4.3.1. Agricultural Contaminants

#### 4.3.1.1. *p,p*- -dichlorodiphenyltrichloroethane

*Occurrence*: Technical grade *p*,*p*'-*dichlorodiphenyltrichloroethane* (DDT) (Figure 8(1)) is a complex mixture of DDT, its isomers and related compounds. As an organochlorine insecticide, DDT, had a major impact on the incidence of malaria and typhus as a cheap and effective method of killing the female *Anopheles* mosquito, which is the malaria parasite vector, and lice, which spread *Rickettsia prowazekii*, the cause of epidemic typhus [691]. Cost-effectiveness analyses shows that DDT is the least expensive, yet effective insecticide for prevention of malaria that kills thousands of people each day [692,693]. Nevertheless, owing to the extensive use of DDT in agriculture to control insects, such as the pink boll worm (*Pectinophora gossypiella*) on cotton, codling moth (*Cydia pomonella*) on deciduous fruit, Colorado potato beetle (*Leptinotarsa decemlineata*), and the European corn borer (*Ostrinia nubilalis*), and DDT's resistance to degradation or metabolism, which results in bioaccumulation in the food chain, it has been largely banned in the early 1970s with the expectation that all use would be stopped [694]. In 2006, however, the World Health Organization (WHO) reversed a 30-year policy by endorsing the use of DDT for malaria control [695]. DDT and its metabolites and degradation products, *p*,*p*- -dichlorodiphenyldichloroethylene (DDE) and *p*,*p*- -dichlorodiphenyldichloroethane (DDD), have been found in human breast milk [696,697], as well as some of the foods, including American cheese, butter, catfish, carrots, summer squash, celery, and salmon [694]. IARC [691] and Smith [696] reported that the mean concentrations of DDT in population have declined in much of the world: from 5000–10,000 μg/kg to around 1000 μg/kg of milk fat or even lower over the last three decades. Although different concentrations are found in different regions, the declines seen in various countries correspond to their restrictions on use of DDT.

*Carcinogenicity:* In some studies, DDT produced liver tumors in rats and mice at doses exceeding 46 mg/kg bw by gavage or 250 mg/kg in diet, as well as increases in incidences of malignant lymphomas and lung neoplasms in mice [68,691,694]. In contrast, US National Cancer Institute [698] bioassays at up to 642 ppm in male rats and 175 ppm in female mice detected no evidence for carcinogenicity of DDT. The DDT metabolites, DDE and DDD, also were hepatocarcinogenic in mice [699].

*Genotoxicity/DNA Binding (Adducts)*: The genotoxicity data on DDT and related compounds were overwhelmingly negative; however, some evidence of DNA damage, chromosome aberrations, and micronuclei formation was reported in human lymphocytes exposed to DDT in vitro [691,694,699,700]. No covalent DNA binding has been reported.

*Metabolism:* Due to its high lipophilicity, DDT, DDE and DDD tend to accumulate in the adipose tissue [694,701,702]. In mammals, including humans, DDT is primarily dehydrochlorinated to DDD, which is further metabolized to easily excreted 2,2-bis-chlorophenyl acetic acid isomers. To a lesser degree, DDT is also converted to DDE, which tends to bioaccumulate in lipid-rich tissues [691,700,702]. In rats, DDT and its metabolites has been shown to induce several CYPs, including CYP2B and CYP3A [703]. DDT can be also biotransformed to methylsulfonyl intermediates, which exhibit toxicity, in particular in adrenal gland [704].

*MoA*: DDT was shown to have a liver tumor promoting effect in mice [705], which, based on mechanistic studies [706], was attributed to its accumulation in the lipid layer of liver cell membranes and reduction of cell-cell communication, thereby diminishing tissue homeostatic control of incipient neoplastic cells. This MoA implies a requirement for a sufficient exposure over time to maintain the interference with intercellular communication throughout the liver. IARC also found strong evidence that DDT acts as endocrine disruptor, is immunosuppressive and can induce oxidative stress, all of these MoAs are operable in humans [691].

