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Pharmacologic and toxicologic studies of N,N-diethyltoluamide
TOXICOLOGY
AND
APPLIED
7, 772-780
PHARMACOLOGY
Pharmacologic
and
(1965)
Toxicologic
Studies
of
N,N-Diethyltoluamidel II.
NNDiethyl-o-toluamide ANTHONY
and
M. AMBROSE~AND
N,N-Diethyl-p-toluamide’ DONALD H. YOST~
Toxicology Division, U. S. Army Environmental Health Agency, Pathology Division, U. S. Army Medical Research Laboratory, Received
August
Edgewood, Maryland, Fort Knox, Kentucky
and
3, 1964
N,N-Diethyl-o-toluamide and lV,N-diethyl-p-toluamide, henceforth referred to as o-DET and p-DET, respectively, are at least two known impurities or end products of reaction occurring in the synthesis of NJ-diethyl-m-toluamide (m-DET), an insect repellent (Smith, 1958) which is intended for use either as a solution in ethanol or isopropanol and/or as an aerosolby direct application to the skin of man and possibly in the treatment of wearing apparel. Since o-DET and p-DET occur as by-products in the synthesis of m-DET either alone or as a mixture of both, in varying amounts, toxicologic studies were undertaken to rule out possible health hazards from the presence of these two isomers, and to serve as a guide in establishing limits of impurities and standards for the widely accepted insect repellent m-DET. Toxicologic studies reported in this paper follow the samegeneralpattern previously describedin the evaluation of m-DET (Ambrose et al., 1959). o-DET and p-DET have the following chemical structure (o- and p- represent substitution of a methyl group for the corresponding isomers):
,CA ‘CsHs (P)
Both isomers are colorless, crystalline solids, practically insoluble in water, and soluble in cottonseed oil and isopropanol to the extent of approximately 12 and 20% respectively; and practically nonvolatile on exposure at room temperature (23-27’C) for 20 days. 1 Presented in part at the Forty-second Annual Meeting of the Federation of American Societies for Experimental Biology, April 14-18, 1958, Philadelphia, Pennsylvania. 2 Present address: Department of Pharmacology, Medical College of Virginia, Richmond, Virginia 23219. 3 Present address: Department of Pathology, Scientific Divisions, Abbott Laboratories, North Chicago, Illinois 60064. 772
TOXICITY
OF
O-
and
~DIETHYLTOLUAMIDE
773
METHODS
The samples of o-DET4 and p-DET5 used in these studies were of the highest purity obtainable and contained, respectively, 90% ortho isomer and 95% para isomer. In administering the above compounds to experimental animals, no allowance or correction was made for percentage composition; both were considered to be 100%. Albino rats of the Wistar-CWL strain were used throughout. Young albino rabbits obtained on the open market were used after about 2 weeks’ observation and acclimatization to laboratory conditions. Acute oral toxicity. Male and female rats weighing 80-120 g each were used. The test materials, as solutions in cottonseed oil, were administered by stomach tube in single oral doses to 2.5 male and 46 female rats for o-DET, and to 24 male and 35 female rats for p-DET. Suitable dilutions with cottonseed oil were made so that the dosage administered was contained in approximately 1 ml, equivalent to approximately 10 ml/kg of cottonseed oil. Larger volumes of oil were intentionally avoided because of possible laxative effects. In the experience of the senior author, doses of cottonseed oil and other edible oils in excess of 10 ml/kg are always accompanied by softening of the stools or slight diarrhea. The LDsO values were calculated by the method of Miller and Tainter ( 1944). Repeated oral doses by intubation. The subacute oral toxicity was studied in male rats weighing 150-207 g at start. Ten male rats were used for each compound. Both compounds were dissolved in cottonseed oil and administered daily as a single dose by gastric intubation. A 10% solution of o-DET and a 12.5% solution of p-DET were used, respectively. The o-DET-treated rats received daily 6 ml/kg (equivalent to 600 mg/kg of o-DET), and the p-DET-treated rats received daily 9 ml/kg (equivalent to 1125 mg/kg of p-DET) for 19 days. Body weight records were kept, and 24 hours after the last dose all surviving rats were autopsied. Gross observations were made, and representative portions of the following organs were removed and preserved in 4% neutralized aqueous formaldehyde solution for histopathologic study: urinary bladder, testes, adrenals, kidneys, spleen, pancreas, liver, small intestine, stomach, heart, lungs, thyroid, and brain. Hematoxylin-eosin stained sections were studied histologically. Repeated oral doses 6y dietary addition. Matched groups of weanling (21-28 days) rats of approximately equal weight (49-53 g) , consisting of 10 rats of each sex, were placed on each of the following dietary levels of o-DET added to the basic diet”: 0.0 (control), 0.01, 0.05, 0.1, 0.5, or l.O%, respectively, for 30 weeks. Similarly, groups of rats were placed on p-DET diets of the same percentage composition. Rats were housed in groups of 5 to a cage. All rats had free access to their respective diets and water at all times. Throughout the entire experimental period, all rats were weighed once a week, and food consumption was determined. Hemoglobin determinations were made at 14 and 28 weeks for rats on 0, 0.5, and 1% diets. After 30 weeks on the respective die& all rats were autopsied and the following organs were removed, weighed, and fixed in 4yo neutralized aqueous formaldehyde solution for histopathologic study: 4 Kindly 5 Kindly 6 Purina
supplied supplied Laboratory
by by
Montrose Chemical Company,
Newark, New Jersey. Hercules Powder Company, Wilmington, Delaware, Chow, Ralston Purina Company, St. Louis, Missouri
774
ANTHONY
M.
AMBROSE
AND
DONALD
H.
