Part 2

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._