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microsome test, of psychopharmaceuticals used in meat production
104(1982)333-337 Elsevier BiomedicalPress
Mutation Research,
333
Investigation, with the Salmonella/microsome test, of psychopharmaceuticals used in meat production Angelika M. Preiss, Michael Scheutwinkel-Reich, Ingrid F011e, Heidemarie G. G r o h m a n n and Hans-Jargen Stan Institut f a r Lebensmittelchemie, Technische Universitgit Berlin, Maller-Breslau-Strasse 10, I000 Berlin 12 (F.R.G.)
(Accepted 26 February 1982) Summary Azaperone, acepromazine, xylazine and diazepam were tested for mutagenic activity. To screen these veterinary drugs we used the Salmonella/microsome test on 5 histidine-auxotrophic strains, with and without metabolic activation. Azaperone and xylazine were found to be weakly mutagenic.
Veterinary psychopharmaceutical drugs are used as tranquilizers to reduce aggressiveness and activity during live-stock breeding. A special application is the treatment of animals during transport to slaughter houses to avoid mortality and loss of meat quality caused by stress reactions. Currently, the following drugs are in use: the butyrophenone azaperone (Heykants et al., 1971; Rauws and Oiling, 1978; Fischer et al., 1975), the benzodiazepine diazepam, the phenothiazine acepromazine, and the xylidinothiazine xylazine (Fischer et al., 1975). The formulae of these drugs are given in Fig. 1. Since residues of these drugs are to be expected in meat destined for human consumption it seemed desirable to investigate their possible mutagenicity, especially that of azaperone which has been detected in kidney and liver of pigs (Olling et al., 1980). Materials and methods
(1) Test substances. All tranquilizers are generous gifts: azaperone (Janssen Pharmaceutica, Beerse, Belgium), acepromazine (Clin-Midy, Paris, France), diazepam (Hoffmann-LaRoche, Grenzach-Wyhlen, F.R.G.), xylazine (Bayer AG, Leverkusen, F.R.G.). Positive control substances, 1,2-benzopyrene, 2-aminoanthracene, and sodium azide were commercially available (Serva, Heidelberg; Merck, Darmstadt, F.R.G.). Nitroquinoline-N-oxide was generously provided by Dr. Beutin (Bundesgesundheitsamt, Berlin). 0165-7992/82/0000-0000/$02.75 © ElsevierBiomedicalPress
334
TABLE 1 MUTAGENIC
ACTIVITY
OF
VETERINARY
PSYCHOPHARMACEUTICALS
IN
Salmonella
typhimurium Drug
Concentration (~g/plate)
R e v e r t a n t s per p l a t e ( m e a n TA1535
TA100
-$9
-$9
Azaperone
+$9
plates)
6
TA1537
TA1538 -$9
-$9
+$9
33
23
102
130
9
13
26
1
26
12
111
131
12
10
10
28
8
97
107
11
10
Control (EtOH) a
+$9
of
TA98
+$9
-$9
+$9
43
37
33
19
39
33
35
19
47
35
41
100
30
13
116
140
9
20
24
65
28
48
500
45
25
115
142
5
19
21
80
28
61
.
.
.
111
-
84
114
-
85
107
29
61
750
.
.
1000
-
1250
41
25
113
1500 2000 2500 Control (DMSO) a Acepromazine
. .
.
.
124 .
.
10 .
.
-
29
. .
-
23
. .
