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An Evaluation of the Toxicity of Certain Sulfonamides for Young Chickens
An Evaluation of the Toxicity of Certain Sulfonamides for Young Chickens PAUL A. MATTIS,* S. E. MCKINNEY, ELIZABETH A. PATCH, AND ETHEL L. WILLIAMS Department of Pharmacology, The Medical Research Division, Sharp and Dohme, Inc., Glenolden, Pa.
T
HE interest aroused by the treatment of coccidiosis in poultry with sulfanilamide (Levine, 1939) led to studies with other compounds in an attempt to find a sulfonamide that would be effective without causing marked toxic manifestations. Certain preliminary studies (Swales, 1944) using sulfamerazine (4-methyl-2sulfanilamidopyrimidine) and other studies using a mixture of sodium sulfamerazine 80 percent, 'Sulfathalidine' (phthalylsulfathiazole) 10 percent and sodium carbonate 10 percent (SS2) (Graham, Hawkins, Swales, Thorp) suggest that this mixture may be of value in the treatment of infections in poultry caused by Eimeria. These findings have prompted a study of the effects on young chickens of a diet containing sulfamerazine or a mixture of sodium sulfamerazine, 'Sulfathalidine' and sodium carbonate (SS2). The only previous study of the toxic effects of sulfamerazine for chickens was devoted primarily to the effects on nervous tissue (Welch etal., 1943). Procedure: Thirty-five chicks, 32 days old, were divided into 5 groups of 7 each and allowed free access to a diet1 for 14
days. One group served as a control; the other 4 groups received the same diet to which had been added the sulfonamides as follows: 0.5 percent or 1.0 percent sulfamerazine, 0.5 percent or 1.0 percent SS2. The birds were weighed, and blood samples taken for the determination of the sedimentation rate, hematocrit reading, total erythrocyte counts, differential leucocytic counts, and concentration of hemoglobin at the beginning, on the seventh day and at the end of the experiment. Determinations of the free and total sulfonamide concentrations in the blood were made at intervals during the test, using the technique of Bratton and Marshall (1939, 1940). All of the control chickens and 5 birds from each of the treated groups were sacrificed on the fourteenth day and the following tissues were taken for histopathological examinations: lung, heart, liver, spleen, kidney, proventriculus, gizzard, duodenum, cecum, bone marrow, lymph node, and in certain birds, thyroid, adrenal, and testis or ovary. Since nervous tissues from chickens receiving sulfamerazine had been studied previously (Welch et al., 1943) no sections of this tissue were taken. Results: During the test, observations were made of the general health and welfare of the birds. Although a few chicks
* Present address: The Department of Pharmacology, University of Florida School of Pharmacy, Gainesville, Fla. 1 Purina 'Startena' and cracked corn. The drugs were added to the 'Startena.' 59
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on May 29, 2015
(Received for publication, August 23, 1945)
60
PAUL A. MATTIS, S. E. MCKINNEY, E. A. PATCH, AND E. L.
WILLIAMS
TABLE 1.—Average weight and percentage increase The data were treated statistically 2 to
in weight of chickens fed a diet containing sulfamerazine (0.5 percent or 1.0 percent) or SS2* (0.5 percent determine if the differences between the or 1.0 percent) weight increments of the various treated Initial Day Wt. in gms.
Wt. in gms.
Percentage increase
196
302
54
181
302
67
176 189 201
261 320 292
51 69 45
* A mixture of sodium sulfamerazine, 80 percent, 'Sulfathalidine,' 10 percent and sodium carbonate, 10 percent.
appeared somewhat unthrifty, all birds ate well, gained weight and showed no manifestations of toxicity during the 14 days of the experiment. Table 1 presents the average initial and final weights of the birds and the average percentage gains in weight during 14 days.
In Table 2 have been tabulated the hematological findings for these birds. Attention may be called to the regularity of the total number of erythrocytes and 2 We are indebted to Mr. J. L. Ciminera for Ms kindness in making the statistical evaluations.
TABLE 2.—Certain hematologicalfindingsin chickens administered sulfonamides in the diet for 14 days
Drug fed
Day
Differential Leucocyte Count*— Erythro- Sedimen- Hemato- HemoPercentage tation crit cytes globin Rate per(in (gm./lOO Basocent millions) (mm./ cc.) Hetero- Lymph- Mono- Eosinohr.) phils phils phils cytes cytes
Control
1st 7th 14th
2.36 2.4 2.52
2 7 2
28 33 32
10.0 10.0 10.9
21 20 19
65 64 64
7 9 11
3 2 2
4 5 4
0.5 percent Sulfamerazine .
