PERILLYL ALCOHOL AS A BACTERICIDE AND YEASTICIDE

Abstract: Perillyl alcohol as an agent, which is toxic to bacteria and yeasts, and a method of killing bacteria and yeasts by treating them with toxic concentrations of perillyl alcohol.

Description: PERILLYL ALCOHOL ' ΑS A BACTERICIDE AND YEASTICIDE TECHNICAL FIELD This invention relates to the technical field of bactericides and yeasticides. BACKGROUND ART During the study of limonene as a hand cleaner, the applicants found that fully oxygenated limonene is a bacteri- cide. A review of the literature revealed that oxygenated limonene contains several oxidation products including: cis and trans-carvepl, trans-p-menth-8-ene-l,2-diol, limonene 1,2-epoxide, limonene 8,9-epoxide, cis and trans-p-mentha-2, 8-dien-l-ol, and perillyl alcohol, as was outlined by Blu ann in Chemical Abstracts, Volume 63, 1965, on page 1819. The applicants found that a principal bactericide generated by the oxidation of limonene is perillyl alcohol which, in bactericidal concentrations, kills bacteria and yeast . Perillyl alcohol is a monocyclic monoterpene similar to limonene . Limonene is not bactericidal . Their chemical formulas follow below . Perillyl Alcohol Limonene It should be noted that the formula for perillyl al¬ cohol is identical to the formula for limonene with the ex¬ ception of a hydroxyl group which replaces a hydrogen atom at carbon 7. Because limonene is not bactericidal, the exchange of a hydroxyl group for a hydrogen atom at carbon 7, was not expected to make the resulting compound (perillyl alcohol) bactericidal. All other monocyclic monoterpenes that have known bactericidal activity are similar to limonene, but have a hydroxyl group or an oxygen atom replacing a hy¬ drogen atom on the benzene ring at carbons 2, 3, 4 or 8 (as opposed carbon 7, 9, or 10) that is appreciated in the struc¬ tures of: carveol, carvone, hydrocarveol, hydrocarvone, pulegone, isopulegol, menthol, menthone, terpinen-4-ol, and a-terpineol which follow. None of the monocyclic monoterpenes that have bactericidal or yeasticidal activity have a hydroxyl group at the carbon 7, 9, or 10 position. Carveol Carvone Di y rccarvecl Dihydrccarvone Puleσcne Menthol Terσinen-4-ol Although it was not expected, the applicants were totally surprised to find that perillyl alcohol is toxic to bacteria and yeast . Perillyl alcohol is an oil with a terpenic aroma. It is insoluble in water, is poorly soluble in propylene glycol, and is almost insoluble in glycerine. Perillyl alcohol is soluble in alcohol and is miscible in oil. It is used as a flavoring agent for cosmetics and perfumes, but heretofore, it has not been used as a bactericide. Perillyl alcohol can be produced by the oxidation of limonene as was demonstrated by Blumann in Chemical Abstracts, Volume 63, 1965 on page 1819, and Bardychev in Chemical Abstracts, Volume 80, 1974, page 359. It can be produced by the acetylation of limonene as described by Ansari, Hifzure R. (German Offen 2,513,910 and Canadian Patent No. 1,077,959) . Walling made perillyl alcohol (Canadian Patent No. 981,695) from betapinene by adding benzyl peroxide to betapinene followed by alkaline hydrolysis to perillyl alcohol. There are several disclosures in the prior art of antimicrobial activity of limonene and other terpenes . Zukerman studies the effect of auto-oxidized d-limonene on bacteria, but found it was weakly bacteriostatic, was un¬ stable, and lost its bacteriostatic effect on keeping as was discussed in Nature 169: 517 (1951) . He never studied perillyl alcohol. Kurita investigated the fungicidal ac¬ tivity of several components of essential oils as was re¬ ported in Biol. Chem., 45(4) , 945-952, 1981, but he never studied the toxic activity of perillyl alcohol against bacteria nor yeasts. Murdock and Allen showed that the germicidal effect of sodium