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  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
gram per kilo); no symptoms observed for about six hours.

September 16: No symptoms.

September 17: Weight, 4,250 grams; injected 18 cc 2 per cent caffein
(0.084 gram per kilo); tetanus and death after two hours. _Autopsy_:
Severe hemorrhagic pneumonia; kidneys pale, other organs normal.

Since two controls survived the same dose in proportion to the body
weight of the animal without showing any symptoms, the assumption is
justified that the lower resistance to caffein was due to the presence
of pneumonia, thus affording additional support to the view that the
toxicity of caffein may be increased in disease.


INJECTION INTO THE PERITONEAL CAVITY.

These experiments were carried out on full-grown and on young subjects.
As in previous experiments, doses of different sizes were employed. A
dose of 0.2 gram per kilo was tried first and then reduced gradually to
0.1 gram per kilo.

 _Cat 10. Female. Weight, 2,970 grams._

October 9, 1909: 1.30 p. m., 30 cc 2 per cent caffein (0.2 gram per
kilo) injected into the peritoneal cavity; urine examined for albumin
and sugar, negative; cat found dead at 2.30 p. m. No urine in the
bladder.

 _Cat 16. Black female. Weight, 2,420 grams._

October 9, 1910: Urine examined for albumin and sugar, negative; 2.30
p. m., 22 cc 2 per cent caffein (0.183 gram per kilo) injected into the
peritoneal cavity; found dead at 3 p. m.

 _Cat 99. Well-fed gray female. Weight, 3 kilos._

June 22, 1911: 3.40 p. m., 15 cc 2 per cent caffein injected into
peritoneal cavity; salivation and marked irritability within one hour
after injection.

June 24: Alive, appetite good.

 _Cat 98. Well-fed black male. Weight, 4,100 grams._

June 22: 3.45 p. m., 20.5 cc 2 per cent caffein (0.1 gram per kilo)
injected into peritoneal cavity; very irritable a few minutes after
injection, no other symptoms.

June 24: No symptoms, appetite good.

 _Cat 93. Black and white. Weight, 1,450 grams._

June 22: 3 p. m., 30 cc 2 per cent caffein (0.137 gram per kilo)
injected into peritoneal cavity; salivation, no other symptoms; under
observation until 6 p. m.

June 23: 9 a. m., no urine, cat showed no symptoms.

June 24: 9 a. m., no symptoms, took nourishment as usual.

 _Cat 87. Well-fed white female. Weight, 2,615 grams._

June 23: 2.45 p. m., 19 cc 2 per cent caffein (0.145 gram per kilo)
injected into peritoneal cavity; became irritable and restless.

June 24: 9 a. m., no symptoms, took nourishment as usual.

 _Cat 97. Gray. Age, 3 months. Weight, 500 grams. Diet, meat._

June 24: 2.25 p. m., 5 cc 2 per cent caffein (0.2 gram per kilo)
injected into peritoneal cavity; 4 p. m., no symptoms.

June 29: Died.

 _Cat 96. Gray and white. Age, 3 months. Weight, 575 grams. Diet, meat._

June 24: 2.20 p. m., 4 cc 2 per cent caffein (0.139 gram per kilo)
injected into peritoneal cavity; 3.55 p. m., no symptoms.

June 30: Died.

 _Cat 95. Black. Age, about 3 months. Weight, 860 grams. Diet, meat._

June 24: 10.15 a. m., 8.6 cc 2 per cent caffein injected into
peritoneal cavity, salivation immediately after injection; 10.25,
convulsions and paralysis; died 10.45 a. m. _Autopsy_: Macroscopical
examination of the organs, negative.

 _Cat 94. Black and white. Weight, 790 grams. Age, about 3 months.
 Diet, meat._

June 24: 10 a. m., 8 cc 2 per cent caffein injected into peritoneal
cavity; 4 p. m. under continual observation since injection, cat very
irritable, respiration more rapid than normal, diarrhea present.

Examination of the above protocols show that a dose of 2 decigrams per
kilo was fatal within one hour to one cat and that a somewhat smaller
dose killed another individual in 30 minutes. Amounts under 0.15
gram per kilo were just sufficient to induce mild symptoms, such as
increased irritability and salivation, which disappeared within a few
hours. In no case were the effects noticeable on the following day.
The experiments on young kittens are especially interesting, as they
proved, contrary to expectation, to be distinctly more resistant than
full grown individuals. The death of Nos. 97 and 96 within five and six
days, respectively, can not be ascribed to caffein, since some of the
controls also died. Moreover, it will be remarked in this connection
that no symptoms appeared in three of the four young kittens after the
administration of a dose which was rapidly fatal to adult cats. The
rapid death of No. 95 after the same dose forms an exception which can
not be accounted for, as macroscopical examination at autopsy proved
negative.


ADMINISTRATION BY MOUTH.

Two decigrams per kilo were given at first, but it was found that this
amount was surely fatal. The dose was therefore reduced to 0.125 gram
per kilo. In all of these experiments caffein was given by means of a
soft rubber catheter slipped over the stem of a funnel which served as
a stomach tube. A 2 per cent aqueous solution was used throughout these
tests except in one case in which caffein was given mixed with the food.

  _Cat 92. Black and white female. Weight, 1,750 grams._

June 10: 12.05 p. m., 14 cc 2 per cent caffein (0.16 gram per kilo)
given by mouth; cat was quiet when tied on holder, struggled only a
little when tube was put into stomach; 12.30 p. m., cat vomited, no
other symptoms.

June 13: Condition good, appetite good.

 _Cat 87. White female. Weight, 2,620 grams. Diet, meat._

June 5: 2.15 p. m., 20 cc 2 per cent caffein (0.15 gram per kilo)
solution administered by mouth through stomach tube; 2.30 p. m., cat
irritable, but no other symptoms; 5 p. m., condition about the same,
except that it was more irritable and showed some stiffness of the
extremities.

June 13: Alive and in good condition, appetite good, not irritable.

 _Cat 91. White female. Weight, 3,050 grams._

June 10: 12 noon, 23 cc (0.15 gram per kilo) of 2 per cent caffein
administered by mouth, cat struggled violently; 1.30, salivation; 1.40
p. m., convulsions; died at 2 p. m. _Autopsy_: Congestion of lungs,
liver, and spleen; heart vessels injected; other organs normal.

 _Cat 88. Black and white female. Weight, 3,260 grams. Diet, meat._

June 5: 2.20 p. m., 25 cc of 2 per cent caffein (0.15 gram per kilo)
given by mouth; 2.45 p. m., cat irritable, no other symptoms (cat did
not vomit after the administration of caffein); 4 p. m., cat found
dead. _Autopsy_: Liver very much congested; heart contracted; body was
still warm at the time of autopsy.

 _Cat 90. White and yellow female. Weight, 2,685 grams. Diet, meat._

June 5: 3.15 p. m., 27 cc of 2 per cent caffein (0.2 gram per kilo)
given by mouth through stomach tube, about half an hour later cat
became irritable and began to salivate; at 4.30 p. m. salivation
became more marked, dyspnoea was well developed, and the cat was
quite restless and had tremors; 5 p. m., short spasms of posterior
extremities, but lay quietly in the cage most of the time; 5.20 p. m.,
convulsions of short duration and death, muscular relaxation followed
immediately after convulsions, no vomiting, diarrhea observed after
administration of caffein, and cat passed about 10 cc of urine.

June 6: 9 a. m., found dead.

 _Cat 89. White and black female. Weight, 2,860 grams. Diet, meat._

June 5: 3.15 p. m., 28.6 cc (0.2 gram per kilo) of 2 per cent caffein
given by mouth through stomach tube, no vomiting observed, nor any
other symptoms; 3.30, found dead. _Autopsy_: Organs normal; liver
congested.

 _Cat 82. Gray female. March 3, weight 2,450 grams; June 6, weight
 2,750 grams. Diet, 150 grams of meat daily._

June 7: Given 0.4125 gram of caffein in 150 grams of meat, did not eat.

June 8: Given 0.4125 gram of caffein in 150 grams of meat, refused to
eat.

June 9: Given 150 grams of meat without caffein, ate all of it.

June 10: No food given.

June 11: No food given.

June 12: Given 0.4125 gram caffein in 150 grams of meat (150 mg per
kilo), ate all of it.

June 13: Found dead. _Autopsy_: Lungs congested, liver congested; other
organs apparently normal.

 _Cat 100. Gray female. Weight, 2,740 grams. Diet, meat._

July 17: 3 p. m., 17 cc of 2 per cent caffein (124 mg per kilo) given
by mouth through stomach tube at 3.20 p. m.; 5 p. m., very irritable,
but no other symptoms.

July 18: Under observation all day, no symptoms.

 _Cat 93. Black and white female. Weight, 1,640 grams._

July 17: 3.30 p. m., 10 cc (0.125 gram per kilo) of 2 per cent caffein
given by mouth through stomach tube.

July 18: Under observation all day, no symptoms.

From the results of the experiments of this series it appears that
0.15 gram caffein per kilo may be fatal within a few hours after its
administration, even if the drug is mixed with a moderate amount of
meat. Experiments 87 and 92 show, however, that this amount may be
borne by some individuals without any serious consequences, as the cats
were under observation for some time after they received caffein, and
no untoward symptoms were noticed in either of them during this time.
It may be remarked that cat No. 92 vomited shortly after it received
caffein. It is practically certain, therefore, that this amount of
caffein in proportion to the weight of the animal will in the great
majority of cases prove fatal, and perhaps in a smaller percentage
of individuals it is surely toxic if it does not escape absorption.
Smaller doses may cause irritability in some individuals, but symptoms
referable to nervous symptoms of muscles were absent, as in experiments
Nos. 93 and 100. The minimum fatal dose of caffein for the cat when
given by mouth is, therefore, 0.15 gram per kilo.

TABLE 9.--_Subcutaneous injection; cats._

  SERIES A.

  -------+--------+---------+-----------------------+-------------------
  Number.| Weight.| Caffein |     Symptoms.         |  Duration of life.
         |        |per kilo.|                       |
  -------+--------+---------+-----------------------+-------------------
         |_Grams._| _Gram._ |                       |
      4  | 1,440  |  0.30   | 65 minutes            | Over 29 hours.
      5  | 1,396  |   .30   |                       | About 2 hours.
  -------+--------+---------+-----------------------+-------------------

  SERIES B.

  -------+--------+---------+-----------------------+-------------------
      3  | 2,854  |  0.25   |                       | 30 minutes.
      6  | 1,645  |   .243  | Few minutes           | 1 hour 30 minutes.
      8  | 1,735  |   .25   | 30 minutes.           | 1 hour.
      9  | 1,960  |   .25   | 1 hour                | 1 hour 45 minutes.
     12  | 1,185  |   .20   | 3 hours               | Less than 18 hours.
     14  | 1,855  |   .20   | 1 hour 20 minutes     |     Do.
     15  | 2,145  |   .20   | 40 minutes.           |     Do.
     19  | 1,100  |   .236  | 15 minutes            |     Do.
     20  |   790  |   .25   |                       | 4 hours 30 minutes.
  -------+--------+---------+-----------------------+--------------------

  SERIES C.

  -------+--------+---------+-----------------------+-------------------
     24  | 1,300  |  0.153  | 1 hour                | Survived.
     17  | 2,620  |   .15   | 15 minutes            | Do.
     23  | 1,645  |   .15   |                       | 1 hour 10 minutes.
      7  | 1,285  |   .155  | 20 minutes            | 1 hour.
      9  | 2,285  |   .14   |                       | 65 minutes.
  -------+--------+---------+-----------------------+-------------------

  SERIES D.

  -------+--------+---------+-----------------------+-------------------
  Number.|Weight. | Caffein |    Symptoms.          | Duration of life.
         |        |per kilo.|                       |
  -------+--------+---------+-----------------------+-------------------
         |_Grams._| _Gram._ |                       |
     13  |    730 |  0.139  | Restlessness          |
     21  |  1,165 |   .138  | None                  |
     25  |    965 |   .103  |   do.                 |
     26  |  1,605 |   .125  |   do.                 |
     27  |  1,625 |   .125  |   do.                 |
     28  |  2,335 |   .128  |   do.                 | Received 2 doses:
         |        |         |                       |  survived.
     40  |  2,710 |   .129  |   do.                 |  Do.
     41  |  1,785 |   .123  |   do.                 |  Do.
     42  |  2,315 |   .112  |   do.                 |  Do.
     38  |  2,325 |   .120  | Mild                  | Died after second
         |        |         |                       |  dose.
  -------+--------+---------+-----------------------+-------------------

  SERIES E.(1)

  -------+--------+---------+-----------------------+-------------------
     43  |  3,225 |  0.124  |                       | 40 minutes.
     48  |  3,050 |   .118  |                       | Died soon after.
     47  |  4,220 |   .084  |                       | Survived.
  (2)47  |  4,250 |   .084  |                       | 2 hours.
  -------+--------+---------+-----------------------+-------------------

  (1) Pathological conditions.

  (2) Two days after first injection.

TABLE 10.--_Injections into peritoneal cavity; cats._

  -------+--------+---------+-----------------------+-------------------
  Number.|Weight. | Caffein |      Symptoms.        |  Duration of life.
         |        |per kilo.|                       |
  -------+--------+---------+-----------------------+-------------------
         |_Grams._| _Gram._ |                       |
     99  |  3,000 |  0.100  | Mild                  | Survived.
     98  |  4,100 |   .100  |   do.(1)              |   Do.
     93  |  1,450 |   .137  | Very mild             |   Do.
     87  |  2,615 |   .145  |   do.                 |   Do.
     97  |    505 |   .200  | None                  | 5 days.
     96  |    575 |   .139  |   do.                 | 6 days.
     95  |    860 |   .200  | 15 minutes            | 30 minutes.
     94  |    790 |   .200  | Diarrhea              | Survived.
     10  |  2,970 |   .200  |                       | 1 hour.
     16  |  2,420 |   .183  |                       | 30 minutes.
  -------+--------+---------+-----------------------+-------------------

  (1) In few minutes.

TABLE 11.--_Administration of caffein by mouth; cats._

  -------+--------+---------+-----------------------+-------------------
  Number.|Weight. | Caffein |      Symptoms.        |  Duration of life.
         |        |per kilo.|                       |
  -------+--------+---------+-----------------------+-------------------
         |_Grams._| _Gram._ |                       |
     91  |  3,050 |  0.15   | 1 hour 40 minutes     | 2 hours.
     88  |  3,260 |   .15   |                       | 1 hour 40 minutes.
     92  |  1,750 |   .16   | 25 minutes            | Survived.
     87  |  2,620 |   .15   | 3 hours               |    Do.
     90  |  2,685 |   .20   | 1 hour 15 minutes     | Less than 18 hours.
     89  |  2,860 |   .20   |                       | 75 minutes.
     82  |  2,450 |   .15   |                       | Less than 24 hours.
    100  |  2,740 |   .124  | 1 hour 40 minutes     | Survived.
     93  |  1,640 |   .125  |                       |    Do.
  -------+--------+---------+-----------------------+-------------------


SUMMARY.

The toxicity of caffein in cats is shown to be the same when given by
mouth as when injected subcutaneously, the minimum fatal doses in both
cases being 0.15 gram per kilo. When introduced by the intraperitoneal
route, caffein is, on the contrary, distinctly less toxic. After the
administration of 0.137 and 0.145 gram caffein per kilo (Nos. 93
and 87) salivation in one cat (No. 93) and irritability and muscular
stiffness in the other were the only effects noticed. These symptoms
were no longer observed the next day and the cats appeared to be
perfectly normal. Experiments with larger doses indicate that the
minimum fatal dose by this method of administration is about 0.2 gram
per kilo.


EXPERIMENTS ON DOGS.

The experiments were carried out on well-fed adult dogs and on puppies,
kept under observation for some time before the drug was administered.
Only those manifesting no signs of abnormality were used for these
tests. Caffein was given by mouth mixed with 10 to 20 grams of meat,
or subcutaneously in 2 per cent aqueous solution. The young animals
received caffein dissolved in milk. The determination of the minimum
toxic or fatal doses when the drug was fed presented considerable
difficulty, as in many instances the ingestion of the drug was closely
followed by vomiting.


ADMINISTRATION BY MOUTH.

SERIES A.

The effective dose in these experiments showed considerable variation.
One dog (No. 38) died after a dose of 0.12 gram caffein per kilo, while
some subjects survived doses of 0.2 and 0.23 gram per kilo. In the 12
experiments given in Table 12, page 62, it will be noticed that from
0.12 to 0.152 gram per kilo proved fatal to three dogs, while three
others survived the same amounts in proportion to the body weight. The
results were the same with larger doses. It may be observed in this
connection that in the case of the five dogs in which vomiting was
noticed some time during the 24 hours following the administration
of caffein, four survived, No. 38 being the exception. The greater
toxicity of caffein in this case is in all probability due to some
morbid process, the presence of which was indicated by the high
temperature of this subject.

That vomiting may avert a fatal issue after larger doses of caffein
is made further probable by experiment on dog No. 48, for which, in
the absence of vomiting, a dose of 0.2 gram of caffein per kilo proved
fatal. On this supposition the discrepancy in the results obtained in
this series may be readily explained. The smallest doses which proved
fatal in these experiments were 0.145 and 0.152 gram per kilo. No. 38,
which died from a dose of 0.12 gram per kilo, may be considered as an
exception, as this subject was not normal. Experiments with caffein on
dogs were made at various other times in this laboratory but failed to
show that smaller doses of caffein, even when vomiting did not occur
after its administration, were fatal, although toxic effects were
observed. The conclusion is therefore justified that the minimum fatal
dose of caffein for the normal dog is about 0.15 gram per kilo when
given by mouth.


SUBCUTANEOUS INJECTION.

SERIES B.

To determine the toxicity of caffein more accurately, especially for
comparison with animals of other species, the subcutaneous method
of administration was also used. The injections were made with a
syringe of 20 cc capacity, the contents of which were introduced into
contiguous areas. The results of experiments on six dogs indicate that
approximately 150 to 160 mg per kilo is the minimum fatal dose, since
such doses proved fatal to two out of the three animals receiving this
amount, while three others which received doses of from 143 to 160 mg
per kilo survived.


EXPERIMENTS ON PUPPIES.

SERIES C.

In these experiments the resistance of young growing puppies to caffein
was studied. Caffein was given by mouth to all the subjects except one,
to which it was administered subcutaneously. The protocols, only a few
of which are given, and the tabulated data of the experiments (p. 62)
show that the age of the animal has a decided influence on the toxicity
of caffein.

 _Dog 11. Weight, 1,260 grams._

August 2: At 10 a. m. given 12.5 cc of 2 per cent of caffein through
stomach tube; 2 p. m., had convulsions, diarrhea, salivation, and
stiffness of limbs.

August 3: Found dead 9 a. m. _Autopsy_: Thoracic viscera apparently
normal; stomach immensely distended and filled with a white, cheesy
mass and some fluid; round worms plentiful in stomach and small
intestine; mucosa of entire intestine congested; contents of lower
intestine congested; liver pale; spleen flabby; kidney congested.

