Toxicity of carbamates for mammals

Bull. Org. mond. Sante 1971, 44, 241-249 Blull. Wld Hlth Org. J Toxicity of Carbamates for Mammals M. VANDEKAR,' R. PLESTINA2 & K. WILHELM 2 Toxicity studies have been carried out with a number of monomethylcarbamates, most of which reached an advanced stage in the World Health Organization insecticide evalua- tion programme. Both quantitative and qualitative distinctions have been found between the carbamates studied, and certain common characteristics that distinguish them in several important aspects from organophosphorus insecticides have been demonstrated. During the last 10 years the toxic properties of carbamates have been studied at the Institute for Medical Research, Zagreb, Yugoslavia.3 In vitro and in vivo studies have been carried out and, in some instances, comparisons with organophosphorus com- pounds have been performed. This paper deals with monomethylcarbamates that entered the WHO insecticide evaluation programme (Table 1) and outlines the results of determinations of acute toxicity, of persistence in the blood, and of the effects of different rates of infusion into the jugular or portal veins of rats. A particular objective of the latter studies was to determine the doses necessary to produce a given symptom (and/or cholinesterase depression) or death. A brief account is also given of studies carried out in volunteers to develop a suitable method for measuring exposure to propoxur. STUDIES IN EXPERIMENTAL ANIMALS Relationship between acute toxicity and anticholin- esterase activity Male rats were usually used for the determination of LD50 values, except for propoxur and m-isopropyl- phenyl methylcarbamate, whose toxicity was deter- mined in female rats. No significant differences in LD50 values between the two sexes were found. Three routes of administration were employed; the results are summarized in Table 1. In addition to typical symptoms of anticholin- esterase poisoning, five carbamates (carbaryl, m-iso- 1 Scientist/Toxicologist, Vector Biology and Control, World Health Organization, Geneva, Switzerland. ' Institute for Medical Research, Yugoslav Academy of Sciences and Arts, Zagreb, Yugoslavia. aThese investigations have been supported in part by research grants from the World Health Organization. propylphenyl methylcarbamate, phenyl methylcar- bamate, 3,5-diisopropylphenyl methylcarbamate, and Landrin f) produced a transient but very pronounced anaesthetic effect immediately following the intra- venous administration of a dose near the LD50 (Wilhelm & Vandekar, 1966). Lethal doses caused deep anaesthesia with severe dyspnoea, and respira- tion ceased within a few minutes. If artificial respira- tion was applied for 2-5 minutes within the first minute of apnoea spontaneous respiration resumed and the animals slowly recovered from anaesthesia and cholinergic symptoms gradually developed. Thus, the pronounced anaesthetic effect with respiratory failure was the most critical sign determining the intravenous toxicity of these five compounds. A simi- lar but much less pronounced anaesthetic effect was. also produced by the intraperitoneal injection of four carbamates (carbaryl, m-isopropylphenyl methylcar- bamate, phenyl methylcarbamate, and 3,5-diisopro- pylphenyl methylcarbamate). However, no such effect was observed when these compounds were admin- istered orally. It should be noted that the narcotic effects de- scribed above were produced only by carbamates of relatively low toxicity and closely resembled those caused by certain organophosphorus compounds of low toxicity (Vandekar, 1957). The mechanism of action is not certain, but Heath (1961) put forward the reasonable hypothesis that, since these effects. are marked only after injection into the blood stream, the concentrations produced are sufficiently high to block nerve conduction and motor end- plates, the narcotic block of conduction being attri- buted to a block in sodium-ion transport across the axon membrane. t Names against which this symbol oppears are identi- fied in the Glossary on pages 445-446. 