ACUTE CAFFEIN INTOXICATION.

The object of these experiments was to determine the resistance to caffein in various species of animals and by various methods of administration. Caffein was therefore given by mouth and injected subcutaneously into the peritoneal cavity, into the muscles, and intravenously. As far as could be judged by appearance, healthy animals were selected for the subjects of the experiments, but as it is impossible to diagnose with any degree of accuracy the condition of the animal while it is alive, post mortem examinations were resorted to in many cases in which the issue of the experiment was fatal. Since the age of the animal may modify toxicity full grown, as well as young, animals were employed for these experiments; diet, race, and season also play an important part in determining the toxicity of a drug and these factors were also taken into account in the present investigation.

EXPERIMENTS ON RABBITS.

Animals of different varieties were used and were given caffein by all of the methods indicated in the preceding paragraph. Some of the rabbits employed in these experiments received oats, others received a diet exclusively of carrots for several days or weeks previous to the administration of caffein. The experiments were conducted at all seasons of the year.

SUBCUTANEOUS INJECTION.

From a study of the literature on the toxicity of caffein it seemed that about 150 mg per kilo is probably the lethal dose for the rabbit when the drug is injected subcutaneously. Preliminary observations were therefore carried out with such a dose, but it was found, on the contrary, that this amount per kilo was hardly sufficient to induce symptoms in the great majority of cases.

Series A.

[Doses of 147 to 167 mg of caffein per kilo were employed in these experiments.]

Rabbit 332. Belgian hare, female. Weight, 1,070 grams. Diet, oats.

March 25: 8.5 cc 2 per cent caffein (158 mg per kilo) injected subcutaneously at 2.15 p. m.; 4 p. m., reflexes increased; 5.45 p. m., increases of reflexes still more marked.

March 26: Rabbit looked normal; no symptoms observed.

Rabbit 331. Belgian hare, female. Weight, 1,170 grams. Diet, oats.

March 25: 2.15 p. m., 9 cc 2 per cent caffein (153 mg per kilo) injected subcutaneously; 4 p. m., reflexes increased; 5.45 p. m., condition the same.

March 26: Rabbit looks normal; no symptoms observed.

Rabbit 328. Belgian hare, female. Weight, 1,200 grams. Diet, oats.

March 25: 9 cc 2 per cent caffein injected subcutaneously (150 mg per kilo); 4 p. m., reflexes increased; 5.45 p. m., reflexes increased but not markedly.

March 26: No symptoms; rabbit looks normal.

Rabbit 322. White female. Weight, 1,065 grams. Diet, oats.

March 17: 8 cc 2 per cent caffein (150 mg per kilo) injected subcutaneously at 11.55 a. m.; 12.55 p. m., reflexes increased, but no tetanus nor any other symptoms.

March 18: Rabbit running around in cage; condition apparently normal.

March 25: Condition of rabbit good.

Rabbit 217. White. Weight, 1,355 grams. Diet, oats.

October 29: 10 cc 2 per cent caffein (147 mg per kilo) injected subcutaneously at 1.51 p. m. 5.15 p. m., rabbit alive; survived.

Rabbit 219. Maltese. Weight, 1,820 grams. Diet, oats.

October 29: 14 cc 2 per cent caffein injected subcutaneously at 1.40 p. m. (153 mg per kilo); 5.15, rabbit alive; survived.

Rabbit 194. White female. Weight, 1,490 grams. Diet, oats.

October 14: 13 cc 2 per cent caffein (174 mg per kilo) injected subcutaneously; increased reflexes and tremors were observed.

October 15: Condition of rabbit good; no symptoms.

Rabbit 191. Brown male. Weight, 1,915 grams. Diet, oats.

October 14: 16 cc 2 per cent caffein (167 mg per kilo) injected subcutaneously; reflexes increased and tremors present.

October 15: Condition of rabbit good.

A study of this series shows that about 150 mg of caffein per kilo caused increased reflexes within one to two hours after injection. When the dose was increased, as in rabbits 194 and 191, the symptoms were more pronounced; 150 mg per kilo may be regarded as the minimum dose which produces symptoms of nervous irritability when caffein is injected subcutaneously. Experiments with larger doses were therefore carried out in order to determine the minimum fatal dose.

Series B.

Approximately 0.2 gram of caffein per kilo was employed in these experiments. Diet and race as possible factors which may influence the toxicity of caffein were made the subject of study in these experiments which were divided into two groups as shown in the table, page 25.

Rabbit 95. Gray and white male. Weight, 1,478 grams. Diet, oats.

February 27: 11.30 a. m., 15 cc 2 per cent caffein (210 mg per kilo) injected subcutaneously; 2.20 p. m., no symptoms, tremors observed when handled, but not marked, reflexes slightly increased, no muscular rigidity nor any other symptoms; 2.45 p. m., rabbit suddenly became very restless, jumped off the table, and had convulsions; 3.45 p. m., rabbit died, rigor mortis set in almost immediately after death.

Rabbit 96. Gray and white male. Weight, 1,585 grams. Diet, oats.

February 27: 16 cc 2 per cent caffein (200 mg per kilo) injected subcutaneously at 3.40 p. m.; increased reflexes observed about one hour after caffein was injected, but no other symptoms.

February 28: Rabbit found dead.

Rabbit 112. Black female. Weight, 875 grams. Diet, oats.

March 18: 9 cc 2 per cent caffein (205 mg per kilo) injected subcutaneously at 3 p. m.; 3.30 p. m., rabbit became restless, reflexes were increased, tremors were observed, but no other symptoms; 4.15 p. m., rabbit had tremors, was handled but this failed to induce tetanus, 10 minutes later tetanus of short duration with recovery occurred.

March 19: 9 a. m., found dead.

Rabbit 119. Yellow white female. Weight, 1,060 grams. Diet, oats.

April 17: 10 cc 2 per cent caffein (188 mg per kilo) injected subcutaneously at 2.10 p. m.

April 18: Rabbit found dead.

Rabbit 195. White female. Weight, 1,300 grams. Diet, carrots, since October 7.

October 14: 13 cc 2 per cent caffein (0.2 gram per kilo) injected subcutaneously at 11.15 a. m.; 2.25 p. m., rabbit had convulsions and died. Note: Ulceration of rectum was noticed.

Rabbit 208. Gray. Weight, 1,068 grams. Diet, carrots, October 7-15, inclusive.

October 15: 10 cc 2 per cent caffein injected subcutaneously at 11 a. m.; 1 p. m., increased reflexes and tremors observed; 3.45 p. m., tremors were marked when rabbit was handled.

October 16: Rabbit found dead. Note: Looked poorly nourished.

Rabbit 247. Belgian hare, female. Weight, 1,295 grams. Diet, oats last 10 days before experiment.

November 10: 11 a. m., urine obtained from the bladder was acid to litmus and did not contain sugar or albumen, 13 cc 2 per cent caffein was injected subcutaneously; 1.30 p. m., 15 cc urine obtained was markedly alkaline to litmus and reduced Fehling's solution; 2.30 p. m., reduction of urine considerable, marked tremors observed but no tetanus.

November 11: 10.30 a. m., 95 cc urine collected gave moderate reduction of Fehling's solution, no symptoms, condition of rabbit seemed to be good.

Rabbit 248. Belgian hare, female. Weight, 1,305 grams. Diet, oats the last 10 days before the experiment.

November 10: 11 a. m., urine markedly acid to litmus, no albumen, no sugar; 13 cc 2 per cent caffein injected subcutaneously; 1.30 p. m., urine was slightly alkaline to litmus, no reduction of Fehling's solution; 2 p. m., reflexes increased; 2.30 p. m., 2 cc urine obtained from bladder, sugar abundant; 4.45 p. m., reflexes increased as before, but no tetanus.

November 11: 10.30 a. m., urine collected showed slight reduction of Fehling's solution; otherwise condition of rabbit was good; rabbit did not show any effects of caffein.

Rabbit 337. Belgian hare. Weight, 1,040 grams. Diet, carrots, March 31 to April 6, inclusive.

April 6: 3 p. m., 11 cc 2 per cent caffein injected subcutaneously in the back (0.211 per kilo); 4.30 p. m., reflexes much exaggerated.

April 7: 8.15 a. m.; condition good, no symptoms.

