%HAIR CONDITIONER80% aqueous Polyvinylpyrrolidone/silicone                   3.0oil (80/20) in glycerol stearate +quaternized ammonium surfactantGlycerol stearate wax   3.5Cetearyl alcohol        3.0Antimicrobicidial mixture of this invention                   0.5Distilled water         90.0Total                   100.0%SYNDET TOILET BAR SOAPStearic acid            36.5Alkyl amide (Armid HT)  5.0Hydrogenated tallow glycerides                   5.0Sodium cocoyl isethionate                   43.0Microbicidal mixture of this invention                   3.0Distilled water         7.5Syndet Flakes           100.0%Perfume                 1.0Citric acid             0.6Water dye solution      2.5Titanium dioxide        0.4______________________________________ *MVE/MA is methyl vinyl ether/maleic anhydride copolymer 
    
    
    
     The advantageous properties of the present mixture are illustrated by the following test data. 
     A. WATER SOLUBILITY 
     The water solubility or insolubility of mixtures of 1,2-dibromo-1,4-dicyano butane (a&#39;) to diazolidinyl urea (b&#39;) in 1% aqueous solutions at room temperature is shown in following Table I. 
     
                       TABLE I______________________________________Weight ratio of (a&#39;) to (b&#39;)______________________________________ 0.5:99.5              soluble 0.8:99.2              soluble 1.0:99.0              soluble 2.0:98.0              soluble 5.0:95.0              soluble10.0:90.0              soluble15.0:85.0              soluble20.0:80.0              soluble22.0:78.0              insoluble______________________________________ 
    
     The above results demonstrate that mixtures of (a) to (b) at 20:80 are still soluble; however, with only a 2% increase in the amount of (a), the mixture becomes insoluble. 
     B. SYNERGISM INDEX OF THE PRESENT MIXTURE 
     Tables II through VI which follow demonstrate the effective synergistic interaction between components (a) and (b) against a broad range of fungi and gram negative and gram positive bacteria. The Synergism Index was determined by the mathematical treatment of data described by Kull et al. in Applied Microbiology, volume 9, pages 538-541 (1961) using the following formula: 
     
         Synergisn Index (SI)=QA/Qa+QB/Qb 
    
     where 
     QA is the quantity of compound (a) in the mixture, producing an endpoint; 
     Qa is the quantity of compound (a) acting alone, producing an endpoint; 
     QB is the quantity of compound (b) in the mixture, producing an endpoint and 
     Qb is the quantity of compound (b) acting alone, producing an endpoint. 
     When SI is equal to 1, a mere additive effect of the components in the mixture is indicated; however, when SI is less than 1, synergism has occurred and when SI is greater than 1, antagonism of the two components is indicated. Although the SI is greater than 1 for control of certain organisms, synergistic control of others is still viable as shown by the following data. 
     According to the above scientifically approved method of determining synergism, the quantity of dibromo-1,4-cyano butane (DBDCB) in the mixture with diazolidonyl urea (DAD) is compared with the quantity of dibromo-1,4-dicyano butane alone which is required to reach the same endpoint, i.e. to produce the same microbiocidal effect as the mixture. 
     In the following Tables II-VI various concentrations of the present mixture were inoculated with the following pathogens. 
     
                       TABLE II______________________________________0.5% DBDCB (0.5:99.5 DBDCB to DAD)Use Level   Organism Oa      Ob    OA    OB    SI______________________________________0.05    AN       1000    3200  2.5   497.5 0.160.05    CAN      400     15000 2.5   497.5 0.040.05    EC       500     1600  2.5   497.5 0.320.05    BC       1500    1250  2.5   497.5 0.400.05    PSA      500     1600  2.5   497.5 0.320.05    SA       500     1600  2.5   497.5 0.320.1     AN       1000    3200  5     995   0.320.1     CAN      400     15000 5     995   0.080.1     EC       500     1600  5     995   0.630.1     BC       1500    1250  S     995   0.800.1     PSA      500     1600  5     995   0.630.1     SA       500     1600  5     995   0.630.2     AN       1000    3200  10    1990  0.630.2     CAN      400     15000 10    1990  0.160.2     EC       500     1600  10    1990  1.260.2     BC       1500    1250  10    1990  1.600.2     PSA      500     1600  10    1990  1.260.2     SA       500     1600  10    1990  1.26______________________________________ 
    
