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Structure—activity relationship within a series of m-diaminobenzene derivatives
25
Mutation Research, 78 (1980) 25--31 © Elsevier/North-Holland Biomedical Press
STRUCTURE--ACTIVITY RELATIONSHIP WITHIN A SERIES OF m-DIAMINOBENZENE DERIVATIVES
MAJDI M. SHAHIN, A. BUGAUT and G. KALOPISSIS L'Oreal Research Laboratories, L 'Oreal, 1, Avenue de St Germain, 93601 Aulnay-sous-Bois (France)
(Received 19 September 1979) (Revision received 19 November 1979) (Accepted 21 November 1979)
Summary We investigated the mutagenicity of m-diaminobenzene (m-phenylenediamine) and four 2,4-diaminoalkylbenzenes (methyl, ethyl, isopropyl and n-butyl) in Salmonella typ hi m ur i um strains TA100, TA1538 and TA98 in the absence and presence of $9 induced by Acoclor 1254. m-Diaminobenzene was the most active mutagen, followed by 2,4-diaminotoluene and 2,4-diaminoethylbenzene, resp. Negative response was observed for both 2,4-diaminoisopropylbenzene and 2,4-diamino-n-butylbenzene. Thus, depending on the size of the substituting alkyl group at the C~ position of 2,4-diaminoalkylbenzene, a decline and loss of mutagenic activity was observed.
m-Diaminobenzene induces frameshift mutations in Salmonella t y p h i m u r i u m strain TA1538 in the presence of liver microsomal fractions from rats pretreated with Aroclor 1254 [4] or with phenobarbital [6]. It also induces frameshift mutations in strain TA98 in the presence of metabolic activation [9]. 2,4Diaminotoluene also induces mutations in Salmonella t yphi m uri um [4,8,9]. Urine from rats treated with 2,4-diaminotoluene, but not urine from rats treated with 2,4-diaminobutylbenzene, reverts Salmonella t yphi m uri um strain TA1538 [ 5]. Our preliminary experiments have shown that the introduction of the methyl group into m-diaminobenzene causes a decrease in mutagenicity, thereby indicating that m-diaminobenzene is more mutagenic in Salmonella t y p h imu r iu m than 2,4-diaminotoluene. We therefore initiated a study of the structure--activitiy relationship to determine the effect of different alkyl groups on the mutagencity of m-diaminobenzene. To ensure that we were testing the mutagenicity of a single component, we tested purified and unpurified samples of the compounds. The first 2 of the 5 compounds are used as hair-dye components [4] and the other 3 are structurally related to hair dyes; conse-
26 quently, information about their mutagenic activity or inactivity in relation to their structures is of interest to us. Materials and methods
Salmonella typhimurium 2 of the strains, TA1538 and TA98, can be used for the detection of mutagens that cause frameshift mutations, whereas TA100 can be used for the detection of mutagens causing base-pair substitutions [2,7]. The strains were kindly provided by Dr. B.N. Ames. Media, preparation of cell samples, and treatment conditions The media, preparation of cell samples, and treatment conditions used in the present studies have been described by Ames [1] and Ames et al. [2,3]. The a m o u n t of $9 mix added was 0.5 ml/plate. Chemicals The 5 c o m p o u n d s (Fig. 1) tested were: m-diaminobenzene, 2,4-diaminotoluene, 2,4-diaminoethylbenzene, 2,4-diaminoisopropylbenzene and 2,4-diaminon-butylbenzene. All were synthesized in our laboratories by catalytic reduction, with H2 Pd/C, from the corresponding m-nitroanilines. After distillation, all gave satisfactory analyses (m.p. and b.p. data recorded in Tables 1--5). Melting points were recorded on a Kofler Thermopan Microscope and are uncorrected. The positive control substance 1,2-diamino-4-nitrobenzene (4-nitro-O-phenylenediamine) was obtained from Merck, Darmstadt (Germany). Dimethyl sulfoxide (DMSO) was obtained from Carlo Erba, Divisione Chimica, Milano (Ita-
NH= @NHz m-Diaminobenzene
NH2 H3C'~ 2,4-Diaminotoluene
H3C.~" J . .NHz H3C~CH'T~ ~ ~'~J" NHz 2,4-Diaminoisopropylbenzene
NH2 HsC'-~'~ 24-
Diaminoethylbenzene
NH2 H'C'~" I ~"~/f~NH z 2.4-Diamino n-butylbenzene
Fig. 1. Chemical structure and names of the compounds tested.
