*2.2. Bisphenol F*

Bisphenol F is an aromatic compound related to bisphenol A through its basic structure containing two phenol groups. The two aromatic rings of bisphenol F are linked through methylene. Generally, bisphenol F comprises several isomers such as 2,2- -, 2,4- - and 4,4- -dihydroxydiphenylmethane. Recently, bisphenol F was detected in several mustard products containing seeds of white mustard (*S. alba*) as well as in various plants of the orchid family (Orchidaceae) (*Coeloglossum viride* var. *bracteatum* (rhizome), *Galeola faberi* (rhizome), *Gastrodia elata* (rhizome), *Tropidia curculioides* (root)) and in the seeds of *Xanthium strumarium,* which are partly used as herbal remedies [95] (Table 3).


**Table 3.** Occurrence of bisphenol F in foods related to mustard.

n.d. not detected.

Bisphenol F is structurally similar to bisphenol A and belongs to one of the main substitutes replacing bisphenol A in the production of epoxy resins and polycarbonates used in the manufacture of adhesives, plastics, coatings and other applications [101]. Thus, the occurrence of bisphenol F is constantly increasing in products leading to consumer exposure from a broad number of sources.

In contrast to bisphenol A, bisphenol F in mustard and other plant species is not derived from synthetic materials, packaging or other sources of contamination, but is most likely formed as a breakdown product of glucosinolates [97].

#### Evaluation of Possible Health Risks

The occurrence of bisphenol F in mustard products is obviously due to the formation from a natural ingredient in the seeds during mustard production. Zoller et al. excludes the origin of bisphenol F from contamination through epoxy resin or other sources like food packaging. In support of this, the 4,4- -isomer was nearly exclusively detected in the samples. A contamination with bisphenol F would most likely lead to the occurrence of all isomers which are commercially used as a mixture of the three isomers, respectively [97].

It was observed that the highest levels of bisphenol F were detected in mild mustard samples compared to spicy ones. Additionally, bisphenol F was detected in mustard produced from seeds of *S. alba* but not from *B. juncea* or *B. nigra* or in mustard flour [97,99]. Therefore, based on differences in composition of the mustard species it is considered that bisphenol F is formed from the glucosinolate glucosinalbin, because it is predominantly found in the seeds of white mustard (*S. alba*) but not in *B. juncea* or *B. nigra*. This possibly can also explain the higher levels of bisphenol F in mild mustard types mainly contain *S. alba* which is milder in taste than other mustard species.

Recent findings from Zoller et al. verified the myrosinase-catalysed formation of 4 hydroxybenzyl isothiocyanate from 4-hydroxy benzyl glucosinolate (sinalbin). In the presence of water, the isothiocyanate is hydrolysed to the intermediate 4-hydroxybenzylalcohol. Subsequently, formation of a carbocation from the isothiocyanate by release of thiocyanate or from the 4-hydroxybenzyl alcohol by protonation and release of water is suggested. A further 4-hydroxybenzyl alcohol molecule could result in dimerization and formation of the 4,4- -isomer of bisphenol F [97]. Additionally, it was pointed out that 4,4- -bisphenol F could also be formed from 4-hydroxybenzylalcohol in the stomach due to its acidic conditions as it was also shown for other glucosinolate breakdown products [102].

After absorption bisphenol F is distributed throughout the body, also including the reproductive organs and the foetus by crossing the placental barrier. As glucuronide or sulfate conjugate, bisphenol F is rapidly excreted mainly via urine and to a lesser extent in faeces, however residues of bisphenol F were still detectable in tissues 96 h after a single dose was administered to rats [103].

So far, no comprehensive evaluation of possible health risks due to the exposure of bisphenol F in humans is available. Subchronic and chronic toxicity studies as well as studies on reproductive and developmental toxicity are still lacking. However, the structural similarity of bisphenol F to bisphenol A suggests comparable biological (adverse) effects and the potential to disrupt the human endocrine system [104–108].

