307
Mutation Research, 68 (1979) 307--312 © Elsevier/North-Holland Biomedical Press
MUTAGENICITY OF A N T H R A Q U I N O N E AND AZO DYES IN AMES'
Salmonella typbimurium TEST S. VENTURINI and M. TAMARO
Institute of Microbioloy, University of Trieste, Trieste (Italy) (Received 8 August 1978) (Revision received 3 July 1979) (Accepted 5 July 1979)
Summary 23 dyes belonging to different chemical classes -- anthraquinones, mono- and bis-azo c o m p o u n d s -- were tested for their mutagenic activity on Ames strains o f Salmonella typhimurium. 5 dyes induced frameshift mutations.
Anthraquinone and azo compounds are industrially important substances largely used as dyes. Anthraquinone derivatives are closely related to anthracycline antitumor antibiotics of k n o w n mutagenicity and carcinogenicity [1-5]. Azo dyes, mono-azo as well as bis-azo derivatives, represent a large part of the total dye~stuffs manufactured, and are primarily used for coloring substrates from foods and textiles through to plastics. It is known that the azo group is linked with carcinogenesis, even when produced by metabolic activation, as it is with certain mammalian and bacterial enzymes [6,7], and some azo compounds axe mutagenic in bacterial tests [8--10]. 23 industrial c o m p o u n d s widely used as textile dyes, and belonging to the above~ited chemical classes (see Table 1 for structures) were tested b y Ames' test for mutagenicity. Substances listed in Table 1 were obtained from A.C.N.A., Bayer, Ciba, Hoechst, I.C.I. and Sandoz. We are aware that these preparations may contain impurities up to 30% of their weight. However no further purification was attempted, because we wanted to test the potential danger that they represent in actual use. Materials and methods
Salmonella typhimurium strains TA1535, TA100, TA1538 and TA98 were obtained from Dr. Bruce N. Ames {University of California, Berkeley). These bacterial tester strains have a mutation in the histidine operon which consequently results in nutritional dependence on histidine.
308
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309 TA1535 and TA100 can be reverted to prototrophy by DNA base-pair substitutions, whereas TA1538 and TA98 are reverted by frameshift mutation. Mutation tests were carried out according to the soft-agar overlay method described by Ames et al. [11,12]. Substances listed in Table 1 were dissolved in dimethylsulfoxide (DMSO) and tested at dose levels of 100, 500 and 1000 pg/ plate. When tests were done for metabolic activation by mammalian microsomal enzymes, 0.5 ml of "$9 mix" was added to the top agar. $9 mix was prepared from liver homogenate of rats treated with Aroclor 1254 plus adequate cofactots, as described by Ames et al. [13]. Positive controls were used for the tester strains as follows: ethyl methanesulfonate (EMS) for TA1535; methyl methanesulfonate (MMS) for TA100 and hycanthone for TA1538 and TA98.
TABLE2 (A) M U T A G E N I C A C T I V I T Y OF A N T H R A Q U I N O N E COMPOUNDS Compound
N u m b e r o f His* r e v e r t a n t s / p l a t e a ~g/plate
I
TA1535
TAI00
--
_
+b
_
+b
_
+b
415 29---O 10__._2
424 31--'O 6__3
463 34"-~ 10___9
+b
TA1538
TA98
C.I. disperse red 15
1000 500 I00
3 3 1
5 4 3
10 7 2
45 40 38
380 27-'-0 5_5
II Violet C i b a c e t 2 R C.I. disperse v i o l e t 1
1000 500 I00
2 1 2
6 4 4
inhib. 8 11 21 3
5 2 2
6 4 2
3 3 1
18 19 21
III Alizarin light b l u e SE C.I. acid b l u e 43
1000 500 100
2 1 1
3 4 1
22 22 25
12 11 13
18 12 18
52 40 38
19 16 13
141 109 96
IV C.I. acld g r e e n 41
1000 500 100
5 4 4
4 4 1
16 11 10
17 17 19
1 2 3
1 7 6
9 9 11
12 12 14
V Light blue A N L C.I. acid b l u e 25
1000 500 100
1 3 4
6 6 7
1 2 10
0 1 7
2 0 7
8 1 9
1 1 0
4 3 1
VI Blue F 3 G A C.I. r e a c t i v e b l u e 2
1000 500 100
17 10 10
14 10 3
21 11 4
4 3 5
1 0 2
2 1 2
4 2 3
5 3 4
VII Light blue B C.I. acid b l u e 41
1000 500 100
1 4 1
1 1 2
2 3 4
5 2 2
3 2 3
6 6 7
3 4 6
4 3 6
VIII Blue F 3 R C.I. disperse b l u e 3
1000 500 100
1 1 1
3 2 1
7 10 12
10 10 9
8 7 4
4 4 4
18 18 15
21 18 16
R e d C i b a c e t 3B
F a s t g r e e n 5G
a Number of revertant colonies per plate, mean of 4 separate expe~ments
after subtracting spontaneous
revertants. b Indicates plates without
($9 mix) stimulated b y A m -
( - - ) o r w i t h ( + ) rat-liver m i c r o s e m a l
clor 1 2 5 4 . V a l u e s u n d e r l i n e d are i n d i c a t i v e o f m u t a g e n i c a c t i v i t y . Inhlb., inhibition of bacterial gro wth.
