FI58423B - DESIGNATION PROCEDURES INNEHAOLLANDE ETT SALT AV 1,6-BIS- (N1-P-CHLOROPHENYLDIGUANIDO-N5) -HEXAN - Google Patents

Description

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Sweden-Sweden (SE) 6 ^ 31/71 (71) KemaNord AB, Box 5052, S-102U2 Stockholm 5, Sweden-Sweden (SE) (72) Ronald Arthur Stephenson, Henley-on-Thames, Oxfordshire, England-England (GB), Bente Lissy Laursen, Ektorp, Ove Henning Mattsson, Täby, Sweden s-Sweden (SE) (7! +) Oy Kolster Ah (5U) Disinfectant containing 1,6-bis- (N -p The present invention relates to a novel disinfectant, which is characterized in that it contains a chlorine-phenyldiguanido-N 2) -hexane salt. This invention relates to a novel disinfectant which is characterized in that it contains Containing 1,6-bis- (N, β-chlorophenyldiguanido-N,.) -hexane and a sequestering aminocarboxylic acid, namely ethylenediaminetetraacetic acid, N-hydroxyethylethylenediaminetetraacetic acid, dihydroacetic acid, diethylenetriaminepentaacetic acid, nitrilotriacetic acid, nitrilotriacetic acid with an antimicrobial quaternary ammonium compound that promotes salt dissolution.

Bis-biguanides are (generally) known to have certain bactericidal properties, and in particular one such compound - (1-bis-1H-β-chlorophenyldiguanido-N 2) hexane, which has the unprotected name "chlorhexidine", has been found to have considerable use against bacteria. as a lethal substance and primarily as a general disinfectant. Unfortunately, however, the known bis-guanidine bases - and most of their salts - are only sparingly soluble in water (the solvent chosen in the preparation of disinfectants) and therefore have only limited use from a commercial point of view.

The new class of chlorhexidine salts can be dissolved in water more easily than most known salts and is more active than all known salts - and 58423 these compounds are therefore more useful especially as disinfectants and generally as bactericides.

The disinfectant according to the invention thus consists of novel chlorhexidine salts, i.e. chlorhexidine sequestrates, and solubilizers, which are quaternary ammonium salts, and suitable carriers.

The following abbreviations are generally used for the sequestering aminocarboxylic acids used:

Ethylenediaminetetraacetic acid: EDTA

N-hydroxyethylethylenediaminetetraacetic acid: HEDTA

Diethylenetriaminepentaacetic acid: DTPA

Nitrilotriacetic acid: NTA

N, with the M-dihydroksietyyliaminoetikkahappo; DAA.

Of these private aminocarboxylic acids, HEDTA and DAA are particularly preferred.

The new chlorhexidine salts - these compounds can be considered as a reaction product between a substantially acidic sequestering aminocarboxylic acid and a basic chlorhexidine (and can therefore be referred to not only as "salts" but also as "sequestrates") - may contain these two compounds (acid and base) differently. acids are usually polyvalent (having two or more acid groups) and chlorhexidine is dibasic, and because not all basic / acidic groups need to be “exhausted.” However, in general (but not always) acidic aminocarboxylic acids tend to act as either monovalent or divalent; as acids (in which either one or two acidic groups have been used).

Thus, for example, a particularly preferred aminocarboxylic acid, HEDTA, acts as either a monovalent or divalent acid and, as far as can be determined, the only salts formed are chlorhexidine mono-HIDTA acetate (where HEDTA is single).

Of the non-salts (sequestrates) obtained using the aminocarboxylic acids listed above, the following are particularly preferred: monochlorohexidine-ethylenediamine tetraacetate (chlorhex / EDTA); monochlorhexidine mononitrilotriacetate (chlorhex / NTA); trichlorhexidine di (diethylenetriamine pentaacetate) (3 chlorhex / 2 DTPA); monochlorohexidine di (N, N-dihydroxyethylaminoacetate) (chlorhex / 2 DAA); and in particular monochlorohexidine mono-N-hydroxyethyl ethylenediamine triacetate (chlorhex / HEDTA) and monochlorhexidine di (N-hydroxyethylethylenediamine triacetate) (chlorhex / 2 HEDTA).

Said sequestrates may be prepared by any of the conventional methods used in the preparation of salts. In particular, they can be prepared by a direct acid / base reaction or they can be prepared by an exchange reaction (or double 58423 decomposition). another salt of the acid and between the second salt of the base.

Although aminocarboxylic acids (and their salts) are generally water soluble, chlorhexidine (and its salts) is not particularly water soluble. Therefore, it is recommended that the reaction be carried out in the presence of a medium in which chlorhexidine is soluble and which is readily miscible with the water present. Such media include alkanols, and particularly suitable alkanols include methanol and ethanol. Thus, the reaction is preferably carried out in aqueous methanol or ethanol.

Of course, both chlorhexidine and the aminocarboxylic acid may be dissolved in an aqueous solution of an alkanol, or the former may be dissolved in a vehicle, and the latter in water, and the two solutions may be mixed.

When the reaction is carried out using a salt of chlorhexidine and a salt of an aminocarboxylic acid (exchange reaction), the former is preferably one formed with it with a common organic acid (e.g. diacetate) and the latter is preferably an alkali metal salt (e.g. sodium salt). Mineral acid salts of chlorhexidine should be avoided as they are very sparingly soluble in water. The salts formed can be isolated by conventional techniques, but for some purposes they do not need to be isolated at all (their aqueous solution can be used as such) and this will be described below.

It has been found that new chlorhexidine sequestrates have higher bactericidal activity than chlorhexidine. Thus, first, the range of chlorhexidine activity can be "extended" at a given concentration, i. chlorhexidine may be inactive against certain bacterial species at a certain concentration, while the corresponding sequestrate is active when used as the same concentration of chlorhexidine base. Second, if a certain concentration of chlorhexidine is required to kill a particular bacterium, a lower concentration of the corresponding sequestrate is sufficient to achieve the same killing effect.

The phrase "suitable carrier" is used herein to exclude the possibility that the nature of the carriers, considered, of course, in relation to the use for which the preparation is to be placed, could be harmful rather than beneficial.

The type of carrier used and thus the nature of the preparation will depend greatly on the purpose for which the preparation is to be used. The incomparably most important use of the preparations is as disinfectants, primarily for use in hospitals, and as such the preparation is usually a liquid, the carrier being e.g. water.

The disinfectants according to the invention generally contain chlorhexidine and an aminocarboxylic acid in stoichiometric molar ratios. Thus, for example, a preparation made from a chlorhex / HEDTA mixture contains these two parts in a molar ratio of 1: 1, while a preparation made from a chlorhex / HEDTA mixture contains these two parts in a molar ratio of 1: 2.

