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The acute oral toxicity in guinea pigs of acetylsalicylic acid, phenacetin, and caffeine, alone and combined
TOXICOLOGY
AND
APPLIED
2,
PHARMACOLOGY
23-32
(1960)
The Acute Oral Toxicity in Guinea Pigs of Acetylsalicylic Acid, Phenacetin, and Caffeine, Alone and Combined ELDON Department
of Pharmacology,
M.
Queen’s Recrived
BOYD
University,
Afrgusf
Kingston,
Ontario,
Canada
19, 1959
Boyd (1959a-d) has shown that the acute oral toxicity in albino rats of a mixture of acetylsalicylic acid, phenacetin, and caffeine is, in general, a summation of the corresponding toxicity of the individual ingredients of the mixture. According to Smith (1958)) there is no corresponding evidence available on any other species except the guinea pig. Loewe et al. (1927) analyzed, by means of isoboles, data upon the threshold (“Schwelle sichtbarer Allgemeinwirkungen”) and lethal (“tijdliche Grenzdosis”) doses of acetylsalicylic acid, phenacetin, and codeine, given orally to guinea pigs, and offered some evidence of antagonism of toxic effects. The presently described work was undertaken to determine if similar antagonism could be found in a mixture of acetylsalicylic acid, phenacetin, and caffeine given orally to guinea pigs. METHODS
The experiments were performed upon young, male, CBL guinea pigs, weighing 30@-500 g. The numbers of animals used are indicated in Table 1. While under close observation, they were placed in metabolism cages, food was withheld for 16 hours prior to oral administration of the drug, and subsequent measurements were made as described by Boyd (1959a). The results are summarized in Tables 1-3. RESULTS
The clinical signs of acute toxicity to acetylsalicylic acid in the guinea pig were the same as reported by Boyd (1959a) in albino rats except that rhinorrhagia did not occur and there was little diarrhea. Death was due to respiratory failure preceded by generalized convulsions or prostration in most animals, occasionally to cardiovascular shock. The LDao t SE 23
or
CHANCES
a The results are expressed b Mean of APC estimate
Water (controls) Acetylsalicylic acid Phenacetin Caffeine APC found APC estimate
Water (controls) Acetylsalicylic acid Phenacetin Caffeine APC found APC estimate
Ihug
A COMPARISON
38 36 37 37 33 -
Food
38 36 37 37 33 -
-I__-~-
N
BODY
intake:
Body
76 2 57 ” 40 -c 26 2 41 r 482
grams
grams
29 11 20 12 14 15
Purina
-c 10 k 17 i 13
!I 13 2 14
weight:
1
AND
2
33 25 21 28
drug
30 23
17 21 25
wt. per 24 hr
___-
4
IN
18 20 23 8 49 21” estimate.
82 -t 73 j, 68 21 65_t 91 ” 71 ”
+37 c 15 + 8221 -3338 -12 c 39 + 6219 + 2-t30
TEMPERATURE
at P < 0.05 and at N = 33 for APC
87-t 7s k 65 & 48-t31 72 lr 69 f
body
$33 2 15 + l-t21 -11 Ik 37 -20 & 34 -6626 -j&29
cr decrease
3
(-)
COLONIC APCa
Days after
AND AND
per kilogram
2 -c -c -c
80-c 17 73 21 23 62 2 33 36 k-31 65 &2S 66 ‘-+ 28
pellets
(+)
-c 12
increase +24 -3322 -18 -24 -11 -11
1 INTAKE,
CAFFEINE,
TABLE WATER
PHENACETIN,
FOOD
ASA,
+15 -14 -8k12 -25 -15 -12
WEIGHT,
as mean & standard deviation. differs from mean of APC actual
IN
~_
GUINEA
GIVEN
82 t 822 68 t 73 3~ 75 -t 75 2
16 8 21 26 16 14
+44 & 16 $11 +- 19 + 4,31 - 4246 +10*21 + 7-26
5
PIGS
$ 3
F
M s g
estimate
found
Phenacetin Caffeine APC found APC estimate
Water (controls) Acetylsalicylic acid
APC
APC
Caffeine
___-
38 36
Water (controls) acid Acetylsalicylic Phenacetin 47 19
1.6 2.1
51 2.6 * 4.3
106.6 102.8
102.3 101.2
103.2 103.2
t 4.1 ?I 2.0
k 2.9 ‘-c 2.2
‘-c 1.5 c+ 1.1
138 -c 52”
temperature:
Colonic
79 IL 69 117 rt 27
124 -c 54
130 & 39 156 -‘49
per kilogram
2
1 (Continued)
35
21 41
-f- 6.8 -r- 2.8
2 2
-..___~
102.2 101.5
103.3 102.9
68 k
45 2 79 2
_______
milliliters
62 -C 53
33
-
1
123 77 2k
intake:
98.9 100.3
Water
37 37
38 36
-
37 33
37
N
Drug
TABLE
“F ~-
body
3
after
drug
102.9 103.2
103.1 101.3
103.3 103.2
153
i t
1.0 1.4
-c 1.5 c 3.1
?I 0.8 rt 1.1
rt 52h
121 t 45 134 rt 37
159 ‘-e 76
132 rfi 2 30 39 155
wt. per 24 hr
Days
103.1 103.0
102.7 102.8
103.2 103.1
143
175 122
151
47
4sh
z!I 0.9 +- 1.9
-c 2.7 ” 1.2
& 1.4 ” 1.2
”
?I 82 2 24
‘-
113 rt-t- 40 42 134
4
--
103.5 103.7
103.4 103.0
103.4 103.8
134
i t
t 2
r 2
t
1.1 1.1
1.2 1.1
1.0 1.0
36
95 71
I? 44 173 2 139 2
134
112 C2 23 32 131
5
3
2 F a
2
2
% 0
8
2
z
26
ELDON
M.
