0278-6915/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd

Fd Chem. Toxw Vol 30, No. 7, pp 635-660, 1992 Pnnted m Great Britain All rights reserved

Review Section A COMPILATION OF GENOTOXICITY AND CARCINOGENICITY DATA ON AROMATIC AMINOSULPHONIC ACIDS R. JUNG, D. STEINLE* and R. ANLIKER* Hoechst AG, Gewerbetoxlkologle, PO Box 800320, D-6230 Frankfurt am Main, Germany and *Ecological and Toxicological Assocmtlon of Dyes and Orgamc Pigments Manufacturers (ETAD), PO Box, CH-4005, Basel, Switzerland (Accepted 16 March 1992)

Summary--A review is presented to evaluate existing information on genotox~city and carcinogenicity testing of various aromatic aminosulphomc acids (AASAs) A great variety of water-soluble azo dyes can form aromatic phenyl- or naphthyl-ammosulphomc acids by ebemlcal and enzymatic reduction. AASAs are also used as intermediates in the synthes~sof azo dyes and azo p~gments and can anse as contaminants in the final products Comparisons have been made with the data available on the corresponding unsulphonated analogues, some of which are known to be genotox~c and/or carcinogemc The vast majority of the AASAs were concluswely non-mutagenic in the Ames test. In most cases the absence of genotoxic~tywas also demonstrated with a variety of other test systems m wtro and m vivo It is concluded that AASAs, m contrast with some of their unsulphonated analogues, generally have no or very low genotoxtc and tumorigemc potentml.

Introduction Epidemiological data have shown an association between occupational exposure to 2-naphthylamme (no. 118 in Tables), benzidme (115), 4-aminobiphenyl (114) and 2-methyl-4-chloroaniline, and the risk of cancer of the urinary bladder. The carcmogenicity effects in humans and animals of a number of lipophilic aromatic amines are well documented, and experimental evidence indicates that this carcinogenicity is dependent on metabolic actwation. On the other hand, several aromatic amines have been shown not to possess tumorigenic actwity even when they are positive in the Ames test, clearly indicating that carcinogenicity is not a common feature for all compounds in this class of chemicals. Aromatic amines are important intermediates m the synthesis of a great variety of products, such as plastic materials, pesticides, rubber chemicals, and colourings, primarily azo colourings. The production and use of the above-mentioned four carcinogenic amines has been abandoned by the major dyestuffs manufacturers, and in many industrialized countries their production and use are controlled by very stringent regulations. 2-Naphthylamine (118) also played an important role as a starting material in the synthesis of key intermediates for colourings, especially aminonaphthalenesulphonic acids. *To whom correspondence should be addressed. Abbreviation: AASA = aromatic armnosulphomc acid.

Alternative synthetic routes to these aminonaphthalenesulphonic acids were developed to circumvent the use of the hazardous 2-naphthylamine (Anliker, 1977). Undoubtedly, substituted aminonaphthalenesulphonic acids, and generally aromatic aminosulphonic acids (AASAs; often also called 'sulphonated aromatic amines') will continue to be major intermedmtes in the manufacture of organic colourings. The seeming structural similarity between AASAs and the unsulphonated analogues gave nse to the suspicion that they might also possess a similar tumorigemc activity. As in other classes of chemicals, some of the existing shortcomings of structure/activity analogies can also be shown with these groups of chemicals (Anliker, 1983). The sulphonic acid closely related to 2-naphthylamine (118), 2aminonaphthalene-1-sulphonic acid (22), did not produce any carcinogenic effects in a chronic feeding study in mice, even at high dose levels of 1000 mg/kg body weight (Della Porta, 1982). Theiss et al. (1981) stud~ed seven 1-amino- and 2-aminonaphthalenesulphonic acids in the lung adenoma test in the strain A mouse using lp administration. The authors concluded that neither l-naphthylamine (117) nor the four sulphonic acid derivatives of l-naphthylamine tested were tumorigenic. However, 2-naphthylamine and two out of the three sulphomc acid derivatives of 2-naphthylamine tested were believed to have produced a statistically significant tumour response. A later paper by the same group (Maronpot et al., 1986) 635

N~

I

88-21-1

C6HTNO3S

~

121--47-I

C6HvNO3S

SO3H

~ S 0 3 H

N~

121-57-3

C6H~NO3S

so3H

+

Structure

No.

