1. Exposure Characterization

1.3. Methods of detection and quantification

Representative methods for the detection and quantification of gentian violet and leucogentian violet are summarized in Table 1.1.

Table 1.1

Representative methods for the detection and quantification of gentian violet and leucogentian violet in various matrices .

1.3.1. Air

No methods for the detection and quantification of gentian violet or leucogentian violet particulates in air were found.

1.3.2. Water

Gentian violet is measured in water for environmental monitoring and to determine the efficiency of physical, chemical, and biological methods to remove, decolourize, or degrade gentian violet in wastewater (Mani & Bharagava, 2016). Ultraviolet-visible absorbance techniques are commonly used to measure the reduction of the purple colour from highly concentrated wastewater samples, while liquid chromatography with spectroscopic or mass spectrometry detection is a more sensitive technique (Tkaczyk et al., 2020). For residue analysis in environmental water samples, pre-treatment procedures are required to concentrate gentian violet residues before analysis. Magnetic, ionic liquid, nanoparticle material, and microextraction techniques such as magnetic solid-phase extraction, dispersive liquid−liquid microextraction, micro-cloud point extraction, and monolithic fibre-based solid-phase microextraction have been used to isolate gentian violet residues from aqueous samples before analysis, with detection limits ranging from 0.03 to 5 μg/L (Šafařík & Šafaříková, 2002; Zhang et al., 2012; Wang et al., 2015; Ghasemi & Kaykhaii, 2016; Moradi Shahrebabak et al., 2020).

1.3.3. Soil

Leucogentian violet has been identified in soil near waste discharged from a dye-manufacturing plant by means of Soxhlet extraction with 2-propanol and analysis by gas chromatography-tandem mass spectrometry (Nelson & Hites, 1980).

1.3.4. Food, beverages, and consumer products

Gentian violet is not permitted for use as a food additive, but numerous methods have been developed to determine residues of gentian violet and its metabolite, leucogentian violet, in animal products as a result of veterinary treatment with gentian violet (WHO, 2014a; Verdon & Andersen, 2017). In gentian violet-treated fish, the major metabolite (leucogentian violet) has a longer residence time (> 79 days) than gentian violet (~5 days) in fish (Thompson et al., 1999). Thus, leucogentian violet is the marker residue used to monitor gentian violet use in aquaculture, and seafood analysis methods must assess both compounds. Many early methods of residue analysis were based on the extraction of muscle with an acidic buffer and acetonitrile, liquid−liquid partitioning, and solid-phase clean-up with alumina, followed by high-performance liquid chromatography (HPLC) separation (Roybal et al., 1990). Several approaches have been used to enable the detection of both the chromatic dye and the colourless leuco base, including electrochemical detection, post-column oxidation of leucogentian violet with lead oxide (Ascari et al., 2012), and simultaneous visible (gentian violet absorbs at 588 nm) and fluorescence (leucogentian violet excitation at 265 nm with emission at 360 nm) detection (Verdon & Andersen, 2017). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have largely replaced HPLC to meet low-concentration regulatory monitoring levels (e.g. 0.5 μg/kg) for direct quantification of the dye and leuco ions (Hurtaud-Pessel et al., 2011; Xu et al., 2012; Andersen et al., 2018; Eich et al., 2020). Some multiresidue LC-MS/MS methods for the detection of therapeutic dyes in seafood include the oxidation of leuco compounds with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to ensure that dye metabolites are also detected (Tarbin et al., 2008; Andersen et al., 2009; Reyns et al., 2014; Dubreil et al., 2019). A method has been developed to extract gentian violet and leucogentian violet from zebrafish using a solid-phase microextraction probe, which detects residues via direct ionization mass spectrometry from the probe (Xiao et al., 2020). [The Working Group noted that the novel method employed in the study of zebrafish (which are not typically eaten) could have applicability in fish species that are consumed by humans.] Additional multidye LC-MS/MS methods that include sensitive quantification of gentian violet (0.09–2 μg/kg) have been applied to the analysis of foods such as dried tofu and sauces (Hu et al., 2020), and beef, chicken, pork, eggs, and milk (Park et al., 2020). High-resolution mass spectrometry has also been used for the detection and quantification of gentian violet and leucogentian violet (Amelin et al., 2017).

Surface-enhanced Raman scattering and direct mass spectrometry techniques have also been used to detect gentian violet. Silver nanoparticle films and pastes have been used to detect gentian violet on the surface of fish skin and in ballpoint pen ink (Alyami et al., 2019; Saviello et al., 2019). A surface-assisted laser desorption/ionization mass spectrometry method has been used to analyse gentian violet in printed supermarket receipts (Gao et al., 2019).

1.3.5. Biological specimens

Methods for the detection and quantification of gentian violet and leucogentian violet in human biological specimens are similar to those used for food (as described in Section 1.3.4). Gentian violet and leucogentian violet have been determined in human urine via extraction of neutralized urine with dichloromethane, extract clean-up with sodium sulfate, and analysis by HPLC with absorbance or electrochemical detection (Sagar et al., 1995). [The Working Group noted that the methods used for gentian violet and leucogentian violet detection in fish described in Section 1.3.4 could be useful for analysing material from humans or experimental animals. For biological specimen analysis, it might be more important to monitor N-demethylated and/or N-oxide metabolites of gentian violet and leucogentian violet.]