BOYD
was 1.19 I 0.073 g per kilogram compared with 0.92 t 0.045 in albino rats. In guinea pigs dying within 12 hours of drug administration, the dominant feature at autopsy was a severe gastroenteritis. When death was delayed beyond 2 days, the pathological findings were as summarized in Table 3. The latter were similar to those reported in albino rats. TABLE A
COMPARISON
2
OF THE INCIDENCE OF CLINICAL SIGNS IN GUINEA PIGS THE ADMINISTRATION OF ASA, PHENACETIN, CAFFEINE, AND APC
P (ChTX3t~
Acetylsalicylic
Clinical
sign’”
Phenacetin
acid 1 Hour
Withdrawal Restlessness Phonation Diarrhea Ataxia Catalepsy Hyperreflexia Dyspnea Tremor Fasciculation Colonic temperature: M. -I- S.D.
in columns temperatures,
drug
APC found
APC estimate
equals found)
administration
38
21
31
46
0.4
38
30
39
34
0.6
6 3
0 0
0
45 0
0
97
0 0
0 0
10 3
45
85
0 0
3 3
0
0
0
0
21
0
0
101.9 0.73
k
96.3
9 t
3.2
after
drug
3 2
9 0 79 24
99.9 -t 1.9
0.4 0.2 <
0.001 0.4
0
1
0.4 0.8
0
1
0.8
0
0
1.0
101.1 2 1.3
99.3 e 1.9
< 0.001
administration
50
9
70
54
32
33
79
0
14
53
12
17
0
4
14
0.2
1:
0
0
4
6
0.7
12
0 64
0 0
4 11
6 33
0.7 0.05
27
0 39
0 4
12 2
0.05 0.4
12 6
0 0
0 0
1.0 1.0
3
0
0
1 .o
7 0 0 0 0 0 101.6 1 .o
a Numbers except colonic
after
Caffeine
56
6 Hours Restlessness Withdrawal Stupor Dyspnea Tremor Ataxia Shivering Hyperreflexia Fasciculation Prolapse of rectum Convulsions Colonic temperature: M. I+ SD.
FOLLOWING
0 0 0 0 2
92.9 6.7
_c
99.9 3.4
+-
101.5
&
2.1
2-6 represent percentage of incidence which are recorded in degrees Fahrenheit,
97.5
0.005 <
+-
<
0.001
0.001
3.7
of
clinical
signs
IN
Inflammation Congestion Necrosis Minor Edema Contraction Congestion Minor Minor
Histopathology: 1. G.I. Tract 2. Liver 3. Kidneys 4. Heart 5. Lungs 6. Spleen 7. Adrenals 8. Pancreas 9. Testes Inflammation Degeneration Minor Minor Edema Minor Congestion Congestion Minor
33 Hypothermia, stupor
mania
APC
PIGS
3 DYIKC
1.87 k 0.074
AND
GUINEA
TABLE CAFFEINE,
SIGNS
1.19 -c 0.073
Acetylsalicylic acid
PATHOLOGICAL
12 Convulsions,
AND
28
CLINICAL
81
OF
Early (< 12 hr) deaths (% of total deaths) Delayed (2 days) deaths (% of total deaths) Premortem signs
LD,,> * SE (g/k)
Ohservatirm
A COMPARISON
37
stupor
I+ 0.02 1
29 Convulsions,
0.230
Caffeine
ADMINISTRATION
Inflammation Edema Minor Congestion Edema Congestion Congestion Minor Minor
FROM
0.040
Inflammation Degeneration Edema Minor Edema Contraction Congestion Minor Minor
19 Hypothermia, stupor, mania
50
APC
PHEXACETIN,
1.33 k
OF ASA,
28
ELDON
M.