preparation
310
The numbers of spontaneous revertants in the presence of DMSO alone + "$9 mix" were: 12 for strain T A 1 5 3 5 , 125 for strain TA100, 8 for strain T A 1 5 3 8 and 21 for strain TA98. Results and discussion
Table 2 shows the results obtained. 2 of the 8 anthraquinone dyes tested for mutagenicity with Salmonella typhimurium strains, Red Cibacet 3B, C.I. disperse red 15 and Alizarin light blue SE, C.I. acid blue 43, acted as frameshift mutagens. Red Cibacet 3B at a 1 0 0 0 pg/plate dose level showed a reversion 20 times that of the spontaneous rate in strain TA98. Only one of the mono-azo compounds, red GTL--C.I. basic red 18, had any mutagenic activity when tested with strains TA98 and T A 1 5 3 8 at 100, 500 and 1000 #g/plate levels (Table 2). Only weak mutagenic activity was detected when Brown 5R, C.I. acid orange 45 was tested both with and without metabolic activation (Table 2). Another bis-azo dye, Giuba black D, C.I. direct black 17, exhibited frameshift mutagenic activity. Matsushima et ai. [14] showed that the addition of riboflavin to the $9 mix was necessary for mutagenic activity of some azo compounds. Therefore,
TABLE 2 (B) M U T A G E N I C A C T I V I T Y O F M O N O - A Z O C O M P O U N D S Compound
a
Number of His + revertants/plate t~g/plate
IX Yellow ERPL C.I. a c i d y e l l o w
TA1535
TA100
--
--
+b
+b
TA1538 --
TA98 +b
_
+b
I000 500 I00
5 3 1
6 6 7
14 8 8
16 9 7
1 0 0
3 1 0
0 0 0
1 2 0
1000 500 I00
4 4 2
10 9 6
9 2 2
14 7 8
1 1 0
2 1 1
2 0 0
3 2 2
1000 500 100
1 1 2
3 3 3
8 4 4
S 4 5
1 1 0
1 1 2
6 5 8
3 3 6
XII Red GTL C.I. b a s l e r e d 18
1000 500 I00
2 1 0
2 2 1
12 7 1
10 6 6
114 105 98
140 117 115
170 140 I00
122 112 104
XIII Red H3B C.I. r e a c t i v e r e d 3
1000 500 100
12 9 0
19 10 10
13 7 2
18 9 8
5 2 0
6 2 1
4 2 2
8 4 3
XIV Red 2BF C.I. r e a c t i v e r e d 42
1000 500 100
10 6 4
13 10 10
14 8 7
14 4 11
3 4 4
11 9 8
4 5 6
15 9 8
XV Fast red BF C.I. a m d r e d 1 6 1
I000 500 i00
25
X Xylene hght yellow C.I. a c i d y e l l o w 17 XI Gold yellow G C.I. r e a c h v e y e l l o w
Explanation
2G
17
as m T a b l e 2 A .
inhlb. mhib.
5
inhib mh~.
6
6
Inhlb.
lnhlb.
inhlb,
6
2
mhib.