     
                       TABLE III______________________________________2% DBDCB (2.0:98.0 DBDCB to DAD)Use Level   Organism Oa      Ob    OA    OB    SI______________________________________0.05    AN       1000    3200  20    490   0.170.05    CAN      400     15000 20    490   0.080.05    EC       500     1600  20    490   0.350.05    BC       1500    1250  20    490   0.410.05    PSA      500     1600  20    490   0.350.05    SA       500     1600  20    490   0.350.1     AN       1000    3200  20    980   0.330.1     CAN      400     15000 20    980   0.120.1     EC       500     1600  20    980   0.650.1     BC       1500    1250  20    980   0.800.1     PSA      500     1600  20    980   0.650.1     SA       500     1600  20    980   0.650.2     AN       1000    3200  40    1960  0.650.2     CAN      400     15000 40    1960  0.230.2     EC       500     1600  40    1960  1.310.2     BC       1500    1250  40    1960  1.590.2     PSA      500     1600  40    1960  1.310.2     SA       500     1600  40    1960  1.31______________________________________ 
    
     
                       TABLE IV______________________________________1% DBDCB (1.0:99.0 DBDCB to DAD)Use Level   Organism Oa      Ob    OA    OB    SI______________________________________0.05    AN       1000    3200  5     495   0.160.05    CAN      400     15000 5     495   0.050.05    EC       500     1600  5     495   0.320.05    BC       1500    1250  5     495   0.400.05    PSA      500     1600  5     495   0.320.05    SA       500     1600  5     495   0.320.1     AN       1000    3200  10    990   0.320.1     CAN      400     15000 10    990   0.090.1     EC       500     1600  10    990   0.640.1     BC       1500    1250  10    990   0.800.1     PSA      500     1600  10    990   0.640.1     SA       500     1600  10    990   0.640.2     AN       1000    3200  20    1980  0.640.2     CAN      400     15000 20    1980  0.180.2     EC       500     1600  20    1980  1.280.2     BC       1500    1250  20    1980  1.600.2     PSA      500     1600  20    1980  1.280.2     SA       500     1600  20    1980  1.28______________________________________ 
    
     
                       TABLE V______________________________________10% DBDCB (10.0:90.0 DBDCB to DAD)Use Level   Organism Oa      Ob    OA    OB    SI______________________________________0.05    AN       1000    3200  50    450   0.190.05    CAN      400     15000 50    450   0.160.05    EC       500     1600  50    450   0.380.05    BC       1500    1250  50    450   0.390.05    PSA      500     1600  50    450   0.380.05    SA       500     1600  50    450   0.380.1     AN       1000    3200  100   900   0.380.1     CAN      400     15000 100   900   0.310.1     EC       500     1600  100   900   0.760.1     BC       1500    1250  100   900   0.790.1     PSA      500     1600  100   900   0.760.1     SA       500     1600  100   900   0.760.2     AN       1000    3200  200   1800  0.760.2     CAN      400     15000 200   1800  0.620.2     EC       500     1600  200   1800  1.530.2     BC       1500    1250  200   1800  1.570.2     PSA      500     1600  200   1800  1.530.2     SA       500     1600  200   1800  1.53______________________________________ 
    