*Human Exposure*: Numerous studies have investigated human exposure to organochloride pesticides such as DDT, specifically due to concerns over its ability to bioaccumulate in the body and persist in the environment. However, due to the ban of DDT and declining levels of DDT and its metabolites in humans, more recent exposure data are scarce. It has been estimated that over 90% of the DDT detected in the general population is derived from food, particularly from meat, fish, poultry, and root and leafy vegetables [691,694]. The highest average daily intake ranging from 24.2 to 27.8 μg/day, was observed in Arctic populations, that consume foods such as seal or whale [694]. Nevertheless, most countries have seen a significant decline in DDT intake, ranging from 20 to 40% [700]. For example, in Europe total dietary exposure to DDT and its metabolites decreased from 0.00627 μg/kg bw/day in 1997 to 0.0051 μg/kg bw/day in 2005, and in US a decline from 0.0213 to 0.0056 μg/kg bw/day was observed from 1984 to 1991 [700]. EFSA also concluded that in most European countries, current EDI values for DDT, which range from 0.005 to 0.03 μg/kg bw/day in adult and children and up to 1 μg/kg bw in breastfed infants, are below the established provisional TDI of 0.01 mg/kg bw.

*Human Effects*: While some positive associations between DDT and cancers of the liver and testis, and non-Hodgkin lymphoma were reported, there seems to be limited evidence that DDT and related compounds from any source increase cancer rates in humans [691,699,707–710], even in agricultural workers [711]. This absence of carcinogenicity may be due to insufficient exposures, although some occupational exposures have been substantial, or it may reflect the fact that most human populations do not display rates of spontaneous liver tumor development that are as high as sensitive rodent models, indicating a low background of initiation available for promotion to tumor formation.

*Risk*: In 2018, IARC [691] upgraded classification for DDT from possibly (Group 2B) to probably carcinogenic to humans (Group 2A) (Table 2), based on sufficient evidence of carcinogenicity in experimental animals and strong mechanistic evidence that MoA for DDT carcinogenicity can operate in humans.

#### 4.3.1.2. Dioxins and Dioxin-Like Compounds

*Occurrence: Dioxins* and related *Dioxin-Like-Compounds* (DLCs) refer to a complex family of chlorinated compounds with similar structures and biological effects. 2,3,7,8- Tetrachloro-*p*-dioxin (TCDD) (Figure 8(2)) is one of the most potent and prominent dioxins in the environment and is often referred synonymously as "dioxin". Dioxin and DLCs, including polychlorinated dibenzo-para-dioxins (PCDDs), dibenzofurans (PCDFs) and the polychlorinated biphenyls (PCBs), are formed by dimerization of chlorophenols produced during the synthesis of chlorophenoxy acetic acid herbicides [275]. The dimerization of 2,4,5-trichlorophenol yields TCDD while the heterodimerization of 2,4,5-trichlorophenol with related phenols such as 2,4-dichlorophenol yields tri- through heptachlorinated diben-

zodioxins and dibenzofurans. Other sources of TCDD include the use of chlorophenol as wood preservatives, use of chlorine in pulp bleaching, incineration of halogen containing materials [712,713]. DLCs usually occur as mixtures and, in order to express the expected biological activity of mixtures by a common dose metric, toxic equivalency factors (TEFs) relative to the activity of TCDD have been developed [275,365,690,714]. Using TEFs and mass concentrations, dioxin toxic equivalents (TEQs) for a source can be calculated. Based on these values, there are at least 7 PCDDs, 10 PCDFs and 12 PCBs that have dioxin-like activity [715,716]. Food-mediated human exposure to TCDD and DLCs occurs when contaminants from the above-described sources are ingested by animals, including fish, which in turn are used as human foods [275,690,717]. Dioxins have also been detected in human milk, ranging from 5 to 15 ng TEQ/kg lipid [717–719]. Levels of TCDD and DCLs in the environment, and consequently in food, have been declining since the late 1970s because of reduced industrial emissions [720].

*Carcinogenicity:* In rodents, several DLCs, including TCDD, induced neoplasia, mainly of the liver [275,690,721–725]. Other target organs and tissues included thyroid gland, lungs and oral mucosa. TCDD acted as tumor promoter when administered with potent tumor initiators, such as nitrosamines [275,365,724].