27 ly). Glucose 6-phosphate was obtained from Fluka AG, Buchs SG (Switzerland). NADP was obtained from Boehringer Mannheim GmbH (W. Germany).
Solvents All compounds tested, as well as the positive controls, were dissolved in DMSO. Fresh solutions were prepared for each experiment. Animals Male Wistar rats weighing 200--250 g were used. The rats were injected intraperitoneally with Aroclor 1254 (diluted in corn oil to a concentration of 200 mg/ml) at a dose of 500 mg/kg. 5 days later, the rats were killed and the liver-microsomal fraction was prepared [3]. Methods o f evaluation A dose--response relationship, which shows progressive increase over the spontaneous background as a function of concentration, is considered to be a
TABLE 1 MUTAGENIC EVALUATION OF m-DIAMINOBENZENE WITHOUT PURIFICATION, AND AFTER P U R I F I C A T I O N BY D I S T I L L A T I O N , IN Salmonella t y p h i m u r i u m S T R A I N S I N T H E A B S E N C E (--) A N D P R E S E N C E (+) O F R A T - L I V E R - M I C R O S O M E A C T I V A T I O N S Y S T E M
Dose/plate
Revertant colonies/plate a
TAI00
Controls, no additions
B E
Controls, DMSO Controls, 1,2-diamino4-nitrobenzene C o n t r o l s , $9 m i x , no NADP
B
E B
E
TA1538
--
+
135 143
133 135
17 18
31 28
44 48
64 60
1 0 0 pl
111
132
18
26
47
60
20 pg 20 ~g
756 707
418 543
2610 2376
2366 2483
2748 2578
2197 2175
5 0 0 #1 500pl
---
135 135
---
15 15
---
52 46
00 00
--
TA98 +
--
+
m-Diaminobenzene without purification
5 10 20 50 100 250 500 1000
#g pg pg #g pg #g ~g #g
115 122 129 118 111 114 122 131
126 132 162 201 250 348 475 587
13 18 15 24 23 29 36 48
117 207 319 771 1300 1894 2610 3054
49 48 43 45 47 44 59 63
124 213 418 843 1397 2213 3060 3356
m-Diaminobenzene purified by distillation BP ( 1 8 m m ) = 1 7 5 ° C MP = 6 2 . 5 - - 6 3 ° C
5 10 20 50 100 250 500 1000
~g #g ~g pg pg pg ~g ~g
100 101 97 89 107 102 106 111
131 122 131 169 174 217 251 293
14 15 15 13 14 16 19 29
84 103 163 278 468 898 1635 2238
38 36 40 42 39 42 47 53
84 111 145 282 423 877 1524 2172
a The numbers of spontaneous controls have not been subtracted. B, controls at the beginning of the daily experiment; E, controls at the end of the experiment.
28 reliable criterion for genetic activity of compounds tested. A 2.5-fold increase over background, when found in repeated experiments, is also considered to be an indication of weak genetic activity. Results and discussion In the absence of $9 mix only the unpurified m-diaminobenzene showed a slight increase in the number of revertant colonies over those of the control values in strain TA1538, whereas the purified sample was not mutagenic in any of the strains used (Table 1). No mutations were induced by the other 4 compounds (unpurified or purified) in the absence of $9 mix (data are not included in this paper). Thus, actually, none of the 5 compounds was mutagenic in the 3 strains of Salmonella typhimurium in the absence of $9 mix. In our studies with m-diaminobenzene in the presence of $9 mix this c o m p o u n d was highly mutagenic in the 2 strains TA1538 and TA98; to a lesser extent it was also mutagenic in TA100, the unpurified sample being more pronounced in its activity (Table 1). All 3 strains exhibited a dose-related response (Table 1). Among the 5 compounds tested, m-diaminobenzene was the most active mutagen (Table 1), followed by 2,4-diaminotoluene (Table 2) and 2,4-diaminoethylbenzene
TABLE 2 MUTAGENIC EVALUATION OF 2,4-DIAMINOTOLUENEWITHOUTPURIFICATION, AND AFTER PURIFICATION BY DISTILLATION, IN Salmonella typhimurium STRAINS IN THE PRESENCE OF RAT-LIVER-MICROSOME ACTIVATIONSYSTEM Dose/plate
Revertant colonies/plate TAIO0