Rabbit 336. Belgian hare. Weight, 1,040 grams. Diet, carrots, March 31 to April 6, inclusive.

April 6: 3 p. m., 11 cc 2 per cent caffein injected subcutaneously into tissues of the back.

April 7: 8.15 a. m., no symptoms, condition good.

Although symptoms appeared in rabbits of Group I (see table, page 25) about the same time after the administration of caffein as in the rabbits of the preceding series all of them terminated fatally 2¼ hours to 24 hours after its administration. Two of these rabbits (Nos. 195 and 208) were fed carrots for several days before the injection of caffein, the others were fed oats. Since symptoms and death appeared in these two rabbits about the same time as in the rest of this group it may be concluded that caffein is not less toxic when carrots are fed than when oats form the exclusive diet. But since rabbit No. 208 was poorly nourished and ulceration of the rectum was observed in No. 195 it is quite possible that caffein might be less toxic in normal rabbits on this diet. This was tested in rabbits Nos. 336 and 337, both of which seemed to be free from abnormality and were well nourished. Since these rabbits survived and manifested mild symptoms only of intoxication it would seem that a carrot diet decreases the toxicity of caffein.

It was suggested, however, that another factor might be the cause of the greater resistance to caffein in these two rabbits, namely, race. This was tested in rabbits 247 and 248, both Belgian hares. Since the toxicity of caffein in these two rabbits was the same as in Nos. 336 and 337, diet as a factor in acute caffein intoxication may be disregarded. The greater resistance to caffein of these four rabbits is in all probability due, therefore, to a difference of race. This suggestion gained additional support from the experiments of the next series.

Series C.

The object of these experiments was to determine the minimum fatal dose for the gray rabbit and to obtain additional evidence as to the toxicity of caffein in the several varieties of rabbits. Eight experiments were performed, in which from 236 to 252 mg per kilo were given. The white rabbits, three in number, received 250, 242, and 238 mg per kilo. All the others (which were Belgian hares) received from 236 to 252 mg per kilo. Two of the white rabbits were fed carrots for one week preceding the injection of caffein. The other was fed oats. Three of the Belgian hares were on a diet of oats, two were fed carrots the week before the experiment with caffein.

Rabbit 122. White, female. Weight, 2,060 grams. Diet, oats.

April 14: 25 cc of 2 per cent caffein (250 mg per kilo) in aqueous solution injected subcutaneously in the back at 1.35 p. m.; 4.30 p. m., tremors, reflexes increased, condition otherwise good.

April 16: 9 a. m., found dead in cage. Autopsy: Liver deeply congested; kidneys congested in cortex and medulla; stomach showed small hemorrhagic areas, perforating ulcers in pyloric portion; small intestine petechiated on mucosa; lungs and spleen normal.

Rabbit 234. White, female. Weight, 1,650 grams. Diet, November 2-9, carrots.

November 9: 10.45 a. m., 20 cc 2 per cent caffein (242 mg per kilo) administered subcutaneously.

November 10: 9 p. m., found dead.

Rabbit 335. Gray hare, female. Weight, 1,170 grams. Diet, March 31 to April 7, carrots.

April 7: 9.30 a. m., 14 cc 2 per cent caffein solution (240 mg per kilo) injected subcutaneously in the back; 10.30, reflexes much increased, rabbit is extremely sensitive.

April 8: 9 a. m., found dead. Autopsy: Liver was congested and contained several coccidiosis nodules; stomach distended with rather dry food mass; mucosa exhibited mild catarrhal inflammation; mucosa of intestines also slightly inflamed.

Rabbit 249. Belgian hare, female. Weight, 1,185 grams. Diet, oats.

November 11: Urine, 5 cc, from bladder acid to litmus, no sugar, no albumin; 11.50 a. m., 14 cc 2 per cent caffein (236 mg per kilo) administered subcutaneously; 3.45 p. m., reflexes increased, hyperæsthesia marked, but no tetanus, even when handled; 30 cc urine collected at 4 p. m., reduction of Fehling's solution considerable.

November 12: 10 a. m., 8 cc urine collected, reduction heavy, only a few cubic centimeters obtained from bladder, did not contain any sugar, general condition of rabbit good, no symptom of caffein intoxication.

Rabbit 321. Yellow, female. Weight, 1,135 grams. Diet, oats.

March 16, 1910: 11.50 a. m., 14 cc 2 per cent caffein (246 mg per kilo) injected subcutaneously in the back; 2 p. m., reflexes increased, is very sensitive, started to run when put on floor, no handling except what was required for removal and return to cage, feces soft.

March 17: 9.30 a. m., condition good, rabbit put on floor, gait normal, but does not care to walk.

March 18: 9 a. m., walks around when put on floor, appetite good, condition seems to be normal.

March 25: 11 a. m., rabbit still alive, condition good.

Rabbit 250. Belgian hare, female. Weight, 1,435 grams. Diet, oats at least two days before the experiment.

November 11: 11 a. m., urine obtained from bladder acid to litmus, no albumin, no sugar; 11.10 a. m., 18 cc, 2 per cent caffein (252 mg per kilo); 3.45 p. m., reflexes and hyperæsthesia, no tetanus; 4 p. m., 60 cc urine, marked reduction of Fehling's solution.

November 12: 10 a. m., condition of rabbit good, no symptoms of caffein intoxication, 80 cc urine collected, sugar considerable, only a few cubic centimeters of urine obtained from bladder, no reduction of Fehling's solution.

Rabbit 834. Belgian hare, female. Weight, 1,270 grams. Diet, carrots, March 31 to April 7.

April 7: 9.30 a. m., 15 cc 2 per cent caffein (240 mg per kilo) injected subcutaneously in the back; 10.30 a. m., reflexes much increased, rabbit extremely sensitive.

April 8: 9 a. m., condition good, no symptoms.

Rabbit 233. White, male. Weight, 1,675 grams. Diet, carrots, November 2 to 9.

November 9: 10.50 a. m., 20 cc 2 per cent caffein (238 mg per kilo) injected subcutaneously, no symptoms observed until 5 p. m., when increased reflexes and hyperæsthesia were noticed, but no tetanus.

November 10: 9 a. m., paralysis of posterior extremities; died at 1 p. m.

Analysis of the results obtained in the experiments of this series and inspection of Table I, page 25, show that all four of the rabbits which survived doses of 236 to 252 mg of caffein per kilo were Belgian hares. Of the four which died one only was a Belgian hare. The other three were white rabbits. Two of these were fed oats; the other two received carrots during seven days preceding the administration of caffein. This diet does not seem to be a factor, therefore, in the toxicity of caffein. Moreover, it may be observed that rabbit No. 122, which was fed oats, died after receiving 250 mg per kilo, while rabbit No. 250 received the same diet and survived the same dose of caffein per kilo.

Experiments 234 and 334 offer another illustration that the toxicity of caffein is not dependent upon diet, since both rabbits were fed carrots, but the same dose of caffein caused only symptoms in one while it proved fatal to the other. It is evident, therefore, that the difference in resistance to caffein shown in these experiments is in all probability due to race, the Belgian hare being more resistant to caffein than rabbits of other varieties. Rabbit No. 335 seems to be an exception, but the post-mortem examination showed the presence of coccidiosis of the liver. As will be shown later, wherever this condition prevailed even smaller doses of caffein proved fatal.

Series D.

To obtain additional evidence regarding the resistance of the various races of rabbits to caffein and to ascertain the smallest dose which is surely fatal to the gray rabbit or Belgian hare was the object of this series of experiments. The diet in all cases consisted of oats, which was given ad libitum excepting to rabbit No. 235, which received carrots for one week previous to the injection of caffein. The doses administered ranged from 267 to 300 mg per kilo and were administered to different varieties of adult rabbits.

Rabbit 253. Brown and black, male. Weight, 1,600 grams. Diet, oats, November 9 to 12.

November 12: 11.30 a. m., urine from bladder acid, no albumen, no sugar; 11.35 a. m., 22 cc 2 per cent caffein (275 mg per kilo) injected subcutaneously; 11.45 a. m., rabbit jumped, off the table, had convulsions, retraction of head and opisthotonos, general tremors, anterior extremities stretched out, posterior extremities almost normal, frequent twitchings; died at 12.15 p. m.

Rabbit 252. Black, female. Weight, 1,335 grams. Diet, oats, November 9 to 12.

