2,4-DIAMINOANISOLE AND ITS SALTS

This substance was considered by previous working groups, in 1977 (IARC,

1978), 1981 (IARC, 1982) and 1987 (IARC, 1987). Since that time, new data have
become available, and these have been incorporated into the monograph and taken into
consideration in the present evaluation.

1. Exposure Data

1.1 Chemical and physical data

1.1.1 Nomenclature
2,4-Diaminoanisole

Chem. Abstr. Serv. Reg. No.: 615-05-4

Chem. Abstr. Name: 4-Methoxy-1,3-benzenediamine
IUPAC Systematic Name: 4-Methoxy-meta-phenylenediamine
Synonyms: 3-Amino-4-methoxyaniline; C.I. 76050; C.I. Oxidation Base 12; 1,3-
diamino-4-methoxybenzene; 4-methoxy-1,3-phenylenediamine; para-methoxy-
meta-phenylenediamine
2,4-Diaminoanisole sulfate

Chem. Abstr. Serv. Reg. No.: 39156-41-7

Chem. Abstr. Name: 4-Methoxy-1,3-benzenediamine, sulfate
IUPAC Systematic Name: 4-Methoxy-meta-phenylenediamine, sulfate
Synonyms: CI 76051; CI oxidation base 12A; 2,4-DAA sulfate; 2,4-diaminoanisole,
hydrogen sulfate; 2,4-diaminoanisole sulphate; 1,3-diamino-4-methoxybenzene
sulphate; 2,4-diamino-1-methoxybenzene sulphate; 4-methoxy-1,3-benzenediamine
sulfate (1:1); 4-methoxy-1,3-benzenediamine sulphate; 4-methoxy-meta-phenylene-
diamine sulfate; para-methoxy-meta-phenylenediamine sulphate; 4-methoxy-meta-
phenylenediammonium sulphate

–621–
622 IARC MONOGRAPHS VOLUME 79

2,4-Diaminoanisole dihydrochloride

Chem. Abstr. Serv. Reg. No.: 614-94-8

Chem. Abstr. Name: 4-Methoxy-1,3-benzenediamine, dihydrochloride
IUPAC Systematic Name: 4-Methoxy-meta-phenylenediamine, dihydrochloride
Synonyms: 2,4-Diaminoanisole hydrochloride

1.1.2 Structural and molecular formulae and relative molecular masses

OCH3

NH2

NH2

C7H10N2O Relative molecular mass: 138.17

OCH3

NH2 OH
O S O

OH
NH2

C7H10N2O.H2SO4 Relative molecular mass: 236.25

OCH3

NH2

2HCl

NH2

C7H10N2O.2HCl Relative molecular mass: 211.07

1.1.3 Chemical and physical properties of the pure substances

2,4-Diaminoanisole

(a) Description: Needles from diethyl ether (Budavari, 2000)

(b) Melting-point: 67.5 °C (Lide & Milne, 1996)
(c) Spectroscopy data: Infrared [prism (2971), grating (15399)], ultraviolet (855)
and nuclear magnetic resonance [proton (10617)], and mass spectral data have
been reported (Sadtler Research Laboratories, 1980; Lide & Milne, 1996).
 2,4-DIAMINOANISOLE AND ITS SALTS 623

(d) Solubility: Soluble in diethyl ether and ethanol (Lide & Milne, 1996)
(e) Stability: Darkens on exposure to light (Budavari, 2000)
2,4-Diaminoanisole sulfate

(a) Description: Off-white to violet powder (Budavari, 2000)

(b) Solubility: Soluble in water and ethanol (Budavari, 2000)
(c) Spectroscopy data: Infrared [prism/grating (51021)], ultraviolet (26381) and
nuclear magnetic resonance [proton (23809)] data have been reported
(Sadtler Research Laboratories, 1980).
2,4-Diaminoanisole dihydrochloride

(a) Description: Crystalline powder (TCI America, 2000)

(b) Solubility: Soluble in water (TCI America, 2000)

1.1.4 Technical products and impurities

Trade names for 2,4-diaminoanisole include Furro L, Pelagol DA, Pelagol Grey L
and Pelagol L.
Trade names for 2,4-diaminoanisole sulfate include BASF Ursol SLA, Durafur
Brown MN, Fouramine BA, Fourrine 76, Fourrine SLA, Furro SLA, Nako TSA,
Pelagol BA, Pelagol Grey, Pelagol Grey SLA, Pelagol SLA, Renal SLA, Ursol SLA
and Zoba SLE.