Zetger (1988) NTP Rep (1986) Shimtzu (1983)

Zelger (1988) NTP Rep (1986) Chung (1981) Chung (1978) Vemtt (1976)

ETAD (1989) + (w) Zeiser (1988) + (w) NTP Rep (1986)

-

-

Ames lest data

Aromatic ammosulphonic acids

m

DNA

m - cyt

Yoshtml (1988)

Dienckx (1989)

Other data 101 C6HvN 62-53-3

No. -

test

Ames

Group 3

C+

m+ c+ m+ mmm+ m+ m c+

Other data

GMA haembmding cyt cyt DNA GMA GMA DNA haembmdiug

m - SAL m - SAL m - SAL

Unsulphonated analogues

Table I Mutagemctty and other toxtcity data on selected aromatic ammosulphomc actds and thetr unsulphonated analogues*

IARC (1987)

Fassma (1990) Btrner (1988) lshtdate (1988) Ishtdate (1988) Yoshimi (1988) Mitchell (1988) Wangenhelm (1988) yon der Hude (1988) Hamson (1987) Zetger (1987)

Shahm (1989) Ishtdate (1988) Zetger (1987)

Z

NH 2

88--44-8

CH,NOS

NH 2

~

137-51-9

C6H?NO6S2

SO3H

S03H

98-37-3

C6HTNO4S

H O 3 S / ~ O H

HO3S

+

Zeiger (1988) ETAD (1987) NTP Rep (1986)

ETAD (1988) Shtmlzu (1983)

m- DNA m - cyt m - DNA m- GMA

c- cell trans

Yoshlml (1988) ETAD (1988) ETAD (1988) ETAD (1986)

Styles(1973)

103 CeHTNO 95-55-6

102 CTHgN 106-49-0

+ (w)

+

c+ m +/-

haembmdm8 cyt

m SAL m + (w) SAL

DNA

[contd]

Hamson (1987) Kirchner (1982)

De Flora (1984) BIBRA (1984)

Birner (1988) Neumann (1988) Pai (1985) Garner (1984) Ce-~trone (1982) c+ c+ mcm+

haembmdmg haembmdmg GMA

NTP Rep (1984) ETAD (1981)

m ÷ (w) SAL m+ SAL

fj3

~s o

f~

o

No

88-23-3

C6H6NCIO4S

N~

5857-94-3

C6H6NC104S

HO

SO3H

6370-23-6

CI ~

CI

CH 3

CsHtlNO3S

HaC ~

NH 2

Structure

SO3H

NH 3

SO3H

Zelger (1988) NTP Rep (1986)

ETAD (1987)

Ames test data Chung (1981)

Aromatic ammosulphomc aods

Table

Other data

1--contd

106 C~H6CINO 95-85-2

105 C6H6CINO 38191-33-2

No 104 CsHHN 95-68-1

+ (w)

Other data SAL SAL SAL DNA haembmdmg DNA

m + (w) SAL m + (w) SAL

m+ cc-l+ Group 3

C+

m+ m+ m+ m+

Unsulphonated analogues Ames test +

Zelger (1988) NTP Rep (1986)

Zelger (1988) NTP Rep (1985a) Shlmlzu (1983) Wdhams (1989) Blrner (1988) Yoshlml (1988) Garner (1984) DETO (1980) IARC (1987)

z

oo

12

1!

10

SO3H

6471-78-9

CsHttNO4 S

NH 2

Cl"

88-51-7

C7HsCINO3S

CH 3

1

NH 2

~

88-53-9

CTHsCINO3 S

12I-t3

Cl ~ / N H a

CH30"

~

SO3H

SO3H

CH 3

ETAD (1986)

ETAD (1986) Shtmlzu (1985)

Chung (1981)

rn DNA m DNA

m- DNA m- cyt

YoshJml (1988) ETAD (1986)

Wflhams (1989) ETAD (1988)

109 C7HsCIN 95-74-9

108 CTHsC1N 7149-75-9

107 CaHItNO 120-71-8

+ (w)

pulm ad

SAL SAL

[contd]

Ennever (1986) NTP-TR-145 (1978) DNA

m+ c

NTP Rep (1989a) Zelger (1987) Ennever (1986)

IARC (1987)

Maronpot (1986) NTP Rep 85-002 (1985) Vaimo (1985) Garner (1984) NTP-TR- 142 (1978)

Zeiger (1988) NTP Rep (1986)

m + (w) SAL mSAL mSAL

Group 2B

C+

C+

C+

C+

c-

m+ m+

~D

> >

o

0

g~

O

15

14

13

No.

