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Some comparative toxicologic and pharmacologic effects of dimethyl sulfoxide as a pesticide solvent
Some Effects
Comparative of
Dimethyl L.
Division
Toxicologic
of Toxicological
and
Sulfoxide
R.
WEISS
Received
as a Pesticide
AND
Evaluation, Washington,
Pharmacologic
I<.
A.
ORZEL
Food and D.C. 20204
Ma!/
25,
Solvent
Drug
Administrution,
1967
The choice of suitable solvents for drugs, pesticides, and other chemical agents is a continuing problem in toxicologic studies. Dimethyl sulfioxide (DMSO) has been considered as a promising solvent for both water-soluble and waterinsoluble compounds. Its solvency for a wide range of chemicals combined with its low acute toxicity to mammals (Brown et ul., 1963) makes it attractive as a solvent for use in acute toxicity studies. The question of whether or not DMSO intluences the toxicity and pharmacology of various drugs and perhaps contributes some biological action per se has been the subject of many investigations. Diluted solutions of DMSO produce tissue and enzyme changes and induce hypothermia in rats exposed to cold ( Altland et al., 1966). A comparison of the acute toxicities in mice of a variety of drugs in DMSO and saline showed no interaction and no significant evidence for an increase in oral absorption or penetration into the central nervous system (Dixon et al., 1965). However, when DMSO was used as a solvent for poorly absorbed quaternary ammonium salts, the acute toxicity of some salts was changed (Rosen et al., 1965). In addition, experiments designed to test the concept that D>4SO enhances the penetration and passage of drugs across biological barriers have produced negative results ( Horita and Weber, 1964). Rosenkrantz et ul. ( 1963) reported the inertness of DMSO and its usefulness as a solvent for steroids. These investigations suggested that many water-insoluble agents, notably pesticides, could be studied for acute toxic effects in animals with DMSO as a vehicle. The purpose of this investigation was to determine whether using Dh4SO as a solvent for pesticides would significantly alter their toxicologic and pharmacologic properties. For this reason, certain toxicologic and pharmacologic effects of DMSO were evaluated. In most previous drug studies, diluted solutions of DMSO in water or saline were used. The main advantages of DMSO as a pesticide solvent when compared with other solvents, corn oil, an aqueous suspension, propylene glycol, dimethyl formamide, and dimethyl acetamide are that (a) DMSO can dissolve most pesticides in high concentrations needed for acute toxicity determination, (b) DIISO has a low toxicity in the volumes needed. The dilution of DMSO minimizes its advantages by reducing its solubilizing characteristics. For this reason, we evaluated pesticide toxicity in undiluted DMSO, and the effects of this preparation on brain cholinesterase inhibition were investigated and compared with those of carbaryl in various other solvents 546
EFFECTS
OF
DhfSO
AS
A
PESTICIDE
547
SOLVENT
when administered orally or intraperitoneally. The acute oral toxicities of thiram, dieldrin, parathion, carbaryl, and a newer carbamate, MC-A-600, were compared when given in DMSO, in corn oil, or as an aqueous suspension to rats. Finally, some pharmacologic effects were studied to confirm the central deprcssant activity reported for DMSO. Rats treated with DMSO were examined for effects on conditioned avoidance response, spontaneous motor activity, and hexobarbital-induced sleeping times. MATERIALS
AND
METHODS
Animals. FDA-bred male Osborne-Mendel rats weighing 200-300 g were used in most of the experiments. Female rats only were used in one acute toxicity study with various pesticides and for the conditioned avoidance work. Animals were fasted 20 hours before treatment and at other times were maintained on Purina Laboratory Chow and water ad libitum. The rats were observed for 24 hours, 5 days, or 7 days, as shown under Results. Chemical and solvents. For the cholinesterase studies, carbaryl ( l-naphthylN-methyl carbamate, 99.5% ) ,l was prepared in the following solvents: NJdimethyl formamide (DMF),” propylene glycol (PG),’ NJ-dimethyl acetamide (DMA) ,:i dimethyl sulfoxide (DMSO, industrial grade, 99% minimum purity),4 and a mixture of commercial corn oil (90% ) and DMF (10% ). In addition to the above vehicles, an aqueous suspending medium (sodium carboxymethylcellulose, 0.5%; sodium chloride, 0.9%; benzyl alcohol, 0.2% v/v; and Tween SO, 0.4% ) was used. A constant volume of solvent ( 1 ml/kg of body weight) was used for intraperitoneal administration. Oral doses were given by stomach tube in a constant volume of 5 ml/kg. For the oral pesticide toxicity study, thiram (tetramethylthiuram disulfide, 99% ),S dieldrin (hexachloroepoxyoctahyro-elzdo,exo-dimethanonaphthalene) ,6 parathion (97),’ carbaryl, and MC-,4800 (4-b enzothienyl N-methyl carbamate, 98% )” were dissolved in DVISO or in the corn oil-DMF mixture, or were suspended in the aqueous medium. Five dosage levels of these pesticides with 10 rats per dose were given in a constant volume (5 ml/kg) of solvent. The LDjo and slope function of each pesticide-solvent combination were calculated by the method of Litchfield and Wilcoxon (1949). For the sleeping time studies, hexobarbital sodium was dissolved in water (distilled) or DblSO and injected intraperitoneally in a constant volume (1 ml/kg). Choline&erase activity. Brain cholinesterase ( ChE) activity was determined by the Warburg manometric method. At a specific time interval after administration of the solvents or carbaryl solutions, the rats were decapitated. Their brains were removed and quickly frozen over dry ice until analyzed. A 10% ’ SevinQ, Union Carbide Chemical Corp., New York, New York. * Fisher Scientific Co., Fair Lawn, New Jersey. 3 K & K Laboratories, Inc., Jamaica, New York. ’ Crown Zellerbach Corp., Camas, Washington. 6 E. I. duPont de Nemours & Co., Wilmington, Delaware. ’ Shell Chemical Corp., Division Shell Oil Co., New York, New York. ’ ThiophosB, American Cyanamid Co., New York, New York. ’ MobamO, Mobil Chemical Co., Division of Socony-Mobil Co., Metuchen.
New
Jersey.
548
I,.
R.
WEISS
AND
H.
A.
ORZEL
bicarbonate buffer. homogenate of rat brain was prepared in Krebs-Ringer pH 7.4. The main compartment of the flasks contained 2 ml of this buffer and 0.8 ml of homogenate. After the flasks were equilibrated for 10 minutes in an chloatmosphere of 5% C02/95% N2 at 37”, 0.4 ml of 0.016 .U acrtylcholine ride” in buffer was tilted in from the side arm. Manometer readings were made at 5-minute intervals for 20 minutes. Brain ChE activity is expressed as microliters of CO2 liberated per milligram of tissue per hour. Student’s 1 kst was used in the statistical evaluation of the results. Controls were untreated and sacrificed at each time interval and analyzed concurrently with brains from experimental animals. Mean values were considered significantly different from the controls at P < 0.05. Spontaneous motor activity. Spontaneous motor activity (SYIA) was measured on jiggle platforms (LVE Model 1547, Lehigh Valley Electronics, Fogelsville, Pennsylvania). The platforms are not affected by perpendicular loading yet are extremely sensitive to movement of the animals. A plastic cage (5 X 7 X 11 inches, lined with an absorbent board floor) is placed directly in the platform with one animal inside. Sudden movements such as turning, running, or jumping will cause the platform to move slightly, thereby making and breaking an electrical contact. Successive contact interruptions are transferred to a transistor-isolation circuit and a counter. Cumulative numerical totals over a period of time provide a measure of SMA. The rats were divided into six groups of ten. Two groups were administered water orally, two groups were given undiluted DMSO, and two groups received 50% DMSO (v/v). Th e counters on 10 platforms were activated 5 minutes after the administration of these liquids, and counts were recorded at the end of each 15minute period for 1 hour in a darkened room. Sequential testing of two similar treated groups was avoided by alternating the order and time of running each group on the platforms. The total counts of each group of ten rats for each 15-minute period were used to evaluate changes in spontaneous motor activity. Statistical differences between groups were measured using the Mann-Whitney U test (Siegel, 1956). Conditioned avoidance response (CAR). Rats were conditioned in a behavioral programming systemlo to avoid an electroshock applied through a floor grid by depressing a lever during the presentation of a s-second warning light signal. Failure to avoid resulted in a 5-second shock (0.15 watt) I1 coupled with the light. During this period, the animal could escape by depressing a lever which terminated the shock. A lo-second intertrial period followed an avoidance or escape response or the shock period. Animals were trained daily for 2 hours until stable performance baselines were established (usually after lo-15 sessions). 8 Nutritional Biochemicals Corp., Cleveland, Ohio. I0 Designed by Massey Dickinson Co., Inc. (Saxonville, Massachusetts), using LVE Model No. 1316 rat test chambers (Lehigh Valley Electronics) and SGS-001 Shock CeneratorScrambldrs (BRS Electronics, Beltsville, h/laryland ). I’ Constant wattage provides a measurement of shock intensity that is better descriptive of the shock delivered than either constant voltage or constant current.
