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Azo Dyes and Human Health: A Review

King-Thom Chung

Department of Biological Sciences, The University of Memphis, Memphis, Tennessee 38152

Corresponding Author Email: kchung@memphis.edu

ABSTRACT

Synthetic azo dyes are widely used in industries. Gerhardt Domagk discovered that the

antimicrobial effect of red azo dye Prontosil was due to the reductively cleaved (azo reduction)

product sulfanilamine. The significance of azo reduction is thus revealed. Azo reduction can be

accomplished by human intestinal microflora, skin microflora, environmental microorganisms, to

a lesser extent by human liver azoreductase, and by non-biological means. Some azo dyes can

be carcinogenic without being cleaved into aromatic amines. However, the carcinogenicity of

many azo dyes is due to their cleaved product such as benzidine. Benzidine induces various

human and animal tumors. Another azo dye component p-phenylenediamine (p-PDA) is a

contact allergen. Many many azo dyes and their reductively cleaved products as well as

chemically related aromatic amines are reported to affect human health, causing allergies and

other human maladies.

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INTRODUCTION

Azo compounds are chemically represented as R–N=N-R’, where –N=N- is the azo group,

and the R or R’ can be either aryl or alkyl compounds. The International Union of Pure and

Applied Chemistry (IUPAC) defines azo compounds as “derivative of diazene (diimide),

HN=NH, wherein both hydrogens are substituted by hydrocarbyl group, e.g. PhN=NPh

azobenzene or diphenyldiazene” (1). The word azo comes from azote, the French name for

nitrogen that is derived from the Greek a (not) zoe (to live). Historically, the emmergence of

azo dyes was an important step in the development of the chemical industry.

Azo dyes are compounds consisting of a diazotized amine coupled to an amine or a

phenol and contain one or more azo linkages. The essential precursors of azo dyes are

aromatic amines.

Azo compounds have vivid colors and comprise about two-thirds of all synthetic dyes

and are by far the most widely used and structurally diverse class of organic dyes in

commerce (2). At least 3,000 azo dyes available in the past and were used in

pharmaceutical and paper industries as well as printing inks, paints, varnish, lacquer, and

wood stains (3). The colorants of synthetic and natural textile fibers, plastics, leather, hair

dyes, waxes, and petroleum are also azo dyes (4). Azo dyes are the largest and most versatile

class of dyes and account for more than 50% of the dyes produced worldwide (5).

Presumably, more than 2,000 different azo dyes are currently used and over 7 x 105 tons of

these dyes are produced worldwide (6). More than 3,000 tons of azo dyes were certified in

1991 by the U.S. Food and Drug Administration (FDA) for use in foods, drugs, and

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cosmetics. These dyes constitute the major group of FDA certified colorants (5).

Azo dyes are stable in light and resistant to microbial degradation or fading away due to

washing. Therefore, azo dyes are not readily removed from waste water by conventional

waste water treatment methods. It has been estimated that about 10% of the dyestuff in the

dyeing process of textiles do not bind to fibers and are, therefore, released to the environment

(5, 7).

Some azo dye components such as benzidine have been linked to cancers of human

bladder. Also, there is a higher incidence of bladder cancer in dye workers exposed to

azo dyes (5). Therefore, azo dyes pose lethal effects, genotoxicity, mutagenicity, and

carcinogenicity to humans as well as animals. Indiscriminate disposal of azo dyes into the

environment especially from the textile industry is a major threat to human health and

environment (8). This paper is to review the effects of azo dyes and their metabolites and

some chemically related compounds.

BRIEF HISTORY

a. Story of Prontosil

The first azo dye used in medicine is Prontosil (sulfamidochrysoidine) (C12H13N5

O2S) (Figure 1), which is a red coal-tar dye with low toxicity and is used on leather. Other

names for this substance include Sulfoamidochrysoidine, Rubiazol, Prontosil, Aseptil Rojo,

Streptocide, and Sulfamidochrysoidine Hydrochloride. Later the name was abbreviated to

Prontosil. Prontosil was first synthesized by Bayer chemists Josel Klarer (1898-1953) and

Fritz Mietzsch (1896-1958) as part of a research program designed to find dyes that might act

as antibacterial drugs for the I. G. Farbenindustrie directed by Gerhardt Domagk (1895-1964).

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Prontosil was found to be effective against Gram-positive cocci but not against enterobacteria.

The antibacterial efficacy of Prontosil in mice was established in the murine model of

Streptococcus pyogenes systemic infections before 1932. In the same year, I. G.

Farbenindustrie obtained a German patent for the medical utility of Prontosil.

Gerhardt Domagk was responsible for finding antibacterial agents, using rats or rabbits

both in vitro and in vivo. Domagk tested thousands of compounds related to azo dyes, and

brought the importance of Prontosil to the world’s attention in 1932.

Domagk conducted an experiment whereby he injected 26 mice with a hemolytic

streptococcal bacterial culture, then injected 12 mice with a single dose of Prontosil an hour

and a half later. The 14 control mice died. The 12 injected mice, on the other hand, survived.

Domagk discovered that Prontosil affected streptococci in vivo but not in vitro and was non-

toxic to mice. A very dramatic but not published story was that Domagk tested this compound

on his four-year-old daughter, Hildegarde, who contracted a streptococcal infection in her

father’s laboratory when she wasaccidently pricked with a needle. After making 14 incisions,

the physician could find no other solution than to recommend to Domagk that they should

have her arm amputated. At this moment Domagk himself intervened in the treatment.

Domagk injected Hildegarde with a dose of Prontosil and she recovered. The results were

finally published in a paper in the February, 1935 issue of the German journal Deutsche

Medizinische Wochenschrift (9). Eventually, the therapeutic effect of Prontosil was well

spread, and many other researchers began to work with Prontosil. French scientist Theresa

Tréfouël (1892-1978) and his group in 1936 discovered that Prontosil was metabolized into

sulfanilamide (p-aminophenyl-sulfonamide) and identified this group as the active component

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of Prontosil (10).

Sulfanilamide is a simple, colorless molecule and was first synthesized by Paul Geimo, a

chemistry student working at the University of Vienna in his 1909 thesis. Sulfanilamide was

patented in Germany and later sold by Bayer as Prontalin. Prontosil was redefined as a prodrug

then. Sulfanilamide became the first oral version of sulfonamide drugs (10, 11). Following

studies discovered that this compound was bacteriostatic by blocking metabolism. The mode of

action of sulfanilamide was to act as an antimetabolite of para-aminobenzoic acid (PABA),

which is a precursor of nucleic acid biosynthesis. Sulfanilamide also interferes in different

metabolic steps in protein synthesis. Sulfanilamide and its derivatives were proved to be

effective against pneumonia, meningitis, blood poisoning, and gonorrhea. Sulfanilamide

empowered medical doctors to treat bacterial infections. Because the active gradient

sulfanilamide contains sulfur in the structure, sulfur drugs gained their names and became the

major antibacterial agents in that era. However, these sulfur drugs are basically aromatic amines.

Examples of sulfur drugs are shown in List 1. These aromatic amines also induce urinary tract

disorders, hematopoietic disorders, porphyria, hypersensitivity reactions as side effects. When

used in large doses, they can cause strong allergic reactions such as Stevens-Johnson syndrome

and epidermal necrolysis (Lyell syndrome). About 3% of the general population developed

adverse reaction. In adult immunodeficiency disease (AIDS) patients, the adverse response can

reach 50%. Most common manifestations of hypersensitivity reactions are rash and hives.

The sulfanilamide was easily linked to other molecules. Sulfanilamide was off patent in

1936. Prontosil has been replaced in clinical use by other newer sulfonamide drugs including

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sulfathiazole, sulfamethoxazole, etc. and soon gave rise to hundreds of second generation

sulfonamide drugs.

Prontosil was investigated by many researchers including Leonard Colebrook (1883-

1967) in England who introduced Prontosil as a cure for puerperal fever (12, 13). Sulfur drugs

were quickly replaced by the newly developed antibiotics that proved to be more effective

against pathogens and diminished the importance of sulfur drugs in chemotherapy. However,

Prontosil remained as a major drug until the 1960s. Prontsoil is still in use extensively for

opportunistic infections in patients of urinary infections and burn therapy. Sulfonamide in

combination with trimethoprim and sulamethoxazole (TMP-SMZ) is an excellent example of

drug synergism. Prontosil’s discovery ushered in the era of antibiotics and had a profound impact

on the pharmaceutical industry, medical history, and human welfare.

The metabolism of lipid soluble Prontosil to sulfanilamide is an azo reduction that can

be accomplished by the liver; water soluble azo dyes are mostly reduced by intestinal

and skin microflora. However, during Domagk’s (or Tréfouël’s) time, the role of intestinal

microflora in the metabolism of xenobiotics including azo dyes was not known.

b. Story of Methyl Yellow (Butter Yellow, DAB)

James A. Miller (1917-2000) and Elizabeth C. Miller (1920-1987) pioneered the study of the

carcinogenicity of a well-known azo dye, Methyl Yellow (p-dimethylaminoazobenzene,

DAB, Butter Yellow) (Fig. 2) and its metabolites. They discovered that only 4-monomethyl-

aminoazobenzene and its parent compound DAB were carcinogenic (14). All other

metabolites including 4-aminoazobenzene, 4’-hydroxy-4-monomethyl-

aminoazobenzene, 4’-hydroxy-4-aminoazobenzene, and the reductive cleaved products N-

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methyl-p-phenylenediamine, p-phenylenediamine (p-PDA), aniline, p-aminophenol and

o-aminophenol were reported not carcinogenic (Fig 2). Nine other compounds, i.e,

4’-hydroxy-4-dimethylaminoazobenzene, 3’-hydroxy-4-dimethyl-amino-azobenzene,

2’-hydroxy-4-dimethylaminoazobenzene, 4-formylamino-azobenzene, 4-hydroxy-

azobenzene, 2, 4’-diamino-5-dimethylaminodiphenyl, 3-dimethyl-aminocarbazole, N, N-

dimethylphenylenediamine (DMPD), and p-hydroquinone may be metabolites, and were also

not carcinogenic. But DMPD was found to be mutagenic in Salmonella typhymurium tester strain

TA1538 in the presence of microsomal activation (15, 16). Azo-reduction of DAB has been

claimed to be a detoxicification (i.e. metabolic deactivation) pathway (17).

Chung reviewed the significance of azo reduction in the mutagenesis and carcinogenesis of

several azo dyes and noticed that azo reduction is an important metabolic activation for

carcinogenesis of many azo dyes (18-20).

SIGNIFICANCE OF AZO REDUCTION

Human exposure to azo dyes may occur through ingestion, inhalation, or skin contact.

Azo dyes are biotransformed inside the body into aromatic amines. Most notable

biotransformation is reductively cleaved into aromatic amines by azo reductases of intestinal

microflora (20). Examples of aromatic amines metabolically produced from azo dyes are

shown in Table 1. A substantial number of environmental microorganisms including

helminths capable of azo dye reduction has been reported (21). Examples of reported

intestinal microorganisms with azo reduction activity are shown in List 2. The azo reductase

activity in a variety of intestinal preparations was affected by various dietary factors such as

cellulose,proteins, fiber, antibiotics, or supplements with live cultures of lactobacilli (20).

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Plazek, et al. (22) reported that carcinogenic amines were also produced from azo dye by

human skin bacteria in vitro. Stingley et al. (23) also showed that azo dyes such as Methyl Red

and Orange II were cleaved by human skin microbiota including many species in the genera of

Staphylococcus, Corynebacterium, Kytococcus, Micrococcus, Dermacoccus, and Kocuria (23).

Several hundred species are residents of the human skin and many of them express azo reductase

activity. These facts should be of significance to those who use tattoo inks, textiles, and

cosmetics (22, 23).

Only a few aerobic bacteria have been found to reduce azo dyes under aerobic conditions.

The enzyme responsible for azo dye reduction has been partially purified and characterized.

There are three distinct groups of azo-reductases that have been described. There are: flavin

dependent NADH preferred azo reductase, flavin dependent NADPH preferred azo reductase,

and flavin free NADPH preferred azo reductase (24). Each enzyme has been purified from

specific microorganism and studied on the characterization and crystal structure of azo

reductase in Bacillus subtilis, Escherichia coli, Enterococcus fecalis, Pseudomonas aeruginosa ,

Streptomyces cereviceae, and Candida zeylanoides (25-31).