1.1.5 Analysis
Methods for the analysis of aromatic amines, including 2,4-diaminoanisole, in inks
of ball-point and fibre-tip pens and watercolour paints, oxidative hair dyes, dyestuff
mixtures and in paper, coloured textiles and leather products have been reported. These
methods include differential pulse voltammetry, gas chromatography–mass spectro-
metry with mass ion detection, thin-layer chromatography, high-performance thin-layer
chromatography and high-performance liquid chromatography with ultraviolet, diode-
array or mass spectrometry detection (Bernabei et al., 1980; Johansson et al., 1981;
Liem & Rooselaar, 1981; Mancini et al., 1981; Gottschalck & Machens, 1982; Ohshima
et al., 1982; Hoogewijs & Massart, 1983; Sardas et al., 1985; Andrisano et al., 1994,
1995; Friedrichs et al., 1995; Verdú et al., 1996; Winkeler, 1996; Bürgi et al., 1997;
Verdú et al., 1997; Anon., 1998a,b; Bürgi et al., 1998; Chen et al., 1998; Mayer et al.,
1998; Planelles et al., 1998; Štancer & Jeretin, 1998; Tomaselli et al., 1998; Wang &
Chen, 1998; Cioni et al., 1999; Kellert et al., 1999; Planelles et al., 1999; Sinha &
Kumar, 1999; Xiao et al., 1999; Yang et al., 2000).
624 IARC MONOGRAPHS VOLUME 79

1.2 Production

2,4-Diaminoanisole was first prepared in 1913 by the reduction of 2,4-dinitro-

anisole with iron and acetic acid (Richter, 1933; Budavari, 2000).
Information available in 2000 indicated that 2,4-diaminoanisole sulfate was
manufactured by one company in China (CIS Information Services, 2000).

1.3 Use

2,4-Diaminoanisole and its sulfate salt have been used in the preparation of dyes,
especially hair and fur dyes, as an intermediate in the production of C.I. Basic Brown 2
and as a corrosion inhibitor for steel (Budavari, 2000). It was used extensively in perma-
nent, oxidative hair dyes until the late 1970s (IARC, 1993).

1.4 Occurrence

1.4.1 Occupational exposure

According to the 1981–83 National Occupational Exposure Survey (National
Institute for Occupational Safety and Health, 2000), about 23 000 workers in the USA
were potentially exposed to 2,4-diaminoanisole or its sulfate salt. They were all hair-
dressers or cosmetologists. The National Institute for Occupational Safety and Health
(1978) estimated in the 1970s that as many as 400 000 workers were potentially exposed
to 2,4-diaminoanisole in the USA. Hairdressers and cosmetologists comprised most of
this group; a relatively small number of fur dyers were probably exposed to higher
concentrations. According to the Finnish Register of Employees Exposed to Carci-
nogens, no workers were exposed to 2,4-diaminoanisole in Finland in 1997 (Savela et
al., 1999).

1.4.2 Environmental occurrence

No data were available to the Working Group.

1.5 Regulations and guidelines

No occupational exposure limits for 2,4-diaminoanisole have been established. It

is classified as a carcinogen in several countries including Finland, Germany, Sweden,
Switzerland and the USA (American Conference of Governmental Industrial
Hygienists, 2000; Deutsche Forschungsgemeinschaft, 2000; UNEP, 2000).
The former European Community (now the European Union) stated that, effective
in 1978, ‘2,4-diaminoanisole (and its salts) must not form part of the composition of
cosmetic products; and Member States should prohibit the marketing of cosmetic
products containing 2,4-diaminoanisole (and its salts)’ (UNEP, 2000).
 2,4-DIAMINOANISOLE AND ITS SALTS 625

2. Studies of Cancer in Humans

Although hairdressers and barbers and similar occupational groups as well as users
of hair dyes may be exposed to 2,4-diaminoanisole, exposure to this compound itself has
not been evaluated in epidemiological studies of cancer risk. The evidence for a carcino-
genic risk of occupational and personal exposure to hair dyes was reviewed in a previous
IARC Monographs volume (IARC, 1993). The risks for haematopoietic neoplasms and
lymphomas were addressed in two recent papers (Correa et al., 2000a,b).

3. Studies of Cancer in Experimental Animals

3.1 Oral administration

Mouse: Groups of 50 male and 50 female B6C3F1 mice, 6 weeks of age, were fed
diets containing 1200 or 2400 mg/kg diet technical-grade 2,4-diaminoanisole sulfate (of
indeterminate purity, with at least one impurity detected by thin-layer chromatography)
for 78 weeks and were observed for a further 18–19 weeks. Groups of 50 animals of
each sex served as matched controls for each concentration group. The mean body-
weight gains of treated and control animals were similar throughout the study, and the
survival rates were comparable among treated and control mice: by the end of the study,
84, 78, 92 and 82% of males and 74, 76, 76 and 78% of females were still alive in the
low-concentration control, high-concentration control, low-concentration and high-
concentration groups, respectively. Among the males, follicular-cell adenomas of the
thyroid were seen in 1/47 low-concentration controls, 0/40 high-concentration controls,
0/46 at the low concentration and 11/45 at the high concentration (p < 0.001); one male
at the low concentration had a follicular-cell carcinoma. Follicular-cell hyperplasia was
found in 12/45 males at the high concentration. Among the females, follicular-cell
adenomas were found in 0/43 low-concentration controls, 0/41 high-concentration
controls, 0/42 at the low concentration and 6/45 at the high concentration (p = 0.017);
follicular-cell carcinomas were found in 2/45 at the high concentration; and follicular-
cell adenomas and carcinomas combined were found in 8/45 at the high concentration
(p = 0.004). Thyroid hyperplasia occurred in 11/42 females at the low concentration
(National Cancer Institute, 1978). [The Working Group noted that no explanation was
provided for having matched controls for each concentration group.]
Rat: Groups of 50 male and 50 female Fischer 344 rats, 6 weeks of age, were fed
diets containing technical-grade 2,4-diaminoanisole sulfate (same sample as used
above) at a concentration of 5000 mg/kg for 78 weeks or 1250 mg/kg of diet for 10
weeks and 1200 mg/kg of diet for 68 weeks, followed by a 29-week observation period.
Groups of 50 (49 for the high-concentration male controls) animals of each sex served
as matched controls for each concentration group. The mean body-weight gains of male
626 IARC MONOGRAPHS VOLUME 79

