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The acute oral toxicity of caffeine
TOXICOLOGY
AND APPLIED
PHARMACOLOGY
The Acute
(1959)
Oral Toxicity M.
ELDON Department
250-257
1,
of Pharmacology, Received
Queen’s
of Caffeine
BOYD~
University,
December
Kingston,
Ontario,
Canada
8, 1958
The signs of caffeine poisoning in man are summarized by Gemmill (1958) as insomnia, nervousness, headache, nausea, vomiting, tachycardia, premature systoles, augmented respiration, and muscular tremors, the fatal dose being estimated at over 0.14 g/kg. Estimates of the oral minimal lethal dose, median lethal dose, or simply “lethal dose” in various laboratory animals, listed by Spector (1956), lie in the range of 0.1-0.36 g/kg. Signs of caffeine poisoning recorded in mice by Fiihner (1932) are excitement, epileptiform convulsions, anorexia, stiff muscles, and death due to respiratory failure. In the rat, Maloney (1935) lists apprehensiveness, tremors, a staggering gait, distressed breathing, and a state of depression, and Holck (1949) adds loss of weight, restlessness, excitement, and confusion. In rabbits, cats, and/or dogs, Salant and Rieger (1909) record weakness, loss of weight, diarrhea, and inflammation of the stomach and intestines; Salant ( 1911)) a fall in blood pressure; and Sollmann and Pilcher (191 l), strychnine-like convulsions and cardiac arrest. The study herein reported was concerned with an assessment of the clinical and pathological signs of poisoning in animals receiving caffeine in the range of the oral median lethal dose. Preliminary experiments on the dog revealed that a dose of 0.200 g/kg produced excitement, anorexia, and a delayed vomiting. Vomiting began some 2 hours after drug administration and continued at intervals for 24 hours, the vomitus being coffee-colored. The remaining work was performed upon the albino rat in which vomiting cannot occur. 1 The author wishes to acknowledge the technical assistance of Jack Coates, Jr., Marion A. Price-Jones, Patricia E. Sheppard, and Howard D. Steele, and the receipt of a grant in aid of this project through Woodard Research. 2.50
TOXICITY
OF CAFFEINE
251
METHOD
Female albino rats were assembled in metabolism cages, one rat per cage, food was withheld for 16 hours, caffeine was administered in aqueous solution by stomach tube, and the observations noted by Boyd (1959) were made. Caffeine BP, USP was dissolved in distilled water immediately before administration in a volume of 2.0 ml per kilogram body weight. The doses of caffeine expressed as grams per kilogram body weight. and number of animals per dose (in parentheses) were: 0.000 (20), 0.160 (8), 0.180 (16), 0.200 (8), 0.220 (8). The animals were 3-6 months of age when used and averaged (-+ S.D.) 203 i 28 g in body weight. RESULTS
The median lethal dose (-+ S.E.) was calculated to be 0.192 -t 0.018 g per kilogram body weight. The oral median lethal dose of caffeine in rats has been reported as L’approximately” 0.200 g/kg by Smith and Hambourger ( 1935)) and as 0.233 -t 0.014 g/kg by Scott and Chen (1944). There were nineteen survivors and twenty-one deaths. Death time was rather uniform at an average (2 S.D.) of 30.0 L 9.6 hours after administration of drug. Clinical signs in the group of rats which recovered were as follows. One hour after drug administration the animals showed lack of curiosity to sound or sight, on mechanical stimulation they showed hyperreflexia, but had little inclination to walk or move. In brief, they were weak, tense, and withdrawn. When moving, they were unsteady on the feet and their movements were ataxic. Some quarter of the animals adopted brief cataleptic stances and about the same number gave evidence of being in pain (phonation on mild mechanical stimulation). By 6 hours, half the rats had developed varying degrees of cataplectic resistance to movement and a red crusting about the inner canthus of the eye. By 12 hours, the eyelids were swollen and inflamed. At 24 hours, 80% of the animals were in the same clinical state, with loose stools or slight diarrhea and perineal soiling; two rats were normal in clinical appearance and two had tremors, pallor, and were moderately excited. Half the rats were normal at 48 hours, two were excited, and two had bouts of clonic convulsions lasting a few seconds each. At 72 hours all of this group of rats had a normal clinical appearance. Data upon measured clinical signs are summarized in Table 1 and indicate that the clinical syndrome described above was accompanied at 24 hours by anorexia, loss of body weight, a tendency to the drinking of less water, and some fluctuation in body temperature.