1st 7th 14th
2.46 2.44 2.22
4 13 4
34 30 28
10.9 10.7 9.9
18 24 10
64 65 81
12 6 6
2 2 1
4 • 3 2
1.0 percent Sulfamerazine
1st 7th 14th
2.44 2.82 2.68
3 2 2
34 34 31
11.3 12.3 11.0
14 21 18
69 60 75
11 9 3
2 3 1
4 7 3
0.5 percent SS2
1st 7th 14th
2.51 2.52 2.30
9 12 4
34 31 28
11.5 10.9 10.3
24 17 17
58 70 76
9 6 5
5 2
4 5 2
1.0 percent SS2
1st 7th 14th
2.50
5 5 5
31 30 27
10.2 10.9 10.2
15 23 11
71 65 82
8 5 5
2 2
4 5 2
2.41
* Each figure represents the counting of 200 cells.
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Control Sulfamerazine 0.5 percent Sulfamerazine 1.0 percent SS2 0.5 percent SS2 1.0 percent
groups were significant when compared with the untreated control group. There was no significant difference between the percentage increases in weight of the treated groups, except for those birds given SS2 (0.5 percent). The greater weight increase of this group was found to be significant, whereas the weight increase of those birds given 0.5 percent sulfamerazine was found to be of borderline significance. Although these chicks fed 1.0 percent sulfamerazine or 1.0 percent SS2 appeared to exhibit a slowing of the growth rate, the difference between their weight increases and those of the control birds was not statistically significant.
14th Day
TOXICITY OF SULFONAMIDES FOR YOUNG CHICKENS
61
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3.—Average concentration of sulfonamides* the concentration of hemoglobin through- (inTABLE mgm. per 100 cc.) in the whole blood of chickens out the test. Treatment of these data by fed a diet containing sulfamerazine (0.5 percent orl.0 means of Fisher's t test shows that there percent) or SS2 (0.5 per cent or 1.0 percent) were no significant differences between the SulfaSulfaSS2 total numbers of erythrocytes at the bemerazine merazine SS2 1.0 0.5 Day 1.0 O.S ginning of the test and at its completion. percent percent percent percent It was found also that there were no sig10.6 29.2 20.8 Ft 15.0 2nd nificant changes in the hemoglobin con1.0 1.2. 1.2 centration, sedimentation rate or volume ct 20.4 11.8 33.1 F 15.0 6th of packed cells during the experiment. 0.4 0.6 1.3 C 0.4 Examination of the differential leuco10.0 . 21.9 24.3 F 14.8 cytic counts from these birds revealed 10th 0.7 0.4 0.2 C 0.4 only a moderate reduction in the per10.3 21.6 29.6 F 14.1 centage of heterophiles with a concomi- 14th 1.6 1.6 1.5 C 1.5 tant increase that may have been either 10.7 21.2 29.1 absolute or relative in the percentage of Average F 14.7 0.9 1.0 1.1 C 0.6 lymphocytes. The proper interpretation of these results is uncertain. * The sulfonamide concentrations have been exin terms of sulfamerazine. It will be seen (Table 3) that there was pressed Ff=Free. CJ=Conjugated. no general tendency toward an increase of the concentration of sulfonamides in the blood with continued feeding of the drug. the sulfonamide concentrations attained In all cases the blood concentrations re- by those chickens receiving a diet conmained relatively constant, the average taining 0.5 percent sulfamerazine or 0.5 sulfonamide concentration for those birds percent SS2 demonstrates that levels in given 1.0 percent of the drug in each case excess of 6 mgm. per 100 cc. were atbeing approximately twice the concentra- tained without evidence of toxicity. tion attained in the chicks receiving the At autopsy none of the birds demon0.5 percent drug diet. strated any gross lesions. HistopathologiThe amount of conjugated sulfonamide cal examinations of the tissues taken from that occurred in these chickens remained these birds revealed the following changes. Controls: Of 7 livers examined, one quite low. The average amount of drugs occurring in the acetylated form for all showed moderate dilatation of the central birds was 5.1 percent of the total with a veins and sinusoids, and 2 showed considerable vacuolation of the central herange of 0 percent to 13.5 percent. patic parenchyma, with moderate metaHawkins and Kline (1945) have shown that 1.0 percent sulfamethazine (4-6 di- plasia of the perivascular islands of hemamethyl-2-sulfanilamidopyrimidine) in the topoietic tissue. Three of 7 kidneys had diet produced an average concentration foci of hematopoietic tissue showing of sulfonamide in the blood of 7.7 mgm. myeloid metaplasia; 2 of these 7 demonper 100 cc. for normal birds and 6.5 mgm. strated mild parenchymatous degeneraper 100 cc. for their infected birds. They tion of the tubular epithelium. The spleens considered sulfamethazine concentrations were essentially normal, except for 2 that in the neighborhood of 6 mgm. per 100 cc. were considered to show mild lymphoid necessary to prevent loss of birds infected hyperplasia, as was true also of one lymph with coccidia. Reference to the data on node of the seven examined. I n one section
62
PAUL A. MATTIS, S. E. MCKINNEY, E. A. PATCH, AND E. L. WILLIAMS
Sulfamerazine—1.0 percent: Three of 5 livers showed vacuolation of the hepatic parenchyma; one had moderately dilated sinusoids and central veins. One of the kidneys exhibited nephrosis with detritus in the lumens of the tubules. Two sections of myocardium had areas of infiltration with small round cells. These differed from the diffuse exudative lesions seen in the former group of chickens. One chick of the 5 examined was found to have an occasional asexual form of Eimeria in the caecal epithelium. Of 2 bone marrow sections examined, one was moderately hyperplastic and the other was normal. All other tissues examined were negative. SS2—0.5 percent: There was moderate vacuolation of hepatic cells in one liver section, dilatation of the sinusoids and central veins in two sections, and occasional areas of hemorrhage in 3 livers.