     
                       TABLE VI______________________________________5% DBDCB (5.0:95.0 DBDCB to DAD)Use Level   Organism Oa      Ob    OA    OB    SI______________________________________0.05    AN       1000    3200  25    475   0.170.05    CAN      400     15000 25    475   0.090.05    EC       500     1600  25    475   0.350.05    BC       1500    1250  25    475   0.400.05    PSA      500     1600  25    475   0.350.05    SA       500     1600  25    475   0.350.1     AN       1000    3200  50    950   0.350.1     CAN      400     15000 50    950   0.190.1     EC       500     1600  50    950   0.690.1     BC       1500    1250  50    950   0.790.1     PSA      500     1600  50    950   0.690.1     SA       500     1600  50    950   0.690.2     AN       1000    3200  100   1900  0.690.2     CAN      400     15000 100   1900  0.380.2     EC       500     1600  100   1900  1.390.2     BC       1500    1250  100   1900  1.590.2     PSA      500     1600  100   1900  1.390.2     SA       500     1600  100   1900  1.39______________________________________ 
    
     PRESERVATIVE ACTIVITY (CHALLENGE TEST) 
     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: 
     
         ______________________________________                 % wt.______________________________________Phase AStearic Acid            5.00Mineral Oil             2.50Cetyl Alcohol           1.00Lareth-5 and Ceteth-5 and Steareth-5                   0.50Glycerol Monostearate and Polyoxyethylene                   1.50StearatePhase BDeionized Water         88.0Triethanolamine 99%     1.0Citric Acid 30% aqueous solution                   0.60Preservative Admixture  qs______________________________________ 
    
     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. 
     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 VII-XVII below. 
     Tables VII-X are in the Standard Emulsion 
     
                       TABLE VII______________________________________1:99 DBDCB/Diazolidinyl UreaTest Level  Organism 48 Hours 7 Days                          14 Days                                21 Days                                      28 Days______________________________________0.05%  AN       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05%  CAN      3,100    70    &lt;10   &lt;10   14,0000.05%  EC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05%  BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05%  PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05%  SA       6,300    &lt;10   &lt;10   &lt;10   &lt;100.10%  AN       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  CAN      100      &lt;10   &lt;10   &lt;10   &lt;100.10%  EC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  SA       80       &lt;10   &lt;10   &lt;10   &lt;100.20%  AN       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  CAN      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  EC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  SA       &lt;10      &lt;10   &lt;10   &lt;10   &lt;10______________________________________Unpreserved ControlOrganism 48 Hours     7 Days    14 Days______________________________________AN       58,000       42,000    420,000CAN      160,000      83,000    470,000EC       1,400,000    3,800,000 3,800,000BC       36,000,000   30,000,000                           18,000,000PSA      120          45,000    45,000SA       7,500,000    93,000    1,600______________________________________Inoculum ConcentrationOrganism      0 Hours  21 Days______________________________________AN            20,000   120,000CAN           3,400,000                  250,000,000EC            6,100,000                  9,000,000BC            230,000  30,000,000PSA           3,500,000                  11,000,000SA            4,000,000                  11,000,000______________________________________ 
    
     
                       TABLE VIII______________________________________1:999 DBDCB/Diazolidinyl UreaTest Level  Organism 48 Hours 7 Days                          14 Days                                21 Days                                      28 Days______________________________________0.05%  AN       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05%  CAN      6,300    60    10    10    22,0000.05%  EC       270      &lt;10   &lt;10   &lt;10   &lt;100.05%  BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05%  PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05%  SA       5,300    &lt;10   &lt;10   &lt;10   &lt;100.10%  AN       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  CAN      200      10    &lt;10   &lt;10   &lt;100.10%  EC       20       &lt;10   &lt;10   &lt;10   &lt;100.10%  BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  SA       400      &lt;10   &lt;10   &lt;10   &lt;100.20%  AN       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  CAN      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  EC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  SA       &lt;10      &lt;10   &lt;10   &lt;10   &lt;10______________________________________Unpreserved ControlOrganism 48 Hours     7 Days    14 Days______________________________________AN       58,000       42,000    420,000CAN      160,000      83,000    470,000EC       1,400,000    3,800,000 3,800,000BC       36,000,000   30,000,000                           18,000,000PSA      120          45,000    45,000SA       7,500,000    93,000    1,600______________________________________Inoculum ConcentrationOrganism      0 Hours  21 Days______________________________________AN            20,000   120,000CAN           3,400,000                  250,000,000EC            6,100,000                  9,000,000BC            230,000  30,000,000PSA           3,500,000                  11,000,000SA            4,000,000                  11,000,000______________________________________ 
    
