Part 3

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.

[B] Survived first dose.

SERIES E.

It was shown in series A that 0.15 caffein per kilo caused symptoms of intoxication. Before concluding, however, that this is the smallest dose which causes symptoms of poisoning, a number of experiments were performed with smaller doses. It was found that in the great majority of cases 0.1 caffein per kilo may cause diuresis, but no nervous or muscular symptoms. In some rabbits, however, even such a dose proved fatal. Post-mortem examinations in these cases showed the presence of coccidiosis of the liver, and it will be recalled that similar observations were made before. It is quite possible, therefore, that coccidiosis of the liver is an important factor in decreasing the resistance to caffein. Experiment 551 (p. 25) shows that other conditions may likewise increase the toxicity of caffein.

_Rabbit 325. White, female. Weight, 1,065 grams. Diet, oats._

March 17: 11 a. m., 6 cc 2 per cent (112 mg per kilo) caffein injected subcutaneously in the back. About 5 cc of urine squeezed out from bladder before injecting caffein.

March 17: 1 p. m., hind legs crossed and stretched out, front legs also extended; rabbit lying stretched out on her belly.

March 17: 5.40 p. m., rabbit still alive, condition somewhat improved.

March 18: 9 a. m., found dead, stiff and cold. _Autopsy_: Hemorrhagic area at point of inoculation; subcutaneous region of both thighs presented a hemorrhagic infiltration of the tissues; liver contained lesions of coccidiosis; other organs apparently normal.

_Rabbit 330. Belgian hare, female. Weight, 935 grams; poorly nourished._

March 18: 3.35 p. m., 5 cc 2 per cent caffein (107 mg per kilo) injected into subcutaneous tissues in the back; 5.30 p. m., no symptoms.

March 19: 9 a. m., no symptoms.

March 25: Weight, 825 grams.

_Rabbit 329. Belgian hare, male. Weight, 775 grams; poorly nourished. Received March 18._

March 18: 3.30 p. m., 4 cc 2 per cent caffein (103 mg per kilo) injected into subcutaneous tissues in the back; 5.30 p. m., no symptoms.

March 19: 9 a. m., no symptoms.

March 25: Rabbit alive in good condition; weight, 825 grams.

_Rabbit 320. Black, male. Weight, 1,040 grams. Diet, oats._

March 17: 11 a. m., 6 cc 2 per cent caffein (115 mg per kilo) injected subcutaneously in the back; only a few drops of urine obtained from bladder before injecting caffein; 1 p. m., rabbit very restless; ran away when placed on floor; cried when touched with a piece of paper; no tremors observed, but rabbit became exhausted and was unable to walk; legs extended out; after running for about a minute dyspnoea was very marked, but rabbit soon raised himself on his legs; 5.40 p. m., rabbit up on his legs.

March 18: 9 a. m., found dead, but still warm. _Autopsy_: Lungs studded with small grayish white nodules, adhesions to costal pleura; probably lesions of coccidiosis; liver studded with coccidiosis nodules. Hemorrhages at point of inoculation.

_Rabbit, 551. Gray, female. Weight, January 26, 1,650 grams. Diet, oats; fed 20 cc of 25 per cent alcohol daily from January 26-31._

January 31: Weight, 1,450 grams; 10.20 a. m., temperature 101.6°; 10.45, a. m., temperature 101.6°; received 7 cc 2 per cent caffein subcutaneously into back; 11.15 a. m., convulsions of short duration; raised himself on posterior legs, anterior legs wide apart; 4.10 p. m., looked normal, not hypersensitive; 4.30 p. m., condition seemed to be good.

February 1: 9 a. m., found dead, was alive at 5.30 p. m. of previous day. _Autopsy_: Lesions found involved thoracic cavity mainly; lungs were hepatized and a fibro plastic exudate caused them to adhere to costal pleura; liver engorged and appeared fatty; no marked lesions affecting digestive tract, a slight catarrh of stomach being the only noticeable feature; kidneys and spleen normal.

TABLE 1.--_Subcutaneous injections of caffein--rabbits._

SERIES A.

----+--------+-------+-------------+--------------+-------+----------- | |Caffein| Appearance | Duration | | No.| Weight.| per | of symptoms | of life. | Diet. | Remarks. | | kilo | in-- | | | ----+--------+-------+-------------+--------------+-------+----------- |_Grams._| _Mg._ | | | | 332| 1,070 | 158 |1 hour |Survived | Oats |Gray. | | | 45 minutes| | | 331| 1,170 | 153 | do. | do. | do. | Do. 328| 1,200 | 150 | do. | do. | do. | Do. 322| 1,065 | 150 |1 hour | do. | do. |White. 217| 1,355 | 147 | | do. | do. | Do. 219| 1,820 | 153 | | do. | do. |Maltese. 194| 1,490 | 174 | | do. | do. |White. 191| 1,915 | 167 | | do. | do. |Light brown. ----+--------+-------+-------------+--------------+-------+------------

SERIES B, GROUP I.

----+--------+-------+-------------+--------------+-------+------------- 95| 1,478 | 210 |2 hours |3 hours |Oats |White. | | | 50 minutes | 10 minutes | | 96| 1,585 | 200 |1 hour |About 18 hours| do. |Gray white. 112| 875 | 205 |30 minutes | do. | do. |Black. 119| 1,060 | 188 | | do. | do. |Yellow white. 195| 1,300 | 200 | |3 hours |Carrots|White. | | | | 10 minutes | | 208| 1,068 | 188 |2 hours |About 24 hours| do. |Gray. ----+--------+-------+-------------+--------------+-------+-------------

SERIES B, GROUP II.