and female rats at the high concentration were lower than those of controls throughout
most of the study. The mortality rates of the treated and control male rats were similar
by the end of the study: 54, 61, 60 and 54% of the animals were still alive in the low-
and high-concentration control and treated groups, respectively. The female rats showed
a significantly accelerated mortality rate, in particular at the high dietary concentration
of the chemical, with 46, 74, 58 and 44% of the animals alive in low- and high-concen-
tration control and low- and high-concentration treated groups, respectively. Malignant
thyroid follicular-cell tumours were found in 2/35 low-concentration male controls, 0/48
high-concentration male controls, 2/47 at the low concentration and 17/49 at the high
concentration (p = 0.001) and in 2/38 low-concentration female controls, 1/45 high-
concentration female controls, 1/46 at the low concentration and 10/49 at the high
concentration (p = 0.006). Eight males and three females at the high concentration but
none of the controls had multiple follicular-cell tumours. The incidence of tumours of
thyroid C-cell origin (adenomas or carcinomas) was significantly increased in male rats,
with 1/35 in low-concentration male controls, 1/48 in high-concentration male controls,
4/47 in males at the low concentration and 10/49 at the high concentration (p = 0.004),
but not in female rats. In males, squamous-cell carcinomas, basal-cell carcinomas or
sebaceous adenocarcinomas of the skin were found in 0/36 low-concentration controls,
0/48 high-concentration controls, 2/48 at the low concentration and 7/49 at the high
concentration (p = 0.007). Preputial or clitoral gland adenomas, papillomas or
carcinomas were found in 0/36 low-concentration control males, 0/48 high-concen-
tration control males, 2/48 at the low concentration, 8/49 at the high concentration (p <
0.003) and in 0/39 low-concentration female controls, 3/50 high-concentration female
controls, 5/49 at the low concentration (p = 0.049) and 8/49 at the high concentration. In
the Zymbal gland, squamous-cell carcinomas or sebaceous adenocarcinomas were found
in 0/36 low-concentration male controls, 0/48 high-concentration male controls, 1/48 at
the low concentration and 8/49 at the high concentration (p = 0.003); and sebaceous
adenocarcinomas were found in 0/39 low-concentration female controls, 0/50 high-
concentration female controls, 0/49 at the low concentration and 7/49 at the high concen-
tration (p = 0.006) (National Cancer Institute, 1978). [The Working Group noted that no
explanation was provided for having matched controls for each concentration group.]
Groups of 40–60 female Fischer 344 rats, 6 weeks of age, were fed a diet containing
2,4-diaminoanisole sulfate [purity not stated] at a concentration of 0 (control), 1200,
2400 or 5000 mg/kg for up to 82–86 weeks. Another 15 rats were fed a diet containing
5000 mg/kg for 10 weeks and were observed up to about 87 weeks. The mean body
weights of rats at the high concentration were lower than those of controls. By 87–94
weeks, follicular-cell adenomas or carcinomas of the thyroid were found in 0/37
controls, 0/47 at the low concentration, 2/33 at the intermediate concentration and 28/40
at the high concentration (21 with adenomas and seven with carcinomas) and in 1/12 rats
treated for 10 weeks. Mammary adenocarcinomas were found in 0/37 controls, 0/47 at
the low concentration, 5/33 at the intermediate concentration and 3/40 at the high
concentration; mammary adenomas were found in only 1/33 rats at the intermediate
 2,4-DIAMINOANISOLE AND ITS SALTS 627

concentration and 1/47 at the low concentration. Carcinomas (squamous- or sebaceous-

cell or mixed) of the clitoral gland were found in 0/37 controls, 8/47 at the low concen-
tration, 15/33 at the intermediate concentration, 9/40 at the high concentration and 1/12
rats treated for 10 weeks (Evarts & Brown, 1980). [The Working Group noted that no
statistical analysis was provided.]

3.2 Skin application

Two studies, one in mice (Burnett et al., 1975) and one in rats (Kinkel & Holzmann,
1973), in which 2,4-diaminoanisole sulfate was applied by skin painting, could not be
evaluated because the test agent represented a mixture of compounds as a hair-dye
formulation.

