The Toxicity of Caffein: An experimental study on different species of animals
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Title: The Toxicity of Caffein: An experimental study on different species of animals
Author: William Salant
J. B. Rieger
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*** START OF THE PROJECT GUTENBERG EBOOK THE TOXICITY OF CAFFEIN: AN EXPERIMENTAL STUDY ON DIFFERENT SPECIES OF ANIMALS ***
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_.
[Illustration: UNITED STATES DEPARTMENT OF AGRICULTURE SEAL]
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.
These observations seemed to be confirmed by Pratt(74) (1868), who
reported that the isolated posterior extremities and muscle fibers of
the toad placed in a solution of caffein (1 grain to a wineglassful
of water) for three minutes were contracted, while controls placed in
distilled water were relaxed. This experiment is, of course, defective,
as normal salt solution should have been used in both cases. When the
muscular fibers previously immersed in caffein solution were placed
under the microscope violent contractions were observed. The same
author administered from 2 to 18 grains at a dose to five healthy young
men. After the administration of 12 grains he noticed mental anguish,
tremors of the hands and arms, and insomnia. Doses under 5 grains had
no marked effect except a diminution in the frequency of the pulse and
wakefulness.
About the same time Amory(4) (1868) published the results of his
studies on the toxicity of caffein in cats, dogs, rabbits, and pigeons.
In all cases very large doses were introduced directly into the stomach
by means of a temporary gastric fistula. Ten grains given in meat to a
dog caused restlessness, but no other symptoms. Doses of 30 grains and
above were invariably fatal. Seventy-three grains given to a cat caused
death within 20 minutes.
From observations on frogs, guinea pigs, rabbits, and on one dog,
Leven(53) (1868) concluded that caffein which he gave in the form of
the citrate in doses of 10 mg to frogs, from 150 to 200 mg to guinea
pigs, and three to four times the latter amount to rabbits, stimulates
the central nervous system and the voluntary, cardiac, and smooth
muscles. He found that 0.9 gram caffein was fatal for a rabbit when
injected subcutaneously, while 1 gram of the citrate was not toxic
for a dog of medium size. Caffein applied directly to muscle fiber
causes tetanus and destroys muscular contractility, while a nerve fiber
similarly treated loses its irritability.
According to Johansen(44) (1869), caffein acts directly on the muscular
fiber. After the subcutaneous injection of 0.02 gram of caffein
into frogs, he observed contraction of the muscles at the site of
injection, then contraction of the anterior extremities, and finally
the posterior extremities become rigid and extended. Johansen observed
muscular rigidity after caffein, even after curara was injected,
or after ligating the vessels, or cutting the nerves which supply
the muscles. He also observed that large doses of caffein diminish
muscular irritability. When cardiac muscle was poisoned with caffein,
microscopical examination showed that the striations disappeared.
Johansen also states that reflexes disappear after caffein poisoning.
He never observed tetanus in frogs, but reported tonic and clonic
convulsions as a result of caffein poisoning in mammals. Somewhat
different effects of caffein in frogs were observed by Buchheim and
Eisenmenger(14) (1870). After the injection of 2 per cent of the
citrate the frogs soon become inactive. He also observed muscular
twitching of the extremities, which gradually increased, with rigidity
of the muscles and opisthotonos, while respiration became slow and
superficial, finally stopping altogether.
Aubert(6) (1872) studied the toxicity of caffein in man and other
animals. After the ingestion of 0.36 gram, he observed dizziness, but
doses of 0.12 and 0.24 gram were without any apparent effect. On the
other hand, a dose of 0.5 gram of caffein was followed by increased
frequency of the pulse, which soon disappeared. After one hour he
noticed dizziness and trembling of the hands, which likewise passed
away soon. The injection of 0.16 gram of a 2 per cent solution of
caffein into the jugular vein of a rabbit weighing 1,090 grams caused
tetanus and death in two and one-half minutes, and 0.12 gram injected
into a rabbit weighing 980 grams caused death in one minute. Much
larger doses could be borne, however, when artificial respiration was
resorted to. A dog which was given 3 grams of caffein survived when
artificial respiration was performed. Aubert reports, on the other
hand, a similar experiment with 0.25 gram of caffein which terminated
fatally.
That caffein may give rise to different effects in various species
of animals was observed for the first time by Bennett.(9) He studied
its action on frogs, mice, rabbits, and cats, and attempted to
determine the minimum fatal dose in rabbits and cats. He also reported
experiments with thein. In his first communication on the subject he
states that the administration of thein to rabbits first increased
and then diminished the frequency of respiration, while the pulse
was decreased in frequency. Caffein, which he apparently thought was
different from thein, caused increased frequency of respiration, while
the pulse was markedly retarded after a preliminary acceleration.
He also noticed congestion of the ears, muscular incoordination,
tetanus, paralysis, diminished reflexes, and contraction of the pupils.
Bennett reported the minimum fatal dose of caffein for a rabbit
weighing 3.25 pounds as being 5.25-5.5 grains. The symptoms in cats
after the administration of toxic doses of thein or of caffein were
great excitement, paralysis alternating with convulsions, and profuse
salivation. The minimum fatal dose for a cat weighing 5 pounds was,
according to Bennett, 6 grains of caffein and 5.5 grains of thein. Only
one experiment on a mouse is reported; the administration of 0.1 grain
proved fatal. The symptoms were the same as those observed in cats and
rabbits after the administration of caffein. The experiments on frogs
indicate that the symptoms were about the same as those previously
described in the case of warm-blooded animals except that the reflexes
are almost completely lost after the subcutaneous injection of doses
of one-sixteenth to one-twelfth of a grain. The latter dose was fatal
for frogs. It would be of interest to know the comparative toxicity of
caffein to frogs and mammals, but unfortunately the weights were not
reported.
Schmiedeberg(79) (1874) noticed that the administration of 20 mg
of caffein to frogs weighing about 45 grams was followed, in _Rana
esculenta_, in about 25 minutes, by increased reflexes, 7 minutes later
by tetanus. Several attacks occurred, but tonic spasms were never
observed. On the contrary, when the same amount of caffein was given to
_Rana temporaria_ weighing 45 grams he noticed a marked diminution of
the reflexes and tonic rigidity of the muscles after 23 minutes; the
reflexes were greatly increased, however, about 24 hours later. The
frogs were under observation for three days, and although symptoms were
still present at the end of this time in the subjects of both species
tetanus was never observed in _Rana temporaria_.
Peretti's(70) (1875) studies on the effects of caffein were confined
chiefly to observations on dogs. He also made observations on a few
rabbits and reported an experiment on one cat to which he administered,
by subcutaneous injection, 0.18 gram of caffein per kilo and noticed
increased frequency in lachrymation and crying. The cat was found
dead the next day. The subcutaneous injection of a rabbit in which
artificial respiration was instituted with 0.36 gram of caffein per
kilo proved fatal soon after the injection without any manifestation of
symptoms. Small doses of caffein, 0.1 gram, given to a rabbit weighing
3,670 grams, failed to produce any visible effects. Doses under 0.1
gram per kilo likewise failed to induce any symptoms in dogs. When
0.1 gram of caffein per kilo was given by mouth or subcutaneously it
was followed by restlessness, salivation, rigidity of hind legs, and
vomiting. In both instances the dogs recovered. The symptoms were more
severe when the dose was increased to 0.185 gram per kilo, but even
in this case the dog recovered. A dose of 0.2 gram per kilo, however,
proved fatal.
Henneguy(36) (1875) experimented on three frogs to which he gave 0.01
gram of caffein citrate subcutaneously. He observed mild stimulation
of the nervous system and of the muscles, as well as increased cardiac
activity. Later, voluntary movement and respiration disappeared and
sensations diminished, but convulsions of the extremities appeared.
Cardiac activity was then diminished, the heart being finally arrested
in systole. Since the motor nerves retained their irritability even
after the reflexes disappeared, he concluded that the loss of motion
was due to the action of caffein on the nerve centers.
Binz(11) (1878) reported experiments on dogs and also made some
observations on man with caffein. The subcutaneous injections of 0.2
gram caffein may prove fatal to dogs, although some survive such a
dose. The toxic dose in man varies from 0.5 to 1.5 grams. Disturbance
of the circulation, such as palpitation of the heart and fullness of
pulse, restlessness, and diarrhea were the symptoms he observed.
Extensive investigations on the action of caffein were carried out by
Leblond(50) (1883), who studied its effect on the circulation in man
and lower animals, and its toxicity in the lower animals alone. Five
to twenty centigrams of caffein and 0.06 to 0.25 gram of salicylate
of soda were dissolved and injected into the muscles of the thigh
of young guinea pigs weighing a little over 300 grams. In the three
experiments reported the death of the animals occurred after 23
minutes, 40 minutes, and 1 hour and 20 minutes. Symptoms appeared in
from 10 to 15 minutes after the injection of caffein. Incoordination of
movements, convulsions, both tonic and clonic, opisthotonos, tremors,
increased frequency of respiration, ataxia, paralysis were the symptoms
observed. It is worthy of note that the appearance of paresis preceded
the convulsions. Diminished sensation was reported in one pig, but no
sensory disturbances nor reflexes had been observed in the other. Two
rabbits, one of which received 0.5 and the other about 0.3 gram of
caffein per kilo with equal parts of salicylate of soda, were injected
subcutaneously into the thigh. Diminished sensation, paresis of the
posterior extremities, hyperexcitability, convulsions, opisthotonos,
dilation of the veins of the ear were observed. Death followed in 1
hour and 23 minutes in one rabbit and in 3 hours and 7 minutes in the
other.
Filehne(22) (1886) experimented with caffein on _Rana esculenta_ and
_Rana temporaria_. The subcutaneous injection of 7 mg of caffein into
_Rana esculenta_ caused tetanus, while 50 mg given by mouth caused
tonic spasms. He further stated that the difference between _Rana
esculenta_ and _Rana temporaria_ as regards the reaction to caffein was
one of degree only.
Amat(3) (1889) reported experiments on three guinea pigs, in which 0.4
to 0.5 gram per kilo injected subcutaneously proved fatal within 38 and
44 minutes. One guinea pig which received 0.1 gram of caffein per kilo
survived. The symptoms observed in the two fatal cases were general
muscular rigidity and convulsions.
Parisot(68) (1890) made a study of the toxicity of caffein on different
species of animals. Unlike most of his predecessors, however, he
reported, at least in some cases, the weight of the animals on which
he worked. After the subcutaneous and intramuscular injections of from
5 to 20 mg of caffein into _Rana temporaria_ weighing from 14 to 16
grams, he noticed increased irritability at first; later, a loss of
reflexes, inability to use the muscles, complete muscular rigidity
resembling rigor mortis, and also cessation of heart action. The effect
of caffein produced in the green frog was analogous to that observed
in strychnin poisoning. Parisot found, however, that muscular rigidity
developed, although very gradually, also in the green frog, but it
set in much later than in frogs of the other species and without
superseding the clonic convulsions. According to Parisot, the muscular
rigidity after caffein persists after the destruction of the brain and
spinal cord, thus showing that it is not of nervous origin. He further
emphasized the difference in the behavior of these two species of frogs
toward caffein by stating that he never observed tetanic convulsions
in the red frog. His experiments also indicate that the green frog is
more resistant to caffein than _Rana temporaria_, as the same doses
which are fatal for the latter were only toxic for _Rana esculenta_.
The number of experiments, however, is too few to justify a positive
conclusion on this point. Parisot also made some experiments on
turtles. The results he obtained show that caffein is at least as toxic
for these animals as for the frogs he experimented upon, 0.33 gram per
kilo (carapace not included in weight) having proved fatal within 24
hours. Two experiments on one pigeon were also reported by the same
observer; two doses of 0.06 gram per kilo given at an interval of four
hours caused mental depression and muscular rigidity, but the pigeon
survived.
Experiments with caffein on the human subject made by Parisot showed
that man is far more susceptible to this substance than the other
animals he investigated. After the ingestion of 0.3 gram of caffein
symptoms of intoxication pointing to cerebral disturbance appeared,
which became more marked when the size of the doses was increased.
It will be noticed that the nature of the action of caffein, whether
it is a nerve or a muscle poison, formed the subject of several
investigations. Binz(11) (1890) brought forward additional evidence
in support of the view that caffein acts primarily on the ganglion
cells, and not on the muscle directly. This he has shown by injecting
0.5 gram into each of two rabbits after cutting the sciatic nerve
on one side; in one case he also resected the obdurator and crural
nerves on the same side. Clonic spasms developed in both subjects soon
after caffein was given, but in each rabbit the side operated upon
remained paralyzed. Baldi(8) (1891) studied the action of caffein on
_Rana esculenta_. After injecting from 4 to 20 mg tetanus, such as
observed in strychnin poisoning, was noticed. Fröhner(26) (1892) made
observations on the comparative toxicity of caffein in domesticated
animals. After the administration of 5 grams of caffein sodium
salicylate by mouth to a dog weighing 10 kilos, he noticed salivation,
restlessness, vomiting, and convulsions as in strychnin poisoning.
Death occurred three hours after the drug was given. On autopsy he
noticed mild inflammation of the mucous membranes of the stomach and
intestines and edema of the lungs; the heart was in diastole. A dose
of 2 grams of caffein sodium salicylate given to the same animal
subcutaneously two days previously provoked only very slight symptoms.
The subcutaneous injection of 10 grams of the same preparation into
a pig weighing 30 kilos caused death in two and a half hours, with
the production of symptoms of disturbance of the nervous system and
of gastrointestinal irritation. The same dose per kilo of body weight
given to a goat likewise caused death in two and a half hours after its
administration. Examination on autopsy revealed inflammation of the
gastrointestinal tract. Similar lesions were found in a horse killed by
100 grams of caffein, in which he also noticed hemorrhage of the mucosa
in the fundus of the stomach.
Gourewitch(28) (1907) conducted experiments with caffein on rabbits,
pigeons, and white rats. It appears from his protocol that single doses
of about 0.2 to 0.25 gram caffein per kilo given subcutaneously proved
to be fatal. He states, however, that the resistance to caffein was
markedly diminished, when its administration was repeated daily, for
much smaller amounts sufficed to cause death in these animals. A dose
of 120 mg of caffein per kilo proved fatal after the third injection.
When the dose was increased to 170 mg per kilo, the animal succumbed to
the effects of caffein after the second injection. His experiments on
the other animals do not indicate the degree of resistance to caffein,
since the weights for some were not given while for the others no
attempt was made to determine the minimum toxic or fatal dose.
Maurel(55) (1907) studied the influence of different methods of
administration on the toxicity of caffein on frogs and rabbits. He
determined the minimum toxic and lethal doses of caffein hydrobromid
which he employed in 1 to 2 per cent solutions. He concluded from his
experiments that the toxicity of caffein when given by mouth is twice
as great for the frog as for the rabbit.
More recently Hale(33) carried out a number of experiments on guinea
pigs in which he determined the toxicity of caffein given in the
form of the citrate and made into a pill with mucilage of acacia and
arrow-root starch. After the pill was dried it was fed to the animal,
due precaution being taken that none of it was lost during feeding.
From experiments on guinea pigs which received doses of 0.3 to 0.6 gram
caffein citrate, the following data have been reported: Three decigrams
per kilo given to one pig was not fatal. Of three pigs which received
0.4 per kilo, one died and two survived. Exactly the same results were
obtained in three others which received 0.5 per kilo. Two guinea pigs,
which received 0.55 and 0.6 per kilo each, died after 15 and 7 hours,
respectively, while another animal survived a dose of 0.45 per kilo.
This review of the literature on the toxicity of caffein, although
bearing evidence of considerable investigation and extending over
three-quarters of a century, is largely qualitative in character. It
appears from the experiments that the main object of the investigations
was to ascertain the nature of the action of caffein, whether it is a
muscle or a nerve poison. The comparative toxicity in different species
of animals by the accurate determination of the toxic and fatal doses
received but little attention. To fill the gap in our knowledge of the
toxic effects of caffein, the present investigation was undertaken.
This, it will be seen, proved to be a most laborious task, because
in the large number of experiments careful observations showed that
individuals of the same species varied considerably in their reaction
to the drug. Numerous other factors, as will be shown, were also found
to play an important part in the determination of the toxicity of
caffein.
ACUTE CAFFEIN INTOXICATION.
The object of these experiments was to determine the resistance to
caffein in various species of animals and by various methods of
administration. Caffein was therefore given by mouth and injected
subcutaneously into the peritoneal cavity, into the muscles, and
intravenously. As far as could be judged by appearance, healthy
animals were selected for the subjects of the experiments, but as it
is impossible to diagnose with any degree of accuracy the condition of
the animal while it is alive, post mortem examinations were resorted
to in many cases in which the issue of the experiment was fatal. Since
the age of the animal may modify toxicity full grown, as well as young,
animals were employed for these experiments; diet, race, and season
also play an important part in determining the toxicity of a drug and
these factors were also taken into account in the present investigation.
EXPERIMENTS ON RABBITS.
Animals of different varieties were used and were given caffein by
all of the methods indicated in the preceding paragraph. Some of the
rabbits employed in these experiments received oats, others received
a diet exclusively of carrots for several days or weeks previous to
the administration of caffein. The experiments were conducted at all
seasons of the year.
SUBCUTANEOUS INJECTION.
From a study of the literature on the toxicity of caffein it seemed
that about 150 mg per kilo is probably the lethal dose for the rabbit
when the drug is injected subcutaneously. Preliminary observations
were therefore carried out with such a dose, but it was found, on the
contrary, that this amount per kilo was hardly sufficient to induce
symptoms in the great majority of cases.
SERIES A.
[Doses of 147 to 167 mg of caffein per kilo were employed in these
experiments.]
_Rabbit 332. Belgian hare, female. Weight, 1,070 grams. Diet, oats._
March 25: 8.5 cc 2 per cent caffein (158 mg per kilo) injected
subcutaneously at 2.15 p. m.; 4 p. m., reflexes increased; 5.45 p. m.,
increases of reflexes still more marked.
March 26: Rabbit looked normal; no symptoms observed.
_Rabbit 331. Belgian hare, female. Weight, 1,170 grams. Diet, oats._
March 25: 2.15 p. m., 9 cc 2 per cent caffein (153 mg per kilo)
injected subcutaneously; 4 p. m., reflexes increased; 5.45 p. m.,
condition the same.
March 26: Rabbit looks normal; no symptoms observed.
_Rabbit 328. Belgian hare, female. Weight, 1,200 grams. Diet, oats._
March 25: 9 cc 2 per cent caffein injected subcutaneously (150 mg per
kilo); 4 p. m., reflexes increased; 5.45 p. m., reflexes increased but
not markedly.
March 26: No symptoms; rabbit looks normal.
_Rabbit 322. White female. Weight, 1,065 grams. Diet, oats._
March 17: 8 cc 2 per cent caffein (150 mg per kilo) injected
subcutaneously at 11.55 a. m.; 12.55 p. m., reflexes increased, but no
tetanus nor any other symptoms.
March 18: Rabbit running around in cage; condition apparently normal.
March 25: Condition of rabbit good.
_Rabbit 217. White. Weight, 1,355 grams. Diet, oats._
October 29: 10 cc 2 per cent caffein (147 mg per kilo) injected
subcutaneously at 1.51 p. m. 5.15 p. m., rabbit alive; survived.
_Rabbit 219. Maltese. Weight, 1,820 grams. Diet, oats._
October 29: 14 cc 2 per cent caffein injected subcutaneously at
1.40 p. m. (153 mg per kilo); 5.15, rabbit alive; survived.
_Rabbit 194. White female. Weight, 1,490 grams. Diet, oats._
October 14: 13 cc 2 per cent caffein (174 mg per kilo) injected
subcutaneously; increased reflexes and tremors were observed.
October 15: Condition of rabbit good; no symptoms.
_Rabbit 191. Brown male. Weight, 1,915 grams. Diet, oats._
October 14: 16 cc 2 per cent caffein (167 mg per kilo) injected
subcutaneously; reflexes increased and tremors present.
October 15: Condition of rabbit good.
A study of this series shows that about 150 mg of caffein per kilo
caused increased reflexes within one to two hours after injection.
When the dose was increased, as in rabbits 194 and 191, the symptoms
were more pronounced; 150 mg per kilo may be regarded as the minimum
dose which produces symptoms of nervous irritability when caffein is
injected subcutaneously. Experiments with larger doses were therefore
carried out in order to determine the minimum fatal dose.
SERIES B.
Approximately 0.2 gram of caffein per kilo was employed in these
experiments. Diet and race as possible factors which may influence the
toxicity of caffein were made the subject of study in these experiments
which were divided into two groups as shown in the table, page 25.
_Rabbit 95. Gray and white male. Weight, 1,478 grams. Diet, oats._
February 27: 11.30 a. m., 15 cc 2 per cent caffein (210 mg per kilo)
injected subcutaneously; 2.20 p. m., no symptoms, tremors observed
when handled, but not marked, reflexes slightly increased, no muscular
rigidity nor any other symptoms; 2.45 p. m., rabbit suddenly became
very restless, jumped off the table, and had convulsions; 3.45 p. m.,
rabbit died, rigor mortis set in almost immediately after death.