1.4. Occurrence and exposure

1.4.1. Environmental occurrence

Gentian violet is not known to occur naturally in the environment. Gentian violet and leucogentian violet production and their use (e.g. during the production of ink cartridges and coloured paper, and during the recycling of printed paper) may result in the release of these compounds into the environment via streams of both industrial and municipal wastewater (Health Canada, 2020; Tkaczyk et al., 2020).

When released into the environment, gentian violet exists in cationic form. Considering its physicochemical properties, gentian violet exists only in the particulate phase in the atmosphere. [The Working Group also noted that the water solubility of gentian violet is several orders of magnitude higher than that of leucogentian violet and that the octanol/water partition coefficient of gentian violet is one order of magnitude higher, which has implications for its fate in the environment.] Particulate-phase gentian violet is removed from the atmosphere by wet and dry deposition and may be susceptible to direct photolysis by sunlight. Gentian violet is expected to be immobile if released into soil. Soils containing organic carbon and clay will adsorb gentian violet’s cationic form more strongly than its neutral counterpart. Volatilization from moist soil is not expected. According to the transformation rates observed during a river die-away test, biodegradation may be an important environmental process in soil and water. If released into water, gentian violet is expected to adsorb on suspended solids and sediment, and the non-adsorbed fraction will exist almost entirely in the cationic form; therefore volatilization from water is not expected. Gentian violet is not expected to undergo hydrolysis in the environment (NCBI, 2013).

Leucogentian violet was detected in a soil sample taken near a bank of the Buffalo River, New York, close to a dyestuff-manufacturing plant (Nelson & Hites, 1980). Theoretical estimations of concentrations of non-sulfonated triarylmethane dyes in surface water (also representing drinking-water) were calculated for three industrial sources in Canada based on the maximum production capacities of these industries: 3.2 × 10⁻⁴ mg/L from the paper-dyeing industry, 9.5 × 10⁻⁴ mg/L from the de-inking industry, and 2.1 × 10⁻⁴ mg/L from the general formulation industry. These conservative estimates were made for gentian violet, malachite green, and two other triarylmethane dyes collectively, assuming that any one of the four dyes could be substituted for another (Health Canada, 2020). In the National Water Pollution Control and Treatment Project in Dong Lin, China, gentian violet concentrations of 0.87 and 0.049 µg/L were found in the water from turtle farming ponds and effluent environmental water, respectively (Zhang et al., 2012). Gentian violet absorbs light at an ultraviolet maximum of 590 nm with potential for direct photolysis. In water, the photoreaction is reported to give para-dimethylamino phenol and 4,4′-bis dimethylamino benzophenone, the leuco and demethylated derivatives of gentian violet. The bioconcentration in aquatic organisms is low, as suggested by the estimated bioconcentration factor of 3 L/kg in fish (NCBI, 2013), but such models may not be appropriate for triarylmethane dyes because of their cationic nature. For these triarylmethanes, partitioning to proteins in the cell membranes is more likely to occur than partitioning to lipids (Health Canada, 2020).

A study was performed to analyse the presence of 16 dyes, which included triarylmethanes and their metabolites such as gentian violet and leucogentian violet, in wild fish in Belgium. Muscle samples were analysed from individual yellow-phased European eels (Anguilla anguilla) from 91 locations in rivers, canals, and lakes sampled between 2000 and 2009. Gentian violet and leucogentian violet were detected in samples from 58.2% and 50.5% of the locations, respectively. The concentrations of gentian violet and leucogentian violet ranged between 0.12 and 2.60 µg/kg (Belpaire et al., 2015). In an earlier study conducted in Germany, gentian violet and leucogentian violet were found in tissue samples from wild eels caught in seven out of eight receiving waters of effluents from municipal sewage treatment plants. The concentrations of gentian violet and leucogentian violet ranged from 0.06 to 6.71 µg/kg (Schuetze et al., 2008).

1.4.2. Occurrence in food and feed

Gentian violet is used in veterinary medicine and in the aquaculture industry for the control of ectoparasites, and fungal and bacterial infections. Residues of both gentian violet and leucogentian violet may be present in muscle and skin after gentian violet treatment. Although gentian violet metabolizes within days of treatment, leucogentian violet persists in fish muscle and skin for months and is considered to be the marker residue (Thompson et al., 1999). [The Working Group noted that in the reports described below, the methods either detected gentian violet and leucogentian violet separately, or detected total residues as the sum of gentian violet and leucogentian violet after leucogentian violet had been oxidized to gentian violet.]

