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Tissue Clearance of Rofenaid® in Chickens and Turkeys
Tissue Clearance of Rofenaid® in Chickens and Turkeys J. F E L L I G , J. W E S T H E I M E R , M . J. W A L S H AND W. L. M A R U S I C H Chemical Research Department, Hoffmann-La Roche Inc., Nutley, New Jersey, 07110 (Received for publication April 2, 1971)
R
STUDY 1 I t was the purpose of this study to determine Rofenaid® tissue levels and tissue clearance following administration to broiler chickens throughout their raising period at the recommended use level of 0.02% in the feed. Day-old Peterson Cross chicks were assembled into 28 uniform groups of 10 birds each (5 females and 5 males) based on body weight. Eleven groups were placed for 8 weeks on a diet free of any additives or medicants except for 0.02% Rofenaid®.
T h e remaining 17 control groups received the same ration b u t without the 0.02% Rofenaid®. These additional unmedicated groups provided an extra source of control tissue for validation of the analytical methods. For the first 5 weeks, the birds received a high-energy broiler starter ration and for the remaining 3 weeks a broiler finisher ration. T h e birds were kept in electrically-heated, thermostatcontrolled batteries for the first 4 weeks and were transferred to regular finishing units for the last 4 weeks. Feed intake, drug intake, and performance of the birds are shown in Table 1. At the end of the feeding period, groups of 10 birds (equal number of males and females) were sacrificed at 0, 1, 2, 3, 4, and 5 days of drug withdrawal and the following tissues collected for analysis: muscle, liver, skin, kidney, and blood plasma. T h e tissues were held in the deep-freeze prior to analysis. T h e tissues from 10 birds were pooled for each t r e a t m e n t and withdrawal time and assays carried out in quadruplicate on these pooled samples. T h e results of the residue assays are also summarized in Table 1. At 0 days of drug withdrawal, the levels of S D M were higher in all tissues than the levels of ormetoprim. T h e highest levels of both compounds were found in kidney (2.75 and 0.43 p.p.m. respectively) and the lowest in skin (0.15 and 0.05 p.p.m., respectively). After 1 day of drug withdrawal, only negligible levels ( < 0 . 1 p.p.m.) of either compound were found in the tissues and a t 2 days of drug withdrawal neither compound could be detected.
1777
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OFENAID®, a combination of sul• fadimethoxine (4-sulfanilamido-2,6dimethoxypyrimidine) and ormetoprim (2,4-diamino-5-(4,5-dimethoxy-2-methylbenzyl)-pyrimidine) in a ratio of 5 : 3 , has been shown to be an effective and safe antibacterial and coccidiostat for broilers and turkeys (Mitrovic et al., 1969a, b, 1971a, b ; Marusich et al., 1969, 1971). T r e a t m e n t of food-producing animals with Rofenaid® has created the need for tissue residue studies capable of accurately measuring the disappearance of both components of the drug from the animal body. A review of these studies forms the subject of the present report. Assays for sulfadimethoxine (SDM) and ormetoprim were carried out in our laboratory by previously described tissue residue procedures sensitive to 0.1 p.p.m. (Fellig and Westheimer, 1968; Fellig et al., 1971). Arsenic was determined by an outside contract laboratory (Hazleton Laboratories, Inc., Falls Church, Virginia), using a standard method (Food Chemicals Codex, 1963).
1778
J. F E L L I G , J. W E S T H E I M E R , M . J. W A L S H AND W. L. MARTJSICH
TABLE 1.—Performance of broiler chickens receiving Rofenaid (Study I) Intake of Rofenaid Diet
Basal Rofenaid 0.02%
Initial wt., g.
8-wk. gain, g.
8-wk. wt., g.
%
gain
Feed intake, g-
Feed conv.
Total, m
37
1,420
1,383
100
2,927
2.116
37
1,455
1,418
102.5
3,009
2.122
£-
Avera
§e
mg.
: daily
Mortal.
