AU1530697A

AU1530697A – Antimicrobial compositions
– Google Patents

AU1530697A – Antimicrobial compositions
– Google Patents
Antimicrobial compositions

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Publication number
AU1530697A

AU1530697A
AU15306/97A
AU1530697A
AU1530697A
AU 1530697 A
AU1530697 A
AU 1530697A
AU 15306/97 A
AU15306/97 A
AU 15306/97A
AU 1530697 A
AU1530697 A
AU 1530697A
AU 1530697 A
AU1530697 A
AU 1530697A
Authority
AU
Australia
Prior art keywords
antimicrobial
ointment
acid
test
weight
Prior art date
1996-09-06
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)

Abandoned

Application number
AU15306/97A
Inventor
Jeffrey F Andrews
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

3M Co

Original Assignee
Minnesota Mining and Manufacturing Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
1996-09-06
Filing date
1997-01-07
Publication date
1998-03-26

1997-01-07
Application filed by Minnesota Mining and Manufacturing Co
filed
Critical
Minnesota Mining and Manufacturing Co

1998-03-26
Publication of AU1530697A
publication
Critical
patent/AU1530697A/en

Status
Abandoned
legal-status
Critical
Current

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Classifications

A—HUMAN NECESSITIES

A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE

A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES

A61K31/00—Medicinal preparations containing organic active ingredients

A61K31/21—Esters, e.g. nitroglycerine, selenocyanates

A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids

A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin

A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms

A—HUMAN NECESSITIES

A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING

A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS

A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids

A01N37/12—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing the group, wherein Cn means a carbon skeleton not containing a ring; Thio analogues thereof

A—HUMAN NECESSITIES

A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE

A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES

A61K9/00—Medicinal preparations characterised by special physical form

A61K9/0012—Galenical forms characterised by the site of application

A61K9/0041—Mammary glands, e.g. breasts, udder; Intramammary administration

A—HUMAN NECESSITIES

A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE

A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics

A61P31/04—Antibacterial agents

Description

ANTIMICROBIAL COMPOSITIONS
Field of the Invention This invention relates to antimicrobial compositions useful for topical application to an animal’s skin to kill infectious microorganisms and moisturize the animal’s skin.
Background of the Invention Lotions, salves and the like can be applied to the skin for various restorative purposes. For example, lotions may be used to condition or moisturize the texture of dry skin, or they may have antimicrobial properties to prevent or to treat infections. Thus, a diverse range of products is potentially available for human and veterinary use.
Of particular importance in the veterinary context is the prevention of mastitis in cows and the moisturizing of cow teats. Cows are susceptible to mastitis, and cow teat skin is susceptible to drying. Currently, various «teat dips» are available for treatment of cow teats to prevent bacterial colonization. Available teat dips, however, include aqueous carriers (or vehicles) which can cause frostbite and skin damage in cold weather. Consequently, the use of teat dips is often interrupted during cold weather, increasing the likelihood of bacterial colonization of cow teats, resulting in bovine mastitis.
There is a continuing demand for effective and inexpensive moisturizing or antimicrobial compositions useful for treating animal and human skin, and in particular for treating cow teats to moisturize and prevent mastitis. There is particular need for such a composition that may be used in cold weather without risk of frostbite.

Summary of the Invention
In a first aspect, the invention involves an antimicrobial ointment including an antimicrobial fatty acid monoester of a polyhydroxy alcohol, an ointment vehicle composition including petrolatum, and a chelating agent.
The polyhydroxy alcohol used to form the antimicrobial monoester preferably is selected from the group consisting of glycerol, propylene glycol and saccharides. The fatty acid used to form the antimicrobial monoester preferably is selected from the group consisting of caproic acid, heptanoic acid, caprylic acid, capric acid, undecanoic acid and lauric acid. Particularly preferred monoesters include glycerol monolaurate and propylene glycol monocaprylate.
The antimicrobial ointment preferably includes at least about 25% by weight of petrolatum and more preferably at least about 50% by weight of petrolatum. The antimicrobial ointment preferably includes between about 0.1% and about 10% by weight of the fatty acid monoester of a polyhydroxy alcohol.
A preferred antimicrobial ointment of the invention also includes at least about 0.1% by weight of a chelating agent, with lactic acid being a preferred chelating agent.
The antimicrobial ointment may include at least about 1% by weight of a viscosity modifying agent. The antimicrobial ointment may also include at least about 0.5% by weight of an inactive emollient. The antimicrobial ointment may include at least about 1% by weight of wax. The antimicrobial ointment may similarly include at least about 0.5% by weight of cetyl alcohol. A preferred antimicrobial ointment of the invention includes