BOYD
The syndrome of intoxication to phenacetin in the guinea pig was also similar to that reported in albino rats by Boyd (1959b) except that exophthalmos, dacryorrhea, and sialorrhea did not appear in guinea pigs. Most of the animals died within 48 hours of respiratory failure preceded by colonic temperatures of 77-80” F. The LDzo ir SE was 1.87 + 0.074 g per kilogram compared with 1.65 2 0.35 in albino rats. Histopathology in guinea pigs dying after 2 days was similar to that reported in albino rats except that renal tubular necrosis was of a minor degree. The LDzo -t SE for caffeine was found to be 0.230 % 0.021 g per kilogram in guinea pigs. Boyd (1959~) reported 0.192 -t 0.018 in albino rats. The clinical and pathological syndrome was similar in the two species. It is of interest that intussusception of the ileum was found in some 10% of albino rats: intussusception and/or prolapse of the rectum was present in 12% of the guinea pigs. The common cause of death was respiratory failure following tonic-clonic convulsions. The APC mixture consisted of 50 g of acetylsalicylic acid, 46 g of phenacetin, and 4 g of caffeine in each 100 g, as used by Boyd (1959d) in albino rats. In guinea pigs the LD,,, ? SE was 1.33 f 0.040 g per kilogram; in albino rats 1.42 -+ 0.01 g per kilogram. The LD;,” -t SE estimated from values for the three ingredients in the same manner as for albino rats was calculated to be 1.47 -+ 0.071 g per kilogram. The LDr,(, found for the APC mixture was some 10% lower (P < 0.05) than was estimated from the sum of proportionate LDnO’s of the ingredients. An estimate of the clinical signs was made in a similar manner and compared with values actually found upon the APC mixture. The comparisons are given in Tables 1 and 2. It may be seen that the intake of water during the second to fourth days was lower than estimated from the three ingredients. Ataxia occurred very commonly in phenacetin intoxication, rarely with the APC mixture. There was less hypothermia, inactivity or withdrawal, ataxia, and shivering, and more restlessness from the APC mixture than was estimated from the values found upon the three ingredients. Thus the syndrome of intoxication of the APC mixture is a composite of that of its ingredients with the signs of intoxication from acetylsalicylic acid tending to dominate the picture. The causes of death from the APC mixture were similar to those noted in albino rats by Boyd (1959d) except that terminal hyperpyrexia was not found in guinea pigs. The histopathologic picture (Table 3) was similar to that recorded in albino rats.
TOXICITY
OF APC
IN
GUINEA
PIGS
29
DISCUSSION
The median lethal dose of acetylsalicylic acid in the guinea pig has not been previously reported (Smith, 1958). It was found during this investigation to be 29% higher (P < 0.001) than in the albino rat (Boyd, 1959a). The guinea pig is therefore no more sensitive than the rat to the acutely toxic effects of oral acetylsalicylic acid. This is in contrast to the sensitivity of the guinea pig to the toxic effects of certain other drugs such as penicillin (e.g., Schneierson and Perlman, 1956) and chlortetracycline (Ambrus et al., 1952). The syndrome of intoxication to acetylsalicylic acid in the guinea pig was similar to that in the albino rat (Boyd, 1959a). Most of the animals died within 12 hours from tonic-clonic convulsions associated with an acute gastroenteritis. Lish et al. (1959) have suggested that gastric irritation produced by acetylsalicylic acid may be associated with its ability to inhibit carbonic anhydrase. It would seem likely that such enzyme inhibition was only part of the mechanism of action since there was considerable necrosis of the gastrointestinal mucosa in the guinea pigs. When death was delayed beyond 48 hours, it was accompanied in addition by evidence of edema and congestion of the liver, lungs, and adrenal glands, and necrosis of the renal tubules. Phenacetin in doses of the range of the oral LDno produced hypothermia, stupor, and respiratory failure, mostly within 48 hours, associated with an acute gastroenteritis, edema, and/or congestion of the lungs, adrenals, pancreas, and to a lesser extent, the renal tubules. The LD,,, did not differ significantly from the corresponding value in albino rats (Boyd, 1959b). Excessive use of phenacetin has occurred in Alemanic Switzerland since World War II and has been suggested as a cause of a corresponding increase in the incidence of chronic interstitial nephritis (Gsell, 1958). Gsell et al. ( 1958) listed amongst the symptoms, lassitude, thirst, loss of weight, and vomiting; these occur in acute phenacetin poisoning as noted herein and by Boyd ( 195913). Scheidegger ( 1958) listed the pathological findings as a shrunken kidney with proliferation of connective tissue, round cell infiltration of the cortex, and calcification of the papillae. Tholen (1958) concluded that phenacetin abuse was a probable cause; Pletscher (1958) was less certain. Studer and Zbinden ( 1955) were unable to reproduce the renal lesions in rats given phenacetin in a dose of 300 mg per kilogram per day orally for over six months. Studer et al. (1958b) found that Saridon (which
30
ELDON
M.
BOYD
contains phenacetin) did not augment renal lesions produced in rats and rabbits by various chemical nephrotoxic agents. Studer et al. (1958a) found that Saridon augmented an interstitial nephritis produced by intravenous injection of Staphylococcus aweus haemolyticus in rats. Phenacetin was found in this study and that of Boyd (1959b) to be able to produce toxic effects on the renal tubules in acutely lethal doses. While these lesions did not appear greater than those seen after acutely lethal doses of drugs such as benzylpenicillin ammonium (Boyd et al., 1959), it is possible, though not necessarily so, that repeated daily use of phenacetin might have adverse effects on the kidneys. The median lethal dose of caffeine was found to be some 20% higher (P = 0.01) in the guinea pig than in the albino rat (Boyd, 19.59~). The syndrome, consisting of excitement, convulsions, gastroenteritis, and edema and/or congestion of the liver, heart, lungs, spleen, and adrenal glands, was similar to that found in albino rats by Boyd (1959~). Addition of signs of toxicity of the three ingredients in proportions in which they were present in the APC mixture yielded, in most instances, figures which were insignificantly different from the corresponding figure obtained by use of the APC mixture directly. On the basis of data upon the median lethal dose, the toxicity of the APC mixture was lo%, greater in guinea pigs, l.SF, less in albino rats (Boyd, 1959d), than the sum of the toxicity of the three ingredients. The central nervous system was stimulated by median lethal doses of acetylsalicylic acid and depressed, through hypothermia, by corresponding doses of phenacetin. The effect of the APC mixture upon the central nervous system was more akin to that of acetylsalicylic acid than that of phenacetin. The APC mixture and its three ingredients produced a gastroenteritis and toxic effects of varying degree upon various organs of the body. SUMMARY The clinical and pathological syndrome of intoxication from median lethal doses of a mixture of acetylsalicylic acid, phenacetin, and caffeine, given orally to guinea pigs, was found to be, in general, similar to a summation of the corresponding syndromes of the three ingredients given separately. Values for the LD,, t SE of the three ingredients and of the mixture were, respectively, 1.19 + 0.073, 1.87 ? 0.074, 0.230 fO.021, and 1.33 & 0.040 g per kilogram body weight. At these doses, all four agents produced a gastroenteritis and toxic effects of varying degree upon several organs, Acetylsalicylic acid and caffeine excited the central nervous system, phenacetin depressed through marked hypothermia, and the mixture usually excited.