5123-63-7

CIoHIsNO3 S

32432-55-6

C9H|4N203S

CH 3

-~ NH 2

/

HO3S ~-

-,~

CH3

N%

3577-63-7

CTHTNOsS

SO3H

H3C ~

HOOC

NH 2

Structure

--

-

--

ETAD (1989)

ETAD (1989)

NTP Rep (1984)

Ames test data

AromaUc ammosulphomc actds Other data

Table l--contd

112 CIoH~sN 91-66-7

I11 CgH 14N2 3102-70-3

-

-

110 C7HTNO2 118-92-3

test

No

mmm-

Group 3

c-

m m m+ m+ m-

SAL SAL DNA

SAL SAL GMA cyt DNA

Other data

Unsulphonated analogues Ames

Zelger (1988) Shlmlzu (1983) Yoshlml (1988)

IARC (1987)

Zelger (1987) NTP Rep (1984b) Mitchell (1988) NTP Rep (1985b) Cesarone (1982) NTP-TR-36 (1978)

z o-

19

18

17

16

I

-

~

H~~

N

SO3H

~

I

117-61-3

CI2HI2N206S2

N

3365-90-0

CI2HI2N206S2

3365--89-7

CI2HuN03S

98-40-8

C~I13NO3S

SO3H

~

~

S%H

NHC2H5

-

ETAD (1989)

Ashby (1982)

ETAD(1989)

¢

cell trans Ashby (1978)

115

115 CI2HI2N 92-87-5

il4 CI2HIIN 92-67-1

113 CgHI3N 94-68-8

c + Group I

c + Group I

c + Group I

IARC (1987)

IARC (1987)

IARC (1987)

[contdl

>

O

)0

O X

o

22

21

81-16-3

CloH9NO3S

SO3H

84-86-6

CIoH9NO3S

SO3H

81-11-8

C14HI4N206S2

SO3H

Structure

2N-Q-c.=c.

No.

SO3H

NH 2

-

Le (1985) Pogodma (1984) Purchase (1978)

Kler (1986) Chung (1981)

NTP Rep (1990) ETAD (1989) Zelger (1987)

Ames test data

Aromatac amlnosulphomc acads

GMA cyt

c e-

m- DNA m - cyt

c

m m

ETAD (1989) ETAD (1989) Pogodma (1984) Della Porta (1982)

D E T O (1980)

ETAD (1989) NTP Rep (1989b)

Other data

Table 1--contd

118 CIoH9N 91-59-8

117 CIoH9N 134-32-7

116 C14HI4N2 621-96-5

No

+

+

test

Ames

c+

Group 3

m-

Group 1

DNA

GMA cyt cyt cyt

mm+ mm+ C-

SAL SAL SAL SAL

m+ m+ m+ m÷

Other data

Unsulphonated analogues

IARC (1987)

IARC (1987)

Fassma (1990) lsh~date (1988) Isbldate (1988) NTP Rep (1988) Vamlo (1985) Brambdla (1985)

lshldate (1988) Zelger (1988) NTP Rep (1986) Kler (1986)

z

bO

26

25

24

23

1 17-26-4

Cl0H9NO6S 2

SO3H

SOaH

86-60-2

Ct0H9NO3S

93-00-5

C)0H9NO3S

~

HO3S~

81-05-0

C1oHgNO3 S

SO3H

NH 2

NH2

-

-

-

-

-

-

Kmr (1986) Purchase (1978) Garner (1977)

Le (1985)

Le (1985)

Le (1985)

C-

c-

cell trans

c pulm ad

c pulm ad

Purchase (1978) Purchase (1978)

Thetss (1981)

Thelss (I981)

118

118

118

118

+

+

C+

C+

C+

C+

Group 1

Group 1

Group I

Group 1

[contd]

IARC (1987)

IARC (1987)

IARC (1987)

IARC (1987)

o

o

o

29

28

27

No.

HO3S I

118-03-6

CIoH9NO9S 3

v

SO3H

~

118-33-2

Cl0H9~O6S2

SO3H

86-65-7

C10HgNO6S 2

~

H03S ~

HO3S

SO3H

Structure

~SO3H

NH 2

NH2

-

-

-

ETAD (1989)

ETAD (1989)

ETAD (1989)

Ames test data

Aromatic ammosulphonlc acids

c +~ pulm ad Theiss (1981)

Other data

Table l---contd

I 18

118

118

No

+

+

Ames test

c+

c+

¢+

Group I

Group 1

Group 1

Other data

Unsulphonated analogues

IARC(1987)

IARC (1987)

IARC (1987)

32

31

30

HOsS~

H03S

90-51-7

CIoH9NO4S

HO

5639-34-9

CtoH9NO4S

CIoH9NO4S 116-63-2

SO3H

~

/OH

OH

ETAD (1990) Freeman (1987)

Chung (1981)

ETAD(1987) Kler (1986) Chung (1981) m + cyt

Gayathn (1980)

