Part 7

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 Brown(81) with ergot, the effect was quite different from those obtained by Meltzer and Auer(58) 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 Maurel(55) 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's(56) experiments show that various substances behave differently in this regard. Thus the toxicity of strychnin, he states, is three times as great when given subcutaneously as when given by mouth and six times that of the minimum fatal dose by vein. It may be remarked, however, that examination of his data shows that his doses are much too large for the rabbit. In experiments with other drugs little or no difference between the two modes of administration was noticed. Thus, digitalin was but slightly more active when given subcutaneously than by mouth, while the toxicity of emetin hydrochlorid was just the same, whichever one of these methods of introducing the substance was used. Differences in the toxicity of substances have also been observed between subcutaneous and intravenous modes of administration, but here, too, the differences for various substances were unequal.

EXPERIMENTS ON GUINEA PIGS.

The toxicity of caffein was studied in a large number of individuals. The experiments were conducted on full-grown animals and were carried out at different seasons of the year in a variety of ways. Special attention was given to diet as a possible factor influencing resistance to caffein, and the effect of different modes of administration on toxicity. Some animals were therefore fed oats, some carrots, others received both hay and oats. Caffein was introduced subcutaneously, intraperitoneally, and by mouth.

SUBCUTANEOUS INJECTION.

SERIES A.

Preliminary experiments carried out on three guinea pigs, which received 360, 300, and 290 mg of caffein per kilo subcutaneously have shown that such doses were rapidly fatal. Two of the animals were seized with convulsions half an hour after the introduction of caffein and died during the attack. The other had tetanus two minutes after the injection of caffein. Repeated attacks followed, which terminated in the death of the animal two and a half hours later. The fatal and toxic doses must therefore be considerably under 0.3 gram of caffein per kilo when introduced by this path and smaller doses were therefore injected. The results are shown in the experiments of the next series.

SERIES B.

Experiments with 2 decigrams per kilo constituted this series.

_Guinea pig 20. Female. Weight, 497 grams. Diet, oats._

April 2: 5 cc 2 per cent caffein injected subcutaneously at 11.30 a. m.; 1.50 p. m., spasm of short duration. Died at 3 p. m., three and one-half hours after injection.

_Guinea pig 38. Brown male. Weight, 570 grams. Diet, carrots and oats week previous to injection._

February 11: 3.50 p. m., 6 cc 2 per cent caffein injected subcutaneously in back (210 mg per kilo); 4.15, reflexes increased, had convulsion of short duration when disturbed; 4.45 p. m., on handling, repeated convulsion and paralysis; 5 p. m., guinea pig lying on his side, respiration difficult and labored.

February 11: 5.05 p. m., guinea pig found dead, 2 hours and 15 minutes after injection.

_Guinea pig 37. Male. Weight, 820 grams. Diet, carrots and oats during week preceding the injection of caffein._

February 11: 3.35 p. m., 8.5 cc 2 per cent caffein injected subcutaneously in the back; 5 p. m., pig very sensitive, anterior extremities paralyzed when handled, frequent spasms of posterior extremities, no symptoms noticed before 5 p. m., although watched all the time; 5.05 p. m., guinea pig on his legs and looked normal. No attack on handling.

February 12: 9 a. m., found dead; died within 18 hours.

_Guinea pig 13. Female. Weight, 618 grams. Diet, oats._

March 29: 2.45, 6 cc 2 per cent caffein injected subcutaneously (0.194 grams per kilo).

March 30: Died at 4 p. m., 25 hours after injection.

_Guinea pig 36. Male. Weight, 850 grams. Fed oats and carrots for one week previous to injection._

February 11: 3.30 p. m., 8.5 cc 2 per cent caffein injected subcutaneously into back; 5 p. m., somewhat more sensitive than normal, no other symptoms, no effect on handling; 5.05 p. m., no symptoms.

February 12: 9 a. m., found dead, about 18 hours after injection.