,ng./kg. 2/170
601.8
10.7
14.3
4/110
Clearance of Rofenaid in IDroiler chickens (Study 1) drawal
Muscle
Kidney
Sk in
Liver
Ormetoprim
SDM
Ormetoprim
SDM
Ormetoprim
SDM
Ormetoprim
0 1 2 3 4 5
0.13 0 0 0 0 0
0.46 0 0 0 0 0
0.35 0 0 0 0 0
0.89 0.09 0 0 0 0
0.05 0 0 0 0 0
0.15 0 0 0 0 0
0.43 0.07 0 0 0 0
S D M
2.75 0 0 0 0.5 0
Ormeto " SDM prim 0.05 0 0 0
1.53 0 0 0
0
0
Each number represents the average from 4 assays on a tissue sample pooled from 10 birds. Values in p.p.m. STUDY 2
This was an extension of Study 1, designed to show whether the administration to broiler chickens of Rofenaid® a t 0.02% in the feed, in combination with either arsanilic acid or 3-nitro-4-hydroxyphenylarsonic acid a t their respective recommended use levels of 0 . 0 1 % and 0.005% in the feed, influenced the tissue clearance pattern. Day-old Peterson Cross chicks were assembled into 36 uniform groups of 12 birds each (6 females and 6 males) based on body weight. Six replicates of 12 birds each were assigned to each treatment group, with 6 replicates maintained on the control ration free of additives or medicants. T h e birds were maintained as described for Study 1. Feed intake, drug intake, and performance of the birds are shown in Table 2. At the end of the feeding period, 4 to 9 birds from each treatment group were sacrificed at 0, 1, 2, 3, 4, and 5 days of drug withdrawal and the following tissues collected for analysis:
muscle, liver, skin and fat, kidney, and blood. T h e tissues were stored in the deepfreeze prior to analysis. T h e assay results for the treatment group receiving Rofenaid® only, shown in Table 3, confirm the findings of Study 1. Measurable amounts of both drugs were found at 0 days of drug withdrawal, the highest levels being in the kidney (SDM 3.3 p.p.m., ormetroprim 1 p.p.m.). At 1 day of drug withdrawal, drug levels, whenever present, dropped below 0.1 p.p.m., and following 2 days of drug withdrawal neither compound was detected. As seen in Tables 4 and 5, tissue levels of Rofenaid® were not affected b y either of the arsenicals and tissue clearance was complete after 2 days of drug withdrawal. Arsenic was detected only in liver and kidney and arsenic levels were unaffected by the simultaneous feeding of Rofenaid®. At 0 days of drug withdrawal, average arsenic levels in liver and kidney were 0.9 and 1.3 p.p.m. respectively in t r e a t m e n t group 3, 1.1 and 1.3 p.p.m. in group 4, 1.0
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days
Plasma
1779
TISSUE CLEARANCE OF ROFENAID
and 1.1 p.p.m. in group 5; and 0.9 and 1.0 p.p.m. in group 6. All treatment groups were free of arsenic within the required drug withdrawal period of 5 days. STUDY 3
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At the end of the 13-week treatment period, ten birds each were sacrificed at 0, 2 and 3 days of drug withdrawal and nine birds each at 4 and 5 days of drug withdrawal. Samples of muscle, liver, skin and fat, kidney, and blood were collected for chemical analysis and stored in the deepfreeze. The results of the residue assays are summarized in Table 7. As had been the case with broiler chickens, levels of SDM at 0 days of drug withdrawal were markedly higher in all tissues than levels
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I t was the purpose here to determine tissue levels and tissue clearance of Rofenaid® in turkeys, following prolonged administration in the feed at the recommended use level of 0.01%. Two-day old Broad Breasted White poults were assembled into 12 uniform groups of 10 birds each (5 females and 5 males) based on body weight. Six replicates of 10 poults were placed for 13 weeks on a diet free of any medicants except for Rofenaid® at a level of 0.01%. The other 6 replicates were fed the control ration without 0.01% Rofenaid®. During the first 4 weeks, the birds were held in electrically-heated, thermostat-controlled batteries with raised wire floors. At the end of this period, the birds were transferred to growing units with raised wire floors and no additional heat. At 8 weeks, the birds were placed into floor pens where they were kept until the end of the experiment. A turkey starter ration with 28% protein was fed for the first 8 weeks, and a turkey grower ration with 25% protein for the last 5 weeks. Feed intake, drug intake and performance of the birds are shown in Table 6.
1780
J. FELLIG, J. WESTHEIMER, M. J. WALSH AND W. L. MARUSICH TABLE 3.—Clearance of Rofenaid in broiler chickens (Study 2)
Withdrawal time, days
Muscle
Bird if
( W o 0
71 101 129 74
0 0 0 0
1
79; 92; 15 459 701; 759; 32 772; 735
0 0 0 0
2
13; 60 68: 161 99; 103 186
3
06 08 08 09
Liver
S D M
0 0 0 0
48 76 61 41
Onneto-
Skin and fat S D M
Ormetoprim 06 06 05 05
g D M
0.30 0.49 0.37 0.33
0.95 1.58 0.95 0.88
0 0 0 0
0 0 0 0
0 0 0 0
0.08 0.07 0.08 0.07
0 0 0 0
0 0.13 0 0
0 0 0 0
0 0. 07 0 0
0 0 0 0
0 0 0.06 0
0 0 0 0
0 0 0 0
603; 607 791 778 770; 631
0 0 0 0
0 0 0 0
0 0 0 0
0.05 0 0 0
0 0 0 0
0 0 0 0
4
454 644 694 727
0 (1 0 0
0 0 (1 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
5
11 159 215 108
0 0
0 0
n 0
n 0
0 II 0 0
0 0 0 0
0 0 0 0
0 0 0 0
1 02
0 05
0
0
0
0
S D M
Ormetoprim
SDM
3.32
0 0 0 0
0.77 1.38 1.13 0.81
0.07
0 0 0 0
0 0 0 0
0
0 0 0 0
0 0 0 0
0
0 0 0 0
0 0 0 0
0
0 0 0 0
0 0 0 0
0
0 0 0 0
0 0 0 0
" Pooled samples.
TABLE 4.—Clearance of Rofenaid in broiler chickens receiving arsanilic acid (Study 2)
Withdrawal time, days
0
1
Muscle
Bird #
64 49 69 115 96; 673 22;674 88; 675 28; 664
Skin and fat
Liver SDM
Ormetopnm
0.23 0.09 0.30 0.17
1.00 0.95 0.64 1.05
0 0 0 0
0 0 0 0
SDM
Ormetopnm
0 0 0 0.06
0, T1 0. 41 0. 31 0. 46
0 0 0 0
Ormetopnm
Kidney*
SDM
Ormeto-
SDM
0.05 0.05 0 0
0 0. 26 0. 28 0. 48
0. 48
3. 10
0 64
3. 60
0 0.13 0 0
— 0 0 0
0 0 0 0
0
I)
0
0.08
2
169; 100; 5; 34;
677 667 668 676
0 0 0 0
0 0 0 0
0.09 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0. 13
0
0
0
3
615; 745; 643; 764;
678 679 680 669
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0.06 0 0 0
0 0 0 0
0
0
0
0
4
657;172 792; 180 642; 177 618; 175
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0.11 0 0 0
0 0 0 0
0
0
0
0
672; 719; 638; 666;
115 118 120 116
0 0 0 0
0 0.06 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0
0
0
0
224; 599; 187; 700;
260 251 255 258
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0
0
0
0
5
6
* Pooled samples.
