Part 1

Issued April 4, 1912.

U.S. DEPARTMENT OF AGRICULTURE,

BUREAU OF CHEMISTRY--BULLETIN No. 148.

H. W. WILEY, CHIEF OF BUREAU.

THE TOXICITY OF CAFFEIN:

AN EXPERIMENTAL STUDY ON DIFFERENT SPECIES OF ANIMALS.

BY

WILLIAM SALANT, _Chief Pharmacological Laboratory, Division of Drugs_,

AND

J. B. RIEGER, _Assistant Chemist_.

WASHINGTON: GOVERNMENT PRINTING OFFICE. 1912.

LETTER OF TRANSMITTAL.

U. S. DEPARTMENT OF AGRICULTURE,

BUREAU OF CHEMISTRY,

_Washington, D. C., November 14, 1911_.

Sir: I have the honor to submit for your approval a manuscript on the toxicity of caffein, which is the first of a series of reports to be made by Dr. Salant on the pharmacology of this drug; the conclusions here reported are, therefore, in some particulars to be regarded as tentative. The data obtained are primarily of use in the execution of the food and drugs act, but are capable of much broader application.

Acknowledgment is made of the assistance rendered by Dr. John R. Mohler, Chief of the Pathological Division, Bureau of Animal Industry, and his assistants, in performing the autopsies recorded in this report. I recommend the publication of the manuscript as Bulletin No. 148 of the Bureau of Chemistry.

Respectfully,

H. W. WILEY, _Chief_.

Hon. JAMES WILSON,

_Secretary of Agriculture_.

This publication may be procured from the Superintendent of Documents, Government Printing Office Washington, D. C., at 15 cents per copy

CONTENTS.

Page. Introduction 5

Historical review of the literature on the toxicity of caffein 9

Acute caffein intoxication 18

Experiments on rabbits 18 Subcutaneous injection 18 Administration by mouth 26 Injection into the peritoneal cavity 28 Intramuscular injection 33 Intravenous injection 37 Summary 42

Experiments on guinea pigs 43 Subcutaneous injection 43 Injection into the peritoneal cavity 47 Administration by mouth 49 Summary 52

Experiments on cats 53 Subcutaneous injection 53 Injection into the peritoneal cavity 56 Administration by mouth 57 Summary 59

Experiments on dogs 60 Administration by mouth 60 Subcutaneous injection 60 Experiments on puppies 61 Summary 62

Chronic caffein intoxication 63 Experiments on rabbits 63 Experiments on dogs 75

Discussion of results 91

General summary and conclusions 95

Bibliography 97

THE TOXICITY OF CAFFEIN.

INTRODUCTION.

Comparative physiology has established the fundamental fact that some properties are common to all forms of living matter. But the same method of inquiry has also led to the recognition of marked differences in the physiological processes of various species of animals. Among the most important investigations which contributed to the knowledge of such variation of function are the studies in comparative metabolism. It is now recognized that metabolism is in some respects quite different in herbivora and in carnivora. Some forms of oxidation are much greater in the rabbit than in cats and dogs. Nuclein metabolism presents important differences in the rabbit and in man, while the mode of neutralizing acid in the body may be cited as another variation in the metabolism of these forms. Perhaps the most striking examples of differences in the metabolism of different organisms is furnished by the results of studies on the fate of certain poisons introduced into the body.

The classical experiments of Bunge and Schmiedeberg(15)[A] on the synthesis of hippuric acid are of interest in this regard. It will be recalled that in the dog the synthesis takes place in the kidney; the rabbit is able to form hippuric acid in the liver as well as in the kidney, while frogs can synthesize hippuric acid even when both of these organs have been removed or excluded from the circulation. Observations on the fate of some of the alcohols of the fatty acid series have likewise shown that these substances may be combined with glycuronic acid in some animals but not in others. According to Thierfelder and Von Mering,(84) tertiary alcohols are combined in this manner in the rabbit but not in the dog. According to Neubauer,(64) the primary and secondary alcohols are so combined in the dog as well as in the rabbit, but to a greater degree in the latter.

[A] The small figures refer to the bibliography at the end of this bulletin.

Pohl(73) found that amyl alcohol is largely eliminated by the lungs in the cat and in the dog. The protocols of his experiments show that 65 per cent of the alcohol given these animals was thus recovered, while he recovered only 22 per cent of this substance in the expired air of the rabbit. Examination of the urine showed the presence of glycuronic acid. Hofmeister's(37) work with tellurium in the dog is of interest in this connection. He made the important discovery that some animals possess the power of methylation as well as of demethylation. Abderhalden and Brahm's(1) experiments with pyridin show that the same is true of young dogs when on a meat diet. His experiments on rabbits with this substance were negative.