1
4
7
311 TABLE 2 (C) M U T A G E N I C A C T I V I T Y O F B I S - A Z O C O M P O U N D S Compound
N u m b e r o f His + r e v e r t a n t s / p l a t e a /zg/Plate
XVI
TA1535
TA100
_
_
+b
+b
TA1538
TA98
_
_
+b
+b
1000 500 100
0 1 0
3 3 1
6 4 4
7 4 3
1 0 0
3 1 1
5 6 4
6 3 2
1000 500 100
3 2 2
4 2 4
13 11 11
16 8 10
5 4 4
6 3 4
3 3 2
7 6 6
1000 500 100
12 9 10
12 8 5
8 8 2
7 8 3
0 1 0
1 1 0
2 1 1
4 5 4
1000 500 100
6 2 1
7 5 3
32 26 26
27 27 26
5_0 44 32
76 69 39
1000 500 100
1 0 0
3 3 1
7 6 1
7 6 2
4 3 1
3 3 2
3 0 0
5 0 3
Black VLN C.I. a c i d b l a c k 26
1000 500 100
5 1 0
4 3 1
7 4 4
6 4 4
2 2 1
4 2 3
8 7 4
6 7 7
XXII Navy blue G C.I. a c i d b l u e 1 2 0
1000 500 100
3 2 2
3 1 0
6 1 4
7 6 1
3 2 3
4 2 2
4 4 2
5 4 3
XXIII Giuba Black D C.I. d~cect b l a c k 17
1000 500 100
6 1 0
8 5 6
42 33 11
51 41 15
81 59 22
120 97 36
98 62 29
190 87 55
Nylosan scarlet F 3 G L C.I. a c i d r e d 111
XVII Fast scarlet N C.I. a c i d r e d 85
XVIII Fast red RS C.I. a c i d r e d 1 1 4
XIX Brown 5R C.I. a c i d o r a n g e 45
XX Fast orange G C.I. a c i d o r a n g e 33
XXI
6.._2 58 51
80 71 59
Explanatlon as in Table 2A.
m o n o ~ z o and bis-azo dyes, which were not detected as mutagens by the Salmonella standard plate method, were also pre-incubated in an activatingmixture prepared according to Matsushirna et al. [14]. None of the tested compounds was mutagenic even after pre-incubation with riboflavin added to $9 mix (data not shown). The two anthraquinone compounds, which gave positive results with the mutagenicity test, both have at the same time an amine and a hydroxyl group that Brown et al. [2] have defined as mutagenic functional groups. Similarly, by comparing the structural formulae of the mono-azo compounds tested, it m a y be inferred that the nitro group, which is present only in the sole mutagenic dye Red GTL, C.I. basic red 18, is a functional group for the mutagenicity of this class of compounds. T w o bis-azo dyes showed mutagenicity without need of metabolic activation (Table 2). This observation contrasts with previous reports that azo derivatives are transformed into active compounds only after reduction of the azo group to an amino group [15]. Tentatively, this discrepancy m a y be explained by the fact that two muta-
312
genic dyes, Brown 5R, C.I. acid orange 45 and Giuba Black D, C.I. direct black 17, are the only t w o c o m p o u n d s among the 8 tested that carry the amino substituent in their aromatic rings. Acknowledgements We thank Mrs. S. Saincich and Ms. C. Gamboz for skillful technical assistance. S. Venturini is the recipient of a fellowship from Anna Villa Rusconi Foundation, Varese (Italy). References 1 P a n / , B., C. M o n t i - B r a g a d i n a n d L. S a m e r , E f f e c t o f excLsion r e p m r s y s t e m o n a n t i b a c t e r i a l a n d m u t a g e m c a c t i v i t y o f d a u n o m y c i n a n d o t h e r i n t e r c a l a t i n g a g e n t s in S t y phi m ur i um , E x p e r i e n t i a , 31 (1975) 787--788. 2 B r o w n , J.P., a n d J . R . B r o w n , M u t a g e n e s l s b y 9 , 1 0 - a n t h r a q u i n o n e d e r i v a t l v e s a n d r e l a t e d c o m p o u n d s m Salmonella t y p h l m u r i u m , M u t a t i o n R e s . , 4 0 ( 1 9 7 6 ) 2 0 3 - - 2 2 4 . 3 D u b o s t , M., P. G a n t e r , R . M o r a l , L. N i n e t , S. P i n n e r t , J. P r e n d ' H o m m e a n d G.S. W e r n e r , U n n o u v e l a n t l b i o t l q u e ~ p r o p r t d t ~ s c y t o s t a t i q u e s : la r u b i d o m y c i n e , C . R . A c a d . Sci., 2 5 7 ( 1 9 6 3 ) 1 8 1 3 . 