*Genotoxicity/DNA Binding (Adducts):* TCDD is not DNA-reactive in vitro or in vivo and does not covalently bind to DNA [365,723,726–728]. Similarly, PCBs are mainly not DNAreactive, although some evidence of DNA damage, SCE and chromosomal aberrations were observed in human lymphocytes [690,729]. PCBs can be metabolically activated to electrophilic quinoid intermediates, and can produce DNA adducts in vitro; however, no DNA adducts were observed in vivo [730].

*Metabolism:* Similar to DDT described in the section above, TCDD and DLCs are highly lipophilic and thus, tend to accumulate in the adipose-rich tissues, liver has been also shown as a primary site of TCDD accumulation in rodents [365,731–735]. TCDD metabolism is very slow and limited and the compound is eliminated mainly unchanged in the feces [734,735], although it induces activities of CYP1A1, CYP1A2, and CYP1B1 enzymes, which are also involved in the hydroxylation of PCDDs and PCBs [736], in mice and rats [365,734]. Rat hepatocytes show greater rate of TCDD metabolism compared to that in guinea pigs, this feature may underly the intraspecies differences in susceptibility to the toxicity of TCDD [737].

*MoA:* Dioxins are not DNA-reactive, but enhance liver tumor development through epigenetic mechanisms mediated by binding to the aryl hydrocarbon receptor leading to toxicity and enhanced cell proliferation [275,724,738–740]. Accordingly, for these events a threshold can be established. For example, NOAELs for hepatocyte proliferation in the NTP bioassays were 0.003 μg/kg at the 14-week interim evaluation and 0.022 μg/kg at 53 weeks for TCDD [721] and between 10 μg and 100 μg for 3,3- ,4,4- -tetrachloroazoxybenzene (PCB 126) [722]. The hepatocarcinogenicity of TCDD in rats was greater in females than in males apparently due to the influence of estrogenic hormones [741], although the specific mechanism(s) has not been elucidated. Induction of oxidative damage can also play a role in carcinogenicity of dioxins. These MoAs are considered to be operational in humans [275].

*Human Exposure:* Mean dietary exposure to all dioxins in adults occurs primarily through consumption of food of animal origin, such as meat, dairy products, eggs and some fish, and is estimated to be 0.3–3 pg/kg bw day [275,365,716,717,742]. In 2010–2021 EDI for PCDDs and PCDFs varied from 0.001 pg TEQ/kg bw/day to 74.31 pg WHO-TEQ/day [743] depending on the country and method used for estimation of intake. Per capita intake of dioxins in US population is estimated to be lower (17 to 24 pg per capita) compared to that of European population (29 to 97 pg per capita) [365]. In nursing infants, dietary intake of dioxins can reach up to 53 pg TEQ/kg bw/day for TCDD [742] and over 150 WHO-TEQ/kg bw/day for PCDDs and PCBs [719]. Due to limitations in use, intake of dioxins has substantially reduced over the years.

*Human Effects:* There is no epidemiological evidence that implicates consumption of low-level DCLs-containing foods in human cancer causation [713,744]. Nevertheless, continuing evaluation of highly exposed individuals is strengthening the observations of increased cancer risk with dioxin exposure, in particular for lung cancer, soft-tissue sarcoma and non-Hodgkin lymphoma, although the increases are small for these relatively high exposures [745,746]. In addition to carcinogenicity, exposure to dioxins is associated with a variety of adverse effects, including dermatological effects (chloracne), cardiovascular diseases, endocrine disorders (diabetes, affected thyroid function), reproductive effects, neurological disorders and an increase in hepatic enzymes [365].

*Risk:* TCDD and DLCs have been classified by IARC [275,690] to be human carcinogens (Group 1) (Table 2) based on sufficient epidemiological information, animal carcinogenicity data and strong mechanistic considerations. In 1998, WHO modified a previously established TDI for TCDD from 10 pg/kg bw to a range of 1–4 pg TEQs kg bw/day [747,748], while the SCF [749] and JECFA [365] established TDI for dioxins of 2 and 2.3 pg/kg bw, respectively. In contrast, the US EPA [750] has proposed that dioxin doses in the range of 1 pg/kg might represent a cancer risk. This assessment was criticized as overly conservative [751–754], and the EPA has yet to issue a reanalysis of the cancer TCDD dose response reassessment.
