Toxicology, 67 (1991) 85--96 Elsevier Scientific Publishers Ireland Ltd.
Acute and subacute inhalation toxicity studies of a new broad spectrum insect repellent, N, N-diethylphenylacetamide R. Vijayaraghavan*, S.S. Rao, M.V.S. Suryanarayana and R.V. Swamy Defence Research and Development Establishment, Gwalior - - 474 002 (India) (Received August 23rd, 1990; accepted January 8th, 1991)
Summary N,N-Diethylphenylacetamide (DEPA) is an inexpensive, long-acting and broad spectrum insect repellent. The acute LCs0 for a 4-h exposure of DEPA aerosol was found to be 1.451 mg 1-l (1.290--1.633) in male and !.375 mg ! -I (1.307--1.447) in female rats. DEPA did not cause delayed deaths. Acute exposure to 0.9 LCs0 revealed that liver might be a target organ for DEPA toxicity. On subacute exposures to 0.2, 0.6 and 0.8 LCs0 for 6 h per day, 5 days a week for 2 weeks, there was no significant change in the 0.2 LCs0 group, as evaluated by the body weight gain and organ body weight ratio. The minimal changes observed in the 0.6 LCs0 group were of reversible type as the animals recovered on cessation of exposure. A massive concentration of 0.8 LCs0 produced lethal effects. The study shows that DEPA has a low mammalian toxicity by inhalation as was found earlier with cutaneous application of the insect repellent.
Key words: N,N-diethylphenylacetamide; Insect repellent; Inhalation; Toxicity
Introduction N,N-Diethylphenylacetamide (DEPA) is a new inexpensive and long-acting insect repellent [1--3]. Extensive field trials indicate that DEPA is as potent as N,Ndiethyl-m-toluamide (Deet) and has a broad spectrum of repellent action [4]. DEPA and its formulations are reported to repel mosquitoes [2,4 6], black flies [4], biting flies [7], cockroaches [8] and land leeches [4]. Acute and subacute dermal and oral toxicity of DEPA has been thoroughly studied and it is reported to be safe [9--11 ]. Low dose of DEPA applied dermally did not produce any adverse effect on foetus and reproduction [ 12]. DEPA is found *Address all correspondence to." R. Vijayaraghavan, Visiting Research Associate, Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, U.S.A. 0300-483X/9 !/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
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nonmutagenic by the Ames test and by the mouse bone marrow micronucleus test [ 13 ]. DEPA is excreted partly unchanged and partly as N-ethylphenylacetamide, phenylacetamide and as conjugated phenylacetic acid [ 11,14---16]. In guinea pigs which mainly excrete DEPA in urine, only 24% of the dermally applied 14C-labelled DEPA was recovered in urine [9] showing that a major portion might have evaporated. Since mosquitoe repellents can also be used as pressurised sprays, data on inhalation toxicity are also required. Since inhalation toxicity of DEPA has not been reported so far, the present investigation has been carried out. Materials and methods Chemicals All the chemicals used for the biochemical studies were of analytical grade (BDH or E. Merck, Bombay, India). Ethylacetate used was of chromatography grade. DEPA was synthesised in our chemistry division [4], characterised spectroscopically [5] and found to be over 99% pure by gas chromatography. Animals Albino rats (Wistar strain), both male and female, weighing 70--100 g (initial weight), were used for the study. They were housed in polypropylene cages, four per cage, with dust-free rice husk as the bedding material. The rice husk was changed every other day. The animals were given pellet diet (Lipton feed, Bombay, India) and water ad libitum except before the determination of LCs0 when they were fasted overnight with only water ad libitum. Inhalation exposure DEPA aerosol was generated using a glass air blast nebuliser, diluted with filtered air in a glass helical assembly, and passed into the animal exposure chamber (Fig. 1). The specially designed leakproof exposure chamber, made of stainless steel with a glass door was of 50 1capacity. The chamber was equipped with ports for monitoring temperature, humidity, pressure and also for sampling chamber air for analysis of particle size and concentration of the test chemical. A small electric bulb (25 W) was fixed at the top for illuminating the chamber. The animals were exposed in small stainless steel wire cages, one per cage inside the chamer (whole body exposure). The chamber was operated dynamically and the exit aerosol was passed through a series of decontamination units to a suction pump and out of the room. The suction pump was so adjusted using a regulator, that the pressure inside the chamber was kept at a pressure of 1 cm H20 below the atmospheric pressure. The calculated t99 i.e. the time required to reach the theoretical concentration, was 8--10 min. The total air passed through the chamber was between 25--35 1 min -I and the animal loading complement (total animal volume x 100/capacity of the exposure chamber) was less than 2.0%. The inhalation chamber temperature was 25--28°C and the relative humidity was 40---60%. Particle size analysis The particle size analysis was carried out using an optical particle counter (5 chan-
86
HEPAFilter
Pump
Regulator
~F1ow
O
Filtered air supply Pump Exhau~ ~
~(
meter
Exposure Chamber
Regulator
DecoDtsm4 na.t/ou ~ t s
~(
Fig. I. Schematicdiagram of dynamicexposure assembly. nel, Royco Particle Monitor, Royco lnc, CA, U.S.A.). Count median diameter (CMD) and geometric standard deviation (a g) were calculated using a 2 cycle logarithmic probability paper. Mass median diameter (MMD) was calculated using the Hatch-Choate quation [ 17] and the aerodynamic median diameter (AMD) was calculated, by multiplying the M M D with the square root of the density of DEPA. Concentration determination The theoretical or nominal concentration was determined from the rate of nebulisation and the total air flow (nebuliser air flow + dilution air flow). The actual concentration of DEPA was determined by gravimetric analysis using 0.2 #m rating polytetrafluorethylene (PTFE) coated Whatman membrane filters. In a second method, 500 ml of the chamber air containing DEPA was trapped with the help of a 50 ml gas tight syringe (10 strokes) in a high-efficiency glass impinger containing ethylacetate. Later, a 0.5 #l aliquot was injected in a Aimil-Nucon gas chromatograph (Nucon Engineers, New Delhi, India) with the following conditions: column - - 15% diethylene glycol succinate (DEGS) on chromosorb W (high performance); detector - - flame ionisation detector; temperature - - oven: 220°C; injector - - 250°C and detector - - 250°C. The retention time of DEPA was found to be 6'42". Standard DEPA solutions were prepared by dissolving known amounts of DEPA in ethylacetate. A 0.5/~! aliquot was injected and a standard curve was plotted. Using the standard curve, the concentration of DEPA in the exposure chamber was calculated.