YOST
testes, kidneys, spleen, liver, and heart. In addition, the following organs were also removed and preserved for histopathologic study: urinary bladder, small intestine, stomach, pancreas, lungs, thyroid, adrenals, brain, and reproductive organs of the female rats. Hematoxylin-eosin sections were examined histologically. Repeated dermal application-rabbits. Two groups of albino rabbits consisting of 5 males and 5 females, weighing 3-5 kg each at start, were used for o-DET. One group received o-DET dissolved in cottonseed oil and the other o-DET in isopropanol. Two similar groups of rabbits were used for p-DET. Ten per cent solutions of the respective compounds dissolved in cottonseed oil and in isopropanol, as the carrier vehicles, were used. As controls, two groups of 5 rabbits each were used for the respective carrier vehicles. Daily applications of 2 ml/kg (equivalent to 200 mg/kg of o-DET or p-DET) were made to the closely clipped, unabraded trunks of each rabbit, 5 days/week for 13 weeks (65 applications). Repeated dermal application--rats. Ten male rats weighing 308-370 g each at start were used. Daily applications of 1.3 ml/rat of a 20% solution of o-DET in isopropanol (equivalent to 260 mg o-DET/rat/day) were made to the closely clipped, unabraded trunks of each rat, 5 days/week for 4 weeks (20 applications). A similar experiment was conducted with p-DET, and the carrier vehicle. For both rabbits and rats, the area inuncted represented approximately 10% of the total body surface. NO restraining devices were used to prevent licking, and all animals were weighed once each week. Twenty-four hours after the last application, 3 rabbits in each group receiving o-DET or p-DET in cottonseed oil and isopropanol, the controls, and all the rats were autopsied. The following organs were preserved for histopathologic study: urinary bladder, gonads, adrenals, kidneys, pancreas, spleen, liver, stomach, small intestine, heart, lungs, thyroid, brain, and skin. OcuZar eflects. The effects of 0-DET and p-DET on mucous membranes were determined in albino rabbits. TWO drops, as a single dose, of a 10yO solution of o-DET or p-DET in cottonseed oil, equivalent to 10 mg of the respective compounds, were instilled into the conjunctival sac of one eye of each of 4 rabbits. The contralateral eye was used as control with cottonseed oil alone. The effects of the compounds on the eye as a whole were observed at 2-hour intervals during the first day of application and at 24, 48, and 72 hours thereafter. RESULTS Acute Oral Toxicity-Rats Any deaths which occurred did so within the first 24 hours. With o-DET, no difference in toxicity was observed between sexes; with p-DET, a slight difference was apparent. However, the difference was not statistically significant. The acute oral LD5,, values, sexes combined, with 95% confidence limits, are 1.21 _t 0.042 for o-DET and 2.3 ? 0.19 g/kg for p-DET. Repeated Oral Doses by Intubation Male rats receiving daily, approximately 50% of the LDjo dose of mg/kg) or P-DET ( 1125 mg/kg) as a single dose for 19 days, showed of adverse effects as judged by mortality, gross appearance, behavior, Upon histopathologic examination of tissues, these rats did not appear
o-DET (600 no evidence and growth. to be signin-
TOXICITY
OF
o-
and
p-DIETHYLTOLUAMIDE
775
cantly affected by the administration of these two compounds as compared to control rats receiving cottonseed oil alone. Repeated
Oral Doses by Dietary
Addition
No difference in appearance and behavior, survival; and food consumption was noted between control rats and rats on the various diet levels of o-DET or fi-DET. Hemoglobin values at 14 and 28 weeks for rats on 0.5 and 1.0% dietary levels of o-DET or p-DET were not different from the controls. In Table 1, data are summarized on average terminal body weight for rats on diets containing varying concentrations of o-DET for 30 weeks. In Table 2, similar data are summarized for $-DET. Body weights of male rats on the various dietary levels of o-DET were not significantly different from those of the control, However, growth of female rats on 0.5 and 1.0% levels of o-DET was significantly decreased. Growth (body weights) of both male and female rats on fi-DET diets (Table 2) was unaffected. Organ-to-body weight ratio data for o-DET are summarized in Table 1. Statistically increased ratios occurred for the liver of both male and female rats on 0.5 and 1.0% ; and for the kidneys of male rats on l.O(& and female rats on 0.5 and l.O$# dietary levels of o-DET. In Table 2, similar data on organ: body weight ratios are summarized for rats on varying dietary levels of p-DET. Statistically significant increased ratios occur for the liver of male rats on the 0.174 and higher levels and for female rats on 0.5 and l.O$% dietary levels of p-DET. No other organs were significantly different from the controls. Histopathologic findings. Limited morphologic changes possibly related to the dietary intake of o-DET or p-DET for 30 weeks appeared to be confined to the kidneys only. The changes were essentially similar for each of the two isomers and varied only with respect to the dietary levels. However, it must be noted that the changesin the kidneys were minimal and not dramatic. As compared with respective controls, focal areas of necrosis of renal tubular epithelium were seen sometimesassociated with limited numbers of lymphoid cells infiltrating the interstitial tissues. In other areas inflammatory cell infiltrates alone were found and, where the lesions were present for sometime, scar tissue had replaced the damaged tubular cells. These changes occurred in the kidneys of rats fed each of the two isomers at the l.OyO dietary level. At the 0.55? level, similar lesionswere observed; however, the number and severity of the lesions were markedly decreasedand, in a few rats, no kidney damage was found. At the 0.1% level, less than one-fourth of the rats had kidney lesions.No lesionswere observed in rats on O.OS$Zand O.Olc/, dietary levels of o-DET or p-DET. Other organs were not significantly different from the controls. Repeated
Dermal
Applicatio-Rabbits
Repeated daily application of 200 mg,/kg of o-DET in solution either in cottonseed oil or isopropanol, for 65 days, to the closely clipped trunks of albino rabbits was without apparent effects. No mortality was encountered other than that which occurred when one male and one female rabbit in each group was destroyed because of accidental injuries sustainedin handling. Throughout the entire courseof I3 weeks, the general appearance and behavior of all rabbits were normal. Growth, as judged
k standard error. differs significantly
10 10 10 10 10 10
Females 0.0 0.01 0.05 0.1 0.5 1 .o
a Mean b Value
9 10 10 10 10 9
Number of rats
Males 0.0 0.01 0.05 0.1 0.5 1 .o
Concentration in diet ((76)
ORGAN:BODY
DATA FOR MALE VARIOUS
from
-’ 5~ z!z Ifr: -” -e
t rt 2 -t & -c
controls,
254.1 245.3 250.2 243.5 232.5 222.2
420.1 395.2 399.0 417.8 397.5 369.3 -
0.75 t 0.04 0.72 f 0.06 0.73 2 0.05 0.77 & 0.01 0.80 & 0.02 0.81 & 0.07
Testes
0.64 0.66 0.64 0.69 0.73 0.78
0.65 0.70 0.66 0.63 0.70 0.80 k k ” 2 2 k
k -c _t 2 2 _t
Kidneys
0.02 0.01 0.02 0.08 o.03b o.oZb
0.02 0.02 0.02 0.02 0.02 0.040
Organ
weight
0.17 0.18 0.17 0.17 0.17 0.18
0.13 0.14 0.14 0.14 0.14 0.12 f & ‘2 & ”
* ?I k 2 c Ik
of body
30 WEEKS
0.05 0.01 0.01 0.01 0.01 0.01
0.01 0.01 0.00 0.01 0.01 0.00
Spleen
as per cent
TABLE 1 AND FF.MALE RATS THAT SURVIWLI FOR APPROXIMATELY CONCENTRATIONS OF NJ-DIETHYL-LJ-TOLUAMIDE (o-DET)
P < 0.05.