Controls, no additions
B E
Controls, DMSO
00 O0
136 124
TA1538 33 32
TA9S 37 40
1 0 0 ttl
126
36
39
Controls, 1,2-diamino4-nitrobenzene
B E
20 pg 20 p g
351 399
2400 2442
2070 2130
C o n t r o l s , $9 m i x , no N A D P
B E
5 0 0 ttl 5 0 0 pl
118 128
25 27
31 28
2,4-Diaminotoluene without purification
5 10 20 50 100 250 500 1000
pg pg ttg ttg Pg ttg #g pg
124 122 153 152 162 177 197 193
47 45 6O 73 115 390 714 1032
53 55 68 103 158 169 500 746
2,4-Diaminotoluene, purified by distillation BP 18 m m = 1 9 0 ° C MP = 9 9 ° C
5 10 20 50 100 250 500 i000
ttg pg pg pg pg ttg pg pg
132 127 142 159 178 216 185 199
46 51 59 111 133 329 678 942
49 60 63 105 143 307 572 818
29 TABLE 3 M U T A G E N I C E V A L U A T I O N OF 2 , 4 - D I A M I N O E T H Y L B E N Z E N E W I T H O U T P U R I F I C A T I O N , AND A F T E R P U R I F I C A T I O N BY D I S T I L L A T I O N , IN Salmonella t y p h i m u r i u m S T R A I N S IN T H E P R E S ENCE OF R A T - L I V E R - M I C R O S O M E A C T I V A T I O N SYSTEM Dose •plate
Revertant c o l o n i e s / p l a t e TA100
Controls, no additions C o n t r o l s , DMSO Controls, 1,2-diamino4-nitrobenzene C o n t r o l s , $9 m i x , no NADP
B E
TA1538
TA98
O0 O0
137 132
38 39
61 58
1 0 0 pl
130
35
60
20 pg 20 pg
383 394
2193 2477
2038 1984
500 pl 5 0 0 ~1
130 140
25 25
44 46
2,4-Diaminoethylbenzene without purification
50 100 200 500 1000 2500 5000 10 0 0 0
pg ~g ~g Dg #g pg ~g ~g
165 184 154 177 169 159 152 161
37 41 62 83 128 260 234 106
72 99 143 270 417 462 284 173
2,4-Diaminoethylbenzene purified b y distillation BP 18 m m = 1 6 5 ° C
50 100 200 500 1000 2500 5000 10 0 0 0
pg pg Dg Pg ~g ~g ~g pg
155 175 165 179 171 175 156 146
39 80 81 189 330 414 259 95
71 97 125 192 482 367 365 208
(Table 3), resp. In contrast, the purified samples of the 2 compounds 2,4-diaminoisopropylbenzene and 2,4-diamino-n-butylbenzene, which were tested over a wide range of concentrations, gave a negative response (Tables 4 and 5). The results of m-diaminobenzene and 2,4-diaminotoluene are in agreement with those obtained previously [4] ; both were mutagenic in Salmonella typhimurium. In our investigations, at all concentrations used, the mutagenic activity of m-diaminobenzene was higher than that of 2,4-diaminotoluene. It is, however, inadequate and very difficult to compare data obtained in different laboratories. In our investigations, to control possible errors during the experimental procedure and to ensure the effectiveness of the system, controls were carried o u t at the beginning and end of each daily experiment (see Tables). In addition, the purified and unpurified materials of each of the compounds were tested on the same day using the same $9 mix and the same cell culture. Furthermore, several samples of each compound, including commercial products when available, were included in our investigations. The same pattern of activity was always observed. 2-Aminoanthracene at different concentrations was also used as a positive control and exhibited the expected responses. As already reported, the present investigations were undertaken to study the structure--activity relationship by investigating whether the addition of certain
3O TABLE 4 MUTAGENIC EVALUATION OF 2,4-DIAMINOISOPROPYLBENZENE WITHOUT PURIFICATION, A N D A F T E R P U R I F I C A T I O N BY D I S T I L L A T I O N , I N S a l m o n e l l a t y p h i m u r i u m S T R A I N S IN T H E PRESENCE OF RAT-LIVER-MICROSOME ACTIVATION SYSTEM
Dose/Plate
Revertant c o l o n i e s / p l a t e TA100
Controls, no a d d i t i o n s
B E
