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; liverengorged 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.3321,0701581 hour 45 minutesSurvivedOatsGray.3311,170153do.do.do.Do.3281,200150do.do.do.Do.3221,0651501 hourdo.do.White.2171,355147do.do.Do.2191,820153do.do.Maltese.1941,490174do.do.White.1911,915167do.do.Light brown.SERIES B, GROUP I.951,4782102 hours 50 minutes3 hours 10 minutesOatsWhite.961,5852001 hourAbout 18 hoursdo.Gray white.11287520530 minutesdo.do.Black.1191,060188do.do.Yellow white.1951,3002003 hours 10 minutesCarrotsWhite.2081,0681882 hoursAbout 24 hoursdo.Gray.SERIES B, GROUP II.2471,2952002.5 hoursSurvivedOatsGray.2481,3052003 hoursdo.do.Do.3371,0402111.5 hoursdo.CarrotsDo.3361,045211do.do.do.Do.SERIES C.1222,0602502 hours 55 minutes1.5 daysOatsWhite.2341,650242About 24 hoursCarrotsDo.3351,1702401 hourdo.do.Gray coccidiosis.2491,1852364 hoursSurvivedOatsGray.3211,1352462 hours 10 minutesdo.do.Yellow.2501,4352524 hours 35 minutesdo.do.Gray.3341,2702401 hourdo.CarrotsDo.2331,6752386 hours 10 minutes26 hoursdo.White.SERIES D.2531,60027510 minutes35 minutesOatsBrown and black.2521,33527030 minutes4 hours 55 minutesdo.Black.3278202922 hours 15 minutesdo.White.3401,465273About 18 hoursdo.White and brown.3411,4502701 hour 25 minutesdo.Do.3261,6452432 hours50 hoursdo.White.2351,87526720 hoursCarrotsGray.3168602672 hours 45 minutes3 hours 20 minutesOatsDo.3951,4102833 hoursSurvivedDo.3951,215275do.About 2 daysOatsDo.3961,475272do.Surviveddo.Do.3961,2452751 hourAbout 18 hoursdo.Do.3971,3752903 daysdo.Do.3981,5702935.5 hoursdo.Do.3991,7253006 hoursdo.Do.SERIES E.3251,0651122 hoursLess than 22 hoursOatsWhite female.330935107NoneSurvivedGray.329775103do.do.Gray male.3201,0401152 hours46 hoursOatsBlack male.5511,45010030 minutesLess than 24 hoursdo.Gray female.
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.2481,1703303 hoursSurvivedOatsGray.2411,3802902 hoursdo.do.White male.2498903253 hoursdo.do.Gray male.SERIES B.239935363SurvivedOatsGray male.254975369About 2 daysdo.Gray female.2671,05034250 minutes1 hourdo.White.2681,100363About 3 hoursdo.Do.4191,6003506 hoursLess thando.Gray male.4201,2503521 hour2 hoursdo.Do.4211,4853516 hoursLess thando.Do.4241,2952932 hours22 hoursdo.White male.4231,205300do.Less thando.Do.4221,440291do.2½ hoursdo.Do.
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 regionwas 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 effectsof 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.