in percent by weight at least about 0.1% glyceryl monolaurate, at least about 0.1% propylene glycol monolaurate, at least about 0.1% lactic acid, and at least about 1% wax. Preferred antimicrobial ointments of the invention produce at least about a 10 5 fold reduction in colony forming units of bacteria in an in vitro bacterial kill test. Similarly, preferred antimicrobial ointments of the invention produce at least about a ten (10) fold reduction in Streptococcus uberis contamination eight hours following application to cow teat skin.
In another aspect, the invention involves a method of producing an antimicrobial composition including the steps of (a) melting a vehicle composition comprising petrolatum, waxes or a mixture thereof; and (b) adding (1) an antimicrobial fatty acid monoester of a polyhydroxy alcohol, and (2) a chelating agent to the melt to form a melt mixture, the melt mixture forming the antimicrobial ointment upon cooling. A preferred antimicrobial ointment produced by this method includes at least about 25% by weight of the vehicle composition and between about 0.1% and about 10% by weight of the monoester. The vehicle composition preferably includes petrolatum.
In another aspect, the invention involves a method of treating a bovine teat comprising the step of applying to skin of the bovine teat an antimicrobial composition including (a) an antimicrobial fatty acid monoester of a polyhydroxy alcohol; (b) a chelating agent; and (c) a vehicle composition, wherein the vehicle composition is effectively water insoluble. The treatment method generally is effective at preventing bacterial colonization of bovine teats as well as at preventing drying of the skin of the teat. The preferred

antimicrobial composition for use in the treatment method includes between about 0.1% and about 10% by weight of the monoester, wherein the polyhydroxy alcohol is selected from the group consisting of glycerol, propylene glycol and saccharides, and wherein the fatty acid is selected from the group consisting of caproic acid, heptanoic acid, caprylic acid, capric acid, undecanoic acid and lauric acid.
The antimicrobial compositions of the invention have significant advantages with respect to moisturizing and skin conditioning capabilities over aqueous teat dips commercially available for preventing bovine mastitis. The antimicrobial compositions are effective against both bacteria and viruses. Furthermore, the products of the invention can be produced with a wide range of consistencies to give the consumer a variety of choices for applying the product. A significant advantage of the compositions is that they can be applied to farm animals during cold weather without risk of frostbite. Other advantages will be apparent from the detailed description and claims.
Description of the Preferred Embodiments The invention involves antimicrobial compositions useful for treating skin to prevent or to cure infection and/or dryness.
In one preferred embodiment, the invention combines an antimicrobial agent and a chelating agent within an ointment vehicle. The antimicrobial ointments include at least one antimicrobial monoester as an antimicrobial agent. The chelating agent is included as a further synergistic component with respect to the antimicrobial properties of the composition. Additional compounds may be added to modify the emollient properties,

the rheology, the aesthetic properties and other aspects of the ointment.
The preferred active antimicrobial agents are fatty acid monoesters of polyhydroxy alcohols. The antimicrobial ointment typically has between 0.01% and 20% by weight of the antimicrobial monoesters, preferably between 0.1% and 10% and more preferably between 0.5% and 5% by weight of the antimicrobial monoesters.
Appropriate polyhydroxy alcohols (i.e., polyols or polyhydric alcohols) suitable for use as a component within the antimicrobial monoesters include glycerol, propylene glycol, polyalkylene glycol such as polyethylene glycol and polypropylene glycol, polyglycerol, and saccharides. Saccharides include monosaccharides such as glucose, disaccharides such as sucrose and polysaccharides such as starch. Preferred polyhydroxy alcohols have less than about eight carbon atoms. Glycerol and propylene glycol are particularly preferred polyhydroxy alcohols for the formation of antimicrobial esters for use in ointments of the invention.
Appropriate fatty acids suitable for use as a component within the antimicrobial monoesters include straight and branched carboxylic acids with greater than about six carbon atoms. The carbon chains of the fatty acids may be saturated, monounsaturated or polyunsaturated. Preferred fatty acids have between about six carbon atoms and about 24 carbon atoms, and more preferably between about 8 carbon atoms and about 16 carbon atoms. Generally, preferred fatty acids include straight chained, saturated fatty acids.
It may be desirable to use a combination of more than one fatty acid monoester of a polyhydroxy alcohol. The combination of two or more monoesters can provide a