     
                       TABLE IX______________________________________1:4 DBDCB/Diazolidinyl UreaTest Level  Organism 48 Hours 7 Days                          14 Days                                21 Days                                      28 Days______________________________________0.05%  AN       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05%  CAN      50       &lt;10   &lt;10   &lt;10   &lt;100.05%  EC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05%  BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05%  PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05%  SA       50       &lt;10   &lt;10   &lt;10   &lt;100.10%  AN       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  CAN      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  EC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  SA       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  AN       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  CAN      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  EC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  SA       &lt;10      &lt;10   &lt;10   &lt;10   &lt;10______________________________________Unpreserved ControlOrganism 48 Hours     7 Days    14 Days______________________________________AN       58,000       42,000    420,000CAN      160,000      83,000    470,000EC       1,400,000    3,800,000 3,800,000BC       36,000,000   30,000,000                           18,000,000PSA      120          45,000    45,000SA       7,500,000    93,000    1,600______________________________________Inoculum ConcentrationOrganism      0 Hours  21 Days______________________________________AN            14,000   20,000CAN           240,000  340,000EC            640,000  6,100,000BC            690000   120,000PSA           1,100,000                  3,500,000SA            1,100,000                  4,000,000______________________________________ 
    
     
                       TABLE X______________________________________1:9 DBDCB/Diazolidinyl UreaTest Level  Organism 48 Hours 7 Days                          14 Days                                21 Days                                      28 Days______________________________________0.05%  AN       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05%  CAN      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05%  EC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05%  BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05%  PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05%  SA       10       &lt;10   &lt;10   &lt;10   &lt;100.10%  AN       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  CAN      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  EC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  SA       100      &lt;10   &lt;10   &lt;10   &lt;100.20%  AN       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  CAN      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  EC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  SA       &lt;10      &lt;10   &lt;10   &lt;10   &lt;10______________________________________Unpreserved ControlOrganism 48 Hours     7 Days    14 Days______________________________________AN       58,000       42,000    420,000CAN      160,000      83,000    470,000EC       1,400,000    3,800,000 3,800,000BC       36,000,000   30,000,000                           18,000,000PSA      120          45,000    45,000SA       7,500,000    93,000    1,600______________________________________Inoculum ConcentrationOrganism      0 Hours  21 Days______________________________________AN            14,000   20,000CAN           240,000  340,000EC            640,000  6,100,000BC            690000   120,000PSA           1,100,000                  3,500,000SA            1,100,000                  4,000,000______________________________________ 
    