CRoHgNO 4384-92-3

120

119

119 C~oH,NO 2834-92-6 Wdlmms (1989)

DNA

m-

[contd]

Kter (1986) Dabney (1985) Garner (1977)

SAL SAL SAL

m mm-

f~

o

P~

e~

o

H

O

HO3S ~

.NH2

3

/

~

CIoH9NO7S2

S

SO3H OH

90-20-0

CIoHgNOTS2

OH

NI-12

SO3H

SO3H

OH

-

-

-

ETAD (1989)

Freeman (1987)

Chung (1980

Ames test data

m

DNA

m + GMA m- cyt

ETAD (1990)

Palmer (1979) Palmer (1979)

Other data

Table I--contd

122 C~0HgNO 606-41-7

CIoHgNO 2834-9 I-5

12l

119

No

test

Ames

Other data

Unsulphonated analogues

+ =posmve response - = negative response w = weak response ~ = quesUonnable results c =carcmogemoty, oral apphcatlon cell trans =cell transformation study haembmdmg= haemoglobin adducts m = genotoxmlty/mutagemcltym cyt = cytogemc~ty/chromosomemutation m DNA = DNA repair/indicator test m GMA = gene mutatton assay m eucaryotm cells (not Ames/Pnval) m SAL = Salmonella/mlcrosome (Ames) test pulm ad = pulmonary adenoma carcmogemclty test *With the exception of the Salmonella/mlcrosome assay, only a selecuon of the available data is shown For human carcinogens, only the IARC classlficaUon ts menUoned IARC classification. Group 1, carcmogemc to humans, Group 2B, possibly carcmogemc to humans, Group 3, not classifiable as to its carcmogemclty to humans

35

34

2007-20-7

CIoH9NO7S2

NH 2

33

H3OS

Structure

No

Aromatic ammosulphomc aods

z

Genotoxlclty and carcmogemcity of AASAs attempted to validate this test system using 65 chemicals together with available results of 2-year carcinogenicity and in vitro mutagemcity tests. The authors concluded that there was a poor agreement between genotoxicity results and the results obtained with strain A mice. Furthermore, results obtained with strain A mice are also not in agreement with results from 2-year carcinogenicity tests on aromatic amines and several other miscellaneous chemicals. Based on these findings, the concern of Theiss et al. expressed in their previous paper (Theiss et a l , 1981) about the possible carcmogenic potential of AASAs is certainly not justified. The aim of this paper ~s to investigate whether the available toxicity data on AASAs involved in dyestuffs chemistry give indications of any genotoxic or carcinogenic potential. The role of aromatic aminosulphonic acids as dyestuff intermediates and metabolites The sulphonated derivatives of the aminonaphthalenes and anilines are particularly important dye intermediates. The sulphonic acid group is used extensively in the dye chemistry to confer water solubility and for its ability to act as a good leaving group in nucleophllic substitutions. It is used almost exclusively for these purposes since it has only a minor effect on the colour of a dye. The amino group is the single most important functional group in dye chemistry. In azo-dye intermediates, it not only provides access to the azo group (by diazotization and coupling), but is also the most widely used auxochrome as can be seen from its presence in so many coupling components. Dye intermediates and similar compounds are an important group of primary metabolites of azo dyes, which constitute over 65% of all dyestuffs. Azo dyes all share one or several azo ( - - N : N - - ) groups, but they constitute a large variety of differing structures with a wide range of physlcochemical properties: from extremely water-soluble dyes to practically insoluble azo pigments, and from anionic and cationic to nonionic dyes. It has already been shown that under anaerobic conditions a variable percentage of the azo groups can be reduced enzymatically to the corresponding sulphonated and unsulphonated aromatic amines in bacteria and in mammalian systems including humans. In bis- and trisazo compounds the metabohc reduction of the azo groups proceeds stepwise under intermediate formation of the partly reduced molecules (i.e. azo-amino compounds). The formation of carcinogenic aromatic amines is assumed to be the major causative factor of the carcinogenic response to a given dye in an animal lifetime bioassay. Typical examples are the benzidine dyes, which are metabolized to the known human carcinogen benzidine. Based on present knowledge, it might be assumed that azo dyes, which are metabolized to a substantial