4.137’
3.5ob
7.41 3.35 10.43 15.05
13.54 16.02 15.01 14.21 9.56 15.70
Terminal body weightn 63)
WEIGHT
3.34 3.39 3.29 3.52 4.06 4.68
I!I c rt ” ” +-
-t k ” 31 k -+
Liver 2.98 3.24 3.15 3.23 3.94 4.54
weight&
0.14 0.07 0.10 0.11 0.07” 0.13b
o.llb
o.otb
0.07 0.09 0.09 0.08
ON DIETS CONTAINING
0.28 0.31 0.30 0.29 0.30 0.30
0.25 0.27 0.25 0.24 0.26 0.25
-c f 2 IL k 2
-c 2 e k & -c
Heart
0.01 0.01 0.01 0.01 0.01 0.00
0.00 0.01 0.01 0.01 0.01 0.01
Q Mean b Value
0.5 1.0
0.1
0.01 0.05
Females 0.0
1.0
0.5
0.05 0.1
0.01
Males 0.0
(%)
Concentration in diet
243.6
9
controls,
228.1
240.2 229.4
10 10
423.4
408.0 405.3 383.5
400.8
400.2
240.3 250.6
10
VARIOUS
FOR MALE
14.17
IL
51 2
f
-c IL
A
k f k
FEMALE
0.69
0.74
0.77 0.76
0.75 0.68
RATS
0.03 0.04
-
-
-
-
f
0.02
” 0.02 r+ 0.05
iI f
IL 0.04
Testes
CONCENTRATIONS
AND
P < 0.05.
6.79
5.10 6.71
8.58
9.53 7.21
9.65
1 I .64 9.04 14.94
I!I 16.75
f
(8)
Terminal body weighta
DATA
9 9
9
10 10
9 8
9
from
WEIGIIT
rats
C standard error. differs significantly
of
Number
ORCAN:BODY
TABLE
2 SURVIVED
0.71
0.66 0.69
0.68
0.67 0.63
0.68
0.64 0.70
0.66 0.65
0.65
0.02
0.02
”
0.03
” 0.02 -e 0.01
r+ 0.00
-+ 0.02 ” 0.02
”
21 0.02 2 0.01
-c 0.02 +- 0.02
2
Kidneys
Organ weight
0.13
0.18
0.18 0.18
0.18
0.16 0.16
0.14
0.14 0.14
0.14 0.14
30
cent
-c 0.01
r+ 0.01 A 0.01
k 0.01
rfr 0.01 f. 0.02
& 0.01
-c 0.00 k 0.01
k 0.01 2 0.02
-c 0.01
Spleen
as per
(#-DET)
FOR APPROXIMATELY
OF N,N-DIETHYL-p-TOLUAMIDE
THAT
of
body
WEEKS
4.04
3.31 3.65
3.28 3.36
3.17
4.22
2.71 3.67
3.23 3.24
3.11
weighta
CONTAINING
-e O.llh
k 0.09 C 0.08”
I!I 0.11 k 0.06
-c 0.07
2~ 0.12”
?I alob ? 0.10”
” 0.12 XL 0.09
2 0.05
Liver
ON DIETS
0.26
0.31
0.29
0.32 0.30
0.25 0.30
0.24
0.27
0.26
0.26 0.25
0.01 0.01
0.01
f
0.01
-c 0.01
_’ 0.01 2 0.01
I+ 0.01 k 0.01
f
f
k
k 0.01 2 0.01
51 0.01
Heart
m
b
Fc:
$ t;
z
2
E y
?
8
3
i;
3
X
778
ANTHONY
M.
AMBROSE
AND
DONALD
H.