However, it is possible to prepare mixtures which contain an "excess" of either component (i.e., either chlorhexidine or arainocarboxylic acid 58423 k acid has been added) and in many cases the use of an excess - especially an excess of aminocarboxylic acid - is preferred and appears to improve somewhat more bactericidal properties. than one might expect. For example, excesses of the aminocarboxylic acid (e.g., 50 moles) produce preparations that function quite satisfactorily.

As mentioned above, the disinfectant according to the invention further contains quaternary ammonium compounds as a solubilizing agent for the chlorhexidine salt. Of the large amounts of quaternary ammonium compounds (QAC) available, the following appear to be recommended for use in the disinfectants of the invention: those having a cation of the general formula: 1 '1' YN-Y2 (IV) 't CH3 (where Y is 8-20 carbon atoms) an alkyl group, a diisobutylphenoxyethoxy-1 ... ethyl group or a diisobutylcresoxyethoxyethyl group, and Y is an aralkyl group, preferably a benzyl group, optionally substituted at its aromatic ring) especially those having a "benzalkonium" cation described in the literature alkyldimethylbenzylammonium ion in which the alkyl group is a well-known "coconut" group which is primarily a mixture of C10-18 alkyl groups such as lauryl, myristyl, etc.) and compounds sold under the tradename Hyamine LOX and Hyamine 1622 in the United States (disclosing diisobutylcresol-ethoxyethyldimethylbenzylammonium and diisobutylphenoxyethoxyethyldimethyl-1-benzyl ethylammonium ions) and myristyldimethylbenzylammonium compounds; those having a cation of the general formula CH + y3-n-ch „(V) f -3 CH3 5 58423 wherein Y ^ is either an alkyl group having 8 to 18 carbon atoms or an alkylbenzyl group in which the alkyl moiety contains 8 to 20 carbon atoms , in particular cetyltrimethylammonium compounds such as bromide, known unprotected as "cetrimide"; those having a cation of the general formulas? H3 + Y ^ -E-CH-

L T5 J

wherein Y 1 and Y 9, which may be the same or different groups, are both alkyl groups having 8 to 18 carbon atoms, especially dideylmethylammonium and dioctyldimethylammonium cations having a cation of the general formula Y6 + Y7-N-CBL - // \ (VII)

8 W

wherein Y 1 is an alkyl group having 8 to 20 carbon atoms; and Y 1 and Y® are the same or different groups and are both hydroxy-substituted alkyl or hydroxyalkoxyalkyl groups, especially an alkyldi (hydroxyethyl) benzylammonium cation, wherein the alkyl group is e.g. a "coconut" group; and those having a cation of the general formula: XX ~ + I (Vili)

X

Y9 wherein Y9 is an alkyl group having 6 to 20 carbon atoms, especially a cetylpyridinium cation ·

Particularly preferred quaternary ammonium cations are benzalkonlum, myristyldimethylbenzyllammonlum, cetyltrimethylammonium and coco (hydroxyethyl) benz8ylammonium ion.

6 58423

It does not seem to matter very much which anion is associated with the quaternary ammonium cat ion. In any case, hydroxides, chlorides and bromides are preferred.

Particularly preferred quaternary anunoniumyhdisteitä use in the compositions of this invention are the following: set yylitr imetyyliammon iumbromid i bent salkoniumhydroksidi myristyylidimetyylibentsyyliammoniumhydroksidi d idekyylidimetyyliammoniumkloridi di-ium chloride isobutyylikresoksietoksietyylidimetyylibent syyliammon di-p isobutyylifenoksietoksietyylidimetyylibentsyyliammoniumkloridi et yl ipyr in iumklor id id i d iokt yylid suck ylamino ium chloride.

The amount of quaternary ammonium compound to be added depends on the sequestrate, mainly the aminocarboxylic acid and its amount, as well as on the quaternary ammonium compound itself. Thus, it is difficult to give valid rules for how much quaternary compound is needed to dissolve a sequestrate. In any case, as a general rule, an effective quaternary compound - e.g., cetrimide (cetyltrimethylammonium bromide) - can dissolve enough sequestrate to give an aqueous solution of 20% by weight to dissolve the self-weight amount of sequestrate (while at lower sequester concentrate concentrations). compound tends to dissolve the sweat more than its own weight). Other quaternary compounds may dissolve only enough sequesterate to give a 5% solution, i.e. they may dissolve only 1/10 of their own weight of sequestrate.

A few general examples of the maximum dissolution of different quaternary ammonium compounds with different chlorhexidine sequestrates are as follows (percentages are based on the weight of the final solution and the resulting solutions are stable): 18% cetrimide dissolves 27% chlorhex / HEDTA, 30% benzalkon dissolve 15% chlorhex / HEDTA, 30% benzalkonium chloride dissolve 15% chlorhex / HEDTA, 20% benzalkonium bromide dissolves 20% chlorhex / HEDTA, 30% dodecyldimethylethylammonium methylsulfate dissolves 30% chlorohex / HEDTA, 30% setrim % cetrimide dissolves 5% chlorhex / EDTA, 20% cetrimide dissolves 30% 3 chlorhex / 2 DTPA.

τ 58423

In any given case, the dissolution efficiency of a particular quaternary compound relative to a particular sequestrate can be readily determined experimentally.

There also appears to be a synergistic effect between the quaternary and the sequestrate compound, with the bactericidal activity of the preparation generally being significantly higher (in some cases considerably higher) than would be expected from a simple summation of the activities of the two components.

The disinfectants of this invention can be prepared simply by mixing the sequestrate with the carrier and the quaternary ammonium compound (and additional sequestrate moiety, as appropriate) in a suitable medium. As mentioned above, the sequestrates formed do not necessarily need to be isolated from their reaction media.

It therefore follows that the disinfectants of this invention can be prepared simply by dissolving chlorhexidine and aminocarboxylic acid in water or aqueous alkanol in the presence of a quaternary ammonium compound.

Since the quaternary ammonium compound is capable of forming salts with aminocarboxylic acids (salts being quaternary ammonium sequestrates), it is also possible to prepare the disinfectant of the invention by dissolving chlorhexidine and the quaternary ammonium sequestrate in water or aqueous alkanol.

The following examples illustrate the invention.