Three of the 5 kidneys examined showed mild nephrosis and occasional foci of small round cell infiltration. The bone marrows from all 5 chickens were examined and found to be normally active. No lesions were observed in any of the other tissues examined SS2—1.0 percent: Only one liver section of 5 examined showed mild vacuolation of the hepatic cells and dilatation of the sinusoids. Another chick had a mild nephrosis and occasional areas of infiltration with small round cells. Except for moderate metaplasia of the hematopoietic foci in one of the chickens all other tissues were uniformly negative. Four bone marrow sections showed normal activity Discussion: Previous investigators have indicated that the administration of sulfonamides, either in the diet or orally in the form of tablets or capsules, has an adverse effect on the normal growth increment of chickens. Levine (1939) stated that sulfanilamide added to the diet of growing chickens in concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 percent by weight prevented a normal weight gain. Sulfonamide concentrations attained under these conditions were not given. Rosenthal (1939) administered sulfanilamide to chickens in doses of 0.5, 1.0, and 2.0 gm. per kg. daily for varying periods and reported either temporary or permanent weight loss. Although no sulfonamide concentrations were reported, the blood levels attained must have been considerable in view of levels obtained in comparable studies by Richardson (1939). Investigations in this laboratory (Welch et al., 1943) with sulfamethylthiazole, sulfadiazine, sulfamerazine and sulfamethazine indicated that, with high blood concentrations, chickens either lost weight or did not gain weight at rates similar to those of untreated control birds. In these studies the average 4 hour blood concen-
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of myocardium there were foci of infiltration that may be described as diffuse exudative myocarditis. Five of the 7 birds were found to have coccidiosis, although none of the lesions found was severe. All other tissues examined were normal. The bone marrows were normal. Experimental Chickens :Sulfamerazine— 0.5 percent: The livers of 5 chickens were examined; considerable vacuolation of the hepatic parenchyma was found in two and no other changes were observed. The kidneys were essentially normal. One of 5 spleens showed moderate lymphoid hyperplasia. Two of the 5 sections of myocardium were found to show small epicardial areas of diffuse exudative myocarditis. Of 5. bone marrows examined all were normally active, one showing a moderate increase in erythropoiesis. The other tissues examined (lymph nodes, pancreas, testes or ovaries, lungs, adrenals, thyroids and gastrointestinal tract) were essentially normal. No lesions of coccidiosis were found in this group of chickens.
TOXICITY or SULFONAMIDES FOR YOUNG CHICKENS
The results presented herein indicate that sulfamerazine or SS2 in the concentrations used produced no alterations considered irreparable in the histomorphology of the organs examined. No significant changes in the weight increments were noted and hematological studies revealed no noteworthy effects on the hematopoietic system. Studies of the biood to determine the presence of methemoglobin were not done, but since high blood concentrations were not maintained for any considerable period and no cyanosis was noted, we felt such studies to be unnecessary. It will be noted that mild nephrosis was found in 5 of the 20 drug-treated birds. Since 2 of the control chickens showed
similar lesions, there exists the possibility that these changes may have been present before the birds were placed on test. Four of the 10 birds given sulfamerazme alone were found to have lesions in the myocardium. I t seems unlikely that these diffuse exudative lesions were due to the sulfonamide treatment since they did not occur in those birds treated with SS2 and since a similar lesion occurred in one of the control birds. Similar lesions of the myocardium have been noted (Stubbs) in chickens that have not been subjected to sulfonamide therapy. The age of the birds is probably the explanation for the irregular occurrence of metaplasia of the hematopoietic tissue in the liver, kidney, spleen and rarely in the pancreas and gastrointestinal tract. No correlation between such metaplasia, activity of the bone marrow, and sulfonamide concentrations could be shown. Attention may be called to the incidence of coccidial lesions in the untreated control chickens. All of these birds showed light infections and no attempt was made to determine the species of Eimeria present. The fact that of the 20 sulfonamide treated chickens only one contained coccidial forms may indicate some therapeutic efficacy for the drugs used. However, since coccidial infections are known to be self-limited, of short duration, and, depending upon the species, may show little or no pathologic change (Tyzzer, 1929-1932), conclusions as to therapeutic efficiency in these experiments are not justified. Conclusions: The addition of sulfamerazine (0.5 percent or 1.0 percent) or SS2 (a mixture of sodium sulfamerazine 'Sulfathalidine' and sodium carbonate) (0.5 percent or 1.0 percent) to the diet for 14 days did not retard significantly the growth rate of young, growing chickens. No significant change in the hematology
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tration for sulfamerazine of a dosage of 0.25 gm. every 12 hours was 41.7 mgm. per 100 c c , a concentration approximately 40 percent greater than that produced by feeding the same drug as 1.0 percent of the diet. Horton-Smith and Taylor (1943) allowed chickens to have access to a saturated solution of sulfamethazine as drinking water for IS days and, although no toxicity was mentioned, there were apparently no noteworthy lesions found at autopsy. On the other hand, Hawkins and Kline, (1945) reported a marked toxic effect in those birds fed 1.0 percent sulfamethazine in the diet as evidenced by their failure to gain weight. No hematologic or pathologic studies were reported. Other workers (Levine, 1940; Delaplane and Stuart, 1941;. Heiman, 1943) have found no toxic effects following the administration of sulfapyridine (0.7 percent) and sulfathiazole in concentrations of from 0.4 percent to 3.3 percent. The reports on sulfaguanidine are conflicting, although Beach (1943) showed an adverse effect on the weight gain of his birds given sulfaguanidine (0.5 percent) in the mash.