Controls, D M S O Controls, 1,2-diamino4-nitrobenzene Controls, $9 m i x , no NADP 2 , 4 - D i a m i n o i s o P r o P ylbenzene without
purification
2,4-Diaminoisopropyl-
b e n z e n e purified b y distillation BP 18 m m = 1 6 6 - - 1 6 8 ° C MP = 4 7 ° C
00 00
TA1538
TA9S
115 123
28 28
48 53
1 0 0 #1
113
29
47
20 p g 20 p g
398 392
2273 2543
2045 2207
5 0 0 pl 5 0 0 pl
120 115
14 16
43 45
5 10 20 50 100 250 500 1000
pg pg pg pg #g ~g pg ~g
109 116 125 132 132 123 131 130
28 26 25 30 43 61 65 64
45 57 51 66 53 73 79 83
5 10 20 50 100 250 500 1000
pg #g pg pg ~g pg pg #g
94 89 107 104 117 115 107 97
21 26 20 21 28 36 40 35
46 35 55 55 52 42 50 54
alkyl groups to the structure of a known mutagen has any effect on its mutagenicity. The results of the purified samples of m-diaminobenzene, 2,4-diaminotoluene and 2,4-diaminoethylbenzene can be seen in Tables 1, 2 and 3. The t w o compounds that have the alkyl groups CH3 and C2Hs in their structures were less mutagenic than m-diaminobenzene; the c o m p o u n d with the ethyl group exhibited lower activity than the c o m p o u n d with the methyl group. A complete removal of the mutagenic activity was achieved in the presence of isopropyl CH3~
jCH-CH3
or
n-butyl (CH3--(CH2)3--)
groups (Tables 4 and 5). Thus depending on the size of the substituting alkyl group in 2,4-diaminoalkylbenzene, a decline and loss of mutagenic activity was observed.
31 TABLE 5 MUTAGENIC EVALUATION OF 2,4-DIAMINOBUTYLBENZENE WITHOUT PURIFICATION, AND A F T E R P U R I F I C A T I O N BY D I S T I L L A T I O N , IN Salmonella t y p h i m u r i u m S T R A I N S IN T H E P R E S ENCE OF RAT-LIVER-MICROSOME ACTIVATION SYSTEM Do s e / p l a t e
Revertant colonies/plate TA100
Controls, no additions Controls, DMSO Controls, 1,2-diamino4-nitrobenzene C o n t r o l s , $9 m i x , no NADP
B E
TA1538
TA98
00 00
116 128
42 32
47 43
100 ~
114
34
47
20 pg 20 pg
345 356
2467 2325
2276 2005
5 0 0 pl 5 0 0 pl
119 129
25 22
28 28
2,4-Diaminobutylbenzene without purification
5O 100 200 500 1000 2500 5000 10 000
pg ~g ~g pg ~g pg ~g pg
191 189 223 202 155 161 125 000
49 48 46 62 65 70 58 00
58 49 53 65 49 56 25 00
2,4-Diaminobutylbenzene purified by d i s t i l l a t i o n BP 1 8 m m = 1 7 0 ° C
50 100 200 500 1000 2500 5000 10 000
~g ~g ~g pg pg pg pg pg
153 201 209 172 166 164 163 076
40 40 38 52 67 55 54 00
49 53 53 52 63 57 15 00
References 1 A m e s , B.N., T h e d e t e c t i o n o f c h e m i c a l m u t a g e n s w i t h e n t e r i c b a c t e r i a , in: A. H o l l a e n d e r ( E d . ) , C h e m i cal M u t a g e n s . P r i n c i p l e s a n d M e t h o d s f o r t h e i r D e t e c t i o n , V o l . 1, P l e n u m , N e w Y o r k , 1 9 7 1 , p p . 2 6 7 - 282. 2 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 t h e d e t e c t i o n a n d classif i c 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 o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 0 ( 1 9 7 3 ) 7 8 2 - - 7 8 6 . 3 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 S a l m o n e l l a / m a m m a l i a n m i 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 Res., 3 1 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 4 A m e s , B.N., H . O . K a m m e n a n d E. Y a m a s a k i , H a i r d y e s are m u t a g e n i c : i d e n t i f i c a t i o n o f a v a r i e t y o f m u t a g e n i c i n g r e d i e n t s , P r o c . Natl. A c a d . Sci. ( U . S . A . ) , 7 2 ( 1 9 7 5 ) 2 4 2 3 - - 2 4 2 7 . 5 C i n k o t a i , K.I., C.C. J o n e s , J . C . T o p h a m a n d P . A . W a t k i n s , E x p e r i e n c e w i t h m u t a g e n i c t e s t s as i n d i c a t o r s o f c a r c i n o g e n i c a c t i v i t y , M u t a t i o n Res., 53 ( 1 9 7 8 ) 1 6 7 - - 1 6 8 . 