647

extent to carcinogenic amines in the living organlsms, should be suspected as potential carcinogens. Conversely, if non-carcinogenic amines are formed, the probabihty that the parent compound has carclnogenic properties becomes very small. It must be emphasized that beside this reductive cleavage of the azo group, other metabolic transformations could occur in the sense of additional activation or deactivation. Genotoxicity data of aromatic aminosulphonic acids As already mentioned, AASAs constitute a large number of potential metabolites especially of anionic dyes. Although few bioassay data on carcinogenicity exist, much information is available on genotoxiclty. This paper compiles a substantial amount of published and unpublished mutagenicity data. Table 1 displays available results of the Salmonella/microsomc test (Ames test), and other genotoxiclty and carcinogenicity data for 35 AASAs. For comparison, some available data on the unsulphonated analogues are also given. In contrast with mutagcnic and carcinogenic amines, such as 4-aminobiphenyl, benzidine and 2naphthylamine, the sulphonated analogues are not mutagenic in the Ames test. In the case of 2-naphthylamine several aminosulphonic acid isomers were non-mutagenic (Le et al., 1985). In a recent publication (Freeman et al., 1987), the results from the Ames and Prival mutagenlcity test of six aminonaphthalenesulphonic acids were reported. Five of them showed a very weak positive response (i.e. 1amino-naphthalene-7-sulphonic acid, 1-amino-8-hydroxynaphthalene-4-sulphomc acid, 2-amino-6-sulphonic acid (24), 2-amino-8-hydroxynaphthalcne-6sulphonic acid 32 (g-acid) and 3-ammo-8-hydroxynaphthalene-6-sulphonlc acid (y-acid)), whereas l-amlno-8-hydroxynaphthalene-3,6-disulphonic acid (34) was negative in both tests. The authors presume that the very weak positive response exhibited by the five amlno-naphthalenesulphonlc acids might be due to impurities since the samples used in their assays were not 100% pure. Such an assumption is supported by tests with highly purified materials. Le et al. (1985) found 2-aminonaphthalene-6-sulphonic acid (24) to be non-mutagenlc in the Salmonella/ microsome test in the presence of hepatic S-9 derived from the rat, hamster, monkey and humans. In the same test, using S-9 derived from the rat, 2amlno-8-hydroxynaphthalcne-6-sulphonic acid (32) (),-acid) (ETAD, 1990) and very recently 3-amino8-hydroxynaphthalene-6-sulphonic acid (J-acid) (ETAD, 1991) gave a negative response (not listed in Table 1). Similar experiences regarding purity were encountered with 2-amino-5-methylbenzenesulphonic acid (5) (4B-acid) and 2-amino-5-chloro-4-methylbenzenesulphonic acid ( l l ) (CLT-acid), which were negative in the Ames test only after thorough

No

HO3S -

-

~

C6HTNSO3

-

Structure

NH 2

Aromattc aminosulphomcacid (hypothetical dye metabohte)

N~N

C.I. 19140 C.I. Acid Yellow 23

NaO3S---~

C.l. Food Yellow 3

C.I. 15985

NaO3S~

C.I, 14600 C.I. Acid Orange 20

SO3Na

COONa

HO ~-----N~SO3Na

HO

Borzelleca (1988a) Borzelleca (1988b) Maekawa (1987)

IARC (1987) NTP-TR-208 (1981)

BIBRA (1988)

Azo dye wRh negaUveresults m caremogemcRyassays IARC (1987) Schach von WIttenau (1983)

Table 2. Ammes from azo dyes with negatwe results m caranogematy assays*

I01

No.

Unsulphonated analogue of amine

l0

CH30

/~.~-"-

. CH3

CaHItNSO 4

HO3S

T

NH~

CoHTNSO3

H03S

NH2

HO

N= N

C.I. 16035 C.I. Food Red 17

NaO3SH 3 C ~

cH3

OCH 3

C.I. Food Red 1

C.L 14700

NaO3S

C.I. 13065 C.I. Acid Yellow 36

NaO3S~__

C.I. 15980 C.I. Food Orange 2

Na03S

HO

NaO3S

HO

SO3Na

NH©

SO3Na

Borzelleca(1989)

IARC (1987) Schach von Wittenau (1983)

Vettoraz~ (1981) ADL (1980)

Vettorazzl (1981)

[contdl

107

101

> >

o

=s

o

21

No

C10H9NSO3

SO3H

NH 2

Structure

AromaUc amlnosulphomcacid (hypotheUcal dye metabohte)

C.I. 20285 C.I. Food Brown 3

NaO3S

N~N

SO3Na

OH

N~N

HO

N~N

C.I. 14720 C.I. Acid Red 14

C.I. 16185 C.I. Acid Red27

NaO3S~

SO3Na

N~-----NS

~

SO3Na

SO3Na

C~OH

HO

HO

SO3Na

Vettorazzi (1981) Drake (1978) Carpamm (1978)

BIBRA (1990) NTP-TR-220 (1982)