In contrast to bisphenol A [104], no health-based guidance values for bisphenol F or legal regulations for foodstuff exist. However, some authorities evaluated the possible health risks to consumers from the natural occurrence of bisphenol F in mustard products. The Swiss authority FSVO (Federal Food Safety and Veterinary Office) carried out a risk assessment and evaluated the ratio between the lowest observed adverse effect level (LOAEL) and the dietary intake of bisphenol F from mustard products. In a very rudimentary approach, it was concluded that a daily intake of 0.67 mg of bisphenol F may be tolerable for a person of 60 kg body weight corresponding to a tolerable daily intake of 11 μg/kg body weight/day. The calculation was based on a LOAEL (Lowest Observed Adverse Effect Level) of 20 mg/kg body weight/day which was the lowest dose tested in a repeated 28-day toxicity study in rats leading to reduced body weight and several changes of blood parameters. Including several extrapolation factors (from LOAEL to NOAEL (No Observed Adverse Effect Level); from subacute to chronic toxicity) and an uncertainty factor of 100, it was assumed that consumption of 80 g mustard with the highest detected level of bisphenol F (8.4 mg/kg) would pass the margin of safety of 1800 which was postulated by the authority [98].

The German Federal Institute for Risk Assessment (BfR) used for its assessment the tolerable daily intake (TDI) of bisphenol A of 4 μg/kg body weight/day which was established by the European Food Safety Authority (EFSA) in 2015 [104] and is currently being re-evaluated [109]. The German authority concluded that even in the case of consuming high amount of mustard (4 g/day) with a maximum bisphenol F content of 6 mg/kg, the daily mean intake of bisphenol F would not exceed the assumed TDI of 4 μg/kg body weight/day. In the case of "normal consumers", the estimated intake quantity is 100 times lower [110]. Therefore, adverse health effects due to the intake of bisphenol F from mustard are not to be expected, although further toxicological studies are needed for a final

evaluation. It should be underlined that bisphenol A substitutes appear to have similar metabolism, potencies and action and may pose similar potential health risks as bisphenol A. Thus, consumption of mustard may be an important source of bisphenol F and continues monitoring of this compound and its relevant sources, either natural or technical-induced, and thorough investigations on its health effects in humans remain important [100,111,112].

#### *2.3. Erucic Acid*

Mustard seeds are regarded as an important source of edible oil, which are used due to its nutty and pungent flavour and its high smoke point (250 ◦C), especially in Eastern and North-Western India [113]. Oil from Brassica plants differ from other vegetable oils mainly due to their significant proportion of long-chain monoenoic fatty acids, eicosenoic and erucic acids. Wendlinger et al. detected up to 19 fatty acids in mustard oils, of which oleic acid (18:1n-9), linoleic acid (18:2n-6), alpha-linolenic acid (18:3n-3), eicosanoic acid (20:1n-9) and erucic acid (22:1n-9) belong to the major ones [114]. Erucic acid is a typical example of a very-long chain mono-unsaturated fatty acid (VLCMFA) with 22 carbon atoms and one double bond between C13 and C14, also referred to as docosenoic acid (C22:1). Further, it belongs to the predominant fatty acid in seed oils from the three commercial cultivated mustard species *B. nigra, B. juncea* and *S. alba* containing levels over 30% erucic acid of total fatty acids (Table 4).


**Table 4.** Erucic acid concentration in mustard products.


**Table 4.** *Cont.*

<sup>a</sup> 2 crop years, <sup>b</sup> total fat content 4%, <sup>c</sup> total fat content 28.8%.

The fatty acid profile and erucic acid content of mustard seeds depend on several factors such as breeding techniques, total oil content, climatic conditions as well as morphological and physiological determinants [116]. Recently, breeding programs has successfully produced canola quality (low, zero erucic acid content) of *B. juncea* and low erucic acid content genotypes are cultivated in few countries [126]. Nevertheless, mustard seeds and mustard oils are one of the food products with the highest erucic acid content that can be found on the market. Based on the low consumption rate of mustard oil in the European Union, mustard seeds and mustard condiment or table mustard remain here the major sources regarding erucic acid intake [127].