 _Dog 10. Weight, 1,650 grams._

July 26: 9.30 a. m., 29 cc of 2 per cent caffein added to 60 cc of milk
offered, but refused, and was therefore fed by mouth through stomach
tube; 10.25 a. m., no symptoms; 11.30 a. m., restlessness, extremities
stiff, post. extremities spread apart, dog shows well-marked symptoms
of caffein poisoning; 12.10 p. m., symptoms more severe, extremities
extended and spread out, is lying flat on belly so that nose touches
floor of the cage; 12.40 p. m., found dead; was alive at 12.10 p. m.
_Autopsy_: Lungs showed hemorrhagic foci in all lobes; heart apparently
normal; liver fatty; stomach and intestines filled with round worms;
spleen and kidney apparently normal.

 _Dog 9. Weight, 3,000 grams._

July 25: 350 mg caffein per kilo; 5 p. m., lying down most of the
time, occasionally walks about in stall; restlessness present, but not
marked; 5.30 p. m., vomit which looked frothy and mucilaginous noticed
on the floor of the stall; no meat particles noticed in vomit, though
searched for; whines occasionally.

July 26: 9 a. m., looks well; no signs of the effect of caffein given
the day previous.

 _Dog 8. Yellow female. Weight, 3,100 grams._

July 22: 10.50 a. m., received 1.1 grams of caffein in 10 grams of meat
(354 mg caffein per kilo); 3 p. m., vomited mucus; gait clumsy; refused
to eat; continually drinking water; very restless; 4 p. m., convulsions
set in at 3.55 p. m.; tonic rigidity of the posterior extremities;
profuse salivation; convulsions were both tonic and clonic in
character, and resembled those seen in rabbits in caffein intoxication;
a striking feature was the duration of the spasm, which began at 3.55
p. m. and kept up for more than two hours.

July 23: Found dead at 9 a. m.

The data recorded in the table and in the protocols of the experiments
of series C show that four out of the seven animals experimented upon
died in less than 24 hours after caffein was fed; three of these
received 300 to 354 mg caffein per kilo, and one received 200 mg
caffein per kilo. No. 8 vomited four hours after caffein was given.
No vomiting was observed in the other three dogs. From 0.300 to 0.350
gram of caffein per kilo may be regarded, therefore, as surely fatal
to young growing puppies. That this is in all probability the minimum
lethal dose appears from the following experiments: No. 9, which
received 350 mg per kilo, vomited one hour after and survived, which
indicates that some of it was probably not absorbed. The amount which
entered the circulation was therefore less than 350 mg per kilo.
Since No. 15, which received 250 mg caffein subcutaneously, likewise
survived, the probabilities are that 300 to 350 mg per kilo were the
minimum fatal doses for these animals. Moreover, No. 12, which received
200 mg caffein per kilo, survived, no vomiting having been observed.
The case of No. 11, in which the same amount of caffein in proportion
to body weight proved fatal, may be explained perhaps by the findings
of the autopsy.

The results obtained in these experiments justify the conclusion that
young growing dogs can stand larger doses of caffein than full-grown
and older dogs.

Attention may also be called here to the difference in the symptoms
produced by caffein in very young and in adult dogs. It was often
noticed in these experiments that the symptoms in older subjects
when given toxic doses of caffein set in rather abruptly and ended in
instantaneous death. We failed to observe this phenomenon after the
administration of large amounts of caffein to very young dogs, in which
tonic and clonic convulsions alternating with paresis were observed.
These symptoms set in rather gradually and lasted several hours (see
experiment No. 8), resembling the rabbit in this regard.


SUMMARY.

The toxicity of caffein for adult dogs is about the same, whether given
by mouth or injected subcutaneously. The resistance of puppies to
caffein is much greater than that of adults.

TABLE 12.--_Administration by mouth; dogs._ (_Series A._)

  ----+--------+-------+-----------+------------------------------------
      |        |Caffein|           |
   No.| Weight.|  per  | Results.  |               Remarks.
      |        | kilo. |           |
  ----+--------+-------+-----------+------------------------------------
      |_Kilos._|_Gram._|           |
   47 |  13.60 | 0.144 |Survived   |Vomiting observed.
   55 |  12.75 |  .200 |  do.      |Stiffness of muscles; no other
      |        |       |           | symptoms.
   56 |   7.95 |  .200 |Found dead |
      |        |       |  next day |
   52 |  13.60 |  .147 |Survived   |
   57 |   6.50 |  .230 |  do.      |Vomited after 1 hour; convulsions
      |        |       |           |  after 1 hour and 45 minutes.
   39 |  23.10 |  .120 |  do.      |Increased frequency of respiration,
      |        |       |           |  thirst, loss of appetite, vomited
      |        |       |           |  rest of day when he drank water,
      |        |       |           |  salivation, restlessness, passed
      |        |       |           |  feces frequently.
   48 |  11.50 |  .174 |  do.      |Vomiting observed.
   48 |  12.00 |  .200 |Found dead |No vomiting observed. Second dose
      |        |       |  next day |  was given 8 days after first.
   54 |  13.40 |  .200 |Survived   |Vomiting observed.
   49 |  13.15 |  .152 |Found dead |
      |        |       |  next day |
   38 |  14.50 |  .120 |  do.      |Symptoms after 1½ hours: Dog had
      |        |       |           |  a temperature of 104° F. before
      |        |       |           |  caffein was given; vomited 3 hours
      |        |       |           |  after caffein was fed.
   18 |  10.80 |  .145 |  do.      |
  ----+--------+-------+-----------+------------------------------------

TABLE 13.--_Subcutaneous injection; dogs._ (_Series B._)

  ----+--------+-------+-----------+------------------------------------
      |        |Caffein|           |
   No.| Weight.|  per  | Results.  |               Remarks.
      |        | kilo. |           |
  ----+--------+-------+-----------+------------------------------------
      |_Kilos._|_Gram._|           |
   62 |   9.30 | 0.161 |Survived   |Restlessness and vomiting one-half
      |        |       |           |  hour after injection.
   61A|  14.00 |  .160 |Found dead |
      |        |       |  next day |
   63 |  12.00 |  .150 |Survived   |Restlessness 1 hour after injection.
   64 |  14.00 |  .150 |  do.      |Restlessness and thirst 45 minutes
      |        |       |           |  after injection.
   59 |   7.20 |  .160 |Died 1 hour|Marked restlessness, but no
      |        |       |  and 20   |  convulsion.
      |        |       |  minutes  |
      |        |       |  after    |
      |        |       | injection.!
   61 |  14.60 |  .143 |Survived   |Symptoms observed in 1½ hours.
  ----+--------+-------+-----------+------------------------------------

TABLE 14.--_Administration by mouth to puppies._ (_Series C._)

  ----+--------+-------+-----------+------------------------------------
      |        |Caffein|           |
   No.| Weight.|  per  | Results.  |               Remarks.
      |        | kilo. |           |
  ----+--------+-------+-----------+------------------------------------
      |_Kilos._|_Gram._|           |
    8 |  3.10  | 0.354 |Found dead |Vomited in 4 hours after feeding;
      |        |       |  next day |  restlessness, loss of appetite,
      |        |       |           |  thirst, incoordination of muscles,
      |        |       |           |  convulsions.
    9 |  3.15  |  .350 |Survived   |Muscular incoordination and
      |        |       |           |  stiffness, restlessness, vomited 1
      |        |       |           |  hour after caffein was given.
   10 | 1.60   |  .350 |Died in    |Convulsion; no vomiting.
      |        |       |  3 hours  |
   11 | 1.26   |  .200 |Found dead |Salivation; convulsions.
      |        |       |  next day |
   12 | 1.28   |  .200 |Survived   |No symptoms.
   15 | 1.20   |  .250 |  do.      |Subcutaneous injection.
   16 | 3.50   |  .300 |Died in    |Convulsions 45 minutes after caffein
      |        |       |  1 hour   |  was fed.
  ----+--------+-------+-----------+------------------------------------




CHRONIC CAFFEIN INTOXICATION.


The object of this study was to ascertain the effect of repeated dosage
when caffein was given daily or at longer intervals. The experiments
were tried on rabbits and on dogs. As in the experiments on acute
intoxication, the animals were under observation for some time in the
laboratory before the administration of caffein was begun in order to
ascertain the presence or absence of abnormality. The relation of diet
to toxicity received some attention, but the question was not studied
exhaustively in the present investigation.


EXPERIMENTS ON RABBITS.

Full-grown adult as well as young rabbits were employed. The diet
consisted either of carrots or of oats; water was given ad libitum. The
rabbits were kept in metal cages in a well-lighted and well-ventilated
room. Unnecessary handling or any other procedure tending to fatigue or
to cause discomfort to the animals was very carefully avoided, since we
had found that such treatment was likely to decrease the resistance of
the rabbit to caffein. The caffein was administered by feeding by mouth
and through a stomach tube, or by the subcutaneous method. In a good
many cases it was given daily, in some at longer intervals.


SERIES A.

The experiments of this series formed a preliminary study for the
purpose of testing the effect of moderate doses. One decigram of
caffein per kilo was given daily for several days; when administered
at longer intervals the dose was increased to 150 to 200 mg per kilo.
It was found that the smaller doses did not produce any symptoms; even
the weights of the animals were not influenced. Doses of medium size
given on two successive days were likewise without any noticeable
effect (Nos. 182, 183, 123, 101). When a third dose of this size was
given within 48 or 24 hours it proved fatal (Nos. 123, 182, and 183).
Exceptionally, however, moderately large doses (for rabbits) may be
given for three consecutive days without fatal issue, as in rabbit No.
101. When given at intervals of two to three days, larger doses, as may
be seen from the protocols, can be administered without causing acute
death (Nos. 173, 181, 201).

The results of the tests of this series point to the absence of
any accumulation and to the possible elimination of moderate doses
of caffein and its products of decomposition within 24 hours or
thereabouts. When the doses are larger the time of its elimination is
apparently longer, as shown by the fact that repetitions of the dose
the next day may be fatal, but when a longer interval is allowed it
may be given without causing death. It will be observed that only one
rabbit of this series survived, but it was extremely emaciated. This
condition has been observed in a number of cases after caffein had been
given for several days. Even when the drug was withdrawn the animals
continued to lose weight. This may be explained by the condition of
the gastro-intestinal canal as found at autopsy. The presence of
inflammation of the mucous membrane of the stomach and intestines, with
ulceration of the mucous membrane of the pylorus in one of the rabbits
(No. 173) of the series, in all probability caused diminution or loss
of appetite, which of itself would tend to cause loss of flesh and
strength and finally death. Protocols of the experiments follow.

 _Rabbit 173._ _Carrots were fed from October 1 to 18 and oats for the
 remainder of the experiment._

  ---------+---------+---------
   Date.   | Weight. | Caffein
           |         |per kilo.
  ---------+---------+---------
           | _Grams._| _Gram._
  Oct. 9   |  1,980  |  0.141
  Oct. 11  |  1,905  |   .190
  Oct. 13  |  1,930  |   .207
  Oct. 16  |  2,005  |  0.220
  Oct. 18  |  1,845  |
  Oct. 20  |  1,740  |   .230
  ---------+---------+--------

October 21: Paralysis of posterior extremities.

October 22: 9 a. m., found dead.

The urine was examined before and after the administration of caffein.
No symptoms were observed after the administration of caffein (5 doses
in 11 days), nor was albumen or sugar found in the urine after any of
the experiments on this rabbit. _Autopsy_: Pyloric mucosa exhibited
several ulcers; small intestines showed slight inflammation; liver
deeply congested; kidneys showed marked inflammation of cortex; other
organs practically normal.

 _Rabbit 181._ _Diet, carrots September 29-October 17, then oats._

  ---------+---------+----------
   Date.   | Weight. | Caffein
           |         |per kilo.
  ---------+---------+----------
           | _Grams._| _Gram._
  Oct. 4   |  1,425  |  0.105
  Oct. 5   |  1,450  |   .100
  Oct. 6   |  1,270  |   .100
  Oct. 7   |  1,210  |   .100
  Oct. 8   |  1,375  |   .130
  Oct. 9   |  1,305  |   .153
  Oct. 11  |  1,370  |  0.175
  Oct. 13  |  1,385  |   .180
  Oct. 16  |  1,345  |   .200
  Oct. 17  |  1,030  |
  Oct. 18  |  1,230  |
  Oct. 20  |  1,215  |
  ---------+---------+----------

Rabbit was markedly emaciated and weak. No albumen or sugar found in
the urine as a result of caffein feeding.

 _Rabbit 182._ _Diet of carrots from September 29._

Received caffein subcutaneously as follows:

  ---------+---------+-----------
   Date.   | Weight. | Caffein
           |         |per kilo.
  ---------+---------+-----------
           | _Grams._|  _Gram._
  Oct. 4   |  1,765  |   0.100
  Oct. 5   |  1,880  |    .100
  Oct. 6   |  1,750  |    .100
  Oct. 7   |  1,710  |    .100
  Oct. 8   |  1,685  |    .135
  Oct. 9   |         |    .150
  Oct. 11  |  1,605  |    .174
  ---------+---------+----------

October 12: 11 a. m., 23 hours after caffein was given, convulsions
with recovery; rabbit died at 1.30 p. m. No sugar was found in the
urine at any time after the administration of caffein. Albumen was
present only in one specimen.

 _Rabbit 183._ _Diet of carrots from September 29._

Received caffein subcutaneously as follows:

  ---------+---------+----------
   Date.   | Weight. | Caffein
           |         |per kilo.
  ---------+---------+----------
           |_Grams._ | _Gram._
  Oct. 4   |  1,385  |   0.100
  Oct. 5   |  1,460  |    .100
  Oct. 6   |  1,385  |    .100
  Oct. 7   |  1,240  |    .122
  Oct. 8   |  1,310  |   0.153
  Oct. 9   |  1,390  |    .142
  Oct. 11  |  1,390  |    .187
  ---------+---------+---------

October 12: 9 a. m., found dead. No albumen was found in the urine.
Only one sample contained sugar.

 _Rabbit 123._ _White, female._ _Diet, oats._

Received caffein subcutaneously as follows:

  ---------+---------+---------
    Date.  | Weight. | Caffein
           |         |  per
           |         |  kilo.
  ---------+---------+---------
           |_Grams._ |_Grams._
   Apr. 14 |  2,350  |   42
   Apr. 16 |  2,250  |   90
   Apr. 17 |  2,325  |   86
   Apr. 20 |  2,126  |  141
   Apr. 21 |  1,965  |  152
   Apr. 22 |  1,876  |  160
  ---------+---------+---------

Rabbit died 30 minutes after last injection of caffein. _Autopsy_:
Stomach exhibited marked inflammation of mucosa. Slight enteritis.
Liver and kidneys were deeply congested and dark colored.

 _Rabbit No. 101, white male._ _Diet, oats._

Received caffein subcutaneously as follows:

  ---------+---------+---------
    Date.  | Weight. | Caffein
           |         |  per
           |         |  kilo.
  ---------+---------+---------
           |_Grams._ |_Grams._
   Mar. 18 |  2,025  |  0.100
   Mar. 19 |  1,970  |   .100
   Mar. 20 |  2,009  |   .100
   Mar. 22 |  1,855  |   .100
   Mar. 23 |  1,738  |   .114
   Mar. 24 |  1,815  |   .166
   Mar. 25 |  1,830  |   .185
   Mar. 26 |  1,710  |   .176
   Mar. 29 |  1,734  |   .219
   Apr. 1  |  1,606  |   .224
  ---------+---------+---------

April 5: Found dead. _Autopsy_: Marked inflammation of gastric mucosa.
Considerable enteritis affecting the whole extent of the intestines;
liver congested and friable; kidneys deeply congested in cortical and
medullary portions; spleen congested, but of normal size; lungs and
heart normal.

Four days, 0.1 per kilo; 10 doses in 14 days.

 _Rabbit 201._ _Diet of carrots begun October 1; October 19, oats._

Subcutaneous injections as follows:

  ---------+---------+---------
    Date.  | Weight. | Caffein
           |         |  per
           |         |  kilo.
  ---------+---------+---------
           |_Grams._ |_Grams._
   Oct. 9  |  1,000  |  0.150
   Oct. 11 |  1,015  |   .180
   Oct. 13 |  1,065  |   .187
   Oct. 16 |  1,065  |   .225
   Oct. 18 |    850  |
   Oct. 20 |    890  |   .111
  ---------+---------+---------

Under observation six hours October 20; no symptoms.

October 23: Died; was much emaciated but did not show any symptoms;
emaciation set in when caffein was withdrawn; urine never contained
sugar or albumen; symptoms observed after second dose only.


SERIES B.

The question whether caffein is cumulative in the rabbit, suggested in
the preceding experiments, was the subject of further investigation
in Series B. Caffein was given by mouth or subcutaneously. Carrots
formed the exclusive diet, a measured amount being given. The rabbits
were kept under observation for two weeks, except Nos. 370 and 373,
records of which were made only for four days before the administration
of caffein was begun. Caffein was given by mouth in experiments of
Groups I and III. Rabbits 292, 293, and 295 received daily 20 cc water
by mouth for four days previous to the administration of caffein,
while in the rabbits of Group II the caffein treatment was preceded
by the injection of 0.8 per cent salt solution subcutaneously. The
object in both cases was to ascertain whether or not the method of
the administration of caffein has any influence on the animal, but
observation made from day to day failed to show any effect of such
treatment. About 1 decigram of caffein per kilo was administered daily,
with occasional intermissions. Later in the course of the experiment
the doses were increased, 0.15 gram per kilo being the maximum dose
given. Rabbit 293 died after the third dose with symptoms of typical
caffein poisoning. The administration of the same dose of caffein was
continued 10 days longer in Nos. 292 and 295. It was omitted on the
seventh, fourteenth, and fifteenth days of the experiment. On the
eighteenth day of the experiment the dose was increased to 150 mg per
kilo and was repeated 2 days later. No. 295 was found dead the next
day. No. 292 survived. Rabbits 313 and 315 may be considered together,
as they were treated alike in every respect. The initial dose of 100
mg caffein per kilo was finally increased to 122 mg. After the twelfth
dose the emaciation was well marked and the rabbits were very weak.
No. 313 was found dead 2 days, and No. 315 3 days, after the last dose
of caffein was given. It should be remarked in this connection that
symptoms of caffein poisoning were never observed in these rabbits.
Death was not due, therefore, primarily to caffein, but the rapid loss
of flesh and strength observed during the last few days suggests that
it was due to malnutrition apparently brought about by caffein.

The results obtained by subcutaneous injection of caffein are given in
the table as Group II. The initial dose of 100 mg per kilo was injected
daily. No. 298 died after the second dose. Nos. 223 and 296 received
this amount daily for 6 days. An intermission of 2 days followed, at
the end of which the same dose was given again. The next day it was
increased to 150 mg per kilo, but no effect was observed; 48 hours
later this dose was repeated. No. 223 was found dead, but its mate
survived. Symptoms of acute caffein intoxication were not observed in
any of these rabbits. It would seem, therefore, that caffein is not
cumulative. This supposition, however, appears somewhat contradictory
in view of the fact that out of the eight rabbits of this series six
died, nor could any cause of death be ascribed other than caffein.
Also the first results of Experiments 293, 370, and 373 might be
considered as indicating that cumulation, though to a moderate extent,
does take place, since in these cases reflexes developed after the
drug was given for some time. But this view is contradicted by the
results of Experiment 371, in which 150 mg per kilo given 5 days after
the daily dosage of caffein was suspended likewise caused increased
reflexes. Cumulation, therefore, does not account for the effects
noted in the other rabbit. It will be observed that rabbit No. 370,
as well as Nos. 371 and 373, had diarrhea for several days. It is
quite possible that the weakened condition rendered the rabbits more
sensitive to the action of the drug. This is made highly probable by
the observations recorded in the experiments on acute intoxication
with caffein in which death occurred after small doses. In such cases
some pathological condition was often disclosed by the autopsy. The
results of this series corroborate, therefore, those of Series A, and
indicate again the absence of cumulative action. The results obtained
are in all probability due to malnutrition and other conditions brought
about by congestion of the viscera and consequent injury to the
gastro-intestinal canal.