252
ELDON
M. BOYD
In the group of rats which died, the clinical syndrome began in a manner similar to that described above. Clinical deterioration was progressive from the tenth hour until death. The animals ate no food and drank no water. Diarrhea became profuse and loss of body weight was marked. TABLE 1 BODY WEIGHT, FOOD INTAKE, WATER INTAKE, URINE OUTPUT, AND COLONIC TEMPERATURE OF ALBINO RATS FOLLOWING ORAL ADMINISTRATION OF CAFFEINE IN THE RANGE OF THE MEDIAN LETHAL DOSE’” Days after
No. of animals
Group
Body Controls Survivors Nonsurvivors
1
weight:
grams
20 19 21 Food
Controls Survivors Nonsurvivors
intake:
Water Controls Survivors Nonsurvivors
grams
intake:
chow
Urine
output: 20 19 21
Controls Survivors
20 19 21
milliliters
or decrease
per
per
milliliters
per
3 (-)
+14*5 -8212 -22 -c 5 kilogram
body
kilogram
kilogram
body
weight
per
24 hr
weight
82 +- 24 79 f 26 per
24
hr
127 f 32 138 f 60 o-+0 body
39 +- 31 26 +- 23 10 -r- 9 temperature:
+13e6 + l&l4 -
93 k 24 46 C 28 O&O
183 2 51 59 & 67 Of0
Color&
Nonsurvivors
(+)
93 t 23 16 & 20 O&O
20 19 21
Controls Survivors Nonsurvivors
increase
+13*4 - t+9 -16&S
20 19 21
caffeine
2
weight
124 -c 25 183 ?I 74 per
24 hr
38 -t- 18 65 ‘- 28 l&3
37 IL 15 82 k 43 -
99.1 k 0.9 99.9 -+ 1.1 94.9 e 4.0
99.6 +- 1.1 100.0 ?I 15 -
“F
98.9 AI 0.8 98.1 2 2.8 93.7 ?z 3.2
a The results are expressed as mean 31 standard deviation. They
became
anuric.
Body
temperature
fell.
These,
plus
the
clinical
signs described above, persisted until death. Two-thirds of the animals died of respiratory failure following tetanic convulsions. The remaining deaths were due to cardiovascular collapse. The gross appearance of the organs at autopsy suggested the presence of extensive inflammation of the gastrointestinal to the liver, kidneys, spleen, adrenals, thymus
tract and toxic damage gland, and lungs. The
TOXICITY
OF CAFFEINE
253
cardiac portion of the stomach, which is lined by stratified squamous epithelium, was distended with gas, and, in some 20% of cases, there were small areas of inflammation at or near its junction with the glandular portion of the stomach. The pyloric or glandular part of the stomach was pale with numerous (4 to 24) small areas of inflammation and hemorrhage. The duodenum, jejunum, and ileum were obviously inflamed in some 40% of the animals which died. The duodenum and jejunum were moderately distended with a yellow-stained fluid which occasionally contained blood. The amount of succus entericus which could be expressed from the ileum was obviously reduced in 2OF of the animals. There were intussusceptions of the ileum in two rats. The cecum was distended with a yellow fluid. The colon appeared normal in most animals, occasionally slightly inflamed. There were areas of pallor over the liver. The kidneys and spleen were pale. The mesentery and omentum were occasionally yellow-stained. The adrenal glands were enlarged and dark red in color and the thymus was reddish mauve, in contrast to its normal white color. The atria of the heart were engorged with blood in a majority of rats. In all animals the lungs were of a reddish-brown color and often obviously enlarged. The ovaries! skeletal muscle of the ventral abdominal wall, submaxillary salivary glands, brain, skin, and homogenized residual carcass appeared normal. Shifts in the wet weight and water levels of organs and tissues are summarized in Table 2. Values in the control