     Tables XI-XII are in a Anion Emulsion 
     
                       TABLE XI______________________________________6:94 DBDCB/Diazolidinyl Urea           48Test Level  Organism Hours   7 Days                         14 Days                               21 Days                                     28 Days______________________________________0.05%  AN       17000   100   &lt;10   &lt;10   &lt;100.05%  CAN      47,000  25,000                         320,000                               230,000                                     6,000,0000.05%  EC       350,000 &lt;10   &lt;10   &lt;10   &lt;100.05%  BC       &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.05%  PSA      &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.05%  SA       6,300   &lt;10   &lt;10   &lt;10   &lt;100.10%  AN       &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.10%  CAN      2,500   2,100 19,000                               70,000                                     80,0000.10%  EC       &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.10%  BC       &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.10%  PSA      &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.10%  SA       590     &lt;10   &lt;10   &lt;10   &lt;100.20%  AN       &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.20%  CAN      &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.20%  EC       &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.20%  BC       &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.20%  PSA      &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.20%  SA       &lt;10     &lt;10   &lt;10   &lt;10   &lt;10______________________________________Unpreserved ControlOrganism 48 Hours     7 Days    14 Days______________________________________AN       180,000      700,000   1,500,000CAN      2,800,000    6,000,000 2,100,000EC       100,000,000  100,000,000                           100,000,000BC       100,000,000  100,000,000                           100,000,000PSA      100,000,000  100,000,000                           100,000,000SA       100,000,000  100,000,000                           76,000,000______________________________________Inoculum ConcentrationOrganism      0 Hours  21 Days______________________________________AN            80,000   150,000CAN           630,000  1,900,000EC            1,900,000                  2,400,000BC            1,900,000                  4,000,000PSA           2,300,000                  3,600,000SA            700,000  2,100,000______________________________________ 
    
     
                       TABLE XII______________________________________12:88 DBDCB/Diazolidinyl UreaTest Level  Organism 48 Hours 7 Days                          14 Days                                21 Days                                      28 Days______________________________________0.05%  AN       17000    100   &lt;10   &lt;10   200.05%  CAN      51,000   1,500 1,000 &lt;10   7,2000.05%  EC       4,800    &lt;10   &lt;10   &lt;10   &lt;100.05%  BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05%  PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05%  SA       50,000   &lt;10   &lt;10   &lt;10   &lt;100.10%  AN       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  CAN      80       &lt;10   &lt;10   &lt;10   200.10%  EC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10%  SA       590      &lt;10   &lt;10   &lt;10   &lt;100.20%  AN       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  CAN      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  EC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20%  SA       &lt;10      &lt;10   &lt;10   &lt;10   &lt;10______________________________________Unpreserved ControlOrganism 48 Hours     7 Days    14 Days______________________________________AN       180,000      700,000   1,500,000CAN      2,800,000    6,000,000 2,100,000EC       100,000,000  100,000,000                           100,000,000BC       100,000,000  100,000,000                           100,000,000PSA      100,000,000  100,000,000                           100,000,000SA       100,000,000  100,000,000                           76,000,000______________________________________Inoculum ConcentrationOrganism      0 Hours  21 Days______________________________________AN            80,000   150,000CAN           630,000  1,900,000EC            1,900,000                  2,400,000BC            1,900,000                  4,000,000PSA           2,300,000                  3,600,000SA            700,000  2,100,000______________________________________ 
    
     Tables XIII-XVI are in a Nonion Emulsion 
     
                                           TABLE XIII__________________________________________________________________________1:49 DBDCB/Diazolidinyl UreaTest     48Level    Organism    Hours        7 Days             14 Days                  21 Days                       28 Days__________________________________________________________________________0.05%    AN   160 &lt;10  &lt;10  &lt;10  300.05%    CAN  43,000        3,400,000             3,400,000                  16000000                       29,000,0000.05%    EC   4,600        &lt;10  &lt;10  &lt;10  &lt;100.05%    BC   &lt;10 &lt;10  &lt;10  &lt;10  &lt;100.05%    PSA  &lt;10 &lt;10  &lt;10  &lt;10  &lt;100.05%    SA   1,100        &lt;10  &lt;10  &lt;10  &lt;100.10%    AN   &lt;10 &lt;10  &lt;10  &lt;10  &lt;100.10%    CAN  &lt;10 320  84,000                  510,000                       33,000,0000.10%    EC   30  &lt;10  &lt;10  &lt;10  &lt;100.10%    BC   &lt;10 &lt;10  &lt;10  &lt;10  &lt;100.10%    PSA  &lt;10 &lt;10  &lt;10  &lt;10  &lt;100.10%    SA   590 &lt;10  &lt;10  &lt;10  &lt;100.20%    AN   &lt;10 &lt;10  &lt;10  &lt;10  &lt;100.20%    CAN  &lt;10 &lt;10  &lt;10  &lt;10  &lt;100.20%    EC   &lt;10 &lt;10  &lt;10  &lt;10  &lt;100.20%    BC   &lt;10 &lt;10  &lt;10  &lt;10  &lt;100.20%    PSA  &lt;10 &lt;10  &lt;10  &lt;10  &lt;100.20%    SA   &lt;10 &lt;10  &lt;10  &lt;10  &lt;10__________________________________________________________________________Unpreserved ControlOrganism  48 Hours   7 Days                   14 Days__________________________________________________________________________AN     150,000    160,000                   160,000CAN    2,400,000  420,000                   420,000EC     100,000,000             100,000,000                   1,000,000BC     100,000,000             100,000,000                   1,000,000PSA    100,000,000             100,000,000                   1,000,000SA     9,700,000  520,000                   630__________________________________________________________________________Inoculum ConcentrationOrganism      0 Hours              21 Days__________________________________________________________________________AN            190,000              26,000CAN           1,900,000              380,000EC            4,600,000              700,000BC            5,200,000              310,000PSA           5,200,000              14,000,000SA            8,400,000              960,000__________________________________________________________________________ 
    