YOST
by body weight, remained unchanged, and food consumption was normal. Xo difference was observed in skin reactions between rabbits treated with the respective vehicles with or without o-DET. Histopathologic changes were observed in the kidneys only, and these consisted of mild interstitial nephritis, which possibly could be related to the effects of o-DET. Rabbits treated similarly with 200 mg/kg of p-DET in cottonseed oil or isopropanol for 65 days reacted essentially the same as rabbits treated with o-DET. ?;o mortality, other than one accidental death, was encountered. General appearance, growth, and food consumption were normal. Histologically, the changes observed in these rabbits were essentially the same as those observed for the rabbits treated with o-DET. However, the inflammatory lesions in the kidneys were somewhat more extensive than those found with o-DET, but in only one instance could be considered severe. Repeated Demal
Application-Rats
Repeated dermal application for 20 days, of 260 mg/kg/day o-DET or p-DET dissolved in isopropanol produced no signs of intoxication, cutaneous reactions, or radical departure from normal appearance and behavior. Histopathologic examinations of the tissues of rats treated with o-DET or p-DET, and the controls, were essentially the same. Thickening and scaliness of the skin, and slight-to-moderate epidermal proliferation were a constant findin,.u Histologic examination did not reveal any significant degree of inflammatory changes occurring in the dermal structure. In the kidneys, no significant differences were noted between the controls and those treated with o-DET or p-DET. Very slight degrees of hydropic change and interstitial infiltration with lymphocytes were noted, but these changes occurred in all groups including the controls. Ocular Effects Ten milligrams o-DET or p-DET as a single dose dissolved in cottonseed oil instilled into the conjunctival sac of the rabbit eye was not irritating. DISCUSSIOK
The experimental results show that o-DET ( LDjo, 1.2 1 g/kg) is slightly more toxic as a single dose than p-DET (LDx), 2.3 g/kg) when administered intragastrically to rats. The LDSo of the meta isomer of NJ-diethyltoluamide (m-DET) with 9570 confidence limits has been reported as 2.0 * 0.17 g/kg (Ambrose et al., 1959). On the basis of the above data obtained on the ortho and para isomers, o-DET appears to be approximately twice as toxic as p-DET and m-DET. With all three isomers, no significant difference in mortality was observed between sexes. Repeated oral doses of approximately 50% of the estimated LD5,,‘s for rats for 19 consecutive days showed no evidence of adverse effects either grossly or on histologic examination of the tissues. Similar studies with m-DET (unpublished data) were also negative. Upon repeated feeding to rats for 30 weeks, o-DET appeared to be slightly more toxic than p-DET. A slight inhibitory effect on growth of female rats only was observed at the 0.5 and 1.0% levels. With p-DET no inhibition in growth was observed. On an organ: body weight basis, the livers of both male and female
TOXICITY
OF
O- and
~DIETHYLTOLUAMIDE
779
rats on 0.5 and 1.0% o-DET and P-DET were significantly heavier than those of the controls. The kidneys of male rats on 1.0% and of female rats on 0.5 and 1.0% o-DET were also significantly heavier. Similar results were obtained with m-DET (Ambrose et aZ., 1959), which would suggest that all three isomers of N,N-diethyltoluamide have a similar stress effect on the liver and kidneys. Renal changes consisting of necrosis of the tubular epithelium, interstitial cellular infiltration and tiny foci of scar tissue in the tubules were observed in rats, particularly on the 1.0% dietary level of o-DET or p-DET. These changes decreased in number and severity at the 0.Sp8 level. They were present in only about one-fourth of the rats at the 0.1% level, and none were found in rats on the O.OSsi, and O.Olc/o dietary levels of o-DET or p-DET. These changes have been considered as minimal and not dramatic. In view of the importance of this study in connection with earlier studies on m-DET (Ambrose et aZ., 1959), the tissues were reviewed by the Genitourinary Section, Forensic Pathology Section, and the Veterinary Section of the Armed Forces Institute of Pathology. While some of the above histopathologic changes were observed in the kidneys of rats on the various dietary levels of o-DET or p-DET, these changes were considered to be of no significance, and unrelated to the compounds fed. Repeated cutaneous application to rabbits (65 applications) caused little or no skin reaction with either o-DET or P-DET in doses of 200 mg/kg,‘day. A mild interstitial nephritis was observed in the kidneys of rabbits, which possibly could be related to the effects of o-DET. With p-DET the inflammatory lesions in the kidneys were more extensive than those observed with o-DET: however, in only one instance could they be considered severe. In rats, 20 applications of 260 mg/rat,/day of either o-DET or p-DET produced no signs of intoxication and only minor or insignificant histologic changes that were also seen in the controls. Similar observations were made with n2-DET (unpublished results). These studies suggest that o-DET and p-DET have little or insignificant irritant effects on rabbit and rat skin and caused little or no discomfort or irritation when placed in contact with mucous membranes of the eyes of rabbits. The present studies suggest that the presence of 554 of o-DET or P-DET as impurities in the insect repellent lV,:V-diethyl-~n-toluamide (m-DET) is probably not a serious health problem. However, of the three isomers studied-the ortho and pava isomers presented in this paper and the meta previously published (Ambrose et al., 1959)-the ortho appears to be the most toxic, and the nzeta and para the least toxic, on single acute oral administration to rats. On cutaneous administration to rabbits and rats no such comparison can be made. SUMMARY Toxicologic data are presented on two isomers of NJ-diethyltoluamide, namely, S,N-diethyl-otoluamide (o-DET) and S,N-diethyl-p-toluamide (p-DET), which appear as impurities in S,Sdiethyl-m-toluamide (m-DET), a new insect repellent. The acute oral LD,,r for rats, both sexes, is of the order of 1.21 t 0.042 g/kg for o-DET and 2.3 & 0.19 g/kg for p-DET. Repeated daily gastric intubation of 507, of the estimated LD,, values to rats for 19 days showed no evidence of adverse effects, either grossly or histologically. Feeding diets containing 0.0, 0.01, 0.05, 0.1, 0.5, and 1.0% (equivalent to 0, 100, 500, lr)~o, 5000, and 10,000 ppm) of o-DET or p-DET for 30 weeks to rats revealed no serious deleterious effects other than a slight depression in growth for female rats on the two highest concentrations
780
ANTHONY
M.
AMBROSE
AND
DONALD
H.
YOST
of o-DET in the diet; hypertrophy of the livers of both male and female rats on 0.5 and 1.0% o-DET or p-DET; and hypertrophy of the kidneys of male rats on 1.07’0 and female rats on 0.5 and 1.0% o-DET. Minimal morphologic changes observed in the kidneys of rats on the various dietary levels of o-DET or p-DET are considered to be insignificant and unrelated to the compounds fed. Other organs were unaffected. Repeated cutaneous application of o-DET or p-DET in doses of 200 mg/kg/day for 65 days to rabbits and 260mg/day to rats for 20 days produced only minor, fleeting and incipient skin reactions and only minor or insignificant microscopic changes in the kidneys only. On mucous membranes of the eye, no irritation, chemosis or conjunctivitis was observed.
ACKNOWLEDGMENTS The authors gratefully T. Salamone and Donald cology Division.
acknowledge K. Huffman,
the technical IJ. S. Army
assistance of Clara V. Miller, and of Richard Medical Corps trainees assigned to the Toxi-
REFERENCES AMBROSE,
studies 97-115. MILLER,
A. M.,
on
HUFFMAN,
NJ-diethyltoluamide.
D. K., and SALAMONE, R. T. I. NJ-diethyl-m-toluamide.
(1959).
Pharmacologic Toxicol. Appl.
L. C., and TAINTER, M. L. (1944). Estimation of the EDs, and of logarithmic-prohit graph paper. Proc. Sot. Ezptl. Biol. Med. 57, 261-264. SMITH, C. N. (1958). Insect repellents. Soap Chem. Specialties 34, 105-112.
its
and toxicologic Phavmacol. 1, error
by
means
AND
APPLIED
7, 772-780
PHARMACOLOGY
Pharmacologic
and
(1965)
Toxicologic
Studies
of
N,N-Diethyltoluamidel II.