87
Acute toxicity studies Various concentrations of DEPA aerosols were generated and the animals were exposed for 4 h for the calculation of LCs0. The chamber temperature, humidity, particle size and the actual concentration were monitored periodically. The animals were observed for mortality up to 14 days. The LCs0 was calculated for male and female rats separately by probit analysis [ 18]. A high concentration (0.9 LCs0) of DEPA was also generated and male rats were exposed for 4 h, for identifying the target organs of DEPA toxicity.
Subacute toxicity studies After calculating the LCs0 for 4 h exposure, the LCs0 for 6 h exposure was calculated mathematically. For subacute exposures the three concentrations used were, 0.2 LCs0 (12 male and 12 female), 0.6 LCs0 (12 male and 12 female) and 0.8 LCs0 (6 male and 6 female). Male and female rats were exposed separately for 6 h per day, 5 days a week for 2 weeks. Twenty-four hours after the last exposure (13th day), 50% of the animals of the 0.2 LCs0 and 0.6 LCs0 groups were sacrificed and the remaining were allowed to recover for a further 15 days and then sacrificed (27th day). In the 0.8 LCs0 group, all surviving animals were sacrificed 24 h after the last exposure (13th day). Temperature, humidity, particle size and the concentration were properly maintained.
Body weight change During the subacute exposures the animals were weighed daily before the exposure. The body weight of the individual animal was converted to percent change of the pre-exposure body weight.
Sample collection The animals were lightly anaesthetised with ether and maximum blood was drawn using heparinised capillary from the retro-orbital plexus. They were sacrificed by cervical dislocation and important organs viz., lung, liver, kidney, heart, spleen and adrenal were dissected out. The organs were weighed after freeing from blood and adhering tissues.
Organ body weight index The individual weights of various organs were converted to percent of body weight and expressed as organ body weight index (OBWI).
Biochemical analysis Plasma was used for the estimation of glutamate pyruvate transaminase (GPT) [19], alkaline phosphatase (ALP) [20], urea [21] and cholesterol [22] after 0.9 LCs0 acute exposure and 0.6 LCs0 subacute exposure.
Statistical analysis The data were analysed by Student's t-test for statistical significance.
88
Results
Particle size and concentration For the generation of aerosols, undiluted DEPA was used. In order not to alter the t99 and the ventilation of the exposure chamber, various concentrations of DEPA aerosols were generated by adjusting the pressure of the air feed to the nebuliser. Table I shows the correlation coefficients, CMD, MMD and AMD of DEPA aerosols calculated using the Royco Particle Counter. A good correlation (r = 0.993) was obtained between the AMD and the pressure. Since the particle size was varying, 1.00---1.625 kg cm -2 pressures were used for generation of DEPA aerosols and then diluted to get the desired concentration. The particle size was also checked using an Andersen Sampler (Andersen Inc, Atlanta, U.S.A.) at 1.5 kg cm -2 pressure and the AMD was found to be 1.8/~m. The concentration was determined from the breathing zone of the animal. Using the gravimetric method, the actual concentration was found to be 55.3 -4- 1.7% of the nominal concentration and by the gas chromatographic analysis the recovery was 57.4 q- 1.4%. For all calculations the actual concentration determined by gas chromatographic method was used. Acute toxicity studies The LCs0 was calculated by probit analysis for a period of 4 h exposure in male and female rats separately using various concentrations (Table II). During the course of the exposure hyperaemia of paws, nasal and periorbital areas was observed. The animals generally died during the exposure or within 60 min after the exposure and thereafter no death was observed during the 14-day observation period.
TABLE 1 COUNT MEDIAN DIAMETER (CMD), MASS MEDIAN DIAMETER AERODYNAMIC MEDIAN DIAMETER (AMD) OF DEPA AEROSOLS PRESSURES S. no.
Pressure (kg cm -2)
r
50th* CMD (~m)
84th*
Geometric standard deviation
(MMD) AND AT VARIOUS
MMD (t~m)
AMD (#m)
0.472 0.581 0.850 1.027 1.206 1.568 1.678
0.474 0.584 0.854 1.032 1.212 1.576 1.686
(o g) I 2 3 4 5 6 7
0.25 0.50 0.75 1.00 1.25 1.50 1.75
0.980 0.971 0.948 0.936 0.927 0.919 0.923
0.410 0.535 0.807 0.980 1.141 1.448 1.530
0.809 0.631 0.920 1.110 1.307 1.705 1.823
1.241 1.175 1.140 1.133 1.145 1.177 1.192
*Percentile.
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TABLE 11 LC50 OF DEPA AEROSOLS IN RATS EXPOSED FOR 4 h Sex
LCso (mg 1-I)
95% confidence limit
LCt50 (mg • rain per m3)
Male Female Combined
1.451 1.375 1.390
1.290---1.633 1.307--1,447 1.267--1.525
348 240 330 000 333 600
Exposure to a massive concentration o f 0.9 LCso (1.306 mg 1-I) of DEPA for 4 h was carried out on a group o f six male rats. No animal died during the exposure but one animal was critical. Following this exposure there was a significant reduction of OBWI of lung and spleen and an increase in liver and adrenal. The relative weight o f kidney and heart were not significantly affected (Table III). Biochemical changes following acute exposure are shown in Table IV. There was a significant increase in GPT, ALP and cholesterol, but there was no change in urea.