TABLE 16.--_Chronic caffein intoxication of rabbits; Series B
on cumulation._

  --------------+--------------------+--------------------+-------------
                |      Group I.      |      Group II.     |  Group III.
         Data.  +------+------+------+------+------+------+------+------
                |  No. |  No. |  No. |  No. |  No. |  No. |  No. |  No.
                | 292. | 293. | 295. | 296. | 223. | 298. | 315. | 313.
  --------------+------+------+------+------+------+------+------+------
  Diet (grams          |      |      |      |      |      |      |
    carrots in         |      |      |      |      |      |      |
    2 days)       1,000| 1,000|   975|   930|   905|   880|   355|   300
  Caffein              |      |      |      |      |      |      |
    administered       |      |      |      |      |      |      |
    (cc) and weight    |      |      |      |      |      |      |
    (grams):           |      |      |      |      |      |      |
                       |      |      |      |      |      |      |
    Mar. 5       {     |      |      |      |      |      |      |
                 {1,410| 1,470| 1,045| 1,040| 1,070|   955|   770|   770
                       |      |      |      |      |      |      |
    Mar. 7       {     |      |      |      |      |      |      |
                 {1,415| 1,360| 1,140| 1,090| 1,095| 1,000|   715|   690
                       |      |      |      |      |      |      |
    Mar. 9       {     |      |      |      |      |      |      |
                 {1,350| 1,270| 1,070| 1,000| 1,055| 1,005|   655|   665
                       |      |      |      |      |      |      |
    Mar. 11      {     |      |      |      |      |      |      |
                 {1,505| 1,465| 1,190| 1,230| 1,285| 1,250|   755|   760
                       |      |      |      |      |      |      |
    Mar. 16      {     |      |      |      |      |      |      |
                 {1,580| 1,460| 1,230| 1,165| 1,170| 1,145|   730|   745
                       |      |      |      |      |      |      |
    Mar. 17      {     |      |      |      |      |      |      |
                 {1,515| 1,415| 1,080| 1,040| 1,115| 1,105|   720|   685
                       |      |      |      |      |      |      |
    Mar. 19      {     |      |      |      |      |      |      |
                 {1,565| 1,570| 1,280| 1,195| 1,235| 1,220|   710|   735
                       |      |      |      |      |      |      |
    Mar. 21      {    7|     7|     6|      |      |      |     4|     4
                 {1,585| 1,530| 1,265| 1,150| 1,215| 1,260|   755|   700
                       |      |      |      |      |      |      |
    Mar. 22      {    7|     7|     6|  (1) |  (1) |  (1) |     4|     4
                 {1,440| 1,315| 1,175| 1,100| 1,045| 1,150|   675|   635
                       |      |      |      |      |      |      |
    Mar. 23      {    7|     7|     6|  (1) |  (1) |  (1) |     4|     4
                 {1,335| 1,140| 1,110| 1,145| 1,190| 1,230|   715|   700
                       |      |      |      |      |      |      |
    Mar. 24      {    7|      |     6|  (1) |  (1) |  (1) |     4|     4
                 {1,310|  (2) | 1,090| 1,115| 1,170| 1,250|   680|   650
                       |      |      |      |      |      |      |
    Mar. 25      {    7|      |     6|  (1) |  (1) |  (1) |     4|     4
                 {1,375|      | 1,035| 1,125| 1,215| 1,215|   695|   685
                       |      |      |      |      |      |      |
    Mar. 26      {    7|      |     6|      |      |      |     4|
                 {1,255|      | 1,095| 1,105| 1,155| 1,150|   675|   695
                       |      |      |      |      |      |      |
    Mar. 27      {     |      |      |      |      |      |      |
                 {     |      |      |      |      |      |      |
                       |      |      |      |      |      |      |
    Mar. 28      {    7|      |     6|   5.5|     6|     6|     4|     4
                 {1,355|      | 1,115| 1,120| 1,160| 1,155|   595|   685
                       |      |      |      |      |      |      |
    Mar. 29      {    7|      |     6|     6|     6|     5|     4|     4
                 {1,385|      | 1,150| 1,155| 1,165|   955|   695|   675
                       |      |      |      |      |      |      |
    Mar. 30      {    7|      |     6|     6|     6|      |     4|     4
                 {1,330|      | 1,075| 1,035| 1,095| Dead.|   630|   610
                       |      |      |      |      |      |      |
    Mar. 31      {    7|      |     6|     6|     6|      |     4|     4
                 {1,325|      | 1,170| 1,110| 1,140|      |   690|   605
                       |      |      |      |      |      |      |
    Apr. 1       {    7|      |     6|     6|     6|      |     4|     4
                 {1,335|      | 1,050| 1,050| 1,120|      |   625|   620
                       |      |      |      |      |      |      |
    Apr. 2       {    7|      |     6|     6|     6|      |     4|     4
                 {1,390|      | 1,125| 1,090| 1,155|      |   695|   625
                       |      |      |      |      |      |      |
    Apr. 3       {     |      |      |      |      |      |      |
                 {     |      |      |      |      |      |   200|
                       |      |      |      |      |      |      |
    Apr. 4       {     |      |      |      |      |      |      |
                 {1,300|      | 1,005| 1,105| 1,080|      |   585|   580
                       |      |      |      |      |      |      |
    Apr. 5       {    7|      |     6|     6|     6|      |     4|     4
                 {1,385|      | 1,090| 1,130| 1,090|      |   655|   630
                       |      |      |      |      |      |      |
    Apr. 6       {    9|      |   7.5|   7.5|     8|      |      |
                 {1,260|      | 1,010| 1,050| 1,110|      |   560|   530
                       |      |      |      |      |      |      |
    Apr. 7       {     |      |      |      |      |      |      |
                 {     |      |      |      |      |      |      |   (3)
                       |      |      |      |      |      |      |
    Apr. 8       {    9|      |   7.5|   7.5|     8|      |      |
                 {1,260|      | 1,000| 1.090| 1.965|      | Dead.|
                       |      |      |      |      |      |      |
    Apr. 9    Survived.|      | Dead.|Surv. | Dead.|      |      | Dead.
  ---------------------+------+------+------+------+------+------+------

  (1) On these days 5 _cc_ of salt solution was administered
  subcutaneously.

  (2) Dead Mar. 23.

  (3) Found dead 9 a. m.

TABLE 17.--_Chronic intoxication of rabbits, series B, Group
IV, on cumulation._

RABBIT, 370.

  ---------+---------+----------+--------+--------+----------+----------
           |         |          |        |        | Caffein  |
    Date.  | Weight. | Carrots. | Water. | Urine. |   by     | Symptoms.
           |         |          |        |        | stomach. |
  ---------+---------+----------+--------+--------+----------+----------
           |         |          |        |        |_Mg per_  |
           | _Grams._| _Grams._ | _cc._  |        |  _kilo._ |
   Aug. 7  |  2,155  |      450 |     50 |   280  |          |
   Aug. 8  |  2,030  |      450 |     25 |   185  |          |
   Aug. 9  |  2,105  |      290 |      0 |   275  |          |
   Aug. 10 |  2,095  |      450 |     30 |   335  |          |
   Aug. 11 |  2,105  |      450 |     65 |   360  |      50  |
   Aug. 12 |  2,125  |      450 |     65 |   220  |      50  |
   Aug. 13 |  2,120  |      350 |     25 |   265  |      50  |
   Aug. 14 |  2,170  |      450 |     35 |   275  |      75  |
   Aug. 15 |  2,175  |      350 |    (?) |   200  |      75  |
   Aug. 16 |  2,170  |      360 |     65 |   250  |      75  |
   Aug. 17 |  2,175  |      310 |     35 |   170  |     100  |
   Aug. 18 |  2,095  |      180 |     40 |   285  |     100  | Severe
           |         |          |        |        |          | diarrhea.
   Aug. 19 |  2,120  |      400 |    (?) |   285  |     125  |    Do.
   Aug. 20 |  2,120  |      400 |    (?) |   310  |     125  | Better.
   Aug. 21 |  2,120  |      400 |     70 |   250  |     125  |    Do.
   Aug. 22 |  2,040  |      400 |     45 |   265  |     150  | Diarrhea
           |         |          |        |        |          | bad.
   Aug. 23 |  2,030  |      370 |     35 |   220  |     150  | Diarrhea
           |         |          |        |        |          | better.
   Aug. 24 |  1,950  |      215 |     40 |   120  |     150  |    Do.
   Aug. 25 |  1,885  |      195 |     35 |    60  |     200  | Reflexes.
   Aug. 26 |         |          |        |        |          | Found
           |         |          |        |        |          |dead at 9.
  ---------+---------+----------+--------+--------+----------+----------

RABBIT, 373.

  ---------+---------+----------+--------+--------+----------+----------
   Aug. 7  |  2,240  |     450  |     50 |    230 |          |
   Aug. 8  |  2,150  |     150  |     30 |    300 |          |
   Aug. 9  |  2,120  |     205  |      0 |    150 |          |
   Aug. 10 |  2,150  |     450  |     15 |    245 |          |
   Aug. 11 |  2,195  |     450  |      5 |    285 |      50  |
   Aug. 12 |  2,160  |     450  |     65 |    325 |      50  |
   Aug. 13 |  2,120  |     300  |     45 |    190 |      50  |
   Aug. 14 |  2,195  |     450  |     40 |    265 |      75  |
   Aug. 15 |  2,215  |     350  |     35 |    200 |      75  |
   Aug. 16 |  2,205  |     310  |     45 |    225 |      75  |
   Aug. 17 |  2,240  |     400  |     40 |    265 |     100  |
   Aug. 18 |  2,255  |     350  |     30 |    320 |     100  |
   Aug. 19 |  2,115  |     185  |    (?) |    170 |     125  | Severe
           |         |          |        |        |          | diarrhea.
   Aug. 20 |  2,115  |     280  |     35 |    195 |     125  | Diarrhea.
           |         |          |        |        |          | better
   Aug. 21 |  2,050  |     175  |     75 |    115 |     125  | Slight
           |         |          |        |        |          | diarrhea.
   Aug. 22 |  2,060  |     180  |     75 |    130 |     150  |
   Aug. 23 |  2,005  |     200  |     75 |    125 |     150  | Reflexes.
   Aug. 24 |  1,990  |     200  |     75 |    150 |     150  | Slight
           |         |          |        |        |          | diarrhea.
   Aug. 25 |  1,950  |     255  |     55 |    132 |     175  | Severe
           |         |          |        |        |          | diarrhea.
   Aug. 26 |  1,870  |     205  |     80 |    140 |    None  |    Do.
   Aug. 27 |  1,830  |     200  |     50 |    140 |     do.  |    Do.
   Aug. 28 |  1,950  |     400  |     25 |    265 |     do.  | Slight
           |         |          |        |        |          | diarrhea.
   Aug. 29 |  1,825  |     400  |      0 |    315 |     do.  | Very weak
           |         |          |        |        |          | and in
           |         |          |        |        |          | poor
           |         |          |        |        |          | condition
   Aug. 30 |  1,850  |          |     10 |    140 |     do.  |
   Aug. 31 |  1,835  |          |        |        |          |
  ---------+---------+----------+--------+--------+----------+----------

RABBIT, 371.

  ---------+---------+----------+--------+--------+----------+----------
   Aug. 7  |  2,240  |    450   |     50 |    300 |          |
   Aug. 8  |  2,260  |    450   |     50 |    225 |          |
   Aug. 9  |  2,310  |    430   |   (?)  |    300 |          |
   Aug. 10 |  2,295  |    450   |     50 |    305 |          |
   Aug. 11 |  2,320  |    450   |     50 |    335 |      50  |
   Aug. 12 |  2,280  |    450   |     70 |    400 |      50  |
   Aug. 13 |  2,300  |    350   |     70 |    255 |      50  |
   Aug. 14 |  2,265  |    425   |     55 |    154 |      75  |
   Aug. 15 |  2,260  |    250   |     40 |    125 |      75  |
   Aug. 16 |  2,295  |    155   |     70 |  Lost  |      75  |
   Aug. 17 |  2,180  |    105   |     70 |    120 |     100  |Severe
           |         |          |        |        |          | diarrhea.
   Aug. 18 |  2,150  |    125   |     70 |    100 |     100  |Diarrhea
           |         |          |        |        |          | better.
   Aug. 19 |  2,075  |    210   |   (?)  |    192 |     100  |Diarrhea
           |         |          |        |        |          | severe.
   Aug. 20 |  2,075  |    280   |     70 |    180 |     100  |Do.
   Aug. 21 |  2,165  |    260   |     50 |    225 |  None    |Diarrhea
           |         |          |        |        |          | better.
   Aug. 22 |  2,105  |    400   |     50 |    275 |   do.    |
   Aug. 23 |  2,080  |    300   |      0 |    145 |   do.    |Diarrhea
           |         |          |        |        |          | severe.
   Aug. 24 |  2,105  |    250   |     15 |    245 |   do.    |Do.
   Aug. 25 |  2,055  |    320   |     10 |    176 |     150  |Reflexes.
   Aug. 26 |  2,040  |    190   |     75 |    250 |     150  |Died at
           |         |          |        |        |          | 1 p. m.,
           |         |          |        |        |     | without having
           |         |          |        |        |     | showed any
           |         |          |        |        |     | symptoms other
           |         |          |        |        |     | than reflexes.
  ---------+---------+----------+--------+--------+----------+----------


SERIES C.

The subjects used in these experiments were rabbits of medium size and
were apparently young or at any rate were not very old. The series
was planned for the study of the possible effect of diet on the
toxicity of caffein when given for some time, and therefore oats were
substituted for carrots, which had been fed in the previous work, as
already stated. Caffein was given by mouth in the usual way, in 1 per
cent solution, 100 mg. per kilo daily. Fourteen rabbits were used for
these tests. Their weights were recorded daily and observations made at
frequent intervals during the day.

The only change noticed in all of the experiments of this series was
progressive loss of weight which set in from 3 to 8 days after the
administration of the drug was begun. The duration of life varied
considerably. No. 382 died after the first dose. No. 389 lived 2
days, No. 386, 3 days, and No. 385, 5 days; No. 390 lived 7 days and
No. 404 lived 20 days after the administration of caffein was begun.
The duration of life in all the others was from 11 to 16 days. The
findings at autopsy are interesting and suggestive as regards the
possible explanation of the effects of repeated dosage of caffein.
In eight of the rabbits there was involvement of the mucous membrane
of the stomach or intestines or of both. Since the same condition of
the gastro-intestinal canal was observed in previous experiments with
caffein when injected subcutaneously, the mere passing of the tube
into the stomach is obviously not the cause of this condition. The
fatal outcome due is therefore, as was suggested above, to inanition
brought about by the condition of the gastro-intestinal canal. Moreover
parallel experiments carried out on rabbits in the same way with
alcohol survived this treatment much longer. Obviously then the passing
of the soft rubber catheter is not the cause of this condition of the
gastro-intestinal canal nor the diet. Rabbits were fed oats exclusively
for several months in this laboratory and thrived. The presence of
pneumonia in the other rabbits of this series may be regarded as
accidental, as it is inconceivable that one or two doses of caffein,
as was the case in Nos. 382 and 389, could predispose the lungs to
infection. The results of these experiments therefore are in harmony
with those of the preceding two series, indicating that caffein does
not accumulate in the body, and that the toxicity of caffein, whether
of the single dose or of repeated doses is the same, on a diet of
carrots or of oats. These results also show that caffein is much more
toxic with repeated dosage. As stated in the historical part of this
bulletin the same view was held by Gourewitch.(28)

 _Rabbit 386._ _Belgian female._

Given 1 cc of 1 per cent caffein for each 100 grams, through stomach
tube.

  --------+--------+----------
   Date.  | Weight.|Treatment.
  --------+--------+----------
          |_Grams._|   _cc._
   Aug. 17|  1,300 |  13.0
   Aug. 18|  1,215 |  12.0
   Aug. 19|  (?)   |   (?)
  --------+--------+----------

August 20: Found dead 9 a. m. _Autopsy_: Lungs slightly congested;
liver engorged and friable; gall cyst well filled.

 _Rabbit 389._ _Black male._

Given 1 cc of 1 per cent caffein for each 100 grams, through stomach
tube.

  --------+--------+----------
   Date.  | Weight.|Treatment.
  --------+--------+----------
          |_Grams._|   _cc._
   Aug. 17|  1,070 |  10.0
   Aug. 18|  1,025 |  10.0
  --------+--------+----------

August 19: Found dead 9 a. m. _Autopsy_: Lungs severely congested and
partially hepatized; liver was engorged; other organs appeared normal.

 _Rabbit 382._ _Belgian female._

On August 17 weighed 1,035 grams; received 1 cc of 1 per cent caffein
for each 100 grams; 10 cc of 1 per cent caffein given in all.

August 18: Found dead 9 a. m. _Autopsy_: Lungs congested and hepatized;
liver engorged; stomach showed numerous petechial hemorrhages on
mucosa; kidneys slightly congested; intestines appeared normal.

 _Rabbit 385._ _Belgian female._

Given 1 cc of 1 per cent caffein for each 100 grams, through stomach
tube.

  --------+--------+----------
   Date.  | Weight.|Treatment.
  --------+--------+----------
          |_Grams._|   _cc._
   Aug. 17|    780 |  8.0
   Aug. 18|    760 |  7.5
   Aug. 19|    755 |  7.5
   Aug. 20|    715 |  7.0
   Aug. 21|    700 |  7.0
  --------+--------+----------

August 22: Found dead 9 a. m. _Autopsy_: Lungs exhibited pneumonic
lesions, with inflammation of adjacent pleura; a fibro-plastic exudate
present around lung; liver showed a coccidial infestation; stomach
distended with ingesta; mucous membrane characterized by a catarrhal
inflammation; contents of small intestine liquid in nature and bile
stained; large intestine somewhat impacted; liver and kidneys seemingly
normal.

 _Rabbit 404._ _White male._

Given 1 cc 1 per cent caffein for each 100 grams.

  ------------+---------+------------
   Date.      | Weight. | Treatment.
  ------------+---------+------------
              |_Grams._ | _cc._
   Aug. 20    |  1,465  |    14.5
   Aug. 21    |  1,475  |    14.5
   Aug. 22(1) |         |
   Aug. 23    |  1,475  |    14.5
   Aug. 24    |  1,400  |    14.0
   Aug. 25(2) |  1,405  |    14.0
   Aug. 26    |  1,415  |    14.0
   Aug. 27    |  1,400  |    14.0
   Aug. 28(1) |         |
   Aug. 29    |  1,310  |    13.0
   Aug. 30    |  1,320  |    13.0
   Aug. 31    |  1,330  |    13.5
   Sept. 1    |  1,335  |    13.5
   Sept. 2    |  1,315  |    13.0
   Sept. 3    |  1,350  |    13.5
   Sept. 4    |  1,335  |    13.5
   Sept. 5    |  1,350  |    13.5
   Sept. 6    |  1,380  |    14.0
   Sept. 7    |  1,375  |    14.0
   Sept. 8    |  1,325  |    13.0
  ------------+---------+------------

  (1) Not fed.

