3.3.2. Chloropropanols

*Occurrence*: *3-Chloro-1,2-propanediol* (CP) (Figure 3(2)) and *1,3-dichloro-2-propanol* (DP) (Figure 3(3)) are formed during the acid hydrolysis of vegetable proteins through the reaction of chloride ions with triglycerides [115,360–362]. Several are present at low levels ranging from 9.6–82.7 μg/kg, in various foods, most notably refined oils, acidhydrolyzed proteins and soy sauces; however, some sauces contain as high as 18 mg/kg or 876 ppm [115,276,361,363–365]. Chloropropanols can be also found in paper-based food contact materials [366,367].

*Carcinogenicity*: In a two-year bioassay in rats, CP produced increases in kidney, Leydig cell, and mammary neoplasms administered at doses up to 400 ppm (29.5 mg/kg bw/day) in drinking water [115,276]. DP produced increases in neoplasms in the liver, kidney and tongue in rats at doses up to 30 mg/kg bw/day [115,365,368,369].

*Genotoxicity/DNA Binding (Adducts):* CP was genotoxic in some in vitro assays, but not in vivo [115,370,371]. In contrast, DP was clearly genotoxic in vivo and in vitro, with or without metabolic activation [115,276,361,365,368]; however, formation of DNA adducts has not been reported. Genotoxicity of DP was attributed to formation of epoxide intermediate [368].

*Biotransformation*: CP is metabolized by alcohol dehydrogenase to chlorolactic acid, while DP is metabolized by CYP2E1, to cytotoxic metabolites, including 1,3-dichloroacetone [115,361].

*MoA*: No clear MoA has been established for carcinogenicity of CP and DP. There is evidence that CP induces tumors by a hormonally mediated and/or cytotoxic MoA [362,372]. Oxidative damage has been also implicated [373]. Nevertheless, genotoxic MoA cannot be excluded for CP and DP [115].

*Human Exposure*: Mean EDI for CP was calculated to range from 0.2 to 3.8 μg/kg bw/day in adults and to be 1.3 μg/kg bw/day in children [276,362]. Mean EDI for DP was estimated to be 7 μg/person/day from soy sauce consumption, and 0.1 μg/person/day from dietary sources other than soy sauce [365,369].

*Human Effects*: No adequate data are currently available to assess the potential carcinogenicity of the chloropropanols in humans [115].

*Risk*: IARC [115] classifies CP and DP as possibly carcinogenic to humans (Group 2B) (Table 2). JECFA [276] set a provisional maximum tolerable daily intake (PMTDI) of 4 μg/kg bw/day for CP, while EFSA [362] established a much lower TDI of 2 μg/kg bw/day. JECFA concluded that no TDI can be estimated for DP based on its effects [365,369]; however, based on calculated MoE, DP in diet was considered to be of low concern for human health [361].

#### 3.3.3. Ethyl Carbamate (Urethane)

*Occurrence*: *Ethyl Carbamate* (EC) (Figure 3(4)), also referred to as urethane, is a fermentation product formed from the reaction of ethanol and carbamyl phosphate [374,375]. It is present as a natural trace constituent in various alcoholic beverages and in fermented food items, including cheese, bread, yogurt, soy sauce and fermented soybean products [374,376–379]. Mean concentrations of EC in some spirits, particularly in stonefruit brandies, have been measured in a range of 4 to 122 μg/kg (or 0.1 to 1400 μg/L), while in foods lower concentrations, ranging from 0.2 to 16 μg/kg, were observed [374,376–378]. EC content in foods can also increase with thermal processing [380].

*Carcinogenicity*: With oral administration to mice, EC up to 600 ppm induced mainly liver, lung, harderian gland, skin, mammary gland, ovaries, blood vessels and forestomach neoplasms [377,381,382]. In rats, oral administration of EC resulted in an increased incidence of Zymbal and mammary gland carcinomas [68,377].