4 Shahin0 M.M., a n d R . C . y o n B o r s t e l , C o m p a r i s o n o f m u t a t i o n i n d u c t i o n A . Q . m r e v e r m o n s y § t e m s o f Saccharomyces cerevisiae and Salmonella t y p h i m u r i u m , M u t a t i o n Res., 53 ( 1 9 7 8 ) 1 - - 1 0 . 5 S h a h m , M.M., a n d R . C . y o n B o r s t e l , G e n e t l c a c t i v i t y o f t h e a n t ] m i c r o b i a l f o o d a d d i t i v e s A F - 2 a n d H1 9 3 m Saccharomyces cerewsiae, M u t a t i o n R e s . , 3 8 ( 1 9 7 6 ) 3 7 9 - - 3 8 0 . 6 P o i n e r , L . A . , J . A . Miller, E.C. Miller a n d K. S a r o , N - B e n z o y l o x y - N - m e t h y l - 4 - a m i n o a z o b e n z e n e : its c a r c i n o g e n i c a c t l v i t y in t h e r a t a n d its r e a c t i o n s w i t h p r o t e i n s a n d n u e l e l c a c i d s a n d t h e i r c o n s t i t u e n t s m w t r o , C a n c e r Res., 2 7 ( 1 9 6 7 ) 1 6 0 0 - - 1 6 1 3 . 7 P r e u s s m a n n , R., H. D r u c k r e y , S. I v a n k o v i c a n d A . V . H o d e n b e r g , C h e m i c a l s t r u c t u r e a n d c a r c l n o g e n i c i t y o f a l i p h a t i c h y d r a z o , a z o a n d a z o x y c o m p o u n d s a n d o f t r i a z e n e s p o t e n t i a l in vivo a l k y l a t i n g a g e n t s , A n n . N . Y . A c a d . Scl., 1 6 3 ( 1 9 6 9 ) 6 9 7 - - 7 1 6 . 8 V e n i t t , S., a n d C.T. Bushell, M u t a g e n i c i t y o f t h e f o o d c o l o u r b r o w n F K a n d c o n s t i t u e n t s in Salmonella t y p h i m u r i u m , M u t a t i o n R e s . , 4 0 ( 1 9 7 6 ) 3 0 9 - - 3 1 6 . 9 Y a h a g , T., M. D e g a w a , Y. S e i n o , T. M a t s u s h i m a , M. N a g a o , T. S u g i m u r a a n d Y. H a s h i m o t o , M u t a g e n i clty of carcinogenic azo dye and their derivatives, Cancer Lett., 1 (1975) 91--96. 1 0 B r o w n , J.P., G.W. R o e h m a n d R . J . B r o w n , M u t a g e n i c l t y t e s t i n g o f c e r t i f i e d f o o d c o l o r s a n d r e l a t e d a z o , x a n t h e n e a n d t r l p h e n y l m e t h a n e d y e s w i t h t h e S a l m o n e l l a / m i c r o s o m e s y s t e m , M u t a t i o n Res., 5 6 (1978) 249--271. 11 A m e s , B.N., F . D . L e e a n d W.E. D u r s t o n , A n i m p r o v e d b a c t e r i a l t e s t s y s t e m f o r d e t e c t i o n a n d classlflc a t i o n o f m u t a g e n s a n d c a r c i n o g e n s , P r e c . N a t i . A c a d . Sci. ( U . S . A . ) , 7 0 ( 1 9 7 3 ) 7 8 2 - - 7 8 6 . 1 2 A m e s , B.N., J. M c C a n n a n d E. Y a m a s a k i , M e t h o d s f o r d e t e c t i n g c a r c i n o g e n s a n d m u t a g e n s w i t h t h e S a l m o n e l l a / m a m m a l i a n m l c r o s o m e m u t a g e n i c i t y t e s t , M u t a t i o n R e s . , 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 1 3 A m e s , B.N., W.E. D u r s t o n , E. Y a m a s a k i a n d F . D . Lee, C a r c i n o g e n s a r e m u t a g e n s , a s i m p l e t e s t c o m b i n i n g liver h o m o g e n a t e f o r a c t i v a t i o n a n d b a c t e r i a f o r d e t e c t i o n , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 0 (1973) 2281--2285. 1 4 M a t s u s h L m a , T., B. T e l c h m a n n , M. S a w a m u r a a n d T. S u g l m u r a , M u t a g e m c l t y o f a z o c o m p o u n d s I m p r o v e d m e t h o d f o r d e t e c t i n g t h e i r m u t a g e n i c i t l e s b y t h e S a l m o n e l l a m u t a t i o n test, M u t a t l o n R e s . , 54 (1978) 220--221. 1 5 G a r n e r , R . C . , a n d C . A . N u t m a n , T e s t i n g o f s o m e a z o d y e s a n d theLr r e d u c t i o n p r o d u c t s f o r m u t a g e m c i t y u s i n g Salmonella t y p h i m u r i u m T A 1 5 3 8 , M u t a t i o n R e s . , 4 4 ( 1 9 7 7 ) 9 - - 1 9 .