6 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 of s o m e a z o d y e s a n d t h e i r 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 n i 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 . 7 M c C a n n , J., N . E . S p i n g a r n , J. K o b o r i a n d B . N . A m e s , D e t e c t i o n o f c a r c i n o g e n s as m u t a g e n s : b a c t e r i a l t e s t e r s t r a i n s w i t h R f a c t o r p l a s m i d s , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 2 ( 1 9 7 5 ) 9 7 9 - - 9 8 3 . 8 S h a h , M.J., R . J . P i e n t a , W.B. L e b h e r z a n d A.W. A n d r e w s , C o m p a r a t i v e s t u d i e s of b a c t e r i a l m u t a t i o n a n d h a m s t e r cell t r a n s f o r m a t i o n i n d u c e d b y 2 , 4 - t o l u e n e d i a m i n e , in: S. W e i n h o u s e , M. F o t i a n d P . A . Bergbauer (Eds.), Proceedings of the 68th Annual Meeting of the American Association of Cancer Research, 18 (1977) 23. 9 Y o s h i k a w a , K., H. U c h i n o a n d H. K u r a t a , S t u d i e s o n m u t a g e n i c i t y o f hai~ d y e , Eisei S h i k e n s k o H o k u , 94 (1976) 28--32.
Mutation Research, 78 (1980) 25--31 © Elsevier/North-Holland Biomedical Press
STRUCTURE--ACTIVITY RELATIONSHIP WITHIN A SERIES OF m-DIAMINOBENZENE DERIVATIVES
MAJDI M. SHAHIN, A. BUGAUT and G. KALOPISSIS L'Oreal Research Laboratories, L 'Oreal, 1, Avenue de St Germain, 93601 Aulnay-sous-Bois (France)
(Received 19 September 1979) (Revision received 19 November 1979) (Accepted 21 November 1979)
Summary We investigated the mutagenicity of m-diaminobenzene (m-phenylenediamine) and four 2,4-diaminoalkylbenzenes (methyl, ethyl, isopropyl and n-butyl) in Salmonella typ hi m ur i um strains TA100, TA1538 and TA98 in the absence and presence of $9 induced by Acoclor 1254. m-Diaminobenzene was the most active mutagen, followed by 2,4-diaminotoluene and 2,4-diaminoethylbenzene, resp. Negative response was observed for both 2,4-diaminoisopropylbenzene and 2,4-diamino-n-butylbenzene. Thus, depending on the size of the substituting alkyl group at the C~ position of 2,4-diaminoalkylbenzene, a decline and loss of mutagenic activity was observed.
m-Diaminobenzene induces frameshift mutations in Salmonella t y p h i m u r i u m strain TA1538 in the presence of liver microsomal fractions from rats pretreated with Aroclor 1254 [4] or with phenobarbital [6]. It also induces frameshift mutations in strain TA98 in the presence of metabolic activation [9]. 2,4Diaminotoluene also induces mutations in Salmonella t yphi m uri um [4,8,9]. Urine from rats treated with 2,4-diaminotoluene, but not urine from rats treated with 2,4-diaminobutylbenzene, reverts Salmonella t yphi m uri um strain TA1538 [ 5]. Our preliminary experiments have shown that the introduction of the methyl group into m-diaminobenzene causes a decrease in mutagenicity, thereby indicating that m-diaminobenzene is more mutagenic in Salmonella t y p h imu r iu m than 2,4-diaminotoluene. We therefore initiated a study of the structure--activitiy relationship to determine the effect of different alkyl groups on the mutagencity of m-diaminobenzene. To ensure that we were testing the mutagenicity of a single component, we tested purified and unpurified samples of the compounds. The first 2 of the 5 compounds are used as hair-dye components [4] and the other 3 are structurally related to hair dyes; conse-
26 quently, information about their mutagenic activity or inactivity in relation to their structures is of interest to us. Materials and methods
