Glycerite of hydrogen peroxide

GLYCERITE A COMPARISON

OF HYDROGEN

PEROXIDE*

OF ITS BACTERIOTOXIC EFFECT ON CLOSTRIDIA OTHER ANTISEPTICS

ETHAN ALLAN BROWN, M.D., RITA

WITH THAT

OF

E. SKIPFINGTON, B.S. AND W. B. KRABEK, M.S.

Boston, Massachusetts HEN it was found that reIativeIy stabIe sohrtions of urea peroxide3 and of hydrogen peroxide6 in substantiaIIy anhydrous gIycero1 couId be prepared, studies were made of its bacterioIogic and toxicoIogic properties. Comparisons of the peroxide soIutions with mercuria1 soIutions4 and with iodine solution9 by a cylinder pIate method have been reported. The physioIogic properties of the peroxide-gIycero1 soIution have been described.lF2 The present paper records the resuIts obtained in comparing the action of glycerite of hydrogen peroxide on CIostridium weIchii and CIostridium tetani with that of thirty-two commercia1 antiseptic soIutions: Aqueous peroxide soIutions have been used to a considerabIe extent and urea peroxide crystaIs to a lesser extent in the treatment of anaerobic infections. Many references in the Iiterature’*16 refer to Iavage with peroxide soIutions. Some workers have injected such soIutions. The Iatter procedure is accompanied by considerabIe danger due to emboIusg or gas formation in the tissues.8 Peroxide soIutions obviousIy shouId not be used in cIosed spaces. MeIeneyll recommends thorough exposure of the affected tissue to the active agent. This hoIds true for a11 topica appIications, regardIess of the type of medication. The poor resuIts reported for some peroxide compositionslo appear to be due, in part at least, to faiIure in foIIowing this recommendation. Another possibIe error is due to the fact that peroxide soIutions in the usua1 concentrations are reIativeIy sIow in their bactericidal action and hence must be in contact continuousIy with the organisms.

W

Many of the commercia1 antiseptic soIutions, such as some of the mercuria1 SOIUhave a rapid bacteriotoxic and tions, sIower (frequentIy much sIower than peroxide) bactericida1 action. As in the other comparison studies previa cup plate technicI” ousIy pubIished, modified by the use of glass or porceIain cyIinders was used. The method was described in the frrst paper of the series4 and consisted essentiaIIy as foIIows: Difco dextrose agar, with and without IO per cent added horse serum, was preinocmated with 0.2 m1. of a twenty-two to twenty-sixhour cuIture of the organism for each 25 m1. of meIted agar. After the poured plate ( IOO mm. diameter) had hardened, sIightIy warmed cyIinders, 8 mm. in outside diameter the rims of which had been paraffined, were pIaced on the agar. The solution to be tested was pIaced in the cyIinders in 0.2 m1. amounts. The pIates were immediateIy incubated anaerobicaIIy13 for eighteen hours. SubcuItures of approximateIy 3 mm. cross section were taken radiaIIy from the edge of the cup and pIaced in modified Brewer’s thiogIycoIIate medium and incubated at 37°C. for at Ieast seven days. After subcuIturing, the distance of the outer edge of the cIear zone, if any, to the outer edge of the cup was measured to the nearest 0.3 mm. and recorded for each cyIinder. At the same time the distance from the edge of the cyIinder to the inner and outer edge of the indentation Ieft by the removal of materia1 for subcuIturing was measured and recorded. Because of space Iimitations the tabIe shows onIy the average dista1 measurement of the subcuIture area cIosest to the cup that showed

* The expenses for this study were defrayed in part by The Asthma

630

Research Foundation,

American

Inc., of Boston,

Mass.

Journal sf Surgery

Brown

of Hydrogen

et aI.-Glycerite

TABLE ACTION

OF

TETANI

IN

ANTISEPTIC THE

SOLUTIONS

PRESENCE

AND

63’

Although solutions of urea peroxide in glycerol are in general more stable than simiIar soIutions in water, some lots of glycerol yield solutions that are less stable than others. Hydrogen peroxide solutions in gIycero1 are apparentIy of short life in the absence of stabilizing agents. It was

grobvth in the subculture tube. This procedure ob\-iousIy favors the antiseptic. At least three, and in some cases more, replicate tests Lvere made. The vaIues in the table have been rounded off to the nearest whoIe number since this is sufficient for comparative purposes. (TabIe I.) BACTERIOTOSIC

Peroxide

I ON

CLOSTRIDIUM

ABSENCE

OF

-

IO

WELCH11

PER

tClostridium

CENT

AND

HORSE

-

\\‘eIchii

CIostricIium

Plain _

1057~ Serum

PIair

r8i

26

20

20

39 33

I 0 :j;

Serum

-_

Glycerite of hydrogen peroxide. ................................ GIycerite of hydrogen peroxide (with urea). ...................... Glycerite of hydrogen peroxide (with oxine). ..................... GIycerite of hydrogen peroxide (with oxine and urea). ............. Tincture of hydrogen peroxide .................................. GIyceroI saturated with oxine ................................

