4.4.1. Alkylated Imidazoles

*Occurrence: 2-methylimidazole* (2-MI) (Figure 9(1)) and *4-methylimidazole* (4-MI) (Figure 9(2)) are formed during fermentation and cooking by ammoniation of simple sugars [115,863–865]. They have been identified as by-products in foods including caramel coloring (Classes III and IV) and caramel-colored syrups, cola, ammoniated molasses, wine, Worcestershire sauce, and soy sauce [115,863,866–871]. 4-MI has been also detected in the milk from cows fed ammoniated forage [872,873]. Alkylated imidazoles can be also formed during thermal processing of natural constituents not containing caramel coloring, thus up to 466 μg/kg of 4-MI and up to 135 μg/kg of 2-MI were detected in roasted barley, malt and cocoa powder [874].

*Carcinogenicity*: Both, 2-MI and 4-MI, were carcinogenic in rodent studies [115,875–878]. Specifically, 2-MI induced thyroid follicular cell hypertrophy in mice and hyperplasia in rats by 15 days [879]. In a 2-year feed study, there was some evidence of carcinogenic activity of 2-MI in male rats based on increased incidences of thyroid gland follicular cell neoplasms and clear evidence of carcinogenic activity in female rats based on increased incidences of thyroid gland follicular cell neoplasms [876,878]. In addition, increased incidences of hepatocellular adenoma in male and female rats may have been related to exposure. In mice, there was some evidence of carcinogenic activity of 2-MI, based on increased incidences of thyroid gland follicular cell adenoma and hepatocellular neoplasms in males and increased incidences of hepatocellular adenoma in females [876,878]. In NTP bioassays, 4-MI fed to groups of male and female mice in diet containing 312 ppm (equivalent to

80 mg/kg bw/day) and greater for 106 weeks, increased incidences of pulmonary alveolar/bronchiolar adenoma in all dosed groups of females, alveolar/bronchiolar carcinoma in males given 1250 ppm, and alveolar/bronchiolar adenoma or carcinoma (combined) in males fed 1250 ppm and in females fed 625 and 1250 ppm [875,877]. In male and female rats fed diets containing 4-MI at up to 2500 ppm to males or 5000 ppm to females (115 and 260 mg/kg bw/day, respectively) for 106 weeks, there was no evidence of carcinogenic activity in males and only equivocal evidence in females based on modest increases in the incidences of mononuclear cell leukemia [875,877].

*Genotoxicity/DNA Binding (Adducts)*: 2-MI and 4-MI were negative in bacterial mutation assays when tested either with or without an exogenous bioactivation system [115,865,880,881]. 2-MI yielded mixed results in vivo for induction of chromosomal damage, as measured by micronucleated erythrocyte frequency and was negative in bone marrow micronucleus tests in rats and mice when administered intraperitoneally three times at 24-h intervals [115]. In a 14-week study of 2-MI; however, a significant exposure-related increase in the frequency of micronucleated erythrocytes was noted in peripheral blood of male and female mice [865,878]. While 4-MI produced SCE, chromosome aberrations and micronuclei induction in human peripheral lymphocytes in vitro [882], no increase in the frequencies of micronucleated erythrocytes was seen in the bone marrow of male rats or mice which were administered 4-MI by intraperitoneal injection, or in peripheral blood samples from male and female mice dosed in feed for 14 weeks [115,865,875,881].

*Metabolism*: 2- and 4-MI are rapidly absorbed and quickly eliminated in the urine mainly unchanged [871,883,884]. Mice cleared 2-MI faster than rats [878]. In rats, 2-MI is distributed to several tissues, including the thyroid [885], while 4-MI is mainly distributed to intestines, liver and kidney [115]. The principal urinary metabolite of 2-MI is the ring oxidized 2-MI, which possesses nucleophilicity [886,887]. Metabolism of 4-MI in rats and mice is similar, and the major metabolite detected in both species was hydroxylated 4-MI [884].