Salmonella typhimurium 2 of the strains, TA1538 and TA98, can be used for the detection of mutagens that cause frameshift mutations, whereas TA100 can be used for the detection of mutagens causing base-pair substitutions [2,7]. The strains were kindly provided by Dr. B.N. Ames. Media, preparation of cell samples, and treatment conditions The media, preparation of cell samples, and treatment conditions used in the present studies have been described by Ames [1] and Ames et al. [2,3]. The a m o u n t of $9 mix added was 0.5 ml/plate. Chemicals The 5 c o m p o u n d s (Fig. 1) tested were: m-diaminobenzene, 2,4-diaminotoluene, 2,4-diaminoethylbenzene, 2,4-diaminoisopropylbenzene and 2,4-diaminon-butylbenzene. All were synthesized in our laboratories by catalytic reduction, with H2 Pd/C, from the corresponding m-nitroanilines. After distillation, all gave satisfactory analyses (m.p. and b.p. data recorded in Tables 1--5). Melting points were recorded on a Kofler Thermopan Microscope and are uncorrected. The positive control substance 1,2-diamino-4-nitrobenzene (4-nitro-O-phenylenediamine) was obtained from Merck, Darmstadt (Germany). Dimethyl sulfoxide (DMSO) was obtained from Carlo Erba, Divisione Chimica, Milano (Ita-
NH= @NHz m-Diaminobenzene
NH2 H3C'~ 2,4-Diaminotoluene
H3C.~" J . .NHz H3C~CH'T~ ~ ~'~J" NHz 2,4-Diaminoisopropylbenzene
NH2 HsC'-~'~ 24-
Diaminoethylbenzene
NH2 H'C'~" I ~"~/f~NH z 2.4-Diamino n-butylbenzene
Fig. 1. Chemical structure and names of the compounds tested.
27 ly). Glucose 6-phosphate was obtained from Fluka AG, Buchs SG (Switzerland). NADP was obtained from Boehringer Mannheim GmbH (W. Germany).
Solvents All compounds tested, as well as the positive controls, were dissolved in DMSO. Fresh solutions were prepared for each experiment. Animals Male Wistar rats weighing 200--250 g were used. The rats were injected intraperitoneally with Aroclor 1254 (diluted in corn oil to a concentration of 200 mg/ml) at a dose of 500 mg/kg. 5 days later, the rats were killed and the liver-microsomal fraction was prepared [3]. Methods o f evaluation A dose--response relationship, which shows progressive increase over the spontaneous background as a function of concentration, is considered to be a
TABLE 1 MUTAGENIC EVALUATION OF m-DIAMINOBENZENE WITHOUT PURIFICATION, AND AFTER P U R I F I C A T I O N BY D I S T I L L A T I O N , IN Salmonella t y p h i m u r i u m S T R A I N S I N T H E A B S E N C E (--) A N D P R E S E N C E (+) O F R A T - L I V E R - M I C R O S O M E A C T I V A T I O N S Y S T E M
Dose/plate
Revertant colonies/plate a
TAI00
Controls, no additions
B E
Controls, DMSO Controls, 1,2-diamino4-nitrobenzene C o n t r o l s , $9 m i x , no NADP
B
E B
E
TA1538
--
+
135 143
133 135
17 18
31 28
44 48
64 60
1 0 0 pl
111
132
18
26
47
60
20 pg 20 ~g
756 707
418 543
2610 2376
2366 2483
2748 2578
2197 2175
5 0 0 #1 500pl
---
135 135
---
15 15
---
52 46
00 00
--
TA98 +
--
+
m-Diaminobenzene without purification
5 10 20 50 100 250 500 1000
#g pg pg #g pg #g ~g #g
115 122 129 118 111 114 122 131
126 132 162 201 250 348 475 587
13 18 15 24 23 29 36 48
117 207 319 771 1300 1894 2610 3054
49 48 43 45 47 44 59 63
124 213 418 843 1397 2213 3060 3356
m-Diaminobenzene purified by distillation BP ( 1 8 m m ) = 1 7 5 ° C MP = 6 2 . 5 - - 6 3 ° C
5 10 20 50 100 250 500 1000
~g #g ~g pg pg pg ~g ~g
100 101 97 89 107 102 106 111
131 122 131 169 174 217 251 293
14 15 15 13 14 16 19 29
84 103 163 278 468 898 1635 2238
38 36 40 42 39 42 47 53
84 111 145 282 423 877 1524 2172
a The numbers of spontaneous controls have not been subtracted. B, controls at the beginning of the daily experiment; E, controls at the end of the experiment.