The metabolism of caffein and theobromin furnish another illustration of differences in the physiological mechanism of animals. Although the substances found in the urine of man, dog, and rabbit after the administration of caffein and theobromin were the same, the quantities varied considerably. According to Krüger and Schmidt,(47) over 14 per cent of the theobromin introduced into the rabbit is eliminated as 7-methyl xanthin in the urine. The dog eliminates only about 0.67 per cent. On the other hand, the amount of tri-methyl xanthin eliminated was only 3 per cent in the dog and not quite 1 per cent in the rabbit.

It appears, therefore, from studies in comparative metabolism, whether endogenous or exogenous, that well-marked physiologic and chemical differences exist in various species of animals. That pharmacological action may likewise vary in different species of animals is shown by the following investigations. According to Guinard,(31) who made an exhaustive study of morphin, the reaction to this alkaloid varies in different forms of life, both qualitatively and quantitatively. He established its narcotic effect in the dog, rabbit, guinea pig, white mice, and rats, while for the cat, horse, ox, sheep, hog, and goat it is, on the contrary, a stimulant. Moreover, there is no evidence of cerebral effect. The stimulating effect of morphin on the nervous system in some animals was also observed by Noe(65) in experiments with this substance on the hedgehog.

Guinard(29), (30) has also shown that morphin has no narcotic effect in the marmot, although this animal is very sensitive to the drug. Two milligrams per kilo were found to be a surely fatal dose for this animal. His experiments on the comparative toxicity of morphin(30), (31) show a considerable range of variation in different species. Thus the fatal dose for the dog was found to be 0.65 per kilo, while 7 mg per kilo is the fatal dose for the horse. About twice the amount is fatal for the ox and 0.2 mg per kilo kills the pig. Experiments with other drugs has shown that a considerable range of variation in resistance exists in animals of different species.

Noe's(65) studies on the comparative toxicity of chloral brought out the interesting fact that the rabbit is more resistant to it than the hedgehog and the latter more resistant than the guinea pig. Perhaps the most striking example of a difference in reaction of the same substance in widely different species is that furnished by apocodein, quinin, and yohimbin. According to Gunn(32) these substances have been found to cause vasodilation in warm-blooded animals, but they constrict the blood vessels of the frog.

Experiments with apomorphin likewise show that the reaction to this substance varies in different species of animals. The resistance of the cat to this drug is, according to Guinard(31), ten times greater than that of the dog, and the latter is more sensitive than the rabbit to the crystalline form of apomorphin when given intravenously. According to Kobert(45) amygdalin is without effect on dogs, but it is poisonous to rabbits. Lapicque(49) found that the toxicity of curara varies in different species of frogs, the dose required to produce paralysis in _Rana esculenta_ being three times greater than in _Bufo vulgaris_. Weir Mitchell(59) pointed out long ago that turtles stand enormous doses of curara. Schmiedeberg's experiments with caffein on _Rana temporaria_ and _Rana esculenta_ (and more recently those of Jacobi and Golowinski(42) with caffein, theobromin, and theophyllin) are also of interest in this connection. These experimenters observed well-marked differences of reaction to methyl-xanthins in these closely allied forms.

Experiments with quinin have shown that the action of this substance differs in some animals. It causes a fall of temperature immediately after its administration in the guinea pig, but frequently produces, at first, a rise of temperature, followed by an unimportant fall, in rabbits, dogs, and man.

The numerous investigations which have been carried out on the effect of atoxyl within recent years have contributed much to the comparative pharmacology of this substance. Although the symptoms and organic changes produced by this substance in a variety of animals present no great differences, the resistance of some has been found to vary; according to Köster(46) it is more toxic for dogs than for rabbits. A number of other substances have been found by various experimenters to vary in toxicity for different species of animals. Cantharadin, phenol, atropin, and strychnin may be mentioned as illustrations.

Pharmacological studies on lower forms of life have also revealed marked variations in the effect of some poisons. Observations made by Danilewski(18) with hydrochinone indicate that solutions of 1 to 100 or 200 are toxic to Celentrates, causing paralysis in these organisms. Echinoderms are killed within one or two hours in 1 to 1,000 or 2,000 solution, while in Vermes even weaker solutions cause tetanus and finally paralysis. The experiments of Drzewina(19) with potassium cyanid are also interesting in this connection. Teleosts placed in 100 cc of sea water containing twentieth-normal potassium cyanid showed signs of asphyxia and died in 10 to 20 minutes. Actinia placed in a solution of sea water containing five times as much potassium cyanid were active on the thirteenth day of the experiment. Similar results were obtained with other marine organisms.