animals were not significantly different from control values reported by Boyd (1959) and have been omitted from Table 2. All of the animals had lost weight at the time of death. Controls sacrificed at the same times had regained body weight lost during the initial period of restriction of food intake. The body weight of rats dying from caffeine poisoning averaged 14.5p’/c less than that of the controls. Part of the loss of body weight was due to reduction in the amount of food and products of digestion of food in the gastrointestinal tract. As shown in Table 2, the relative distribution of weight had shifted in the bodies of these animals. There was a considerable loss of weight in the tissues of the gastrointestinal tract, in the liver. and in the spleen. The adrenal glands and lungs gained weight, absolutely and relatively to autopsy body weight. Changes in the absolute weight of heart, ovaries, and residual carcass (the residuum of the body after removal of the organs listed in Table 2) were so small that these
254
ELDON
M. BOYD
tissues comprised a relatively greater proportion of autopsy body weight than was the case in the control rats. Most of the organs of the body were more dry than normal. Drying was particularly evident in mesentery-omentum, the submaxillary salivary TABLE
2
MEAN SHIFTS~ IN THE WET WEIGHTb AND WATER LEVEL@ OF ORGANS AND TISSUES OF ALBINO RATS AT AUTOPSY FOLLOWING DEATH DUE TO ORAL ADMINISTRATION OF A LETHAL Organ or tissue
Spleen Liver Jejunum Colon Submaxillary glands Pyloric stomach Ileum Cecum Duodenum Thymus gland Kidneys Cardiac stomach Mesentery-omentum Skin Brain Abdominal wall muscle Residual carcass Ovaries Heart Lungs Adrenal glands
DOSE
OF CAFFEINE Water level
Wet weight
-58.0 -43 .o
(
+ -
-27.3 -26.3
(
+ 4.5 -12.6
(0.6)
-25.3
(
-21.5
(0.001)
-24.4
(
+ +
-21.9 -15.8
(
2.2 8.3
(0.1)
(‘3.8)
(0.4)
-
1.7
-
2.6
(0.4)
-
2.5 3s
(0.6)
-
3.1
-27.2 -20.1
-
3.2
-
1.4
+
1.2 0.5
(0.9) (0.9)
+
6.5
(0.1)
+
6.6
-14.6
(0.05) (0.02)
(0.02)
(0.2) (0.2) (0.6) (0.8)
-15.3
3.1 6.4
(0.4) (0.3) (0.01) (
0.0
(0.1)
-
8.8
(1.0) (0.025)
+10.3 +16.7
(
-
9.4
(0.02)
(0.025)
-15.4
+20.1
(0.001)
-
2.9
(0.1)
+47.4
(0.005)
-11.8
(
+67.9
(0.001)
+11.9
(0.005)
(0.005)
0 Expressed as a mean percentage change from the controls, 100 [ (xd - xc) /xc], where Fa! is the mean of the drug-treated is the mean of the controls. In parentheses is the probability (P) equals zero by a t test. b Calculated as a percentage of body weight at autopsy. O Calculated as grams water per 100 g dry weight of tissue.
specifically as animals and Fc that xd - zc
glands, the skin, the ovaries, the colon, the lungs, the liver, skeletal muscle of the ventral abdominal wall, and residual carcass. A significant increase in water level was found in the adrenal glands, possibly in the spleen. Histopathologic observations were as follows. Capillaries and veins of the lamina propria were dilated and engorged with blood in all areas of
TOXICITY
255
OF CAFFEINE
the gastrointestinal tract from the pyloric stomach to the cecum. panying the congestion there were varying degrees of necrosis glandular epithelium and some hemorrhage. The goblet cells were ous and enlarged. The sinusoids of the liver were distended with
I
I
2
3
4
5
6
Accomof the numerpacked
7
DAYS FIG. 1. Percentage changes in body weight, food put, and colonic temperature of a rat which survived in a dose of 0.22Og per kilogram body weight.