According to the European Food Safety Authority reports published between 2015 and 2020, few Member States (one to four) reported one or two samples that were non-compliant for the presence of gentian violet and leucogentian violet in their national veterinary drug residue control plan (EFSA, 2015, 2016, 2017, 2018, 2019, 2020). In the European Rapid Alert System for Food and Feed, very few notifications of non-compliant samples associated with imports or trade between Member States have been reported. Since 2005, 15 notifications of gentian violet or leucogentian violet residue violations have been made by EU Member States in eel, salmon, tilapia, rainbow trout, catfish, pangasius, and sturgeon (caviar). Residue concentrations have typically ranged from 0.8 to 6.6 µg/kg, although two high-concentration (41.1 and 654.6 µg/kg) samples were reported for eel from Indonesia in 2006 (European Commission, 2020).

In a study of processed fish and shrimp samples in Korean local markets, gentian violet was detected (168.4 µg/kg) in 1 of 67 eel samples tested. It was not detected in the other 186 processed fish and shrimp samples, which originated from the Republic of Korea, China, Thailand, Viet Nam, Norway, Peru, and the Russian Federation, or were of unknown origin (Lee et al., 2010). Among fish obtained from a local market in China, 7.15 µg/kg of gentian violet was detected in tilapia; none was detected in carp, sea cucumber, or seashell (Xu et al., 2012). Among 20 salmon and shrimp samples purchased from different markets in China, 1.2 µg/kg of gentian violet and 2.5 µg/kg of leucogentian violet were detected in one salmon muscle sample (Tao et al., 2011). Leucogentian violet (0.6–1.0 µg/kg) was detected in 5 out of 208 samples of rainbow trout obtained from local fish retailers and supermarkets in Turkey (Kaplan et al., 2014). In the Russian Federation, 5.3 µg/kg of gentian violet was detected in black caviar (Amelin et al., 2017). Gentian violet and leucogentian violet residues have also been reported for samples tested in the USA, Canada, and Jordan (Table 1.2; WHO, 2014a; Gammoh et al., 2019). In the EU and USA, respectively, 3% and 6% of reported veterinary drug violations detected in finfish in 2001–2008 and 2001–2006, respectively, were due to the detection of gentian violet. The concentrations detected in the EU and the USA did not differ (Love et al., 2011).

Table 1.2

Detection and quantification of gentian violet and leucogentian violet in aquaculture products available on the international market^a.

In a screening study of 19 commercially available processed animal products (salmon feed ingredients) from central Europe, leucogentian violet was detected in one poultry blood-meal sample (Nácher-Mestre et al., 2016).

1.4.3. Occupational exposure

Occupational exposure to gentian violet is expected to occur via dermal contact during paper dyeing, via inhalation of dust or aerosols produced during the formulation of dye or ink, or during the filling of containers such as ink cartridges and ballpoint pens (ECHA, 2012). [The Working Group noted that occupational exposure to gentian violet and leucogentian violet may occur through dermal contact and inhalation at workplaces where the compounds are produced or applied (see Sections 1.1.2 and 1.2.2).] In a survey conducted in the USA in 1981–83, 75 632 people were estimated to be potentially occupationally exposed to gentian violet: 69% of them working in health services, 12% in printing and publishing, and 8% in agricultural services (NIOSH, 2017). [The Working Group noted that it is unclear whether these percentages reflect modern exposure patterns, given the age of the study.]

1.4.4. Exposure in the general population

The predominant source of exposure to dye substances in the triarylmethanes group is from the use of products that contain them that are available to consumers (Health Canada, 2020). Exposure of the general population can potentially occur during the use of the consumer products described in Section 1.1.2, such as ballpoint and marker pens (orally by sucking or via dermal contact), topical treatments for animals (inhalation or dermal), coloured paper, hair dye, aquarium fish treatments, or through the consumption of contaminated drinking-water or residue-containing fish (Table 1.2). A screening assessment performed by Health Canada suggested exposure via drinking-water to be the main route of exposure to gentian violet. A potential dose of 0.0001 mg/kg body weight (bw) per day was estimated for the Canadian general population on the basis of predicted surface water concentrations as a result of environmental release by the paper de-inking industry. Other exposure scenarios considered, but not included in the estimation because of lower estimated exposures, were surface water due to industrial release from paper dyeing in paper mills and production facilities, and consumer “down-the-drain” releases, consumption via food, and the use of consumer products such as paper products, mixtures, or manufactured items in which gentian violet is used as a pigment (Health Canada, 2020).

[The Working Group noted that despite the multitude of sources, no quantitative exposure data were available.]

2. Cancer in Humans

No data were available to the Working Group.

3. Cancer in Experimental Animals

3.1. Gentian violet

See Table 3.1.

Table 3.1

Studies of carcinogenicity with gentian violet in experimental animals.

