**4. Epigenetic Carcinogens or Carcinogens with Uncertain Mode of Action and Related Chemicals Present in Food**

This section provides an overview of food-derived carcinogens that are typically negative in genotoxicity assays in vitro and in vivo, and which facilitate neoplastic development through molecular and cellular mechanisms other than direct DNA reactivity. This section also includes carcinogens that do not have enough mechanistic data for classification. Chemical structures of carcinogens and related chemicals discussed in this section are provided in Figures 6–10.

#### *4.1. Phytotoxins*

In 2018, FDA announced a ban on seven synthetically derived agents, including methyl eugenol, myrcene, pulegone, benzophenone, ethyl acrylate, pyridine and styrene for use as flavoring substances [610]. The majority of these substances have natural counterparts, discussed in this manuscript.

**Figure 6.** Chemical structures of non-DNA-reactive carcinogenic phytochemicals present in foods.

#### 4.1.1. β-Myrcene

*Occurrence: β-myrcene* (Figure 6(1)) is an acyclic monoterpene, which occurs naturally in a variety of plants, including verbena, lemongrass, bay, rosemary, basil, cardamon and is a constituent in many fruits, vegetables and beverages, such as citrus peel oils and juices, pineapple, celery, carrot, beer, white wine, and many others [611–613]. The highest levels of β-myrcene, up to 10 g/kg dry weight, were reported in hops [611]. It is also widely used as a flavor and fragrance material.

*Carcinogenicity:* Oral administration of β-myrcene by gavage up to 1000 mg/kg bw, 5 days a week, induced a significant increase in liver tumors (adenomas and carcinomas) in male and female mice. In rats, increased incidences of renal tubular adenomas and carcinomas were reported [611,614–616].

*Genotoxicity/DNA Binding (Adducts)*: β-myrcene lacks genotoxicity and mutagenicity in vitro and in vivo [611,613,614,616–619], accordingly, no covalent DNA binding was reported.

*Metabolism*: Metabolism of β-myrcene involves oxidation of the carbon–carbon double bond (Figure 6(1)) to an epoxide intermediate, which after hydrolysis gives rise to diol conjugates, 10-hydroxylinalool and 7-methyl-3-methyleneoct-6-ene-1,2-diol, that were detected in the urine of rabbits and rats [611,620–622]. Diols are further oxidized to corresponding aldehydes and hydroxy acids [613]. These reactions are likely metabolized by CYPs [619], and β-myrcene was shown to inhibit activity of CYP2B1 in vitro and induce CYP2B1/B2 in vivo [623,624].

*MoA*: The mechanism of tumor induction by β-myrcene remains largely unknown [612]. Analyses of cancer data by FEMA [619,622] suggested that hepatocarcinogenesis in mice and renal tumors in rats are secondary to cytotoxicity of β-myrcene at high carcinogenic doses, and in the kidney are related to the chronic progressive nephropathy and possibly unusual nephrosis. While it is structurally similar to another terpene, d-limonene, which

is known to bind to α2u-urinary globulin producing nephropathy, IARC [611] concluded that β-myrcene did not meet all of the criteria to explain its carcinogenicity by a α2uglobulin-associated mechanism. Histopathologic assessment of the kidneys from rats chronically dosed with β-myrcene confirmed that due to complex renal pathology, α2uglobulin nephropathy cannot be the sole MoA of carcinogenicity [625].

*Human Exposure*: Daily per capita intake of β-myrcene in US was calculated to be 3 μg/kg bw/day [622,626]. Another, more recent FDA estimation suggested an EDI of 1.23 μg/kg bw/day [612]. In Europe, estimated per capita intake was calculated to be 4.8 μg/kg bw/day [616].

*Human Effects*: No findings on human carcinogenicity are available [611].

*Risk*: IARC [611] classifies β-myrcene as possibly carcinogenic to humans (Group 2B) (Table 2). Safety assessment of β-myrcene by FEMA concluded that MoA of carcinogenicity in rodents is not relevant to humans and rodent carcinogenicity is not indicative of a health risk [619,622]. JECFA and EFSA [616,626] concluded that at estimated current dietary intake, β-myrcene would not pose a safety concern. Despite these conclusions, FDA recently withdrew authorization for use of a synthetic form of myrcene as a food additive due to its carcinogenicity in accordance with the Delaney Clause [610,612,627].