EFFECTS
OF
DMSO
AS
A
PESTICIDE
549
SOLVEKT
RESULTS
Tables 1 and 2 show the relative acute toxicities following intraperitoneal and oral administration of DMSO, DMA, DMF, and PG, undiluted and diluted 50% v/v in dstilled water. Mortalities occurring within a 24-hour and 5-day period are reported to distinguish between early and delayed toxicity. The studies on comparative acute toxicities between the undiluted and diluted solvents take into consideration such physical factors as hygroscopic effects and the liberation of considerable heat of solution which are present with the undiluted solvents. Since differences in the specific gravities of the six liquids are small, dosages are expressed in volume per body weight (ml/kg). The LD60 values of the solvents were not determined; these values have been reported elsewhere (Sanderson, 1959; Davis and Jenner, 1959; Spiegel and Noseworthy, 1963; Willson et al., 1965). The volume dose of the diluted solvents is expressed as a quantity of pure solvent for comparison. In Table 1, mortality following intraperitoneal administration is observed for DMA at 1 ml/kg, for DMF at 2 ml/kg, and for DMSO and PG at 8 &kg. Dilution of DMF and DMSO had a negligible effect on the acute toxicity; dilution of DMA and PG reduced the delayed toxicity. As expected, corn oil and water are nontoxic at the highest dose studied (8 ml/kg). TABLE TOXICITY~
OF v.4~10~
SOLVENTS
1
ADMINISTERED
INTRAPERIToKEALLY
Dose Observation period
Solvent Corn
oil
DMA Dilutedb DMF Dilutedb DMSO Dilutedb Propylene
glycol
Dilutedb Distilled
water
24 hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days
a Expressed as a mortality fraction. b Fifty percent, v/v, in distilled water.
(ml/kg
TO RATS
body
weight)
1
2
o/6
O/6
O/S
O/S
O/S O/6
O/S
O/6 Oh3
S/6
-
6/f-3 O/6 l/6 O/6
-
-
6 16
-
0 /6 “/6 O/6
O/6 O/6 O/6
-
I/S -
-
-
O/6
O/6
O/S
O/S
-
-
O/6
‘J/6
O/S
O/6
4
-
O/‘-5 3/6
-
O/S
O/f5
O/6
O/6
a/s -
O/6
-
P/6 O/S
-
O/6 O/6 0 /6
-
8
O/6 O/6 -
O/6 O/6
O/6 216 O/6
l/6 O/6 416
O/6 O/6 O/S
O/6
Observation period
Solvent Corn
oil
DMA Diluted* DMF Dilutedb DMSO Diluted* Propylene
glycol
Diluted* Distilled
water
2-L hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days “~4 hourr; 5 days 24 hours 5 days 24 hours 5 days
40 O/6 O/6
6p
S/G -
6 /e
o/s O/S
a Expressed as a mortality fraction. ’ Fifty percent, v/v, in distilled water.
Table 2 shows the results of administering single oral doses of these solvents, undiluted and diluted 50/50 with distilled water. Mortalities occurred with DMA and DMF at 5 ml/kg, and with DMSO and PG at 20 ml/kg. With the exception of DMA, the dilution of these solvents did not alter the toxicity. A delayed toxicity was found with DMA, DMF, DMSO, and PG by each route. In common with other solvents, a favorable characteristic of DMSO as a pesticide solvent for acute toxicity studies is that it has no effect on brain cholinesterase activity after intraperitoneal and oral administration. Tables 3 and 4 show the inertness of DMSO and other solvents. No significant effect on rat brain cholinesterase activity was observed at 1 ml/kg, intraperitoneally, or 5 ml/kg, orally, with any of the vehicles commonly used as pesticide solvents. When animals were treated intraperitoneally or orally with carbaryl in a constant volume of each solvent (Tables 3 and 4, respectively), solvents differed significantly in the degree and duration of cholinesterase inhibition. DMF caused a greater depression of cholinesterase activity and increased the duration of inhibition, when compared with the same intraperitoneal dose of carbaryl in the other solvents (Table 3). As shown in Table 4, oral administration of carbaryl in DMF or the aqueous suspension produced a longer lasting cholinesterase inhibition than carbaryl in the other solvents, compared with control values. Maximum inhibition was obtained with carbaryl in corn oil in the shortest time (0.5
Carbaryl
o E~presstd
Carbaryl
administration b Figures
+
Propylene
+
DMSO
~IJWJI
of solvent significantly
as microliters
of CO,
(1 mg/kg) or carbaryl different from control,
liberated
7.30 7.17
7.20 7.13
6.99
Carharyl
7.33 6.78
7.33 6.75
0. lrr 0.20
AVTII-ITP
f f
f f
f
f
per (25 mg/kg) P < 0.05.
milligram
0.09 0.26
0.29 0.20
0.16
0.29
l 0.1% 2~ 0.12
f It
Control
+
IO”,)
CHOLINESTER.~SE
7.20
Carbaryl
(9Orr/,,
BHAIN
DMF
+
Treatment
oil-DMF
Carbaryl
D?dA
+
Corn
-
It.41
TABLE
0.19
0.15
f
f0.S
f
f
f
f
in the
solvent.
per
0.14b
0.35b
0.19
O.llb
0.91
zk 0.17*
f
zk 0.326
*
Hour
hour.
INTHAPERITONE.~L
of tissue
4.99
7.11
5.31
7.28
3.76
7.08
4.33
7.51
4.54
7.31
0.5
AFTER
Values
5.69
7.23
4.68
6.67
4.09
6.42
4.71
7.71
5.14
7.01
3
0.31
0.10”
0.24
0.13b
0.33*
0.19
f
f
are
means
& standard
7.32 5.74
0.22
7.30 6.07
5.54
6.87
5.71
7.05
5.97
7.05
0.35
0.16”
0.35
0.19h
0.17
0.25
error
zt 0.34”
f
zk 0.28 zt 0.l.P
f
f
f
f
Ik 0.17b
f
Y Hours
OF C.~RB~RYI,IN
o.tzlb
31 O.lY*
f
f
f
f
f0.14
*
f
1 Hour
.~DMIXIHTHATION
of
7. 09
10 brains
6.58
6.67
6.84
~OLvEKTs
0.24 0.17*
0.18
0.21
0.20
following
xk 0.1”
31 0.33
f
zk 0. ov
* f
f
f
* 0.16 zk 0.18
6 Hours
6.GG 5.94
6.71
7.34
7.08 6.79
V.4a1or:s
0.14
0.20
0.11
0.14
0.28 0.2”
rt 0.24
zt 0.28
f
It
* f
f 0.34 zk 0.09
f
*
iatraperitone;~l
6.99
7.37
7.01
G.55
7.14 6.89
7.24 6 52
6.51
6.85
24 Hours
5
g
:
5
2
z
a-
%
~ r 2
g
f2 2 m
g
f0.17
f
7.01
6.89
” Expressed or carbaryl ten animals s Figures c Percent
0.07
f
0.19
0.40
0.28
o.20
f
0.5
0.W
f f
6.93
0.22 0.36”
o.39
5.55
0.13
f
0.15 o.31*
f
f f
f0.17 f 0.27*
f 0.19 f0.14"
0.10
f
f
Hour
AFTER
5.51
6.84
7.10 5.08
7.30 5.91
7.65 4.86
7.68 4.06
ACTIVITY OR.~L
5.24
f
o. 2”*
7.26 4.30
0.19
+0.20*
f
6.61 f 0.33 5 52 fo.57*
4.15
+
7.36
0.19
3~0.24 f 0.5lb
7.08 5.11
7.11 4.52
6.95 -5.24
4 46
0.16
0.21
0.29*
+
f
+ 0.24*
-I 0.18
o.3Yb
+
0 19
“3
f
+
0 27
0. “5
25' f 0.31 f 0.3"P “2’
0.09 0.20
5.92
6.89
7.03
f f
0.12 0.18
IO
7 36 f 0.97 6 7.5 xt 0 96
II Iti
18
1 I“ zk 0.31 *to 23
62' + 0, 2c2 zt 0.27
f 0 37 f0 16
* f
7 fl4 f
7.31
7.50 7.43
7. 49 5.06
6.74 6.96
7.01 7.09
24 Hours
of the solvent (5 ml/kg) mortality. \Yhen le%s than
0.21 0 23"
zk 0.25 6 98 + 0.33
6.83
7
7.03 5.74
0.17 0.116
+ +
f f
3~ 0.15
6.63
0.22
f
6.59
_____-
6 Hours
SOLVENTS
6.97 6.45
0.21*
VARIOUS
* 0.19 x!z 0.45*
*
f
7 80 f
7.17 5.93
7.08
f o.29
f0.16
5.34
6.64 5.74
7.03
fO.16
+ o.38*
3 Hours
OF CARBAKYLIN
4.92
6.73
1 Hour
ADMINISTRATION
4
as mean f standard error in microliters of CO2 liberated per milligram of tissue per hour after oral administration (50 mg/kg) in the solvent: each figure represents the brains from 10 animals except when otherwise indicated hy survived, a new group was given carbaryl in the appropriate solvent. significantly different from control, P < 0.05. mortality.