broader range of antimicrobial activity relative to a single monoester. Preferred combinations include two or more monoesters of straight chained, saturated fatty acids, with between about six carbon atoms and about twelve carbon atoms. Particularly preferred saturated fatty acids for incorporation into the monoesters include caproic, heptanoic, caprylic, pelargonic, capric, undecanoic, lauric, myristic, and palmitic. Further description of useful fatty acid monoesters may, for example, in U.S. Patent 5,378,731 and references therein. The antimicrobial compositions of the invention contain a nonaqueous vehicle, or carrier, that is preferably water insoluble. The nonaqueous vehicles generally may include organic and inorganic, oils and waxes. Preferred nonaqueous vehicles include ointment vehicles, such as hydrocarbons, especially petrolatum. Preferred antimicrobial ointments have at least about 25% by weight of petrolatum and more preferably at least about 50% by weight of petrolatum. The present inventor has found that the antimicrobial monoesters when used in a petrolatum vehicle generally are as effective as currently available aqueous products.
Suitable chelating agents for use in the antimicrobial compositions of the present invention include those selected from the group consisting of ethylenedia ine tetraacetic (EDTA) acid and its salts, hydroxyalkanoic acids such as lactic acid and citric acid, polyphosphoric acid and its salts, acidic sodium hexametaphosphate (commercially available as SPORIX acidic sodium hexametaphosphate, from International Sourcing, Inc., Upper Saddle River, N.J.), and mixtures thereof. Lactic acid is a preferred chelating agent because it may advantageously function as both a chelator and a

moisturizing agent in the composition of the present invention. The antimicrobial composition generally includes an amount greater than about 0.1% by weight of chelating agent per quantity of antimicrobial ointment, preferably between 0.1% and 5% and more preferably between about 0.5% to about 2% by weight of chelating agent.
The antimicrobial compositions of the invention may contain emollients or moisturizers in addition to the antimicrobial fatty acid monoesters of polyhydroxy alcohols, which may also act as emollients, and the chelating agents, which may also act as a moisturizers (e.g., lactic acid). A variety of typical emollients are suitable for use in the invention. Preferred emollients include cetyl alcohol, stearyl alcohol, lanolin, lanolin derivatives, isostearic acid, esters of isostearic acid, isoεtearyl alcohol, ethylene glycol esters, propylene glycol esters, emollient glycerides and the like. An antimicrobial ointment of the invention, when an emollient is used, generally includes greater than about 1% by weight of emollients, preferably between about 2% and 25% and more preferably between about 5% and 15% by weight of emollients.
The antimicrobial compositions of the invention may also include viscosity modifiers as inert ingredients. The viscosity modifiers can be used to alter the properties of the ointment vehicle to be more suitable for a particular application. Some of the viscosity modifiers are also suitable for use as an ointment vehicle. The viscosity modifiers are used to alter the viscosity and more generally the rheologic properties imparted to the antimicrobial ointment by the ointment vehicle.
Organic solvents may be used as viscosity modifiers as well as beeswax, paraffin, carnauba wax,

polyethylene glycol, ethylene glycol monostearates, ethylene glycol distearates, mineral oil, and synthetic spermaceti wax. The antimicrobial compositions, when containing viscosity modifiers, generally include at least about 1% by weight of viscosity modifier and preferably between about 2% and about 20% by weight of viscosity modifier. The viscosity may be adjusted with such agents to produce a product for application in a particularly desired way. The antimicrobial ointment optionally may include fragrance, fillers and other compounds to alter the cost and aesthetics of the ointment, including materials such
TM as FINSOLV TN , which are useful for improving spreadability and texture. The antimicrobial compositions, including ointments, of the invention have a variety of uses for application to skin. For example, the antimicrobial compositions may be applied to human skin to prevent infections while moisturizing the skin. Similarly, the antimicrobial compositions may be used as a first aid product to treat cuts, scratches, abrasions and the like on animals and humans. The composition may be used as a hoof moisturizer for horses and as a treatment for skin infections on dogs. A preferred use of the antimicrobial composition involves application of the antimicrobial composition to cow teats to prevent bacterial colonization of teat skin while moisturizing the teat. Colonization of teat skin with bacteria is correlated with the development of bovine mastitis. An additional preferred use of the antimicrobial composition of the invention involves application to bovine skin to prevent or cure the viral infection Bovine Herpes Mammallitis.

