3.5.3.2. Trans-2 hexenal

*Occurrence*: *Trans-2-hexenal* (2-HEX) is a 6-carbon aliphatic unsaturated aldehyde (Figure 5(8)), which accounts for over 65% of total annual volume of unsaturated aldehydes used as flavor ingredients [548,549]. 2-HEX has been identified in a variety of plant species, including peppers, tomatoes and potatoes, and is referred to as leaf aldehyde [576]. The highest content of 2-HEX was reported in bananas, which contain approximately 32 ppm or 76 mg/kg [548,577].

*Carcinogenicity*: 2-HEX has not been tested for carcinogenicity in a 2-year bioassay [578]; however, based on structural similarities with *trans*,*trans*-2,4-hexadienal (Figure 5(7,8)), it can be expected to produce forestomach tumors in rodents when administered by oral gavage [548]. Some evidence of tumorigenicity was found in rats and mice that received three intraperitoneal injections of 2-HEX at the total dose of 150 mg/kg bw 18-month after the exposure [579]. Specifically, higher incidences of leukemia, liver and kidney tumors were described in mice, while in rats, tumors of parotid gland and lungs were reported.

*Genotoxicity/DNA Binding (Adducts)*: 2-HEX was genotoxic in several assays in vitro producing DNA damage, mutagenicity, clastogenicity and aneugenicity [548,553,578,580]. No induction of gene mutations, direct DNA strand breaks or micronucleus formation was reported in rodents in vivo [553,579]. In humans, non-smoking volunteers that consumed three to six bananas per day for 3 days showed at least a doubling of micronuclei in exfoliated buccal cells [581]. Rinsing the oral cavity with water containing 10 ppm 2-HEX produced a more pronounced effect. Subsequent experiments in rats [575,582] concluded that such exposures posed a negligible risk except in very special situations. 2-HEX has been also shown to form adducts with DNA and proteins [548,553]. Specifically, cyclic 1,*N*2-propanodeoxyguanosine adducts were detected in vitro and in several tissues of rats, including forestomach, esophagus, liver and kidneys, following oral doses of up to 500 mg/kg bw; however, the covalent binding index was calculated to be extremely low (0.06) [583–586]. Using a physiologically based in silico model developed in rats, formation of 2-HEX DNA adducts in humans from current levels of dietary intake was predicted to be three orders of magnitude lower compared to endogenous DNA adduct levels [587].

*Biotransformation*: 2-HEX is readily oxidized to *trans*-2-hexenoic acid in vitro by mouse cytosolic fraction and isoenzymes of rat aldehyde dehydrogenase [548,553]. Conjunction with GSH is a major mechanism involved in detoxication [548,549,587].

*MoA*: While DNA adduct formation can be related to mutagenicity of 2-HEX in vitro, EFSA concluded that based on the in vivo findings, concern for genotoxicity for this compound can be ruled out [553]. Induction of oxidative damage, exacerbated by GSH depletion, can potentially play an important role in carcinogenicity [588].

*Human Exposure*: Combined daily per capita intake of 2-HEX from foods was calculated to be 2390 μg/person per day or 31–165 μg/kg bw/day, with majority of exposures occurring with consumption of bananas [548,575]. An EDI based on the maximized survey-derived daily intake of 2-HEX as a flavoring substance was calculated to be 409 and 2800 μg/capita per day (or 0.007 and 0.05 mg/kg bw per day) in US and Europe, respectively [578].

*Human Effects*: Data assessing association of human cancer risk with 2-HEX exposure are currently lacking.

*Risk*: JECFA [549] and EFSA [578] concluded that 2-HEX would not pose a safety concern at the current levels of intake as a flavoring substance. Based on low covalent binding of 2-HEX, estimated cancer risk of 1–5 per 10<sup>7</sup> lives was considered to be negligible; however, under certain circumstances, utilization of 2-HEX as a flavoring agent or fungicide can increase cancer risk to 2–6 per 10<sup>4</sup> lives [575,582].