The results of these experiments, as observed in five guinea pigs, indicate that two decigrams of caffein per kilo of animal produce symptoms within a half to about two and a quarter hours after injection. Death followed in two guinea pigs 70 minutes to 1 hour after the first manifestations of symptoms. Two others died during the night, while one lived 25 hours after the injection of caffein. Even 2 decigrams caffein per kilo weight might therefore be fatal to the guinea pig. Experiments carried out later have shown, however, that the resistance to caffein is appreciably greater in some guinea pigs. This is indicated by the following experiments, in which doses of 0.2 to 0.24 gram caffein per kilo were administered by the same path.

SERIES C.

_Guinea pig 66. Yellow and dark brown male. Weight, 510 grams. Diet, oats._

October 4: 5 cc 2 per cent caffein (0.2 gram per kilo) injected subcutaneously in the back at 3 p. m.; 5 p. m., no symptoms.

October 5: 9 a. m., alive; condition good.

October 9: Found dead. _Autopsy_: Congestion of liver, kidney, and small intestine.

_Guinea pig 65. White and black male. Weight, 510 grams. Diet, oats._

October 4: 5 cc 2 per cent caffein (0.2 gram per kilo) injected subcutaneously in the back at 3 p. m.; 5 p. m., no symptoms.

October 5: 9 a. m., condition good.

_Guinea pig 60. White and gray female. Weight, 320 grams. Diet, oats._

October 3: 2.25 p. m., 3.5 cc 2 per cent caffein (0.219 gram per kilo) injected subcutaneously in the back; 3.40 p. m., convulsion with recovery; 3.50 p. m., frequent spasms with paralysis, especially of anterior extremities; 5.30 p. m., tetanus when removed from cage and put on floor.

October 4: 8.50 a. m., found dead. _Autopsy_: Congestion of small intestines, lungs, liver.

_Guinea pig 57. White and gray female. Weight, 350 grams. Diet, oats._

October 3: 2.15 p. m., 3.5 cc 2 per cent caffein injected subcutaneously in the back (0.2 gram per kilo); 3.40 p. m., convulsions with recovery; 5.30 p. m., no marked symptoms.

October 4: 8.50 a. m., alive, active.

October 6: Found dead at 9 a. m. _Autopsy_: Congestion of lungs and liver; kidneys petechiated; severe gastro-enteritis.

_Guinea pig 68. Yellow male. Weight, 785 grams. Diet, oats._

October 6: 11.35 a. m., 7.8 cc 2 per cent caffein (0.2 gram per kilo) injected subcutaneously; 12 noon, reflexes increased markedly; 4.20 p. m., reflexes the same as at 12 noon.

October 7: 9 a. m., dead. _Autopsy_: Lungs congested; liver congested and fatty; spleen congested, kidney showed hemorrhagic spots; gastric mucosa necrotic; small portion of small intestine inflamed.

_Guinea pig 69. White male. Weight, 585 grams. Diet, oats._

October 6: 11.40 a. m., 5.8 cc 2 per cent caffein injected subcutaneously; 12 noon, reflexes increased, but not as much as in No. 68; 4.20 p. m., guinea pig hypersensitive, reflexes increased more than at 12 noon.

October 7: 9 a. m., alive.

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

_Guinea pig 61. Brown and black female. Weight, 330 grams. Diet, oats._

October 3: 4 p. m., 4 cc 2 per cent caffein (240 mg per kilo) injected subcutaneously; 5.30 p. m., reflexes increased; runs, but drags posterior extremities.

October 4: 8.50 a. m., found dead.

_Guinea pig 62. White, yellow, and black female. Weight, 335 grams. Diet, oats._

October 3: 4.05 p. m., 4 cc 2 per cent caffein (238 mg per kilo) injected subcutaneously in the back; 5 p. m., convulsions; 5.20 p. m., convulsions, alternating with paralysis of anterior and posterior extremities.

October 4: 8.50 a. m., found dead.