ADL (1980) Clode (1987)

Azo dye with negative results m carcmogenlcRyassays

Table 2 - - - c o n t d

107

No

amine

Unsulphonated analogue of

o~

37

36

CsHIINSO3

c~

~ C ~ s 0 3

NH2

CsHIINSO3

CH3

C H 3 ~

NI-I2

H

SO3H

NaOsS

i~c

NaO3S

CH3

N

O3Na ~

C.I. 14700 C.I. Food Red 1

c~

HO

~

NaO3S

N

HO

C.I. 16255 C.I. Acid Red 18

C.I. 14815 C.I. Food Red 2

NaO3S

SO3Na

HO

SO3Na

IARC(1987) SchachyonWRtenau(1983)

Vettorazzl(1981)

Brantom(1988) BIBRA(1986) Mason(1974)

[conta]

123

123

~

r.~

0

g~ e~

o

g

38

!1

NO.

HO3S

HOsS

HOsS

"CH3

C IoHIoN2SO3

NH 2

CTI'IgNSOs

CHs

CTI~NSO3CI

Cl

N~

Structure

Aromattc amlnosulphonicacid (hypothetical dye metabohte)

N

HO

NaO3S---O

C.I. Food Black 1

SO3Na

COONa

NaO3S

HO

SO3Na

NHCOCH 3

BIBRA (1989b) Vettorazzl (1981) Drake (1977) Gaunt (1972)

BIBRA(1982a)

BIBRA (1989a) IARC (1987) NTP-TR-225 (1982)

Azo dye with negattve results in carcinogemcttyassays

C.I. 28440

C.I. 15850 C.I. Pigment Red 57

N

O3Na

C.I. Pigment Red 53:1

C.I. 15585:1

N

SO3Na

CHs--~

CI

OH

Table 2---contd

124

102

No 108

2nllne

Unsulphonated analogue of

Z

CIoH9NSO4

H O 3 S / ~

NH2

OH

t

C.I. 16035 C.I. FoodRed 17

H3C

C.I. 15985 C.I. FoodYellow3

NaO3S ~N'---N ~

HO

N_~_NHO~

C.I. 15980 C.I. FoodOrange2

NaO3S~

SO3Na

SO3Na

SO3Na

Borzelleca (1989)

BIBRA (1988) IARC(1987) NTP-TR-208(1981)

Vettorazzt(1981)

[cored]

!19

r~

>.

o

o

33

No. 39

HO3S

CI0HgNS207

NH2

CIoHgNS207

HO3S ~ O H

Structure H03S

Aromaticammosulphomcacid (hypotheucaldye metabohte)

SO3H

NaO3S

NaO3S - - ~

N = . N - ~

C.I. 16255 C.I. Acid Red 18

N~ N

C.I. 16185 CT Acid Red 27

N

N

SO3Na

S03Na

SO3Na

HO SO3Na

C.I. 16230 C.I. Food Orange 4

Na03S

HO

IARC (1987) Clode (I987)

Zeiger (1987) NTP-TR-211 (1987) IARC (1987)

Brantom (1987) BIBRA (1986) Mason (1974)

Azo dye with negativeresults m carcinogenicttyassays

Table 2 - - c o n t d

119

119

No.

Unsulphonated analogue of amine

o~

41

40

j,.~ NH2

/NH 2

CIoH9NSO4

SO3H

~

OH

C toH9NS04

SO3H

OH

NaO3S

N

C.I. 14700 C.I. Food Red 1

C.I. 14815 C.I. Food Red 2

• SO3Na

HO

N

HO

NaO3S

C.I. 14720 C.I. Acid Red 14

SO3Na

OH ~

SO3Na

SO3Na

Vettorazzl (1981)

Schachyon Wittenau(1983)

IARC (1987)

BIBRA(1990) NTP-TR-220(1982)

[contd]

119

119

u.

o'J

o

o

o

HO3S

SO2I-IN. OH

OH

CloH1oN2S207

H2N

C12HI4N2S20s

SOsH

CITHI6N209S 3

H O s S / ~

H,C----~

Structure

So3NH2H

©

©

~

~

OH

NH2

C.I. 17200 C.I. Acid Red 33

NaOsS

N~N

N

SOsNa

SO3Na

NHCOCH3

C.I. 18050 C.I. Food Red 10

NaO3S

N

HO

~

SO3Na

_ NHO2S

C.I. Acid Red 106

C.I. 18110

NaOsS

N~-N ~

HO

~ CI-I~

BIBRA(1982b) BIBRA(1982c)

VettorazzJ(1981) WHO(1977)

IARC/WHO(1981)

Azo dye withnegativeresultsin carcmogemcltyassays

127

126

125

No

amine

Unsulphonated analogueof

*ForaromaUcammosulphomcacids withnumbers 1-35and for unsulphonatedanalogueswithnumbers101-122,see Table 1 (mutagemcltyand othertoxicitydata of selectedaromaUcammosulfomcacidsand their unsulphonatcdanalogues).