From these data it is evident that the toxicity of a substance may vary considerably in different forms of life. It has been shown also by some investigations cited by Salant(78) that the action of drugs may be modified by different conditions in the environment as well as in the subject of the experiment. The recognition of the importance of these factors in determining pharmacological action has contributed much to the elucidation of the mechanism by which drugs and other substances produce physiological effects in the body. Moreover, such knowledge has often enhanced the therapeutic value of pharmaco-dynamic agents and has frequently served to avert effects of an undesirable character in man and domestic animals. The results obtained in one species of animals under a particular condition do not admit, therefore, of universal application. Furthermore, the nature of the action of a drug can only be partly learned from the manifestation of its acute effects. Equally important, therefore, especially in studies on toxicity, are the changes produced in chronic intoxication.

That the acute effects of a substance can hardly be considered a correct estimate of its toxicity is shown by the evidence obtained in experiments on tolerance and cumulative action of drugs; for the toxicity of a substance may diminish when the substance is given steadily for a long time if the body acquires tolerance for it. Arsenic, morphin, and cannabis indica may be cited as illustrations of drugs, the toxicity of which decreases with repeated doses, while digitalis and lead show a tendency to increased toxicity when similarly administered. Moreover the acute and chronic effects are sometimes qualitatively different. According to Igersheimer(41) the symptoms in acute atoxyl intoxication are nausea, vomiting, and diarrhea. These symptoms are absent in chronic intoxication, in which trophic disturbances of the skin and inflammation of the mucous membranes were the effects produced. That the acute action of atoxyl differs from the chronic effects was likewise shown by experiments on animals. The studies of von Anrep(5) on chronic atropin intoxication are of interest in this connection, as he found that after 10 to 15 injections of atropin there is no manifestation of symptoms such as is observed in acute intoxication, while the effects on the circulation are also less marked, the acceleration of the pulse being less than after the same dose in a normal subject not accustomed to its use. When the administration of atropin is continued for a longer time its usual effects on the pulse disappear altogether; there is, on the contrary a decreased frequency of the pulse. If atropin has been administered for from two to three weeks, respiration is likewise affected.

HISTORICAL REVIEW OF THE LITERATURE ON THE TOXICITY OF CAFFEIN.

Caffein was discovered in 1820 by Runge,(77) Pelletier,(60) and Robiquet(75) and was first analyzed by Dumas and Pelletier,(20) but its exact percentage composition was determined by Pfaff and Liebig,(71), (72) while to Herzog (13), (18) belongs the credit of having established that it is basic. Strecker(82) prepared caffein synthetically by heating theobromin silver and methyl iodid in a closed tube for 24 hours. Soon after its discovery in coffee Oudry(67) reported the presence of a substance in tea which he called "thein." Its identity with caffein was established 15 years later by Jobst(43) and also by Mulder.(62), (63) According to Brill,(13) Mulder (1838) was also the first to perform experiments with caffein on animals. After the administration of one-half grain to a pregnant rabbit he observed loss of appetite and kyphosis. The rabbit aborted but recovered from the effects of caffein. It has since been made the subject of numerous investigations which were carried out on a variety of animals. Observations with caffein were also made on the human subject. About four years after Mulder published his results, Lehmann(51) (1842) reported experiments on a number of people who were given caffein. The administration of from 2 to 10 grains of the alkaloid was followed by headache, palpitation of the heart, increased frequency and irregularity of the pulse, tinnitus aurium, photopsia, insomnia, and even delirium. Similar experiments reported by Frerichs(25) (1846) indicate that in doses of 25 grains it may induce severe symptoms about 15 minutes after its administration. He also observed circulatory as well as nervous symptoms and vomiting.

According to Albers(2) (1852), 4.5 grains of caffein citrate injected subcutaneously into the thigh of a rabbit was soon followed by diminished motion and tremors of the operated thigh. Other symptoms reported were spasms of the facial muscles, increased respiratory movements, and mental confusion. Of interest in this connection are the experiments of Cogswell(17) (1852) on frogs. He concluded that in point of destructive action on the tissues, caffein is far superior to morphin and may be compared to strychnin and coniin, its action on the nervous system he believed to be principally confined to the effect on the brain and spinal cord.

Lehmann(52) (1853) observed increased frequency of heart action after the administration of 4 grains, which were given with a normal diet to an adult man. When the dose was doubled the frequency of the pulse was still more increased, heart action became stronger, and tremors and confusion of thought with excitement of the imagination made their appearance. There was also an increased desire to micturate.