#### 2.3.1. Toxicological Effects

Like other long-chain fatty acids, erucic acid is transported to the tissues either bound to serum albumin or in esterified form incorporated in lipoproteins. The heart and skeletal muscles primarily utilize fatty acids as energy-providing substrates mainly through mitochondrial ß-oxidation. However, the capacity for mitochondrial ß-oxidation is reduced for long chain fatty acids (>18 carbon atoms). In this case, erucic acid is likewise sparsely oxidised by the mitochondrial β–oxidation system, probably due to the poor utilization of erucoyl-CoA as substrate by the mitochondrial acyl-CoA dehydrogenase. Furthermore, erucic acid also appears to inhibit the overall rate of fatty acid oxidation, by the mitochondria. It is worth noting, that both the capacity for ß-oxidation and inhibition of the tricarboxylic acid cycle differs between species. Rats compared to pigs seem to have a lower capacity for ß-oxidation and higher ability to inhibit oxidation of tricarboxylic acid-cycle intermediates [128,129].

Therefore, high exposure of erucic acid leads to accumulation of triacylglycerols in heart and other tissues. It has been shown that the lipidosis is reversible and transient during prolonged exposure, probably due to the increased peroxisomal chain shortening mediated by peroxisome proliferator-activated receptors. However, myocardial lipidosis is the most common and sensitive effect associated with short-term, and to a lesser extent, sub-chronic exposure of erucic acid in all animal species examined. With respect to liver tissue, the presence of erucic acid appears to induce the peroxisomal β–oxidation system, resulting in no accumulation of erucic acid and in reduced inhibition of mitochondrial ß-oxidation. Consequently, the liver is able to export erucic acid as very low-density lipoprotein (VLDL) [130–133].

Given what is known about the metabolism of erucic acid, it seems reasonable to expect that humans would also be susceptible to myocardial lipidosis following exposure to high levels of erucic acid. Clouet et al. observed a very low capacity in human heart

mitochondria for the direct utilization of erucic acid as a substrate for energy requirements. Furthermore, activation of fatty acids due to the transfer of acyl groups from Coenzyme A (CoA) to carnitine, which belongs to the preliminary step of their beta-oxidation, was also reduced with high erucic acid exposure [134].

Imamura et al. showed that higher levels of docosenoic (22:1) and nervonic (24:1) acids in plasma phospholipids from diverse dietary sources, were associated with higher incidence of congestive heart failure in two independent cohorts, assuming possible cardiac toxicity of long-chain monounsaturated fatty acids [135]. In contrast, Matsumoto et al. described a lower incidence of coronary heart disease with increased levels of erucic acid in erythrocytes [136].

According to EFSA, treatment with Lorenzo's oil, which is a mixture of omega-9 fatty acids (oleic and erucic) to normalize the accumulation of very long chain fatty acids in adrenal and cerebral tissues in patients with adrenoleukodystrophy, led to undesirable effects on the hematopoietic system at doses of 100 mg/kg body weight per day [127].

So far, no reliable information is available regarding the development of myocardial lipidosis after high intake of erucic acid in humans. Epidemiological studies show no clear association between cardiac disease in humans and a diet high in erucic acid. One has to notice, that erucic acid metabolism is relatively complex in respect of differences in organ dependent metabolism of long chain fatty acids including lipid incorporation, chain shortening and elongation.

The EFSA Panel on Contaminants in the Food Chain (CONTAM) noted, that erucic acid-induced myocardial lipidosis observed in several animal species may also be relevant in humans, especially with the background that myocardial lipidosis is associated with cardiac insufficiency [127,137]. Therefore, oils, including high erucic acid content, are considered undesirable for human consumption. In 2016, EFSA conducted a comprehensive toxicological review and risk assessment of erucic acid in food and feed and established a tolerable daily intake (TDI) for erucic acid of 7 mg/kg body weight. The TDI is based on the observed development of myocardial lipidosis in juvenile rats which were treated for 7 days with 1 g erucic acid/kg body weight, and in neonatal piglets that received 1.1 g/kg erucic acid for 14 days [131,132]. Higher doses of erucic acid resulted in adverse effects on liver, kidney, skeletal muscles and caused changes in body and testis weight. Moreover, higher erucic acid intake was accompanied by mitochondrial damage and disorganisation of myofibrils as well as higher incidence of myocardial necrosis and fibrosis. No conclusion on genotoxicity and carcinogenicity could be made by EFSA due to limited data available. A single generation reproductive study was performed in rats and guinea pigs where doses of erucic acid up to 7500 mg/kg body weight/day were not associated with any adverse reproductive or developmental effects [127].
