3.1.1.2. Estragole

*Occurrence: Estragole* (1-methoxy-4-(2-propenyl)-benzene) (Figure 1(2)) is a natural constituent of a number of aromatic plants and their essential oil fractions including among others tarragon, sweet basil, sweet fennel and anise star [77,78,82,108]. As a flavoring agent it is used at maximum levels of 50 ppm [79].

*Carcinogenicity:* Estragole and its 1- -hydroxy metabolite were hepatocarcinogenic in mice when administered in diet at doses up to 600 mg/kg bw for 12 months [91,92]. In mice susceptibility to estragole carcinogenicity was strain specific [91]. In rats, estragole administered by gavage up to 600 mg/kg bw, 5 days/week for 3 months showed evidence of carcinogenic activity, increasing incidences of cholagiocarcinomas and hepatocellular adenomas [109].

*Genotoxicity/DNA Binding (Adducts):* Genotoxicity and DNA binding of estragole has been reported [78,79,85,96,98,110–112]. However, it was primarily negative in in vitro tests [109], likely due to inadequate bioactivation [25].

*Biotransformation*: With regard to metabolism, studies in rats indicate that the proximate carcinogen, the 1- -hydroxy metabolite, was produced in minimal amounts at doses in the range of 1–10 mg/kg bw/day [79]. In humans, this metabolite appears to be produced at an even lower rate [113]. These considerations would argue for the existence of a practical threshold for carcinogenic risk in human population [114].

*MoA:* Formation of DNA adducts and genotoxicity are considered to underly carcinogenicity of estragole [79,108].

*Human Exposure*: Based on the annual production volume for flavoring substances, the per capita intake of estragole in the US is 5 μg/day [78], while other sources estimated average baseline exposures to estragole from food intake to range from 500 to 5000 μg/day, with an average exposure of 1000 μg/person/day [108].

*Human Effects*: No evidence for human carcinogenicity of estragole is available [108].

*Risk*: The Expert Panel of the Flavor and Extract Manufacturers' Association (FEMA), concluded that based on the fact that genotoxic and carcinogenic effects of estragole are dose dependent, present dietary exposures to estragole do not pose a significant cancer risk to humans [79]. However, JECFA indicated that further research is required to assess potential human risk from low-level exposures [78]. Analyses of cancer responses in rodents demonstrated that thresholds for estragole carcinogenicity were well above the levels normally associated with human consumption [114]. Based on the carcinogenic potency, the European Medical Agency (EMA) [108] calculated an ADI for adults of 52 μg/person/day.

## 3.1.1.3. Methyl Eugenol

*Occurrence*: *Methyl eugenol* (ME) (1,2-dimethoxy-4-(2-propenyl)benzene) (Figure 1(3)) occurs in a variety of plants, including nutmeg, sweet basil, tarragon, allspice and pimento [77–79,82,115]. Both ME and eugenol (Figure 1(3,4)) were found in juice from oranges treated on the tree with rind-injuring abscission agents used to loosen the fruit for mechanical harvesting [116]. As a flavoring agent, ME was used in the past at a maximum level of 50 ppm [79]; however, since 2008, ME has been banned for direct addition to foods in Europe (Regulation EC No. 1334/2008) [106].

*Carcinogenicity*: In a 2-year study, with ME administered to rats and mice of both sexes at doses up to 150 mg/kg bw by gavage, 5 days/week for 105 weeks, chemicalrelated increases in liver neoplasms occurred in all dosed groups of rats [79,115,117]. In the glandular stomach, mucosal atrophy, an early indication of potential neoplasia, was increased at all doses in rats and malignant gastric neuroendocrine tumors were observed in high dose group in male mice. In rats, gastric neuroendocrine cell hyperplasia was evident at 6 months and neuroendocrine tumors occurred in the high dose group. Other neoplasms with increased incidence included forestomach squamous cell papilloma or carcinoma, renal tubule adenomas, malignant mesotheliomas, mammary gland fibroadenomas and fibromas of the subcutaneous tissue [115,117].

*Genotoxicity/DNA Binding (Adducts)*: ME tested generally negative in genotoxicity tests in vitro and in vivo [115,117]. However, it induced chromosomal aberrations and UDS in vitro [82,115] and formed DNA adducts in human hepatocytes [98] and the livers of rats [118], turkey and chicken fetuses [85,86]. Moreover, correlation between formation of DNA adducts and tumor formation has been shown for ME, and a threshold for tumors was calculated at 1020.1 molecules/kg/day [119]. Results of PBPK modelling for rats and humans support validity of linear extrapolation of ME tumor data from rodents to humans [120]. However, the application of this log/linear plot for extrapolation is not uniformly accepted [121].