Blood Ormeto-
SDM
0 0 0 0
0.12 1.02 0.80 1.16
0.08 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0.09 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
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0.35 0. 57 0. 6+ 0. 48
Blood
Kidney* Ormetoprim
1781
TISSUE CLEARANCE OF ROFENAID
TABLE 5.—Clearance of Rofenaid in broiler chickens receiving 3-nitro-4-hydroxyphenylarsonic acid {Study Z)
Withdrawal time, days
Ormeto- SDM 0
131 117 591 196
p.p.m. Skin and fat
Muscle
Birds #
Ormeto- SDM
0 0 0 0
0.26 0.41 0.33 0.33
0.39 0.36 0 0
0. 53 0. 88 1. 21 1. 66
0.30 0 0 0.18
0. 18 0. 23 0. 21 0 . 22
710; 740; 742; 786;
953 954 955 841
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0. 05 0
0 0 0 0
0 0 0 0
2
596; 21; 157; 167;
946 947 948 956
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
3
757; 751; 695; 645;
958 959 960 941
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
4
601; 185 799; 186 715; 182 682; 184
0 0 0
0 0 0
—
—
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
300; 24; 121; 173;
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0
n 0
5
6
92 97 91 96
598;244 193; 247 183; 242 996; 248
Ormeto-
SDM
0.43
4.00
0.58
4.60
0
0
0
0.13
0
0
0
0
0
0
0
0
0
0
0
0
0 0
0
0
0
0
—
0
0
—
Ormeto- SDM 0.13 0 0 0
0.62 1.02 1.04 0.82
0 0 0 0
0 0 0 0
—.
0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0
0 0 0 0
— 0
0
* Pooled samples.
of ormetoprim. Sulfonamide levels were highest in the kidney, where they averaged 6.7 p.p.m., followed by the liver with 1.3 p.p.m., blood 1.0 p.p.m., muscle 0.65 p.p.m., and skin and fat 0.52 p.p.m. Levels of ormetoprim at 0 days of drug withdrawal were highest in kidney (average 0.40 p.p.m.) and liver (average 0.14 p.p.m.) and marginal only in the other tissues. No ormetoprim could be detected after 2 days of drug withdrawal. At 2 and
3 days of drug withdrawal, SDM could be detected only in a few isolated samples and drug levels in each case were well below 0.1 p.p.m. No SDM could be detected after 3 days of drug withdrawal. CONCLUSIONS
The above longed broiler
results of the studies described indicate that following the proadministration of Rofenaid® to chickens at the recommended use
TABLE 6.—Performance of turkeys receiving Rofenaid at 0.01% in feed for 13 weeks Intake of Rofenaid Diet
^r birds
Initial wei ht ? > g-
Final wei ht S >
•
r
I
'
„ Gain
Basal
40
56
4,280
4,224
100
Rofenaid 0.01%
48
55
4,600
4,545
107.6
Feed consumed
, Conv. v
>
9,906
2.34
10,502
2.31
Total mg.
1,050.2
Average daily mg.
mg./kg.
11.54
4.96
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1
Blood
Kidney*
Ormeto- SDM
1782
J. FELLIG, J. WESTHEIMEE, M. J. WALSH AND W. L. MARTJSICH TABLE 7.—Clearance of Rofenaid in turkeys p.p.,m.
Withdrawal time, days
Muscle
Bird#
Liver
Ormetoprim
SDM
Ormetoprim
Kidney
Skin and fat
SDM
Ormetoprim
SDM
Ormetoprim
SDM
204 226 647 173 710 728
0.06 0. 08 0 0. 05 0 0
0.8 0.8 0.7 0.8 0.4 0.4
0.05 0.2 0.1 0.07 0.2 0.2
1.5 1.9 1.5 1.5 0.9 0.6
0 0 0 0 0.05 0.08
0.4 0.7 0.7 0.6 0.4 0.3
0.4 0.5 0.4 0.4 0.3 0.4
2
345 327 268 257 950, 948 693
0 0 0. 06 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0.05 0 0 0
0 0 0 0 0 0
0 0 0 0.08 0 0.08
0 O 0 0 0 0
0.05 0 0 0 0 0
3
161, 212 233, 151 711 949 758 274
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0.05
0
0
— 0
— 0
0 0 0
0.06 0 0
0 0 0
0 0 0
— 0
— 0
0
0.05
634 740, 654 692 341, 244 271 299
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
259, 255 726, 326 750 755, 281 639 213
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
—
—
4
5
—
0 0 0 0 0
4.4 6.0 7.2 10.0 4.8 7.6
0 0 0 0 0
SDM
0 0 0 0 0.2 0
1.0 1.3 0.9 1.0 0.9 1.1
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0
0 0 0
—
—
0 0
0 0
0 0
0 0
— 0 0 0
—
0 0 0
level, the birds were clear of the drug 2 days after cessation of treatment. The combined administration of Rofenaid® and either arsanilic acid or 3-nitro-4hydroxy-phenylarsonic acid at their respective recommended use levels did not influence tissue levels or time required for clearance of the drugs. Also, following the prolonged administration of Rofenaid® to turkeys at the recommended use level, the birds were clear of the drug 2 days after cessation of treatment. It is concluded from these data that a 2-day drug withdrawal period will ensure the clearance of the components of Rofenaid® from the tissues of broilers and turkeys following the prolonged feeding of the drug at the recommended use levels.