3.1.1. Mouse

Oral administration (feed)

In a study of chronic toxicity and carcinogenicity that complied with Good Laboratory Practice (GLP) (NCTR, 1984; Littlefield et al., 1985), a total of 720 male and 720 female B6C3F₁ mice (age, approximately 4–5 weeks) were given feed containing gentian violet (purity, 99%; methyl violet, 1%) at a concentration of 0, 100, 300, or 600 ppm [approximately equivalent to 0, 12.5, 33.9, and 66.1 mg/kg bw per day for males, and 0, 14.3, 37.5, and 71.4 mg/kg bw per day for females] for the control group and the groups at the lowest, intermediate, and highest dose, respectively, for up to 24 months. The feed containing gentian violet was certified to be within 10% of the target dose. For the mice treated for 24 months, there were 192 males and 192 females in the control group and 96 males and 96 females in each group treated with gentian violet. For the mice treated for 12 or 18 months, there were 48 males and 48 females in the control group and 24 males and 24 females in each group treated with gentian violet. Mortality was very low until approximately 450 days (15 months), after which there was a significant positive dose-related trend in males (P = 0.01288, Cochran–Armitage test) and females (P = 0.00005, Cochran–Armitage test), with mortality being significantly higher in all treated groups of females compared with controls. At study termination, survival was 167/192, 83/96, 77/96, and 74/96 in males, and 167/192, 69/96, 70/96, and 35/96 in females, for the control group and the groups at the lowest, intermediate, and highest dose, respectively. Treatment with gentian violet did not influence the terminal body weights of males or females. Complete necropsies and histopathological examinations were performed.

In male mice at 24 months, there was a significant positive trend in the incidence of hepatocellular adenoma [P < 0.001, Cochran–Armitage trend test] and of hepatocellular carcinoma (P < 0.001, trend test), with a significant increase in the incidence of hepatocellular adenoma at the intermediate and highest dose [P < 0.01 and P < 0.001, respectively, one-tailed Fisher exact test], and of hepatocellular carcinoma at the highest dose [P < 0.01, one-tailed Fisher exact test]. The incidence of Harderian gland adenoma was also significantly increased at the intermediate and highest dose (P < 0.05 and [P = 0.0362], respectively, one-tailed Fisher exact test). At 12 or 18 months, no treatment-associated neoplasms were reported in males.

In female mice at 24 months, there was a significant positive trend in the incidence of hepatocellular adenoma and of hepatocellular carcinoma (both P < 0.001, Cochran–Armitage trend test), with a significant increase in the incidence of hepatocellular adenoma [P < 0.01 and P < 0.001, one-tailed Fisher exact test] and of hepatocellular carcinoma (both P < 0.001, one-tailed Fisher exact test) at the intermediate and highest dose, respectively, when compared with controls. Treatment with gentian violet caused a significant positive trend in the incidence of Harderian gland adenoma (P = 0.001, Cochran–Armitage trend test), with the incidence being significantly higher at the lowest, intermediate, and highest dose [P < 0.05, P < 0.001, and P < 0.005, respectively, one-tailed Fisher exact test] than in controls. Significant positive trends in the incidence of type A reticulum cell sarcoma [histiocytic sarcoma] were reported for the urinary bladder, ovaries, uterus, and vagina [P < 0.0005, P = 0.009, P < 0.001, P = 0.001, respectively, Cochran–Armitage trend test], with a significant increase in incidence (urinary bladder, P < 0.05 and P < 0.01; ovaries, P = 0.036 and P = 0.04; uterus, P < 0.01 and P < 0.001; and vagina, P = 0.04 and P < 0.001, Fisher exact test) at the intermediate and highest dose, respectively. At 18 months, a significant positive trend in the incidence of hepatocellular adenoma (P = 0.002, Cochran–Armitage trend test) was observed, with the increase being significant (P = 0.005, one-tailed Fisher exact test) at the highest dose. At 12 months, treatment with gentian violet did not cause a significant increase in the incidence of tumours in female mice.

Regarding non-neoplastic lesions observed at 24 months, exposure to gentian violet caused a significant positive trend and an increase in the incidence of erythropoiesis in the spleen and atrophy of the ovaries in females treated with gentian violet compared with controls. [The Working Group noted that this was a well-conducted study that complied with GLP, males and females were used, the duration of exposure and observation was adequate, and a high number of mice per group was used.]

3.1.2. Rat

(a) Oral administration

In a study in rats [age and strain not reported], oral administration [regimen not reported] of 4:4′:4′′-hexamethyltriaminotriphenylmethane [gentian violet, purity not reported] for more than 300 days caused gastric papilloma and adenomatous proliferation in the hepatic tissue (Kinosita, 1940). [The Working Group noted that the study lacked details on study design and primary data and was considered inadequate for the evaluation of the carcinogenicity of gentian violet in experimental animals.]

(b) Transplacental and perinatal exposure, followed by oral administration (feed)

