Part 11

The question whether caffein is cumulative in the rabbit, suggested in the preceding experiments, was the subject of further investigation in Series B. Caffein was given by mouth or subcutaneously. Carrots formed the exclusive diet, a measured amount being given. The rabbits were kept under observation for two weeks, except Nos. 370 and 373, records of which were made only for four days before the administration of caffein was begun. Caffein was given by mouth in experiments of Groups I and III. Rabbits 292, 293, and 295 received daily 20 cc water by mouth for four days previous to the administration of caffein, while in the rabbits of Group II the caffein treatment was preceded by the injection of 0.8 per cent salt solution subcutaneously. The object in both cases was to ascertain whether or not the method of the administration of caffein has any influence on the animal, but observation made from day to day failed to show any effect of such treatment. About 1 decigram of caffein per kilo was administered daily, with occasional intermissions. Later in the course of the experiment the doses were increased, 0.15 gram per kilo being the maximum dose given. Rabbit 293 died after the third dose with symptoms of typical caffein poisoning. The administration of the same dose of caffein was continued 10 days longer in Nos. 292 and 295. It was omitted on the seventh, fourteenth, and fifteenth days of the experiment. On the eighteenth day of the experiment the dose was increased to 150 mg per kilo and was repeated 2 days later. No. 295 was found dead the next day. No. 292 survived. Rabbits 313 and 315 may be considered together, as they were treated alike in every respect. The initial dose of 100 mg caffein per kilo was finally increased to 122 mg. After the twelfth dose the emaciation was well marked and the rabbits were very weak. No. 313 was found dead 2 days, and No. 315 3 days, after the last dose of caffein was given. It should be remarked in this connection that symptoms of caffein poisoning were never observed in these rabbits. Death was not due, therefore, primarily to caffein, but the rapid loss of flesh and strength observed during the last few days suggests that it was due to malnutrition apparently brought about by caffein.

The results obtained by subcutaneous injection of caffein are given in the table as Group II. The initial dose of 100 mg per kilo was injected daily. No. 298 died after the second dose. Nos. 223 and 296 received this amount daily for 6 days. An intermission of 2 days followed, at the end of which the same dose was given again. The next day it was increased to 150 mg per kilo, but no effect was observed; 48 hours later this dose was repeated. No. 223 was found dead, but its mate survived. Symptoms of acute caffein intoxication were not observed in any of these rabbits. It would seem, therefore, that caffein is not cumulative. This supposition, however, appears somewhat contradictory in view of the fact that out of the eight rabbits of this series six died, nor could any cause of death be ascribed other than caffein. Also the first results of Experiments 293, 370, and 373 might be considered as indicating that cumulation, though to a moderate extent, does take place, since in these cases reflexes developed after the drug was given for some time. But this view is contradicted by the results of Experiment 371, in which 150 mg per kilo given 5 days after the daily dosage of caffein was suspended likewise caused increased reflexes. Cumulation, therefore, does not account for the effects noted in the other rabbit. It will be observed that rabbit No. 370, as well as Nos. 371 and 373, had diarrhea for several days. It is quite possible that the weakened condition rendered the rabbits more sensitive to the action of the drug. This is made highly probable by the observations recorded in the experiments on acute intoxication with caffein in which death occurred after small doses. In such cases some pathological condition was often disclosed by the autopsy. The results of this series corroborate, therefore, those of Series A, and indicate again the absence of cumulative action. The results obtained are in all probability due to malnutrition and other conditions brought about by congestion of the viscera and consequent injury to the gastro-intestinal canal.