Mutation Research, 68 (1979) 307--312 © Elsevier/North-Holland Biomedical Press
MUTAGENICITY OF A N T H R A Q U I N O N E AND AZO DYES IN AMES'
Salmonella typbimurium TEST S. VENTURINI and M. TAMARO
Institute of Microbioloy, University of Trieste, Trieste (Italy) (Received 8 August 1978) (Revision received 3 July 1979) (Accepted 5 July 1979)
Summary 23 dyes belonging to different chemical classes -- anthraquinones, mono- and bis-azo c o m p o u n d s -- were tested for their mutagenic activity on Ames strains o f Salmonella typhimurium. 5 dyes induced frameshift mutations.
Anthraquinone and azo compounds are industrially important substances largely used as dyes. Anthraquinone derivatives are closely related to anthracycline antitumor antibiotics of k n o w n mutagenicity and carcinogenicity [1-5]. Azo dyes, mono-azo as well as bis-azo derivatives, represent a large part of the total dye~stuffs manufactured, and are primarily used for coloring substrates from foods and textiles through to plastics. It is known that the azo group is linked with carcinogenesis, even when produced by metabolic activation, as it is with certain mammalian and bacterial enzymes [6,7], and some azo compounds axe mutagenic in bacterial tests [8--10]. 23 industrial c o m p o u n d s widely used as textile dyes, and belonging to the above~ited chemical classes (see Table 1 for structures) were tested b y Ames' test for mutagenicity. Substances listed in Table 1 were obtained from A.C.N.A., Bayer, Ciba, Hoechst, I.C.I. and Sandoz. We are aware that these preparations may contain impurities up to 30% of their weight. However no further purification was attempted, because we wanted to test the potential danger that they represent in actual use. Materials and methods
Salmonella typhimurium strains TA1535, TA100, TA1538 and TA98 were obtained from Dr. Bruce N. Ames {University of California, Berkeley). These bacterial tester strains have a mutation in the histidine operon which consequently results in nutritional dependence on histidine.
308
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E
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£
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U
U
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z
M
•
E
Z
309 TA1535 and TA100 can be reverted to prototrophy by DNA base-pair substitutions, whereas TA1538 and TA98 are reverted by frameshift mutation. Mutation tests were carried out according to the soft-agar overlay method described by Ames et al. [11,12]. Substances listed in Table 1 were dissolved in dimethylsulfoxide (DMSO) and tested at dose levels of 100, 500 and 1000 pg/ plate. When tests were done for metabolic activation by mammalian microsomal enzymes, 0.5 ml of "$9 mix" was added to the top agar. $9 mix was prepared from liver homogenate of rats treated with Aroclor 1254 plus adequate cofactots, as described by Ames et al. [13]. Positive controls were used for the tester strains as follows: ethyl methanesulfonate (EMS) for TA1535; methyl methanesulfonate (MMS) for TA100 and hycanthone for TA1538 and TA98.
TABLE2 (A) M U T A G E N I C A C T I V I T Y OF A N T H R A Q U I N O N E COMPOUNDS Compound
N u m b e r o f His* r e v e r t a n t s / p l a t e a ~g/plate
I
TA1535
TAI00
--
_
+b
_
+b
_
+b
415 29---O 10__._2
424 31--'O 6__3
463 34"-~ 10___9
+b
TA1538
TA98
C.I. disperse red 15
1000 500 I00
3 3 1
5 4 3
10 7 2
45 40 38
380 27-'-0 5_5
II Violet C i b a c e t 2 R C.I. disperse v i o l e t 1
1000 500 I00
2 1 2
6 4 4
inhib. 8 11 21 3
5 2 2
6 4 2
3 3 1
18 19 21
III Alizarin light b l u e SE C.I. acid b l u e 43
1000 500 100
2 1 1
3 4 1
22 22 25
12 11 13
18 12 18
52 40 38
19 16 13
141 109 96
IV C.I. acld g r e e n 41
1000 500 100
5 4 4
4 4 1
16 11 10
17 17 19
1 2 3
1 7 6
9 9 11
12 12 14
V Light blue A N L C.I. acid b l u e 25
1000 500 100
1 3 4
6 6 7
1 2 10
0 1 7
2 0 7
8 1 9
1 1 0
4 3 1
VI Blue F 3 G A C.I. r e a c t i v e b l u e 2
1000 500 100
17 10 10
14 10 3
21 11 4
4 3 5
1 0 2
2 1 2
4 2 3
5 3 4
VII Light blue B C.I. acid b l u e 41
1000 500 100
1 4 1
1 1 2
2 3 4
5 2 2
3 2 3
6 6 7
3 4 6
4 3 6
VIII Blue F 3 R C.I. disperse b l u e 3
1000 500 100
1 1 1
3 2 1
7 10 12
10 10 9
8 7 4
4 4 4
18 18 15
21 18 16
R e d C i b a c e t 3B
F a s t g r e e n 5G
a Number of revertant colonies per plate, mean of 4 separate expe~ments
after subtracting spontaneous
revertants. b Indicates plates without
($9 mix) stimulated b y A m -
( - - ) o r w i t h ( + ) rat-liver m i c r o s e m a l
clor 1 2 5 4 . V a l u e s u n d e r l i n e d are i n d i c a t i v e o f m u t a g e n i c a c t i v i t y . Inhlb., inhibition of bacterial gro wth.