PART I: Preparation of chlorhexidine sequestrates

Example 1

Mono (chlorhexidine) mono (ethylenediaminetetraacetate) 0.625 g (0.001 mol) of chlorohexidine diacetate was dissolved in 25 ml of distilled water and added to a solution of 0.336 g (0.001 mol) of disodium ethylenediaminetetraacetate in 10 ml. A fine white precipitate was obtained which was filtered, washed with distilled water and dried to give mono (chlorhexidine) mono (ethylenediamine tetraacetate) melting at 235 ° C (this salt contains 1 mole of chlorhexidine cation and 1 mole of EDTA anion). .

8 58423

In order to ensure that the salt had actually formed, comparative experiments were made for possible mixtures that might be present. Chemical experiments showed that the product did not contain sodium or acetate ions, but contained a chlorhexidine cation and an EDTA anion. Chlorhexidine has a melting point of 127 ° C, EDTA has a melting point of 2U2 ° C and a physical mixture of the two in mole ratios melts at 215 ° C.

The marks obtained from the differential calibration also confirmed the the presence of salt.

Simple examples showed that the solubility of this salt was very low and of the order of 0.02% w / w in water at 20 ° C. To further determine the solubility, small amounts of product weighing 0.01, 0.02 and 0.03 g were weighed into beakers and 100 g of distilled water was added. These mixtures were heated to 100 ° C with stirring, the resulting solutions were made up to 100 g to allow water loss due to evaporation and then cooled to room temperature. Ko. a small crystal of the salt was added and after 2k hours it was observed whether all the salt was dissolved, in which case the solubility was marked as the concentration of the solution in which no clear precipitate remained. By this technique the water solubility was found to be 0.02% w / w at 20 ° C.

Example 2

Mono- (chlorhexidine) mono (hydroxyethylethylenediamine triacetate: L)

Method A

2.66 g of chlorhexidine base and 1.1 ± 0 g of hydroxyethylethylenediamine triacetic acid were dissolved together in 50 ml of distilled water under heating. Thus, the acid and base were present in an approximately equimolar ratio. The resulting solution was allowed to cool and after a few hours a white precipitate settled to the bottom. The precipitate was filtered, washed and dried to give mono (chlorhexide: Lni) mono (hydroxyethylethylenediamine triacetate). The pH of the mother liquor was 7.3 while the salt had a melting point of 173 ° C and a solubility of 0.2% w / w at 20 ° C as measured by the technique described in Example 1.

Method B

25 ml of a 0.01 M solution of chlorhexidine in methanol (i.e. 0.1263 g of chlorhexidine) was added to 0.0695 g of HEDTA in 35 ml of distilled water. Thus, the acid and base were present in equimolar ratios. This solution was evaporated to half volume with a steam bath, then allowed to cool slowly and, after standing for 2 hours, crystallization was started by scraping the wall of the vessel with a glass rod. The precipitate was filtered off and dried to give the desired salt.

58423

Method C

1 g of chlorhexidine diacetate was dissolved in 25 ml of methylated industrial spirit, and the solution was added to a solution of 0.1 g of HEDTA in 3.2 ml of normal sodium hydroxide and 5 ml of distilled water. Thus, the acid and base were present in equimolar ratios. The precipitate formed on standing was filtered, washed with distilled water and dried to give the desired compound.

In all cases, the differential separation calorimeter indicated that salt formation had occurred. This was confirmed by melting points as follows:

The melting points:

HEDTA 156 ° C

Chlorhexidine 127 ° C

Salt (methods A and B) 173 ° C

(method C) 177 ° C

Physical mixture of acid and base 12U ° C

Example 3

Mono (chlorhexidine) di (hydroxyethylethylenediamine triacetate) 1.33 g of chlorhexidine and 1.0 g of HEDTA were dissolved together in 25 ml of distilled water with heating. Thus, the molar ratio of base to acid was 1: 2. The solution was cooled to give a white precipitate which was filtered, washed and dried to give mono (chlorhexidine) di (hydroxyethylethylenediamine triacetate). The pH of the solution was 6.1, the salt had a melting point of 172 ° C and its solubility in water was 0.3% w / w at 20 ° C as measured by the method of Example 1.

Example H

Mono (chlorhexidine) mono (nitrilotriacetate) 25 ml of a solution of 0.0U-M chlorhexidine diacetate in distilled water was added to 25 ml of a solution of 0.1 + M disodium nitrilotriacetate in distilled water.

Thus, the acid and base were present in equimolar ratios. A fine oily precipitate formed which initially remained suspended in the liquid but solidified on standing. This precipitate was filtered, washed with distilled water and dried to give mono (chlorhexidine) mono (nitrilotriacetate).

10 6 84 2 3

The solubility of the salt in water at 20 ° C was 0.3% w / w as measured by the method described in Example 1.

The differential separation calorimeter confirmed salt formation and this was further confirmed by melting point experiments to obtain the following melting points:

NTA 225 ° C

Chlorhexidine 127 ° C

Salt 176 ° C

NTA and chlorhexidine

physical mixture 129 ° C

Example 5

Tri (chlorhexidine) di (diethylenetriamine pentaacetate) 0.69 g of diethylenetriamine pentaacetic acid was dissolved in 100 ml of distilled water under heating. 1.33 g of chlorhexidine were dissolved in this solution with heating and stirring. Thus, base and acid were present in a molar ratio of 3 * 2. After cooling, an oil separated, but after two days at room temperature all the oil had turned into white crystals. The crystals were recrystallized from water, dried at 100 ° C to constant weight to give tri (chlorohexidine) di (diethylenetriamine pentaacetate). This compound had a melting point of 157 ° C and a solubility in water of 0.2 i / w / v at 20 ° C as measured by the method of Example 1.

Example 6

Mono (chlorhexidine) di (dihydroxyethylaminoacetate) 100 ml of a solution of chlorhexidine diacetate in 2 [mu] l of distilled water were added to a solution of 1.234 g of the monosodium salt of dihydroxyethylaminoacetic acid (corresponding to 1.0432 g of dihydroxyethylacetate) in acetic acid. Thus, the chlorhexidine cation and the dihydroxyethylaminoacetic acid anion were present in a molar ratio of 1: 2. The precipitate was filtered, washed and dried to give mono (chlorhexidine) di (dihydroxyethylaminoacetate). The solubility of this salt in water as measured by the method of Example 1 was 0.2 i / v / w at 20 ° C.