3.3 Administration with known carcinogens or modifying factors

Rat: In an evaluation of the promoting effect of 2,4-diaminoanisole sulfate on

thyroid carcinogenesis, groups of 21 male Wistar rats, 7 weeks of age, were given an
intraperitoneal injection of 2.1 g/kg bw N-nitrosobis(2-hydroxypropyl)amine
(NBHPA) in water at the start of the study followed by a diet containing 0.5% 2,4-dia-
minoanisole sulfate for 19 weeks; other groups either received the intraperitoneal
injection of NBHPA with no 2,4-diaminoanisole sulfate, the diet containing 2,4-diami-
noanisole sulfate with no NBHPA or a control diet. The total observation period was
20 weeks. 2,4-Diaminoanisole sulfate alone did not cause thyroid tumours, but
NBHPA alone caused thyroid follicular-cell adenomas in 6/21 (28%) rats and carci-
nomas in 1/21 (4%) rats. The combination of NBHPA and 2,4-diaminoanisole sulfate
increased the incidence of thyroid adenomas to 20/21 (95%) and that of carcinomas to
9/21 (42%) (p < 0.05; χ2 test). The incidence of hyperplasia of the thyroid follicular
epithelium was also increased in the group given the combination (Kitahori et al.,
1989).
In a study to assess the synergistic effect of three thyroid carcinogens, 2,4-diamino-
anisole sulfate, N,N′-diethylthiourea (see monograph in this volume) and 4,4′-thio-
dianiline, groups of 20–21 male Fischer 344/Crj rats, 6 weeks of age, were fed a diet
containing 610 mg/kg 2,4-diaminoanisole sulfate for 52 weeks alone or in combination
with 200 mg/kg N,N′-diethylthiourea and 46 mg/kg 4,4′-thiodianiline. After 52 weeks of
treatment, the rats were killed, necropsied and evaluated for tumour incidences. 2,4-Dia-
minoanisole sulfate alone did not induce thyroid tumours but significantly (p < 0.01)
increased the incidences of thyroid follicular-cell tumours caused by the combination of
the other two agents. 2,4-Diaminoanisole sulfate did not induce liver tumours or lung
tumours but may have increased the incidences of these tumours produced by 4,4′-thio-
dianiline (Hasegawa et al., 1991). [From the study design, the Working Group
concluded that it was not possible to assess the synergistic effect of 2,4-diaminoanisole
628 IARC MONOGRAPHS VOLUME 79

sulfate, if any, on the incidence of thyroid gland tumours induced by N,N′-diethylthio-

urea and/or 4,4′-thiodianiline.]

4. Other Data Relevant to an Evaluation of Carcinogenicity

and Its Mechanisms

4.1 Absorption, distribution, metabolism and excretion

4.1.1 Humans
After application of [14C]2,4-diaminoanisole at 4 μg/cm2 (3–15 cm2 per individual
[exact number of individuals not given]) to the ventral forearm of male volunteers for
24 h, the skin penetration was estimated to be 3.9 ± 0.9%, as determined by excretion
of radiolabel in the urine (Marzulli et al., 1981).

4.1.2 Experimental systems

After application of [14C]2,4-diaminoanisole at 4 μg/cm2 (3–15 cm2 per animal
[exact number of animals not given]) to the abdomen of male and female rhesus
monkeys (Macaca mulatta) for 24 h, the skin penetration was estimated to be
4.7 ± 4.3%, as determined by excretion of radiolabel in the urine (Marzulli et al., 1981).
Dermal absorption of [14C]2,4-diaminoanisole from three hair-dye formulations contai-
ning 0.6–1.8% by female Sprague-Dawley rats varied from 0.26 to 1.1% of the admi-
nistered dose (Hofer & Hruby, 1983).
After intraperitoneal injection of 50 mg/kg bw [14C]2,4-diaminoanisole to rats, 85%
of the radiolabel was excreted in the urine and 9% in the faeces after 48 h. The major
metabolites were 4-acetylamino-2-aminoanisole, 2,4-diacetylaminoanisole and 2,4-
diacetylaminophenol, and were excreted in the urine both free and as glucuronides and
sulfates (Grantham et al., 1979). After administration by gavage of [14C]2,4-diamino-
anisole [dose not specified] to 18 male and female Sprague-Dawley rats, 49.9 ± 6.9 and
52.1 ± 4.8% of the applied dose were recovered in the urine and faeces, respectively,
over 5 days. As 5.6 ± 1.7% of the orally administered radiolabel was eliminated in the
bile within 3 h, the radiolabel found in the faeces might have originated from absorbed
material (Hofer & Hruby, 1983).
The metabolism and covalent binding of [14C]2,4-diaminoanisole to cellular macro-
molecules in vitro and in vivo were shown to be cytochrome P450-dependent.
Incubation of rat liver and kidney microsomes with radiolabelled 2,4-diaminoanisole in
the presence of NADPH and oxygen led to the formation of products that were bound
covalently to microsomal protein. Inhibitors of cytochrome P450 enzymes in vivo and
in vitro decreased the binding; pretreatment with phenobarbital increased binding; and
pretreatment with β-naphthoflavone had no effect. More [14C-ring]-labelled than [14C-
 2,4-DIAMINOANISOLE AND ITS SALTS 629

methyl]-labelled 2,4-diaminoanisole was bound; when the hydrogens in the methyl

group were replaced by deuterium, both the binding and the mutagenicity of 2,4-dia-
minoanisole increased. Liver microsomes catalysed irreversible binding to endogenous
microsomal RNA; no binding to purified calf thymus DNA was detected (Dybing et al.,
1979a). When 10–200 mg/kg bw [3H]2,4-diaminoanisole were injected into rats, the
label was bound covalently to liver and kidney proteins. No covalent binding to hepatic
RNA or DNA was detected (Dybing et al., 1979b).