116
-
-
112
-
-
2
15
4
20
7
23
32
97
27
61
31
16
126
129
6
12
16
45
23
41 41
1
26
21
127
123
4
13
8
45
23
10
24
13
122
120
3
10
15
53
24
52
100
37
26
123
138
5
10
16
44
26
54
500
14
21
123
136
1
12
2
41
20
33
1000
7
0
31
61
0
0
4
30
0
22
2500
0
0
0
0
0
0
0
0
0
0
Diazepam
1
30
13
138
121
5
12
11
48
27
36
10
21
18
122
119
3
11
14
44
26
31
100
22
19
127
131
5
11
17
40
24
36
500
26
14
103
128
3
7
17
42
26
36
1000
31
24
112
121
2
9
15
47
24
35 43
Xylazine a
1
29
13
127
120
5
11
14
45
28
10
30
13
143
115
4
12
14
43
26
39
100
31
17
145
124
6
7
14
46
27
43
500
31
22
156
113
6
7
16
43
19
38
1000
41
20
156
104
5
12
15
58
27
35
2500
73
31
181
116
6
10
16
56
27
35
5000
65
39
197
119
5
10
23
65
26
35
6000
67
47
208
-
7000
78 76
42
215 200
73 83
61
183 167
8000 9000 10000
-
-
-
25
64
26
-
-
-
25 25
65 73
25 26
-
-
-
32 39
76 76
23 30
-
335 TABLE 1 (continued) Drug
Concentration (/zg/plate)
TA1535
TA100
TA1537
TA1538
TA98
- $ 9 +$9
- $ 9 +$9
- $ 9 +$9
- $ 9 +$9
- $ 9 +$9
720 1400 2400
-
780 1200 1800
0.4 0.8
. .
. .
0.8 2 4
-
128 252 368
1
-
2 5
-
480 811 -
4 10 20
NaN 3
Nitroquinoline-Noxide
NH 2-anthracene
Benzo[a]pyrene
Revertants per plate (mean of 6 plates)
-
-
. .
. .
. .
-
800 2600 5300
-
87 138 381
-
660 1300 2350
-
378 620 1506
-
35 85 121
-
71 137 309
. .
. .
-
250 710 1400
m
. .
183 392 -
-
-
-
680 2000 5150 125 240 436
aMean of 12 plates.
(2) Bacterial strains. T h e h i s t i d i n e - a u x o t r o p h i c s t r a i n s o f Salmonella typhimurium T A 1 5 3 5 , T A 1 5 3 7 , T A 1 5 3 8 , T A 9 8 a n d T A I 0 0 w e r e o b t a i n e d f r o m D r . B.N. Ames (Berkeley, CA).
(3) $9 rat-liver homogenate.
T h e $9 mix was p r o d u c e d f r o m W i s t a r rats t h a t h a d
b e e n t r e a t e d w i t h A r o c l o r 1254 ( E h r e n s d o r f e r , A u g s b u r g , F . R . G . ) 5 d a y s b e f o r e d e c a p i t a t i o n . T h e p r e p a r a t i o n w a s p e r f o r m e d a c c o r d i n g t o A m e s et al. (1975). T h e p r o t e i n c o n t e n t w a s f o u n d t o b e 5 m g / p l a t e , a c c o r d i n g t o L o w r y et al. (1951).
(4) Solution of compounds.
A z a p e r o n e w a s d i s s o l v e d in e t h a n o l ( E t O H ; M e r c k ,
D a r m s t a d t , F . R . G . ) u p t o t h e c o n c e n t r a t i o n o f 2.5 m g / 1 0 0 / x l . All t h e o t h e r d r u g s w e r e d i s s o l v e d in d i m e t h y l s u l f o x i d e ( D M S O ; M e r c k , D a r m s t a d t , F . R . G . ) u p t o 5 m g / 1 0 0 #1 o r 10 m g / 1 0 0 #1 r e s p e c t i v e l y .
(5) Assay procedure.
T h e m u t a g e n i c i t y t e s t w a s p e r f o r m e d a c c o r d i n g t o A m e s et
al. (1975) b u t w i t h d u e r e g a r d t o t h e r e c o m m e n d a t i o n s
of de Serres and Shelby
(1979).
