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.

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.

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:

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:

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:

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:

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:

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.

Table 17.—Chronic intoxication of rabbits, series B, Group IV, on cumulation.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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; 1.30p. 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:

¹ Diarrhea.