2634 - 241 M. VANDEKAR, R. PLESTINA & K. WILHELM Table 1 Acute toxicity of monomethylcarbamates and their inhibition of AChE Methylcarbamate 1 -naphthyl o-isopropylphenyl o-isopropoxyphenyl m-isopropylphenyl 2-chloro-4,5-dimethylphenyl phenyl 3,5-diisopropylphenyl 3,4,5-trimethylphenyl 4- benzothienyl m-cym-5-yl Common _ or proprietary name carbaryl propoxur carbanolate Landrin Mobam promecarb Male rats Intravenous Intravenous Intraperitoneal LDso EDso LDso (mg/kg) (mg/kg) (mg/kg) 41.9 a 66.0 a 10.6 3.15 3.00 13.6 a 29.7 a 31.8 a 24.8 5.30 1.42 0.334 0.168 0.144 1.5 0.707 0.189 200 b 142 b 30.0 14.2 11.2 357 b 267 b 94.4 40.8 27.2 Oral LD50 (mg/kg) 250 374 c 100 39.2 c 60.0 c 540 c 1 000 C 236 c 336 125 Bovine erythrocyte AChE 150 (M) 1.5 x 10-6 1.2 x 10-6 7.0 x 10-7 5.6 x 10-8 7.3 x 10-8 8.8 x 10-5 1.9 x 10-7 9.0 x 10-7 8.8 x 10-8 a Pronounced anaesthetic effect observed. b Slight anaesthetic effect observed. c Data from the Medical Research Council Laboratories, Carshalton, England. Simeon (1966) compared the acute toxicity for rats of 9 monomethyl carbamates, administered in a single intravenous injection, with their anticholin- esterase activity determined on purified bovine ery- throcyte cholinesterase (see Table 1), the enzyme being incubated with a given concentration of inhi- bitor for 30 minutes at 25°C and its activity sub- sequently measured titrigraphically at the same tem- perature and at pH 7.4. Good correlation was found between the 1,s and the intravenous LD50 values, the correlation coefficient being 0.89. Such a degree of correlation should be regarded as par- ticularly high in view of the small number of com- pounds compared and the relatively narrow range of their Is0 values (8.8 x 10-5 to 5.6 X 10-8 M) and LDte values (3.0-136.0 mg/kg) and in view of the fact that in some instances the correlation was likely to be vitiated to a certain extent by the presence of the narcotic effect described above. These results indicate that, unlike their (nonsynergized) insecti- cidal activity (Fukuto et al., 1962), the acute toxicity of carbamates for rats follows their anticholin- esterase activity. In mammals, the rate of meta- bolism of this type of carbamate is, apparently, not a factor in determining their LD50 values, and their toxicity can therefore be predicted from the degree to which they inhibit cholinesterase in vitro. Symptoms and persistence of inhibitor in the body A straight comparison of the type and duration of symptoms was carried out with 15 monomethyl- carbamates by injecting groups of4 rats intravenously with a single i.v.-LD50 dose (Wilhelm & Vandekar, 1966; Wilhelm, unpublished data). The intensity of different cholinergic symptoms varied consider- ably from one compound to the other, indicating marked differences in their distribution in the body following intravenous injection. At the same time, differences in the duration of cholinergic symptoms were recorded: with some compounds symptoms ceased within 45 minutes, while with others they lasted for more than 2-3 hours. These differences led the authors to study the persistence of the inhibitor in the body. This was carried out by determining the anticholinesterase activity of rat serum at given intervals after the intravenous injec- tion of a single sublethal dose of carbamate. A pro- cedure analogous to that used in the study of some dimethyl phosphate esters was employed (Vandekar & Heath, 1957), the anticholinesterase activity of rat serum being estimated on human erythrocyte ChE in vitro. It has been found that the carbamates inducing symptoms of short duration (e.g., propoxur, m-isopropylphenyl methylcarbamate, carbanolate, and promecarb) persisted in the blood for only 1-2 242 TOXICITY OF CARBAMATES FOR MAMMALS hours, while those inducing prolonged symptoms (e.g., carbaryl, o-isopropylphenyl methylcarbamate, phenyl methylcarbamate, and Landrin t) persisted in the blood for more than 6 hours. Similar results were obtained when carbamates were administered orally in equitoxic doses (Wilhelm, unpublished data): compounds inducing symp- toms of prolonged duration (4-5 hours), such as carbaryl, persisted in the blood for a considerably longer period than did those inducing symptoms of shorter duration (about 2 hours), such as propoxur. As described below, it was possible, by using a similar method in studies on human volunteers, to determine the presence of free inhibitor in the blood for several hours after the ingestion of rela- tively