63
64
PAUL A. MATTIS, S. E. MCKINNEY, E. A. PATCH, AND E. L. WILLIAMS
REFERENCES
Beach, J. R., 1943. Sulfaguanidine for cecal coccidiosis. Vet. Med. 38:147. Bratton, A. C , and E. K. Marshall, Jr., 1939. A new coupling component for sulfanilamide determinations. J. Biol. Chem. 128: 537-550. Delaplane, J. P., and H. O. Stuart, 1941. Chemotherapeutic value of sulfathiazole in preventing and treating infectious coryza (hemophilus gallinarum infection) in chickens. J. Am. Vet. M. A. 99:41-^2. Graham, Robert. Personal communication. Hawkins, Philip A., and E. E. Kline, 1945. Treatment of cecal coccidiosis with sulfamethazine. Poultry Science 24: 277-281. Hawkins, Philip A. Personal communication.
Heiman, Victor, 1943. Sulfathiazole for cold in chickens. Vet. Med. 38:26-28. Horton-Smith, C , and E. L. Taylor, 1943. Saturated solution of sulphamethazine as a substitute for drinking water in the treatment of caecal coccidiosis in chickens—a preliminary note. Vet. Rec. (London) 55: 109-110. Levine, P. P., 1939. The effect of sulfanilamide on the course of experimental avian coccidiosis. Cornell Vet. 29:309-320. , 1940. Effect of sulfapyridine on experimental avian coccidiosis. J. Parasitol. 26: 233235. Marshall, E. K., Jr., J. T. Litchfield, Jr., and H. J. White, 1940. The comparative therapeutic activity of sulfanilamide, sulfapyridine, and diaminosulfone in streptococcus infections in mice. J. Pharmacol, and Exp. Therap, 69:89-102. Richardson, Arthur P., 1939. Cyanosis in chickens and mice induced by sulfanilamide. Bull. Johns Hopkins Hosp. 45:445-455. Rosenthal, Sanford M., 1939. Studies on Chemotherapy. VIII. Some toxic effects of repeated administration of sulfanilamide and sulfanilyl sulfanilamide ("Di-sulfanilamide") to rabbits and chickens. Pub. Health Rep. 54: 95-106. Stubbs, E. L. Personal communication. Swales, W. E., 1944. On the chemotherapy of caecal coccidiosis (Eirheria Tenella) of chickens. Canad. J. Research, D 22:131-140. Swales, W. E. Personal communication. Thorp, W. T. S. Personal communication. Tyzzer, E. E., 1929. Coccidiosis in gallinaceous birds. Am. J. Hyg. 10: 269-282. Tyzzer, E. E., H. Theiler, and E. E. Jones, 1932. Coccidiosis in gallinaceous birds. I I . A comparative study of species of Eimeria of the chicken. Am. J. Hyg. 15:319-393. Welch, A. D., Paul A. Mattis, A. R. Latven, W. M. Benson, and Ethol H . Shiels, 1943. Sulfamerazine (2-sulfanilamido-4-methylpyrimidine) I. A comparison of sulfamerazine with sulfadiazine on the basis of absorption, excretion and toxicity. J. Pharmacol, and Exper. Therap. 77: 357-391.
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of young chickens was observed after 14 days on such diets. The average concentrations of free sulfonamides in the whole blood of chickens given diets containing 0.5 percent sulfamerazine, 1.0 percent sulfamerazine, 0.5 percent SS2, or 1.0 percent SS2 were respectively: 14.7, 29.1, 10.7 and 21.2 mgm. per 100 cc. The average amount of acetylated drug remained low, not exceeding 9.0 percent of the total sulfonamide concentration determined. No striking pathologic, changes were found in any of the chickens that had been sacrificed after 14 days on the various diets containing these sulfonamides. It appears likely, from the data presented, that the administration, for therapeutic purposes, of sulfamerazine or SS2 in a dosage of 1 percent or less of the diet, should not give rise to serious toxic manifestations in chickens.