  (2) Reflexes.

September 9: Found dead 9 a. m. _Autopsy_: Both lungs showed extensive
pneumonia, with adhesions to pleura; pleuritis and pericarditis very
marked; large amount of fibrous exudate in pleural cavity; pyloric end
of stomach slightly congested; liver congested; other organs normal.

 _Rabbit 393._ _Belgian._

Given 1 cc of 1 per cent caffein to each 100 grams, through stomach
tube.

  ------------+---------+------------
   Date.      | Weight. | Treatment.
  ------------+---------+------------
              | _Grams._|  _cc._
   Aug. 17    |  950    |   9.5
   Aug. 18    |  910    |   9.0
   Aug. 19    |  895    |   9.0
   Aug. 20    |  910    |   9.0
   Aug. 21    |  905    |   9.0
   Aug. 22(1) |         |
   Aug. 23    |  825    |   8.0
   Aug. 24    |  870    |   8.5
   Aug. 25    |  835    |   8.5
   Aug. 26    |  780    |   8.0
   Aug. 27    |  765    |   7.5
   Aug. 28(1) |         |
   Aug. 29    |  710    |
   Aug. 30(2) |         |
   Aug. 31(1) |         |
  ------------+---------+------------

  (1) Not fed.

  (2) Condition very poor; not fed.

September 1: Found dead. _Autopsy_: Lungs congested and adhering to the
pleura; extensive inflammation of pleura; liver slightly enlarged and
congested; mucosa of stomach and small intestines slightly congested;
other organs normal.

 _Rabbit 390._ _Belgian, male._

Given 1 cc of 1 per cent caffein to each 100 grams through stomach tube.

  ------------+---------+------------
   Date.      | Weight. | Treatment.
  ------------+---------+------------
              | _Grams._|   _cc._
   Aug. 17    |  1,490  |   15.0
   Aug. 18    |  1,370  |   14.0
   Aug. 19    |  1,365  |   13.5
   Aug. 20    |  1,340  |   13.5
   Aug. 21    |  1,265  |   12.5
   Aug. 22(1) |         |
   Aug. 23    |  1,120  |   11.0
  ------------+---------+------------

  (1) Not fed.

August 24: Found dead 9 a. m. _Autopsy_: Heart and lungs appeared
normal; abdominal viscera showed no apparent pathologic change other
than coccidial infection of the liver and fullness of the blood
vessels.

 _Rabbit 392._ _Maltese, female._

Given 1 cc of 1 per cent caffein to each 100 grams through stomach tube.

  ------------+---------+-----------
   Date.      | Weight. | Treatment.
  ------------+---------+-----------
              | _Grams._|   _cc._
   Aug. 17    |  1,265  |   12.5
   Aug. 18    |  1,275  |   12.5
   Aug. 19    |  1,240  |   12.5
   Aug. 20    |  1,220  |   12.0
   Aug. 21    |  1,245  |   12.5
   Aug. 22(1) |         |
   Aug. 23    |  1,180  |   12.0
   Aug. 24    |  1,190  |   12.0
   Aug. 25    |  1,155  |   11.5
   Aug. 26    |  1,140  |   11.5
   Aug. 27    |  1,140  |   11.5
   Aug. 28(1) |         |
   Aug. 29    |  1,115  |   11.0
   Aug. 30    |  1,080  |   11.0
   Aug. 31    |  1,020  |   10.0
   Sept. 1    |    995  |   10.0
   Sept. 2    |    930  |    9.0
  ------------+---------+-----------

   (1) Not fed.

Died at 3 p. m. September 2. _Autopsy_: The stomach and small
intestines showed numerous small hemorrhagic spots; a thick coating of
mucus surrounded the contents of the stomach; the other organs were
apparently normal.

 _Rabbit 403._ _Black._

Given 1 cc of 1 per cent caffein for each 100 grams.

  ------------+---------+-----------
   Date.      | Weight. | Treatment.
  ------------+---------+-----------
              | _Grams._|   _cc._
   Aug. 20    |  1,640  |    16.5
   Aug. 21    |  1,640  |    16.5
   Aug. 22(1) |         |
   Aug. 23    |  1,490  |    15.0
   Aug. 24    |  1,515  |    15.0
   Aug. 25    |  1,475  |    15.0
   Aug. 26    |  1,390  |    14.0
   Aug. 27    |  1,330  |    13.5
   Aug. 28(1) |         |
   Aug. 29    |  1,130  |    11.5
   Aug. 30    |  1,055  |    10.5
  ------------+---------+-----------

  (1) Not fed.

August 31: Found dead at 3 p. m. _Autopsy_: Extensive gastroenteritis;
liver enlarged and congested; spleen slightly congested; peritoneum
thickened and congested; other organs normal.

 _Rabbit 884._ _Black, female._

Given 1 cc of 1 per cent caffein for each 100 grams through stomach
tube.

  ------------+---------+-----------
   Date.      | Weight. | Treatment.
  ------------+---------+-----------
              | _Grams._|   _cc._
   Aug. 16    |  1,195  |   12.0
   Aug. 17    |  1,205  |   12.0
   Aug. 18    |  1,140  |   11.5
   Aug. 19    |  1,180  |   12.0
   Aug. 20    |  1,145  |   11.5
   Aug. 21    |  1,145  |   11.5
   Aug. 22(1) |         |
   Aug. 23    |  1,005  |   10.0
   Aug. 24    |  1,035  |   10.5
   Aug. 25    |    990  |   10.0
   Aug. 26    |    960  |    9.5
   Aug. 27    |    955  |    9.5
   Aug. 28(1) |         |
   Aug. 29    |    870  |    9.0
   Aug. 30(2) |    850  |    8.5
   Aug. 31    |    810  |    8.0
   Sept. 1    |    740  |    7.5
  ------------+---------+-----------

  (1) Not fed.

  (2) Poor condition, mucus from rectum.

September 2: Found dead at 9 a. m. _Autopsy_: The mucosa of stomach
showed numerous hemorrhagic spots; the first portion of the small
intestines was slightly congested; the other organs were apparently
normal in appearance.

 _Rabbit 383._ _Belgian, female._

Given 1 cc of 1 per cent caffein for each 100 grams through stomach
tube.

  -----------+---------+------------
    Date.    | Weight. | Treatment.
  -----------+---------+------------
             | _Grams._|   _cc._
  Aug. 16    |   995   |    10.0
  Aug. 17    | 1,005   |    10.0
  Aug. 18    |   990   |    10.0
  Aug. 19    |   895   |     9.0
  Aug. 20    |   945   |     9.5
  Aug. 21    |   965   |     9.5
  Aug. 22(1) |         |
  Aug. 23    |   875   |     9.0
  Aug. 24    |   855   |     8.5
  Aug. 25    |   850   |     8.5
  Aug. 26    |   785   |     8.0
  Aug. 27    |   710   |     7.0
  -----------+---------+------------

  (1) Not fed.

August 28: Found dead at 9 a. m. _Autopsy_: Lungs, heart, and spleen
apparently normal; liver infected with coccidia; stomach apparently
normal; walls of small intestines injected; colon marked congestion and
hemorrhagic; kidneys hemorrhagic.

 _Rabbit 387._ _Belgian male._

Given 1 cc of 1 per cent caffein for each 100 grams through stomach
tube.

  -----------+---------+------------
    Date.    | Weight. | Treatment.
  -----------+---------+------------
             | _Grams._|   _cc._
  Aug. 17    |  1,260  |    12.5
  Aug. 18    |  1,340  |    13.0
  Aug. 19    |  1,335  |    13.0
  Aug. 20    |  1,300  |    13.0
  Aug. 21    |  1,325  |    13.0
  Aug. 22(1) |         |
  Aug. 23    |  1,205  |    12.0
  Aug. 24    |  1,200  |    12.0
  Aug. 25    |  1,285  |    12.5
  Aug. 26    |  1,185  |    12.0
  Aug. 27    |  1,255  |    12.5
  Aug. 28(1) |         |
  Aug. 29    |  1,115  |    11.0
  Aug. 30    |  1,135  |    11.5
  Aug. 31    |  1,175  |    12.0
  Sept. 1    |  1,050  |    10.5
  Sept. 2    |    900  |     9.0
  -----------+---------+------------

  (1) Not fed.

September 3, found dead. _Autopsy_: Stomach and small intestines showed
numerous hemorrhagic spots; thick coating of mucus surrounded the
contents of the stomach; bladder was greatly distended with urine; the
other organs were apparently normal.

 _Rabbit 388._ _Belgian male._

Given 1 cc of 1 per cent caffein for each 100 grams, through stomach
tube.

  -----------+---------+------------
    Date.    | Weight. | Treatment.
  -----------+---------+------------
             | _Grams._|   _cc._
  Aug. 17    |  1,080  |     10.0
  Aug. 18    |  1,115  |     11.0
  Aug. 19    |  1,150  |     11.5
  Aug. 20    |  1,130  |     11.5
  Aug. 21    |  1,120  |     11.0
  Aug. 22(1) |         |
  Aug. 23    |  1,020  |     10.0
  Aug. 24    |    985  |     10.0
  Aug. 25    |    960  |      9.5
  Aug. 26    |    900  |      9.0
  Aug. 27    |    875  |      9.0
  Aug. 28(1) |         |
  -----------+---------+------------

  (1) Not fed.

August 29, found dead 9 a. m. _Autopsy_: Heart and lungs normal; liver
and kidneys engorged; stomach normal; intestines showed a catarrhal
inflammation, though not severe; spleen normal; walls of colon somewhat
injected.

 _Rabbit 391._ _Belgian._

Given 1 cc of 1 per cent caffein to each 100 grams through stomach tube.

  -----------+--------+-----------
     Date.   | Weight.|Treatment.
  -----------+--------+-----------
             |_Grams._|  _cc._
   Aug. 17   |   940  |   9.5
   Aug. 18   |   950  |   9.5
   Aug. 19   |   955  |   9.5
   Aug. 20   |   935  |   9.5
   Aug. 21   |   945  |   9.5
   Aug. 22(1)|        |
   Aug. 23   |   835  |   8.5
   Aug. 24   |   805  |   8.0
   Aug. 25   |   800  |   8.0
   Aug. 26   |   765  |   7.5
   Aug. 27(2)|   690  |   7.0
   Aug. 28(1)|        |
   Aug. 29   |   565  |   5.5
  -----------+--------+-----------

  (1) Not fed.

   (2) Poor condition.

August 30, found dead 9 a. m. _Autopsy_: Heart injected; lungs normal;
liver affected slightly with coccidiidea; stomach normal in appearance;
small intestines normal, but colon considerably inflamed; kidneys
slightly engorged; other organs normal.

 _Rabbit 402._ _Black female._

Given 1 cc of 1 per cent caffein to each 100 grams.

  -----------+--------+-----------
     Date.   | Weight.|Treatment.
  -----------+--------+-----------
             |_Grams._|  _cc._
   Aug. 20   | 2,030  |   20.0
   Aug. 21   | 1,950  |   19.5
   Aug. 22(1)|        |
   Aug. 23   | 1,955  |   19.5
   Aug. 24   | 1,905  |   19.0
   Aug. 25   | 1,890  |   19.0
   Aug. 26   | 1,780  |   18.0
   Aug. 27   | 1,765  |   17.5
   Aug. 28(1)|        |
   Aug. 29   | 1,630  |   16.5
   Aug. 30   | 1,540  |   15.5
   Aug. 31   | 1,510  |   15.0
   Sept. 1   | 1,425  |   14.0
  -----------+--------+-----------

  (1) Not fed.

September 2, found dead 9 a. m. _Autopsy_: The lungs were badly
congested, the posterior lobe of the right lung showing hepatization;
the liver was considerably enlarged and congested; the mucous membrane
of the stomach and small intestines was congested and showed numerous
hemorrhagic spots; the kidneys showed slight congestion; all other
organs normal.


SERIES D.

The evidence brought forth in the preceding pages regarding cumulation
of caffein naturally suggests the question whether or not the body
acquires a tolerance for it. This question has already been answered in
the affirmative by Gourewitch,(28) but owing to the method he used for
the identification of caffein and the few experiments made his results
are not conclusive. The experiments of series A, B, and C might be
regarded as indicating that tolerance for caffein is not acquired by
the rabbit. It was noticed, however, that the rabbit apparently does
tolerate increasingly larger doses under certain conditions, as the
following experiments show:

 _Rabbit 223._ _Belgian hare, male._

October 22: Weight, 1,520 grams; 15 cc 2 per cent caffein injected
subcutaneously at 2 p. m.

November 1: 10.30 a. m., weight, 1,510 grams; 17 cc 2 per cent caffein
injected subcutaneously (225 mg per kilo), reflexes observed, but no
tetanus.

November 4: 10.30 a. m., weight 1,535 grams; 19 cc 2 per cent caffein
injected subcutaneously at 2.40 p. m.; 4.40 p. m., no symptoms.

November 8: Weight, 1,425 grams; 20 cc 2 per cent caffein (285 mg per
kilo) injected at 11.45 p. m.; 5 p. m., no symptoms.

November 17: Weight, 1,325 grams; 22 cc 2 per cent caffein injected at
2.55 p. m. (329 mg per kilo), no symptoms.

November 18: Rabbit in good condition.

 _Rabbit 224. Belgian hare, female. Diet, carrots._

October 18: Weight, 1,935 grams; 11.20 a. m., 15 cc 2 per cent caffein
(155 mg per kilo) injected.

November 1: Weight, 1,780 grams; 20 cc 2 per cent caffein (224 mg per
kilo) injected subcutaneously, reflexes increased, muscle tremors
present, but no other symptoms.

November 4: Weight, 1,710 grams; 21.5 cc 2 per cent caffein (252 mg per
kilo) injected.

November 8: Weight, 1,435 grams; 22.5 cc 2 per cent caffein or 314 mg
per kilo injected at 11.40 p. m.; 5 a. m., no symptoms.

November 17: Weight, 1,340 grams; 24 cc 2 per cent caffein (358 mg per
kilo) injected subcutaneously.

November 18: 9 a. m., rabbit died.

 _Rabbit 226. Gray male. Diet, carrots._

October 28: Weight, 1,045 grams; 10 cc 2 per cent caffein injected
subcutaneously at 1.50 p. m.; 4.30 p. m., tremors observed, but no
other symptoms.

October 29: Rabbit in good condition.

November 1: Weight, 950 grams; 10.55 a. m., 11 cc 2 per cent caffein
injected subcutaneously (231 mg per kilo).

November 4: Weight, 930 grams; 2.50 p. m., 12 cc 2 per cent caffein
injected subcutaneously (258 mg caffein per kilo).

November 6: Weight, 945 grams; 11.45 a. m., 15 cc 2 per cent caffein
(313 mg per kilo) injected subcutaneously.

November 17: Rabbit still alive; weight, 890 grams.

The results of these experiments indicate that when sufficient time is
allowed between two successive injections, susceptibility to caffein
is not increased. The rabbit, on the contrary, seems to acquire a
tolerance for the drug, for the fourth dose was 15 per cent larger than
the minimum fatal dose of caffein. This is in all probability due to
the better elimination of caffein and its products of decomposition and
to recovery from the deleterious effects of each dose, made possible by
long intervals between injections.

The results of these experiments may be briefly summed up by stating
that subminimum doses of caffein given to the rabbit daily or at
intervals (not too long) do not produce any symptoms such as were
observed in acute caffein intoxication, namely, increased reflexes
and convulsions, or increased rate of respiration, thus showing that
it is not cumulative. But evidence of undoubted summation of effect
was adduced to show that if the administration of subminimum doses of
caffein be continued daily for a period of 11 to 18 days the result
is fatal. Tolerance, however, may be acquired, although to a limited
extent only, provided sufficiently long intervals between injections
are allowed to give time for repair of the injury done by the drug and
to develop a mechanism for its better decomposition and elimination.
Furthermore, the evidence just given indicates that the elimination
of subminimum doses of caffein and its products of decomposition
is probably accomplished within 24 hours or thereabouts. That the
elimination of larger doses is not accomplished in this interval is
made probable by the following experiment:

 _Gray rabbit 455. Female. Diet, oats._

October 12: Weight, 1,185 grams; 3.30 p. m., 11.5 cc 2 of per cent
caffein injected into the lumbar muscles; 3 p. m., reflexes increased.

October 13: 10 a. m., rabbit weighed 1,070 grams; no symptoms of
caffein poisoning, reflexes normal; 10.30 a. m., 10 cc 2 per cent
caffein injected into the lumbar muscles; 11.30 a. m., rabbit jumped
off the table, had convulsions, and died.


EXPERIMENTS ON DOGS.

Having gained some information respecting the effects of repeated doses
of caffein on rabbits, it was of interest to find out how carnivora
reacted to the drug when similarly administered. A number of dogs
were used for the purpose. Considerable variation in the mode of
experimentation, as will appear later, was allowed.

Since the condition of the animal, its age, environment, or diet might
be factors influencing toxicity, tests were made on full-grown and
on young growing dogs whose food was varied. The subjects of the
experiment were kept under observation for a few days to several weeks
before the administration of caffein was begun, in order to determine
whether or not any morbid condition existed, as well as to ascertain
whether the new environment had any effect on these animals. Caffein
was given chiefly by mouth, but the subcutaneous method was also
employed during a portion of the experimental period in some dogs. The
initial dose, which varied for different individuals, was maintained
for a variable length of time. It was then progressively increased,
in most cases until the death of the animal. With larger doses the
intervals between successive injections were also increased.


SERIES A.

Six dogs were used in this series. Caffein was administered by mouth
for periods of six days to five weeks. It was given daily or at
intervals of two, and sometimes of three, days. In a few instances
the drug was withheld for four or even for seven days, and its
administration was resumed at the end of this time. The initial dose
in these experiments varied approximately between 40 and 140 mg per
kilo. The doses were then increased gradually, and thus the maximum
resistance of the subject to caffein was tested. The diet consisted
either exclusively of meat or largely of carbohydrates with a minimum
amount of meat to give flavor to the food.

 _Dog 11. Female._

Diet consisted of rice, 250 grams; cane sugar, 250 grams; meat, 50
grams; cracker meal, about 100 grams. Caffein was given by mouth daily
or at intervals of one day, when the dose did not exceed 1.5 grams.
Before the dose was increased to 2 grams, or approximately 0.213
gram caffein per kilo, an interval of two days was allowed. Symptoms
were noticed the next day. An interval of two days was therefore
allowed again at the end of which the same dose was repeated. It will
be remarked that there were no symptoms this time, and the general
condition of the dog seemed to be good. Two grams of caffein were,
therefore, given daily during the next two days without any untoward
effects; the dose was then increased to 2.5 grams. Even after this
enormous quantity no symptoms were observed except slight tremors. When
this dose was repeated 26 hours later, it proved fatal. No albumin
or sugar was found in the urine, although the dog was fed on a very
liberal carbohydrate diet. The following is a complete record of the
experiment.

April 20: Urine acid, no albumin, no sugar.

April 21: Urine free from sugar.

April 22: Urine free from sugar. 1 gram caffein given in the afternoon.