The differential ordering calorimeter showed salt formation and this was confirmed by melting point size 11a to give the following melting points:

Dihydroxyethylaminoacetic acid 180 ° C

Chlorhexidine 127 ° C

Salt 86 ° C

Physical mixture 107 ° C

11 S 8 4 2 3

Part II:

Preparations containing quaternary ammonium compounds Example 7

Mono (chlorhexidine) mono (HEDTA) / Cetrimide a) Preparation of a mixture of li +, 3 g of chlorhexidine, 20 kg of cetyltrimethylammonium bromide and 7.8 g of hydroxyethylethylenediamine triacetic acid (HEDTA) were reacted in 57.5 g of water with stirring until was dilutable with water in all ratios and contained a mixture of mono (chlorhexidine) mono- (hydroxyethylethylenediamine triacetate) and cetyltrimethylammonium bromide.

b) Bacterial Toxicity Test (Antiseptic Test)

The antibacterial activity of the solution obtained in a) was tested according to the British Standard Specification 2 ^ 62 variant.

A particularly stringent bacterial resistance test against the highly resistant gram-negative bacterium Pseudomonas pyocyane in the presence of hard water and organic matter is given below (see also "Method for laboratory evaluation of disinfectant activity of Quaternary ammonium compounds by suspension test procedure", published by the British Standard Institution, British Standards House, SWl London, i960).

(i) Materials Nutrient solution Use nutrient solution No 2 containing:

Lab-lemco (trademark of proteinaceous substance) 10 g

Peptonia 10 g

Sodium chloride 5 g

Distilled water up to 1000 ml ....... 2

This mixture was kept in an autoclave for 15 min. At an overpressure of 1 kg / cm (121 ° C). The final pH is about 7.

12 b84 2 3

inactivator

Nutrient solution with added 0,5 l »lecithin (ex ovo) (- a substance obtained from egg browns and a combination of choline, glycerol, phosphoric acid and various fatty acids) and 2 ^ Lubrol V. (a trademark for long-chain fatty acids). anhydrous condensation product of alcohol and ethylene oxide). If necessary, adjust the pH to 7 »2.

bacterial Species

Pseudomonas aeruginosa (pyo cry ane a) NCTC No. 6749 maintained on nutrient toagar.

(ii) Preparation of the culture

The culture of the organism was prepared from agar in nutrient solution and germinated at 37 ° C for 24 hours. Daily subcultures were continued and cultures between the third and seventh generations were used for the experiment. The solution culture of Pseudomonas pyooyanea was aseptically filtered before use or shaken, allowed to settle for 15 min. and the supernatant was used for the experiment.

Viljelylaimennus

One part of the solution culture was diluted with 24 parts of distilled water, i.e.

4 ml of solution culture + $ 6 ml of distilled water. The average number of bacteria at such dilution is about 5 * 10 organisms / ml.

Experimental Procedure

A series of two-fold dilutions of the antibacterial preparations to be tested were prepared in distilled water and 2.5 ml of these were transferred to sterile test tubes. From time zero, 2.5 ml of diluted bacterial culture suspensions containing 20 # of bovine serum were added to each test tube containing the antibacterial preparation to be tested.

After 5 minutes, a looped (approximately 0.01 ml) mixture of bacterial culture and antibacterial preparation was transferred from each tube to any medium formed by the inactivator solution in a new set of sterile test tubes. A total of 10 min. after one loop of the original mixture of bacterial culture and antibacterial preparation was transferred to a similar new test tube set containing medium formed by the inactivator solution. The inactivator stopped the more activity of the antibacterial agent and allowed the bacteria to grow again.

13 58423

All tubes containing the inactivator solution inoculated with the loop-transferred bacteria were germinated at 37 ° C for 48 hours and examined for bacterial growth. · The result was marked positive when growth occurred and negative when no growth occurred. Minimum concentration of bactericidal agent to give a negative input of 5 min. after the touch test, can be seen and confirmed for 10 min. by contact test and this concentration was recorded as a lethal dilution of that bacterial toxin · Tubes with negative results were further cultured in nutrient solution to ensure that the bacteria were indeed dead and that no bacteriostasis had occurred.

The results are expressed as the minimum concentration tested to obtain 100 killing power in 5 minutes at 21 ° C.

By way of example, the mean of several experiments with benzalkonium chloride-BPC was as follows:

Dilution Transfer in minutes 5 10 1-666 - 1,500 ppm - - 1-1000 - 1,000 "+ - 1-1353 - 750 + - 1-2000 - 500" + + 100 killing power concentration - 1500 ppm. It should be noted that the actual number can be anywhere from 1000 to 1500 ppm, and thus the lethal concentrations shown in the following experiments are subject to substantially the same errors.

The concentration of the various mixtures prepared according to the examples can be determined (expressed in parts per million of total organic material (bis-biguanide plus quaternary ammonium compound + aminocarboxylic acid)) and thus, knowing the relative amounts of components, the lethal dilution ·

Score:

The results are shown in Table I below. To make comparisons, lethal dilutions with chlorhexidine digluconate and a typical quaternary ammonium sequester mono (myristyldimethylbenzylammonium) mono (HEDTA) were also tested.

lit 58423

Table I

Lethal dilutions

Test substance a) bis- b) quaternary c) acid organic biguanidine amino (ppm) substance base (ppm) nium compound a) + b) + c) (cation + anion) ppm) ppm

Disinfectant according to the invention 60 90 30 100

Chlorhexidine Digluconate (Comparison) 200 to 150,350 QAC Sequestrate (Comparison) - 1060 ititO 1500 These results demonstrate the superiority of the preparation of this invention as a bactericide over chlorhexidine digluconate and QAC sequestrate.

Example 8 1 + 2.1 + g to 2b, 2% a (w / w) of benzalkonium hydroxide (a compound of formula IV in which the benzalkonium ion in the alkyl moiety is an alkyl-ethyl-ethyl-benzyl ammonium having from 8 to 18 carbon atoms) 1 +, 38 g of ethylenediaminetetraacetic acid (EDTA) was added to the solution. The mixture was diluted to a total volume of 97 ml and 0.1+ g of solid chlorhexidine was added. The resulting preparation consisted of a clear aqueous solution.

Example 9

Mono (chlorhexidine) mono (HEDTA) / Cetrimide

A solution of 12.6 g of chlorhexidine, 18.8 g of cetyltrimethylammonium bromide and 9.2 g of hydroxyethylethylenediaminetetraacetic acid in 300 g of water was heated to 60 ° C and 8 parts of glycerol were added with stirring. A fully gelatinized mixture was obtained which could be used for application to the skin. Example 10

Mono (chlorohexidine i) -mono (HEDTA) / s etrimide a) Preparation of the mixture

A solution of 17.6 g of chlorhexidine, 18.2 g of cetyltrimethylammonium bromide and 11.2 g of hydroxyethylethylenediaminetetraacetic acid in 53 g of water was prepared.

b) Bactericidal effect

The antibacterial effects of the mixture prepared in a) were tested in the same manner as in Example 7 · The results are shown in Table II below.

is 5,8423

Table II Lethal dilution

Bacterial toxin a) ppm bis- b) ppm quaternary c) ppm ppm organic biguanidine amino acid substance a) + b) + c) base nium compound

Mixture prepared in a) 75 75 45 195 These results are to be compared with those shown for chlorhexidine digluconate and QAC sequestrate in Table 1 above, where it is found that the preparation of the present invention is significantly superior as both bacterial agents as a bactericide.