_Rabbit 96. Gray and white male. Weight, 1,585 grams. Diet, oats._
February 27: 16 cc 2 per cent caffein (200 mg per kilo) injected
subcutaneously at 3.40 p. m.; increased reflexes observed about one
hour after caffein was injected, but no other symptoms.
February 28: Rabbit found dead.
_Rabbit 112. Black female. Weight, 875 grams. Diet, oats._
March 18: 9 cc 2 per cent caffein (205 mg per kilo) injected
subcutaneously at 3 p. m.; 3.30 p. m., rabbit became restless, reflexes
were increased, tremors were observed, but no other symptoms; 4.15 p. m.,
rabbit had tremors, was handled but this failed to induce tetanus,
10 minutes later tetanus of short duration with recovery occurred.
March 19: 9 a. m., found dead.
_Rabbit 119. Yellow white female. Weight, 1,060 grams. Diet, oats._
April 17: 10 cc 2 per cent caffein (188 mg per kilo) injected
subcutaneously at 2.10 p. m.
April 18: Rabbit found dead.
_Rabbit 195. White female. Weight, 1,300 grams. Diet, carrots, since
October 7._
October 14: 13 cc 2 per cent caffein (0.2 gram per kilo) injected
subcutaneously at 11.15 a. m.; 2.25 p. m., rabbit had convulsions and
died. _Note_: Ulceration of rectum was noticed.
_Rabbit 208. Gray. Weight, 1,068 grams. Diet, carrots, October 7-15,
inclusive._
October 15: 10 cc 2 per cent caffein injected subcutaneously at 11
a. m.; 1 p. m., increased reflexes and tremors observed; 3.45 p. m.,
tremors were marked when rabbit was handled.
October 16: Rabbit found dead. _Note_: Looked poorly nourished.
_Rabbit 247. Belgian hare, female. Weight, 1,295 grams. Diet, oats
last 10 days before experiment._
November 10: 11 a. m., urine obtained from the bladder was acid to
litmus and did not contain sugar or albumen, 13 cc 2 per cent caffein
was injected subcutaneously; 1.30 p. m., 15 cc urine obtained was
markedly alkaline to litmus and reduced Fehling's solution; 2.30 p. m.,
reduction of urine considerable, marked tremors observed but no tetanus.
November 11: 10.30 a. m., 95 cc urine collected gave moderate reduction
of Fehling's solution, no symptoms, condition of rabbit seemed to be
good.
_Rabbit 248. Belgian hare, female. Weight, 1,305 grams. Diet, oats the
last 10 days before the experiment._
November 10: 11 a. m., urine markedly acid to litmus, no albumen,
no sugar; 13 cc 2 per cent caffein injected subcutaneously; 1.30 p. m.,
urine was slightly alkaline to litmus, no reduction of Fehling's
solution; 2 p. m., reflexes increased; 2.30 p. m., 2 cc urine obtained
from bladder, sugar abundant; 4.45 p. m., reflexes increased as before,
but no tetanus.
November 11: 10.30 a. m., urine collected showed slight reduction of
Fehling's solution; otherwise condition of rabbit was good; rabbit did
not show any effects of caffein.
_Rabbit 337. Belgian hare. Weight, 1,040 grams. Diet, carrots, March
31 to April 6, inclusive._
April 6: 3 p. m., 11 cc 2 per cent caffein injected subcutaneously in
the back (0.211 per kilo); 4.30 p. m., reflexes much exaggerated.
April 7: 8.15 a. m.; condition good, no symptoms.
_Rabbit 336. Belgian hare. Weight, 1,040 grams. Diet, carrots, March
31 to April 6, inclusive._
April 6: 3 p. m., 11 cc 2 per cent caffein injected subcutaneously into
tissues of the back.
April 7: 8.15 a. m., no symptoms, condition good.
Although symptoms appeared in rabbits of Group I (see table, page 25)
about the same time after the administration of caffein as in the
rabbits of the preceding series all of them terminated fatally 2¼
hours to 24 hours after its administration. Two of these rabbits (Nos.
195 and 208) were fed carrots for several days before the injection of
caffein, the others were fed oats. Since symptoms and death appeared
in these two rabbits about the same time as in the rest of this group
it may be concluded that caffein is not less toxic when carrots are
fed than when oats form the exclusive diet. But since rabbit No. 208
was poorly nourished and ulceration of the rectum was observed in No.
195 it is quite possible that caffein might be less toxic in normal
rabbits on this diet. This was tested in rabbits Nos. 336 and 337, both
of which seemed to be free from abnormality and were well nourished.
Since these rabbits survived and manifested mild symptoms only of
intoxication it would seem that a carrot diet decreases the toxicity of
caffein.
It was suggested, however, that another factor might be the cause of
the greater resistance to caffein in these two rabbits, namely, race.
This was tested in rabbits 247 and 248, both Belgian hares. Since the
toxicity of caffein in these two rabbits was the same as in Nos.
336 and 337, diet as a factor in acute caffein intoxication may be
disregarded. The greater resistance to caffein of these four rabbits
is in all probability due, therefore, to a difference of race. This
suggestion gained additional support from the experiments of the next
series.
SERIES C.
The object of these experiments was to determine the minimum fatal
dose for the gray rabbit and to obtain additional evidence as to
the toxicity of caffein in the several varieties of rabbits. Eight
experiments were performed, in which from 236 to 252 mg per kilo were
given. The white rabbits, three in number, received 250, 242, and 238
mg per kilo. All the others (which were Belgian hares) received from
236 to 252 mg per kilo. Two of the white rabbits were fed carrots for
one week preceding the injection of caffein. The other was fed oats.
Three of the Belgian hares were on a diet of oats, two were fed carrots
the week before the experiment with caffein.
_Rabbit 122. White, female. Weight, 2,060 grams. Diet, oats._
April 14: 25 cc of 2 per cent caffein (250 mg per kilo) in aqueous
solution injected subcutaneously in the back at 1.35 p. m.; 4.30 p. m.,
tremors, reflexes increased, condition otherwise good.
April 16: 9 a. m., found dead in cage. _Autopsy_: Liver deeply
congested; kidneys congested in cortex and medulla; stomach showed
small hemorrhagic areas, perforating ulcers in pyloric portion; small
intestine petechiated on mucosa; lungs and spleen normal.
_Rabbit 234. White, female. Weight, 1,650 grams. Diet, November 2-9,
carrots._
November 9: 10.45 a. m., 20 cc 2 per cent caffein (242 mg per kilo)
administered subcutaneously.
November 10: 9 p. m., found dead.
_Rabbit 335. Gray hare, female. Weight, 1,170 grams. Diet, March 31 to
April 7, carrots._
April 7: 9.30 a. m., 14 cc 2 per cent caffein solution (240 mg per
kilo) injected subcutaneously in the back; 10.30, reflexes much
increased, rabbit is extremely sensitive.
April 8: 9 a. m., found dead. _Autopsy_: Liver was congested and
contained several coccidiosis nodules; stomach distended with rather
dry food mass; mucosa exhibited mild catarrhal inflammation; mucosa of
intestines also slightly inflamed.
_Rabbit 249. Belgian hare, female. Weight, 1,185 grams. Diet, oats._
November 11: Urine, 5 cc, from bladder acid to litmus, no sugar,
no albumin; 11.50 a. m., 14 cc 2 per cent caffein (236 mg per
kilo) administered subcutaneously; 3.45 p. m., reflexes increased,
hyperæsthesia marked, but no tetanus, even when handled; 30 cc urine
collected at 4 p. m., reduction of Fehling's solution considerable.
November 12: 10 a. m., 8 cc urine collected, reduction heavy, only a
few cubic centimeters obtained from bladder, did not contain any sugar,
general condition of rabbit good, no symptom of caffein intoxication.
_Rabbit 321. Yellow, female. Weight, 1,135 grams. Diet, oats._
March 16, 1910: 11.50 a. m., 14 cc 2 per cent caffein (246 mg per kilo)
injected subcutaneously in the back; 2 p. m., reflexes increased, is
very sensitive, started to run when put on floor, no handling except
what was required for removal and return to cage, feces soft.
March 17: 9.30 a. m., condition good, rabbit put on floor, gait normal,
but does not care to walk.
March 18: 9 a. m., walks around when put on floor, appetite good,
condition seems to be normal.
March 25: 11 a. m., rabbit still alive, condition good.
_Rabbit 250. Belgian hare, female. Weight, 1,435 grams. Diet, oats at
least two days before the experiment._
November 11: 11 a. m., urine obtained from bladder acid to litmus, no
albumin, no sugar; 11.10 a. m., 18 cc, 2 per cent caffein (252 mg per
kilo); 3.45 p. m., reflexes and hyperæsthesia, no tetanus; 4 p. m., 60
cc urine, marked reduction of Fehling's solution.
November 12: 10 a. m., condition of rabbit good, no symptoms of caffein
intoxication, 80 cc urine collected, sugar considerable, only a few
cubic centimeters of urine obtained from bladder, no reduction of
Fehling's solution.
_Rabbit 834. Belgian hare, female. Weight, 1,270 grams. Diet, carrots,
March 31 to April 7._
April 7: 9.30 a. m., 15 cc 2 per cent caffein (240 mg per kilo)
injected subcutaneously in the back; 10.30 a. m., reflexes much
increased, rabbit extremely sensitive.
April 8: 9 a. m., condition good, no symptoms.
_Rabbit 233. White, male. Weight, 1,675 grams. Diet, carrots, November
2 to 9._
November 9: 10.50 a. m., 20 cc 2 per cent caffein (238 mg per kilo)
injected subcutaneously, no symptoms observed until 5 p. m., when
increased reflexes and hyperæsthesia were noticed, but no tetanus.
November 10: 9 a. m., paralysis of posterior extremities; died at
1 p. m.
Analysis of the results obtained in the experiments of this series and
inspection of Table I, page 25, show that all four of the rabbits which
survived doses of 236 to 252 mg of caffein per kilo were Belgian hares.
Of the four which died one only was a Belgian hare. The other three
were white rabbits. Two of these were fed oats; the other two received
carrots during seven days preceding the administration of caffein.
This diet does not seem to be a factor, therefore, in the toxicity of
caffein. Moreover, it may be observed that rabbit No. 122, which was
fed oats, died after receiving 250 mg per kilo, while rabbit No. 250
received the same diet and survived the same dose of caffein per kilo.
Experiments 234 and 334 offer another illustration that the toxicity
of caffein is not dependent upon diet, since both rabbits were fed
carrots, but the same dose of caffein caused only symptoms in one
while it proved fatal to the other. It is evident, therefore, that
the difference in resistance to caffein shown in these experiments is
in all probability due to race, the Belgian hare being more resistant
to caffein than rabbits of other varieties. Rabbit No. 335 seems to
be an exception, but the post-mortem examination showed the presence
of coccidiosis of the liver. As will be shown later, wherever this
condition prevailed even smaller doses of caffein proved fatal.
SERIES D.
To obtain additional evidence regarding the resistance of the various
races of rabbits to caffein and to ascertain the smallest dose which
is surely fatal to the gray rabbit or Belgian hare was the object of
this series of experiments. The diet in all cases consisted of oats,
which was given ad libitum excepting to rabbit No. 235, which received
carrots for one week previous to the injection of caffein. The doses
administered ranged from 267 to 300 mg per kilo and were administered
to different varieties of adult rabbits.
_Rabbit 253. Brown and black, male. Weight, 1,600 grams. Diet,
oats, November 9 to 12._
November 12: 11.30 a. m., urine from bladder acid, no albumen, no
sugar; 11.35 a. m., 22 cc 2 per cent caffein (275 mg per kilo)
injected subcutaneously; 11.45 a. m., rabbit jumped, off the table,
had convulsions, retraction of head and opisthotonos, general tremors,
anterior extremities stretched out, posterior extremities almost
normal, frequent twitchings; died at 12.15 p. m.
_Rabbit 252. Black, female. Weight, 1,335 grams. Diet, oats, November
9 to 12._
November 12: 11.30 a. m., 18 cc 2 per cent caffein (270 mg per kilo)
injected subcutaneously. Urine obtained from bladder before injection,
acid, no albumen, no sugar, color normal, tremors and great excitement
noticed about 12 noon; 4.30 p. m., when handled, showed unusual
restlessness and excitement followed by convulsions with opisthotonos;
occasional twitching, condition bad. Died 4.35 p. m.
_Rabbit 327. White, female. Weight, 820 grams. Diet, oats, March 8 to
16._
March 16: 11.45 a. m., 12 cc 2 per cent caffein (292 mg per kilo)
injected subcutaneously in the back; 2 p. m., found dead, but was still
warm. _Autopsy_: Hemorrhagic area at point of injection into spinal
muscles; subcutaneous abdominal region exhibited a large area of cheesy
purulent material; liver and spleen were engorged; bladder filled;
intestines normal.
_Rabbit 340. White and brown male. Weight, 1,465 grams. Diet, oats._
March 30: 3.20 p. m., 20 cc of 2 per cent caffein (273 mg per kilo)
injected subcutaneously in back.
March 31: 9 a. m., found dead.
_Rabbit 341. White and brown. Weight, 1,450 grams. Diet, oats._
March 30: 3.20 p. m., 20 cc 2 per cent caffein (270 mg per kilo)
injected subcutaneously in back; 4.40 p. m., found in dying condition,
had convulsions; 4.45 p. m., dead.
_Rabbit 326. White, male. Weight, 1,645 grams. Diet, oats, March 8 to
16._
March 16, 1910: 12 noon, 20 cc 2 per cent caffein (243 mg per kilo)
injected subcutaneously in the back; 2 p. m., tremors marked,
hypersensitive, started to run when put on floor; rabbit was not
handled any more than was required for his removal from and return to
cage.
March 17: 9.30 a. m., tremors still present and marked, otherwise
general condition good; no other symptoms.
March 18: 9.30 a. m., no appetite, tremors still present, general
condition poor; died about 2 p. m.
_Rabbit 235. Belgian hare, male. Weight, 1,870 grams. Diet, carrots,
November 2 to 9._
November 10: 11.05 a. m., 25 cc 2 per cent caffein (267 mg per kilo)
injected subcutaneously; reflexes increased and tremors, but no tetanus
observed; found dead next morning.
_Rabbit 316. Belgian hare, female. Weight, 860 grams. Diet, oats,
March 8 to 16._
March 16, 1910: 11.40 a. m., 12 cc 2 per cent caffein (267 mg per kilo)
injected subcutaneously in the back; 2.15 p. m., reflexes somewhat
increased, but not markedly so; walked when put on floor; gait clumsy
and slow; tremors of head observed; 2.35 p. m., rabbit lying in his
cage, posterior extremities extended and rigid, anterior extremities
flexed, head retracted; is still breathing; occasional spasms observed.
Rabbit died at 3 p. m. _Autopsy_: No lesion at point of injection in
dorsal spinal muscles; liver and spleen engorged; intestines injected;
other organs apparently normal.
_Rabbit 395. Belgian hare, male. Weight, 1,410 grams._
August 18: 1 p. m., 20 cc 2 per cent caffein (283 mg per kilo) injected
subcutaneously in the back; 4 p. m., reflexes markedly increased;
5 p. m., reflexes about the same, but no tetanus.
August 19, 9.15 a. m.: Reflexes increased markedly.
August 21, weight, 1,215 grams. Given 275 mg per kilo of caffein; no
symptoms observed.
August 23, found dead. _Autopsy_: Liver greatly engorged; stomach
fairly well distended and mucous membrane in a slightly inflammatory
condition; contents of small intestine liquid in nature, but walls of
same appeared normal; other organs normal in appearance.
_Rabbit 396. Belgian hare, female. Weight, 1,475 grams. Diet, oats._
August 18: 1 p. m., 20 cc 2 per cent caffein (272 mg per kilo) injected
subcutaneously in the back; 4 p. m., reflexes increased markedly;
5 p. m., reflexes increased markedly but no tetanus.
August 19: 10.30 a. m., reflexes still increased very markedly; rabbit
jumps when touched.
August 21: Weight, 1,245 grams. Injected subcutaneously 275 mg of
caffein per kilo; reflexes increased, posterior extremities stiff over
hour later.
August 22: 9 a. m., found dead. _Autopsy_: Thoracic organs normal in
appearance; stomach distended and mucous membrane affected with a
catarrhal inflammation; contents of stomach were covered with a shiny
mucus; contents of small intestine liquid in nature and bile stained;
liver showed a coccidial infestation; kidneys and spleen normal in
appearance.
_Rabbit 397. Belgian hare, male. Weight, 1,375 grams. Diet, oats._
August 19: 10.30 a. m., 20 cc 2 per cent caffein (290 mg per kilo)
injected subcutaneously in the back.
August 22: 9 a. m., found dead. _Autopsy_: Stomach distended with
ingesta; mucous membrane exhibited a catarrhal inflammation with
excessive secretions; major portion of intestines showed a condition
similar to that of stomach, contents consisting mainly of a shiny
mucus; liver enlarged; other organs apparently normal.
_Rabbit 398, Belgian hare, female. Weight, 1,570 grams. Diet, oats._
August 19: 10.30 a. m., 23 cc 2 per cent caffein (293 mg per kilo)
injected subcutaneously in the back; 4 p. m., found dead. _Autopsy_:
Thoracic organs seemingly normal; mucous membrane of stomach exhibited
a catarrhal inflammation generally; large intestines somewhat impacted
but walls appeared normal; other organs normal.
_Rabbit 399, Belgian hare, male. Weight, 1,725 grams. Diet, oats._
August 19: 10.30 a. m., 26 cc 2 per cent caffein (300 mg per kilo)
injected subcutaneously in the back; found dead at 4.30 p. m.
_Autopsy_: Lungs slightly congested; liver engorged and friable; gall
cyst well filled; stomach exhibited catarrhal gastritis; injection of
mesenteries and intestines; kidney showed marked cortical congestion.
The results of the experiments of this series likewise indicate that
the Belgian hare is more resistant to caffein than the rabbits of other
varieties. Thus, of the four gray rabbits (Nos. 235, 316, 395, and
396), which received 267 to 283 mg of caffein per kilo, two died and
two lived,[B] one of which, 396, showed the presence of coccidiosis of
the liver. On the other hand it will be observed that the black and
white rabbits which received from 270 to 275 mg of caffein per kilo all
died from the effects of the drug; one within 1 hour and 25 minutes
and another within 50 hours after the administration of the caffein,
while No. 340 died in the night. Furthermore it will be noted that of
the last three rabbits of this series, which were Belgian hares and
received 290, 293, and 300 mg of caffein, two died six hours after the
injection, while the other, No. 397, lived three days. The minimum
fatal dose of caffein for Belgian hares is, therefore, about 290 to 300
mg per kilo when injected subcutaneously, which is about 50 per cent
greater than for rabbits of other varieties.
[B] Survived first dose.
SERIES E.
It was shown in series A that 0.15 caffein per kilo caused symptoms of
intoxication. Before concluding, however, that this is the smallest
dose which causes symptoms of poisoning, a number of experiments were
performed with smaller doses. It was found that in the great majority
of cases 0.1 caffein per kilo may cause diuresis, but no nervous or
muscular symptoms. In some rabbits, however, even such a dose proved
fatal. Post-mortem examinations in these cases showed the presence
of coccidiosis of the liver, and it will be recalled that similar
observations were made before. It is quite possible, therefore,
that coccidiosis of the liver is an important factor in decreasing
the resistance to caffein. Experiment 551 (p. 25) shows that other
conditions may likewise increase the toxicity of caffein.
_Rabbit 325. White, female. Weight, 1,065 grams. Diet, oats._
March 17: 11 a. m., 6 cc 2 per cent (112 mg per kilo) caffein injected
subcutaneously in the back. About 5 cc of urine squeezed out from
bladder before injecting caffein.
March 17: 1 p. m., hind legs crossed and stretched out, front legs also
extended; rabbit lying stretched out on her belly.
March 17: 5.40 p. m., rabbit still alive, condition somewhat improved.
March 18: 9 a. m., found dead, stiff and cold. _Autopsy_: Hemorrhagic
area at point of inoculation; subcutaneous region of both thighs
presented a hemorrhagic infiltration of the tissues; liver contained
lesions of coccidiosis; other organs apparently normal.
_Rabbit 330. Belgian hare, female. Weight, 935 grams; poorly
nourished._
March 18: 3.35 p. m., 5 cc 2 per cent caffein (107 mg per kilo)
injected into subcutaneous tissues in the back; 5.30 p. m., no symptoms.
March 19: 9 a. m., no symptoms.
March 25: Weight, 825 grams.
_Rabbit 329. Belgian hare, male. Weight, 775 grams; poorly nourished.
Received March 18._
March 18: 3.30 p. m., 4 cc 2 per cent caffein (103 mg per kilo)
injected into subcutaneous tissues in the back; 5.30 p. m., no symptoms.