TABLE 16.--_Chronic caffein intoxication of rabbits; Series B on cumulation._

+--------------------+--------------------+------------- | Group I. | Group II. | Group III. Data. +------+------+------+------+------+------+------+------ | No. | No. | No. | No. | No. | No. | No. | No. | 292. | 293. | 295. | 296. | 223. | 298. | 315. | 313. --------------+------+------+------+------+------+------+------+------ Diet (grams | | | | | | | carrots in | | | | | | | 2 days) 1,000| 1,000| 975| 930| 905| 880| 355| 300 Caffein | | | | | | | administered | | | | | | | (cc) and weight | | | | | | | (grams): | | | | | | | | | | | | | | Mar. 5 { | | | | | | | {1,410| 1,470| 1,045| 1,040| 1,070| 955| 770| 770 | | | | | | | Mar. 7 { | | | | | | | {1,415| 1,360| 1,140| 1,090| 1,095| 1,000| 715| 690 | | | | | | | Mar. 9 { | | | | | | | {1,350| 1,270| 1,070| 1,000| 1,055| 1,005| 655| 665 | | | | | | | Mar. 11 { | | | | | | | {1,505| 1,465| 1,190| 1,230| 1,285| 1,250| 755| 760 | | | | | | | Mar. 16 { | | | | | | | {1,580| 1,460| 1,230| 1,165| 1,170| 1,145| 730| 745 | | | | | | | Mar. 17 { | | | | | | | {1,515| 1,415| 1,080| 1,040| 1,115| 1,105| 720| 685 | | | | | | | Mar. 19 { | | | | | | | {1,565| 1,570| 1,280| 1,195| 1,235| 1,220| 710| 735 | | | | | | | Mar. 21 { 7| 7| 6| | | | 4| 4 {1,585| 1,530| 1,265| 1,150| 1,215| 1,260| 755| 700 | | | | | | | Mar. 22 { 7| 7| 6| (1) | (1) | (1) | 4| 4 {1,440| 1,315| 1,175| 1,100| 1,045| 1,150| 675| 635 | | | | | | | Mar. 23 { 7| 7| 6| (1) | (1) | (1) | 4| 4 {1,335| 1,140| 1,110| 1,145| 1,190| 1,230| 715| 700 | | | | | | | Mar. 24 { 7| | 6| (1) | (1) | (1) | 4| 4 {1,310| (2) | 1,090| 1,115| 1,170| 1,250| 680| 650 | | | | | | | Mar. 25 { 7| | 6| (1) | (1) | (1) | 4| 4 {1,375| | 1,035| 1,125| 1,215| 1,215| 695| 685 | | | | | | | Mar. 26 { 7| | 6| | | | 4| {1,255| | 1,095| 1,105| 1,155| 1,150| 675| 695 | | | | | | | Mar. 27 { | | | | | | | { | | | | | | | | | | | | | | Mar. 28 { 7| | 6| 5.5| 6| 6| 4| 4 {1,355| | 1,115| 1,120| 1,160| 1,155| 595| 685 | | | | | | | Mar. 29 { 7| | 6| 6| 6| 5| 4| 4 {1,385| | 1,150| 1,155| 1,165| 955| 695| 675 | | | | | | | Mar. 30 { 7| | 6| 6| 6| | 4| 4 {1,330| | 1,075| 1,035| 1,095| Dead.| 630| 610 | | | | | | | Mar. 31 { 7| | 6| 6| 6| | 4| 4 {1,325| | 1,170| 1,110| 1,140| | 690| 605 | | | | | | | Apr. 1 { 7| | 6| 6| 6| | 4| 4 {1,335| | 1,050| 1,050| 1,120| | 625| 620 | | | | | | | Apr. 2 { 7| | 6| 6| 6| | 4| 4 {1,390| | 1,125| 1,090| 1,155| | 695| 625 | | | | | | | Apr. 3 { | | | | | | | { | | | | | | 200| | | | | | | | Apr. 4 { | | | | | | | {1,300| | 1,005| 1,105| 1,080| | 585| 580 | | | | | | | Apr. 5 { 7| | 6| 6| 6| | 4| 4 {1,385| | 1,090| 1,130| 1,090| | 655| 630 | | | | | | | Apr. 6 { 9| | 7.5| 7.5| 8| | | {1,260| | 1,010| 1,050| 1,110| | 560| 530 | | | | | | | Apr. 7 { | | | | | | | { | | | | | | | (3) | | | | | | | Apr. 8 { 9| | 7.5| 7.5| 8| | | {1,260| | 1,000| 1.090| 1.965| | Dead.| | | | | | | | Apr. 9 Survived.| | Dead.|Surv. | Dead.| | | Dead. ---------------------+------+------+------+------+------+------+------

(1) On these days 5 _cc_ of salt solution was administered subcutaneously.