_Guinea pig 70. White and brown male. Weight, 545 grams. Diet, oats._

October 7: 3 p. m., 6.5 cc 2 per cent caffein (238 mg per kilo) aqueous solution injected subcutaneously; 3.50 p. m., reflexes increased.

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

_Guinea pig 71. Brown and white male. Weight, 540 grams. Diet, oats._

October 7: 3 p. m., 6.5 cc 2 per cent caffein solution (0.24 gram per kilo) injected subcutaneously; 3.45 p. m., reflexes increased, tetanus.

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

_Guinea pig 72. Brown and white male. Weight, 560 grams. Diet, oats._

October 7: 3 p. m., 6.5 cc 2 per cent caffein (0.232 gram per kilo) aqueous solution administered by subcutaneous injection; 3.35 p. m., reflexes increased.

October 10: found dead. _Autopsy_: Nos. 70, 71, 72 showed congestion of organs.

The reaction to caffein in the experiments of this series (C) showed considerable variation. The appearance of symptoms, as well as the final outcome of the experiments, differed markedly in a number of cases, notwithstanding the fact that the conditions were the same; thus the administration of 0.2 gram per kilo to guinea pigs, all of which received the same diet, induced no symptoms in two of the animals (Nos. 66 and 65), while marked symptoms were observed in the other four; in two of these the symptoms appeared in one hour and a quarter after injection, and in two others (Nos. 68 and 69), mild symptoms only appeared in 20 or 25 minutes. The last two were under observation for 4 hours longer, but there was no visible change in their condition. The duration of life in all of these guinea pigs, as indicated in the table, likewise varied. Two (Nos. 60 and 68) died during the night after they received caffein, one survived (No. 65), and three others (Nos. 57, 66, and 69) lived 2½, 5, and 9 days, respectively. Experiments with larger doses likewise showed differences in the behavior of these animals toward caffein, but they were not quite so marked. As shown in the table, symptoms appeared in from 35 minutes to 1.5 hours after injection. The duration of life was less than 1 day in two pigs, about twice as long in two others, and in one case between 2 and 3 days.

A comparison made with results obtained in the preceding series shows a striking difference in the resistance to caffein. As 2 decigrams per kilo proved more rapidly fatal to the guinea pig than the larger doses employed in the later experiments, this difference in the resistance to caffein may be due to several factors. As pointed out in the experiments on rabbits, age might be an important factor influencing the toxicity of caffein. Unfortunately, no accurate data were available on the age of the guinea pigs, but they were all apparently full grown, although they differed in weight considerably. The difference in their ages was in all probability not very great. Moreover, it will be observed that the resistance in series B and C differed in animals of approximately the same weight. This is evident on comparing experiments Nos. 20, 38, and 13 of series B with Nos. 65, 66, and 69 of the next series. Again, further inspection and analysis of these tables show no difference in the toxicity, although there may be considerable difference in the weight, from which it may be concluded that the animals were of about the same age or that this plays no part in the resistance to caffein in the guinea pig.

Diet is another factor which should be taken into consideration in this connection. The recent work of Hunt(39) indicates that this may influence the resistance of animals to some poisons. Our experiments, however, fail to show any difference in the toxicity of the caffein in guinea pigs, whether fed oats, carrots, or both, for different results were obtained on the same diet, and there seemed to be little or no difference in the toxicity of caffein when the diet was different. Other explanations suggest themselves to account for the results obtained. Seasonal changes have been assigned by a number of investigators as a cause of variation in the resistance to drugs. According to Focke,(24) frogs are more susceptible to digitalis in the spring than in the summer, while Moschkowitsch(61) and Edmunds(21) reported the very opposite results. Schmiedeberg's(80) observations on strophantin in frogs were in harmony with those of Edmunds(21) and Moschkowitsch.(61) Similar results were reported with guinea pigs. Harrington's(34) experiments indicate that stimulation of the vagus is less effective from October to January than from February to April, when they are also much more susceptible to operative procedure. Hunt found that the resistance of guinea pigs to aceto nitril is about twice as great in the summer months as it is in January and February.