March 19: 9 a. m., no symptoms.
March 25: Rabbit alive in good condition; weight, 825 grams.
_Rabbit 320. Black, male. Weight, 1,040 grams. Diet, oats._
March 17: 11 a. m., 6 cc 2 per cent caffein (115 mg per kilo) injected
subcutaneously in the back; only a few drops of urine obtained from
bladder before injecting caffein; 1 p. m., rabbit very restless; ran
away when placed on floor; cried when touched with a piece of paper; no
tremors observed, but rabbit became exhausted and was unable to walk;
legs extended out; after running for about a minute dyspnoea was very
marked, but rabbit soon raised himself on his legs; 5.40 p. m., rabbit
up on his legs.
March 18: 9 a. m., found dead, but still warm. _Autopsy_: Lungs studded
with small grayish white nodules, adhesions to costal pleura; probably
lesions of coccidiosis; liver studded with coccidiosis nodules.
Hemorrhages at point of inoculation.
_Rabbit, 551. Gray, female. Weight, January 26, 1,650 grams. Diet,
oats; fed 20 cc of 25 per cent alcohol daily from January 26-31._
January 31: Weight, 1,450 grams; 10.20 a. m., temperature 101.6°;
10.45, a. m., temperature 101.6°; received 7 cc 2 per cent caffein
subcutaneously into back; 11.15 a. m., convulsions of short duration;
raised himself on posterior legs, anterior legs wide apart; 4.10 p. m.,
looked normal, not hypersensitive; 4.30 p. m., condition seemed to be
good.
February 1: 9 a. m., found dead, was alive at 5.30 p. m. of previous
day. _Autopsy_: Lesions found involved thoracic cavity mainly; lungs
were hepatized and a fibro plastic exudate caused them to adhere to
costal pleura; liver engorged and appeared fatty; no marked lesions
affecting digestive tract, a slight catarrh of stomach being the only
noticeable feature; kidneys and spleen normal.
TABLE 1.--_Subcutaneous injections of caffein--rabbits._
SERIES A.
----+--------+-------+-------------+--------------+-------+-----------
| |Caffein| Appearance | Duration | |
No.| Weight.| per | of symptoms | of life. | Diet. | Remarks.
| | kilo | in-- | | |
----+--------+-------+-------------+--------------+-------+-----------
|_Grams._| _Mg._ | | | |
332| 1,070 | 158 |1 hour |Survived | Oats |Gray.
| | | 45 minutes| | |
331| 1,170 | 153 | do. | do. | do. | Do.
328| 1,200 | 150 | do. | do. | do. | Do.
322| 1,065 | 150 |1 hour | do. | do. |White.
217| 1,355 | 147 | | do. | do. | Do.
219| 1,820 | 153 | | do. | do. |Maltese.
194| 1,490 | 174 | | do. | do. |White.
191| 1,915 | 167 | | do. | do. |Light brown.
----+--------+-------+-------------+--------------+-------+------------
SERIES B, GROUP I.
----+--------+-------+-------------+--------------+-------+-------------
95| 1,478 | 210 |2 hours |3 hours |Oats |White.
| | | 50 minutes | 10 minutes | |
96| 1,585 | 200 |1 hour |About 18 hours| do. |Gray white.
112| 875 | 205 |30 minutes | do. | do. |Black.
119| 1,060 | 188 | | do. | do. |Yellow white.
195| 1,300 | 200 | |3 hours |Carrots|White.
| | | | 10 minutes | |
208| 1,068 | 188 |2 hours |About 24 hours| do. |Gray.
----+--------+-------+-------------+--------------+-------+-------------
SERIES B, GROUP II.
----+--------+-------+-------------+--------------+-------+-----------
247| 1,295 | 200 |2.5 hours |Survived |Oats |Gray.
248| 1,305 | 200 |3 hours | do. | do. | Do.
337| 1,040 | 211 |1.5 hours | do. |Carrots| Do.
336| 1,045 | 211 | do. | do. | do. | Do.
----+--------+-------+-------------+--------------+-------+-----------
SERIES C.
----+--------+-------+-------------+--------------+-------+------------
122| 2,060 | 250 |2 hours |1.5 days |Oats |White.
| | | 55 minutes | | |
234| 1,650 | 242 | |About 24 hours|Carrots| Do.
335| 1,170 | 240 |1 hour | do. | do. |Gray
| | | | | |coccidiosis.
249| 1,185 | 236 |4 hours |Survived |Oats |Gray.
321| 1,135 | 246 |2 hours | do. | do. |Yellow.
| | | 10 minutes | | |
250| 1,435 | 252 |4 hours | do. | do. |Gray.
| | | 35 minutes | | |
334| 1,270 | 240 |1 hour | do. |Carrots| Do.
233| 1,675 | 238 |6 hours |26 hours | do. |White.
| | | 10 minutes | | |
----+--------+-------+-------------+--------------+-------+------------
SERIES D.
----+--------+-------+-------------+--------------+-------+-----------
253| 1,600 | 275 |10 minutes |35 minutes |Oats |Brown and
| | | | | | black.
252| 1,335 | 270 |30 minutes |4 hours | do. |Black.
| | | | 55 minutes | |
327| 820 | 292 | |2 hours | do. |White.
| | | | 15 minutes | |
340| 1,465 | 273 | |About 18 hours| do. |White and
| | | | | | brown.
341| 1,450 | 270 | |1 hour | do. | Do.
| | | | 25 minutes | |
326| 1,645 | 243 |2 hours |50 hours | do. |White.
235| 1,875 | 267 | |20 hours |Carrots|Gray.
316| 860 | 267 |2 hours |3 hours |Oats | Do.
| | | 45 minutes | 20 minutes | |
395| 1,410 | 283 |3 hours |Survived | | Do.
395| 1,215 | 275 | do. |About 2 days |Oats | Do.
396| 1,475 | 272 | do. |Survived | do. | Do.
396| 1,245 | 275 |1 hour |About 18 hours| do. | Do.
397| 1,375 | 290 | |3 days | do. | Do.
398| 1,570 | 293 | |5.5 hours | do. | Do.
399| 1,725 | 300 | |6 hours | do. | Do.
----+--------+-------+-------------+--------------+-------+-----------
SERIES E.
----+--------+-------+-------------+--------------+-------+------------
325| 1,065 | 112 |2 hours |Less than | Oats |White female.
| | | | 22 hours | |
330| 935 | 107 |None |Survived | |Gray.
329| 775 | 103 |do. | do. | |Gray male.
320| 1,040 | 115 |2 hours |46 hours | Oats |Black male.
551| 1,450 | 100 |30 minutes |Less than | do. |Gray female.
| | | | 24 hours | |
----+--------+-------+-------------+--------------+-------+------------
ADMINISTRATION BY MOUTH.
These experiments were carried out on two varieties of rabbits, the
white and the gray. The diet consisted chiefly of oats, but in a few
cases carrots formed the exclusive diet. Food and water were given ad
libitum. A 2 per cent solution of caffein was administered through a
stomach tube. Since the resistance to most drugs is commonly supposed
to be greater when given by mouth than when administered by any
other path, doses of 175 to 200 mg per kilo were fed in a series of
preliminary experiments, all of which were performed on gray rabbits
weighing from 865 to 1,135 grams, and which were fed carrots for
several days previous to the experiment. Three of the rabbits survived,
two without showing any symptoms; in the other case paralysis of the
posterior extremities was observed five hours after he received caffein
and he was found dead the next morning. Unfortunately no autopsy was
performed. The low resistance to caffein of this animal was probably
due to some abnormal condition which developed about the time of the
experiment, since this rabbit received 325 mg of caffein per kilo
two weeks previously and increased reflexes only were observed as a
result of this treatment. Hence 200 mg of caffein per kilo can not
be considered the toxic dose when fed by mouth. In the following
experiments larger doses were therefore given.
SERIES A.
_Rabbit 248. Belgian hare. Weight, 1,170 grams. Diet, oats._
November 17: 1.20 p. m., 19.5 cc 2 per cent caffein (330 mg per kilo)
administered by the mouth; 4.30 p. m., somewhat hypersensitive.
November 19: No symptoms; at 9 a. m., urine collected, no reduction of
Fehling's solution; rabbit survived.
_Rabbit 241. White male. Weight, 1,380 grams. Diet, oats._
November 17: 1.15 p. m., 20 cc 2 per cent caffein (290 mg per kilo)
administered by the mouth; 4.30 p. m., some hypersensitiveness, but no
other symptoms.
November 18: 9 a. m., urine collected, no reduction of Fehling's
solution; no symptoms; rabbit survived.
_Rabbit 249. Belgian hare. Weight, 890 grams. Diet, oats._
November 17: 1.30 p. m., 14.5 cc 2 per cent caffein (325 mg per kilo)
administered; 4.30 p. m., hypersensitiveness; no other symptoms.
November 18: 10 a. m., no symptoms; urine collected, no reduction;
rabbit survived.
SERIES B.
The object of these experiments was to determine the minimum fatal dose
of caffein in the two varieties of rabbits, the white and the gray. All
of the animals selected were approximately of the same weight.
_Rabbit 239. Belgian hare, male. Weight, 935 grams. Diet, oats._
November 19: 4 p. m., 17 cc 2 per cent caffein (363 mg per kilo)
administered by mouth, followed by 10 cc of 0.9 per cent salt solution.
November 20: Urine examined, no sugar found, no symptom noticed at any
time after injection.
_Rabbit 254. Belgian hare, female. Weight, 975 grams. Diet, oats._
November 19: 4.05 p. m., 18 cc 2 per cent caffein (369 mg per kilo)
administered by mouth, followed by 10 cc of 0.9 per cent salt solution.
November 20: 9 a. m., rabbit found dead.
_Rabbit 267. White. Weight, 1,050 grams. Diet, oats._
November 23: 12.10 p. m., 18 cc 2 per cent caffein (342 mg per
kilo) given by mouth, followed by 18 cc salt solution; 1 p. m.,
increased reflexes, tremors marked but no tetanus; 1.05 p. m., rabbit
stretched on abdomen, posterior extremities in extended position and
paralyzed, soon after clonic spasms set in, which recurred about every
minute; 1.14 p. m., tetanus and death. _Autopsy_: Liver showed fatty
degeneration; slight inflammation of stomach and intestines; other
organs normal.
_Rabbit 268. White. Weight 1,100 grams. Diet, oats._
November 23: 20 cc 2 per cent caffein (363 mg per kilo) administered
by mouth, followed by 20 cc salt solution; 1.15 p. m., somewhat
hypersensitive; 4.30 p. m., tremors fairly marked, no urine passed,
about 2 cc of bloody looking urine obtained from bladder, which
contained albumen and a considerable amount of glycogen; rabbit died.
_Rabbit 419, Belgian hare, male. Weight, 1,600 grams. Diet, oats._
September 26: 10 a. m., 28 cc 2 per cent caffein (350 mg per kilo)
given by mouth; reflexes increased at 4 p. m.; 6 p. m., reflexes still
increased, no other symptoms.
September 27: 9 a. m., found dead. _Autopsy_: Lungs, liver, and kidneys
congested; other organs normal.
_Rabbit 420. Belgian hare, male. Weight, 1,250 grams. Diet, oats._
September 26: 10 a. m., 22 cc 2 per cent caffein (352 mg per kilo)
given by mouth; 11.35 a. m., convulsions; 12 noon, found dead.
_Autopsy_: Liver showed very extensive coccidiosis; no other lesions.
_Rabbit 421. Belgian hare, male. Weight, 1,485 grams. Diet, oats._
September 26: 10 a. m., 26 cc 2 per cent caffein (351 mg per kilo)
administered by mouth; 4 p. m., reflexes increased; 6 p. m., reflexes
as before, no tetanus observed.
September 27: 9 a. m., rabbit found dead. _Autopsy_: Congestion of
lungs and kidneys; liver congested and slightly fatty.
_Rabbit 424. White, male. Weight, 1,295 grams. Diet, oats._
September 26: 2 p. m., 19 cc 2 per cent caffein (293 mg per kilo)
administered by mouth; 4 p. m., reflexes increased, no other symptoms;
6 p. m., no change since 4 p. m.
September 27: 12 noon, convulsions and death. _Autopsy_: Congestion of
the lungs; no other lesions.
_Rabbit 423. White, male. Weight, 1,205 grams. Diet, oats._
September 26: 2 p. m., 18 cc 2 per cent caffein administered by mouth;
4 p. m., reflexes increased, no tetanus; 6 p. m., condition unchanged
since 4 p. m.
September 27: 9 a. m., found dead. _Autopsy_: Lungs, liver, and kidneys
congested; other organs normal.
_Rabbit 422. White, male. Weight, 1,440 grams. Diet, oats._
September 26: 2 p. m., 21 cc 2 per cent caffein (291 mg per kilo) given
by mouth; reflexes increased at 4 p. m.
September 27: 3 p. m., alive, no symptoms; 4 p. m., convulsions with
recovery, this was soon followed by a violent attack of tetanus, which
lasted about one minute and was succeeded by paralysis; rabbit died
at 4.30. _Autopsy_: Liver slightly congested; a small portion of the
intestine showed congestion and edema; other organs normal.
A study of these experiments shows also considerable variation in the
toxicity of caffein when given by mouth. In some cases a dose of 300
mg per kilo, and even less, caused death, as in rabbits 423 and 424.
In other rabbits, however, approximately the same doses of caffein
produced increased reflexes only. The same symptoms were produced in
Nos. 248 and 249 after the administration of 325-330 mg of caffein
per kilo, while another rabbit (No. 239) survived a dose of 363 mg
per kilo. That this is exceptional, however, appears from the result
of the following experiments on rabbits Nos. 419, 420, and 421, all
of which died after receiving 350 mg of caffein per kilo, and rabbits
267 and 268, to which doses of 363 and 342 mg, respectively, per kilo
proved fatal. It will be observed further that the gray rabbits are
more resistant to caffein than the white animals, as 350 mg per kilo
was the smallest fatal dose for rabbits 419, 420, and 421, all of which
were gray rabbits, while a dose of 290 mg per kilo was fatal for some
of the white rabbits. Again, it will be noticed that of the two gray
rabbits, Nos. 254 and 239, which received the largest doses in these
experiments, namely, 369 and 363 mg, respectively, one survived. The
largest doses given to the white rabbits were 363 and 342 mg caffein
per kilo. Both of these died from the effects of the drug. It may be
concluded, therefore, that the minimum toxic dose for the gray rabbit
is about 325 mg of caffein per kilo, and the minimum fatal dose is at
least 350 mg per kilo. It is to be remarked in this connection that
post-mortem examination showed extensive coccidiosis in rabbit 420
and fatty liver in No. 421, while the macroscopical examination of
the organs of Nos. 424 and 423 failed to show the presence of such
abnormalities. Since, as was observed in the section on subcutaneous
injection and elsewhere in this investigation, pathological changes are
apt to decrease the resistance to caffein, it is quite possible that
350 mg per kilo is not the minimum fatal dose for the normal rabbit.
Indeed, the experiment on rabbit 239 lends support to this view, thus
furnishing additional evidence of difference in the resistance to
caffein in the two varieties of rabbits.
TABLE 2.--_Administration of caffein by mouth._
SERIES A.
------+--------+-------+-----------+--------------+-------+-----------
Rabbit| |Caffein| | Duration | |
No. | Weight.| per | Symptoms | of life. | Diet. |Remarks.
| | kilo. | | | |
------+--------+-------+-----------+--------------+-------+-----------
|_Grams._| _Mg._ | | | |
248 | 1,170 | 330 |3 hours |Survived |Oats |Gray.
| | | 10 minutes| | |
241 | 1,380 | 290 |2 hours | do. | do. |White male.
| | | 15 minutes| | |
249 | 890 | 325 |3 hours | do. | do. |Gray male.
------+--------+-------+-----------+--------------+-------+-----------
SERIES B.
------+--------+-------+-----------+--------------+-------+-----------
239 | 935 | 363 | |Survived |Oats |Gray male.
254 | 975 | 369 | |About 2 days | do. |Gray female.
267 | 1,050 | 342 |50 minutes |1 hour | do. |White.
| | | | 4 minutes | |
268 | 1,100 | 363 | |About 3 hours | do. | Do.
419 | 1,600 | 350 |6 hours |Less than | do. |Gray male.
| | | | 24 hours | |
420 | 1,250 | 352 |1 hour |2 hours | do. | Do.
| | | | 35 minutes | |
421 | 1,485 | 351 |6 hours |Less than | do. | Do.
| | | | 24 hours | |
424 | 1,295 | 293 |2 hours |22 hours | do. | White male.
423 | 1,205 | 300 | do. |Less than | do. | Do.
| | | | 19 hours | |
422 | 1,440 | 291 | do. |2½ hours | do. | Do.
------+--------+-------+-----------+--------------+-------+------------
INJECTION INTO THE PERITONEAL CAVITY.
In a number of experiments caffein was introduced into the peritoneal
cavity. Rabbits of different varieties receiving a diet of oats or
carrots were employed for this purpose; food and water were given ad
libitum. The minimum doses required to induce symptoms or cause death
in these animals were determined; tests with caffein were also made on
young rabbits in order to study the influence of age on the resistance
to this substance. The results are shown in the following experiments:
SERIES A.
In this series large doses were administered, approximating 0.3 gram
per kilo.
_Rabbit 71. Gray female. Weight, 1,659 grams._
January 20: 2.20 p. m., 25 cc aqueous solution 2 per cent caffein (300
mg per kilo) were injected into the peritoneal cavity; 3.45 p. m., when
doors of cage were opened rabbit had spasm of short duration.
January 21: Rabbit found dead.
_Rabbit 72. Gray and white. Weight, 1,402 grams._
January 21: 11.17 a. m., 20.2 cc (aqueous solution) of 2 per cent
caffein (300 mg per kilo) injected into peritoneal cavity from burette;
11.25 a. m., paralysis; 11.30 a. m., rabbit had convulsion when picked
up from the floor, followed by several spasms later; 11.35 a. m.,
typical tetanus; 12.30 noon, found dead.
_Rabbit 61. Black female. Weight, 2,143 grams._
January 19: 40 cc 2 per cent caffein, aqueous solution (300 mg per
kilo), injected into peritoneal cavity from burette; tetanus when about
30 cc were injected; when removed from holder, repeated and violent
convulsions, terminating in death.
SERIES B.
The object of these experiments was to determine the minimum lethal
dose; 0.2 to 0.15 gram of caffein per kilo was injected into the
rabbits of this series.
_Rabbit 69. White female. Weight, 1,714 grams._
January 20: 10.15 a. m., 6 cc 2 per cent caffein, aqueous solution,
injected into peritoneal cavity. No symptoms, under observation for 45
minutes, rabbit defecated rather copiously; feces were soft; 11 a. m.,
6 cc 2 per cent caffein, aqueous solution, injected into peritoneal
cavity, no symptoms, under observation for 40 minutes; 11.40, 6 cc 2
per cent caffein injected into peritoneal cavity; 11.45, rabbit is
restless, reflexes increased.
_Rabbit 70. Gray and white female. Weight, 1,487 grams._
January 20: 1.30 p. m., 15 cc 2 per cent aqueous solution of caffein
(0.2 gram per kilo) injected into the peritoneal cavity; 2.20 p. m., no
symptoms.
January 30: About 2 p. m. rabbit died.
February 1: _Autopsy_: Cirrhosis of the liver; enteritis of small
intestines; stomach and kidneys normal.
_Rabbit 93. Maltese, male. Weight, 1,197 grams._
March 2: 11.30 a. m., 12 cc of 2 per cent caffein (200 mg per kilo)
injected into peritoneal cavity; 11.35 a. m., while being released from
holder, tetanus occurred, repeated attacks later, clonic convulsions
with tonic rigidity of posterior extremities during the attacks as
well as during intervals, anterior extremities were relaxed during the
intervals between the attacks, opisthotonos of cervical region but
kyphosis in lumbar region were observed, no salivation nor dilatation
of the pupils; 2 p. m., rabbit died.
_Rabbit 92. Yellow female. Weight, 1,388 grams._
February 25, 4.15 p. m., 14 cc 2 per cent caffein (0.2 gram per kilo)
injected into peritoneal cavity; 4.20 p. m., restlessness and increased
reflexes, rabbit found stretched out in cage, but raised himself on his
legs again; 4.45, general tremor when touched.
February 26: 9 a. m., rabbit found dead. _Autopsy_: Hemorrhage into
abdominal muscles at site of injection; hemorrhage also in walls of
stomach opposite similar spot in abdominal wall.
_Rabbit 309. Belgian hare, female. Weight, 1,500 grams. Diet, oats._
March 2: 2.05 p. m., 2 per cent caffein solution (0.2 gram per kilo)
injected into peritoneal cavity; 2.25 p. m., found dead, no urine found
in bladder.