benzoate against yeast was enhanced by the orange peel oil and d-limonene, as was out¬ lined in Food Technology, Vol. 14, No. 9, 1960, pages 441-5. They never studied the antimicrobial activity of perillyl alcohol against bacteria nor yeast. Kellner et al demon¬ strated that several ethereal oils and some of these con¬ stituents have antimicrobial activity as was reported in Arnei ittel-Forsσhung, 5, 224-9, 1955.' He confirmed that limonene is not bactericidal. He never studied perillyl alcohol for bactericidal activity. Gauvreau showed a means of producing disinfecting compositions in U.S. Patent No. 3,595,975 by combining cetyl pyridinium hydrochloride with terpenes to form antiseptics, but he never studied the use of perillyl alcohol alone nor in combination with cetyl pyridinium hydrochloride. The active ingredient in his dis¬ infecting compositions was cetyl pyridinium hydrochloride (and not t.he terpenes) . A. Morel revealed the sterilizing action of carveol, dihydrocarveol, and their ozonization productions in Comp. Rend. Soc. Biol. Volume 115, pages 536-8 (1934) . He never studied the bactericidal effect of perillyl alcohol. It should be pointed out that drugs which are bacteri¬ cidal are usually not f ngicidal , and drugs which are fungi¬ cidal are usually not bactericidal. In addition, drugs which are bactericidal frequently promote the growth of yeast- Table A, which follows, exemplifies the bactericidal and -1- f ngicidal activity of several commonly used antibacterial, antiyeast, and antifungal antibiotics. TABLE A ANTIBIOTICS ANTIBIOTIC ACTIVITY AGAINST A. Anti-bacterial Gm + Bact G - Bact A F Bact Yeast Fungi G_m + Bact = Gram positive Bacteria, On - Bact = Gram Negative Bacteria, A F Bact = Acid Fast Bacteria, Y = Kills Organism, N = No Activity Against Organism It should be noted in the table above that none of the antibacterial antibiotics kill fungi, and none of the anti¬ fungal nor anti -yeast antibiotics kill bacteria. Thus , an antifungal antibiotic is not expected to kill bacteria and an antibacterial antibiotic is not expected to kill fungi or yeasts . Antifungal antibiotics do not necessarily kill yeast and anti-yeast antibiotics do not necessarily kill fungi . DISCLOSURE OF THE INVENTION This invention relates to the use of perillyl alcohol as a bactericide and yeasticide. Perillyl alcohol is an oil which is available commercially, but heretofore, it has not been recognized as a bactericide. It is slightly viscous and when applied, readily adheres to glass, metal, wood, cloth, rope, book covers, paper, paint, cement, ceramics, plastic, plant surfaces, skin, mucus membranes, and teeth, leaving an oily film. Because it is not soluble in water, its adherence to surfaces allows prolonged exposure and makes perillyl alcohol an ideal agent to kill bacteria and yeast regardless of whether they infect plants, animals or humans . The exact method of killing bacteria and yeast is unknown, but it is thought that perillyl alcohol kills bac¬ teria and yeast by lysing the cell membrane of the organ¬ isms which is lethal to the organisms. In practice, any surface, on which it is desirable to kill or prevent the growth of bacteria and yeast , is treated with bactericidal concentrations of perillyl alcohol by swab¬ bing, wiping, painting, washing, brushing, spraying, or any other direct application technique. Alternatively, perillyl alcohol can be incorporated in creams, ointments, tinctures, gels, suppositories, paints, sprays, aerosols, toothpastes, solutions, emulsions, soaps, scrubs, mouthwashes, or anti¬ septics, and applied anywhere it is desirable to kill or pre¬ vent the growth of bacteria and yeast . BEST MODE FOR CARRYING OUT THE INVENTION The following examples are illustrative of the best mode of carrying out the invention. They are, obviously, not to be construed as limitative