NNDiethyl-o-toluamide ANTHONY
and
M. AMBROSE~AND
N,N-Diethyl-p-toluamide’ DONALD H. YOST~
Toxicology Division, U. S. Army Environmental Health Agency, Pathology Division, U. S. Army Medical Research Laboratory, Received
August
Edgewood, Maryland, Fort Knox, Kentucky
and
3, 1964
N,N-Diethyl-o-toluamide and lV,N-diethyl-p-toluamide, henceforth referred to as o-DET and p-DET, respectively, are at least two known impurities or end products of reaction occurring in the synthesis of NJ-diethyl-m-toluamide (m-DET), an insect repellent (Smith, 1958) which is intended for use either as a solution in ethanol or isopropanol and/or as an aerosolby direct application to the skin of man and possibly in the treatment of wearing apparel. Since o-DET and p-DET occur as by-products in the synthesis of m-DET either alone or as a mixture of both, in varying amounts, toxicologic studies were undertaken to rule out possible health hazards from the presence of these two isomers, and to serve as a guide in establishing limits of impurities and standards for the widely accepted insect repellent m-DET. Toxicologic studies reported in this paper follow the samegeneralpattern previously describedin the evaluation of m-DET (Ambrose et al., 1959). o-DET and p-DET have the following chemical structure (o- and p- represent substitution of a methyl group for the corresponding isomers):
,CA ‘CsHs (P)
Both isomers are colorless, crystalline solids, practically insoluble in water, and soluble in cottonseed oil and isopropanol to the extent of approximately 12 and 20% respectively; and practically nonvolatile on exposure at room temperature (23-27’C) for 20 days. 1 Presented in part at the Forty-second Annual Meeting of the Federation of American Societies for Experimental Biology, April 14-18, 1958, Philadelphia, Pennsylvania. 2 Present address: Department of Pharmacology, Medical College of Virginia, Richmond, Virginia 23219. 3 Present address: Department of Pathology, Scientific Divisions, Abbott Laboratories, North Chicago, Illinois 60064. 772
TOXICITY
OF
O-
and
~DIETHYLTOLUAMIDE
773
METHODS
The samples of o-DET4 and p-DET5 used in these studies were of the highest purity obtainable and contained, respectively, 90% ortho isomer and 95% para isomer. In administering the above compounds to experimental animals, no allowance or correction was made for percentage composition; both were considered to be 100%. Albino rats of the Wistar-CWL strain were used throughout. Young albino rabbits obtained on the open market were used after about 2 weeks’ observation and acclimatization to laboratory conditions. Acute oral toxicity. Male and female rats weighing 80-120 g each were used. The test materials, as solutions in cottonseed oil, were administered by stomach tube in single oral doses to 2.5 male and 46 female rats for o-DET, and to 24 male and 35 female rats for p-DET. Suitable dilutions with cottonseed oil were made so that the dosage administered was contained in approximately 1 ml, equivalent to approximately 10 ml/kg of cottonseed oil. Larger volumes of oil were intentionally avoided because of possible laxative effects. In the experience of the senior author, doses of cottonseed oil and other edible oils in excess of 10 ml/kg are always accompanied by softening of the stools or slight diarrhea. The LDsO values were calculated by the method of Miller and Tainter ( 1944). Repeated oral doses by intubation. The subacute oral toxicity was studied in male rats weighing 150-207 g at start. Ten male rats were used for each compound. Both compounds were dissolved in cottonseed oil and administered daily as a single dose by gastric intubation. A 10% solution of o-DET and a 12.5% solution of p-DET were used, respectively. The o-DET-treated rats received daily 6 ml/kg (equivalent to 600 mg/kg of o-DET), and the p-DET-treated rats received daily 9 ml/kg (equivalent to 1125 mg/kg of p-DET) for 19 days. Body weight records were kept, and 24 hours after the last dose all surviving rats were autopsied. Gross observations were made, and representative portions of the following organs were removed and preserved in 4% neutralized aqueous formaldehyde solution for histopathologic study: urinary bladder, testes, adrenals, kidneys, spleen, pancreas, liver, small intestine, stomach, heart, lungs, thyroid, and brain. Hematoxylin-eosin stained sections were studied histologically. Repeated oral doses 6y dietary addition. Matched groups of weanling (21-28 days) rats of approximately equal weight (49-53 g) , consisting of 10 rats of each sex, were placed on each of the following dietary levels of o-DET added to the basic diet”: 0.0 (control), 0.01, 0.05, 0.1, 0.5, or l.O%, respectively, for 30 weeks. Similarly, groups of rats were placed on p-DET diets of the same percentage composition. Rats were housed in groups of 5 to a cage. All rats had free access to their respective diets and water at all times. Throughout the entire experimental period, all rats were weighed once a week, and food consumption was determined. Hemoglobin determinations were made at 14 and 28 weeks for rats on 0, 0.5, and 1% diets. After 30 weeks on the respective die& all rats were autopsied and the following organs were removed, weighed, and fixed in 4yo neutralized aqueous formaldehyde solution for histopathologic study: 4 Kindly 5 Kindly 6 Purina
supplied supplied Laboratory
by by
Montrose Chemical Company,
Newark, New Jersey. Hercules Powder Company, Wilmington, Delaware, Chow, Ralston Purina Company, St. Louis, Missouri
774
ANTHONY
M.
AMBROSE
AND
DONALD
H.