The invention may be illustrated by way of the following examples.
EXAMPLES
The following examples demonstrate the antimicrobial effectiveness and the skin conditioning of the products of the invention.
A test ointment (nonaqueous teat dip) is prepared from the following ingredients given in percents by weight:
Ingredient % / wt
Lauricidin TM 1.0
Propylene Glycol Monocaprylate 1.0
Lactic Acid 2.0
Cetyl Alcohol 3.0
Lanolin 10.0
White Beeswax 5.0
TM
Finsolv TN 4.0
Ungerer Mask DD 49978™ 0.5
White petrolatum 73.5 Lauricidin TM (glyceryl monolaurate) was purchased from Med-Chem Laboratories Inc., Galena, IL. The propylene glycol monocaprylate was purchased from Uniche a North America, Chicago, IL. The white beeswax was purchased from ACROS Organics, Pittsburgh, PA. Finsolv TN™ is a mixture of alkyl benzoates with the alkyl groups having chain lengths ranging between C12 to C15 purchased from
Finetex, Inc., Spencer, NC. Ungerer Mask DD 49978 TM is a fragrance purchased from Ungerer, Inc. , Lincoln Park, NJ. The white petrolatum was purchased from Pennsylvania Refining Co., Butler, PA.
To produce the test ointment, the white petrolatum and the lanolin were added to a mixing tank. The mixing tank was heated. Mixing was begun when the white

petrolatum and the lanolin were partially melted. After mixing was begun, the other ingredients were added sequentially in the following order: Finsolv TN™, cetyl alcohol, Lauricidin TM, propylene glycol monocaprylate, white beeswax and lactic acid. Heating and mixing was continued while these other ingredients were added. The mixture was heated to 160°F ± 10°F (71°C±6°C) to produce a clear liquid melt. Heating was stopped and mixing was continued while the mixture cooled. When the mixture had cooled to 120°F – 130°F (48.9°C – 54.4°C), the fragrance
(Ungerer Mask DD 49978 TM) was added while mixing continued. When the batch had cooled to about 115°F
(46.1°C), the mixture was poured into ointment jars.
To evaluate the antimicrobial effectiveness of the test ointment described above, both in vitro tests and tests on cow teats were performed. These tests are described below. Example 1 and Comparative Examples 1-3
These examples involve in vitro tests of the antimicrobial effectiveness of the test ointment of the example. The test was performed with two different types of bacteria, Staphylococcus aureus (gram positive, ATCC #25923) and Escherichia coli (gram negative, ATCC #25922) . For comparison with the test ointment of the invention, three commercially available aqueous udder balms were also
TM tested. Dr. Naylor is distributed by H.W. Naylor Co.,
Inc., Morris, NY, Ken Ag TM is distributed by Ness & Clark,
Inc. , Ashland, OH, and 4 Seasons TM is distributed by Mills Fleet Farm, Appleton, WI. The comparisons of antimicrobial activity involve a bacterial count after exposure to the antimicrobial ointment for 0 minutes and for 20 minutes at 37°C.