*Genotoxicity/DNA Binding (Adducts)*: Genotoxicity and clastogenicity of EC has been demonstrated in vitro and in vivo [377,382–384]. The formation of etheno DNA adducts was reported in the liver [385], lung [386] and other organs [387] in rats and mice.

*Biotransformation*: EC is metabolized predominantly by CYP2E1 to reactive metabolites, vinyl carbamate and vinyl carbamate epoxide [377,382,388–390]. Formation of vinyl carbamate was also reported after incubation of human liver and lung microsomes with EC [391,392], suggesting similarities in metabolism of EC between humans and rodents.

*MoA*: Formation of reactive metabolite and consequent transition mutations in *Kras* oncogene is considered as major mechanism involved in tumorigenesis of EC [377,388,393]. Other potential MoAs may involve proinflammatory signaling, mitochondrial dysfunction and ROS formation [376].

*Human Exposure*: Under normal dietary habits, excluding alcoholic beverages, the EDI was in the range of 0.01 to 0.02 μg/kg bw/day, or an average of 0.015 μg/kg bw/day [378,394]. Mean EC intake from diet and alcoholic beverages rages from 0.015 to 0.065 μg/kg bw/day [374].

*Human Effects*: No epidemiologic studies are currently available to assess hazardous effects of EC in humans [377,378].

*Risk:* IARC [377] evaluated EC as probably carcinogenic to humans (Group 2A) (Table 2). JECFA [378] and EFSA [379] concluded that exposure to EC in food, excluding alcoholic beverages, poses low concern for human health. Nevertheless, health concern exists for consumers of alcoholic beverages, and mitigation measures should the implemented to reduce levels of EC in certain spirits. Schlatter and Lutz [395] calculated a virtually safe dose for EC of 0.02 to 0.08 μg/kg bw/day, which represents negligible risk to human health. Nevertheless, consumption of alcoholic beverages, in particular stone-fruit distillates, increases cancer risk to approximately 0.01%.

#### *3.4. Heat-Generated Carcinogens*

Heating and combustion of organic materials (e.g., tobacco) is well established to generate carcinogens. With respect to foods, three major types are acrylamide, heterocyclic amines and polycyclic aromatic hydrocarbons [396].

**Figure 4.** Chemical structures of DNA-reactive heat-generated carcinogens and related chemicals present in foods. Asterisks indicate sites of activation.

#### 3.4.1. Acrylamide

*Occurrence: Acrylamide* (AC) (propen-2-amide) is an unsaturated amide (Figure 4(1)), which is formed in heated foods, especially those rich in carbohydrates, by a reaction of reducing sugars with asparagine [397–399] and consequently is present in a variety of food products, notably baked or fried foods [68,400–403]. Efforts to reduce AC formation in foods have been active. For example, asparaginase has been proposed for use in food manufacture to convert asparagine to aspartic acid, thereby depleting one of the precursors of AC formation [403–405].

*Carcinogenicity:* AC was tested for carcinogenicity in rats by oral administration. In males, it increased the incidences of peritoneal mesotheliomas found in the region of the testis and of follicular adenomas of the thyroid gland. In females, thyroid follicular tumors, mammary gland tumors, glial tumors of the central nervous system, oral cavity papillomas, uterine adenocarcinomas and clitoral gland adenomas were increased. In four screening bioassays in mice, AC, given either orally or intraperitoneally, increased both the incidence and multiplicity of lung tumors in all experiments [402,406,407]. In a two-year rodent carcinogenicity bioassay [408], AC produced clear evidence of carcinogenic activity in rats and mice. Specifically, administration of up to 50 ppm AC in drinking water resulted in increased incidences of thyroid gland and heart tumors in rats of both sexes, of malignant mesotheliomas and cancer in the pancreatic islets in male rats and of cancers in the clitoral gland, liver, mammary gland, skin, and mouth or tongue in female rats. Increased incidences of cancer in the harderian gland, lung, and stomach were observed in male and female mice, in addition, female mice also had increased incidences of cancer in the mammary gland, skin, and ovary.