YOST
testes, kidneys, spleen, liver, and heart. In addition, the following organs were also removed and preserved for histopathologic study: urinary bladder, small intestine, stomach, pancreas, lungs, thyroid, adrenals, brain, and reproductive organs of the female rats. Hematoxylin-eosin sections were examined histologically. Repeated dermal application-rabbits. Two groups of albino rabbits consisting of 5 males and 5 females, weighing 3-5 kg each at start, were used for o-DET. One group received o-DET dissolved in cottonseed oil and the other o-DET in isopropanol. Two similar groups of rabbits were used for p-DET. Ten per cent solutions of the respective compounds dissolved in cottonseed oil and in isopropanol, as the carrier vehicles, were used. As controls, two groups of 5 rabbits each were used for the respective carrier vehicles. Daily applications of 2 ml/kg (equivalent to 200 mg/kg of o-DET or p-DET) were made to the closely clipped, unabraded trunks of each rabbit, 5 days/week for 13 weeks (65 applications). Repeated dermal application--rats. Ten male rats weighing 308-370 g each at start were used. Daily applications of 1.3 ml/rat of a 20% solution of o-DET in isopropanol (equivalent to 260 mg o-DET/rat/day) were made to the closely clipped, unabraded trunks of each rat, 5 days/week for 4 weeks (20 applications). A similar experiment was conducted with p-DET, and the carrier vehicle. For both rabbits and rats, the area inuncted represented approximately 10% of the total body surface. NO restraining devices were used to prevent licking, and all animals were weighed once each week. Twenty-four hours after the last application, 3 rabbits in each group receiving o-DET or p-DET in cottonseed oil and isopropanol, the controls, and all the rats were autopsied. The following organs were preserved for histopathologic study: urinary bladder, gonads, adrenals, kidneys, pancreas, spleen, liver, stomach, small intestine, heart, lungs, thyroid, brain, and skin. OcuZar eflects. The effects of 0-DET and p-DET on mucous membranes were determined in albino rabbits. TWO drops, as a single dose, of a 10yO solution of o-DET or p-DET in cottonseed oil, equivalent to 10 mg of the respective compounds, were instilled into the conjunctival sac of one eye of each of 4 rabbits. The contralateral eye was used as control with cottonseed oil alone. The effects of the compounds on the eye as a whole were observed at 2-hour intervals during the first day of application and at 24, 48, and 72 hours thereafter. RESULTS Acute Oral Toxicity-Rats Any deaths which occurred did so within the first 24 hours. With o-DET, no difference in toxicity was observed between sexes; with p-DET, a slight difference was apparent. However, the difference was not statistically significant. The acute oral LD5,, values, sexes combined, with 95% confidence limits, are 1.21 _t 0.042 for o-DET and 2.3 ? 0.19 g/kg for p-DET. Repeated Oral Doses by Intubation Male rats receiving daily, approximately 50% of the LDjo dose of mg/kg) or P-DET ( 1125 mg/kg) as a single dose for 19 days, showed of adverse effects as judged by mortality, gross appearance, behavior, Upon histopathologic examination of tissues, these rats did not appear
o-DET (600 no evidence and growth. to be signin-
TOXICITY
OF
o-
and
p-DIETHYLTOLUAMIDE
775
cantly affected by the administration of these two compounds as compared to control rats receiving cottonseed oil alone. Repeated
Oral Doses by Dietary
Addition
No difference in appearance and behavior, survival; and food consumption was noted between control rats and rats on the various diet levels of o-DET or fi-DET. Hemoglobin values at 14 and 28 weeks for rats on 0.5 and 1.0% dietary levels of o-DET or p-DET were not different from the controls. In Table 1, data are summarized on average terminal body weight for rats on diets containing varying concentrations of o-DET for 30 weeks. In Table 2, similar data are summarized for $-DET. Body weights of male rats on the various dietary levels of o-DET were not significantly different from those of the control, However, growth of female rats on 0.5 and 1.0% levels of o-DET was significantly decreased. Growth (body weights) of both male and female rats on fi-DET diets (Table 2) was unaffected. Organ-to-body weight ratio data for o-DET are summarized in Table 1. Statistically increased ratios occurred for the liver of both male and female rats on 0.5 and 1.0% ; and for the kidneys of male rats on l.O(& and female rats on 0.5 and l.O$# dietary levels of o-DET. In Table 2, similar data on organ: body weight ratios are summarized for rats on varying dietary levels of p-DET. Statistically significant increased ratios occur for the liver of male rats on the 0.174 and higher levels and for female rats on 0.5 and l.O$% dietary levels of p-DET. No other organs were significantly different from the controls. Histopathologic findings. Limited morphologic changes possibly related to the dietary intake of o-DET or p-DET for 30 weeks appeared to be confined to the kidneys only. The changes were essentially similar for each of the two isomers and varied only with respect to the dietary levels. However, it must be noted that the changesin the kidneys were minimal and not dramatic. As compared with respective controls, focal areas of necrosis of renal tubular epithelium were seen sometimesassociated with limited numbers of lymphoid cells infiltrating the interstitial tissues. In other areas inflammatory cell infiltrates alone were found and, where the lesions were present for sometime, scar tissue had replaced the damaged tubular cells. These changes occurred in the kidneys of rats fed each of the two isomers at the l.OyO dietary level. At the 0.55? level, similar lesionswere observed; however, the number and severity of the lesions were markedly decreasedand, in a few rats, no kidney damage was found. At the 0.1% level, less than one-fourth of the rats had kidney lesions.No lesionswere observed in rats on O.OS$Zand O.Olc/, dietary levels of o-DET or p-DET. Other organs were not significantly different from the controls. Repeated
Dermal
Applicatio-Rabbits
Repeated daily application of 200 mg,/kg of o-DET in solution either in cottonseed oil or isopropanol, for 65 days, to the closely clipped trunks of albino rabbits was without apparent effects. No mortality was encountered other than that which occurred when one male and one female rabbit in each group was destroyed because of accidental injuries sustainedin handling. Throughout the entire courseof I3 weeks, the general appearance and behavior of all rabbits were normal. Growth, as judged
k standard error. differs significantly
10 10 10 10 10 10
Females 0.0 0.01 0.05 0.1 0.5 1 .o
a Mean b Value
9 10 10 10 10 9
Number of rats
Males 0.0 0.01 0.05 0.1 0.5 1 .o
Concentration in diet ((76)
ORGAN:BODY
DATA FOR MALE VARIOUS
from
-’ 5~ z!z Ifr: -” -e
t rt 2 -t & -c
controls,
254.1 245.3 250.2 243.5 232.5 222.2
420.1 395.2 399.0 417.8 397.5 369.3 -
0.75 t 0.04 0.72 f 0.06 0.73 2 0.05 0.77 & 0.01 0.80 & 0.02 0.81 & 0.07
Testes
0.64 0.66 0.64 0.69 0.73 0.78
0.65 0.70 0.66 0.63 0.70 0.80 k k ” 2 2 k
k -c _t 2 2 _t
Kidneys
0.02 0.01 0.02 0.08 o.03b o.oZb
0.02 0.02 0.02 0.02 0.02 0.040
Organ
weight
0.17 0.18 0.17 0.17 0.17 0.18
0.13 0.14 0.14 0.14 0.14 0.12 f & ‘2 & ”
* ?I k 2 c Ik
of body
30 WEEKS
0.05 0.01 0.01 0.01 0.01 0.01
0.01 0.01 0.00 0.01 0.01 0.00
Spleen
as per cent
TABLE 1 AND FF.MALE RATS THAT SURVIWLI FOR APPROXIMATELY CONCENTRATIONS OF NJ-DIETHYL-LJ-TOLUAMIDE (o-DET)
P < 0.05.