intake, oral
water intake, administration
urine outof caffeine
erythrocytes and the hepatic cells were shrunken and pale-stained. The brush border of cells lining the renal proximal convoluted tubules appeared necrotic, red blood cells were packed in capillaries in the region of the loops of Henle, and cells lining the distal convoluted tubules had swollen to seal off the lumen. The periphery of the spleen appeared contracted, with erythrocytes
256
ELDON
M. BOYD
forced out of the sinuses and into the central sinuses where they were tightly packed, compressing the cords of Billroth and reticulum of the red pulp. There was a deficiency of zymogenic granules in the acinar glands of the pancreas and a deficiency of blood in the capillaries of the islets of Langerhans. The thymus gland appeared normal except for occasional areas of necrosis of the reticular cells. Sinusoids of the zona glomerulosa and zona fasciculata of the adrenal cortex were full of packed red blood cells. Capillaries in cardiac muscle of the auricles and ventricles and in the alveolar walls of the lung were dilated and filled with packed blood cells. The remaining organs appeared normal histologically. Rats which survived the initial toxic effects of these doses of caffeine were observed for 6 weeks. There were no delayed deaths in this period. Recovery was accompanied by a marked increase in urine output and water intake, as exemplified in Fig. 1. There was some increase in appetite and gain in body weight. All measurements were returning toward normal by the end of 1 week. SUMMARY The oral median lethal dose of caffeine in albino rats was estimated to be (2S.E.) The clinical signs of poisoning from 0.192 + 0.018 g per kilogram body weight. these doses were schizophreniform withdrawal, hyperreflexia, vertigo, ataxia, evidence of pain, cataleptic stances, cataplexy, diarrhea, anuria, anorexia, adipsia, hypothermia, blepharitis, and loss of body weight. Death occurred in (&SD.) 30 -C 9.6 hours and was immediately due to respiratory failure following tetanic convulsions or to cardiovascular collapse. Autopsy revealed the presence of a fulminating gastroenteritis, congestion of the lungs, hepatitis, nephritis, toxic effects upon the heart, spleen, pancreas, thymus gland, adrenal glands, and dehydration of many organs and tissues. Survival was characterized by a marked polydipsia and polyuria. REFERENCES
E. M. (1959). The acute oral toxicity of acetylsalicylic acid. Toxicol. Phurmucol. 1, 229-239. F~~HNER, H. (1932). Beitrage zur vergleichenden Pharmakologie. 1. Die giftigen und tijdlichen Gaben einiger Substanzen ftir Friische und MLuse. Arch. exptl. Pathol. Phamnukol. Naunyn-Schmiedeberg’s 166, 437-471. In Pharmacology in Medicine (V. A. GEMMILL, C. L. (1958). The xanthines. Drill, ed.), 2nd edition, pp. 304-305. McGraw-Hill, New York. HOLCK, H. G. 0. (1949). Dosage of drugs for rats. In The Rat in Laboratory Investigation (E. J. Farris and J. Q. Griffith, Jr., eds.), 2nd edition, p. 328. Lippincott, Philadelphia. MALONEY, A. H. (1935). Contradictory actions of caffeine, coramine, and metrazol. Quart. J. Exptl. Physiol. 26, 155-166.
BOYD,
Apjl.
