The toxicity of four concentrations of DMSO

TOXICOLOGY

AND APPLIEDPHARMACOLOGY

The

Toxicity

15,275-281(1969)

of Four

Concentrations

ELMER G.~ORTHLEYAND

C.DONALD

of DMSO SCHOTT

Medical Research Laboratory, Edgewood Arsenal, Maryland 21010 Received September 16,1968

The Toxicity of Four Concentrationsof DMSO. WORTHLEY, E. G., and ScHorr, C. D. (1969).Toxicol. Appl. Pharmucol. l&275-281. Thetoxicity of dimethyl sulfoxide (DMSO) to mice wasmeasuredby giving ip dosesin concentrationsof 25,50,75, or 100%. It wasfound that the LDSOincreased somewhatasthe concentration decreased,but the increasewassignificant only at the interval between50 and 25%. The LD50 valuesfound for the abovefour concentrationswere 15.4,13.3,11.9,and 10.9g/kg, respectively.

Dimethyl sulfoxide has been added to the scientist’s repertory of vehicles for dissolving or suspending drugs for topical or parenteral administration to animals. Dimethyl sulfoxide is regarded as nontoxic when administered topically, (Horita and Weber, 1964; Kelley, 1965; Kligman, 1965; McDermot et al., 1965; Pisanu and Cerimele, 1965 ; Scherbel et al., 1965 ; Stoughton, 1965 ; Stoughton and Fritsch, 1964), but small doses of the undiluted material are toxic when administered by other routes (Brown et al., 1963 ; Mallach and Etzler, 1965 ; Rosenkrantz et al., 1963 ; Sommer and Tauberger, 1964; Willson et al., 1965; Worthley and Schott, 1966). We have found it to be an excellent solvent at nontoxic concentrations and have used it as a suspending agent for crude drug samples in animal toxicity screening experiments (Worthley et al., 1967; Worthley and Schott, 1967, 1969). One of our reasons for selecting this solvent was that it does not significantly alter the toxicity of the materials with which it is mixed (McDermot et al., 1965; Braude and Monroe, 1965; Dixon et al., 1964,1965). In ancillary studies, we noted that the ip LD50 of DMSO in mice ranged from 10 to 20 g/kg. Toxicity data reported elsewhere are also inconsistent (Table 1). This study was conducted to ascertain whether the concentration of DMSO in a solution affects its toxicity. METHODS

Replicate

groups ‘of 10 male, Swiss albino, cesarean-derived, 20-30 g mice were

given dosesof DMSO at various concentrations (at 0.1 Briggsian log dose intervals) ip and were retained for observation (up to 5 days) until death or until normal activity

returned. The room temperature was 24” to 28”, with the relative humidity ranging from 20% to 30 % during the day and from 60% to 70% during the night. After administration of the DMSO, the mice were put into clear polyethylene cubicles (7 x 7 x 7 in.), one mouse per cubicle. After observation in the cubicle (without food and water) for approximately 8 hours, the mice were transferred to small plastic, metal-topped cages (6 x 11 x 5 in.), one per cage. Food and water were available ad Iibitum in the 275

276

WORTHLEY

AND

TABLE TOXICITY

OF DIMETHYL

Solution (% DMSO)

Sex

30 (water) 30 (water) 40 (water) 50 (saline) 50 (saline) 50 (saline) 50 (saline) 50 (saline) 50(saline) 60 (water) 80 (water) 100 100 loo loo 100 100 100 100 100 100 100

Both Both Both Both Both Both Both Both Both Both Both Both Female Both Both Female Both Female Both Both Both Both

SCHOTT

1

IN THE MOUSE

SULFOXIDE

Administration route iv* ip” Topical iv iv ip ip d

OsrCal Topical Topical Topical iv iv iv SC OsrCal Oral Oral Oral Oral

AS REPORTED IN THE LITERATURE

LD50 DMSO (g/W 7.2 14.7 >22.0 8.9 11.0

17.7 20.1 16.0 14.0 >37.0 (49.0 c91.0 7.6 5.8 3.8 13.9 20.5 16.5 21.4 22.0 24.2 23.1

Reference Farrant (1964) Fat-rant (1964) Smith et al. (1968) Caujolle et al. (1964) Caujolle et al. (1967) Caujolle et al. (1964) Caujolle et al. (1967) Caujolle et al. (1967) Caujolle et al. (1967) Smith et al. (1968) Smith et al. (1968) Smith et al. (1968) Sommeret al. (1964) Willson et al. (1965) Rosenkrantz et al. (1963) Sommeret al. (1965) Rosenkrantz et af. (1963) Sommerand Tauberger(1965) Willson et al. (1965) Brown et al. (1963) Mallach and Etzler (1965) Mallach and Etzler (1965)

u DMSO was diluted with either water or isotonic saline solution. b Intravenous. c Intraperitoneal. d Subcutaneous.