November 12: 11.30 a. m., 18 cc 2 per cent caffein (270 mg per kilo) injected subcutaneously. Urine obtained from bladder before injection, acid, no albumen, no sugar, color normal, tremors and great excitement noticed about 12 noon; 4.30 p. m., when handled, showed unusual restlessness and excitement followed by convulsions with opisthotonos; occasional twitching, condition bad. Died 4.35 p. m.

Rabbit 327. White, female. Weight, 820 grams. Diet, oats, March 8 to 16.

March 16: 11.45 a. m., 12 cc 2 per cent caffein (292 mg per kilo) injected subcutaneously in the back; 2 p. m., found dead, but was still warm. Autopsy: Hemorrhagic area at point of injection into spinal muscles; subcutaneous abdominal region exhibited a large area of cheesy purulent material; liver and spleen were engorged; bladder filled; intestines normal.

Rabbit 340. White and brown male. Weight, 1,465 grams. Diet, oats.

March 30: 3.20 p. m., 20 cc of 2 per cent caffein (273 mg per kilo) injected subcutaneously in back.

March 31: 9 a. m., found dead.

Rabbit 341. White and brown. Weight, 1,450 grams. Diet, oats.

March 30: 3.20 p. m., 20 cc 2 per cent caffein (270 mg per kilo) injected subcutaneously in back; 4.40 p. m., found in dying condition, had convulsions; 4.45 p. m., dead.

Rabbit 326. White, male. Weight, 1,645 grams. Diet, oats, March 8 to 16.

March 16, 1910: 12 noon, 20 cc 2 per cent caffein (243 mg per kilo) injected subcutaneously in the back; 2 p. m., tremors marked, hypersensitive, started to run when put on floor; rabbit was not handled any more than was required for his removal from and return to cage.

March 17: 9.30 a. m., tremors still present and marked, otherwise general condition good; no other symptoms.

March 18: 9.30 a. m., no appetite, tremors still present, general condition poor; died about 2 p. m.

Rabbit 235. Belgian hare, male. Weight, 1,870 grams. Diet, carrots, November 2 to 9.

November 10: 11.05 a. m., 25 cc 2 per cent caffein (267 mg per kilo) injected subcutaneously; reflexes increased and tremors, but no tetanus observed; found dead next morning.

Rabbit 316. Belgian hare, female. Weight, 860 grams. Diet, oats, March 8 to 16.

March 16, 1910: 11.40 a. m., 12 cc 2 per cent caffein (267 mg per kilo) injected subcutaneously in the back; 2.15 p. m., reflexes somewhat increased, but not markedly so; walked when put on floor; gait clumsy and slow; tremors of head observed; 2.35 p. m., rabbit lying in his cage, posterior extremities extended and rigid, anterior extremities flexed, head retracted; is still breathing; occasional spasms observed. Rabbit died at 3 p. m. Autopsy: No lesion at point of injection in dorsal spinal muscles; liver and spleen engorged; intestines injected; other organs apparently normal.

Rabbit 395. Belgian hare, male. Weight, 1,410 grams.

August 18: 1 p. m., 20 cc 2 per cent caffein (283 mg per kilo) injected subcutaneously in the back; 4 p. m., reflexes markedly increased; 5 p. m., reflexes about the same, but no tetanus.

August 19, 9.15 a. m.: Reflexes increased markedly.

August 21, weight, 1,215 grams. Given 275 mg per kilo of caffein; no symptoms observed.

August 23, found dead. Autopsy: Liver greatly engorged; stomach fairly well distended and mucous membrane in a slightly inflammatory condition; contents of small intestine liquid in nature, but walls of same appeared normal; other organs normal in appearance.

Rabbit 396. Belgian hare, female. Weight, 1,475 grams. Diet, oats.

August 18: 1 p. m., 20 cc 2 per cent caffein (272 mg per kilo) injected subcutaneously in the back; 4 p. m., reflexes increased markedly; 5 p. m., reflexes increased markedly but no tetanus.

August 19: 10.30 a. m., reflexes still increased very markedly; rabbit jumps when touched.

August 21: Weight, 1,245 grams. Injected subcutaneously 275 mg of caffein per kilo; reflexes increased, posterior extremities stiff over hour later.

August 22: 9 a. m., found dead. Autopsy: Thoracic organs normal in appearance; stomach distended and mucous membrane affected with a catarrhal inflammation; contents of stomach were covered with a shiny mucus; contents of small intestine liquid in nature and bile stained; liver showed a coccidial infestation; kidneys and spleen normal in appearance.

Rabbit 397. Belgian hare, male. Weight, 1,375 grams. Diet, oats.

August 19: 10.30 a. m., 20 cc 2 per cent caffein (290 mg per kilo) injected subcutaneously in the back.

August 22: 9 a. m., found dead. Autopsy: Stomach distended with ingesta; mucous membrane exhibited a catarrhal inflammation with excessive secretions; major portion of intestines showed a condition similar to that of stomach, contents consisting mainly of a shiny mucus; liver enlarged; other organs apparently normal.

Rabbit 398, Belgian hare, female. Weight, 1,570 grams. Diet, oats.

August 19: 10.30 a. m., 23 cc 2 per cent caffein (293 mg per kilo) injected subcutaneously in the back; 4 p. m., found dead. Autopsy: Thoracic organs seemingly normal; mucous membrane of stomach exhibited a catarrhal inflammation generally; large intestines somewhat impacted but walls appeared normal; other organs normal.

Rabbit 399, Belgian hare, male. Weight, 1,725 grams. Diet, oats.

August 19: 10.30 a. m., 26 cc 2 per cent caffein (300 mg per kilo) injected subcutaneously in the back; found dead at 4.30 p. m. Autopsy: Lungs slightly congested; liver engorged and friable; gall cyst well filled; stomach exhibited catarrhal gastritis; injection of mesenteries and intestines; kidney showed marked cortical congestion.

The results of the experiments of this series likewise indicate that the Belgian hare is more resistant to caffein than the rabbits of other varieties. Thus, of the four gray rabbits (Nos. 235, 316, 395, and 396), which received 267 to 283 mg of caffein per kilo, two died and two lived,[B] one of which, 396, showed the presence of coccidiosis of the liver. On the other hand it will be observed that the black and white rabbits which received from 270 to 275 mg of caffein per kilo all died from the effects of the drug; one within 1 hour and 25 minutes and another within 50 hours after the administration of the caffein, while No. 340 died in the night. Furthermore it will be noted that of the last three rabbits of this series, which were Belgian hares and received 290, 293, and 300 mg of caffein, two died six hours after the injection, while the other, No. 397, lived three days. The minimum fatal dose of caffein for Belgian hares is, therefore, about 290 to 300 mg per kilo when injected subcutaneously, which is about 50 per cent greater than for rabbits of other varieties.

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 dyspnœa 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 kiloAppearance of symptoms inDuration of life. Diet. Remarks.

Grams. Mg.

332 1,070 1581 hour 45 minutesSurvived OatsGray.

331 1,170 153do.do.do.Do.

328 1,200 150do.do.do.Do.

322 1,065 1501 hourdo.do.White.

217 1,355 147do.do.Do.

219 1,820 153do.do.Maltese.

194 1,490 174do.do.White.

191 1,915 167do.do.Light brown.

SERIES B, GROUP I.

95 1,478 2102 hours 50 minutes3 hours 10 minutesOatsWhite.

96 1,585 2001 hourAbout 18 hoursdo.Gray white.

112 875 20530 minutesdo.do.Black.

119 1,060 188do.do.Yellow white.

195 1,300 2003 hours 10 minutesCarrotsWhite.

208 1,068 1882 hoursAbout 24 hoursdo.Gray.

SERIES B, GROUP II.

247 1,295 2002.5 hoursSurvivedOatsGray.

248 1,305 2003 hoursdo.do.Do.

337 1,040 2111.5 hoursdo.CarrotsDo.

336 1,045 211do.do.do.Do.

SERIES C.

122 2,060 2502 hours 55 minutes1.5 daysOatsWhite.

234 1,650 242About 24 hoursCarrotsDo.

335 1,170 2401 hourdo.do.Gray coccidiosis.

249 1,185 2364 hoursSurvivedOatsGray.

321 1,135 2462 hours 10 minutesdo.do.Yellow.