TOXICITY
OF CAFFEINE
257
(1911). The effect of caffein on the circulation. /. Pharmacol. Exptl. 3, 468-469. SALANT, W., and RIEGER, J. B. (1909). The toxicity of caffein. J. Phavmacol. Exptl. Tkerap. 1, 572-574. SCOTT, C. C., and CHEN, K. K. (1944). Comparison of the action of l-ethyl theobromine and caffeine in animals and man. J. Pkarmacol. Exptl. Therap. 82, 89-97. SMITH, P. K., and HAMBOURGER, W. E. (1935). Antipyretic and toxic effects of combinations of acetanilid with sodium bromide and with caffeine. /. Pkamacol. Ezptl. Therap. 55, 200-205. SOLLMANN, T., and PILCHER, J. D. (1911). The actions of caffein on the mammalian circulation. I. The persistent effects of caffein on the circulation. J. Pkarmacol. Erptl. Therap. 3, 19-92. SPECTOR, W. S. (1956). Handbook of Toxicology, Vol. I, pp. 56-57. Saunders, Philadelphia.
SALANT,
Therap.
W.
AND APPLIED
PHARMACOLOGY
The Acute
(1959)
Oral Toxicity M.
ELDON Department
250-257
1,
of Pharmacology, Received
Queen’s
of Caffeine
BOYD~
University,
December
Kingston,
Ontario,
Canada
8, 1958
The signs of caffeine poisoning in man are summarized by Gemmill (1958) as insomnia, nervousness, headache, nausea, vomiting, tachycardia, premature systoles, augmented respiration, and muscular tremors, the fatal dose being estimated at over 0.14 g/kg. Estimates of the oral minimal lethal dose, median lethal dose, or simply “lethal dose” in various laboratory animals, listed by Spector (1956), lie in the range of 0.1-0.36 g/kg. Signs of caffeine poisoning recorded in mice by Fiihner (1932) are excitement, epileptiform convulsions, anorexia, stiff muscles, and death due to respiratory failure. In the rat, Maloney (1935) lists apprehensiveness, tremors, a staggering gait, distressed breathing, and a state of depression, and Holck (1949) adds loss of weight, restlessness, excitement, and confusion. In rabbits, cats, and/or dogs, Salant and Rieger (1909) record weakness, loss of weight, diarrhea, and inflammation of the stomach and intestines; Salant ( 1911)) a fall in blood pressure; and Sollmann and Pilcher (191 l), strychnine-like convulsions and cardiac arrest. The study herein reported was concerned with an assessment of the clinical and pathological signs of poisoning in animals receiving caffeine in the range of the oral median lethal dose. Preliminary experiments on the dog revealed that a dose of 0.200 g/kg produced excitement, anorexia, and a delayed vomiting. Vomiting began some 2 hours after drug administration and continued at intervals for 24 hours, the vomitus being coffee-colored. The remaining work was performed upon the albino rat in which vomiting cannot occur. 1 The author wishes to acknowledge the technical assistance of Jack Coates, Jr., Marion A. Price-Jones, Patricia E. Sheppard, and Howard D. Steele, and the receipt of a grant in aid of this project through Woodard Research. 2.50
TOXICITY
OF CAFFEINE
251
METHOD
Female albino rats were assembled in metabolism cages, one rat per cage, food was withheld for 16 hours, caffeine was administered in aqueous solution by stomach tube, and the observations noted by Boyd (1959) were made. Caffeine BP, USP was dissolved in distilled water immediately before administration in a volume of 2.0 ml per kilogram body weight. The doses of caffeine expressed as grams per kilogram body weight. and number of animals per dose (in parentheses) were: 0.000 (20), 0.160 (8), 0.180 (16), 0.200 (8), 0.220 (8). The animals were 3-6 months of age when used and averaged (-+ S.D.) 203 i 28 g in body weight. RESULTS
The median lethal dose (-+ S.E.) was calculated to be 0.192 -t 0.018 g per kilogram body weight. The oral median lethal dose of caffeine in rats has been reported as L’approximately” 0.200 g/kg by Smith and Hambourger ( 1935)) and as 0.233 -t 0.014 g/kg by Scott and Chen (1944). There were nineteen survivors and twenty-one deaths. Death time was rather uniform at an average (2 S.D.) of 30.0 L 9.6 hours after administration of drug. Clinical signs in the group of rats which recovered were as follows. One hour after drug administration the animals showed lack of curiosity to sound or sight, on mechanical stimulation they showed hyperreflexia, but had little inclination to walk or move. In brief, they were weak, tense, and withdrawn. When moving, they were unsteady on the feet and their movements were ataxic. Some quarter of the animals adopted brief cataleptic stances and about the same number gave evidence of being in pain (phonation on mild mechanical stimulation). By 6 hours, half the rats had developed varying degrees of cataplectic resistance to movement and a red crusting about the inner canthus of the eye. By 12 hours, the eyelids were swollen and inflamed. At 24 hours, 80% of the animals were in the same clinical state, with loose stools or slight diarrhea and perineal soiling; two rats were normal in clinical appearance and two had tremors, pallor, and were moderately excited. Half the rats were normal at 48 hours, two were excited, and two had bouts of clonic convulsions lasting a few seconds each. At 72 hours all of this group of rats had a normal clinical appearance. Data upon measured clinical signs are summarized in Table 1 and indicate that the clinical syndrome described above was accompanied at 24 hours by anorexia, loss of body weight, a tendency to the drinking of less water, and some fluctuation in body temperature.