Stuhlmann and Falck(83) (1857) were the first to make a study of the toxicity of caffein on animals of different species. The administration of 0.5 gram of caffein subcutaneously or per rectum in rabbits induced tremors, tonic and clonic convulsions, paralysis, and increased frequency of respiration at first followed by violent dyspnoea. On autopsy he noticed congestion of the organs and in two of the three rabbits experimented upon punctiform hemorrhages of the brain with congestion of the meninges were found. In the other rabbit anemia of the brain was observed. Experiments on cats were carried out by subcutaneous, intravenous, and rectal injections. The symptoms observed after the administration of 0.5 to 0.7 gram of caffein were the same as in rabbits except that the cats developed diarrhea when caffein was given and no anatomic lesions were found on autopsy. The effect of caffein on dogs indicated that in subjects of medium weight a dose of 0.5 gram given by mouth might produce restlessness and increased frequency of respiration, while the injection of the same amount intravenously into such animals may cause death. Large, full-grown dogs, however, survived an intravenous injection of 2 grams of caffein, showing symptoms of incoordination, salivation, and frequent defecation. These investigators also made observations on caffein, using pigeons and other birds; 0.5 to 0.1 gram introduced into the stomach caused vomiting, diarrhea, tonic, but more frequently clonic, convulsions, incoordination, tremors, paresis, and paralysis.

In a few, but not in all of the birds, there was at first increased frequency of respiration followed by dyspnoea and circulatory disturbances. These amounts of caffein proved fatal in all of the experiments on birds. Inflammation of the intestinal mucosa and congestion of the meninges were the only changes found on autopsy. Stuhlmann and Falck also studied the effects of caffein on fishes and toads. Mitscherlich(60) (1858) fed 0.4 gram of caffein with bread to a rabbit and noticed lowered temperature, fatigue, convulsions, first increased then decreased frequency of respiration, and on autopsy congestion of all the viscera. He also reported observations on two frogs, one of which was given one-sixteenth of a grain of caffein in a pill with bread. It was administered to the other frog in aqueous solution, but the mode of administration was not published. The symptoms observed were in the main the same as in rabbits. In pigeons 0.125 gram introduced into the stomach caused severe vomiting, muscular incoordination, tonic rigidity of the limbs, and retraction of the head. Respiration was increased in frequency. Death followed within 3 hours and 15 minutes.

From a series of experiments on frogs which Hoppe(38) carried out (1858) by applying one-fourth of a grain of caffein to the muscles of the back, he concluded that caffein causes paralysis of the nerves, spinal cord, and brain, sensation being paralyzed before movement. The injurious action of caffein proceeds, according to Hoppe, from the spinal cord. This was based on experiments on two frogs, _Rana esculenta_, in which the right leg was amputated, the nerve being left intact, while the nerve of the other leg was ligated. At the end of 30 minutes paralysis was more marked on the right than on the left side. In another frog of the same species he resected the femoral nerve on the right side; about 1½ hours after the administration of caffein convulsions were observed. The left leg was rigid, but the right was relaxed.

Voit(85) (1860) ligated the vessels of the right lower extremity, cut the nerves of the left leg, and introduced a few drops of caffein solution into the stomach. Shortly afterwards tetanus of the right leg occurred on touching the back of the animal; the left leg was motionless. Later the entire body exhibited tetanic convulsions. From this and similar experiments Voit concluded that caffein acts first and principally on the central nervous system, and that caffein is also poisonous to nerve and muscle fibers, as they die when a solution of caffein is applied to them. The action of caffein, according to Voit, is similar in great part to that of strychnin. The effect on the blood vessels is particularly interesting, as Voit observed dilatation of the vessels, due as he thought to muscular paralysis, and also transudation and congestion of the capillaries.

Kurzak(48) (1860) made a study of the comparative toxicity of caffein in frogs and rabbits and came to the conclusion that the lethal dose for frogs is about one-seventh of that for rabbits. Caffein citrate in the form of crystals was administered in both cases by mouth. The doses given to frogs were 1 to 1.5 grains. He observed convulsions and increased respiratory activity at first; after one hour respiration diminished and voluntary muscular activity disappeared. Even on the second day convulsions were sometimes noticed. Death occurred at the end of the first or second day. Experiments on only two rabbits were reported, 0.8 gram of caffein citrate causing the death of one at the end of 13 hours. The symptoms noticed were the same practically as in frogs, but it is interesting to observe that ecchymosis of the mucous membranes of the stomach near the cardia was the only lesion found on autopsy. Several experiments made on different days on the other rabbit indicated that the toxic dose exceeded 0.5 gram, while smaller doses caused but very mild symptoms.

According to Gentilhomme(27) (1867), after caffein the reflexes are at first diminished and then disappear altogether. Death is produced by stiffness and immobility of all the muscles, particularly of the muscles of respiration, thus causing asphyxia. He furthermore held that caffein has no effect on cardiac or smooth muscle fiber, its action being specific on voluntary muscle fiber, contractions of which he observed under the microscope, thus differing completely from strychnin, which is a nerve poison.