4.1.3 Comparison of animals and humans

The amount of 2,4-diaminoanisole absorbed after dermal application is of the same
order of magnitude in humans, monkeys and rats. No data were available on meta-
bolism in humans to allow a comparison with data from experiments with rats.

4.2 Toxic effects

4.2.1 Humans
No data were available to the Working Group.

4.2.1 Experimental systems

The oral LD50 of 2,4-diaminoanisole sulfate in an oil-in-water emulsion in rats was
> 4000 mg/kg bw; the intraperitoneal LD50 of a solution in dimethyl sulfoxide was
372 mg/kg bw (Burnett et al., 1977).
Male and female B6C3F1 mice and Fischer 344 rats were fed diets containing
0.075–0.58% 2,4-diaminoanisole sulfate for 4 weeks, followed by a 2-week obser-
vation period. One male rat each at 0.075, 0.125 and 0.58% and one male mouse at
the highest dietary concentration died. No gross abnormalities were noted in either
rats or mice (National Cancer Institute, 1978).
In a study of the effects of 2,4-diaminoanisole sulfate on thyroid function, groups of
15 male Wistar rats were given the compound in the diet at a concentration of 0.5% or
were painted daily with a 5% solution on a 5 × 4-cm area of dorsal skin for up to 6
weeks. Five rats from each group were killed at weeks 1, 3 and 6. The mean thyroid
weight of rats fed 2,4-diaminoanisole sulfate was significantly increased (60%) from
week 1, and was markedly greater (2.5-fold) than the control value at week 6. In the
group treated in the diet, the mean serum concentration of thyroid-stimulating hormone
was markedly higher than that in the control group at weeks 1 and 3 (10- and 16-fold,
respectively), but was only slightly elevated (2.5-fold) at week 6. Similarly, reductions
in the serum concentrations of thyroxine and triiodothyronine noted at earlier times were
less pronounced at week 6. In contrast to dietary administration, cutaneous application
of 2,4-diaminoanisole sulfate did not have a significant effect on thyroid organ weight
or function (Kitahori et al., 1989).
630 IARC MONOGRAPHS VOLUME 79

In an experiment in which groups of five male Iva:Siv50 rats were treated with 2,4-
diaminoanisole sulfate at a dietary concentration of 0.25% for up to 8 weeks, the serum
concentration of thyroid-stimulating hormone was increased by 68% after 1 week and
that of triiodothyronine was decreased at weeks 1, 2, 4 and 8 (Zbinden, 1988).

4.3 Reproductive and prenatal effects

4.3.1 Humans
No information was available on persons exposed to 2,4-diaminoanisole alone. The
evidence for reproductive disorders due to exposure of hairdressers to chemicals was
evaluated from a literature review for the years 1985–93. Associations with menstrual
disorders and spontaneous abortions were found in some epidemiological studies on
hairdressers, but no association was found in other studies. It was concluded that there
is little evidence for an increased incidence of reproductive disorders among hair-
dressers. None of the evidence related specifically to 2,4-diaminoanisole (Kersemaekers
et al., 1995).

4.3.2 Experimental systems

No studies were available in which 2,4-diaminoanisole was tested alone.
Three commercially available hair-dye formulations, containing 0.02, 2 or 4%
2,4-diaminoanisole sulfate and several aromatic amine derivatives among their consti-
tuents, were tested for teratogenicity in groups of 20 mated female Charles River CD
rats. Each formulation was mixed with an equal volume of hydrogen peroxide and
applied topically to a shaved site on the dorsoscapular region at a dose of 2 mL/kg bw
on days 1, 4, 7, 10, 13, 16 and 19 of gestation. The dams were killed on day 20 of
gestation. There was no significant increase in the incidence of soft-tissue anomalies in
the living fetuses, but minor skeletal changes were seen in nine of 169 live fetuses in
three of 20 litters of dams given the formulation containing the highest concentration
of 2,4-diaminoanisole sulfate (4%). On comparison with the three negative control
groups, this finding was found to be statistically significant (p < 0.05 to p < 0.01) but
was considered by the authors not to be biologically significant (Burnett et al., 1976).

4.4 Effects on enzyme induction/inhibition and gene expression

No data were available to the Working Group.

4.5 Genetic and related effects

4.5.1 Humans
No data were available to the Working Group.
 2,4-DIAMINOANISOLE AND ITS SALTS 631

4.5.2 Experimental systems (see Table 1 for references)

The results obtained with 2,4-diaminoanisole sulfate trihydrate and with 2,4-diami-
noanisole dihydrochloride are listed separately, but this distinction is not made in the
summary of the results given below.

(a) DNA damage

2,4-Diaminoanisole induced DNA double-strand breaks in primary rat hepatocytes
in culture and in liver cells of rats. No change in DNA viscosity was seen in liver cells
of rats that received 2,4-diaminoanisole by intraperitoneal injection. DNA damage was
produced in liver and brain, but not stomach, colon, kidney, bladder, lung or bone-
marrow cells of mice as measured in the Comet assay.
2,4-Diaminoanisole induced unscheduled DNA synthesis in HeLa cells with and
without S9.