Other environmental microorganisms including fungi, protozoa, and cestode Ascaris

lumbncoides and the nematode Moniezia expansa have also been reported to reduce azo dyes

(32, 33). Chemical reducing agents such as sodium hydrosulfite, sodium dithionite, zero-valence

iron (Feo) and electron sources for biological reactions such as reduced flavin adenine

nucleotides (FADH) as well as reduced nicotinamide adenine dinucleotide (NADH) are also able

to reduce azo dyes (34-36).

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Recently, Hong and Gu (37) demonstrated that azo reduction by Shewanella strains was

coupled to the oxidation of electron donors and linked to the electron transport and energy

conservation in cell membranes. Anaerobic azo reduction by bacteria was shown to be capable of

coupling the transformation of toxic organic substances to the reduction of azo compounds

simultaneously. It would be ideal for bioremediation of environments contaminated with azo

dyes and other toxic organic chemicals (37).

Several studies have shown that oxidation of organic compounds using Fenton’s reagents

(H202, Fe++) is efficient to degrade organic compounds like azo dyes (38); Guivarch et al. (39)

also demonstrated the degradation of azo dyes in water by electron-Fenton processes. Tantak and

Chaudhari (40) also demonstrated degradation of azo dyes by sequential Fenton’s oxidation and

aerobic biological treatment of wastes. Konstantonow and Albanis (41) reported that TiO2

assisted photocatalytic degradation of azo dyes in aqueous solution. These processes were

reported to achieve effective oxidative degradation and mineralization of azo dyes. These

processes produced some intermediates such as hydroxylated derivatives, naphthoquinone,

phenolic compounds, organic acids, and other toxic products. Small quantities of aromatic

amines are also detected in the residues. These toxic wastes including aromatic amine remain to

be an environmental problem.

Examples of aromatic amines would be released from azo dyes upon reduction are listed in

Table 1.

CARCINOGENICITY OF AZO DYES

Some azo dyes can be carcinogenic directly without being cleaved into aromatic amines

(14). The following are examples (List 3).

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1.4-Aminoazobenzene (Cas No-60-09-3)

4-Aminoazobenzene is also called Aniline Yellow or 4-phenylazoaniline and was first

produced in 1861 by C. Mene. Aniline Yellow is used in microscopy for vital staining, in

pyrotechnics for yellow colored smoke, in yellow pigments and in inks including inks for inkjet

printers. It is also used in insecticides, lacquers, varnishes, waxes, oil stains and styrene resins. It

is also an intermediate in the synthesis of other dyes such as Chrysoidine, Indulines, Solid

Yellow and Acid Yellow. It has high hepatocarinogenicity to male mice when given as a single

intraperitoneal injection as low as 0.027 to 0.15 μmole/g body weight but not in female mice

(42). This dye also induces liver tumors in rats by oral administration and epidermal tumors by

application to the skin (43).

2. o-Aminoazotoluene (Cas No. 97-56-3)

o-Aminoazotoluene is also known as C.I. Solvent Yellow 3 or Fast Garnet GBC base. In

hamsters, o-aminoazotoluene produced tumors in urinary bladder, gall bladder, lung, and liver. It

can also induce tumors in urinary bladder, gall bladder, and liver in dogs and rats (44). 3.

Methyl Yellow (Cas No. 97-56-3)

MethyYellow (Butter Yellow) was used as a food additive in 1918 but was quickly

removed from the food additive list in the same year because it was discovered to be a strong

cancer agent (14, 45, 46).

4. Methyl Yellow Derivatives

Methyl Yellow derivatives were carcinogenic to rats. 3’-methyl-4-monomethylamino-

azobenezene and 3’-methyl-4-dimethylaminoazobenzene were nearly twice as active 4-DAB,

and 4-ethylmethylaminoazobenzene had the same activity as 4-DAB. Both 3’-nitro- and 3-

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chloro-4-dimethylaminoazobenzene had about the same activity as 4-DAB. However, since the

3’-nitro derivatives were incompletely absorbed, their real activity appears to be about one and

half that of 4-DAB. 2’-Nitro and 2-chloro-4-dimethylaminoazobenzenes were about one half to

one third as active, and 4’-chloro-4-dimethylaminoazobenzene was approximately one fourth as

active as the parent dye (14)

5. Sudan Azo Dyes

Sudan azo dyes such as Sudan 1 (1-phenylazo-2-naphthol) (Cas No. 842-07-9) is also

called CI Solvent Yellow 14 and Solvent Orange R. It is an intense orange-red solid that is to

colorize waxes, oils, petrol, solvents, and polishes. Sudan I has also been adopted for coloring

various foodstuffs, especially curry powder and chili powder, although the use of Sudan I in

foods is now banned in many countries because it is reported as a carcinogen (47). Sudan I is

still used in some orange-colored smoke formulations and as a coloring for cotton refuse (cotton

waste) used in chemistry experiments.

Sudan II (Cas No. 3118-97-6) {1-[(2, 4-dimethylphenyl)azo]-2-naphthalenol} or [1-(2, 4-

xylylazo)-2-naphthol], also known as Solvent Orange 7, is a member of the Sudan family of

hydrophobic fat-staining dye. Sudan dyes are a group of lipid soluble solvent dyes often called

lysochromes. They are diazo dyes. Sudan II is used for certain solvents and may also be used to

stain some proteins bound lipids in paraffin sections. By studying the cellular regions where the

dye is sequestered, Sudan II has been used to evaluate how certain toxins interact with

membranes. Sudan II is considered a hazardous substance according to Osaha 29 CFR

1920.1200, although it is not reported as a carcinogen.

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Sudan III (Cas No 85-96-9) [1-(4-(phenyldiazenyl)phenyl]azonaphthalen-2-ol] is a

lysochrome diazo dye. Its other names are Sudan Red BK, Fat Ponceau G, Cerasin Red, C.I.

26100, Solvent Red 23, Sudan Red, Sudan Red III, Sudan V, Sudan Red B, Sudan G, Scarlet

B, and Tony Red. It is used to color nonpolar substances like oils, fats, waxes, greases,

various hydrocarbon products, and acrylic emulsions. Its main use is as a fuel dye in the

United States of America mandated by the IRS to distinguish low-taxed heating oil from

automotive diesel fuel and by the EPA to mark fuels with higher sulfur content. It is reported as

a carcinogen (48).

Sudan IV (C24H20N4O) (Cas No. 85-83-6) [1-{2-methyl-4-(2-methylphenyldiazenyl)

phenyl}azonapthalen-2-ol] is also called Sudan R, C.I. Solvent Red 24, C.I. 26105, Lipid

Crimson, Oil Red, Oil Red BB, Fat Red B, Oil Red IV, Scarlet Red, Scarlet Red N.F, and

Scarlet Red Scharlach. Sudan IV is a fat-soluble diazo dye used for the staining of lipids,

triglycerides and lipoproteins on frozen paraffin sections. Sudan IV is considered an illegal

dye, mainly because of its harmful effect over a long period of time. It is a carcinogen and

was ruled unsafe in the 1995 food safety regulations report. Susan dyes are often found as

food contaminants and are illegally used (49-51). Sudan I, Sudan III, and Sudan IV have been

classified as category 3 carcinogens by IARC (51).

6. Para Red (Cas No. 7410-10-2)

Para Red {1-[(E)-(4-nitrophenyl)diazenyl]-2-naphthol} is also called Paranitraniline Red,

Pigment Red 1, and C.I. 12070. It is used to dye cellulose fabrics. The dye can be washed away

easily cellulose fabrics if not dyed correctly. Acidic and basic stages both occur during the

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standard formation of Para Red, and acidic or basic by products may be present in the final

product.

In the United Kingdom (UK), the Para Red is not permitted in food. The UK's Food Standard

Agency Standards (FSA) stated that, "The Agency’s independent scientific experts have advised

that, although there are very limited data available, it would be prudent to assume that it could be

a genotoxic carcinogen."

7. Other Azo Dyes

Many azo dyes of the organic colorants were listed as carcinogens in the safety review of

the use of certain azo dyes in cosmetic products adopted by the Opinion of the Scientific

Committee on Cosmetic and non-Food Products Intended for Consumer Concerning

(SCCNFP) during the 19th plenary meeting of 27 February, 2002 (52). Azo dyes listed as

carcinogens are shown in List 4. Although the above named compounds were listed as

carcinogens, some of them are limited evidence in experimental animals, and inadequate

evidence in humans for their carcinogenicities (IARC category 3). Other carcinogenic azo

dyes might not be included in the SCCNFP list. For those listed as carcinogens, the author did

not know whether their carcinogenicity was due to the dye itself or due to the cleaved

products, aromatic amines. If the carcinogenicity is due to the cleaved aromatic amines, then t

hose dyes should be classified into the section of carcinogenicity of azo dye metabolites (VI).

Other azo dyes are not discussed above. Examples are: M-methyl-4-aminoazo-

benzene (MAB) is a hepatocarcinogen in mice (53), 6-dimethylphenylazobenthiazole (6BT) is

also a rat hepatocarcinogen (18, 54). Ponceau 3R is a human carcinogen (55). Orasol Navy Blue

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2R (Cas No. 61969-42-4) and thiodiphenyl-4, 4’-diazo-bis-salicyclic were both mutagenic in the

Salmonella/mammalian microsome system (18, 56) and were possible carcinogens.

Brown and De Vito predicted the carcinogenicity of azo dyes by pointing out that azo dyes

with structures containing free aromatic amine groups that could be metabolically oxidized

without azo reduction. These azo dyes that may be activated via direct oxidization of the azo

linkage to highly reactive electrophilic diazonium salts. Each mechanism may be compound

specific (57).

CARCINOGENICITY OF AZO DYES METABOLITES

Carcinogenicity or the adverse effects of the majority of azo dyes were probably due to the

cleaved components, aromatic amines. Chung and Cerniglia in 1992 (19) postulated that azo

dyes contain benzidine or p-phenylenediamine (p-PDA) as the mutagenic moieties. Therefore,

this author will address primarily the carcinogenicity of benzidine, p-PDA and some aromatic

amines structurally related to benzidine and p-PDA.

1. Carcinogenicity of benzidine and its congener

Benzidine has been identified as a carcinogen that can cause human urinary bladder cancer

(57-59). Benzidine has also been reported to induce cancer of genitourinary tract (58), pancreas,

liver (58), gallbladder (58), bile duct (58), lung (58), large intestine (58), stomach (58),

lymphopoies (58), and renal cell (58) as well as non-Hodgkin’s lymphoma (58, 59). Notable azo

dyes that contain benzidine moiety in their structure were used in industry intensively and are

probably still being used in different parts of the world. According to IACR’s classification, all

azo dyes metabolized to benzidine were classified as a category 1 carcinogen (60). Examples of

azo dyes that released benzidine after reduction are indicated in List 5.

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Benzidine and its congeners such as 3, 3’,-dimethylbenzidine (o-tolidine), 3, 3’-

dimethooxy-benzidine (o-dianisidine), and 3, 3’- dichlorobenzidine are the starting materials for

the synthesis of azo dyes referred to as benzidine-based or benzidine congener-based dyes.

Examples of benzidine-based dyes are Direct Blue 6, Direct Brown 9, Direct Black 38, etc. The

production of benzidine-based dyes has significantly decreased during the last century.

Although the National Institute of Occupational Safety and Health (NIOSH) and the

Environmental Protection Agency (EPA) of the U. S. A listed many potentially available derived

dyes, only a few are found in commercial use today (58).

The major company producing 3, 3’-dimethylbenzidine in the United States stopped

production in 1975. Imports appeared to be the major source of 3, 3’-dimethylbenzidine.