*Genotoxicity/DNA Binding (Adducts)*: AC is genotoxic and forms DNA adducts in target as well as non-target tissues of carcinogenicity, including the liver, lung, kidney, leucocytes and, testes in mice and in the liver, brain, thyroid, leukocytes and testes or mammary gland in rats [37,409]. In a dose–response study, a 0.1 μg/kg bw was established as a NOAEL for DNA adduct formation [410], suggesting plausibility of thresholds for carcinogenicity. In rats dosed with AC at 2 or 15 mg/kg bw for up to 28 days, DNA synthesis was increased in target tissues, but not in a non-target tissue, the liver [411]. In mice, administration of AC at 7.5, 15 and 30 mg/kg bw/day by gavage for 28 days produced a significant increase in micronuclei formation in the peripheral blood and an increase in *gpt* mutation frequencies in testes and lungs [412]. In addition, AC induced DNA strand breaks in various tissues in rats and mice [413–415].

*Biotransformation*: AC has two potentially reactive sites, a conjugated double bond and an amide group (Figure 4(1)) [399]. In vivo, AC is epoxidized at its double bond to glycidamide (GA) [416]. Both, AC and GA, are reactive and while there is some evidence for genotoxicity of AC [417], GA appears to be the DNA-reactive metabolite of AC [418]. GA reacts readily with DNA [407,419,420], forming purine adducts [410,421]. In rats, following administration of AC at 3 mg/kg bw, the majority of metabolites excreted in urine were AC-GSH conjugates, while a substantial proportion of the remainder consisted of two GA-derived mercapturic acids [422]. GA and dihydroxypropionamide were not detected at this dose level. The metabolism of AC in humans was investigated in a study in which male volunteers were administered 3 mg/kg AC orally. At 24 h, urine contained a third of the administered dose, and the majority of metabolites was derived from direct conjugation of AC with GSH [422]. GA, dihydroxypropionamide and one unidentified metabolite were also detected in urine. This study indicated both similarities and differences in the metabolism of AC between humans and rodents.

*MoA*: The carcinogenicity of AC may result from a combination of DNA reactivity and increased cell proliferation in target tissues. However, non-genotoxic MoA, such as alterations of calcium signaling, might be more relevant for tumorigenicity of AC, since evidence of its genotoxicity are weak [25]. In addition, some studies provide evidence of oxidative DNA damage by AC, as well as epigenetic modifications which might be involved in tumorigenesis [423–425]. Several possible MoAs have been reviewed [409].

*Human Exposure:* Dietary exposure to AC results from consumption of foods preparation of which involves cooking at high temperatures (e.g., French fries and potato chips), other exposure routes may involve dermal contact and inhalation. EDI of AC in several European populations was estimated to range from 0.4 to 1.9 μg/kg bw/day [425], with average dietary intake of 0.5 μg/kg bw/day in adults worldwide [426].

*Human Effects:* Numerous epidemiologic studies have examined the relationship of dietary consumption of AC and risk for cancers of the kidney, large bowel, urinary bladder, oral cavity and pharynx, esophagus, larynx, breast, and ovary [406,427]. No evidence of any association was found. Individual susceptibility, however, may be related to genetic polymorphisms in enzymes involved in activation and detoxification of AC [428]. Two cohort mortality studies were conducted among workers exposed to AC. The first showed no significant excess of cancer but was of small size, short duration of exposure and short latency. In the other study, in one plant in the Netherlands and three in the US, a nonsignificant increase was found in deaths from pancreatic cancer, but there was no trend with increasing exposure. In a prospective study, increased risks were found for postmenopausal endometrial and ovarian cancer with increasing dietary AC intake, particularly among never-smokers. Risk of breast cancer was not associated with AC intake [429].