(b) Mutation and allied effects

2,4-Diaminoanisole caused frameshift mutations in Salmonella typhimurium in the
presence of exogenous metabolic activation from liver microsomes from uninduced and
induced mice, rats and rabbits and from humans. It produced gene mutations and chro-
mosomal aberrations in rodent cells in vitro.
2,4-Diaminoanisole was metabolized to mutagenic products by ram seminal
vesicle microsomes or purified prostaglandin H synthase (Robertson et al., 1983;
Sarkar et al., 1992). The reaction product of 2,4-diaminoanisole and hydrogen
peroxide was mutagenic in Salmonella in the presence and absence of an exogenous
metabolic activation system (Watanabe et al., 1989).
2,4-Diaminoanisole induced mutation and mitotic recombination in Saccharomyces
cerevisiae. It was not mutagenic to Neurospora crassa in a single study.
2,4-Diaminoanisole did not transform Syrian hamster embryo cells in culture in a
single study.
Urine of phenobarbital-induced rats treated with 2,4-diaminoanisole was mutagenic
in the presence, but not the absence, of rat liver microsomes. If rats were treated with
β-naphthoflavone, methylcholanthrene, or 2,3,7,8-tetrachlorodibenzo-para-dioxin, the
mutagenicity of their urine in the presence of microsomes was decreased. Treatment of
the urine with β-glucuronidase increased mutagenic activity in the presence and absence
of liver microsomes (Reddy et al., 1980).
2,4-Diaminoanisole produced sex-linked recessive lethal mutations, but not
somatic cell recombination, in Drosophila melanogaster. It produced sister chromatid
exchanges, but not micronuclei, in mouse bone-marrow cells. It did not produce domi-
nant lethal mutations in rats or altered sperm morphology in mice.
 632
Table 1. Genetic and related effects of 2,4-diaminoanisole, its sulfate trihydrate and its dihydrochloride

Test system Resulta Doseb Reference

(LED/HID)
Without With
exogenous exogenous
metabolic metabolic
system system

2,4-Diaminoanisole

IARC MONOGRAPHS VOLUME 79

Salmonella typhimurium TA100, TA1535, TA1537, reverse – – 500 μg/plate Bruce & Heddle (1979)
mutation
Salmonella typhimurium TA100, reverse mutation – – 60 de Giovanni-Donnelly (1981)
Salmonella typhimurium TA100, reverse mutation NT + 10 μg/plate Parodi et al. (1981)
Salmonella typhimurium TA100, TA102, TA98, reverse mutation NT (+)c 13.26 Sarkar et al. (1992)
Salmonella typhimurium TA1537, reverse mutation – + 3 Prival et al. (1980)
Salmonella typhimurium TA98, reverse mutation (+) + 3 Prival et al. (1980)
Salmonella typhimurium TA1538, reverse mutation NT + 10 Ames et al. (1975); Dybing &
Aune (1977); Dybing et al.
(1979a)
Salmonella typhimurium TA1538, reverse mutation NT + 5 Dybing & Thorgeirsson
(1977); Aune & Dybing
(1979)
Salmonella typhimurium TA1538, reverse mutation NT + 1.4 Aune et al. (1980a)
Salmonella typhimurium TA1538, TA98, reverse mutation – + 10 de Giovanni-Donnelly (1981)
Salmonella typhimurium TA1538, reverse mutation NT + 60 μg/plate Mohn et al. (1982)
Salmonella typhimurium TA98, reverse mutation – + 19 μg/plate Yoshikawa et al. (1977)
Salmonella typhimurium TA98, reverse mutation – + 20 μg/plate Bruce & Heddle (1979)
Salmonella typhimurium TA98, reverse mutation NT + 10 μg/plate Aune et al. (1980b)
Salmonella typhimurium TA98, reverse mutation NT + 1.25 μg/plate Parodi et al. (1981)
Salmonella typhimurium TA98, reverse mutation – +d 50 μg/plate Maack et al. (1986)
Neurospora crassa, forward mutation, spot test, ad-3 locus – NT 400 μg/plate Ong (1978)
Gene mutation, Chinese hamster V79 cells, Hprt locus in vitro – – 552 Fassina et al. (1990)
Table 1 (contd)

