3.5.3.1. Malondialdehyde, 4-Hydroxynonenal, Crotonaldehyde, *trans,trans*-2,4-hexadienal

*Occurrence:* Several α,β-unsaturated aldehydes, including *malondialdehyde* (MDA) (Figure 5(4)), in its enolic form, *4-hydroxynonenal* (HNE) (Figure 5(5)) and *crotonaldehyde* (CA) (Figure 5(6)), occur as contaminants in food, especially edible oils [548,551,552]. *Trans,trans-2,4-hexadienal* (Figure 5(7)) is used as a flavoring agent [548,549,553] and was detected in a variety of food products, including olives, caviar, chicken and beef [115].

*Carcinogenicity:* The α,β-unsaturated aldehydes that have been tested in standard 2-year bioassays in rodents include CA [554], MDA [555], and *trans*,*trans*-2,4-hexadienal [556,557]. In the rat, CA administered at 0.6 and 6.0 mM/L in drinking water for 113 weeks was associated with development of neoplastic nodules of the liver [554] in conjunction with

overt hepatotoxicity (necrosis, fibrosis, cholestasis, and inflammation). In the same strain of rats, the incidence of thyroid follicular cell neoplasms was increased following 103 weeks of administration of MDA at 100 mg/kg bw/day (5 days/week) by oral gavage, in addition, pancreatic islet cell adenomas were observed in male rats in the group that received 50 mg MDA/kg [555,558]. Dosing of rats and mice with *trans*,*trans*-2,4-hexadienal by gavage in com oil at dosages greater than 45 mg/kg, 5 days/week, for up to 105 weeks resulted in an increased incidence of squamous-cell papillomas and carcinomas of the forestomach in both species [115,556]. In a neonatal mouse model, no tumors were observed after administration of CA, MDA and HNE via intraperitoneal injections up to 3000 nmol [559].

*Genotoxicity/DNA Binding (Adducts)*: CA [552,560,561], HNE [562], MDA [551,563], and *trans*,*trans*-2,4-hexadienal [115,557,560,561] have been shown to be genotoxic, especially in vitro [548]. Unsaturated aldehydes are considered to be strong alkylating agents, and as such they can covalently bind to DNA and proteins. In particular, formation of DNA adducts was detected in vitro and in vivo in multiple tissues of rats and mice after exposure to CA [552], MDA [551], and *trans*,*trans*-2,4-hexadienal [115,556]. DNA adducts of CA have been also detected in exposed humans [552,564]. Another lipid peroxidation product, HNE, reacts with DNA chemically and can form DNA adducts [565,566], however it may be too reactive with proteins for DNA adducts to be formed in vivo if administered directly. Thus, in serum-containing medium HNE was not mutagenic to cultured cells, whereas a protected form was [567]. Depletion of GSH and resulting oxidative stress are thought to be prerequisites for formation of DNA adducts by unsaturated aldehydes [548].

*Biotransformation*: The oxidation of fatty acids leads to formation of hydroperoxides, which in turn, decompose in a terminal reaction to form aldehydes from the methyl terminus of the fatty acid chain [548]. The levels of hydroperoxides formed are often estimated by assay of thiobarbituric acid-reactive substances [568,569], but more precise measurement techniques are available [570]. Detoxication of unsaturated aldehydes occurs primarily through reactions with GSH, yielding metabolites that are excreted in the urine of rats and humans [548,552].

*MoA:* In addition to direct DNA-reactivity and mutagenicity, oxidative stress and immunomodulation might also play a role in the carcinogenic MoA of α,β-unsaturated aldehydes [115,548,552]. Based on the findings in vitro and in vivo, EFSA ruled out genotoxicity concern for *trans*,*trans*-2,4-hexadienal [557].

*Human Exposure:* Humans are exposed to α,β-unsaturated aldehydes from food and alcoholic beverages, as well as endogenously, particularly in some disease states [548,571]. Levels of dietary exposure are low, particularly in the case of the flavoring agent *trans*,*trans*-2,4-hexadienal whose per capita intake is estimated at 100 μg/kg of bw/day [549].

*Human Effects:* Several epidemiological studies provided little evidence of a positive association between CA exposure with the lung [572,573], oral cavity, stomach, and colon [574] cancer risks in humans [552].

*Risk:* IARC [115,552] classified CA and *trans*,*trans*-2,4-hexadienal as possibly carcinogenic to humans (Group 2B), while MDA was considered to be not classifiable as to its carcinogenicity to humans (Group 3) [558] (Table 2). In a risk assessment of CA [575], a conclusion was made that based on an analysis of the doses which produced DNA adducts, use of the hepatocellular tumor data from the rat drinking water carcinogenicity study is likely to overestimate human cancer risk. This is indicative of a practical threshold for the genotoxic and carcinogenic effect of CA. The JECFA evaluation [549] of the NOAEL for *trans*,*trans*-2,4-hexadienal of 15 mg/kg bw/day was based on the NTP Report [556] and was estimated to be >100,000 times its current EDI when used as a flavoring agent, thus JECFA concluded that this flavoring agent does not pose a safety concern. FEMA reaffirmed α,β-unsaturated aldehydes as GRAS, based on the lack of evidence of potential hazard to human health at concentrations present in food [548].