Aqueous suspension + Carbaryl
f f
7.09
Propylene + Carbaryl 7.15
7.39 7.05
DMSO + Carbaryl
0.12 0.23
f +
6.92 7 34
DMF + Carbaryl
7.24 I!r 0.29 6 81 + 0.18 + 0.19 zk 0.19
I@)
Control
CHOLINESTERASIP
7.51 6.89
(!XJ”$,
BRAIN
DMA + Carbaryl
glycol
Treatment
Corn oil-DMF + Carbaryl
--
RAT
TABLE
EFFECTS
OF
DMSO
AS
A
PESTICIDE
553
SOLVENT
hour) after oral administration, whereas DMSO and the aqueous suspension gave similar inhibition between 0.5 and 3 hours. Possibly carbaryl is more rapidly and completely absorbed in corn oil than in other vehicles. The oral dose of carbaryl (50 mg/kg) is well below the lethal dose level. The volumes of DMA and DMF alone (5 mg/kg) were tolerated during the first 24 hours, but deaths were noted at the 6- and 24-hour intervals after administration of D){F or DMA with carbaryl, indicating some enhancement of toxicity. Data are presented in Table 5 on the acute oral toxicity of thiram, dieldrin, parathion, carbaryl, and MC-A-600 when administered as solutions in DMSO and corn oil and as suspensions in water. Animals treated with an anticholinesTABLE ,~CVTTE ORAL
TOXICITY
OF
\:a~rous
5
PESTIVIDES
IX
DIFFERENT
Mho (mg/kg)Pesticide
Solvents
95yc ~__
Thiram (7 days)
DMSO Aqueous suspension Corn oil
790 560
Dieldrin (7 days)
Parathion (24 hours)
Carbaryl (24 hours)
MC-A-600 (24 hours)
a Only female b Significantly
DMSO Aqueous suspension Corn oil
confidence
limits
(715-1400)
(1.29-3.96) (1.50-5.35) (1.37-2.25)
(52m 134) (56 101)
1.61 I.77
(1.36-1.99)
71 64
(51-93)
1 .40
(1.13-l.
2.10 1.80
(1 72-2 .56) (l12s-e.57)
1 25 1.50
(1.01-1.55) (1.00-2.2.5)
4.70
(3.9x-~
1.21
(0.98-l.
1.65
(1.27-2.15)
1 “1 ” y-2
(1.19-4.52)
1.18 1.31 1.56
(0.96-l. (1.13-l. (I. 31-l.
83
DMSO Aqueous suspension Corn oil
5.55)”
DMSO aqueous suspension Corn oil
290
DMSO Aqueous suspension Corn oil
70 110
(93
130)
109
(76
158)
rats were different,
Slope function950/; confidence limits 2.26 2.83 1.76
1000
(4671335) (329-952)
SOL~ECXTS"
255 “30
used in this experiment. P < 0.05. from other
(227- 37 1) (228. “86) (Isl-~wl)
(56- 87)
solvents
(1.38-2.27)
(1.04-l.
74)
51))
40)
45) 52) 86)
in the same group.
terase (parathion, carbaryl, and MC-A-600) were observed for 24 hours, as deaths are infrequent after this period. With thiram and dieldrin, deaths usually occur after the first 2448 hours, and animals treated with these pesticides were observed for 7 days. In the dieldrin study, deaths occurred with corn oil and the aqueous suspension during the first 2 days, whereas with DMSO most deaths occurred at 3 to 7 days. With thiram in each of the solvents, a delayed toxicity was seen after 3 days. In one case, parathion in corn oil, the toxicity was significantly decreased. With parathion, the onset of toxic signs of anticholinesterase poisoning was slower when the solvent was corn oil rather than DMSO or the aqueous suspension. Deaths from parathion intoxication occurred more rapidly with these latter solvents, but differences were not noted with MC-A-600 and
554
L.
R.
WEISS
AND
R.
A.
ORZEL
carbaryl in corn oil, DMSO, or the aqueous suspension. While these variations appear to be solvent related, the LD5,, values and slope function do not indicate that different solvent systems have any effects of practical significance on the toxicity of these pesticides. A depressant effect after the administration of DMSO to animals has been described (Brown et al., 1963; Braude and Monroe, 1965; Willson et aI., 1965). Results in Tables 6, 7, and 8 show the nature of this depression. Spontaneous motor activity was measured in six groups of ten rats. Two groups were treated orally (1.0 ml/kg) with water and two groups received undiluted DMSO. TABLE EFFECTS
OF DMSO
6
ON SPONTANEOUS
Cumulative
MOTOR
counts
ACTIVITY
per time
period
(minutes) -
Solventa
Water
DMSO
DMSO
(5ooj,
a Orally, b Figures
o-15
15-30
30-45
G-60
I II
35,368 25.736
26 I583 23,889
23,569 22,747
18.671 20 ( 245
I
22.175 31.678
17,796* 13.581*
10,644*
II I II
24, m? 25,911
“8,149
15,043
22,477
28,847
30,999
28,761
PJ”P
v/v)
Total
8.51’?*
R.98lh
11,964”
104,191
92.617 59.1w 66,204b x9 ( 947
114.518
1.0 ml/kg, ten rats per group for each solvent and tested as separate groups (I and II). are significantly different from water and DMSO (5Oc7,,) groups: P < 0.0.5.
Another two groups were given DMSO (1.0 ml/kg) diluted with water, 50% v/v. The cumulative counts and significance of ten rats for consecutive 15-minute periods are listed in Table 6. After the first 15 minutes, a distinct reduction in activity was measured in the groups treated with undiluted DMSO; this reduction became more pronounced at the end of 45 and 60 minutes. However, administration of the same dose of diluted DMSO did not cause a change in activity from the water-treated controls. Table 7 compares the effects of water and DMSO as solvents for hexobarbital sodium in control and DMSO-pretreated rats. The sleeping times for each group of animals were not significantly different, indicating that after 24 hours, a large oral dose of undiluted DMSO (10 ml/kg) TABLE EFFECT
OF DMSO
Group Control DMSOc
PRETREATMEKT
Hexobarbital” 26.2 28.4
7
OR HEXOB.~RBITAL
in water f
0.7
f
1.21
SLEEPIKG
Hexobarbital” 26.7 27.9
TIME“
in DMSO 3~ 1.15 xk I.93
a Values are in minutes, means f standard error; N = 20 per group. * Hexobarbital, 100 mg/kg; solvent, 1 ml/kg, administered intraperitoneallg. c Oral, 10 ml/kg, 24-hour pretreatment.
1~ RA’I’S
EFFECTS
OF
DMSO
AS
A
TABLE THE EFFECTS
OF DMSO
First Rat NO.
&4%
PESTICIDE
5.55
8
ON THE CONDITIONED
AVOIDANCE
RESPONSE~*~
hour
E%,
Second
NE%
Treatment, oral (5 ml/kg)
8
81
19
0
33
75 94
8.5 5
0 1
DMSO DMSO DMSO Hz0 Hz0
II
23
0
Hz0
1
96
4
0
27
72
523
0
41 19
SOLVENT
’ Only female rats were used in this experiment. b Responses during first hour (no treatment) and second A?0 = avoidance, EyO = escape, and NELjb = no escape.
hour
.49,
hour
WL
NE%;.