of the invention since various other embodiments can be readily evolved in view of the teachings provided herein. Example 1 In accordance with this invention, perillyl alcohol was studied for its toxicity against bacteria and yeast . The organisms tested included the bacteria: Staphylococcus aureus 53T, and Staphylococcus aureus 54T; Staphylococcus epider- idis 10, Streptococcus mutans which causes dental plaque. Streptococcus faecalis 15, Bacillus subtilis ATCC 6633, Escherichia coli, Xanthomonas campestris pv vesicatoria, a plant pathogen, Salmonella 14 (para B) , Pseudomonas aeriginosa ATCC 115, and Pseudomonas cepacia GM 36; Mycobacteria fortuitum ATCC 6841, an acid fast pathogen, and the yeast: Candida albicans, a common cause of skin, mouth, and vaginal infections. The minimal bactericidal concentra¬ tion of perillyl alcohol needed to kill these bacteria and yeast is outlined in Table B below. The perillyl alcohol used in the tests was obtained from Aldrich Chemical Company, Milwaukee, Wisconsin, Catalogue Number 21,839-1 and Lot Number 2029 JE-KE. B . YEAST . 1. Candida albicans 0.005 0.0025 0.0025 * Indicates strains of Staphylococcus aureus associated with toxic shock. The standard assay used to test the toxic activity of perillyl alcohol against the different bacteria and yeast was as follows: various dilutions of perillyl alcohol were prepared in an appropriate broth medium for each test strain. An inoculum of lθδ colony-forming units (CFR/ l) was used. Each test was incubated at the proper temperature for each organism and subcultured (0.01 m. ) at 10 minutes, 60 minutes, and 24 hours onto agar media free of perillyl alcohol. Re¬ sults were expressed as the toxic concentration, i.e., the lowest concentration of perillyl alcohol (ml perillyl alcohol/total ml of test) killing at least 99.99% of the bacterial inoculum. The activity of perillyl alcohol against Mycobacteria was assayed using undiluted oil, and oil diluted up to 1:8000 in Proskauer Beck liquid medium. Each test was inoculated with lθ6 CFU/ml of Mycobacteria and incubated at 37°C in 7ό CO2 in air. At various time intervals, each test was shaken vigorously and a 0.01 aliquot removed. This was subcultured onto Dubos oleic acid-albumin agar plates to determine the number of viable Mycobacteria remaining. Each test was sampled in this fashion after incubation for 10 minutes, 60 minutes, 24 hours, 1, 2, 3 and 4 weeks. The toxic concentra¬ tion was defined as the lowest concentration of oil killing at least 99.99% of the original inoculum. Details of each assay are presented in Table C which follows. TABLE C Test conditions used to assay the toxic activity of Perillyl Alcohol : SUBCULTURE INCUBATION ORGANISM BROTH MEDIUM AGAR MEDIUM CONDITIONS 1. Sta p hylococcus. Mueller-Hinto 5% sheep blood air at 37°C Bacillus, and Enterobacteriaceae 2. Streptococcus Todd-Hewitt 5% sheep blood 10% C f- ml m air at 37°C 3. Xanthomas Mueller-Hinton blood agar a r at 3 7°C . Mycobacteria Proskauer Beck Dubos oleic 7% CO 2 at liquid medium acid-albumin 37 C C agar 5. Candida albicans Sabouraud 5% sheeD blood air at- 37°C dextrose EXAMPLE 2 PLAQUE INHIBITION BY PERILLYL ALCOHOL Perillyl alcohol was effective in killing all the dif¬ ferent strains of bacteria and yeast tested, including Streptococcus mutans. Its ability to kill Streptococcus mutans makes it very effective in inhibiting plaque formation on surfaces. The best method of demonstrating the plaque in¬ hibiting properties of a substance is by the Streptococcus Mutans Plaque Inhibition Test, which is explained below. In the laboratory small glass rods were immersed in perillyl alcohol and quickly removed simulating the painting or rinsing of the teeth with perillyl alcohol. The glass rods were allowed to dry by hanging to allow any excess non- adherent or excess amount of the perillyl