Results
and
discussion
The mutation
rates i n d u c e d by the d r u g s tested at several c o n c e n t r a t i o n s are
s u m m a r i z e d i n T a b l e 1 t o g e t h e r w i t h r e s u l t s f r o m a series o f c o n t r o l s . A f t e r t h e f i r s t screening the test series w e r e p e r f o r m e d in a s t a n d a r d c o n c e n t r a t i o n r a n g e b e g i n n i n g w i t h 1 #g p e r p l a t e a n d e n d i n g a t t h e l i m i t o f s o l u b i l i t y . T h i s s t a n d a r d c o n c e n t r a t i o n
336 CH3
~
F " - - ~ C - - ( C H~)3--N
N
N"--'F'F', ~-~
NN ell3
Azaperone
Xylazine
~H3 //O I. CH3-"-C 0"
N ] /CH3 CH?--CH~--CH?-- N \CH~
Acepromazine
II
CH~
@
CI ~ " ~ " ~ C = N /
Di a zepam
Fig. l. Structural formulae of the drugs tested.
range includes 1, 10, 100, 500, 1000 or 1250, 2500 and 5000/~g per plate. For this test, 5 tester strains were used: TA1535, TAI00, TA1537, TA1538 and TA98. In the case of elevated numbers of revertants with any tester strain at one concentration, smaller concentration intervals were applied. Azaperone exhibited mutagenic activity, after metabolic activation, in the tester strains TA1538 and TA98 which detect frameshift mutations. In the concentration range up to 750 tzg per plate a clear dose-response relationship was evident. At higher concentrations, toxic effects may inhibit a further increase in the number of revertants. The results with TA1538 and TA98 are confirmed with TA1537, although the latter tester strain was not used at all concentration steps. When the 2 tester strains TA1535 and TAI00 were used, no mutagenic effects were observed. The 2 drugs acepromazine and diazepam did not show any mutagenic activity under the experimental conditions described. For acepromazine, limited test reliability was found owing to its cytotoxicity starting for all tester strains at concentrations higher than 500/~g per plate. The limitation of test reliability for diazepam, however, was caused by its low solubility in top agar: diazepam precipitates in concentrations higher than 1 mg per plate. Our results on the non-mutagenicity of diazepam in the Ames assay are in accord with those reported by Waskell (1978) obtained with the tester strains TA98 and TAI00. Assaying xylazine, we found the results difficult to interpret. Using
337
TA1535, we observed a doubling of the spontaneous mutation rate with and without metabolic activation. In both test series a clear dose-response relationship was observed, but it should be taken into account that the slope of the dose-response curve is rather shallow and the doubling of the spontaneous mutation rate occurred at fairly high concentrations. The other tester strain, TA100, which detects mainly base-pair substitution, showed only a slight increase in the mutation rate without metabolic activation but the doubled spontaneous mutation rate was not reached. After metabolic activation the same tester strain was not influenced by xylazine. With the other tester strains only the revertants of TA1538 approached twice the spontaneous mutation rate at the highest concentration. This effect is restricted in the test series without metabolic activation. Summarizing our results, we consider azaperone and xylazine as weakly mutagenic in the Ames assay, whereas diazepam and acepromazine did not show any mutagenic effect in this test. As a consequence of our results, further investigations on the mutagenicity of azaperone and xylazine are desirable. In addition, the metabolism of these compounds should be elucidated in order that metabolites may be subjected to mutagenicity testing.
Acknowledgements We thank Prof. B.N. Ames for kindly furnishing us with the tester strains, Mr. C. Boiselle for helpful discussions, and Mr. U. Wawzon for technical assistance.