TOXICITY
OF APC
IN
GUINEA
PIGS
31
ACKNOWLEDGMENTS The author wishes to acknowledge the assistance Price-Jones, Patricia E. Sheppard, and H. D. Steele project through Woodard Research.
of J. Coates, Jr., Marion A. and of a grant in aid of the
REFERENCES AMBRUS, C. M., SIDERI, C. N., JOHNSON, G. C., and HARRISON, J. W. E. (1952). Toxicity of penicillin and aureomycin in guinea pigs. Antibiotics G Chemotherapy 2, 521-527. BOYD, E. M. (1959a). The acute oral toxicity of acetylsalicylic acid. Tozicol. dppl. Pharntacol. 1, 229-239. BOYD, E. M. (1959b). The acute oral toxicity of phenacetin. Toxicol. Appl. Phannacol. 1, 240-249. Boun, E. M. (1959~). The acute oral toxicity of caffeine. Toxicol. Appl. Pharmacol. 1, 250-237. BOYD, E. M. (1959d). The acute oral toxicity of a mixture of acetylsalicylic acid, phenacetin, and caffeine. Toxicol. Appl. Pharmacol. 1, 258-266. BOYD, E. M., BROUGHTON, R. J., and JAMES, J. (1959). The acute oral toxicity of benzylpenicillin ammonium. Antibiotics G Chemotherapy in press. GSELL, 0. (1958). PrimPr chronisch interstitielle Nephritis in Beziehung zum Phenacetinabusus. B1d1. schweiz. Akad. med. Wiss. 14, 124-130. GSELL, O., VON RECXENRERG, H. K., and MIESCHER, P. (1958). Primary chronic interstitial nephritis (pyelonephritis) Clinical, experimental and aetiological observations. Ger. Med. Monthly 3, 37-43. Lrsrr, P. M., DUNGAN, K. W., and ROBBINS, S. R. (1959). Gastrointestinal pharmacology of antipyretic-analgetic agents. I. Effects on acid secretion and ulcer formation in the Shay rat. Arch. intern. pkarmacodynumie 119, 389.397. LOEWE, S., K#ER, E., and MUISCHNEIC, H. (1927). fiber Kombinationswirkungen. VII. Mitteilung: Grundlagen der Priifung von Drei-Pharmakamischungen.-Anwendung auf Phenazetin-Azetylsalizylsaure-Codeinmischungen. Arch. exptl. Pathol. Pharmacol. Naunyn-Schmiedeberg’s 120, 25.40. PLETSCHER, A. (1958). ijber die Toxikologie des Phenacetins. Bull. schweis. Akad. med. Wiss. 14, 10@111. SCHEIDEC~ER, S. (1958). Pathologisch-anatomischer Beitrag zur Frage der Chronischen interstitiellen Nephritis in Anschluss an Abusus von phenacetinhaltigen Analgetica. Bull. schweiz. Akad. med. Wiss. 14, 139-153. SCHNEIERSOPIT, S. S., and PERLMAN, E. (1956). Toxicity of penicillin for the Syrian hamster. PYOC. Sot. Exptl. Biol. Med. 81, 229-230. SMITH, P. K. (1958j. Acetophenetidin. A Critical Bibliographic Review, pp. 132-134. Interscience, New York. STUDER, A., and ZBINDEN, G. (1955). Experimenteller Beitrag zur Frage von Nierenschaden bei Abusus von phenacetinhaltigen Schmerzmitteln. Expevientia 11, 450-45 1. STUDER, A., ZBINDEN, G., and FUST, B. (1958a). Weitere tierexperimentelle Untersuchungen zur Frage Schmerzmittelmissbrauch und interstitielle Nephritis. Schweiz. mfd. Wochschv. 88, 469-470.
32
ELDON
M.
BOYD
STUDER, A., ZBINDEN, G., SCHXRER, K., and FUST, R. (195%). Tierexperimentclle Untersuchungen zur Frage der interstitiellen Nephritis bei Missbrauch phenacetinhaltiger Schmerzmittel. Bull. schweis. Akad. med. Win. 14, 154.165. TH~~LEN, H. (1958). Chronisch interstitielle Nephritis und Abusus phenacetinhaltiger Medikamente. Bull. schweiz. Akad. need. Wiss. 14, 134-138.