95
5
0
X9
11
0
84 87
16 13
0 0
Ql 97
9
0
3
0
(treatment)
in terms
of percent:
showed no residual effects. Oral doses of undiluted DMSO (l-5 ml/kg) after 1-4 hour pretreatments and daily for I4 days (2 ml/kg) were also without effect on hexobarbital-induced sleeping times in rats (unpublished data). In Table 8, the effects on the conditioned avoidance response (CAR) of rats treated orally sh ow no blockage of this response. While the with DMSO or water (5 ml/kg) generally higher avoidance levels and uniformity of avoidance responses between animals at the second hour (treatment) is a possible reflection of a “warm up” phenomenon, the fact remains that DMSO did not depress the CAR during the second hour period in which the animals reach their “normal avoidance levels.” DISCUSSION
The results of acute toxicity studies following intraperitoneal and oral doses of DMSO to rats indicate its potential usefulness as a solvent for pesticides. Toxicologists working with pesticides and drugs need a vehicle with good polar and nonpolar solubility characteristics and the relative inertness of water and corn oil. For acute toxicity work, DMSO has certain properties that satisfy these criteria and others that limit its usefulness. DMSO is rapidly absorbed and uniformly distributed in all tissues, and because of its short half-life is excreted largely unchanged (Hucker et al., 1963). These properties may explain the low toxicity and apparent lack of pharmacologic effects. The data show that DMSO has a lower acute toxicity than DMA and DMF. The use of undiluted DMSO as a vehicle for pesticides does not enhance or decrease the pharmacologic or toxicologic properties of the pesticides used in these experiments. The results suggest that DMSO does not interact or enhance the activity of the anticholinesterases in the brains of rats. The reported unique ability of DMSO to penetrate tissues, increase the permeability of tissues to drugs and pesticides, and enhance the absorption of chemicals following oral administration does not seem to be important from the standpoint of acute pesticide toxicity. The LDsO values, slope functions, and mortality dose-response curves were generally similar for each pesticide whether in DMSO, in corn oil, or as an aqueous suspension. In these oral and intraneritoneal experiments, ob-
L.
556
R. WEISS
AND
R.
A.
ORZEI.
servations demonstrated that significant individual variations may occur as a result of solvent action on the toxicity of a pesticide. It should be pointed out that certain physicochemical and pharmacologic effects of DMSO prohibit its undiluted use intravenously (Willson et al., 1965; DiStefano and Klahn, 1965). However, diluted DMSO has no effect on the oral or intravenous toxicity of a variety of drugs in mice (Dixon et al., 1965). The results with pesticides and undiluted DMSO confirm earlier reports (Ben et al., 1964; Feinman et al., 1964) that systemically administered diluted DMSO, in general, does not materially influence drug toxicity or cellular penetration. The hypothesis that DMSO can alter pharmacologic or toxicologic effects is not supported. The question whether undiluted DMSO as a solvent could alter the effects of drugs and pesticides on the central nervous system was partially answered by the brain cholinesterase experiments. The depressant effects of DMSO reported by others do not appear to be related to an action on the central nervous system. Our work indicated that DMSO, 5 mg/kg orally, has no significant effect on the conditioned avoidance response in rats. The depressant effects on spontaneous motor activity after oral administration of DMSO probably were induced by a localized action in the viscera since diluted DVlSO did not produce these changes and hexobarbital sleeping times were unchanged with undiluted DMSO. However, additional central nervous system studies seem warranted with D;\/ISO (diluted and undiluted) before concluding that this solvent does not affect experimental drug and pesticide investigations. For this reason, adequate control studies with DMSO should be conducted whenever this solvent is used, to determine any variations due to drug-solvent interactions. SUMMARY The acute oral and intraperitoneal toxicities of dimethyl sulfoxide (DMSO ), dimethyl acetamide (DMA), dimethyl formamide (DMF), and propylene glycol (PC), diluted and undiluted, were compared in rats after 24-hour and s-day periods. The results indicate that DMSO is less toxic than DMA and DMF. The toxicities of DMSO and DMF were unchanged by dilution with water. A delayed toxicity was noted with DMSO, DMA, DMF, and PG by each route. None of these solvents influenced brain cholinesterase activity. Some significant differences in the inhibition of cholinesterase by carbaryl in the various solvents were observed. The acute oral toxicities of thiram, dieldrin, carbaryl, MC-A-600, and parathion were generally similar whether administered in DMSO, in corn oil, or as an aqueous suspension. Some differences in the times of the onset of signs of toxicity were noted, Undiluted DMSO produced a depression of spontaneous motor activity without an effect on hexobarbital sleeping time or the conditioned avoidance response. The depression was abolished by dilution. Because of the low acute toxicity and apparent lack of interaction, DMSO is a useful solvent for studying the effects of pesticides in animals. ACKNOWLEDGMENTS The authors appreciate J. Bryant, and R. Lee,
the technical assistance Jr., in these studies.
provided
by R. E. Brodie,
J. R. Reeves,
III,
REFERENCES AL-I-LAND, P. D., HIGHMAN, sulfoxide in rats exposed 123, 853-859.
B., and PARKER, M. ( 1966). to cold: tissues and enzyme
Induction changes.
of hypothermia by dimethyl Proc. SOC. Exptl. Biol. Med.
EFFECTS
OF
DMSO
AS
A
PESTICIDE
SOLVENT
557
BEN, M.,
DIXON, K. L., A~AMSON, K. H., FEINXIAN, H., and RALL, D. P. ( 1964). Toxicity of various drugs using dimethyl sulfoxide (DMSO) as a solvent. Pharmacologist 6, 189. BRAU~E, M. C., and MONROE, R. R. (1965). Dimethyl sulfoxide: interactive effects with alphaglucochloralose. Current Therup. Res. 7, 502-509. BROWN, V. K., ROBINSON, V., and STEVENSON, D. E. (1963). A note on the toxicity and solvent properties of dimethyl sulfoxide. J. Phurm. Phurmucol. 15, 688-692. DAVIS, K. J., and JENNEH, P. M. (1959). Toxicity of three drug solvents. Toxicol. Appl. Phurmucol. 1, 576-578. DISTEFANO, V., and KLAHN, J. J. ( 1965). Oh servations on the pharmacology and hemolytic activity of dimethyl sulfoxide. Toxicol. Appl. Phurmacol. 7, 660-666. DIXON, R. L., ADAIUSON, R. H., BEN, M., and RALL, D. P. ( 1965 ). Apparent lack of interaction between dimethyl sulfoxide (DMSO) and a variety of drugs. Proc. Sot. Exptl. Biol. Med. 118, 756-759. FEIN~\IAN, H., BEN, M., and LEVIN, R. (1964). Toxicity of dimethyl sulfoxide in primates. Pharmacologist 6, 188. HORITA, A., and WEBEH, L. J. ( 1964). Skin penetrating property of drugs dissolved in dimethyl sulfoxide and other vehicles. Life Sci. 3, 1389-1395. HUCKER, H. B., AH~ZAD, P. M., and MILLEH, E. A. (1966). Absorption, distribution and metabolism of dimethyl sulfoxide in the rat, rabbit and guinea pig. J. Phurmucol. Exptl. Therup. 154, 176184. LITCHFIELD, J. T., JH., and WILCOXON, F. ( 1949). A simplified method of evaluating doseeffect experiments. J. Pharmucol. Exptl. Therup. 96, 99-113. ROSEN, H., BLUSIENTHAL, A., PANASEVICH, R., and MCCALLUM, J. (1965). Dimethyl sulfoxide (DMSO) as a solvent in acute toxicity determinations. Proc. Sot. Exptl. Biol. Med. 120,
511-514. ROSENKRANTZ, H., HADIDIAN, Z., SEAY, H., and MASON, H. M. (1963). Dimethyl sulfoxide: its steroid solubility and endocrinologic and pharmacologic-toxicologic characteristics. Cancer Chemotherapy Rept. 31, 7-24. SANDERSON, D. M. ( 1959). A note on glycerol formal as a solvent in toxicity testing. 1. Phurm. Pharmucol. 11, 150-156. SIEGEL, S. (1956). NonpuTumetric Statistics for the Behavioral Sciences, p. 312. McGrawHill, New York. SPIEGEL, A. J., and NOSEWOHTHY, M. M. (1963). Use of nonaqueous solvents in parenteral products. J. Phurm. Sci. 52, 917-927. WILLSON, J. E., BROWN, D. E., and T~XMENS, E. K. (1965). A toxicologic study of dimethyl sulfoxide. Toricol. Appl. Pharmacol. 7, 104-112.