small doses of carbamate. Relationship between symptoms and cholinesterase activity In a series of experiments, brain and plasma ChE activity was determined in rats to which propoxur was administered intramuscularly at different dos- ages, the animals being sacrificed at the moment the first symptoms appeared or at a given time after injection (Plestina & Vandekar, 1966). At dosages that did not produce any noticeable symptoms (0.25-1.0 mg/kg), the activity of both brain and plasma ChE was reduced by varying amounts, down to about 60% of the normal level. The dose at which a very slight tremor occurred (2.0 mg/kg) reduced the brain and plasma ChE activities to 470 and 49% of the normal levels, respectively, the animals being sacri- ficed immediately after this sign had been observed. At higher dosages (10.0 and 50.0 mg/kg) the degree of inhibition of both brain and plasma cholinesterase closely followed the severity of the symptoms that were produced, the brain ChE usually showing 5-15% greater inhibition than the plasma ChE. In another series of experiments the relationship between the brain and plasma cholinesterase activi- ties and the degree of symptoms was studied during the infusion of propoxur into the jugular vein (Plestina, unpublished data). Different rates of infusion, ranging from 0.04 to 0.80 i.v.-LD50/hour, were used. At given time intervals the animals were sacrificed and their brain and plasma ChE levels were determined spectrophotometrically (Ellman et al., 1961), the analytical procedure being com- pleted within 12 minutes after the animals had been decapitated. Three main symptoms (tremor, muscle fasciculations, and salivation) were regularly recorded and were arbitrarily classified according to 5 degrees of intensity. The mean brain and plasma ChE activi- ties in 4-5 animals, expressed as percentages of the mean levels in 40 normal animals, are shown in Fig. 1. These results show good correlation between the activities of the two enzymes and good agreement between the degree of enzyme depression and the Fig. 1 Effect of propoxur on cholinesterase activity in rats * 1--I- w ) DURATION OF INFUSION (min). * brain cholinesterase O plasma cholinesterase * Propoxur was infused into the jugular vein at rates of 0.04, 0.08, 0.20, 0.40, and 0.80 i.v.-LDso/h. Each point represents the mean of the levels in 4-5 animals. 243 M. VANDEKAR, R. PLESTINA & K. WILHELM intensity of symptoms, the onset of symptoms being recorded only after the brain ChE activity dropped to about 50% of normal. It may be noted that during the second hour of infusion a steady state was reached so far as enzyme inhibition and severity of symptoms were concerned. Ratio between LD50 and ED50 values It has been shown from the kinetics of the inhi- bition of cholinesterase by monomethylcarbamates and organophosphorus compounds that the LD50 is likely to be a much greater multiple of the dose causing signs of poisoning for the carbamates than for the organophosphates. The expected difference was demonstrated by a comparison of LD50 and ED5, values, intravenous and intramuscular, of a number of monomethylcarbamates and two organophos- phorus compounds (Vandekar et al., 1965). The slightest evoked tremor was selected as the most reliable sign of poisoning in rats from which to estimate the ED50 values. Carbamates gave much greater LD5O/ED5o ratios (20-35 for intravenous administration, cf. Table 1) than the two organo- phosphorus compounds studied, paraoxon and its dimethyl homologue, which gave the ratios 4.2 and 2.5, respectively. Similarly, in experiments per- formed on dogs with m-isopropylphenyl methylcar- bamate and later with propoxur and promecarb (Svetlicic, personal communication) a high LD50/ ED50 ratio was observed, the initial symptoms including both tremor and increased salivation. Goldberg et al. (1963) have reported for m-iso- propylphenyl methylcarbamate a similar (25: 1) ratio between the intraperitoneal LD50 and the dose causing behavioural changes in 50% of a group of specially trained rats. Tolerated dose at different rates of infusioni into general and portal circulation Proceeding along the lines described above, the tolerated dose of carbamates at different rates of infu- sion was compared with that of organophosphorus