preparation
310
The numbers of spontaneous revertants in the presence of DMSO alone + "$9 mix" were: 12 for strain T A 1 5 3 5 , 125 for strain TA100, 8 for strain T A 1 5 3 8 and 21 for strain TA98. Results and discussion
Table 2 shows the results obtained. 2 of the 8 anthraquinone dyes tested for mutagenicity with Salmonella typhimurium strains, Red Cibacet 3B, C.I. disperse red 15 and Alizarin light blue SE, C.I. acid blue 43, acted as frameshift mutagens. Red Cibacet 3B at a 1 0 0 0 pg/plate dose level showed a reversion 20 times that of the spontaneous rate in strain TA98. Only one of the mono-azo compounds, red GTL--C.I. basic red 18, had any mutagenic activity when tested with strains TA98 and T A 1 5 3 8 at 100, 500 and 1000 #g/plate levels (Table 2). Only weak mutagenic activity was detected when Brown 5R, C.I. acid orange 45 was tested both with and without metabolic activation (Table 2). Another bis-azo dye, Giuba black D, C.I. direct black 17, exhibited frameshift mutagenic activity. Matsushima et ai. [14] showed that the addition of riboflavin to the $9 mix was necessary for mutagenic activity of some azo compounds. Therefore,
TABLE 2 (B) M U T A G E N I C A C T I V I T Y O F M O N O - A Z O C O M P O U N D S Compound
a
Number of His + revertants/plate t~g/plate
IX Yellow ERPL C.I. a c i d y e l l o w
TA1535
TA100
--
--
+b
+b
TA1538 --
TA98 +b
_
+b
I000 500 I00
5 3 1
6 6 7
14 8 8
16 9 7
1 0 0
3 1 0
0 0 0
1 2 0
1000 500 I00
4 4 2
10 9 6
9 2 2
14 7 8
1 1 0
2 1 1
2 0 0
3 2 2
1000 500 100
1 1 2
3 3 3
8 4 4
S 4 5
1 1 0
1 1 2
6 5 8
3 3 6
XII Red GTL C.I. b a s l e r e d 18
1000 500 I00
2 1 0
2 2 1
12 7 1
10 6 6
114 105 98
140 117 115
170 140 I00
122 112 104
XIII Red H3B C.I. r e a c t i v e r e d 3
1000 500 100
12 9 0
19 10 10
13 7 2
18 9 8
5 2 0
6 2 1
4 2 2
8 4 3
XIV Red 2BF C.I. r e a c t i v e r e d 42
1000 500 100
10 6 4
13 10 10
14 8 7
14 4 11
3 4 4
11 9 8
4 5 6
15 9 8
XV Fast red BF C.I. a m d r e d 1 6 1
I000 500 i00
25
X Xylene hght yellow C.I. a c i d y e l l o w 17 XI Gold yellow G C.I. r e a c h v e y e l l o w
Explanation
2G
17
as m T a b l e 2 A .
inhlb. mhib.
5
inhib mh~.
6
6
Inhlb.
lnhlb.
inhlb,
6
2
mhib.