#### 4.1.2. Pulegone

*Occurrence*: *Pulegone* (PUL) ((R)-5-methyl-2-(1-methylethylidine)cyclohexanone) (Figure 6(2)) is a naturally occurring monoterpene ketone found in a variety of plants, in particular mint species, such as *Nepeta cataria* (catnip), *Mentha piperita*, and pennyroyal and is used as a flavoring agent [628–631].

*Carcinogenicity:* Oral administration of PUL to mice at up to 150 mg/kg bw in corn oil by gavage, 5 days/week for 105 weeks, significantly increased incidences of hepatocellular adenoma in both sexes and incidences of hepatoblastoma in male mice [628,632]. In rats, increased incidences of urinary bladder neoplasms were observed in females only, while no evidence of carcinogenic activity was observed in males.

*Genotoxicity/DNA Binding (Adducts)*: PUL was not genotoxic or mutagenic in vitro and in vivo [628,629,631–633]. Genotoxicity studies with herbal preparations containing PUL, such as peppermint oil, also yielded negative result [631]. No covalent DNA binding has been reported, although reactive metabolites of PUL can covalently bind to proteins [634].

*Metabolism*: PUL is metabolized by different pathways, including hydroxylation in the 9-position to toxic metabolite menthofuran or in the 5-position to piperitenone; reduction of the carbon-carbon double bond, which results in formation of menthone and isomenthone; or conjugation with GSH [631,635,636]. Hydroxylation to menthofuran involves multiple CYPs, including human CYP2E1, CYP1A2, CYP2C19 and CYP3A4 [637–639]. The major metabolites of PUL detected in humans were 10-hydroxypulegone, 8- and 1 hydroxymenthone, and menthol [640]. Metabolism of PUL to menthofuran can result in formation of reactive metabolites, in particular, epoxide pulegone 8-aldehyde (γ-ketoenal) and *p*-cresol, that can bind to proteins and deplete GSH levels [628,631,634,639,641].

*MoA:* An epigenetic MoA for PUL-induced urinary bladder tumors in female rats was proposed to involve chronic exposure to high concentrations resulting in excretion and accumulation of PUL and its cytotoxic metabolites, particularly piperitenone, in the urine, leading to urothelial cytotoxicity and sustained regenerative urothelial cell proliferation eventually resulting in development of urothelial tumors [642]. In addition, toxicity of menthofuran and covalent binding of its metabolites to proteins can lead to chronic regenerative cell proliferation, which can contribute to liver and urinary bladder carcinogenesis [628,630,631].

*Human Exposure:* Dietary exposure to PUL results primarily from ingestion of products flavored with spearmint or peppermint oil, such as confectionery, chewing gum, as well as alcoholic and non-alcoholic beverages [630,631]. JECFA [629] estimated an intake for PUL of approximately 2 μg/person/day or 0.04 μg/kg bw/day in Europe and 12 μg/person/day or 0.03 μg/kg bw per day in USA. The European Commission (Regulation EC No. 1334/2008) [106] set a limit of 20 mg/kg for PUL and menthofuran in foods and beverages.

*Human Effects:* No epidemiological studies linking PUL to human cancer risk have been conducted [628].

*Risk:* IARC [628] concluded that PUL was possibly carcinogenic to humans (Group 2B) (Table 2) based on sufficient evidence for carcinogenicity in experimental animals but inadequate evidence in humans. JECFA [629] found no safety concern when PUL is used as a flavoring agent. EMA [631] suggested that MoA for tumor induction in rodents is not relevant for carcinogenicity risk in humans, and recommended an acceptable exposure limit of 0.75 mg/kg bw/day.

#### *4.2. Mycotoxins*

Fumonisin B1 and Fusarin C are the major toxins derived from *Fusarium* fungi species, *Fusarium verticilloides* (also known as *moniliforme*) and *proliferatum*, which are common contaminants on crops, in particular corn [247,276,305,643].