alcohol to drip off as would be expected to occur when excess is applied to the teeth. The amount which adhered to the glass rods was tested for its antiplaque activity against Streptococcus mutans simulating the application of perillyl alcohol to the teeth and then the antiplaque activity of the perillyl alcohol remaining on the teeth was determined. As expected, the perillyl alcohol which remained adherent to the glass rods showed excellent antiplaque activity by killing Streptococcus mutans. This strongly supports the finding that perillyl alcohol can be tasted in the mouth for 3-4 hours after only drops are applied to the teeth and are not removed by saliva. It is this adherence to the teeth that gives prolonged con¬ tact with Streptococcus mutans, the etiologic agent which causes plaque, and makes perillyl alcohol so effective in inhibiting plaque formation on teeth. It is recognized that plaque has different degrees of adherence to teeth according to the quantity and quality of the substances which are incorporated in the plaque. As plaque is removed from the teeth, Streptococcus mutans , the etiologic agent which causes plaque can be cultured from the plaque. Vigorous rinsing of the glass rods to remove plaque simulates the brushing of teeth to remove plaque. Lightly adherent plaque is removed with the first wash, and more strongly adherent plaque is removed with the second wash, and very strongly adherent plaque is removed only by the third wash. The number of colonies of Streptococcus mutans which can be cultured from each washing corresponds to the amount of plaque removed at each washing, and when no Streptococcus mutans remains on the glass rods after three rinses, it shows that plaque is not present and proves an effective antiplaque agent (or antimicrobial) has been used. This laboratory assay is generally accepted by oral microbiologists as most nearly simulating the deposition of Streptococcus mutans generated plaque on teeth and allows a method for testing the inhibition of plaque formation on teeth. Table D below shows the antiplaque activity of perillyl alcohol and compares its plaque inhibiting activity with controls of water and glycerol. TABLΞ D Effect of various agents on the in vitro plaque development by Streptococcus πutar.s Colony Forming Units of s.. mutans Growth of s. r.utar.s cr. er ml. in: Subculture of Rod to: .iG :.G The method to determine the in vitro plaque development by Streptococcus mutans was as follows: Small glass rods (2mm diameter, 1 cm length) were immersed in distilled water, glycerol, and perillyl alcohol diluted in glycerol, after which they were removed and allowed to dry by hanging on ■sterile floss overnight. Streptococcus mutans (10 4 CFU/ml) was inoculated into individual sterile tubes containing 4.5 ml Todd-Hewitt broth with 5% sucrose, after which the rods were suspended in the medium. After 24 hours incubation at 37°C in 10% CO- in air, the rods were removed and the number of CFU/ml in the broth was determined by dilution plate counts. The rods were dried on sterile filter paper and placed in a sterile tube to which 3 ml of physiologic saline was added. Each tube was mixed vigorously (Vortex Genie Mixer, Scientific Products, Evanston, 111.) for 5 seconds, the saline was removed, and the CFU/ml were determined by dilution plate counts (wash #1) . Each rod was dried again on sterile filter paper, was placed in a second sterile tube to which 3 ml of physiologic saline was added and mixed vigorously (Vortex) for 30 seconds. The CFU/ml in this second saline (wash #2) was determined by dilution plate counts. After a third drying on filter paper, each rod was placed in a third tube, to which 3 ml of physiologic saline was added and the tube was vigorously mixed (Vortex) for three minutes. The CFU/ml in this third saline (wash #3) was determined by