     
                       TABLE XIV______________________________________1:20 DBDCB/Diazolidinyl UreaTest           48Level Organism Hours   7 Days                        14 Days                              21 Days                                    28 Days______________________________________0.05% AN       90      &lt;10   &lt;10   &lt;10   600.05% CAN      180     220   3,600 320,000                                    120,000,0000.05% EC       1,500   &lt;10   &lt;10   &lt;10   &lt;100.05% BC       &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.05% PSA      &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.05% SA       1,200   &lt;10   &lt;10   &lt;10   &lt;100.10% AN       &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.10% CAN      &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.10% EC       &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.10% BC       &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.10% PSA      &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.10% SA       &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.20% AN       &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.20% CAN      &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.20% EC       &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.20% BC       &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.20% PSA      &lt;10     &lt;10   &lt;10   &lt;10   &lt;100.20% SA       &lt;10     &lt;10   &lt;10   &lt;10   &lt;10______________________________________Unpreserved ControlOrganism 48 Hours     7 Days    14 Days______________________________________AN       150,000      160,000   160,000CAN      2,400,000    420,000   420,000EC       100,000,000  100,000,000                           1,000,000BC       100,000,000  100,000,000                           1,000,000PSA      100,000,000  100,000,000                           1,000,000SA       9,700,000    520,000   630______________________________________Inoculum ConcentrationOrganism      0 Hours  21 Days______________________________________AN            190,000  26,000CAN           1,900,000                  380,000EC            4,600,000                  700,000BC            5,200,000                  310,000PSA           5,200,000                  14,000,000SA            8,400,000                  960,000______________________________________ 
    
     
                       TABLE XV______________________________________6:94 DBDCB/Diazolidinyl UreaTest           48Level Organism Hours    7 Days                         14 Days                               21 Days                                     28 Days______________________________________0.05% AN       30       &lt;10   &lt;10   &lt;10   200.05% CAN      &lt;10      20    380   36,000                                     90,0000.05% EC       230      &lt;10   &lt;10   &lt;10   &lt;100.05% BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05% PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05% SA       71,000   &lt;10   &lt;10   &lt;10   &lt;100.10% AN       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10% CAN      &lt;10      &lt;10   &lt;10   &lt;10   130,0000.10% EC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10% BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10% PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10% SA       60       &lt;10   &lt;10   &lt;10   &lt;100.20% AN       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20% CAN      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20% EC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20% BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20% PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20% SA       &lt;10      &lt;10   &lt;10   &lt;10   &lt;10______________________________________Unpreserved ControlOrganism 48 Hours      7 Days   14 Days______________________________________AN       160,000       40,000   40,000CAN      4,400,000     960,000  1,000,000EC       100,000,000   1,000,000                           1,000,000BC       100,000,000   1,000,000                           1,000,000PSA      100,000,000   1,000,000                           1,000,000SA       27,000,000    9,1000   6,500______________________________________Inoculum ConcentrationOrganism      0 Hours  21 Days______________________________________AN            190,000  26,000CAN           1,900,000                  380,000EC            4,600,000                  700,000BC            5,200,000                  310,000PSA           5,200,000                  14,000,000SA            8,400,000                  960,000______________________________________ 
    
