Mutation Research, 277 (1992) 201-220 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1110/92/$15.00
201
MUTREV 07322
Mutagenicity of azo dyes: Structure-activity relationships King-Thom
Chung
a
and Carl E. Cerniglia b
Department of Biology, Memphis State University, Memphis, TN 38152, USA and h Microbiology Division, National Center for Toxicological Research, Jefferson, AR 72079, USA (Received 12 March 1991) (Revision received 20 February 1992) (Accepted 25 February 1992)
Keywords." Azo dyes, mutagenicity; Structure-activity relationships, azo reduction; Azoreductases; p-Phenylenediamine; Benzidine moieties
Summary Azo dyes are extensively used in textile, printing, leather, paper making, drug and food industries. Following oral exposure, azo dyes are metabolized to aromatic amines by intestinal microflora or liver azoreductases. Aromatic amines are further metabolized to genotoxic compounds by mammalian microsomal enzymes. Many of these aromatic amines are mutagenic in the Ames Salmonella/microsomal assay system. The chemical structure of many mutagenic azo dyes was reviewed, and we found that the biologically active dyes are mainly limited to those compounds containing p-phenylenediamine and benzidine moieties. It was found that for the phenylenediamine moiety, methylation or substitution of a nitro group for an amino group does not decrease mutagenicity. However, sulfonation, carboxylation, deamination, or substitution of an ethyl alcohol or an acetyl group for the hydrogen in the amino groups leads to a decrease in the mutagenic activity. For the benzidine moiety, methylation, methoxylation, halogenation or substitution of an acetyl group for hydrogen in the amino group does not affect mutagenicity, but complexation with copper ions diminishes mutagenicity. The mutagenicity of benzidine or its derivatives is also decreased when in the form of a hydrochloride salt with only one exception. Mutagenicity of azo dyes can, therefore, be predicted by these structure-activity relationships.
I. Introduction Azo dyes, produced by the diazotization of aromatic amines, are widely used in the textile,
Correspondence: Dr. King-Thom Chung, Department of Biology, Memphis State University, Memphis, TN 38152, USA.
Abbreviation: DAB : methyl yellow: butter yellow, 4-dimethylaminoazobenzene : N,N-dimethyl-4-aminoazobenzene: dimethylaminoazobenzene.
leather, printing, paper and food industries [36]. Currently, there are at least 3000 azo dyes in use. Some of them are contained in various mouthwashes, drugs, cosmetics, and other consumer goods and pharmaceutical products [25]. In the textile industry, azo dyes are extensively used for the coloring of wool, cotton, polyester clothing and for various substrates [4,12]. In the food industry, azo dyes are used as food colorants [23,67]. The food colorants in the United States certified since 1906 are Amaranth, Erythrosine,
202
Indigotine, Light Green, Naphthol Yellow, Orange I and Ponceau 3R. Of these 7, 3 are azo dyes: Amaranth, Orange I and Ponceau 3R. In
the past 30 years, many food, drug and cosmetic (FD & C) colors have been banned from commercial use as food colorants [43].
TABLE 1 MUTAGENIC AZO DYES: CHEMICAL STRUCTURES AND REQUIREMENTS FOR METABOLIC ACTIVATION COMMON NAME
CHEMICAL STRUCTURE
METABOLIC ACTIVATION
REFERENCE
AZO DYES CONTAINING THE NITRO-GROUPS NeO
OzN
Alizarin Yellow G G
~ N f N
Brown, eL al. 1978
~ O H
OtN~NfN~OH H0
Acid Alizarin Yellow R
CzHs s/ \ c:Hj, I÷((cHa) X.
Red GTL o,,~"6-~..,_~6-'~ (C.I. BasicRed 18) ~ ~ OH Naw
2RB
Brown, eL al. 1978
.~. _
Venturini and Tamaro, 1979
OH CI .
• N --
.~. CO
N e s t m a n n , et. al., 1981
OIN~ 2
CI AZO DYES CONTAINING BENZENEAMINES AND RELATED COMPOUNDS Chrysoidin (C.I. Index 11270)
\ -~,=.
CH3 Sudan IV
~N=N
~
w~
~.