dilu¬ tion plate counts. After a fourth drying, each rod was rolled on the surface of a 5% sheep blood agar plate and was then placed in a tube of 9 ml Todd-Hewitt broth after which the blood agar plate and the broth were incubated 24 hours at 37°C in 10% C0 2 in air. Results of this assay were inter¬ preted as follows: if organisms were recovered from wash #1 only this was considered weak attachment of the organisms to the rod. Moderately attached organisms were recovered in wash #2 and strongly attached organisms were recovered in wash #3. Growth on the blood agar or Todd-Hewitt broth sub¬ cultures was considered to be due to very strongly attached organisms which were not killed by the antimicrobial on the rods. Results of this assay are demonstrated in ' Table D above. It should be noted (in Table D) that the highest dilution of perillyl alcohol to completely prevent strong attached organisms and very strongly attached organisms was a 25% solution (vol/final vol) of perillyl alcohol in glycerol. EXAMPLE 3 FORMULATIONS WHICH INCORPORATE PERILLYL ALCOHOL AS A COMPOUND TO KILL BACTERIA AND YEAST The following formulations are prepared using perillyl alcohol in liquids, gels, soaps, paints, pastes, creams, ointments, suppositories, tampons, aerosols, and emulsions. When bacteria and yeast are treated with perillyl alcohol containing formulations, the formulations kill or prevent the growth of bacteria and yeast . A. LIQUIDS CHEMICAL % OF OTAL RANC-1 ACTION 1. SOLUTIONS CR SPRAYS a. Perillyl Alcohol 6.0% 0.1-50% bactericide Corn Oil 94.0% 50-99.9% diluent 100.0% b. Perillyl Alcohol 1.0% 0.1-50% bactericide Ethyl Alcohol 99.0% 50-99.9% diluent 100.0% 2. MOUTKWASK a. Perillyl Alcohol 50.0% 0.1-50% yeasticide Flavor 2.0% 1-5% flavor Ethyl Alcohol 43.0% 45-98.9% diluent 100.0% B. DENTIFRICE 1. LIQUID Liquid soap concentrate Saccharin Clove Oil Cinr.ar.cn Oil Feoterr.int Oil Ethyl Alcohol Colcr Perillyl Alcohol z-cz-αe GΞL CHEMICAL % OF TOTAL RANGE ACTION Sodium onofluorophosphate 0.8% 0.5-1.5% antiplaque Perillyl Alcohol 50.0% 1-50% bactericide Kvdrated silica xercgel 10.0% 8-15% abrasive Kvdrated thickening silica 8.5% 5-10% binder Sorbitol 70% solution 18.8% 5-73.3% humectant Polyethylene glycol 32 5.0% 3-7% bodying ager.t Sodium lauryl sulfate 1.5% 1-2% surfactant Carbcxyr.ethyl cellulose gum 1.0% 0.5-2% binder S 0 alcohol 1.0% 0.5-2% stabilizer Flavor 3.0% 2-4% flavor Saccharin 0.2% 0.1-0.5% flavor F D ' C Green 42 0.1% 0.1-0.5% colcr F D & C Yellow =10 0-1% 0.1-0.5% colcr 1C0.0% ASTΞ CHEMICAL % OF TOTAL RANGE ACTIT Sodium monofluorophosphate 0.8% ■ 0.5-1.5% ar.ticlaαue Perillyl Alcohol 50. C% 1-50% bactericide Dicalciu phosphate dihydrate 22.0% 20.4-30% abrasive Water 16.0% 11.1-69.5% diluent Glycerine 5.1% 4.5-12.5% bodying ager.t Flavor 2.0% 2-3% flavor Sodium lauryl sulfate 1.5% 1-2% surfac t an t Carboxy ethyl cellulose gum 1.4% 0.5-2.0% binder Tetrascdium pyrophosphate 1.0% 0.5-2.0% binder Sodium saccharin 0.2% 0.1-c.5% flavor 100.0% C OINTMENTS S SUPPOSITORIES WITH AND WITHOUT HYDROCORTISONE 1. OINTMENT WITH HYDROCORTISONE CHEMICAL % OF TOTAL RANGE ACTION Perillyl Alcohol 1.0% 0.1-15.0% bactericide Polyethylene glycol 3350 59.0% 48.5-59.7% bodying age: & emulsifie: Polyethylene glycol 400 39.0% 31.5-39.7% bodying age: & emulsifie; Hvdrocortisone 1.0% 0.5-5. os ant - 100.0% infla matcrv 2. OINTMENT WITHOUT KYDROCOR: :SONΞ CHEMICAL _ Γ ^ O ^ll RANG; ACTI3N Perillyl Alcohol 1.0% o.ι-i5.c% yeasticice Polyethylene glycol 3350 59.5% 51.0-59.95- bcdying age: ' emulsifie: Polyethylene glycol 400 39.5% 34.0-39.95% bodying age: 100.0% & emulsifie: 3. SUPPOSITORY WITHOUT HYDROCORTISONE Perillyl Alcohol 6.0% 0.1-15% bactericid; ■ o.oxvethvier.e σivcol 56.5% 51.0-59.95% bodvino arer.t ύ emulsifier Polyethylene glycol 2 2 ~ - 37.5% 34.0-39.95% bcdyir.