28 reliable criterion for genetic activity of compounds tested. A 2.5-fold increase over background, when found in repeated experiments, is also considered to be an indication of weak genetic activity. Results and discussion In the absence of $9 mix only the unpurified m-diaminobenzene showed a slight increase in the number of revertant colonies over those of the control values in strain TA1538, whereas the purified sample was not mutagenic in any of the strains used (Table 1). No mutations were induced by the other 4 compounds (unpurified or purified) in the absence of $9 mix (data are not included in this paper). Thus, actually, none of the 5 compounds was mutagenic in the 3 strains of Salmonella typhimurium in the absence of $9 mix. In our studies with m-diaminobenzene in the presence of $9 mix this c o m p o u n d was highly mutagenic in the 2 strains TA1538 and TA98; to a lesser extent it was also mutagenic in TA100, the unpurified sample being more pronounced in its activity (Table 1). All 3 strains exhibited a dose-related response (Table 1). Among the 5 compounds tested, m-diaminobenzene was the most active mutagen (Table 1), followed by 2,4-diaminotoluene (Table 2) and 2,4-diaminoethylbenzene
TABLE 2 MUTAGENIC EVALUATION OF 2,4-DIAMINOTOLUENEWITHOUTPURIFICATION, AND AFTER PURIFICATION BY DISTILLATION, IN Salmonella typhimurium STRAINS IN THE PRESENCE OF RAT-LIVER-MICROSOME ACTIVATIONSYSTEM Dose/plate
Revertant colonies/plate TAIO0
Controls, no additions
B E
Controls, DMSO
00 O0
136 124
TA1538 33 32
TA9S 37 40
1 0 0 ttl
126
36
39
Controls, 1,2-diamino4-nitrobenzene
B E
20 pg 20 p g
351 399
2400 2442
2070 2130
C o n t r o l s , $9 m i x , no N A D P
B E
5 0 0 ttl 5 0 0 pl
118 128
25 27
31 28
2,4-Diaminotoluene without purification
5 10 20 50 100 250 500 1000
pg pg ttg ttg Pg ttg #g pg
124 122 153 152 162 177 197 193
47 45 6O 73 115 390 714 1032
53 55 68 103 158 169 500 746
2,4-Diaminotoluene, purified by distillation BP 18 m m = 1 9 0 ° C MP = 9 9 ° C
5 10 20 50 100 250 500 i000
ttg pg pg pg pg ttg pg pg
132 127 142 159 178 216 185 199
46 51 59 111 133 329 678 942
49 60 63 105 143 307 572 818
29 TABLE 3 M U T A G E N I C E V A L U A T I O N OF 2 , 4 - D I A M I N O E T H Y L B E N Z E N E W I T H O U T P U R I F I C A T I O N , AND A F T E R P U R I F I C A T I O N BY D I S T I L L A T I O N , IN Salmonella t y p h i m u r i u m S T R A I N S IN T H E P R E S ENCE OF R A T - L I V E R - M I C R O S O M E A C T I V A T I O N SYSTEM Dose •plate
Revertant c o l o n i e s / p l a t e TA100
Controls, no additions C o n t r o l s , DMSO Controls, 1,2-diamino4-nitrobenzene C o n t r o l s , $9 m i x , no NADP
B E
TA1538
TA98
O0 O0
137 132
38 39
61 58
1 0 0 pl
130
35
60
20 pg 20 pg
383 394
2193 2477
2038 1984
500 pl 5 0 0 ~1
130 140
25 25
44 46
2,4-Diaminoethylbenzene without purification
50 100 200 500 1000 2500 5000 10 0 0 0
pg ~g ~g Dg #g pg ~g ~g
165 184 154 177 169 159 152 161
37 41 62 83 128 260 234 106
72 99 143 270 417 462 284 173
2,4-Diaminoethylbenzene purified b y distillation BP 18 m m = 1 6 5 ° C
50 100 200 500 1000 2500 5000 10 0 0 0
pg pg Dg Pg ~g ~g ~g pg
155 175 165 179 171 175 156 146
39 80 81 189 330 414 259 95
71 97 125 192 482 367 365 208