*Biotransformation:* Similar to other ABs discussed above, ME is bioactivated by CYP1A2 through hydroxylation at the 1 position (Figure 1(3)) to produce reactive 1- -hydroxymethyle ugenol, followed by sulfation. Other metabolic pathways include oxidation of the 2- ,3- double bond to form ME-2,3-oxide and O-demethylation followed by spontaneous rearrangement to form eugenol quinone methide [68,79,115].

*MoA:* DNA-binding of 1- -hydroxy ME metabolite most likely underlies MoA for the several types of ME-induced neoplasms [115,122]. In rat liver, ME rapidly induced preneoplastic lesions indicating tumor initiating activity [118]. In addition, based on mechanistic studies of other chemicals that have induced gastric neuroendocrine tumors [123], the mucosal atrophy may have produced decreased hydrochloric acid production which stimulates gastrin production leading to neuroendocrine cell proliferation, and eventually to neuroendocrine neoplasia.

*Human Exposure*: The overall EDI of ME in US from dietary sources was estimated to be 0.77 μg/kg bw/day, with basil, nutmeg and allspice being primary sources of exposure [79]. JECFA calculated mean per-capita dietary exposure to ME of 80.5 μg/day in US and 9.6 μg/day in Europe [78]. The total dietary intake of food containing ME was calculated to be 66 μg/kg bw/day for regular consumers [122].

*Human Effects*: No epidemiological studies evaluating evidence of human carcinogenicity from ME are available [68,115].

*Risk:* ME has been classified by IARC [115] as possibly carcinogenic to humans (Group 2B) (Table 2) based on sufficient evidence for carcinogenicity in animals. While FEMA concluded that present exposures to ME do not pose significant risk to human health [79], estimated MoE based on the dose–response modelling ranges from 100 to 800, suggesting that the dietary intake of ME is of high concern [122]. In 2018, the FEMA Expert Panel removed ME from the FEMA Generally Recognized as Safe (GRAS) list, citing the need for additional data to clarify the relevance of DNA adducts formed by ME in humans [124].

#### 3.1.1.4. *α*- and *β*-asarone

*Occurrence*: Propenylic phenylpropenes, *α-* and *β-asarone* ((E)-/(Z)-1,2,4-trimethoxy-5-prop-1-enylbenzene) (Figure 1(5,6)), are constituents of essential oils (e.g., calamus oil) which are present in certain plants such as *Acorus* spp. and *Aarum* spp. and are used as flavoring agents [125,126]. *β-asarone* content varies with the source of the plant; Indian plant oil is approximately 75–95% β-asarone, whereas European is 5–10% [127,128].

*Carcinogenicity*: When fed to rats for 2 years at doses up to 2000 mg/kg bw, *β-asarone* induced leiomyosarcomas of the small intestine of males but not females [126–128]. Feeding Indian calamus oil at 0.05% and greater produced intestinal tumors in male and female rats, while feeding European calamus oil induced leiomyosarcomas and additionally, liver neoplasms at 1% and greater. Hepatocarcinogenicity of *α-* and *β-asarone* was also reported following oral administration or intraperitoneal injections to mice [91,129].

*Genotoxicity/DNA Binding (Adducts)*: In the in vitro genotoxicity assays, *α-* and *βasarone* produced conflicting results, while in vivo mutagenicity data is limited [128]. Nevertheless, positive results in the in vitro mutagenicity assays were obtained in the presence of bioactivating systems or in metabolically competent cell lines, including human Hepa-G2

cells [94,126,129–131]. Asarones also induced SCE, UDS and DNA breaks in vitro [126,132]. Both isomers produced DNA adducts in rat hepatocytes [133] and in avian embryos [86].

*Biotransformation*: In rat hepatocytes, the major metabolite of asarones was 2,4,5 trimethoxycinnamic acid, which was not genotoxic [131]. In rat and human liver microsomes epoxide-derived side-chain diols were the major metabolites, and the major bioactivation pathway for *α-asarone* was considered to be 3- -hydroxylation of propenylic side chain by CYP1A2, while for β-asarone, epoxidation by CYP3A4 prevails [126,134–136]. O-demethylation catalyzed by CYP1A1, 2A6, 2B6, and 2C19 was a minor reaction.

*MoA:* The mutagenicity and DNA binding of side chain epoxides formed during bioactivation of asarones suggests that this intermediate is responsible for carcinogenic effects, at least in the liver [129,136]. The MoA for induction of the intestinal tumors remains undetermined.