(2) Dead Mar. 23.

(3) Found dead 9 a. m.

TABLE 17.--_Chronic intoxication of rabbits, series B, Group IV, on cumulation._

RABBIT, 370.

+---------+----------+--------+--------+----------+---------- | | | | | Caffein | Date. | Weight. | Carrots. | Water. | Urine. | by | Symptoms. | | | | | stomach. | ---------+---------+----------+--------+--------+----------+---------- | | | | |_Mg per_ | | _Grams._| _Grams._ | _cc._ | | _kilo._ | Aug. 7 | 2,155 | 450 | 50 | 280 | | Aug. 8 | 2,030 | 450 | 25 | 185 | | Aug. 9 | 2,105 | 290 | 0 | 275 | | Aug. 10 | 2,095 | 450 | 30 | 335 | | Aug. 11 | 2,105 | 450 | 65 | 360 | 50 | Aug. 12 | 2,125 | 450 | 65 | 220 | 50 | Aug. 13 | 2,120 | 350 | 25 | 265 | 50 | Aug. 14 | 2,170 | 450 | 35 | 275 | 75 | Aug. 15 | 2,175 | 350 | (?) | 200 | 75 | Aug. 16 | 2,170 | 360 | 65 | 250 | 75 | Aug. 17 | 2,175 | 310 | 35 | 170 | 100 | Aug. 18 | 2,095 | 180 | 40 | 285 | 100 | Severe | | | | | | diarrhea. Aug. 19 | 2,120 | 400 | (?) | 285 | 125 | Do. Aug. 20 | 2,120 | 400 | (?) | 310 | 125 | Better. Aug. 21 | 2,120 | 400 | 70 | 250 | 125 | Do. Aug. 22 | 2,040 | 400 | 45 | 265 | 150 | Diarrhea | | | | | | bad. Aug. 23 | 2,030 | 370 | 35 | 220 | 150 | Diarrhea | | | | | | better. Aug. 24 | 1,950 | 215 | 40 | 120 | 150 | Do. Aug. 25 | 1,885 | 195 | 35 | 60 | 200 | Reflexes. Aug. 26 | | | | | | Found | | | | | |dead at 9. ---------+---------+----------+--------+--------+----------+----------

RABBIT, 373.

+---------+----------+--------+--------+----------+---------- Aug. 7 | 2,240 | 450 | 50 | 230 | | Aug. 8 | 2,150 | 150 | 30 | 300 | | Aug. 9 | 2,120 | 205 | 0 | 150 | | Aug. 10 | 2,150 | 450 | 15 | 245 | | Aug. 11 | 2,195 | 450 | 5 | 285 | 50 | Aug. 12 | 2,160 | 450 | 65 | 325 | 50 | Aug. 13 | 2,120 | 300 | 45 | 190 | 50 | Aug. 14 | 2,195 | 450 | 40 | 265 | 75 | Aug. 15 | 2,215 | 350 | 35 | 200 | 75 | Aug. 16 | 2,205 | 310 | 45 | 225 | 75 | Aug. 17 | 2,240 | 400 | 40 | 265 | 100 | Aug. 18 | 2,255 | 350 | 30 | 320 | 100 | Aug. 19 | 2,115 | 185 | (?) | 170 | 125 | Severe | | | | | | diarrhea. Aug. 20 | 2,115 | 280 | 35 | 195 | 125 | Diarrhea. | | | | | | better Aug. 21 | 2,050 | 175 | 75 | 115 | 125 | Slight | | | | | | diarrhea. Aug. 22 | 2,060 | 180 | 75 | 130 | 150 | Aug. 23 | 2,005 | 200 | 75 | 125 | 150 | Reflexes. Aug. 24 | 1,990 | 200 | 75 | 150 | 150 | Slight | | | | | | diarrhea. Aug. 25 | 1,950 | 255 | 55 | 132 | 175 | Severe | | | | | | diarrhea. Aug. 26 | 1,870 | 205 | 80 | 140 | None | Do. Aug. 27 | 1,830 | 200 | 50 | 140 | do. | Do. Aug. 28 | 1,950 | 400 | 25 | 265 | do. | Slight | | | | | | diarrhea. Aug. 29 | 1,825 | 400 | 0 | 315 | do. | Very weak | | | | | | and in | | | | | | poor | | | | | | condition Aug. 30 | 1,850 | | 10 | 140 | do. | Aug. 31 | 1,835 | | | | | ---------+---------+----------+--------+--------+----------+----------

RABBIT, 371.