The bacterial cultures were prepared by suspending the bacteria in tryptic soy broth and incubating the suspensions at 37°C for 24 hours. The suspensions were designed to have an initial inoculum count of between 10 to 108 CFUs/ml, which was later confirmed with plate counts. (CFU = colony forming unit). Then, a 0.1 ml aliquot of the 24 hour bacterial culture suspension was added to a centrifuge tube containing 10 mis of antimicrobial ointment to form an ointment bacterial suspension (OBS) . Prior to the addition of the bacterial culture suspension, the antimicrobial ointment was softened in a water bath at 37°C to assist with the mixing of the OBS. The OBS in the centrifuge tube was vortexed for 20 seconds and returned to the water bath after aliquots were removed.
A 1.0 ml aliquot of OBS was removed from the centrifuge tube both immediately after vortexing and 20 minutes after vortexing. Serial dilutions were performed with each 1.0 ml aliquot. To perform the first dilution, the 1.0 ml aliquot was placed in a test tube containing
9.0 mis of Letheen Broth TM to form a l:io dilution. Two additional serial dilutions were made by first placing a 1 ml aliquot of the 1:10 dilution into 9.0 is of Letheen
Broth TM to form a 1:100 diluti»on and subsequently placing a 1 ml aliquot of the 1:100 dilution into 9.0 mis of Letheen Broth™ to form the 1:1000 dilution. Letheen
Broth TM is a neutralizing agent to prevent further multiplication of bacteria, sold by Difco Laboratories.
The letheen broth was warmed prior to mixing to 37°C to maintain optimum mixing. The tubes were vortexed.
Following vortexing, 0.1 ml of solution from each of the serial diluted tubes separately was spread on tryptic soy agar plate. The inoculated plates were

incubated at 37°C for forty-eight hours . Following incubation , the bacterial colonies on the plates were counted and compared to the initial inoculum count . The results of the tests are shown in Table 1. TABLE 1
IN-VITRO BACTERIAL KILL ASSAY RESULTS
Active Inoculant Zero 20
Product Ingredient ( 8 ) Bacter a M Count Minutes Minutes 2.7 X 108 7.6 1.9 X

