C01 GentianViolet 129

GENTIAN VIOLET,
LEUCOGENTIAN VIOLET,
MALACHITE GREEN,
LEUCOMALACHITE GREEN,
AND CI DIRECT BLUE 218
VOLUME 129
This publication represents the views and expert

opinions of an IARC Working Group on the
Identification of Carcinogenic Hazards to Humans,
which met remotely, 22 February to 5 March 2021
LYON, FRANCE - 2022
IARC MONOGRAPHS
ON THE IDENTIFICATION
OF CARCINOGENIC HAZARDS
TO HUMANS
GENTIAN VIOLET AND
LEUCOGENTIAN VIOLET
1. Exposure Characterization para-rosaniline chloride, methyl violet 10B,

methylrosanilium chloride, aniline violet
(ECHA, 2020a; NCBI, 2020).
1.1 Identification of the agent
Gentian violet is a cationic triphenylmethane (b) Structural and molecular formulae, and
dye. Leucogentian violet, the leuco base or relative molecular mass
reduced form of gentian violet, is formed by
the chemical or enzymatic reduction of gentian
violet. Gentian violet and its leuco base are
H3 C CH3
susceptible to oxidation−reduction and demeth- N
+
ylation reactions. Cl
-
1.1.1 Gentian violet

(a) Nomenclature
Chem. Abstr. Serv. Reg. No.: 548-62-9

H3 C CH3
Chem. Abstr. Serv. name: N-[4-[bis[4- N N
(dimethylamino)phenyl]methylene]-2,5- CH3 CH3
cyclohexadien-1-ylidene]-N-methylmetha-
naminium chloride (1 : 1)
EC No.: 208-953-6 Molecular formula: C25H30ClN3
IUPAC systematic name: [4-[bis[4-(dimethyl- Relative molecular mass: 407.98
amino)phenyl]methylidene]cyclo-hexa-2,5-
dien-1-ylidene]-dimethylazanium chloride; (c) Chemical and physical properties of the
(4-[4,4-bis(dimethylamino)benzhydrylidene] pure substance
cyclohexa-2,5-dien-1-ylidene)dimethylam-
monium chloride; tris(4-(dimethylamino) Description: green to very dark green powder;
phenyl)methylium chloride dark purple in solution
Synonyms: CI Basic Violet 3, CI 42555, Boiling point: 631.92 °C (ECHA, 2020a)
basic violet, crystal violet, hexamethyl-
43
IARC MONOGRAPHS – 129
Melting point: 205–215 °C (decomposes) 1.1.2 Leucogentian violet

(NCBI, 2013); 198 °C (ECHA, 2020a)
(a) Nomenclature
Density: 1.19 g/cm3 at 20 °C (OEHHA, 2019)
Solubility: 4000 mg/L at 25 °C, and 10–50 g/L Chem. Abstr. Serv. Reg. No.: 603-48-5
at 27 °C and pH 3.07, in water (ECHA, 2020a); Chem. Abstr. Serv. name: leucocrystal violet
soluble in ethanol and chloroform (NCBI, EC No.: 210-043-9
2013)
IUPAC systematic name: 4-[bis[4-(dimethyl-
Vapour pressure: 1.02 × 10−13 mm Hg amino)phenyl]methyl]-N,N-dimethylaniline
[1.36 × 10−14 kPa] at 25 °C (estimated) (NCBI,
Synonyms: leucocrystal violet, leuco Basic
2013); 0 Pa at 25 °C (ECHA, 2020a)
Violet 3, crystal violet leucobase, 4,4′,4′′-tris-
Auto-ignition temperature: > 190 °C (United (dimethylamino)triphenylmethane, 4,4′,4′′-
States Pharmacopeia, 2014) methylidynetris-N,N-dimethyl-benzenamine,
Stability and reactivity: stable under normal 4,4′,4′′-methylidynetris-N,N-dimethyl-
conditions; light-sensitive; incompatible with aniline, tris[para-(dimethylamino)phenyl]
strong oxidizing agents, reducing agents, methane, N,N,N′,N′,N′′,N′′-hexamethyl-4,4′,4′′-
and strong acids (United States Pharmaco- methylidynetrianiline (NCBI, 2020).
peia, 2014; Mani & Bharagava, 2016)
Octanol/water partition coefficient (P): log (b) Structural and molecular formulae, and
Kow = 0.51 (NCBI, 2013) relative molecular mass
Henry’s law constant: 3.06 × 10−16 atm m3 mol−1
[3.10 × 10−10 Pa m3 mol−1] (estimated) at 25 °C
(NLM, 2020) H3 C CH3
N
Ultraviolet maximum: 590 nm (water)
(NCBI, 2013).
(d) Impurities
Gentian violet is composed primarily of
hexamethyl-para-rosaniline (crystal violet)
with impurities of pentamethyl-para-rosani- H3 C CH3
N N
line and tetramethyl-para-rosaniline (Cooksey,
CH3 CH3
2017). The purity of gentian violet may range
from > 76% to < 90% (w/w) (ECHA, 2012).
The composition of commercial gentian violet
is typically > 96% hexamethyl-para-rosani- Molecular formula: C25H31N3
line, < 4% pentamethyl-para-rosaniline, < 4% Relative molecular mass: 373.53
tetramethyl-para-rosaniline, and a trace amount
of trimethyl-para-rosaniline (OEHHA, 2019).
(c) Chemical and physical properties of the
Unreacted reagents such as Michler’s ketone or
pure substance
Michler’s base may also be present (Cooksey,
2017). Description: white to very pale lavender
powder
44
Gentian violet and leucogentian violet
Boiling point: decomposition at 227.8 °C, reaction, leucogentian violet is produced from
before reaching the boiling point (ECHA, the condensation of N,N-dimethylaniline
2020b) with formaldehyde, reaction with additional
Melting point: 175–177 °C (NCBI, 2013); N,N-dimethylaniline, and oxidation in the
176.8 °C (ECHA, 2020b) presence of chloranil and a catalyst such as
Density: 1.141 g/cm3 at 19.6 °C (ECHA, 2020b) (dihydrodibenzotetraaza[14]annulene) iron, a
vanadium or molybdenum compound, or a
Solubility: 1.3 mg/L at 20 °C and pH 7.4–8.7 nitrous gas (Gessner & Mayer, 2000).
in water (ECHA, 2020b); 0.6 mg/mL in
ethanol (NCBI, 2013) (b) Production volume
Vapour pressure: 1.95 × 10−5 Pa at 20 °C India and China are the largest producers of
(ECHA, 2020b) gentian violet (ECHA, 2012). [No information was
Stability and reactivity: stable under normal found on production volumes in these countries.]
conditions; light- and air-sensitive; carbon In the USA, the production volumes of gentian
and nitrogen oxides and hydrogen chloride violet were reported to be between > 500 000
may form from thermal decomposition and 1 million pounds [> 227–454 tonnes] per
(Chemical Book, 2017; ECHA, 2020b). year in 1986 and 1990, and between 10 000 and
Octanol/water partition coefficient (P): log 500 000 pounds [between 4.54 and 227 tonnes]
Kow = 5.9 (ECHA, 2020b) per year in 1994, 1998, and 2002 (NCBI, 2013).
Ultraviolet maximum: 260 nm (Merck, 2021). Gentian violet is not produced in the European
Union (EU), but the EU imports 210–230 tonnes
of gentian violet per year (ECHA, 2012). In 2020,
(d) Impurities
gentian violet was available from 36 suppliers
Leucogentian violet is available with a purity in China, 15 suppliers in the USA, 9 suppliers
ranging from 98% to > 99%. in India, and 2 suppliers in Europe (Chemical
Register, 2020a).
1.2 Production and use (c) Uses
1.2.1 Gentian violet Gentian violet has been in use for more than
a century as a dye or pigment, biological stain,
(a) Production process
and topical antiseptic. It has numerous diverse
Several methods are reported to produce applications because of its colouring and medic-
gentian violet, each resulting in different inal properties.
compositions of the N-methylated para-rosani- The deep blue-violet colour of gentian violet
line dye components (Gessner & Mayer, 2000; is used to dye numerous textiles including silk,
Cooksey, 2017). High-purity hexamethyl-pa- cotton, wool, and nylon. Gentian violet is also
ra-rosaniline is produced from the conden- used as a dye for paper and as a pigment for ball-
sation of N,N-dimethylaniline with Michler’s point pen and printer ink, paint, plastic, gasoline,
ketone (4,4-bis(dimethylamino)benzophenone), varnish, oil, and wax (Gessner & Mayer, 2000;
which is an intermediate generated from the ECHA, 2012; Mani & Bharagava, 2016). Gentian
reaction of carbonyl dichloride (phosgene) with violet can be used in food-packaging materials.
dimethylaniline (ECHA, 2012; Cooksey, 2017). Gentian violet is used to mark locations on the
Gentian violet can also be generated from the skin for body piercings (Skellie, 2020) and has
oxidation of leucogentian violet. In a “one-pot” also been used as a hair dye (Diamante et al.,
45
2009). [The Working Group noted that more blood transfusion-associated Chagas disease,
than 100 posts and videos can be found online and Leishmania), nematodes (pinworms), and
describing the use of gentian violet as a cheap some viral infections (oral hairy leukoplakia),
source of home-made hair dye.] and may contribute to the inhibition of angio-
Gentian violet is used in clinical and bacteri- genesis and tumour growth (Maley & Arbiser,
ological laboratories as a stain for biological spec- 2013). The antimicrobial properties of gentian
imens, because it permits visualization of cellular violet also have applications in veterinary medi-
and histological morphology, and to distinguish cine. Gentian violet has been used in poultry feed
Gram-positive from Gram-negative bacteria; to inhibit the growth of moulds and fungi, as a
gentian violet is the primary purple stain used in topical treatment for bacterial and fungal infec-
the Gram staining method (Boyanova, 2018). It is tions of the skin and eyes in livestock, and as an
used in surgery as a skin-marking dye (Granick immersion-bath treatment for fungal and para-
et al., 1987) and in chromoendoscopy to stain sitic infections in fish, including Ichthyophthirius
the gastrointestinal tract to distinguish lesions multifiliis, the protozoan that causes white spot
from normal tissue (Singh et al., 2020). It is used disease (WHO, 2014a). Although gentian violet is
to detect the presence of bacteria in countless restricted for use in aquaculture, it is a common
biological assays and is also a pH indicator, with treatment for diseases in aquarium fish. Gentian
a colour change from yellow at pH 0.0 to blue-vi- violet is also used in aerosol sprays, in combi-
olet at pH 2.0 (Cooksey, 2017). nation with antibiotics or insecticides, for the
The antibacterial, antifungal, and anthel- treatment of skin and hoof diseases in animals
mintic properties of gentian violet have resulted (Christodoulopoulos, 2009; Mutebi et al., 2016).
in numerous applications in medicine (Maley
& Arbiser, 2013). As a topical treatment, 1.2.2 Leucogentian violet
gentian violet is effective against Gram-positive
bacteria including Staphylococcus aureus and (a) Production process
Streptococcus, and has been used for the treat- Leucogentian violet is produced by the
ment of eczema, impetigo, and to prevent infec- condensation of formaldehyde with N,N-
tion and promote the healing of wounds, burns, dimethylaniline to form 4,4′-methylene-bis
inflammation resulting from radiotherapy, and (N,N-dimethylaniline), which is reacted with
the umbilical stumps of infants. Importantly, additional N,N-dimethylaniline to yield the
gentian violet has been effectively used to treat leuco base of gentian violet (Gessner & Mayer,
methicillin-resistant Staphylococcus aureus 2000).
infections of the dermis, middle ear, chest
cavity, nostrils, and vascular grafts. For decades, (b) Production volume
washing affected areas with a dilute solution of Leucogentian violet is manufactured in and/
gentian violet has been used to treat fungal infec- or imported to the European Economic Area in
tions; notably, oral, oesophageal, vulvovaginal a volume of between 1 and 10 tonnes per annum
(Watson & Calabretto, 2007), nipple, and cath- (ECHA, 2020b). In 2020, leucogentian violet was
eter infections caused by Candida. Coating inva- available from 22 suppliers in China, 5 suppliers
sive medical devices (e.g. catheters) with gentian in the USA, 2 suppliers in India, and 1 supplier
violet reduces the adherence of pathogenic in Canada (Chemical Register, 2020b). [Data on
organisms to biofilms, which may lead to infec- quantities produced and used elsewhere in the
tion. Finally, gentian violet has been used against world were not found by the Working Group.]
protozoa (e.g. Trypanosoma cruzi, which cause
46
(c) Uses Leucogentian violet is a metabolite resulting