The major sources of the 3, 3’-dimethylbenzidine released into the environment is the reduction

of 3, 3’-dimethylbendine-based azo dyes including Acid Black 209, Acid Red 114, Direct Black

154, etc. It was reported that administration of dihydrochloride salt of 3, 3’-dimethylbenzidine in

drinking water induced adenoma or carcinoma of the Zymbal gland and liver, adenoma of basal-

cell, carcinoma, adenoma or papilloma of squamous-cell or carcinoma of perputial and clitoral

glands and adenomatous polyps of large intestine in rats of both sexes (61). Exposure of rats (of

unspecified sex) to 3, 3’-dimethylbenzidine by subcutaneous injection caused primarily

carcinoma of the Zymbal-gland (58). 3, 3’-Dimethyl-benzidine is reasonably anticipated to be a

human carcinogen (60).

3, 3’-Dichlorobenzidine induced carcinomas of the sebaceous gland (58), lung tumor (58),

sarcomas (58), tumor of lower jaw (58), and Zymbal gland tumors (58) in mouse. In rats (58), 3,

3’-dichlorobenzidine induced Zymbal gland tumors, skin tumors, mammary gland tumors ,

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intestinal tumors, bladder tumors, tumors of hamematopoietic system, salivary gland tumors ,

liver tumors, thyroid tumors, leukemia, sarcomas, Sebaceous tumors, bone tumors. In dogs,

3, 3’-dichlorobenzidine caused lung tumors and bladder tumors (58). 3, 3’-Dichlorobenzidine

may also cause human bladder cancer (63).

3, 3’-Dimethoxybenzidine and 3, 3’-dimethoxybenzidine-hydrochloride are used as an

intermediate in the production of dyes and pigments. 3, 3’-Dimethoxybenzidine has been

reported to have damaging effects on the liver, kidneys, spleen, bladder, endocrine, and

gastrointestinal in animal studies. Increased incidences of tumors in several organs have been

reported in rats orally exposed to 3, 3'-dimethoxy-benzidine or its salt (58). EPA has classified

3, 3'-dimethoxy-benzidine as a Group 2B, probably human carcinogen (64).

4-Aminobiphenyl [(1, 1’-biphenyl)-amine] (Cas No. 92-67-1) has been reported to be

metabolically released by deamination of benzidine (58, 65). 4-Aminobiphenyl can be present in

tobacco smoke (66), in fumes of cooking oils (67), and in contaminated food dyes (68). It is a

potent human carcinogen and induces human bladder cancer (69, 70).

4-Aminobiphenyl also causes cancer in mice. Bladder and liver tumors have also been observed

in rabbits and dogs following oral administration of 4-aminobiophenyl. Mammary gland and

intestinal tumors have been reported in rats exposed by subcutaneous injection (69, 70).

3, 3’,-5, 5’-Tetramethylbenzidine (TMB) is a widely used chromogen (71) and

is not mutagenic by the Ames test (61, 72) and did not induce formation of tumors in a single-

arm study of rats (73). TMB seems to be the only benzidine analogues that is neither mutagenic

nor carcinogenic and has been used as a replacement for carcinogenic compounds such as

benzidine (74) and o-phenylenediamine (75).

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The nitro derivatives of benzidine were usually more mutagenic than that of

benzidine in Salmonella typhimurium tester strainTA98 without S9 (76). The addition of a

sulfonic acid group to benzidine molecule reduced the mutagenicity. When both sides of

the azo linkage of azo dyes were sulfonated, the compounds were usually not carcinogenic in

any animal species (57).

1-Amino-2-naphthol can be released by the 1-amino-2-naphthol based azo dyes such as

Lithol Red and Orange II (18). 1-Amino-2-naphthol has been reported to be non-mutagenic

(16, 18); but Dillion, et al. (77) reported that 1-amino-2-naphthol was mutagenic to

Salmonella typhimurium tester strain TA100, not to strain TA98. Bonser et al. (78) also

reported that the formation of papilloma and carcinoma and an unusually high incidence

of squamous metaplasia in the surgically implanted mouse bladder in the pellets contained

1-amino-2-naphthol. The Health and Environmental Effects Profiles for 1-amino-2-naphthol

and 1-amino-2-naphthol hydrochloride reported that existing data are insufficient to determine

a Reference Dose (RfD) or carcinogenic potency factor for 1-amino-2-naphthol and

1-amino-2-naphthol hydrochloride (79).

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2. Carcinogenicity of p-phenylenediamine (p-PDA)

Initially, p-PDA was postulated to be a mutagenic component in several azo dyes (21).

However, p-PDA was reported to be non-mutagenic (80); Shahin, et al. (81-83) reported

it to be only a weak mutagen. Chung, et al. (84) found it to be weakly mutagenic to

Salmonella tester strain TA98 with metabolic activation. Lin and Solodar (80) also reported

that p-PDA became mutagenic only after it was oxidized. Watanabe, et al. (85) discovered

that p-PDA became strongly mutagenic in Salmonella typhimurium tester strain TA1538 in

the presence of microsomal fraction following oxidation by H2O2. So it can be concluded that

pure fresh p-PDA is not mutagenic, but it easily becomes mutagenic after oxidation.

p-PDA is a component in hair dye, which is a public concern. Sontag (86) pointed out that

p-PDA increased the formation of liver tumors in mice. Rollison, et al. (87) reported that

there is an association between personal hair dye use and non-Hodgkin's lymphoma, multiple

myeloma, acute leukemia, and bladder cancer in at least one well-designed study. Those

associations were not consistently observed across studies. A formal meta-analysis was not

possible due to the heterogeneity of the exposure assessment across the studies. Bolt and

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Golka (88) pointed out that carcinogenicity of p-PDA is under debate. The authors suggested

that since earlier exposures could have an impact decades later, the possibility of bladder

cancer in hairdressers who have intensively worked with permanent hair dyes during earlier

decades prior to 1980s should be taken into account.

Recently, Turesky, et al. (89) reported that hair dye p-PDA could be contaminated with

the carcinogenic 4-aminobiphenyl. So the reported carcinogenicity and mutagenicity of

p-PDA might be due to the contaminant, not due to p-PDA itself. It is interesting to note that

DMPD is highly mutagenic (15, 16) but has not been reported to be carcinogenic (14). So

the carcinogenicity of Methyl Yellow (DAB) studied by Miller and Miller (14) may be

activated via direct oxidation of the azo linkage to highly reactive electrophilic diazonium,

which is a free radical that induces carcinogenesis (57, 90). Therefore, azo reduction of

Methyl Yellow to generate p-PDA or N-methyl-p-phenylenediamine, both of which were

neither mutagenic nor carcinogenic, was regarded as a process of detoxification (14). p-PDA

is supplemented by other aniline analogues or derivatives such as 2,5-diaminohydroxy-

ethylbenzene and 2, 5-diaminotoluene in its uses. 2, 5-Diaminotoluenes itself is often used as

a substitute for p-PDA. No adequate information to indicate that both 2, 5-diaminohydroxy-

ethylbenzene and 2, 5-diaminotoluene were genotoxic

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p-PDA is usually mixed with H2O2 for hair dyeing. Oxidized p-PDA becomes a

diaminophenazines, which are extremely mutagenic (91) The reported contamination of

p-PDA with carcinogenic 4-aminobiphenyl, would put p-PDA as a high risk compound to

humans for commercial use (89).

3. Carcinogenicity of some other monocyclic aromatic amines (MAAs)

a. Aniline (Cas No. 62-53-2)

Aniline is the prototype of aromatic amine and a primary compound of industrial

chemistry. About 2.3 million tons were produced in 1996. Aniline can also be released from

the reduction of Orange G and 1-phenylazo-2-naphthol. Aniline is used in the synthesis of

polyurethane, diphenylmethane-3, 4-diisocyanate (MDI), rubber, dyes, pesticides, fibers, and

pharmaceuticals (92). Aniline has been reported to induce tumors in the spleen of rats but

does not induce bladder cancer in humans or animals. Carcinogenicity of aniline is now

proved to be due to the contaminant β-naphthylamine (93).

b. p-Nitroaniline (Cas No.100-01-6)

p-Nitroaniline is also called 4-nitroaniline or 1-amino-4-nitrobenzene with the formula

C6H6N2O2 and is commonly used as an intermediate in the synthesis of dyes, antioxidants,

pharmaceuticals, gasoline, medicines for chickens, and as a corrosion inhibitor.

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p-Nitroaniline can be released from Para Red. p-Nitroaniline was reported to be mutagenic in

the presence or absence of S9 mix activation in Salmonella typhimurium tester strain TA98,

but the results were negative for all other strains (77). P-Nitroaniline was also reported to

induce tumors in B6C3F1 mice (94). The Dutch Expert Committee on Occupational

Standards of the Health Council of the Minister of Social Affairs and Employment, the Health

Council of the Netherlands, evaluates and judges the carcinogenic properties of substances to

which workers are occupationally exposed. The committee recommends classifying

p-nitroaniline as a suspected human carcinogen (95).

c. 2, 4-Dimethylaniline

Another aniline derivative, 2, 4-dimethylaniline (Cas No. 95-68-1) is also called

m-xylidine and 2, 4-xylidine. Osano, et al. showed that 2, 4-dimethylaniline showed

teratogenic properties in developing Xenopus frogs (96). 2, 4-Dimethylaniline was

mutagenic and carcinogenic (97). 2, 4-Dimethylaniline can be released from 1-[(2, 4-

dimethyphenyl)azo]-2-naphthalenol by azo reduction.

d. o-Toluidine (Cas No. 95-53-4)

o-Toluidine is also called 2-methylaniline and is used as an intermediate in dye, rubber, and

pharmaceutical products (98, 99). It was first synthesized in 1844 and was suspected to be a

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carcinogen before 1921 (100). Up to 1954, o-toluidine was still not considered as a cause of

cancer. Richter (101) reviewed historical aspects of o-toluidine in detail. Numerous publications

proved that o-toluidine induced urinary bladder cancer in animals and humans (94 ,99, 102, 103).

The German Commission for the Investigation of Health Hazards of Chemical Compounds in the

Work Area classifies o-toluidine as a proven human bladder carcinogen (104). The IARC also

upgraded o-toluidine from group 2A to group 1 carcinogen for humans in 2010 (105).

Many monocyclic aromatic amines (MAAs) are genotoxic and impose hazards to human

health. The mutagenicity of more than 80 of these amines has been reviewed by Chung, et al.

(106) with primary emphasis on evaluation by the Ames Salmonella/microsome testing system.

Many of these amines are mutagenic in Salmonella tester strains TA98 and TA100, but S9 mix is

required for activity (106). 2, 4-Diaminotoluene, m-phenylenediamine (1, 3-diaminobenzene)

and a few amines containing a nitro-group including 4-nitro-o- phenylenediamine (4-nitro-2-

aminoaniline), 2-nitro-p-phenylenediamine (4-nitro-1,4-diaminobenzene), 2-amino-4-nitrophenol

(4-nitro-2-aminophenol), 2-amino-5-nitrophenol (3-nitro-6-aminophenol), m-nitroaniline, 4-

nitro-2-amino-6-methylaniline, 4-nitro-2-amino-6β-hydroxyethylaniline, 4-nitro-2-amino-6β-

hydroxypropylaniline, 4-nitro-2-amino-6-isopropylaniline, 2-nitro-6-methyl-p-

phenylenediamine, 2-nitro-6β-hydroxylethyl-p-phenylenediamine, 4-amino-3-nitro-6-

methoxylaniline, 4-amino-3-nitro-6-fluoroaniline,4-amino-3-nitro-6-chloroaniline, 4-nitro-o-

phenylenediamine are direct mutagens. Among these mutagens, the carcinogenicity of the

following compounds are noticed:

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i. 2, 4-Diaminotoluene (Cas No. 95-80-7)

2, 4-Diaminotoluene has been reported to cause tumor in rats and mice. Most tumors are

hepatocellular carcinoma, fibroma, mammary gland tumors, and kidney carcinoma (107). But no

epidemiological studies evaluated the relationship between human cancer specifically and 2, 4-

diaminotoluene. Based on sufficient evidence of carcinogenicity from studies on experimental

animals, the National Institute for Occupational Safety and Health (NIOSH) of the U. S. A. listed

2, 4-diaminotoluene as a potential human occupational carcinogen.

ii. 2-Nitro-p-phenylenediamine (Cas No. 5307-14-2).