7.4

.2 X

Bronopol
4 Seasons Chloroxylenol S. Aureus 6.0 X 10 8
TNTC TNTC E. Coli 1.2 X 10c TNTC
<10CFϋ/gm Test Glyceryl S. Aureus 6.2 X 1010 8.4 X 105 <10CFU/gm ointment Monolaurate, Propylene E. Coli 1.9 X 1010 <10CFU/gm <10CFU/gm Glycol Monocaprylate, Lactic Acid TNTC=Too numerous to count The test ointment had superior antimicrobial properties compared with the aqueous udder balms. Example 2 and Comparative Example 4 These examples were designed to test the relatively long term (eight hour) antimicrobial effectiveness of the test ointment in use on cow teats specifically against Streptococcus uberis. For comparison (Comparative Example 4) , the test was also performed with an aqueous, commercial teat dip, Lauricare TM from 3M, Saint Paul, MN. Cows were selected for normal teat size and structure. At the start of the test teats were washed and rinsed with alcohol. All teats then were dipped to about 3/4 length in a bacterial solution of S. uberis at a concentration of 1.0x10 7 bacteri.a/ml i.n mi.lk suspension. The teats were allowed to dry for ten minutes. Then, ointment vehicle without germicide (monoesters) was added to control teats (left side) and test ointment was added to test teats (right side) . Similarly, other cows were treated with Lauricare TM teat dip, an aqueous based product, on test teats (right side) with no treatment on the control teats (left side) . Eight hours after treating the teats, the number of colony forming units (CFU's) recovered from the test and control teats was determined by first scraping the teats separately with a spatula with the residue placed into a centrifuge tube. Next, each teat was rinsed with 16 mis of letheen broth, which was placed into a separate centrifuge tube. Then, each teat was scrapped again with any residue being placed into the same centrifuge tube with the first scrapings. The materials in the tubes were appropriately diluted, and the diluent was plated onto sheep blood agar. The inoculated plates were incubated at 35-37°C for 48 hours. The colonies were counted on plates having between 30 and 300 colonies per plate to determine the CFU's per ml. The log reduction in bacteria from the test teats relative to bacteria from the control teats was calculated for each set of teats. The results from the rinse tests and the scrapping tests were averaged together. For each cow the results for the two left (control) teats and two right (test) teats also were averaged together. The results of the experiments, presented as percent reductions and log reductions in contamination, with the TM test ointment and with Lauricare are presented in Table TABLE 2 LONG TERM (8 HR. ) RESIDUAL GERMICIDAL EFFECTS OF AGAINST STREP. UBERIS ON TEAT SKIN Product Percent Reduction Log Reduct:ion Cow#l Cow#2 Cow#3 Cow#l Cow#2 CθW#3 Test Ointment 98.12 93.96 96.66 1.73 1.22 1.47 TM Lauricare Teat Dip 81.40 96.13 97.70 0.73 1.41 1.63 The test ointment had comparable effectiveness as the aqueous teat dip against these bacteria under realistic conditions. Example 3 This example demonstrates effectiveness of the test ointment against the Bovine Herpes Mammillitis virus. The Bovine Herpes Mammillitis virus (strain New York 1, VR-845) was obtained from the American Tissue Type Collection, Rockville, MD. The test medium was Eagles™ minimal essential medium (sold by Gibco-BRL, Grand Island, NY) supplemented with 2% (v/v) fetal bovine serum heat inactivated by heating to 56°C for 30 minutes. The medium was also supplemented with 100 units/ml penicillin (sold by USB, Cleveland, OH), 10 μg/ml gentamicin and 2.5 μg/ml Fungizone™ (Amphotericin B sold by Sigma, St. Louis, MO) . The virus was grown to a sufficiently high titer. Then, the cell debris was removed, and aliquots of the virus were stored at -70°C until use. Viruses were exposed to the test ointment in suspension for exposure times of 1 minute (23°C) or 20 minutes (40°C) . A l.Og quantity of test ointment was placed in a test tube along with a 0.5ml aliquot of virus suspension. For the 1 minute exposure, the mixture was vortexed for the entire one minute. For the 20 minute exposure, the mixture was vortexed for one minute and every four minutes thereafter. The mixture was held at 40°C during exposure except during vortexing. Immediately following the exposure period, the mixture was titred using a series of 10-fold dilutions using Eagle's TM minimal essential medium. The series of dilutions were assayed for infectivity. Several controls were used. A virus control included a 0.5 ml aliquot of virus suspension mixed with l.Og of test media. Cytotoxicity controls included l.Og of test ointment mixed with 0.5 ml of Eagle's TM minimal essential medium. A series of dilutions of the cytotoxicity control mixture was challenged with stock virus and assayed for infectivity in order to determine the dilution of the test ointment which has virucidal activity. The experiments with the cytotoxicity control mixture were performed in the same way as the test ointments experiments. Infectivity was determined using bovine kidney cell lines obtained from ViroMed Laboratories, Inc., Cell Culture Division. Cultures were grown and used as monolayers in disposable tissue culture labware. Dilutions of test mixtures and controls were inoculated into bovine kidney cell cultures in quadruplicate. The cells were inoculated with 100 μl of each dilution and incubated at 36-37°C in 5.1-6.5% C02. The cells were observed for seven days for the presence of virus and/or cytotoxic effects. Bovine Herpes Mammillitis virus produces a typical cytopathic effect on bovine kidney cells. The method of Karber was used to calculate 50 percent end points, TCID5o (TCID=Tissue Culture Infectivity Dose) , which is the dose that results in infection of 50% of the cells. Percent reduction values are obtained as follows: %Reduction = 1 - (TCID50/TCID50 virus control) 100 (ASTM Standards on Materials and Environmental Microbiology, 1994, E1052-85; Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections, E.H. Lennette and N.J. Schmidt, editors, Fifth Edition, 1979, p 32-35) . The results of the one minute and 20 minutes exposure tests are displayed in Table 3. The titre of the virus control in both tests was 6.75 logn). Infectivity was not detected in either test for the test ointment samples. This puts a lower limit on the titre of <1.5 logio and an upper limit on the percent reduction of >99.999%.

TABLE 3
One Minute Exposure Period 20 Minute Exposure
Period
Dilution Virus Control Test Sample: Virus Control Test
Sampleβ
(CELL CONTROL) 0000 0000 0000
0000 ιo»2 ++++ oooo ++++ oooo lO»3 ++++ oooo ++++ oooo lO»4 ++++ oooo ++++ oooo lO»5 ++++ oooo ++++ oooo lO»6 ++++ oooo ++++ oooo lO»7 0+00 oooo +000 oooo

Percent Reduction NA >99.999% NA ≥99.999% Key:
(+) = positive for the presence of test virus
(0) = no test virus recovered and/or no cytotoxicity present
(T) = Cytototoxicity present
The results of the cytotoxicity control tests are displayed in Table 4. Again the samples containing the diluted test ointment eliminated all signs of virus activity.