Leucogentian violet is used as a precursor in from the veterinary use of gentian violet for the
the production of gentian violet dye (Gessner & treatment of fish and poultry. Residues of leuco-
Mayer, 2000). Leucogentian violet has been used gentian violet may be found in fatty muscle and
as a chromogenic reagent for several analytical skin (WHO, 2014a).
applications. Leucogentian violet is colourless
and reacts quickly with oxidizers and free radi- 1.3 Methods of detection and
cals to yield gentian violet, which is strongly quantification
coloured. The reaction can be readily observed
by visualization or spectrophotometric analysis. Representative methods for the detection and
Leucogentian violet is used in forensic analysis quantification of gentian violet and leucogentian
to enhance blood-impression evidence from violet are summarized in Table 1.1.
fingerprints and footwear. Fixation with a
5-sulfosalicylic acid solution denatures proteins 1.3.1 Air
in the blood, allowing leucogentian violet to react
with haem on the surface of the print. In the No methods for the detection and quanti-
presence of hydrogen peroxide, haem catalyses fication of gentian violet or leucogentian violet
the oxidation of leucogentian violet to gentian particulates in air were found.
violet, producing the characteristic purple
colour that results in enhanced print visualiza- 1.3.2 Water
tion (Spence & Asmussen, 2003; Bossers et al., Gentian violet is measured in water for envi-
2011). Although other forensic dyes react with ronmental monitoring and to determine the
proteins and amino acids, the haem-sensitive efficiency of physical, chemical, and biological
reaction of leucogentian violet indicates the pres- methods to remove, decolourize, or degrade
ence of blood. In analytical chemistry, the oxida- gentian violet in wastewater (Mani & Bharagava,
tion reaction of leucogentian violet to gentian 2016). Ultraviolet-visible absorbance techniques
violet has been used for sensitive spectrophoto- are commonly used to measure the reduction
metric determination of hypochlorite, hydrogen of the purple colour from highly concentrated
peroxide, iodine/iodide, and metals (Borges & wastewater samples, while liquid chromatog-
Reis, 2011). In a method for antimony determi- raphy with spectroscopic or mass spectrometry
nation, based on the reaction of antimony (III) detection is a more sensitive technique (Tkaczyk
with potassium iodate under acidic conditions et al., 2020). For residue analysis in environ-
to generate iodine, iodine oxidizes leucogentian mental water samples, pre-treatment procedures
violet to enable colorimetric detection (Tiwari are required to concentrate gentian violet resi-
et al., 2006). Leucogentian violet has also been dues before analysis. Magnetic, ionic liquid,
used as a radiochromic indicator to enable the nanoparticle material, and microextraction
measurement of radiation exposure by dosime- techniques such as magnetic solid-phase extrac-
ters. Free radical production from gamma-radi- tion, dispersive liquid−liquid microextraction,
ation on a matrix can cause radiolytic oxidation micro-cloud point extraction, and monolithic
of leucogentian violet, which generates a visible fibre-based solid-phase microextraction have
measure of radiation exposure (Dhevi et al., been used to isolate gentian violet residues from
2020). aqueous samples before analysis, with detec-
tion limits ranging from 0.03 to 5 μg/L (Šafařík
47
48

Table 1.1 Representative methods for the detection and quantification of gentian violet and leucogentian violet in various
matrices
Sample matrix Sample preparation Analytical Agent LOD Reference

technique (unless otherwise stated)
Water
Drinking- and river Magnetic SPE Vis GV 0.5–1.0 μg/L Šafařík & Šafaříková
water spectrophotometry (2002)
Pond and effluent water TC-IL-DLLME using 1-octyl- HPLC-UV GV 0.030 μg/L Zhang et al. (2012)
3-methylimidazolium
hexafluorophosphate
Waste- and tap water MCPE using Triton X-114 UV–vis GV 5.1 μg/L Ghasemi & Kaykhaii
spectrophotometry 17.6 μg/L (LOQ) (2016)
Aquaculture water Monolithic fibre SPME, HPLC-vis/FLD GV 0.14 μg/L Wang et al. (2015)
evaporation, and reconstitution in 0.46 μg/L (LOQ)
methanol LGV 0.013 μg/L
0.043 μg/L (LOQ)
Soil
River sediment and soil Soxhlet extraction with GC-MS LGV NR Nelson & Hites (1980)
2-propanol
Food
Dried tofu, chili sauce, Extraction with MeOH/ACN, LC-MS/MS GV 0.03 μg/kg Hu et al. (2020)
seafood sauce, and purification with d-SPE using 0.09 μg/kg (LOQ)
tomato sauce PSA, GCB, alumina, and C18
filtration
Beef, pork, chicken, egg, Extraction with ACN/acetic LC-MS/MS GV, LGV 2 μg/kg (LOQ) Park et al. (2020)
milk, flatfish, eel, and acid, anhydrous sodium sulfate,
shrimp purification with d-SPE using C18
and PSA filtration
Trout and shrimp Extraction with HAH, ACN/ LC-MS/MS GV 0.15 µg/kg (CCα) Eich et al. (2020)
ascorbic acid, anhydrous 0.19 µg/kg (CCβ)
magnesium sulfate, and heated LGV 0.27 µg/kg (CCα)
ultrasonic treatment 0.43 µg/kg (CCβ)
Trout, salmon, and Extraction with ACN, magnesium LC-MS/MS Sum of 0.02 µg/kg (CCα) Dubreil et al. (2019)
prawns sulfate, filtration, oxidation GV + LGV
with DDQ, evaporation, and
reconstitution in ACN/ascorbic
acid
Fish blood and extracts Extraction with ACN, alumina- SALDI-TOF-MS GV 0.1 pg/mL Gao et al. (2019)
SPE, and TiO2 nanoflake
dispersion
Table 1.1 (continued)
Trout, salmon, catfish, Extraction with HAH, ACN, LC-MS/MS GV < 0.5 μg/kg Andersen et al. (2018)
tilapia, shrimp, Arctic magnesium sulfate, evaporation, < 1.0 µg/kg (LOQ) Hurtaud-Pessel et al. (2011)
char, barramundi, eel, reconstitution in ACN/ascorbic 0.13 µg/kg (CCα)
frog legs, hybrid striped acid, and filtration 0.17 μg/kg (CCβ)
bass, pompano, scallops, LGV < 0.5 μg/kg
sea bream, smoked < 1.0 µg/kg (LOQ)
trout, dried shrimp, 0.42 μg/kg (CCα)
and highly processed 0.54 μg/kg (CCβ)
canned eel and dace
products; the canned
products contained oil,
salt, sugar, flavourings,
spices, sauces, and/or
preservatives
Trout, shrimp, humpback Extraction with ACN and water, HPLC-HR-TOF-MS GV 0.01 μg/L Amelin et al. (2017)
salmon, carp, mackerel, and filtration 0.04 μg/L (LOQ)
caviar, and crawfish LGV 0. 1 μg/L
0.4 μg/L (LOQ)
Eel Extraction with ACN, sodium LC-MS/MS Sum of < 0.01 μg/kg Reyns et al. (2014)
acetate, oxidation with DDQ, GV + LGV 0.25 μg/kg (LOQ)
evaporation, McIlvaine buffer
pH 6.5/ACN, CBA and SCX-SPE,

evaporation, reconstitution in
ammonium acetate buffer pH 4.5/
ACN, and filtration
Salmon and shrimp Extraction with citrate buffer/ GV 0.248 μg/kg (CCα) Ascari et al. (2012)
ACN, LLE with dichloromethane, 0.335 μg/kg (CCβ)
SCX-SPE, filtration, post-column LGV (detected 0.860 μg/kg (CCα)
oxidation with PbO2 as GV) 1.162 μg/kg (CCβ)
Silver carp, crucian carp, Extraction with HAH/p-TSA/ UPLC-MS/MS GV 0.15 μg/kg Xu et al. (2012)
tilapia, mandarin fish, ammonium acetate/ACN, LLE 0.50 μg/kg (LOQ)
bream, and sea cucumber with dichloromethane, diethylene LGV 0.15 μg/kg
glycol, ACN, evaporation, 0.50 μg/kg (LOQ)
reconstitution in ACN, MCAX-
SPE, evaporation, reconstitution
in ammonium acetate/ACN/
formic acid, and filtration
49
50

Salmon Extraction with ammonium LC-MS/MS Sum of 1.4 μg/kg (CCα) Tarbin et al. (2008)
acetate buffer pH 4.5, ACN, GV + LGV 2.4 μg/kg (CCβ)
d-SPE with alumina, LLE with
dichloromethane, formic acid,
oxidation with DDQ, and SCX-
SPE
Biospecimens
Human urine SPE HPLC-ECD GV 0.5 μg/L Sagar et al. (1995)
ACN, acetonitrile; CBA, cation exchange cartridges; CCα, decision limit: the concentration level at which there is probability α (usually defined as 1% for non-authorized substances)
that a blank sample will give a signal at this level or higher; CCβ, detection capability: the concentration level at which there is a probability β (usually defined as 5%) that the method
will give a result lower than CCα; DDQ, 2,3-dichloro-5,6-dicyanobenzoquinone; d-SPE, dispersive solid-phase extraction; ECD, electrochemical detection; GC, gas chromatography;
GCB, graphitized carbon black; GV, gentian violet; HAH, hydroxylamine hydrochloride; HPLC, high-performance liquid chromatography; HR-TOF, high-resolution quadrupole
time-of-flight; LC, liquid chromatography; LGV, leucogentian violet; LLE, liquid–liquid extraction; LOD, limit of detection; LOQ, limit of quantification; MCAX, mixed-mode cation
exchange; MCPE, micro-cloud point extraction; MeOH, methanol; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NR, not reported; PbO2, lead (II) oxide; PSA, primary
secondary amine; p-TSA, para-toluenesulfonic acid; SALDI-TOF, surface-assisted laser desorption/ionization time-of-flight; SCX, strong cation exchange; SPE, solid-phase extraction;
SPME, solid-phase microextraction; TC-IL-DLLME, temperature-controlled ionic liquid dispersive liquid–liquid microextraction; TiO2, titanium dioxide; UPLC, ultra-performance
liquid chromatography; UV, ultraviolet; vis, visible light; vis/FLD, visible light and fluorescence detection.
& Šafaříková, 2002; Zhang et al., 2012; Wang (Verdon & Andersen, 2017). Liquid chromatog-
et al., 2015; Ghasemi & Kaykhaii, 2016; Moradi raphy-tandem mass spectrometry (LC-MS/MS)
Shahrebabak et al., 2020). methods have largely replaced HPLC to meet
low-concentration regulatory monitoring levels
1.3.3 Soil (e.g. 0.5 μg/kg) for direct quantification of the
dye and leuco ions (Hurtaud-Pessel et al., 2011;
Leucogentian violet has been identified in Xu et al., 2012; Andersen et al., 2018; Eich et al.,
soil near waste discharged from a dye-manu- 2020). Some multiresidue LC-MS/MS methods
facturing plant by means of Soxhlet extraction for the detection of therapeutic dyes in seafood
with 2-propanol and analysis by gas chroma- include the oxidation of leuco compounds with
tography-tandem mass spectrometry (Nelson & 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to
Hites, 1980). ensure that dye metabolites are also detected
(Tarbin et al., 2008; Andersen et al., 2009; Reyns
1.3.4 Food, beverages, and consumer et al., 2014; Dubreil et al., 2019). A method has
products been developed to extract gentian violet and
Gentian violet is not permitted for use as leucogentian violet from zebrafish using a solid-
a food additive, but numerous methods have phase microextraction probe, which detects
been developed to determine residues of gentian residues via direct ionization mass spectrometry
violet and its metabolite, leucogentian violet, in from the probe (Xiao et al., 2020). [The Working
animal products as a result of veterinary treat- Group noted that the novel method employed in
ment with gentian violet (WHO, 2014a; Verdon the study of zebrafish (which are not typically
& Andersen, 2017). In gentian violet-treated fish, eaten) could have applicability in fish species that
the major metabolite (leucogentian violet) has a are consumed by humans.] Additional multidye
longer residence time (> 79 days) than gentian LC-MS/MS methods that include sensitive quan-
violet (~5 days) in fish (Thompson et al., 1999). tification of gentian violet (0.09–2 μg/kg) have
Thus, leucogentian violet is the marker residue been applied to the analysis of foods such as
used to monitor gentian violet use in aquacul- dried tofu and sauces (Hu et al., 2020), and beef,
ture, and seafood analysis methods must assess chicken, pork, eggs, and milk (Park et al., 2020).
both compounds. Many early methods of residue High-resolution mass spectrometry has also
analysis were based on the extraction of muscle been used for the detection and quantification of
with an acidic buffer and acetonitrile, liquid− gentian violet and leucogentian violet (Amelin
liquid partitioning, and solid-phase clean-up et al., 2017).
with alumina, followed by high-performance Surface-enhanced Raman scattering and
liquid chromatography (HPLC) separation direct mass spectrometry techniques have also
(Roybal et al., 1990). Several approaches have been used to detect gentian violet. Silver nano-
been used to enable the detection of both the particle films and pastes have been used to detect
chromatic dye and the colourless leuco base, gentian violet on the surface of fish skin and in
including electrochemical detection, post- ballpoint pen ink (Alyami et al., 2019; Saviello
column oxidation of leucogentian violet with et al., 2019). A surface-assisted laser desorption/
lead oxide (Ascari et al., 2012), and simulta- ionization mass spectrometry method has been
neous visible (gentian violet absorbs at 588 nm) used to analyse gentian violet in printed super-
and fluorescence (leucogentian violet excitation market receipts (Gao et al., 2019).
at 265 nm with emission at 360 nm) detection
51
1.3.5 Biological specimens in the environment.] Particulate-phase gentian