1
4
7
311 TABLE 2 (C) M U T A G E N I C A C T I V I T Y O F B I S - A Z O C O M P O U N D S Compound
N u m b e r o f His + r e v e r t a n t s / p l a t e a /zg/Plate
XVI
TA1535
TA100
_
_
+b
+b
TA1538
TA98
_
_
+b
+b
1000 500 100
0 1 0
3 3 1
6 4 4
7 4 3
1 0 0
3 1 1
5 6 4
6 3 2
1000 500 100
3 2 2
4 2 4
13 11 11
16 8 10
5 4 4
6 3 4
3 3 2
7 6 6
1000 500 100
12 9 10
12 8 5
8 8 2
7 8 3
0 1 0
1 1 0
2 1 1
4 5 4
1000 500 100
6 2 1
7 5 3
32 26 26
27 27 26
5_0 44 32
76 69 39
1000 500 100
1 0 0
3 3 1
7 6 1
7 6 2
4 3 1
3 3 2
3 0 0
5 0 3
Black VLN C.I. a c i d b l a c k 26
1000 500 100
5 1 0
4 3 1
7 4 4
6 4 4
2 2 1
4 2 3
8 7 4
6 7 7
XXII Navy blue G C.I. a c i d b l u e 1 2 0
1000 500 100
3 2 2
3 1 0
6 1 4
7 6 1
3 2 3
4 2 2
4 4 2
5 4 3
XXIII Giuba Black D C.I. d~cect b l a c k 17
1000 500 100
6 1 0
8 5 6
42 33 11
51 41 15
81 59 22
120 97 36
98 62 29
190 87 55
Nylosan scarlet F 3 G L C.I. a c i d r e d 111
XVII Fast scarlet N C.I. a c i d r e d 85
XVIII Fast red RS C.I. a c i d r e d 1 1 4
XIX Brown 5R C.I. a c i d o r a n g e 45
XX Fast orange G C.I. a c i d o r a n g e 33
XXI
6.._2 58 51
80 71 59
Explanatlon as in Table 2A.
m o n o ~ z o and bis-azo dyes, which were not detected as mutagens by the Salmonella standard plate method, were also pre-incubated in an activatingmixture prepared according to Matsushirna et al. [14]. None of the tested compounds was mutagenic even after pre-incubation with riboflavin added to $9 mix (data not shown). The two anthraquinone compounds, which gave positive results with the mutagenicity test, both have at the same time an amine and a hydroxyl group that Brown et al. [2] have defined as mutagenic functional groups. Similarly, by comparing the structural formulae of the mono-azo compounds tested, it m a y be inferred that the nitro group, which is present only in the sole mutagenic dye Red GTL, C.I. basic red 18, is a functional group for the mutagenicity of this class of compounds. T w o bis-azo dyes showed mutagenicity without need of metabolic activation (Table 2). This observation contrasts with previous reports that azo derivatives are transformed into active compounds only after reduction of the azo group to an amino group [15]. Tentatively, this discrepancy m a y be explained by the fact that two muta-
312
genic dyes, Brown 5R, C.I. acid orange 45 and Giuba Black D, C.I. direct black 17, are the only t w o c o m p o u n d s among the 8 tested that carry the amino substituent in their aromatic rings. Acknowledgements We thank Mrs. S. Saincich and Ms. C. Gamboz for skillful technical assistance. S. Venturini is the recipient of a fellowship from Anna Villa Rusconi Foundation, Varese (Italy). References 1 P a n / , B., C. M o n t i - B r a g a d i n a n d L. S a m e r , E f f e c t o f excLsion r e p m r s y s t e m o n a n t i b a c t e r i a l a n d m u t a g e m c a c t i v i t y o f d a u n o m y c i n a n d o t h e r i n t e r c a l a t i n g a g e n t s in S t y phi m ur i um , E x p e r i e n t i a , 31 (1975) 787--788. 2 B r o w n , J.P., a n d J . R . B r o w n , M u t a g e n e s l s b y 9 , 1 0 - a n t h r a q u i n o n e d e r i v a t l v e s a n d r e l a t e d c o m p o u n d s m Salmonella t y p h l m u r i u m , M u t a t i o n R e s . , 4 0 ( 1 9 7 6 ) 2 0 3 - - 2 2 4 . 3 D u b o s t , M., P. G a n t e r , R . M o r a l , L. N i n e t , S. P i n n e r t , J. P r e n d ' H o m m e a n d G.S. W e r n e r , U n n o u v e l a n t l b i o t l q u e ~ p r o p r t d t ~ s c y t o s t a t i q u e s : la r u b i d o m y c i n e , C . R . A c a d . Sci., 2 5 7 ( 1 9 6 3 ) 1 8 1 3 . 