252
ELDON
M. BOYD
In the group of rats which died, the clinical syndrome began in a manner similar to that described above. Clinical deterioration was progressive from the tenth hour until death. The animals ate no food and drank no water. Diarrhea became profuse and loss of body weight was marked. TABLE 1 BODY WEIGHT, FOOD INTAKE, WATER INTAKE, URINE OUTPUT, AND COLONIC TEMPERATURE OF ALBINO RATS FOLLOWING ORAL ADMINISTRATION OF CAFFEINE IN THE RANGE OF THE MEDIAN LETHAL DOSE’” Days after
No. of animals
Group
Body Controls Survivors Nonsurvivors
1
weight:
grams
20 19 21 Food
Controls Survivors Nonsurvivors
intake:
Water Controls Survivors Nonsurvivors
grams
intake:
chow
Urine
output: 20 19 21
Controls Survivors
20 19 21
milliliters
or decrease
per
per
milliliters
per
3 (-)
+14*5 -8212 -22 -c 5 kilogram
body
kilogram
kilogram
body
weight
per
24 hr
weight
82 +- 24 79 f 26 per
24
hr
127 f 32 138 f 60 o-+0 body
39 +- 31 26 +- 23 10 -r- 9 temperature:
+13e6 + l&l4 -
93 k 24 46 C 28 O&O
183 2 51 59 & 67 Of0
Color&
Nonsurvivors
(+)
93 t 23 16 & 20 O&O
20 19 21
Controls Survivors Nonsurvivors
increase
+13*4 - t+9 -16&S
20 19 21
caffeine
2
weight
124 -c 25 183 ?I 74 per
24 hr
38 -t- 18 65 ‘- 28 l&3
37 IL 15 82 k 43 -
99.1 k 0.9 99.9 -+ 1.1 94.9 e 4.0
99.6 +- 1.1 100.0 ?I 15 -
“F
98.9 AI 0.8 98.1 2 2.8 93.7 ?z 3.2
a The results are expressed as mean 31 standard deviation. They
became
anuric.
Body
temperature
fell.