References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 31,347-364. de Serres, F.J., and M.D. Shelby (1979) Recommendations on data production and analysis using the SalmoneUa/microsome mutagenicity assay, Mutation Res., 64, 159-165. Fischer, A., L. Kotter and W. Kreuzer (1975) Zur Problematik des Einsatzes von Psychopharmaka bei Schlachttieren, Forschungsbericht der Deutschen Forschungsgemeinschaft, R0ckst~tnde in Fleisch und Fleischerzeugnissen, pp. 20-31. Heykants, J., J. Symoens and R. Marsboom (1971) On the distribution and metabolism of azaperone (R1929) in the rat and pig, Arzneim.-Forsch. (Drug Res.), 21, 1357-1358. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein measurement with the foline phenol reagent, J. Biol. Chem., 193, 265-275. Oiling, M., R.W. Stephany and A.G. Rauws (1980) Tranquilizer residues in slaughter pigs, in: A. van Miert (Ed.), Trends in Veterinary Pharmacology and Toxicology, Elsevier, Amsterdam, pp. 335-336. Rauws, A.G., and M. Olling (1978) Residues of azaperone and azaperol in slaughter pigs, J. Vet. Pharmacol. Ther., 1, 57-62. Waskell, L. (1978) A study of the mutagenicity of anesthetics and their metabolites, Mutation Res., 57, 141-153.
Mutation Research,
333
Investigation, with the Salmonella/microsome test, of psychopharmaceuticals used in meat production Angelika M. Preiss, Michael Scheutwinkel-Reich, Ingrid F011e, Heidemarie G. G r o h m a n n and Hans-Jargen Stan Institut f a r Lebensmittelchemie, Technische Universitgit Berlin, Maller-Breslau-Strasse 10, I000 Berlin 12 (F.R.G.)
(Accepted 26 February 1982) Summary Azaperone, acepromazine, xylazine and diazepam were tested for mutagenic activity. To screen these veterinary drugs we used the Salmonella/microsome test on 5 histidine-auxotrophic strains, with and without metabolic activation. Azaperone and xylazine were found to be weakly mutagenic.
Veterinary psychopharmaceutical drugs are used as tranquilizers to reduce aggressiveness and activity during live-stock breeding. A special application is the treatment of animals during transport to slaughter houses to avoid mortality and loss of meat quality caused by stress reactions. Currently, the following drugs are in use: the butyrophenone azaperone (Heykants et al., 1971; Rauws and Oiling, 1978; Fischer et al., 1975), the benzodiazepine diazepam, the phenothiazine acepromazine, and the xylidinothiazine xylazine (Fischer et al., 1975). The formulae of these drugs are given in Fig. 1. Since residues of these drugs are to be expected in meat destined for human consumption it seemed desirable to investigate their possible mutagenicity, especially that of azaperone which has been detected in kidney and liver of pigs (Olling et al., 1980). Materials and methods
(1) Test substances. All tranquilizers are generous gifts: azaperone (Janssen Pharmaceutica, Beerse, Belgium), acepromazine (Clin-Midy, Paris, France), diazepam (Hoffmann-LaRoche, Grenzach-Wyhlen, F.R.G.), xylazine (Bayer AG, Leverkusen, F.R.G.). Positive control substances, 1,2-benzopyrene, 2-aminoanthracene, and sodium azide were commercially available (Serva, Heidelberg; Merck, Darmstadt, F.R.G.). Nitroquinoline-N-oxide was generously provided by Dr. Beutin (Bundesgesundheitsamt, Berlin). 0165-7992/82/0000-0000/$02.75 © ElsevierBiomedicalPress
334
TABLE 1 MUTAGENIC
ACTIVITY
OF
VETERINARY
PSYCHOPHARMACEUTICALS
IN
Salmonella
typhimurium Drug
Concentration (~g/plate)
R e v e r t a n t s per p l a t e ( m e a n TA1535
TA100
-$9
-$9
Azaperone
+$9
plates)
6
TA1537
TA1538 -$9
-$9
+$9
33
23
102
130
9
13
26
1
26
12
111
131
12
10
10
28
8
97
107
11
10
Control (EtOH) a
+$9
of
TA98
+$9
-$9
+$9
43
37
33
19
39
33
35
19
47
35
41
100
30
13
116
140
9
20
24
65
28
48
500
45
25
115
142
5
19
21
80
28
61
.
.
.
111
-
84
114
-
85
107
29
61
750
.
.
1000
-
1250
41
25
113
1500 2000 2500 Control (DMSO) a Acepromazine
. .
.
.