Race might also be thought of as an important factor in this connection. Since the guinea pigs used at different seasons of the year were of several varieties, there is no reason to suppose, however, that the varieties experimented upon in the summer were more resistant than those used in the winter and spring. It is highly probable, therefore, that the greater resistance to caffein of the guinea pigs of series C than those of series B was due to seasonal variation.

Doses of 0.20 to 0.24 gram caffein per kilo weight, therefore, may be regarded as the minimum fatal dose for the guinea pig, depending upon the season. Since 0.2 gram per kilo proved to be rapidly fatal in series B, this quantity was perhaps not the minimum fatal dose for the guinea pig at the season during which the experiments were made. Additional tests with smaller doses were therefore carried out during February and March. The results are shown in series D.

SERIES D.

_Guinea pig 49. Male. Weight, 510 grams. Diet, oats for 1 month previous to experiment._

March 17: 3 p. m., 4 cc 2 per cent caffein (0.16 gram per kilo) were injected subcutaneously; 4.40 p. m., reflexes increased; 5.40 p. m., no symptoms.

March 18: 9 a. m., found dead, died in less than 18 hours. _Autopsy_: Hemorrhage into abdominal cavity; liver and spleen unduly congested; intestines injected; hemorrhagic area at point of injection.

_Guinea pig 40. Male. Weight, 630 grams. Diet, oats and carrots one week previous to injection._

February 12: 11 a. m., 5 cc 2 per cent caffein (0.158 gram per kilo) injected subcutaneously into back.

February 13: 1 p. m., still alive.

February 14: 9 a. m., found dead.

_Guinea pig 45. Female. Weight, 435 grams. Diet, oats for about one month previous to injection._

March 17: 3 p. m., 3.5 cc of 2 per cent caffein injected subcutaneously in the back (0.160 gram per kilo); 4.35 p. m., no symptoms; 5.40 p. m., no symptoms.

_Guinea pig 39. Male. Weight, 820 grams. Diet, oats and carrots._

February 12: 11 a. m., 6 cc (0.15 gram per kilo) 2 per cent caffein injected subcutaneously in back.

February 14: 9 a. m., alive; seemed to be in good condition; found dead at 1 p. m.

_Guinea pig 41. Weight, 660 grams. Diet, oats and carrots one week previous to injection._

February 12: 11 a. m., 5 cc (0.15 gram per kilo) 2 per cent caffein injected subcutaneously.

February 14: 2 p. m., pig alive; apparently normal.

February 18: Guinea pig still alive and apparently in good condition.

_Guinea pig 46. Female. Weight, 470 grams. Diet, oats about one month previous to experiment._

March 17: 3.15 p. m., 4 cc (0.170 gram per kilo) 2 per cent caffein injected into back subcutaneously; 4.35 p. m., reflexes increased, tremors on handling marked; 5.40 p. m., no change, symptoms about as before.

March 18: 2.30 p. m., no symptoms.

The experiments of this series (D) likewise showed a considerable difference in the resistance of the individual guinea pigs. Nos. 41, 45, and 46 survived; the rest of the pigs died within 18 hours to 2 days after the administration of caffein. Since an autopsy was held on one only, it is impossible to assign a cause for the variation in the toxicity of caffein in these guinea gigs, as the diet and the other conditions under which the experiments were conducted were the same. It was found in the experiments on cats and rabbits that the presence of morbid processes tends to increase the toxicity of caffein. The observations of Ophüls(66) are of interest in this connection. He found spontaneous lesions of the kidney and liver in a large proportion of guinea pigs examined. The greater susceptibility to caffein of guinea pigs Nos. 39, 40, 49, is probably due therefore to some pathological change which increased its toxicity. About 0.2 to 0.24 gram per kilo may therefore be regarded as the minimum lethal dose for the normal guinea pig when caffein is introduced subcutaneously, the minimum toxic dose being about 150-160 mg per kilo.