compounds. Thus, it was shown that slowing the rate of intravenous infusion of the three tested car- bamates (propoxur, promecarb, and m-isopropyl- phenyl methylcarbamate) led to a large increase in the doses producing death, animals reaching, in the course of infusion, a " stage of equilibrium " of symptom intensity, which lasted for 10 or more hours (Vandekar & Fajdetic, 1966). This is illus- trated in Fig. 2, in which results obtained with propoxur are compared with those obtained with two organophosphorus compounds, paraoxon and Fig. 2 Doses of propoxur, paraoxon, and the dimethyl homologue of paraoxon causing death of rats * 24r 22 20 19 -16 -J ..14 12 w 10 6 4 2 o 0 C 5 0 0 0 0 . 0 0 0 0 'B.0 o0 1 2 3 4 5 6 7 RATE OF INFUSION (LD50/h) propoxur dimethyl homologue of paraoxon paraoxon 8 * The compounds were infused into the jugular vein at different rates. Each point represents the mean of the levels observed in 6 animals. The levels are expressed in terms of i.v.-LD5o values, and all were determined in the same laboratory. its dimethyl homologue. While a large increase in the tolerance of propoxur was produced when the rate of infusion was reduced to less than 3 LD,0/hour, the rate of infusion of the dimethyl phosphate had to be reduced to less than 2.0 LD50/hour and that of the diethyl phosphate to less than 0.1 LD50/hour to produce an increase in the dose tolerated. It was possible to make a further distinction between carbamates and organophosphorus com- pounds in this type of experiment. Whereas a decrease in the rate of infusion of carbamates was accompanied by a remarkable increase in the lethal dose, no increase in the dose necessary to produce the first symptoms was recorded at the rates of infu- sion employed. Vandekar & Fajdetid (1966) showed, for a number of carbamates injected into the jugular vein of the rat, that the ratio between the doses producing death and those producing the first notice- 244 TOXICITY OF CARBAMATES FOR MAMMALS able symptoms (tremor or fasciculations) became larger as the rate of infusion was reduced, reaching values around 50. Similar experiments with paraoxon, in which the rates ofinfusion were 0.059-0.29 mg/kg/h, revealed constant ratios between the doses causing death and those producing the first symptoms, the first symptoms being observed only after about half of the lethal dose had been injected. Tolerance of the monomethylcarbamates was also studied at different rates of infusion into the portal circulation, and this was compared with the toler- ance observed when they were infused into the jugular vein (Plestina, unpublished data). The results obtained with propoxur and promecarb are shown in Fig. 3. It may be noted that the amount tolerated until the moment of appearance of the first cholinergic symptoms was approximately the IIA UC same for both compounds, regardless of the rate and route of infusion. The amount of carbamate required to kill the rats was dependent on both the rate and the route of infusion, larger amounts being tolerated by infusion via the portal vein, particularly at the lower rates of infusion. It should be pointed out, however, that much more pronounced differ- ences between the two routes of infusion were observed in similar studies with organophosphorus compounds belonging to the group of direct ChE inhibitors: the amounts tolerated via the portal vein were about five times those tolerated via the jugular vein. These results indicate that, with the carbamates studied, the role of the liver, while demonstrable, is relatively small and that the main factor contributing to the large increase in tolerance at lower rates of infusion is probably the rapid Fig. 3 Lethal and symptom-producing doses of propoxur (top) and promecarb (bottom) for the rat * - C -J%1; C]cK LL w H 4 CJ 0 0 0 6 - 4 2 E11F f - -r .26 '10 8 .2 3 1 2 3 4 6 8 10 12 RATE OF INFUSION (i.v.LD50 /h) * The black columns indicate doses producing first symptoms in, and the white columns doses causing death of, rats subjected to ntrajugular infusion. Diagonally shaded columns indicate doses producing first symptoms in, and stippled columns doses causing death of, rats subjected to intraportal infusion. Each dose represents the mean for 3-6 rats. 245 I I.I M. VANDEKAR, R. PLESTINA & K. WILHELM reactivation of the inhibited cholinesterase, which "competes " with the inhibition process during infusion. The fact that the dermal toxicity of a number of monomethylcarbamates has been shown to be markedly lower than their toxicity when administered by other routes (Gaines, 1960, 1969) can be attributed mainly to the same phenomenon. STUDIES IN VOLUNTEERS Useful as they are, experiments on laboratory animals are of limited use in evaluating methods for measuring exposure to a compound (or the extent to which it is absorbed), nor are they of value in certain other areas, such as clarifying the significance of blood cholinesterase levels in relation to different conditions of exposure to a given com- pound. Field studies on persons exposed to insectici- dal carbamates and studies in volunteers may provide the answer to such practical problems. Some studies in volunteers that have been carried out with the particular objective of evaluating methods for mea- suring blood cholinesterase levels in persons exposed to propoxur (Vandekar, unpublished report to WHO, 1966; Plestina, 1968; Plestina, unpublished data) will be briefly described. Propoxur (95 % pure, recrystallized before use) was given orally to healthy volunteers-investi- gators who were actively involved in the study- either in a single dose or in several doses over a certain period of time, usually 2 hours-the latter imitating to a certain extent the occupational expo- sure to insecticides. Different methods for cholin- esterase assay were compared; signs and symptoms, if any, were correlated to cholinesterase activity; the persistence of the inhibitor in the blood was studied; and the excretion of phenol derivatives in the urine was determined. Single dose A 42-year-old male volunteer (90 kg body weight) ingested 1.5 mg of propoxur per kg of body weight about 2 hours after his usual " Continental " break- fast. This dose was similar to that given to two male subjects by Dawson et al. (1964) in a study undertaken to develop a quantitative method for determining the metabolites of this insecticide ex- creted in urine. As shown in Fig. 4, a rapid fall in erythrocyte cholinesterase activity but no depres- sion in plasma cholinesterase was observed, both enzymes being determined spectrophotometrically (Ellman et al., 1961). This is consistent with the difference observed in the affinity of propoxur for Fig. 4 Effect of propoxur on cholinesterase levels in man * 0-4 1-- 2: I.- u 4 --- PLASMA ChE -0- ERYTHROCYTE ChE * The cholinesterase levels were determined in a single subject following the administration of 1.5 mg of propoxur per kg of body weight. the two enzymes in vitro, the 10 values for erythro- cyte and plasma cholinesterase being 4.6 x 10-' M and 2.3 x 10-5 M, respectively (Wilhelm, 1967). The lowest erythrocyte cholinesterase level (27.0 %Y of normal) was observed 15 minutes after ingestion. No signs were observed at that time, but moderate discomfort, described as " pressure in the head ", was present. Blurred vision and nausea developed 3 minutes later, and 20 minutes after ingestion the subject was pale and his face was sweating; his pulse rate was 140/min (before ingestion it was 76) and his blood pressure was 175/95 mm Hg (before ingestion it was 135/90). Within the next 10 minutes pronounced nausea, with repeated vomiting and profuse sweating, developed. These symptoms lasted, with no change in intensity, from about the 30th until about the 45th minute; during this period erythro- cyte cholinesterase activity recovered from a level of 50.4% to one of 55.5 % of its normal value. One hour after ingestion the subject was feeling better and his sweating was less pronounced, but he still felt nauseated and tired. His pulse and blood pres- sure were found to be normal 10 minutes later, and 2 hours after ingestion he was feeling well and he had a complete lunch and dinner without discomfort. The rapid disappearance of symptoms was consistent with the further rapid recovery of erythrccyte cholinesterase activity. The persistence of inhibitor in the blood was determined by means of the procedure described 246 TOXICITY OF CARBAMATES FOR MAMMALS above. The inhibition of erythrocyte cholinesterase in vitro by plasma samples collected 0.5, 2.0, and 7.5 