124 .
.
10 .
.
-
29
. .
-
23
. .
116
-
-
112
-
-
2
15
4
20
7
23
32
97
27
61
31
16
126
129
6
12
16
45
23
41 41
1
26
21
127
123
4
13
8
45
23
10
24
13
122
120
3
10
15
53
24
52
100
37
26
123
138
5
10
16
44
26
54
500
14
21
123
136
1
12
2
41
20
33
1000
7
0
31
61
0
0
4
30
0
22
2500
0
0
0
0
0
0
0
0
0
0
Diazepam
1
30
13
138
121
5
12
11
48
27
36
10
21
18
122
119
3
11
14
44
26
31
100
22
19
127
131
5
11
17
40
24
36
500
26
14
103
128
3
7
17
42
26
36
1000
31
24
112
121
2
9
15
47
24
35 43
Xylazine a
1
29
13
127
120
5
11
14
45
28
10
30
13
143
115
4
12
14
43
26
39
100
31
17
145
124
6
7
14
46
27
43
500
31
22
156
113
6
7
16
43
19
38
1000
41
20
156
104
5
12
15
58
27
35
2500
73
31
181
116
6
10
16
56
27
35
5000
65
39
197
119
5
10
23
65
26
35
6000
67
47
208
-
7000
78 76
42
215 200
73 83
61
183 167
8000 9000 10000
-
-
-
25
64
26
-
-
-
25 25
65 73
25 26
-
-
-
32 39
76 76
23 30
-
335 TABLE 1 (continued) Drug
Concentration (/zg/plate)
TA1535
TA100
TA1537
TA1538
TA98
- $ 9 +$9
- $ 9 +$9
- $ 9 +$9
- $ 9 +$9
- $ 9 +$9
720 1400 2400
-
780 1200 1800
0.4 0.8
. .
. .
0.8 2 4
-
128 252 368
1
-
2 5
-
480 811 -
4 10 20
NaN 3
Nitroquinoline-Noxide
NH 2-anthracene
Benzo[a]pyrene
Revertants per plate (mean of 6 plates)
-
-
. .
. .
. .
-
800 2600 5300
-
87 138 381
-
660 1300 2350
-
378 620 1506
-
35 85 121
-
71 137 309
. .
. .
-
250 710 1400
m
. .
183 392 -
-
-
-
680 2000 5150 125 240 436
aMean of 12 plates.
(2) Bacterial strains. T h e h i s t i d i n e - a u x o t r o p h i c s t r a i n s o f Salmonella typhimurium T A 1 5 3 5 , T A 1 5 3 7 , T A 1 5 3 8 , T A 9 8 a n d T A I 0 0 w e r e o b t a i n e d f r o m D r . B.N. Ames (Berkeley, CA).
(3) $9 rat-liver homogenate.
T h e $9 mix was p r o d u c e d f r o m W i s t a r rats t h a t h a d
b e e n t r e a t e d w i t h A r o c l o r 1254 ( E h r e n s d o r f e r , A u g s b u r g , F . R . G . ) 5 d a y s b e f o r e d e c a p i t a t i o n . T h e p r e p a r a t i o n w a s p e r f o r m e d a c c o r d i n g t o A m e s et al. (1975). T h e p r o t e i n c o n t e n t w a s f o u n d t o b e 5 m g / p l a t e , a c c o r d i n g t o L o w r y et al. (1951).
(4) Solution of compounds.
A z a p e r o n e w a s d i s s o l v e d in e t h a n o l ( E t O H ; M e r c k ,
D a r m s t a d t , F . R . G . ) u p t o t h e c o n c e n t r a t i o n o f 2.5 m g / 1 0 0 / x l . All t h e o t h e r d r u g s w e r e d i s s o l v e d in d i m e t h y l s u l f o x i d e ( D M S O ; M e r c k , D a r m s t a d t , F . R . G . ) u p t o 5 m g / 1 0 0 #1 o r 10 m g / 1 0 0 #1 r e s p e c t i v e l y .