Subacute toxicity studies The coefficient of variation of the exposure concentrations, within-day and between-day were less than 10%. Following subacute exposure to 0.2 LCs0 and 0.6 LC50 there was no mortality. In the 0.8 LCs0 group, out of the six males exposed, one died after the second exposure and the remaining five survived the subsequent exposures. However, in the females, following 0.8 LCs0 exposure, three died after the second exposure and two after the third exposure. The remaining one survived for the rest of the exposures. The body weight changes (percent changes from initial values) of male and female rats exposed to DEPA aerosols are shown in Fig. 2 and Fig. 3, respectively. There was no significant difference in the body weight gain o f control and 0.2 LC50 groups of male and female rats. Following exposure to 0.6 LC50, the body weight gain was significantly reduced but the slope of the curve was only slightly less than the control. In the 0.8 LCs0 group particularly, the lone survived female rat showed minimal weight gain. The OBWI of male and female rats following subacute exposure is also shown in Table III. The 0.2 LCs0 group did not show any significant difference from the control. A significant increase in the liver weight was observed both in male and in female exposed to 0.6 LCs0 and 0.8 LCs0. The liver weight was normalised in the 0.6 LCs0 group which was allowed to recover. A significant increase in the kidney weight was also observed following exposure to 0.6 LCs0. The other groups also showed a tendency of increased kidney weight. Normalisation occurred in those animals which were allowed to recover. Plasma GPT, cholesterol and urea of male rats exposed to 0.6 LCs0 did not show any significant change when compared to control.
90
0.705 ± 0.023
± 44±
0.139 0.097* 0.146 b 0.094**
4.612 ± 0.213 6.214 ± 0.068** 3.868 ± 0.155 c 5.976
4.546 ± 0.345
4.539 5.142 4.446 6.934
5.162 4- 0.250*
4.374 4- 0.201
Liver
± 44±
0.021 0.019"* 0.024 b 0.018
0.851 ± 0.027 0.919 ± 0.019 0.761 4- 0.012 0.906
0.823 4- 0.047
0.787 0.987 0.838 0.862
0.760 4- 0.019
0.797 4- 0.023
Kidney
44± 4-
0.014 0.028 0.009 0.008
0.377 + 0.010 0.386 4- 0.008 0.363 4- 0.009 0.391
0.379 4- 0.011
0.372 0.379 0.375 0.387
0.361 4- 0.010
0.366 4- 0.010
Heart
Mean ± S.E. of six animals (male 0.8 LCso, five animals and female 0.8 LCso; one animal only. Statistically significant from control vs. 0.2 LCso, 0.6 LCso or 0.8 LCso; *P < 0.05; **P < 0.001. Statistically significant from 0.6 LCso vs. recovery; ap < 0.05; bp < 0.01; cp < 0.001.
Subacute exposure 0.2 LC5o 0.679 ± 0.029 0.6 LC5o 0.680 a- 0.045 Recovery 0.571 4- 0.026 a 0.8 LC5o 0.639
Female rat Control
0.017 0.037 0.020 b 0.018
0.542 ± 0.011**
Acute exposure 0.9 LC5o
± 444-
0.697 4- 0.019
Male r l t Control
Subacute exposure 0.2 LC50 0.687 0.6 LC5o 0.778 Recovery 0.573 0.8 LC5o 0.642
Lung
Group
4± 44-
0.051 0.022 0.061 0.093
0.802 ± 0.063 0.977 ± 0.042 0.716 4- 0.012 0.601
0.849 4- 0.116
0.778 0.821 0.799 0.585
0.442 4- 0.024**
0.755 4- 0.044
Spleen
4± 44-
0.001 0.001 0.001 0.002
0.027 ± 0.001 0.026 4- 0.001 0.031 4- 0.002 0.038
0.028 ± 0.002
0.024 0.025 0.025 0.027
0.040 4- 0.004**
0.023 ± 0.001
Adrenal
CHANGES IN O R G A N BODY W E I G H T I N D E X OF RATS F O L L O W I N G ACUTE A N D SUBACUTE EXPOSURE TO DEPA AEROSOLS
TABLE 111
T A B L E IV C H A N G E S IN B L O O D P A R A M E T E R S F O L L O W I N G A C U T E A N D S U B A C U T E E X P O S U R E TO DEPA A E R O S O L S IN M A L E R A T S Group
Glutamate pyruvate transaminase (I.U. 1-1)
Alkaline phosphatase (1.U. I -I)
Urea (mg d1-1)
Cholesterol (mg dl -])
Control
12.1 4- 1.1
226.7 4- 27.7
46.7 4- 3.2
96.3 4- 3.5
Acute exposure 0.9 LCs0
33.3 4- 10.1"
370.8 4- 35.9*
46.0 4- 3.1
120.4 4- 6.1"
Subacute exposure 0.6 LC.so
10.2 4- 0.9
--
38.6 4- 2.5
103.6 4- 4.1
Mean 4- S.E. of six animals. *Statistically significant from control, P < 0.05.
DEPA 8ubacute Exposure (Male Rat) 226
200Q d Y
w 176I
~18oe
T 126"
o
100 r
T 76 001 0
I
I
I
I
I
8
10
16 Days
20
28
30
Fig. 2. Body weight changes during and after exposure to DEPA aerosols in male rats.., control; +, 0.2 LCs0; *, 0.6 LCs0; x, 0.8 LCs0.
92
DEPA 8ubacute Exposure (Female Rat) 226 S o d 200y w
• 176I
I
t 100"
• 128 I
f • 100 o n t
r I
O
78
6 0
0 8
~ 10
16
20
26
30
Days Fig. 3. Body weight changes during and after exposure to DEPA aerosols in female rats.., control; +, 0.2 LCs0; *, 0.6 LCs0; x, 0.8 LCs0.
Discussion
In the present study, the generated particles were well within the respirable range recommended for the rodent species [23,24]. All the other parameters required for the inhalation exposure were also well within the recommended standards [25,26]. The acute LCs0 for 4 h exposure was found to be 1.451 mg 1-l (348 240 mg • min per m 3) in male rats and 1.375 mg 1 -] (330 000 mg- min per m 3) in female rats. LCs0 for 4 h exposure of N,N-diethyl-m-toluamide (Deet) and dimethylphthalate (DMP) are more than 5.0 mg 1-1 [27--29] showing that acute toxicity of DEPA is more than that of Deet or DMP. The dose mortality curve of DEPA inhalation is very steep. This may indicate that even at high sublethal doses mortality will be minimal and, hence, greater margin of safety of the compound [ 30]. It is possible that DEPA does not have any specific target for toxic effects and in lethal concentrations, the death may be due to a general failure of all the systems. During acute exposures to high doses, animals generally died during the exposure and there were no delayed deaths. Deet is reported to cause delayed deaths [29]. A possible explanation of the differential lethality may be that DEPA is metabolised quickly and eliminated [ 11,14,15] and does not produce any residual effects.