**Figure 7.** Chemical structures of non-DNA-reactive carcinogenic mycotoxins and related chemicals present in foods.

#### 4.2.1. Fumonisins

*Occurrence: Fumonisin B*<sup>1</sup> (FB1) (Figure 7(1)), is the most prevalent member of fumonisins class. It has the chemical structure of a substituted 2-amino-icosane diester, which has features in common with the sphingoid base backbone of sphingolipids [644,645]. The

highest concentrations of FB1, which range from 310 to up to 23,800 μg/kg, were reported in maize and maize-based cereal products [276].

*Carcinogenicity*: In female mice, oral administration of FB1 caused an increase in hepatocellular adenomas and carcinomas. In the study in male rats, an increase in cholangocarcinomas and hepatocellular carcinomas were observed, while in the other rat study, FB1 induced renal tubule carcinomas in males exposed to up to 100 ppm [247,646,647]. FB1 was reported to have liver cancer initiating activity, as evidenced by induction of preneoplastic foci in rats by 7 weeks of dosing [648]. Some studies suggest that it also has tumor promoting activity [649,650]. For example, FB1 administered in diet had promotional activity on liver tumors initiated by AFB1 and *N*-methyl-*N*- -nitro-*N*-nitrosoguanidine in trout [651].

*Genotoxicity/DNA Binding (Adducts):* The structure of FB1 (Figure 7(1)) lacks features that confer DNA reactivity. Accordingly, it was not mutagenic in bacteria; however, a positive result was reported in a luminescence induction assay in the absence of metabolic activation [247,647]. The compound did not induce DNA repair synthesis in the liver cells of rats in vitro or in vivo and no evidence for DNA adduct formation with oligonucleotides in vitro was found [247]. However, evidence for induction of DNA damage by FB1 was reported in rat brain glioma cells and human fibroblasts in vitro, and in spleen and liver cells isolated from exposed rats [652–654]. In addition, FB1 caused DNA fragmentation in rat liver and kidney [655]. Positive results were obtained in micronucleus assays in vitro with human-derived hepatoma (HepG2) cells but not with rat hepatocytes [656]. In bone marrow of mice, an increase in formation of micronuclei was found after intraperitoneal injection of FB1 [657]. Positive results were obtained in chromosomal aberration assays with rat hepatocytes.

*Metabolism*: There is little or no evidence that fumonisins are metabolized in vivo or in vitro [247,646,647]. Nevertheless, FB1 induced CYP1A activity in hepatoma cell line, and CYP2E activity in rats, while inhibiting CYP2C11 and CYP1A2 enzymes [658,659]. Liver and kidney retain most absorbed material [660]. Hydrolyzed FB1 is more toxic compared to the parental form, and recently, hydrolyzed metabolites were detected in the kidney and liver of rats administered FB1 by intraperitoneal injections [661].

*MoA:* One postulated MoA for FB1 carcinogenicity involves disruption of sphingolipid metabolism, either through inhibition of ceramide synthesis [662] or due to changes in polyunsaturated fatty acid and phospholipid pools [649], leading to alteration of signaling pathways that control cell behavior and DNA synthesis [247,645,660,663–666]. Such perturbations produce alterations in cell turnover. Another proposed MoA involves oxidative stress, which is likely to mediate DNA damage observed in some assays [276,291,652,654,660]. In support of this hypothesis, FB1 was demonstrated to increase lipid peroxidation in rat kidney and liver and decrease levels of antioxidant enzymes [667,668]. In addition, dosing of rats with 100 μg/kg bw for 12 weeks resulted in downregulation of hepatic antioxidant genes [655].

*Human Exposure:* In Europe and North America, EDI to FB1 ranges from 0.01 to 0.2 μg/kg bw/day, while in other countries with different climate, cultivation practices and higher consumption of maize and maze-based products, EDI levels are much higher, reaching up to 354.9 μg/kg bw/day in South America and Africa, and up to 740 μg/kg bw/day in China [660]. The highest levels of chronic dietary exposure, ranging from 0.18 to 3.9 μg/kg bw/day for FB1 and from 0.27 to 6.4 μg/kg bw/day for total fumonisins were reported in children, with cakes, cookies and pies, cereal-based foods and cereal grain being the main contributors [276]. JECFA reported that in adults, mean chronic exposures to FB1 did not exceed 0.56 μg/kg bw/day for FB1 and 0.82 μg/kg bw/day for total fumonisins, respectively.