+---------+----------+--------+--------+----------+---------- Aug. 7 | 2,240 | 450 | 50 | 300 | | Aug. 8 | 2,260 | 450 | 50 | 225 | | Aug. 9 | 2,310 | 430 | (?) | 300 | | Aug. 10 | 2,295 | 450 | 50 | 305 | | Aug. 11 | 2,320 | 450 | 50 | 335 | 50 | Aug. 12 | 2,280 | 450 | 70 | 400 | 50 | Aug. 13 | 2,300 | 350 | 70 | 255 | 50 | Aug. 14 | 2,265 | 425 | 55 | 154 | 75 | Aug. 15 | 2,260 | 250 | 40 | 125 | 75 | Aug. 16 | 2,295 | 155 | 70 | Lost | 75 | Aug. 17 | 2,180 | 105 | 70 | 120 | 100 |Severe | | | | | | diarrhea. Aug. 18 | 2,150 | 125 | 70 | 100 | 100 |Diarrhea | | | | | | better. Aug. 19 | 2,075 | 210 | (?) | 192 | 100 |Diarrhea | | | | | | severe. Aug. 20 | 2,075 | 280 | 70 | 180 | 100 |Do. Aug. 21 | 2,165 | 260 | 50 | 225 | None |Diarrhea | | | | | | better. Aug. 22 | 2,105 | 400 | 50 | 275 | do. | Aug. 23 | 2,080 | 300 | 0 | 145 | do. |Diarrhea | | | | | | severe. Aug. 24 | 2,105 | 250 | 15 | 245 | do. |Do. Aug. 25 | 2,055 | 320 | 10 | 176 | 150 |Reflexes. Aug. 26 | 2,040 | 190 | 75 | 250 | 150 |Died at | | | | | | 1 p. m., | | | | | | without having | | | | | | showed any | | | | | | symptoms other | | | | | | than reflexes. ---------+---------+----------+--------+--------+----------+----------

SERIES C.

The subjects used in these experiments were rabbits of medium size and were apparently young or at any rate were not very old. The series was planned for the study of the possible effect of diet on the toxicity of caffein when given for some time, and therefore oats were substituted for carrots, which had been fed in the previous work, as already stated. Caffein was given by mouth in the usual way, in 1 per cent solution, 100 mg. per kilo daily. Fourteen rabbits were used for these tests. Their weights were recorded daily and observations made at frequent intervals during the day.

The only change noticed in all of the experiments of this series was progressive loss of weight which set in from 3 to 8 days after the administration of the drug was begun. The duration of life varied considerably. No. 382 died after the first dose. No. 389 lived 2 days, No. 386, 3 days, and No. 385, 5 days; No. 390 lived 7 days and No. 404 lived 20 days after the administration of caffein was begun. The duration of life in all the others was from 11 to 16 days. The findings at autopsy are interesting and suggestive as regards the possible explanation of the effects of repeated dosage of caffein. In eight of the rabbits there was involvement of the mucous membrane of the stomach or intestines or of both. Since the same condition of the gastro-intestinal canal was observed in previous experiments with caffein when injected subcutaneously, the mere passing of the tube into the stomach is obviously not the cause of this condition. The fatal outcome due is therefore, as was suggested above, to inanition brought about by the condition of the gastro-intestinal canal. Moreover parallel experiments carried out on rabbits in the same way with alcohol survived this treatment much longer. Obviously then the passing of the soft rubber catheter is not the cause of this condition of the gastro-intestinal canal nor the diet. Rabbits were fed oats exclusively for several months in this laboratory and thrived. The presence of pneumonia in the other rabbits of this series may be regarded as accidental, as it is inconceivable that one or two doses of caffein, as was the case in Nos. 382 and 389, could predispose the lungs to infection. The results of these experiments therefore are in harmony with those of the preceding two series, indicating that caffein does not accumulate in the body, and that the toxicity of caffein, whether of the single dose or of repeated doses is the same, on a diet of carrots or of oats. These results also show that caffein is much more toxic with repeated dosage. As stated in the historical part of this bulletin the same view was held by Gourewitch.(28)