_Rabbit 307. Belgian hare, female. Weight, 1,320 grams. Diet, oats._
March 2: 12 noon, urine obtained from bladder, clear amber colored, no
albumin, no reduction; 12.06 p. m., 10 cc of 2 per cent caffein (0.151
gram per kilo) injected into peritoneal cavity; 1.30 p. m., rabbit
placed on floor, runs around but anterior and posterior extremities
soon extended, in tonic condition; 2.25 p. m., reflexes increased,
paralysis of extremities, dyspnoea; 4.50 p. m., about 100 cc urine
collected, no albumin, reduction of Fehling's solution moderate.
March 3: 9.30 a. m., posterior extremities extended and rigid,
anterior extremities paralyzed, respiration less frequent and deeper
than normal. Rabbit died at 11.50 a. m.; urine collected since 4.50
p. m. previous day gave very heavy reduction of Fehling's solution.
_Autopsy_: Animal in good condition; in the left axillary region was
observed a hemorrhage into the subcutaneous and muscular tissue of
that region. The ventral portion of the large colon, in contact with
the ventral abdominal wall, showed a hemorrhagic area about one-half
inch in length, such as might be produced by a puncture or bruise of
the colon through the abdominal wall. A small portion of the small
intestine adjacent to the colon was affected in a similar manner. All
internal organs were apparently normal.
_Rabbit 308. Belgian hare, female. Weight, 1,350 grams. Diet, oats._
March 2: 11.45 a. m., urine obtained from bladder, no albumen, no
reduction; 11.50 a. m., 10 cc 2 per cent caffein (0.15 gram per kilo)
injected into peritoneal cavity; 1.30 p. m., not very active, no
abnormal symptoms otherwise; 3.30 p. m., rabbit looked depressed, made
very little attempt to move about, remained in one position most of
the time when placed on floor; 4.30 p. m., 180 cc urine collected, no
albumen, reduction of Fehling's solution moderate.
March 3: 9.30 a. m., rabbit looks normal, is able to walk but is easily
fatigued when made to walk about or when placed on his side, followed
by paralysis of anterior extremities, posterior extremities apparently
normal, about 90 cc of urine collected at noon was free from albumen,
did not reduce Fehling's solution.
March 4: 11 a. m., lying on his side in cage, anterior extremities
limp, posterior extremities extended and rigid, is in dying condition.
March 5: 9 a. m., found dead. _Autopsy_: Liver engorged; spleen
congested, but not enlarged; kidneys, some congestion in cortex;
stomach filled, mucosa thickened and easily pulled off; petechial
hemorrhages on serosa of colon.
SERIES C.
The experiments of this series were made to determine the minimum toxic
dose.
_Rabbit 295. Belgian hare, female. Weight, 1,205 grams. Diet,
carrots._
March 1: 10.40 a. m., 6 cc 2 per cent caffein (0.1 gram per kilo)
injected into peritoneal cavity; about 2 cc of urine obtained before
injecting caffein; 2 p. m. 100 cc urine, bloody in appearance,
collected, a moderate quantity of albumen present, no reduction; 3.40
p. m., no symptoms.
March 4: 2 p. m., rabbit looks well.
_Rabbit 293. Belgian hare, female. Weight, 1,605 grams. Diet, carrots._
March 1: Urine from bladder clear, alkaline; 11.55 a. m., 8 cc 2 per
cent caffein (0.1 gram per kilo) injected into peritoneal cavity;
3 p. m., 90 cc urine normal in color collected, no albumen, no
reduction; 3.40 p. m., no symptoms.
March 4: 1.15 p. m., rabbit looks normal.
_Rabbit 292. Belgian hare, male. Weight, 1,595 grams. Diet, carrots._
March 1: 10.10 a. m., 8 cc 2 per cent caffein (0.1 gram per kilo)
solution injected into peritoneal cavity; 10.40 a. m., rabbit urinated,
reflexes increased, but no other symptoms; 10.50 a. m., no urine
obtained from bladder; 2 p. m., 105 cc of clear pale urine collected;
no albumen, no reduction; 3.40 p. m., no symptoms.
March 4: 2 p. m., rabbit looks well, urine collected, did not contain
sugar.
_Rabbit 298. Belgian hare, female. Weight, 1,205 grams. Diet, carrots._
March 1: 4.06 p. m., 7.5 cc 2 per cent caffein solution (0.125 gram
per kilo) injected into peritoneal cavity, urine obtained from bladder
immediately after injection, no albumen, no reduction; 5.30 p. m.,
reflexes increased, rabbit was able to run around, but became paralyzed
soon; 5.40 p. m., rabbit is again able to run around.
March 3: 10 a. m., anterior extremities paralyzed, is able to use
posterior extremities.
March 4: 1 p. m., rabbit looks normal.
_Rabbit 223. Belgian hare, male. Weight, 1,165 grams. Diet, carrots._
March 1: 3.50 p. m., urine obtained from bladder clear, amber colored,
no albumen, no sugar; 3.52 p. m., 7.5 cc 2 per cent caffein (125 mg per
kilo) injected into peritoneal cavity; 5.40 p. m., rabbit makes little
attempt to run when put on the floor, weakness of extremities marked.
March 4: 1.15 p. m., rabbit normal.
SERIES D.
The object of the experiments of this series was to study the effect of
age on the resistance to caffein. Half-grown rabbits were, therefore,
used in the following experiments.
_Rabbit 310. Belgian hare, female. Weight, 880 grams. Diet, oats._
March 2: 3.25 p. m., 9 cc 2 per cent caffein (0.2 gram per kilo)
injected into peritoneal cavity.
March 3: 9.30 a. m., no symptoms, rabbit looks normal.
March 4: 11 a. m., posterior extremities abducted, walked when placed
on the floor, made no attempt to change attitude when placed on its
side, remained some time in this position.
March 5: 9 a. m., found dead. _Autopsy_: Liver showed areas of
degeneration; kidneys congestion and petechial hemorrhage on cortex;
small and large intestines, inflammation marked; bladder distended.
_Rabbit 75. Gray and white, female. Weight, 842 grams._
January 25: 3 p. m., 8.5 cc 2 per cent caffein solution (0.2 gram per
kilo) injected into peritoneal cavity; 3.15 p. m., anterior extremities
weak and reflexes increased.
January 27: Rabbit paralyzed but is able to turn over when placed on
back.
_Rabbit 74. Gray and white, female. Weight, 692 grams._
January 25: 3 p. m., 7 cc 2 per cent caffein (0.2 gram per kilo)
solution injected into peritoneal cavity; 3.15 p. m., reflexes
increased and anterior extremities paralyzed.
January 27: Rabbit recovered and is able to walk about in the room.
_Rabbit 312, maltese, female. Weight, 740 grams. Diet, oats._
March 3: 11.47 a. m., urine obtained from bladder, appearance normal,
no albumen, no reduction of Fehling's solution; 11.50 a. m., 7.5 cc 2
per cent caffein (0.2 gram per kilo) injected into peritoneal cavity;
2.30 p. m. anterior extremities paralyzed, posterior extremities rigid
and extended; 5 p. m. (about), rabbit died.
_Rabbit 311. Belgian hare, female. Weight, 650 grams. Diet, oats._
March 3: 11.26 a. m., urine obtained from bladder normal in appearance,
albumen considerable, reduction of Fehling's solution none; 11.27 a. m.,
6 cc 2 per cent caffein solution (0.2 gram per kilo) injected into
peritoneal cavity; 2.30 p. m., rabbit seemed to be normal, no symptoms
had developed; urine collected contained a large amount of sugar,
reduction was very heavy, but no albumen was found.
March 4: 11 a. m., condition good, moves about when put on floor; gait,
normal.
_Rabbit 78. Yellow and white. Weight 659 grams._
January 26: 1.30 p. m., 8.5 cc 2 per cent caffein (250 mg per kilo)
injected into peritoneal cavity, under observation the rest of the
afternoon, no symptoms.
January 27: 4 p. m., no symptoms developed.
_Rabbit 317. Belgian hare, female. Weight 635 grams. Diet oats._
March 15: 10.35 a. m., 8 cc 2 per cent caffein (0.252 gram per kilo)
injected into peritoneal cavity; 12 noon, marked abduction of hind
legs, was unable to walk after a little exertion, rabbit died between
12.30 and 12.50 p. m. _Autopsy_. Right lung hepatized and showed
adhesions to costal and mediastinal pleura; liver studded with nodules
of coccidiosis; spleen congested; stomach filled, mucosa normal;
intestines injected; colon hemorrhagic on serosa in ventral region,
near point of injection; kidneys normal.
_Rabbit 323. White, female. Weight 820 grams. Diet oats._
March 15: 10.45 a. m., 10 cc 2 per cent caffein (250 mg per kilo)
injected into peritoneal cavity; 12 noon, reflexes increased, hind legs
abducted but is able to walk, symptoms are mild; 1.40 p. m., tremors,
weakness, and abduction of head and legs much more marked than at 12
noon.
March 16: Condition good.
March 17: Condition good, recovery apparently complete.
Since the experiments of Series A, which were intended as preliminary
tests, have shown that 0.3 gram of caffein per kilo when introduced
into the peritoneal cavity is rapidly absorbed and is fatal, much
smaller doses were employed in subsequent trials with the drug. This
is shown in series B, which may be divided into two groups. Group I,
consisting of rabbits 69, 70, 92, 93, and 309, which received 0.2
gram of caffein per kilo, and Group II, Nos. 307 and 308, into which
0.15 gram of caffein per kilo was injected. Three rabbits of Group I
(Nos. 92, 93, 309) died from the effects of caffein; rabbit 309 twenty
minutes after injection, and rabbits Nos. 92 and 93, twenty hours and
two and one-half hours, respectively, after the administration of
caffein. In both of these rabbits symptoms appeared within five minutes
after the injections were made. Rabbits 69 and 70, it will be noticed,
survived the same amount of caffein in proportion to body weight as was
given to the other members of this group. Increased peristalsis and
the distribution of the dose may account for the greater resistance
of rabbit No. 69. The case of rabbit No. 70 is evidently one of
exceptional resistance to caffein, since both the rabbits of Group II
died from the effects of a much smaller dose, namely, 0.15 gram of
caffein per kilo. Moreover, macroscopical examination at the autopsy of
Nos. 307 and 308 failed to show any lesions which might tend to lessen
the resistance to caffein.
That a dose of 0.15 gram per kilo is therefore in all probability the
minimum fatal dose for the rabbit when injected into the peritoneal
cavity appears from the results of the experiments in series C, in
which smaller doses, 0.125 gram of caffein per kilo caused mild
symptoms only, while 0.1 gram per kilo rarely induced any symptoms.
It may be remarked that the rabbits of series C were fed carrots
while rabbits Nos. 307 and 308 received oats. Their resistance to
caffein may be different, but, as was pointed out in the earlier part
of this investigation, diet does not seem to influence the toxicity
of the single dose of caffein. Doses of 150 and of 100 to 125 mg per
kilo, when injected into the peritoneal cavity, may be considered,
respectively, as the minimum fatal and minimum toxic doses for the gray
rabbit. Analysis of the experiments in series D shows much greater
resistance to caffein than in the other rabbits which received it
intraperitoneally. Thus, after the administration of 0.2 gram per kilo
to each of five rabbits, no effect was observed in two cases (Nos.
310, 311), while in two others (Nos. 74, 75) symptoms developed, but
they survived. Only one rabbit, No. 312, died from the effects of this
dose; the autopsy showed the presence of degeneration of the liver and
petechial hemorrhages on the cortex of the kidneys in the case of No.
310, which was probably the cause of death rather than the caffein.
Two decigrams of caffein can not be considered, therefore, the fatal
dose for rabbits. This is further corroborated by the results obtained
in experiments with larger doses. Rabbit 78, which received 257 mg per
kilo, failed to show any symptoms. The same amount in proportion to
body weight in No. 323 caused mild symptoms only, while the rapid death
of rabbit No. 317 after the same dose of caffein may be explained by
the lesion found at autopsy, thus affording additional evidence that
disease may decrease the resistance to caffein. It will be observed
that all the members of this series were young rabbits and, as will
be shown later, young animals of other species are likewise more
resistant to caffein than adult animals. Similar results were obtained
by von Anrep, who observed that atropin is less toxic in young than in
full-grown animals.
Observations were also made on the diuretic effect of caffein when
injected into the peritoneal cavity. The results shown in the following
table indicates the stimulating effect on renal secretion whether
the diet consisted of oats or of carrots. The urine of some rabbits
contained moderate amounts of sugar after from 0.2 to 0.15 gram of
caffein per kilo was given; albumen was observed in one case, but in
none of the others. In rabbit No. 311 albumin was found before the
injection of caffein, but none in the urine which was collected three
hours after caffein was injected.
_Effect of caffein on renal secretion._
-----+--------+---------+-------+--------------------+---------
No. | Weight.| Caffein | Urine.| Time. | Diet.
| |per kilo.| | |
-----+--------+---------+-------+--------------------+---------
|_Grams._| _Gram._ | _cc._ | |
307 | 1,320 | 0.150 | 100 | 4.5 hours | Oats.
308 | 1,305 | .150 | 180 | do. | Do.
295 | 1,205 | .100 | 100 | 2 hours 20 minutes | Carrots.
293 | 1,605 | .100 | 90 | 3 hours | Do.
292 | 1,595 | .100 | 105 | 4 hours | Do.
-----+--------+---------+-------+--------------------+---------
NOTE.--The amount of urine secreted in three hours by control
rabbits, on a carrot diet, varied between 35 and 50 cc, the average
weight of the animals being a little above 1,600 grams. The secretion
of urine on an oat diet was much less for an equal period of time.
TABLE 3.--_Intraperitoneal injections._
SERIES A.
----+--------+-------+-------------+--------------+-------------------
| |Caffein| Time of | Duration |
No.| Weight.| per | appearance | of life. | Remarks.
| | kilo. | of symptoms.| |
----+--------+-------+-------------+--------------+-------------------
|_Grams._|_Gram._| | |
71| 1,659 | 0.3 |85 minutes |24 hours |Gray.
61| 2,143 | .3 | |At the end of |Black.
| | | | injection |
72| 1,402 | .3 |8 minutes |1.25 hour |Gray and white.
----+--------+-------+-------------+--------------+-------------------
SERIES B, GROUP I.
----+--------+-------+-----------+--------------+---------------------
70| 1,487 | 0.2 | |10 days |Gray and white.
93| 1,492 | .2 |5 minutes |2.5 hours |Maltese; given second
| | | | | dose after 3 days,
| | | | | died 2.5 hours later.
69| 1,492 | .2 |About |Survived |White.
| | | 5 minutes | |
92| 1,388 | .2 |5 minutes |24 hours |Yellow.
309| 1,500 | .2 | |20 minutes |Belgian; oats.
----+--------+-------+-----------+--------------+---------------------
SERIES B, GROUP II.
----+--------+-------+-------------+--------------+-------------------
308| 1,350 | 0.15 |3 hours and |About 2.5 days|Belgian; oats.
| | | 40 minutes| |
307| 1,320 | .15 |1 hour |24 hours | Do.
| | | 24 minutes| |
----+--------+-------+-------------+--------------+-------------------
SERIES C.
----+--------+-------+-------------+--------------+-------------------
223| 1,165 | 0.125|2 hours |Survived |Belgian; carrots.
293| 1,605 | .1 | | do. | Do.
295| 1,205 | .1 | | do. | Do.
292| 1,595 | .1 | | do. | Do.
298| 1,205 | .125|1.5 hours | do. | Do.
----+--------+-------+-------------+--------------+-------------------
SERIES D.
----+--------+-------+-------------+--------------+-------------------
310| 880 | 0.2 |2 days(1) |About |Belgian; oats.
| | | | 2.5 days(1)|
311| 650 | .2 | |Survived | Do.
312| 740 | .2 |40 minutes |4.5 hours |Maltese; oats.
78| 659 | .257|15 minutes |Survived |Yellow and white;
| | | | | oats.
75| 842 | .2 | do. | do. |Gray and white.
74| 692 | .2 | do. | do. |
317| 635 | .252|1 hour |About 2 hours |Belgian; oats.
| | | 25 minutes| |
323| 820 | .25 |1 hour |Survived |White; oats.
| | | 15 minutes| |
----+--------+-------+-------------+--------------+-------------------
(1) Not due to caffein.
INTRAMUSCULAR INJECTION.
Well-fed rabbits, which received a diet exclusively of oats, were used
for these experiments. The injections were made into the lumbar or into
the gluteal muscles.
SERIES A.
In this series the caffein was injected into the gluteal muscles.
_Rabbit 284. Brown and white, female. Weight, 1,100 grams._
December 14: 2 p. m., 11 cc 2 per cent caffein injected into the
gluteal muscles (0.2 gram per kilo), under observation until 5 p. m.,
had frequent convulsions; at 5 p. m. in a comatose condition. Rabbit
was found dead the next morning.
_Rabbit 286, white and black, female. Weight, 1,315 grams._
December 15: 2.30 p. m., 13 cc 2 per cent caffein injected into the
gluteal muscles (0.1977 gram per kilo), tremors and increased reflexes
observed during the next two hours, but no other symptoms.
December 17: Rabbit alive.
_Rabbit 285, yellow and white, female. Weight, 1,385 grams._
December 14: 10.15 a. m., 14 cc 2 per cent caffein injected into
the gluteal muscles (0.2 gram per kilo), general tremors, but no
convulsions observed. Rabbit survived.
December 17: Rabbit still alive.
_Rabbit 287. Belgian hare, female. Weight, 1,140 grams._
December 15: 2.15 p. m., 11 cc of 2 per cent caffein injected into the
gluteal muscles; 2.30 p. m., tonic contractions of posterior limbs.
Paralysis and death at 2.40 p. m.
SERIES B.
In series B the caffein was injected into the lumbar muscles.
_Rabbit 307. Belgian hare, female. Weight, 1,175 grams._
February 16: 11.05 a. m., 8 cc 2 per cent caffein injected (0.136 gram
per kilo) into the lumbar muscles; under observation until 4 p. m., no
symptoms; 4 p. m., allowed to walk on the floor; after walking a short
distance loss of coordination and paralysis of posterior extremities;
5.20 p. m., found dead.
_Rabbit 306. Belgian hare, female. Weight, 1,860 grams._
February 16: 11 a. m., 12.5 cc 2 per cent caffein injected into the
lumbar muscles; 12 noon, no symptoms; 2 p. m., walked about 10 feet,
exhaustion and paralysis; 3 p. m. found dead.
_Rabbit 181. Belgian hare. Weight, 1,230 grams._ (Was experimented on
some time previously.)
February 16: 10.55 a. m., 8 cc 2 per cent caffein injected into the
lumbar muscles; (0.130 gram per kilo); 12 noon, no symptoms; 2 p. m.,
no symptoms; 3 p. m., put on the floor, walked about 10 feet and was
exhausted, posterior extremities paralyzed; 4 p. m., found dead.
SERIES C.
In the fall of the same year additional experiments were carried out
with doses ranging from 100 to 200 milligrams of caffein per kilo,
which were injected into the lumbar muscles. The results are given in
the following abbreviated protocols:
_Rabbit 425. Belgian hare. Weight 1,520 grams._
September 27: 10.30 a. m., 7.5 cc 2 per cent caffein injected into the
lumbar muscles; 2 p. m., reflexes increased.
September 28: Rabbit normal.
October 5: Weight, 1,620 grams; 2.50 p. m., 10 cc 2 per cent caffein
injected into lumbar muscles; 3.05 p. m., reflexes increased.
October 13: Weight, 1,520 grams; 10.30 a. m., 10 cc 2 per cent caffein
(131 mg per kilo) injected; 11 a. m., no symptoms; 11.30 a. m.,
reflexes much increased.
October 14: Alive, no symptoms.
_Rabbit 426. Belgian hare, female. Weight, 1,425 grams._
September 27: 7 cc 2 per cent caffein injected into the lumbar muscles
at 10.30 a. m.; 2 p. m., reflexes increased.
September 28: Rabbit normal.
October 5: Weight, 1,425 grams; 2.55 p. m., 9 cc 2 per cent caffein
injected into lumbar muscles; 3.05 p. m., reflexes increased.
October 13: Weight, 1,405 grams; 10.30 a. m., 10 cc 2 per cent caffein
(142 mg per kilo) injected; 11 a. m., no symptoms; 11.30 a. m.,
reflexes increased.
October 14: Rabbit alive, no symptoms.
_Rabbit 427. Belgian hare, female. Weight, 1,780 grams._
September 27: 9 cc 2 per cent caffein injected into the lumbar muscles;
2 p. m., reflexes increased.
September 28: Rabbit normal.
October 5: Weight, 1,850 grams; 3 p. m., 11.5 cc 2 per cent caffein
injected into lumbar muscles; 3.10 p. m., reflexes increased.
October 13: Weight, 1,830 grams; 10.40 a. m., 14 cc 2 per cent caffein
(153 mg per kilo) injected into lumbar muscles; 11 a. m., no symptoms;
11.30 a. m., reflexes increased.