T
HE interest aroused by the treatment of coccidiosis in poultry with sulfanilamide (Levine, 1939) led to studies with other compounds in an attempt to find a sulfonamide that would be effective without causing marked toxic manifestations. Certain preliminary studies (Swales, 1944) using sulfamerazine (4-methyl-2sulfanilamidopyrimidine) and other studies using a mixture of sodium sulfamerazine 80 percent, 'Sulfathalidine' (phthalylsulfathiazole) 10 percent and sodium carbonate 10 percent (SS2) (Graham, Hawkins, Swales, Thorp) suggest that this mixture may be of value in the treatment of infections in poultry caused by Eimeria. These findings have prompted a study of the effects on young chickens of a diet containing sulfamerazine or a mixture of sodium sulfamerazine, 'Sulfathalidine' and sodium carbonate (SS2). The only previous study of the toxic effects of sulfamerazine for chickens was devoted primarily to the effects on nervous tissue (Welch etal., 1943). Procedure: Thirty-five chicks, 32 days old, were divided into 5 groups of 7 each and allowed free access to a diet1 for 14
days. One group served as a control; the other 4 groups received the same diet to which had been added the sulfonamides as follows: 0.5 percent or 1.0 percent sulfamerazine, 0.5 percent or 1.0 percent SS2. The birds were weighed, and blood samples taken for the determination of the sedimentation rate, hematocrit reading, total erythrocyte counts, differential leucocytic counts, and concentration of hemoglobin at the beginning, on the seventh day and at the end of the experiment. Determinations of the free and total sulfonamide concentrations in the blood were made at intervals during the test, using the technique of Bratton and Marshall (1939, 1940). All of the control chickens and 5 birds from each of the treated groups were sacrificed on the fourteenth day and the following tissues were taken for histopathological examinations: lung, heart, liver, spleen, kidney, proventriculus, gizzard, duodenum, cecum, bone marrow, lymph node, and in certain birds, thyroid, adrenal, and testis or ovary. Since nervous tissues from chickens receiving sulfamerazine had been studied previously (Welch et al., 1943) no sections of this tissue were taken. Results: During the test, observations were made of the general health and welfare of the birds. Although a few chicks
* Present address: The Department of Pharmacology, University of Florida School of Pharmacy, Gainesville, Fla. 1 Purina 'Startena' and cracked corn. The drugs were added to the 'Startena.' 59
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on May 29, 2015
(Received for publication, August 23, 1945)
60
PAUL A. MATTIS, S. E. MCKINNEY, E. A. PATCH, AND E. L.
WILLIAMS
TABLE 1.—Average weight and percentage increase The data were treated statistically 2 to
in weight of chickens fed a diet containing sulfamerazine (0.5 percent or 1.0 percent) or SS2* (0.5 percent determine if the differences between the or 1.0 percent) weight increments of the various treated Initial Day Wt. in gms.
Wt. in gms.
Percentage increase
196
302
54
181
302
67
176 189 201
261 320 292
51 69 45
* A mixture of sodium sulfamerazine, 80 percent, 'Sulfathalidine,' 10 percent and sodium carbonate, 10 percent.
appeared somewhat unthrifty, all birds ate well, gained weight and showed no manifestations of toxicity during the 14 days of the experiment. Table 1 presents the average initial and final weights of the birds and the average percentage gains in weight during 14 days.
In Table 2 have been tabulated the hematological findings for these birds. Attention may be called to the regularity of the total number of erythrocytes and 2 We are indebted to Mr. J. L. Ciminera for Ms kindness in making the statistical evaluations.
TABLE 2.—Certain hematologicalfindingsin chickens administered sulfonamides in the diet for 14 days
Drug fed
Day
Differential Leucocyte Count*— Erythro- Sedimen- Hemato- HemoPercentage tation crit cytes globin Rate per(in (gm./lOO Basocent millions) (mm./ cc.) Hetero- Lymph- Mono- Eosinohr.) phils phils phils cytes cytes
Control
1st 7th 14th
2.36 2.4 2.52
2 7 2
28 33 32
10.0 10.0 10.9
21 20 19
65 64 64
7 9 11
3 2 2
4 5 4
0.5 percent Sulfamerazine .
1st 7th 14th
2.46 2.44 2.22
4 13 4
34 30 28
10.9 10.7 9.9
18 24 10
64 65 81
12 6 6
2 2 1
4 • 3 2
1.0 percent Sulfamerazine
1st 7th 14th
2.44 2.82 2.68
3 2 2
34 34 31
11.3 12.3 11.0
14 21 18
69 60 75
11 9 3
2 3 1
4 7 3
0.5 percent SS2
1st 7th 14th
2.51 2.52 2.30
9 12 4
34 31 28
11.5 10.9 10.3
24 17 17
58 70 76
9 6 5
5 2
4 5 2
1.0 percent SS2
1st 7th 14th
2.50
5 5 5
31 30 27
10.2 10.9 10.2
15 23 11
71 65 82
8 5 5
2 2
4 5 2
2.41
* Each figure represents the counting of 200 cells.