In a study of chronic toxicity and carcinogenicity that complied with GLP (NCTR, 1988; Littlefield et al., 1989), groups of male and female Fischer 344 rats (F₀ generation) (180 controls and 90 treated rats per group) were given feed containing gentian violet (purity, 99%; methyl violet, 1%) at a concentration of 0, 100, 300, or 600 ppm, for the control group, and the groups at the lowest, intermediate, and highest dose, respectively, for at least 80 days. While still receiving treated feed, female rats were mated with males that were receiving the same doses of gentian violet. Two offspring (F₁ generation) of each sex were randomly selected from each litter and three rats allocated per cage as weanlings [age, not reported] to the study of chronic toxicity and carcinogenicity. The F₁ rats were exposed to the same doses as their respective F₀ parents for up to 24 months. [These dose levels were approximately equivalent to 0, 4.3, 11.4, and 22.9 mg/kg bw per day for male F₁ rats, and 0, 5.7, 14.3, and 28.6 mg/kg bw per day for female F₁ rats.] The feed containing gentian violet was certified to be within 10% of the target dose. For the interim evaluation at 24 months, there were 180 F₁ males and 180 F₁ females in the control group and 90 F₁ males and 90 F₁ females in each dose group. For the interim evaluation at 12 or 18 months, there were 15 F₁ males and 15 F₁ females in each group. Mortality was significantly increased in male rats at the intermediate dose, and there was a significant dose-related increase in mortality in female rats, with the increase in mortality being significant for females at the intermediate and highest dose. Survival was 121/180, 60/90, 47/90, and 55/90 in males, and 121/180, 56/90, 36/90 and 31/90 in females, for the control group and the groups at the lowest, intermediate, and highest dose, respectively. At 24 months, the terminal body weights of male and female rats receiving gentian violet at the highest dose were significantly lower than those of the controls [with the final mean body weights being 92% and 86% of those of the male and female control rats, respectively]. Complete necropsies and histopathological examinations were performed.

In male rats at 24 months, there was a significant positive trend in the incidence of hepatocellular adenoma (P < 0.01, Peto trend test), with incidence being significantly increased at the intermediate and highest dose (both P < 0.01, Peto test and Bonferroni correction). Such a significant positive trend was also observed for the incidence of follicular cell adenocarcinoma of the thyroid gland (P < 0.01, Peto trend test), with the incidence being significantly increased in rats at the lowest and the highest dose (P < 0.05 and P < 0.01, respectively, Peto test and Bonferroni correction). There was a significant positive trend in the incidence of follicular cell adenoma or adenocarcinoma (combined) of the thyroid gland [P < 0.05, Cochran–Armitage trend test], with incidence being significantly increased at the highest dose [P < 0.01, one-tailed Fisher exact test]. Mesothelioma of the testis or epididymis was observed with an incidence of 3%, 2%, 6%, and 9% in the control group and in the groups receiving the lowest, intermediate, and highest dose, respectively [statistical analysis of the incidence of mesothelioma could not be performed, because the incidence was not reported as the number of rats with lesions per number of rats examined microscopically]. At 12 or 18 months, treatment did not cause a significant increase in the incidence of tumours in male rats. However, mesothelioma of the testis or epididymis was observed at 18 months with an incidence of 0%, 0%, 13%, and 13% in the control group and in groups at the lowest, intermediate, and highest dose, respectively [statistical analysis of the incidence of mesothelioma could not be performed because the incidence was not reported as the number of rats with lesions per number of rats examined microscopically].

In female rats, at 24 months, there was a significant positive trend in the incidence of follicular cell adenocarcinoma of the thyroid gland (P < 0.01, Peto trend test), and a significant increase in incidence at the two higher doses (P < 0.05 and P < 0.01, respectively, Peto test and Bonferroni correction). There was a significant positive trend in the incidence of follicular cell adenoma or adenocarcinoma (combined) of the thyroid gland [P < 0.01, Cochran–Armitage trend test], with a significant increase in incidence at the two higher doses [P < 0.01 and P < 0.001, respectively, one-tailed Fisher exact test]. Adenomas and adenocarcinomas of the clitoral gland were also observed with an incidence of 12%, 6%, 18%, and 33% in the control group and in groups at the lowest, intermediate, and highest dose, respectively [statistical analysis of the incidence of adenoma or adenocarcinoma (combined) of the clitoral gland could not be performed because the incidence was not reported as the number of rats with lesions per number of rats examined microscopically]. At 18 months, there was a significant positive trend in the incidence of mononuclear cell leukaemia (P < 0.05, Cochran–Armitage trend test), with a significant increase in incidence in females at the highest dose (P < 0.01, one-tailed Fisher exact test). At 12 months, no treatment-associated neoplasms were reported in females.

Regarding non-neoplastic lesions observed at 24 months, most were reported in the liver. Gentian violet caused a significant positive trend in the incidence and an increase in the incidence of hepatocyte regeneration and of mixed cell foci in all treated groups of male and female rats. Other lesions listed below also showed at least a significant positive trend in incidence, with incidence being significantly increased in one or two dose groups. In males, these other non-neoplastic lesions included clear cell foci, eosinophilic foci, basophilic foci, cytoplasmic vacuolization, and centrilobular necrosis of the liver, follicular cysts of the thyroid gland, red pulp hyperplasia of the spleen, and hyperplasia of the mesenteric lymph nodes. In females, these other non-neoplastic lesions included eosinophilic foci, haematopoietic cell proliferation, centrilobular fatty change and necrosis, and bile duct hyperplasia of the liver, and hyperplasia of the bone marrow. [The Working Group noted that this was a well-conducted study that complied with GLP, males and females were used, the duration of exposure and observation was adequate, and a high number of rats per group was used.]

4. Mechanistic Evidence

4.1. Absorption, distribution, metabolism, and excretion

4.1.1. Humans

No data were available to the Working Group.