Dietary administration of 2-nitro-p-phenylenediamine was reported to be carcinogenic to

female B6C3F1 mice, causing an increased incidence of hepatocellular neoplasms, primary

adenomas. There was no convincing evidence for the carcinogenicity of the

compound in Fischer 344 rats in both sexes or in male B6C3F1 mice (108).

iii. 2-Amino-4-nitrophenol (Cas No. 99-57-0)

The use of 2-amino-4-nitrophenol in cosmetic products is prohibited in the Commission

of the European Economic Community (109). According to the National Toxicological

Program (NTP) of the U. S. A. toxicology and carcinogenesis studies, there was some

evidence of increase incidences of renal cortical (tubular cell) adenomas by treatment of

2-amino-4-nitrophenol for male F344/N rats. The incidences of renal tubular cell hyperplasia

were also increased in male rats exposed to 2-amino-4-nitrophenol; but there was no evidence

of carcinogenic activity of 2-amino-4-nitrophenol for female F344/N rats or female B6C3

mice (110).

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iv. 4-Nitro-o-phenylenediamine (Cas No. 99-56-9)

The SCCCNFP/SCCP is of the opinion that the use of 4-nitro-o-phenylenediamine

itself as an oxidative hair dye substance at a maximum concentration of 0.5% in the finished

cosemetic product (after mixing with hydrogen peroxide) does not pose a risk to the health of

the consumer, yet it has a sensitizing potential. 4-Nitro-o-phenylenediamine itself is not

genotoxic in vivo. However, studies on genotoxicity/mutagenicity in hair dye formulations

should be undertaken following the relevant SCCCNFP/SCCP opinions and in accordance

with its “Notes of Guidance.”

2, 4-Diaminoanisole, 2, 5-diaminoanisole, o-PDA (1, 2-diaminobenzene),

2, 4-diaminoethoxybenzene, 2, 4-diaminopropoxybenzene, 2, 4-diaminoethylbenzene, 2, 4-

isopropoxybenzene, 4-Nβ-hydroxyethylamino-3-nitroanisole (3-nitro-4-Nβ-hydroxyethyl

aminoanisole), 4-amino-3-nitro-phenoxyethanol, p-hydroxy-m-PDA(2, 4-diaminophenol),

4-amino-3-nitro-6-methylaniline, 4-amino-3-nitro-6-isopropylaniline,

4-amino-3-nitro-5β- hydroxymethylaniline, 4-amino-3-nitro-5-methylaniline, 4-amino-3-

nitro-5β-hydroxypropylanilne, 4-amino-3-nitro-5-isopropylaniline, 4-amino-3-nitro-5, 6-

dimethylaniline, and 4-amino-3-nitro-2, 5-dimethylaniline were mutagenic in the

Salmonella tester strain TA98, but S9 mix is required for activity (106). Among the above

mentioned compounds, 2, 4-diaminoanisole (Cas No. 615-05-04) is identified as a carcinogen,

and o-PDA is suspected to be carcinogenic.

Sufficient evidence exists for the carcinogenicity of 2, 4-diaminoanisole sulfate in

experimental animals (111). When administered in the diet, 2, 4-diaminoanisole sulfate

increased the incidences of thyroid follicular cell adenomas in mice of both sexes and thyroid

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follicular cell carcinomas in female mice. When administered in the diet, 2, 4-diaminoanisole

sulfate increased the incidences of squamous cell carcinomas, basal cell carcinomas, or

sebaceous adenocarcinomas of the skin and its associated glands; malignant thyroid follicular

cell tumors; and preputial or clitoral gland adenomas, papillomas, or carcinomas in rats of

both sexes, and thyroid C-cell adenomas or carcinomas and Zymbal gland squamous cell

carcinomas or sebaceous adenocarcinomas in male rats (112). When administered in the diet

to female rats, 2,4-diaminoanisole sulfate induced mammary adenocarcinomas and

carcinomas of the clitoral gland and increased the incidences of follicular cell adenomas or

carcinomas and C-cell carcinomas of the thyroid (111). The IARC Working Group reported

that there were no adequate data available to evaluate the carcinogenicity of 2, 4-

diaminoanisole sulfate in humans. Therefore, 2, 4-diaminoanisole is classified as carcinogenic

to humans (Group 2B) in IARC volume 79 (113).

v. o-Phenylenediamine (o-PDA)

o-Phenylenediamine was found to be genotoxic in vitro and in vivo (114) and in Salmonella

mutagenicity test. Administration of o-phenylenediamine-dihydrochloride in drinking water to

F344/DuCrj rats and Crj BDF mice of both sexes for two years induced hepatocellular adenomas

in rats in both sexes and in female mice, and hepatocellular adenomas in male mice (114). o-

Phenylenediamine dihydrochloride causes both local reactions and systemic damages to humans.

Local actions include severe dermatitis and urticarial in the eye. o-phenylenediamine

dihdrochloride induces chemosis, lacrimation, exophthalmos, ophthalmia, and even permanent

blindness. Systematical damages include asthma, gastritis, rise in blood pressure, transudation

into serious cavities, vertigo, tremors, convulsions, and comas (115). o-PDA is first oxidized to

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the reactive o-quinone diimine, which reacts with another molecule of o-PDA to form 2, 3-

diaminophenazine, which is found to be mutagenic in Salmonella typhymurium strain TA100

with metabolic activation (116). In subsequent reactions, trimer and higher oligomers can be

formed from diaminophenazine. A trimer of o-PDA is known as Bandrowski’s base, which is

extremely mutagenic in reverting strain TA1538 (117). o-PDA can be used to make

tiabendazole, pyrazinamide, morinamide, chemizole, chlormidazole, and other pharmaceuticals

(118). For many applications, o-PDA has been replaced by safer chemicals such as 3, 3’, 5, 5’-

tetramethylbenzidine (119).

vi. m-Phenylenediamine (m-PDA)

m-PDA) is also called 1,3-PDA and is used in the preparation of various polymers

including aramid fibers, epoxy resin, wire aramid coating and polyurea clastomers, and as an

accelerator and for adhesive resins and components of dyes such as Basic Brown, Basic Orange

2, Direct Black 38, and Developed Black BH. It also used as a coupling agent in hair-dye. m-

PDA is used in large quantities in the United States (120). Because no data are available for

humans, and there is inadequate evidence of carcinogenicity in animals,

m-PDA is not considered as a human carcinogen. But the oxidation products are highly

mutagenic (91)

vii. Others

As the author postulated the nitro-group containing monocyclic aromatic amines are direct

mutagens, but 4-amino-3-nitro-6-isopropylaniline, 4-amino-3-nitro-5β-hydroxymethylaniline, 4-

amino-3-nitro-5-methylaniline, 4-amino-3-nitro-5β-hydroxypropylanilne, 4-amino-3-nitro-5-

isopropylaniline, 4-amino-3-nitro-5, 6-dimethylaniline, and 4-amino-3-nitro-2,5-dimethylaniline

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are containing nitro-group in the structure but still require S9 activation for mutagenic activity. It

is possible that the number and position of amino and/or nitro groups are crucial for their

mutagenicities (106).

Several parameters such as lowest unoccupied molecular orbital energy (Elumo), highest

occupied orbital energy (Ehomo), and hydrophobicity are important. However, what factors

determine the minimum requirement for the compound to be mutagenic and what factors

determine the extent of mutagenicity are not known (106). Since mutagenicity and

carcinogenicity are intimately related, those mutagenic monocyclic aromatic amines (MAAs) are

likely to be carcinogenic.

ALLERGENICITY

Besides the use as a hair dye component, p-PDA is used in engineering polymers and

composites (121). The Center of Disease Control (CDC) of the United States lists p-PDA as a

contact allergen. p-PDA induced throat irritation (pharynx and larynx), bronchial asthma,

and/or sensitization dermatitis {(NIOSH, Registry of Toxic Effects of Chemical Substances

(RTECS) entry for p- PDA.} Likewise, the potential effects of the o-phenylenediamine may

cause eye irritation, skin irritation, dermatitis, and an allergic reaction. Another malady is

methemoglobinemia, which is characterized by dizziness, drowsiness, headache, shortness of

breath, cyanosis, rapid heart rate, chocolate-brown colored blood, and liver damage.

m-PDA may cause sensitization reactions, eye irritation and injury, skin irritation, dermatitis,

blackened skin, and bronchial asthma. Other symptoms include allergic skin reactions, irritation

of mucous membranes, coughing, burning sensation, runny nose, sore throat,

methemoglobinemia, cyanosis, headache, dizziness, drowsiness, mental confusion, pulmonary

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edema, kidney and liver damage, central nervous system effects, and conjunctivitis. Eye contact

may cause discomfort, tearing, blurring of vision, reddening, partial clouding of the corneas, and

swelling of the eye and surrounding tissues. Exposure may also result in mucous membrane and

respiratory tract irritation. When this compound is heated, it will decompose to oxides of

nitrogen. Some workers 30 to 50 years old who were exposed to m-PDA for 5-10 years

complained of dysuria. A scratch test with m-PDA produced positive reactions in 8 % of the

people who also suffered from eosinophiluria and had urinary m-PDA levels of 0.3 to 40 μg/100

ml. Cystoscopy revealed edema of the mucosa, polypous swellings and infiltration of the area of

triangle and cervix of urinary bladder. Effects were also observed in people exposed to

hyperreflexia, hyporeflexia, anisoreflexia, skin hyperesthesia and pathological changes in

kidneys and liver. The eosinophilic character of these alterations was confirmed cytologically

(69).

Kleniewska and Maibach (122) studied the allergenicity of 16 aminobenzenes

including p-PDA, p-toluidine, p-sulfanilic acid, p-aminobenzoic acid, and p-nitroaniline.

Their structure-function relationships using sensitizations for 24 occlusive patches in guinea

pigs were examined. Activating chemical groups -NH2 ,-CH3 and -OH were more potent

sensitizers than compounds with deactivating groups -SO3H, -COOH, and -NO2.

Benzidine is acutely toxic to humans by ingestion. Symptoms include cyanosis,

headache, mental confusion, nausea, and vertigo. Dermal exposure may cause skin

rashes and irritation as well as bladder injury (69, 123, 124). Exposure effects on the blood,

liver, kidney, and central nervous system from oral exposure of benzidine to animals have

been reported (123).

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4-Aminoazobenzene is present in yellow pigments it is also found in insecticides

lacquers, varnishes, waxes, oil stains, and pyrotechnics for yellow smoke, It can cause skin

allergenic reaction. Symtoms of exposure to 4-aminobiphenyl include redness, swelling,

itching, and fluid-filled blisters.

o-Aminoazotoluene can cause eczema of the hands and arms. When this compound is

heated to decomposition, o-aminoazotoluene emits toxic fumes of carbon monoxide, carbon

dioxide, and nitrogen oxides (125).

Exposure to Methyl Yellow through inhalation, skin absorption, ingestion, and/or skin

contact may induce an enlarged liver, kidney disturbance, contact dermatitis, cough, wheezing,

dyspnea, bloody sputum, bronchial secretions, frequent urination, and hematuria. 1-Amino-2-

napththol-4-sulfonic acid may cause eye and skin irritation as well as gastrointestinal

disturbance.

Tartrazine is a certified food color, mainly yellow, and can cause allergies to humans.

Human exposure to tartrazine is usually by ingestion or cutaneous contact. Symptoms appear

after a period of time ranging from a few minutes to 14 hours. Approximately 360,000

Americans, less than 0.12% of the general population (126), are affected by tartrazine.

According to the FDA, tartrazine causes hives in fewer than 1 in 10,000 people, or 0.01%

(127). It is not clear how many individuals are sensitive or intolerant to tartrazine, but the

University of Guelph estimates that it is one to 10 out of 10, 000 people in Canada. The

advice to deal with tartrazine sensitivity is to avoid tartarazine totally (128).

There is no evidence that it had an effect on most people with asthma (130). McCann, et

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al. (130) reported that a mixture of tartrazine, Ponceau 4R (E124), Sunset Yellow FCF,

carmoisine, and sodium benzoate may cause hyperactivity in children. However, an

independent review of the study concluded that the clinical significance of these observations

remained unclear (131). In 1994, Rowe and Rowe conducted a study at the University of

Melbourne and suggested that children previously identified as hyperactive might exhibit an

increase in irritability, restlessness, and sleep disturbance after ingesting tartrazine (132).