recommended use level of 0.02% in feed. All tissues were found to be clear of sulfadimethoxine and ormetoprim 2 days after cessation of treatment. When Rofenaid® at the same dose level was administered to broiler chickens in combination with either arsanilic acid at 0.01% in the feed or with 3-nitro-4-hydroxyphenylarsonic acid at 0.005% in the feed, tissue levels of the drugs were unaffected, as was the time required for clearance of the tissues. Rofenaid® was administered to turkeys for a period of 13 weeks at the recommended use level of 0.01% in the feed. All tissues were clear of sulfadimethoxine and ormetoprim 2 days after drug withdrawal.
SUMMARY
Fellig, J., and J. Westheimer 1968. Determination of sulfadimethoxine in animal tissues. J. Agric. Food Chem. 16: 738-740. Fellig, J., J. Westheimer, M. J. Walsh and R. A.
REFERENCES
Rofenaid® was administered to broiler chickens for a period of 8 weeks at the
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0
Blood Ormetoprim
1783
T I S S U E CLEARANCE o r ROFENAID
Saperstein, 1971. Determination of ormetoprim in animal tissues. J. Agric. Feed Chem. in press. Food Chemicals Codex, National Academy of Sciences—National Research Council 1963. Arsenic test, pg. 47. Marusich, W. L., E. Ogrinz, M. Brand and M. Mitrovic, 1969. Safety and compatibility of sulfadimethoxine potentiated mixture (Ro 5-0013), a new broad spectrum coccidiostat-antibacterial, in chickens. Poultry Sci. 48: 217-222. Marusich, W. L., E. F. Ogrinz, B. Hecht and M. Mitrovic, 1971. Safety of sulfadimethoxine potentiated mixture (Rofenaid®), a new broad spectrum coccidiostat antibacterial, in turkeys. Poultry Sci. SO: 512-517.
Mitrovic, M., E. G. Schildknecht and G. Fusiek, 1969a. Anticoccidial activity of sulfadimethoxine potentiated mixture (Ro 5-0013) in chickens. Poultry Sci. 48: 210-216. Mitrovic M., G. Fusiek and E. G. Schildknecht, 1969b. Antibacterial activity of sulfadimethoxine potentiated mixture (Ro 5-0013) in chickens. Poultry Sci. 48: 1151-1155. Mitrovic, M., G. Fusiek and E. G. Schildknecht, 1971a. Antibacterial activity of sulfadimethoxine potentiated mixture (Rofenaid®) in turkeys. Poultry Sci. 50: 525-529. Mitrovic, M., E. G. Schildknecht and G. Fusiek, 1971b. Anticoccidial activity of sulfadimethoxine potentiated mixture (Rofenaid®) in turkeys. Poultry Sci. 50:517-525.
A N N E T T E C. R E Y N O L D S , A. V. D E G U Z M A N 0 AND R. E . CLEGG
Department of Biochemistry, Kansas State University, Manhattan, Kansas 66502 (Received for publication April 3, 1971)
INTRODUCTION
A
PREVIOUS
ported (Counsell et al., 1962) f as twelve to
investigation
demon-
s t r a t e d t h a t oral administration of
triparanol
(Mer-20)
to
laying
hens
fifteen
times
that
of
triparanol
given
orally. SC-10644 g also has been reported to effectively lower plasma cholesterol b y
caused serum cholesterol to rise a n d re-
50%
duced egg formation (Nelson a n d Clegg,
orally for two weeks. Cholestyramine, a
1962).
bile acid sequestrant, administered as 1 %
This
contrasted
rise with
in
serum
results
cholesterol
obtained
from
in normal r a t s when administered
of the diet inhibits plasma rise
1959;
( T e n n e n t et al., 1959). Diethylstilbestrol
Blohm a n d Mackenzie, 1959; and
50%
in
cholesterol-fed
cholesterol
r a t s b y other investigators (Blohm et al.,
cockerels
Kayser, 1960). T h e effectiveness of SC-
causes a rise in liver a n d plasma cho-
12937 e in lowering plasma cholesterol in
lesterol and blood lipid in chickens ( E n -
hypercholesterolemic
tenman et al., 1940; Linsay et al., 1946).
rats
has
been
re-
Effects " Contribution 114, Dept. of Biochemistry, Kansas State Agricultural Experiment Station. b Supported in part by H E 12294-01, National Institutes of Health, Bethesda, Md. c Present address: Dept. of Biochem., Univ. of Santo Tomas, Manila, Philippines. e 20, 25-diazacholestenol dihydrochloride
of
compounds 10644,
and
four
hypocholesterolemic
(triparanol,
SC-12937,
cholestyramine)
on
SC-
serum
f Unpublished data, Ranney, R. E., and Cook, D. L. (1962). G. D. Searle and Co., Chicago. g 2-(l-pyrrolidinyl) ethyl triphenylmethane-4 carboxylate hydrochloride
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Influence of Some Hypocholesterolemic Compounds on the Serum Protein Patterns and Serum Cholesterol of Normal and Diethylstilbestrol-Treated Cockerelsa,b
R
STUDY 1 I t was the purpose of this study to determine Rofenaid® tissue levels and tissue clearance following administration to broiler chickens throughout their raising period at the recommended use level of 0.02% in the feed. Day-old Peterson Cross chicks were assembled into 28 uniform groups of 10 birds each (5 females and 5 males) based on body weight. Eleven groups were placed for 8 weeks on a diet free of any additives or medicants except for 0.02% Rofenaid®.
