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1 THE BACTERIOLOGICAL EVALUATION OF SOME NEW WATER SOLUBLE ORGANO-MERCURY COMPOUNDS, JOHN H. WALDO, H. A. SHONLE AND H. M. POWELL Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana Received for publication December 1, 1930 INTRODUCTION The therapeutic use of organo-mercury compounds as bactericides is dependent in large measure on their solubility in water and their toxicity. Two methods for increasing the solubility of such compounds have been used. Introduction of acid- or nitrogen-containiing groups gives increased solubility, but the former cause a loss of germicidal action and the latter give products having much greater toxicity than the acid derivatives. Neither of these procedures has therefore given a satisfactory solution to the problem. A chemical combination which would produce increased water solubility and lower toxicity would do much toward bettering the application of organo-mercury compounds in the therapeutic field. In 1922 Kharasch2 developed a method whereby organometallic compounds can be made soluble by condensing them with mercapto-carboxylic or sulfonic acids. When mercurials of the type RHgX, where X is an inorganic radical, react with a mercapto acid, HSR' where R' is a group 1 The chemical preparation of these compounds has been presented in abstract before the Division of Medicinal Products of the American Chemical Society at the Detroit meeting, September, 1927, by M. S. Kharasch, H. A. Shonle and John H. Waldo and at the Atlanta meeting, April, 1930, by John H. Waldo and H. M. Powell. The authors wish to express their appreciation to Drs. G. H. A. Clowes and M. S. Kharasch and Mr. W. A. Jamieson, for advice and suggestions in the conduct of this work. 2 U. S. Patent 1,589,599, reissue

2 324 JOHN H. WALDO, H. A. SHONLE AND H. M. POWELL containing an alkyl or aryl carboxylic or sulfonic acid, a double decomposition occurs: RHgX + HSR' = RHgSR' + HX The new compound RHgSR' is soluble in NaHCO8 solution and forms with the alkali metals soluble salts whose solutions are in general stable and do not give an immediate precipitate of HgS with (NH4)2S. In collaboration with Kharasch, Cohen (1929) investigated alkyl mercurithioglycollic acids and found them to possess marked bactericidal properties. In collaboration with Kharasch an extended series of mercury compounds of the general formula RHgSR' were prepared and tested in this laboratory.3 This series might be expected to contain members which would be very active germicides and yet possess the low toxicity of mercury compounds containing carbonyl groups. In the selection of a substance therapeutically valuable as a germicidal agent, such factors as solubility, stability, lack of marked acid or basic reactions, absence of inhibition of cell growth and low toxicity must be considered. Also effective bactericidal and bacteriostatic action against both vegetative and spore formers especially in the presence of adequate organic matter must be shown. In evaluating a series of germicides, other factors being equal, that compound having maximum destructive effect on bacteria and minimum toxicity would be the most desirable for therapeutic use. For these reasons due attention was given to toxic properties and it was decided to adopt a preliminary standard test, as outlined by Reddish (1927) for comparative bactericidal tests. To avoid the criticism which might be offered against bactericidal tests in watery media, an additional test, modifying this technique by using normal horse serum as diluent instead of distilled water, has been carried out. It was first determined that the compounds used did not precipitate the proteins of ' U. S. Patent 1,672,615.

3 WATER SOLUBLE ORGANO-MERCURY COMPOUNDS 325 normal serum, a characteristic frequently common to organomercury compounds. BIOLOGICAL EVALUATION The compounds were first evaluated by the standard Reddish test, and then by the following modification, which consisted in planting 0.5 cc. of twenty-four-hour broth culture into 5 cc. quantities of germicide dilution4 made with undiluted normal horse serum. After a ten-minute exposure at 20 C., a 3-mm. loop of this material was subcultured to broth and readings were made after forty-eight hours incubation. Tubes showing no growth at this time produced normal growth when planted with 0.02 to 0.01 cc. of old untreated culture. For comparative purposes, three commercial germicidal preparations were selected and submitted to test by identical technique. A test with highly virulent organisms was introduced to determine the effect of these germicides on bacteria directly cultured from the blood stream of animals. Pneumococci (type I) of such a virulence that a hundred millionth to a billionth cubic centimeter of a broth culture was fatal to mice were planted in 0.5 cc. doses into 5 cc. quantities of aqueous germicidal dilution. After an exposure of thirty minutes transplants were made into broth and in addition 0.1 cc. quantities were injected into mice. The presence of viable organisms in the germicide mixture was determined by growth in the subculture tubes and by death of the injected mice. There was complete agreement between these two tests. It was noted that organisms directly cultured from an animal are much more resistant to a germicide than organisms passed through several subcultures in media. It appears that the figures representing the toxicity of organomercury compounds should represent not alone the acute or immediate degree of toxicity, but should be representative of any later effects as well. For this reason, toxicity figures recorded in the table were determined after seven days observation, and the M.L.D. was determined from animals surviving after that time. It has been noted that following such a period of observation, I The original germicides were 1 per cent water solutions.

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6 328 JOHN H. WALDO, H. A. SHONLE AND H. M. POWELL surviving animals suffer no such ill effects as latent mercury poisoning. Toxicity was determined by intravenous injection, and three well known commercial preparations were used as controls. The data thus obtained are tabulated in table 1. It was found that in the presence of serum the bactericidal action of these mercurials was not materially lower than their action in the presence of water. This is in distinct contrast to the general observation that the presence of serum tends to reduce the effectiveness of germicides. Table 1 shows that one member, ethyl-mercuri-thiosalicylic acid, is quite superior in all three properties recorded. It combines the greatest bactericidal action both in the general test GURMICIDE TABLE 2 Bacteriostatic value 24 HOuRs 48 HOuRs 72 HOuRS Staph. B. Staph. B. Staph. B. aureus typhosu8 aureus typhomts aureus typho8u8 C2H^HgSKii COOH... 1: 3, 000, 000 1:1, 000,0001: 2, 000, 00011: 700, 00011:1, 000, 00011: 700, 000 HgCI2... 1:100,000 1:1, 160,000 1:50,000 1:100,000 1:50,000 1:60,000 Germicide B... 1:1,000,000 1:60,000 1:700,000 1:50,000 1:600,000 1:50,000 and against virulent organism with a low degree of toxicity. It has also been demonstrated to possess a minimum hemolytic action toward red blood cells. This work will be more specifically outlined in conjunction with more extensive tests and will be reported upon at a later date. For this reason bacteriostatic tests were carried out on the above product, and comparison made with mercuric chloride and a commercial mercurial germicide. A 3-mm. loop of twenty-fourhour broth culture was planted into 5 cc. quantities of the indicated dilutions of germicide made in broth and incubated at 37.50C. Readings for growth were made at the end of twentyfour, forty-eight and seventy-two hours.

7 WATER SOLUBLE ORGANO-MERCURY COMPOUNDS 329 SUMMARY 1. The animal toxicity and bacteriostatic and bactericidal action of some new organo-mercury germicides has been presented. 2. One of the group, ethyl-mercurithiosalicylic acid has been shown to possess properties indicating particular value as a germicide. REFERENCES COHEN, S. J The pharmacology and toxicology of some new organomercury compounds. Jour. Pharmacol. and Exper. Therap., 35, 343. REDDISH, G. F Examination of disinfectants. Amer. Jour. Pub. Health, 17, 320. Downloaded from on April 26, 2018 by guest

RICINOLEATE UPON BACTERIA

A COMPARATIVE STUDY OF THE ACTION OF SODIUM RICINOLEATE UPON BACTERIA From the Division of Laboratories and Research, New York State Department of Health, Albany Received for publication, May 14, 1928