*MoA:* The MoA of 2-MI for induction of thyroid neoplasms likely involves interference with thyroid homeostasis, as described for several other chemicals [888]. 2-MI produced exposure-related reduction in thyroxine (T4) and increases in thyroid-stimulating hormone (TSH) in rats and had a lesser effect on T4 in mice [878]. The decrease in T4 can be attributed to increased hepatic UDP-glucuronyl transferase activity [876], which would lead to increased conjugation and excretion of T4. In response to T4 reduction, pituitary production of TSH increases which stimulates function and growth of the thyroid [888]. Likewise, the MoA of 2-MI for the liver tumors appears to involve a trophic effect on the liver reflected by liver weight and enzyme increases [876]. Both of these MoAs represent adaptive effects [889], which would be anticipated to be reversible. Nevertheless, IARC concluded that relevance of such tumor response in animals to humans cannot be excluded [115]. The MoA of 4-MI in induction of lung tumors in mice remains unclear, but does not involve genotoxicity, cytotoxicity or mitogenicity [115,890,891].

*Human Exposure:* The overall EDI for 4-MI for US population ranges from 0.13 to 0.51 μg/kg bw/day, with cola-type carbonated beverages being the highest contributor [892]. The average dietary intake for 4-MI in Europe was estimated to be between 0.4 to 3.7 μg/kg bw/day [893]. EDI of 4-MI from caramel colors ranges from 6 to 11 μg/kg bw/day for Class III and 7 to 9 μg/kg bw/day for Class IV [871].

*Human Effects:* No epidemiologic studies assessing human cancer risk of 2-MI or 4-MI were found [115].

*Risk:* IARC classifies 2-MI and 4-MI as possibly carcinogenic to humans (Group 2B) (Table 2). JECFA limits level of 4-MI to 200 and 250 mg/kg in caramel colors Classes III and IV, respectively [894]. EFSA suggest ADI of 300 mg/kg bw/day for all classes of caramel color [870]. FDA and EFSA concluded that at levels present in caramel colors 4-MI is not expected to be a concern to human health [871].

#### 4.4.2. Furan

*Occurrence*: *Furan* (oxacyclopentadiene) (Figure 9(3)) is a volatile contaminant formed in some foods during heat treatment techniques such as canning and jarring where the furan cannot escape [895]. The sources for the formation of furan include the oxidation of polyunsaturated fatty acids or the decomposition of carbohydrates or amino acids, but the relative contributions of these processes in actual foods is not known [896,897]. Analysis of approximately 300 food samples found furan levels ranging from nondetectable (below the limits of detection of the method) to 175 ppb [898]. Particularly high levels were found in foods that are roasted (e.g., coffee, cocoa, nuts, toasted bread, popcorn) or heated in closed containers (e.g., canned food, ready meals and baby food) [896–899].

*Carcinogenicity:* In rodent carcinogenicity studies [68,900,901], furan, administered to rats by gavage at 8 mg/kg bw, 5 days/week, induced a high incidence of cholangiocarcinomas in both males and females, at lower doses these tumors were reclassified as cholangiofibrosis [896,902]. In addition, incidences of mononuclear cell leukemia were increased in both sexes, and in males, a high incidence of hepatocellular neoplasms was also produced, while in females the incidence was moderate. In mice, hepatocellular neoplasms were induced at 8 mg/kg bw/day, 5 days/week [900]. In female mice, increased incidence of hepatocellular altered foci and hepatocellular tumors were preceded by a dosedependent increase in cell proliferation [903]. At lower dosages (up to 2 mg/kg bw), furan administered to male rats by gavage induced malignant mesotheliomas in the epididymis and testicular tunics, dose-related increases in the incidence of mononuclear cell leukemia and dose-related increasing trend in the incidence of hepatocellular adenomas [902].

*Genotoxicity/DNA Binding (Adducts):* Furan produced some genotoxicity in bacterial and mammalian cells in vitro [896,897,901], and chromosomal aberrations in mice, but was negative in SCE, in the mouse bone marrow erythrocyte micronucleus assay, and did not induce UDS in rats or mice [900,904,905]. In Big Blue rats, furan produced mainly negative responses in micronucleus or mutagenicity assays; however, some DNA damage was reported in the comet assay only at cytotoxic doses [906]. DNA strand breaks reported in rat liver but not in the bone marrow were associated with oxidative stress [907]. DNA-protein crosslinking were observed in turkey embryos after dosing with furan [908]. A major metabolite of furan, cis-2-butene-1,4-dial, is a direct acting mutagen [909], which can covalently bind to DNA in vitro [910], but not in vivo [911]. Thus, some DNA binding observed in liver and kidney DNA was attributed to other furan metabolites [912].