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Control Sulfamerazine 0.5 percent Sulfamerazine 1.0 percent SS2 0.5 percent SS2 1.0 percent
groups were significant when compared with the untreated control group. There was no significant difference between the percentage increases in weight of the treated groups, except for those birds given SS2 (0.5 percent). The greater weight increase of this group was found to be significant, whereas the weight increase of those birds given 0.5 percent sulfamerazine was found to be of borderline significance. Although these chicks fed 1.0 percent sulfamerazine or 1.0 percent SS2 appeared to exhibit a slowing of the growth rate, the difference between their weight increases and those of the control birds was not statistically significant.
14th Day
TOXICITY OF SULFONAMIDES FOR YOUNG CHICKENS
61
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3.—Average concentration of sulfonamides* the concentration of hemoglobin through- (inTABLE mgm. per 100 cc.) in the whole blood of chickens out the test. Treatment of these data by fed a diet containing sulfamerazine (0.5 percent orl.0 means of Fisher's t test shows that there percent) or SS2 (0.5 per cent or 1.0 percent) were no significant differences between the SulfaSulfaSS2 total numbers of erythrocytes at the bemerazine merazine SS2 1.0 0.5 Day 1.0 O.S ginning of the test and at its completion. percent percent percent percent It was found also that there were no sig10.6 29.2 20.8 Ft 15.0 2nd nificant changes in the hemoglobin con1.0 1.2. 1.2 centration, sedimentation rate or volume ct 20.4 11.8 33.1 F 15.0 6th of packed cells during the experiment. 0.4 0.6 1.3 C 0.4 Examination of the differential leuco10.0 . 21.9 24.3 F 14.8 cytic counts from these birds revealed 10th 0.7 0.4 0.2 C 0.4 only a moderate reduction in the per10.3 21.6 29.6 F 14.1 centage of heterophiles with a concomi- 14th 1.6 1.6 1.5 C 1.5 tant increase that may have been either 10.7 21.2 29.1 absolute or relative in the percentage of Average F 14.7 0.9 1.0 1.1 C 0.6 lymphocytes. The proper interpretation of these results is uncertain. * The sulfonamide concentrations have been exin terms of sulfamerazine. It will be seen (Table 3) that there was pressed Ff=Free. CJ=Conjugated. no general tendency toward an increase of the concentration of sulfonamides in the blood with continued feeding of the drug. the sulfonamide concentrations attained In all cases the blood concentrations re- by those chickens receiving a diet conmained relatively constant, the average taining 0.5 percent sulfamerazine or 0.5 sulfonamide concentration for those birds percent SS2 demonstrates that levels in given 1.0 percent of the drug in each case excess of 6 mgm. per 100 cc. were atbeing approximately twice the concentra- tained without evidence of toxicity. tion attained in the chicks receiving the At autopsy none of the birds demon0.5 percent drug diet. strated any gross lesions. HistopathologiThe amount of conjugated sulfonamide cal examinations of the tissues taken from that occurred in these chickens remained these birds revealed the following changes. Controls: Of 7 livers examined, one quite low. The average amount of drugs occurring in the acetylated form for all showed moderate dilatation of the central birds was 5.1 percent of the total with a veins and sinusoids, and 2 showed considerable vacuolation of the central herange of 0 percent to 13.5 percent. patic parenchyma, with moderate metaHawkins and Kline (1945) have shown that 1.0 percent sulfamethazine (4-6 di- plasia of the perivascular islands of hemamethyl-2-sulfanilamidopyrimidine) in the topoietic tissue. Three of 7 kidneys had diet produced an average concentration foci of hematopoietic tissue showing of sulfonamide in the blood of 7.7 mgm. myeloid metaplasia; 2 of these 7 demonper 100 cc. for normal birds and 6.5 mgm. strated mild parenchymatous degeneraper 100 cc. for their infected birds. They tion of the tubular epithelium. The spleens considered sulfamethazine concentrations were essentially normal, except for 2 that in the neighborhood of 6 mgm. per 100 cc. were considered to show mild lymphoid necessary to prevent loss of birds infected hyperplasia, as was true also of one lymph with coccidia. Reference to the data on node of the seven examined. I n one section
62
PAUL A. MATTIS, S. E. MCKINNEY, E. A. PATCH, AND E. L. WILLIAMS
Sulfamerazine—1.0 percent: Three of 5 livers showed vacuolation of the hepatic parenchyma; one had moderately dilated sinusoids and central veins. One of the kidneys exhibited nephrosis with detritus in the lumens of the tubules. Two sections of myocardium had areas of infiltration with small round cells. These differed from the diffuse exudative lesions seen in the former group of chickens. One chick of the 5 examined was found to have an occasional asexual form of Eimeria in the caecal epithelium. Of 2 bone marrow sections examined, one was moderately hyperplastic and the other was normal. All other tissues examined were negative. SS2—0.5 percent: There was moderate vacuolation of hepatic cells in one liver section, dilatation of the sinusoids and central veins in two sections, and occasional areas of hemorrhage in 3 livers.