Comparative of
Dimethyl L.
Division
Toxicologic
of Toxicological
and
Sulfoxide
R.
WEISS
Received
as a Pesticide
AND
Evaluation, Washington,
Pharmacologic
I<.
A.
ORZEL
Food and D.C. 20204
Ma!/
25,
Solvent
Drug
Administrution,
1967
The choice of suitable solvents for drugs, pesticides, and other chemical agents is a continuing problem in toxicologic studies. Dimethyl sulfioxide (DMSO) has been considered as a promising solvent for both water-soluble and waterinsoluble compounds. Its solvency for a wide range of chemicals combined with its low acute toxicity to mammals (Brown et ul., 1963) makes it attractive as a solvent for use in acute toxicity studies. The question of whether or not DMSO intluences the toxicity and pharmacology of various drugs and perhaps contributes some biological action per se has been the subject of many investigations. Diluted solutions of DMSO produce tissue and enzyme changes and induce hypothermia in rats exposed to cold ( Altland et al., 1966). A comparison of the acute toxicities in mice of a variety of drugs in DMSO and saline showed no interaction and no significant evidence for an increase in oral absorption or penetration into the central nervous system (Dixon et al., 1965). However, when DMSO was used as a solvent for poorly absorbed quaternary ammonium salts, the acute toxicity of some salts was changed (Rosen et al., 1965). In addition, experiments designed to test the concept that D>4SO enhances the penetration and passage of drugs across biological barriers have produced negative results ( Horita and Weber, 1964). Rosenkrantz et ul. ( 1963) reported the inertness of DMSO and its usefulness as a solvent for steroids. These investigations suggested that many water-insoluble agents, notably pesticides, could be studied for acute toxic effects in animals with DMSO as a vehicle. The purpose of this investigation was to determine whether using Dh4SO as a solvent for pesticides would significantly alter their toxicologic and pharmacologic properties. For this reason, certain toxicologic and pharmacologic effects of DMSO were evaluated. In most previous drug studies, diluted solutions of DMSO in water or saline were used. The main advantages of DMSO as a pesticide solvent when compared with other solvents, corn oil, an aqueous suspension, propylene glycol, dimethyl formamide, and dimethyl acetamide are that (a) DMSO can dissolve most pesticides in high concentrations needed for acute toxicity determination, (b) DIISO has a low toxicity in the volumes needed. The dilution of DMSO minimizes its advantages by reducing its solubilizing characteristics. For this reason, we evaluated pesticide toxicity in undiluted DMSO, and the effects of this preparation on brain cholinesterase inhibition were investigated and compared with those of carbaryl in various other solvents 546
EFFECTS
OF
DhfSO
AS
A
PESTICIDE
547
SOLVENT
when administered orally or intraperitoneally. The acute oral toxicities of thiram, dieldrin, parathion, carbaryl, and a newer carbamate, MC-A-600, were compared when given in DMSO, in corn oil, or as an aqueous suspension to rats. Finally, some pharmacologic effects were studied to confirm the central deprcssant activity reported for DMSO. Rats treated with DMSO were examined for effects on conditioned avoidance response, spontaneous motor activity, and hexobarbital-induced sleeping times. MATERIALS
AND
METHODS
Animals. FDA-bred male Osborne-Mendel rats weighing 200-300 g were used in most of the experiments. Female rats only were used in one acute toxicity study with various pesticides and for the conditioned avoidance work. Animals were fasted 20 hours before treatment and at other times were maintained on Purina Laboratory Chow and water ad libitum. The rats were observed for 24 hours, 5 days, or 7 days, as shown under Results. Chemical and solvents. For the cholinesterase studies, carbaryl ( l-naphthylN-methyl carbamate, 99.5% ) ,l was prepared in the following solvents: NJdimethyl formamide (DMF),” propylene glycol (PG),’ NJ-dimethyl acetamide (DMA) ,:i dimethyl sulfoxide (DMSO, industrial grade, 99% minimum purity),4 and a mixture of commercial corn oil (90% ) and DMF (10% ). In addition to the above vehicles, an aqueous suspending medium (sodium carboxymethylcellulose, 0.5%; sodium chloride, 0.9%; benzyl alcohol, 0.2% v/v; and Tween SO, 0.4% ) was used. A constant volume of solvent ( 1 ml/kg of body weight) was used for intraperitoneal administration. Oral doses were given by stomach tube in a constant volume of 5 ml/kg. For the oral pesticide toxicity study, thiram (tetramethylthiuram disulfide, 99% ),S dieldrin (hexachloroepoxyoctahyro-elzdo,exo-dimethanonaphthalene) ,6 parathion (97),’ carbaryl, and MC-,4800 (4-b enzothienyl N-methyl carbamate, 98% )” were dissolved in DVISO or in the corn oil-DMF mixture, or were suspended in the aqueous medium. Five dosage levels of these pesticides with 10 rats per dose were given in a constant volume (5 ml/kg) of solvent. The LDjo and slope function of each pesticide-solvent combination were calculated by the method of Litchfield and Wilcoxon (1949). For the sleeping time studies, hexobarbital sodium was dissolved in water (distilled) or DblSO and injected intraperitoneally in a constant volume (1 ml/kg). Choline&erase activity. Brain cholinesterase ( ChE) activity was determined by the Warburg manometric method. At a specific time interval after administration of the solvents or carbaryl solutions, the rats were decapitated. Their brains were removed and quickly frozen over dry ice until analyzed. A 10% ’ SevinQ, Union Carbide Chemical Corp., New York, New York. * Fisher Scientific Co., Fair Lawn, New Jersey. 3 K & K Laboratories, Inc., Jamaica, New York. ’ Crown Zellerbach Corp., Camas, Washington. 6 E. I. duPont de Nemours & Co., Wilmington, Delaware. ’ Shell Chemical Corp., Division Shell Oil Co., New York, New York. ’ ThiophosB, American Cyanamid Co., New York, New York. ’ MobamO, Mobil Chemical Co., Division of Socony-Mobil Co., Metuchen.
New
Jersey.
548
I,.
R.
WEISS
AND
H.
A.
ORZEL
bicarbonate buffer. homogenate of rat brain was prepared in Krebs-Ringer pH 7.4. The main compartment of the flasks contained 2 ml of this buffer and 0.8 ml of homogenate. After the flasks were equilibrated for 10 minutes in an chloatmosphere of 5% C02/95% N2 at 37”, 0.4 ml of 0.016 .U acrtylcholine ride” in buffer was tilted in from the side arm. Manometer readings were made at 5-minute intervals for 20 minutes. Brain ChE activity is expressed as microliters of CO2 liberated per milligram of tissue per hour. Student’s 1 kst was used in the statistical evaluation of the results. Controls were untreated and sacrificed at each time interval and analyzed concurrently with brains from experimental animals. Mean values were considered significantly different from the controls at P < 0.05. Spontaneous motor activity. Spontaneous motor activity (SYIA) was measured on jiggle platforms (LVE Model 1547, Lehigh Valley Electronics, Fogelsville, Pennsylvania). The platforms are not affected by perpendicular loading yet are extremely sensitive to movement of the animals. A plastic cage (5 X 7 X 11 inches, lined with an absorbent board floor) is placed directly in the platform with one animal inside. Sudden movements such as turning, running, or jumping will cause the platform to move slightly, thereby making and breaking an electrical contact. Successive contact interruptions are transferred to a transistor-isolation circuit and a counter. Cumulative numerical totals over a period of time provide a measure of SMA. The rats were divided into six groups of ten. Two groups were administered water orally, two groups were given undiluted DMSO, and two groups received 50% DMSO (v/v). Th e counters on 10 platforms were activated 5 minutes after the administration of these liquids, and counts were recorded at the end of each 15minute period for 1 hour in a darkened room. Sequential testing of two similar treated groups was avoided by alternating the order and time of running each group on the platforms. The total counts of each group of ten rats for each 15-minute period were used to evaluate changes in spontaneous motor activity. Statistical differences between groups were measured using the Mann-Whitney U test (Siegel, 1956). Conditioned avoidance response (CAR). Rats were conditioned in a behavioral programming systemlo to avoid an electroshock applied through a floor grid by depressing a lever during the presentation of a s-second warning light signal. Failure to avoid resulted in a 5-second shock (0.15 watt) I1 coupled with the light. During this period, the animal could escape by depressing a lever which terminated the shock. A lo-second intertrial period followed an avoidance or escape response or the shock period. Animals were trained daily for 2 hours until stable performance baselines were established (usually after lo-15 sessions). 8 Nutritional Biochemicals Corp., Cleveland, Ohio. I0 Designed by Massey Dickinson Co., Inc. (Saxonville, Massachusetts), using LVE Model No. 1316 rat test chambers (Lehigh Valley Electronics) and SGS-001 Shock CeneratorScrambldrs (BRS Electronics, Beltsville, h/laryland ). I’ Constant wattage provides a measurement of shock intensity that is better descriptive of the shock delivered than either constant voltage or constant current.