October 14: Rabbit alive, no symptoms.
_Rabbit 453. Belgian hare, male. Weight, 1,160 grams._
October 12: 3.45 p. m., 11.5 cc 2 per cent caffein in aqueous solution
injected into lumbar muscles; 4.15 p. m., reflexes increased;
4.30 p. m., paralyzed.
October 13: 9 a. m., found dead. _Autopsy_: Gastric mucosa
hemorrhagic; liver darkened; other organs normal.
_Rabbit 455. Belgian hare, gray, female. Weight, 1,185 grams._
October 12: 3.30 p. m., 11.5 cc 2 per cent caffein injected into the
lumbar muscles; 4 p. m., reflexes increased.
October 13: Rabbit weighed 1,070 grams, no symptom of caffein
poisoning, reflexes normal; 10.30 a. m., 10 cc 2 per cent caffein
injected into the lumbar muscles; 11.30 a. m., jumped off the table,
had attack of convulsions and died. _Autopsy_: Findings same as in No.
453.
_Rabbit 428. Belgian hare, gray, male. Weight, 1,650 grams._
October 5: 4 p. m., 14.8 cc 2 per cent caffein (0.18 gram per kilo)
injected into the lumbar muscles.
October 6: Found dead.
_Rabbit 429. Belgian hare, male. Weight, 1,340 grams._
October 5: 4 p. m., 13.5 cc 2 per cent caffein (0.2 gram per kilo)
injected into lumbar muscles.
October 8: Rabbit found dead.
SERIES D.
Further experiments making injections into both the lumbar and the
gluteal muscles, were made in this series.
_Rabbit 577. Gray male. Weight, 1,380 grams._
February 14: 3 p. m. 14 cc 2 per cent caffein injected into the gluteal
muscles of the right side; 3.10 p. m., restless, jumped off the table
and walked about, reflexes increased; 3.45 p. m., passed 30 cc clear,
straw-colored urine; 4.45 p. m., allowed to walk about, ran across the
room, about 20 feet, looked tired, stretched himself out on the floor,
then raised himself and walked about showing no disturbance of gait.
February 15: 9 a. m., found dead.
_Rabbit 578. Gray, female. Weight, 1,670 grams._
February 14: 3.05 p. m., 18 cc 2 per cent caffein solution injected
into the gluteal muscles of the right side; 3.15 reflexes increased,
but not restless; 5 p. m., allowed to walk about, no symptoms observed.
February 15: Found dead.
_Rabbit 579. White and gray, male. Weight, 1,490 grams._
February 14: 3.15 p. m., 15 cc 2 per cent caffein solution injected
into the gluteal muscles of the right side; put in cage; 3.30 p. m.,
reflexes increased; 5 p. m., taken out of cage and allowed to walk
across the room, no special symptoms noticed.
February 23: Still alive.
_Rabbit 580. Gray male. Weight, 1,510 grams._
February 14: 3.35 p. m., 15 cc 2 per cent caffein solution injected
into lumbar muscles.
February 23: Still alive, in good condition.
_Rabbit 581. Gray female. Weight, 1,680 grams._
February 14: 3.45 p. m., 17 cc 2 per cent caffein solution injected
into the lumbar muscles of the right side; 4 p. m., reflexes increased;
4.15 p. m., jumped off the table and had wild convulsions, became very
restless, walked about the laboratory; 4.25 p. m., had convulsions
occasionally; 4.30 p. m., extremities extended and quite rigid;
4.35 p. m., convulsions and death.
_Rabbit 582. Gray male. Weight, 1,870 grams._
February 14: 4.15 p. m., 18 cc 2 per cent caffein solution injected
into the lumbar muscles of right side; 5 p. m., reflexes increased;
walked about in the room, then rested; 5.15 p. m., had short spasm when
handled.
February 23: Alive; good condition.
The data presented in these experiments show that the toxicity of
caffein when injected into the muscles of the lumbar regions is the
same as when injected into the gluteal muscles. The rabbits of series
A received approximately 0.2 gram caffein per kilo and two died as
a result of this treatment. The other two survived but symptoms of
caffein intoxication were observed.
In series B smaller doses proved fatal, from which it would appear
that caffein is more toxic when injected into the lumbar muscles.
Further observations, however, failed to corroborate the results
obtained in this series. Thus, in series C, 130 to 150 mg of caffein
per kilo injected into the lumbar muscles produced mild symptoms
only. Experiments with larger doses showed that 0.180 gram caffein
per kilo may cause death. It will be noticed, on the other hand, that
rabbit No. 455 survived a dose of 0.2 gram per kilo. New experiments
were therefore carried out in which the same amounts of caffein in
proportion to the weight of the animals were injected into the lumbar
muscles as into the gluteal muscles. As shown in the experiments of
series D, one rabbit (No. 581) died shortly after caffein was injected
into the lumbar muscles; two recovered. Two of the three which received
injections into the gluteal muscles were found dead the next day; one
recovered. Post-mortem examination failed to indicate the presence
of any abnormalities. The rate of absorption of caffein from the
gluteal and from the lumbar muscles seems to be, therefore, the same,
or not to differ very much. The observations of Auer and Meltzer(7)
are of interest in this connection. According to their investigations
adrenalin is more rapidly absorbed from the lumbar than from the
gluteal muscles. This is in all probability due to the greater delicacy
of the test they employed (since they judged the rate of absorption
by the effect of adrenalin on blood pressure) as well as to the much
greater activity of the substance.
TABLE 4.--_Intramuscular injections._
SERIES A.
----+--------+-------+----------+-----------+----------+--------------
| |Caffein| | | |
No.| Weight.| per | Symptoms | Duration | Site of | Remarks.
| | kilo | after-- | of life. |injection.|
----+--------+-------+----------+-----------+----------+--------------
|_Grams._|_Gram._| | | |
284| 1,100 | 0.200 | 3 hours |Less than | Gluteal |White and
| | | | 20 hours | | brown female.
286| 1,315 | .1977| 2 hours |Survived | do. |White and
| | | | | | black female.
285| 1,385 | .200 | Present | do. | do. |Yellow and
| | | | | | white female.
287| 1,140 | .210 |15 minutes|25 minutes | do. |Gray female.
----+--------+-------+----------+-----------+----------+--------------
SERIES B.
----+--------+-------+----------+-----------+----------+-------------
307| 1,175 | 0.136 | 5 hours |6 hours, | Lumbar |Gray female.
| | | | 20 minutes| |
306| 1,860 | .134 | 3 hours |4 hours | do. | Do.
181| 1,230 | .130 | 4 hours |5 hours | do. |Gray.
----+--------+-------+----------+-----------+----------+-------------
SERIES C.
----+--------+-------+----------+-----------+----------+-------------
425| 1,520 | 0.131 | 1 hour |Survived | Lumbar |Gray.
426| 1,405 | .142 |30 minutes| do. | do. |Gray female.
427| 1,830 | .153 |50 minutes| do. | do. | Do.
453| 1,160 | .200 |30 minutes|Less than | do. |Gray male.
| | | | 20 hours | |
455| 1,185 | .200 | do. |Survived | do. |Gray female.
428| 1,650 | .180 | |Less than | do. |Gray male.
| | | | 20 hours | |
429| 1,340 | .200 | | do. | do. | Do.
----+--------+-------+----------+-----------+----------+-------------
SERIES D.
----+--------+-------+----------+-----------+----------+-------------
577| 1,380 | 0.200 |10 minutes|Less than | Gluteal |Gray male.
| | | | 18 hours | |
578| 1,670 | .210 | do. | do. | do. | Do.
579| 1,490 | .200 |15 minutes|Survived | do. |White and
| | | | | | gray male.
580| 1,510 | .200 | | do. | Lumbar |Gray male.
581| 1,680 | .200 |15 minutes|50 minutes | do. | Do.
582| 1,870 | .192 |45 minutes|Survived | do. | Do.
----+--------+-------+----------+-----------+----------+-------------
Examination of Table 4 shows that 14 rabbits received from 180 to 210
mg caffein per kilo. The appearance of symptoms in these rabbits varied
considerably. In some increased reflexes could be noticed in 10 to
15 minutes after the injection of caffein; in others it was delayed
2 or 3 hours. It might be added that the onset of symptoms occurred
in many cases very soon after the administration of the drug--on an
average about 10 to 30 minutes after the drug was injected. After
smaller doses were administered by injection into the lumbar muscles
the appearance of symptoms was delayed several hours in some cases. The
duration of life in these 14 rabbits varied considerably. Eight of them
died within 1 to 20 hours; six survived. About 0.2 gram caffein per
kilo may be regarded as the minimum fatal dose, while the minimum toxic
dose is somewhere between 130 and 150 mg per kilo.
INTRAVENOUS INJECTION.
These experiments were carried out on well-fed, full-grown gray
rabbits. The diet for several days preceding the experiments consisted
of oats or carrots, which were given ad libitum. The injections were
made into the ear veins from a burette or by means of a syringe, the
temperature of the caffein solution being about 40° C. Attention
was also directed to the effect of the rate of injection and of the
concentration on the toxicity. The minimum toxic as well as lethal
doses were determined as shown in the following experiments.
SERIES A.
In these experiments the rate of injection was about 1 cc of 2 per cent
caffein solution per minute.
_Rabbit 194. White, female. Weight, 1,310 grams._
October 19: Injected 7.5 cc 2 per cent solution caffein (115 mg
per kilo) into the ear vein. Rabbit showed stiffness; paralysis of
extremities appeared soon after.[C] Rabbit survived.
[C] Time of injection inadvertently omitted, but was probably not
slower than in the other cases of this series.
_Rabbit 556. Gray, female. Weight, 1,635 grams._
January 31: 2 p. m., 11 cc 2 per cent caffein (134 mg per kilo)
injected into ear vein, in about 11 minutes; 2.10 p. m., convulsions,
rabbit remained lying on its side; during the rest of the hour it had
convulsions occasionally; 3.20 p. m., convulsions and died. Rabbit did
not urinate after the injection of caffein.
_Rabbit 557. Gray, female. Weight, 1,580 grams._
January 31: 2.30 to 2.37 p. m., 7 cc 2 per cent caffein injected from
the burette at the rate of 1 cc per minute; 2.37 p. m., flow of liquid
ceased, veins were engorged and bled freely, injection was continued
by means of a syringe; 2 cc 2 per cent caffein injected in two
minutes; injections discontinued as convulsions appeared; 2.50 p. m.,
rabbit raised itself but fell over; 3.10 p. m., rabbit assumed normal
attitude, walked about the floor without manifesting any signs of the
effects of caffein; 4.30 p. m., walked about, gait normal, condition
seemed to be good.
February 1: 2 p. m., condition good, appetite good, total amount of
caffein injected, 9 cc 2 per cent solution, or 114 mg per kilo.
_Rabbit 558. Gray, female. Weight, 1,590 grams._
January 31: 3 p. m., given 8 cc 2 per cent caffein in eight minutes;
3.10 p. m., violent convulsions; 3.20 p. m., rabbit was stretched out
on his abdomen, extremities extended, urinated; 4.30 p. m., looked
normal; was able to walk about.
February 1: 2 p. m., condition good, appetite good.
_Rabbit 292. Belgian hare, male. Weight, 1,770 grams._
February 18: 4.26 to 4.39 p. m., 12.5 cc warm caffein solution (0.141
gram per kilo) injected into ear vein, convulsion followed when this
quantity was injected, tonic rigidity of limbs followed soon after;
4.52 p. m., condition unchanged, rabbit on floor, limbs stretched out,
and lying on abdomen.
_Rabbit 294. Belgian hare, female. Weight, 1,350 grams. Carrot diet
for about 10 days before the experiment._
February 19: 12.20 p. m., 5 cc 2 per cent caffein (74 mg per kilo)
injected into ear vein in five minutes, edema of the ear, other ear
used, 3.5 cc injected in 10 minutes, repeated convulsions; 1.25 p. m.,
rabbit still alive, frequent attacks of convulsions; 2.30 p. m., found
dead. Total amount injected in 15 minutes, 8.5 cc, or 0.126 gram per
kilo.
It will be observed in the preceding experiments that symptoms of
severe intoxication were present in all of the six rabbits, but only
two of these (Nos. 294 and 556) died from the effects of caffein. Of
those which survived, three received doses of 100 to 114 mg caffein
per kilo, and another (No. 292) received 141 mg of caffein per kilo.
The death of rabbits Nos. 294 and 556 may be regarded therefore as a
case of exceptionally low resistance to caffein.
SERIES B.
Doses of 160 to 200 mg caffein per kilo were employed in these
experiments. The rate of injection was 1 cc per minute, with the
exception of Experiment 254, in which 10.8 cc 2 per cent caffein were
introduced in 17 minutes and 25 seconds.
_Rabbit 562. Gray female. Weight, 1,650 grams. Diet, oats._
February 1: Injection began at 3 p. m., injected 10 cc in 12 minutes;
3.01 p. m. to 3.09 p. m., 3 cc injected, convulsions; 3.09 p. m. to
3.14 p. m., 3 cc injected, followed by violent convulsions, marked
opisthotonos; 4.30 p. m., rabbit died; total quantity injected, 16 cc.
_Rabbit 561. Gray female. Weight, 1,450 grams. Diet, oats._
February 1: Injection began at 11.40 a. m.; 11.48, rabbit struggled,
7 cc 2 per cent caffein injected; 11.50, convulsions, 10 cc 2 per
cent caffein total amount injected; 11.55 a. m., injections stopped;
injections resumed 11.58, violent convulsions, injections discontinued,
total quantity received, 14.5 cc 2 per cent caffein solution;
1.30 p. m., found dead, did not urinate, 25 cc urine found in the
bladder.
_Rabbit 560. Gray male. Weight, 1,620 grams. Diet, oats._
February 1: Injection began 11 a. m.; 11.10 a. m., 7 cc 2 per cent
caffein injected, rabbit struggled; 1 cc was injected during the
next three minutes, rabbit struggled but there were no convulsions,
injection stopped; resumed at 11.15 a. m. and continued 10 minutes,
8 cc 2 per cent caffein introduced during this time; total amount
caffein injected, 16 cc; reflexes markedly increased; 12 noon, tetanic
convulsions off and on until 2 p. m., then remained stretched out on
abdomen, extremities extended.
February 2: 9 a. m., found dead.
_Rabbit 559. Gray female. Weight, 1,875 grams. Diet, oats._
January 31: 4 p. m., convulsions after injection of 9 cc 2 per cent
caffein in 14 minutes; 4.08 p. m., convulsions after injection of 7 cc
caffein in 8 minutes; 4.10 to 4.12 p. m., injected 2 cc more, rabbit
lying stretched out on abdomen, extremities extended; total amount of
caffein injected, 18 cc (190 mg per kilo).
February 1: 2 p. m., condition good, walked about, appetite good,
passed 155 cc dark, reddish-brown urine since 5.30 p. m. previous day.
_Rabbit 279. Gray and white female. Weight, 1,320 grams._
February 24: 10.09 a. m., 6 cc 2 per cent caffein passed rapidly
into jugular vein; 10.15 a. m., involuntary twitching of muscles
of legs, but no other symptoms; 10.23 to 10.26, 3 cc of 2 per cent
caffein injected; 10.27 to 10.28, 2 cc 2 per cent caffein injected,
convulsions; 10.29, convulsions stopped; 10.32, convulsions; 11 a. m.,
rabbit lying on its side, anterior extremities paralyzed, posterior
extremities contracted, no clonic convulsions, breathed deeper and
more slowly than normal; 11.10 a. m., rabbit died, had no convulsions
immediately before death; amount of caffein injected, 11 cc 2 per cent
solution, or 0.166 gram per kilo.
_Rabbit 254. Belgian hare, female. Weight, 1,285 grams. Diet, oats._
November 12: 1.30-1/3 to 1.47¾ p. m., received 10.8 cc 2 per
cent caffein from burette into ear vein, after injection of 6.2 cc
dyspnoea, 6.7 cc struggling, convulsions; at 1.50½ p. m., released
from holder, paralysis especially marked in the anterior extremities;
1.50 p. m., recovered, survived; total amount injected, 10.8 cc 2 per
cent caffein in 17 minutes and 25 seconds, or 0.16 gram caffein per
kilo.
_Rabbit 255. Belgian hare, male. Weight, 1,105 grams. Diet, oats._
November 12: 2.31¾ to 2.35¼ p. m., received 3.7 cc; from 2.37-1/6
to 2.46-1/6 p. m., 5 cc injected; after injection of 6.1 cc convulsions
followed by dyspnoea, then continuous struggling; when 8.3 cc were
injected rabbit had another convulsion; 2.47 p. m., tonic contraction
of anterior extremities; amount injected, 8.7 cc (158 mg per kilo) in
15 minutes and 35 seconds.
_Rabbit 567. Gray female. Diet, oats._
February 6: Injection began at 4.11 p. m.; 4.18, convulsions after
injection of 5 cc 2 per cent caffein; 4.21, convulsion after total
injection of 8 cc; 4.24 p. m., injection resumed and 2 cc more
introduced; 4.28 p. m., convulsions, injected 2 cc more; total caffein
injected, 12 cc, or 162 mg per kilo; 4.40 p. m., rabbit paralyzed in
posterior extremities; 5 p. m., found dead.
In the eight experiments comprising series B rabbits Nos. 567, 254,
279, and 255, which may be designated as Group II, received doses of
162, 160, 166, and 158 mg, respectively. Nos. 562, 561, 560, and 559,
which may be designated as Group I, received about 200 mg caffein per
kilo. In Group II, which received the smaller doses, one (No. 254)
survived. This may be regarded as exceptional, since, as was shown
in the experiments of the preceding series, even smaller doses may
be fatal. About 160 mg per kilo is, therefore, the smallest surely
fatal dose. This might be regarded as a contradiction of the results
obtained for rabbit No. 559, but it will be noticed that in this case
diuresis was very marked. The results of experiments Nos. 294 and 255
are of interest in this connection, since they indicate that a moderate
difference in the rate of injection is without any effect on the
toxicity of caffein. The greater resistance to caffein of rabbit No.
559 is in all probability due, therefore, to increased diuresis.
SERIES C.
In these experiments the minimum toxic dose was determined. The
conditions were the same as in the experiments of the other series.
_Rabbit 293. Belgian hare, female. Weight, 1,610 grams. Diet, oats._
February 18: 3.40 to 3.43 p. m., 4 cc 2 per cent warm caffein solution
injected into ear vein, convulsions when 3 cc were injected, repeated
attacks; 4 p. m., raised itself on legs, but fell over immediately and
lay stretched on abdomen.
February 19: 9 a. m., rabbit looked normal, apparently recovered.
_Rabbit 227. White male. Weight, 2,320 grams._
October 26: 3.29¼ to 3.37½ p. m., injected into ear from burette
6.7 cc 2 per cent caffein, no symptoms; experiment discontinued;
survived.
_Rabbit 563. Gray female. Weight, 1,650 grams. Diet, oats._
February 6: Injection began at 1.02 p. m., injected 3.5 cc 2 per cent
caffein (42 mg per kilo) in four minutes, 0.6 cc more within the
next two and one-half minutes, total amount injected 4.1 cc; 1.10
p. m., hypersensitive, some disturbance of muscular coordination;
restlessness; 1.35. p. m., reflexes decreased, urinated and walked
about, gait normal. Under observation for several days; no symptoms
noted.
_Rabbit 564. Gray female. Weight, 1,515 grams._
February 6: Injection began at 1.26 p. m., 3.5 cc 2 per cent caffein
(46 mg per kilo) injected at the rate of 1 cc per minute; 1.30 p. m.,
reflexes increased; 1.34 p. m., marked paresis of the extremities,
rabbit stretched out on abdomen, legs abducted and partly extended,
able to hop about but gait disturbed, no untoward symptoms noticed,
under observation for several days after experiment.
_Rabbit 565. Gray female. Weight, 1,545 grams. Diet, oats._
February 6: Started to inject at 3.40 p. m., received 2.5 cc 2 per
cent caffein intravenously in two minutes or 32 mg per kilo, under
observation all afternoon, no symptoms.
_Rabbit 566. Gray female. Weight, 1,900 grams. Diet oats._
February 6: Injection began at 3.05 p. m., received 3 cc 2 per cent
caffein intravenously in three minutes or 31 mg per kilo, no symptoms
observed.
These experiments show that a dose of about 50 mg per kilo when
injected intravenously produces mild symptoms, such as increased
reflexes. In the four experiments with this amount of caffein these
effects were observed in each case. In the experiments in which
smaller quantities, 30 mg per kilo, were given intravenously there
was no manifestation of symptoms. A dose not over 50 mg per kilo
may, therefore, be regarded as the minimum toxic dose when injected
intravenously under the conditions stated.
SERIES D.
A 0.5 per cent caffein solution was used in these experiments in order
to test the effect of concentration on its toxicity; the rate of
injection was 1 cc per minute.