α acεr.t 100.0% & emulsifier 4. SUPPOSITCRY WITH KYDRCCCRTISONE CHEMICAL % OF TOTAL RANGE ACTION Perillyl Alcohol l.o% 0.1-15% yeasticide Polyethylene glycol 1000 74.0% 60.0-75.2% bodying acar.t & emulsifier Poivethvlene glycol 3250 " ' " 24.0% 20.0-24.2% bodying ager.t ' emulsifier Hvdrocortisone 1.0% 0.5-5.0% anti-inflam a 100.0% D. CREA-MS WITHOUT HYDROCORTISONE CHEMICAL 1. Perillyl Alcohol Cetyl alcohol Arlacel 165 ** Scrbitol 70% solution Water 2. Perillyl Alcohol Spermaceti wax Scrbitan moncstearate* Polyethylene 20 Scrbitan mcnostearate* Water 100.0% Ξ. CREAMS WITH HYDRCCORTIΞONΞ CHEMICAL % OF 1. Perillyl Alcohol c.1-15.0% yeasticide Cetyl alcohol 12.0-13.0% thickener Arlacel 165 ** 3.5-7.5% em 1sifier Scrbitol 70% solution 3.5-3.0% humectant Hydrocortisone 0.5-5.0% anti-inflar-. Water 46.5-30.4% diluent * Cr :da, Inc., 51 Madison Ave. , New York, New York 10010 ** GΓ'cεroi moncstearate and poiyoxyethylene εtearate IC: of America (Formerly Atlas Chemical Industries) , Wi Lmmgton, Delaware 19899 G. AEROSOLS WITHOUT HYDROCORTISONE 1. Perillyl Alcohol 6.0% 0.5-50% bactericide Ethyl alcohol 94.0% 50-99.5% diluent 100.0% Pressurized nitrogen propellant at 100-125 psig CHEMICAL % OF TOTAL RANGE ACTION 2. Perillyl Alcohol 10.0% 0.5-50.0% yeasticide Scvbean Oil 90.0% 50.0-99.5% diluent 100.0% Pressurized nitrogen propellant at 100-125 psig H. AEROSOL WITH HYDROCORTIΞONΪ 1. Perillyl Alcohol Soybean oil Hydrocortisone Pressurized nitrogen propellant at 100-125 psig OIL IN WATER EMULSION CHEMICAL 1. Perillyl Alcohol 2. Corn oil Arlacel 40** Tween 40 3. Water 100.0% Fe * t 2 to 70°C Heat 3 to 72°C. Add 3 to 2 with continuous agitation. When 3 and 2 cool to 40°C, add 1 with continuous agitation until room temperature is reached. J. OIL IN WATER EMULSION WITH SOAP (BACTERICIDAL SOAP) CHEMICAL % OF TOTAL RANGE ACTION 1. Perillyl Alcohol 10.0% 0.1-25% bactericide 2. Corn oil 21.0% 20.0-40.0% oil Arlacel 40** 2.0% 1.0-3.0% emulsifier Tween 40 3.0% 2.0-4.0% emulsifier Liquid soap concentrate 3.5% 2.5-5.0% surfactant 3. Water 60.5% 23-74.4% diluent 100.0% Heat 2 to 70°C. Heat 3 to 72°C. Add 2 to 2 with continuous agitation. When 3 and 2 cool to 40°C, add 1 with continuous acitaticn until room temoerature is reached. WATER IN OIL EMULSION CHEMICAL 1. Perillyl Alcohol 2. Arlacel 186** Soybean oil Ceresin wax Beeswax Tween SO 3. Water Heat 2 to 70 C C. Heat 3 to 72°C. Add 3 to 2 with continuous agitation. When 3 and 2 cool to 40°C, add 1 with continuous agitation until room temperature is reached. PAINT 1. ENAMEL CHEMICAL Ferillyl Alcohol Titanium dioxide Calcium carbonate Silicate Soya al yd resin Mineral soirits 2. LATEX CHEMICAL Perillyl Alcohol Titanium dioxide Silicate Calcium carbonate Vinyl acrylic resin solids Glycol Water While only certain preferred embodiments of this invention have been shown and described by way of illustration, many modifications will occur to those skilled in the art and it is, therefore, desired that it be understood that it is intended herein, to cover all such modifications that fall within the true soirit and scoce of this invention.

Claims: CLAIMS 1. A broad spectrum antimicrobial agent toxic to bacteria and yeasts wherein the toxic agent is perillyl alcohol. 2. Λ method of killing bacteria or yeast wherein the improvement comprises treating bacteria or yeast in their habitat with toxic concentrations of perillyl alcohol. 3. The method of Claim 2 wherein the bacteria are selected from the group consisting of Staphylococcus, Bacillus, Enterobacteriaceae, Streptococcus, Xanthomonas, and Mycobacteria. 4. The method of Claim 2 wherein the yeast is Candida Albicans.

Inventors: CHASTAIN DOYLE E, SANDERS CHRISTINE C, SANDERS W EUGENE JR, CHASTAIN, DOYLE, E., SANDERS, CHRISTINE, C., SANDERS, W., EUGENE, JR.
Applicants: CHASTAIN DOYLE E, CHASTAIN, DOYLE, E.
Application year: 1992
Main classification: A61K31
IPC codes: A01N31, A61L2