Garner and Nutman, 1977 Sole and Chipman, 1986
+
Brown, et. al., 1978
CH$ ~ N : N
OH
OCH
Guiba Black D
~
NH3 ~"
(C.I. D i r e c t B l a c k 17)
+
Brown, et. al., 1978 Chung, et. al., 1981
+
Chung, et. al., 1978 Brown and Dietrich, 1983 Prival, et. al., 1984
tt0
=0
CHs
Methyl Red %CH3
Me*. l
"'''
Venturini and Tamaro, 1979
203
TABLE
1 (continued)
Dimethylaminoazobenzene (DAB, Methyl Yellow, Butter
@N=N
@
N/CH)
Yellow)
~ 3
+
N - Methyl - 4 - Amlnoazobenzene 4 - A m l n o a z o b e n z e n e (AB) (p - A m i n o a z o b e n z e n e ) (C.I. S o l v e n t Yellow) C.L l l 0 0 0
@
'
"
@
NIC M.N~
Yahagi, et. al., 1975 Brown, et. al., 1978 Chung, et. al., 1981 Brown and Dietrich, 1983 Ashby, et. al., 1983
1976
+
Yahagi, et. al.,
'%
+
Yahagi, et. al., 1975
N(¢M~,
+
Yahag/, et. al., 1975
~
.~.
Yahagl, et. al., 1975
2 - M e t h y l - DAB
~
-
O - Aminoazotoluene(o. AT)
~
- ¢~ N .
3 - M e t h o x y - AB
~
N., ~
N%
+
Yahagl, et. a l . , , 975
N-OIl .AB
~ , -
s . s @
~{0s}
+
Yahagl, et. al., 1975
N . A c e t o x y . MAB
~O~-NoM~s/~'
__
Yahagi, et. ,l., 1975
4"
Yahagt, et. al., 1975
m.~
Yahag/, et. al., 1975
÷
Yahagi, e t . al.t 1 975
±
Y.h.
c s ~. ~ N
0CH~
0C0CH
, .ethoxyo
.hooy,
OB - MAP, (4' - MC - N . O11- MAB)
,@
a4i0-C 4 ' - MC - N - A c e t o x y - MAB
@/c,,.. o t -
NsN
0 -- C - - ~ i
0
CH~
~.,o.~@...~r/o 4 ' - M C - N - B e n z o y l o x y - MAB
~ ' / 0
-~ - ~
,e
.l,1976
N - O H - MAB
±
4' - M e . N - OH - MAB
3' - Me - DAB
H,C~,_N.M@
N(~4~~
.l,1,,6
"~
Mori, et. al., 1980 Mori, et. al., 1983
÷
Mori,et.al.,19S0
HIc 3' - Me - MAB
3'- Me. AB
~N.M@NH,
204
Approximately 6.8 × 105 kg of Amaranth was consumed per year in over 60 countries [18]. The use of this colorant is currently prohibited in the United States [18]. But some other countries are still using this colorant in their food industries.
Orange I and Ponceau 3R were also banned for use in food. These dyes are added to soft drinks, cereals, gelatin desserts, candies, hot dogs, ice creams, drugs and cosmetics [23,67]. Interestingly, a considerable amount of food colorants are con-
TABLE 1 (continued) CH~
3
Mo
NAc
+
Mor, e , a ,
-t"
Mori, et. al., 1980
+
Mori, et. al. 1980 Mori, et. al. 1983
+
Morl, et. al., 1980
"l--
Mori, et.
(css,
~
Mori, et. aL, 1980
(C~)1
~
Mori, et. al., 1980
+
Mori, et. al., 1980
.~.
Sori, et. al., 1980
COCH! CH|
3' - Me . A B - NAC
~O.~- M.~.-~K~coc, $ sonic
3 ' - C H 2 O H - DAB
~).-~.
N~ .