_Rabbit 569. Gray male. Weight, 1,475 grams. Diet, oats._
February 6: 11.50 a. m. to 12.01 p. m., injected 10 cc 0.5 per cent
caffein; 12.03 to 12.12 p. m., injected 10 cc of 0.5 per cent caffein;
12.13 to 12.26 p. m., injected 10 cc of 0.5 per cent caffein, total
amount injected, 30 cc; 12.20, passed 35 cc of urine; 12.30, increased
reflexes, but no convulsions; 4 p. m., reflexes increased.
February 11: Alive, condition good.
_Rabbit 574. Gray female. Weight, 1,555 grams. Diet, oats._
February 8: 10.25 to 10.33 a. m., injected 4 cc of 0.5 per cent caffein
in salt solution, injection discontinued for five minutes; 10.38 to
11.10, injected 30 cc, total amount of caffein solution received, 34
cc; 11.55 a. m., very sensitive; reflexes markedly increased.
February 9: Alive, condition good.
_Rabbit 571. Gray female. Weight, 1,530 grams. Diet, oats._
February 7: Injection 3.18 to 3.50 p. m., received 30 cc in 32 minutes,
not hypersensitive; 3.55, restlessness and weakness of extremities;
4.10 p. m., control of anterior extremities impaired, distinctly
paretic but tried to walk about, died the same afternoon.
_Rabbit 568. Gray male. Weight, 1,605 grams. Diet, oats._
February 7: Injection 10.53 to 11.01 a. m., injected 10 cc 0.5 per cent
caffein; 11.03, injection resumed after two minutes interval; 11.14,
received 10 cc 0.5 per cent caffein intravenously in 11 minutes; 11.16,
injection resumed; 11.35, received 12 cc 0.5 per cent caffein, total
amount of caffein solution received, 32 cc; 12.30 p. m., urinated 14
cc of bloody urine; 12.55 p. m., convulsions and death a few minutes
later. Autopsy showed congestion of viscera, but no other lesions.
_Rabbit 570. Gray female. Weight, 1,225 grams. Diet, oats._
February 7: 2.06 to 2.35 p. m., injected 24.5 cc 0.5 per cent caffein,
reflexes increased but no convulsions, paresis especially marked in
the anterior extremities; 3 p. m., passed urine which was normal in
appearance, reflexes not increased but rabbit was weak.
February 9: Found dead. _Autopsy_: Liver, spleen, and kidneys
congested; large intestines hemorrhagic; omentum congested and showed
the presence of small caseous nodules; liver showed adhesion to
diaphragm; viscera presented the appearance of intraabdominal infection.
Of the five rabbits of this series three died as a result of the
administration of caffein. The other two which survived showed mild
symptoms only, such as increased reflexes, but no evidence of severe
poisoning such as was observed after the injection of the same doses of
caffein in series A when a 2 per cent solution of caffein was injected.
Convulsions were noticed in one case only (No. 568); paresis in two
cases (Nos. 570 and 571). The nervous symptoms even in this group,
therefore, were much milder than in series A. The percentage of death,
however, was greater than in series A, in which the concentration of
caffein was four times as great. It is quite probable that the strain
on the heart due to the sudden increase in volume of the blood and
its dilution might be an important factor in increasing the toxicity
of caffein. It is conceivable that doses just sufficiently large to
depress the normal heart may cause paralysis of an already overstrained
organ.
SERIES E.
In the two experiments of this series the rate of injection as a
possible factor influencing the toxicity of caffein was tested. A 2
per cent caffein solution was injected at the rate of 1 cc in two and
one-half to three minutes.
_Rabbit 572. Gray male. Weight, 1,770 grams. Diet, oats._
February 8: Injection began at 3 p. m., discontinued at 3.37 p. m., and
resumed at 3.38 p. m.; rabbit was restless; injection finished at 3.52
p. m. Total quantity received, 17.4 cc 2 per cent caffein intravenously
in 52 minutes; struggled intermittently during the injection; anterior
legs paralyzed.
February 9: Found dead.
_Rabbit 573. Gray male. Weight, 1,810 grams. Diet, oats._
February 8: Started to inject at 1.35 and discontinued at 2.27 p. m.;
received 18 cc 2 per cent caffein intravenously in 52 minutes; reflexes
markedly increased soon after; 2.45, passed bloody urine; 4.30 p. m.
reflexes increased; no other symptoms.
February 9: 9 a. m., found dead.
It will be observed that some retardation of the onset of symptoms
was caused by slower injection, but the final result was the same as
when the injections were made more rapidly. It is quite probable,
therefore, that a much slower rate of injection may lessen considerably
the toxicity of caffein.
From the results of the experiments by intravenous injection summarized
in the table, it appears that the minimum toxic dose for rabbits of a
2 per cent caffein solution, injected at the rate of 1 cc per minute,
is about 50 mg per kilo. Twice the dose induces severe symptoms and may
be fatal; 160 mg per kilo are surely fatal. If the rate of injection
is diminished, the toxicity of caffein is lessened, but this effect
is not marked unless the injections are very slow. Dilution of the
caffein solution suppresses to some extent the nervous symptoms, but
the toxicity, on the contrary, seems to be increased.
TABLE 5.--_Intravenous injections._
SERIES A.
----+--------+-------+-----------+-----------+-------+---------------
| |Caffein| | Duration | |
No.|Weight. | per | Symptoms. | of life. | Diet. | Remarks.
| | kilo.| | | |
----+--------+-------+-----------+-----------+-------+---------------
|_Grams._| _Mg._ | | | |
194| 1,310 | 114 |Present |Survived |Oats |White female.
556| 1,635 | 134 |10 minutes |20 minutes | do. |Gray female.
557| 1,580 | 114 |Present |Survived | do. | Do.
558| 1,590 | 100 | do. | do. | do. | Do.
292| 1,770 | 141 | do. | do. | do. | Do.
194| 1,350 | 126 | do. |10 minutes |Carrots| Do.
----+--------+-------+-----------+-----------+-------+---------------
SERIES B, GROUP I.
----+--------+-------+-----------+-----------+-------+---------------
562| 1,650 | 200 | |1½ hours |Oats |Gray female.
561| 1,450 | 200 | | do. | do. | Do.
560| 1,620 | 200 |Present |Less than | do. | Do.
| | | | 24 hours | |
559| 1,875 | 190 | do. |Survived | do. | Do.
----+--------+-------+-----------+-----------+-------+---------------
SERIES B, GROUP II.
----+--------+-------+-----------+-----------+-------+---------------
279| 1,320 | 166 | |1 hour | |Gray and white
| | | | | | female.
254| 1,285 | 160 | |Survived |Oats |Gray female.
567| | 162 | |About 45 | | Do.
| | | | minutes | |
255| | 158 | |Died | |
----+--------+-------+-----------+-----------+-------+---------------
SERIES C.
----+--------+-------+-----------+-----------+-------+---------------
293| 1,610 | 500 |Present |Survived |Oats |Gray female.
227| 2,320 | 570 |None | do. | |White male.
563| 1,650 | 500 |Present | do. | do. |Gray female.
564| 1,515 | 460 | do. | do. | | Do.
565| 1,545 | 320 |None | do. | do. | Do.
566| 1,900 | 310 | do. | do. | do. | Do.
----+--------+-------+-----------+-----------+-------+---------------
SERIES D.
----+--------+-------+-----------+-----------+-------+---------------
569| 1,475 | 100 |Present |Survived |Oats |Gray male.
574| 1,555 | 112 | do. | do. | do. | Gray female.
571| 1,530 | 100 | do. |About | do. | Do.
| | | | 2 hours | |
568| 1,605 | 100 | |20 minutes | do. |Gray male.
570| 1,225 | 100 | |Less than | do. | Do.
| | | | 20 hours | |
----+--------+-------+-----------+-----------+-------+---------------
SERIES E.
----+--------+-------+-----------+-----------+-------+---------------
572| 1,770 | 200 |Present |About |Oats |
| | | | 24 hours | |
573| 1,810 | 200 | | do. | do. |
----+--------+-------+-----------+-----------+-------+---------------
SUMMARY.
The results of the experiments on rabbits show considerable
variation in the toxicity of the single dose. Individuals differed
so widely in their resistance to this drug that the same experiments
had to be repeated many times with each method of administration
before satisfactory conclusions could be drawn. This is strikingly
illustrated in the experiments by intravenous injection in which a
dose of nearly 0.2 gram per kilo was not fatal. Similar instances of
exceptional resistance or of sensitiveness to caffein were observed
when it was given in other ways. A comparison of the toxicity of
caffein administered by different methods in this investigation shows
well-marked differences in its activity, although they are not quite
so striking as similar experiments with other alkaloids reported by
several observers. The toxicity of caffein in these experiments on
the rabbit indicates that it is greatest when given by vein and least
when given by mouth. The ratio of the minimum toxic doses by these
two methods of introduction of caffein was about 7.1; the relation
of the minimum fatal dose was about 3.1. The toxicity when given
subcutaneously is about 15 to 20 per cent greater than when given
by mouth. The difference between the intramuscular and subcutaneous
injection is even more marked. The toxicity of caffein when injected
into the muscles is about midway between that administered by the
subcutaneous and intraperitoneal routes, and is about half that
injected intravenously. Meltzer and Auer,(58) who experimented with a
number of drugs found that the intramuscular method of administration
is as effective as the intravenous, fluorescin forming the only
exception according to their observations. In the experiments of
Sollman and Brown(81) with ergot, the effect was quite different from
those obtained by Meltzer and Auer(58) with the drugs they used.
It is quite possible that the result obtained with ergot is merely
illustrative of a difference in the behavior of various substances
in this regard. This appears probable on account of the difference
in the rate of absorption for various substances. Thus, according to
Achard, Gaillard, and Ribot (Compt. rend. Soc. biol., 1907, _62_: 90),
absorption from the peritoneal cavity varies with the concentration of
the solution and the size of the molecule. The smaller the molecule and
the greater the concentration the more rapid the absorption. That the
rate of absorption from the intramuscular tissues is unequal and varies
for different substances appears from the experiments of Meltzer and
Auer.(58) The difference was very striking between intramuscular and
subcutaneous administration of curara or adrenalin; the results were
somewhat different with morphin and with fluorescin. As shown in their
protocols, the onset of the symptoms after the intramuscular injection
of morphin was sooner than after subcutaneous injection, but in time
the difference diminishes and disappears altogether. The absorption
of fluorescin is much faster when the intramuscular path is used than
when given subcutaneously, but the writers state that the rate falls
far behind that of the intravenous administration. The difference
in toxicity we observed between feeding by mouth and subcutaneous
injection, although distinct, was not very great. It was much less than
Maurel(55) obtained with the hydrobromid of caffein in the rabbit.
Whether this difference between his results and ours is due to the use
of the pure alkaloid in our experiments and the hydrobromid employed by
Maurel can not be stated at present with any degree of accuracy. It is
hoped that the work in progress in the laboratory will throw some light
on the subject in the near future. But Maurel's(56) experiments show
that various substances behave differently in this regard. Thus the
toxicity of strychnin, he states, is three times as great when given
subcutaneously as when given by mouth and six times that of the minimum
fatal dose by vein. It may be remarked, however, that examination of
his data shows that his doses are much too large for the rabbit. In
experiments with other drugs little or no difference between the two
modes of administration was noticed. Thus, digitalin was but slightly
more active when given subcutaneously than by mouth, while the toxicity
of emetin hydrochlorid was just the same, whichever one of these
methods of introducing the substance was used. Differences in the
toxicity of substances have also been observed between subcutaneous and
intravenous modes of administration, but here, too, the differences for
various substances were unequal.
EXPERIMENTS ON GUINEA PIGS.
The toxicity of caffein was studied in a large number of individuals.
The experiments were conducted on full-grown animals and were carried
out at different seasons of the year in a variety of ways. Special
attention was given to diet as a possible factor influencing resistance
to caffein, and the effect of different modes of administration on
toxicity. Some animals were therefore fed oats, some carrots, others
received both hay and oats. Caffein was introduced subcutaneously,
intraperitoneally, and by mouth.
SUBCUTANEOUS INJECTION.
SERIES A.
Preliminary experiments carried out on three guinea pigs, which
received 360, 300, and 290 mg of caffein per kilo subcutaneously have
shown that such doses were rapidly fatal. Two of the animals were
seized with convulsions half an hour after the introduction of caffein
and died during the attack. The other had tetanus two minutes after the
injection of caffein. Repeated attacks followed, which terminated in
the death of the animal two and a half hours later. The fatal and toxic
doses must therefore be considerably under 0.3 gram of caffein per kilo
when introduced by this path and smaller doses were therefore injected.
The results are shown in the experiments of the next series.
SERIES B.
Experiments with 2 decigrams per kilo constituted this series.
_Guinea pig 20. Female. Weight, 497 grams. Diet, oats._
April 2: 5 cc 2 per cent caffein injected subcutaneously at 11.30 a. m.;
1.50 p. m., spasm of short duration. Died at 3 p. m., three and
one-half hours after injection.
_Guinea pig 38. Brown male. Weight, 570 grams. Diet, carrots and oats
week previous to injection._
February 11: 3.50 p. m., 6 cc 2 per cent caffein injected
subcutaneously in back (210 mg per kilo); 4.15, reflexes increased, had
convulsion of short duration when disturbed; 4.45 p. m., on handling,
repeated convulsion and paralysis; 5 p. m., guinea pig lying on his
side, respiration difficult and labored.
February 11: 5.05 p. m., guinea pig found dead, 2 hours and 15 minutes
after injection.
_Guinea pig 37. Male. Weight, 820 grams. Diet, carrots and oats during
week preceding the injection of caffein._
February 11: 3.35 p. m., 8.5 cc 2 per cent caffein injected
subcutaneously in the back; 5 p. m., pig very sensitive, anterior
extremities paralyzed when handled, frequent spasms of posterior
extremities, no symptoms noticed before 5 p. m., although watched all
the time; 5.05 p. m., guinea pig on his legs and looked normal. No
attack on handling.
February 12: 9 a. m., found dead; died within 18 hours.
_Guinea pig 13. Female. Weight, 618 grams. Diet, oats._
March 29: 2.45, 6 cc 2 per cent caffein injected subcutaneously (0.194
grams per kilo).
March 30: Died at 4 p. m., 25 hours after injection.
_Guinea pig 36. Male. Weight, 850 grams. Fed oats and carrots for one
week previous to injection._
February 11: 3.30 p. m., 8.5 cc 2 per cent caffein injected
subcutaneously into back; 5 p. m., somewhat more sensitive than normal,
no other symptoms, no effect on handling; 5.05 p. m., no symptoms.
February 12: 9 a. m., found dead, about 18 hours after injection.
The results of these experiments, as observed in five guinea pigs,
indicate that two decigrams of caffein per kilo of animal produce
symptoms within a half to about two and a quarter hours after
injection. Death followed in two guinea pigs 70 minutes to 1 hour
after the first manifestations of symptoms. Two others died during the
night, while one lived 25 hours after the injection of caffein. Even
2 decigrams caffein per kilo weight might therefore be fatal to the
guinea pig. Experiments carried out later have shown, however, that the
resistance to caffein is appreciably greater in some guinea pigs. This
is indicated by the following experiments, in which doses of 0.2 to
0.24 gram caffein per kilo were administered by the same path.
SERIES C.
_Guinea pig 66. Yellow and dark brown male. Weight, 510 grams.
Diet, oats._
October 4: 5 cc 2 per cent caffein (0.2 gram per kilo) injected
subcutaneously in the back at 3 p. m.; 5 p. m., no symptoms.
October 5: 9 a. m., alive; condition good.
October 9: Found dead. _Autopsy_: Congestion of liver, kidney, and
small intestine.
_Guinea pig 65. White and black male. Weight, 510 grams. Diet, oats._
October 4: 5 cc 2 per cent caffein (0.2 gram per kilo) injected
subcutaneously in the back at 3 p. m.; 5 p. m., no symptoms.
October 5: 9 a. m., condition good.
_Guinea pig 60. White and gray female. Weight, 320 grams. Diet, oats._
October 3: 2.25 p. m., 3.5 cc 2 per cent caffein (0.219 gram per kilo)
injected subcutaneously in the back; 3.40 p. m., convulsion with
recovery; 3.50 p. m., frequent spasms with paralysis, especially of
anterior extremities; 5.30 p. m., tetanus when removed from cage and
put on floor.
October 4: 8.50 a. m., found dead. _Autopsy_: Congestion of small
intestines, lungs, liver.
_Guinea pig 57. White and gray female. Weight, 350 grams. Diet, oats._
October 3: 2.15 p. m., 3.5 cc 2 per cent caffein injected
subcutaneously in the back (0.2 gram per kilo); 3.40 p. m., convulsions
with recovery; 5.30 p. m., no marked symptoms.
October 4: 8.50 a. m., alive, active.
October 6: Found dead at 9 a. m. _Autopsy_: Congestion of lungs and
liver; kidneys petechiated; severe gastro-enteritis.
_Guinea pig 68. Yellow male. Weight, 785 grams. Diet, oats._
October 6: 11.35 a. m., 7.8 cc 2 per cent caffein (0.2 gram per kilo)
injected subcutaneously; 12 noon, reflexes increased markedly;
4.20 p. m., reflexes the same as at 12 noon.
October 7: 9 a. m., dead. _Autopsy_: Lungs congested; liver congested
and fatty; spleen congested, kidney showed hemorrhagic spots; gastric
mucosa necrotic; small portion of small intestine inflamed.
_Guinea pig 69. White male. Weight, 585 grams. Diet, oats._
October 6: 11.40 a. m., 5.8 cc 2 per cent caffein injected
subcutaneously; 12 noon, reflexes increased, but not as much as in No.
68; 4.20 p. m., guinea pig hypersensitive, reflexes increased more than
at 12 noon.
October 7: 9 a. m., alive.
October 15: 9 a. m., found dead.
_Guinea pig 61. Brown and black female. Weight, 330 grams. Diet, oats._
October 3: 4 p. m., 4 cc 2 per cent caffein (240 mg per kilo) injected
subcutaneously; 5.30 p. m., reflexes increased; runs, but drags
posterior extremities.
October 4: 8.50 a. m., found dead.
_Guinea pig 62. White, yellow, and black female. Weight, 335 grams.
Diet, oats._
October 3: 4.05 p. m., 4 cc 2 per cent caffein (238 mg per kilo)
injected subcutaneously in the back; 5 p. m., convulsions; 5.20 p. m.,
convulsions, alternating with paralysis of anterior and posterior
extremities.
October 4: 8.50 a. m., found dead.
_Guinea pig 70. White and brown male. Weight, 545 grams. Diet, oats._
October 7: 3 p. m., 6.5 cc 2 per cent caffein (238 mg per kilo) aqueous
solution injected subcutaneously; 3.50 p. m., reflexes increased.
October 9: 9 a. m., found dead.
_Guinea pig 71. Brown and white male. Weight, 540 grams. Diet, oats._
October 7: 3 p. m., 6.5 cc 2 per cent caffein solution (0.24 gram per
kilo) injected subcutaneously; 3.45 p. m., reflexes increased, tetanus.
October 9: 9 a. m., found dead.
_Guinea pig 72. Brown and white male. Weight, 560 grams. Diet, oats._
October 7: 3 p. m., 6.5 cc 2 per cent caffein (0.232 gram per kilo)
aqueous solution administered by subcutaneous injection; 3.35 p. m.,
reflexes increased.
October 10: found dead. _Autopsy_: Nos. 70, 71, 72 showed congestion of
organs.
The reaction to caffein in the experiments of this series (C) showed
considerable variation. The appearance of symptoms, as well as the
final outcome of the experiments, differed markedly in a number of
cases, notwithstanding the fact that the conditions were the same;
thus the administration of 0.2 gram per kilo to guinea pigs, all of
which received the same diet, induced no symptoms in two of the animals
(Nos. 66 and 65), while marked symptoms were observed in the other
four; in two of these the symptoms appeared in one hour and a quarter
after injection, and in two others (Nos. 68 and 69), mild symptoms only
appeared in 20 or 25 minutes. The last two were under observation for
4 hours longer, but there was no visible change in their condition.
The duration of life in all of these guinea pigs, as indicated in the
table, likewise varied. Two (Nos. 60 and 68) died during the night
after they received caffein, one survived (No. 65), and three others
(Nos. 57, 66, and 69) lived 2½, 5, and 9 days, respectively.
Experiments with larger doses likewise showed differences in the
behavior of these animals toward caffein, but they were not quite so
marked. As shown in the table, symptoms appeared in from 35 minutes to
1.5 hours after injection. The duration of life was less than 1 day in
two pigs, about twice as long in two others, and in one case between 2
and 3 days.