4 Shahin0 M.M., a n d R . C . y o n B o r s t e l , C o m p a r i s o n o f m u t a t i o n i n d u c t i o n A . Q . m r e v e r m o n s y § t e m s o f Saccharomyces cerevisiae and Salmonella t y p h i m u r i u m , M u t a t i o n Res., 53 ( 1 9 7 8 ) 1 - - 1 0 . 5 S h a h m , M.M., a n d R . C . y o n B o r s t e l , G e n e t l c a c t i v i t y o f t h e a n t ] m i c r o b i a l f o o d a d d i t i v e s A F - 2 a n d H1 9 3 m Saccharomyces cerewsiae, M u t a t i o n R e s . , 3 8 ( 1 9 7 6 ) 3 7 9 - - 3 8 0 . 6 P o i n e r , L . A . , J . A . Miller, E.C. Miller a n d K. S a r o , N - B e n z o y l o x y - N - m e t h y l - 4 - a m i n o a z o b e n z e n e : its c a r c i n o g e n i c a c t l v i t y in t h e r a t a n d its r e a c t i o n s w i t h p r o t e i n s a n d n u e l e l c a c i d s a n d t h e i r c o n s t i t u e n t s m w t r o , C a n c e r Res., 2 7 ( 1 9 6 7 ) 1 6 0 0 - - 1 6 1 3 . 7 P r e u s s m a n n , R., H. D r u c k r e y , S. I v a n k o v i c a n d A . V . H o d e n b e r g , C h e m i c a l s t r u c t u r e a n d c a r c l n o g e n i c i t y o f a l i p h a t i c h y d r a z o , a z o a n d a z o x y c o m p o u n d s a n d o f t r i a z e n e s p o t e n t i a l in vivo a l k y l a t i n g a g e n t s , A n n . N . Y . A c a d . Scl., 1 6 3 ( 1 9 6 9 ) 6 9 7 - - 7 1 6 . 8 V e n i t t , S., a n d C.T. Bushell, M u t a g e n i c i t y o f t h e f o o d c o l o u r b r o w n F K a n d c o n s t i t u e n t s in Salmonella t y p h i m u r i u m , M u t a t i o n R e s . , 4 0 ( 1 9 7 6 ) 3 0 9 - - 3 1 6 . 9 Y a h a g , T., M. D e g a w a , Y. S e i n o , T. M a t s u s h i m a , M. N a g a o , T. S u g i m u r a a n d Y. H a s h i m o t o , M u t a g e n i clty of carcinogenic azo dye and their derivatives, Cancer Lett., 1 (1975) 91--96. 1 0 B r o w n , J.P., G.W. R o e h m a n d R . J . B r o w n , M u t a g e n i c l t y t e s t i n g o f c e r t i f i e d f o o d c o l o r s a n d r e l a t e d a z o , x a n t h e n e a n d t r l p h e n y l m e t h a n e d y e s w i t h t h e S a l m o n e l l a / m i c r o s o m e s y s t e m , M u t a t i o n Res., 5 6 (1978) 249--271. 11 A m e s , B.N., F . D . L e e a n d W.E. D u r s t o n , A n i m p r o v e d b a c t e r i a l t e s t s y s t e m f o r d e t e c t i o n a n d classlflc a t i o n o f m u t a g e n s a n d c a r c i n o g e n s , P r e c . N a t i . A c a d . Sci. ( U . S . A . ) , 7 0 ( 1 9 7 3 ) 7 8 2 - - 7 8 6 . 1 2 A m e s , B.N., J. M c C a n n a n d E. Y a m a s a k i , M e t h o d s f o r d e t e c t i n g c a r c i n o g e n s a n d m u t a g e n s w i t h t h e S a l m o n e l l a / m a m m a l i a n m l c r o s o m e m u t a g e n i c i t y t e s t , M u t a t i o n R e s . , 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 1 3 A m e s , B.N., W.E. D u r s t o n , E. Y a m a s a k i a n d F . D . Lee, C a r c i n o g e n s a r e m u t a g e n s , a s i m p l e t e s t c o m b i n i n g liver h o m o g e n a t e f o r a c t i v a t i o n a n d b a c t e r i a f o r d e t e c t i o n , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 0 (1973) 2281--2285. 1 4 M a t s u s h L m a , T., B. T e l c h m a n n , M. S a w a m u r a a n d T. S u g l m u r a , M u t a g e m c l t y o f a z o c o m p o u n d s I m p r o v e d m e t h o d f o r d e t e c t i n g t h e i r m u t a g e n i c i t l e s b y t h e S a l m o n e l l a m u t a t i o n test, M u t a t l o n R e s . , 54 (1978) 220--221. 1 5 G a r n e r , R . C . , a n d C . A . N u t m a n , T e s t i n g o f s o m e a z o d y e s a n d theLr r e d u c t i o n p r o d u c t s f o r m u t a g e m c i t y u s i n g Salmonella t y p h i m u r i u m T A 1 5 3 8 , M u t a t i o n R e s . , 4 4 ( 1 9 7 7 ) 9 - - 1 9 .