93
Exposure to massive concentrations of DEPA caused excessive salivation, hyperaemia of paws, nasal and periorbital areas, tremors and convulsions. Similar findings were reported when massive doses of DEPA were administered orally [9,16]. Exposure to massive concentrations revealed that the liver is affected. Lung and kidney are only slightly affected. Rao et al. [9] also reported that administration of 2.0 LDs0 of DEPA orally damaged the liver. Toxic hepatitis was reported with other insect repellents like Deet and DMP [31 ]. Increase of plasma GPT and ALP also revealed that the liver is affected while the kidney is not, since there was no significant change in plasma urea level. Short-term subacute toxicity studies were carried out to assess the damages caused by DEPA in sublethal doses. The animals were exposed to 0.2 LCs0, 0.6 LCs0 and 0.8 LCs0 (for a 6-h period) for 5 days a week for 2 weeks. Exposures to high concentrations were also used as low doses of 1/12 LDs0 were reported to cause negligible effects even if DEPA was applied dermally or given orally for 3 ~ 4 months [9,101. Lethality was not observed in the animals exposed up to 0.6 LCs0 subacutely and even in 0.8 LCs0 the deaths were observed only up to 3 days. This may perhaps indicate that resistance to the toxic effects of DEPA might have developed by continuous exposure to the chemical. Rao et al. [32] recently observed that on daily dermal application of DEPA in rats for 7 days in different doses, the proportion of N-ethylphenylacetamide and phenylacetamide in relation to the unmetabolised DEPA excreted in urine was increased, indirectly indicating enhancement of drug metabolising enzyme activity of the animals. The body weight gains of the animals exposed to 0.2 LCs0 in both sexes did not show any significant difference from those of the controls. The body weights of animals exposed to 0.6 and 0.8 LCs0 were significantly lower than the control. The pattern of growth was uniform during the exposure period and also during the recovery period of the animals exposed to 0.2 LCs0 and 0.6 LCs0. Only in the 0.8 LCs0 group both in males and females, there was a fall in the body weight up to 2 days post exposure, thereafter it started to increase. The sudden fall in the weight following exposure might be due to stress. Rao et al. [ 10] also reported a fall in the body weight of male rats given DEPA orally at 2/3 LDs0 dose for 7 days. A sex difference is also observed in the toxicity of DEPA. The LCs0 was lower in female than in male. This finding is in agreement with the cutaneous LDs0 of DEPA in rats [ 11 ]. Metabolic capacity of microsomes of female rats is lower as compared to males for dealkylation [ 33 ]. This may perhaps be the reason for the greater toxicity of DEPA in females compared to males by inhalation exposure. Increased OBWI of liver and kidney in both male and female rats exposed to 0.6 LCs0 and 0.8 LCs0 has been a constant finding. The organ weights were normalised in the animals that were allowed to recover. Increased liver weight was also reported in male and female rats given DEPA orally at 2/3 LDs0 for 7 days [ 10]. Macko and Bergmann [29] in their studies also reported an increased liver weight in both male and female rats given various doses of Deet. Plasma cholesterol, GPT, and urea of male rats exposed subacutely to 0.6 LCs0 of DEPA did not show any significant change, even though isolated changes were observed in rats administered DEPA, percutaneously or orally [ 10,12 ]. Exposure to 94
subacute dose of 0.2 LCs0 does not cause any significant alterations and the changes recorded following subacute exposure to 0.6 LCs0 seem to be of a reversible type. The study shows that DEPA has a low mammalian toxicity by the inhalation route as was found earlier with cutaneous application of the insect repellent [ll,14].
Acknowledgements The authors are grateful to Brig (Dr.) KM Rao, Director, Defence R&D Establishment, for the encouragement and to Dr. P.K. Ramachandran, Emeritus Scientist for the valuable suggestions and for critically reviewing the manuscript.