Example 11 a) Preparation of a mixture 15 g of HEDTA di- (myristyldimethylbenzylammonium) eu and 8 g of chlorhexidine were dissolved in 77 g of water to give an aqueous solution.

(b) Bacterial toxicity tests

The antibacterial activity of the mixture obtained in a) was tested in the same manner as in Example 7 and the results are shown in Table HI below *

Table lii Lethal dilution

Bacterial toxin a) ppm bis- b) ppm quaternary o) ppm ppm organic biguanidine amino acid substance base compound a) + b) + o) (cationic anion)

Mixture prepared in a) 53 71 29 153 These results must be compared with those shown for chlorhexidine digluconate and QAC sequestrate in Table I above, where it is found that the preparation of the present invention is significantly better as a bactericide than these two conventional substances.

Example 12

Mono (chlorhexidine) di (EDTA) / myristyldimethylbenzylammonium hydroxide, 367.5 g (1 mole) of myristyldimethylbenzylammonium chloride was dissolved in the smallest possible amount of isopropyl alcohol. A saturated solution of 5 g (1 mole) of potassium hydroxide in absolute ethanol was added with stirring. After a few hours, the potassium chloride precipitate that had formed was filtered off and washed with isopropyl alcohol, and the washings were returned to the solution. The resulting alcoholic solution contained myryldimethylbenzylammonium hydroxide *. This solution was divided into ten equal portions, each containing 0.1 mol (34 * 9 g) of quaternary ammonium hydroxide.

To prepare an aqueous solution of the di- (myristyldimethylbenzylammonium) salt of 15% w / w EDTA, 14.6 g (0.05 moles) of solid ethylenediaminetetraacetic acid was dissolved in warm water and 1 part of the myristyldimethylbenzoxydiamine ammonium hydroxide prepared above was added thereto. The resulting solution containing 47 * 7 g (0.05 mol) of the di- (myristyldimethylbenzylammonium) salt of KDTA was diluted to 3 * 8 ml.

100 ml of this 15-36 solution contained 15 g of di- (myristyldimethylbenzylammonium) ethylenediaminetetraacetate, i.e. * 10.44 g of quaternary ammonium cation and 4 * 56 g of dibasic SDTA anion ).

The molecular weight of the chlorhexidine base is 505 °, so when preparing a chlorhexidine salt containing 1 mole of chlorhexidine base per 2 moles of SDTA, the ratio of anion to cation should be 584 * 505 (excluding hydrogen ions) 4-di g of dibasic anion, dissolves 505 * 584 x 4.56 to 4.12 g of chlorhexidine.

Thus, 2 any 15-36 solution of di- (myristyldimethylbenzylammonium) ethylenediaminetetraacetate at 40 ° C was added to 0.08 g of chlorhexidine to give a clear solution * Crystals formed on cooling *

Example 13

Mono (chlorohydeidine) -di (HBOTA) / nyristyldimethylbenzylammonium hydroxide) Using essentially the same procedure as in Example 12, a mono (myriathyldimethyl-benzammonium) salt of hydroxyethylethylenediaminetetraacetic acid was prepared by taking one of the fractions containing 0.1 m g (0.1 mol) of an aqueous solution of HEDTA, and then diluting the solution to 416 ml * This 15 wt.% solution of quaternary ammonium sequester contained 8.18% quaternary ammonium cation and 6.82 36 monobasic HBDTA anion.

To prepare the kono (chlorhexidine) di (HBI) TA) salt, it is necessary to add chlorhexidine at a molecular weight ratio of 505 parts of chlorhexidine to 2 x 278 parts of HEDTA measured on the free acid or 277 parts measured on the monobasic anion.

1T 5,8423 Thus, 124 g of chlorhehexidine was dissolved in 2 ml of a 15- # solution of monomyristyldimethylbenzyldiammonium mono (HEDTA) -8 [mu] l to give the required solution of mono (chlorhexidine) -di (hydroxyethyl ethylene dlamlin triacetate).

This solution could be diluted with distilled water to give, for example, a solution containing 820 ppm of quaternary ammonium cation, 628 ppm of monobasic HEDTA anion and 620 ppm of chlorhexidine.

Example 14

Mono (chlorhexidine) mono (HEDTA) (myriethyldimethylbenzylammonium hydroxide) a) Preparation of the mixture

In the same manner as in Example 12, di- (myristyl-dimethylbenzylammonium) -mono (by hydroxyethylethylenediamine triacetate) was prepared using 0.1 mol of quaternary ammonium hydroxide and 13-9 g (0.05 mol) of HEDTA and diluted to 314 ml to give a weight of 134 ml. -, »solution containing 10.6 # of quaternary ammonium cation and 4» 4 # of two basic HEDTA anions.

By taking 2 ml of this 13-wt. mono (kloorihek8idiini) mono (hydroksletyylietyleenidiamilnitri acetate). The result was a clear solution that could be further diluted to a clear solution containing 1060 ppm quaternary ammonium cation, 440 ppm HEDTA anion, and 800 ppm chlorhexidine.

(b) Bacterial toxicity tests

The antibacterial activity of the mixture prepared in a) above was tested in the same manner as in Example 7 above. * For comparative purposes, lethal dilutions of chlorhexidine digluconate and di (myristyldimethylbenzylammonium) mono (hydroxyethylethylenediamine triacetate) were also tested. The results are shown in Table iv below.

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Example 15

Mono (chlorhexidine) di (HEDTA) / mythiyldimethylbenzylammonium hydroxide a) Preparation of the mixture

In the same manner as in Example 14, a mono (chlorhexidine) di (hydroxyethylethylenediamine triacetate) containing one mole of chlorhehequidine per two moles of HEDTA anion was prepared by taking 2 ml of 13 μl of di (myristyldimethylbenzylamethyl) amineethyleneoxy and adding 0.08 g of chlorhexidine. The result was a clear solution which could be further diluted with water.

(b) Bacterial toxicity tests

The antibacterial activity of the mixture prepared in a) was tested in the same manner as in Example 7. The results are mentioned in Table V below.