Tartrazine is a permitted food coloring in Canada because Canada found that existing

scientific evidence does not show that this synthetic food color is unsafe in the general

population (133). The European Food Safety Authority allows for tartrazine to be

used in processed cheese, canned fruit or vegetables, processed fish, or fishery

products, and wine-based drinks (134). The use of tartrazine was banned in Norway and

Austria but the ban was overturned by a European Union directive (135).

Sulfanilamide, the metabolite of Prontosil studied by Gerhardt Domagk is a sulfonamide

drug (sulfur drug) that is the basis of several groups of drugs including child antibacterial

pediazole, antimicrobial sulfacetamide, sulfadiazine, sulfadimidine, sulfafurazole sulfisomidine,

sulfadoxine, sulfamethoxazole, sulfamoxole, sulfadimethoxine antidiabetic agents sulfonylureas,

diuretics, anticonvulsants, dermatologicals, and antiretrovirals (136) (List 1). Although these

synthesized sulfur drugs were not generated from azo dyes any more, there are basically

aromatic amines. Maladies include urinary tract disorders, haemopoietic discorders, porphyria,

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and hypersensitivity reactions when taking these drugs. Allergies to sulfonamide are common.

The most common symtoms are rash and hives as well as life-threatening manifestations of

hypersensitivity including Stevens–Johnson syndrome, toxic epidermal necrolysis (also known

as Lyell syndrome) agranulocytosis, lymolytic anemia, thrombocytopenia, fulminant hepatic

necrosis and acute pancreatitis are reported (136, 137)

2-Amino-p-cresol, a metabolite produced from azo dye Disperse Yellow was discovered

by Stahlmann, et al. (138) to be a strong allergen causing a marked increase in lymph node

weight and cell proliferation, accompanied by a relative decrease of T-cells and relative

increases in B-cell and IA cells in a modified local lymph node assay protocols in NMRI

mice. On the other hand, the other metabolite of Disperse Yellow, 3, 4-aminoacetanilide

led to an increase in lymph node weight and cellularity at a higher concentration of 30%,

with no consistent changes in the phenotypic analysis, indicating that 4-aminoacetanilide was

a weak sensitizer.

Disperse Blue 106 and Disperse Blue 124 have been shown to cause an allergic contact

dermatitis to a variety of garments, which include underwear, blouses, pants, swimming suits,

pantyhose, shoulder pads, and materials used of leggings and body suits (139-14). Exposure to

consumers can prove fatal when these chemicals come in contact with the skin as they might

generate incurable diseases .

OTHER MALADIES.

Trypan Blue has been shown to be carcinogenic and teratogenic (142). The reduced

amaranth (FD & C Red #2) and reduced Sunset Yellow (FD & C Yellow # 6) were reported to

induce cytoxicity when incubated with repair deficient Escherichia coli strain in the absence of

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hepatic enzymes (143). Amaranth was banned to use in food in the USA in 1976 but is still a

certified food colorant for other countries. Sunset Yellow is a certified food color. Various

genotoxic related illnesses of workers in textile dyeing plants have been frequently reported in

various countries (144). Yoshida and Miyakawa (145) disclosed that occupational exposure to

benzidine dyes (for kimoto painting) might have possibly resulted in bladder cancer among

kimoto painters in Japan. Usha reported (146) that eczema, contact dermatitis, ashma, chronic

bronchitis, tuberculosis, hematoma, bladder cancer, and irritation to eyes were common among

workers of textile industries in Sanganer, India. Pelclova, et al. (147) also reported that a high

incidence of chromosomal aberrations in 42 rotogravure printing plant workers. Morikawa, et al.

(148) reported triple primary cancers including kidney, urinary bladder, and liver in dye workers.

There may be more instances of multiple diseases related to workers in azo dye industries that

have not been reported.

DISCUSSION

It was estimated in the 1980’s that nearly 280,000 tons of textile dyes were annually

discharged into industrial effluents worldwide (149). Azo dyes make up approximately 70% of

weights of all dyestuffs used, the largest group of the most used synthetic dyes released into the

environment (6, 150-152). Microbial decolorization of azo dyes have been extensively reviewed

elsewhere (152). However, these dyes remain difficult to be completely degraded. Residue azo

dyes and their degradation products still damage water quality (152, 153). Degradation products

are toxic to aquatic organisms, allergenic, mutagenic, and carcinogenic to humans and render the

water unfit for its intended use. For example, Bae and Freeman (154) demonstrated that C. I.

Direct Blue 218 was very toxic to daphnids with a 48-h LC50 between 1.90 and 100 mg/L. Azo

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dyes decrease the passage of light penetration and gas dissolution in lakes, rivers, and other

bodies of water. The ecological impact and environmental damage have been reviewed

elsewhere (5, 154, 155); therefore, they are not discussed in this article.

The whole life-cycle of azo dyes in colored clothes is an unavoidable source of human

exposure. The textile fibers are not necessarily allergenic; rather, the dyes used to color the

fabrics or formalin finishing resins added to make them wrinkle-resistant, shrink-proof, or easily

laundered are responsible for direct contact (156). The most common sensitizers include the

Disperse Dye application class of azo dyes, which are loosely held in the fibers and are easily

rubbed off.

CONCLUSION

Azo dyes preceded the discovery of sulfur drug medicine and facilitated the development of

the chemical industry. Azo dyes and their cleaved products, aromatic amines, are carcinogenic,

mutagenic, allergenic, and also cause various human maladies in addition to being harmful

pollutants to our environment. If we can effectively restrict the use of azo dyes and control the

spread of pollution of azo dyes and their toxic aromatic amines in our environment, we can

certainly drastically reduce the incidence of cancer and other relevant human diseases.

Regulation, prevention, and research for industrial substitution are urgently called for by this

author. Further, we should invest more in the study of the mechanisms, remedies, and prevention

of those maladies induced by azo dyes and their metabolites in order to protect our health and

environment

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REFERENCES

1. IUPAC, Compendium of Chemical Terminology, 2nd ed. (the “Gold Book”) 1997. Online

corrected version (2009) “azo compounds”.

2. Mock GH, Freeman H. Dye Application, Manufacture of Dye Intermediates and Dyes. In

Kent and Riegel’s, Handbook of Industrial Chemistry and Biotechnology, 11th ed. Chapter

13. 2007; pp. 449-590.

3. Meyer U. Biodegradation of systemic organic colorants. In Microbial Degradation of

Xenobiotics and Recalcitrant Compounds. FEMS Symp. Vol.12. Lesinger, T., Cook,

A.M.,Hutter, R. M., and Nuesch, J., Eds., Academic Press, New York, 1981: 371.

4. Anon Ecological and Toxicological Association of Dyes and Pigments

Manufacturers, Textile Chemists and Colorist, “German Ban of Use of Certain Azo

Compounds in Some Consumer Goods.ETAD InformationNotice No 6, 1996: 28: 11.-13.

5. Puvaneswari N. Muthukrishnan J. Gunasekkaren P. Toxicity assessment and

microbial degradation of azo dyes. Indian J.Exper Biol. 2006:44: 618-626.

6. Mossvi S. Kher X. Madamar D. Isolation, characterization and decoloration of textile dyes by

a mixed bacterial consortium. Dyes and Pigm. 2007: 7(3):723-729.

7. Hildenbrand S. F. Schmahl FW. Wodarz R. Kimmel R. Dartsch PC. Azo Dyes and

carcinogenic aromatic amines in cell cultures Int. Arch. Occup. Enviro Health.

1999:72(3): M052-M056.

8. Correia VM. Stephenson TS. Judd SJ. Characterization of textile wastewaters - a review.

Environ. Technol 1994:15:917-929.

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9. Domagk G. Ein Beitrag zur Chemotherapie der bakteriellen infektionen.

Dtsch. med. Wochenschr. 1935:61: 250-253.

10. Tréfouël, JT. Nitti F. et D. Bovet D. 1. “Activité du p-aminophénylsulfamide

surlʾinfection streptococcique expérimentar de la souris et du lapin”. C. R. Soc. Biol.

1935:120: 23, novembre, 1935. p. 756.

11. Hörlein, H. Deutsches Reich Patentschrift Nr. 226239, May 18, 1909.

12. Turk JL. Leonard Colebrook: The chemotherapy and control of streptococcal infections.

J Royal Soc Med. 1994: 87 (12): 727–728. PMC 1294976. PMID 7853294.edit

13. Ellis H. "Leonard Colebrook and the treatment of puerperal sepsis". Brit J. Hosp

Medici.(London, England : 2005: 72 (2): 109. PMID 21378618.edit

14. Miller JA. Miller EC. The carcinogenicity of certain derivatives of

p-dimethylaminoazobenzene. J. Exp. Med. 1948: 87(2): 139-156.

15. Chung KT. Fulk GE. Andrews AW. The mutagenicity of Methyl Orange

and metabolites produced by intestinal anaerobes. Mutat. Res. 1978: 58:375-379.

16. Chung KT. Fulk GE. Andrews AW. Mutagenicity testing of some commonly used

dyes. Appl Environ Microbiol. 1981:42:641-648.

17. Miller JA. Miller EC. Finger GC. Further studies on carcinogenicity of dyes related to

4-aminoazobenzene :the requirements for an unsubstituted 2-position. 1957:Cancer Res.

1957: 17:387-398.

18. Chung KT. The significance of azo-reduction in the mutagenesis and carcinogenesis of

azo dyes. Mutat Res. 1983: 114:269-281.

19. Chung KT. Cerniglia CE. Mutagenicity of azo dyes: structure-activity relationships. Mutat.

35
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

Res. 1992: 277: 201-220.

20. Chung KT Stevenson SE. Jr. Cerniglia CE.The reduction of azo dyes by intestinal

microflora. Crit Rev. Microbiol. 1992:18:175-190.

21. Chung KT. Stevens SE. Jr. Degradation of azo dyes by environmental microorganisms

and helminths. Environ Toxicol Chem. 1993:12:2121-2132

22. Platzek T. Lang G. Grohmann G. Gi U-S. Baltes W. Formation of a carcinogenic

aromatic amine from an azo dye by human skin bacteria in vitro. Hum Exper Toxicol.

1999: 19(8):552-559.

23. Stingley RL. Zou, W. Heinze TM. Chen H. Cerniglia CE. Metabolism of azo dyes by

human skin microbiota. J Med Microbiol. 2010: 59:108-114.

24. Feng J. Cerniglia CE. Chen H. Toxicological significance of azo dye metabolism by

human intestinal microflora. Front in Biosc. Elite 2012: 4:568-586.

25. Morokutti A. Lyskowski A. Sollner S. Pointner E. Fitzpatrick TB. Kratky C. Gruber K.

Macheroux P. Structure and function of YcnD from Bacillus subtilis, a flavin-containing

oxidoreductase. Biochem. 2005: 44:13724-33.

26. Ito K. Nakanishi M. Lee WC. Sasaki H. Zenno S. Saigo K. Y. Kitade Y. Tanokura M.

Three-dimensional structure of AzoR from Escherichia coli. An oxidereductase conserved

in microorganisms. J Biol Chem. 2006: 28: 20567-76.

27. Liu ZJ. Chen H. Shaw N., Hopper, SL. Chen L. Chen S. Cerniglia CE. Wang. BC.

Crystal structure of an aerobic FMN-dependent azoreductase (AzoA) from Enterococcus

faecalis. Arch. Biochem. Biophys.2007: 463:68-77.

28. Chen H. Xu H. Kweon O. S. Chen S. Cerniglia CE. .Functional role of tryptophan of

36
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

Enterococcus faecalis azoreductase (AzoA) as resolved by structural and mutational

analysis. Microbiol. 2008:154:2659-67.

29. Wang CJ. Hagemeier C. Rahman N., E. Lowe, E. Noble M. Coughtrie M. Sim E.

Westwood I. Molecular cloning, characterization and ligand-bound structure of an

azoreductase from Pseudomonas aeruginosa. J. Mol. Biol.2007: 373:1213-28.

