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 Hale³³ for acetanilid. Is it not possible that caffein may similarly be affected by poisons circulating within the body? Indeed the recent work of Loeb²³ 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.

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.

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.

Table 7.—Injection into peritoneal cavity; guinea pigs.

Table 8.—Caffein by mouth; guinea pigs.