3.1.1.1. Safrole

*Occurrence: Safrole* (SAF) (4-allyl-1,2-methylenedioxy-benzene) (Figure 1(1)), the prototype compound of the AB group, is present in *Sassafras*, nutmeg, cinnamon, sweet basil and star anise [77,82,87]. Until 1960, the beverage root beer contained approximately 30 ppm safrole it being made from the root of *Sassafras albidum* which contains about 85% safrole in the essential oil from its root bark [88].

*Carcinogenicity*: Dietary administration of SAF at up to 5000 mg/kg body weight (bw) to mice and rats caused increases in the incidences of hepatocellular carcinoma or cholangiocarcinoma [78,89,90]. SAF carcinogenicity in mice was strain specific [91]. The hepatocarcinogenicity of 1- -hydroxy SAF metabolites has been also demonstrated [91,92].

*Genotoxicity/DNA Binding (Adducts)*: Genotoxicity tests yielded inconclusive results, being generally negative in vitro, although, some genotoxicity was observed in vivo [78,93,94]. SAF also induced in vitro chromosomal aberrations sister chromatid exchange (SCE), unscheduled DNA synthesis (UDS) and DNA damage [90]. Nevertheless, guanine derivative SAF-DNA adducts were isolated from the livers of multiple species, including rats, mice [82,90,95,96], chicken and turkey [85,86] and humans [97,98].

*Biotransformation:* SAF undergoes bioactivation primarily on its side chain (Figure 1(1)) to form a hydroxy metabolite which is subsequently sulfated [99,100]. These reactions involve several cytochrome P450 (CYP) enzymes, especially CYP1A2 [83] and sulfotransferase [101]. Genotoxic effects of SAF are likely mediated by metabolites, 1- -hydroxysafrole and 1- -sulfoxysafrole [77]. A number of hydroxylated metabolites have been isolated from human urine [102].

*MoA*: SAF was considered to be a genotoxic carcinogen, based on its ability to induce formation of DNA adducts [90].

*Human Exposure*: Humans may ingest SAF with edible spices, such as sassafras, cinnamon, nutmeg, mace, star anise, ginger, black and white pepper, and from chewing betel quid [68]. An Estimated Daily Intake (EDI) for SAF was reported to be 300 μg/person/day [82,90]. JECFA [78] estimated the intake of safrole to be around 879 μg/person/day.

*Human Effects:* Most of the evidence that SAF may be carcinogenic to humans comes from studies of individuals who chew betel quid, which is known to contain up to 15 mg/g SAF. Thus, SAF-like DNA adducts have been reported in oral squamous cell cancers [97,103] and hepatocellular carcinoma [104] isolated from users of betel quid. Betel quid users are known to have an increased risk for oral cancer development [103].

*Risk*: SAF was classified by the IARC [87] as possibly carcinogenic to humans (Group 2B) (Table 2). Reflecting these concerns, JECFA did not allocate an Acceptable Daily Intake (ADI) [78]. The direct addition of SAF to food is prohibited in the USA (21 CFR § 189.180) [105] and Europe (Regulation EC No. 1334/2008) [106]. Nevertheless, exposure to SAF continues to occur [107].