Three of the 5 kidneys examined showed mild nephrosis and occasional foci of small round cell infiltration. The bone marrows from all 5 chickens were examined and found to be normally active. No lesions were observed in any of the other tissues examined SS2—1.0 percent: Only one liver section of 5 examined showed mild vacuolation of the hepatic cells and dilatation of the sinusoids. Another chick had a mild nephrosis and occasional areas of infiltration with small round cells. Except for moderate metaplasia of the hematopoietic foci in one of the chickens all other tissues were uniformly negative. Four bone marrow sections showed normal activity Discussion: Previous investigators have indicated that the administration of sulfonamides, either in the diet or orally in the form of tablets or capsules, has an adverse effect on the normal growth increment of chickens. Levine (1939) stated that sulfanilamide added to the diet of growing chickens in concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 percent by weight prevented a normal weight gain. Sulfonamide concentrations attained under these conditions were not given. Rosenthal (1939) administered sulfanilamide to chickens in doses of 0.5, 1.0, and 2.0 gm. per kg. daily for varying periods and reported either temporary or permanent weight loss. Although no sulfonamide concentrations were reported, the blood levels attained must have been considerable in view of levels obtained in comparable studies by Richardson (1939). Investigations in this laboratory (Welch et al., 1943) with sulfamethylthiazole, sulfadiazine, sulfamerazine and sulfamethazine indicated that, with high blood concentrations, chickens either lost weight or did not gain weight at rates similar to those of untreated control birds. In these studies the average 4 hour blood concen-
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of myocardium there were foci of infiltration that may be described as diffuse exudative myocarditis. Five of the 7 birds were found to have coccidiosis, although none of the lesions found was severe. All other tissues examined were normal. The bone marrows were normal. Experimental Chickens :Sulfamerazine— 0.5 percent: The livers of 5 chickens were examined; considerable vacuolation of the hepatic parenchyma was found in two and no other changes were observed. The kidneys were essentially normal. One of 5 spleens showed moderate lymphoid hyperplasia. Two of the 5 sections of myocardium were found to show small epicardial areas of diffuse exudative myocarditis. Of 5. bone marrows examined all were normally active, one showing a moderate increase in erythropoiesis. The other tissues examined (lymph nodes, pancreas, testes or ovaries, lungs, adrenals, thyroids and gastrointestinal tract) were essentially normal. No lesions of coccidiosis were found in this group of chickens.
TOXICITY or SULFONAMIDES FOR YOUNG CHICKENS
The results presented herein indicate that sulfamerazine or SS2 in the concentrations used produced no alterations considered irreparable in the histomorphology of the organs examined. No significant changes in the weight increments were noted and hematological studies revealed no noteworthy effects on the hematopoietic system. Studies of the biood to determine the presence of methemoglobin were not done, but since high blood concentrations were not maintained for any considerable period and no cyanosis was noted, we felt such studies to be unnecessary. It will be noted that mild nephrosis was found in 5 of the 20 drug-treated birds. Since 2 of the control chickens showed
similar lesions, there exists the possibility that these changes may have been present before the birds were placed on test. Four of the 10 birds given sulfamerazme alone were found to have lesions in the myocardium. I t seems unlikely that these diffuse exudative lesions were due to the sulfonamide treatment since they did not occur in those birds treated with SS2 and since a similar lesion occurred in one of the control birds. Similar lesions of the myocardium have been noted (Stubbs) in chickens that have not been subjected to sulfonamide therapy. The age of the birds is probably the explanation for the irregular occurrence of metaplasia of the hematopoietic tissue in the liver, kidney, spleen and rarely in the pancreas and gastrointestinal tract. No correlation between such metaplasia, activity of the bone marrow, and sulfonamide concentrations could be shown. Attention may be called to the incidence of coccidial lesions in the untreated control chickens. All of these birds showed light infections and no attempt was made to determine the species of Eimeria present. The fact that of the 20 sulfonamide treated chickens only one contained coccidial forms may indicate some therapeutic efficacy for the drugs used. However, since coccidial infections are known to be self-limited, of short duration, and, depending upon the species, may show little or no pathologic change (Tyzzer, 1929-1932), conclusions as to therapeutic efficiency in these experiments are not justified. Conclusions: The addition of sulfamerazine (0.5 percent or 1.0 percent) or SS2 (a mixture of sodium sulfamerazine 'Sulfathalidine' and sodium carbonate) (0.5 percent or 1.0 percent) to the diet for 14 days did not retard significantly the growth rate of young, growing chickens. No significant change in the hematology
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tration for sulfamerazine of a dosage of 0.25 gm. every 12 hours was 41.7 mgm. per 100 c c , a concentration approximately 40 percent greater than that produced by feeding the same drug as 1.0 percent of the diet. Horton-Smith and Taylor (1943) allowed chickens to have access to a saturated solution of sulfamethazine as drinking water for IS days and, although no toxicity was mentioned, there were apparently no noteworthy lesions found at autopsy. On the other hand, Hawkins and Kline, (1945) reported a marked toxic effect in those birds fed 1.0 percent sulfamethazine in the diet as evidenced by their failure to gain weight. No hematologic or pathologic studies were reported. Other workers (Levine, 1940; Delaplane and Stuart, 1941;. Heiman, 1943) have found no toxic effects following the administration of sulfapyridine (0.7 percent) and sulfathiazole in concentrations of from 0.4 percent to 3.3 percent. The reports on sulfaguanidine are conflicting, although Beach (1943) showed an adverse effect on the weight gain of his birds given sulfaguanidine (0.5 percent) in the mash.