EFFECTS
OF
DMSO
AS
A
PESTICIDE
549
SOLVEKT
RESULTS
Tables 1 and 2 show the relative acute toxicities following intraperitoneal and oral administration of DMSO, DMA, DMF, and PG, undiluted and diluted 50% v/v in dstilled water. Mortalities occurring within a 24-hour and 5-day period are reported to distinguish between early and delayed toxicity. The studies on comparative acute toxicities between the undiluted and diluted solvents take into consideration such physical factors as hygroscopic effects and the liberation of considerable heat of solution which are present with the undiluted solvents. Since differences in the specific gravities of the six liquids are small, dosages are expressed in volume per body weight (ml/kg). The LD60 values of the solvents were not determined; these values have been reported elsewhere (Sanderson, 1959; Davis and Jenner, 1959; Spiegel and Noseworthy, 1963; Willson et al., 1965). The volume dose of the diluted solvents is expressed as a quantity of pure solvent for comparison. In Table 1, mortality following intraperitoneal administration is observed for DMA at 1 ml/kg, for DMF at 2 ml/kg, and for DMSO and PG at 8 &kg. Dilution of DMF and DMSO had a negligible effect on the acute toxicity; dilution of DMA and PG reduced the delayed toxicity. As expected, corn oil and water are nontoxic at the highest dose studied (8 ml/kg). TABLE TOXICITY~
OF v.4~10~
SOLVENTS
1
ADMINISTERED
INTRAPERIToKEALLY
Dose Observation period
Solvent Corn
oil
DMA Dilutedb DMF Dilutedb DMSO Dilutedb Propylene
glycol
Dilutedb Distilled
water
24 hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days
a Expressed as a mortality fraction. b Fifty percent, v/v, in distilled water.
(ml/kg
TO RATS
body
weight)
1
2
o/6
O/6
O/S
O/S
O/S O/6
O/S
O/6 Oh3
S/6
-
6/f-3 O/6 l/6 O/6
-
-
6 16
-
0 /6 “/6 O/6
O/6 O/6 O/6
-
I/S -
-
-
O/6
O/6
O/S
O/S
-
-
O/6
‘J/6
O/S
O/6
4
-
O/‘-5 3/6
-
O/S
O/f5
O/6
O/6
a/s -
O/6
-
P/6 O/S
-
O/6 O/6 0 /6
-
8
O/6 O/6 -
O/6 O/6
O/6 216 O/6
l/6 O/6 416
O/6 O/6 O/S
O/6
Observation period
Solvent Corn
oil
DMA Diluted* DMF Dilutedb DMSO Diluted* Propylene
glycol
Diluted* Distilled
water
2-L hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days 24 hours 5 days “~4 hourr; 5 days 24 hours 5 days 24 hours 5 days
40 O/6 O/6
6p
S/G -
6 /e
o/s O/S
a Expressed as a mortality fraction. ’ Fifty percent, v/v, in distilled water.
Table 2 shows the results of administering single oral doses of these solvents, undiluted and diluted 50/50 with distilled water. Mortalities occurred with DMA and DMF at 5 ml/kg, and with DMSO and PG at 20 ml/kg. With the exception of DMA, the dilution of these solvents did not alter the toxicity. A delayed toxicity was found with DMA, DMF, DMSO, and PG by each route. In common with other solvents, a favorable characteristic of DMSO as a pesticide solvent for acute toxicity studies is that it has no effect on brain cholinesterase activity after intraperitoneal and oral administration. Tables 3 and 4 show the inertness of DMSO and other solvents. No significant effect on rat brain cholinesterase activity was observed at 1 ml/kg, intraperitoneally, or 5 ml/kg, orally, with any of the vehicles commonly used as pesticide solvents. When animals were treated intraperitoneally or orally with carbaryl in a constant volume of each solvent (Tables 3 and 4, respectively), solvents differed significantly in the degree and duration of cholinesterase inhibition. DMF caused a greater depression of cholinesterase activity and increased the duration of inhibition, when compared with the same intraperitoneal dose of carbaryl in the other solvents (Table 3). As shown in Table 4, oral administration of carbaryl in DMF or the aqueous suspension produced a longer lasting cholinesterase inhibition than carbaryl in the other solvents, compared with control values. Maximum inhibition was obtained with carbaryl in corn oil in the shortest time (0.5
Carbaryl
o E~presstd
Carbaryl
administration b Figures
+
Propylene
+
DMSO
~IJWJI
of solvent significantly
as microliters
of CO,
(1 mg/kg) or carbaryl different from control,
liberated
7.30 7.17
7.20 7.13
6.99
Carharyl
7.33 6.78
7.33 6.75
0. lrr 0.20
AVTII-ITP
f f
f f
f
f
per (25 mg/kg) P < 0.05.
milligram
0.09 0.26
0.29 0.20
0.16
0.29
l 0.1% 2~ 0.12
f It
Control
+
IO”,)
CHOLINESTER.~SE
7.20
Carbaryl
(9Orr/,,
BHAIN
DMF
+
Treatment
oil-DMF
Carbaryl
D?dA
+
Corn
-
It.41
TABLE
0.19
0.15
f
f0.S
f
f
f
f
in the
solvent.
per
0.14b
0.35b
0.19
O.llb
0.91
zk 0.17*
f
zk 0.326
*
Hour
hour.
INTHAPERITONE.~L
of tissue
4.99
7.11
5.31
7.28
3.76
7.08
4.33
7.51
4.54
7.31
0.5
AFTER
Values
5.69
7.23
4.68
6.67
4.09
6.42
4.71
7.71
5.14
7.01
3
0.31
0.10”
0.24
0.13b
0.33*
0.19
f
f
are
means
& standard
7.32 5.74
0.22
7.30 6.07
5.54
6.87
5.71
7.05
5.97
7.05
0.35
0.16”
0.35
0.19h
0.17
0.25
error
zt 0.34”
f
zk 0.28 zt 0.l.P
f
f
f
f
Ik 0.17b
f
Y Hours
OF C.~RB~RYI,IN
o.tzlb
31 O.lY*
f
f
f
f
f0.14
*
f
1 Hour
.~DMIXIHTHATION
of
7. 09
10 brains
6.58
6.67
6.84
~OLvEKTs
0.24 0.17*
0.18
0.21
0.20
following
xk 0.1”
31 0.33
f
zk 0. ov
* f
f
f
* 0.16 zk 0.18
6 Hours
6.GG 5.94
6.71
7.34
7.08 6.79
V.4a1or:s
0.14
0.20
0.11
0.14
0.28 0.2”
rt 0.24
zt 0.28
f
It
* f
f 0.34 zk 0.09
f
*
iatraperitone;~l
6.99
7.37
7.01
G.55
7.14 6.89
7.24 6 52
6.51
6.85
24 Hours
5
g
:
5
2
z
a-
%
~ r 2
g
f2 2 m
g
f0.17
f
7.01
6.89
” Expressed or carbaryl ten animals s Figures c Percent
0.07
f
0.19
0.40
0.28
o.20
f
0.5
0.W
f f
6.93
0.22 0.36”
o.39
5.55
0.13
f
0.15 o.31*
f
f f
f0.17 f 0.27*
f 0.19 f0.14"
0.10
f
f
Hour
AFTER
5.51
6.84
7.10 5.08
7.30 5.91
7.65 4.86
7.68 4.06
ACTIVITY OR.~L
5.24
f
o. 2”*
7.26 4.30
0.19
+0.20*
f
6.61 f 0.33 5 52 fo.57*
4.15
+
7.36
0.19
3~0.24 f 0.5lb
7.08 5.11
7.11 4.52
6.95 -5.24
4 46
0.16
0.21
0.29*
+
f
+ 0.24*
-I 0.18
o.3Yb
+
0 19
“3
f
+
0 27
0. “5
25' f 0.31 f 0.3"P “2’
0.09 0.20
5.92
6.89
7.03
f f
0.12 0.18
IO
7 36 f 0.97 6 7.5 xt 0 96
II Iti
18
1 I“ zk 0.31 *to 23
62' + 0, 2c2 zt 0.27
f 0 37 f0 16
* f
7 fl4 f
7.31
7.50 7.43
7. 49 5.06
6.74 6.96
7.01 7.09
24 Hours
of the solvent (5 ml/kg) mortality. \Yhen le%s than
0.21 0 23"
zk 0.25 6 98 + 0.33
6.83
7
7.03 5.74
0.17 0.116
+ +
f f
3~ 0.15
6.63
0.22
f
6.59
_____-
6 Hours
SOLVENTS
6.97 6.45
0.21*
VARIOUS
* 0.19 x!z 0.45*
*
f
7 80 f
7.17 5.93
7.08
f o.29
f0.16
5.34
6.64 5.74
7.03
fO.16
+ o.38*
3 Hours
OF CARBAKYLIN
4.92
6.73
1 Hour
ADMINISTRATION
4
as mean f standard error in microliters of CO2 liberated per milligram of tissue per hour after oral administration (50 mg/kg) in the solvent: each figure represents the brains from 10 animals except when otherwise indicated hy survived, a new group was given carbaryl in the appropriate solvent. significantly different from control, P < 0.05. mortality.