(CS~,
HOHIC
3 ' - CH2OH- MAB
~--.,N--~-C~
HOHIC 3' " C H 2 O H " AB
3' - C H O - D A B
4~M'N'-4~NH,
S .C ~ " ~ " ~~H . . ~ ~ " - ~ /
aL, 1980
,q
H0¢ 3'- COOH-DAB
3'-
~NffiN--~N
9 H0C COOH-MAB
~,-N,M.-.~
,:~.H3
CH1
3'- Me- 4'- OH- DAB
so - ~ > -
M,. - - ~ ) - - N I C S
CHtCI
~~
ClCH:I
3, 3' - B i s ( C h l o r o m e t h y l ) a z o b e n z e n e
~
_
NfM_ ~ _
3'" CH 2OAc "DAB
,
201
MUTREV 07322
Mutagenicity of azo dyes: Structure-activity relationships King-Thom
Chung
a
and Carl E. Cerniglia b
Department of Biology, Memphis State University, Memphis, TN 38152, USA and h Microbiology Division, National Center for Toxicological Research, Jefferson, AR 72079, USA (Received 12 March 1991) (Revision received 20 February 1992) (Accepted 25 February 1992)
Keywords." Azo dyes, mutagenicity; Structure-activity relationships, azo reduction; Azoreductases; p-Phenylenediamine; Benzidine moieties
Summary Azo dyes are extensively used in textile, printing, leather, paper making, drug and food industries. Following oral exposure, azo dyes are metabolized to aromatic amines by intestinal microflora or liver azoreductases. Aromatic amines are further metabolized to genotoxic compounds by mammalian microsomal enzymes. Many of these aromatic amines are mutagenic in the Ames Salmonella/microsomal assay system. The chemical structure of many mutagenic azo dyes was reviewed, and we found that the biologically active dyes are mainly limited to those compounds containing p-phenylenediamine and benzidine moieties. It was found that for the phenylenediamine moiety, methylation or substitution of a nitro group for an amino group does not decrease mutagenicity. However, sulfonation, carboxylation, deamination, or substitution of an ethyl alcohol or an acetyl group for the hydrogen in the amino groups leads to a decrease in the mutagenic activity. For the benzidine moiety, methylation, methoxylation, halogenation or substitution of an acetyl group for hydrogen in the amino group does not affect mutagenicity, but complexation with copper ions diminishes mutagenicity. The mutagenicity of benzidine or its derivatives is also decreased when in the form of a hydrochloride salt with only one exception. Mutagenicity of azo dyes can, therefore, be predicted by these structure-activity relationships.
I. Introduction Azo dyes, produced by the diazotization of aromatic amines, are widely used in the textile,
Correspondence: Dr. King-Thom Chung, Department of Biology, Memphis State University, Memphis, TN 38152, USA.
Abbreviation: DAB : methyl yellow: butter yellow, 4-dimethylaminoazobenzene : N,N-dimethyl-4-aminoazobenzene: dimethylaminoazobenzene.
leather, printing, paper and food industries [36]. Currently, there are at least 3000 azo dyes in use. Some of them are contained in various mouthwashes, drugs, cosmetics, and other consumer goods and pharmaceutical products [25]. In the textile industry, azo dyes are extensively used for the coloring of wool, cotton, polyester clothing and for various substrates [4,12]. In the food industry, azo dyes are used as food colorants [23,67]. The food colorants in the United States certified since 1906 are Amaranth, Erythrosine,
202
Indigotine, Light Green, Naphthol Yellow, Orange I and Ponceau 3R. Of these 7, 3 are azo dyes: Amaranth, Orange I and Ponceau 3R. In
the past 30 years, many food, drug and cosmetic (FD & C) colors have been banned from commercial use as food colorants [43].
TABLE 1 MUTAGENIC AZO DYES: CHEMICAL STRUCTURES AND REQUIREMENTS FOR METABOLIC ACTIVATION COMMON NAME
CHEMICAL STRUCTURE
METABOLIC ACTIVATION
REFERENCE
AZO DYES CONTAINING THE NITRO-GROUPS NeO
OzN
Alizarin Yellow G G
~ N f N
Brown, eL al. 1978
~ O H
OtN~NfN~OH H0
Acid Alizarin Yellow R
CzHs s/ \ c:Hj, I÷((cHa) X.
Red GTL o,,~"6-~..,_~6-'~ (C.I. BasicRed 18) ~ ~ OH Naw
2RB
Brown, eL al. 1978
.~. _
Venturini and Tamaro, 1979
OH CI .
• N --
.~. CO
N e s t m a n n , et. al., 1981
OIN~ 2
CI AZO DYES CONTAINING BENZENEAMINES AND RELATED COMPOUNDS Chrysoidin (C.I. Index 11270)
\ -~,=.
CH3 Sudan IV
~N=N
~
w~
~.