violet is removed from the atmosphere by wet
Methods for the detection and quantifica- and dry deposition and may be susceptible to
tion of gentian violet and leucogentian violet in direct photolysis by sunlight. Gentian violet is
human biological specimens are similar to those expected to be immobile if released into soil. Soils
used for food (as described in Section 1.3.4). containing organic carbon and clay will adsorb
Gentian violet and leucogentian violet have gentian violet’s cationic form more strongly than
been determined in human urine via extrac- its neutral counterpart. Volatilization from moist
tion of neutralized urine with dichloromethane, soil is not expected. According to the transfor-
extract clean-up with sodium sulfate, and mation rates observed during a river die-away
analysis by HPLC with absorbance or electro- test, biodegradation may be an important envi-
chemical detection (Sagar et al., 1995). [The ronmental process in soil and water. If released
Working Group noted that the methods used into water, gentian violet is expected to adsorb
for gentian violet and leucogentian violet detec- on suspended solids and sediment, and the
tion in fish described in Section 1.3.4 could be non-adsorbed fraction will exist almost entirely
useful for analysing material from humans or in the cationic form; therefore, volatilization
experimental animals. For biological specimen from water is not expected. Gentian violet is not
analysis, it might be more important to monitor expected to undergo hydrolysis in the environ-
N-demethylated and/or N-oxide metabolites of ment (NCBI, 2013).
gentian violet and leucogentian violet.] Leucogentian violet was detected in a soil
sample taken near a bank of the Buffalo River,
1.4 Occurrence and exposure New York, close to a dyestuff-manufacturing
plant (Nelson & Hites, 1980). Theoretical estima-
1.4.1 Environmental occurrence tions of concentrations of non-sulfonated triaryl-
Gentian violet is not known to occur natu- methane dyes in surface water (also representing
rally in the environment. Gentian violet and drinking-water) were calculated for three indus-
leucogentian violet production and their use trial sources in Canada based on the maximum
(e.g. during the production of ink cartridges production capacities of these industries:
and coloured paper, and during the recycling of 3.2 × 10−4 mg/L from the paper-dyeing industry,
printed paper) may result in the release of these 9.5 × 10−4 mg/L from the de-inking industry,
compounds into the environment via streams and 2.1 × 10−4 mg/L from the general formu-
of both industrial and municipal wastewater lation industry. These conservative estimates
(Health Canada, 2020; Tkaczyk et al., 2020). were made for gentian violet, malachite green,
When released into the environment, gentian and two other triarylmethane dyes collectively,
violet exists in cationic form. Considering its assuming that any one of the four dyes could be
physicochemical properties, gentian violet exists substituted for another (Health Canada, 2020).
only in the particulate phase in the atmosphere. In the National Water Pollution Control and
[The Working Group also noted that the water Treatment Project in Dong Lin, China, gentian
solubility of gentian violet is several orders of violet concentrations of 0.87 and 0.049 µg/L
magnitude higher than that of leucogentian were found in the water from turtle farming
violet and that the octanol/water partition coef- ponds and effluent environmental water, respec-
ficient of gentian violet is one order of magni- tively (Zhang et al., 2012). Gentian violet absorbs
tude higher, which has implications for its fate light at an ultraviolet maximum of 590 nm with
potential for direct photolysis. In water, the
52
photoreaction is reported to give para-dimeth- residue (Thompson et al., 1999). [The Working
ylamino phenol and 4,4′-bis(dimethylamino) Group noted that in the reports described below,
benzophenone, the leuco and demethylated the methods either detected gentian violet and
derivatives of gentian violet. The bioconcentra- leucogentian violet separately, or detected total
tion in aquatic organisms is low, as suggested by residues as the sum of gentian violet and leuco-
the estimated bioconcentration factor of 3 L/kg gentian violet after leucogentian violet had been
in fish (NCBI, 2013), but such models may not be oxidized to gentian violet.]
appropriate for triarylmethane dyes because of According to the European Food Safety
their cationic nature. For these triarylmethanes, Authority reports published between 2015 and
partitioning to proteins in the cell membranes is 2020, few Member States (one to four) reported
more likely to occur than partitioning to lipids one or two samples that were non-compliant
(Health Canada, 2020). for the presence of gentian violet and leuco-
A study was performed to analyse the pres- gentian violet in their national veterinary drug
ence of 16 dyes, which included triarylmethanes residue control plan (EFSA, 2015, 2016, 2017,
and their metabolites such as gentian violet and 2018, 2019, 2020). In the European Rapid Alert
leucogentian violet, in wild fish in Belgium. System for Food and Feed, very few notifi-
Muscle samples were analysed from individual cations of non-compliant samples associated
yellow-phased European eels (Anguilla anguilla) with imports or trade between Member States
from 91 locations in rivers, canals, and lakes have been reported. Since 2005, 15 notifications
sampled between 2000 and 2009. Gentian of gentian violet or leucogentian violet residue
violet and leucogentian violet were detected violations have been made by EU Member States
in samples from 58.2% and 50.5% of the loca- in eel, salmon, tilapia, rainbow trout, catfish,
tions, respectively. The concentrations of gentian pangasius, and sturgeon (caviar). Residue
violet and leucogentian violet ranged between concentrations have typically ranged from 0.8
0.12 and 2.60 µg/kg (Belpaire et al., 2015). In an to 6.6 µg/kg, although two high-concentration
earlier study conducted in Germany, gentian (41.1 and 654.6 µg/kg) samples were reported
violet and leucogentian violet were found in for eel from Indonesia in 2006 (European
tissue samples from wild eels caught in seven out Commission, 2020).
of eight receiving waters of effluents from munic- In a study of processed fish and shrimp
ipal sewage treatment plants. The concentrations samples in Korean local markets, gentian
of gentian violet and leucogentian violet ranged violet was detected (168.4 µg/kg) in 1 of 67 eel
from 0.06 to 6.71 µg/kg (Schuetze et al., 2008). samples tested. It was not detected in the other
186 processed fish and shrimp samples, which
1.4.2 Occurrence in food and feed originated from the Republic of Korea, China,
Thailand, Viet Nam, Norway, Peru, and the
Gentian violet is used in veterinary medicine Russian Federation, or were of unknown origin
and in the aquaculture industry for the control of (Lee et al., 2010). Among fish obtained from
ectoparasites, and fungal and bacterial infections. a local market in China, 7.15 µg/kg of gentian
Residues of both gentian violet and leucogentian violet was detected in tilapia; none was detected
violet may be present in muscle and skin after in carp, sea cucumber, or seashell (Xu et al.,
gentian violet treatment. Although gentian violet 2012). Among 20 salmon and shrimp samples
metabolizes within days of treatment, leuco- purchased from different markets in China,
gentian violet persists in fish muscle and skin 1.2 µg/kg of gentian violet and 2.5 µg/kg of
for months and is considered to be the marker leucogentian violet were detected in one salmon
53
muscle sample (Tao et al., 2011). Leucogentian 1.4.4 Exposure in the general population
violet (0.6–1.0 µg/kg) was detected in 5 out of
208 samples of rainbow trout obtained from The predominant source of exposure to dye
local fish retailers and supermarkets in Turkey substances in the triarylmethanes group is from
(Kaplan et al., 2014). In the Russian Federation, the use of products that contain them that are
5.3 µg/kg of gentian violet was detected in black available to consumers (Health Canada, 2020).
caviar (Amelin et al., 2017). Gentian violet and Exposure of the general population can poten-
leucogentian violet residues have also been tially occur during the use of the consumer
reported for samples tested in the USA, Canada, products described in Section 1.1.2, such as ball-
and Jordan (Table 1.2; WHO, 2014a; Gammoh point and marker pens (orally by sucking or via
et al., 2019). In the EU and USA, respectively, 3% dermal contact), topical treatments for animals
and 6% of reported veterinary drug violations (inhalation or dermal), coloured paper, hair
detected in finfish in 2001–2008 and 2001–2006, dye, aquarium fish treatments, or through the
respectively, were due to the detection of gentian consumption of contaminated drinking-water or
violet. The concentrations detected in the EU residue-containing fish (Table 1.2). A screening
and the USA did not differ (Love et al., 2011). assessment performed by Health Canada
In a screening study of 19 commercially avail- suggested exposure via drinking-water to be
able processed animal products (salmon feed the main route of exposure to gentian violet. A
ingredients) from central Europe, leucogentian potential dose of 0.0001 mg/kg body weight (bw)
violet was detected in one poultry blood-meal per day was estimated for the Canadian general
sample (Nácher-Mestre et al., 2016). population on the basis of predicted surface
water concentrations as a result of environmental
release by the paper de-inking industry. Other
1.4.3 Occupational exposure exposure scenarios considered, but not included
Occupational exposure to gentian violet is in the estimation because of lower estimated
expected to occur via dermal contact during exposures, were surface water due to industrial
paper dyeing, via inhalation of dust or aerosols release from paper dyeing in paper mills and
produced during the formulation of dye or ink, production facilities, and consumer “down-the-
or during the filling of containers such as ink drain” releases, consumption via food, and the
cartridges and ballpoint pens (ECHA, 2012). [The use of consumer products such as paper prod-
Working Group noted that occupational expo- ucts, mixtures, or manufactured items in which
sure to gentian violet and leucogentian violet may gentian violet is used as a pigment (Health
occur through dermal contact and inhalation at Canada, 2020).
workplaces where the compounds are produced [The Working Group noted that despite the
or applied (see Sections 1.1.2 and 1.2.2).] In a multitude of sources, no quantitative exposure
survey conducted in the USA in 1981–83, 75 632 data were available.]
people were estimated to be potentially occupa-
tionally exposed to gentian violet: 69% of them
1.5 Regulations and guidelines
working in health services, 12% in printing
and publishing, and 8% in agricultural services 1.5.1 Exposure limits and guidelines
(NIOSH, 2017). [The Working Group noted that
it is unclear whether these percentages reflect Gentian violet is listed by the European
modern exposure patterns, given the age of the Chemicals Agency as a carcinogen (Category 2)
study.] and as a carcinogen (Category 1B) when the
54
Table 1.2 Detection and quantification of gentian violet and leucogentian violet in aquaculture products available on the
international marketa
Country Country of Agent Year Sample type No. of samples No. of positive Concentration (µg/kg) Reference
reported origin tested samples (%)
Mean ± SD Range
Canada – GV and LGV 2008−2009 Tilapia, salmon, 135 6 (4.4) 2.48 ± 2.32 0.64–5.60 WHO (2014a)
and shrimp
GV and LGV 2009−2010 NA 484 0 NA NA
GV and LGV 2010−2011 Tilapia, perch, 542 11 (2.0) 1.92 ± 1.69 0.50–4.30
shrimp, milkfish,
and catfish
GV and LGV 2011−2012 Bass and prawn 396 2 (0.5) 2.23 ± 2.02 0.80–3.65
GV and LGV 2012−2013 Perch and dried 269 3 (1.1) 3.06 ± 2.07 0.98–5.12
fish maw
USA – GV and LGV 2004 NA 622 0 NA NA WHO (2014a)
GV and LGV 2005 NA 536 0 NA NA
GV and LGV 2006 NA 588 0 NA NA
GV and LGV 2007 Eel, catfish, and 686 3+ (0.4)b NR 2.5–26.9
shrimp
Jordan Viet Nam GV Pangasius 27 17 (62) 11.7 0.362–41.3 Gammoh
LGV Pangasius 27 5 (18) 5.26 0.178–10.58 et al. (2019)
United Arab GV Pangasius 27 8 (29) 4.4 0.945–10.6
Emirates LGV Pangasius 27 NA NA NA
China GV Tilapia 27 11 (40) 4.6 1.24–9.48

LGV Tilapia 27 2 (7) 2.1 1.29–2.81
Argentina GV Argentine hake 20 NA NA NA
LGV Argentine hake 20 NA NA NA
USA GV Pacific hake 20 NA NA NA
LGV Pacific hake 20 NA NA NA
All GV Pangasius, tilapia, 121 36 (30) 6.9 0.362–41.3
countries Argentine hake,
above, and Pacific hake
reported by LGV Pangasius, tilapia, 121 7 (5.7) 3.2 0.178–10.58
Jordan Argentine hake,
and Pacific hake
55
56

Country Country of Agent Year Sample type No. of samples No. of positive Concentration (µg/kg) Reference
reported origin tested samples (%)
Mean ± SD Range
Republic of China Sum of GV + Eel 7 1 (14) 168.4 NA Lee et al.
Korea Republic LGV Fish and shrimp 246 0 NA NA (2010)
of Korea,
China,
Thailand,
Viet Nam,
Norway,
Peru, the
Russian
Federation
China China GV Tilapia NR NR 7.15 NR Xu et al.
GV Carp, sea NR 0 NA NA (2012)
cucumber, and
seashell
China China GV Salmon and 20 1 (5) 1.2 NA Tao et al.
LGV shrimp 1 (5) 2.5 NA (2011)
Turkey Turkey LGV Rainbow trout 208 5 (2.4) [0.70] 0.52–1.0 Kaplan et al.
(2014)
Russian Russian GV Sturgeon caviar 1 1 (NA) 5.3 NA Amelin et al.
Federation Federation? (2017)
GV, gentian violet; LGV, leucogentian violet; NA, not applicable; NR, not reported; SD, standard deviation.
a Monitoring of gentian violet and leucogentian violet by Canada and the USA, in frozen fish imported to Jordan, and in aquaculture products sold in local markets in China, the
Republic of Korea, Turkey, and the Russian Federation.