4.1.2. Experimental systems

The absorption, distribution, metabolism, and excretion of gentian violet has been reviewed in Docampo & Moreno (1990), WHO (2014b), and OEHHA (2019).

(a) In vivo

Radiolabelled gentian violet was administered orally to rats and mice. Male and female Fischer 344 rats treated by gavage were given a single dose of [¹⁴C]-labelled gentian violet (4.8 mg/kg bw for males, 5.2 mg/kg bw for females). The distribution of the [¹⁴C]-labelled dye was measured in the liver, kidney, fatty tissue, gonads, muscle, urine, and faeces at 2, 4, 14, 24, and 36 hours after administration. Maximal residue levels were found at 4 hours in the liver, kidney, muscle, and gonads; a plateau was reached in fatty tissue after 24 hours. The depletion half-lives in male and female livers were 14.5 and 17.0 hours, respectively. The recovery values for males and females (males/females) were 2.2/2.2% and 72.9/63.8% of the single gentian violet dose in the urine and the faeces, respectively. In bile collected from cannulated 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 and B6C3F₁ mice by gavage. Maximal residue levels were found in fatty tissues of females of both species, and a statistically significant sex difference (P < 0.01) was noted. Residue levels in kidney and muscle tissues from both species, and in mouse livers, also showed sex differences. The recovery values for males and females (males/females) were 2.2%/1.6% and 65.5%/72.8% in the urine and the faeces of rats, respectively, and 5.9%/8.1% and 65.9%/67.4% in the urine and faeces of mice (McDonald et al., 1984a; NCTR, 1989).

Regarding the metabolism of gentian violet, McDonald & Cerniglia (1984) showed that leucogentian violet was excreted in the faeces collected from a female Fischer 344 rat that was given [¹⁴C]-labelled gentian violet by gavage for 4 days. The metabolites of gentian violet were also analysed in mice and rats by NCTR (1989) and identified as three demethylated metabolites (pentamethyl para-rosaniline and N,N,N′,N′- and N,N,N′,N′′-tetramethyl para-rosanilines) and two reduced metabolites (leucogentian violet and leuco-pentamethyl para-rosaniline). A summary of the proposed metabolism of gentian violet and leucogentian violet is provided in Fig. 4.1.

Fig. 4.1

Metabolic pathways for gentian violet and leucogentian violet

(b) In vitro

In bacteria, McDonald & Cerniglia (1984) demonstrated that gentian violet was transformed to leucogentian violet after incubation under anaerobic conditions with microflora isolated from human faeces, and from the intestinal contents of rats and chickens.

When metabolized by rat liver microsomes, gentian violet appears to undergo one-electron reduction by cytochrome P450 to produce a carbon-centred free radical (Harrelson & Mason, 1982). This carbon-centred radical can be formed by photoreduction of gentian violet after exposure to visible light (Docampo et al., 1988).

McDonald et al. (1984b) studied the metabolism of gentian violet in the presence of liver microsomes obtained from both sexes of four mouse strains, three rat strains, hamster, guinea-pig, and chicken: the main metabolites identified were pentamethyl para-rosaniline and the isomeric N,N,N′,N′- and N,N,N′,N′′-tetramethyl para-rosanilines. Comparable patterns of demethylated metabolites were observed between species. [The Working Group noted that information about the relative amounts of the different metabolites, including leucogentian violet, was sparse.]

4.2. Evidence relevant to key characteristics of carcinogens

This section summarizes the evidence for the key characteristics of carcinogens (Smith et al., 2016), including whether gentian violet (and leucogentian violet) is electrophilic or can be metabolically activated to an electrophile; is genotoxic; or induces oxidative stress. Insufficient data were available for the evaluation of other key characteristics of carcinogens.

4.2.1. Is electrophilic or can be metabolically activated to an electrophile

Through measurement of sedimentation and viscosity, it was shown that gentian violet binds externally to the surface of the DNA helix, with a high degree of preference for two adjacent A−T base pairs, and that it induces severe bending accompanied by unwinding of the DNA helix (Müller & Gautier, 1975; Wakelin et al., 1981).

The ability of gentian violet to bind to bovine haemoglobin was demonstrated in vitro by Liu et al. (2013) using several spectroscopic and molecular modelling methods. A change in the spatial conformation of bovine haemoglobin was observed after binding of gentian violet (Liu et al., 2013).

4.2.2. Is genotoxic

Table 4.1, Table 4.2, Table 4.3, and Table 4.4 summarize the available studies of the genetic and related effects of gentian violet.

Table 4.1

Genetic and related effects of gentian violet in human primary cells and human cell lines in vitro.

Table 4.2

Genetic and related effects of gentian violet in non-human mammals in vivo.

Table 4.3

Genetic and related effects of gentian violet in non-human mammalian cells in vitro.

Table 4.4

Genetic and related effects of gentian violet in non-mammalian experimental systems.

(a) Humans

(i) Exposed humans

No data were available to the Working Group.

(ii) Human primary cells and human cell lines in vitro

See Table 4.1.

In human primary cells in vitro, a single concentration of gentian violet induced an increase in chromosomal aberration in cultured primary human peripheral blood lymphocytes from healthy donors (Au et al., 1978; Hsu et al., 1982), and from healthy individuals and patients with β-thalassaemia (Krishnaja & Sharma, 1995).