Test system Resultb Dosec Reference

(LED/HID)
Without With
exogenous exogenous
metabolic metabolic
system system

2,4-DIAMINOANISOLE AND ITS SALTS

Cell transformation, Syrian hamster embryo cells, focus assay – NT 50 Pienta & Kawalek (1981)
Unscheduled DNA synthesis, human HeLa cells in vitro + + 138 Loprieno et al. (1983)
Gene mutation, mouse lymphoma L5178Y cells in vitro, Tk locus + NT 19 Palmer et al. (1977)
DNA damage, Sprague-Dawley rat liver cells treated in vivo +e 91.2 ip × 1 Parodi et al. (1981)
(alkaline elution assay)
DNA damage, Sprague-Dawley rat liver cells treated in vivo – 50 ip × 1 Brambilla et al. (1985)
(DNA viscosity)
Sister chromatid exchange, male mouse bone-marrow cells in vivo + 12 ip × 1 Parodi et al. (1983)
Micronucleus formation, rat bone-marrow cells in vivo – 500 po × 2 Hossack & Richardson
(1977)
Micronucleus formation, (C57BL/6 × C3H/He)F1 mouse bone- – 500 ip × 5 Bruce & Heddle (1979)
marrow cells in vivo
Dominant lethal mutation, rats in vivo – 20 ip × 3/week; Burnett et al. (1977)
8 weeks
Sperm morphology, (C57BL/6 × C3H/He)F1 mice in vivo – 500 ip × 5 Bruce & Heddle (1979)
2,4-Diaminoanisole sulfate trihydrate
Salmonella typhimurium TA100, reverse mutation – (+) 333 μg/plate Dunkel et al. (1985)
Salmonella typhimurium TA100, reverse mutation – + 100 μg/plate Zeiger et al. (1988)
Salmonella typhimurium TA97, reverse mutation (+) + 33 μg/plate Zeiger et al. (1988)
Salmonella typhimurium TA1535, reverse mutation – – 10 000 μg/plate Dunkel et al. (1985)
Salmonella typhimurium TA1535, reverse mutation – – 3333 μg/plate Zeiger et al. (1988)
Salmonella typhimurium TA1537, reverse mutation – + 10 μg/plate Dunkel et al. (1985)
Salmonella typhimurium TA1538, reverse mutation NT +f 0.87 μg/plate Robertson et al. (1983)

633
 634
Table 1 (contd)

Test system Resultb Dosec Reference

(LED/HID)
Without With
exogenous exogenous
metabolic metabolic
system system

IARC MONOGRAPHS VOLUME 79

Salmonella typhimurium TA1538, reverse mutation – + 1 μg/plate Dunkel et al. (1985)
Salmonella typhimurium TA98, reverse mutation – + 3.3 μg/plate Dunkel et al. (1985)
Salmonella typhimurium TA98, reverse mutation – + 10 μg/plate Reddy et al. (1980)
Salmonella typhimurium TA98, reverse mutation + + 1 μg/plate Zeiger et al. (1988)
Escherichia coli WP2 uvrA, reverse mutation – – 10 000 μg/plate Dunkel et al. (1985)
Saccharomyces cerevisiae, mitotic recombination (growing cells) + NT 500 Mayer & Goin (1980)
Drosophila melanogaster, somatic recombination (w/w+ locus) – 118 feed Rodriguez-Arnaiz &
Hernández Aranda (1994)
Drosophila melanogaster, sex-linked recessive lethal mutations + 1180 feed Blijleven (1977)
DNA strand breaks, rat hepatocytes in vitro (alkaline elution) + NT 708 Storer et al. (1996)
Gene mutation, mouse lymphoma L5178Y cells, Tk locus in vitro + + 2 Mitchell et al. (1988)
Gene mutation, mouse lymphoma L5178Y cells, Tk locus in vitro + + 3.9 Myhr & Caspary (1988)
Chromosomal aberrations, Chinese hamster lung fibroblasts + NT 60 Ishidate (1988)
in vitro
Urine from rats (50 mg/kg ip × 1), Salmonella typhimurium TA98 – + 25 μL urine/ Reddy et al. (1980)
mutagenicityg plate
Dominant lethal mutation, Holtzman albino rats in vivo – 40 ip × 3/week; Sheu & Green (1979)
10 weeks
2,4-Diaminoanisole dihydrochloride
Salmonella typhimurium TA100, TA1535, reverse mutation NT – 1000 μg/plate Shahin et al. (1980)
Salmonella typhimurium TA1537, TA97, reverse mutation NT + 50 μg/plate Shahin et al. (1980, 1983,
1985)
Salmonella typhimurium TA1538, TA98, reverse mutation NT + 10 μg/plate Shahin et al. (1980)
Table 1 (contd)

Test system Resultb Dosec Reference

(LED/HID)
Without With
exogenous exogenous
metabolic metabolic

2,4-DIAMINOANISOLE AND ITS SALTS

system system

Salmonella typhimurium TA98, reverse mutation – + 10 μg/plate Venitt et al. (1984)

Salmonella typhimurium TA1538, reverse mutation – + 10 μg/plate Loprieno et al. (1982)
Salmonella typhimurium TA98, reverse mutation (fluctuation test) NT + 0.33 Venitt et al. (1984)
Salmonella typhimurium TA98, reverse mutation NT + 2.5 μg/plate Loprieno et al. (1982)
Salmonella typhimurium TA98, reverse mutation – + 10 μg/plate Watanabe et al. (1989)
Salmonella typhimurium TA98, reverse mutation (in the presence + + 0.03 μg/plate Watanabe et al. (1989)
of H2O2)
Saccharomyces cerevisiae D4, mitotic gene conversion – + 1055h Loprieno et al. (1982)
Schizosaccharomyces pombe, forward mutation ade locus + + 528 Loprieno et al. (1982)
Drosophila melanogaster, sex-linked recessive lethal mutations + 3165 feed Blijleven (1982)
Gene mutation, Chinese hamster V79 cells, Hprt locus in vitro + – 844i Loprieno et al. (1982)
Chromosomal aberrations, Chinese hamster ovary (CHO) cells + + 50 Darroudi et al. (1982)
in vitro
Unscheduled DNA synthesis, human HeLa cells in vitro + + 176 Loprieno et al. (1983)
DNA strand breaks, ddY mice (liver and brain) in vivo (Comet + 200 po × 1 Sasaki et al. (1999)
assay)
Micronucleus formation, male mouse bone-marrow cells in vivo – 60 ip × 1 Morita et al. (1997)
Urine from rats (100 mg/kg bw po or ip), mutation in Salmonella – + 100 μL urine/ Shahin et al. (1980)
typhimurium TA1538, TA98 plate
Urine from rats (120 mg on skin), mutation in Salmonella – + 100 μL urine/ Shahin et al. (1980)
typhimurium TA1538, TA98 plate