(5) Assay procedure.
T h e m u t a g e n i c i t y t e s t w a s p e r f o r m e d a c c o r d i n g t o A m e s et
al. (1975) b u t w i t h d u e r e g a r d t o t h e r e c o m m e n d a t i o n s
of de Serres and Shelby
(1979).
Results
and
discussion
The mutation
rates i n d u c e d by the d r u g s tested at several c o n c e n t r a t i o n s are
s u m m a r i z e d i n T a b l e 1 t o g e t h e r w i t h r e s u l t s f r o m a series o f c o n t r o l s . A f t e r t h e f i r s t screening the test series w e r e p e r f o r m e d in a s t a n d a r d c o n c e n t r a t i o n r a n g e b e g i n n i n g w i t h 1 #g p e r p l a t e a n d e n d i n g a t t h e l i m i t o f s o l u b i l i t y . T h i s s t a n d a r d c o n c e n t r a t i o n
336 CH3
~
F " - - ~ C - - ( C H~)3--N
N
N"--'F'F', ~-~
NN ell3
Azaperone
Xylazine
~H3 //O I. CH3-"-C 0"
N ] /CH3 CH?--CH~--CH?-- N \CH~
Acepromazine
II
CH~
@
CI ~ " ~ " ~ C = N /
Di a zepam
Fig. l. Structural formulae of the drugs tested.
range includes 1, 10, 100, 500, 1000 or 1250, 2500 and 5000/~g per plate. For this test, 5 tester strains were used: TA1535, TAI00, TA1537, TA1538 and TA98. In the case of elevated numbers of revertants with any tester strain at one concentration, smaller concentration intervals were applied. Azaperone exhibited mutagenic activity, after metabolic activation, in the tester strains TA1538 and TA98 which detect frameshift mutations. In the concentration range up to 750 tzg per plate a clear dose-response relationship was evident. At higher concentrations, toxic effects may inhibit a further increase in the number of revertants. The results with TA1538 and TA98 are confirmed with TA1537, although the latter tester strain was not used at all concentration steps. When the 2 tester strains TA1535 and TAI00 were used, no mutagenic effects were observed. The 2 drugs acepromazine and diazepam did not show any mutagenic activity under the experimental conditions described. For acepromazine, limited test reliability was found owing to its cytotoxicity starting for all tester strains at concentrations higher than 500/~g per plate. The limitation of test reliability for diazepam, however, was caused by its low solubility in top agar: diazepam precipitates in concentrations higher than 1 mg per plate. Our results on the non-mutagenicity of diazepam in the Ames assay are in accord with those reported by Waskell (1978) obtained with the tester strains TA98 and TAI00. Assaying xylazine, we found the results difficult to interpret. Using
337
TA1535, we observed a doubling of the spontaneous mutation rate with and without metabolic activation. In both test series a clear dose-response relationship was observed, but it should be taken into account that the slope of the dose-response curve is rather shallow and the doubling of the spontaneous mutation rate occurred at fairly high concentrations. The other tester strain, TA100, which detects mainly base-pair substitution, showed only a slight increase in the mutation rate without metabolic activation but the doubled spontaneous mutation rate was not reached. After metabolic activation the same tester strain was not influenced by xylazine. With the other tester strains only the revertants of TA1538 approached twice the spontaneous mutation rate at the highest concentration. This effect is restricted in the test series without metabolic activation. Summarizing our results, we consider azaperone and xylazine as weakly mutagenic in the Ames assay, whereas diazepam and acepromazine did not show any mutagenic effect in this test. As a consequence of our results, further investigations on the mutagenicity of azaperone and xylazine are desirable. In addition, the metabolism of these compounds should be elucidated in order that metabolites may be subjected to mutagenicity testing.
Acknowledgements We thank Prof. B.N. Ames for kindly furnishing us with the tester strains, Mr. C. Boiselle for helpful discussions, and Mr. U. Wawzon for technical assistance.
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