A comparison made with results obtained in the preceding series shows
a striking difference in the resistance to caffein. As 2 decigrams per
kilo proved more rapidly fatal to the guinea pig than the larger doses
employed in the later experiments, this difference in the resistance
to caffein may be due to several factors. As pointed out in the
experiments on rabbits, age might be an important factor influencing
the toxicity of caffein. Unfortunately, no accurate data were available
on the age of the guinea pigs, but they were all apparently full grown,
although they differed in weight considerably. The difference in their
ages was in all probability not very great. Moreover, it will be
observed that the resistance in series B and C differed in animals of
approximately the same weight. This is evident on comparing experiments
Nos. 20, 38, and 13 of series B with Nos. 65, 66, and 69 of the next
series. Again, further inspection and analysis of these tables show
no difference in the toxicity, although there may be considerable
difference in the weight, from which it may be concluded that the
animals were of about the same age or that this plays no part in the
resistance to caffein in the guinea pig.
Diet is another factor which should be taken into consideration in
this connection. The recent work of Hunt(39) indicates that this may
influence the resistance of animals to some poisons. Our experiments,
however, fail to show any difference in the toxicity of the caffein
in guinea pigs, whether fed oats, carrots, or both, for different
results were obtained on the same diet, and there seemed to be
little or no difference in the toxicity of caffein when the diet was
different. Other explanations suggest themselves to account for the
results obtained. Seasonal changes have been assigned by a number of
investigators as a cause of variation in the resistance to drugs.
According to Focke,(24) frogs are more susceptible to digitalis in the
spring than in the summer, while Moschkowitsch(61) and Edmunds(21)
reported the very opposite results. Schmiedeberg's(80) observations
on strophantin in frogs were in harmony with those of Edmunds(21) and
Moschkowitsch.(61) Similar results were reported with guinea pigs.
Harrington's(34) experiments indicate that stimulation of the vagus is
less effective from October to January than from February to April,
when they are also much more susceptible to operative procedure. Hunt
found that the resistance of guinea pigs to aceto nitril is about twice
as great in the summer months as it is in January and February.
Race might also be thought of as an important factor in this
connection. Since the guinea pigs used at different seasons of the year
were of several varieties, there is no reason to suppose, however,
that the varieties experimented upon in the summer were more resistant
than those used in the winter and spring. It is highly probable,
therefore, that the greater resistance to caffein of the guinea pigs of
series C than those of series B was due to seasonal variation.
Doses of 0.20 to 0.24 gram caffein per kilo weight, therefore, may
be regarded as the minimum fatal dose for the guinea pig, depending
upon the season. Since 0.2 gram per kilo proved to be rapidly fatal
in series B, this quantity was perhaps not the minimum fatal dose for
the guinea pig at the season during which the experiments were made.
Additional tests with smaller doses were therefore carried out during
February and March. The results are shown in series D.
SERIES D.
_Guinea pig 49. Male. Weight, 510 grams. Diet, oats for 1 month
previous to experiment._
March 17: 3 p. m., 4 cc 2 per cent caffein (0.16 gram per kilo) were
injected subcutaneously; 4.40 p. m., reflexes increased; 5.40 p. m., no
symptoms.
March 18: 9 a. m., found dead, died in less than 18 hours. _Autopsy_:
Hemorrhage into abdominal cavity; liver and spleen unduly congested;
intestines injected; hemorrhagic area at point of injection.
_Guinea pig 40. Male. Weight, 630 grams. Diet, oats and carrots one
week previous to injection._
February 12: 11 a. m., 5 cc 2 per cent caffein (0.158 gram per kilo)
injected subcutaneously into back.
February 13: 1 p. m., still alive.
February 14: 9 a. m., found dead.
_Guinea pig 45. Female. Weight, 435 grams. Diet, oats for about one
month previous to injection._
March 17: 3 p. m., 3.5 cc of 2 per cent caffein injected subcutaneously
in the back (0.160 gram per kilo); 4.35 p. m., no symptoms; 5.40 p. m.,
no symptoms.
_Guinea pig 39. Male. Weight, 820 grams. Diet, oats and carrots._
February 12: 11 a. m., 6 cc (0.15 gram per kilo) 2 per cent caffein
injected subcutaneously in back.
February 14: 9 a. m., alive; seemed to be in good condition; found dead
at 1 p. m.
_Guinea pig 41. Weight, 660 grams. Diet, oats and carrots one week
previous to injection._
February 12: 11 a. m., 5 cc (0.15 gram per kilo) 2 per cent caffein
injected subcutaneously.
February 14: 2 p. m., pig alive; apparently normal.
February 18: Guinea pig still alive and apparently in good condition.
_Guinea pig 46. Female. Weight, 470 grams. Diet, oats about one month
previous to experiment._
March 17: 3.15 p. m., 4 cc (0.170 gram per kilo) 2 per cent caffein
injected into back subcutaneously; 4.35 p. m., reflexes increased,
tremors on handling marked; 5.40 p. m., no change, symptoms about as
before.
March 18: 2.30 p. m., no symptoms.
The experiments of this series (D) likewise showed a considerable
difference in the resistance of the individual guinea pigs. Nos. 41,
45, and 46 survived; the rest of the pigs died within 18 hours to 2
days after the administration of caffein. Since an autopsy was held on
one only, it is impossible to assign a cause for the variation in the
toxicity of caffein in these guinea gigs, as the diet and the other
conditions under which the experiments were conducted were the same.
It was found in the experiments on cats and rabbits that the presence
of morbid processes tends to increase the toxicity of caffein. The
observations of Ophüls(66) are of interest in this connection. He found
spontaneous lesions of the kidney and liver in a large proportion of
guinea pigs examined. The greater susceptibility to caffein of guinea
pigs Nos. 39, 40, 49, is probably due therefore to some pathological
change which increased its toxicity. About 0.2 to 0.24 gram per kilo
may therefore be regarded as the minimum lethal dose for the normal
guinea pig when caffein is introduced subcutaneously, the minimum toxic
dose being about 150-160 mg per kilo.
Experiments were also conducted to determine the largest dose which
does not produce any visible effects. In a number of tests with from
100 to 120 mg caffein per kilo (series E, see Table 6, p. 51) no
manifestation of nervous or muscular disturbance nor any departure
from the normal in respiratory activity was observed. Such quantities
may be regarded as the largest doses which are surely safe for these
animals. It is quite possible, therefore, that the greater variation in
the toxicity of caffein observed in these experiments is due to morbid
conditions. Moreover, there is some evidence that caffein increases
the toxicity of certain poisons, as shown by Hale(33) for acetanilid.
Is it not possible that caffein may similarly be affected by poisons
circulating within the body? Indeed the recent work of Loeb(23) makes
this supposition highly probable. This investigator found that caffein
and adrenalin injected together produce myocarditis in the rabbit. It
is conceivable that the combined action of caffein and some preexisting
poison may cause changes which terminate in the death of the animal.
The delayed death of guinea pigs after the administration of caffein
observed in this and other series may probably be accounted for in this
way.
Experiment 57 lends some support to this view. The condition of the
kidneys and the presence of a severe gastro-enteritis are sufficient to
account for the death of this case. Again the frequent association of
gastro-enteritis and congestion of the organs in caffein intoxication
found in different animals makes it highly probable that these lesions
were caused by caffein.
INJECTION INTO THE PERITONEAL CAVITY.
The experiments were carried out with different doses. All the guinea
pigs in this series were kept on a uniform diet, consisting of oats.
Most of them were of average size and there were no wide variations
in their weights. The experiments of series A with the smallest doses
were conducted in March and April; all the other experiments it will be
noticed were made in October.
SERIES A.
_Guinea pig 41. Weight, 700 grams. Diet, oats._
April 1: 3.30 p. m., 4.5 cc 2 per cent caffein (130 mg per kilo)
injected into peritoneal cavity. 5.35 p. m., symptoms present but no
tetanus.
April 2: Found dead about 2 p. m., duration of life about 22 hours.
_Autopsy_: Subcutaneous hemorrhage at the point of inoculation; serious
exudate on visceral and parietal peritoneum with marked inflammation of
peritoneum; portions of intestines showed slight enteritis.
_Guinea pig 49. Male. Weight, 370 grams. Diet, oats._
April 1: 3.15 p. m., 2.5 cc 2 per cent caffein (135 mg per kilo)
injected into the peritoneal cavity; 5.30 p. m., symptoms present;
reflexes increased, but no tetanus. Guinea pig survived.
_Guinea pig 47. Female. Weight, 550 grams. Diet, oats since about
February 4._
March 17: 3.30 p. m., 3.5 cc 2 per cent caffein (127 mg per kilo)
injected into peritoneal cavity; 4.35 p. m., increased irritability
present, but not marked; 5.40 p. m., symptoms about the same as before.
March 18: 2.30 p. m., condition good; no symptoms. Survived.
_Guinea pig 50. Female. Weight, 290 grams. Diet, oats._
April 1: 3.30 p. m., 2 cc 2 per cent caffein (138 mg per kilo) injected
into peritoneal cavity; 5.35 p. m., symptoms present; reflexes much
increased, but no tetanus. Survived.
SERIES B.
_Guinea pig 51. Yellow female. Weight, 415 grams._
October 1: 9.50 a. m., 3 cc (144 mg per kilo) 2 per cent caffein
injected into peritoneal cavity; 4.30 p. m., no symptoms, although
under observation all day.
October 3: 2 p. m., alive.
_Guinea pig 52. White male. Weight, 450 grams._
October 1: 9.45 a. m., 3.5 cc, 2 per cent caffein (155 mg per kilo),
injected into peritoneal cavity; 4.30 p. m., no symptoms developed
since injection.
October 3: 2 p. m., alive.
_Guinea pig 58. Brown and white male. Weight, 490 grams._
October 1: 9.45 a. m., 4 cc, 2 per cent caffein (163 mg per kilo),
injected into peritoneal cavity; 4.30 p. m., no symptoms developed
since injection.
October 3: 2 p. m., alive.
October 8: Found dead. _Autopsy_: Congestion of lungs, spleen, liver,
kidneys, and small intestines.
SERIES C.
_Guinea pig 59. Gray and white. Weight, 375 grams. Diet, oats._
October 3: 2 p. m., 3.75 cc (0.2 gram per kilo) injected into
peritoneal cavity; 2.15 p. m., reflexes increased but not markedly; 4
p. m., reflexes still more increased; no other symptoms; 5.30 p. m., no
symptoms.
October 4: 8.50 a. m., guinea pig alive and active.
_Guinea pig 58. Brown and white. Weight, 380 grams. Diet, oats._
October 3: 2 p. m., 3.8 cc caffein (0.2 gram per kilo), 2 per cent
solution, injected into peritoneal cavity; 2.10 p. m., hind legs
extended, then tetanus; attack lasted a few seconds, after which pig
raised himself on his legs, but reflexes remained much exaggerated; 4
p. m. to 5.30 p. m., no symptoms of caffein intoxication.
October 4: 8.50 a. m., guinea pig alive and active.
_Guinea pig 56. Gray and white male. Weight, 440 grams. Diet, oats._
October 1: 11.30 a. m., received 4.6 cc of 2 per cent caffein solution
(0.2 gram per kilo) into abdominal cavity; 11.45 a. m., stiffness and
rigidity of posterior extremities, reflexes increased; 12.30 p. m.,
hind legs paralyzed, reflexes increased; 4.35 p. m., no symptoms,
guinea pig in good condition.
October 3: Still alive in good condition.
October 14: Died. _Autopsy_: Anterior lobe of right lung hepatized.
Small portion of small intestine edematous. Other organs normal.
_Guinea pig 55. White and yellow male. Weight, 690 grams. Diet, oats._
October 1: 11.30 a. m., received 6.5 cc of 2 per cent solution caffein
(188 mg per kilo) into peritoneal cavity; 11.40 a. m., stiffness in
all extremities, reflexes markedly increased; 12.30 p. m., reflexes
increased, anterior and posterior extremities paralyzed; 3 p. m., found
dead.
SERIES D.
_Guinea pig 67. Gray and yellow male. Weight, 330 grams. Diet,
oats._
October 5: 11.25 a. m., 4 cc of 2 per cent caffein injected into
peritoneal cavity (240 mg per kilo); 11.30 a. m., tetanus--survived,
convulsions off and on. Death at 2.55 p. m. _Autopsy_: Severe
gastroenteritis; kidney petechiated; congestion of lungs and liver.
_Guinea pig 63. Gray and white male. Weight, 340 grams. Diet, oats._
October 5: 11.20 a. m., 4 cc of 2 per cent caffein (235 mg per kilo)
injected into peritoneal cavity.
October 14: Alive and in good condition.
_Guinea pig 64. Brown and black female. Weight, 305 grams._
October 5: 11.35 a. m., 3.8 cc 2 per cent solution caffein (250 mg per
kilo) injected into peritoneal cavity; 11.40 a. m., tetanus--survived,
convulsions off and on, died at 4.15 p. m. _Autopsy_: Findings exactly
the same as in No. 67.
Examination of the results of the experiments by intraperitoneal
injections showed that 0.2 gram caffein per kilo was toxic in
two guinea pigs (Nos. 59 and 58). Severe symptoms were observed
within 15 minutes in No. 56 and within one hour in No. 55 after the
administration of approximately the same dose of caffein. One of
these died within three and one-half hours; the other, No. 56, made
a good recovery from the acute effects. This amount of caffein may
be regarded, therefore, as the minimum toxic dose for the guinea pig
when injected into the peritoneal cavity. This is corroborated by
the experiments of series B in which smaller doses failed to show
any muscular, nervous, or respiratory symptoms, nor were there any
after effects noticed, as all of them survived and were kept under
observation for some time. The guinea pigs of series A, however, seem
to contradict these results. It will be remarked that appreciably
smaller doses induced symptoms in all of them, and one case terminated
fatally. The seasonal variation, as already pointed out, is in all
probability likewise responsible for the difference in the resistance
between the guinea pigs of series A and B. Tests were made also to
determine the minimum fatal dose. For this purpose the experiments of
series D were performed. The resistance of No. 63 in this series is
quite striking. We are unable to explain such a discrepancy in the
results obtained under practically uniform conditions. The minimum
fatal dose of caffein, when injected into the peritoneal cavity, is
therefore about 240 to 250 milligrams per kilo. These amounts, it will
be observed, were rapidly fatal, in striking contrast to the results
obtained when such doses were injected subcutaneously. This is probably
due to a better absorption from the peritoneal cavity than from the
subcutaneous tissues.
ADMINISTRATION BY MOUTH.
All the guinea pigs in these experiments were kept on a diet of hay and
oats and were of large size. The tests were made with different doses
of caffein in order to determine the limits of toxicity when the drug
was administered by mouth.
_Guinea pig 129. White and black male. Weight, 855 grams. Diet,
oats and hay._
June 6: 2.20 p. m., 12 cc of 2 per cent caffein (0.28 gram per kilo)
by mouth; 3 p. m., reflexes increased; 5 p. m., reflexes still more
increased; no other symptoms.
June 7: 9 a. m., found dead; guinea pig passed 75 cc urine, which was
almost colorless. _Autopsy_: Heart and blood vessels injected; lungs
congested; small intestines congested; other organs apparently normal.
_Guinea pig 130. Black and brown male. Weight, 800 grams. Diet, oats
and hay._
June 6: 2.30 p. m., 12 cc of 2 per cent caffein (0.3 gram per kilo)
administered by mouth; 3 p. m., reflexes increased; 5 p. m., increase
of reflexes greater than at 3 p. m.
June 7: 9 a. m., found dead; only a few cubic centimeters of urine
passed since 4 p. m. _Autopsy_: Heart and blood vessels injected; lungs
congested; small intestines congested slightly.
_Guinea pig 181. White and yellow male. Weight, 860 grams. Diet, oats
and hay._
June 6: 2.40 p. m., 12 cc 2 per cent caffein administered by mouth; 3
p. m., reflexes increased; 5 p. m., reflexes still more marked.
June 7: 9 a. m., found dead, pig passed about 5 cc urine since 4 p. m.
of previous day. _Autopsy_: Same as in No. 130.
_Guinea pig 136. White and black male. Weight, 1,000 grams. Diet, oats
and hay._
June 9: 4 p. m., 7.5 cc 2 per cent caffein solution injected
subcutaneously into the back; 4.50 p. m., reflexes increased.
June 10: 9.30 a. m., more sensitive than normal guinea pigs, but
reflexes not quite so marked as at 5 p. m. previous day, about 15
cc urine passed since caffein was injected, reduction of Fehling's
solution considerable, no albumin.
June 13: Alive and in good condition. Appetite good.
(NOTE.--Parallel test with urine from two guinea pigs which
did not receive caffein failed to show reduction of Fehling's solution.)
_Guinea pig 137. White and brown male. Weight, 925 grams. Diet, oats
and hay._
June 9: 4 p. m., 7 cc 2 per cent solution caffein injected
subcutaneously; 4.50 p. m., reflexes increased.
June 10: Reflexes less marked than at 5 p. m. previous day, but is
more sensitive than normal guinea pig, about 10 cc urine passed since
injection of caffein, moderate amount of reduction of Fehling's
solution.
June 13: Guinea pig alive, appetite good, condition good.
June 16: 9 a. m., found dead.
_Guinea pig 135. White and black male. Weight, 955 grams. Diet, hay
and oats._
June 9: 3 p. m., 7.5 cc 2 per cent caffein solution given by mouth
through stomach tube; 4.50 p. m., reflexes increased.
June 10: Reflexes less than on previous day and less marked than in No.
136, a few cubic centimeters dirty brown urine collected but could not
be tested for reduction.
June 13: Condition good, appetite good.
June 16: 9 a. m., found dead.
_Guinea pig 134. White and brown male. Weight, 740 grams. Diet, hay
and oats._
June 9: 2.55 p. m., 6 cc warm 2 per cent caffein solution given by
mouth through stomach tube; 4.50 p. m., reflexes increased.
June 10: 9.30 a. m., reflexes much less than day before, increase
slight, a few cubic centimeters of urine passed since injection of
caffein, looked brown and dirty, could not be tested for reducing
substances.
June 13: Guinea pig alive, appetite good, condition good.
June 14: 9 a. m., found dead.
_Guinea pig 128. White and black male. Weight, 1,075 grams. Diet, hay
and oats._
June 7: 10 a. m., 11 cc 2 per cent caffein by mouth through stomach
tube; 11.10 a. m., no symptoms, no urine passed; 1 p. m., increased
reflexes, about 15 cc (estimated) urine passed; 4 p. m., reflexes
increased, still more urine passed (about 20 cc); 4.50 p. m., tetanus,
frequent attacks, then paralysis and death at 4.58 p. m. _Autopsy_:
Lungs congested; blood vessels of heart injected; intestines slightly
congested; fatty liver.
_Guinea pig 126. White and gray male. Weight, 980 grams. Diet, oats
and hay._
June 7: 9.40 a. m., 9.8 cc 2 per cent caffein given by mouth through
stomach tube; 10 a. m., no symptoms; 11.10 a. m., no urine passed,
reflexes increased; 1 p. m., more sensitive than before; 4 p. m.,
increase of reflexes more marked, no urine passed; 4.45 p. m., about 15
cc urine collected; 5 p. m., no change.
June 8: 9 a. m., reflexes about the same as 5 p. m. previous day, no
urine passed since 4.45 p. m. previous day, considerable reduction
of Fehling's solution, much more than urine of guinea pig No. 127;
11.05 a. m., convulsions; 12 noon, still alive and stretched out on
abdomen; died at 1 p. m. _Autopsy_: Lungs badly congested; heart and
blood vessels injected; blood vessels of kidney and of small intestines
injected; liver engorged with blood; a few necrotic spots in stomach.
_Guinea pig 127. White, black, and brown male. Weight, 760 grams.
Diet, oats and hay._
June 7: 9.50 a. m., 7.6 cc 2 per cent caffein by mouth through stomach
tube; 10 a. m., no symptoms; 11.10 a. m., reflexes increased, no urine
passed; 1 p. m., very sensitive; 4 p. m., sensitiveness increased,
about 20 cc urine passed; 5 p. m., no change.
June 8: 9 a. m., reflexes about the same as 5 p. m. previous day; 9.30
a. m., guinea pig passed 30 cc urine since he received caffein, urine
showed a moderate amount of reduction; 12 noon, convulsions; died at
2.30 p. m. _Autopsy_: Lungs congested; blood vessels of heart and of
intestines injected; numerous necrotic spots in stomach; other organs
apparently normal.
Examination of the protocols shows that the absorption of caffein from
the gastro-intestinal canal was quite rapid, symptoms having been
observed as early as 20 minutes after its introduction. The duration
of life, it will be remarked, varied with the size of the dose. When
approximately 3 decigrams per kilo were fed, all the animals died in
the night. They lived, therefore, less than 18 hours. Two decigrams
per kilo were likewise fatal, but the duration of life was longer.
To decide whether or not this is the smallest fatal dose, smaller
amounts were fed. It seemed at first that about 150 mg per kilo was the
smallest toxic dose, and about 200 mg per kilo the minimum fatal dose.