b Probably an underestimate.
Michler’s ketone or Michler’s base impurity is or Singapore (Health Canada, 2018; NZ EPA,
present at 0.1% or more (ECHA, 2012). It is classi- 2019; HSA, 2020). United States Food and Drug
fied as a substance of very high concern (ECHA, Administration regulations require that hair
2012). Gentian violet is very toxic to aquatic life dyes containing gentian violet are accompanied
(acute H400 and chronic H410), is harmful if by a cautionary statement for skin and eye irri-
swallowed (H302), causes serious eye damage tation, with instructions to perform a skin patch
(H318), and is suspected of causing cancer (H350) test before use (Diamante et al., 2009).
(ECHA, 2020a). No stand-alone regulations were found for
The Joint Food and Agriculture Organization leucogentian violet.
of the United Nations/WHO Expert Committee
on Food Additives (JEFCA) concluded that there 1.5.2 Reference values for biological
is no acceptable daily intake or maximum residue monitoring of exposure
limit for gentian violet and its marker leucogen-
tian violet (WHO, 2014a). Gentian violet is not No reference values for biological monitoring
authorized for use as a veterinary drug in the of gentian violet or leucogentian violet exposure
Australia, Brazil, Canada, Chile, the EU, New were found.
Zealand, or the UK, and there is zero tolerance
for residues of gentian violet in food for human
consumption (Verdon & Andersen, 2017; Health 2. Cancer in Humans
Canada, 2019). In the USA, gentian violet is not
permitted for use in animal feeds or as a veteri- No data were available to the Working Group.
nary drug for food-producing animals (US FDA,
2007). Gentian violet and leucogentian violet are
not permitted for use as food additives or in food 3. Cancer in Experimental Animals
packaging in the USA (US FDA 2020, 2021). In
Canada, gentian violet is not permitted for use 3.1 Gentian violet
in animal feeds or in aquaculture production
(Health Canada, 2018). See Table 3.1.
In food products derived from animals where
gentian violet is prohibited for use, there is zero 3.1.1 Mouse
tolerance for residues of gentian violet and/or
its metabolite leucogentian violet, which is the Oral administration (feed)
marker residue that indicates the use of gentian In a study of chronic toxicity and carcino-
violet (WHO, 2014a). Reference points for action genicity that complied with Good Laboratory
range from 0.5 to 2.0 μg/kg, as determined by the Practice (GLP) (NCTR, 1984; Littlefield et al.,
detection capabilities of the analytical methods 1985), a total of 720 male and 720 female B6C3F1
used in national and international residue moni- mice (age, approximately 4–5 weeks) were given
toring programmes for each compound, or for feed containing gentian violet (purity, 99%;
the sum of gentian violet and leucogentian violet methyl violet, 1%) at a concentration of 0, 100,
residues (Verdon & Andersen, 2017). 300, or 600 ppm [approximately equivalent to
Gentian violet is not permitted for use as a hair 0, 12.5, 33.9, and 66.1 mg/kg bw per day for
dye in the European Economic Area (European males, and 0, 14.3, 37.5, and 71.4 mg/kg bw per
Commission, 2009), and it is not approved for day for females] for the control group and the
any cosmetic use in Canada, New Zealand, groups at the lowest, intermediate, and highest
57
58

Table 3.1 Studies of carcinogenicity with gentian violet in experimental animals
Study design Route Incidence of Significance Comments

Species, strain (sex) Purity tumours
Age at start Vehicle
Duration Dose(s)
Reference No. of animals at start
No. of surviving animals
Full carcinogenicity Oral Liver Principal strengths: complied with GLP;
Mouse, B6C3F1 (M) Purity, 99% (impurity, 1% Hepatocellular adenoma used males and females; adequate duration
~4–5 wk methyl violet) 17/183, 14/92, [P < 0.001, Cochran−Armitage trend test; of exposure and observation; high number of
24 mo Feed 20/93*, 37/93** *P < 0.01, **P < 0.001, one-tailed Fisher mice per group
Littlefield et al. 0, 100, 300, 600 ppm exact test] Other comments: the incidence of
(1985) 192, 96, 96, 96 Hepatocellular carcinoma hepatocellular adenoma or carcinoma
167, 83, 77, 74 (combined) was not reported
27/183, 15/92, P < 0.001, trend test; *P < 0.01, one-tailed
17/93, 33/93* Fisher exact test
Harderian gland: adenoma
7/187, 7/92, *P < 0.05, one-tailed Fisher exact test;
10/94*, 9/89** [**P = 0.0362, one-tailed Fisher exact test];
[NS], Cochran−Armitage trend test
Duration Dose(s)
Full carcinogenicity Oral Liver Principal strengths: complied with GLP;
Mouse, B6C3F1 (F) Purity, 99% (impurity, 1% Hepatocellular adenoma used males and females; adequate duration
~4–5 wk methyl violet) 8/185, 8/93, [P < 0.001, Cochran−Armitage trend test; of exposure and observation; high number of
24 mo Feed 36/93*, 20/95** *P < 0.01, **P < 0.001; one-tailed Fisher mice per group
Littlefield et al. 0, 100, 300, 600 ppm exact test] Other comments: the incidence of
(1985) 192, 96, 96, 96 Hepatocellular carcinoma hepatocellular adenoma or carcinoma
167, 69, 70, 35 (combined) was not reported
7/185, 5/93, [P < 0.001, Cochran−Armitage trend test,
30/93*, 73/95* trend test; *P < 0.001, one-tailed Fisher
exact test]
8/186, 11/93*, P = 0.001, Cochran−Armitage trend test;
18/89**, *P < 0.05, **P < 0.001, ***P < 0.005, one-
15/94*** tailed Fisher exact test
Bladder: reticulum cell sarcoma type A [histiocytic
sarcoma]
0/188, 2/92, [P < 0.005, Cochran−Armitage trend test;
3/89*, 5/91** *P < 0.05, **P < 0.01; one-tailed Fisher
exact test]

Ovaries: reticulum cell sarcoma type A [histiocytic
sarcoma]
0/178, 1/90, [P = 0.009, Cochran−Armitage trend test;
3/89*, 5/89** *P = 0.036, **P = 0.04; one-tailed Fisher
exact test]
Uterus: reticulum cell sarcoma type A [histiocytic sarcoma]
6/90*, 12/93** *P < 0.01, **P < 0.001; one-tailed Fisher
exact test]
Vagina: reticulum cell sarcoma type A [histiocytic sarcoma]
1/182, 1/90, [P = 0.001, Cochran−Armitage trend test;
4/88*, 8/87** *P = 0.04, **P < 0.001; one-tailed Fisher
exact test]
59
60

Duration Dose(s)
Full carcinogenicity Oral Liver Principal strengths: complied with GLP; used
Mouse, B6C3F1 (M) Purity, 99% (impurity, 1% Hepatocellular adenoma males and females
~4–5 wk methyl violet) 3/48, 0/24, [NS] Principal limitations: small number of mice
18 mo Feed 2/24, 2/22 per treated group
Littlefield et al. 0, 100, 300, 600 ppm Hepatocellular carcinoma Other comments: the incidence of
(1985) 48, 24, 24, 24 hepatocellular adenoma or carcinoma
5/48, 1/24, [NS]
NR (combined) was not reported
2/24, 2/22
2/47, 2/24, 2/23, [NS]
0/21
Full carcinogenicity Oral Liver Principal strengths: complied with GLP; used
Mouse, B6C3F1 (F) Purity, 99% (impurity, 1% Hepatocellular adenoma males and females
~4–5 wk methyl violet) 3/47, 0/22, 3/24, [P = 0.002, Cochran−Armitage trend test; Principal limitations: small number of mice
18 mo Feed 8/24* *P = 0.005, one-tailed Fisher exact test] per treated group
Littlefield et al. 0, 100, 300, 600 ppm Hepatocellular carcinoma Other comments: the incidence of
(1985) 48, 24, 24, 24 hepatocellular adenoma or carcinoma
1/47, 0/22, 1/24, [NS]
3/24
2/46, 2/21, 3/23, [NS]
1/23
Uterus: reticulum cell sarcoma type A [histiocytic sarcoma]
0/47, 0/22, 1/24, [NS]
1/24
Bladder: reticulum cell sarcoma type A [histiocytic
sarcoma]
0/47, 1/22, 1/24, [NS]
0/23
Vagina: reticulum cell sarcoma type A [histiocytic sarcoma]
0/46, 0/22, 1/23, [NS]
0/22
Duration Dose(s)
Full carcinogenicity Oral Liver: hepatocellular adenoma Principal strengths: complied with GLP; used
Mouse, B6C3F1 (M) Purity, 99% (impurity, 1% 0/48, 2/24, [NS] males and females
~4–5 wk methyl violet) 0/24, 0/24 Principal limitations: small number of mice
12 mo Feed per treated group
NCTR (1984) 0, 100, 300, 600 ppm Other comments: the incidence of
48, 24, 24, 24 hepatocellular adenoma or carcinoma
Full carcinogenicity Oral Harderian gland: adenoma Principal strengths: complied with GLP; used
Mouse, B6C3F1 (F) Purity, 99% (impurity, 1% 2/48, 0/24, [NS] males and females
~4–5 wk methyl violet) 1/24, 0/24 Principal limitations: small number of mice
12 mo Feed Vagina: reticulum cell sarcoma type A [histiocytic sarcoma] per treated group
NCTR (1984) 0, 100, 300, 600 ppm Other comments: the incidence of
0/45, 1/23, 0/24, [NS]
48, 24, 24, 24 hepatocellular adenoma or carcinoma
0/23

61
62

Duration Dose(s)
Full carcinogenicity Transplacental and Liver: hepatocellular adenoma Principal strengths: complied with GLP;
Rat, F344 (M) perinatal exposure, 1/179, 2/90, P < 0.01, Peto trend test; *P < 0.01, Peto used males and females; adequate duration of
NR (weanling) followed by oral 3/88*, 4/89* test and Bonferroni correction exposure and observation; high number of rats
24 mo administration (feed) One rat at 100 ppm had a hepatocellular per group
NCTR (1988) Purity, 99% (impurity, 1% carcinoma
methyl violet)
Thyroid gland
Feed
Follicular cell adenoma
0, 100, 300, 600 ppm
180, 90, 90, 90 1/163, 0/84, [NS]
121, 60, 47, 55 0/74, 2/79
Follicular cell adenocarcinoma
1/163, 4/84*, P < 0.01, Peto trend test; *P < 0.05,
2/74, 5/79** **P < 0.01, Peto test and Bonferroni
correction
Follicular cell adenoma or adenocarcinoma (combined)
2/74, 7/79* *P < 0.01, one-tailed Fisher exact test]
Testis and epididymis: mesothelioma
3%, 2%, 6%, 9% NR, incidence reported only as percentage
Multiple organs: mononuclear cell leukaemia
104/180, 66/90, NS
69/90, 51/90
Duration Dose(s)
Full carcinogenicity Transplacental and Liver: hepatocellular adenoma Principal strengths: complied with GLP;
Rat, F344 (F) perinatal exposure, 0/170, 1/90, NS used males and females; adequate duration of
NR (weanling) followed by oral 2/83, 1/87 exposure and observation; high number of rats
24 mo administration (feed) Thyroid gland per group
NCTR (1988) Purity, 99% (impurity, 1% Follicular cell adenoma
methyl violet)
1/159, 2/83, [NS]
Feed
3/76, 3/77
0, 100, 300, 600 ppm
180, 90, 90, 90 Follicular cell adenocarcinoma
121, 56, 36, 31 1/159, 1/83, P < 0.01, Peto trend test; *P < 0.05,
4/76*, 6/77** **P < 0.01, Peto test and Bonferroni
correction
Follicular cell adenoma or adenocarcinoma (combined)
7/76*, 9/77** *P < 0.01, **P < 0.001, one-tailed Fisher
exact test]
Multiple organs: mononuclear cell leukaemia
77/171, 38/90, NS

45/87, 40/87
Clitoral gland: adenoma or adenocarcinoma (combined)
12%, 6%, 18%, NR, incidences reported only as
33% percentages
63
64