4.137’
3.5ob
7.41 3.35 10.43 15.05
13.54 16.02 15.01 14.21 9.56 15.70
Terminal body weightn 63)
WEIGHT
3.34 3.39 3.29 3.52 4.06 4.68
I!I c rt ” ” +-
-t k ” 31 k -+
Liver 2.98 3.24 3.15 3.23 3.94 4.54
weight&
0.14 0.07 0.10 0.11 0.07” 0.13b
o.llb
o.otb
0.07 0.09 0.09 0.08
ON DIETS CONTAINING
0.28 0.31 0.30 0.29 0.30 0.30
0.25 0.27 0.25 0.24 0.26 0.25
-c f 2 IL k 2
-c 2 e k & -c
Heart
0.01 0.01 0.01 0.01 0.01 0.00
0.00 0.01 0.01 0.01 0.01 0.01
Q Mean b Value
0.5 1.0
0.1
0.01 0.05
Females 0.0
1.0
0.5
0.05 0.1
0.01
Males 0.0
(%)
Concentration in diet
243.6
9
controls,
228.1
240.2 229.4
10 10
423.4
408.0 405.3 383.5
400.8
400.2
240.3 250.6
10
VARIOUS
FOR MALE
14.17
IL
51 2
f
-c IL
A
k f k
FEMALE
0.69
0.74
0.77 0.76
0.75 0.68
RATS
0.03 0.04
-
-
-
-
f
0.02
” 0.02 r+ 0.05
iI f
IL 0.04
Testes
CONCENTRATIONS
AND
P < 0.05.
6.79
5.10 6.71
8.58
9.53 7.21
9.65
1 I .64 9.04 14.94
I!I 16.75
f
(8)
Terminal body weighta
DATA
9 9
9
10 10
9 8
9
from
WEIGIIT
rats
C standard error. differs significantly
of
Number
ORCAN:BODY
TABLE
2 SURVIVED
0.71
0.66 0.69
0.68
0.67 0.63
0.68
0.64 0.70
0.66 0.65
0.65
0.02
0.02
”
0.03
” 0.02 -e 0.01
r+ 0.00
-+ 0.02 ” 0.02
”
21 0.02 2 0.01
-c 0.02 +- 0.02
2
Kidneys
Organ weight
0.13
0.18
0.18 0.18
0.18
0.16 0.16
0.14
0.14 0.14
0.14 0.14
30
cent
-c 0.01
r+ 0.01 A 0.01
k 0.01
rfr 0.01 f. 0.02
& 0.01
-c 0.00 k 0.01
k 0.01 2 0.02
-c 0.01
Spleen
as per
(#-DET)
FOR APPROXIMATELY
OF N,N-DIETHYL-p-TOLUAMIDE
THAT
of
body
WEEKS
4.04
3.31 3.65
3.28 3.36
3.17
4.22
2.71 3.67
3.23 3.24
3.11
weighta
CONTAINING
-e O.llh
k 0.09 C 0.08”
I!I 0.11 k 0.06
-c 0.07
2~ 0.12”
?I alob ? 0.10”
” 0.12 XL 0.09
2 0.05
Liver
ON DIETS
0.26
0.31
0.29
0.32 0.30
0.25 0.30
0.24
0.27
0.26
0.26 0.25
0.01 0.01
0.01
f
0.01
-c 0.01
_’ 0.01 2 0.01
I+ 0.01 k 0.01
f
f
k
k 0.01 2 0.01
51 0.01
Heart
m
b
Fc:
$ t;
z
2
E y
?
8
3
i;
3
X
778
ANTHONY
M.
AMBROSE
AND
DONALD
H.