Experiments were also conducted to determine the largest dose which does not produce any visible effects. In a number of tests with from 100 to 120 mg caffein per kilo (series E, see Table 6, p. 51) no manifestation of nervous or muscular disturbance nor any departure from the normal in respiratory activity was observed. Such quantities may be regarded as the largest doses which are surely safe for these animals. It is quite possible, therefore, that the greater variation in the toxicity of caffein observed in these experiments is due to morbid conditions. Moreover, there is some evidence that caffein increases the toxicity of certain poisons, as shown by Hale(33) for acetanilid. Is it not possible that caffein may similarly be affected by poisons circulating within the body? Indeed the recent work of Loeb(23) makes this supposition highly probable. This investigator found that caffein and adrenalin injected together produce myocarditis in the rabbit. It is conceivable that the combined action of caffein and some preexisting poison may cause changes which terminate in the death of the animal. The delayed death of guinea pigs after the administration of caffein observed in this and other series may probably be accounted for in this way.

Experiment 57 lends some support to this view. The condition of the kidneys and the presence of a severe gastro-enteritis are sufficient to account for the death of this case. Again the frequent association of gastro-enteritis and congestion of the organs in caffein intoxication found in different animals makes it highly probable that these lesions were caused by caffein.

INJECTION INTO THE PERITONEAL CAVITY.

The experiments were carried out with different doses. All the guinea pigs in this series were kept on a uniform diet, consisting of oats. Most of them were of average size and there were no wide variations in their weights. The experiments of series A with the smallest doses were conducted in March and April; all the other experiments it will be noticed were made in October.

SERIES A.

_Guinea pig 41. Weight, 700 grams. Diet, oats._

April 1: 3.30 p. m., 4.5 cc 2 per cent caffein (130 mg per kilo) injected into peritoneal cavity. 5.35 p. m., symptoms present but no tetanus.

April 2: Found dead about 2 p. m., duration of life about 22 hours. _Autopsy_: Subcutaneous hemorrhage at the point of inoculation; serious exudate on visceral and parietal peritoneum with marked inflammation of peritoneum; portions of intestines showed slight enteritis.

_Guinea pig 49. Male. Weight, 370 grams. Diet, oats._

April 1: 3.15 p. m., 2.5 cc 2 per cent caffein (135 mg per kilo) injected into the peritoneal cavity; 5.30 p. m., symptoms present; reflexes increased, but no tetanus. Guinea pig survived.

_Guinea pig 47. Female. Weight, 550 grams. Diet, oats since about February 4._

March 17: 3.30 p. m., 3.5 cc 2 per cent caffein (127 mg per kilo) injected into peritoneal cavity; 4.35 p. m., increased irritability present, but not marked; 5.40 p. m., symptoms about the same as before.

March 18: 2.30 p. m., condition good; no symptoms. Survived.

_Guinea pig 50. Female. Weight, 290 grams. Diet, oats._

April 1: 3.30 p. m., 2 cc 2 per cent caffein (138 mg per kilo) injected into peritoneal cavity; 5.35 p. m., symptoms present; reflexes much increased, but no tetanus. Survived.

SERIES B.

_Guinea pig 51. Yellow female. Weight, 415 grams._

October 1: 9.50 a. m., 3 cc (144 mg per kilo) 2 per cent caffein injected into peritoneal cavity; 4.30 p. m., no symptoms, although under observation all day.

October 3: 2 p. m., alive.

_Guinea pig 52. White male. Weight, 450 grams._

October 1: 9.45 a. m., 3.5 cc, 2 per cent caffein (155 mg per kilo), injected into peritoneal cavity; 4.30 p. m., no symptoms developed since injection.

October 3: 2 p. m., alive.

_Guinea pig 58. Brown and white male. Weight, 490 grams._