T h e remaining 17 control groups received the same ration b u t without the 0.02% Rofenaid®. These additional unmedicated groups provided an extra source of control tissue for validation of the analytical methods. For the first 5 weeks, the birds received a high-energy broiler starter ration and for the remaining 3 weeks a broiler finisher ration. T h e birds were kept in electrically-heated, thermostatcontrolled batteries for the first 4 weeks and were transferred to regular finishing units for the last 4 weeks. Feed intake, drug intake, and performance of the birds are shown in Table 1. At the end of the feeding period, groups of 10 birds (equal number of males and females) were sacrificed at 0, 1, 2, 3, 4, and 5 days of drug withdrawal and the following tissues collected for analysis: muscle, liver, skin, kidney, and blood plasma. T h e tissues were held in the deep-freeze prior to analysis. T h e tissues from 10 birds were pooled for each t r e a t m e n t and withdrawal time and assays carried out in quadruplicate on these pooled samples. T h e results of the residue assays are also summarized in Table 1. At 0 days of drug withdrawal, the levels of S D M were higher in all tissues than the levels of ormetoprim. T h e highest levels of both compounds were found in kidney (2.75 and 0.43 p.p.m. respectively) and the lowest in skin (0.15 and 0.05 p.p.m., respectively). After 1 day of drug withdrawal, only negligible levels ( < 0 . 1 p.p.m.) of either compound were found in the tissues and a t 2 days of drug withdrawal neither compound could be detected.
1777
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OFENAID®, a combination of sul• fadimethoxine (4-sulfanilamido-2,6dimethoxypyrimidine) and ormetoprim (2,4-diamino-5-(4,5-dimethoxy-2-methylbenzyl)-pyrimidine) in a ratio of 5 : 3 , has been shown to be an effective and safe antibacterial and coccidiostat for broilers and turkeys (Mitrovic et al., 1969a, b, 1971a, b ; Marusich et al., 1969, 1971). T r e a t m e n t of food-producing animals with Rofenaid® has created the need for tissue residue studies capable of accurately measuring the disappearance of both components of the drug from the animal body. A review of these studies forms the subject of the present report. Assays for sulfadimethoxine (SDM) and ormetoprim were carried out in our laboratory by previously described tissue residue procedures sensitive to 0.1 p.p.m. (Fellig and Westheimer, 1968; Fellig et al., 1971). Arsenic was determined by an outside contract laboratory (Hazleton Laboratories, Inc., Falls Church, Virginia), using a standard method (Food Chemicals Codex, 1963).
1778
J. F E L L I G , J. W E S T H E I M E R , M . J. W A L S H AND W. L. MARTJSICH
TABLE 1.—Performance of broiler chickens receiving Rofenaid (Study I) Intake of Rofenaid Diet
Basal Rofenaid 0.02%
Initial wt., g.
8-wk. gain, g.
8-wk. wt., g.
%
gain
Feed intake, g-
Feed conv.
Total, m
37
1,420
1,383
100
2,927
2.116
37
1,455
1,418
102.5
3,009
2.122
£-
Avera
§e
mg.
: daily
Mortal.
,ng./kg. 2/170
601.8
10.7
14.3
4/110
Clearance of Rofenaid in IDroiler chickens (Study 1) drawal
Muscle
Kidney
Sk in
Liver
Ormetoprim
SDM
Ormetoprim
SDM
Ormetoprim
SDM
Ormetoprim
0 1 2 3 4 5
0.13 0 0 0 0 0
0.46 0 0 0 0 0
0.35 0 0 0 0 0
0.89 0.09 0 0 0 0
0.05 0 0 0 0 0
0.15 0 0 0 0 0
0.43 0.07 0 0 0 0
S D M
2.75 0 0 0 0.5 0
Ormeto " SDM prim 0.05 0 0 0
1.53 0 0 0
0
0
Each number represents the average from 4 assays on a tissue sample pooled from 10 birds. Values in p.p.m. STUDY 2
This was an extension of Study 1, designed to show whether the administration to broiler chickens of Rofenaid® a t 0.02% in the feed, in combination with either arsanilic acid or 3-nitro-4-hydroxyphenylarsonic acid a t their respective recommended use levels of 0 . 0 1 % and 0.005% in the feed, influenced the tissue clearance pattern. Day-old Peterson Cross chicks were assembled into 36 uniform groups of 12 birds each (6 females and 6 males) based on body weight. Six replicates of 12 birds each were assigned to each treatment group, with 6 replicates maintained on the control ration free of additives or medicants. T h e birds were maintained as described for Study 1. Feed intake, drug intake, and performance of the birds are shown in Table 2. At the end of the feeding period, 4 to 9 birds from each treatment group were sacrificed at 0, 1, 2, 3, 4, and 5 days of drug withdrawal and the following tissues collected for analysis:
