J. B. Rieger

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.

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.
No.	Weight.	Caffein per kilo	Appearance of symptoms in	Duration of life.	Diet.	Remarks.
	Grams.	Mg.
332	1,070	158	1 hour 45 minutes	Survived	Oats	Gray.
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 50 minutes	3 hours 10 minutes	Oats	White.
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 10 minutes	Carrots	White.
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 55 minutes	1.5 days	Oats	White.
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 10 minutes	do.	do.	Yellow.
250	1,435	252	4 hours 35 minutes	do.	do.	Gray.
334	1,270	240	1 hour	do.	Carrots	Do.
233	1,675	238	6 hours 10 minutes	26 hours	do.	White.
SERIES D.
253	1,600	275	10 minutes	35 minutes	Oats	Brown and black.
252	1,335	270	30 minutes	4 hours 55 minutes	do.	Black.
327	820	292		2 hours 15 minutes	do.	White.
340	1,465	273		About 18 hours	do.	White and brown.
341	1,450	270		1 hour 25 minutes	do.	Do.
326	1,645	243	2 hours	50 hours	do.	White.
235	1,875	267		20 hours	Carrots	Gray.
316	860	267	2 hours 45 minutes	3 hours 20 minutes	Oats	Do.
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 22 hours	Oats	White female.
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 24 hours	do.	Gray female.

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 No.	Weight.	Caffein per kilo	Symptoms.	Duration of life.	Diet.	Remarks.
	Grams.	Mg.
248	1,170	330	3 hours	Survived	Oats	Gray.
241	1,380	290	2 hours	do.	do.	White male.
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.
268	1,100	363		About 3 hours	do.	Do.
419	1,600	350	6 hours	Less than	do.	Gray male.
420	1,250	352	1 hour	2 hours	do.	Do.
421	1,485	351	6 hours	Less than	do.	Do.
424	1,295	293	2 hours	22 hours	do.	White male.
423	1,205	300	do.	Less than	do.	Do.
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.

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.

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 Meltzer⁷ 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.

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.

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: 1.30¹/₃ 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 2.37¹/₆ to 2.46¹/₆ 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.

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 Brown⁸¹ with ergot, the effect was quite different from those obtained by Meltzer and Auer⁵⁸ 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 Maurel⁵⁵ 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's⁵⁶ 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 Hunt³⁹ 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 Moschkowitsch⁶¹ and Edmunds²¹ reported the very opposite results. Schmiedeberg's⁸⁰ observations on strophantin in frogs were in harmony with those of Edmunds²¹ and Moschkowitsch.⁶¹ Similar results were reported with guinea pigs. Harrington's³⁴ 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 OphÜls⁶⁶ 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 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.

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.

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

Table 8.—Caffein by mouth; guinea pigs.

¹ Subcutaneous injection for comparison.