AND
APPLIED
2,
PHARMACOLOGY
23-32
(1960)
The Acute Oral Toxicity in Guinea Pigs of Acetylsalicylic Acid, Phenacetin, and Caffeine, Alone and Combined ELDON Department
of Pharmacology,
M.
Queen’s Recrived
BOYD
University,
Afrgusf
Kingston,
Ontario,
Canada
19, 1959
Boyd (1959a-d) has shown that the acute oral toxicity in albino rats of a mixture of acetylsalicylic acid, phenacetin, and caffeine is, in general, a summation of the corresponding toxicity of the individual ingredients of the mixture. According to Smith (1958)) there is no corresponding evidence available on any other species except the guinea pig. Loewe et al. (1927) analyzed, by means of isoboles, data upon the threshold (“Schwelle sichtbarer Allgemeinwirkungen”) and lethal (“tijdliche Grenzdosis”) doses of acetylsalicylic acid, phenacetin, and codeine, given orally to guinea pigs, and offered some evidence of antagonism of toxic effects. The presently described work was undertaken to determine if similar antagonism could be found in a mixture of acetylsalicylic acid, phenacetin, and caffeine given orally to guinea pigs. METHODS
The experiments were performed upon young, male, CBL guinea pigs, weighing 30@-500 g. The numbers of animals used are indicated in Table 1. While under close observation, they were placed in metabolism cages, food was withheld for 16 hours prior to oral administration of the drug, and subsequent measurements were made as described by Boyd (1959a). The results are summarized in Tables 1-3. RESULTS
The clinical signs of acute toxicity to acetylsalicylic acid in the guinea pig were the same as reported by Boyd (1959a) in albino rats except that rhinorrhagia did not occur and there was little diarrhea. Death was due to respiratory failure preceded by generalized convulsions or prostration in most animals, occasionally to cardiovascular shock. The LDao t SE 23
or
CHANCES
a The results are expressed b Mean of APC estimate
Water (controls) Acetylsalicylic acid Phenacetin Caffeine APC found APC estimate
Water (controls) Acetylsalicylic acid Phenacetin Caffeine APC found APC estimate
Ihug
A COMPARISON
38 36 37 37 33 -
Food
38 36 37 37 33 -
-I__-~-
N
BODY
intake:
Body
76 2 57 ” 40 -c 26 2 41 r 482
grams
grams
29 11 20 12 14 15
Purina
-c 10 k 17 i 13
!I 13 2 14
weight:
1
AND
2
33 25 21 28
drug
30 23
17 21 25
wt. per 24 hr
___-
4
IN
18 20 23 8 49 21” estimate.
82 -t 73 j, 68 21 65_t 91 ” 71 ”
+37 c 15 + 8221 -3338 -12 c 39 + 6219 + 2-t30
TEMPERATURE
at P < 0.05 and at N = 33 for APC
87-t 7s k 65 & 48-t31 72 lr 69 f
body
$33 2 15 + l-t21 -11 Ik 37 -20 & 34 -6626 -j&29
cr decrease
3
(-)
COLONIC APCa
Days after
AND AND
per kilogram
2 -c -c -c
80-c 17 73 21 23 62 2 33 36 k-31 65 &2S 66 ‘-+ 28
pellets
(+)
-c 12
increase +24 -3322 -18 -24 -11 -11
1 INTAKE,
CAFFEINE,
TABLE WATER
PHENACETIN,
FOOD
ASA,
+15 -14 -8k12 -25 -15 -12
WEIGHT,
as mean & standard deviation. differs from mean of APC actual
IN
~_
GUINEA
GIVEN
82 t 822 68 t 73 3~ 75 -t 75 2
16 8 21 26 16 14
+44 & 16 $11 +- 19 + 4,31 - 4246 +10*21 + 7-26
5
PIGS
$ 3
F
M s g
estimate
found
Phenacetin Caffeine APC found APC estimate
Water (controls) Acetylsalicylic acid
APC
APC
Caffeine
___-
38 36
Water (controls) acid Acetylsalicylic Phenacetin 47 19
1.6 2.1
51 2.6 * 4.3
106.6 102.8
102.3 101.2
103.2 103.2
t 4.1 ?I 2.0
k 2.9 ‘-c 2.2
‘-c 1.5 c+ 1.1
138 -c 52”
temperature:
Colonic
79 IL 69 117 rt 27
124 -c 54
130 & 39 156 -‘49
per kilogram
2
1 (Continued)
35
21 41
-f- 6.8 -r- 2.8
2 2
-..___~
102.2 101.5
103.3 102.9
68 k
45 2 79 2
_______
milliliters
62 -C 53
33
-
1
123 77 2k
intake:
98.9 100.3
Water
37 37
38 36
-
37 33
37
N
Drug
TABLE
“F ~-
body
3
after
drug
102.9 103.2
103.1 101.3
103.3 103.2
153
i t
1.0 1.4
-c 1.5 c 3.1
?I 0.8 rt 1.1
rt 52h
121 t 45 134 rt 37
159 ‘-e 76
132 rfi 2 30 39 155
wt. per 24 hr
Days
103.1 103.0
102.7 102.8
103.2 103.1
143
175 122
151
47
4sh
z!I 0.9 +- 1.9
-c 2.7 ” 1.2
& 1.4 ” 1.2
”
?I 82 2 24
‘-
113 rt-t- 40 42 134
4
--
103.5 103.7
103.4 103.0
103.4 103.8
134
i t
t 2
r 2
t
1.1 1.1
1.2 1.1
1.0 1.0
36
95 71
I? 44 173 2 139 2
134
112 C2 23 32 131
5
3
2 F a
2
2
% 0
8
2
z
26
ELDON
M.