25

‘5

22

21

21

26

2

.\lercresin, tincture r : 1000 ..................................... Mercurochrome aqueous 2 yO. .................................. Mercurochrome, surgica1 z %. .................................. Metaphen, tincture I : 200 ...................................... Metaphen,aqueous 1:500 ...................................... Merthiolate, tincture I : 1000. .................................. MerthioIate,aqueous I:IOOO ................................... Merthiolate, glycerite I : IOOO ................................... lMersepta1, tincture I : 500 ...................................... Merseptal, aqueous 1: 1500 ..................................... Merbromin,surgicaIzC;;, ....................................... Merbromin, aqueous 2 ?;, ..................................... Iodine, tincture USP 7%. ..................................... Iodine, tincture miId 2y0 ....................................... Biodone (aqueous iodine) 2 yo. .................................. Iodine,aqueousN.F.2L7, ...................................... Iodine, gIycerite 2y0 ........................................... Zephiran, tincture I : IOOO ...................................... Zephiran,aqueous I:IOOO ...................................... Phemerol, tincture I : 500. ...................................... PhemeroI,aqueous I:IOOO ..................................... CepacoI, tincture I : 4000. ..................................... Ceepryn,tincture I:500 ....................................... Ceepryn,aqueous r:rooo ...................................... Acriffavine (I : 1000)........................................... S.T.37 I:IOOO ................................................ AzochIoramide (1:3300) ....................................... Tenicide ..................................................... Auralgan ..................................................... Otosmosan ................................................... Otomide ..................................................... LysoI (1:64) ..................................................

39 34

I0

4’ 37 35 3

19 8

42 3’

9 4

21

18

17 27

14

28

3: 35 4

9 3 5

10 5 7 i

3 3 5 3 26 ‘3 6” 9

II

39 21

19

:I 3 3 : 16 4 II 0

* The commercia1 antiseptics were used fuI1 strength except where the concentratron t SubcuIture zones were measured radiaIIy in mm. from the edge of the cyIinder.

Ma.v, I 949

Tetani

-. ..--

SoIution *

.._____.~

CLOSTRIDIUM SERUM

7 ;

6 25 5 4 34 6

IS grven

4 7 ‘3 3 4 ‘4 5

parentheses.

632

Brown

et aI.-GIycerite

found that the addition of small amounts of oxine (S-hydroxyquinoIine) resuIted in greater stabiIity with a11 Iots of gIycero1 and permitted the use of concentrated hydrogen peroxide (90 per cent) in making stabIe soIutions. The soIutions tested, with their concentrations, are shown in the tabIe and wiI1 not be reIisted here, except for the peroxide compositions and some Iesser known soIutions which are mixtures. The composition of the peroxide soIutions was as foIIows: GIycerite of hydrogen peroxide: hydrogen peroxide 1.5 per cent in gIycero1 GIycerite of hydrogen peroxide with urea: urea peroxide 4 per cent in gIycero1 Glycerite of hydrogen peroxide with oxine: hydrogen peroxide 1.5 per cent; oxine 0.1 per cent in gIycero1 Glycerite of hydrogen peroxide with oxine and urea: urea peroxide 4 per cent: oxine 0.1 per cent in gIycero1 Tincture of hydrogen peroxide: urea peroxide 4 per cent; oxine 0.1 per cent in aIcohoI 50 per cent; acetone I0 per cent; water 40 per cent The compositions of the those shown on the respective

foIIowing IabeIs.

are

Otomide: suIfaniIamide 5 per cent; urea IO per cent; chIorbutano1 3 per cent; gIycero1 AuraIgan: antipyrine 0.87 Gm.; benzocaine 0.23 Gm.; gIycero1 0.463 fI. oz. Otosmosan: urea 2.4 Gm.; suIfathiazoIe 2.02 Gm.; benzocaine 0.20 Gm.; gIycero1 20.0 Gm. Tenicide: aIcoho1 56 per cent; chIoroform 8 per cent; iodine (total) 1.2 per cent; zinc suIfocarboIate, saIicyIic acid, formaIdehyde, thymo1 and camphor COMMENT

The contro1 soIutions, “tincture soIvent” (50 per cent aIcoho1, IO per cent acetone, 40 per cent water) gIycero1 and aIcoho1 70 per cent gave no zones under the experimenta1 conditions. With the exception of U.S.P. tincture of iodine which was essentiaIIy equa1 in potency, the gIycerite of hydrogen peroxide compositions were far more effective in bactericida1 action than any of the other thirty-two soIutions. This is to be ex-