YOST
by body weight, remained unchanged, and food consumption was normal. Xo difference was observed in skin reactions between rabbits treated with the respective vehicles with or without o-DET. Histopathologic changes were observed in the kidneys only, and these consisted of mild interstitial nephritis, which possibly could be related to the effects of o-DET. Rabbits treated similarly with 200 mg/kg of p-DET in cottonseed oil or isopropanol for 65 days reacted essentially the same as rabbits treated with o-DET. ?;o mortality, other than one accidental death, was encountered. General appearance, growth, and food consumption were normal. Histologically, the changes observed in these rabbits were essentially the same as those observed for the rabbits treated with o-DET. However, the inflammatory lesions in the kidneys were somewhat more extensive than those found with o-DET, but in only one instance could be considered severe. Repeated Demal
Application-Rats
Repeated dermal application for 20 days, of 260 mg/kg/day o-DET or p-DET dissolved in isopropanol produced no signs of intoxication, cutaneous reactions, or radical departure from normal appearance and behavior. Histopathologic examinations of the tissues of rats treated with o-DET or p-DET, and the controls, were essentially the same. Thickening and scaliness of the skin, and slight-to-moderate epidermal proliferation were a constant findin,.u Histologic examination did not reveal any significant degree of inflammatory changes occurring in the dermal structure. In the kidneys, no significant differences were noted between the controls and those treated with o-DET or p-DET. Very slight degrees of hydropic change and interstitial infiltration with lymphocytes were noted, but these changes occurred in all groups including the controls. Ocular Effects Ten milligrams o-DET or p-DET as a single dose dissolved in cottonseed oil instilled into the conjunctival sac of the rabbit eye was not irritating. DISCUSSIOK
The experimental results show that o-DET ( LDjo, 1.2 1 g/kg) is slightly more toxic as a single dose than p-DET (LDx), 2.3 g/kg) when administered intragastrically to rats. The LDSo of the meta isomer of NJ-diethyltoluamide (m-DET) with 9570 confidence limits has been reported as 2.0 * 0.17 g/kg (Ambrose et al., 1959). On the basis of the above data obtained on the ortho and para isomers, o-DET appears to be approximately twice as toxic as p-DET and m-DET. With all three isomers, no significant difference in mortality was observed between sexes. Repeated oral doses of approximately 50% of the estimated LD5,,‘s for rats for 19 consecutive days showed no evidence of adverse effects either grossly or on histologic examination of the tissues. Similar studies with m-DET (unpublished data) were also negative. Upon repeated feeding to rats for 30 weeks, o-DET appeared to be slightly more toxic than p-DET. A slight inhibitory effect on growth of female rats only was observed at the 0.5 and 1.0% levels. With p-DET no inhibition in growth was observed. On an organ: body weight basis, the livers of both male and female
TOXICITY
OF
O- and
~DIETHYLTOLUAMIDE
779
rats on 0.5 and 1.0% o-DET and P-DET were significantly heavier than those of the controls. The kidneys of male rats on 1.0% and of female rats on 0.5 and 1.0% o-DET were also significantly heavier. Similar results were obtained with m-DET (Ambrose et aZ., 1959), which would suggest that all three isomers of N,N-diethyltoluamide have a similar stress effect on the liver and kidneys. Renal changes consisting of necrosis of the tubular epithelium, interstitial cellular infiltration and tiny foci of scar tissue in the tubules were observed in rats, particularly on the 1.0% dietary level of o-DET or p-DET. These changes decreased in number and severity at the 0.Sp8 level. They were present in only about one-fourth of the rats at the 0.1% level, and none were found in rats on the O.OSsi, and O.Olc/o dietary levels of o-DET or p-DET. These changes have been considered as minimal and not dramatic. In view of the importance of this study in connection with earlier studies on m-DET (Ambrose et aZ., 1959), the tissues were reviewed by the Genitourinary Section, Forensic Pathology Section, and the Veterinary Section of the Armed Forces Institute of Pathology. While some of the above histopathologic changes were observed in the kidneys of rats on the various dietary levels of o-DET or p-DET, these changes were considered to be of no significance, and unrelated to the compounds fed. Repeated cutaneous application to rabbits (65 applications) caused little or no skin reaction with either o-DET or P-DET in doses of 200 mg/kg,‘day. A mild interstitial nephritis was observed in the kidneys of rabbits, which possibly could be related to the effects of o-DET. With p-DET the inflammatory lesions in the kidneys were more extensive than those observed with o-DET: however, in only one instance could they be considered severe. In rats, 20 applications of 260 mg/rat,/day of either o-DET or p-DET produced no signs of intoxication and only minor or insignificant histologic changes that were also seen in the controls. Similar observations were made with n2-DET (unpublished results). These studies suggest that o-DET and p-DET have little or insignificant irritant effects on rabbit and rat skin and caused little or no discomfort or irritation when placed in contact with mucous membranes of the eyes of rabbits. The present studies suggest that the presence of 554 of o-DET or P-DET as impurities in the insect repellent lV,:V-diethyl-~n-toluamide (m-DET) is probably not a serious health problem. However, of the three isomers studied-the ortho and pava isomers presented in this paper and the meta previously published (Ambrose et al., 1959)-the ortho appears to be the most toxic, and the nzeta and para the least toxic, on single acute oral administration to rats. On cutaneous administration to rabbits and rats no such comparison can be made. SUMMARY Toxicologic data are presented on two isomers of NJ-diethyltoluamide, namely, S,N-diethyl-otoluamide (o-DET) and S,N-diethyl-p-toluamide (p-DET), which appear as impurities in S,Sdiethyl-m-toluamide (m-DET), a new insect repellent. The acute oral LD,,r for rats, both sexes, is of the order of 1.21 t 0.042 g/kg for o-DET and 2.3 & 0.19 g/kg for p-DET. Repeated daily gastric intubation of 507, of the estimated LD,, values to rats for 19 days showed no evidence of adverse effects, either grossly or histologically. Feeding diets containing 0.0, 0.01, 0.05, 0.1, 0.5, and 1.0% (equivalent to 0, 100, 500, lr)~o, 5000, and 10,000 ppm) of o-DET or p-DET for 30 weeks to rats revealed no serious deleterious effects other than a slight depression in growth for female rats on the two highest concentrations
780
ANTHONY
M.
AMBROSE
AND
DONALD
H.
YOST
of o-DET in the diet; hypertrophy of the livers of both male and female rats on 0.5 and 1.0% o-DET or p-DET; and hypertrophy of the kidneys of male rats on 1.07’0 and female rats on 0.5 and 1.0% o-DET. Minimal morphologic changes observed in the kidneys of rats on the various dietary levels of o-DET or p-DET are considered to be insignificant and unrelated to the compounds fed. Other organs were unaffected. Repeated cutaneous application of o-DET or p-DET in doses of 200 mg/kg/day for 65 days to rabbits and 260mg/day to rats for 20 days produced only minor, fleeting and incipient skin reactions and only minor or insignificant microscopic changes in the kidneys only. On mucous membranes of the eye, no irritation, chemosis or conjunctivitis was observed.
ACKNOWLEDGMENTS The authors gratefully T. Salamone and Donald cology Division.
acknowledge K. Huffman,
the technical IJ. S. Army
assistance of Clara V. Miller, and of Richard Medical Corps trainees assigned to the Toxi-
REFERENCES AMBROSE,
studies 97-115. MILLER,
A. M.,
on
HUFFMAN,
NJ-diethyltoluamide.
D. K., and SALAMONE, R. T. I. NJ-diethyl-m-toluamide.
(1959).
Pharmacologic Toxicol. Appl.
L. C., and TAINTER, M. L. (1944). Estimation of the EDs, and of logarithmic-prohit graph paper. Proc. Sot. Ezptl. Biol. Med. 57, 261-264. SMITH, C. N. (1958). Insect repellents. Soap Chem. Specialties 34, 105-112.
its
and toxicologic Phavmacol. 1, error
by
means