30. Liger D. Graille M. Zhou CZ. Leulliot N. Quevillon-Cheruel S. Blondeau K. Janin J. van

Tilbeurgh H. Crystal structure and functional characterization of yeast YLR011wp, an

enzyme with NAD(P)H-FMN and ferric iron reductase activities. J Biol Chem. 2004:

279:34890-7

31. Martins MAM. Cardoso MH. Queiroz MJ. Ramalho MT. Campus AMO. Biodegradation

of azo dyes by the yeast Candida zeylanoides in batch aerated cultures. Chemosphere

1999:38: 2456-2460.

32. Douch PGC. Azo and nitro-reductase of the cestode Moniezia expansa. Localization of

the enzyme activities and optimum assay conditions. Xenobi. 1975:5: 773-780.

33. Douch PGC. Blair SSB. The metabolism of foreign compounds in the cestode, Moniezia

expensa , and the nematode Ascaris lumbricoides var suum. Xenobi.1975: 5:279-292.

34. Weber EJ. Iron-mediated reductive transformation-investigation of reaction mechanism.

Environ. Sci. Technol., 1996: 29:1163-1170.

35. Keck EK. Klein Kudlich M. Stolz A. Knackmuss HJ. Mattes. R. Reduction of azo dyes by

redox mediators originating in the naphthalene sulfonic acid degradation pathway of

Sphingomonas sp. strain BN6. Appl Environ Microbiol.1997: 63(9):3684–3690.

36. Nam S. Renganatham V. Non-enzymatic reduction azo-dyes by NADH. Chemospher

37
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

2000:40:351-357.

37. Hong Y-G. Gu JD. Physiology and biochemistry of reduction of azo compounds by

Shewanella strains relevant to electron transport chain. Appl Microbiol Biotechnol. 2010:

88:837-843.

38. Aaron JJ. Oturan MA. New photochemical and electrochemical methods for degradation

of pesticides in aqueous media: environmental application. Tr J Chem. 2001: 25:509-520.

39. Guivarch E. Trevin S. Lahitte C. Oturan MA. Degradation of azo dyes in water by electro-

Fenton process. Environ Chem Lett. 2003:1:38-44.

40. Tantak NP. Chaudhari S. 2006. Degradation of azo dyes by sequential Fenton’s oxidation

and aerobic biological treatment. J. Hazard Materials B. 2006:136 :698-705.

41. Konstantinou IK. Albanis TA. TiO2-assisted photocatalytic degradation of azo dyes in

aqueous solution : kinetic and mechanistic investigations: a review. Appl Catlysis B:

Environmental.2004: 49:1-14.

42. Delclos KB. Tarpley WG. Miller EC. Miller. JA. 4-aminoazobenzene and N, N-dimethyl-

4-aminoazobenzene as equipotent hepatic carcinogens in male C57BL/6 X C3H/He F1

mice and characterization of N-(Deoxyguanosin-8-yl)-4-aminoazobenzene as the major

persistent hepatic DNA-bound dye in these mice. Cancer Res. 1984:44:2540-2550

43. IARC. Some aziridines, N-, S- & O-mustards and selenium. IARC monographs on

the evaluation of the carcinogenic risk of chemicals to man. 1975: 9:1-268.

44. Nelson AA. Woodard G. Tumors of the urinary bladder, gall bladder, and liver in dogs

fed o-aminoazotoluene or p-dimethylaminoazobenzene. J Natl Canc Inst. 1953:13:1497-

1509.

38
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

45. Levine WG. Metabolism of azo dyes: implication for detoxification and activation. Drug

Metab Rev. 1991:23(3/4):253-309,

46. Opie EL. The Pathogenesis of tumors of the liver produced by Butter Yellow. The J.

Exper Med. 1944:80 (3): 231–246.

47. Stiborová M. Martínek V. Rýdlová H. Hodek P. Frei. E. Sudan I is a potential carcinogen

for human: evidence for its metabolic activation and detoxication by human recombinant

cytochrome P450 1A1 and liver microsomes. Cancer Res. 2002: 62 (20): 5678- 84.

48. Refat NA. Ibrahim ZS. Moustafa GG. Sakamoto KQ. Ishizuka M. FujitaS The induction

of cytochrome P450 1A1 by Sudan dyes". J. Biochem. Mol. Toxicol. 2008: 22 (2): 77

49. Han D. Yu M. Knopp D. Nissner R. Wu M. Deng A. Development of highly sensitive and

specific-enzyme-linked immunosorbent assay for detection of Sudan I and food samples.

. Agri Food Chem. 2007:53: 6424-6430.

50. He I.Su Y. Fang B. Shen X. Zeng Z. Liu Y. Determination of Sudan dyes residues in eggs

by liquid chromatography and gas chromatography-mass spectrometry. Anal. Chim. Acta

2007:594:139-146.

51. Calbiani F. Carer, M. Elviri L. Mangia A. Zagnoni I. 2004. Accurate mass measurements

for the confirmation of Sudan azo-dyes in hot chilli products by capillary liquid

chromatography-electrospray tandem quadrupole orthogonal-acceleration time of

flight mass spectrometry. J. Chromatogr. A. 2004: 1058-127-135.

52. Safety Review of the Use of Certain Azo-Dyes in Cosmetic Products adopted by the

SCCNFP SCCNFP/0495/01, final Opinion of the Scientific Committee on Cosmetic

Products an non-Food Products Intended for Consumer Concerning (SCCNFP) during

39
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

the 19th plenary meeting of 27 February, 2002.

53. Delclos KB. Miller EC. Miller JA. Liem A. Sulfuric acid esters as major ultimate

electrophilic and hepatocarcinogenic metabolites of 4-aminoazobenzene and its N-methtyl

derivatives in infant male C57BL/66J x C3H/HeJF1 (B6C3F1) mice. Carcinogenesis

1986:7(2):277-87.

54. Ashby J. Lefevre PA. Callander RD. The possible role of azoreduction in the bacterial

mutagenicity of 4-dimethylaminoazobenzene (DAB) and two of its analogue (6BT and 5I).

Mutat Res. 1983: 116:271-279.

55. IARC, International Agency for Research on Cancer, IARC Monograph on the

evaluation of carcinogenic risk of chemicals to man, some aromatic azo compounds. WHO

monogr. Ser. 1974:8:199-206.

56. Nestmann ER. Kowbel DJ. Wheat JA. Mutagenicity of in Salmonella of used by defense

personnel for detection of liquid chemical warfare agent. Carcinogenesis 1981:2:879-883.

57. Brown MA. De Vito SC. Predicting azo dye toxicity. Crit Rev Environ Sci Technol.1993:

23(3): 249-324.

58. Chung K-T. Carcinogenicity, allergenicity, and lupus-inducibility of arylamines. Front

BioSci. Elite. 2016:29-39.

59. Chung, K-T. Occurrence, uses, and carcinogenicity of arylamines. Front in Biosci.

Elite. 2015:7: 367-393.

60. IARC. IARC Monographs on the Evaluation of carcinogenic Risks to Humans.

Some aromatic amines, organic dyes, and related exposures. 2010: 99:1-692.

40
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

61. Chung K-T. Chen S-C. Claxton LD. Review of Salmonella typhimurium mutagenicity of

benzidine, benzidine analogues, and benzidine based dyes. Mutat Res. 2006:12: 58-76.

62. NTP. Toxicology and carcinogenesis studies of 3, 3’-dimethylbenzidine dihydrochloride

(Cas No. 612-82-) in 344/N rats drinking water studies. Technical Report Series. No 390,

Research Triangle Park, NC. National Toxicology Program, 231pp.Toxicol.1991:1: 475-490.

63. "3, 3'-Dichlorobenzidine". U.S. Environmental Protection Agency, Integrated Risk

Information System. 7 March 2011. Accessed 3, May 2011.

64. U.S. Environmental Protection Agency (EPA). Health Effects Assessment Summary Tables.

FY 1997 Update. Solid Waste and Emergency Response, Office of Emergency and Remedial

Response, Cincinnati, OH. EPA/540/R-97-036.

65. Beyerbach A. Rothman N. Bhatnagar VK. Kashyap R. Sabbioni G. Hemoglobin adducts

in workers exposed to benzidine and azo dyes. Carcinogenesis 2006: 27 (8): 1600-1606.

66. Hoffman D. Djordjevic MV. I. Hoffman I. The changing cigarette. Prevent Med. 1997: 26:

427-434.

67. Chiang T. A. Pei-Fen W. Ying LS. Wang LF. Ko Y-C. Mutagenicity and aromatic amine

content of fumes from heated cooking oil produced in Taiwan. Fd Chem Toxicol. 1999:37(2-

3): 125-134.

68. Richfield-Frantz N. Bailey JE. Jr. Bailey CJ. Determination of unsulfonated aromatic amines

in FD&C Yellow No. 6 by the diazotization and coupling procedure followed by reversed-

phase high-performance liquid chromatography. J. Chromat., 1985:3:109-123.

69. Sittig M. Hanbook of Toxic and harzardous chemicals and carcinogens. 2nd ed.

1985.Noyes Publications, Park Ridge, NJ.

41
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

70. U.S. Department of Health and Human Services. The Newest Report on Carcinogens,1998

Summary. Public Health Service, National Toxicology Program. 1998.

71. Frey A. Meckelein B. Extermest D. Schmidt MA. 2000. A stable and highly

sensitive 3, 3’,5, 5’-tetramethylbenzidine-based substrate reagent for enzyme-linked

immunosorbent assays. J Immunol Methods 2000: 233:47-56.

72. Chung K-T. Chen S-C. Wong T-Y. Li, Y-S. Wei C-I. M. W. Chou MW. Mutagenicity

studies of benzidine and its analogs: Structure-activity relationships. Toxicol Sci. 2000:56

(2): 351–356. doi:10.1093/toxsci/56.2.351. PMID 10910993.

73. Holland VR. Saunders BC. Rose FL.Walpole AL. A safer substitute for benzidine in the

detection of blood. Tetrahedron, 1974:30: 3299-3302.

74 Yang J. Wang H. H. Zhang H. One spot synthesis of silver nanoplates and charge-transfer

complex nanofibers. J Phy Chem. C. 2008:112 (34): 13065–13069.

75. Deshpande SS. Enzyme Immunoassays: From Concept to Product Development. New

York: Chapman & Hall. 1996. p. 169.

76. Prival MJ. Bell SJ. Mitchell VD. Peiperl MD. V. Vaughan VL. Mutagenicity of benzidine

and benzidine-congener dyes and selected monoazo dyes in a modified Salmonella assay.

1984: 136: 33-37.

77. Dillon D. Combes R. Zeiger E. Activation by caecal reduction of the azo dye D and C Red

no.9 to bacterial mutagen. Mutagenesis 1994:9: 295-299.

78. Bonser GM. Clayson DB. Jull JW. The potency of 2-methylcholanthrene relative to other

carcinogens on bladder implantation. Br J Cancer 1963:17(2): 235-241.

79. U. S. EPA Health and Environmental Effects Profile for 1-Amino-2-naphthol and

42
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

1- amino-2-naphtol hydrochloride. 1986. US. Environmental Protection Agency,

Washington, D/ C. EPAS/600/X-87/029(NTISPB89120315).

80. Lin GHY. Solodar WE. Structure-activity relationship studies on the mutagenicity of some

azo dyes in the Salmonella/microsome assay. Mutagenesis 1988:3(4): 311-315

81. Shahin MM. Andrillon P. Goetz N. Bore P. Bugaut A. Kalopissis G. Studies on the

mutagenicity of p-phenylenediamine in Salmonella typhimurium. Mutat Res. 1979:68: 327-

336

82. Shahin MM. Mutagenic evaluation of nitroanilines and nitroaminophenol in Salmonella

typhimurium. Internatl J Cosmet Sci. 1985: 7: 277-289.

83. Shahin M. The importance of analyzing structure-activity relationships in mutagenicity

studies. Mutat Res. 1989: 22:165-221.

84. Chung K-T. Murdock C. Stevens SE. Jr. Li Y-S. Huang TS. Wei C-I. Chou MW.

Mutagenicity and toxicity studies of p-phenylenediamine and its derivatives. Toxicol. Letters

1995:81: 23-32.