of Hydrogen

Peroxide

petted somewhat because of the oxidizing nature of the soIutions and the anaerobic character of the organisms. That this activity was not due to the presence of free oxygen in the space above the pIate is indicated by positive subcuItures. If aerobic conditions were present due to free oxygen, it wouId be expected that no growth wouId appear. Some visibIe growth was apparent in a11 the pIates. The presence of serum appeared to have Iess effect on the bacteriotoxic activity of the majority of soIutions than was the case with aerobic cuItures. The strain of CI. tetani used proved to be more susceptibIe to a11 the antiseptics but more particuIarIy to the peroxides than was CI. weIchii. AI1 the mercuria1 soIutions showed effects of essentiaIIy the same order. The cationic soIutions showed the usua1 Iow degree of activity. The point has been made14 that tests using agar are not suitabIe for the evaIuation of quaternary ammonium saIts. Since both agar and tissue have an eIectric charge of the same sign it wouId seem that the use of agar wouId give a better indication of the activity of cationic substances on massive tissue areas than the use of methods which do not use materia1 with a charge of the same sign as that of the tissue on which it is to be used. The formation of a surface Mm on skinI with viabIe organisms underneath wouId seem to bear this out. Otomide containing a 5 per cent suIfaniIamide showed a considerabIe zone with CI. tetani, a much smaIIer one with CI. weIchii. The iodine soIutions showed more activity with CI. tetani than with CI. weIchii. SUMMARY

In cyIinder pIate tests on Cl. weIchii and CI. tetani gIycerite of hydrogen peroxide soIutions showed much greater activity than did thirty-two other antiseptic soIutions with the exception of U.S.P. tincture of iodine. TweIve mercuria1 soIutions showed Iess activity than did the peroxide American

Journal

of Surgery

Brown

et aI.-GIycerite

solutions and were of about equal nctivit.y when compared with each other. Seven cationic solutions faiIed to demonstrate activity in severa cases and were of sIight effect in other cases. Otomide, a soIution containing SuIfaniIamide, produced a zone of considerabIe size with CI. tetani. REFERENCES I. Bnow~, E. A. Studies on tissue tolerance to a new glycerol peroxide antiseptic solution. Ann. Allergy, 4: 33, 1946. 2. BROWN, E. A. Glycerite of hydrogen peroxide: a correIative review of Iaboratory and cIinica1 data. Ohio State hf. J., 42: 600, 1946. 3. BROWN, E. A., ARRAMSON, H. A., GORIN, M. H., KAuFFhfANN, H. 0. and SHANLEY, E. S. The stability of solutions of urea peroxide in glycerol, isopropano1 and ethylene gIyco1. J. Am. Pbarm. A. (Scient. Ed.), 35: 304, 1946. 4. BROWN, E. A., KRABEK, W. and SKIFFINGTOK, RITA E. GIycerite of hydrogen peroxide. I. C:omparison of its bacteriotoxic action with that of mercurial solutions. J. Bact., 53: 793, 1947. 5. BROWY, E. A., KRABEK, W. B. and SKIFFINGTON, RITA E. Glycerite of hydrogen peroxide. II. Comparison of its bacteriotoxic action with that of solutions containing Iodine. Journal Lancer, 67: 405, 1947. 6. BROWN, E. A., KRABEK, W. B., SKIFFINGTON,RITA 1:. and CRT-ICKSHANK,G. A. The effect of oxine

May,

1949

of Hydrogen

Peroxide

613

i8-h~tlrosycl~linolinc) on the stability of glyceritc of hydrogen peroxide. J. .4n1. I’bnrni. A. (Scirn~. Ed.), 37: 34. 1947. 7. COLE, LESI 1~. TetaIlus. Bril. A\f. J., 2: jjo, 194.1. 8. DELBET, P. Etude stir la therapeutique des plaies de guerre. Bull. Acad. de m&f., I’uri.s, 73: 678, IgIj.

9. GRAF, A. Umspritzungen mit HzOz bei Gasbrand. Miincben. med. Wcbnscbr., 86: 28, ,939. IO. K\;EPL,M., HAUSER, E., CALDUXLL, G. and OCHSNER, A. Experimenta studies on urea peroxide with CIostridium welchii in vitro and in viva. Neu; Orleans hf. C” S. J., 96: 294, 1944. I I. ~IELENEIY,F. \I’. The important anaerobic infections and the use of zinc peroxide in their contro1. U. S. Nav. M. Bull., 40: 53, ,942. DOROTIIX A. 12. ~IILLER, B. F., ABRAMS, R., MUBER, and KLEIN, hl. Formation of invisibh, nonperceptible tiIms on hands by- cationic soaps. Proc. Sot. Exper. Bio1.e Med., 54: 174, 1943. ‘3. MORTON, H. E. An improved technique for growing micro-organisms under anerobic conditions. J. Uact., 46: 373, 1943. 14. QUISNO, R., GI~BY, I. W., FOTER, ht. J. The Effect of agar upon the germicidal potency of the quaternary ammonium saIts. J. Am. Pbnrm. A. (Scient. Ed.), 35: 317, 1946. 15. RUEHLE, G. L. A. and BREWER, C. i\l. United States Food and Drug Administration hlcthods of Testing Antiseptics and Disinfectants. U. S. Dept. Agr. Cir. 198, 1931. 16. WHITE, J. H. Report of a case of gas gangrene treated with oxygen injection and peroxide. J. Oklabomcr hf. A., 21: p), 1928.