April 23: 9 a. m., dog was very thirsty, drank a large quantity of
water, urine did not reduce Fehling's solution.

April 24: 2.30 p. m., 1 gram caffein, no sugar in urine.

April 25: 1 gram caffein administered.

April 26: Weight, 10.6 kilos, urine collected in the morning, no sugar;
4.10 p. m., 1.5 grams caffein.

April 27: 1.5 grams caffein; 1.30 p. m., diet as before, no sugar in
urine.

April 28: Weight, 10.2 kilos, no caffein, no sugar in urine.

April 30: Weight, 10.4 kilos, no sugar in urine; 4.20 p. m., 2 grams
caffein.

May 1: Urine examined, sugar absent, weight 10 kilos, vomited, sick,
tremors observed, drank 500 cc water at one time, appetite poor.

May 2: No caffein, drank 150 cc water.

May 3: Urine, no sugar, moderate quantity of albumen present; 12
noon, 2 grams caffein given by mouth, weight 10.3 kilos; 2 p. m.,
urine, sugar negative, condition of dog good, no symptoms of caffein
intoxication.

May 4: 10 a. m., about 10 cc thick, dark-colored mucilaginous urine
found in collecting bottle; albumin a little more than a trace,
decidedly less than on May 3, no sugar, condition of dog pretty good
except for slight muscular tremors; 4 p. m., 2 grams caffein by mouth
(as usual).

May 5: Urine not examined, no symptoms; 4 p. m., 2 grams caffein.

May 6: Urine not examined; 2.30 p. m., 2.5 grams caffein given by
mouth; 4 p. m., slight tremor, no other symptoms.

May 7: No examination of urine, no symptoms observed; 4 p. m., 2.5
grams caffein.

May 8: 9 a. m., found dead, urine collected since last dose of caffein
was given did not contain any sugar or albumin, the amount of caffein
fed to this dog was 18 grams in 18 days. _Autopsy_: Post-mortem
examination showed marked enteritis with hemorrhagic spots on the
mucosa; liver and kidneys congested and dark colored; lungs congested;
thyroid gland was greatly enlarged and congested.

 _Dog 28_.

April 30: Weight, 6.8 kilos; the diet consisted of 250 grams rice,
250 grams sugar, 100 grams cracker meal, and 100 grams of meat. On
May 3 his weight was 7 kilos. He received 1 gram of caffein by mouth
at 12 noon. At 2 p. m. he vomited and tremors were observed. The
next day, May 4, tremors were still present though less pronounced.
Examination of the urine for sugar and albumin was negative; on May 4,
1 gram caffein was given again and repeated on May 5. On this date his
general condition was not good--dog had no appetite and refused to take
caffein. As the dog lost 10 per cent of his weight he was put on a meat
diet exclusively and the dose of caffein was reduced to 0.5 gram. He
became sick after the second dose, and the administration of caffein
was therefore discontinued. It was resumed after five days and the
caffein was administered in increasing amounts, i. e., on May 18, 0.5
gram; May 19, 0.5; May 20, 1; May 21, 1 gram in two doses of 0.5 each,
given at intervals of one hour; May 22, 1 gram. Dog became irritable,
but no other symptoms were observed. The administration of caffein
was omitted the next day. On the following day when the same dose of
caffein was given there was again marked irritability and tremors. The
experiment was therefore discontinued.

 _Dog 22. Male bulldog._

June 24: Dog weighed 13.7 kilos. Diet consisted of meat exclusively;
1 gram caffein was given by mouth; diarrhea developed; no caffein was
given for three days.

June 28: Dog weighed 13.6 kilos, 1.5 grams caffein given at 10 a. m.

June 30: 1.75 grams caffein administered.

July 2: Dog weighed 13.5 kilos; 2 grams caffein or 0.15 gram per kilo,
caused well-marked thirst, but did not produce any other symptoms.

 _Dog 20. Female_.

May 12: Weight, 7.7 kilos. Fed liberal carbohydrate diet, consisting of
rice, 100 grams; sugar 100 grams; meat and cracker meal, a sufficient
quantity to flavor the food.

May 14: Weight, 7.7 kilos. Examination of urine for albumin and sugar
gave negative results. Urine was acid to litmus.

May 17: Weight, 7.4 kilos. Three hours after it was fed the dog
received 0.5 gram caffein by mouth. The test of the urine the next day
for sugar was negative, but a trace of albumin was present. It will
be noticed that the doses were increased gradually and that symptoms
were observed only after the fourth dose of 0.1 gram per kilo. Later
meat was substituted for the carbohydrate diet and the administration
of caffein was stopped for four days. At the end of this period 100
mg caffein per kilo was fed daily for five days, and the dose was
then very gradually increased. Diarrhea occurred twice, but no other
symptoms, the second attack having lasted a few days. The following is
a complete record of the experiment:

May 19: 0.5 gram caffein 11.45 a. m.

May 20: 0.75 gram caffein 12.45 a. m.

May 21: 0.75 gram caffein 12 noon; no sugar, no albumin in urine.

May 22: 0.75 gram caffein; urine, same condition found; no symptoms.

May 23: Weight, 7.5 kilos; no caffein.

May 24: 0.75 gram caffein; tremors very marked.

May 25: No caffein.

May 26: 0.75 gram caffein.

May 27: 0.75 gram caffein.

May 28: 0.75 gram caffein.

May 29: 1 gram caffein in two doses of 0.75 and 0.25 gram.

May 30: No caffein.

May 31: No caffein; meat diet exclusively.

June 1: No caffein; meat diet exclusively.

June 2: No caffein; no sugar, no albumin in urine.

June 3: Weight, 7.6 kilos; 0.75 gram caffein; no sugar in urine.

June 4: Weight, 7.3 kilos; 0.75 gram caffein; no sugar in urine.

June 5: Weight, 7.5 kilos; 0.8 gram caffein; drank 500 cc water; ate
200 grams meat.

June 6: Weight, 7.4 kilos; 0.8 gram caffein; 500 cc urine; drank 500 cc
water; ate 200 grams meat; no symptoms.

June 7: Weight, 7.7 kilos; 0.8 gram caffein 10 a. m.; 400 cc urine, 500
cc water, 200 grams meat.

June 8: Weight, 7.5 kilos; 0.9 gram caffein, 450 cc urine, 1 p. m.; 200
grams meat, 500 cc water.

June 9: Weight, 7.6 kilos; 0.9 gram caffein, 1 p. m.; 500 cc water, 200
grams meat and bone dust; diarrhea and restlessness all afternoon.

June 10: Weight, 7.6 kilos; 1 gram caffein, 500 cc water, 200 grams
meat, 480 cc urine.

June 11: Weight, 8 kilos; 1 gram caffein, 470 cc urine, 500 cc water,
200 grams meat.

June 12: Weight, 7.8 kilos; 1 gram caffein, 710 cc urine, 500 cc water,
200 grams meat.

June 13: 450 cc urine, 500 cc water, 300 grams meat.

June 14: Weight, 7.9 kilos; 1.2 grams caffein, 500 cc water, 300 grams
meat, 490 cc urine.

June 15: Weight 7.8 kilos, 500 cc water, 300 grams meat, 550 cc urine.

June 16: Weight 8.0 kilos, 1.2 gram caffein, 500 cc water, 300 grams
meat, bone dust added to check diarrhea.

June 17: 500 cc water, 300 grams meat, 450 cc urine, diarrhea
continues, bone dust added.

June 18. Weight 7.8 kilos, 1.3 gram caffein, 300 grams meat, 500 cc
water, 300 cc urine.

June 19: Dog very thirsty, drank 1 liter of water and ate 350 grams of
meat; 960 cc urine passed during the past 24 hour.

June 21: Weight 7.5 kilos, 1.5 grams caffein given at 10 a. m. At 2
p. m. convulsions and death. This dog received a total of 21.15 grams
caffein in 25 doses during a period of 35 days, which amounts to an
average of 85 mg per kilo daily.

 _Dog 19._ _Female fox terrier._

May 13: Weight 6.4 kilos. Diet consisted of rice, 100 grams; sugar,
100 grams; and a sufficient quantity of meat and cracker meal to give
flavor to the food. Examination of the urine showed a trace of albumin
but no sugar. The urine was acid to litmus. Two days later the urine
was alkaline to litmus. There was still a small amount of albumin but
no sugar.

May 17: 0.5 gram caffein was given by mouth. Examination of the urine
collected the next day still showed the presence of albumin and the
absence of reducing substances. The dog had tremors. Caffein was,
therefore, not administered.

May 19: 0.5 gram caffein was given by mouth.

May 20: 0.75 gram caffein was fed at 12.45 p. m. The dog vomited
during the night and tremors were observed the next morning. The urine
collected was examined for albumin and sugar, but neither was found.

May 21: 12 noon, 0.75 gram caffein was fed. The dog weighed 6 kilos,
which therefore represented a loss of 0.4 kilo. Grew abnormally thirsty
and lost appetite, but no other symptoms of caffein poisoning were
observed.

May 22: The dog was again given 0.75 gram caffein at 12 noon. The
examination of the urine for albumin and sugar gave negative results.
The dog died at 4.15 p. m. The fatal dose for this dog was therefore
0.125 gram caffein per kilo, and the total amount of caffein ingested
in six days amounted to 3.25 grams, or 0.54 gram per day, which makes
90 mg per kilo.

 _Dog 21._ _White female bull._

This dog was kept on a diet exclusively of meat, and was given water
ab libitum. From 0.5 to 0.6 gram of caffein was administered daily for
seven days; the doses were then increased and were given at longer
intervals. No symptoms of the effects of caffein were observed until a
dose of 1.5 gram was fed, when diarrhea was noticed on the next day. In
the following record the details of the experiment are given:

  ----------+---------+----------
    Date.   | Weight. | Caffein.
  ----------+---------+----------
            | _Kilos._| _Grams._
  June 7    |   12.5  |    0.5
  June 8    |   12.5  |     .5
  June 9    |   12.5  |     .5
  June 10   |   12.3  |     .6
  June 11   |   12.3  |     .6
  June 12   |   12.3  |     .6
  June 13   |   12.3  |     .6
  June 14   |   12.3  |     .8
  June 16   |   12.7  |    0.8
  June 18   |   12.9  |    1.0
  June 21   |   13.4  |    1.2
  June 24   |   13.3  |    1.5
  June 25   |   (1)   |     .0
  June 27   |   13.5  |    1.5
  June 30   |   13.5  |    1.75
  ----------+---------+----------

  (1) Diarrhea.

July 2: 11.30 a. m., 2.0 gram caffein fed by mouth; 1.30 p. m.,
tetanus, dog died. The total amount of caffein fed to dog No. 21 out
of the 25 days of the experiment was 14.45 grams, or an average of 578
mg per day, which amounts to about 42 to 43 mg per kilo of body weight.

Notwithstanding the diversity in the method of experimentation,
there was a striking uniformity in some of the results obtained. All
the experiments of the series showed absence of cumulative action
of caffein. The experimental evidence presented indicates that
moderately large doses may be given at intervals of about 24 hours
without inducing any symptoms of nervous or any other disturbance.
This is illustrated in the tests on dog 11, which were preliminary
in character. In this subject 100 to 150 mg of caffein per kilo were
ingested daily for several days without showing any changes. Later
in the course of the experiment, after larger doses were given, mild
symptoms only, such as tremors, were observed. Additional evidence
of the absence of cumulative action of caffein was furnished by the
results of the following experiments:

Dog 23 received 142 mg of caffein per kilo on three successive days.
His general condition indicated that these amounts of caffein were
toxic, but he survived. In another series of tests, made after he was
allowed to rest a few days, he again failed to show any cumulation of
the drug, as he survived this time a series of tests of longer duration
than the first.

A much better illustration of the absence of cumulative action of the
drug is furnished by the experiments on dog No. 20. In this case 100 to
125 mg of caffein per kilo, given on 10 consecutive days, did not cause
any marked effects. Diarrhea and restlessness were the only symptoms
observed. These experiments therefore show that the elimination and
decomposition of caffein are apparently effected by the body within
twenty-four hours or thereabouts.

Experiments on dog 19, however, form an exception--the third dose of
125 mg caffein per kilo having proved fatal. The very low protein
content of the diet of this dog suggests itself as a possible cause of
the lower resistance to caffein of this subject. But it may be observed
that the same diet was furnished to dog 20, which stood such amounts of
caffein much longer. The presence of a trace of albumin in the urine
of dog 19 is likewise inadmissible as a cause of the difference in
the toxicity of caffein in this dog, for the urine of dog 20 likewise
contained a trace of albumin. The alkaline reaction of the urine,
together with the fact that the first dose of only 60 mg of caffein per
kilo induced symptoms of toxicity, suggests the presence of an abnormal
condition which in all probability was the cause of the death of this
subject under the conditions indicated.

In a large number of experiments on caffein performed in this
laboratory it has been observed that symptoms due to caffein often
disappeared when the administration of the same dose of the drug
was continued. Thus dog 19 vomited when the amount of caffein was
increased to 125 mg per kilo. When this amount was repeated the next
day there was no vomiting. Similar observations were made on dogs
11 and 23, also on other dogs. No. 22 developed diarrhea at first;
when the administration of caffein was resumed several days later,
however, there was no diarrhea. In other experiments performed in
this laboratory, symptoms of nervous irritability induced by caffein
disappeared on continued treatment.

It was interesting, therefore, to inquire whether resistance to caffein
would be increased by the continued administration of progressively
larger amounts of the drug. When doses of 150 and over were fed, the
intervals allowed were usually longer than 24 hours. Two and sometimes
three days were permitted to elapse between two successive doses. This
was done in order to allow time for recovery from possible changes
induced by larger doses of caffein, and thus prevent the summation of
effect. In the experiments considered, therefore, Nos. 11, 23, 20, and
19, the toxicity of caffein does not seem to be greater than in the
experiments on acute caffein intoxication in the dog. It was thought,
however, that the large initial doses or the quick change to large
doses when the amounts used in the beginning were small, might have
something to do with failure to induce a marked degree of tolerance.
The experiment on dog 21 was therefore carried out by giving from 40
to 60 mg per kilo for eight days, and then increasing the dose, but
tolerance could not be induced, as is shown in the protocol to the
experiment.


SERIES B.

According to the studies of Chittenden,(16) low protein diet improves
the general metabolism of the body, fatigue is diminished, and
bodily vigor, therefore, correspondingly increased. The expectation
is, therefore, justified that the defense of the organism against
deleterious substances introduced into the body is much improved
by such a diet, thus increasing its resistance to poisons. Hunt's
experiment on this subject, also quoted by Chittenden, lends support
to this view. He found that mice fed on carbohydrates chiefly, or on
foods containing only a small amount of protein, were more resistant
to acetonitril. It was interesting, therefore, to inquire whether the
toxicity of caffein differs under similar conditions of diet.

A fixed diet of the same calorific value was provided for all dogs
of this series, but the protein content for three of the animals was
approximately one-third of the amount usually fed to dogs. Caffein was
at first administered subcutaneously, but all the dogs on a low protein
diet developed abscesses at the site of injection, while none of those
on high protein diet showed a local reaction. Feeding by mouth was then
begun and continued throughout the experiment in each case. The initial
dose was 50 mg per kilo, which was given daily for seven to nine days.
It was then increased progressively by 25 mg per kilo; 75 mg per kilo
were administered for one to two days, 100 mg for two to three days,
125 mg for one to two days, 150 mg for one to two days, and a single
dose of 175 mg. It will be remarked that sometimes an interval of one
day had to be allowed during which no caffein was fed.

 _Dog 30. Black and tan hound, male._

The dog was under observation for about eight weeks before the
experiment was begun and had received a high protein diet. He was then
given 50 mg caffein for nine consecutive days. On the tenth day the
dose was increased to 75 mg per kilo. As no symptoms developed, this
dose was increased to 100 mg per kilo, and was fed one day apart. It
was then raised to 125 mg per kilo. For the first time since the drug
was fed, symptoms appeared; they were noticed a few hours after feeding
and persisted during the next day. Although the appetite was good,
no caffein was given on this day. On the following day this dose was
repeated. As the symptoms were not serious, 150 mg per kilo were given
daily for the next three days, until 175 mg per kilo was reached. This
dose proved fatal within six hours. Record of experiment follows:

October 9: Weight, 9 kilos, on full nitrogen diet, received daily 0.724
gram nitrogen per kilo or 87 calories per kilo, received 18 grams meat
per kilo, 4 grams lard per kilo, 3 grams carbohydrates per kilo, bone
dust, ad libitum.

November 3: Weight, 9.10 kilos.

November 10: Weight, 9 kilos.

November 20: Weight, 9.55 kilos.

November 29: Weight, 8.70 kilos.

December 6, 7, 8, and 9: Received subcutaneously 22 cc 2 per cent
caffein. Condition good, site of injection normal.

December 10, 11, 12, 13, and 14: Received 0.4375 gram caffein by
mouth equal to 0.050 gram per kilo, no symptoms, appetite and general
condition good.

December 15: 11.30 a. m., received 0.6563 gram caffein by mouth, or
0.75 gram per kilo, no symptoms, appetite good, condition excellent.

December 16: 11 a. m., received 0.870 gram caffein by mouth, or 0.1
gram per kilo, weight 8.70 kilos, no symptoms.

December 17: No caffein given.

December 18: Received 0.870 gram caffein, or 0.1 gram per kilo, no
symptoms.

December 20: 2.45 p. m., received 1.0875 grams caffein, or 0.125 per
kilo; 4 p. m., ate food readily, seemed very uncomfortable and sick.

December 21: 9 a. m., stiffness in muscles, but no other symptoms,
appetite good, no caffein given.

December 22: 11 a. m., received 1.0875 grams caffein, or 0.125 gram per
kilo; 3 p. m., depressed in spirits and sick, but no other symptoms
observed.

December 23: 11.30 a. m., received 1.305 grams caffein, or 0.150 gram
caffein per kilo; 1.30 p. m., apparently quite sick, but no other
symptoms, had good appetite.

December 24: 10 a. m., received 0.175 gram caffein per kilo; 4 p. m.,
when about to be fed fell over and died; no autopsy.

The total amount of caffein given dog 30 was 11.3458 grams,
administered for a period of eighteen days. The average daily amount
per kilo was therefore 72 mg. The feces became offensive when the
amounts of caffein were increased to 75 mg per kilo. It will be
observed that in this dog the appetite was uniformly good until the day
of his death. Whether or not this is the cause of his resistance to
caffein will be discussed later.

 _Dog 32. White, male, young._

Although he was growing rapidly this dog's weight was constant, but
he looked anemic. He received a high protein diet until December 3,
when the rations were increased by one-third. This dog was under
observation from October 26 to December 6 when the administration of
caffein was begun. He then received 50 mg caffein per kilo daily for
nine days consecutively without showing any effects, when the dose was
increased to 75 mg per kilo, then to 100 mg per kilo. This dose was
further increased to 150 mg per kilo without causing symptoms, which
was repeated the next day. No symptoms having been observed after such
amounts of caffein, 175 mg per kilo were fed. This dose, however,
proved fatal within two hours. Record of experiment follows:

October 26: Weight, 6.90 kilos.

November 3: Weight, 6.90 kilos.

November 10: Weight, 6.90 kilos.

November 20: Weight, 6.90 kilos.

November 29: Weight, 6.55 kilos.