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9
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33
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graphic identification of urinary metabolites of insect repellent N,N-diethylphenylacetamide on inhalation exposure in rats. J. Chromatogr. Biomed. Appl., 493 (1989) 210. S.S. Rao, D.K. Jaiswal and P.K. Ramachandran, Distribution and metabolism of insect repellent N,N-diethylphenylacetamide on oral exposure in rats. Toxicol. Lett., in press. T. Hatch and S.P. Choate, Statistical description of the size properties of non-uniform particulate substances. J. Franklin Inst., 207 (1929) 369. D.J. Finney, Probit Analysis, Cambridge University Press, London, 1977. S. Reitman and S. Frankel, A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol., 28 (1957) 56. O.A. Bessey, O.H. Lowry and M.J. Brock, A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J. Biol. Chem., 164 (1946) 321. D.R. Wybenga, J.D. Giorgio and V.J. Pileggi, Manual and automated methods for urea nitrogen measurement in whole serum. Clin. Chem., 17 (1971) 891. D. Watson, A simple method for the determination of serum cholesterol, Clin. Chim. Acta, 5 (1960) 637. O.G. Raabe, H. Yeh, G.J. Newton, R.F. Phalen and D.J. Valasquez, Deposition of inhaled monodisperse aerosols in small rodents, in Inhaled Particles IV, Pergamon Press, New York, 1976. T.R. Lewis, P.E. Marrow, R.O. McClellan, O.G. Raabe, G.L. Kennedy, B.A. Schwetz, T.J. Goehl, J.H. Roycroft and R.S. Chhabra, Contemporary issues in toxicology; Establishing aerosol exposure concentration for inhalation toxicity studies. Toxicol. Appl. Pharmacol., 99 (1989) 377. H.N. MacFarland, Designs and operational characteristics of inhalation exposure equipment. A review. Fundam. Appl. Toxicol., 3 (1983) 603. R.O. McClellan, B.B. Boeeker and J.A. Lopes, Inhalation toxicology. Considerations in the design and operation of laboratories, in A.S. Tegeris and Md. Laurel (Eds), Toxicology Laboratory Design and Management for the 80's and Beyond, S. Karger, Basel, 1984, p. 170. N.I. Sax, Dangerous Properties of Industrial materials, Von Nostrand Co., New York, 1985. R.J. Lewis Sr and R.L. Tatken, Registry of Toxic Effects of Chemical Substances, US. Dept. Health Human Services, N1OH, Cincinnati, OH, Vol. l, 1979, p. 808. J.A. Macko and J.D. Bergmann, Inhalation Toxicities of N,N-diethyl-m-toluamide, US. Army Environ. Hyg. Agency, Study No. 75-51-0034-80, 1979. C.D. Klassen, Principles of toxicology, In: A. Goodman Gilman, T.W. Rail, A.S. Nies and P. Taylor (Eds.), The Pharmacological Basis of Therapeutics, 8th Edn., Pergamon Press, New York, 1990, p. 49. G.A. Konovalov and A.N. Romanov, The early haemodialysis in the treatment of intoxication with repellents DETA, benphthalate and dimethylphthalate (Russian), Anest. Reanim., 2 (1980) 54. S.S. Rao, R.V. Swamy, C.D. Raguveeran, M.K. Agrawal, S. Prakash, K.M. Rao and P.K. Ramachandran, Recent work on insect repellent N,N-diethylphenylacetamide: insect and mammalian toxicity and metabolic aspects, In: Entomology for Defence Services, symposium proceedings, Defence R&D Establishment, Gwalior, In press. S. El Defrawy El Masry and G.J. Mannering, Sex dependent differrences in drug metabolism in the rat. Drug Metab. Dispos., 2 (1974) 279.
Acute and subacute inhalation toxicity studies of a new broad spectrum insect repellent, N, N-diethylphenylacetamide R. Vijayaraghavan*, S.S. Rao, M.V.S. Suryanarayana and R.V. Swamy Defence Research and Development Establishment, Gwalior - - 474 002 (India) (Received August 23rd, 1990; accepted January 8th, 1991)
Summary N,N-Diethylphenylacetamide (DEPA) is an inexpensive, long-acting and broad spectrum insect repellent. The acute LCs0 for a 4-h exposure of DEPA aerosol was found to be 1.451 mg 1-l (1.290--1.633) in male and !.375 mg ! -I (1.307--1.447) in female rats. DEPA did not cause delayed deaths. Acute exposure to 0.9 LCs0 revealed that liver might be a target organ for DEPA toxicity. On subacute exposures to 0.2, 0.6 and 0.8 LCs0 for 6 h per day, 5 days a week for 2 weeks, there was no significant change in the 0.2 LCs0 group, as evaluated by the body weight gain and organ body weight ratio. The minimal changes observed in the 0.6 LCs0 group were of reversible type as the animals recovered on cessation of exposure. A massive concentration of 0.8 LCs0 produced lethal effects. The study shows that DEPA has a low mammalian toxicity by inhalation as was found earlier with cutaneous application of the insect repellent.
Key words: N,N-diethylphenylacetamide; Insect repellent; Inhalation; Toxicity
Introduction N,N-Diethylphenylacetamide (DEPA) is a new inexpensive and long-acting insect repellent [1--3]. Extensive field trials indicate that DEPA is as potent as N,Ndiethyl-m-toluamide (Deet) and has a broad spectrum of repellent action [4]. DEPA and its formulations are reported to repel mosquitoes [2,4 6], black flies [4], biting flies [7], cockroaches [8] and land leeches [4]. Acute and subacute dermal and oral toxicity of DEPA has been thoroughly studied and it is reported to be safe [9--11 ]. Low dose of DEPA applied dermally did not produce any adverse effect on foetus and reproduction [ 12]. DEPA is found *Address all correspondence to." R. Vijayaraghavan, Visiting Research Associate, Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, U.S.A. 0300-483X/9 !/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
85
nonmutagenic by the Ames test and by the mouse bone marrow micronucleus test [ 13 ]. DEPA is excreted partly unchanged and partly as N-ethylphenylacetamide, phenylacetamide and as conjugated phenylacetic acid [ 11,14---16]. In guinea pigs which mainly excrete DEPA in urine, only 24% of the dermally applied 14C-labelled DEPA was recovered in urine [9] showing that a major portion might have evaporated. Since mosquitoe repellents can also be used as pressurised sprays, data on inhalation toxicity are also required. Since inhalation toxicity of DEPA has not been reported so far, the present investigation has been carried out. Materials and methods Chemicals All the chemicals used for the biochemical studies were of analytical grade (BDH or E. Merck, Bombay, India). Ethylacetate used was of chromatography grade. DEPA was synthesised in our chemistry division [4], characterised spectroscopically [5] and found to be over 99% pure by gas chromatography. Animals Albino rats (Wistar strain), both male and female, weighing 70--100 g (initial weight), were used for the study. They were housed in polypropylene cages, four per cage, with dust-free rice husk as the bedding material. The rice husk was changed every other day. The animals were given pellet diet (Lipton feed, Bombay, India) and water ad libitum except before the determination of LCs0 when they were fasted overnight with only water ad libitum. Inhalation exposure DEPA aerosol was generated using a glass air blast nebuliser, diluted with filtered air in a glass helical assembly, and passed into the animal exposure chamber (Fig. 1). The specially designed