Table V Lethal dilution

Bacterial ppm kva- ppm kva- ppm ppm chloro ppm ak- ^ chloro- Organic toxin ternary residue- HKDTA rhexative rhexic acid cross amry anion dine constituents total dinium multidium ammonium + quaternary - ppm distation of the cation from the weight of the ammonium-cation parts

Preparation of (a) above 400 283 117 108 391 28 308

Comparing these results with the results of the quaternary ammonium sequestrate and chlorhexldine dgluconate shown in Table IV above, it is found that the preparation of the present invention is clearly superior to both of these conventional bactericides.

Example 16

Hono (chlorhexidine) -mono- and di (HEDTA / nyristyldimethylbenzyl-1) ammonium hydroxide [a) Preparation of the mixture Using exactly the same procedure as in Example 14, mixtures were prepared from 13 £ v / v di (myristyldimethylbenzylammonium) 2 3 of oxyethylethylenediamine triacetate) and chlorhexidine base corresponding to mixtures of mono (chlorhexidine) mono (HEDTA) salt and mono (chlorohexidine) di (HEDTA) salt with an excess of di (quaternary ammonium) HBDTA) salt · When in Example 14 the mono (chlorhexidine) mono (HEDTA) salt was prepared from 15 Ree quaternary ammonium HEDTA salt and Θ Ree from chlorhexidine and in Example 15 the mono (chlorhexidine) di (HEDTA) salt was prepared from 13 Arms quaternary ammonium salt and $ 4 chlorhexidine, in this example the following mixtures were prepared: a) 13 ^ quaternary HEDTA + 1 ^ chlorohexidine (i.e. · less than 1: 2) h) 13 "" ♦ £ 2 N (i.e. less than 1: 2) c) 13 "" + 6 # "(i.e. · more than 1: 2 but less than 1: 1) b) Bacterial toxicity tests

The antibacterial activity of the mixtures prepared in a) above was tested in the same manner as in Example 7 · The results are shown in Table VI below · 21 58423 • H 5 rt o J3 4 »o 00: oJ H co

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Example 17

Mono (chlorhexidine) di (HEDTA) / didecyldimethylammonium chloride a) Preparation of the mixture

A series of mixtures of chlorhexidine di (hydroxyethylethylenediamine triacetate) and didecyldimethylammonium chloride were prepared.

The di (HEDTA) salt of chlorhexidine can be prepared by the method of Example 5 and the solid redissolved in water »but in this example. 0.2 v / v solution of the salt directly by dissolving 0.954 g of chlorhexidine and 1.046 g of HEDTA 1000 in any distilled water »

Didecyldimethylammonium chloride is commercially available in the form of a concentrated solution containing 5 ° C. salt and 50 water. A 0.2 .mu.l solution of this salt was prepared by dissolving 4.0 g of this machine enthrant 1000 in any distilled water.

The two solutions were then mixed in the following volume ratios;

Solution of quaternary ammonium salt Chlorhexidine di (HEDTA) A. (comparison) 100 56 0 B. 90 56 10? 6 C. 70 56 50 i> D. 50 1> 50 56 e. 50 io 70 i * p. 10 90 i> G. 0 100 (b) Bacterial toxicity tests

The antibacterial activity of the solutions A-G prepared in a) above was tested in the same manner as in Example 7 above. The lethal dilution of chlorhexidine digluconate was also tested as a control. The results are shown in Table VII below and are also shown graphically in Figure 1 of the accompanying drawings.

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58423 2b

Example 18

Mono (chlorhexidine) -di (HEDTA) / Hyamine 10X

a) Preparation of the mixture

A solution of 0.2% w / w chlorhexidine di (hydroxyethylethylenediamine triacetate) was prepared by dissolving 0.954 chlorhexidine base and 1.046 g of HEDTA in 1000 ml of distilled water, and a 0.2% w / v Hyamine 10X solution was also prepared. isobutylcresoxyethoxyethyldimethylbenzylammonium chloride) in distilled water.

The two solutions were then mixed in the following proportions:

Solution Mono (chlorhexidine) Hyamine 10X

di (HEDTA): a A 100% 0 Jfc B 75 & 25 * C 50% 50 * D 25 * 75 * E (comparison) 0 96 100 96

In all cases, clear, stable solutions were obtained.

(b) Bacterial toxicity tests

The antibacterial activity of the mixtures prepared in a) was tested in the same manner as in Example 7 · The results obtained are shown in Table VIII below and also graphically in Figure 2 of the accompanying drawings.

Table Vili Deadly dilutions

Solution Bacterial- Chlorine-hexane- HEDTA- Quaternary- Chloride poison codidine cation anion cross ammonium anion total concentration ppm ppm sodium cation ppm vyye ppm nia ppm A 300 143 157 0 0 B 400 143 157 92 Θ C 500 119 131 230 20 L> 1000 119 131 690 60 E (comparison) »1000 0 0» 920 »80 These results show that mono (chlorhexidine) di- (HEDTA) is not only a much better bactericide than Hyamine 10X, but also that chlorohexidine sequestrate and mixtures of a quaternary ammonium compound show synergism - as in Example 17 »the curve of lethal dilutions of mixtures of the two compounds is always below line A, which shows the results that would be obtained if the bactericidal effects of the two compounds were additive (experiments showed that the lethal dilution of the quaternary ammonium compound alone was about 2500 ppa and line A is plotted on this basis).

Example 19

Mono (chlorhexidine) -di (HEDTA) / Hyamine 1622 a) Preparation of the mixture

The $ 0.2 w / w solution of chlorhexidine di (HEDTA) prepared in Example 30 was mixed with $ 0.2 w / w Hyaaine 1622 solution (American trade name for diisobutylphenoxyethoxyethyldimethylbenzylammonium chloride) in the following proportions:

Solution Mono (chlorhequeidine) - Hyaaine 1622 di (HEDTA): a A 100 i> 0 B 75 & 25 # C 50 # 50 56 D 25 # 75 ^ E (comparison) 0 £ 100 et seq.

In all cases, clear, stable solutions were obtained.

(b) Bacterial toxicity tests

The antibacterial activity of the mixtures prepared in a) was tested in the same manner as in Example 7. The results obtained are shown in Table IX below, and also graphically in Figure 3 of the accompanying drawings.

