3.5.1. Ethylene Oxide

*Occurrence*: *Ethylene oxide* (EtO) (Figure 5(1)) is primarily used as an intermediate in the production of ethylene glycol [275]. However, in some countries, including USA, Canada and India, EtO has been also used as a fumigant for its disinfectant properties, and hence it may be present as a residue on contaminated foods including spices, nuts, sesame seeds, dry fruits and vegetables, milk powder and cereal, at various concentrations exceeding 0.05 mg/kg and even reaching 1800 mg/kg in some herbal teas [475–477]. Due to known hazardous effects, use of EtO in food production in Europe is prohibited (Regulation (EC) No 1107/2009) [478], and maximum residue levels are established between 0.02 and 0.1 mg/kg (Regulation (EC) No 396/2005) [479]. Such regulations led to a recent recall of a variety of products containing EtO contaminated sesame seeds or locust bean gum, including bread, sauces, ice cream and other fermented milk products [480,481]. In addition to exogenous sources, EtO can be also formed endogenously as a result of lipid peroxidation reactions, metabolic activity of microbiota or following metabolism of ethylene [482–484]. Endogenous levels of EtO in humans were estimated to range from 0.13 to 6.9 ppb [485].

*Carcinogenicity*: EtO is a multisite carcinogen in rodents, with target organs including the hematopoietic system in mice and rats, the lung, Harderian gland, mammary gland, and uterus in mice, and the brain and mesothelium in rats [68,275,486–489]. Oral administration of up to 30.5 mg/kg bw of EtO through gavage twice a week produced an increase in the forestomach squamous cell carcinoma in rats [490].

*Genotoxicity/DNA Binding (Adducts)*: EtO exhibited genotoxic and mutagenic effects, albeit weak, in experimental systems in vitro and in vivo, moreover, cytogenic alterations and DNA damage, including chromosomal aberrations, SCE, *hprt* mutations, micronucleus

formation, and DNA single-strand breaks were reported in peripheral blood of humans with occupational exposures [275,487–489,491–495]. As a direct alkylating agent, EtO covalently binds to DNA, predominantly at the *N*7 position of guanine [487,496–499].

*Biotransformation*: EtO can be either hydrolyzed, spontaneously or in the presence of microsomal epoxide hydrolase, to ethylene glycol with subsequent conversion to oxalic acid, formic acid and carbon dioxide, or conjugate with GSH to form mercapturic and thiodiacetic acids [275,488,500]. Several studies implied that polymorphism in human GST genotype, in particular GSTT1, might underly the difference in susceptibility to adverse effects of EtO [501–504].

*MoA*: While carcinogenicity of EtO is attributed to its genotoxicity and mutagenicity [275,489], formation of *N*7-guanine DNA adducts are not likely to contribute to the carcinogenic MoA. These adducts are not pro-mutagenic and are steadily repaired not leading to accumulation of abasic sites [497,505]. Hence, mutagenicity of EtO was attributed to minor adducts, *N*3-2-hydroxyethyladenine and O6-2-hydroxyethylguanine [506]. Due to high repair rate of DNA adducts formed by alkylating agents, existence of thresholds for EtO genotoxicity it plausible [489,507]. Several studies also attempted to use dose–response data for genotoxicity endpoints to estimate safe exposure levels to EtO [508,509].

*Human Exposure*: Exposures to EtO occur predominantly through inhalation, while dietary exposures are negligible [275]. Thus, EDI from all food sources amounts to 10 μg/person/day, which is lower than endogenous production of EtO by bacteria (15–20 μg/day) [510]. Average per capita consumption of EtO with spices was estimated to range from 0.21 μg/person/day in New Zealand to 1.6 μg/person/day in US [511].

*Human Effects:* There is no evidence of adverse health effects related to the consumption contaminated foods, mainly due to the rapid breakdown of EtO. While some epidemiological studies report association between occupational exposure (primarily through inhalation) to EtO and higher risk of lympho-haematopoietic [512] and breast cancers [513], the evidence is limited and not supported by meta-analysis studies [68,275,487,489,514–516].

*Risk:* Despite only limited evidence of carcinogenicity in humans, IARC [275] classified EtO as human carcinogen (Group 1) (Table 2), based on the mechanistic evidence of its genotoxicity in workers. While the NTP and the US EPA [68,517] reached the same conclusion, systemic analyses of carcinogenicity and toxicity data conducted by several authors suggests that such classification grossly overestimates the risk of EtO [489,516]. One study estimated that cancer risk from consumption of EtO with contaminated spices was negligible [511]. The German Federal Institute for Risk Assessment also established that EtO intake at or below 0.037 μg/kg bw/day should be considered of low concern [518].