85. Watanabe T. Hirayama T. Fukui S. The mutagenic modulating of p-phenylenediamine on

the oxidation of o- or m-phenylenediamine with hydrogen peroxide in the Salmonella test.

Mutat Res. 1990:245: 15-22.

86. Sontag JM. Carcinogenicity of substituted benzenediamine (phenylenedamine) on rats and

mice. J Natl Canc Inst. 1981: 66: 591-602.

87. Rollison DE. Helzlsouer KJ. Pinney SM. Personal hair dye use and cancer: a systematic

literature review and evaluation of exposure assessment in studies published since 1992. J

Toxicol Environ.Health. Part B, Critical reviews 2006: 9 (5): 413–39.

43
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

88. Bolt HM. K. Golk K. The debate on carcinogenicity of permanent hair dyes: new insights.

Crit Rev Toxicol. 2007: 37(6): 521-636.

89. Turesky RJ. Freeman JP. Holland RD. Nestorick DM. Miller DW. Ratnasinghe RL.

Kadlubar FF. Identification of aminobiphenyl derivatives in commercial hair dyes.

Chem Res Toxicol. 2003: 16: 1162-1173.

90. Collier SW. Storm JE. Jr. Bronaugh RL. Reduction of azo dyes during in vitro percutaneous

absorption. Toxico Appl Pharmacol. 1993: 118-73-79.

91. Watanabe T. Hirayama T. Fukui. The mutagenic modulating effect of

p-phenylenediamine on the oxidation of o- or m-phenylenediamine with hydrogen

peroxide in the Salmonella test. Mutat Res. 1990: 245:125-22.

92. Bomhard EM. Herbold BA. Genotoxic activities of aniline and its metabolites and their

relationship to the carcinogenicity of aniline in the spleen of rats. Crit Rev Toxicol. 2005:

35:783-835.

93. Kahl T. Schröder K-W. Lawrence FR. Marshall WJ. Höke H. Jäckh R. "Aniline" in

Ullmann's Encyclopedia of Industrial Chemistry, John Wiley & Sons: New York. 2007.

94. NTP. Toxicology and carcinogenesis studies of p-nitroaniline (CAS No. 100-06-1)

in B6F1 mice (gavage studies). Natl Toxicol Program Tech Rep Ser. 1993:418:1-203.

95. p-Nitroaniline: Evaluation of the carcinogenicity and genotoxicity. The Hague: Health

Council of the Netherlands, publication no. Health Council of the Netherlands

2008/08OSH ISBN 978-90-5549-694-5.

96. Osano O. Oladimeji OO. Kraak MH. Admiraal W. Teratogenic effects of amitraz,

2,4-dimethylaniline, and paraquat on developing frog (Xenopus) embryos. Arch Contam

44
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

Toxicol. 2002: 43: 42-49.

97. Yoshimi N. Sugie S. Iwata H. Niwa K. Mori H. Hasida C. Shimizu H. The genotoxicity

of a variety of aniline derivatives in a DNA repair test with primary cultured rat

hepatocytes. Mutat. Res. 1988: 206: 183-191.

98. Danflora N. The genetic toxicology of ortho-toluidine. Mutat. Res. 1991:258: 207-236.

99. Sellers C. Markowitz S. Reevaluating the carcinogenicity of ortho-toluidine:a new

conclusion and its implications. Regu Toxicol Pharmacol. 1992: 16: 301-317.

100. Anon: Cancer of the bladder among workers in aniline factories. International

Labor Office, Studies and Reports Series F. No.1 1920: Genena, Switzerland.

101. Richter E. Biomonitoring of human exposure to arylamines-historical and future aspects

with special emphasis on ortho-toluidine. Front in Biosci. Elite,2015:7: 305-321.

102. IARC. Ortho-toluidine. IARC Monogr. Eval. Carcinog. Risks Hum. 2000: 77: 267-322.

103. Carreon T. Hein, M. Viet SM. Hanley KW. Ruder AM. Ward EM. Increased bladder

cancer risk among workers exposed to o-toluidine and aniline. A reanalysis. Occup

Environ Med. 2010:67:348-350.

104. EurekAlert. The Global Source for Science News, Public Releaser, 19 July, 2006.

Deutsche Forschungsgemeinschaft. DFG presents. The 2006 List of MAK and BAT

Values List. Focus on health protection during pregnancy. Wiley-VCH Verlag

GmbH, D-69451. Weinheim, Germany.

105. IARC. International Agency for Research on Cancer: some aromatic amines organic

Dyes and related exposures. IARC Monographs on the Evaluation of Carcinogenic Risks

to Humans. 2010:1-692. Lyon, France.

45
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

106. Chung K-T. Kirkovsaky L. Kirkovsky A. Purcell WP. Review of mutagenicity of

monocyclic aromatic amines: quantitative structure-activity relationships. Mutat. Res.

1997: 387:1-16.

107. Morton LO. Youssef AF. Lloyd E. Kiopes AL. Goldsworth TL. Fort FL. Evaluation of

carcinogenic responses in the Eker rat following short-term exposure to selected

nephrotoxins and carcinogens. Toxicol Pathol. 2002:30 (5): 559-564.

108. National Toxicology Program (NTP). Abstract for TR-169-2-Nitro-p-phenlenediamine

(CASRN 5307-14-2), Reported date, 1979. Bioassay of 2-Nitro-p-phenylenediamine for

possible carcinogenicity (CAS No. 5307-14-2).

109. Commission of the European Communitites (1991) Commission Directive 91/184/EEC

of 12 March 1991. Off 1 Eu Commun. 1991: L91: 59-62.

110. NTP toxicology and carcinogenesis studies of 2-amino-4-nitrophenol (Cas No. 99-57-0)

in F344/N rats and B6C3F1 mice (Gavage studies). Natl. Toxicol Program Tech Rep Ser.

1988. 339: 1-170.

111. IARC. Some Aromatic Amines, Anthraquinones and Nitroso Compounds and Inorganic

Fluorides Used in Drinking-water and Dental Preparations. Monographs on the

Evaluation of the Carcinogenic Risk of Chemicals to Humans. Lyon, France.

1982:27:103–117.

112. Bioassay of 2, 4-diaminoanisole sulfate for possible carcinogenicity Tech Rep Ser No.

84; DHEW Publ. No. (NIH) 1978: 78-1334. National Cancer Institute. Washington DC,

Government Printing Office.

113. IARC. Monographs on the Evaluation of Carcinogenic Risks to Humans. Some

46
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

Thyrotropic Agents. Lyon, France. 2001:79:1-780

114. Matsumoto M. Suzuki M. Kano M. Aiso S. Yamazaki K. Fukushima S.

Carcinogenicity of ortho-phenylenediamine dihydrochloride in rats and mice by

two-year drinking water treatment. Arch Toxicol. 2012: 86 (5): 791-804.

115. Gosselin EE. Smith RP. Hodge HC. Clinical Toxicology of Commercial Products. 5th

ed. Baltimore: Williams and Wilkins. 1984:.P-11-210.

116. Srrift AF. Arce GT. Krahn DF. O’Neil RM. Reynolds VL. Evaluation of carbendazime

for gene mutations in the Salmonella/Ames plate incorporation assay: the role of

aminophenazine impurities. Mutat Res.1994: 321: 43-56.

117. Ames BN. Kammen H. Yamasaki E. Hair dyes are mutagenic identification of a variety

of mutagenic ingredients. Proc Natl Acad Sci. U.S.A. 1975:72: 2423-2427.

118. Renault J. Baron M. Mailliet P. Giorgirenaul S. Paoletti C. Cros S. Heterocyclic Quinones

2. Quinoxaline-5, 6-(and 5, 8-)-diones-potential antitumoral Agents. Eur J Med Chem.

1981:16: 545-550.

119. Deshpande SS. Enzyme Immunoassays: From Concept to Product Development. New

York: Chapman & Hall. 1996:P 169. ISBN 978-0-412-05601-7.

120. 1, 3-Benzenediamine from HSDB 5384 NIH. U.S. National Library of Medicine Toxinet.

DataBasehttp://toxnet.nih/gov.gov/cgi.bin/sis.search/r?dbs+hsdb:@tem+@rel+108- 45-2

121. Thyssen JP. White JM and European Society of Contact Dermatitis. Epidemiological

data on consumer allergy to p-phenylenediamine. Contact Dermat. 2008:59: 327-343.

122. Kleniewska, D. Maibach H. 1980. Allergenicity of aminobenzene compounds:structure-

function relationships. Derm Beruf Umwelt. 1980:28: 11-13.

47
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

123. ATSDR. Agency for Toxic Substances and Disease Registry. Toxicological Profile for

Benzidine. U.S. Public Health Service, U.S. Department of Health and Human Services,

Atlanta, GA. 1995.

124. U.S. Environmental Protection Agency. Integrated Risk Information System (IRIS)

on Benzidine .National Center for Environmental Assessment, Office of Research and

Development, Washington, DC. 1999.

125. NTP. .Toxicology and Carcinogenesis Studies of Naphthalene (Cas No. 91-20-3) in

B6C3F1 Mice (inhalation studies). U.S. Department of Health and Human Services

Public Service, National Institutes of Health Technical Report Series 1992:No. 410.

126. Elhkim MO. Héraud F. Bemrah N. Gauchard F. Lorino T. Lambre C. Fre’my JM. Poul

JM. New considerations regarding the risk assessment on Tartrazine: An update

toxicological assessment, intolerance reactionsand maximum theoretical daily intake in

France. Reg Toxicol. Pharmacol. 2007:47 (3): 308–316.

127. United States Food and Drug Administration. “Does FDA& C Yellow No. 5 cause

any allergic reactions?” Archived from the original on 2007-10-09. Retrieved 2007-10-

20.

128. Dipalma JR. Tartrazine sensitivity. American Family Physician. 1990:42 (5): 1347-50.

129. USA. "Tartrazine exclusion for allergic... [Cochrane Database Syst Rev. 2001] .

Ncbi.nlm.nih.gov. 2014-01-24. Retrieved 2014-02-07.

130. McCann D. Barrett A. Cooper A. Crumpler D. Dalen L. Grimshaw K. Kitchin E. Lok K.

Porteous L. Prince E. Sonuga-Barke E. Warner JO. Stevenson J Food additives and

hyperactive behavior in 3-year old and 8/9-year-old children in the community: a

48
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

randomized, double-blinded, placebo-controlled trial. The Lancet 2007:370 (9598):

1560–1567.

131. EFSA. Assessment of the results of the study by McCann et al. (2007) on the effect

of some colours and sodium benzoate on children’s behaviour. The EFSA J 2008:

660:1-53.

132. Rowe KS. Rowe KJ. Synthetic food coloring and behavior: a dose response in a double-

blind, placebo controlled, repeated-measures study. J. Pediatri. 1994:12: 691- 698.

133. Table III of section B.16.100

(http://law.lois.justice.gc.ca/eng/regulations/C.R.C%2C_c.870/page158.html#docCont).

Food Drug Regulations.

134. Further details can be found on the EFSA food additives database page on tartrazine.

(http://webgate:ec.europa.eu/sanco_foods/main/?event=substance.view&identifier+7

135. EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) (November

2009). "Scientific Opinion on the re-evaluation Tartrazine (E 102)". In European Food

Safety Authority. EFSA Journal (European Food Safety Authority) 2009:7 (11): 1331–

1382. doi:10.2903/j.efsa.2009.1331. Retrieved 2011-10-09. "The Panel concludes that

The present dataset does not give reason to revise the ADI of 7.5 mg/kg bw/day."

136. "Sulfa Drugs Allergy - Sulfa Bactrim Drug Allergies". Allergies.about.com. Retrieved

17, January 2014.

137. Harrison's Principles of Internal Medicine, 13th Ed. McGraw-Hill Inc. 1994: 604.

138. Stahlmann R. Wegner M. Riecke K. Kruse M. T. Platzek T. Sensitizing potential of four

textile dyes and some of their metabolites in a modified local lymph node assay. Toxicol.

49
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT

2006:219(1-3): 113-123.

139. Joe EK. Allergic contact dermatitis to textile dyes. Dermat Online J. 2007:7(1): 9.

140. Mathur N. Bhatnagar P. Sharma P. Review of the mutagenicity of textile products.

Universal J Environ ResTechnol. 2012:2(2):1-18.