muscle, liver, skin and fat, kidney, and blood. T h e tissues were stored in the deepfreeze prior to analysis. T h e assay results for the treatment group receiving Rofenaid® only, shown in Table 3, confirm the findings of Study 1. Measurable amounts of both drugs were found at 0 days of drug withdrawal, the highest levels being in the kidney (SDM 3.3 p.p.m., ormetroprim 1 p.p.m.). At 1 day of drug withdrawal, drug levels, whenever present, dropped below 0.1 p.p.m., and following 2 days of drug withdrawal neither compound was detected. As seen in Tables 4 and 5, tissue levels of Rofenaid® were not affected b y either of the arsenicals and tissue clearance was complete after 2 days of drug withdrawal. Arsenic was detected only in liver and kidney and arsenic levels were unaffected by the simultaneous feeding of Rofenaid®. At 0 days of drug withdrawal, average arsenic levels in liver and kidney were 0.9 and 1.3 p.p.m. respectively in t r e a t m e n t group 3, 1.1 and 1.3 p.p.m. in group 4, 1.0
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days
Plasma
1779
TISSUE CLEARANCE OF ROFENAID
and 1.1 p.p.m. in group 5; and 0.9 and 1.0 p.p.m. in group 6. All treatment groups were free of arsenic within the required drug withdrawal period of 5 days. STUDY 3
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At the end of the 13-week treatment period, ten birds each were sacrificed at 0, 2 and 3 days of drug withdrawal and nine birds each at 4 and 5 days of drug withdrawal. Samples of muscle, liver, skin and fat, kidney, and blood were collected for chemical analysis and stored in the deepfreeze. The results of the residue assays are summarized in Table 7. As had been the case with broiler chickens, levels of SDM at 0 days of drug withdrawal were markedly higher in all tissues than levels
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I t was the purpose here to determine tissue levels and tissue clearance of Rofenaid® in turkeys, following prolonged administration in the feed at the recommended use level of 0.01%. Two-day old Broad Breasted White poults were assembled into 12 uniform groups of 10 birds each (5 females and 5 males) based on body weight. Six replicates of 10 poults were placed for 13 weeks on a diet free of any medicants except for Rofenaid® at a level of 0.01%. The other 6 replicates were fed the control ration without 0.01% Rofenaid®. During the first 4 weeks, the birds were held in electrically-heated, thermostat-controlled batteries with raised wire floors. At the end of this period, the birds were transferred to growing units with raised wire floors and no additional heat. At 8 weeks, the birds were placed into floor pens where they were kept until the end of the experiment. A turkey starter ration with 28% protein was fed for the first 8 weeks, and a turkey grower ration with 25% protein for the last 5 weeks. Feed intake, drug intake and performance of the birds are shown in Table 6.
1780
J. FELLIG, J. WESTHEIMER, M. J. WALSH AND W. L. MARUSICH TABLE 3.—Clearance of Rofenaid in broiler chickens (Study 2)
Withdrawal time, days
Muscle
Bird if
( W o 0
71 101 129 74
0 0 0 0
1
79; 92; 15 459 701; 759; 32 772; 735
0 0 0 0
2
13; 60 68: 161 99; 103 186
3
06 08 08 09
Liver
S D M
0 0 0 0
48 76 61 41
Onneto-
Skin and fat S D M
Ormetoprim 06 06 05 05
g D M
0.30 0.49 0.37 0.33
0.95 1.58 0.95 0.88
0 0 0 0
0 0 0 0
0 0 0 0
0.08 0.07 0.08 0.07
0 0 0 0
0 0.13 0 0
0 0 0 0
0 0. 07 0 0
0 0 0 0
0 0 0.06 0
0 0 0 0
0 0 0 0
603; 607 791 778 770; 631
0 0 0 0
0 0 0 0
0 0 0 0
0.05 0 0 0
0 0 0 0
0 0 0 0
4
454 644 694 727
0 (1 0 0
0 0 (1 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
5
11 159 215 108
0 0
0 0
n 0
n 0
0 II 0 0
0 0 0 0
0 0 0 0
0 0 0 0
1 02
0 05
0
0
0
0
S D M
Ormetoprim
SDM
3.32
0 0 0 0
0.77 1.38 1.13 0.81
0.07
0 0 0 0
0 0 0 0
0
0 0 0 0
0 0 0 0
0
0 0 0 0
0 0 0 0
0
0 0 0 0
0 0 0 0
0
0 0 0 0
0 0 0 0
" Pooled samples.
TABLE 4.—Clearance of Rofenaid in broiler chickens receiving arsanilic acid (Study 2)
Withdrawal time, days
0
1
Muscle
Bird #
64 49 69 115 96; 673 22;674 88; 675 28; 664
Skin and fat
Liver SDM
Ormetopnm
0.23 0.09 0.30 0.17
1.00 0.95 0.64 1.05
0 0 0 0
0 0 0 0
SDM
Ormetopnm
0 0 0 0.06
0, T1 0. 41 0. 31 0. 46
0 0 0 0
Ormetopnm
Kidney*
SDM
Ormeto-
SDM
0.05 0.05 0 0
0 0. 26 0. 28 0. 48
0. 48
3. 10
0 64
3. 60
0 0.13 0 0
— 0 0 0
0 0 0 0
0
I)
0
0.08
2
169; 100; 5; 34;
677 667 668 676
0 0 0 0
0 0 0 0
0.09 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0. 13
0
0
0
3
615; 745; 643; 764;
678 679 680 669
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0.06 0 0 0
0 0 0 0
0
0
0
0
4
657;172 792; 180 642; 177 618; 175
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0.11 0 0 0
0 0 0 0
0
0
0
0
672; 719; 638; 666;
115 118 120 116
0 0 0 0
0 0.06 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0
0
0
0
224; 599; 187; 700;
260 251 255 258
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0
0
0
0
5
6
* Pooled samples.