hours after ingestion indicated the presence of propoxur in the plasma at molar concentrations of 5.Ox 10-', 3.2x 10-7, and 1.5 x 10-7, respectively. The method described by Dawson et al. (1964) was used to determine phenol derivatives in the urine, and the results are shown in Table 2. The amount of o-isopropoxyphenol excreted in the urine over a 24-hour period corresponded to about 45% Table 2 Excretion of phenol derivatives by a human subject following a dose of 1.5 mg of propoxur per kg of body weight Volume of urine ConcentrationTime after ingestion (ml) in urine('4g/ml) Pre-exposure value 55 20.0 1 h 50 min 108 177.5 4 h 45 min 107 195.6 7 h 10 min 250 37.8 8 h 35 min 196 21.3 8 h 35 min to 24 h 790 24.3 of the total dose of propoxur ingested. As abundant vomiting started 23 minutes after ingestion it may be assumed that a relatively large proportion of the ingested dose did not reach the circulation. Excre- tion was very rapid, 81 % of the total amount excreted being found in the first 2 samples, which were collected within 43/4 hours after ingestion. In another experiment a single dose of 0.36 mg/kg produced a rapid fall in erythrocyte cholinesterase to 57% of the normal level within 10 minutes, short-lasting (about 5 minutes) stomach discomfort, blurred vision, and moderate facial redness and sweating. The cholinesterase recovered to its normal value within 3 hours. Repeated doses In a number of experiments carried out to study the effect of storage of inhibited blood samples under various conditions, volunteers took 5 doses of either 0.15 mg/kg or 0.20 mg/kg at half-hourly intervals. In each subject a symptomless depression of erythrocyte cholinesterase to about 60% of the normal level was observed, recovery being rapid after the cessation of dosing (Fig. 5). Similarly, pronounced-and, as a rule, symptomless-daily depression and reactivation of cholinesterase was observed in persons who were occupationally exposed to this insecticide (Vandekar et al., 1968). Fig. 5 Comparison of cholinesterase levels as determined spectrophotometrically and tintometrically * 120 5x0.20 mg/kg---i; I 100 80 60F I.n a 2 TIME (h) 3 4 51 A erythrocyte cholinesterase A whole-blood cholinesterase determined spectrophotometrically A plasma cholinesterase o whole-blood cholinesterase, determined tintometrically * The levels were measured in a single human subject who was given 5 doses of 0.20 mg of propoxur per kg of body weight at 30-min intervals. u-.. 247 a-0 1_ 248 M. VANDEKAR, R. PLESTINA & K. WILHELM It may also be noted in Fig. 5 that whole-blood cholinesterase levels determined by the tintometric method, a field method designed originally for deter- mining exposure to organophosphorus compounds (Edson, 1958), were in reasonably good agreement with the erythrocyte cholinesterase levels determined by the Ellman spectrophotometric method. A slight adjustment was, however, made to render the tinto- metric method suitable for determining the degree of cholinesterase inhibition in persons exposed to carbamates: to diminish the reactivation of carba- moylated enzyme, the substrate was added to the reaction mixture immediately after the addition of the blood sample. The usefulness of methods for determining the degree of exposure to propoxur has been discussed by Wright et al. (1969) on the basis of experience gained in a number of village-scale and large-scale field trials carried out with this compound. The studies in volunteers have shown clearly that, while a single relatively small oral dose (0.36 mg/kg) of propoxur may produce symptoms of short dura- tion, higher doses may be tolerated without symp- toms (although there is appreciable inhibition of erythrocyte cholinesterase) if they are divided into portions that are taken within a relatively brief period. CONCLUSIONS Certain common characteristics of monomethyl- carbamates distinguish them in several important respects from organophosphorus compounds. The differences between the two groups of compounds are attributable to the fact that the inhibition of cholinesterase produced by carbamates is of short duration in comparison with that produced by organophosphorus compounds. Although carba- mates of low toxicity induce symptoms of somewhat longer duration than do more toxic ones when applied at equilethal dosages, there is a rapid recovery in cholinesterase activity and a rapid disap- pearance of symptoms after the cessation of expo- sure, in marked contrast to poisoning by organo- phosphorus compounds. Being direct inhibitors of cholinesterase, carba- mates produce rapid inhibition in the early stages of exposure; it is, however, difficult to produce a severe degree of inhibition, because the rate of reactivation of the inhibited cholinesterase ap- proaches that of inactivation. Thus, as demon- strated by infusion experiments and LD50/ED50 ratios, the lethal dose of a carbamate is a consider- ably greater multiple of the dose causing the first signs of poisoning than is the case for organophos- phorus compounds. Consequently, in cases of occu- pational overexposure to carbamates, an early warn- ing of poisoning-the appearance of slight but unmistakable symptoms-may be expected long be- fore a dangerous dose is absorbed. At a certain rate of absorption-for propoxur, about 0.30 mg/kg/h orally-exposure may be continued for long periods without danger of poisoning. REFERENCES Dawson, J. A., Heath, D. F., Rose. J. A., Thain, E. M. & Ward, J. B. (1964) Bull. Wid Hlth Org., 30, 127 Edson, E. F. (1958) Wld Crops, 10, 49 Ellman, G. L., Courtney, K. D., Andres, V., Jr & Feather- stone, R. M. (1961) Biochem. Pharmacol., 7, 88 Fukuto, T. R., Metcalf, R. L., Winton, M. Y. & Roberts, P. A. (1962) J. econ. Ent., 55, 341 Gaines, T. B. (1960) Toxicol. appl. Pharmacol., 2, 88 Gaines, T. B. (1969) Toxicol. appl. Pharmacol., 14, 515 Goldberg, M. E., Johnson, H. E., Knaak, J. B. & Smyth, H. F., Jr (1963) J. Pharmacol. exp. Ther., 141, 244 Heath, D. F. (1961) Organophosphorous poisons, Oxford, Pergamon Plegtina, R. (1968) Prilog poznavanju toksicnih svojstava o-izopropoksifenilmetilkarbamata, Zagreb (M.Sc. Thesis) Plegtina, R. & Vandekar, M. (1966) Studies in the toxi- cology ofN-methylcarbamates: IV. Symptoms as related to cholinesterase activity. In: XVth International Congress on Occupational Health, Vienna, 19-24 September 1966, Vienna, vol. 2, p. 525 Simeon, V. (1966) Studies in the toxicology of N-methyl- carbamates: IL. Correlation between anticholinesterase activity and acute toxicity. In: XVth International Congress on Occupational Health, Vienna, 19-24 Sep- tember 1966, Vienna, vol. 2, p. 521 Vandekar, M. (1957) Nature (Lond.), 179, 154 Vandekar, M. & Fajdetic, T. (1966) Studies in the toxicology of N-methylcarbamates: III. Tolerance of carbamates at different rates of intravenous in- fusion. In: XVth International Congress on Occupational Health, Vienna, 19-24 September 1966, Vienna, vol. 2, p. 529. TOXICITY OF CARBAMATES FOR MAMMALS 249 Vandekar, M. & Heath, D. F. (1957) Biochem. J., 67, 202 Vandekar, M., Hedayat, S., Plegtina, R. & Ahmady, G. (1968) Bull. Wid Hlth Org., 38, 609 Vandekar, M., Reiner, E., Svetli6ic, B. & Fajdetid, T. (1965) Brit. J. industr. Med., 22, 317 Wilhelm, K. (1967) Inhibitorni u6inak nekih karbamatnih insekticida na eritrocitnu i serumsku kolinesterazu eovjeka. In: II jugosl. kongres za med. rada, Sadrdfaji saopeenja, Split, 1967 Wilhelm, K. & Vandekar, M. (1966) Studies in the toxicology of N-methylcarbamates: L Comparative toxicity tests and estimation of persistence of inhibitor in the body. In: XVth International Congress on Occu- pational Health, Vienna, 19-24 September 1966, Vienna, vol. 2, p. 517 Wright, J. W., Fritz. R. F., Hocking, K. S., Babione, R., Gratz, N. G., Pal, R., Stiles, A. R. & Vandekar, M. (1969) Bull. Wld Hlth Org., 40, 67 DISCUSSION BARNES: Did any of the volunteers who were given carbamates complain of muscle weakness? VANDEKAR: Yes. Both volunteers who complained of symptoms mentioned tiredness and weakness; one of them specifically described it as " muscle weakness ". HOLAN: The symptoms that occurred in the human vol- unteers sound psychologically similar to those that are produced by acute anxiety. Was a double blind study carried out or did the subjects know that they were taking the pesticide? VANDEKAR: Profuse sweating, nausea, and vomiting are also observed in some spray-men who are over-exposed in the field. They cannot be attributed to acute anxiety. The volunteer was given no information on the bio- chemical findings until after his recovery.