News
Carcinogenicity of some aromatic amines and related
compounds
In May–June, 2020, a Working Group
of 19 scientists from nine countries
met remotely at the invitation of the
International Agency for Research
on Cancer (IARC) to finalise their
evaluation of the carcinogenicity
of aniline (and aniline hydrochlo­
ride), o-anisidine (and o-anisidine
hydrochloride), o-nitroanisole, and
cupferron. These assessments will be
published in Volume 127 of the IARC
Monographs.1
A n i l i n e , o- a n i s i d i n e , a n d
o-nitroanisole were classified as
“probably carcinogenic to humans”
(Group 2A). These evaluations
were based on “strong” evidence
that they belong, on the basis
of mechanistic considerations,
to a class of aromatic amines for
which several members (including
o-toluidine, 2-naphthylamine,
and 4-aminobiphenyl) have been
classified as “carcinogenic to humans”
(Group 1). 2 Aniline, o-anisidine,
and o-nitroanisole are concordant
with other aromatic amines in this
mechanistic class with respect to
the mechanism of bioactivation
to electrophiles, genotoxicity, and
target organs of carcinogenicity
in chronic animal bioassays. The
Group 2A classification also applies
to aniline hydrochloride and
o-anisidine hydrochloride, which
exist in equilibrium with the parent
compounds in the body. For all agents,
data were sparse regarding exposure
levels in workers and in the general
population; however, the available
data indicate that exposures are
higher in occupational situations than
in other scenarios.
Aniline is a high-production-
volume chemical manufactured and
used primarily in China, Europe, and
the USA. It is used in the synthesis
of isocyanates (for polyurethane
production), rubber-processing
chemicals, dyes and pigments (used
www.thelancet.com/oncology Published online June 25, 2020 https://doi.org/10.1016/S1470-2045(20)30375-2 1
in textiles, leather, and tattoo inks3),
pharmaceuticals, and pesticides.
Tobacco smoke is a main source of
exposure to aniline in the general
population, with measurements
indicating higher concentrations
in sidestream than in mainstream
smoke. Aniline has also been detected
in drinking water. In humans, aniline
is readily absorbed by the dermal,
oral, and inhalation routes, and is
excreted in the urine primarily as
N-acetyl-para-aminophenol. In
experimental animals, including
rodents, rabbits, cats, dogs, pigs, and
sheep, aniline metabolism is similar
to that in humans. There is “strong”
evidence in experimental systems that
aniline exhibits key characteristics of
carcinogens. Aniline is metabolically
activated to electrophiles, is
genotoxic, induces oxidative stress,
and alters cell proliferation, cell death,
or nutrient supply. In parallel to an
established metabolic activation
paradigm for carcinogenic aromatic
amines, aniline undergoes cytochrome
P450-catalysed N-hydroxylation to
phenylhydroxylamine in dogs, rats,
and in other experimental metabolic-
activation studies. Further activation
by O-acetylation, and spontaneous
heterolysis of the N-acetoxy
metabolite, are then expected to give
a DNA-reactive electrophilic nitrenium
ion. The N-acetoxy derivative of
aniline, prepared synthetically,
forms DNA adducts, including at the
C8 position of guanine.4 In humans,
data were available on adducts with
haemoglobin, but not with DNA.
Aniline undergoes DNA binding in
the liver, spleen, and kidney of rats.
It induces DNA damage in human
cell lines in vitro and in rats and mice,
and it is consistently clastogenic
in various mammalian tests. The
evidence for carcinogenicity in
experimental animals was “sufficient”
on the basis of the increased
incidence of malignant neoplasms
in two independent studies in one
species. Like the aromatic amine
o-toluidine, 2 aniline hydrochloride
caused malignant tumours of the
spleen and mesothelioma of the
tunica vaginalis of the testes in male
F344 rats. 5,6 The splenic tumours
included fibrosarcomas or sarcomas
(combined; also observed in females),
as well as haemangiosarcomas; 5,6
these tumour types also occurred
in other organs within the body
cavities.6 The evidence in humans was
“inadequate” as aniline’s effects could
not be differentiated from those of
other bladder carcinogens in the most
informative studies (in occupational
cohorts).
o-Anisidine is used mainly as
a chemical intermediate in the
synthesis of azo pigments and dyes
for consumer products, textiles, and
paper. o-Anisidine has been detected
in consumer products, in tattoo
inks, in tobacco smoke, and in urine
samples from the general population.