leakproof exposure chamber, made of stainless steel with a glass door was of 50 1capacity. The chamber was equipped with ports for monitoring temperature, humidity, pressure and also for sampling chamber air for analysis of particle size and concentration of the test chemical. A small electric bulb (25 W) was fixed at the top for illuminating the chamber. The animals were exposed in small stainless steel wire cages, one per cage inside the chamer (whole body exposure). The chamber was operated dynamically and the exit aerosol was passed through a series of decontamination units to a suction pump and out of the room. The suction pump was so adjusted using a regulator, that the pressure inside the chamber was kept at a pressure of 1 cm H20 below the atmospheric pressure. The calculated t99 i.e. the time required to reach the theoretical concentration, was 8--10 min. The total air passed through the chamber was between 25--35 1 min -I and the animal loading complement (total animal volume x 100/capacity of the exposure chamber) was less than 2.0%. The inhalation chamber temperature was 25--28°C and the relative humidity was 40---60%. Particle size analysis The particle size analysis was carried out using an optical particle counter (5 chan-
86
HEPAFilter
Pump
Regulator
~F1ow
O
Filtered air supply Pump Exhau~ ~
~(
meter
Exposure Chamber
Regulator
DecoDtsm4 na.t/ou ~ t s
~(
Fig. I. Schematicdiagram of dynamicexposure assembly. nel, Royco Particle Monitor, Royco lnc, CA, U.S.A.). Count median diameter (CMD) and geometric standard deviation (a g) were calculated using a 2 cycle logarithmic probability paper. Mass median diameter (MMD) was calculated using the Hatch-Choate quation [ 17] and the aerodynamic median diameter (AMD) was calculated, by multiplying the M M D with the square root of the density of DEPA. Concentration determination The theoretical or nominal concentration was determined from the rate of nebulisation and the total air flow (nebuliser air flow + dilution air flow). The actual concentration of DEPA was determined by gravimetric analysis using 0.2 #m rating polytetrafluorethylene (PTFE) coated Whatman membrane filters. In a second method, 500 ml of the chamber air containing DEPA was trapped with the help of a 50 ml gas tight syringe (10 strokes) in a high-efficiency glass impinger containing ethylacetate. Later, a 0.5 #l aliquot was injected in a Aimil-Nucon gas chromatograph (Nucon Engineers, New Delhi, India) with the following conditions: column - - 15% diethylene glycol succinate (DEGS) on chromosorb W (high performance); detector - - flame ionisation detector; temperature - - oven: 220°C; injector - - 250°C and detector - - 250°C. The retention time of DEPA was found to be 6'42". Standard DEPA solutions were prepared by dissolving known amounts of DEPA in ethylacetate. A 0.5/~! aliquot was injected and a standard curve was plotted. Using the standard curve, the concentration of DEPA in the exposure chamber was calculated.
87
Acute toxicity studies Various concentrations of DEPA aerosols were generated and the animals were exposed for 4 h for the calculation of LCs0. The chamber temperature, humidity, particle size and the actual concentration were monitored periodically. The animals were observed for mortality up to 14 days. The LCs0 was calculated for male and female rats separately by probit analysis [ 18]. A high concentration (0.9 LCs0) of DEPA was also generated and male rats were exposed for 4 h, for identifying the target organs of DEPA toxicity.
Subacute toxicity studies After calculating the LCs0 for 4 h exposure, the LCs0 for 6 h exposure was calculated mathematically. For subacute exposures the three concentrations used were, 0.2 LCs0 (12 male and 12 female), 0.6 LCs0 (12 male and 12 female) and 0.8 LCs0 (6 male and 6 female). Male and female rats were exposed separately for 6 h per day, 5 days a week for 2 weeks. Twenty-four hours after the last exposure (13th day), 50% of the animals of the 0.2 LCs0 and 0.6 LCs0 groups were sacrificed and the remaining were allowed to recover for a further 15 days and then sacrificed (27th day). In the 0.8 LCs0 group, all surviving animals were sacrificed 24 h after the last exposure (13th day). Temperature, humidity, particle size and the concentration were properly maintained.
Body weight change During the subacute exposures the animals were weighed daily before the exposure. The body weight of the individual animal was converted to percent change of the pre-exposure body weight.
Sample collection The animals were lightly anaesthetised with ether and maximum blood was drawn using heparinised capillary from the retro-orbital plexus. They were sacrificed by cervical dislocation and important organs viz., lung, liver, kidney, heart, spleen and adrenal were dissected out. The organs were weighed after freeing from blood and adhering tissues.
Organ body weight index The individual weights of various organs were converted to percent of body weight and expressed as organ body weight index (OBWI).
Biochemical analysis Plasma was used for the estimation of glutamate pyruvate transaminase (GPT) [19], alkaline phosphatase (ALP) [20], urea [21] and cholesterol [22] after 0.9 LCs0 acute exposure and 0.6 LCs0 subacute exposure.
Statistical analysis The data were analysed by Student's t-test for statistical significance.
88
Results
Particle size and concentration For the generation of aerosols, undiluted DEPA was used. In order not to alter the t99 and the ventilation of the exposure chamber, various concentrations of DEPA aerosols were generated by adjusting the pressure of the air feed to the nebuliser. Table I shows the correlation coefficients, CMD, MMD and AMD of DEPA aerosols calculated using the Royco Particle Counter. A good correlation (r = 0.993) was obtained between the AMD and the pressure. Since the particle size was varying, 1.00---1.625 kg cm -2 pressures were used for generation of DEPA aerosols and then diluted to get the desired concentration. The particle size was also checked using an Andersen Sampler (Andersen Inc, Atlanta, U.S.A.) at 1.5 kg cm -2 pressure and the AMD was found to be 1.8/~m. The concentration was determined from the breathing zone of the animal. Using the gravimetric method, the actual concentration was found to be 55.3 -4- 1.7% of the nominal concentration and by the gas chromatographic analysis the recovery was 57.4 q- 1.4%. For all calculations the actual concentration determined by gas chromatographic method was used. Acute toxicity studies The LCs0 was calculated by probit analysis for a period of 4 h exposure in male and female rats separately using various concentrations (Table II). During the course of the exposure hyperaemia of paws, nasal and periorbital areas was observed. The animals generally died during the exposure or within 60 min after the exposure and thereafter no death was observed during the 14-day observation period.
TABLE 1 COUNT MEDIAN DIAMETER (CMD), MASS MEDIAN DIAMETER AERODYNAMIC MEDIAN DIAMETER (AMD) OF DEPA AEROSOLS PRESSURES S. no.