44

43

42

No.

Aromattcammosuiphomcacid (hypotbeucaldye metabohte)

Table 2--contd

Z e~

657

Genotox~clty and carcmogeniclty of AASAs purification. Both substances were also negative in the micronucleus assay (ETAD, 1988). The two compounds reported by Freeman et al. (1987) to be weakly positive m the Ames test (i.e. lamino-naphthalene-7-sulphonic acid and 1-amino-8hydroxynaphthalene-4-sulphonic acid) are not listed as mutagemc in Table 1 of this review in view of the uncertainty regarding the purity of the samples tested. Unlike benzidme (115) and 2-aminonaphthalene (118), aniline (101) and p-ammotoluene (102) have only weak (aniline) or no significant carcinogenic potential m animal studies and are not mutagemc in the Ames test. The sulphonated analogues of aniline and p-aminotoluene are therefore, as expected, not mutagenic in the Ames test. In some cases, methyl and chlorine subst~tuents m the amline molecule considerably influence the genotoxic or carcmogemc potency. Several of these chemicals are mutagenic m the Ames test. The sulphonated derivatwes, however, as shown in Table 1, were not mutagenic m the Ames test. In addition to the results from the Ames test, some of the compounds gwen in Table 1 have been studied in other tests on genotoxiclty (m vivo and m vitro) including cell transformation assays. The outcome of all these studies reflects, with only few exceptions, the absence of genotoxicity for AASAs. 2-Ammonaphthalene-1,5-disulphonic acid (26) and the disulphonlc acid derivatives of benzldine, (18) and (19), were not active in cell transformation assays m vitro, whereas the unsulphonated carclnogemc amines induced cell transformation m vitro In contrast to 2-aminonaphthalene (118), which has been reported to cause D N A damage (positwe m several UDS tests), the l-sulphonic acid analogue (22) was negatwe in th~s test system. 2-Aminonaphthalene induced gene conversion in Saccharomyces cerewsiae with and without metabolic activation. 2-Naphthylamine-l-sulphonic acid (22) did not produce gene conversion m trp or ade genes of S. cerevistae strata D4 (Della Porta et al., 1982). Supportive evidence of non-genotoxlclty for AASAs also comes from mutagenicity studies with azo dyes usmg reductive assay conditions. Structures of Direct Black 19 analogues with unsulphonated p-phenyleneblsazo moieties were active m the Salmonella/microsome assay, whereas the sulphonated derivatives were not (Lm and Solodar, 1988). To summarize the results, in general aromatic phenyl- and naphthyl-aminosulphonic acids are nonmutagenic to Salmonella. Other tests on genotoxiclty have also been considered. The data compiled show no or very low mutagenic potentml. In addition, three AASAs were negative in feeding studies on carcmogenicity. The available data indicate that sulphonation of phenyl- and naphthylamines decreases toxicity considerably. Where comparable data on sulphonated and unsulphonated analogues exist, loss

of genotoxlc and/or carcinogenic suiphonation is observed.