141. Pratt M. Taraska V. Disperse blue dyes 106 and 124 are common causes of textile

dermatitis and should serve as screening allergens for this condition. Am J Contact

Dermat. 2000:11: 30-41.

142. Hartman CP. Fulk GE. Andrews AW. Azo reduction of trypan blue a known carcinogen

by a cell-free extract of a human intestinal anaerobes. Mutat Res. 1978: 58(2-3): 125-

132.

143. Sweeney EA. Chipman JK. Forsythe SJ. Evidence for direct-acting oxidative

genotoxicity by reduction products of azo dyes. Environ Health Perspect. 1994:102:

119-122.

144. Dönbak L. Rencüzogullari E. Topaktas M. Sahin G. A biomonitoring study on the

workers from textile dyeing plants. Russian J. Genet. 2006:42: 613-618.

145. Yoshida O. Miyakawa M. Etiology of bladder cancer: Metabolic aspects. In:Analytical

and Experimental Epidemiology of Cancer” Proceedings of the Third International

Symposium on the Princess Takmutsu Cancer Research Fund Japan.1973.

146. Usha M. Impact analysis of industries in Sanganer. P. G. Diploma Field Study Report

Submitted to Indira Gandhi Center for HEEPS, University of Rajasthan, Jaipur

India.1989.

147. Pelclova D. Rossner P. Pickova J. Chromosome aberrations in rotogravure printing plant

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workers. Mutat Res. 1990: 245:299-303.

148. Moirkawa Y. Shiomi K. Ishihara Y. Matsuura N. Triple primary cancers involving

kidney, urinary bladder and liver in a dye workers. Am J Indus Med. 1997:3:44-49.

149. Jin XC. Liu GQ. Xu ZH. Tao WY. Decolourization of a dye effluent by Aspergillus

fumigatus XC6. Appl Microbiol Biotechnol. 2007:74: 239-243.

150. Saratable RC. Saratable JG. Chang DS. Govindwar SP. Bacterial decolorization and

degradation of azo dyes: a review. J. Taiwan Inst Chem Engineers 2011:42(1)138-157.

151.Kusic H. Juretic D. Koprivance N. Marion V. Božié AL. Photooxidation processes for

Azo dye in aqueous media: Modeling of degradation kinetics and ecological parameters

evaluation. J Hazard Material 2011:185(2-3): 1558-1568. ISSN 0304-3894.

152. Pandy, A., P. Singh and L. Lyengaer L. Bacterial decolorization and degradation of

azo dyes. Internat Biodeter Biodegrad. 2007: 59:73-84.

153. Banat IM. Nigam P. Singh D. Marchant R. Microbial decolorization of textile dye

decontaining effluents: a review. Biosources Technol. 1996: 58(3):217-227.

154. Bae JS. Freemann HS. Aquartic toxicity evaluation of new direct dyes to the Daphnia

magna. Dyes Pigment. 2007:73(11): 1937-1945.

155. Chequer FMD. Dorta DJ. deOlivera DP. 2011. Azo dyes and their metabolites: does the

discharge of the azo dye into water bodies represent human and ecological risks? In

Advances in Treating Textile Effluent. Prof. Peter Hauser (ed.) 2011:1-23.

Available from http://www.intechopen.com/books/advances-treating-textile-

effulent/azo-dyes-and-their metabolites.does-the-discharge-of-the azo-dye-into-water-

bodies-the represent-human and ecological risk

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156 Chang JS. Chou C. Chen SY. Decolorization of azo dyes with immobilized

Psudomonas luteola. Process Biochem. 2001: 36(8-9):757-763.

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Table. 1. Examples of Aromatic Amines Metabolically Produced from Azo Dyes

Names of aromatic amines Sources of Azo Dyes

4-Aminobenzenesulfonic acid Ponceau BS, Methyl Orange, Orange II,

Ponceau S

1-Amino-2-Naphthol Acid Red 88, Orange II, Para Red,Ponceau BS

, Lithol

Red, 1-Phenylazonaphthol

Aniline Orange G, 1-Phenylazo-2-naphthol

4-Aminoazobenzene, Methyl Yellow

Benzidine Congo Red, Direct Blue 6, Direct Black 38,

Direct Brown 95

2,5-Diaminobenzenesulfonic acid Ponceau BS, Ponceau S

2,4-Dimethylaniline 1-[2, 4-(Dimethylphenyl)azo]-2-naphthalenol

N, N-Dimethyl-p-phenelenediamine (p-PDA) Methyl Orange, Methyl Red, Methyl Yellow

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p-Nitroaniline Para Red

p-Phenylenediamine (p-PDA) 4’-Hydroxy-4-aminoazobenzene, 4-

Aminoazobenzene

4’-Hydroxy-4-monomethylaminoazbenzene,

Sudan IV

Sulphanilinic Sunset Yellow, Tartrazine

Toluidine 1-[2-Methyl-4-[(2-

methylphenyl)azo]phenylazo-2-

naphthaleneol

2, 4, 5-Trimethyaniline Ponceau 3 R

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___

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List. 1. Examples of Sulfonamides (Sufur Drugs)________________________

Pediazole, Sulacetyamide, Sulfadiazine, Sulfadimidine, Sulfurazole, Sulfisomidine

(aka sulfaisodimidine), Sulfadoxine, Sulamethoxazole, Sulfamoxole, Sufanitrane

Sulfadimethoxine, Sulfamethoxypyridazone, Sulfmetoxydiazine. Sulfadoxine,

Sulfamedopyrazine, Acetohexamide, Carbutamide, Chlorpropamide, Glibenclamide (also known

as Glyburide), Glibormuride, Gliclazide, Glyclopyramidine, Glimepiride, Gliprizide.

Gliquidone, Glisoxepide ,Tolazamide, Tolbutamide. Acetazolamide, Bumetanide,Chlorthalidone,

Clopamide, Dorzolamide, Flurosemide, Indapamide, Hydrochlorothiazide (HCT, HCTZ, HZT),

Mefruside, Metolazone, Xipamide. Ethoxazolamide, Sultiame, Topiramate,

Zonisamide.Mafenide, Antiretrovirals, Amprenavir, Durunnavir, Delavirdine,(non-nucleoside

reverse transcriptas inhibitor), Fosamprenavir (protease inhibitor), Tipranavir (Protease

inhibitor). Stimulant, Azabon, Apricoxib (COX-2 inbitor), Bosentan (endothelin receptor

antagonist), Celecoxib (COX-2inhibitor), Dofetilide (class III antiarrthy themic), Dronedarone

(Class III antiarrthythemic), Ibutilide ( Class III antiarrthythemic), Parecoxib (COX-2 inhibitor),

Probenecid (PBN), Sotalol (beta blocker), Sulfasalazine (SSZ), Sumatriptan (alpha blocker),

Udenafil (PDE5 inhibitor).

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List. 2. Reported Intestinal Microorganisms with Azo Reduction Activity

___________________________________________________________________________

Acidaminonococcus fermentans, Acerobacter aerogenes, Bacteriodes vulgatus, Bacteroides

distasonis, Bacteroides fragilis, Bacteroides ovatus , Bacteroides thetaiotaomicron,

Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium infantis,

Butyrivibrio sp., Citrobacter sp., Clostridium nexile, Clostridium clostridiiforme, Clostridium

paraputrificum, Clostridium ramosum, Clostridium perfringens, Clostridium difficile,

Coprococcus catus, Enterococcus faecalis, Escherichia coli, Eubacterium sp., Eubacterium

aerofaciens, Eubacterium biforme, Eubacterium hadrum, Fusobacterium prausnitzii,

Fusobacterium sp., Lactobacilluscatenaforme, Peptostreptococcus productus, Pneumococcus

sp., Proteus vulgaris,Proteus sp., Pseudomonas aeruginosa, Pseudomonas pyocyanea,

Rumonococcus bromii, Salmonella paratyphi, Salmonella typhimurium, Shigella dysenteriae,

Staphylococcus aureus, Streptococcus faecalis, Streptococcus haemolyticus, Veillonella parvula,

etc. (20, 21).

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List. 3. Azo dyes whose Carcinogenicities are not due to their Cleaved

Products

4-Aminoazobenzene, o-Aminoazotoluene, 3’-Methyl-4-monomethylaminoazobenzene,

Methyl Yellow, 3’-Methyl-4-dimethylaminoazobenzene, 4-Ethylmethylaminoazobenezene,

Sudan I, Sudan II, Sudan III, Sudan IV, Para Red, Ponceau 3R, , Orasol Navy Blue 2RB,

6-(p-Dimethylaminophenyl)azobenzothiazole3’-Nitro-4-dimethylaminoazobenzene,

2’-Nitro-4-dimethylaminoazobenzene, 4’-Chloro-4-dimethylaminoazobenzene, 3’-Chloro-4-

dimethylaminoazobenzene. Thiodiphenyl-4, 4’-diazobissalicyclic Acid

________________________________________________________________

List. 4. Azo Dyes listed as Carcinogens

___________________________________________________________________________Sol

vent Yellow 1 (Cas No. 60-09-3, also called p-(phenylazo)aniline; p-aminoazobenzene; Solvent

Yellow 2 (Cas No. 60-11-7), also called 4-(dimethylamino)azobenzol; Solvent Yellow 3 (Cas

No. 97-56-3); Pigment Orange 5 (Cas No. 3468-63-1); Solvent Orange 2 (Cas No. 2646-17-5);

Pigment Red 3 (Cas No. 24525-85-6); Solvent Red 80 (Cas No. 6358-53-8), commonly called

Citrus Red 2; Pigment Red 53 (Cas No. 2092-56-0), also called D&C Red No. 8, Pigment 53:1,

barium salt (Cas No. 5160-02-1); Acid Red 26 (Cas No. 3761-53-3), also called Ponceau 26;

xylidine; Ponceau 2R; Acid Dye (Cas No. 3564-09-8), also called Ponceau 3R; Direct Red 28

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(Cas. No. 573-58-0), also called Congo Red; Direct Blue 6 (Cas No. 2602-46-2); Acid Red 114

(Cas No. 6459-94-5); Direct Blue 14 (Cas No. 72-57-1); Direct Blue 53 (Cas No. 314-13-6);

Direct Blue 15 (Cas No. 2429-74-5); Direct Blue 218 (Cas No.28407-37-6); Direct Brown 95

(Cas No. 16071-86-6); Direct Black 38 (Cas No.1937-37-7); Basic Red 9 (Cas No. 479-73-2);

Basic Red 9 hydrochloride (Cas No. 569-61-9); Disperse Blue 1 (Cas No. 2475-45-8); Pigment

Yellow 34 (Cas No. 1344-37-2); and Pigment Red 104 (Cas No. 1256-85-8).

_______________________________________________________________

List. 5. Examples of Azo Dyes that Released Benzidine after Azo Reduction

___________________________________________________________________________

Acid Black 29, Acid Black 232, Acid Black 94, Acid Orange 45, Acid Red 85, Azoic Diazo

Component 112, Direct Black 4, Direct Black 29, Direct Black 38, Direct Blue 2, Direct Blue

6, Direct Brown 1, Direct Brown 1:2, Direct Brown 2, Direct Briwn 6, Direct Brown 25,

Direct Brown 27, Direct Brown 31, Direct Brown 33, Direct Brown 51, Direct Brown 59,

Direct Brown 74, Direct Brown 79, Direct Brown 95, Direct Brown 101, Direct Brown 154,

Direct Dye, Direct Green 1, Direct Green 6, Direct Green 8, Direct Green 8:1, Direct Orange

1, Direct Orange 8, Direct Red 1, Direct Red 10, Direct Red 13, Direct Red, Direct Red 28,

Direct Red 37, Direct Violtet 1, Direct Violet 4, Direct Violet 12, Direct Violet 22, Direct

Yellow 1, Direct Yellow 24, Mordant Red 57, Direct Red 44.

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Figure. 1. Chemical Structures of Prontosil and Sulfanilamide

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Figure. 2. Chemical structures of Methyl Yellow (p-Dimethylaminoazobenzine) and its

Metabolites

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