Table IX

Solution Bacterial- Chlorohex- HEDTA- Quaternary Chloride toxin coccidian anion ammonium cation anion total ion, ppm nia ppm ppm lightness ppm ppm A 250 119 151 0 0 B 500 177 198 115 10 c 600 143 157 276 24 D> 1000> 119> 131 Λ 690> 60 E (comparison) »1000 0 0» 920 »80 The wedding results show that mono (chlorhexidine) -di (HEDTA) is not only a much better bactericide than Hyamine 1622, but also that chlorhexidine sequestrate and mixtures of the quaternary ammonium compound show synergism - as in Example 17, the lethal dilution curve B of the mixtures of the two compounds is always below line A, which shows the results that would be obtained if the bactericidal effects of the two compounds were additive (experiments showed that the quaternary compound alone the lethal dilution was about 2500 ppm and line A is drawn from this).

Symbol 20

Mono (chlorhexidine) di (HEDTA) / cetylpyridinium chloride a) Preparation of mixtures

To a 0.2% w / v solution of chlorhexidine di (HEDTA) prepared in Example 18 was stirred a 0.2% w / w solution of cetylpyridinium chloride in the following proportions.

Solution Mono (chlorhexidine) Cetylpyridinium chloride di (HEDTA) A 100 £ 0 i »B 75 i> 25 * C 50 # 50 $ D 25 i» 75 * E (comparison) 0 <j> 100 £

In all cases, clear, stable solutions were obtained.

(b) Bacterial toxicity tests

The antibacterial activity of the mixtures prepared in a) was tested in the same manner as in Example 7 · The results obtained are shown in Table X below and also graphically in Figure 4 of the accompanying drawings *

Table X

Solution Bacterial- Chlorohexane- HEDTA anion Quaternary- Chloride- poison coconut cation, ppm cross-ammonium anion female concentration, ppm multi-potassium ppm ppm thione ppm A 300 143 157 0 0 B 400 143 157 90 10 C 600 143 157 270 30 D 600 72 78 40 5 45 E (v ^ r1jai-> 1000 0 0> 900> 100 The wedding results show that mono (chlorhexidine) di (HEDTA) is not only a much better bactericide than cetylpyridinium chloride, but also that chlorhexidine sequestrate and synergies occur in the mixture of a quaternary ammonium compound - as in Example 17, curve B showing lethal dilutions of mixtures of the two compounds is always below line A, which shows the results that would be obtained if the bactericidal effects of these two compounds were additive (experiments showed that methylpyridinium chloride alone about 2500 ppm 27 5 8423 and line A is drawn based on this).

Example 21

Mixtures of chlorhexidine HEDTA sequestrates and cocoedihydroxyethylbenzylammonium hydroxide and HEDTA salt ) to distilled water. Thus, the molar ratio of ammonium cation to HEDTA anion in solution was 2: 1. A similar solution (Solution II) was prepared containing an additional 0.42 g of chlorhexidine. The two solutions were then mixed in the following proportions:

The resulting solution

Solution Weight- Weight- ^ Weight- # Weight- # HEDTA- part part chloroammonium anion solution solution hexidation cation ta II II AIO 0 0.769 0.277 B 5 1 0.07 0.769 0.277 C 2 1 0, 14 0.769 0.277

Dl 1 0.21 0.769 0.277

El 2 0.28 0.769 0.277 PO 1 0.42 0.769 0.277 b) Bacterial toxicity tests

The antibacterial activity of the mixtures prepared in a) above was tested in the same manner as in Example 7. Chlorhexidine diguconate was also tested for comparison. The results are shown in Table XI below.

Table XI Lethal dilutions

Solutions Bacterial ppm quaternary ppm HEDTA ppm chlorine ppm glucomoxins total anion hexidine ninate, ammonium cation ppm onion A 750 551 199 0 0 B 704 485 175 44 0 C 528 343 123 62 0 D 400 245 88 67 0 E 380 221 79 80 0 P 350 198 52 100 0

Chlorhexyl 660 0 0 376 284 zidine digluconate 26 584 23 These results show the superiority of preparations containing quaternary ammonium cations, HBDTA anions and chlorhexidine cations, both chlorhexidine digluconate and quaternary ammonium sequestrates.

Example 22

Chlorhexidine - Mixtures of HSDTA Sequestrate and Dioctyldimethylammonium Chloride a) Preparation of Mixtures Using the same technique as in Example 21, the following solutions were prepared:

Solution I

Dioctyldimethylammonium chloride 0.63 g HEDTA 0.28 g

Distilled water up to 100 ml

Solution II

Dioctyldimethylammonium chloride 0.64 g HEDTA 0.28 g

0.5 g of chlorhexidine

Distilled water up to 100 ml The two solutions were then mixed in the following proportions:

The resulting solution

Solution Part by weight Part by weight by weight of chlorine by weight of ammonium by weight of solution I of solution II of hexidine of the cation HEDTA anion AIO 0 0.569 0.28 B 7 1 0.063 0.569 0.28 C 3 1 0.125 0.569 0 , 28

Dl 1 0.25 0.569 0.28

El 2 0.33 0.569 0.28 F 0 1 0.5 0.569 0.28 b) Bactericidal experiments

The antibacterial activity of the mixtures prepared in a) above was tested in the same manner as in Example 7. · For comparison, chlorhexidine digluconate was also tested. The results are shown in Table XII below.

29 58423

Table XII Lethal dilutions

Solution Bacterial toxin Quaternary HEDTA Chlorhexane Total gluconate ammonium cation ** anion dine cation anion, pp ppm ppm ppm n ppm ppm A 750 500 250 oo B 485 500 150 55 0 C 516 500 150 66 0 D 421 217 108 96 0 E 425 200 100 125 0 F 598 167 85 148 0

Chlorhexidine digluconate 660 0 0 576 284 These results show the superiority of preparations containing quaternary ammonium cations, HEDTA anions and chlorhexidine cations to both chlorhexidine digluconate and quaternary ammonium sequestrate.

Example 25

Mixtures of Elohexidine -HEDTA - Sequestrate and Cetyltrimethylammonium Bromide

As discussed above, mono (chlorhexidine) mono (HEBTA) is soluble in water at 20 ° C in an amount of only 0.2% and mono (chlorhexidine) di (HEDTA) in an amount of only about 0.5%. This is a serious disadvantage from a commercial point of view and this example shows that the solubility of these chlorhexidine sequestrates can be greatly increased by the addition of cetyltrimethylammonium bromide and thus produce a commercially superior product.

Mixtures of chlorohexidine and HEDTAx were added to water and cetyltrimethylammonium bromide (cetrimide) was added until a clear solution was obtained. Alternatively, solutions can be prepared by adding solid chlorhexyl dilnequestrate to water and adding ammonium compound until dissolution occurs.

In this way, the following solutions were prepared!