activity

on

Mechanistic considerations It has been repeatedly reported (Combes and Haveland-Smith, 1982; Garner and Nutman, 1977) that sulphonat~on renders amines less mutagenic and non-carcinogemc, and azo dyes based on AASAs non-carcinogenic (see Table 2). It appears to be a general rule that highly sulphonated azo dyes are poorly absorbed from the gut (Walker, 1970), and therefore become b~oavailable to a significant extent only after azo reducuon to the corresponding ammosulphonic acids. The degree of absorption from the gut is dependent on the polarity of the molecule, and sulphonation increases the polarity of the molecule For sulphonated amines, significant absorption has been observed (for review see Walker, 1970). Monosulphonated amines (e.g. sulphanilic acid, naphthlomc acid) are absorbed to a large extent, possibly m the zwitterionic form. Unlike aromatic amines, however, ring substitution by sulphonation of azo dyes based on AASAs prevents the activation of amino groups by liver metabolism (Garner and Nutman, 1977). Lin and Solodar (1988) beheve that sulphonation (as in the case of p-phenylenediamine) Increases the electronegatlvity of the metabolized molecule and thus decreases the electrophilicity required to attack D N A bases. Recently, Levme (1991) m h~s review on metabohsm of azo dyes explained that highly charged sulphonated azo dyes resist bacterial enzymic attack, and are poorly absorbed from the intestinal tract thus providing poor access to the hver endogenous metabolism As m the case of the non-carcmogemc, non-mutagenic 2-naphthylammo-l-sulphomc acid, pharmacokmetic studies indicate a rapid elimination of unchanged aminosulphonic acid after oral or iv administration. No biotransformation or cleavage of the sulphonic group was detected (Della Porta, 1982). As a more general conclusion to a study on the structural bas~s of the mutagemc~ty of phenylazoanihne dyes, Rosenkranz and Klopman (1989) reported that sulphonation appears to interfere with a structure in the azo dyes that is required for oxidation of the amino group A negatwe inductive effect of the sulphomc group may influence azo reduction or amine oxidation Combes and Haveland-Smlth (1982) suggested that the sulphonlc group may exert an unfavourable electromc influence on the activation of the 4-amino-Natom by exerting a strong negative inductive effect. The negative inductive effect of the sulphonic acid group on azo reduction has been demonstrated; Walker and Ryan (1971) showed that analogues of Red 2G and Red 10B sulphonated in the p-position of the phenyl ring were reduced by cell-free extracts

658

R. JUNG et al.

of Streptococcus faecahs two to three times more rapidly than the parent compound, owing to the substitution with an electron-withdrawing group.

Absence of evidence of the carcinogenic potential of aromatic aminosulphonic acids Few carcinogenicity studies have been carried out on substituted amlnonaphthalene- and aminobenzenesulphonic acids, probably because these compounds are expected to be non-carcinogenic. However, the existence of a considerable number of negative bioassays of azo dyes, which are metabolized to such aods, give additional evidence of their low chronic toxicity and their non-carcinogenic (or at most low carcinogenic) potential. It has been demonstrated that azo dyes are metabolized in the organism to aromatic amines by reductive cleavage of the azo group in bacterial systems both in animals and humans. However, the rate of this reaction is very much dependent on the individual structure and can differ widely. This has been shown by Watabe et al. (1980) in their study on the reduction of four azo dyes by micro-organisms from human faeces. The formation of AASA metabolites is certainly not always quantitative. There are several factors influencing the rate of formation such as other competing metabolic processes, rate of absorption, route of admimstratlon, low capacity of the reductlve enzymes, lack of availability of the dye to these enzymes, steric hindrance, etc. Table 2 lists azo dyes for which adequate negative 2-year bioassays have been done. With a few exceptions, the dyes can hypothetically form only AASAs (also compiled in Table 2) on reductive cleavage of the azo group. Considering the very high doses applied in most of the studies shown in Table 2, it can be assumed that substantial quantities of the corresponding AASAs have been formed in the gastro-intestinal tract and/or in the liver. The lack of tumorigenic response in these studies is indicative of a very low or even absent carcinogenic potential of these metabolltes. This conclusion is supported by the genotoxicity data summarized in Table 1. It is generally accepted that genotoxicity is a primary event in the carcinogenic process. Therefore the absence of genotoxiclty with amlnosulphonic acid supports the conclusion that these compounds do not act as genotoxlc carcinogens. Table 1 also includes negative carcmogeniclty bioassay data for several aminosulphonic acids. In the case of benzidine and 2-naphthylamine, it has been shown experimentally that sulphonation of the parent carcinogenic amine leads to non-carcinogenic substances. Although there is a long history of production of a large number of AASAs in the dyestuff manufacturing Industry, there are no indications of any carcinogenic risks from these compounds. In

contrast with these findings, bladder cancer cases resulting from exposure to 2-naphthylamine and benzidine are well known. An interesting observation in this connection was made by Kumar et aL (1981), who found in their immunological studies a correlation between increased lymphocyte reactivity and bladder cancer risk in workers exposed to 2-naphthylamme (118). Workers exposed to 2-naphthylamine-l-sulphonic acid (22), but not to 2-naphthylamine, had lymphocytes with a normal range of reactivity, which is in keeping with the suggestion that this compound is non-carcinogenic.

Conclusion The evaluation of available information and data on AASAs, either in use for dyestuff manufacture or being formed by reductlve cleavage of azo dyes, shows that these compounds, unlike some known carcinogenic aromatic amines, generally have no or very low genotoxic effect and hence no or minimal tumorigenic potential. In addition, considering the low exposure to AASAs prevailing in the techmcal application and use of azo dyes, there is not likely to be any significant toxic risk if AASAs are formed by metabolic cleavage in the organism or in the environment, or occur as micro-contaminants in colourmgs.

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