250 1,435 2524 hours 35 minutesdo.do.Gray.

334 1,270 2401 hourdo.CarrotsDo.

233 1,675 2386 hours 10 minutes26 hoursdo.White.

SERIES D.

253 1,600 27510 minutes35 minutesOatsBrown and black.

252 1,335 27030 minutes4 hours 55 minutesdo.Black.

327 820 2922 hours 15 minutesdo.White.

340 1,465 273About 18 hoursdo.White and brown.

341 1,450 2701 hour 25 minutesdo.Do.

326 1,645 2432 hours50 hoursdo.White.

235 1,875 26720 hoursCarrotsGray.

316 860 2672 hours 45 minutes3 hours 20 minutesOatsDo.

395 1,410 2833 hoursSurvivedDo.

395 1,215 275do.About 2 daysOatsDo.

396 1,475 272do.Surviveddo.Do.

396 1,245 2751 hourAbout 18 hoursdo.Do.

397 1,375 2903 daysdo.Do.

398 1,570 2935.5 hoursdo.Do.

399 1,725 3006 hoursdo.Do.

SERIES E.

325 1,065 1122 hoursLess than 22 hours OatsWhite female.

330 935 107NoneSurvivedGray.

329 775 103do.do.Gray male.

320 1,040 1152 hours46 hours OatsBlack male.

551 1,450 10030 minutesLess than 24 hoursdo.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 kiloSymptoms.Duration of life. Diet. Remarks.

Grams. Mg.

248 1,170 3303 hoursSurvivedOatsGray.

241 1,380 2902 hoursdo.do.White male.

249 890 3253 hoursdo.do.Gray male.

SERIES B.

239 935 363SurvivedOatsGray male.

254 975 369About 2 daysdo.Gray female.

267 1,050 34250 minutes1 hourdo.White.

268 1,100 363About 3 hoursdo.Do.

419 1,600 3506 hoursLess thando.Gray male.

420 1,250 3521 hour2 hoursdo.Do.

421 1,485 3516 hoursLess thando.Do.

424 1,295 2932 hours22 hoursdo.White male.

423 1,205 300do.Less thando.Do.

422 1,440 291do.2½ hoursdo.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, dyspnœa; 4.50 p. m., about 100 cc urine collected, no albumin, reduction of Fehling's solution moderate.

March 3: 9.30 a. m., posterior extremities extended and rigid, anterior extremities paralyzed, respiration less frequent and deeper than normal. Rabbit died at 11.50 a. m.; urine collected since 4.50 p. m. previous day gave very heavy reduction of Fehling's solution. Autopsy: Animal in good condition; in the left axillary region was observed a hemorrhage into the subcutaneous and muscular tissue of that region. The ventral portion of the large colon, in contact with the ventral abdominal wall, showed a hemorrhagic area about one-half inch in length, such as might be produced by a puncture or bruise of the colon through the abdominal wall. A small portion of the small intestine adjacent to the colon was affected in a similar manner. All internal organs were apparently normal.

Rabbit 308. Belgian hare, female. Weight, 1,350 grams. Diet, oats.

March 2: 11.45 a. m., urine obtained from bladder, no albumen, no reduction; 11.50 a. m., 10 cc 2 per cent caffein (0.15 gram per kilo) injected into peritoneal cavity; 1.30 p. m., not very active, no abnormal symptoms otherwise; 3.30 p. m., rabbit looked depressed, made very little attempt to move about, remained in one position most of the time when placed on floor; 4.30 p. m., 180 cc urine collected, no albumen, reduction of Fehling's solution moderate.

March 3: 9.30 a. m., rabbit looks normal, is able to walk but is easily fatigued when made to walk about or when placed on his side, followed by paralysis of anterior extremities, posterior extremities apparently normal, about 90 cc of urine collected at noon was free from albumen, did not reduce Fehling's solution.

March 4: 11 a. m., lying on his side in cage, anterior extremities limp, posterior extremities extended and rigid, is in dying condition.

March 5: 9 a. m., found dead. Autopsy: Liver engorged; spleen congested, but not enlarged; kidneys, some congestion in cortex; stomach filled, mucosa thickened and easily pulled off; petechial hemorrhages on serosa of colon.

Series C.

The experiments of this series were made to determine the minimum toxic dose.

Rabbit 295. Belgian hare, female. Weight, 1,205 grams. Diet, carrots.

March 1: 10.40 a. m., 6 cc 2 per cent caffein (0.1 gram per kilo) injected into peritoneal cavity; about 2 cc of urine obtained before injecting caffein; 2 p. m, 100 cc urine, bloody in appearance, collected, a moderate quantity of albumen present, no reduction; 3.40 p. m., no symptoms.

March 4: 2 p. m., rabbit looks well.

Rabbit 293. Belgian hare, female. Weight, 1,605 grams. Diet, carrots.

March 1: Urine from bladder clear, alkaline; 11.55 a. m., 8 cc 2 per cent caffein (0.1 gram per kilo) injected into peritoneal cavity; 3 p. m., 90 cc urine normal in color collected, no albumen, no reduction; 3.40 p. m., no symptoms.

March 4: 1.15 p. m., rabbit looks normal.

Rabbit 292. Belgian hare, male. Weight, 1,595 grams. Diet, carrots.

March 1: 10.10 a. m., 8 cc 2 per cent caffein (0.1 gram per kilo) solution injected into peritoneal cavity; 10.40 a. m., rabbit urinated, reflexes increased, but no other symptoms; 10.50 a. m., no urine obtained from bladder; 2 p. m., 105 cc of clear pale urine collected; no albumen, no reduction; 3.40 p. m., no symptoms.

March 4: 2 p. m., rabbit looks well, urine collected, did not contain sugar.

Rabbit 298. Belgian hare, female. Weight, 1,205 grams. Diet, carrots.

March 1: 4.06 p. m., 7.5 cc 2 per cent caffein solution (0.125 gram per kilo) injected into peritoneal cavity, urine obtained from bladder immediately after injection, no albumen, no reduction; 5.30 p. m., reflexes increased, rabbit was able to run around, but became paralyzed soon; 5.40 p. m., rabbit is again able to run around.

March 3: 10 a. m., anterior extremities paralyzed, is able to use posterior extremities.

March 4: 1 p. m., rabbit looks normal.

Rabbit 223. Belgian hare, male. Weight, 1,165 grams. Diet, carrots.

March 1: 3.50 p. m., urine obtained from bladder clear, amber colored, no albumen, no sugar; 3.52 p. m., 7.5 cc 2 per cent caffein (125 mg per kilo) injected into peritoneal cavity; 5.40 p. m., rabbit makes little attempt to run when put on the floor, weakness of extremities marked.

March 4: 1.15 p. m., rabbit normal.

Series D.

The object of the experiments of this series was to study the effect of age on the resistance to caffein. Half-grown rabbits were, therefore, used in the following experiments.

Rabbit 310. Belgian hare, female. Weight, 880 grams. Diet, oats.

March 2: 3.25 p. m., 9 cc 2 per cent caffein (0.2 gram per kilo) injected into peritoneal cavity.

March 3: 9.30 a. m., no symptoms, rabbit looks normal.

March 4: 11 a. m., posterior extremities abducted, walked when placed on the floor, made no attempt to change attitude when placed on its side, remained some time in this position.

March 5: 9 a. m., found dead. Autopsy: Liver showed areas of degeneration; kidneys congestion and petechial hemorrhage on cortex; small and large intestines, inflammation marked; bladder distended.

Rabbit 75. Gray and white, female. Weight, 842 grams.

January 25: 3 p. m., 8.5 cc 2 per cent caffein solution (0.2 gram per kilo) injected into peritoneal cavity; 3.15 p. m., anterior extremities weak and reflexes increased.

January 27: Rabbit paralyzed but is able to turn over when placed on back.

Rabbit 74. Gray and white, female. Weight, 692 grams.

January 25: 3 p. m., 7 cc 2 per cent caffein (0.2 gram per kilo) solution injected into peritoneal cavity; 3.15 p. m., reflexes increased and anterior extremities paralyzed.

January 27: Rabbit recovered and is able to walk about in the room.

Rabbit 312, maltese, female. Weight, 740 grams. Diet, oats.