In rats, o-anisidine is readily absorbed,
widely distributed to tissues, and
excreted primarily via the urine as
N-acetyl-2-methoxyaniline. There is
“strong” evidence in experimental
systems that o-anisidine exhibits
key characteristics of carcinogens.
o-Anisidine is metabolically activated
to electrophiles, is genotoxic, and
alters cell proliferation, cell death,
or nutrient supply. o-Anisidine is
metabolised to electrophiles by
human and other mammalian hepatic
microsomes in vitro to form DNA
adducts, with N-(deoxyguanosin-8-
yl)-2-methoxyaniline as the major
form.7 In the urinary bladder of rats,
o-anisidine induces this DNA adduct
and causes DNA damage. 8 It is
mutagenic in base-pair substitution
strains of bacteria. The evidence
for carcinogenicity in experimental
animals was “sufficient”. In F344 rats
Lancet Oncol 2020
Published Online
June 25, 2020
https://doi.org/10.1016/
S1470-2045(20)30375-2
For more on the IARC
Monographs see http://
monographs.iarc.fr/
Upcoming meetings
Sept 14–18, 2020, volume 126:
Opium;
Nov 3–10, 2020, volume 128:
Acrolein, Crotonaldehyde, and
Arecoline;
Feb 22–March 5, 2021,
volume 129: Gentian Violet,
Leucogentian Violet, Malachite
Green, Leucomalachite Green,
and C I Direct Blue 218
IARC Monograph Working
Group Members
DM DeMarini (USA) – Meeting
Chair; T Carreón-Valencia (USA);
WM Gwinn (USA); NB Hopf
(Switzerland); MS Sandy (USA) –
Subgroup Meeting Chairs;
T Bahadori (USA) ; GM Calaf
(Chile); G Chen (Canada);
A de Conti (USA); L Fritschi
(Australia); M Gi (Japan);
PD Josephy (Canada); J Kirkeleit
(Norway); K Kjaerheim (Norway);
S Langouët (France);
DM McElvenny (UK); CM Sergi
(Canada); LT Stayner (USA);
T Toyoda (Japan)
Declaration of interests
All Working Group members
declare no competing interests
Invited Specialists
None
Representatives
None
Observers
None
IARC Secretariat
L Benbrahim-Tallaa; F Chung;
IA Cree; F El Ghissassi; Y Grosse
(Responsible Officer); KZ Guyton;
H Mattock; K Müller;
MK Schubauer-Berigan;
E Suonio; MC Turner
Declaration of interests
All secretariat declare no
competing interests.
For the Preamble to the IARC
Monographs see https://
monographs.iarc.fr/wp-content/
uploads/2020/04/127-
participants.pdf
 News
For IARC declarations of
interests see https://
monographs.iarc.fr/wp-content/
uploads/2020/04/Short-list-of-
participants_127-1_June.pdf
Disclaimer
The views expressed are those of
the authors and do not
necessarily represent the
decisions, policy, or views of their
respective institutions.
2 www.thelancet.com/oncology Published online June 25, 2020 https://doi.org/10.1016/S1470-2045(20)30375-2
and B6C3F1 mice exposed via the diet,
o-anisidine hydrochloride increased
the incidence of transitional cell
carcinoma of the urinary bladder. 9
In experimental animals, the urinary
bladder is a common target organ of
carcinogenicity for other aromatic
amines administered orally, including
o-toluidine and 2-naphthylamine in
rats, and 4-aminobiphenyl in dogs
and mice.2 o-Anisidine hydrochloride
also induced renal pelvis transitional
cell carcinoma and thyroid tumours
in male rats.9 The evidence in humans
was “inadequate”, consisting solely
of a bladder cancer case series with
concomitant exposure to other
bladder carcinogens.
o-Nitroanisole is used primarily as
an industrial precursor for o-anisidine.
o-Nitroanisole has been detected in
drinking water, and may contaminate
water and soil, but no data were found
on occupational or general-population
exposure levels. In orally exposed rats,
o-nitroanisole is readily absorbed,
widely distributed to tissues, and
excreted primarily via the urine. There
is “strong” evidence in experimental
systems that o-nitroanisole exhibits
key characteristics of carcinogens.
o-Nitroanisole is metabolically
activated to electrophiles, is genotoxic,
and alters cell proliferation, cell death,
or nutrient supply. o-Nitroanisole
is metabolised to the aromatic
amine o-anisidine, and is activated
by human hepatic extracts to form
N-(2-methoxyphenyl)hydroxylamine-
derived DNA adducts in vitro.10 In the
urinary bladder of rats, o-nitroanisole
induces this DNA adduct and causes
DNA damage.10,11 It is also mutagenic
in mammalian cells and in base-
pair substitution strains of bacteria.