     
                       TABLE XVI______________________________________12:88 DBDCB/Diazolidinyl UreaTest           48Level Organism Hours    7 Days                         14 Days                               21 Days                                     28 Days______________________________________0.05% AN       70       &lt;10   &lt;10   &lt;10   100.05% CAN      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05% EC       150      &lt;10   &lt;10   &lt;10   &lt;100.05% BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05% PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05% SA       8,700    &lt;10   &lt;10   &lt;10   &lt;100.10% AN       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10% CAN      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10% EC       10       &lt;10   &lt;10   &lt;10   &lt;100.10% BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10% PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10% SA       60       &lt;10   &lt;10   &lt;10   &lt;100.20% AN       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20% CAN      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20% EC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20% BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20% PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.20% SA       &lt;10      &lt;10   &lt;10   &lt;10   &lt;10______________________________________Unpreserved ControlOrganism 48 Hours      7 Days   14 Days______________________________________AN       160,000       40,000   40,000CAN      4,400,000     960,000  1,000,000EC       100,000,000   1,000,000                           1,000,000BC       100,000,000   1,000,000                           1,000,000PSA      100,000,000   1,000,000                           1,000,000SA       27,000,000    9,1000   6,500______________________________________Inoculum ConcentrationOrganism      0 Hours  21 Days______________________________________AN            190,000  26,000CAN           1,900,000                  380,000EC            4,600,000                  700,000BC            5,200,000                  310,000PSA           5,200,000                  14,000,000SA            8,400,000                  960,000______________________________________ 
    
     
                       TABLE XVII______________________________________Shampoo2:98 DBDCB/Diazolidinyl UreaTest           48Level Organism Hours    7 Days                         14 Days                               21 Days                                     28 Days______________________________________0.025% AN       70,000   &lt;10   &lt;10   &lt;10   &lt;100.025% CAN      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.025% EC       460      &lt;10   &lt;10   &lt;10   &lt;100.025% BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.025% PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.025% SA       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05% AN       2,400    &lt;10   &lt;10   &lt;10   &lt;100.05% CAN      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05% EC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05% BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05% PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.05% SA       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10% AN       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10% CAN      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10% EC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10% BC       &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10% PSA      &lt;10      &lt;10   &lt;10   &lt;10   &lt;100.10% SA       &lt;10      &lt;10   &lt;10   &lt;10   &lt;10______________________________________Unpreserved ControlOrganism 48 Hours      7 Days   14 Days______________________________________AN       160,000       1800000  40,000CAN      80            2300     3,000EC       30,000        4100     40BC       1,000,000     1,000,000                           1,000,000PSA      1,000,000     1,000,000                           10,000SA       &lt;10           &lt;10      &lt;10______________________________________Inoculum ConcentrationOrganism       0 Hours   21 Days______________________________________AN             110,000   70,000CAN            460,000   5,700,000EC             25,000,000                    170,000BC             74,000    3,800,000PSA            3,900,000 2,300,000SA             120,000   1,800,000______________________________________ 
    
     The results shown in Tables VII-XVII demonstrate that the compositions of the invention are completely effective against the tested organisms in comparison to the unpreserved controls. 
     As indicated above, it is only within the critical weight ratios and concentration ranges of (a) and (b) which are proscribed by this invention that the synergistic antimicrobial effect of the mixture can be increased at least 3 fold over that of the individual components when used alone at the same dosage level. 
     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: