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structure. From their refractive indices they appear t o be chiefly cycloparaffins. Any cycloolefins which may exist as intermediates are apparently hydrogenated by the hydrogen set free in their formation from simple olefins to yield the corresponding cycloparaffins.
Literature Cited (1) Cross, R., Handbook of Petroleum, Asphalt and Natural Gas, Kansas City, Mo., Kansas City Testing Lab., 1931. (2) Davis, H. El., and Daugherty, J. P., IND.ENQ.CREM.,Anal. Ed.,
( 5 ) Hague, E. N., and Wheeler, R. V., Fuel, 8, 560 (1929).
VOL. 30, NO. 5
J. Ckem. Soc., 1929,378;
( 6 ) Hugel and Artichevitch, Ann. combustibles liquides, 3, 985 (1928). (7) KatB, T., J. Soc. Chem. I n d . Japan, 36, Suppl. Binding, 228 (1933). (8) Leslie, E. H., and Potthoff, E. H., IND. ENG.CHEM.,18, 776 (1926). (9) Loveless, A. W. T., Ibid., 18, 826 (1926). (10) Rice, F. O., J. Am. Chem. SOC.,55, 3035 (1933). (11) Walker, H. j. p h y s . Chem., 31,961 (1927). (12) Wheeler, R. V., and Wood, W. L., J. Chem. Soc., 1930, 1819.
w.,
4. 193 (1932).
(3) Faragher,’ W. F., Morrell, J. C., and Levine, I. M., IND.ENQ. CHEM.,Anal. Ed., 2, 18 (1930). (4) Frey, F. E., and Hepp, H. J., IND.ENG.CHEM.,25, 441 (1933).
RECEIVED November 6, 1938. Dissertation submitted in partial fulfillment of the requirements for the degree of doctor of philosophy in the Department of Chemistry, Graduate College, State University of Iowa, in June, 1934.
Toxicological Studies of Derris Chronic Toxicity of Derris’ ANTHONY M. AMBROSEa AND HARVEY B. HAAG Food Research Division, Bureau of Chemistry and Soils, United States Department of Agriculture, and Department of Pharmacology, Medical College of Virginia, Richmond, Va.
Studies are reported upon the chronic toxicity of derris (Derris elliptica) for rabbits, dogs, and rats. For rabbits, 60 mg. per kg. and above showed signs of cumulative toxicity. I n adult dogs on diets containing 0.04 per cent derris, no symptoms of toxicity were observed. I n young growing dogs on the same diet, the most pronounced effect was the stunting of growth. Derris diets containing less than 0.0312 per cent derris had no demonstrable effect on growth of rats. As the concentration of derris in the diet was increased, the inhibition in growth was more marked. Pathologic studies on the tissues of dogs and rats indicated that derris in all concentrations studied was somewhat injurious, the liver being the only organ consistently affected.
D
URIKG the last decade a vast amount of information on the toxicity of derris (Derris elliptica) for various insects has been published. Roark ( I S ) published an extensive bibliography on the subject which was consulted by the authors in the preparation of this and previous papers from this laboratory. Derris and derris preparations in one form or another have gained widespread use as an insecticide
in the control of various plant insects (4-8, 11, 16); as a remedial agent for combating mites, fleas, and body lice on animals (3, 6, 11, I d , 17); and as a killing agent for roaches. and house flies (3,6, 10, 11, l a ) . I n all these studies derris and derris preparations were found toxic to both sucking and chewing insects. The literature on the toxicology and pharmacology of derris was discussed in previous papers of this series ( 1 ) . In considering the usefulness of a n insecticide, it is most important t h a t due attention be given to the question of whether or not prolonged ingestion of the material in minute is, i n amounts approaching those which quantities-that might be present on sprayed fruits or vegetables-will have any untoward or deleterious effect upon the animal economy. While it is the general opinion that derris would be noninjurious in the quantities that might be present upon sprayed foods, no experimental evidence has come to the authors’ attention bearing out this belief. I n an attempt to throw some light upon this problem, rabbits, dogs, and white rats have been maintained on small daily doses of derris for periods of 1 t o 8 months. The sample of Devris elliptica root used in these studies contained 9.6 per cent rotenone and 28.6 per cent total carbon tetrachloride extractives. The average powdered derris root on the American market contains about 4.5 per cent rotenone and from 14 to 18 per cent total extractives. A sample unusually high in active principles was selected for these studies. The sample and analysis were furnished by the Bureau of Entomology and Plant Quarantine, United States Department of Agriculture. 1 This is the fourth of a series of papers on the toxicology of Derris elltptzca and its constltuents. The others rtppeared in J. Pharmacol., 43, 193 (1931), and in IND. ENQ.CHEM.,28. 815 (1936), and 29, 429 (1937). 2 Present address, United States Department of Agriculture, Stanfordi University Medical School, Ban Francisco, Calif.
INDUSTRIAL AND ENGINEERING CHEMISTRY
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Rabbits Twenty-one young rabbits, weighing approximately 1.5
kg. each, were divided into seven groups of three rabbits each. To the animals in six of these groups were given daily doses of 10, 20, 30, 60, 80, or 100 mg. of derris per kg. of body weight, administered by stomach tube as a water suspension of the ground root. The animals were fed ad libitum on a diet of Purina Rabbit Chow and were weighed every other day. Chemical analyses of the urine for sugar and albumin were made at frequent intervals throughout the experiments. Rabbits surviving the 30-day experimental period wcre kept under observation an additional 30 days. TABLEI. TION OF
EFFECTON RABBITSOF DAILYORALADMINISTRASUBLETHAL DOSESOF DERRISOVER A PERIODOF 30 DAYS
No. of RabGroup bii,s
r
1 (control) 2 3 4 5 6
3
Daily Doses iMg./ko. 0
3 3 3 3 3
7
3
Change i n weight
Results 30-day feeding period
30-day postfeedin observation perioi
% +12.0
All survived
All survived
10 20 30 60 80
+ 111.9 1.5 + + 11.5 +- 39 .. 83
All survived All survived All survived All.survived 1 died after 7 days
All survived All survived All survived 1 died after 3 days 2 survived
100
....
ldiedafter 5days 1 died after 11 days 1 died after 17 days
..... . . .
593
tributed, the duration of the experiment, concentrations of derris in the diet, and quantities of food and of derris ingested. The animals were fed once a day, at which time careful note was made as to food consumption and general appearance. The dogs were weighed at intervals of 2 weeks, and a t similar intervals blood studies were made which included estimation of blood sugar, nonprotein nitrogen, and hemoglobin, as well as enumeration of erythrocytes and leucocytes. The urine was likewise examined for sugar and albumin. It had been previously established by feeding experiments and by analysis of the basal diet used3 that from 15 to 20 grams per kg. of body weight, plus an adequate supply of vitamins, constituted a diet sufficient for maintenance and growth. Table I1 shows that the average daily food consumption in all groups was approximately 20 grams per kg. of body weight, which assured proper maintenance and growth. Hence any effect noted could not be ascribed to a lowered or inadequate food intake. Dogs in groups 2, 3, 4, and 5 showed no characteristic symptoms of intoxication, no radical changes in body weight compared with their initial weight, and no deviation in food consumption. Dogs started on higher concentrations of derris (above 0.04 per cent) refused to eat, and the experiments were discontinued. The two dogs in group 4 cast litters of five and six pups which at birth appeared normal in every respect. All the pups in one litter died within 30 days, probably as a result of worms or pneumonia; those of the other survived and developed normally, and a t weaning age were discarded. Two pups (group 8) out of a litter of six, were placed a t weaning age on a diet containing 0.04 per cent of derris, and two (group 7) were used as controls. The two pups on the experimental diet showed no signs of intoxication but did not grow as well as the controls. At the close of the experiment the significant difference observed in the control pups over those on the experimental diet was that the control pups were almost twice as large as those on the experimental diet. The average increase in weight of the control pups was 473 per cent, as compared to 375 per cent for the pups in group 8, on 0.04 per cent derris diet. While the number of experiments on young growing dogs is too small to warrant any definite conclusions, the desirability of extending the observation is definitely indicated. Each of three dogs (group 6) was given 60 mg. (total) of powdered derris root in capsules daily. Higher doses were not tolerated but produced almost immediate emesis. Table
Table I records the results of this experiment. There were no appartmt changes in the general appearance or well being of the rabbits of groups 2, 3, and 4 (receiving 10, 20, and 30 mg. of de’rris, respectively) as judged by activity, food consumption, and increase in body weight, when compared with the controls of group 1. At the close of the experiment there was an average increase of 11 per cent in body weight of all animals in the first four groups. I n the fifth group (receiving 60 mg. of derris) there was some indication of a toxic effect. The animals in this group were not so active, and the food consumption was about 10 per cent less than hhat of the first four groups. When the administration of derris was terminated a t the end of the thirtieth day, two of the animals showed a slight increase in weight; the third showed a loss and died a few days later. In the sixth group of rabbits (receiving; 80 mg. of derris) there was definite evidence of a toxic effect; food consumption was reduced to about 50 per cent of tkat of the first four groups. All showed a decrease in body weight, and one animal died after 7 days; the rea Balra, a brand of dog food compounded by The Valentine Company, maining 1;wo lived beyond the 30-day observation period. Richmond, Va., having the following composition: protein, 33 per cent; In the larjt group (receiving 100 mg. of derris) one fatality fat, 11; carbohydrates, 39; ash and salt, 7.2; PzOs,0.65; and an adequate occurred ttfter the fifth day, and within 17 days the remaining supply of vitamins. two animiils had died. Analysis of urine showed no sugar or albumin throughout the entire period of the experiment. TABLE11. EFFECTON DOGSOF DAILYORALADMINISTRATION OF SMALL These results suggest that derris in daily DOSESOF DERRISOVER EXTENDED PERIODS doses of 60 mg. per kg. of body weight and Derris Administrationa NO. hv. Daily Daily above is not promptly eliminated or detoxified of Feeding -Av. WeightFood ConIn consumpby the rabbit, indicating that in these doses it Group Dogs Period Before After sumption“ diet tionb Kg. Kg. Grarns/kg. % Mg./kg. Dags produces a “cumulative” effect. This is cerAdults tainly true for the group of rabbits receiving 80 0.0 0.0 18 180 10.9 11.5 1 (control) to 100 mg. 0.04 8.0 20 4.2 3.6 130 2
Dogs In the next experiment thirteen adult dogs and four )growing pups (litter mates) were used. I n one experiment (group 6) derris was administered in capsules; in all other experiments it was intimately mixed with the diet. Table 11 shows the manner in which the dogs were dis-
3
4 5
240 190 190 190
9.8 7.7 7.7 7.6
9.1 8.1 9.8
19
21 22
.. 7.8 Pups 22 1.9 10.9 2 240 7 (control) 22 2.0 9.5 8 2 240 0 Group 6 was fed table scraps; all others received Balra. b Based on food eaten,. C Ground derris root in capsules, 60 mg. per day. 6
0.04 0.02 0.013
7.6 4.2 2.9 7.8
0.0
0.0 8.8
c
0.04
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I1 shows that dogs in this group received approximately the same amount of derris per kg. of body weight as dogs in groups 2 , 3 , and 8. This experiment was conducted in order to assure the daily consumption of a definite amount of derris, an amount larger than might be found theoretically in a diet consisting entirely of sprayed fruits and vegetables. The objection which might be raised to this particular experiment is that derris so administered is not completely disintegrated, and that the dogs were not exposed to the complete effects of the substance. However, this is believed not to be the case, since the capsules were administered one-half hour before the animals were fed, assuring ample time for disintegration of the capsules and their contents. Fatal doses of derris administered in capsules were of the same order as when administered in aqueous suspension (1). The only difference observed was in the survival time. When administered in susDension, derris was somewhat more rapid in its action than when administered in capsules.
* 1
vc
1,. 3, AO. 5
group of rats served as control, and the remaining seven groups were placed on the basal diet containing 0.0078, 0.0156, 0.0312, 0.0625, 0.125, 0.25, and 0.5 per cent derris, respectively. The basal diet was the same as for dogs and was the one used in the stock rat colony a t the Medical College of Virginia for several years. The food was placed in specially constructed cups in order to minimize spilling and wastage. The weights of the animals and food were recorded every 5 days. The growth curves for rats receiving derris, as well as for the controls, are shown in Figure 1. From these curves i t is apparent that rats on diets containing 0.0078 and 0.0156 per cent derris grew as well as the controls. As the concentration of derris was increased above 0.0156 per cent, the growth rates became progressively lower. After 110 days the group of rats on 0.0625 per cent derris all died within a few days of one another. This was rather unexpected, because up to the time of their death all had appeared normal except for inhibition in growth. However, on autopsy the pathologist diagnosed the cause of death as pneumonia, probably as a partial result of the lowered resistance of the animals,. produced by the toxic effects of the derris in the diet. Animals on diets containing 0.125, 0.25, and 0.5 per cent derris lived only a few days. After 150 days all rats in each of the surviving groups were killed and autopsies performed. Gross changes were not apparent in any of the animals. Microscopic changes are reported under “Pathological Studies.” These studies clearly indicated that rats on diets containing relatively high concentrations of derris did not grow a t the normal rate. This decreased growth rate could be ascribed either to a smaller food intake or to the toxic action of the derris. T o illustrate this point, when rats which had been on experimental diets high in derris were placed on a basal diet, they showed an immediate increase in food consumption and body weight. When the basal diet was later replaced by the respective experimental diets, there was an immediate drop in food consumption, followed by loss in body weight. It appears, therefore, that while a decreased food intake was a contributing factor in lowering the growth rate, derris skiould be considered as having played a definite role. This is further indicated by the fact that an amount of food equivalent to that consumed by rats on 0.125 per cent derris was sufficient to maintain a slight growth in control animals, whereas in animals on the experimental diet there was no increase in body weight.
Pathological Studies I
I
I
FIGURE1. EFFECTOF CONTINUED INGESTION DERRISDIETS ON GROWTH CURVES O F R.4TS
OF
Blood studies in all dogs throughout the experimental period showed no important departures from normal. Urinary analyses for sugar and albumin were consistently negative. On the termination of the feeding experiments, the animals were killed, and macroscopical and microscopical examinations of the organs were made, No characteristic macroscopic changes were observed in the viscera. I n dogs of group 2 numerous small intestinal worms were found, suggesting that derris has no vermifugelike action. Significant microscopic changes will be discussed under “Pathological Studies,”
White Rats I n this series of experiments rats weighing 45 to 60 grams each were divided into eight groups of five rats each. One
The results are based on studies of the tissues of fifteen dogs (two used as control and thirteen receiving various quantities of derris) as outlined in Table 11, and of seventeen rats, including three controls and three groups receiving 0.0078, 0.0156, and 0.0312 per cent of derris as part of a fixed ration. Dogs were killed by intravenous injection of approximately 5 cc. of a saturated solution of magnesium sulfate and potassium chloride. The rats were killed by a sharp blow on the head. The bodies were opened immediately, and specimens were taken from the various organs (spleen, liver, kidneys, heart, lungs, stomach, intestines) and placed in 10 per cent formalin solution without delay. Sections for microscopic examination were made by the paraffin method, with hematoxylin and eosin staining. DOQS. The only organ which showed consistent changes was the liver. These changes were a mild to a moderate increase in periportal lymphocytes, and in five cases an intense vasoconstriction of the hepatic veins together with a marked hepatic congestion and a dilatation of the perivenous lymphatics.
MAY, 1938
INDUSTRIAL AND ENGINEERING CHEMISTRY
.RATS. The main changes were in the liver; they consisted of a mild to moderate periportal lymphatic infiltration, some hyperemia, and an increasing number of focal necrotic areas with an increasing dosage of derris. These foci were scattered in the midzones of the hepatic lobules. The kidneys revealed moderate glomerular and intertubular hyperemia, but the degree was unrelated to derris dosage.
Interpretation In all dogs the liver was mildly affected by the derris. In almost all experiments in which derris was mixed with the food, there was hyperemia of the liver, the sinuses being dilated with blood. In five of these animals the congestion was more intense and was accompanied by a tight constriction of the hepatic veins. When the drug was fed in capsules, these changes were not found, but in two of the three animals fatty changes occurred in the liver. There is not sufficient evidence on which to base an explanation for these differences, but the occasional presence of a small periportal lymphocytic infiltration might implicate the intestines as the source of the irritant. In rats the changes are to be interpreted as an absorption of a mild irritant via the portal vein, which, however, in larger doses becomes sufficiently potent to cause foci of necrosis in the liver lobules. These foci appear in the midzones of the lobules, thus resembling those cases in which infectious disease is the usual cause. The increasing size and number of foci with increasing dose of derris would seem to incriminate derris as the cause of focal necrosis in these rats, and also possibly as the cause of changes in the dogs. This evidence also strengthens the possibility that ingestion of derris was the cause of changes in the internal organs of the dogs. These observations indicate that rabbits can tolerate 30 mg. of derris per kg. of body weight daily for a period of 30 days without any deleterious effect on growth. In larger amounts (80 and 100 mg. per kg.) there is evidence that derris can exert a “cumulative” toxic effect, as shown by inhibition of growth and by death, where the daily administered dose was approximately 15 per cent of the acute fatal dose. This is more readily demonstrated in growing rats by a marked inhibition in growth as compared to normal, on diets containing above 0.0312 per cent of derris. Older and heavier rats placed on diets containing 0.125 and 0.240 per cent derris were better able to withstand the higher doses than were growing rats. However, at these high levels the diet proved fatal within 60 days as compared to approximately 30 days for young rats. It might be of interest to note that rats on 0.0312 per cent derris showed only a slight inhibition of growth and ate approximately 30 mg. of derris per kg. of body weight per day, in contrast to 8 mg. per kg. per day for growing dogs on a diet containing 0.04 per cent derris. According to these results, 0.56 to 2.1 grams of derris might be taken daily by a 70-kg. man without serious injury; 4.5 grams or about one teaspoonful per day might cause death eventually and, with this sublethal dose, there might be an undermining of the health with a predisposition to intercurrent disease, especially when considered in the light of suggestive results on young growing dogs. Roark ( 1 4 , basing his opinion upon studies made on the arsenic content of cabbage treated with lead arsenate, is of the opinion that cabbage similarly treated with derris would not contain more than 25 parts per million. It seems unlikely, therefore, that in fruits or vegetables dusted or sprayed with derris preparations, the concentration of derris could ever attain that employed in the present studies, in which amounts of from 78 to 400 parts of derris were present per million parts of diet. Also, experimental evidence (1, 9, 16)
595
indicates that exposure to the sun and atmosphere quickly causes derris to lose much of its toxicity. However, the effects of oxidation products or other compounds resulting from atmospheric exposure of derris have not been investigated.
Summary Derris administered orally to rabbits in amounts up to 30 mg. per kg. of body weight daily for a period of 30 days produced no demonstrable effects upon growth. In amounts of 60 mg. and above, a distinct “cumulative” toxic effect was observed. On two growing dogs a diet containing 0.04 per cent derris had a stunting effect as compared with litter-mate controls. Adult dogs tolerated similar amounts for periods up to 240 days without manifesting any gross changes in appearance, food consumption, or weight. Likewise, no significant alterations were observed in the blood and urine. Rats maintained on diets containing 0.0078 and 0.0156 per cent derris grew as well as the controls. Rats on 0.0312 per cent derris showed only a slight inhibition in growth; the inhibition was more pronounced as the concentration of derris was increased. On diets containing 0.125, 0.25, and 0.5 per cent derris, the animals did not live. Although the decreased growth rate might have been due partly to lowered food intake, the toxic effect of the derris in the diet should not be overlooked. Pathological studies indicated that derris in all the concentrations used was somewhat injurious to dogs and rats, the liver being the only organ consistently affected. In rats the first effect was a periportal irritation, followed by midzone focal necrosis with larger doses. In dogs the effect in the majority of cases was a periportal irritation, accompanied in most of these cases by vasoconstriction of the hepatic veins, and passive congestion. Further studies of a wider scope are needed before derris or any of its oxidation or decomposition products may be pronounced wholly innocuous when present in minute quantities on fruits or vegetables subjected to dusting or spraying with insecticides containing derris as the active ingredient.
Acknowledgment The writers wish to express their thanks to Frank L. Apperly of the Department of Pathology, Medical College of Virginia, for his cooperation in making the pathological studies here reported.
Literature Cited Ambrose and Haag, IND.ENQ.CHEM.,28,815 (1936); 29, 429 (1937). Campbell, Sullivan, and Jones, Soap, 10 (3),81, 85,87,103,105, 107 (1934). Davidson, U.S. Dept. Agr., Bull. 1228 (1924). Flippance, Gard. Bull. Straits Settlements, 2, 246 (1920). Fulmer, Ontario Dept. Agr., Bul2. 351 (1930). Hamilton J. Econ. Entomol., 26,555 (1933). Huckett, Ibid., 27,440 (1934). Huckett and Hervey, Ibid., 28, 602 (1935). Jones, Gersdorff, Gooden, Campbell, and Sullivan, Ibid., 26,451 (1933). Lapparent, Rev. Zoo2. A g r . , 30, 145 (1934). McIndoo and Sievers, U. S. Dept. Agr., Bull. 1201 (1924). McIndoo, Sievers, and Abbott, J. Agr. Research, 17,177 (1919). Roark, private communication. Roark, U. S. Dept. Agr., Misc. Publ. 120 (1932). Van der Laan, Indische Mercuur, 58, 257 (1935). Walker and Anderson, J . Econ. Entomol., 28,603 (1935). Wells, Bishopp, and Laake, Ibid., 15,90 (1922). RECEIVED December 6, 1937.