March 3: 11.47 a. m., urine obtained from bladder, appearance normal, no albumen, no reduction of Fehling's solution; 11.50 a. m., 7.5 cc 2 per cent caffein (0.2 gram per kilo) injected into peritoneal cavity; 2.30 p. m. anterior extremities paralyzed, posterior extremities rigid and extended; 5 p. m. (about), rabbit died.

Rabbit 311. Belgian hare, female. Weight, 650 grams. Diet, oats.

March 3: 11.26 a. m., urine obtained from bladder normal in appearance, albumen considerable, reduction of Fehling's solution none; 11.27 a. m., 6 cc 2 per cent caffein solution (0.2 gram per kilo) injected into peritoneal cavity; 2.30 p. m., rabbit seemed to be normal, no symptoms had developed; urine collected contained a large amount of sugar, reduction was very heavy, but no albumen was found.

March 4: 11 a. m., condition good, moves about when put on floor; gait, normal.

Rabbit 78. Yellow and white. Weight 659 grams.

January 26: 1.30 p. m., 8.5 cc 2 per cent caffein (250 mg per kilo) injected into peritoneal cavity, under observation the rest of the afternoon, no symptoms.

January 27: 4 p. m., no symptoms developed.

Rabbit 317. Belgian hare, female. Weight 635 grams. Diet oats.

March 15: 10.35 a. m., 8 cc 2 per cent caffein (0.252 gram per kilo) injected into peritoneal cavity; 12 noon, marked abduction of hind legs, was unable to walk after a little exertion, rabbit died between 12.30 and 12.50 p. m. Autopsy. Right lung hepatized and showed adhesions to costal and mediastinal pleura; liver studded with nodules of coccidiosis; spleen congested; stomach filled, mucosa normal; intestines injected; colon hemorrhagic on serosa in ventral region, near point of injection; kidneys normal.

Rabbit 323. White, female. Weight 820 grams. Diet oats.

March 15: 10.45 a. m., 10 cc 2 per cent caffein (250 mg per kilo) injected into peritoneal cavity; 12 noon, reflexes increased, hind legs abducted but is able to walk, symptoms are mild; 1.40 p. m., tremors, weakness, and abduction of head and legs much more marked than at 12 noon.

March 16: Condition good.

March 17: Condition good, recovery apparently complete.

Since the experiments of Series A, which were intended as preliminary tests, have shown that 0.3 gram of caffein per kilo when introduced into the peritoneal cavity is rapidly absorbed and is fatal, much smaller doses were employed in subsequent trials with the drug. This is shown in series B, which may be divided into two groups. Group I, consisting of rabbits 69, 70, 92, 93, and 309, which received 0.2 gram of caffein per kilo, and Group II, Nos. 307 and 308, into which 0.15 gram of caffein per kilo was injected. Three rabbits of Group I (Nos. 92, 93, 309) died from the effects of caffein; rabbit 309 twenty minutes after injection, and rabbits Nos. 92 and 93, twenty hours and two and one-half hours, respectively, after the administration of caffein. In both of these rabbits symptoms appeared within five minutes after the injections were made. Rabbits 69 and 70, it will be noticed, survived the same amount of caffein in proportion to body weight as was given to the other members of this group. Increased peristalsis and the distribution of the dose may account for the greater resistance of rabbit No. 69. The case of rabbit No. 70 is evidently one of exceptional resistance to caffein, since both the rabbits of Group II died from the effects of a much smaller dose, namely, 0.15 gram of caffein per kilo. Moreover, macroscopical examination at the autopsy of Nos. 307 and 308 failed to show any lesions which might tend to lessen the resistance to caffein.

That a dose of 0.15 gram per kilo is therefore in all probability the minimum fatal dose for the rabbit when injected into the peritoneal cavity appears from the results of the experiments in series C, in which smaller doses, 0.125 gram of caffein per kilo caused mild symptoms only, while 0.1 gram per kilo rarely induced any symptoms. It may be remarked that the rabbits of series C were fed carrots while rabbits Nos. 307 and 308 received oats. Their resistance to caffein may be different, but, as was pointed out in the earlier part of this investigation, diet does not seem to influence the toxicity of the single dose of caffein. Doses of 150 and of 100 to 125 mg per kilo, when injected into the peritoneal cavity, may be considered, respectively, as the minimum fatal and minimum toxic doses for the gray rabbit. Analysis of the experiments in series D shows much greater resistance to caffein than in the other rabbits which received it intraperitoneally. Thus, after the administration of 0.2 gram per kilo to each of five rabbits, no effect was observed in two cases (Nos. 310, 311), while in two others (Nos. 74, 75) symptoms developed, but they survived. Only one rabbit, No. 312, died from the effects of this dose; the autopsy showed the presence of degeneration of the liver and petechial hemorrhages on the cortex of the kidneys in the case of No. 310, which was probably the cause of death rather than the caffein.

Two decigrams of caffein can not be considered, therefore, the fatal dose for rabbits. This is further corroborated by the results obtained in experiments with larger doses. Rabbit 78, which received 257 mg per kilo, failed to show any symptoms. The same amount in proportion to body weight in No. 323 caused mild symptoms only, while the rapid death of rabbit No. 317 after the same dose of caffein may be explained by the lesion found at autopsy, thus affording additional evidence that disease may decrease the resistance to caffein. It will be observed that all the members of this series were young rabbits and, as will be shown later, young animals of other species are likewise more resistant to caffein than adult animals. Similar results were obtained by von Anrep, who observed that atropin is less toxic in young than in full-grown animals.

Observations were also made on the diuretic effect of caffein when injected into the peritoneal cavity. The results shown in the following table indicates the stimulating effect on renal secretion whether the diet consisted of oats or of carrots. The urine of some rabbits contained moderate amounts of sugar after from 0.2 to 0.15 gram of caffein per kilo was given; albumen was observed in one case, but in none of the others. In rabbit No. 311 albumin was found before the injection of caffein, but none in the urine which was collected three hours after caffein was injected.

Effect of caffein on renal secretion.

No. Weight.Caffein per kilo.Urine.Time. Diet.

Grams. Gram.cc.

307 1,320 0.150 100 4.5 hours Oats.

308 1,305 .150 180 do.Do.

295 1,205 .100 100 2 hours 20 minutes Carrots.

293 1,605 .100 90 3 hoursDo.

292 1,595 .100 105 4 hoursDo.

Note.—The amount of urine secreted in three hours by control rabbits, on a carrot diet, varied between 35 and 50 cc, the average weight of the animals being a little above 1,600 grams. The secretion of urine on an oat diet was much less for an equal period of time.

Table 3.—Intraperitoneal injections.

SERIES A.

No. Weight.Caffein per kiloTime of appearance of symptoms inDuration of life. Remarks.

Grams. Gram.

71 1,659 0.3 85 minutes24 hoursGray.

61 2,143 .3 At the end of injectionBlack.

72 1,402 .3 8 minutes1.25 hourGray and white.

SERIES B, GROUP I.

70 1,487 0.210 daysGray and white.

93 1,492 .2 5 minutes2.5 hoursMaltese; given second dose after 3 days, died 2.5 hours later.

69 1,492 .2 About 5 minutesSurvivedWhite.

92 1,388 .2 5 minutes24 hoursYellow.

309 1,500 .2 20 minutesBelgian; oats.

SERIES B, GROUP II.

308 1,350 0.15 3 hours and 40 minutesAbout 2.5 daysBelgian; oats.

307 1,320 .15 1 hour 24 minutes24 hours Do.

SERIES C.

223 1,165 0.1252 hoursSurvivedBelgian; carrots.

293 1,605 .1 do. Do.

295 1,205 .1 do. Do.

292 1,595 .1 do. Do.

298 1,205 .1251.5 hours do. Do.

SERIES D.

310 880 0.2 2 days¹About 2.5 days¹Belgian; oats.

311 650 .2 Survived Do.

312 740 .2 40 minutes4.5 hoursMaltese; oats.

78 659 .25715 minutesSurvivedYellow and white; oats.

75 842 .2 do. do.Gray and white.

74 692 .2 do. do.

317 635 .2521 hour 25 minutesAbout 2 hoursBelgian; oats.

323 820 .25 1 hour 15 minutesSurvivedWhite; oats.

¹ Not due to caffein.

INTRAMUSCULAR INJECTION.