Duration Dose(s)
Full carcinogenicity Transplacental and Liver: hepatocellular adenoma Principal strengths: complied with GLP; used
Rat, F344 (M) perinatal exposure, 0/15, 1/15, 0/15, [NS] males and females
NR (weanling) followed by oral 0/14 Principal limitations: small number of rats per
18 mo administration (feed) Thyroid gland group
Littlefield et al. Purity, 99% (impurity, 1% Follicular cell adenoma
(1989) methyl violet)
0/15, 0/15, 1/15, [NS]
Feed
1/15
0, 100, 300, 600 ppm
15, 15, 15, 15 Follicular cell adenoma or adenocarcinoma (combined)
NR 0/15, 0/15, 1/15, [NS]
1/15
Testis and epididymis: malignant mesothelioma
0%, 0%, 13%, NR, incidences reported only as
13% percentages
Full carcinogenicity Transplacental and Thyroid gland Principal strengths: complied with GLP; used
Rat, F344 (F) perinatal exposure, Follicular cell adenocarcinoma males and females
NR (weanling) followed by oral 0/15, 1/11, 0/10, [NS] Principal limitations: small number of rats per
18 mo administration (feed) 0/14 group
Littlefield et al. Purity, 99% (impurity, 1% Follicular cell adenoma or adenocarcinoma (combined)
(1989) methyl violet)
0/15, 1/11, 0/10, [NS]
Feed
0/14
0, 100, 300, 600 ppm
15, 15, 15, 15 Multiple organs: mononuclear cell leukaemia
NR 0/15, 2/11, 2/10, [P < 0.05, Cochran−Armitage trend test;
6/14* *P < 0.01, one-tailed Fisher exact test]
F, female; GLP, Good Laboratory Practice; M, male; mo, month; NR, not reported; NS, not significant; ppm, parts per million; wk, week.
dose, respectively, for up to 24 months. The hepatocellular adenoma and of hepatocellular
feed containing gentian violet was certified to carcinoma (both P < 0.001, Cochran–Armitage
be within 10% of the target dose. For the mice trend test), with a significant increase in the
treated for 24 months, there were 192 males incidence of hepatocellular adenoma [P < 0.01
and 192 females in the control group and 96 and P < 0.001, one-tailed Fisher exact test] and
males and 96 females in each group treated of hepatocellular carcinoma (both P < 0.001,
with gentian violet. For the mice treated for one-tailed Fisher exact test) at the intermediate
12 or 18 months, there were 48 males and 48 and highest dose, respectively, when compared
females in the control group and 24 males and with controls. Treatment with gentian violet
24 females in each group treated with gentian caused a significant positive trend in the inci-
violet. Mortality was very low until approxi- dence of Harderian gland adenoma (P = 0.001,
mately 450 days (15 months), after which there Cochran–Armitage trend test), with the incidence
was a significant positive dose-related trend in being significantly higher at the lowest, inter-
males (P = 0.01288, Cochran–Armitage test) mediate, and highest dose [P < 0.05, P < 0.001,
and females (P = 0.00005, Cochran–Armitage and P < 0.005, respectively, one-tailed Fisher
test), with mortality being significantly higher exact test] than in controls. Significant positive
in all treated groups of females compared with trends in the incidence of type A reticulum cell
controls. At study termination, survival was sarcoma [histiocytic sarcoma] were reported for
167/192, 83/96, 77/96, and 74/96 in males, and the urinary bladder, ovaries, uterus, and vagina
167/192, 69/96, 70/96, and 35/96 in females, [P < 0.0005, P = 0.009, P < 0.001, P = 0.001,
for the control group and the groups at the respectively, Cochran–Armitage trend test],
lowest, intermediate, and highest dose, respec- with a significant increase in incidence (urinary
tively. Treatment with gentian violet did not bladder, P < 0.05 and P < 0.01; ovaries, P = 0.036
influence the terminal body weights of males or and P = 0.04; uterus, P < 0.01 and P < 0.001; and
females. Complete necropsies and histopatho- vagina, P = 0.04 and P < 0.001, Fisher exact test) at
logical examinations were performed. the intermediate and highest dose, respectively.
In male mice at 24 months, there was a signif- At 18 months, a significant positive trend in the
icant positive trend in the incidence of hepato- incidence of hepatocellular adenoma (P = 0.002,
cellular adenoma [P < 0.001, Cochran–Armitage Cochran–Armitage trend test) was observed,
trend test] and of hepatocellular carcinoma with the increase being significant (P = 0.005,
(P < 0.001, trend test), with a significant increase one-tailed Fisher exact test) at the highest dose.
in the incidence of hepatocellular adenoma at At 12 months, treatment with gentian violet did
the intermediate and highest dose [P < 0.01 and not cause a significant increase in the incidence
P < 0.001, respectively, one-tailed Fisher exact of tumours in female mice.
test], and of hepatocellular carcinoma at the Regarding non-neoplastic lesions observed at
highest dose [P < 0.01, one-tailed Fisher exact 24 months, exposure to gentian violet caused a
test]. The incidence of Harderian gland adenoma significant positive trend and an increase in the
was also significantly increased at the interme- incidence of erythropoiesis in the spleen and
diate and highest dose (P < 0.05 and [P = 0.0362], atrophy of the ovaries in females treated with
respectively, one-tailed Fisher exact test). At 12 or gentian violet compared with controls. [The
18 months, no treatment-associated neoplasms Working Group noted that this was a well-con-
were reported in males. ducted study that complied with GLP, males and
In female mice at 24 months, there was a females were used, the duration of exposure and
significant positive trend in the incidence of
65
observation was adequate, and a high number of evaluation at 24 months, there were 180 F1 males
mice per group was used.] and 180 F1 females in the control group and 90
F1 males and 90 F1 females in each dose group.
3.1.2 Rat For the interim evaluation at 12 or 18 months,
there were 15 F1 males and 15 F1 females in each
(a) Oral administration group. Mortality was significantly increased in
In a study in rats [age and strain not reported], male rats at the intermediate dose, and there was
oral administration [regimen not reported] of a significant dose-related increase in mortality in
4:4′:4′′-hexamethyltriaminotriphenylmethane female rats, with the increase in mortality being
[gentian violet, purity not reported] for more significant for females at the intermediate and
than 300 days caused gastric papilloma and highest dose. Survival was 121/180, 60/90, 47/90,
adenomatous proliferation in the hepatic tissue and 55/90 in males, and 121/180, 56/90, 36/90 and
(Kinosita, 1940). [The Working Group noted 31/90 in females, for the control group and the
that the study lacked details on study design and groups at the lowest, intermediate, and highest
primary data and was considered inadequate for dose, respectively. At 24 months, the terminal
the evaluation of the carcinogenicity of gentian body weights of male and female rats receiving
violet in experimental animals.] gentian violet at the highest dose were signifi-
cantly lower than those of the controls [with the
(b) Transplacental and perinatal exposure, final mean body weights being 92% and 86% of
followed by oral administration (feed) those of the male and female control rats, respec-
In a study of chronic toxicity and carcino- tively]. Complete necropsies and histopatholog-
genicity that complied with GLP (NCTR, 1988; ical examinations were performed.
Littlefield et al., 1989), groups of male and female In male rats at 24 months, there was a signif-
Fischer 344 rats (F0 generation) (180 controls icant positive trend in the incidence of hepato-
and 90 treated rats per group) were given feed cellular adenoma (P < 0.01, Peto trend test), with
containing gentian violet (purity, 99%; methyl incidence being significantly increased at the
violet, 1%) at a concentration of 0, 100, 300, or intermediate and highest dose (both P < 0.01,
600 ppm, for the control group, and the groups Peto test and Bonferroni correction). Such a
at the lowest, intermediate, and highest dose, significant positive trend was also observed for
respectively, for at least 80 days. While still the incidence of follicular cell adenocarcinoma of
receiving treated feed, female rats were mated the thyroid gland (P < 0.01, Peto trend test), with
with males that were receiving the same doses of the incidence being significantly increased in rats
gentian violet. Two offspring (F1 generation) of at the lowest and the highest dose (P < 0.05 and
each sex were randomly selected from each litter P < 0.01, respectively, Peto test and Bonferroni
and three rats allocated per cage as weanlings correction). There was a significant positive
[age, not reported] to the study of chronic toxicity trend in the incidence of follicular cell adenoma
and carcinogenicity. The F1 rats were exposed to or adenocarcinoma (combined) of the thyroid
the same doses as their respective F0 parents for gland [P < 0.05, Cochran–Armitage trend test],
up to 24 months. [These dose levels were approx- with incidence being significantly increased at
imately equivalent to 0, 4.3, 11.4, and 22.9 mg/kg the highest dose [P < 0.01, one-tailed Fisher exact
bw per day for male F1 rats, and 0, 5.7, 14.3, and test]. Mesothelioma of the testis or epididymis
28.6 mg/kg bw per day for female F1 rats.] The was observed with an incidence of 3%, 2%, 6%,
feed containing gentian violet was certified to be and 9% in the control group and in the groups
within 10% of the target dose. For the interim receiving the lowest, intermediate, and highest
66
dose, respectively [statistical analysis of the inci- test). At 12 months, no treatment-associated

dence of mesothelioma could not be performed, neoplasms were reported in females.
because the incidence was not reported as the Regarding non-neoplastic lesions observed
number of rats with lesions per number of rats at 24 months, most were reported in the liver.
examined microscopically]. At 12 or 18 months, Gentian violet caused a significant positive trend
treatment did not cause a significant increase in in the incidence and an increase in the incidence
the incidence of tumours in male rats. However, of hepatocyte regeneration and of mixed cell foci
mesothelioma of the testis or epididymis was in all treated groups of male and female rats.
observed at 18 months with an incidence of 0%, Other lesions listed below also showed at least a
0%, 13%, and 13% in the control group and in significant positive trend in incidence, with inci-
groups at the lowest, intermediate, and highest dence being significantly increased in one or two
dose, respectively [statistical analysis of the inci- dose groups. In males, these other non-neoplastic
dence of mesothelioma could not be performed lesions included clear cell foci, eosinophilic foci,
because the incidence was not reported as the basophilic foci, cytoplasmic vacuolization, and
number of rats with lesions per number of rats centrilobular necrosis of the liver, follicular cysts
examined microscopically]. of the thyroid gland, red pulp hyperplasia of the
In female rats, at 24 months, there was a spleen, and hyperplasia of the mesenteric lymph
significant positive trend in the incidence of nodes. In females, these other non-neoplastic
follicular cell adenocarcinoma of the thyroid lesions included eosinophilic foci, haematopoi-
gland (P < 0.01, Peto trend test), and a signifi- etic cell proliferation, centrilobular fatty change
cant increase in incidence at the two higher doses and necrosis, and bile duct hyperplasia of the
(P < 0.05 and P < 0.01, respectively, Peto test and liver, and hyperplasia of the bone marrow. [The
Bonferroni correction). There was a significant Working Group noted that this was a well-con-
positive trend in the incidence of follicular cell ducted study that complied with GLP, males and
adenoma or adenocarcinoma (combined) of females were used, the duration of exposure and
the thyroid gland [P < 0.01, Cochran–Armitage observation was adequate, and a high number of
trend test], with a significant increase in inci- rats per group was used.]
dence at the two higher doses [P < 0.01 and
P < 0.001, respectively, one-tailed Fisher exact
test]. Adenomas and adenocarcinomas of the
3.2 Leucogentian violet
clitoral gland were also observed with an inci- No studies were available to the Working
dence of 12%, 6%, 18%, and 33% in the control Group.
group and in groups at the lowest, interme-
diate, and highest dose, respectively [statistical
analysis of the incidence of adenoma or adeno- 3.3 Evidence synthesis for cancer in
carcinoma (combined) of the clitoral gland could experimental animals
not be performed because the incidence was not
reported as the number of rats with lesions per 3.3.1 Gentian violet
number of rats examined microscopically]. At The carcinogenicity of gentian violet has been
18 months, there was a significant positive trend assessed in male and female mice exposed by
in the incidence of mononuclear cell leukaemia oral administration (in the feed) in one study, in
(P < 0.05, Cochran–Armitage trend test), with male and female rats exposed in utero, followed
a significant increase in incidence in females at by lactational exposure and oral administration
the highest dose (P < 0.01, one-tailed Fisher exact
67
(in the feed) in another study, and in rats exposed 3.3.2 Leucogentian violet
by oral administration in a third study.
In one study that complied with GLP (NCTR, No studies were available to the Working
1984; Littlefield et al., 1985), male and female Group.
B6C3F1 mice were treated with gentian violet
in the feed for up to 24 months. Gentian violet
caused a significant increase, with a significant 4. Mechanistic Evidence
positive trend, in the incidence of hepatocellular
adenoma and hepatocellular carcinoma in males 4.1 Absorption, distribution,
and females at 24 months, and of hepatocellular metabolism, and excretion
adenoma in females at 18 months. In female
mice, gentian violet caused significant increases, 4.1.1 Humans
and significant positive trends, in the incidence
No data were available to the Working Group.
of histiocytic sarcoma for the urinary bladder,
ovaries, uterus, and vagina at 24 months. In males
and females, there was a significant increase in 4.1.2 Experimental systems
the incidence of Harderian gland adenoma at The absorption, distribution, metabolism,
24 months. and excretion of gentian violet has been reviewed
In one study that complied with GLP (NCTR, in Docampo & Moreno (1990), WHO (2014b),
1988; Littlefield et al., 1989), male and female and OEHHA (2019).
Fischer 344 rats were exposed to gentian violet in
utero, followed by lactational exposure and oral (a) In vivo
administration (in the feed), for up to 24 months. Radiolabelled gentian violet was administered
In male and female rats, gentian violet caused orally to rats and mice. Male and female Fischer
a significant increase, and significant positive 344 rats treated by gavage were given a single dose
trend, in the incidence of follicular cell adeno- of [14C]-labelled gentian violet (4.8 mg/kg bw for
carcinoma of the thyroid gland and follicular cell males, 5.2 mg/kg bw for females). The distribu-
adenoma or adenocarcinoma (combined) of the tion of the [14C]-labelled dye was measured in
thyroid gland at 24 months. In females, gentian the liver, kidney, fatty tissue, gonads, muscle,
violet caused a significant increase, and signif- urine, and faeces at 2, 4, 14, 24, and 36 hours
icant positive trend, in the incidence of mono- after administration. Maximal residue levels
nuclear cell leukaemia at 18 months. In males, were found at 4 hours in the liver, kidney, muscle,
gentian violet caused a significant increase, and and gonads; a plateau was reached in fatty tissue
a significant positive trend, in the incidence of after 24 hours. The depletion half-lives in male
hepatocellular adenoma at 24 months. and female livers were 14.5 and 17.0 hours, respec
A study in rats, where gentian violet was tively. The recovery values for males and females
given by oral administration, was considered (males/females) were 2.2/2.2% and 72.9/63.8% of
inadequate for the evaluation of the carcinogen- the single gentian violet dose in the urine and the
icity of gentian violet in experimental animals faeces, respectively. In bile collected from cannu-
(Kinosita, 1940). lated rats, 5.7–6.4% of the single oral dose was
recovered (McDonald et al., 1984a; NCTR, 1989).
Radiolabelled ([14C]) gentian violet was also
administered in multiple doses (twice per day for
7 days) to both male and female Fischer 344 rats
68
and B6C3F1 mice by gavage. Maximal residue mouse strains, three rat strains, hamster, guin-
levels were found in fatty tissues of females of ea-pig, and chicken: the main metabolites iden-
both species, and a statistically significant sex tified were pentamethyl para-rosaniline and the
difference (P < 0.01) was noted. Residue levels in isomeric N,N,N′,N′- and N,N,N′,N′′-tetramethyl
kidney and muscle tissues from both species, and para-rosanilines. Comparable patterns of
in mouse livers, also showed sex differences. The demethylated metabolites were observed
recovery values for males and females (males/ between species. [The Working Group noted
females) were 2.2%/1.6% and 65.5%/72.8% in that information about the relative amounts of
the urine and the faeces of rats, respectively, the different metabolites, including leucogentian
and 5.9%/8.1% and 65.9%/67.4% in the urine and violet, was sparse.]
faeces of mice (McDonald et al., 1984a; NCTR,
1989).
Regarding the metabolism of gentian violet,
4.2 Evidence relevant to key
McDonald & Cerniglia (1984) showed that characteristics of carcinogens
leucogentian violet was excreted in the faeces This section summarizes the evidence for
collected from a female Fischer 344 rat that was the key characteristics of carcinogens (Smith
given [14C]-labelled gentian violet by gavage for et al., 2016), including whether gentian violet
4 days. The metabolites of gentian violet were (and leucogentian violet) is electrophilic or can
also analysed in mice and rats by NCTR (1989) be metabolically activated to an electrophile; is
and identified as three demethylated metabolites genotoxic; or induces oxidative stress. Insufficient
(pentamethyl para-rosaniline and N,N,N′,N′- and data were available for the evaluation of other key
N,N,N′,N′′-tetramethyl para-rosanilines) and characteristics of carcinogens.
two reduced metabolites (leucogentian violet and
leuco-pentamethyl para-rosaniline). A summary
4.2.1 Is electrophilic or can be metabolically
of the proposed metabolism of gentian violet and
leucogentian violet is provided in Fig. 4.1. activated to an electrophile
Through measurement of sedimentation and
(b) In vitro viscosity, it was shown that gentian violet binds
In bacteria, McDonald & Cerniglia (1984) externally to the surface of the DNA helix, with
demonstrated that gentian violet was trans- a high degree of preference for two adjacent A−T
formed to leucogentian violet after incubation base pairs, and that it induces severe bending
under anaerobic conditions with microflora accompanied by unwinding of the DNA helix
isolated from human faeces, and from the intes- (Müller & Gautier, 1975; Wakelin et al., 1981).
tinal contents of rats and chickens. The ability of gentian violet to bind to bovine
When metabolized by rat liver microsomes, haemoglobin was demonstrated in vitro by Liu
gentian violet appears to undergo one-electron et al. (2013) using several spectroscopic and
reduction by cytochrome P450 to produce a molecular modelling methods. A change in the
carbon-centred free radical (Harrelson & Mason, spatial conformation of bovine haemoglobin
1982). This carbon-centred radical can be formed was observed after binding of gentian violet (Liu
by photoreduction of gentian violet after expo- et al., 2013).
sure to visible light (Docampo et al., 1988).
McDonald et al. (1984b) studied the metab-
olism of gentian violet in the presence of liver
microsomes obtained from both sexes of four
69
Fig. 4.1 Metabolic pathways for gentian violet and leucogentian violet