*Human Effects:* Epidemiological evidence shows a link between exposure to *F. moniliforme* contaminated corn and esophageal and hepatocellular cancer [247,305,669–672] but these reports do not indicate the specific compounds involved. Others [673,674] investigated FB1 specifically, but were not able to find significant association between exposure and cancer risk [276].

*Risk*: IARC [247] evaluated FB1 as possibly carcinogenic to humans (Group 2B) (Table 2) based on sufficient evidence for carcinogenicity in experimental animals and inadequate evidence in humans. JECFA [276,647] established a PMTDI of 2 μg/kg bw/day for FB1 alone or in combination with other fumonisins, and recommended to reduce exposures to fumonisins, especially in the areas where maize is consumed at higher levels.

#### 4.2.2. Fusarin C

*Occurrence*: *Fusarin C* (FC) (Figure 7(2)) belongs to 2-pyrrolidinone metabolites produced by various species of the fungus *Fusarium*, including *Fusarium moniliforme* and *oxysporum* [305,675]. FC has been detected in corn and maize grain in concentrations ranging from 28 to 83 mg/kg [305,676,677]. While unstable to heat, FC may survive cooking process [678].

*Carcinogenicity:* FC induced papillomas and carcinomas of the oesophagus and forestomach in mice and rats when administered by oral gavage at 0.5 or 2 mg twice a week to mice or rats, respectively [305]. FC did not act as a promoter in rat liver [679].

*Genotoxicity/DNA Binding (Adducts)*: FC was genotoxic in vitro in the presence of exogenous bioactivation, producing mutagenicity, SCE, chromosomal aberration and micronuclei formation [305,680,681]. Only marginal effect was observed in UDS assay in rat hepatocytes [682]. Currently, no in vivo genotoxicity studies with FC were reported. While crude extracts of *Fusarium moniliforme* produced direct mutagenicity in bacteria as well as positive results in NPL assays, no DNA adducts were measured by NPL assay with pure FC [683,684].

*Metabolism*: FC accumulates mainly in the intestines, stomach and liver after administration by gavage to rats. Studies utilizing rat liver microsomal enzymes showed that FC is metabolized by carboxyesterase to water-soluble fusarin PM1, while monooxygenase is involved in the FC conversion to a mutagenic metabolite [305,685,686]. Hydroxylation at the 1-position resulted in production of two genotoxic metabolites, fusarin Z, which was the most potent mutagen in vitro, and fusarin X [687].

*MoA*: The role of mutagenic effects of FC in the development of cancer is not clear. One study suggested that FC may act as an estrogenic agonist in vitro [688]; however, no effects on mammary glands were detected in carcinogenicity studies [305].

*Human Exposure:* Major source of exposure to FC is maize and maize grain [305,676,677]. Currently, no data on dietary intake levels of FC in humans have been reported.

*Human Effects:* FC has been suggested to be responsible for the high incidence of esophageal cancer in China [680] and South Africa [689]. Changes in the staple diet of Black South Africans from sorghum to maize (corn), on which the fungus grows more easily, has been associated with the epidemic of squamous carcinoma of the esophagus in that area [671].

*Risk:* IARC [305] classifies FC, similar to other toxins derived from *Fusarium moniliforme* as possibly carcinogenic to humans (Group 2B) (Table 2).

#### *4.3. Environmental, Agricultural and Industrial Contaminants*

A variety of industrial contaminants and chemicals used in crop protection and production have caused cancers in experimental models and have been considered to be likely human carcinogens [67,275,690,691]. Traces of these chemicals can contaminate food, albeit at extremely low levels. The cancer risk that such exposures might pose has been a matter of debate.

**Figure 8.** Chemical structures of non-DNA-reactive carcinogenic contaminants in food.