Blood Ormeto-
SDM
0 0 0 0
0.12 1.02 0.80 1.16
0.08 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0.09 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
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0.35 0. 57 0. 6+ 0. 48
Blood
Kidney* Ormetoprim
1781
TISSUE CLEARANCE OF ROFENAID
TABLE 5.—Clearance of Rofenaid in broiler chickens receiving 3-nitro-4-hydroxyphenylarsonic acid {Study Z)
Withdrawal time, days
Ormeto- SDM 0
131 117 591 196
p.p.m. Skin and fat
Muscle
Birds #
Ormeto- SDM
0 0 0 0
0.26 0.41 0.33 0.33
0.39 0.36 0 0
0. 53 0. 88 1. 21 1. 66
0.30 0 0 0.18
0. 18 0. 23 0. 21 0 . 22
710; 740; 742; 786;
953 954 955 841
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0. 05 0
0 0 0 0
0 0 0 0
2
596; 21; 157; 167;
946 947 948 956
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
3
757; 751; 695; 645;
958 959 960 941
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
4
601; 185 799; 186 715; 182 682; 184
0 0 0
0 0 0
—
—
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
300; 24; 121; 173;
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0
n 0
5
6
92 97 91 96
598;244 193; 247 183; 242 996; 248
Ormeto-
SDM
0.43
4.00
0.58
4.60
0
0
0
0.13
0
0
0
0
0
0
0
0
0
0
0
0
0 0
0
0
0
0
—
0
0
—
Ormeto- SDM 0.13 0 0 0
0.62 1.02 1.04 0.82
0 0 0 0
0 0 0 0
—.
0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0
0 0 0 0
— 0
0
* Pooled samples.
of ormetoprim. Sulfonamide levels were highest in the kidney, where they averaged 6.7 p.p.m., followed by the liver with 1.3 p.p.m., blood 1.0 p.p.m., muscle 0.65 p.p.m., and skin and fat 0.52 p.p.m. Levels of ormetoprim at 0 days of drug withdrawal were highest in kidney (average 0.40 p.p.m.) and liver (average 0.14 p.p.m.) and marginal only in the other tissues. No ormetoprim could be detected after 2 days of drug withdrawal. At 2 and
3 days of drug withdrawal, SDM could be detected only in a few isolated samples and drug levels in each case were well below 0.1 p.p.m. No SDM could be detected after 3 days of drug withdrawal. CONCLUSIONS
The above longed broiler
results of the studies described indicate that following the proadministration of Rofenaid® to chickens at the recommended use
TABLE 6.—Performance of turkeys receiving Rofenaid at 0.01% in feed for 13 weeks Intake of Rofenaid Diet
^r birds
Initial wei ht ? > g-
Final wei ht S >
•
r
I
'
„ Gain
Basal
40
56
4,280
4,224
100
Rofenaid 0.01%
48
55
4,600
4,545
107.6
Feed consumed
, Conv. v
>
9,906
2.34
10,502
2.31
Total mg.
1,050.2
Average daily mg.
mg./kg.
11.54
4.96
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1
Blood
Kidney*
Ormeto- SDM
1782
J. FELLIG, J. WESTHEIMEE, M. J. WALSH AND W. L. MARTJSICH TABLE 7.—Clearance of Rofenaid in turkeys p.p.,m.
Withdrawal time, days
Muscle
Bird#
Liver
Ormetoprim
SDM
Ormetoprim
Kidney
Skin and fat
SDM
Ormetoprim
SDM
Ormetoprim
SDM
204 226 647 173 710 728
0.06 0. 08 0 0. 05 0 0
0.8 0.8 0.7 0.8 0.4 0.4
0.05 0.2 0.1 0.07 0.2 0.2
1.5 1.9 1.5 1.5 0.9 0.6
0 0 0 0 0.05 0.08
0.4 0.7 0.7 0.6 0.4 0.3
0.4 0.5 0.4 0.4 0.3 0.4
2
345 327 268 257 950, 948 693
0 0 0. 06 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0.05 0 0 0
0 0 0 0 0 0
0 0 0 0.08 0 0.08
0 O 0 0 0 0
0.05 0 0 0 0 0
3
161, 212 233, 151 711 949 758 274
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0.05
0
0
— 0
— 0
0 0 0
0.06 0 0
0 0 0
0 0 0
— 0
— 0
0
0.05
634 740, 654 692 341, 244 271 299
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
259, 255 726, 326 750 755, 281 639 213
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
—
—
4
5
—
0 0 0 0 0
4.4 6.0 7.2 10.0 4.8 7.6
0 0 0 0 0
SDM
0 0 0 0 0.2 0
1.0 1.3 0.9 1.0 0.9 1.1
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0
0 0 0
—
—
0 0
0 0
0 0
0 0
— 0 0 0
—
0 0 0
level, the birds were clear of the drug 2 days after cessation of treatment. The combined administration of Rofenaid® and either arsanilic acid or 3-nitro-4hydroxy-phenylarsonic acid at their respective recommended use levels did not influence tissue levels or time required for clearance of the drugs. Also, following the prolonged administration of Rofenaid® to turkeys at the recommended use level, the birds were clear of the drug 2 days after cessation of treatment. It is concluded from these data that a 2-day drug withdrawal period will ensure the clearance of the components of Rofenaid® from the tissues of broilers and turkeys following the prolonged feeding of the drug at the recommended use levels.