BOYD
was 1.19 I 0.073 g per kilogram compared with 0.92 t 0.045 in albino rats. In guinea pigs dying within 12 hours of drug administration, the dominant feature at autopsy was a severe gastroenteritis. When death was delayed beyond 2 days, the pathological findings were as summarized in Table 3. The latter were similar to those reported in albino rats. TABLE A
COMPARISON
2
OF THE INCIDENCE OF CLINICAL SIGNS IN GUINEA PIGS THE ADMINISTRATION OF ASA, PHENACETIN, CAFFEINE, AND APC
P (ChTX3t~
Acetylsalicylic
Clinical
sign’”
Phenacetin
acid 1 Hour
Withdrawal Restlessness Phonation Diarrhea Ataxia Catalepsy Hyperreflexia Dyspnea Tremor Fasciculation Colonic temperature: M. -I- S.D.
in columns temperatures,
drug
APC found
APC estimate
equals found)
administration
38
21
31
46
0.4
38
30
39
34
0.6
6 3
0 0
0
45 0
0
97
0 0
0 0
10 3
45
85
0 0
3 3
0
0
0
0
21
0
0
101.9 0.73
k
96.3
9 t
3.2
after
drug
3 2
9 0 79 24
99.9 -t 1.9
0.4 0.2 <
0.001 0.4
0
1
0.4 0.8
0
1
0.8
0
0
1.0
101.1 2 1.3
99.3 e 1.9
< 0.001
administration
50
9
70
54
32
33
79
0
14
53
12
17
0
4
14
0.2
1:
0
0
4
6
0.7
12
0 64
0 0
4 11
6 33
0.7 0.05
27
0 39
0 4
12 2
0.05 0.4
12 6
0 0
0 0
1.0 1.0
3
0
0
1 .o
7 0 0 0 0 0 101.6 1 .o
a Numbers except colonic
after
Caffeine
56
6 Hours Restlessness Withdrawal Stupor Dyspnea Tremor Ataxia Shivering Hyperreflexia Fasciculation Prolapse of rectum Convulsions Colonic temperature: M. I+ SD.
FOLLOWING
0 0 0 0 2
92.9 6.7
_c
99.9 3.4
+-
101.5
&
2.1
2-6 represent percentage of incidence which are recorded in degrees Fahrenheit,
97.5
0.005 <
+-
<
0.001
0.001
3.7
of
clinical
signs
IN
Inflammation Congestion Necrosis Minor Edema Contraction Congestion Minor Minor
Histopathology: 1. G.I. Tract 2. Liver 3. Kidneys 4. Heart 5. Lungs 6. Spleen 7. Adrenals 8. Pancreas 9. Testes Inflammation Degeneration Minor Minor Edema Minor Congestion Congestion Minor
33 Hypothermia, stupor
mania
APC
PIGS
3 DYIKC
1.87 k 0.074
AND
GUINEA
TABLE CAFFEINE,
SIGNS
1.19 -c 0.073
Acetylsalicylic acid
PATHOLOGICAL
12 Convulsions,
AND
28
CLINICAL
81
OF
Early (< 12 hr) deaths (% of total deaths) Delayed (2 days) deaths (% of total deaths) Premortem signs
LD,,> * SE (g/k)
Ohservatirm
A COMPARISON
37
stupor
I+ 0.02 1
29 Convulsions,
0.230
Caffeine
ADMINISTRATION
Inflammation Edema Minor Congestion Edema Congestion Congestion Minor Minor
FROM
0.040
Inflammation Degeneration Edema Minor Edema Contraction Congestion Minor Minor
19 Hypothermia, stupor, mania
50
APC
PHEXACETIN,
1.33 k
OF ASA,
28
ELDON
M.