(Table 3), resp. In contrast, the purified samples of the 2 compounds 2,4-diaminoisopropylbenzene and 2,4-diamino-n-butylbenzene, which were tested over a wide range of concentrations, gave a negative response (Tables 4 and 5). The results of m-diaminobenzene and 2,4-diaminotoluene are in agreement with those obtained previously [4] ; both were mutagenic in Salmonella typhimurium. In our investigations, at all concentrations used, the mutagenic activity of m-diaminobenzene was higher than that of 2,4-diaminotoluene. It is, however, inadequate and very difficult to compare data obtained in different laboratories. In our investigations, to control possible errors during the experimental procedure and to ensure the effectiveness of the system, controls were carried o u t at the beginning and end of each daily experiment (see Tables). In addition, the purified and unpurified materials of each of the compounds were tested on the same day using the same $9 mix and the same cell culture. Furthermore, several samples of each compound, including commercial products when available, were included in our investigations. The same pattern of activity was always observed. 2-Aminoanthracene at different concentrations was also used as a positive control and exhibited the expected responses. As already reported, the present investigations were undertaken to study the structure--activity relationship by investigating whether the addition of certain
3O TABLE 4 MUTAGENIC EVALUATION OF 2,4-DIAMINOISOPROPYLBENZENE WITHOUT PURIFICATION, A N D A F T E R P U R I F I C A T I O N BY D I S T I L L A T I O N , I N S a l m o n e l l a t y p h i m u r i u m S T R A I N S IN T H E PRESENCE OF RAT-LIVER-MICROSOME ACTIVATION SYSTEM
Dose/Plate
Revertant c o l o n i e s / p l a t e TA100
Controls, no a d d i t i o n s
B E
Controls, D M S O Controls, 1,2-diamino4-nitrobenzene Controls, $9 m i x , no NADP 2 , 4 - D i a m i n o i s o P r o P ylbenzene without
purification
2,4-Diaminoisopropyl-
b e n z e n e purified b y distillation BP 18 m m = 1 6 6 - - 1 6 8 ° C MP = 4 7 ° C
00 00
TA1538
TA9S
115 123
28 28
48 53
1 0 0 #1
113
29
47
20 p g 20 p g
398 392
2273 2543
2045 2207
5 0 0 pl 5 0 0 pl
120 115
14 16
43 45
5 10 20 50 100 250 500 1000
pg pg pg pg #g ~g pg ~g
109 116 125 132 132 123 131 130
28 26 25 30 43 61 65 64
45 57 51 66 53 73 79 83
5 10 20 50 100 250 500 1000
pg #g pg pg ~g pg pg #g
94 89 107 104 117 115 107 97
21 26 20 21 28 36 40 35
46 35 55 55 52 42 50 54
alkyl groups to the structure of a known mutagen has any effect on its mutagenicity. The results of the purified samples of m-diaminobenzene, 2,4-diaminotoluene and 2,4-diaminoethylbenzene can be seen in Tables 1, 2 and 3. The t w o compounds that have the alkyl groups CH3 and C2Hs in their structures were less mutagenic than m-diaminobenzene; the c o m p o u n d with the ethyl group exhibited lower activity than the c o m p o u n d with the methyl group. A complete removal of the mutagenic activity was achieved in the presence of isopropyl CH3~
jCH-CH3
or
n-butyl (CH3--(CH2)3--)
groups (Tables 4 and 5). Thus depending on the size of the substituting alkyl group in 2,4-diaminoalkylbenzene, a decline and loss of mutagenic activity was observed.
31 TABLE 5 MUTAGENIC EVALUATION OF 2,4-DIAMINOBUTYLBENZENE WITHOUT PURIFICATION, AND A F T E R P U R I F I C A T I O N BY D I S T I L L A T I O N , IN Salmonella t y p h i m u r i u m S T R A I N S IN T H E P R E S ENCE OF RAT-LIVER-MICROSOME ACTIVATION SYSTEM Do s e / p l a t e
Revertant colonies/plate TA100
Controls, no additions Controls, DMSO Controls, 1,2-diamino4-nitrobenzene C o n t r o l s , $9 m i x , no NADP
B E
TA1538
TA98
00 00
116 128
42 32
47 43
100 ~
114
34
47
20 pg 20 pg
345 356
2467 2325
2276 2005
5 0 0 pl 5 0 0 pl
119 129
25 22
28 28
2,4-Diaminobutylbenzene without purification
5O 100 200 500 1000 2500 5000 10 000
pg ~g ~g pg ~g pg ~g pg
191 189 223 202 155 161 125 000
49 48 46 62 65 70 58 00
58 49 53 65 49 56 25 00
2,4-Diaminobutylbenzene purified by d i s t i l l a t i o n BP 1 8 m m = 1 7 0 ° C
50 100 200 500 1000 2500 5000 10 000
~g ~g ~g pg pg pg pg pg
153 201 209 172 166 164 163 076
40 40 38 52 67 55 54 00
49 53 53 52 63 57 15 00
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