These,
plus
the
clinical
signs described above, persisted until death. Two-thirds of the animals died of respiratory failure following tetanic convulsions. The remaining deaths were due to cardiovascular collapse. The gross appearance of the organs at autopsy suggested the presence of extensive inflammation of the gastrointestinal to the liver, kidneys, spleen, adrenals, thymus
tract and toxic damage gland, and lungs. The
TOXICITY
OF CAFFEINE
253
cardiac portion of the stomach, which is lined by stratified squamous epithelium, was distended with gas, and, in some 20% of cases, there were small areas of inflammation at or near its junction with the glandular portion of the stomach. The pyloric or glandular part of the stomach was pale with numerous (4 to 24) small areas of inflammation and hemorrhage. The duodenum, jejunum, and ileum were obviously inflamed in some 40% of the animals which died. The duodenum and jejunum were moderately distended with a yellow-stained fluid which occasionally contained blood. The amount of succus entericus which could be expressed from the ileum was obviously reduced in 2OF of the animals. There were intussusceptions of the ileum in two rats. The cecum was distended with a yellow fluid. The colon appeared normal in most animals, occasionally slightly inflamed. There were areas of pallor over the liver. The kidneys and spleen were pale. The mesentery and omentum were occasionally yellow-stained. The adrenal glands were enlarged and dark red in color and the thymus was reddish mauve, in contrast to its normal white color. The atria of the heart were engorged with blood in a majority of rats. In all animals the lungs were of a reddish-brown color and often obviously enlarged. The ovaries! skeletal muscle of the ventral abdominal wall, submaxillary salivary glands, brain, skin, and homogenized residual carcass appeared normal. Shifts in the wet weight and water levels of organs and tissues are summarized in Table 2. Values in the control animals were not significantly different from control values reported by Boyd (1959) and have been omitted from Table 2. All of the animals had lost weight at the time of death. Controls sacrificed at the same times had regained body weight lost during the initial period of restriction of food intake. The body weight of rats dying from caffeine poisoning averaged 14.5p’/c less than that of the controls. Part of the loss of body weight was due to reduction in the amount of food and products of digestion of food in the gastrointestinal tract. As shown in Table 2, the relative distribution of weight had shifted in the bodies of these animals. There was a considerable loss of weight in the tissues of the gastrointestinal tract, in the liver. and in the spleen. The adrenal glands and lungs gained weight, absolutely and relatively to autopsy body weight. Changes in the absolute weight of heart, ovaries, and residual carcass (the residuum of the body after removal of the organs listed in Table 2) were so small that these
254
ELDON
M. BOYD
tissues comprised a relatively greater proportion of autopsy body weight than was the case in the control rats. Most of the organs of the body were more dry than normal. Drying was particularly evident in mesentery-omentum, the submaxillary salivary TABLE
2
MEAN SHIFTS~ IN THE WET WEIGHTb AND WATER LEVEL@ OF ORGANS AND TISSUES OF ALBINO RATS AT AUTOPSY FOLLOWING DEATH DUE TO ORAL ADMINISTRATION OF A LETHAL Organ or tissue
Spleen Liver Jejunum Colon Submaxillary glands Pyloric stomach Ileum Cecum Duodenum Thymus gland Kidneys Cardiac stomach Mesentery-omentum Skin Brain Abdominal wall muscle Residual carcass Ovaries Heart Lungs Adrenal glands
DOSE
OF CAFFEINE Water level
Wet weight
-58.0 -43 .o
(
+ -
-27.3 -26.3
(
+ 4.5 -12.6
(0.6)
-25.3
(
-21.5
(0.001)
-24.4
(
+ +
-21.9 -15.8
(
2.2 8.3
(0.1)
(‘3.8)
(0.4)
-
1.7
-
2.6
(0.4)
-
2.5 3s
(0.6)
-
3.1
-27.2 -20.1
-
3.2
-
1.4
+
1.2 0.5
(0.9) (0.9)
+
6.5
(0.1)
+
6.6
-14.6
(0.05) (0.02)
(0.02)
(0.2) (0.2) (0.6) (0.8)
-15.3
3.1 6.4
(0.4) (0.3) (0.01) (
0.0
(0.1)
-
8.8
(1.0) (0.025)
+10.3 +16.7
(
-
9.4
(0.02)
(0.025)
-15.4
+20.1
(0.001)
-
2.9
(0.1)
+47.4
(0.005)
-11.8
(
+67.9
(0.001)
+11.9
(0.005)
(0.005)
0 Expressed as a mean percentage change from the controls, 100 [ (xd - xc) /xc], where Fa! is the mean of the drug-treated is the mean of the controls. In parentheses is the probability (P) equals zero by a t test. b Calculated as a percentage of body weight at autopsy. O Calculated as grams water per 100 g dry weight of tissue.