Pressure (kg cm -2)
r
50th* CMD (~m)
84th*
Geometric standard deviation
(MMD) AND AT VARIOUS
MMD (t~m)
AMD (#m)
0.472 0.581 0.850 1.027 1.206 1.568 1.678
0.474 0.584 0.854 1.032 1.212 1.576 1.686
(o g) I 2 3 4 5 6 7
0.25 0.50 0.75 1.00 1.25 1.50 1.75
0.980 0.971 0.948 0.936 0.927 0.919 0.923
0.410 0.535 0.807 0.980 1.141 1.448 1.530
0.809 0.631 0.920 1.110 1.307 1.705 1.823
1.241 1.175 1.140 1.133 1.145 1.177 1.192
*Percentile.
89
TABLE 11 LC50 OF DEPA AEROSOLS IN RATS EXPOSED FOR 4 h Sex
LCso (mg 1-I)
95% confidence limit
LCt50 (mg • rain per m3)
Male Female Combined
1.451 1.375 1.390
1.290---1.633 1.307--1,447 1.267--1.525
348 240 330 000 333 600
Exposure to a massive concentration o f 0.9 LCso (1.306 mg 1-I) of DEPA for 4 h was carried out on a group o f six male rats. No animal died during the exposure but one animal was critical. Following this exposure there was a significant reduction of OBWI of lung and spleen and an increase in liver and adrenal. The relative weight o f kidney and heart were not significantly affected (Table III). Biochemical changes following acute exposure are shown in Table IV. There was a significant increase in GPT, ALP and cholesterol, but there was no change in urea.
Subacute toxicity studies The coefficient of variation of the exposure concentrations, within-day and between-day were less than 10%. Following subacute exposure to 0.2 LCs0 and 0.6 LC50 there was no mortality. In the 0.8 LCs0 group, out of the six males exposed, one died after the second exposure and the remaining five survived the subsequent exposures. However, in the females, following 0.8 LCs0 exposure, three died after the second exposure and two after the third exposure. The remaining one survived for the rest of the exposures. The body weight changes (percent changes from initial values) of male and female rats exposed to DEPA aerosols are shown in Fig. 2 and Fig. 3, respectively. There was no significant difference in the body weight gain o f control and 0.2 LC50 groups of male and female rats. Following exposure to 0.6 LC50, the body weight gain was significantly reduced but the slope of the curve was only slightly less than the control. In the 0.8 LCs0 group particularly, the lone survived female rat showed minimal weight gain. The OBWI of male and female rats following subacute exposure is also shown in Table III. The 0.2 LCs0 group did not show any significant difference from the control. A significant increase in the liver weight was observed both in male and in female exposed to 0.6 LCs0 and 0.8 LCs0. The liver weight was normalised in the 0.6 LCs0 group which was allowed to recover. A significant increase in the kidney weight was also observed following exposure to 0.6 LCs0. The other groups also showed a tendency of increased kidney weight. Normalisation occurred in those animals which were allowed to recover. Plasma GPT, cholesterol and urea of male rats exposed to 0.6 LCs0 did not show any significant change when compared to control.
90
0.705 ± 0.023
± 44±
0.139 0.097* 0.146 b 0.094**
4.612 ± 0.213 6.214 ± 0.068** 3.868 ± 0.155 c 5.976
4.546 ± 0.345
4.539 5.142 4.446 6.934
5.162 4- 0.250*
4.374 4- 0.201
Liver
± 44±
0.021 0.019"* 0.024 b 0.018
0.851 ± 0.027 0.919 ± 0.019 0.761 4- 0.012 0.906
0.823 4- 0.047
0.787 0.987 0.838 0.862
0.760 4- 0.019
0.797 4- 0.023
Kidney
44± 4-
0.014 0.028 0.009 0.008
0.377 + 0.010 0.386 4- 0.008 0.363 4- 0.009 0.391
0.379 4- 0.011
0.372 0.379 0.375 0.387
0.361 4- 0.010
0.366 4- 0.010
Heart
Mean ± S.E. of six animals (male 0.8 LCso, five animals and female 0.8 LCso; one animal only. Statistically significant from control vs. 0.2 LCso, 0.6 LCso or 0.8 LCso; *P < 0.05; **P < 0.001. Statistically significant from 0.6 LCso vs. recovery; ap < 0.05; bp < 0.01; cp < 0.001.
Subacute exposure 0.2 LC5o 0.679 ± 0.029 0.6 LC5o 0.680 a- 0.045 Recovery 0.571 4- 0.026 a 0.8 LC5o 0.639
Female rat Control
0.017 0.037 0.020 b 0.018
0.542 ± 0.011**
Acute exposure 0.9 LC5o
± 444-
0.697 4- 0.019
Male r l t Control
Subacute exposure 0.2 LC50 0.687 0.6 LC5o 0.778 Recovery 0.573 0.8 LC5o 0.642
Lung
Group
4± 44-
0.051 0.022 0.061 0.093
0.802 ± 0.063 0.977 ± 0.042 0.716 4- 0.012 0.601
0.849 4- 0.116
0.778 0.821 0.799 0.585
0.442 4- 0.024**
0.755 4- 0.044
Spleen
4± 44-
0.001 0.001 0.001 0.002
0.027 ± 0.001 0.026 4- 0.001 0.031 4- 0.002 0.038
0.028 ± 0.002
0.024 0.025 0.025 0.027
0.040 4- 0.004**
0.023 ± 0.001
Adrenal
CHANGES IN O R G A N BODY W E I G H T I N D E X OF RATS F O L L O W I N G ACUTE A N D SUBACUTE EXPOSURE TO DEPA AEROSOLS
TABLE 111
T A B L E IV C H A N G E S IN B L O O D P A R A M E T E R S F O L L O W I N G A C U T E A N D S U B A C U T E E X P O S U R E TO DEPA A E R O S O L S IN M A L E R A T S Group
Glutamate pyruvate transaminase (I.U. 1-1)
Alkaline phosphatase (1.U. I -I)
Urea (mg d1-1)
Cholesterol (mg dl -])
Control
12.1 4- 1.1
226.7 4- 27.7
46.7 4- 3.2
96.3 4- 3.5
Acute exposure 0.9 LCs0
33.3 4- 10.1"
370.8 4- 35.9*
46.0 4- 3.1
120.4 4- 6.1"
Subacute exposure 0.6 LC.so
10.2 4- 0.9
--
38.6 4- 2.5
103.6 4- 4.1
Mean 4- S.E. of six animals. *Statistically significant from control, P < 0.05.