635
 636
Table 1 (contd)

Test system Resultb Dosec Reference

(LED/HID)
Without With
exogenous exogenous

IARC MONOGRAPHS VOLUME 79

metabolic metabolic
system system

Urine from rats (100 mg/kg bw po or ip), mutation in Salmonella – – 300 μL urine/ Shahin et al. (1980)
typhimurium TA100 plate
Urine from rats (120 mg on skin), mutation in Salmonella – – 300 μL urine/ Shahin et al. (1980)
typhimurium TA100 plate

a
+, positive; (+), weak positive; –, negative; NT, not tested
b
LED, lowest effective dose; HID, highest ineffective dose; in-vitro tests, μg/mL; in-vivo tests, mg/kg bw per day; ip, intraperitoneal; po, oral
c
Activation with prostaglandin H synthase
d
Extracts from mammary tissue of lactating rats and human mammary tumour cell lines
e
At 4 h after treatment; increase not significant at 24 h
f
Positive with an exogenous metabolic activation system from a 9000 × g supernatant of rat liver (S9) and, at 10-fold higher dose, with ram seminal
vesicle microsomes
g
Test carried out with 2,4-diaminoanisole disulfate
h
A threefold higher concentration was negative in the absence of metabolic activation.
i
1055 μg/mL was negative in the presence of metabolic activation.
 2,4-DIAMINOANISOLE AND ITS SALTS 637

4.6 Mechanistic considerations

2,4-Diaminoanisole is metabolically activated to covalently protein-bound

products in rat liver and kidney, but no covalent binding to hepatic DNA was detected.
The data on genotoxicity indicate that 2,4-diaminoanisole is genotoxic.
Short-term oral treatment of rats with high doses of diaminoanisole sulfate leads
to the development of thyroid tumours and concomitant alterations in thyroid
hormone function. The serum concentrations of thyroid-stimulating hormone were
elevated and those of thyroxine and triiodothyronine were lowered during the first few
weeks after the beginning of treatment. These alterations in hormone concentrations
tended to normalize after 6 weeks. The alterations in thyroid hormone homeostasis are
presumed to be involved in the induction of thyroid tumours by 2,4-diaminoanisole,
but a genotoxic mechanism cannot be excluded.

5. Summary of Data Reported and Evaluation

5.1 Exposure data

2,4-Diaminoanisole is an aromatic amine which was used extensively in hair dyes

and in the dyeing of furs until the late 1970s.

5.2 Human carcinogenicity data

Although epidemiological studies have been conducted on professional and

personal users of hair dyes, none made specific mention of 2,4-diaminoanisole.

5.3 Animal carcinogenicity data

2,4-Diaminoanisole sulfate was tested by dietary administration in one experiment

in mice and in two experiments in one strain of rats. Thyroid gland adenomas or
carcinomas were induced in mice and rats. Tumours of the skin and of the preputial,
clitoral and Zymbal glands were also induced in rats. 2,4-Diaminoanisole sulfate was
tested for its promoting effects by dietary administration in two strains of rats. In one
study in rats, it promoted thyroid gland tumours induced by N-nitrosobis(2-hydroxy-
propyl)amine.

5.4 Other relevant data

About 2–4% of a dermal dose of 2,4-diaminoanisole is absorbed by humans,

monkeys and rats. The compound is completely absorbed after oral administration to
rats, extensively metabolized to free and conjugated acetylated and oxidized products
638 IARC MONOGRAPHS VOLUME 79

and thereafter excreted in equal percentages of the applied dose in urine and faeces.
The substance is metabolically activated to covalently protein-bound products in rat
liver and kidney, but no covalent binding to hepatic DNA was detected. Short-term
administration of high oral doses to rats induced thyroid enlargement (goitre) and alte-
rations in thyroid hormone homeostasis.
2,4-Diaminoanisole is genotoxic in vitro, producing gene mutations and chromo-
somal damage. It was mutagenic in bacteria in the presence or absence of a microsomal
fraction from the livers of uninduced rats, mice, rabbits or humans. It produced chromo-
somal aberrations and sister chromatid exchange in rodent cells in vitro, mitotic
recombination in yeast and mutations in insects. The results of most tests in mammals
in vivo were negative.

5.5 Evaluation
There is inadequate evidence in humans for the carcinogenicity of 2,4-
diaminoanisole.
There is sufficient evidence in experimental animals for the carcinogenicity of 2,4-
diaminoanisole.

Overall evaluation

2,4-Diaminoanisole is possibly carcinogenic to humans (Group 2B).

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