Au et al. (1978) also showed that gentian violet induced an increase in chromosomal aberrations in HeLa cells.

(b) Experimental systems

(i) Non-human mammals in vivo

See Table 4.2.

After injection of gentian violet in the tail veins of B6C3F₁ mice up to a dose of 8 mg/kg bw, no DNA damage was observed in splenic lymphocytes (Aidoo et al., 1990). Gentian violet also failed to induce chromosomal aberrations in bone marrow erythrocytes of Swiss albino mice that received the substance via drinking-water for 4 weeks up to a dose of 8 mg/kg (Au et al., 1979).

(ii) Non-human mammalian cells in vitro

See Table 4.3.

Aidoo et al. (1990) showed that gentian violet induced DNA damage (nucleoid sedimentation) in cultured lymphocytes from the spleens of B6C3F₁ mice and caused weak gene amplification in SV40-transformed Chinese hamster embryo (CO60) cells. Gentian violet induced DNA strand breaks in whole-blood samples collected from Sprague-Dawley rats (Díaz Gómez & Castro, 2013). When the rats were treated with antioxidants (α-tocopherol, lipoic acid, or N-acetylcysteine) before the blood samples were collected, the genotoxic effects induced by gentian violet were significantly decreased (Díaz Gómez & Castro, 2013). Gentian violet did not induce gene mutations at the hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus of Chinese hamster ovary (CHO) CHO-K1-BH4 cells, but caused a slight increase at the glutamic-pyruvate transaminase (Gpt) locus of CHO AS52 cells (the increase was observed only at very toxic concentrations, and was not reproduced with different gentian violet batches) (Aidoo et al., 1990).

Au et al. (1978) demonstrated that gentian violet induced mitotic anomalies. Gentian violet consistently induced chromosomal aberrations in various cell lines: Mus musculus mouse fibroblast L cells, a fibroblast cell line derived from Peromyscus eremicus, and a fibroblast cell line derived from the Indian muntjac (Muntiacus muntjak) (Au et al., 1978). It also induced chromosomal aberrations in CHO cells (Au et al., 1978, 1979; Au & Hsu, 1979). The cytogenic effect observed in CHO cells decreased in the presence of the S9 metabolic activation system (Au et al., 1979).

(iii) Non-mammalian experimental systems

See Table 4.4.

At low concentrations, gentian violet binds to double-stranded DNA at AT-rich regions, while it binds at all available sites at high concentrations (Fox et al., 1992).

Cornell K-strain chicken embryos treated with gentian violet did not show sister-chromatid exchange (Au et al., 1979; Bloom, 1984).

In one study performed on Drosophila melanogaster, gentian violet did not induce mutations in a sex-linked recessive lethal assay (Mason et al., 1992).

Gentian violet induced DNA damage in Bacillus subtilis (Fujita et al., 1976; Choudhary et al., 2004). Grigg et al. (1984) observed that gentian violet induced DNA strand breaks in Escherichia coli B strain.

An overwhelming majority of the data show that gentian violet did not induce mutagenicity with or without metabolic activation in Salmonella typhimurium strains TA1535, TA100, TA1538, TA97, TA98, TA1537, YG1041, and YG1042 (Shahin & Von Borstel, 1978; Au et al., 1979; Bonin et al., 1981; Levin et al., 1982; Thomas & MacPhee, 1984; Hass et al., 1986; Aidoo et al., 1990; Malachová et al., 2006; Ackerman et al., 2009). However, a few authors reported a mutagenic effect without metabolic activation in TA98, TA100, and TA1535 (Fujita et al., 1976; Bonin et al., 1981), with metabolic activation in TA98 and TA100 (Fujita et al., 1976; Ayed et al., 2017), as well as in TA97 and TA104 (Aidoo et al., 1990). Malachová et al. (2006) described a mutagenic effect of crystal [gentian] violet with metabolic activation in TA98, which was associated with a cytotoxic effect. In E. coli strains, gentian violet caused mutagenicity with and without metabolic activation (Thomas & MacPhee, 1984; Hass et al., 1986), and induced mutagenicity without metabolic activation in a study by Fujita et al. (1976) (not tested with metabolic activation). In the Rosenkranz repairable DNA assay, gentian violet gave positive results in E. coli strains W3110 polA⁺ and P3478 polA^– (Au et al., 1979; Levin et al., 1982), but negative results in the Saccharomyces cerevisiae XV185-14C strain (Shahin & Von Borstel, 1978).

4.2.3. Induces oxidative stress

As already mentioned above (Section 4.1.2.b), gentian violet can lead to the formation of a carbon-centred free radical, either by photoreduction (Reszka et al., 1986; Docampo et al., 1988) or by enzymatic reaction (Harrelson & Mason, 1982).