DEPA 8ubacute Exposure (Male Rat) 226
200Q d Y
w 176I
~18oe
T 126"
o
100 r
T 76 001 0
I
I
I
I
I
8
10
16 Days
20
28
30
Fig. 2. Body weight changes during and after exposure to DEPA aerosols in male rats.., control; +, 0.2 LCs0; *, 0.6 LCs0; x, 0.8 LCs0.
92
DEPA 8ubacute Exposure (Female Rat) 226 S o d 200y w
• 176I
I
t 100"
• 128 I
f • 100 o n t
r I
O
78
6 0
0 8
~ 10
16
20
26
30
Days Fig. 3. Body weight changes during and after exposure to DEPA aerosols in female rats.., control; +, 0.2 LCs0; *, 0.6 LCs0; x, 0.8 LCs0.
Discussion
In the present study, the generated particles were well within the respirable range recommended for the rodent species [23,24]. All the other parameters required for the inhalation exposure were also well within the recommended standards [25,26]. The acute LCs0 for 4 h exposure was found to be 1.451 mg 1-l (348 240 mg • min per m 3) in male rats and 1.375 mg 1 -] (330 000 mg- min per m 3) in female rats. LCs0 for 4 h exposure of N,N-diethyl-m-toluamide (Deet) and dimethylphthalate (DMP) are more than 5.0 mg 1-1 [27--29] showing that acute toxicity of DEPA is more than that of Deet or DMP. The dose mortality curve of DEPA inhalation is very steep. This may indicate that even at high sublethal doses mortality will be minimal and, hence, greater margin of safety of the compound [ 30]. It is possible that DEPA does not have any specific target for toxic effects and in lethal concentrations, the death may be due to a general failure of all the systems. During acute exposures to high doses, animals generally died during the exposure and there were no delayed deaths. Deet is reported to cause delayed deaths [29]. A possible explanation of the differential lethality may be that DEPA is metabolised quickly and eliminated [ 11,14,15] and does not produce any residual effects.
93
Exposure to massive concentrations of DEPA caused excessive salivation, hyperaemia of paws, nasal and periorbital areas, tremors and convulsions. Similar findings were reported when massive doses of DEPA were administered orally [9,16]. Exposure to massive concentrations revealed that the liver is affected. Lung and kidney are only slightly affected. Rao et al. [9] also reported that administration of 2.0 LDs0 of DEPA orally damaged the liver. Toxic hepatitis was reported with other insect repellents like Deet and DMP [31 ]. Increase of plasma GPT and ALP also revealed that the liver is affected while the kidney is not, since there was no significant change in plasma urea level. Short-term subacute toxicity studies were carried out to assess the damages caused by DEPA in sublethal doses. The animals were exposed to 0.2 LCs0, 0.6 LCs0 and 0.8 LCs0 (for a 6-h period) for 5 days a week for 2 weeks. Exposures to high concentrations were also used as low doses of 1/12 LDs0 were reported to cause negligible effects even if DEPA was applied dermally or given orally for 3 ~ 4 months [9,101. Lethality was not observed in the animals exposed up to 0.6 LCs0 subacutely and even in 0.8 LCs0 the deaths were observed only up to 3 days. This may perhaps indicate that resistance to the toxic effects of DEPA might have developed by continuous exposure to the chemical. Rao et al. [32] recently observed that on daily dermal application of DEPA in rats for 7 days in different doses, the proportion of N-ethylphenylacetamide and phenylacetamide in relation to the unmetabolised DEPA excreted in urine was increased, indirectly indicating enhancement of drug metabolising enzyme activity of the animals. The body weight gains of the animals exposed to 0.2 LCs0 in both sexes did not show any significant difference from those of the controls. The body weights of animals exposed to 0.6 and 0.8 LCs0 were significantly lower than the control. The pattern of growth was uniform during the exposure period and also during the recovery period of the animals exposed to 0.2 LCs0 and 0.6 LCs0. Only in the 0.8 LCs0 group both in males and females, there was a fall in the body weight up to 2 days post exposure, thereafter it started to increase. The sudden fall in the weight following exposure might be due to stress. Rao et al. [ 10] also reported a fall in the body weight of male rats given DEPA orally at 2/3 LDs0 dose for 7 days. A sex difference is also observed in the toxicity of DEPA. The LCs0 was lower in female than in male. This finding is in agreement with the cutaneous LDs0 of DEPA in rats [ 11 ]. Metabolic capacity of microsomes of female rats is lower as compared to males for dealkylation [ 33 ]. This may perhaps be the reason for the greater toxicity of DEPA in females compared to males by inhalation exposure. Increased OBWI of liver and kidney in both male and female rats exposed to 0.6 LCs0 and 0.8 LCs0 has been a constant finding. The organ weights were normalised in the animals that were allowed to recover. Increased liver weight was also reported in male and female rats given DEPA orally at 2/3 LDs0 for 7 days [ 10]. Macko and Bergmann [29] in their studies also reported an increased liver weight in both male and female rats given various doses of Deet. Plasma cholesterol, GPT, and urea of male rats exposed subacutely to 0.6 LCs0 of DEPA did not show any significant change, even though isolated changes were observed in rats administered DEPA, percutaneously or orally [ 10,12 ]. Exposure to 94
subacute dose of 0.2 LCs0 does not cause any significant alterations and the changes recorded following subacute exposure to 0.6 LCs0 seem to be of a reversible type. The study shows that DEPA has a low mammalian toxicity by the inhalation route as was found earlier with cutaneous application of the insect repellent [ll,14].
Acknowledgements The authors are grateful to Brig (Dr.) KM Rao, Director, Defence R&D Establishment, for the encouragement and to Dr. P.K. Ramachandran, Emeritus Scientist for the valuable suggestions and for critically reviewing the manuscript.
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