cage. Time of injection, reactions displayed, and time of death were recorded. Rectal temperatures of some of the mice given the 75 % DMSO were also measured. Reagent grade DMSO (CH&SO was volumetrically diluted with isotonic saline solution until 10 %, 25 %, 50 %, and 75 % solutions of DMSO were obtained. Each solution was cooled to room temperature before use. Specific gravity values were graphed

as presented in a paper by Kenttamaa and Lindberg (1960) and determined by interpolation. The specific gravity values obtained were as follows: 100 % DMSO, 1.095 ; 75% DMSO, 1.095; 50% DMSO, 1.07; 25% DMSO, 1.03; and 10% DMSO, 1.01. The LD50 calculations were done by the Bliss method (Bliss, 1952). The test for statistical significance of the differences in LD50 as reported herein is a modification of the t test.1 Doses higher than 100 ml/kg, or 2.5 ml/25 g mouse, were not administered because the volume alone would have affected the mice. Results are given in terms of weight of DMSO unless otherwise indicated. 1 The least significant difference is calculated instead of t (Davies, 1961).

TOXICITY

OF FOUR

CONCENTRATIONS

OF DMSO

277

RESULTS

The reactions displayed by mice that were given DMSO ip followed a consistent pattern which is summarized as follows: some initial discomfort at the site of administration (not apparent at the 50 % and 25 % concentrations), a small, immediate rise in rectal temperature (1” rise with 75 y0 DMSO), followed by marked hypothermia (5” drop in 30 min), depression (decreased locomotor activity, decreased movement of vibrissae, decreased rearing, etc.), tremors, lack of limb coordination when made to walk, flaccid muscle tone, and dyspnea. Immediately before death, the following signs

40

4

8

12

16

20

24

FIG. 1. Effect of saline dilutions of DMSO on survival.

were noted: loss of righting reflex, breathing difficulties (dyspnea, hypopnea, and apnea), micturition, cessation of breathing, and heart failure. The animals were considered dead when the heart stopped. The animals that died an hour or more after dosing became sluggish, but they did not appear to be in trouble until 15-20 min before death. Once the righting reflex was lost, death followed quickly. DetaiIed mortality data are given in Table 2 and Fig. 1 and summarized in Table 3. The toxicity of DMSO decreased with dilution, and the final slopes of the toxicitydilution curves presented in Fig. 1 are similar.* The differences in LDSO are significant except between the 50 % and 75 % dilution and the 75 % and 100 % dilution. Time to 2 The slope and standard deviation (in parentheses) of each curve are as follows: 100% DMSO survivalcurve,16.4(4.6);75~DMSO,18.6(5.5);50~DMSO,22.4(6.9),and25~DMSO,11.7(2.8).

278

WORTHLEY

AND

SCHOTT

death was longer for the low concentration (Fig. 2). The mechanism of death and overt signs displayed were the same for each dilution, but the severity and duration of effects varied. TABLE

2

MORTALITY FRACTIONS IN MICE

GIVEN VARIOUS DILUTIONS SULFOXIDE INTRAPERITONEALLY

DIMETHYL

10% DMSO Dose (g/kg) -10.1 -

MF”

25 % DMSO __-__ Dose (g/b) MF”

o/10 -

21.9 17.4 13.8 11.0 8.8

50 % DMSO -~Dose (g/kg) MF”

lo/lo 7/10 2110 l/10 o/10

17.7 14.0 12.2 8.9 -

lo/lo S/l0 l/l0 O/l0 -

AND DOSES OF

75 % DMSO Dose k/W

MF”

16.7 13.2 10.6 8.4 -

lo/lo S/l0 2110 O/l0 -

100% DMSO Dose (g/kg)

MF”

13.8 11.0 8.7 6.9

lo/lo 4110 l/l0 o/10

4 Mortality fraction. TABLE TOXICITY

3

OF SALINE

DILUTIONS SULFOXIDE’

OF DIMETHYL

DMSO Volume 100 75 50 25 10

%

Weight

%

100 76.7 52.3 26.8 10.1

LD50 k/Wb 10.9 11.9 13.3 15.4 >lO.l

(9.9-l 1.9) (10.9-12.9) (12.7-13.9) (13.8-17.2)

a Data from Table 2 were used for LDSO calculations (Bliss method). b Expressed in terms of DMSO present in solution by weight and administered ip to mice: refer to Methods section. c 95 % confidence limits. DISCUSSION