CH 3 CH 3 CH 3 CH 3
CH 3 CH 3
+
+ N N N N
N N H3 C CH 3
CH 3 H3 C CH 3
H3C
Microbial metabolism
Reduction O
Nocardia corallina Michler’s ketone

NADPH
ra N
N
f lo H3C CH 3
H3C CH 3 ro
ic
Carbon-centred free radical m Gentian violet Demethylation in a cell-free system
n al
n
e s ti t io
i nt d uc Oxidation/demethylation
an Re (microsomes)
um
H
CH 3 CH 3
CH 3 CH 3 CH 3 CH 3
N N NH
+
H3 C CH 3 N N N
+
H3C H3 C CH 3
N
H3 C CH 3 N N
n
H3C CH 3 H3C
io
ct
du
Leucogentian violet
Re
Nitrogen-centred free radical

Pentamethyl para-rosaniline
Demethylation (methyl violet)
Oxidation/demethylation Oxidation/demethylation
CH 3
CH 3
N NH
H3C
CH 3 CH 3
HN NH+ N NH 2
+
CH 3 H 3 C
N
H3 C CH 3
Leuco-pentamethyl para-rosaniline N N
H3 C CH 3 H3 C CH 3
Demethylation
N,N,Nʹ,Nʺ-Tetramethyl para-rosaniline N,N,Nʹ,Nʹ-Tetramethyl para-rosaniline
Leuco-tetramethyl para-rosaniline
Demethylation
Demethylation
Trimethyl para-rosaniline isomers
Further demethylation/deamination and

Demethylation
mineralization products
Dimethyl para-rosaniline isomers
Demethylation
CH 3
NH
+ H2 N NH
H2N
Further deamination
and mineralization
products
NH 2 NH 2
N-Methyl para-rosaniline para-Rosaniline (CI Basic Red 9)
Chemicals with structures in boxes were identified in the tissues of rats and mice treated with gentian violet. Reduced metabolites (leucogentian
violet and leuco-pentamethyl para-rosaniline) were predominant. Arrows with solid lines indicate observed metabolic pathways; arrows with
dashed lines indicate proposed pathways. Michler’s ketone has been classified by IARC in Group 2B (possibly carcinogenic to humans).
NADPH, reduced form of nicotinamide adenine dinucleotide phosphate.
Created by the Working Group.
70
4.2.2 Is genotoxic embryo (CO60) cells. Gentian violet induced

DNA strand breaks in whole-blood samples
Table 4.1, Table 4.2, Table 4.3, and Table 4.4 collected from Sprague-Dawley rats (Díaz Gómez
summarize the available studies of the genetic & Castro, 2013). When the rats were treated
and related effects of gentian violet. with antioxidants (α-tocopherol, lipoic acid,
(a) Humans or N-acetylcysteine) before the blood samples
were collected, the genotoxic effects induced
(i) Exposed humans by gentian violet were significantly decreased
No data were available to the Working Group. (Díaz Gómez & Castro, 2013). Gentian violet
(ii) Human primary cells and human cell lines did not induce gene mutations at the hypo-
in vitro xanthine-guanine phosphoribosyltransferase
(Hprt) locus of Chinese hamster ovary (CHO)
See Table 4.1.
CHO-K1-BH4 cells, but caused a slight increase
In human primary cells in vitro, a single
at the glutamic-pyruvate transaminase (Gpt)
concentration of gentian violet induced an
locus of CHO AS52 cells (the increase was
increase in chromosomal aberration in cultured
observed only at very toxic concentrations, and
primary human peripheral blood lymphocytes
was not reproduced with different gentian violet
from healthy donors (Au et al., 1978; Hsu et al.,
batches) (Aidoo et al., 1990).
1982), and from healthy individuals and patients
Au et al. (1978) demonstrated that gentian
with β-thalassaemia (Krishnaja & Sharma, 1995).
violet induced mitotic anomalies. Gentian violet
Au et al. (1978) also showed that gentian violet
consistently induced chromosomal aberrations
induced an increase in chromosomal aberrations
in various cell lines: Mus musculus mouse fibro-
in HeLa cells.
blast L cells, a fibroblast cell line derived from
(b) Experimental systems Peromyscus eremicus, and a fibroblast cell line
derived from the Indian muntjac (Muntiacus
(i) Non-human mammals in vivo muntjak) (Au et al., 1978). It also induced
See Table 4.2. chromosomal aberrations in CHO cells (Au et al.,
After injection of gentian violet in the tail 1978, 1979; Au & Hsu, 1979). The cytogenic effect
veins of B6C3F1 mice up to a dose of 8 mg/kg observed in CHO cells decreased in the presence
bw, no DNA damage was observed in splenic of the S9 metabolic activation system (Au et al.,
lymphocytes (Aidoo et al., 1990). Gentian violet 1979).
also failed to induce chromosomal aberrations in
bone marrow erythrocytes of Swiss albino mice (iii) Non-mammalian experimental systems
that received the substance via drinking-water See Table 4.4.
for 4 weeks up to a dose of 8 mg/kg (Au et al., At low concentrations, gentian violet binds to
1979). double-stranded DNA at AT-rich regions, while
it binds at all available sites at high concentra-
(ii) Non-human mammalian cells in vitro tions (Fox et al., 1992).
See Table 4.3. Cornell K-strain chicken embryos treated
Aidoo et al. (1990) showed that gentian violet with gentian violet did not show sister-chromatid
induced DNA damage (nucleoid sedimenta- exchange (Au et al., 1979; Bloom, 1984).
tion) in cultured lymphocytes from the spleens In one study performed on Drosophila mela-
of B6C3F1 mice and caused weak gene amplifi- nogaster, gentian violet did not induce mutations
cation in SV40-transformed Chinese hamster
71
72

Table 4.1 Genetic and related effects of gentian violet in human primary cells and human cell lines in vitro
End-point Tissue, cell line Resultsa Concentration Comments Reference

(LEC or HIC)
Without With
metabolic metabolic
activation activation
Chromosomal HeLa cells (cervical + NT 5 μg/mL 5-h treatment; purity, NR Au et al. (1978)
aberration cancer)
Chromosomal Blood peripheral + NT 5 μg/mL 5-h treatment; purity, NR Au et al. (1978)
aberration lymphocytes
Chromosomal Blood peripheral + NT 20 μg/mL Only one dose tested; purity, Hsu et al. (1982)
aberration lymphocytes NR
Chromosomal Blood peripheral + NT 1 μg/mL Only one dose tested; purity, Krishnaja & Sharma
aberration lymphocytesb NR (1995)
h, hour; HIC, highest ineffective concentration; LEC, lowest effective concentration; NR, not reported; NT, not tested.
a +, positive.
b Lymphocytes collected from two groups (i.e. healthy individuals and patients with β-thalassaemia). Level of chromatid aberration in lymphocytes was similar in these two groups.
Table 4.2 Genetic and related effects of gentian violet in non-human mammals in vivo
End-point Species, Tissue, cell Resultsa Dose Route, duration, Comments Reference
strain (sex) type (LED or HID) dosing regimen
DNA damage Mouse, Spleen, – 8 mg/kg Injection in tail vein, Aidoo et al.
(nucleoid B6C3F1 (NR) lymphocytes 1 h before collection (1990)
sedimentation)
Chromosomal Mouse, Swiss Bone marrow, – 8 mg/kg Drinking-water, 4 wk GV dissolved at 20 Au et al. (1979)
aberration albino (NR) erythrocytes and 40 μg/mL, and
consumed dose
calculated to be 4
and 8 mg/kg
GV, gentian violet; h, hour; HID, highest ineffective dose; LED, lowest effective dose; NR, not reported; wk, week.
a –, negative.
Table 4.3 Genetic and related effects of gentian violet in non-human mammalian cells in vitro
End-point Species, tissue, cell line Resultsa Concentration Comments Reference

(LEC or HIC)
Without With
metabolic metabolic
DNA damage (nucleoid Mouse, B6C3F1, spleen, + NT 1 μg/mL 1-h treatment Aidoo et al. (1990)
sedimentation) lymphocytes
DNA strand breaks Rat, blood, leukocytes + NT 250 µg/mLb 24 and 48 h Díaz Gómez &
(comet assay) Castro (2013)
Gene amplification Chinese hamster, embryo, (+) NT 0.125 μg/mL 5-h treatment; weak DNA Aidoo et al. (1990)
C060 (SV40-transformed) amplification observed
Gene mutation (Hprt Chinese hamster, ovary, – – 1 μg/mL 5-h treatment Aidoo et al. (1990)
locus) CHO-K1-BH4
Gene mutation (Gpt locus) Chinese hamster, ovary, (+) – 1.5 μg/mL 5-h treatment; increase Aidoo et al. (1990)
CHO-AS52 observed only at very toxic
concentrations, and not always
reproduced
Mitotic anomaliesc Chinese hamster, ovary, CHO + NT 10 μg/mL 2-h treatment; purity, NR Au et al. (1978)
Chromosomal aberration Mouse, fibroblast L cells + NT 5 μg/mL 5-h treatment; purity, NR Au et al. (1978)
Chromosomal aberration Peromyscus eremicus, NR, + NT 5 μg/mL 5-h treatment; purity, NR Au et al. (1978)
fibroblasts
Chromosomal aberration Indian muntjac, NR, + NT 5 μg/mL 5-h treatment; purity, NR Au et al. (1978)
fibroblasts

Chromosomal aberration Chinese hamster, ovary, CHO + NT 5 μg/mL 5-h treatment; purity, NR Au et al. (1978)
Chromosomal aberration Chinese hamster, ovary, CHO + + 5 μg/mL (−S9); 5-h treatment; S9 decreased the Au et al. (1979)
10 μg/mL (+S9) clastogenic effect; purity, NR
Chromosomal aberration Chinese hamster, ovary, CHO + NT 10 μM [4 µg/mL] Only one dose tested; purity, NR Au & Hsu (1979)
CHO, Chinese hamster ovary; Gpt, glutamic-pyruvate transaminase; h, hour; HIC, highest ineffective concentration; Hprt, hypoxanthine-guanine phosphoribosyltransferase; LEC,
lowest effective concentration; NR, not reported; NT, not tested; S9, 9000 × g supernatant.
a +, positive; –, negative; (+), positive in a study of limited quality.
b Calculated from the data provided in the article (50 μL of gentian violet solution at 0.0245 M for 2 mL of blood; the relative molecular mass of gentian violet is 408).
c Mitotic anomalies include increase in mitotic index, metaphase : anaphase ratio and frequency of anaphase abnormalities (chromatin bridges, lagging chromosomes, chromosome
fragments, and sticky chromosomes).