*Metabolism*: Furan undergoes oxidation by CYP2E1 resulting in ring scission and formation of the α-unsaturated dicarbonyl cis-2-butene-1,4-dial [886,913,914], which is likely the toxic metabolite [915].

*MoA:* The MoA of furan carcinogenicity is uncertain, but likely involves chronic toxicity with increased cell proliferation, which results from binding of furan and cis-2 butene-1,4-dial to GSH and proteins [896,897]. Nevertheless, in vivo DNA reactivity has not been rigorously excluded. In addition, there is evidence that oxidative stress and epigenetic alterations play a role [896,916–918].

*Human Exposure:* As mentioned above, furan is formed in a variety of heat-treated foods by thermal degradation of natural food constituents. Mean dietary exposure to furan in Europe may be as high as 1.23 and 1.01 μg/kg bw/day for adults and 3- to 12 month-old infants, respectively [896]. In the US, FDA [919] calculations estimated that mean daily furan exposures ranged from 0.26 μg/kg bw/day for adults to 0.41 μg/kg/day for infants consuming baby food and 0.9 μg/kg/day for those consuming infant formula. EDI calculated based on the Dortmund Nutritional and Anthropometric Longitudinally Designed (DONALD) study for consumers of commercially jarred foods ranged between 0.182 and 0.688 μg/kg/day [920].

*Human Effects:* Currently, data on effects if furan in humans is limited, and no association with carcinogenicity of furan in humans has been investigated.

*Risk:* IARC [901] classified furan as possibly carcinogenic to humans (Group 2B) (Table 2). EFSA [896] concluded that exposure to furan indicates health concern, due to

uncertainties regarding the MoA of furan carcinogenicity; however, current furan exposures are lower than established MoE of concern.

#### *4.5. Food Additives*

Food additives are added to food in order to improve or maintain certain characteristics, such as taste, texture, appearance or safety. Some are added to foods directly, while others migrate into foods in trace amounts during packaging, storage or handling [921]. According to FDA regulations established in late 1950s, any direct food additive with a carcinogenic potential should not be added to food; however, current advanced understanding of different mechanisms involved in chemical carcinogenesis puts relevance of such regulations under scrutiny [922,923].

**Figure 10.** Chemical structures of non-DNA-reactive carcinogenic food additives.

Monocyclic Phenolics, Synthetic and Natural

*Occurrence:* The synthetic phenolic antioxidants *butylated hydroxyanisole* (BHA) (Figure 10(1)), *butylated hydroxytoluene* (BHT) (Figure 10(2)), *tert-butylhydroquinone* (BHQ) (Figure 10(3)), and *hydroquinone* (HQ) (Figure 10(4)) are widely used as food additives to prevent oxidation of lipids [193,849,924–928]. HQ (Figure 10(4)) can also occurs naturally in food, mainly as a glucose conjugate, 4-hydroxyphenyl-P-glucopyranoside, known as arbutin, but can be also found in the free form [558,929]. Foods rich in arbutin include wheat cereal, bread, coffee and pears, with wheat products and pears having the highest levels of arbutin, 10 and 15 ppm, respectively [929].

*Carcinogenicity:* When fed in the diet, BHA elicited increased forestomach neoplasms in rats, hamsters and mice at doses greater than 2% [193,926,930,931]. BHT produced an increase in mouse lung neoplasms at doses of 0.75% [193,927,931]. BHQ produced no neoplasms in rats or mice when fed up to 5000 ppm, in spite of the positive genotoxicity findings; however, in a 6-week feeding study, preneoplastic lesions and papillomas were observed in forestomach of rats [932,933]. In two-year rodent bioassays, HQ administration by gavage at 25 or 50 mg/kg in water led at both doses to increases in renal tubular adenomas in male rats and mononuclear cell leukemias in female rats [558,934]. While there was no evidence of carcinogenic activity in male mice administered 50 or 100 mg HQ/kg bw in water, 5 days/week by gavage, there was some evidence of carcinogenic activity in female mice, as shown by increases in hepatocellular neoplasms, mainly adenomas, and thyroid follicular cell adenoma at the same doses [558,934].