4.4. Data relevant to comparisons across agents and end-points

The mechanistic characteristics common to carcinogens (the 10 key characteristics of carcinogens) can be investigated through biochemical and cell-based assays run by the United States Environmental Protection Agency (US EPA) and the United States National Institutes of Health Toxicity Forecaster/Toxicology in the 21st Century (ToxCast/Tox21) high-throughput screening programmes (Chiu et al., 2018; Guyton et al., 2018). Since 2017, the IARC Monographs have described the results of high-throughput screening assays to compare activity across agents and other in vitro and in vivo evidence relevant to the key characteristics.

Of the five compounds included in IARC Monographs Volume 129, three have been evaluated in at least some of the US EPA and United States National Institutes of Health high-throughput screening assays: gentian violet (CAS No. 548-62-9), malachite green (malachite green chloride, CAS No. 569-64-2, and malachite green oxalate, CAS No. 2437-29-8), and leucomalachite green (CAS No. 129-73-7) (US EPA, 2020a, b, c, d). Table 4.5 summarizes findings for assay end-points mapped to key characteristics for the compounds evaluated. Details of the specific assays (and end-points) run for each chemical in this volume and the mapping to the key characteristics can be found in the Supplementary Material (Annex 1, Supplementary material for Section 4, web only; available from: https://publications.iarc.fr/603). It is important to note that some assays either lacked, or had uncharacterized and generally low, xenobiotic metabolism, limiting observations primarily to effects elicited by parent compounds. The strengths of the high-throughput screening battery of assays are the standardization of the protocols applied across compounds, allowing comparisons across compounds and the evaluation of specificity of assay end-points to the key characteristics, and ultimately to the apical outcome of carcinogenesis (Becker et al., 2017; Chiu et al., 2018; Watford et al., 2019). The 299 ToxCast/Tox21 assay end-points mapped to key characteristics interrogated in this and other monographs are initially described in Chiu et al. (2018), with the most up-to-date mapping described in detail in IARC Monographs Volume 123 (IARC, 2019). All ToxCast/Tox21 data were downloaded from the US EPA CompTox Chemicals Dashboard 10th Release (US EPA, 2021) between 2 and 19 October 2020 or on 24 February 2021 (malachite green oxalate).

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 129^a .

The individual assessments for each compound are included in the corresponding monographs in the present volume.

4.4.1. Gentian violet

Results were available for 280 assay end-points (out of the 299 that were mapped to key characteristics) for gentian violet (US EPA, 2020a). Gentian violet was considered active in 126 assay end-points, including the one assay end-point mapped to “is electrophilic or can be metabolically activated to an electrophile”, 10 of the 12 assay end-points mapped to “is genotoxic”, 2 of the 5 mapped to “induces epigenetic alterations”, 8 of 16 end-points mapped to “induces oxidative stress”, 27 of the 90 assay end-points mapped to “modulates receptor-mediated effects”, and 78 of 109 end-points mapped to “alters cell proliferation, cell death, or nutrient supply”.

Assays within the “is genotoxic” key characteristic provide measurements of DNA damage or repair in human liver (HepG2), kidney (HEK293T), and intestinal (HCT-116) cell lines, as well as a CHO cell line (CHO-K1) and a chicken lymphoblast cell line (DT40). Gentian violet (purity, > 90%) elicited TP53 activation measured through reporter assays in HCT-116 and HepG2 cells. Gentian violet was considered active, as measured by phosphorylated histone H2AX (γH2AX) assay detecting H2AX protein phosphorylation, consistent with DNA double-strand breaks in a CHO cell line (CHO-K1). Gentian violet was also considered active as measured by assays using DT40 chicken lymphoblastoid cell lines deficient for the DNA-repair genes REV3 and KU70/RAD54. Gentian violet was not considered active as determined by the ATAD5-luc assay in HEK293T cells, which measures levels of ATAD5 protein that localize to the site of stalled replication forks resulting from DNA damage in replicating cells. It is important to note that both positive (e.g. etoposide, 5-fluorouridine, tetra-N-octylammonium bromide, and mitomycin C) and negative (dimethyl sulfoxide) controls are run concurrently, and subsequent analyses and activity calls are normalized against data for positive and negative controls run on the same plates (Hsieh et al., 2019).

4.4.2. Leucogentian violet

Leucogentian violet has not been evaluated in high-throughput screening assays.

4.4.3. Summary

Gentian violet has been evaluated in ToxCast or Tox21 assays with end-points mapped to key characteristics of carcinogens. It was active in a significant fraction of mapped end-points in which it had been tested (45%). Specifically, gentian violet was considered active in most of the “is genotoxic” assay end-points. It was also considered active for a variety of the assay end-points mapped to the following key characteristics: induces epigenetic alterations, induces oxidative stress, modulates receptor-mediated effects, and alters cell proliferation, cell death, or nutrient supply. Relevant to findings in other sections, gentian violet was considered active in an assay measuring thyroid receptor antagonism in GH3, a rat pituitary gland cell line, and was considered to give negative results in an assay measuring thyroid hormone receptor-agonist activity in the same cell line. Gentian violet was considered to give negative results in an assay measuring thyroid hormone receptor-mediated transcription in HepG2 cells. Leucogentian violet has not been evaluated in high-throughput screening assays.

5. Summary of Data Reported

6. Evaluation and Rationale

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