Well-fed rabbits, which received a diet exclusively of oats, were used for these experiments. The injections were made into the lumbar or into the gluteal muscles.

Series A.

In this series the caffein was injected into the gluteal muscles.

Rabbit 284. Brown and white, female. Weight, 1,100 grams.

December 14: 2 p. m., 11 cc 2 per cent caffein injected into the gluteal muscles (0.2 gram per kilo), under observation until 5 p. m., had frequent convulsions; at 5 p. m. in a comatose condition. Rabbit was found dead the next morning.

Rabbit 286, white and black, female. Weight, 1,315 grams.

December 15: 2.30 p. m., 13 cc 2 per cent caffein injected into the gluteal muscles (0.1977 gram per kilo), tremors and increased reflexes observed during the next two hours, but no other symptoms.

December 17: Rabbit alive.

Rabbit 285, yellow and white, female. Weight, 1,385 grams.

December 14: 10.15 a. m., 14 cc 2 per cent caffein injected into the gluteal muscles (0.2 gram per kilo), general tremors, but no convulsions observed. Rabbit survived.

December 17: Rabbit still alive.

Rabbit 287. Belgian hare, female. Weight, 1,140 grams.

December 15: 2.15 p. m., 11 cc of 2 per cent caffein injected into the gluteal muscles; 2.30 p. m., tonic contractions of posterior limbs. Paralysis and death at 2.40 p. m.

Series B.

In series B the caffein was injected into the lumbar muscles.

Rabbit 307. Belgian hare, female. Weight, 1,175 grams.

February 16: 11.05 a. m., 8 cc 2 per cent caffein injected (0.136 gram per kilo) into the lumbar muscles; under observation until 4 p. m., no symptoms; 4 p. m., allowed to walk on the floor; after walking a short distance loss of coordination and paralysis of posterior extremities; 5.20 p. m., found dead.

Rabbit 306. Belgian hare, female. Weight, 1,860 grams.

February 16: 11 a. m., 12.5 cc 2 per cent caffein injected into the lumbar muscles; 12 noon, no symptoms; 2 p. m., walked about 10 feet, exhaustion and paralysis; 3 p. m. found dead.

Rabbit 181. Belgian hare. Weight, 1,230 grams. (Was experimented on some time previously.)

February 16: 10.55 a. m., 8 cc 2 per cent caffein injected into the lumbar muscles; (0.130 gram per kilo); 12 noon, no symptoms; 2 p. m., no symptoms; 3 p. m., put on the floor, walked about 10 feet and was exhausted, posterior extremities paralyzed; 4 p. m., found dead.

Series C.

In the fall of the same year additional experiments were carried out with doses ranging from 100 to 200 milligrams of caffein per kilo, which were injected into the lumbar muscles. The results are given in the following abbreviated protocols:

Rabbit 425. Belgian hare. Weight 1,520 grams.

September 27: 10.30 a. m., 7.5 cc 2 per cent caffein injected into the lumbar muscles; 2 p. m., reflexes increased.

September 28: Rabbit normal.

October 5: Weight, 1,620 grams; 2.50 p. m., 10 cc 2 per cent caffein injected into lumbar muscles; 3.05 p. m., reflexes increased.

October 13: Weight, 1,520 grams; 10.30 a. m., 10 cc 2 per cent caffein (131 mg per kilo) injected; 11 a. m., no symptoms; 11.30 a. m., reflexes much increased.

October 14: Alive, no symptoms.

Rabbit 426. Belgian hare, female. Weight, 1,425 grams.

September 27: 7 cc 2 per cent caffein injected into the lumbar muscles at 10.30 a. m.; 2 p. m., reflexes increased.

September 28: Rabbit normal.

October 5: Weight, 1,425 grams; 2.55 p. m., 9 cc 2 per cent caffein injected into lumbar muscles; 3.05 p. m., reflexes increased.

October 13: Weight, 1,405 grams; 10.30 a. m., 10 cc 2 per cent caffein (142 mg per kilo) injected; 11 a. m., no symptoms; 11.30 a. m., reflexes increased.

October 14: Rabbit alive, no symptoms.

Rabbit 427. Belgian hare, female. Weight, 1,780 grams.

September 27: 9 cc 2 per cent caffein injected into the lumbar muscles; 2 p. m., reflexes increased.

September 28: Rabbit normal.

October 5: Weight, 1,850 grams; 3 p. m., 11.5 cc 2 per cent caffein injected into lumbar muscles; 3.10 p. m., reflexes increased.

October 13: Weight, 1,830 grams; 10.40 a. m., 14 cc 2 per cent caffein (153 mg per kilo) injected into lumbar muscles; 11 a. m., no symptoms; 11.30 a. m., reflexes increased.

October 14: Rabbit alive, no symptoms.

Rabbit 453. Belgian hare, male. Weight, 1,160 grams.

October 12: 3.45 p. m., 11.5 cc 2 per cent caffein in aqueous solution injected into lumbar muscles; 4.15 p. m., reflexes increased; 4.30 p. m., paralyzed.

October 13: 9 a. m., found dead. Autopsy: Gastric mucosa hemorrhagic; liver darkened; other organs normal.

Rabbit 455. Belgian hare, gray, female. Weight, 1,185 grams.

October 12: 3.30 p. m., 11.5 cc 2 per cent caffein injected into the lumbar muscles; 4 p. m., reflexes increased.

October 13: Rabbit weighed 1,070 grams, no symptom of caffein poisoning, reflexes normal; 10.30 a. m., 10 cc 2 per cent caffein injected into the lumbar muscles; 11.30 a. m., jumped off the table, had attack of convulsions and died. Autopsy: Findings same as in No. 453.

Rabbit 428. Belgian hare, gray, male. Weight, 1,650 grams.

October 5: 4 p. m., 14.8 cc 2 per cent caffein (0.18 gram per kilo) injected into the lumbar muscles.

October 6: Found dead.

Rabbit 429. Belgian hare, male. Weight, 1,340 grams.

October 5: 4 p. m., 13.5 cc 2 per cent caffein (0.2 gram per kilo) injected into lumbar muscles.

October 8: Rabbit found dead.

Series D.

Further experiments making injections into both the lumbar and the gluteal muscles, were made in this series.

Rabbit 577. Gray male. Weight, 1,380 grams.

February 14: 3 p. m. 14 cc 2 per cent caffein injected into the gluteal muscles of the right side; 3.10 p. m., restless, jumped off the table and walked about, reflexes increased; 3.45 p. m., passed 30 cc clear, straw-colored urine; 4.45 p. m., allowed to walk about, ran across the room, about 20 feet, looked tired, stretched himself out on the floor, then raised himself and walked about showing no disturbance of gait.

February 15: 9 a. m., found dead.

Rabbit 578. Gray, female. Weight, 1,670 grams.

February 14: 3.05 p. m., 18 cc 2 per cent caffein solution injected into the gluteal muscles of the right side; 3.15 reflexes increased, but not restless; 5 p. m., allowed to walk about, no symptoms observed.

February 15: Found dead.

Rabbit 579. White and gray, male. Weight, 1,490 grams.

February 14: 3.15 p. m., 15 cc 2 per cent caffein solution injected into the gluteal muscles of the right side; put in cage; 3.30 p. m., reflexes increased; 5 p. m., taken out of cage and allowed to walk across the room, no special symptoms noticed.

February 23: Still alive.

Rabbit 580. Gray male. Weight, 1,510 grams.

February 14: 3.35 p. m., 15 cc 2 per cent caffein solution injected into lumbar muscles.

February 23: Still alive, in good condition.

Rabbit 581. Gray female. Weight, 1,680 grams.

February 14: 3.45 p. m., 17 cc 2 per cent caffein solution injected into the lumbar muscles of the right side; 4 p. m., reflexes increased; 4.15 p. m., jumped off the table and had wild convulsions, became very restless, walked about the laboratory; 4.25 p. m., had convulsions occasionally; 4.30 p. m., extremities extended and quite rigid; 4.35 p. m., convulsions and death.

Rabbit 582. Gray male. Weight, 1,870 grams.

February 14: 4.15 p. m., 18 cc 2 per cent caffein solution injected into the lumbar muscles of right side; 5 p. m., reflexes increased; walked about in the room, then rested; 5.15 p. m., had short spasm when handled.