TABLE 4
Dilution Cytotoxicity Control: Neutralization Control: Test Substance + Bovine Herpes Mammillitis
(CELL CONTROL) oooo oooo

-3
10 oooo ++++
10 -4 oooo ++++
TCD5o 0. 1ml <ιo1,5 Neutralized at ≤IO1 , 5 Key: (+) = positive for the presence of test virus (0) = no test virus recovered and/or no cytotoxicity present (T) = Cytototoxicity present Example 4 This example demonstrates the effectiveness of the test ointment for improving the skin condition of a cow's teat. Cows in the study were selected for having normal teats and udders. Teat on a cow's right side were treated with the test ointment while the adjacent teats on the left side were untreated. Five cows were included in the study each with two test teats and two control teats. In other words, ten test teats and ten control teats were involved in the study. The test ointment is applied twice daily following milking. The tests were performed during a relatively cold period. The temperature of the barn was about 40°F-50°F (4.4°C-10.0°C) with low humidity. There was a tendency for drying under these conditions. Barrel Dryness and Teat End Score were determined for both the treated and the control cow teats. In the evaluation, each teat is given a numerical score, and the scores are added to produce an overall result. The evaluator is unaware of which teats received the product and which teats are the control teats. In the Teat Barrel Test, the teat barrel is evaluated by palpation to determine the degree of dryness and roughness, and the numerical score is assigned as follows: 1 = normal soft skin 2 = mild amount of dry feeling and some roughness 3 = moderate loss of moisture and ability to feel edges of skin flakes 4 = skin feels very dry, possibly some cracking and serum exudation The Teat End Keratin Evaluation involves a numerical evaluation of each teat based on the following numerical scale: 1 = none or small amount of soft keratin present 2 = small increase in amount and hardness of keratin 3 = significant increase in amount and hardening of keratin 4 = large amount of very hard keratin present The results of the tests are presented in Tables 5 and 6. TABLE 5 BARREL DRYNESS Start 1 Week 2 Weeks 3 Weeks Right side treatment 22 18 12 12 Left side control 21 19 19 18 TABLE 6 TEAT END SCORE irt l Week 2 Weeks 3 Weeks Right side treatment 18 14 20 17 Left side control 20 16 13 17 The dryness test (Table 5) demonstrates significant improvement over time for the treated teat compared with little improvement shown for the control teats. The teat end scores showed approximately similar results for the treated and the control teats within the variability of the results. Other embodiments of the invention are within the scope of the appended claims. Claims (11) WHAT IS CLAIMED IS:What is claimed is: 1. An antimicrobial ointment comprising: (a) an antimicrobial fatty acid monoester of a polyhydroxy alcohol; (b) a chelating agent; and (c) an ointment vehicle composition comprising petrolatum. 2. The antimicrobial ointment of claim 1, wherein said polyhydroxy alcohol is selected from the group consisting of glycerol, propylene glycol and saccharides. 3. The antimicrobial ointment of claim 1, wherein said fatty acid is selected from the group consisting of caproic acid, heptanoic acid, caprylic acid, capric acid, undecanoic acid and lauric acid. 4. The antimicrobial ointment of claim 1, wherein said ester comprises glycerol monolaurate. 5. The antimicrobial ointment of claim 1, wherein said ester comprises propylene glycol monocaprylate. 6. The antimicrobial ointment of claim 1, wherein said antimicrobial ointment comprises at least about 50% by weight of said petrolatum. 7. The antimicrobial ointment of claim 1, wherein said antimicrobial ointment comprises between about 0.1% and about 10% by weight of said fatty acid monoester of a polyhydroxy alcohol. 8. The antimicrobial ointment of claim 1, wherein said antimicrobial ointment comprises at least about 0.1% by weight of said chelating agent. 9. The antimicrobial ointment of claim 1, wherein said antimicrobial ointment comprises at least about 0.1% by weight of lactic acid. 10. The antimicrobial ointment of claim l, wherein said antimicrobial ointment comprises in percent by weight at least about 0.1% glyceryl monolaurate, at least about 0.1% propylene glycol monolaurate, at least about 0.1% lactic acid, and at least about 1% wax. 11. A method of treating a bovine teat comprising the step of applying to skin of said bovine teat an antimicrobial composition comprising: (a) an antimicrobial fatty acid monoester of a polyhydroxy alcohol; (b) a chelating agent; and (c) a vehicle composition wherein said vehicle composition is effectively water insoluble. 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