Macroscopic examination of the organs, however, threw some doubt on
this supposition, for well-marked lesions were noticed in all of the
guinea pigs which received 0.2 gram per kilo. It is quite possible,
therefore, that the minimum fatal dose may be somewhat higher, as we
have reason to believe that, at least in some pathologic conditions,
the susceptibility to caffein is increased. The presence of fatty
changes in the liver of No. 128 and the rapid death in this case
lends especial support to this view. Hence, the minimum fatal dose is
probably greater than 0.2 gram per kilo for the normal guinea pig. The
doses employed for the tests on guinea pigs Nos. 129, 130, and 131 may
be considered therefore the minimum fatal dose for these animals. It
will be also remarked that macroscopical examination of the organs of
these animals failed to reveal the presence of severe lesions. That the
minimum toxic dose is probably much smaller than 0.28 gram per kilo
is indicated by the experiments on guinea pigs Nos. 135 and 134, in
which 0.15 gram caffein per kilo induced mild symptoms in from two to
three hours. Both of these, however, and also No. 137 died four to six
days after the drug was fed. As already pointed out, caffein may be
a factor in the delayed death of guinea pigs which received moderate
doses of it. That this supposition may also be true for guinea pigs
Nos. 134, 135, and 137 is indeed made probable by the observation that
after moderate amounts of caffein symptoms may persist in the guinea
pig for about 24 hours, and also by the fact that the secretion of
urine in these animals was very scanty, as shown in the preceding
record of the experiments; this means slow elimination of caffein and
its products of decomposition. It is conceivable that the presence of
toxic amounts of caffein in the body for a considerable length of time
would induce changes that ultimately lead to the death of the animal or
that morbid processes are set up by the combined action of caffein and
some preexisting poison. Since some guinea pigs, however, survived the
doses indicated, it is more probable that such changes would be brought
about by caffein in the presence of a preexisting poison. The death of
these pigs, and also of No. 137 several days later, is difficult to
account for on any other theory than the one suggested. Were it not
for the fact that controls, that is, animals of the same lot which had
not received caffein survived all of the experimental animals, changed
conditions of environment or accident might be considered the cause of
death in the guinea pigs of the last series.
TABLE 6.--_Subcutaneous injection of guinea pigs._
SERIES A.
------+--------+-------+----------+----------+--------+------+--------
Number| |Caffein|Appearance| Duration | | |
of pig|Weight. | per | of | of | Diet. |Month.|Remarks.
| | kilo. | symptoms.| life | | |
------+--------+-------+----------+----------+--------+------+--------
|_Grams._|_Gram._| | | | |
18 | 500 | 0.300 |2 minutes |2 hours |Carrots |March |Female.
| | | |40 minutes| | |
15 | 548 | .290 | |30 minutes| Oats | do. | Do.
14 | 442 | 360 |15 minutes| do. | do. | do. | Do.
------+--------+-------+----------+----------+--------+------+--------
SERIES B.
------+--------+-------+----------+----------+--------+------+--------
20 | 497 | 0.200 |2 hours |3 hours | Oats |April |Female.
| | |20 minutes|30 minutes| | |
38 | 570 | .210 |25 minutes|2 hours | Carrots|Febru |Male.
| | | |15 minutes| | -ary |
37 | 820 | .200 |1 hour |Less than |Carrots | do. | Do.
| | |25 minutes| 18 hours |and oats| |
13 | 618 | .194 |25 hours | |Oats |March |Female.
36 | 850 | .200 | 1 hour |18 hours |Carrots |Febru |Male.
| | |30 minutes| |and oats| -ary |
------+--------+-------+----------+----------+--------+------+--------
SERIES C.
------+--------+-------+----------+----------+--------+-------+-------
66 | 510 | 0.200 |None |5 days |Oats |October|Male.
65 | 510 | .200 | do. |Survived | do. | do. | Do.
60 | 320 | .219 |1 hour |Within | do. | do. |Female.
| | |15 minutes| 18 hours| | |
57 | 350 | .200 | do. |About | do. | do. | Do.
| | | | 2-12 days| | |
68 | 785 | .200 |25 minutes|Less than | do. | do. |Male.
| | | | 22 hours| | |
69 | 585 | .200 |20 minutes|9 days | do. | do. | Do.
61 | 330 | .240 |1 hour |Less than | do. | do. |Female.
| | |30 minutes| 24 hours| | |
62 | 335 | .238 |1 hour | do. | do. | do. | Do.
70 | 545 | .238 |50 minutes|About 2 | do. | do. |Male.
| | | | days | | |
71 | 540 | .240 |45 minutes| do. | do. | do. | Do.
72 | 560 | .232 |35 minutes|About 3 | do. | do. | Do.
| | | | days | | |
------+--------+-------+----------+----------+--------+-------+-------
SERIES D.
------+--------+-------+----------+----------+--------+-------+-------
49 | 510 | 0.160 |1 hour |Less than |Oats |March |Male.
| | |40 minutes| 18 hours | | |
40 | 630 | .158 | |Less than |Oats and|Febru | Do.
| | | | 2 days |carrots.| -ary |
45 | 435 | .160 | None |Survived |Oats |March |Female.
39 | 820 | .150 | |2 days |Oats and|February|Male.
| | | | |carrots.| |
41 | 660 | .150 | |Survived | do. | do. |
46 | 470 | .170 |1 hour | do. |Oats (?)|March |Female.
| | |20 minutes| | | |
------+--------+-------+----------+----------+--------+-------+-------
SERIES E.
------+--------+-------+----------+------------+--------+--------+----
19 | 556 | 0.100 | |Survived | Oats |April |
42 | 490 | .120 | None | do. | do. |February|
43 | 430 | .116 | do. | do. | do. | do. |
44 | 535 | .112 | do. | do. | do. | do. |
97 | 330 | .100 | do. | do. | do. |November|
98 | 520 | .100 | do. |About 3 days|Carrots | do. |
------+--------+-------+----------+------------+--------+--------+----
TABLE 7.--_Injection into peritoneal cavity; guinea pigs._
SERIES A.
------+--------+-------+----------+----------+--------+------+--------
Number| |Caffein|Appearance| Duration | | |
of pig|Weight. | per | of | of | Diet. |Month.|Remarks.
| | kilo. | symptoms.| life | | |
------+--------+-------+----------+----------+--------+------+--------
|_Grams._|_Gram._| | | | |
41 | 700 | 0.130 |2 hours |22 hours |Oats |April |Male.
49 | 370 | .135 |2 hours |Survived | do. | do. | Do.
| | |15 minutes| | | |
47 | 550 | .127 |1 hour | do. | do. |March |Female.
50 | 290 | .138 |2 hours | do. | do. |April | Do.
------+--------+-------+----------+----------+--------+------+--------
SERIES B.
------+--------+-------+----------+----------+-------+-------+-------
51 | 415 | 0.144 | None |Survived |Oats |October|Female.
52 | 450 | .155 | do. | do. | do. | do. |Male.
53 | 490 | .163 | do. | do. | do. | do. | Do.
------+--------+-------+----------+----------+-------+-------+-------
SERIES C.
------+--------+-------+----------+----------+-------+-------+--------
59 | 375 | 0.200 |15 minutes|Survived |Oats |October|Gray and
| | | | | | | white.
58 | 380 | .200 |10 minutes| do. | do. | do. |
56 | 440 | .200 |15 minutes|14 days | do. | do. |Male.
55 | 690 | .188 | 1 hour |3 hours | do. | do. | Do.
| | | |30 minutes| | |
------+--------+-------+----------+----------+-------+-------+--------
SERIES D.
------+--------+-------+----------+----------+-------+-------+--------
67 | 330 | 0.240 |5 minutes |30 minutes|Oats |October|Male.
63 | 340 | .235 | |Survived | do. | do. | Do.
64 | 305 | .250 |25 minutes|4 hours | do. | do. |Female.
| | | |40 minutes| | |
------+--------+-------+----------+----------+-------+-------+--------
TABLE 8.--_Caffein by mouth; guinea pigs._
SERIES J.
------+--------+-------+----------+----------+-------+-------+--------
Number| |Caffein|Appearance| Duration | | |
of pig|Weight. | per | of | of | Diet. | Month.|Remarks.
| | kilo. | symptoms.| life | | |
------+--------+-------+----------+----------+-------+-------+--------
|_Grams._|_Gram._| | | | |
129 | 855 | 0.280 |40 minutes|Less than |Hay and|June |Male.
| | | | 8 hours| oats | |
130 | 800 | .300 |30 minutes|Less than | do. | do. | Do.
| | | | 18 hours| | |
131 | 860 | .280 |20 minutes| do. | do. | do. | Do.
135 | 955 | .150 |1 hour 50 |6 days | do. | do. | Do.
| | | minutes | | | |
134 | 740 | .160 |3 hours |4 days | do. | do. | Do.
(1)137| 925 | .150 |50 minutes|6 days | do. | do. | Do.
(1)136| 1,000 | .150 | do. |Survived | do. | do. | Do.
126| 980 | .200 |20 minutes|27 hours | do. | do. | Do.
127| 760 | .200 |1 hour |28 hours | do. | do. | Do.
128| 1,075 | .200 |3 hours |7 hours | do. | do. | Do.
------+--------+-------+----------+----------+--------+------+--------
(1) Subcutaneous injection for comparison.
SUMMARY.
A survey of the results obtained in experiments on guinea pigs shows
that the mode of introduction of caffein exerts but little influence
on its toxicity. On careful analysis it will be observed that the
rate of absorption after the administration of caffein by mouth,
subcutaneously, or intraperitoneally is about the same for the
time of appearance of symptoms. The persistence of the symptoms of
caffein intoxication observed in these experiments for 24 hours after
administration points to slow elimination, which may be expected, owing
to the fact that the guinea pigs passed but little urine and caffein
is not diuretic for these animals. The prolonged presence of caffein
in the body probably exerts a harmful influence or after effect, which
may account for the delayed death of some animals many days after a
single dose of caffein was given. Among the factors which undoubtedly
influence toxicity, season should be considered, while the presence
of a diseased condition undoubtedly tends to decrease the resistance
of the guinea pig to caffein. Diet was without any influence on the
toxicity of the single dose of caffein.
EXPERIMENTS ON CATS.
These experiments were performed on well-fed animals which were kept
under observation for several days before the tests with caffein
were made. The diet consisted of meat exclusively. In some cases the
urine was examined for albumin and sugar before caffein was given. No
tests with caffein were made if large amounts of albumin were found.
It may be remarked that sugar was never found in cats before the
administration of caffein, but that considerable amounts of it were
found in some cases after it was given. Studies by various modes of
administration were made, by subcutaneous injection, intraperitoneally,
or by mouth. Attention was also directed to the resistance to caffein
in young cats, several experiments on kittens being made with this
object in view.
SUBCUTANEOUS INJECTION.
Rost stated that caffein is eliminated in the urine unchanged after
its introduction into the body and that the amounts found varied with
different species of animals. In the rabbit the amount eliminated was
about 21 per cent; in the dog about 8 per cent; and in the cat somewhat
less than 2.5 per cent. It would appear, therefore, that the cat
decomposes caffein more readily than the rabbit or dog; its resistance
consequently ought to be greater than that of the other animals.
Moderately large doses were accordingly employed in the preliminary
experiments (series A), but the results obtained, as shown in the
protocols, indicated that caffein is fully as toxic for the cat as
for the rabbit or dog. The doses were then decreased and experiments
were performed in order to ascertain the smallest toxic as well as the
smallest fatal dose.
SERIES A.
Three decigrams of caffein per kilo were administered in these
experiments. The results are shown in the following protocols:
_Cat 4. Black and white. Weight, 1,440 grams._
May 26: 10.05 a. m., 22 cc 2 per cent caffein (0.3 gram per kilo)
injected subcutaneously; 11.10 a. m., copious salivation, cat
irritable, muscular stiffness present, but no tetanus; 11.45 a. m., cat
restless, convulsions, attacks of short duration, no paralysis observed
after the convulsions, pupils dilated; 4.45 p. m., cat quiet, slight
paralysis present.
May 27: Cat exhausted.
May 28: Found dead.
_Cat 5. Black and white male. Weight, 1,396 grams._
June 3: 10 a. m. 21 cc of 2 per cent caffein (0.3 gram per kilo)
injected subcutaneously; 12 noon, found dead.
Although there was considerable difference in the duration of life
following the injection of the same dose of caffein per kilo, the final
outcome was the same, as both cats died from the effects of the drug.
One died within 2 hours and the other lived more than 30 hours after
its administration. Three decigrams of caffein per kilo is, therefore,
surely fatal to these animals. Tests made with smaller doses are shown
in the following experiments:
SERIES B.
In these experiments the doses employed ranged between 0.20 and 0.25
gram caffein per kilo.
_Cat 3. Black and white female. Weight, 2,854 grams. Well fed._
June 4: 10.30 a. m., 35 cc 2 per cent caffein (0.25 gram per kilo)
injected subcutaneously; 11 a. m., found dead.
_Cat 6. Black and white. Weight, 1,645 grams._
June 3: 20 cc 2 per cent caffein (0.243 gram per kilo) injected
subcutaneously at 3 p. m., cat grew very irritable in a few minutes;
about 4. p. m. reflexes decidedly increased; 5 p. m., cat paralyzed.
June 4: Cat found dead.
_Cat 8. Weight, 1,735 grams._
October 7: 4 p. m., 22 cc 2 per cent caffein (0.25 gram per kilo)
injected subcutaneously in the back; 4.30 p. m., cat irritable,
salivation profuse, convulsions; died at 5.30 p. m.; no urine passed
after caffein was given.
_Cat 9. Weight, 1,960 grams._
October 7: 3.45 p. m., 25 cc 2 per cent caffein (0.25 gram per kilo)
injected subcutaneously in the back; 4.45 p. m., cat very irritable,
repeated attacks of convulsions, salivation copious; died at
5.30 p. m.; cat did not urinate after injection of caffein.
_Cat 12. Striped kitten. Weight, 1,185 grams._
October 9: Urine examined, no albumin, no sugar; 1.45 p. m., 12 cc 2
per cent caffein administered; 5 p. m., cat alive, no symptoms except
salivation and general irritability.
October 10: 10.30 a. m., found dead. About 15 cc urine collected, but
no examination made.
_Cat 14. Black. Weight, 1,855 grams._
October 8: 1.40 p. m., 18.5 cc 2 per cent caffein (0.2 gram per kilo);
3 p. m., cat became restless about 10 minutes after caffein was
injected; cried persistently and moved about in cage, no convulsions,
cat urinated about 15 cc, cat defecated.
October 9: 9 a. m., cat found dead in cage. Urine gave very heavy
reduction of Fehling's solution (much more than was obtained from
urine of rabbits); 20 cc urine analyzed contained 4.65 per cent sugar.
_Autopsy_: Lungs deeply congested; liver marked fatty infiltration
and degeneration; spleen normal; kidneys pale and anemic; intestines
normal; stomach normal.
_Cat 15. Striped. Weight, 2,145 grams._
October 8: 2 p. m., 22 cc (0.2 gram per kilo) 2 per cent caffein
injected subcutaneously; 2.30 p. m., cat irritable, restless, trying to
get out of cage, crying persistently; 2.40, convulsions lasting about
two minutes, then cat raised itself and made attempts to get out of
cage, no salivation, cat urinated about 10 cc and defecated.
October 9: 9 a. m., cat found dead in cage, about 10 cc of urine
contained enormous quantities of sugar. _Autopsy_: Lungs severely
congested; liver showed marked fatty degeneration; spleen normal;
kidneys slightly pale and anemic; intestines mildly congested; stomach
normal.
_Cat 19. White. Weight, 1,100 grams._
October 20: 13 cc of 2 per cent caffein (0.236 gram per kilo). About
15 minutes later cat became irritable, reflexes increased, persistent
crying, stiffness of extremities, diarrhea present; 4.30 p. m.,
stiffness of muscles, coordination much disturbed, walked with great
difficulty; 4.30 p. m., no new symptoms, persistent crying continued.
October 21: Found dead.
_Cat 20. White kitten. Weight, 790 grams._
October 20: 11.35 a. m., 10 cc 2 per cent caffein (0.25 gram per kilo)
given subcutaneously; 12 noon, convulsions followed by paralysis; 1.30
p. m., still breathing, apparently in comatose condition, lay on its
side, dyspnoea, profuse salivation; 4 p. m., convulsions and death.
The results of the experiments of series B show that a dose of even 0.2
caffein per kilo is very toxic for the cat. Symptoms appeared in one
animal 40 minutes after the injection of caffein. Some of them were
found dead 18 hours after injection, which means that the duration
of life was probably a great deal less since there was evidence
that they had been dead for some time. Death occurred quite soon
after larger doses were injected. Cat No. 3 died 30 minutes after it
received caffein. The amounts employed in these experiments can not be
considered therefore as the minimum fatal doses. Smaller doses were
then tried, as shown in the experiments of the next series.
SERIES C.
Experiments were performed on five cats which received from 140 to 155
mg per kilo, as follows:
_Cat 24. Striped. Weight, 1,300 grams._
October 25: 10 a. m., 50 cc urine, albumin moderate amount--no sugar;
10 cc caffein injected subcutaneously at 12 noon; 12.30, irritable,
cried persistently, no appetite; 4 p. m., no convulsions, but
persistent crying.
October 27: Cat was still alive.
_Cat 17. Weight, 2,620 grams._
October 12: 9.30 a. m., 65 cc urine collected; more than a trace of
albumin present, no reduction of Fehling's solution; 3 p. m., 20 cc 2
per cent caffein (150 mg per kilo) injected subcutaneously; 3.15 p. m.,
irritable and restless.
October 13: 9 a. m., about 15 cc urine collected, reduction of
Fehling's solution marked; osazone test also positive.
_Cat 23. Black and white. Weight, 1,645 grams._
October 25: 10 a. m., 140 cc urine collected (since October 23), small
amount of albumin present, no sugar.
October 27: 9 a. m., no albumin; no sugar in urine; 11.50 a. m., 12.5
cc caffein injected subcutaneously (0.15 gram per kilo); 1 p. m.,
convulsions and death.
_Cat 7. Striped kitten. Weight, 1,285 grams._
October 11: Urine collected, no albumin, no sugar; 9.50 a. m., 10 cc 2
per cent caffein injected subcutaneously in the back; 10.10, violent
convulsions lasting about 30 seconds; 10.20, convulsions of shorter
duration; 10.30 convulsions; 10.35, convulsions lasting a few seconds;
urine passed about 10.20, contained a moderate amount of albumin, but
there was no reduction of Fehling's solution; 10.45, profuse salivation
and paralysis; died about 10.50.
_Cat 39. Yellow. Weight, 2,285 grams._
April 13: 2.40 p. m., 16 cc 2 per cent caffein (0.14 gram per kilo)
injected subcutaneously in the back; 3.45 p. m., cat died.
Of the five experiments of this series three died after doses of 140,
150, and 155 mg per kilo. The other two showed symptoms of toxicity,
but survived a dose of 150 mg per kilo which indicated that the minimum
fatal dose was probably reached. To test this supposition smaller doses
were administered, as shown in the following experiments.
SERIES D.
Ten cats were used for this series of experiments, and the doses
administered varied between 103 and 139 mg per kilo. The results shown
in the appended table (p.58) indicate that about 120 to 140 mg of
caffein per kilo may induce mild symptoms in some cases. The conclusion
may be safely drawn therefore that 150 mg per kilo is approximately the
minimum fatal dose for the cat when the drug is given subcutaneously.
That smaller doses are, however, by no means to be regarded as always
safe is shown in the following experiments.
SERIES E.
_Cat 43. Weight, 3,225 grams._[D]
[D] Cat probably old; had been in the laboratory for several weeks
before the experiment. Gained in weight 175 grams.
September 14: 10.20 a. m., 20 cc 2 per cent caffein (0.124 gram per
kilo) injected into the back; 11 a. m., tetanus and death. _Autopsy_:
Lungs congested; liver congested and showed hemorrhagic spots in
capsules and fatty degeneration; kidneys slightly congested; other
organs normal.
_Cat 48. Black female. Weight, 3,050 grams._
September 14: 18 cc 2 per cent caffein (0.118 gram per kilo) injected
subcutaneously in the back; 10.30 a. m., violent convulsions and death.
_Autopsy_: Lungs congested in spots showing numerous petechia; liver
congested; spleen congested; other organs normal.
The diminished resistance to caffein of cats Nos. 43 and 48 might
be due to the pathologic changes found on autopsy, for evidence is
not wanting that the toxicity of drugs might be greatly altered
under pathological conditions. Hunt(40) has shown that resistance to
acetonitril is considerably diminished in chronic alcoholism. This
seems to be true also of other drugs under abnormal conditions. Smaller
doses of atropin(78) are required in lead poisoning than under normal
conditions to produce the same results. The following experiment is of
interest in this connection, for in this case a much smaller dose than
was given in experiments Nos. 43 and 48 produced the typical symptoms
of caffein poisoning and proved to be fatal.
_Cat 47, black and white male. Weight, 4,220 grams._
September 15: Received subcutaneously 18 cc 2 per cent caffein (0.084
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