December 3: Put into cage, diet increased one-third.

December 6, 7, 8, 9: Weight 6.30 kilos; 12.30 p. m., received 16 cc 2
per cent caffein by subcutaneous injection in back, no symptoms of any
kind noticed, site of injection normal.

December 10, 14: 0.05 gram caffein per kilo.

December 15: Received 0.4725 gram caffein by mouth, no symptoms.

December 16: Received 0.655 gram caffein, 0.100 gram per kilo.

December 17: No caffein given.

December 18: Received 0.655 gram caffein daily, 0.100 gram per kilo, no
symptoms.

December 20: Received 0.8188 gram caffein, 0.125 gram per kilo, no
symptoms, appetite good.

December 21: Received 0.9825 gram caffein, 0.150 gram per kilo,
somewhat uncomfortable, no other symptoms.

December 22: Received 0.9825 gram caffein, 0.150 gram per kilo, no
symptoms except some uneasiness.

December 23: 9 a. m., no symptoms, appetite good; 11.30 a. m.,
received 1.146 grams caffein, 0.1759 gram per kilo; 1.30 p. m., died
while making an effort to get out of cage, tonic contraction of limbs
observed before death.

The amount of caffein received during the entire experimental period
was 9.2223 grams, or an average per day approximately of 80 mg per
kilo, and therefore 10 per cent more than dog No. 30 received. It will
be observed that the appetite in dog No. 32 was likewise uniformly
good, and that he received a very high protein diet which was also of a
very high calorific value.

_Autopsy (dog 32)._--Stomach presented a severe inflammation of
the mucosa, especially in the fundus and pyloric portions. The
gastritis was more marked in pyloric portion, and the inflammatory
condition extended along the whole course of small intestines, which
presented numerous hemorrhagic areas, and a thick catarrhal exudate
on the mucosa. The large intestine contained quite a large number of
parasites, probably round worms. The liver was enlarged and the gall
cyst well filled. The spleen was also considerably engorged, kidneys
appeared normal, other organs all appeared normal.

 _Dog 31. Black spaniel, male._

This dog had been under observation one month previous to the
experiments with caffein. The usual initial dose was then administered
for nine days. There were no signs of local irritation when the drug
was given subcutaneously, but symptoms of toxicity were present. These
disappeared, however, when the drug was administered by mouth. The
dose was therefore increased to 75 mg per kilo. This, as will be seen,
proved fatal within six hours. High nitrogen diet, same as No. 30.

November 3: Weight 10.250 kilos.

November 10: Weight, 10.25 kilos.

November 20: Weight, 10.30 kilos.

December 1: Put in cage.

December 6, 7, 8, 9: Weight, 10.20 kilos; received 26 cc 2 per cent
caffein subcutaneously, site of injection normal.

December 6: Very restless and excited, whined when handled as though
muscles were sore, appeared to be sick.

December 10-14: Condition good, received 0.51 gram caffein by mouth
daily, no noteworthy symptoms, appetite continues good, somewhat
restless at intervals.

December 15: 11.30 a. m., received 0.765 gram caffein per mouth
(0.075 gram per kilo); 2 p. m., depressed in spirit, seemed sick and
uncomfortable; 4.15 p. m., when about to feed, animal jumped up, then
fell back dead.

_Autopsy (dog 31)_: Lungs congested; heart filled with blood and
contained small amount of blood-stained fluid in pericardial sac. Liver
deeply congested, soft and friable; gall bladder distended with bile;
kidneys showed inflammation of cortex; spleen pale, normal in size
and consistency; stomach practically empty, the mucosa of the pyloric
portion exhibited severe gastritis, with thick catarrhal exudate. This
catarrhal inflammation extended through the duodenum; remaining portion
of small intestine showed mild inflammation; large intestine appeared
practically normal. The total amount of caffein received by dog 31
during 10 days was 5.395 mg, or a daily average of 53.9 mg per kilo.
This unusually low resistance to caffein (which was practically the
only case in all the experiments on dogs presented in this research)
suggests the presence of some abnormal condition. The bloody exudate
in the pericardial cavity indicating pericarditis, which is likely to
induce secondary changes of cardiac muscle, may be considered as a
possible cause of the increased toxicity of caffein in this case.

 _Dog 29. Male fox terrier, black._

This dog was kept on a low nitrogen diet for nearly five weeks before
the feeding of caffein was begun. The administration of 50 mg of
caffein per kilo was then carried on for eight days without showing any
symptoms of toxicity. The usual increase of dose was then given--75
mg per kilo--which was followed by a manifestation of symptoms.
Further increase, however, to 100 mg per kilo had no visible effect.
Nevertheless it was considered advisable to suspend the feeding of
caffein for one day. The same amounts were then repeated on two
consecutive days. No symptoms having been observed, 125 mg per kilo
were given. As symptoms of toxicity and especially loss of appetite
were observed, the dog was not given any caffein the next day. Since
his appetite had now improved, the experiment with larger doses was
resumed. Death followed after the second dose of 150 mg per kilo.
Protocol follows:

Weight, 9.90 kilos. One-third nitrogen diet. Receives 0.269 gram
nitrogen per kilo (88.269 calories per kilo).

November 3: Weight, 9.85 kilos.

November 10: Weight, 9.55 kilos.

November 12: Weight, 9.40 kilos.

November 29: Weight, 9.85 kilos.

December 6: Weight, 9.90 kilos; 11.35 a. m., received 25 cc 2 per
cent caffein solution by subcutaneous injection in back; 4 p. m., no
symptoms, appetite good.

December 7-9: Received 25 cc caffein 2 per cent solution--subcutaneous
injection, no symptoms, area of injection inflamed and swollen.

December 10, 13: Site of injection showed increased inflammation,
received 0.495 gram caffein (50 mg per kilo) in 30 grams meat daily
without showing any symptoms.

December 14: 12 noon, received 0.7425 gram caffein by mouth (0.075 per
kilo); 2.30 p. m., restless and uneasy.

December 15: 11.30 a. m., received 0.7425 gram caffein by mouth;
2 p. m., depressed in spirits, although continues to have good
appetite.

December 16: Weight, 9.50 kilos; 3.15 p. m., received 0.9509 gram
caffein by mouth; 4.50 p. m., no symptoms.

December 17: Animal rested.

December 18: Received 0.950 gram caffein by mouth, no symptoms.

December 19: Received 0.9509 gram caffein by mouth, no symptoms.

December 20: 2.45 p. m., received 1.1875 grams caffein (0.125 gram per
kilo); 4 p. m., restless and quite sick; ate only a little food.

December 21: 9 a. m., still uncomfortable, allowed to rest, no caffein
given, gradually recovered appetite.

December 22: 11 a. m., received 1.875 grams caffein; 3 p. m., seemed
sick, but showed no other symptoms, appetite fair.

December 23: 9 a. m., showed no symptoms from the day before, ate food
gradually, seemed sick; 11.30 a. m., received 1.425 grams caffein
(0.150 gram per kilo); 1.30 p. m., looked and behaved as if very sick,
no other symptoms; 3.45 p. m., in attempting to get out of box fell over
on back, had convulsions, whined, dyspnoea, died within 30 seconds.

_Autopsy_: Stomach exhibited mild inflammation; small intestine
inflamed and hemorrhagic areas on mucosa; liver engorged and friable;
spleen normal; kidneys slightly congested; other organs appeared
normal. The total amount of caffein fed to Dog 29 was 12.135 grams,
which was given in 18 days. The average daily amount per kilo was
therefore 67.68 mg.

 _Dog 28. Male fox terrier._

Low nitrogen diet was begun about four weeks before the feeding of
caffein; 50 mg of caffein was then fed for seven consecutive days.
Partial loss of appetite was observed after the first dose. As the
experiment progressed the desire for food steadily diminished, and the
feces became fetid. Symptoms of intoxication manifested themselves
early in the experiment, and vomiting occurred after the fourth
dose. The dog was then put on a diet exclusively of meat. After an
intermission of 10 days 109 mg caffein per kilo were given. Since there
were no symptoms, the following day the amount was increased to 125 mg
per kilo. This dose proved fatal within 16 to 20 hours.

This dog was stout and strong, weight 12.25 kilos, received daily 0.269
gram nitrogen per kilo (88.269 calories per kilo).

November 3: Weight, 11.75 kilos.

November 10: Weight, 11.95 kilos.

November 20: Weight, 11.20 kilos. All through this period had been kept
in a cold, poorly ventilated room, put in a warm room, with bedding and
good ventilation.

November 29: Weight, 11.95 kilos.

December 1: Put in a cage; weight, 11.95 kilos.

December 6: Weight, 11.95 kilos; 11.45 a. m., received 0.050 gram
caffein per kilo; then received 30 cc 2 per cent caffein (0.6 gram) in
practically one subcutaneous injection; 4.30 p. m., ate only part of
food.

December 7: 10.25 a. m., received 30 cc 2 per cent caffein by
subcutaneous injection (0.6 gram, or 50 mg, per kilo); 1.45 p. m.,
seemed sensitive to touch, no desire for food, depressed in spirit.

December 8: 11.40 a. m., received 30 cc 2 per cent caffein by
subcutaneous injection (50 mg per kilo); 1 p. m., depressed in spirit,
hind legs seemed somewhat stiff, no desire for regular food, site of
injection inflamed.

December 9: 10.50 a. m., received 30 cc 2 per cent caffein by
subcutaneous injection (50 mg per kilo); 2.30 p. m., had vomited, no
desire for regular food.

December 10: Inflammation of site of injection, and swelling very
pronounced; 2 p. m., received 0.5975 gram caffein, or 50 mg per kilo,
with 30 grams of meat, refused regular food.

December 11, 12: Received 0.5975 gram caffein by mouth, no symptoms
except refusal of regular food, feces fetid.

December 13-22: Put on meat diet exclusively, high temperature, no
caffein, weight 10 kilos, appetite good, feces fetid.

December 22: 12 a. m., weight 11 kilos, received 1.2 grams caffein by
mouth (0.109 gram per kilo); 4 p. m., no symptoms.

December 23: 11.30 a. m., received 1.375 grams caffein (0.125 gram per
kilo) had vomited food of the day before, but could notice no caffein
or capsules in vomit; 4.30 p. m., no symptoms, seemed in good spirits,
appetite good, had no meat to feed with, so was given low nitrogen
feed, of which he ate about one-fourth.

December 24: 9 a. m., found dead, stiff, and cold. The most striking
effect of caffein in this dog is the increased intestinal putrefaction.
The feces were still fetid 10 days after the administration of caffein
was stopped.

_Autopsy, dog 28_: Stomach partially filled with an undigested food
mass; mucosa showed severe inflammation; small intestines presented
a hemorrhagic enteritis along whole extent; large intestine also
exhibited mild inflammation; liver was engorged; spleen appeared
normal; kidneys slightly congested in cortical portion; other organs
appeared normal.

_Dog 24. White and tan male_: Was put on low protein diet six weeks
before experiments with caffein were begun. The initial dose of 50 mg
per kilo was then administered on eight consecutive days. The only
symptoms observed during this period of caffein administration were
those of intestinal putrefaction. Fetid feces were noticed already
after the first dose of caffein was injected. When the second dose of
75 mg of caffein was repeated, mild symptoms appeared, but none have
been observed even with increased amounts of caffein.

One-third nitrogen diet. Received daily 0.269 gram nitrogen per kilo
(88.269 calories per kilo).

October 26: Weight 11.15 kilos. Food consisted of 5 grams cracker meal
per kilo; meat, 3 grams per kilo; lard, 2 grams per kilo; tapioca,
10.69 grams per kilo. Kept in a cold, damp room with poor ventilation
until November 20.

November 3: Weight, 11 kilos.

November 10: Weight, 10.75 kilos.

November 20: Weight, 10.55 kilos; changed to a warm room, with bedding
and good ventilation.

November 29: Weight, 10.85 kilos.

December 1: Put into a cage.

December 6: Weight, 10.90 kilos; 11.25 a. m., received 28 cc 2 per
cent caffein subcutaneously in side, below the shoulders, area washed
with alcohol and ether, approximately 50 mg per kilo administered, no
symptoms.

December 7: 10.15 a. m., received 28 cc 2 per cent caffein injected
subcutaneously; feces soft and very fetid; 1 p. m., depressed in
spirit, eyes dull.

December 9: 10.45 a. m., received 25 cc 2 per cent caffein solution
subcutaneously, feces still fetid, site of injection inflamed and
swollen, no other symptoms.

December 10: Inflammation of area of injection more pronounced;
2 p. m., given 0.5449 gram caffein and 30 grams of meat; 4 p. m., fed,
no symptoms, feces fetid.

December 11: 12 a. m., given 0.5459 gram caffein and 30 grams of meat, no
symptoms, feces fetid.

December 12, 13: Given 0.5459 gram caffein daily, without noticing any
symptoms.

December 14: 12 a. m., received 0.817 gram caffein (75 mg per kilo); 2.30
p. m., restless and uncomfortable, no other symptoms.

December 15: 11.30 a. m., received 0.8175 gram caffein by mouth;
2 p. m., depressed in spirit, acted as though sick, no other symptoms.

December 16: Weight, 11 kilos; 11 a. m., received 0.100 gram caffein
per kilo (1.100 grams) by mouth, no symptoms.

December 17: Rested.

December 18: 2.30 p. m., received 1.100 grams caffein by mouth; 4 p. m.,
no symptoms.

December 19: 12 noon, received 1.100 grams caffein by mouth; 4.15 p. m.,
no symptoms.

December 20: 2.45 p. m., given 1.375 grams caffein (0.125 gram per
kilo); 3.45 p. m., vomited--one of the capsules being found intact,
the other broken open; 4 p. m., given regular diet, containing 1.3757
grams caffein in capsules, ate most of this during the night, whined at
intervals, coordination disturbed, appeared very sick, but exhibited no
other symptoms.

December 21: 9 a. m., found dead, stiff, and cold.

The total amount of caffein received by dog 24 was between 10.109 and
11.484 grams. As one of the capsules vomited was intact and the other
broken open, the amount was probably about 10.75 grams. The fatal dose
in this case was undoubtedly less than 185 mg per kilo--somewhere
between 125 and 185 mg. Autopsy showed heart in diastole; posterior
lobe of right lung deeply congested; liver engorged; gall cyst filled;
spleen appeared normal; stomach well filled with semifluid mass;
pyloric portion of stomach exhibited a severe inflammation of mucosa;
mucosa of duodenum greatly inflamed and showed hemorrhagic areas and
catarrhal exudate; remainder of small intestine also exhibited mild
inflammation; kidneys deeply engorged, mesentery injected.

A comparison of the fatal doses of caffein in the experiments on high
and low protein diet does not show much difference in the resistance to
caffein, since 175 mg per kilo proved fatal to Nos. 30 and 32, while
No. 29 died after receiving 150 mg per kilo, and No. 24 received 125
to 185 mg per kilo. Moreover, No. 28, which was changed from low to
high protein diet, succumbed when given 125 mg per kilo. Observations
made during the experimental period indicate, however, greater toxicity
of caffein in the subjects on low protein diet. Dog 30 showed the
effects of the drug when the dose was increased to 125 mg of caffein
per kilo, while in No. 32, 150 mg per kilo were received without any
manifestation of symptoms. Dog 31, which was likewise on a high protein
diet, is evidently an exception, and its low resistance to caffein
may be accounted for by the condition found at autopsy. In other dogs
on low protein diet symptoms of intoxication appeared early in the
experiment. In Nos. 29 and 24 it was observed as soon as the amount of
caffein was increased to 75 mg per kilo. In dog 28 the first dose of
caffein 50 mg per kilo was toxic. The symptoms of gastro-intestinal
disturbance were especially marked after caffein on low protein diet.
This may seem to contradict the results of experiments on dogs 11
and 20, in which larger doses of caffein failed to induce symptoms
of intoxication. But it should be observed that the diet, which
consisted almost exclusively of carbohydrates, was given only during
the administration of caffein, while in the experiments of series B
the subjects received a low protein diet for several weeks before the
administration of caffein was begun, and it was continued through
the entire caffein period. It will be remarked that the absence of
cumulative action in the experiments of the preceding series was also
observed in dogs on high as well as on low protein diet. The appearance
of symptoms after smaller doses of caffein in the latter experiments
might suggest cumulative action, but since these symptoms disappeared
on continued administration of the substance cumulation is clearly
not indicated. The gastrointestinal lesions observed on post-mortem
examination were, it will be recalled, also found in rabbits similarly
treated. The explanation suggested probably applies also in the case of
dogs.


SERIES C.

As already pointed out in the experiments on acute toxicity of caffein,
young growing dogs are probably more resistant to caffein than adults.
That this may also hold true in chronic caffein intoxication seemed
indicated by the following experiments.

 _Dog 33. Black female puppy. Weight, 4 kilos. Had been continuously
 on a meat diet._

December 22: 2.30 p. m., received 0.69 gram of caffein (0.172 gram per
kilo); 3.15 p. m., no symptoms except that feces were fetid.

December 23: 11.30 a. m., received 0.79 gram of caffein (0.197 gram per
kilo); 1.30 p. m., no symptoms.

December 24: 11 a. m., received 0.87 gram of caffein (0.2009 gram per
kilo); 4 p. m., no symptoms.

It will be observed that the only effect produced in dog 33 by feeding
caffein was increased intestinal putrefaction, although 2.37 grams
of caffein were given in three days. Additional data on the effects
of the age of animals on the resistance to caffein seemed desirable.
The following experiments were therefore carried out. Six puppies of
the same litter were weaned when 7 to 8 weeks old and put on a milk
diet. Three of them received this diet throughout the experimental
period. Meat was substituted in the other three a few days before the
administration of caffein was begun, and was continued until the end of
the experiment. Caffein was given by mouth; the initial dose, which was
administered for several days and then gradually increased, being 160
to 200 mg for each dog, except one, which received only 100 mg per kilo
for several days and then an increased amount.

An intermission of a few days (during which no caffein was given) was
allowed. This was done on account of some studies carried on at the
same time on the effect of caffein on certain constituents of the urine.

PUP NO. 1.