recommended use level of 0.02% in feed. All tissues were found to be clear of sulfadimethoxine and ormetoprim 2 days after cessation of treatment. When Rofenaid® at the same dose level was administered to broiler chickens in combination with either arsanilic acid at 0.01% in the feed or with 3-nitro-4-hydroxyphenylarsonic acid at 0.005% in the feed, tissue levels of the drugs were unaffected, as was the time required for clearance of the tissues. Rofenaid® was administered to turkeys for a period of 13 weeks at the recommended use level of 0.01% in the feed. All tissues were clear of sulfadimethoxine and ormetoprim 2 days after drug withdrawal.
SUMMARY
Fellig, J., and J. Westheimer 1968. Determination of sulfadimethoxine in animal tissues. J. Agric. Food Chem. 16: 738-740. Fellig, J., J. Westheimer, M. J. Walsh and R. A.
REFERENCES
Rofenaid® was administered to broiler chickens for a period of 8 weeks at the
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0
Blood Ormetoprim
1783
T I S S U E CLEARANCE o r ROFENAID
Saperstein, 1971. Determination of ormetoprim in animal tissues. J. Agric. Feed Chem. in press. Food Chemicals Codex, National Academy of Sciences—National Research Council 1963. Arsenic test, pg. 47. Marusich, W. L., E. Ogrinz, M. Brand and M. Mitrovic, 1969. Safety and compatibility of sulfadimethoxine potentiated mixture (Ro 5-0013), a new broad spectrum coccidiostat-antibacterial, in chickens. Poultry Sci. 48: 217-222. Marusich, W. L., E. F. Ogrinz, B. Hecht and M. Mitrovic, 1971. Safety of sulfadimethoxine potentiated mixture (Rofenaid®), a new broad spectrum coccidiostat antibacterial, in turkeys. Poultry Sci. SO: 512-517.
Mitrovic, M., E. G. Schildknecht and G. Fusiek, 1969a. Anticoccidial activity of sulfadimethoxine potentiated mixture (Ro 5-0013) in chickens. Poultry Sci. 48: 210-216. Mitrovic M., G. Fusiek and E. G. Schildknecht, 1969b. Antibacterial activity of sulfadimethoxine potentiated mixture (Ro 5-0013) in chickens. Poultry Sci. 48: 1151-1155. Mitrovic, M., G. Fusiek and E. G. Schildknecht, 1971a. Antibacterial activity of sulfadimethoxine potentiated mixture (Rofenaid®) in turkeys. Poultry Sci. 50: 525-529. Mitrovic, M., E. G. Schildknecht and G. Fusiek, 1971b. Anticoccidial activity of sulfadimethoxine potentiated mixture (Rofenaid®) in turkeys. Poultry Sci. 50:517-525.
A N N E T T E C. R E Y N O L D S , A. V. D E G U Z M A N 0 AND R. E . CLEGG
Department of Biochemistry, Kansas State University, Manhattan, Kansas 66502 (Received for publication April 3, 1971)
INTRODUCTION
A
PREVIOUS
ported (Counsell et al., 1962) f as twelve to
investigation
demon-
s t r a t e d t h a t oral administration of
triparanol
(Mer-20)
to
laying
hens
fifteen
times
that
of
triparanol
given
orally. SC-10644 g also has been reported to effectively lower plasma cholesterol b y
caused serum cholesterol to rise a n d re-
50%
duced egg formation (Nelson a n d Clegg,
orally for two weeks. Cholestyramine, a
1962).
bile acid sequestrant, administered as 1 %
This
contrasted
rise with
in
serum
results
cholesterol
obtained
from
in normal r a t s when administered
of the diet inhibits plasma rise
1959;
( T e n n e n t et al., 1959). Diethylstilbestrol
Blohm a n d Mackenzie, 1959; and
50%
in
cholesterol-fed
cholesterol
r a t s b y other investigators (Blohm et al.,
cockerels
Kayser, 1960). T h e effectiveness of SC-
causes a rise in liver a n d plasma cho-
12937 e in lowering plasma cholesterol in
lesterol and blood lipid in chickens ( E n -
hypercholesterolemic
tenman et al., 1940; Linsay et al., 1946).
rats
has
been
re-
Effects " Contribution 114, Dept. of Biochemistry, Kansas State Agricultural Experiment Station. b Supported in part by H E 12294-01, National Institutes of Health, Bethesda, Md. c Present address: Dept. of Biochem., Univ. of Santo Tomas, Manila, Philippines. e 20, 25-diazacholestenol dihydrochloride
of
compounds 10644,
and
four
hypocholesterolemic
(triparanol,
SC-12937,
cholestyramine)
on
SC-
serum
f Unpublished data, Ranney, R. E., and Cook, D. L. (1962). G. D. Searle and Co., Chicago. g 2-(l-pyrrolidinyl) ethyl triphenylmethane-4 carboxylate hydrochloride
Downloaded from http://ps.oxfordjournals.org/ by guest on May 3, 2015
Influence of Some Hypocholesterolemic Compounds on the Serum Protein Patterns and Serum Cholesterol of Normal and Diethylstilbestrol-Treated Cockerelsa,b