Aqueous suspension + Carbaryl
f f
7.09
Propylene + Carbaryl 7.15
7.39 7.05
DMSO + Carbaryl
0.12 0.23
f +
6.92 7 34
DMF + Carbaryl
7.24 I!r 0.29 6 81 + 0.18 + 0.19 zk 0.19
I@)
Control
CHOLINESTERASIP
7.51 6.89
(!XJ”$,
BRAIN
DMA + Carbaryl
glycol
Treatment
Corn oil-DMF + Carbaryl
--
RAT
TABLE
EFFECTS
OF
DMSO
AS
A
PESTICIDE
553
SOLVENT
hour) after oral administration, whereas DMSO and the aqueous suspension gave similar inhibition between 0.5 and 3 hours. Possibly carbaryl is more rapidly and completely absorbed in corn oil than in other vehicles. The oral dose of carbaryl (50 mg/kg) is well below the lethal dose level. The volumes of DMA and DMF alone (5 mg/kg) were tolerated during the first 24 hours, but deaths were noted at the 6- and 24-hour intervals after administration of D){F or DMA with carbaryl, indicating some enhancement of toxicity. Data are presented in Table 5 on the acute oral toxicity of thiram, dieldrin, parathion, carbaryl, and MC-A-600 when administered as solutions in DMSO and corn oil and as suspensions in water. Animals treated with an anticholinesTABLE ,~CVTTE ORAL
TOXICITY
OF
\:a~rous
5
PESTIVIDES
IX
DIFFERENT
Mho (mg/kg)Pesticide
Solvents
95yc ~__
Thiram (7 days)
DMSO Aqueous suspension Corn oil
790 560
Dieldrin (7 days)
Parathion (24 hours)
Carbaryl (24 hours)
MC-A-600 (24 hours)
a Only female b Significantly
DMSO Aqueous suspension Corn oil
confidence
limits
(715-1400)
(1.29-3.96) (1.50-5.35) (1.37-2.25)
(52m 134) (56 101)
1.61 I.77
(1.36-1.99)
71 64
(51-93)
1 .40
(1.13-l.
2.10 1.80
(1 72-2 .56) (l12s-e.57)
1 25 1.50
(1.01-1.55) (1.00-2.2.5)
4.70
(3.9x-~
1.21
(0.98-l.
1.65
(1.27-2.15)
1 “1 ” y-2
(1.19-4.52)
1.18 1.31 1.56
(0.96-l. (1.13-l. (I. 31-l.
83
DMSO Aqueous suspension Corn oil
5.55)”
DMSO aqueous suspension Corn oil
290
DMSO Aqueous suspension Corn oil
70 110
(93
130)
109
(76
158)
rats were different,
Slope function950/; confidence limits 2.26 2.83 1.76
1000
(4671335) (329-952)
SOL~ECXTS"
255 “30
used in this experiment. P < 0.05. from other
(227- 37 1) (228. “86) (Isl-~wl)
(56- 87)
solvents
(1.38-2.27)
(1.04-l.
74)
51))
40)
45) 52) 86)
in the same group.
terase (parathion, carbaryl, and MC-A-600) were observed for 24 hours, as deaths are infrequent after this period. With thiram and dieldrin, deaths usually occur after the first 2448 hours, and animals treated with these pesticides were observed for 7 days. In the dieldrin study, deaths occurred with corn oil and the aqueous suspension during the first 2 days, whereas with DMSO most deaths occurred at 3 to 7 days. With thiram in each of the solvents, a delayed toxicity was seen after 3 days. In one case, parathion in corn oil, the toxicity was significantly decreased. With parathion, the onset of toxic signs of anticholinesterase poisoning was slower when the solvent was corn oil rather than DMSO or the aqueous suspension. Deaths from parathion intoxication occurred more rapidly with these latter solvents, but differences were not noted with MC-A-600 and
554
L.
R.
WEISS
AND
R.
A.
ORZEL
carbaryl in corn oil, DMSO, or the aqueous suspension. While these variations appear to be solvent related, the LD5,, values and slope function do not indicate that different solvent systems have any effects of practical significance on the toxicity of these pesticides. A depressant effect after the administration of DMSO to animals has been described (Brown et al., 1963; Braude and Monroe, 1965; Willson et aI., 1965). Results in Tables 6, 7, and 8 show the nature of this depression. Spontaneous motor activity was measured in six groups of ten rats. Two groups were treated orally (1.0 ml/kg) with water and two groups received undiluted DMSO. TABLE EFFECTS
OF DMSO
6
ON SPONTANEOUS
Cumulative
MOTOR
counts
ACTIVITY
per time
period
(minutes) -
Solventa
Water
DMSO
DMSO
(5ooj,
a Orally, b Figures
o-15
15-30
30-45
G-60
I II
35,368 25.736
26 I583 23,889
23,569 22,747
18.671 20 ( 245
I
22.175 31.678
17,796* 13.581*
10,644*
II I II
24, m? 25,911
“8,149
15,043
22,477
28,847
30,999
28,761
PJ”P
v/v)
Total
8.51’?*
R.98lh
11,964”
104,191
92.617 59.1w 66,204b x9 ( 947
114.518
1.0 ml/kg, ten rats per group for each solvent and tested as separate groups (I and II). are significantly different from water and DMSO (5Oc7,,) groups: P < 0.0.5.
Another two groups were given DMSO (1.0 ml/kg) diluted with water, 50% v/v. The cumulative counts and significance of ten rats for consecutive 15-minute periods are listed in Table 6. After the first 15 minutes, a distinct reduction in activity was measured in the groups treated with undiluted DMSO; this reduction became more pronounced at the end of 45 and 60 minutes. However, administration of the same dose of diluted DMSO did not cause a change in activity from the water-treated controls. Table 7 compares the effects of water and DMSO as solvents for hexobarbital sodium in control and DMSO-pretreated rats. The sleeping times for each group of animals were not significantly different, indicating that after 24 hours, a large oral dose of undiluted DMSO (10 ml/kg) TABLE EFFECT
OF DMSO
Group Control DMSOc
PRETREATMEKT
Hexobarbital” 26.2 28.4
7
OR HEXOB.~RBITAL
in water f
0.7
f
1.21
SLEEPIKG
Hexobarbital” 26.7 27.9
TIME“
in DMSO 3~ 1.15 xk I.93
a Values are in minutes, means f standard error; N = 20 per group. * Hexobarbital, 100 mg/kg; solvent, 1 ml/kg, administered intraperitoneallg. c Oral, 10 ml/kg, 24-hour pretreatment.
1~ RA’I’S
EFFECTS
OF
DMSO
AS
A
TABLE THE EFFECTS
OF DMSO
First Rat NO.
&4%
PESTICIDE
5.55
8
ON THE CONDITIONED
AVOIDANCE
RESPONSE~*~
hour
E%,
Second
NE%
Treatment, oral (5 ml/kg)
8
81
19
0
33
75 94
8.5 5
0 1
DMSO DMSO DMSO Hz0 Hz0
II
23
0
Hz0
1
96
4
0
27
72
523
0
41 19
SOLVENT
’ Only female rats were used in this experiment. b Responses during first hour (no treatment) and second A?0 = avoidance, EyO = escape, and NELjb = no escape.
hour
.49,
hour
WL
NE%;.