February 23: Alive; good condition.

The data presented in these experiments show that the toxicity of caffein when injected into the muscles of the lumbar regions is the same as when injected into the gluteal muscles. The rabbits of series A received approximately 0.2 gram caffein per kilo and two died as a result of this treatment. The other two survived but symptoms of caffein intoxication were observed.

In series B smaller doses proved fatal, from which it would appear that caffein is more toxic when injected into the lumbar muscles. Further observations, however, failed to corroborate the results obtained in this series. Thus, in series C, 130 to 150 mg of caffein per kilo injected into the lumbar muscles produced mild symptoms only. Experiments with larger doses showed that 0.180 gram caffein per kilo may cause death. It will be noticed, on the other hand, that rabbit No. 455 survived a dose of 0.2 gram per kilo. New experiments were therefore carried out in which the same amounts of caffein in proportion to the weight of the animals were injected into the lumbar muscles as into the gluteal muscles. As shown in the experiments of series D, one rabbit (No. 581) died shortly after caffein was injected into the lumbar muscles; two recovered. Two of the three which received injections into the gluteal muscles were found dead the next day; one recovered. Post-mortem examination failed to indicate the presence of any abnormalities. The rate of absorption of caffein from the gluteal and from the lumbar muscles seems to be, therefore, the same, or not to differ very much. The observations of Auer and 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.

SERIES A.

No. Weight.Caffein per kiloSymptoms after—Duration of life. Site of injection. Remarks.

Grams. Gram.

284 1,100 0.200 3 hoursLess than 20 hours GlutealWhite and brown female.

286 1,315 .1977 2 hoursSurvived do.White and black female.

285 1,385 .200 Present do. do.Yellow and white female.

287 1,140 .210 15 minutes25 minutes do.Gray female.

SERIES B.

307 1,175 0.136 5 hours6 hours, 20 minutes LumbarGray female.

306 1,860 .134 3 hours4 hours do. Do.

181 1,230 .130 4 hours5 hours do.Gray.

SERIES C.

425 1,520 0.131 1 hourSurvived LumbarGray.

426 1,405 .14230 minutes do. do.Gray female.

427 1,830 .15350 minutes do. do. Do.

453 1,160 .20030 minutesLess than 20 hours do.Gray male.

455 1,185 .200 do.Survived do.Gray female.

428 1,650 .180Less than 20 hours do.Gray male.

429 1,340 .200 do. do. Do.

SERIES D.

577 1,380 0.20010 minutesLess than 18 hours GlutealGray male.

578 1,670 .210 do. do. do. Do.

579 1,490 .20015 minutesSurvived do.White and gray male.

580 1,510 .200 do. LumbarGray male.

581 1,680 .20015 minutes50 minutes do. Do.

582 1,870 .19245 minutesSurvived do. Do.

Examination of Table 4 shows that 14 rabbits received from 180 to 210 mg caffein per kilo. The appearance of symptoms in these rabbits varied considerably. In some increased reflexes could be noticed in 10 to 15 minutes after the injection of caffein; in others it was delayed 2 or 3 hours. It might be added that the onset of symptoms occurred in many cases very soon after the administration of the drug—on an average about 10 to 30 minutes after the drug was injected. After smaller doses were administered by injection into the lumbar muscles the appearance of symptoms was delayed several hours in some cases. The duration of life in these 14 rabbits varied considerably. Eight of them died within 1 to 20 hours; six survived. About 0.2 gram caffein per kilo may be regarded as the minimum fatal dose, while the minimum toxic dose is somewhere between 130 and 150 mg per kilo.

INTRAVENOUS INJECTION.

These experiments were carried out on well-fed, full-grown gray rabbits. The diet for several days preceding the experiments consisted of oats or carrots, which were given ad libitum. The injections were made into the ear veins from a burette or by means of a syringe, the temperature of the caffein solution being about 40° C. Attention was also directed to the effect of the rate of injection and of the concentration on the toxicity. The minimum toxic as well as lethal doses were determined as shown in the following experiments.

Series A.

In these experiments the rate of injection was about 1 cc of 2 per cent caffein solution per minute.

Rabbit 194. White, female. Weight, 1,310 grams.

October 19: Injected 7.5 cc 2 per cent solution caffein (115 mg per kilo) into the ear vein. Rabbit showed stiffness; paralysis of extremities appeared soon after.[C] Rabbit survived.

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 dyspnœa, 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 dyspnœa, then continuous struggling; when 8.3 cc were injected rabbit had another convulsion; 2.47 p. m., tonic contraction of anterior extremities; amount injected, 8.7 cc (158 mg per kilo) in 15 minutes and 35 seconds.

Rabbit 567. Gray female. Diet, oats.

February 6: Injection began at 4.11 p. m.; 4.18, convulsions after injection of 5 cc 2 per cent caffein; 4.21, convulsion after total injection of 8 cc; 4.24 p. m., injection resumed and 2 cc more introduced; 4.28 p. m., convulsions, injected 2 cc more; total caffein injected, 12 cc, or 162 mg per kilo; 4.40 p. m., rabbit paralyzed in posterior extremities; 5 p. m., found dead.

In the eight experiments comprising series B rabbits Nos. 567, 254, 279, and 255, which may be designated as Group II, received doses of 162, 160, 166, and 158 mg, respectively. Nos. 562, 561, 560, and 559, which may be designated as Group I, received about 200 mg caffein per kilo. In Group II, which received the smaller doses, one (No. 254) survived. This may be regarded as exceptional, since, as was shown in the experiments of the preceding series, even smaller doses may be fatal. About 160 mg per kilo is, therefore, the smallest surely fatal dose. This might be regarded as a contradiction of the results obtained for rabbit No. 559, but it will be noticed that in this case diuresis was very marked. The results of experiments Nos. 294 and 255 are of interest in this connection, since they indicate that a moderate difference in the rate of injection is without any effect on the toxicity of caffein. The greater resistance to caffein of rabbit No. 559 is in all probability due, therefore, to increased diuresis.

Series C.

In these experiments the minimum toxic dose was determined. The conditions were the same as in the experiments of the other series.

Rabbit 293. Belgian hare, female. Weight, 1,610 grams. Diet, oats.

February 18: 3.40 to 3.43 p. m., 4 cc 2 per cent warm caffein solution injected into ear vein, convulsions when 3 cc were injected, repeated attacks; 4 p. m., raised itself on legs, but fell over immediately and lay stretched on abdomen.

February 19: 9 a. m., rabbit looked normal, apparently recovered.

Rabbit 227. White male. Weight, 2,320 grams.

October 26: 3.29¼ to 3.37½ p. m., injected into ear from burette 6.7 cc 2 per cent caffein, no symptoms; experiment discontinued; survived.

Rabbit 563. Gray female. Weight, 1,650 grams. Diet, oats.

February 6: Injection began at 1.02 p. m., injected 3.5 cc 2 per cent caffein (42 mg per kilo) in four minutes, 0.6 cc more within the next two and one-half minutes, total amount injected 4.1 cc; 1.10 p. m., hypersensitive, some disturbance of muscular coordination; restlessness; 1.35. p. m., reflexes decreased, urinated and walked about, gait normal. Under observation for several days; no symptoms noted.

Rabbit 564. Gray female. Weight, 1,515 grams.

February 6: Injection began at 1.26 p. m., 3.5 cc 2 per cent caffein (46 mg per kilo) injected at the rate of 1 cc per minute; 1.30 p. m., reflexes increased; 1.34 p. m., marked paresis of the extremities, rabbit stretched out on abdomen, legs abducted and partly extended, able to hop about but gait disturbed, no untoward symptoms noticed, under observation for several days after experiment.

Rabbit 565. Gray female. Weight, 1,545 grams. Diet, oats.

February 6: Started to inject at 3.40 p. m., received 2.5 cc 2 per cent caffein intravenously in two minutes or 32 mg per kilo, under observation all afternoon, no symptoms.

Rabbit 566. Gray female. Weight, 1,900 grams. Diet oats.

February 6: Injection began at 3.05 p. m., received 3 cc 2 per cent caffein intravenously in three minutes or 31 mg per kilo, no symptoms observed.