specifically as animals and Fc that xd - zc
glands, the skin, the ovaries, the colon, the lungs, the liver, skeletal muscle of the ventral abdominal wall, and residual carcass. A significant increase in water level was found in the adrenal glands, possibly in the spleen. Histopathologic observations were as follows. Capillaries and veins of the lamina propria were dilated and engorged with blood in all areas of
TOXICITY
255
OF CAFFEINE
the gastrointestinal tract from the pyloric stomach to the cecum. panying the congestion there were varying degrees of necrosis glandular epithelium and some hemorrhage. The goblet cells were ous and enlarged. The sinusoids of the liver were distended with
I
I
2
3
4
5
6
Accomof the numerpacked
7
DAYS FIG. 1. Percentage changes in body weight, food put, and colonic temperature of a rat which survived in a dose of 0.22Og per kilogram body weight.
intake, oral
water intake, administration
urine outof caffeine
erythrocytes and the hepatic cells were shrunken and pale-stained. The brush border of cells lining the renal proximal convoluted tubules appeared necrotic, red blood cells were packed in capillaries in the region of the loops of Henle, and cells lining the distal convoluted tubules had swollen to seal off the lumen. The periphery of the spleen appeared contracted, with erythrocytes
256
ELDON
M. BOYD
forced out of the sinuses and into the central sinuses where they were tightly packed, compressing the cords of Billroth and reticulum of the red pulp. There was a deficiency of zymogenic granules in the acinar glands of the pancreas and a deficiency of blood in the capillaries of the islets of Langerhans. The thymus gland appeared normal except for occasional areas of necrosis of the reticular cells. Sinusoids of the zona glomerulosa and zona fasciculata of the adrenal cortex were full of packed red blood cells. Capillaries in cardiac muscle of the auricles and ventricles and in the alveolar walls of the lung were dilated and filled with packed blood cells. The remaining organs appeared normal histologically. Rats which survived the initial toxic effects of these doses of caffeine were observed for 6 weeks. There were no delayed deaths in this period. Recovery was accompanied by a marked increase in urine output and water intake, as exemplified in Fig. 1. There was some increase in appetite and gain in body weight. All measurements were returning toward normal by the end of 1 week. SUMMARY The oral median lethal dose of caffeine in albino rats was estimated to be (2S.E.) The clinical signs of poisoning from 0.192 + 0.018 g per kilogram body weight. these doses were schizophreniform withdrawal, hyperreflexia, vertigo, ataxia, evidence of pain, cataleptic stances, cataplexy, diarrhea, anuria, anorexia, adipsia, hypothermia, blepharitis, and loss of body weight. Death occurred in (&SD.) 30 -C 9.6 hours and was immediately due to respiratory failure following tetanic convulsions or to cardiovascular collapse. Autopsy revealed the presence of a fulminating gastroenteritis, congestion of the lungs, hepatitis, nephritis, toxic effects upon the heart, spleen, pancreas, thymus gland, adrenal glands, and dehydration of many organs and tissues. Survival was characterized by a marked polydipsia and polyuria. REFERENCES
E. M. (1959). The acute oral toxicity of acetylsalicylic acid. Toxicol. Phurmucol. 1, 229-239. F~~HNER, H. (1932). Beitrage zur vergleichenden Pharmakologie. 1. Die giftigen und tijdlichen Gaben einiger Substanzen ftir Friische und MLuse. Arch. exptl. Pathol. Phamnukol. Naunyn-Schmiedeberg’s 166, 437-471. In Pharmacology in Medicine (V. A. GEMMILL, C. L. (1958). The xanthines. Drill, ed.), 2nd edition, pp. 304-305. McGraw-Hill, New York. HOLCK, H. G. 0. (1949). Dosage of drugs for rats. In The Rat in Laboratory Investigation (E. J. Farris and J. Q. Griffith, Jr., eds.), 2nd edition, p. 328. Lippincott, Philadelphia. MALONEY, A. H. (1935). Contradictory actions of caffeine, coramine, and metrazol. Quart. J. Exptl. Physiol. 26, 155-166.
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