BOYD
The syndrome of intoxication to phenacetin in the guinea pig was also similar to that reported in albino rats by Boyd (1959b) except that exophthalmos, dacryorrhea, and sialorrhea did not appear in guinea pigs. Most of the animals died within 48 hours of respiratory failure preceded by colonic temperatures of 77-80” F. The LDzo ir SE was 1.87 + 0.074 g per kilogram compared with 1.65 2 0.35 in albino rats. Histopathology in guinea pigs dying after 2 days was similar to that reported in albino rats except that renal tubular necrosis was of a minor degree. The LDzo -t SE for caffeine was found to be 0.230 % 0.021 g per kilogram in guinea pigs. Boyd (1959~) reported 0.192 -t 0.018 in albino rats. The clinical and pathological syndrome was similar in the two species. It is of interest that intussusception of the ileum was found in some 10% of albino rats: intussusception and/or prolapse of the rectum was present in 12% of the guinea pigs. The common cause of death was respiratory failure following tonic-clonic convulsions. The APC mixture consisted of 50 g of acetylsalicylic acid, 46 g of phenacetin, and 4 g of caffeine in each 100 g, as used by Boyd (1959d) in albino rats. In guinea pigs the LD,,, ? SE was 1.33 f 0.040 g per kilogram; in albino rats 1.42 -+ 0.01 g per kilogram. The LD;,” -t SE estimated from values for the three ingredients in the same manner as for albino rats was calculated to be 1.47 -+ 0.071 g per kilogram. The LDr,(, found for the APC mixture was some 10% lower (P < 0.05) than was estimated from the sum of proportionate LDnO’s of the ingredients. An estimate of the clinical signs was made in a similar manner and compared with values actually found upon the APC mixture. The comparisons are given in Tables 1 and 2. It may be seen that the intake of water during the second to fourth days was lower than estimated from the three ingredients. Ataxia occurred very commonly in phenacetin intoxication, rarely with the APC mixture. There was less hypothermia, inactivity or withdrawal, ataxia, and shivering, and more restlessness from the APC mixture than was estimated from the values found upon the three ingredients. Thus the syndrome of intoxication of the APC mixture is a composite of that of its ingredients with the signs of intoxication from acetylsalicylic acid tending to dominate the picture. The causes of death from the APC mixture were similar to those noted in albino rats by Boyd (1959d) except that terminal hyperpyrexia was not found in guinea pigs. The histopathologic picture (Table 3) was similar to that recorded in albino rats.
TOXICITY
OF APC
IN
GUINEA
PIGS
29
DISCUSSION
The median lethal dose of acetylsalicylic acid in the guinea pig has not been previously reported (Smith, 1958). It was found during this investigation to be 29% higher (P < 0.001) than in the albino rat (Boyd, 1959a). The guinea pig is therefore no more sensitive than the rat to the acutely toxic effects of oral acetylsalicylic acid. This is in contrast to the sensitivity of the guinea pig to the toxic effects of certain other drugs such as penicillin (e.g., Schneierson and Perlman, 1956) and chlortetracycline (Ambrus et al., 1952). The syndrome of intoxication to acetylsalicylic acid in the guinea pig was similar to that in the albino rat (Boyd, 1959a). Most of the animals died within 12 hours from tonic-clonic convulsions associated with an acute gastroenteritis. Lish et al. (1959) have suggested that gastric irritation produced by acetylsalicylic acid may be associated with its ability to inhibit carbonic anhydrase. It would seem likely that such enzyme inhibition was only part of the mechanism of action since there was considerable necrosis of the gastrointestinal mucosa in the guinea pigs. When death was delayed beyond 48 hours, it was accompanied in addition by evidence of edema and congestion of the liver, lungs, and adrenal glands, and necrosis of the renal tubules. Phenacetin in doses of the range of the oral LDno produced hypothermia, stupor, and respiratory failure, mostly within 48 hours, associated with an acute gastroenteritis, edema, and/or congestion of the lungs, adrenals, pancreas, and to a lesser extent, the renal tubules. The LD,,, did not differ significantly from the corresponding value in albino rats (Boyd, 1959b). Excessive use of phenacetin has occurred in Alemanic Switzerland since World War II and has been suggested as a cause of a corresponding increase in the incidence of chronic interstitial nephritis (Gsell, 1958). Gsell et al. ( 1958) listed amongst the symptoms, lassitude, thirst, loss of weight, and vomiting; these occur in acute phenacetin poisoning as noted herein and by Boyd ( 195913). Scheidegger ( 1958) listed the pathological findings as a shrunken kidney with proliferation of connective tissue, round cell infiltration of the cortex, and calcification of the papillae. Tholen (1958) concluded that phenacetin abuse was a probable cause; Pletscher (1958) was less certain. Studer and Zbinden ( 1955) were unable to reproduce the renal lesions in rats given phenacetin in a dose of 300 mg per kilogram per day orally for over six months. Studer et al. (1958b) found that Saridon (which
30
ELDON
M.
BOYD
contains phenacetin) did not augment renal lesions produced in rats and rabbits by various chemical nephrotoxic agents. Studer et al. (1958a) found that Saridon augmented an interstitial nephritis produced by intravenous injection of Staphylococcus aweus haemolyticus in rats. Phenacetin was found in this study and that of Boyd (1959b) to be able to produce toxic effects on the renal tubules in acutely lethal doses. While these lesions did not appear greater than those seen after acutely lethal doses of drugs such as benzylpenicillin ammonium (Boyd et al., 1959), it is possible, though not necessarily so, that repeated daily use of phenacetin might have adverse effects on the kidneys. The median lethal dose of caffeine was found to be some 20% higher (P = 0.01) in the guinea pig than in the albino rat (Boyd, 19.59~). The syndrome, consisting of excitement, convulsions, gastroenteritis, and edema and/or congestion of the liver, heart, lungs, spleen, and adrenal glands, was similar to that found in albino rats by Boyd (1959~). Addition of signs of toxicity of the three ingredients in proportions in which they were present in the APC mixture yielded, in most instances, figures which were insignificantly different from the corresponding figure obtained by use of the APC mixture directly. On the basis of data upon the median lethal dose, the toxicity of the APC mixture was lo%, greater in guinea pigs, l.SF, less in albino rats (Boyd, 1959d), than the sum of the toxicity of the three ingredients. The central nervous system was stimulated by median lethal doses of acetylsalicylic acid and depressed, through hypothermia, by corresponding doses of phenacetin. The effect of the APC mixture upon the central nervous system was more akin to that of acetylsalicylic acid than that of phenacetin. The APC mixture and its three ingredients produced a gastroenteritis and toxic effects of varying degree upon various organs of the body. SUMMARY The clinical and pathological syndrome of intoxication from median lethal doses of a mixture of acetylsalicylic acid, phenacetin, and caffeine, given orally to guinea pigs, was found to be, in general, similar to a summation of the corresponding syndromes of the three ingredients given separately. Values for the LD,, t SE of the three ingredients and of the mixture were, respectively, 1.19 + 0.073, 1.87 ? 0.074, 0.230 fO.021, and 1.33 & 0.040 g per kilogram body weight. At these doses, all four agents produced a gastroenteritis and toxic effects of varying degree upon several organs, Acetylsalicylic acid and caffeine excited the central nervous system, phenacetin depressed through marked hypothermia, and the mixture usually excited.