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PAUL A. MATTIS, S. E. MCKINNEY, E. A. PATCH, AND E. L. WILLIAMS
REFERENCES
Beach, J. R., 1943. Sulfaguanidine for cecal coccidiosis. Vet. Med. 38:147. Bratton, A. C , and E. K. Marshall, Jr., 1939. A new coupling component for sulfanilamide determinations. J. Biol. Chem. 128: 537-550. Delaplane, J. P., and H. O. Stuart, 1941. Chemotherapeutic value of sulfathiazole in preventing and treating infectious coryza (hemophilus gallinarum infection) in chickens. J. Am. Vet. M. A. 99:41-^2. Graham, Robert. Personal communication. Hawkins, Philip A., and E. E. Kline, 1945. Treatment of cecal coccidiosis with sulfamethazine. Poultry Science 24: 277-281. Hawkins, Philip A. Personal communication.
Heiman, Victor, 1943. Sulfathiazole for cold in chickens. Vet. Med. 38:26-28. Horton-Smith, C , and E. L. Taylor, 1943. Saturated solution of sulphamethazine as a substitute for drinking water in the treatment of caecal coccidiosis in chickens—a preliminary note. Vet. Rec. (London) 55: 109-110. Levine, P. P., 1939. The effect of sulfanilamide on the course of experimental avian coccidiosis. Cornell Vet. 29:309-320. , 1940. Effect of sulfapyridine on experimental avian coccidiosis. J. Parasitol. 26: 233235. Marshall, E. K., Jr., J. T. Litchfield, Jr., and H. J. White, 1940. The comparative therapeutic activity of sulfanilamide, sulfapyridine, and diaminosulfone in streptococcus infections in mice. J. Pharmacol, and Exp. Therap, 69:89-102. Richardson, Arthur P., 1939. Cyanosis in chickens and mice induced by sulfanilamide. Bull. Johns Hopkins Hosp. 45:445-455. Rosenthal, Sanford M., 1939. Studies on Chemotherapy. VIII. Some toxic effects of repeated administration of sulfanilamide and sulfanilyl sulfanilamide ("Di-sulfanilamide") to rabbits and chickens. Pub. Health Rep. 54: 95-106. Stubbs, E. L. Personal communication. Swales, W. E., 1944. On the chemotherapy of caecal coccidiosis (Eirheria Tenella) of chickens. Canad. J. Research, D 22:131-140. Swales, W. E. Personal communication. Thorp, W. T. S. Personal communication. Tyzzer, E. E., 1929. Coccidiosis in gallinaceous birds. Am. J. Hyg. 10: 269-282. Tyzzer, E. E., H. Theiler, and E. E. Jones, 1932. Coccidiosis in gallinaceous birds. I I . A comparative study of species of Eimeria of the chicken. Am. J. Hyg. 15:319-393. Welch, A. D., Paul A. Mattis, A. R. Latven, W. M. Benson, and Ethol H . Shiels, 1943. Sulfamerazine (2-sulfanilamido-4-methylpyrimidine) I. A comparison of sulfamerazine with sulfadiazine on the basis of absorption, excretion and toxicity. J. Pharmacol, and Exper. Therap. 77: 357-391.
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of young chickens was observed after 14 days on such diets. The average concentrations of free sulfonamides in the whole blood of chickens given diets containing 0.5 percent sulfamerazine, 1.0 percent sulfamerazine, 0.5 percent SS2, or 1.0 percent SS2 were respectively: 14.7, 29.1, 10.7 and 21.2 mgm. per 100 cc. The average amount of acetylated drug remained low, not exceeding 9.0 percent of the total sulfonamide concentration determined. No striking pathologic, changes were found in any of the chickens that had been sacrificed after 14 days on the various diets containing these sulfonamides. It appears likely, from the data presented, that the administration, for therapeutic purposes, of sulfamerazine or SS2 in a dosage of 1 percent or less of the diet, should not give rise to serious toxic manifestations in chickens.