The final slope of the survival curves was similar at all concentrations tested (Fig. l), indicating that the concentration of DMSO played a small but significant role in the LD50. The major effect of dilution of DMSO was to increase the dose at which deaths began to occur. Since time to death increased at low concentrations (Fig. 2), it appears plausible to attribute the lesser toxicity of the more dilute systems to repair of potentially lethal interactions, elimination, metabolic alteration, and other detoxification of DMSO processes that would reduce the level of DMSO accumulating at sites critical for survival.

TOXICITY

OF FOUR

CONCENTRATIONS

OF DMSO

279

OQse of DhlsO (g/kg)

FIG. 2. Average time to death of mice receiving isotonic saline dilutions of DMSO.

These ideas are supported by some preliminary evidence that indicates that animals can tolerate higher doses of DMSO when administered in divided doses 10 min apart. The observation that differences in LD50 become statistically significant at DMSO concentrations of less than 50 y0 is similar to findings previously reported in the literature. Several papers (Caujolle et al., 1967; Sulzberger et al., 1967; Scherbel et al., 1967; Smith et al., 1968) indicate that skin irritation decreases markedly when DMSO is applied topically in concentrations below 70 %. Sulzberger et al. (1967) and Scherbel et al. (1967) stated that these differences are probably due to a nonlinear decrease in irritation as compared with the concentration of DMSO. The DMSO molecule may become hydrated or interact with other molecules, changing its effective size and electric charge-properties that could alter membrane penetration. Kenttamaa and Lindberg (1960) reported that concentrations of DMSO above 75 % behave as ideal solutions and have approximately the same specific gravity. It is of some interest that changes in toxicity were observed as we and other investigators (Sulzberger et al., 1967; Scherbel et al., 1967; Smith et al., 1968) approached this concentration. The heat of solution liberated when DMSO and water are mixed may be one reason for the difference in toxicity (Kenttamaa and Lindberg, 1960; Drunkard and Kivelson, 1958). We noted irritation at the site of administration when the higher concentrations of DMSO were given, but rectal temperature did not rise more than 1-2” (75 % DMSO). Although this rise in body temperature would not be expected to be lethal by itself, it could contribute ‘materially to altering membrane permeability and absorption of DMSO, thus enhancing toxicity.

280

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ACKNOWLEDGMENTS The authors wish to thank Sp4 G. A. Hauptmann and Sp5 A. W. Westling for their excellent technical assistance during this investigation. The authors are also indebted to Mrs. Pamela R. Schott for compiling a bibliography on DMSO, part of which is included in this paper, and Crown Zellerbach Corporation, Camas, Washington, for generously supplying the reagent grade DMSO. We also appreciate the helpful suggestions made by Dr. B. Papirmeister, Dr. Leslie, and Mr. F. J. Vocci in preparation of the manuscript. REFERENCES BLISS,C. E. (1952). The Statistics of Bioassay. Academic Press, New York. BRAUDE, M. C., and MONROE, R. R. (1965). Dimethylsufoxide: interactive effects with a-glucochloralose. Current Therap. Res. 7, 502-509. BROWN, V. K., ROBINSON,J., and STEVENSON,D. E. (1963). A note on the toxicity and solvent properties of dimethyl sulfoxide. J. Pharm. Pharmacol. l&688-692. CAUJOLLE, F., CAUJOLLE, D., QUISSOU, H. B., and CALVET,M. M. (1964).Toxicite et aptitudes pharmacologiquesdu DMSO. Acad. Sci. 258,2224-2226. CAUIOLLE,F., CAUJOLLE, D., CROS,S. B., and CALVET,M. M. (1967).Limits of toxic and teratogenictoleranceof dimethyl sulfoxide. Ann. N. Y. Acad. Sci. 141 (Art. l), 110-125. DAVIES,0. L. (1961).StatisticalMethodsin ResearchandProduction,p. 65. Hafner New York. DIXON,R. L., ADAMSON,R. H., BEN,M., and RALL, D. B. (1964).Toxicity of various drugs usingdimethyl sufoxide(DMSO) asa solvent. Pharmacologist6,189. DIXON,R. L., ADAMSON,R. H., BEN,M., and RALL,D. B. (1965).Apparent lack of interaction betweendimethyl sulfoxide (DMSO) and a variety of drugs. Proc. Sot. Exptl. Biol. Med. 118,7x-759.

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