73
74

Table 4.4 Genetic and related effects of gentian violet in non-mammalian experimental systems
Test system End-point Resultsa Concentration Comments Reference

(species, strain) (LEC or HIC)
Without With
metabolic metabolic
Cornell K-strain chicken Sister-chromatid − NA 100 μg/embryo Test solution applied to inner shell Au et al. (1979)
(NR), embryo exchange membrane (after removing the
portion of the shell overlying the
air cell)
Purity, NR
Cornell K-strain chicken Sister-chromatid − NA “Amounts in the Air-cell method (test solution Bloom (1984)
(NR), embryo exchange range of 10–100 μL is dropped onto the inner shell
are used” membrane after removing the
portion of the shell overlying
the air cell) [No more details
given on the amount of GV used
(10–100 μL)]
Purity, NR
Drosophila melanogaster Sex-linked recessive – NA 500 ppm (feed) or Purity, 92% Mason et al. (1992)
lethal assay 550 ppm (injected)
Bacillus subtilis (rec assay) DNA damage, + NT 2 mg/0.02 mL Only one dose tested Fujita et al. (1976)
differential toxicity Purity, NR
Bacillus subtilis BD224 (rec DNA damage, NT + 200 μg/plate Purity, 80–95% Choudhary et al. (2004)
assay) differential toxicity
Escherichia coli B DNA strand breaks + NT ~10 µM [4 µg/mL] Purity, NR Grigg et al. (1984)
Salmonella typhimurium Reverse mutation – NT 4 μg/plate Purity, NR Shahin & Von Borstel
TA1535, TA100 (base (1978)
substitution, at GC)
TA98, TA1538 (1978)
(frameshift +1)
TA1537 (frameshift −1) (1978)
Salmonella typhimurium Reverse mutation – – 50 μg/plate Purity, NR Au et al. (1979)
TA1535, TA100 (base
TA98 (frameshift +1)
TA1537 (frameshift −1)
Without With
metabolic metabolic
Salmonella typhimurium Reverse mutation + + 16 μg/plate Data provided for only one dose; Fujita et al. (1976)
TA98 (frameshift +1) purity, NR
Salmonella typhimurium Reverse mutation - + 16 μg/plate Data provided for only one dose; Fujita et al. (1976)
TA100 (base substitution, purity, NR
at GC)
Salmonella typhimurium Reverse mutation (+) – 0.32 μg/plate Reproducible increase observed Bonin et al. (1981)
TA1535 (base substitution, only in TA1535 at middle dose of
at GC) 0.32 μg/plate; purity, 97%
Salmonella typhimurium Reverse mutation – – 3.2 μg/plate Purity, 97% Bonin et al. (1981)
TA100 (base substitution,
at GC)
TA98, TA1538 (frameshift
+1)
Salmonella typhimurium Reverse mutation – – 50 μg/plate Purity, NR Levin et al. (1982)
at GC)

Salmonella typhimurium Reverse mutation – – 50 μg/plate Purity, NR Levin et al. (1982)
Salmonella typhimurium Reverse mutation – 50 μg/plate Purity, NR Levin et al. (1982)
Salmonella typhimurium Reverse mutation – – 0.5 μg/plate Purity, NR Thomas & MacPhee
TA1535 (base substitution, (1984)
at GC)
Salmonella typhimurium Reverse mutation – – 50 μg/plate Purity, NR Hass et al. (1986)
at GC)
Salmonella typhimurium Reverse mutation – –b 50 μg/plate Purity, NR Hass et al. (1986)
Salmonella typhimurium Reverse mutation – – 50 μg/plate Purity, NR Hass et al. (1986)
75
76

Without With
metabolic metabolic
Salmonella typhimurium Reverse mutation – – 10 μg/plate Purity, > 97% or > 99% Aidoo et al. (1990)
at GC)
Salmonella typhimurium Reverse mutation – – 10 μg/plate Purity, > 97% or > 99% Aidoo et al. (1990)
Salmonella typhimurium Reverse mutation – (+)c 0.5 μg/plate Increase slightly > 2-fold for GV Aidoo et al. (1990)
TA97 (frameshift −1) purity, 97%; increase < 2-fold for
purity, 99%
Salmonella typhimurium Reverse mutation – (+)c 0.5 μg/plate Increase slightly > 2-fold for GV Aidoo et al. (1990)
TA104 (base substitution, purity, 99%; increase < 2-fold for
at AT) purity, 97%
Salmonella typhimurium Reverse mutation – – 25 μg/plate Purity, NR Malachová et al. (2006)
at GC)
Salmonella typhimurium Reverse mutation – (+) 25 μg/plate Purity, NR; GV was highly toxic Malachová et al. (2006)
TA98 (frameshift +1) at 25 μg/plate
YG1041 (frameshift)
YG1042 (base substitution)
Salmonella typhimurium Reverse mutation – – 100 μg/plate Purity, NR Ackerman et al. (2009)
TA100-lux (base
Salmonella typhimurium Reverse mutation – – 100 μg/plate Purity, NR Ackerman et al. (2009)
TA98-lux (frameshift +1)
Salmonella typhimurium Reverse mutation NT (+) 100 μg/plate Analytical grade; fold-increase Ayed et al. (2017)
TA98 (frameshift +1) slightly lower than 2; only one
dose tested
Escherichia coli DG1669 Reverse mutation + + 25 μg/plate Purity, NR Thomas & MacPhee
(frameshift) (1984)
Escherichia coli WP2s (base Reverse mutation + + 5 μmol/L Purity, NR Hass et al. (1986)
substitution, at AT) [2 µg/mL]
Escherichia coli WP2 Reverse mutation + NT 80 μg/plate Purity, NR Fujita et al. (1976)
Escherichia coli W3110 Rosenkranz + + 10 μg/plate Purity, NR Au et al. (1979)
polA+, mutant p3478 polA- repairable DNA assay
Without With
metabolic metabolic
Escherichia coli W3110 Rosenkranz + + 10 μg/plate Purity, NR Levin et al. (1982)
polA+, mutant p3478 polA- repairable DNA assay
Saccharomyces cerevisiae Rosenkranz – NT 8 μg/plate Purity, NR Shahin & Von Borstel
XV185-14C repairable DNA assay (1978)
GV, gentian violet; HIC, highest ineffective concentration; LEC, lowest effective concentration; NA, not applicable; NR, not reported; NT, not tested; ppm, parts per million.
a +, positive; –, negative; (+), positive in a study of limited quality.
b Hass et al. (1986) also reported that the metabolite of GV, leucogentian violet, was positive in Salmonella typhimurium TA98 at 50 μg/plate in the presence of metabolic activation.
Purity was not reported.

c Aidoo et al. (1990) also showed that the major metabolites of GV, pentamethyl-para-rosaniline, N,N,N′,N′-tetramethyl-para-rosaniline, and N,N,N′,N″-tetramethyl-para-rosaniline,
induced a dose-related increase in the number of mutant colonies in Salmonella typhimurium TA97, which reached 1.5-, 1.7-, and 1.4-fold, respectively, compared with the control.

77
in a sex-linked recessive lethal assay (Mason 1988) or by enzymatic reaction (Harrelson &
et al., 1992). Mason, 1982).
Gentian violet induced DNA damage in
Bacillus subtilis (Fujita et al., 1976; Choudhary
et al., 2004). Grigg et al. (1984) observed that
4.3 Other relevant evidence
gentian violet induced DNA strand breaks in 4.3.1 Humans
Escherichia coli B strain.
An overwhelming majority of the data Several studies using patch tests showed that
show that gentian violet did not induce muta- gentian violet was among the least active sensi-
genicity with or without metabolic activation tizers of several tested drugs, because contact
in Salmonella typhimurium strains TA1535, hypersensitivity was rarely observed with gentian
TA100, TA1538, TA97, TA98, TA1537, YG1041, violet (Bajaj et al., 1982; Pasricha & Gupta, 1982;
and YG1042 (Shahin & Von Borstel, 1978; Bajaj & Gupta, 1986; Mahaur et al., 1987).
Au et al., 1979; Bonin et al., 1981; Levin et al., Bielicky & Novák (1969) observed that, in
1982; Thomas & MacPhee, 1984; Hass et al., patients with eczema, gentian violet induced
1986; Aidoo et al., 1990; Malachová et al., 2006; sensitization. Moreover, cross-sensitization be-
Ackerman et al., 2009). However, a few authors tween crystal violet and malachite green was
reported a mutagenic effect without metabolic possible, as the probable determinant groups for
activation in TA98, TA100, and TA1535 (Fujita sensitization are -N(CH3)2 and -N(C2H5)2.
et al., 1976; Bonin et al., 1981), with metabolic
activation in TA98 and TA100 (Fujita et al., 1976; 4.3.2 Experimental systems
Ayed et al., 2017), as well as in TA97 and TA104 No data were available to the Working Group.
(Aidoo et al., 1990). Malachová et al. (2006)
described a mutagenic effect of crystal [gentian]
violet with metabolic activation in TA98, which 4.4 Data relevant to comparisons
was associated with a cytotoxic effect. In E. coli across agents and end-points
strains, gentian violet caused mutagenicity with
and without metabolic activation (Thomas & The mechanistic characteristics common to
MacPhee, 1984; Hass et al., 1986), and induced carcinogens (the 10 key characteristics of carcin-
mutagenicity without metabolic activation in a ogens) can be investigated through biochemical
study by Fujita et al. (1976) (not tested with meta- and cell-based assays run by the United States
bolic activation). In the Rosenkranz repairable Environmental Protection Agency (US EPA)
DNA assay, gentian violet gave positive results in and the United States National Institutes of
E. coli strains W3110 polA+ and P3478 polA– (Au Health Toxicity Forecaster/Toxicology in the
et al., 1979; Levin et al., 1982), but negative results 21st Century (ToxCast/Tox21) high-throughput
in the Saccharomyces cerevisiae XV185-14C screening programmes (Chiu et al., 2018; Guyton
strain (Shahin & Von Borstel, 1978). et al., 2018). Since 2017, the IARC Monographs
have described the results of high-throughput
screening assays to compare activity across
4.2.3 Induces oxidative stress
agents and other in vitro and in vivo evidence
As already mentioned above (Section 4.1.2.b), relevant to the key characteristics.
gentian violet can lead to the formation of a Of the five compounds included in IARC
carbon-centred free radical, either by photore- Monographs Volume 129, three have been
duction (Reszka et al., 1986; Docampo et al., evaluated in at least some of the US EPA and
78
United States National Institutes of Health high- 4.4.1 Gentian violet

throughput screening assays: gentian violet (CAS
No. 548-62-9), malachite green (malachite green Results were available for 280 assay
chloride, CAS No. 569-64-2, and malachite green end-points (out of the 299 that were mapped to
oxalate, CAS No. 2437-29-8), and leucomalachite key characteristics) for gentian violet (US EPA,
green (CAS No. 129-73-7) (US EPA, 2020a, b, 2020a). Gentian violet was considered active in
c, d). Table 4.5 summarizes findings for assay 126 assay end-points, including the one assay
end-points mapped to key characteristics for end-point mapped to “is electrophilic or can be
the compounds evaluated. Details of the specific metabolically activated to an electrophile”, 10
assays (and end-points) run for each chemical in of the 12 assay end-points mapped to “is geno-
this volume and the mapping to the key char- toxic”, 2 of the 5 mapped to “induces epige-
acteristics can be found in the Supplementary netic alterations”, 8 of 16 end-points mapped
Material (Annex 1, Supplementary material for to “induces oxidative stress”, 27 of the 90 assay
Section 4, web only; available from: https://www. end-points mapped to “modulates receptor-me-
publications.iarc.fr/603). It is important to note diated effects”, and 78 of 109 end-points mapped
that some assays either lacked, or had unchar- to “alters cell proliferation, cell death, or nutrient
acterized and generally low, xenobiotic metabo- supply”.
lism, limiting observations primarily to effects Assays within the “is genotoxic” key charac-
elicited by parent compounds. The strengths of teristic provide measurements of DNA damage
the high-throughput screening battery of assays or repair in human liver (HepG2), kidney
are the standardization of the protocols applied (HEK293T), and intestinal (HCT-116) cell lines,
across compounds, allowing comparisons across as well as a CHO cell line (CHO-K1) and a
compounds and the evaluation of specificity of chicken lymphoblast cell line (DT40). Gentian
assay end-points to the key characteristics, and violet (purity, > 90%) elicited TP53 activation
ultimately to the apical outcome of carcino- measured through reporter assays in HCT-116
genesis (Becker et al., 2017; Chiu et al., 2018; and HepG2 cells. Gentian violet was considered
Watford et al., 2019). The 299 ToxCast/Tox21 active, as measured by phosphorylated histone
assay end-points mapped to key characteristics H2AX (γH2AX) assay detecting H2AX protein
interrogated in this and other monographs are phosphorylation, consistent with DNA double-
initially described in Chiu et al. (2018), with the strand breaks in a CHO cell line (CHO-K1).
most up-to-date mapping described in detail in Gentian violet was also considered active as
IARC Monographs Volume 123 (IARC, 2019). measured by assays using DT40 chicken lympho-
All ToxCast/Tox21 data were downloaded from blastoid cell lines deficient for the DNA-repair
the US EPA CompTox Chemicals Dashboard genes REV3 and KU70/RAD54. Gentian violet
10th Release (US EPA, 2021) between 2 and 19 was not considered active as determined by
October 2020 or on 24 February 2021 (malachite the ATAD5-luc assay in HEK293T cells, which
green oxalate). measures levels of ATAD5 protein that localize
The individual assessments for each to the site of stalled replication forks resulting
compound are included in the corresponding from DNA damage in replicating cells. It is
monographs in the present volume. important to note that both positive (e.g. etopo-
side, 5-fluorouridine, tetra-N-octylammonium
bromide, and mitomycin C) and negative (di-
methyl sulfoxide) controls are run concurrently,
and subsequent analyses and activity calls are
79
80