_Rabbit 386._ _Belgian female._

Given 1 cc of 1 per cent caffein for each 100 grams, through stomach tube.

+--------+---------- Date. | Weight.|Treatment. --------+--------+---------- |_Grams._| _cc._ Aug. 17| 1,300 | 13.0 Aug. 18| 1,215 | 12.0 Aug. 19| (?) | (?) --------+--------+----------

August 20: Found dead 9 a. m. _Autopsy_: Lungs slightly congested; liver engorged and friable; gall cyst well filled.

_Rabbit 389._ _Black male._

Given 1 cc of 1 per cent caffein for each 100 grams, through stomach tube.

+--------+---------- Date. | Weight.|Treatment. --------+--------+---------- |_Grams._| _cc._ Aug. 17| 1,070 | 10.0 Aug. 18| 1,025 | 10.0 --------+--------+----------

August 19: Found dead 9 a. m. _Autopsy_: Lungs severely congested and partially hepatized; liver was engorged; other organs appeared normal.

_Rabbit 382._ _Belgian female._

On August 17 weighed 1,035 grams; received 1 cc of 1 per cent caffein for each 100 grams; 10 cc of 1 per cent caffein given in all.

August 18: Found dead 9 a. m. _Autopsy_: Lungs congested and hepatized; liver engorged; stomach showed numerous petechial hemorrhages on mucosa; kidneys slightly congested; intestines appeared normal.

_Rabbit 385._ _Belgian female._

Given 1 cc of 1 per cent caffein for each 100 grams, through stomach tube.

+--------+---------- Date. | Weight.|Treatment. --------+--------+---------- |_Grams._| _cc._ Aug. 17| 780 | 8.0 Aug. 18| 760 | 7.5 Aug. 19| 755 | 7.5 Aug. 20| 715 | 7.0 Aug. 21| 700 | 7.0 --------+--------+----------

August 22: Found dead 9 a. m. _Autopsy_: Lungs exhibited pneumonic lesions, with inflammation of adjacent pleura; a fibro-plastic exudate present around lung; liver showed a coccidial infestation; stomach distended with ingesta; mucous membrane characterized by a catarrhal inflammation; contents of small intestine liquid in nature and bile stained; large intestine somewhat impacted; liver and kidneys seemingly normal.

_Rabbit 404._ _White male._

Given 1 cc 1 per cent caffein for each 100 grams.

+---------+------------ Date. | Weight. | Treatment. ------------+---------+------------ |_Grams._ | _cc._ Aug. 20 | 1,465 | 14.5 Aug. 21 | 1,475 | 14.5 Aug. 22(1) | | Aug. 23 | 1,475 | 14.5 Aug. 24 | 1,400 | 14.0 Aug. 25(2) | 1,405 | 14.0 Aug. 26 | 1,415 | 14.0 Aug. 27 | 1,400 | 14.0 Aug. 28(1) | | Aug. 29 | 1,310 | 13.0 Aug. 30 | 1,320 | 13.0 Aug. 31 | 1,330 | 13.5 Sept. 1 | 1,335 | 13.5 Sept. 2 | 1,315 | 13.0 Sept. 3 | 1,350 | 13.5 Sept. 4 | 1,335 | 13.5 Sept. 5 | 1,350 | 13.5 Sept. 6 | 1,380 | 14.0 Sept. 7 | 1,375 | 14.0 Sept. 8 | 1,325 | 13.0 ------------+---------+------------

(1) Not fed.

(2) Reflexes.