  ---------+---------+--------+-------------+--------------------------
           |         |        | Treatment   |
   Date.   | Weight. | Food   | (2 per cent |         Symptoms.
           |         |(milk). |  caffein).  |
  ---------+---------+--------+-------------+--------------------------
           | _Grams._|  _cc._ |     _cc._   |
  Apr. 21  |   1,450 |   300  |      10.0   | No symptoms.
  Apr. 22  |   1,520 |   300  |      10.0   |    Do.
  Apr. 23  |   1,450 |   250  |      10.0   |    Do.
  Apr. 24  |   1,375 |   250  |      10.0   |    Do.
  Apr. 25  |   1,420 |   250  |      10.0   |    Do.
  Apr. 26  |   1,390 |   250  |      None.  |
  Apr. 27  |   1,400 |   250  |      None.  |
  Apr. 28  |   1,405 |   250  |      None.  |
  Apr. 29  |   1,420 |   250  |      None.  | Passed worms.
  Apr. 30  |   1,430 |   250  |      None.  |   Do.
  May 1    |   1,450 |   250  |      10.0   | No symptoms.
  May 2    |   1,515 |   250  |      15.0   |   Do.
  May 3    |   1,475 |   250  |      15.0   |   Do.
  May 4    |   1,495 |   250  |      15.0   |   Do.
  May 5    |   1,515 |   250  |      22.0   | Seems dull and whines.
  May 6    |   1,535 |   250  |      20.0   | Whines.
  May 7    |   1,525 |   250  |      20.0   | No symptoms.
  May 8    |   1,530 |   250  |      20.0   |    Do.
  May 9    |   1,500 |   250  |      23.0   | Diarrhea; passed worms;
           |         |        |             | tremor and rigidity of
           |         |        |             | legs; whines.
  May 10   |   1,490 |   250  |      None.  | Completely recovered from
           |         |        |             | the effects of 9th.
  May 11   |   1,535 |   250  |      25.0   | Can not balance itself;
           |         |        |             |   continually vomiting.
  May 12   |   1,460 |   300  |      None.  | Recovered from effects.
  May 13   |   1,475 |   350  |      None.  | In good condition.
  May 14   |   1,545 |   250  |      None.  |
  May 15   |   1,550 |   250  |      None.  |
  May 16   |   1,555 |   250  |      None.  |
  May 17   |   1,560 |   250  |      25.0   | Salivated in cage;
           |         |        |             | stiffness of muscles.
  May 18   |   1,450 |   250  |      None.  | Weak and stiff; diarrhea.
  May 19   |   1,500 |   250  |      None.  | No symptoms.
  May 20   |   1,565 |   250  |      None.  |
  May 21   |   1,545 |   250  |      None.  |
  May 22   |   (1)   |   250  |      None.  |
  May 23   |   1,595 |   250  |      27.0   | Tremors; gait clumsy; in-
           |         |        |             | coordination of movements.
  May 24   |   1,495 |   250  |      27.0   | Diarrhea; vomited; weak
           |         |        |             | and stiff; found dead
           |         |        |             | 9 a. m. 25th.
  ---------+---------+--------+-------------+--------------------------

(1) Sunday.

_Autopsy_: Marked pulmonary congestion; liver very pale; heart wall
injected; slight inflammation of stomach and intestines.

PUP NO. 2.

  ---------+---------+---------+------------+---------------------
           |         |  Food   | Treatment  |
   Date.   | Weight. | (milk). |(2 per cent |    Symptoms.
           |         |         | caffein).  |
  ---------+---------+---------+------------+---------------------
           | _Grams._  |   _cc._   |    _cc._     |
   Apr. 21 |  1,350  |    300  |      5.0   | No symptoms.
   Apr. 22 |  1,240  |    300  |      5.0   |     Do.
   Apr. 23 |  1,250  |    200  |      7.5   |     Do.
   Apr. 24 |  1,205  |    200  |      7.0   |     Do.
   Apr. 25 |  1,220  |    200  |      7.0   |     Do.
   Apr. 26 |  1,210  |    200  |    None.   |
   Apr. 27 |  1,210  |    200  |    None.   |
   Apr. 28 |  1,205  |    200  |    None.   |
   Apr. 29 |  1,200  |    200  |    None.   | Passed worms.
   Apr. 30 |  1,210  |    200  |    None.   |
   May 1   |  1,220  |    200  |     10.0   | No symptoms.
   May 2   |  1,220  |    200  |     10.0   |     Do.
   May 3   |  1,235  |    200  |     10.0   |     Do.
   May 4   |  1,235  |    200  |     10.0   |     Do.
   May 5   |  1,235  |    200  |     17.0   | Whines.
   May 6   |  1,250  |    200  |     17.0   |     Do.
   May 7   |  1,235  |    200  |     15.0   | Diarrhea
           |         |         |            | and worms.
   May 8   |  1,250  |    200  |     15.0   | Diarrhea.
   May 9   |  1,165  |    200  |     18.0   | Little or
           |         |         |            | no symptoms.
   May 10  |  1,235  |    200  |     None.  | No symptoms.
   May 11  |  1,300  |    200  |      20.0  | Salivated in cage;
           |         |         |            | refused to eat;
           |         |         |            | draws up hind legs.
   May 12  |  1,200  |    200  |     None.  | Recovered.
   May 13  |  1,215  |    200  |     None.  | In good condition.
   May 14  |  1,280  |    200  |     None.  |
   May 15  |  1,300  |    200  |     None.  |
   May 16  |  1,310  |    200  |     None.  |
   May 17  |  1,310  |    200  |      20.0  | Salivated in cage;
           |         |         |            | stiff; all symptoms.
   May 18  |  1,250  |    200  |     None.  | Weak and stiff.
   May 19  |  1,245  |    200  |     None.  | No symptoms.
   May 20  |  1,310  |    200  |     None.  |
   May 21  |  1,325  |    200  |     None.  |
   May 22  |  1,325  |    200  |     None.  |
   May 23  |  1,325  |    200  |      22.0  | Somewhat stiff.
   May 24  |  1,315  |    200  |      22.0  | Restless; scratches
           |         |         |            | eyes; sick.
  ---------+---------+---------+------------+---------------------

PUP NO. 3.

  ---------+---------+---------+------------+------------------------
   Apr. 21 |  1,215  |    300  |    None.   |
   Apr. 22 |  1,220  |    300  |    None.   |
   Apr. 23 |  1,220  |    200  |    None.   |
   Apr. 24 |  1,200  |    200  |    None.   |
   Apr. 25 |  1,205  |    200  |    None.   |
   Apr. 26 |  1,195  |    200  |    None.   |
   Apr. 27 |  1,200  |    200  |    (1)     |
   Apr. 28 |  1,215  |    200  |    None.   |
   Apr. 29 |  1,220  |    200  |    None.   |
   Apr. 30 |  1,200  |    200  |    None.   |
   May 1   |  1,225  |    200  |     10.0   | No symptoms.
   May 2   |  1,230  |    200  |     10.0   |     Do.
   May 3   |  1,235  |    200  |     10.0   | Coughs and whines.
   May 4   |  1,245  |    200  |     10.0   | Passed worms.
   May 5   |  1,270  |    200  |     17.0   | Eyes appear dim
           |         |         |            | and is continually
           |         |         |            | scratching them.
   May 6   |  1,260  |    200  |     17.0   | Appears restless and
           |         |         |            | draws up hind legs
           |         |         |            | when walking.
   May 7   |  1,240  |    200  |     15.0   | Eyes dim; passed
           |         |         |            | worms; diarrhea.
   May 8   |  1,265  |    200  |     15.0   | Coughing continually;
           |         |         |            | very restless.
   May 9   |  1,240  |    200  |     18.0   | 12 noon; salivated in
           |         |         |            | cage; passed worms;
           |         |         |            | diarrhea; foaming at
           |         |         |            | mouth; can not balance
           |         |         |            | himself; rigidity and
           |         |         |            | tremor of hind legs.
           |         |         |            | 2.15, found dead.
  ---------+---------+---------+------------+------------------------

  (1) Urine squeezed from bladder.

_Autopsy_: Severe pulmonary congestion; catarrhal gastritis; mild
enteritis with small hemorrhagic areas on mucosa.

PUP NO. 4.

  ---------+--------+----------+-----------+----------------------------
           |        |          |Treatment  |
    Date.  | Weight.|  Food.   |(2 per cent|           Symptoms.
           |        |          | caffein). |
  ---------+--------+----------+-----------+----------------------------
           |_Grams._| _Milk_   |   _cc._   |
           |        | _(cc)._  |           |
   Apr. 28 |  1,670 |    300   |           |
   Apr. 29 |  1,670 |    300   |           |
   Apr. 30 |  1,670 |    300   |           |
   May 1   |  1,690 |    300   |           |
   May 2   |  1,690 |    300   |           |
   May 3   |        |    300   |           |
   May 4   |  1,720 |    300   |           |
   May 5   |  1,735 |    300   |           |
           |        |          |           |
           |        |_Meat_    |           |
           |        |(_grams_).|           |
   May 6   |  1,760 |     60   |           |
   May 7   |  1,745 |     80   |           |
   May 8   |  1,710 |    180   |           |
   May 9   |  1,750 |    180   |           |
   May 10  |  1,750 |    180   |           |
   May 11  |  1,755 |    180   |    10.0   |No symptoms.
   May 12  |  1,730 |    180   |    None.  |
   May 13  |  1,785 |    180   |    None.  |
   May 14  |  1,835 |    115   |    10.0   |   Do.
   May 15  |  1,820 |    115   |    10.0   |   Do.
   May 16  |  1,835 |    115   |    10.0   |Passed worms.
   May 17  |  1,860 |    115   |    10.0   |Feces soft and black.
   May 18  |  1,855 |    115   |    15.0   |Stiff; loss of appetite.
   May 19  |  1,770 |    115   |    15.0   |Loss of appetite.
   May 20  |  1,755 |    115   |    15.0   |   Do.
   May 21  |  1,780 |    115   |    17.0   |Restless.
   May 22  |        |    115   |    17.0   |Feces soft and black.
   May 23  |  1,785 |    115   |    17.0   |Loss of appetite.
   May 24  |  1,795 |    115   |           |Loss of appetite; threw up
           |        |          |           |   worms.
   May 25  |  1,630 |    115   |    20.0   |Loss of appetite; worms;
           |        |          |           |   cough; diarrhea.
   May 26  |  1,600 |    115   |    23.0   |Weak; no appetite; diarrhea;
           |        |          |           |   cough.
   May 27  |        |          |           |Found dead, 9 a. m.
  ---------+--------+----------+-----------+----------------------------

_Autopsy._--Lung uniformly congested; liver deeply congested; heart
muscle pale with hemorrhagic areas; kidneys pale with hemorrhagic spots
on surface and in cortex; slight catarrhal inflammation of stomach and
the small intestines.

PUP NO. 5.

  ---------+--------+----------+-----------+----------------------------
           |        |          |Treatment  |
    Date.  | Weight.|  Food.   |(2 per cent|           Symptoms.
           |        |          | caffein). |
  ---------+--------+----------+-----------+----------------------------
           |_Grams._| _Milk_   |   _cc._   |
           |        | _(cc)._  |           |
   Apr. 28 |  1,745 |    300   |           |
   Apr. 29 |  1,745 |    300   |           |
   Apr. 30 |  1,750 |    300   |           |
   May 1   |  1,765 |    300   |           |
   May 2   |  1,765 |    300   |           |
   May 3   |        |    300   |           |
   May 4   |  1,490 |    300   |           |
   May 5   |  1,805 |    300   |           |
           |        |_Meat_    |           |
           |        |(_grams_).|           |
           |        |          |           |
   May 6   |  1,815 |     60   |           |
   May 7   |  1,825 |     80   |           |
   May 8   |  1,770 |    180   |           |
   May 9   |  1,795 |    180   |           |
   May 10  |  1,805 |    180   |           |
   May 11  |  1,800 |    180   |    10.0   |No symptoms.
   May 12  |  1,720 |    180   |    None.  |
   May 13  |  1,815 |    180   |    None.  |
   May 14  |  1,845 |    115   |    10.0   |   Do.
   May 15  |  1,830 |    115   |    10.0   |   Do.
   May 16  |  1,815 |    115   |    10.0   |Loss of weight; no other
           |        |          |           | symptoms.
   May 17  |  1,830 |    115   |    15.0   |No symptoms.
   May 18  |  1,835 |    115   |    15.0   |Stiffness.
   May 19  |  1,825 |    115   |    15.0   |No symptoms.
   May 20  |  1,850 |    115   |    15.0   |A little stiff.
   May 21  |  1,835 |    115   |    17.0   |No symptoms.
   May 22  |        |    115   |    17.0   |
   May 23  |  1,820 |    115   |    17.0   |   Do.
   May 24  |  1,835 |    115   |    20.0   |   Do.
   May 25  |  1,840 |    115   |    20.0   |Feces soft and black.
   May 26  |  1,820 |    115   |    23.0   |
   May 27  |  1,840 |    115   |    25.0   |A little stiff.
   May 28  |  1,830 |    115   |    25.0   |
   May 29  |        |    115   |    None.  |
   May 30  |        |    115   |    None.  |
   May 31  |  1,770 |    115   |    None.  |
   June 1  |  1,765 |    115   |    25.0   |Diarrhea; stiff in hind
           |        |          |           | legs.
   June 2  |  1,750 |    115   |    27.5   |Diarrhea and worms.
   June 3  |  1,635 |          |    27.5   |Paralyzed; vomited;
           |        |          |           | died at 3 p. m.
  ---------+--------+----------+-----------+----------------------------

PUP NO. 6.

  ---------+--------+----------+-----------+----------------------------
           |        |          |Treatment  |
    Date.  | Weight.|  Food.   |(2 per cent|           Symptoms.
           |        |          | caffein). |
  ---------+--------+----------+-----------+----------------------------
           |_Grams._| _Milk_   |   _cc._   |
           |        | _(cc)._  |           |
   Apr. 28 |        |    300   |           |
   Apr. 29 |  1,280 |    300   |           |
   Apr. 30 |  1,290 |    300   |           |
   May 1   |  1,315 |    300   |           |
   May 2   |  1,330 |    300   |           |
   May 3   |        |    300   |           |
   May 4   |  1,360 |    300   |           |
   May 5   |  1,365 |    300   |           |
           |        |_Meat_    |           |
           |        |_(grams)._|           |
           |        |          |           |
   May 6   |  1,395 |     60   |           |
   May 7   |  1,365 |     80   |           |
   May 8   |  1,340 |    180   |           |
   May 9   |  1,380 |    180   |           |
   May 10  |  1,400 |    180   |           |
   May 11  |  1,425 |    180   |    14.5   |No symptoms.
   May 12  |  1,470 |    180   |    None.  |
   May 13  |  1,485 |    180   |    None.  |
   May 14  |  1,510 |    115   |    14.5   |   Do.
   May 15  |  1,500 |    115   |    14.5   |   Do.
   May 16  |  1,485 |    115   |    14.5   |Passed worms.
   May 17  |  1,480 |    115   |    14.5   |
   May 18  |  1,485 |    115   |    19.5   |Feces soft and black;
           |        |          |           |   almost diarrhea.
   May 19  |  1,495 |    115   |    19.5   |
   May 20  |  1,500 |    115   |    19.5   |Scratches her eyes and
           |        |          |           |   chases her tail.
   May 21  |  1,500 |    115   |    17.0   |
   May 22  |        |    115   |    17.0   |
   May 23  |  1,470 |    115   |    17.0   |
   May 24  |  1,465 |    115   |    20.0   |
   May 25  |  1,450 |    115   |    20.0   |Feces soft and black.
   May 26  |  1,450 |    115   |    23.0   |Diarrhea and worms.
   May 27  |  1,355 |    115   |    23.0   |Refused to eat all food.
   May 28  |  1,270 |    115   |    23.0   |Threw up worms, stiff, and
           |        |          |           |   has skin over both eyes.
   May 29  |        |          |           |Found dead.
  ---------+--------+----------+-----------+----------------------------

Highest amount of caffein given, 362 mg per kilo. No autopsy.

Examination of the results obtained in the experiments of series C
shows that young and growing dogs tolerate large amounts of caffein.
In four subjects of this series, Nos. 1, 2, 3, and 6, no effect was
observed when moderately large amounts (160 to 200 mg per kilo of
caffein) were fed. Symptoms were noticed only when these amounts of
caffein were increased from 50 to 60 per cent. The other two dogs,
Nos. 4 and 5, of this series were less resistant, however, to caffein,
as 0.16 gram of the drug per kilo induced well-marked symptoms.
Since these were fed meat, while Nos. 1, 2, and 3 received milk, the
difference in toxicity may be due to the diet employed, but No. 6,
which likewise received a meat diet, failed to show the effects of
caffein when 200 mg per kilo were fed. On the other hand, it should
be noticed that No. 1 died after receiving 360 mg per kilo, No. 2
survived a dose of 334 mg, while No. 3 died after a dose of 322 mg
per kilo of caffein. The fatal doses for Nos. 4, 5, and 6 were 287,
335, and 300 mg per kilo, respectively. Although the differences are
too small to justify any definite conclusion regarding the effect of
a milk diet or of a meat diet on the toxicity of caffein, the results
nevertheless suggest a reasonable possibility that caffein is more
toxic to young dogs when on an exclusively meat diet than when fed
milk. It is perfectly evident, however, that the resistance to caffein
in either case is very great, almost twice that of adult subjects.
As shown in series A and B, 125 to 175 mg per kilo proved fatal to
all but two animals in these experiments, while symptoms of toxicity
appeared after much smaller doses. In other respects the behavior of
young dogs toward caffein was the same as that of the adult. In neither
case was cumulation nor tolerance observed under the conditions of
these experiments. The findings at autopsy were likewise similar,
as gastro-enteritis was the chief lesion observed on macroscopic
examination. It might be mentioned, however, in this connection, that
the symptoms of caffein intoxication in young dogs often presented
marked differences from those observed in those of more advanced age.
The resemblance of the effects of caffein in young puppies and in
rabbits was very striking. In both, the tonic with clonic convulsions
were observed after a sufficient quantity of caffein was administered.
In the dogs which were fully grown a large dose of caffein was usually
followed by tonic convulsions and almost instantaneous death.

Moderately large amounts of caffein fed daily to puppies for several
days--in some cases as long as 10 days--induced mild symptoms only. No
cumulative effect was observed in any of the experiments of series C.
There seems to be tolerance of certain functions toward caffein, but no
general tolerance of the body could be obtained in these experiments.
Caffein is apparently less toxic for adult dogs on high than on low
protein diet. In young and growing dogs caffein is somewhat less
toxic when milk, rather than meat, forms the exclusive diet. Some
pathological conditions apparently increase the toxicity of caffein
also in dogs. The symptoms of caffein intoxication observed in young
dogs are in some respects different from those in full grown and older
animals, and resemble those noticed in rabbits.




DISCUSSION OF RESULTS.


It was pointed out at some length in the introduction that the
toxicity of some drugs may not be the same for all forms of life.
This observation was also made by some investigators who experimented
with caffein on different species of animals. Thus Maurel(55) stated
that caffein is twice as toxic for the frog as for the rabbit when
administered by mouth. Fröhner's(26) experiments, on the other hand,
made on domestic animals, failed to show great differences in the
toxicity of caffein. According to this observer, horses seem to be
more susceptible than cattle, goats, and swine, the minimum toxic dose
being the same for all of these, while the resistance of the dog to
caffein is about midway between that of the horse and the other animals
mentioned. It may be remarked, however, that Fröhner made only 13
experiments. That these data are inadequate for the formation of any
conclusions as to the toxicity of caffein is evident since the most
striking effect of caffein observed in the work herein reported was the
comparatively wide range of variation in the resistance of individuals
of the same species to this drug. This was found to be the case even
when the conditions of experimentation were approximately uniform, and
was observed whatever the mode of administration of the drug employed.
The toxicity for different individuals also varied in acute as well
as in chronic intoxication. It is for this reason that the number of
tests employed were often quite large, for no conclusions of any value
could be drawn without averaging the results of a sufficiently large
number of experiments. Furthermore, it is to be borne in mind that the
action of a drug may differ according to the mode of its introduction
into the body and that different species of animals may vary in this
regard. This is especially true of some substances when given by mouth,
the range in toxicity for certain species of animals being much
greater when thus administered than when injected subcutaneously or
intravenously.