TOXICITY
OF APC
IN
GUINEA
PIGS
31
ACKNOWLEDGMENTS The author wishes to acknowledge the assistance Price-Jones, Patricia E. Sheppard, and H. D. Steele project through Woodard Research.
of J. Coates, Jr., Marion A. and of a grant in aid of the
REFERENCES AMBRUS, C. M., SIDERI, C. N., JOHNSON, G. C., and HARRISON, J. W. E. (1952). Toxicity of penicillin and aureomycin in guinea pigs. Antibiotics G Chemotherapy 2, 521-527. BOYD, E. M. (1959a). The acute oral toxicity of acetylsalicylic acid. Tozicol. dppl. Pharntacol. 1, 229-239. BOYD, E. M. (1959b). The acute oral toxicity of phenacetin. Toxicol. Appl. Phannacol. 1, 240-249. Boun, E. M. (1959~). The acute oral toxicity of caffeine. Toxicol. Appl. Pharmacol. 1, 250-237. BOYD, E. M. (1959d). The acute oral toxicity of a mixture of acetylsalicylic acid, phenacetin, and caffeine. Toxicol. Appl. Pharmacol. 1, 258-266. BOYD, E. M., BROUGHTON, R. J., and JAMES, J. (1959). The acute oral toxicity of benzylpenicillin ammonium. Antibiotics G Chemotherapy in press. GSELL, 0. (1958). PrimPr chronisch interstitielle Nephritis in Beziehung zum Phenacetinabusus. B1d1. schweiz. Akad. med. Wiss. 14, 124-130. GSELL, O., VON RECXENRERG, H. K., and MIESCHER, P. (1958). Primary chronic interstitial nephritis (pyelonephritis) Clinical, experimental and aetiological observations. Ger. Med. Monthly 3, 37-43. Lrsrr, P. M., DUNGAN, K. W., and ROBBINS, S. R. (1959). Gastrointestinal pharmacology of antipyretic-analgetic agents. I. Effects on acid secretion and ulcer formation in the Shay rat. Arch. intern. pkarmacodynumie 119, 389.397. LOEWE, S., K#ER, E., and MUISCHNEIC, H. (1927). fiber Kombinationswirkungen. VII. Mitteilung: Grundlagen der Priifung von Drei-Pharmakamischungen.-Anwendung auf Phenazetin-Azetylsalizylsaure-Codeinmischungen. Arch. exptl. Pathol. Pharmacol. Naunyn-Schmiedeberg’s 120, 25.40. PLETSCHER, A. (1958). ijber die Toxikologie des Phenacetins. Bull. schweis. Akad. med. Wiss. 14, 10@111. SCHEIDEC~ER, S. (1958). Pathologisch-anatomischer Beitrag zur Frage der Chronischen interstitiellen Nephritis in Anschluss an Abusus von phenacetinhaltigen Analgetica. Bull. schweiz. Akad. med. Wiss. 14, 139-153. SCHNEIERSOPIT, S. S., and PERLMAN, E. (1956). Toxicity of penicillin for the Syrian hamster. PYOC. Sot. Exptl. Biol. Med. 81, 229-230. SMITH, P. K. (1958j. Acetophenetidin. A Critical Bibliographic Review, pp. 132-134. Interscience, New York. STUDER, A., and ZBINDEN, G. (1955). Experimenteller Beitrag zur Frage von Nierenschaden bei Abusus von phenacetinhaltigen Schmerzmitteln. Expevientia 11, 450-45 1. STUDER, A., ZBINDEN, G., and FUST, B. (1958a). Weitere tierexperimentelle Untersuchungen zur Frage Schmerzmittelmissbrauch und interstitielle Nephritis. Schweiz. mfd. Wochschv. 88, 469-470.
32
ELDON
M.
BOYD
STUDER, A., ZBINDEN, G., SCHXRER, K., and FUST, R. (195%). Tierexperimentclle Untersuchungen zur Frage der interstitiellen Nephritis bei Missbrauch phenacetinhaltiger Schmerzmittel. Bull. schweis. Akad. med. Win. 14, 154.165. TH~~LEN, H. (1958). Chronisch interstitielle Nephritis und Abusus phenacetinhaltiger Medikamente. Bull. schweiz. Akad. need. Wiss. 14, 134-138.