Solution Chlorhexane-HEDTA- Chlorhexidine Cetyltrimethylammonium dione cation anion and HEDTAt molar bromide weight to weight ratio A 14.0 7.7 1 * 1 20.0 B 18.0 9.2 1x1 + excess 17, 6 C 7.2 7.1 1 * 2 20.7 D 5.5 8.5 1: 2 -f surplus 11.1 30 58423

All these solutions were clear and stable, b) Bactericidal experiments

The antibacterial activity of the solutions prepared in a) was tested in Example 7 by the antieptic test described above. The species Pseudomonas pyocyanea was used in these experiments: A) Standard in hard water (V.H.0 composition) t 10 ^ seta CaClgöEgO (w / v) 17f5 ml 10 tnen MGSO ^ HgO (w / v volume) 5.0 ml

Distilled water 3 »300 ml 2 This is kept in an autoclave at an overpressure of 1 kg / om for 15 min.

B) in distilled water with the addition of 10 l »of bovine serum; and C) Standard in hard water (V.E.0. composition) with the addition of 10 # bovine serum.

Chlorhexidine digluconate was also tested for comparison. The results are shown in Table XIII below.

Table XIII Lethal Dilutions A) Standard hard water, no serum

Solution ppm chlorine ppm HEDTA ppm gluco- ppm cetrimide hexidine anion nate cation A 80 45 0 114 B 200 100 0 289 C 100 100 0 288 D 80 120 0 162

Chlorhexidine digluconate 570 0 430 0 B) Distilled water, 10 l »serum

Solution ppm chlorine ppm HEDTA- ppm gluco- ppm cetrimide hexidine anion nate cation A 130 70 0 185 B 100 50 0 145 C 50 50 0 144 L 60 90 0 121

Chlorhexidine digluconate 285 0 215 0 31 5 8423 C) Standard hard water, 10 # serum

Solution ppm chlorine ppm HEDTA ppm gluco ppm with cetrimide hexidine anion nate cation A 320 180 0 456 B 760 390 0 1097 C 390 380 0 1124 D 200 300 0 405

Chlorhexidine digluconate 1,145 0 655 0 These results show that by adding cetyltrimethylammonium bromide and a sequestering aminocarboxylic acid to mixtures containing chlorhexidine, the amount of chlorhexidine required to produce a particular bactericidal effect can be reduced. Given that HEDTA and cetrimide are both significantly cheaper than chlorhexidine, this makes it possible to significantly reduce the cost of the antibacterial preparation required to achieve a certain effect. Alternatively, the efficacy of a bactericidal preparation that can be prepared at a certain cost can be greatly increased.

Example 24

Mixtures of chlorhexidine-DTPA sequestrate and cetrimide (a) Preparation of mixtures Using the same procedure as in Example 23, the following mixtures of chlorhexidine-DTPA sequestrate and cetrimide were prepared:

Solution Chlorhexa DTPA anion Cetrimide din cation weight # weight 10%, weight # A 10.5 7.5 15.0 B 13.4 6.5 19.5

The resulting solutions were clear.

(b) Bacterial toxicity tests

The antibacterial activity of the solutions prepared in a) was tested in the same manner as in Example 23: A) Standard in hard water (VH0. composition) without serum, B) Distilled water with 10 # bovine serum, and C) Standard in hard water (V .Hf0. Composition) with 10 # bovine serum.

Chlorhexidine digluconate was also tested for comparison. The results are shown in Table XIVa.

32 5842 3

Table XIV Lethal Dilutions A) Standard hard water, no serum

Solution ppm chlorhex- ppm DTPA anion ppm gluconate ppm cetrimide zidine cation A 60 40 o 86 B 67 35 0 9Θ

Chlorhexidine digluconate 570 0 430 0 B) Distilled water, 10 l »serum

Solution ppm chlorhexi- ppm DTPA anion ppm gluconate ppm cetridine cation midi A 90 60 0 129 B 100 50 0 147

Chlorohexane 285 0 215 0 dine digluconate C) Standard hard water, 10 $ 6 serum

Solution ppm chlorhexane ppm DTPA anion ppm gluconate ppm cetridine cation mide A 300 200 0 430 B 670 330 0 980

Chlorhexyl 1145 0 855 0 din digluconate

As in Example 23, these results show that the efficacy of a bactericidal mixture containing chlorhexidine can be greatly increased by adding cetrimide and DTPA and thus saving costs. Similar results were obtained using mixtures of chlorhexidine NTA sequestrate and cetrimide.

Example 25 Efficacy of the Preparations of the Invention Against Various Bacteria To demonstrate that the efficacy of the formulations of the present invention is not limited to Pseudomonas pyocyandea * The killing efficacy of Example 23 Solution A and Example 24 Solution A and chlorhexidine digluconate was tested against a variety of bacteria. The experiments were performed in the same manner as in Example 7 in distilled water containing 10 bovine sera, and the bacteria used were Beaver: 33 5 8 4 2 3 1) Pseudomonas pyocyanea N.C.T.C. 6749 2) Proteus vulgaris N.C.T.C. 4635 3) Salmonella typhl N.C.T.C. 3590 4) Escherichia coli N.C.T.C. 8196 5) Staphylococcus aureus N.C.T.C. 3750 The results obtained are shown in Table XV below.

Table XV Lethal dilution ppm Chlorhexidine sequestrate

Solution l) pyo. 2} p. 3) typhi 4) E.coll 3) Staph.

Example 23

Solution A 200 300 100 150 200

Example 24

Solution A 150 350 100 200 100

Chlorhexidine digluconate 500 750 300 300 500 These results show that the preparations of this invention are clearly superior to chlorhexidine digluconate against a variety of bacteria.

Example 26

To confirm the results obtained with the E. coli species shown in Example 25, experiments were performed on the bactericidal properties of the preparations of this invention against E. coli by the British Standard 3286: 1960 method described above in standard hard water (W.H.O. composition) containing 10 # bovine serum. The solutions used were as follows, with chlorhexidine digluconate as a reference:

Solution weight # chloro weight # weight # weight # weight # weight # rhexidine HEDTA DTPA NTA gluconate cetrimide cation anion anion anion anion A 13.0 7.2 0 0 0 18.9 B 13.6 0 6.5 0 0 11.3 C 14.4 0 0 5.1 0 15.4

Chlorohexyl 11.4 0 0 0 8.6 0 dine digluconate (comparison)

Preparations were tested at concentrations corresponding to 100, 150, 200, 250, 300, 350, 400, 500, 600 and 800 ppm chlorhexidine sequestrate or digluconate.

The results, shown in Table XVI below, are expressed as the number of survivors per million implanted bacteria.

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