Maurel's(56) investigations are of interest in this connection, as his
work embraces a systematic study of the toxicity of a large number
of substances in the rabbit, pigeon, and frog when given by mouth,
subcutaneously, intravenously, or when injected into the muscles.
According to this investigator the range of variation of the toxicity
of a substance is widest when given by mouth. Potassium sulphocyanid,
for example, is about 2.5 times as toxic for the frog as for the
rabbit when given by mouth. Quinin hydrobromid is three times as toxic
for the frog as for the pigeon, while for the rabbit it is twice as
toxic as for the pigeon. When given by hypodermic injection the toxic
dose per kilo weight is practically the same for all three species.
The difference of resistance according to the mode of administration
is even more marked for spartein sulphate. When given by mouth the
toxicity for the rabbit is six times as great as for the frog, but
when injected subcutaneously the toxic dose is about the same for the
rabbit and for the frog. The relation of the mode of administration
to toxicity is further shown in the following substances: For the
rabbit the minimum fatal dose per kilo of quinin hydrobromid is 1.5
grams administered by mouth, 0.5 gram when injected subcutaneously,
and 0.07 gram by the intravenous path, while strychnin sulphate is
twice as toxic administered intravenously as subcutaneously, and six
times as toxic as when administered by mouth. The mode of introduction,
however, does not always affect the toxicity of a substance. This is
made evident by the action of strychnin on frogs in which, according
to Maurel(56), the toxic dose is the same whether given by mouth or
injected into the subcutaneous tissues. This appears to hold true also
for other animals as demonstrated by the experiments of Hatcher(35) on
the cat, in which he observed that strychnin is as readily absorbed
from a full stomach as from the subcutaneous tissues. These findings
are extremely interesting, especially in view of Maurel's(57) work
on the subject, according to which he finds that a substance is much
less toxic when given by mouth than when administered by hypodermic
injection or intravenously. That this generalization does not admit,
however, of universal application is made evident by the work of
various experimenters. Claude Bernard(10) observed that curara is
as poisonous for the pigeon when given by mouth as when injected
subcutaneously, while Zalesky(86) found that samandarin is more
toxic for frogs when introduced into the stomach than by injection
into the lymph sacs. Our experiments with caffein likewise show that
Maurel's generalization does not always hold good, since it was found
in experiments with gray rabbits that the minimum fatal dose is but
moderately greater by mouth than by the subcutaneous path.

Equally interesting is the observation of the writer, that in the
guinea pig the difference in the toxicity between the subcutaneous
and intraperitoneal injections is very slight, while in the cat the
toxicity of caffein is the same whether given by mouth or injected into
the subcutaneous tissues, and is markedly less when injected into the
peritoneal cavity. The experiments on dogs show considerable variation
of effective dose when given by mouth, but the interesting observation
was made that the toxic dose by mouth may be smaller in some cases
than the average dose by subcutaneous injection. If the resistance to
caffein by subcutaneous injection of the different species of animals
experimented upon in the present research be compared, it will be
noticed that the gray rabbit or Belgian hare, which is more resistant
than the other varieties employed, stands more caffein in proportion to
the weight of the body than the other animals.

Although the minimum fatal dose was found to be somewhat larger for
the guinea pig than for the gray rabbit when caffein was injected
intraperitoneally, it was on the contrary smaller by other paths of
introduction, and approximated quite closely the minimum fatal dose for
rabbits of the other varieties. Cats as well as dogs were found to be
distinctly less resistant to caffein than the herbivora.

There are a number of factors far more important than zoological
differences which influence the toxicity of caffein. Some of these are
age, season, and pathologic conditions. As these factors have already
been dwelt upon in their appropriate places, further discussion might
seem unnecessary, but owing to their importance in determining the
action of a drug, emphasis is desirable. Especially is this the case
with pathological conditions in relation to toxicity. While no positive
proof of diminished resistance to caffein in pathological conditions
was obtained by subjecting the suggestion to experimental test, it was
observed in these experiments on rabbits that death occurred in some
individuals after small doses which are usually not even toxic. The
findings at autopsy indicate the presence of pathological conditions.
The same was observed in some experiments on cats and dogs. It is
extremely probable, therefore, that disease modifies the reaction of
the organism to caffein as well as to other drugs.(78)

That the resistance to drugs may vary according to the age of the
subject has been maintained by a number of pharmacologists. According
to Guinard,(30) young dogs, rabbits, and guinea pigs are very
susceptible to morphin, resembling children in this regard.[E] The
minimum fatal dose for these animals is about one-third less than
for the adult. This is not true, however, for the young of other
species. Cats under 15 days of age tolerate twice the toxic dose of
morphin for the adult cat. Young beeves and goats are likewise more
resistant to this alkaloid than adults. On the other hand, according
to Livon,(54) young guinea pigs are more sensitive to alkaloids than
adults. The toxicity of caffein, as shown in the present investigation,
was found to be less in the young than in the adult. In dogs the young
subjects are in some instances almost twice as resistant as adults. The
difference was found to be less in cats and rabbits than in dogs, but
it was quite marked.

  [E] A case of accidental poisoning reported recently by Wichura
  (Münich. Méd. Woch., 1911, No. 30, p. 1618) throws some doubt on the
  accepted view that the susceptibility of young children to morphin is
  greater than that of adults. Wichura also found that the therapeutic
  doses of codein preparations ordinarily recommended for children in
  pleuritic cough are not effective in this condition.

The effect of season on the toxicity of drugs has been discussed in the
section on the experiments on guinea pigs, which were more resistant
to caffein in the fall than in February and March. The effect of
season seems to vary with the animal, but it may also differ with the
substance employed. In Noe's(65) studies on this subject cantharidin
was found to be more toxic for the hedgehog in November than in July.
The effect of season was different for morphin, as it was observed that
the resistance of the hedgehog was greater at the end of the summer
than earlier in the season.

The relation of diet to toxicity of drugs has been studied by
Hunt.(39) His experiments indicate that this is an important factor
in the resistance to acetonitril. The studies here reported on the
effect of diet on toxicity of caffein in rabbits were confined to
experiments with oats and carrots and do not show any modification of
the resistance to caffein. The question of diet in chronic intoxication
in dogs, however, suggests that in these animals diet may affect the
toxicity of caffein, although the data on this subject are far from
satisfactory. There is nevertheless sufficient evidence to suggest that
a high protein diet for the adult dog tends to greater resistance of
the animal to caffein and similarly the growing dog tolerates larger
quantities of caffein on a milk diet than on a diet of meat.

This brings us to a consideration of the behavior of caffein in chronic
intoxication. Although in both rabbits and dogs absence of cumulation
was evident, in other respects decided differences in the resistance
to caffein were observed. While the rabbit tolerates more than twice
the single dose of caffein per kilo for the dog, the result is quite
different in repeated dosage of the drug, the rabbit succumbing to
continued administration of much smaller doses of the drug than the
dog. This is probably due to lesions of the gastro-intestinal canal
caused by caffein which occasions loss of appetite much more readily
in the rabbit than in the dog. The abundant energy reserve in the dog
makes it possible for this animal to stand inanition much longer than
the rabbit and other herbivora. The difference in the behavior of
the rabbit and dog toward caffein is interesting as showing complete
reversal of resistance in acute and chronic intoxication. From the
statement in the introduction it is evident that the size of the
single toxic or lethal dose of a substance is in no wise an index of
the active degree of its toxicity. The experiments with caffein here
reported furnish additional evidence that this is true, at least for
the rabbit.




GENERAL SUMMARY AND CONCLUSIONS.


The toxicity of caffein in the rabbit varies with the mode of its
administration, being least when given by mouth and greatest by
intravenous administration. The toxicity is from 15 to 20 per cent
greater by subcutaneous injections than by mouth, but is about half
of that when injected into the peritoneal cavity. No difference was
observed in the toxicity of caffein whether administered into gluteal
or into the lumbar muscles. When introduced by this route the toxicity
was found to be less by one-third than when it is injected into the
peritoneal cavity, but is about 30 per cent more toxic than the
subcutaneous injections. White or black rabbits were found to be less
resistant to caffein than gray rabbits.

The resistance of the guinea pig to caffein, as of the rabbit,
is greatest when given by mouth. The minimum fatal dose is less
by intraperitoneal injections, but greater than by subcutaneous
injections, thus differing from the rabbit in this regard. The adult
cat is less resistant than the guinea pig or rabbit to caffein. The
minimum lethal dose by mouth is the same as by subcutaneous, and is
less than by intraperitoneal, injection. The minimum fatal dose for
dogs was found to be the same by mouth as by subcutaneous injection and
is almost the same as for the cat. The toxicity of caffein varies in
the guinea pig according to season of the year.

Age is likewise a factor in the toxicity of caffein, young animals
being more resistant than the full-grown and older animals; this was
shown in experiments on rabbits, cats, and dogs. The symptoms of
caffein poisoning also were different in puppies and in full-grown
dogs. Different diets, such as carrots and oats, did not influence
the resistance of rabbits and guinea pigs to caffein. Low protein
diet tends to decrease resistance to caffein in dogs. Young growing
dogs are less resistant to caffein on a meat than on a milk diet.
Caffein is not cumulative in the rabbit or dog, even if administered
for a considerable length of time. Some degree of tolerance may be
induced in the rabbit under certain conditions, but not in dogs under
the conditions of the experiments made in this investigation. The
possibility, however, that dogs may acquire tolerance for caffein
is not excluded. Although the rabbit tolerates a much larger single
dose of caffein than the dog, it was found, in experiments on chronic
intoxication that the rabbit is less resistant to caffein than the
dog. The toxicity of caffein is probably increased under pathological
conditions, since comparatively smaller doses were fatal to rabbits,
cats, and dogs, when marked lesions not due to caffein were found at
autopsy. Glycosuria was observed in rabbits, guinea pigs, and cats when
caffein was given in sufficient amounts.

TABLE 18.--_Acute caffein intoxication: Table showing average
minimum toxic and minimum fatal doses for adult animals._

  Table headings:

  SC: Subcutaneously.
  BM: By mouth.
  IP: Intraperitoneally.
  IM: Intramuscular.
  IV: Intravenous.

  -------------+------+-------------------------------------------------
               |Effect|                 Dose per kilo (grams)
      Animal.  |of    +---------+-----------+-----------+-------+-------
               |dose. |    SC   |     BM    |     IP    |  IM   |  IV
  -------------+------+---------+-----------+-----------+-------+-------
  Rabbit, gray |{Toxic|     0.15|      0.325|      0.100|   0.13| 0.05
               |      |         |           |    -0.125 |  -0.15|
               |{Fatal|      .30|       .350|       .150|    .20| 0.10
               |      |         |           |           |       |  -.16
  Rabbit, white|{Toxic|         |           |           |       |
    or black   |{Fatal|      .20|       .290|           |       |
               |      |         |           |           |       |
  Guinea pig   |{Toxic|0.15- .16|       .150|       .200|       |
               |{Fatal| .20- .24|0.280- .300| .240- .250|       |
               |      |         |           |           |       |
  Cat          |{Toxic| .12- .14|       .125| .125- .150|       |
               |{Fatal|      .15|       .150| .180- .200|       |
               |      |         |           |           |       |
  Dog          |{Toxic|         | .100- .120|           |       |
               |{Fatal| .15- .16| .140- .150|           |       |
  -------------+------+---------+-----------+-----------+-------+-------

NOTE.--The doses given in this table are approximate.




CAFFEIN BIBLIOGRAPHY.


 1. ABDERHALDEN and BRAHM. Zts. Physiol. Chem., 1909,
 =62=: 133.

 2. ALBERS. Deut. Klin., 1852, =5=: 577.

 3. AMAT. Diss. Paris, 1889.

 4. AMORY. Boston Med. Surg. J., 1868, =1=: 261.

 5. VON ANREP. Arch. Ges. Phys., 1880, =21=: 185.

 6. AUBERT. Pflüger's Arch., 1872, =5=: 589.

 7. AUER and MELTZER. J. Pharm. Exper. Ther., 1911,
 =2=: 402.


 8. BALDI. Terapia Moderna, 1891, =5=: 617.

 9. BENNETT. Brit. Med. J., 1874, =2=: 674.

 10. BERNARD, CLAUDE. Leçons sur les substances toxiques,
 Paris, 1857, p. 292.

 11. BINZ. Arch. Exper. Path. Pharm., 1878, =9=: 31.

 12. ---- Ibid., 1891, =28=: 197.

 13. BRILL. Diss. Marburg, 1861.

 14. BUCHHEIM and EISENMENGER. Beitr. Anat. Physiol.,
 1870, =5=: 115.

 15. BUNGE and SCHMIEDEBERG. Arch. Exper. Path.
 Pharm., 1876, =6=: 233.


 16. CHITTENDEN. The Nutrition of Man, 1907.

 17. COGSWELL. Lancet (London), 1852, =2=: 488.


 18. DANILEWSKI. Arch. Exper. Path. Pharm., 1894, =35=: 105.

 19. DRZEWINA. Compt. rend. Soc. biol., 1911, =70=: 772.

 20. DUMAS and PELLETIER. Ann. chim. phys., 1823,
 =24=: 182.


 21. EDMUNDS. J. Amer. Med. Assoc., 1907, =48=: 1744.


 22. FILEHNE. Arch. Phys., 1886, p. 72.

 23. FLEISCHER and LOEB. Archives of Internal
 Medicine, 1909, =3=: 78.

 24. FOCKE. Arch. Pharm., 1903, =241=: 678.

 25. FRERICHS. Handwörterbuch Phys., 1846, =3=: 721.

 26. FRÖHNER. Monats. prakt. Tierh., 1892, =3=: 529.


 27. GENTILHOMME. Bull. Soc. Med. Reims, 1867, =5=: 93.

 28. GOUREWITCH. Arch. Exper. Path. Pharm., 1907, =57=: 314.

 29. GUINARD. Compt. rend. Soc. biol., 1900, =2= (2d ser.):
 727.

 30. ----. Compt. rend., 1893, =113=: 520.

 31. ----. La Morphine et l'apomorphine, Paris, 1903.

 32. GUNN. Arch. Int. Pharm. Ther., 1909, =19=: 319.


 33. HALE. U. S. Public Health and Marine-Hospital Service.
 Hyg. Lab. Bul. 53, p. 43.

 34. HARRINGTON. Amer. J. Phys., 1898, =1=: 385.

 35. HATCHER. J. Amer. Med. Assoc., 1910, =60=: 746.

 36. HENNEGUY. Diss. Montpellier, 1875.

 37. HOFMEISTER. Arch. Exper. Path. Pharm., 1894, =33=: 198.

 38. HOPPE. Écho Méd. Neuchâtel, 1858.

 39. HUNT. U. S. Public Health and Marine-Hospital Service.
 Hyg. Lab. Bul. 69, p. 51.

 40. ----. Ibid., Bul. 33.


 41. IGERSHEIMER and STAMI. Arch. Exper. Path.
 Pharm., 1909, =61=: 18.

 42. JACOBI and GOLOWINSKI. Arch. Exper. Path.
 Pharm., Supplement Bd. 1908, p. 286.

 43. JOBST. Ann. Pharm., 1838, =25=: 63.

 44. JOHANSEN. Diss. Dorpat, 1869.


 45. KOBERT. Lehrbuch der Intoxicationen, 1902, =1=: 24.

 46. KÖSTER. Arch. Gesamt. Phys., 1910, =136=: 17.

 47. KRÜGER and SCHMIDT. Ber. d. chem. Ges., 1899,
 =32=: 2677.

 48. KURZAK. Zts. Aerzte zu Wien, 1860, n. f., =3=: 625.


 49. LAPICQUE. Compt. rend. Soc. biol., 1910, =68=: 1007.

 50. LEBLOND. Diss. Paris, 1883.

 51. LEHMANN, C. G. Lehrbuch für physiolische Chemie, 1842,
 =1=: 336.

 52. LEHMANN, J. Ann. chim. pharm., 1853, =87=: 205.

 53. LEVEN. Arch. Phys., 1868, =1=: 180.

 54. LIVON. Compt. rend. Soc. biol., 1897, =4=: 979.


 55. MAUREL. Compt. rend. Soc. biol., 1907, =62=: 897.

 56. -----. Ibid., 1909, =66=: 782.

 57. -----. Ibid., 1910, =69=: 5.

 58. MELTZER and AUER. J. Exper. Med., 1905, =7=: 59.

 59. MITCHELL. J. Phys., 1862, =5=: 109.

 60. MITSCHERLICH. Diss. Berlin, 1859.

 61. MOSCHKOWITSCH. Arch. Pharm., 1903, =241=: 358.

 62. MULDER. J. prakt. Chem., 1838, =15=: 280.

 63. ----. Poggendorff's Ann. Physik Chem., 1838, =43=: 180.


 64. NEUBAUER. Arch. Exper. Path. Pharm., 1901, =46=: 133.

 65. NOE. Arch. Int. Pharm. Ther., 1904, =12=: 160.


 66. OPHÜLS. Proc. Soc. exper. biol. med., 1911, =8=: 75.

 67. OUDRY. Nouvelle Bibliothek Médicale, 1827; Geiger's
 Magazin Pharm., 1827, =19=: 49.


 68. PARISOT. Diss. Paris, 1890.

 69. PELLETIER. J. Pharm., 1826, =12=: 229; also quoted by
 Brill, 3.

 70. PERETTI. Diss. Bonn, 1875.

 71. PFAFF. Schweigger-Seidel, 1831, =1=: 87.

 72. PFAFF and LIEBIG. Ann. Pharm., 1832, =1=: 17.

 73. POHL. Arch. Exper. Path. Pharm., Suppl. Bd., 1908, p. 427.

 74. PRATT. Boston Med. Surg. J., 1868, =2=: 82.


 75. ROBIQUET. Dict. Tech., Paris, 1823, =4=: 59.

 76. ROST. Diss. Heidelberg, 1895.

 77. RUNGE. Neuste phyto-chem. Entdeckungen, Breslau, 1820.


 78. SALANT. U. S. Dept. Agr., Bureau of Chemistry Cir. 81.

 79. SCHMIEDEBERG and BUNGE. Arch. Exper. Path.
 Pharm., 1874, =2=: 62.

 80. -----. Ibid., 1910, =62=: 296.

 81. SOLLMAN and BROWN. J. Amer. Med. Assoc., 1905,
 =45=: 229.

 82. STRECKER. Ann. Chem. Pharm., 1861, =118=: 151

 83. STUHLMANN and FALCK. Arch. path. Anat. Phys.,
 1857, =11=: 324.


 84. THIERFELDER and VON MERING. Zts. physiol. Chem.,
 1885, =9=: 511.


 85. VOIT. Untersuch. über den Einfluss des Kochsalzes, des Kaffes
 München, 1860.


 86. ZALESKY. Hoppe-Seyler Med. Chem. Untersuch., Berlin, 1866, 85-116.

       *       *       *       *       *

  Transcriber's Notes

  Obvious typographical errors have been corrected, but variations in
  spelling, punctuation and hyphenation have been retained.

  Rabbit 396. The date August 15 has been corrected to August 19.

  The data for Rabbit 123 is presented as printed in the original,
  although it is likely that the Caffein per kilo. values should be
  preceded by a decimal point (or that the heading should be Mg.).

  Standard footnotes are identied alphabetically thus [A]. Notes to the
  tables are placed directly under the tables and identified numerically
  thus (1).  Numeric references within the text refer to the bibliography.

  The headings of Table 18 have been abbreviated for clarity on small
  screens.

  Italics are shown thus _italic_ and bold thus =bold=.