Table 4.5 Summary of results of ToxCast/Tox21 high-throughput screening assays linked to key characteristics of
carcinogens for agents reviewed in IARC Monographs Volume 129a
Key characteristic No. of positive results out of the number of assays

(total number of assays mapped to
characteristic)b Gentian violet Malachite green chloride Malachite green oxalate Leucomalachite green
(CAS No. 548-62-9) (CAS No. 569-64-2) (CAS No. 2437-29-8) (CAS No. 129-73-7)
1. Is electrophilic or can be metabolically 1 out of 1c NT 1 out of 1 0 out of 1
activated (2)
2. Is genotoxic (12) 10 out of 12 1 out of 2 8 out of 9 2 out of 10
4. Induces epigenetic alterations (5) 2 out of 5 5 out of 5 1 out of 1 0 out of 5
5. Induces oxidative stress (16) 8 out of 16 4 out of 10 3 out of 4 4 out of 13
6. Induces chronic inflammation (47) 0 out of 47 0 out of 46 0 out of 1 1 out of 47
8. Modulates receptor-mediated effects (98) 27 out of 90 17 out of 50 22 out of 32 13 out of 69
10. Alters cell proliferation, death, or 78 out of 109 40 out of 63 56 out of 58 24 out of 91
nutrient supply (119)
Total hits out of total no. of assays 126 out of 280 67 out of 176 91 out of 106 44 out of 236
evaluated
CAS, Chemical Abstracts Service; NT, not tested; Tox21, Toxicology in the 21st Century programme; ToxCast, Toxicity Forecaster programme.
a No high-throughput screening data were available for leucogentian violet (CAS No. 603-48-5) and CI Direct Blue 218 (CAS No. 28407-37-6).
b Seven of the 10 key characteristics have mapped high-throughput screening assay end-points, as described by Chiu et al. (2018). The mapping file with findings for IARC Monographs
Volume 129 chemicals is available in the Supplementary Material (Annex 1, Supplementary material for Section 4, web only; available from: https://www.publications.iarc.fr/603). No
assay end-points in ToxCast or Tox21 were determined to be applicable to the evaluation of three key characteristics including causes immortalization, alters DNA repair or causes
genomic instability, and is immunosuppressive.
c Indicates the number of positive results out of the number of assays tested for that chemical.
normalized against data for positive and nega- or enzymatic reduction of gentian violet. Gentian
tive controls run on the same plates (Hsieh et al., violet is widely used as a textile dye, a pigment for
2019). consumer and industrial products (inks, papers,
and coatings), as a biological stain (Gram stain),
4.4.2 Leucogentian violet and for cosmetic purposes (hair dyes and body
piercing). The antibacterial, antifungal, and
Leucogentian violet has not been evaluated in anthelmintic properties of gentian violet make
high-throughput screening assays. it an important agent in human medicine as
an antiseptic to prevent infection and promote
4.4.3 Summary wound healing, and as a topical treatment for
Gentian violet has been evaluated in ToxCast fungal and bacterial infections. Gentian violet
or Tox21 assays with end-points mapped to also has several veterinary applications for the
key characteristics of carcinogens. It was active treatment of fungal and parasite disease in fish,
in a significant fraction of mapped end-points disinfection of aquariums, topical treatment for
in which it had been tested (45%). Specifically, bacterial and fungal infections in livestock, and
gentian violet was considered active in most the prevention of growth of mould and fungi in
of the “is genotoxic” assay end-points. It was poultry feeds. Leucogentian violet is a precursor
also considered active for a variety of the assay in the production of gentian violet dye, and is
end-points mapped to the following key charac- used as an analytical reagent to enhance blood
teristics: induces epigenetic alterations, induces impression evidence in forensic analysis, for
oxidative stress, modulates receptor-mediated laboratory determination of anions and metal
effects, and alters cell proliferation, cell death, ions, and as a radiochromic indicator in dosim-
or nutrient supply. Relevant to findings in other eters to detect radiation exposure. As gentian
sections, gentian violet was considered active in violet may be used to control fish diseases, resi-
an assay measuring thyroid receptor antagonism dues of its major metabolite, leucogentian violet,
in GH3, a rat pituitary gland cell line, and was might be found in treated fish or shellfish, and
considered to give negative results in an assay have a longer residence time than the parent
measuring thyroid hormone receptor-agonist compound.
activity in the same cell line. Gentian violet was Gentian violet may be released into the envi-
considered to give negative results in an assay ronment from waste discharged by textile mills
measuring thyroid hormone receptor-mediated and by other industrial processing, and persists
transcription in HepG2 cells. Leucogentian in soil and aquatic species primarily as leucogen-
violet has not been evaluated in high-throughput tian violet.
screening assays. Overall, data on exposure to gentian violet
and leucogentian violet are sparse. The poten-
tial for occupational exposure to gentian violet
5. Summary of Data Reported and leucogentian violet exists through dermal
contact and inhalation at workplaces where the
compound is produced or applied; however, no
5.1 Exposure characterization current data on exposed occupational popula-
Gentian violet is a cationic triphenylmethane tions or occupational exposure levels were
dye. The reduced form of gentian violet is leuco- identified.
gentian violet, which can be formed by chemical In the general population, exposure can occur
through contact with textiles, paper, and inks
81
containing gentian violet, medicinal or orna- 5.3.2 Leucogentian violet

mental fish treatment, cosmetic application for
hair dyeing and body piercing, and through the No studies were available to the Working
consumption of drinking-water, fish, or shellfish Group.
containing residues of gentian violet and leuco-
gentian violet. One study indicated that drink- 5.4 Mechanistic evidence
ing-water may be an important route of exposure
to gentian violet. No data on absorption, distribution, metab-
Gentian violet is listed by the European olism, or excretion in humans were available.
Chemicals Agency as a carcinogen (Category 2) In mice and rats, orally administered gentian
and is a substance of very high concern. Gentian violet is distributed to the liver, kidney, and
violet is not authorized for use as a veterinary fatty tissue, and is excreted primarily in faeces.
drug or for cosmetic applications in many coun- Various demethylated and reduced metabolites
tries, and there is zero tolerance for residues of have been detected in rats and mice, and in vitro
gentian violet, or its marker leucogentian violet, experiments using microsomal preparations
in food for human consumption. from different species. Bacteria have been shown
to transform gentian violet to the metabolite
leucogentian violet, but data from mammalian
5.2 Cancer in humans species are sparse.
No data were available to the Working Group. For gentian violet, the mechanistic evidence
is suggestive but incoherent across studies in
experimental systems, and no data in humans
5.3 Cancer in experimental animals were available. Regarding the key characteristics
of carcinogens, gentian violet binds to isolated
5.3.1 Gentian violet DNA and to haemoglobin, but no data on DNA
Exposure to gentian violet caused an increase adducts were available. Gentian violet induced
in the incidence of malignant neoplasms in both chromosomal aberrations in human primary
sexes of two species (mouse and rat). cells and in various cultured mammalian cell
In B6C3F1 mice exposed to gentian violet in lines in a few studies. It was considered active in
the feed, there was a significant positive trend various high-throughput in vitro assays indica-
and significant increase in the incidence of tive of DNA damage including TP53 activation
hepatocellular carcinoma in males and females, and γH2AX. However, it did not induce DNA
and of histiocytic sarcoma of the urinary bladder, damage or chromosomal aberrations in orally
ovaries, uterus, and vagina in females in a study exposed mice in the few studies available. In
that complied with Good Laboratory Practice rodent cells in vitro, it induced DNA damage but
(GLP). not gene mutations. It gave negative results in tests
In Fischer 344 rats exposed to gentian violet in chicken embryos and in Drosophila melano-
in utero, followed by lactational exposure and gaster, and largely negative results across various
oral administration (in the feed), there was a Salmonella typhimurium strains, including
significant positive trend and significant increase TA1535, TA100, TA1538, TA97, TA98, TA1537,
in the incidence of follicular cell adenocarcinoma TA104, YG1041, and YG1042. In Escherichia coli
of the thyroid gland in males and females, and of strains, gentian violet caused mutagenicity with
mononuclear cell leukaemia in females in a study and without metabolic activation. For other key
that complied with GLP.
82
characteristics of carcinogens, there is a paucity available. The mechanistic evidence is limited

of available data. for gentian violet, based on suggestive but inco-
For leucogentian violet, data were scarce. herent evidence in experimental systems perti-
nent to key characteristics of carcinogens. The
sufficient evidence for cancer in experimental
6. Evaluation and Rationale animals is based on an increase in the incidence
of malignant neoplasms in males and females of
two species in two studies that comply with GLP.
6.1 Cancer in humans
Leucogentian violet was evaluated as Group 3
There is inadequate evidence in humans because the evidence regarding cancer in humans
regarding the carcinogenicity of gentian violet. and in experimental animals, as well as mecha-
There is inadequate evidence in humans nistic evidence, is inadequate, since no studies
regarding the carcinogenicity of leucogentian were available.
violet.
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GENTIAN VIOLET,

This publication represents the views and expert

LYON, FRANCE - 2022

1. Exposure Characterization para-rosaniline chloride, methyl violet 10B,

1.1.1 Gentian violet

Chem. Abstr. Serv. Reg. No.: 548-62-9

Melting point: 205–215 °C (decomposes) 1.1.2 Leucogentian violet

(c) Uses Leucogentian violet is a metabolite resulting

IARC MONOGRAPHS – 129

Sample matrix Sample preparation Analytical Agent LOD Reference

Gentian violet and leucogentian violet

IARC MONOGRAPHS – 129

1.3.5 Biological specimens in the environment.] Particulate-phase gentian

Gentian violet and leucogentian violet

IARC MONOGRAPHS – 129

Republic of Korea, Turkey, and the Russian Federation.

IARC MONOGRAPHS – 129

Study design Route Incidence of Significance Comments

Gentian violet and leucogentian violet

IARC MONOGRAPHS – 129

Gentian violet and leucogentian violet

IARC MONOGRAPHS – 129

Gentian violet and leucogentian violet

IARC MONOGRAPHS – 129

dose, respectively [statistical analysis of the inci- test). At 12 months, no treatment-associated

Fig. 4.1 Metabolic pathways for gentian violet and leucogentian violet

Nocardia corallina Michler’s ketone

Nitrogen-centred free radical

Further demethylation/deamination and

N-Methyl para-rosaniline para-Rosaniline (CI Basic Red 9)

4.2.2 Is genotoxic embryo (CO60) cells. Gentian violet induced

IARC MONOGRAPHS – 129

End-point Tissue, cell line Resultsa Concentration Comments Reference

End-point Species, tissue, cell line Resultsa Concentration Comments Reference

Gentian violet and leucogentian violet

fragments, and sticky chromosomes).

IARC MONOGRAPHS – 129

Test system End-point Resultsa Concentration Comments Reference

Gentian violet and leucogentian violet

IARC MONOGRAPHS – 129

Purity was not reported.

Gentian violet and leucogentian violet

United States National Institutes of Health high- 4.4.1 Gentian violet

IARC MONOGRAPHS – 129

Key characteristic No. of positive results out of the number of assays

containing gentian violet, medicinal or orna- 5.3.2 Leucogentian violet

characteristics of carcinogens, there is a paucity available. The mechanistic evidence is limited

6.2 Cancer in experimental animals References

There is sufficient evidence in experimental Ackerman J, Sharma R, Hitchcock J, Hayashi T, Nagai Y,

Tarbin JA, Chan D, Stubbings G, Sharman M (2008). dsstoxdb/results?search=DTXSID7031531#inv it

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