*Risk:* Based on sufficient evidence for carcinogenicity in experimental animals and inadequate evidence in humans, IARC [402] classified AC as "probably carcinogenic to humans" (Group 2A) (Table 2). JECFA [403] concluded that estimated MoE for cancer events of 310 for general population and 78 for population with high exposure indicates a human health concern. EFSA [425] also concluded that although the epidemiological studies have

not demonstrated AC to be a human carcinogen, MoE indicates a concern for neoplastic effects based on animal evidence. However, based on the dietary intakes [400,425], exposures to AC are mainly at or below those considered acceptable for AFB1, which is a more potent carcinogen in animal models (Table 2). It would therefore seem unlikely that a significant risk exists for the general population. Furthermore, an expert panel convened by the German Federal Agency of Consumer Health Protection and Veterinary Medicine opined that while AC was a genotoxic carcinogen, it was likely to show a non-linear dose– response curve with respect to carcinogenic effect [430]. In support of this, Baum et al. [431] have shown that at concentrations added to human blood which are comparable to those achieved by intake from food, AC preferentially reacts with protein components of blood, and is 'quenched' without affecting DNA in lymphocytes.

#### 3.4.2. Heterocyclic Amines

*Occurrence: Heterocyclic Amines* (HCAs) are generated in meats cooked at high temperatures which produce protein decomposition [4,305,396,432]. A variety of different HCAs have been identified, representing several structural types reflecting the chemistry of their formation. The major subclass of HCAs, aminoimidazoazaarenes, which is the most abundant in food, includes *2-amino-3-methylimidazo [4,5-f]quinoline* (IQ) (Figure 4(2)), *2-amino-3,4-dimethylimidazo [4,5-f]quinoline* (MeIQ) (Figure 4(3)), *2-amino-3,8-dimethylimidazo [4,5-f]quinoline* (MelQx) (Figure 4(4)), *2-amino-3,4,8-trimethylimidazo[4,5-f]quinoline* (diMeIQx) (Figure 4(5)), and *2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine* (PhIP) (Figure 4(6)) [433].

*Carcinogenicity:* HCAs are potent multisite carcinogens in several species [68,305]. Specifically, oral administration of IQ, MeIQ and MeIQx to rats or mice caused increases in the incidences of tumors in the liver, small and large intestine, forestomach, lung, Zymbal gland, skin, mammary and clitoral glands, as well as lymphomas and leukemias. In the case of MeIQx, hepatic neoplastic lesions were observed only at high doses, indicating possibility of thresholds [45]. In rats, carcinogenicity of IQ in several target tissues was potentiated with high fat diet [434]. IQ was also shown to induce hepatocellular carcinomas in cynomolgus monkeys after chronic dosage of 10 or 20 mg/kg for 5 days/week. PhIP administered orally caused lymphoma in male rats and in mice of both sexes. Moreover, several cancers associated with the Western diet, specifically carcinoma of prostate gland, adenocarcinoma of the small intestine and colon and mammary gland carcinoma were observed in rats orally exposed to PhIP. Neonatal mice are also extremely sensitive to carcinogenic HCAs [68,305]. The order of carcinogenic potencies of selected HCAs is as follows: IQ > MeIQ > MeIQx > PhIP (Table 2).

*Genotoxicity/DNA Binding (Adducts)*: HCAs are potent genotoxic mutagens in various systems in vitro and in vivo [68,305,435,436], including human cells [437]. IQ, MeIQ, MeIQx and PhIP were shown to induce DNA damage and chromosomal aberrations, SCE, micronucleus formation and UDS. HCAs have been shown to form DNA and protein adducts in vitro and in vivo in various species, including humans [305,438–443]. Data from studies investigating formation of PhIP DNA adducts reported that in human tissues at dietarily relevant exposures DNA adducts form with greater efficiency compared to rodents [436]. There was a liner correlation between the dose and DNA-binding of some HCAs in the liver [25].