These experiments show that a dose of about 50 mg per kilo when injected intravenously produces mild symptoms, such as increased reflexes. In the four experiments with this amount of caffein these effects were observed in each case. In the experiments in which smaller quantities, 30 mg per kilo, were given intravenously there was no manifestation of symptoms. A dose not over 50 mg per kilo may, therefore, be regarded as the minimum toxic dose when injected intravenously under the conditions stated.

Series D.

A 0.5 per cent caffein solution was used in these experiments in order to test the effect of concentration on its toxicity; the rate of injection was 1 cc per minute.

Rabbit 569. Gray male. Weight, 1,475 grams. Diet, oats.

February 6: 11.50 a. m. to 12.01 p. m., injected 10 cc 0.5 per cent caffein; 12.03 to 12.12 p. m., injected 10 cc of 0.5 per cent caffein; 12.13 to 12.26 p. m., injected 10 cc of 0.5 per cent caffein, total amount injected, 30 cc; 12.20, passed 35 cc of urine; 12.30, increased reflexes, but no convulsions; 4 p. m., reflexes increased.

February 11: Alive, condition good.

Rabbit 574. Gray female. Weight, 1,555 grams. Diet, oats.

February 8: 10.25 to 10.33 a. m., injected 4 cc of 0.5 per cent caffein in salt solution, injection discontinued for five minutes; 10.38 to 11.10, injected 30 cc, total amount of caffein solution received, 34 cc; 11.55 a. m., very sensitive; reflexes markedly increased.

February 9: Alive, condition good.

Rabbit 571. Gray female. Weight, 1,530 grams. Diet, oats.

February 7: Injection 3.18 to 3.50 p. m., received 30 cc in 32 minutes, not hypersensitive; 3.55, restlessness and weakness of extremities; 4.10 p. m., control of anterior extremities impaired, distinctly paretic but tried to walk about, died the same afternoon.

Rabbit 568. Gray male. Weight, 1,605 grams. Diet, oats.

February 7: Injection 10.53 to 11.01 a. m., injected 10 cc 0.5 per cent caffein; 11.03, injection resumed after two minutes interval; 11.14, received 10 cc 0.5 per cent caffein intravenously in 11 minutes; 11.16, injection resumed; 11.35, received 12 cc 0.5 per cent caffein, total amount of caffein solution received, 32 cc; 12.30 p. m., urinated 14 cc of bloody urine; 12.55 p. m., convulsions and death a few minutes later. Autopsy showed congestion of viscera, but no other lesions.

Rabbit 570. Gray female. Weight, 1,225 grams. Diet, oats.

February 7: 2.06 to 2.35 p. m., injected 24.5 cc 0.5 per cent caffein, reflexes increased but no convulsions, paresis especially marked in the anterior extremities; 3 p. m., passed urine which was normal in appearance, reflexes not increased but rabbit was weak.

February 9: Found dead. Autopsy: Liver, spleen, and kidneys congested; large intestines hemorrhagic; omentum congested and showed the presence of small caseous nodules; liver showed adhesion to diaphragm; viscera presented the appearance of intraabdominal infection.

Of the five rabbits of this series three died as a result of the administration of caffein. The other two which survived showed mild symptoms only, such as increased reflexes, but no evidence of severe poisoning such as was observed after the injection of the same doses of caffein in series A when a 2 per cent solution of caffein was injected. Convulsions were noticed in one case only (No. 568); paresis in two cases (Nos. 570 and 571). The nervous symptoms even in this group, therefore, were much milder than in series A. The percentage of death, however, was greater than in series A, in which the concentration of caffein was four times as great. It is quite probable that the strain on the heart due to the sudden increase in volume of the blood and its dilution might be an important factor in increasing the toxicity of caffein. It is conceivable that doses just sufficiently large to depress the normal heart may cause paralysis of an already overstrained organ.

Series E.

In the two experiments of this series the rate of injection as a possible factor influencing the toxicity of caffein was tested. A 2 per cent caffein solution was injected at the rate of 1 cc in two and one-half to three minutes.

Rabbit 572. Gray male. Weight, 1,770 grams. Diet, oats.

February 8: Injection began at 3 p. m., discontinued at 3.37 p. m., and resumed at 3.38 p. m.; rabbit was restless; injection finished at 3.52 p. m. Total quantity received, 17.4 cc 2 per cent caffein intravenously in 52 minutes; struggled intermittently during the injection; anterior legs paralyzed.

February 9: Found dead.

Rabbit 573. Gray male. Weight, 1,810 grams. Diet, oats.

February 8: Started to inject at 1.35 and discontinued at 2.27 p. m.; received 18 cc 2 per cent caffein intravenously in 52 minutes; reflexes markedly increased soon after; 2.45, passed bloody urine; 4.30 p. m. reflexes increased; no other symptoms.

February 9: 9 a. m., found dead.

It will be observed that some retardation of the onset of symptoms was caused by slower injection, but the final result was the same as when the injections were made more rapidly. It is quite probable, therefore, that a much slower rate of injection may lessen considerably the toxicity of caffein.

From the results of the experiments by intravenous injection summarized in the table, it appears that the minimum toxic dose for rabbits of a 2 per cent caffein solution, injected at the rate of 1 cc per minute, is about 50 mg per kilo. Twice the dose induces severe symptoms and may be fatal; 160 mg per kilo are surely fatal. If the rate of injection is diminished, the toxicity of caffein is lessened, but this effect is not marked unless the injections are very slow. Dilution of the caffein solution suppresses to some extent the nervous symptoms, but the toxicity, on the contrary, seems to be increased.

Table 5.—Intravenous injections.

SERIES A.

No. Weight.Caffein per kiloSymptoms.Duration of life. Diet. Remarks.

Grams. Mg.

194 1,310 114PresentSurvivedOatsWhite female.

556 1,635 13410 minutes20 minutes do.Gray female.

557 1,580 114PresentSurvived do. Do.

558 1,590 100 do. do. do. Do.

292 1,770 141 do. do. do. Do.

194 1,350 126 do.10 minutesCarrots Do.

SERIES B, GROUP I.

562 1,650 2001½ hoursOatsGray female.

561 1,450 200 do. do. Do.

560 1,620 200PresentLess than 24 hours do. Do.

559 1,875 190 do.Survived do. Do.

SERIES B, GROUP II.

279 1,320 1661 hourGray and white female.

254 1,285 160SurvivedOatsGray female.

567 162About 45 minutes Do.

255 158Died

SERIES C.

293 1,610 500PresentSurvivedOatsGray female.

227 2,320 570None do.White male.

563 1,650 500Present do. do.Gray female.

564 1,515 460 do. do. Do.

565 1,545 320None do. do. Do.

566 1,900 310 do. do. do. Do.

SERIES D.

569 1,475 100PresentSurvivedOatsGray male.

574 1,555 112 do.do. do. Gray female.

571 1,530 100 do.About 2 hours do. Do.

568 1,605 10020 minutes do.Gray male.

570 1,225 100Less than 20 hours do. Do.

SERIES E.

572 1,770 200PresentAbout 24 hoursOats

573 1,810 200 do. do.

SUMMARY.

The results of the experiments on rabbits show considerable variation in the toxicity of the single dose. Individuals differed so widely in their resistance to this drug that the same experiments had to be repeated many times with each method of administration before satisfactory conclusions could be drawn. This is strikingly illustrated in the experiments by intravenous injection in which a dose of nearly 0.2 gram per kilo was not fatal. Similar instances of exceptional resistance or of sensitiveness to caffein were observed when it was given in other ways. A comparison of the toxicity of caffein administered by different methods in this investigation shows well-marked differences in its activity, although they are not quite so striking as similar experiments with other alkaloids reported by several observers. The toxicity of caffein in these experiments on the rabbit indicates that it is greatest when given by vein and least when given by mouth. The ratio of the minimum toxic doses by these two methods of introduction of caffein was about 7.1; the relation of the minimum fatal dose was about 3.1. The toxicity when given subcutaneously is about 15 to 20 per cent greater than when given by mouth. The difference between the intramuscular and subcutaneous injection is even more marked. The toxicity of caffein when injected into the muscles is about midway between that administered by the subcutaneous and intraperitoneal routes, and is about half that injected intravenously. Meltzer and Auer,⁵⁸ who experimented with a number of drugs found that the intramuscular method of administration is as effective as the intravenous, fluorescin forming the only exception according to their observations. In the experiments of Sollman and 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.