The evidence for carcinogenicity in
experimental animals was “sufficient”.
o-Nitroanisole increased the incidence
of malignant tumours of the urinary
bladder in experimental animals, as
do the aromatic amines o-anisidine,9
o-toluidine, 2 2-naphthylamine, 2
and 4-aminobiphenyl.2 In male and
female F344 rats, o-nitroanisole
induced urinary bladder sarcoma
and transitional cell carcinoma, and
mononuclear cell leukaemia. Other
tumours included kidney transitional
cell carcinoma in male F344 rats and
malignant liver tumours in male
B6C3F1 mice.12 The evidence for cancer
in humans was “inadequate” as no
data were available.
Cupferron is a reagent used to
separate copper, iron, tin, vanadium,
and thorium from other metals.
Suppliers of cupferron are based
mainly in China and the USA. No
recent data on levels of occupational
or general-population exposures to
cupferron were found. Cupferron was
classified as “possibly carcinogenic to
humans” (Group 2B) on the basis of
“sufficient” evidence of carcinogenicity
in experimental animals and “strong”
evidence in experimental systems that
cupferron exhibits key characteristics
of carcinogens (it is genotoxic).
Cupferron increased the incidence of
malignant neoplasms in two rodent
species. In F344 rats, cupferron induced
haemangiosarcoma of the circulatory
system, hepatocellular carcinoma,
and forestomach squamous-cell
carcinoma in male and female rats, and
mesothelioma of the body cavities in
male rats. In B6C3F1 mice, cupferron
induced haemangiosarcoma of the
circulatory system in male and female
mice, and hepatocellular carcinoma in
female mice.13 The evidence for cancer
in humans was “inadequate” as no data
were available.
We declare no competing interests.
IARC Monographs Vol 127 group
International Agency for Research on Cancer, Lyon,
France
1 International Agency for Research on Cancer.
Volume 127: Some aromatic amines and
related compounds. IARC Working Group.
Lyon, France; May 25 to June 12, 2020.
IARC Monogr Identif Carcinog Hazards Hum
(in press).
2 International Agency for Research on Cancer.
Chemical agents and related occupations.
IARC Monogr Eval Carcinog Risks Hum 2012;
100F: 1–599. http://publications.iarc.fr/123
(accessed June 22, 2020).
3 European Chemicals Agency. Background
document to the Opinion on the Annex XV
dossier proposing restrictions on substances
in tattoo inks and permanent make up. 2019.
https://echa.europa.eu/documents/
10162/23665416/rest_tattoo_inks_bd_
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4 Králík A, Linhart I, Váňa L, Moulisová A.
Identification of new DNA adducts of
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28: 1317–25.
5 United States Environmental Protection
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104-Week chronic toxicity study in rats:
Aniline hydrochloride. Final report. Vols I and
II. Prepared by Haskell Laboratory. National
Technical Reports Library-National Technical
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No. OTS00000721. 1982. https://ntrl.ntis.
gov/NTRL/ (accessed June 22, 2020).
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Rýdlová H, Schmeiser HH, Frei E.
Identification of a genotoxic mechanism for
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pollutant and suspected human carcinogen
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8 Iatropoulos MJ, Duan J-D, Schmuck G,
Williams GM. The urinary bladder carcinogen
propoxur does not produce genotoxic effects
in the urinary bladder of Wistar male rats.
Exp Toxicol Pathol 2015; 67: 453–58.
9 National Cancer Institute. Bioassay of
o-anisidine hydrochloride for possible
carcinogenicity.
Natl Cancer Inst Carcinog Tech Rep Ser 1978;
89: 1–149.
10 Stiborová M, Miksanová M, Smrček S, et al.
Identification of a genotoxic mechanism for
2-nitroanisole carcinogenicity and of its
carcinogenic potential for humans.
Carcinogenesis 2004; 25: 833–40.
11 Toyoda T, Cho YM, Akagi J, et al. Early
detection of genotoxic urinary bladder
carcinogens by immunohistochemistry for
γ-H2AX. Toxicol Sci 2015; 148: 400–08.
12 National Toxicology Program. NTP
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o-nitroanisole (CAS No. 91-23-6) in
F344 rats and B6C3F1 mice (feed studies).
Natl Toxicol Program Tech Rep Ser 1993;
416: 1–482.
13 National Cancer Institute. Bioassay of
cupferron for possible carcinogenicity
(CAS No. 135-20-6).
Natl Cancer Inst Carcinog Tech Rep Ser 1978;
100: 1–131.