*Biotransformation*: The bioactivation of HCAs involves mainly *N*-hydroxylation, usually by CYP1A2 [444], and subsequent acetylation by *N*-acetyltransferase type 2 [445], leading to formation of a reactive nitrenium ion, as with other aromatic amines. Nitrenium ion primarily binds to C8 atom of guanine bases [432,436]. Genetic polymorphism of these enzymes in humans might play a role in susceptibility to genotoxicity and carcinogenicity of HCAs [446]. For example, high levels of DNA damage were observed in cell cultures with rapid acetylation [447] and individuals with rapid acetylator phenotype are believed to have higher risk of certain cancers after exposure to HCAs compared to slow-acetylators [432,445].

*MoA:* Carcinogenicity of HCA most likely results from formation of DNA adducts which lead to mutations in proto-oncogenes and tumor-suppressor genes, including *K-ras*, *Ha-ras*, *Apc*, *β-catenin*, and *TP53* [68,432].

*Human Exposure:* Human intake of HCAs is estimated to range from 0.001 to 0.017 μg/kg bw/day [448] with some intakes as high as 1900 ng [449]. The average lifetime time-weighted consumption of HCAs for US population is estimated to be approximately 0.009 μg/kg/day, with PhIP comprising two thirds of the intake [450].

*Human Effects:* HCAs are reasonably anticipated to be human carcinogens [68,432]. They have been implicated in causing cancers of the breast [451], colon and rectum [449], stomach and esophagus [452], and lung [453]. Estimated consumption by humans of at least one HCA, PhIP, was associated with increased levels of DNA adducts in breast [454] and prostate tissues [455] of patients with cancers at those sites. While consumption of cooked or grilled meat has been associated with various types of cancers, the data do not definitively implicate HCAs as the causative component of these associations [449].

*Risk:* IARC [305] classifies HCAs as either possible (Group 2A) (e.g., IQ) or probable (Group 2B) (e.g., MeIQ, MeIQx, PhIP) human carcinogens (Table 2). An upper-bound risk for US population from dietary exposures to HCAs was estimated to be 28,000 cancers, with PhIP accounting for almost half (46%) of the total risk [448]. The consumption of cooked meat and fish was the primary contributor to cancer risk in humans. Nevertheless, currently no regulations targeting reduction of exposure to HCAs exist [68].

#### 3.4.3. Polycyclic Aromatic Hydrocarbons

*Occurrence: Polycyclic Aromatic Hydrocarbons* (PAHs) is a group of compounds composed of two or more fused aromatic rings, which are present in many foods, either from deposition from air pollution or formed during cooking processes such as with char broiling of meats [275,378,456,457]. Heating of food above 350–400 ◦C leads to formation of PAHs, notably, the prototypical PAH, *benzo[a]pyrene* (BaP) (Figure 4(7)). BaP is found in smoked foods, charcoal-broiled steaks and ground meats [458–460]. The highest levels of BaP are found in grilled meats, at up to 4 ng BaP/g of cooked meat, [460].

*Carcinogenicity:* A variety of PAHs produced sufficient evidence of carcinogenicity in experimental animals [68,461]. In particular, BaP produced tumors in multiple species, including mouse, rat, hamster, guinea pig, rabbit, duck, newt, and monkey, following exposure by many different routes [68,275]. When administered orally, either via gavage or with diet, BaP at dosages up to 14 mg/kg bw/day increased incidences of tumors in lymphoid and haematopoeitic systems and in several organs in mice, including the lung, forestomach, liver, oesophagus and tongue [68,275,378]. Administration of BaP to rats by gavage for two years, produced liver tumors and tumors of forestomach at the lowest dose of 10 mg/kg bw and higher [462]. PAH mixtures, in particular creosote oils, coal-tar pitches, shale oils, anthracene oils, and certain bitumens have been shown to induce skin tumors in mice upon topical application [461,463].