*Human Exposure*: The primary source of human exposure to asarones is through the consumption of alcoholic beverages such as bitters, liqueurs and vermouths, in which levels of calamus oil have been detected up to 0.35 mg/kg [128]. While no regulations for the use of *α-asarone* are currently in place, limits of 0.1 and 1 mg/kg are set for *β-asarone* in food and alcoholic beverages, respectively [126]. Nevertheless, some alcoholic drinks can contain up to 4.96 mg/kg of *β-asarone* [128]. Based on limited British data, maximum EDI for *β-asarone* is approximately 115 μg/day or 2 μg/kg bw/day [127,128].

*Human Effects*: No epidemiological studies investigating association of asarones with human cancer risk has been reported; however, some in vitro studies indicate anticarcinogenic properties of *β-asarone* [137,138].

*Risk:* JECFA and the Scientific Committee on Food (SCF) concluded that the existence of a threshold cannot be assumed for *β-asarone* due to its genotoxicity [127,128]. Accordingly, an ADI for nutritional exposure could not be derived. Committees recommended that calamus oil used in foods should have the lowest practicable levels of *β-asarone*. Calamus oil and its extracts are prohibited from use in the USA (21 CFR § 189.110) [139].

#### 3.1.2. Aristolochic Acids

*Occurrence: Aristolochic acid I* (AAI) (8-methoxy-6-nitrophenanthro[3,4-d]-l,3-dioxole-5-carboxylic acid) (Figure 1(7)) is one of a group of about 14 AAs known to be present in plants belonging to the family *Aristolochiaceae* (Birthwort family). Species known to contain AAs include *A. contorta*, *A. debilis*, *A. fangchi*, and *A. manshuriensis* [15,68,140–142].

*Carcinogenicity*: AAI, either purified or as a mixture with AAII (Figure 1(8)), was carcinogenic in rats and mice after oral exposure producing tumors predominantly in the forestomach and in the kidneys [15,68,140,143,144]. Other target organs of carcinogenicity include lung, uterus and lymphatic system in female mice and urinary bladder, thymus, small intestine and pancreas in rats. In addition, extracts from *Aristolochia* plants, *A. manshuriensis* and *A. fructus* induced forestomach and kidney tumors in rats when administered orally [15].

*Genotoxicity/DNA Binding (Adducts)*: AAI and AAII, have been found to be genotoxic in vitro and in vivo [141,145,146] and to form DNA adducts in vitro and in rodent tissues [141,147–151], as well as in humans urothelial tissues of patients with Chinese herb nephropathy, Balkan endemic nephropathy or urothelial cancer [152,153]. The major AAspecific DNA adducts were 7-(deoxyadenosin-*N*6-yl)aristolactam and 7-(deoxyguanosin-*N*2-yl)aristolactam [141]. Adducts of deoxyguanosine and deoxyadenosine were found in animal studies in both target (forestomach) and non-target tissues (glandular stomach, liver, lung, and bladder). In addition, AAs can bind to codon 61 of the *ras* oncogene and to purines in the *p53* tumor suppressor gene [68,141,153].

*Biotransformation:* Bioactivation of AAI occurs by nitro reduction in the presence of NAD(P)H quinone oxidoreductase and CYP1A2 [154] leading to formation of a nitrenium ion which, by rearrangement reactions, forms adducts on both deoxyguanosine and deoxyadenosine, the latter being biologically more stable [155].

*MoA*: Covalent binding to DNA and resulting mutagenicity is the predominant MoA of AAI carcinogenicity [15,68]. The most frequently observed mutation is a single *TP53* mutation (A to T transversion), consistent with the presence of persistent AAI-adenine adducts in DNA of exposed patients [141,153,156].

*Human Exposure*: AAs are present in herbal products and several teas made from *Aristolochia* plants [68,157] and in wild ginger used by North American Indians [158]. A combined EDI for AAI and AAII was calculated to be 1.7 × <sup>10</sup><sup>−</sup>3–30 <sup>μ</sup>g/kg bw/day [142].

*Human Effects*: Consumption of herbal supplements containing AAs has been linked to nephropathy [159] and cases of urothelial cancer [160,161]. Among patients with AA nephropathy, the rate of urothelial cancer is much higher compared to the prevalence of transitional-cell carcinoma of the urinary tract [68].

*Risk*: Based on the evidence that AA-specific DNA adducts and *TP53* mutations have been described in humans, IARC [15] upgraded classification of AAI from probable human carcinogen (Group 2A) to human carcinogen (Group 1) (Table 2). MoEs for kidney tumor formation calculated based on the rodent data were below 10,000 indicating risk to humans [142]. The US Food and Drug Administration (FDA) advised consumers in 2001 to discontinue use of botanical products that contain AA; however, exposure to AA continues despite its known hazards [162].

#### 3.1.3. Glucosides