Garner and Nutman, 1977 Sole and Chipman, 1986
+
Brown, et. al., 1978
CH$ ~ N : N
OH
OCH
Guiba Black D
~
NH3 ~"
(C.I. D i r e c t B l a c k 17)
+
Brown, et. al., 1978 Chung, et. al., 1981
+
Chung, et. al., 1978 Brown and Dietrich, 1983 Prival, et. al., 1984
tt0
=0
CHs
Methyl Red %CH3
Me*. l
"'''
Venturini and Tamaro, 1979
203
TABLE
1 (continued)
Dimethylaminoazobenzene (DAB, Methyl Yellow, Butter
@N=N
@
N/CH)
Yellow)
~ 3
+
N - Methyl - 4 - Amlnoazobenzene 4 - A m l n o a z o b e n z e n e (AB) (p - A m i n o a z o b e n z e n e ) (C.I. S o l v e n t Yellow) C.L l l 0 0 0
@
'
"
@
NIC M.N~
Yahagi, et. al., 1975 Brown, et. al., 1978 Chung, et. al., 1981 Brown and Dietrich, 1983 Ashby, et. al., 1983
1976
+
Yahagi, et. al.,
'%
+
Yahagi, et. al., 1975
N(¢M~,
+
Yahag/, et. al., 1975
~
.~.
Yahagl, et. al., 1975
2 - M e t h y l - DAB
~
-
O - Aminoazotoluene(o. AT)
~
- ¢~ N .
3 - M e t h o x y - AB
~
N., ~
N%
+
Yahagl, et. a l . , , 975
N-OIl .AB
~ , -
s . s @
~{0s}
+
Yahagl, et. al., 1975
N . A c e t o x y . MAB
~O~-NoM~s/~'
__
Yahagi, et. ,l., 1975
4"
Yahagt, et. al., 1975
m.~
Yahag/, et. al., 1975
÷
Yahagi, e t . al.t 1 975
±
Y.h.
c s ~. ~ N
0CH~
0C0CH
, .ethoxyo
.hooy,
OB - MAP, (4' - MC - N . O11- MAB)
,@
a4i0-C 4 ' - MC - N - A c e t o x y - MAB
@/c,,.. o t -
NsN
0 -- C - - ~ i
0
CH~
~.,o.~@...~r/o 4 ' - M C - N - B e n z o y l o x y - MAB
~ ' / 0
-~ - ~
,e
.l,1976
N - O H - MAB
±
4' - M e . N - OH - MAB
3' - Me - DAB
H,C~,_N.M@
N(~4~~
.l,1,,6
"~
Mori, et. al., 1980 Mori, et. al., 1983
÷
Mori,et.al.,19S0
HIc 3' - Me - MAB
3'- Me. AB
~N.M@NH,
204
Approximately 6.8 × 105 kg of Amaranth was consumed per year in over 60 countries [18]. The use of this colorant is currently prohibited in the United States [18]. But some other countries are still using this colorant in their food industries.
Orange I and Ponceau 3R were also banned for use in food. These dyes are added to soft drinks, cereals, gelatin desserts, candies, hot dogs, ice creams, drugs and cosmetics [23,67]. Interestingly, a considerable amount of food colorants are con-
TABLE 1 (continued) CH~
3
Mo
NAc
+
Mor, e , a ,
-t"
Mori, et. al., 1980
+
Mori, et. al. 1980 Mori, et. al. 1983
+
Morl, et. al., 1980
"l--
Mori, et.
(css,
~
Mori, et. aL, 1980
(C~)1
~
Mori, et. al., 1980
+
Mori, et. al., 1980
.~.
Sori, et. al., 1980
COCH! CH|
3' - Me . A B - NAC
~O.~- M.~.-~K~coc, $ sonic
3 ' - C H 2 O H - DAB
~).-~.
N~ .
(CS~,
HOHIC
3 ' - CH2OH- MAB
~--.,N--~-C~
HOHIC 3' " C H 2 O H " AB
3' - C H O - D A B
4~M'N'-4~NH,
S .C ~ " ~ " ~~H . . ~ ~ " - ~ /
aL, 1980
,q
H0¢ 3'- COOH-DAB
3'-
~NffiN--~N
9 H0C COOH-MAB
~,-N,M.-.~
,:~.H3
CH1
3'- Me- 4'- OH- DAB
so - ~ > -
M,. - - ~ ) - - N I C S
CHtCI
~~
ClCH:I
3, 3' - B i s ( C h l o r o m e t h y l ) a z o b e n z e n e
~
_
NfM_ ~ _
3'" CH 2OAc "DAB
,