*Genotoxicity/DNA Binding (Adducts):* The synthetic phenolics are nongenotoxic [924,931,935,936], although BHQ has yielded some positive findings in vitro, but not in vivo [937,938]. No DNA binding was detected in the target tissue, forestomach, of rats administered BHA or its metabolites up to 1000 mg/kg [939]. HQ tested positive in some in vitro and in vivo genotoxicity assays [344,558,940], but did not form adducts with DNA in vivo [345,346].

*Metabolism:* BHA is mainly metabolized to glucuronide and sulphate conjugates or is demethylated to free phenols, including BHQ [926]. BHQ can further undergo either oxidation to a quinone metabolite or GSH conjugation, as has been shown in rats [941]. Metabolism of BHT in vitro, using mouse microsomes, produced quinone methides, while in vivo, it is oxidized at one or both *tert*-butyl group(s) by microsomal oxygenase, followed by conjugation with glucuronide [927]. HQ is metabolized mainly to sulfate and glucuronide conjugates, but a small percentage may be converted to 1,4-benzoquinone, which can be either conjugated with GSH or form DNA adducts in vitro [558].

*MoA:* The MoAs of BHA and BHT involve epigenetic mechanisms ultimately leading to promotion of background neoplasia [924,930,933,942]. A lifetime dose–response study of tumor promoting effect of BHA in the rat forestomach identified positive effects at 6000 ppm and above, and a NOAEL at 3000 ppm [943]. In the case of BHT, its MoA appears to involve infiltration of monocytes into the pulmonary alveoli and stimulation of proliferation of type II pneumocytes [944,945]. The MoA for the kidney tumor induction in male rats by HQ has been suggested to involve cytotoxicity leading to increased cell proliferation and exacerbation of chronic progressive nephropathy [946–949].

*Human Exposure:* The mean daily intake of BHA varies from 2 to 300 μg/kg bw/day, depending on consumer age, region and estimate methods, and the main sources of exposure, similar to other phenolics, include baked goods, snacks and processed potato products [849,924,926]. EFSA reports [927] a mean dietary exposure to BHT is in the range of 10 to 30 μg/kg bw/day for adults and 10 to 90 μg/kg bw/day for children, while JECFA [849] calculated EDI to be between 700 and 990 μg/kg bw. The EDI for BHQ ranges from 4 to 140 μg/kg bw/day based on poundage data and from 370 to 690 μg/kg bw/day, based on model diets [849]. EFSA [928] determined that exposure to BHQ as a food additive averages to approximately 5 μg/kg bw/day in adults and 257 μg/kg bw/day in children.

*Human Effects:* No epidemiological study has implicated BHA or BHT as human carcinogens [193]. To the contrary, studies has suggested an inverse relationship with cancers of gastrointestinal tract [950,951], which is consistent with demonstrated anticarcinogenicity of monocyclic phenolics against a variety of DNA-reactive carcinogens in animal models [345,951,952]. No evidence for human carcinogenicity was found in studies of occupational exposures or dermatological use of HQ [558,953]. No study of cancer risk with dietary exposures was reported.

*Risk:* IARC [193,558] classified BHA as possibly carcinogenic to humans (Group 2B), while BHT and HQ were considered as not classifiable as to human carcinogenicity (Group 3) (Table 2). BHA and BHT are not considered to pose carcinogenic risks to humans based on a threshold-dependent MoAs that are not relevant to potential human exposure levels [930]. Human exposures are not expected to exceed the ADI values of 0.5, 0.3 and 0.7 mg/kg bw, which have been allocated by JECFA to BHA, BHT and BHQ, respectively [849,954]. EFSA [926–928] arrived at a similar conclusion after re-evaluating ADIs for BHA, BHT and BHQ to be 1, 0.25 and 0.7 mg/kg bw/day, respectively, and comparing them to the mean dietary intakes in children and adult populations.
