2.2.4. Genotoxicity and Carcinogenicity of Alkenylbenzenes Required Restrictions for Their Use in Foods

Due to their genotoxic properties, the use of safrole as a flavoring substance for human food has been prohibited in the USA since 1960 [140]. Moreover, the use of methyleugenol as a flavoring substance in food was also forbidden in the USA by the United States (U.S.) Food and Drug Administration (FDA) in 2018 [141]. By contrast, the use of estragole in foods is not restricted in the USA [9,31]. The U.S. FDA approved *trans*-anethole as a food additive [95]. Isoeugenol is also approved as a flavoring substance in food in the USA [142]. The same applies for eugenol [116,143]. Moreover, the use of eugenol is also permitted in other regions, including Australia, Indonesia, and the European Union (EU) [116]. Besides this, eugenol and isoeugenol are also approved as fish anesthetic, e.g., in Australia, New Zealand and Finland, but not in the EU or the USA [144,145]. In this context, it is not surprising that residues of both substances were also found in the fillet tissue of freshwater fish previously exposed to this compound [146]. Finally, the use of myristicin in food products is not regulated in the USA [147]. Moreover, there are currently no specific guidelines or laws concerning the production or sale of synthetic myristicin or myristicin isolated from natural sources [147]. This also applies to the EU and other regions/countries in the world. The aforementioned information shows that the use of different alkenylbenzenes—some of which have genotoxic potential, e.g., estragole or *trans*-anethole—is not adequately regulated in the USA. Moreover, the use of most alkenylbenzenes is currently not regulated in most other regions of the world, including Asia, Africa, and South America.

In 2001 and 2002, respectively, the Scientific Committee on Food (SCF) of the EC evaluated safrole, methyleugenol, and estragole and concluded that these compounds are genotoxic carcinogens and suggested restrictions for their use in foods [98,109,111]. Based on the SCF's recommendations, the EC prohibited the addition of pure safrole, methyleugenol, and estragole as a flavoring substance to food and established maximum levels for these substances—when naturally present in corresponding ingredients in certain compound foodstuffs, such as soups and sauces or non-alcoholic beverages [148]. Thus, in the EU, estragole, methyleugenol, safrole, and *beta*-asarone shall not be added as such to food (see Annex III Part A of Regulation (EC) No 1334/2006). Further EU restrictions apply to these alkenylbenzenes (in Annex III Part B of Regulation (EC) No 1334/2006). The maximum levels of estragole, methyleugenol, and safrole, naturally present in flavorings and food ingredients with flavoring properties or in certain food compounds to which flavorings and/or food ingredients with flavoring properties have been added, have been defined by the EU Parliament and the Council. Accordingly, estragole may not be present in amounts greater than 50 mg/kg food in dairy products, processed fruits, vegetables (including mushrooms, fungi, roots, tubers, pulses, and legumes), nuts and seeds, and fish products. Non-alcoholic beverages may not contain more than 10 mg estragole per kg. As for methyleugenol, soups and sauces may not contain more than 60 mg/kg;

dairy products and ready to eat savories, no more than 20 mg/kg; meat preparations and meat products, including poultry and game, no more than 15 mg/kg; fish preparations and fish products, no more than 10 mg/kg; and non-alcoholic beverages, no more than 1 mg methyleugenol/kg. In addition, even up to 25 mg/kg safrole may be present in soups and sauces; 15 mg safrole/kg in meat preparations and meat products, including poultry and game; safrole is still permitted in fish preparations and fish products. In non-alcoholic beverages, 1 mg safrole/kg shall not be exceeded. Furthermore, the content of beta-asarone, a major constituent of *Calamus* oils, is legally restricted in Europe for alcoholic beverages to a maximum of 1 mg/kg (see Annex II Part B of Regulation (EC) No 1334/2006). The tetraploid form *of Acorus calamus* L. shall not be used as a source for the production of flavorings and food ingredients with flavoring properties (see Annex IV Part A of Regulation (EC) No 1334/2006). In addition, according to the abovementioned regulation, "the maximum levels shall not apply where a compound food contains no added flavorings and the only food ingredients with flavoring properties which have been added are fresh, dried or frozen herbs and spices". However, the usage of other structurally related (see Table 1) and potentially toxic alkenylbenzenes, such as elemicin or apiol, is, so far, not regulated in the EU, whereas some derivatives, such as eugenol, isoeugenol, and *trans*anethole are listed as authorized flavoring compounds in Regulation (EU) No 872/2012.