95
5
0
X9
11
0
84 87
16 13
0 0
Ql 97
9
0
3
0
(treatment)
in terms
of percent:
showed no residual effects. Oral doses of undiluted DMSO (l-5 ml/kg) after 1-4 hour pretreatments and daily for I4 days (2 ml/kg) were also without effect on hexobarbital-induced sleeping times in rats (unpublished data). In Table 8, the effects on the conditioned avoidance response (CAR) of rats treated orally sh ow no blockage of this response. While the with DMSO or water (5 ml/kg) generally higher avoidance levels and uniformity of avoidance responses between animals at the second hour (treatment) is a possible reflection of a “warm up” phenomenon, the fact remains that DMSO did not depress the CAR during the second hour period in which the animals reach their “normal avoidance levels.” DISCUSSION
The results of acute toxicity studies following intraperitoneal and oral doses of DMSO to rats indicate its potential usefulness as a solvent for pesticides. Toxicologists working with pesticides and drugs need a vehicle with good polar and nonpolar solubility characteristics and the relative inertness of water and corn oil. For acute toxicity work, DMSO has certain properties that satisfy these criteria and others that limit its usefulness. DMSO is rapidly absorbed and uniformly distributed in all tissues, and because of its short half-life is excreted largely unchanged (Hucker et al., 1963). These properties may explain the low toxicity and apparent lack of pharmacologic effects. The data show that DMSO has a lower acute toxicity than DMA and DMF. The use of undiluted DMSO as a vehicle for pesticides does not enhance or decrease the pharmacologic or toxicologic properties of the pesticides used in these experiments. The results suggest that DMSO does not interact or enhance the activity of the anticholinesterases in the brains of rats. The reported unique ability of DMSO to penetrate tissues, increase the permeability of tissues to drugs and pesticides, and enhance the absorption of chemicals following oral administration does not seem to be important from the standpoint of acute pesticide toxicity. The LDsO values, slope functions, and mortality dose-response curves were generally similar for each pesticide whether in DMSO, in corn oil, or as an aqueous suspension. In these oral and intraneritoneal experiments, ob-
L.
556
R. WEISS
AND
R.
A.
ORZEI.
servations demonstrated that significant individual variations may occur as a result of solvent action on the toxicity of a pesticide. It should be pointed out that certain physicochemical and pharmacologic effects of DMSO prohibit its undiluted use intravenously (Willson et al., 1965; DiStefano and Klahn, 1965). However, diluted DMSO has no effect on the oral or intravenous toxicity of a variety of drugs in mice (Dixon et al., 1965). The results with pesticides and undiluted DMSO confirm earlier reports (Ben et al., 1964; Feinman et al., 1964) that systemically administered diluted DMSO, in general, does not materially influence drug toxicity or cellular penetration. The hypothesis that DMSO can alter pharmacologic or toxicologic effects is not supported. The question whether undiluted DMSO as a solvent could alter the effects of drugs and pesticides on the central nervous system was partially answered by the brain cholinesterase experiments. The depressant effects of DMSO reported by others do not appear to be related to an action on the central nervous system. Our work indicated that DMSO, 5 mg/kg orally, has no significant effect on the conditioned avoidance response in rats. The depressant effects on spontaneous motor activity after oral administration of DMSO probably were induced by a localized action in the viscera since diluted DVlSO did not produce these changes and hexobarbital sleeping times were unchanged with undiluted DMSO. However, additional central nervous system studies seem warranted with D;\/ISO (diluted and undiluted) before concluding that this solvent does not affect experimental drug and pesticide investigations. For this reason, adequate control studies with DMSO should be conducted whenever this solvent is used, to determine any variations due to drug-solvent interactions. SUMMARY The acute oral and intraperitoneal toxicities of dimethyl sulfoxide (DMSO ), dimethyl acetamide (DMA), dimethyl formamide (DMF), and propylene glycol (PC), diluted and undiluted, were compared in rats after 24-hour and s-day periods. The results indicate that DMSO is less toxic than DMA and DMF. The toxicities of DMSO and DMF were unchanged by dilution with water. A delayed toxicity was noted with DMSO, DMA, DMF, and PG by each route. None of these solvents influenced brain cholinesterase activity. Some significant differences in the inhibition of cholinesterase by carbaryl in the various solvents were observed. The acute oral toxicities of thiram, dieldrin, carbaryl, MC-A-600, and parathion were generally similar whether administered in DMSO, in corn oil, or as an aqueous suspension. Some differences in the times of the onset of signs of toxicity were noted, Undiluted DMSO produced a depression of spontaneous motor activity without an effect on hexobarbital sleeping time or the conditioned avoidance response. The depression was abolished by dilution. Because of the low acute toxicity and apparent lack of interaction, DMSO is a useful solvent for studying the effects of pesticides in animals. ACKNOWLEDGMENTS The authors appreciate J. Bryant, and R. Lee,
the technical assistance Jr., in these studies.
provided
by R. E. Brodie,
J. R. Reeves,
III,
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EFFECTS
OF
DMSO
AS
A
PESTICIDE
SOLVENT
557
BEN, M.,
DIXON, K. L., A~AMSON, K. H., FEINXIAN, H., and RALL, D. P. ( 1964). Toxicity of various drugs using dimethyl sulfoxide (DMSO) as a solvent. Pharmacologist 6, 189. BRAU~E, M. C., and MONROE, R. R. (1965). Dimethyl sulfoxide: interactive effects with alphaglucochloralose. Current Therup. Res. 7, 502-509. BROWN, V. K., ROBINSON, V., and STEVENSON, D. E. (1963). A note on the toxicity and solvent properties of dimethyl sulfoxide. J. Phurm. Phurmucol. 15, 688-692. DAVIS, K. J., and JENNEH, P. M. (1959). Toxicity of three drug solvents. Toxicol. Appl. Phurmucol. 1, 576-578. DISTEFANO, V., and KLAHN, J. J. ( 1965). Oh servations on the pharmacology and hemolytic activity of dimethyl sulfoxide. Toxicol. Appl. Phurmacol. 7, 660-666. DIXON, R. L., ADAIUSON, R. H., BEN, M., and RALL, D. P. ( 1965 ). Apparent lack of interaction between dimethyl sulfoxide (DMSO) and a variety of drugs. Proc. Sot. Exptl. Biol. Med. 118, 756-759. FEIN~\IAN, H., BEN, M., and LEVIN, R. (1964). Toxicity of dimethyl sulfoxide in primates. Pharmacologist 6, 188. HORITA, A., and WEBEH, L. J. ( 1964). Skin penetrating property of drugs dissolved in dimethyl sulfoxide and other vehicles. Life Sci. 3, 1389-1395. HUCKER, H. B., AH~ZAD, P. M., and MILLEH, E. A. (1966). Absorption, distribution and metabolism of dimethyl sulfoxide in the rat, rabbit and guinea pig. J. Phurmucol. Exptl. Therup. 154, 176184. LITCHFIELD, J. T., JH., and WILCOXON, F. ( 1949). A simplified method of evaluating doseeffect experiments. J. Pharmucol. Exptl. Therup. 96, 99-113. ROSEN, H., BLUSIENTHAL, A., PANASEVICH, R., and MCCALLUM, J. (1965). Dimethyl sulfoxide (DMSO) as a solvent in acute toxicity determinations. Proc. Sot. Exptl. Biol. Med. 120,
511-514. ROSENKRANTZ, H., HADIDIAN, Z., SEAY, H., and MASON, H. M. (1963). Dimethyl sulfoxide: its steroid solubility and endocrinologic and pharmacologic-toxicologic characteristics. Cancer Chemotherapy Rept. 31, 7-24. SANDERSON, D. M. ( 1959). A note on glycerol formal as a solvent in toxicity testing. 1. Phurm. Pharmucol. 11, 150-156. SIEGEL, S. (1956). NonpuTumetric Statistics for the Behavioral Sciences, p. 312. McGrawHill, New York. SPIEGEL, A. J., and NOSEWOHTHY, M. M. (1963). Use of nonaqueous solvents in parenteral products. J. Phurm. Sci. 52, 917-927. WILLSON, J. E., BROWN, D. E., and T~XMENS, E. K. (1965). A toxicologic study of dimethyl sulfoxide. Toricol. Appl. Pharmacol. 7, 104-112.