*Genotoxicity/DNA Binding (Adducts):* PAHs, including BaP, are mutagenic and genotoxic in a variety of test systems, both in vitro and in vivo [47,275,378]. Reactive metabolites of PAHs can covalently bind to DNA, predominantly at the *N*2 position of desoxyguanosine [378]. A linear correlation between DNA adduct formation and mutagenicity was reported, providing evidence against the existence of thresholds for BaP effects [25].

*Biotransformation*: BaP and other PAHs with appropriate structures are bioactivated to oxides and dihydrodiols, which in turn are oxidized to diol epoxides, in a multi-step, inducible pathway involving CYP and epoxide hydrolase microsomal enzyme systems [464,465]. The dihydrodiol epoxide intermediate(s) (e.g., benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (Figure 4(8))) form stable and depurinating DNA adducts, which are primarily responsible for the mutagenic and carcinogenic action of BaP and other PAHs [47,275]. PAHs lacking the structural basis for formation of epoxides which can open and generate relatively stable carbonium ions with appropriate conformations of their hydroxyl groups are at most weakly carcinogenic [466]. For PAHs with lower ionization potential, the one-electron oxidation pathway, which results in the formation of unstable DNA adducts, might also be important [47].

*MoA:* The formation of DNA adducts by reactive metabolites, oxides and diol epoxides, is considered to be an initiating event in the development of tumors caused by BaP and some other PAHs [47,68]. These adducts were shown to induce mutations in oncogenes and tumor suppressor genes, such as *K-ras* and *TP53,* in humans and rodents [275]. However, due to poor quantitative relationship between levels of DNA adduct in target tissues and tumor formation, other factors involved in MoA should be considered. For example, induction of oxidative stress [467], immunosuppression [468], alterations of cell cycle [469] and epigenetic modifications [470] might also contribute to carcinogenic effects of PAHs.

*Human Exposure:* Food contaminated with PAHs, either from environmental sources or during processing and cooking, is the major source of exposure in non-smokers [47,457]. JECFA [378] reported EDI for BaP to be in a range of 0.0006 to 2.04 μg/kg bw/day, and for other PAHs EDI varies from 0.0001 to 0.015 μg/kg bw/day. Intake of PAHs in children is approximately double of the intake in adults [378]. EFSA [47] identified cereals and cereal products as well as seafood as the two highest contributors to the dietary exposure to PAHs. The European Union legislation (Regulations EC No. 835/2011 and No. 2020/1255) provides specific regulations for maximum levels of PAHs in various foods, which, depending on the product, ranges from 1 to 10 μg/kg for BaP and from 1 to 50 μg/kg for all PAHs [457,471,472]. In US, no maximum limits for PAHs in foods has been established, with exception of maximum permissible level of BaP in bottled water of 0.0002 mg/L [68,457].

*Human Effects:* No epidemiological studies on association between exposure to the individual PAHs and human cancers have been conducted, and data on the carcinogenic effects of PAHs in humans is available only for mixtures [68,275,461,463]. Thus, studies of smokers and consumers of certain meat products uncovered evidence of the carcinogenicity of BaP and other PAHs in humans. Lung cancer has been shown to be induced in humans by cigarette smoke, and by exposures to roofing tar and coke oven emissions, all of which contain mixtures of PAH [68]. A recent case control study reported an association between exposure to BaP in the diet and an increased risk for colorectal adenoma [473].

*Risk*: IARC [275] concluded that BaP is a human carcinogen (Group 1) based on the biological plausibility of mechanism of carcinogenicity in humans (Table 2). In a study of human intake in Korea, a possible excess cancer risk ascribed to PAHs using the cancer potency of BaP was calculated to be 2.3 cases per 100,000 persons [474]. Based on the MoE of 25,000 (mean) and 10,000 (high), and a human exposure estimate of 0.004 (mean) to 0.01 (high) μg/kg bw/day, JECFA [378] concluded that the estimated oral intakes of PAHs were of low concern for human health. Similarly, EFSA [47] established that MoE of 17,900 for BaP is indicative of low concern for consumer health at the average estimated dietary exposures; however, for high-level consumers potential concern exists.
