2.3.2. Evaluation of Toxicological Effects

Since it was reported that consumption of oils rich in erucic acid are accompanied with the onset of myocardial lipidosis and heart lesions in a number of species, erucic acid content in edible oils consumed by humans was restricted to certain levels by various regulatory agencies.

The Australia New Zealand Food Standards Code (FSANZ) has considered erucic acid as natural toxicant and set a maximum level of 20 g/kg (2%) in edible oils [138]. This also applies for the European Union. Commission Regulation (EU) 2019/1870 sets a maximum limit of 20 g/kg (2%) for erucic acid in vegetable oils and fats placed on the market for the final consumer or for use as an ingredient in food, whereas the maximum permitted level for mustard oil is 50 g/kg (5%).

In its scientific opinion, EFSA concludes that the 95th percentile of dietary exposure levels of erucic acid is especially high in infants and other children. For highly exposed children, this may pose an elevated health risk [127]. It has to be noted, that stricter levels apply to infant formula and follow-on formula (Commission Delegated Regulation (EU) 2019/828). The use of mustard (or the oil) as ingredient in baby food is not usual, however due to its numerous functional properties the utilization of small amounts cannot be excluded.

For mustard as condiment a maximum level of 35 g/kg (3.5%) was established, however "with acceptance from the competent authority, the maximum level does not apply to mustard oil locally produced and consumed". In the European Union level, no maximum permitted level for erucic acid is set for mustard seeds or powder, which may contribute to a high exposure of the undesirable substance. In contrast to Asiatic countries, use of mustard oil in Europe's cuisines is rare and mustard is mainly consumed in the form of prepared (table) mustard.

The U.S. Food and Drug Administration (FDA) banned mustard oil in pure form by publishing an Import Alert specifically stating that "Expressed mustard oil is not permitted for use as a vegetable oil. It may contain 20 to 40% erucic acid, which has been shown to cause nutritional deficiencies and cardiac lesions in test animals" [139].

According to the literature search on erucic acid content in mustard seeds and products derived from (Table 4), the findings indicate that both seeds and oils represent relevant sources for human intake. Wendlinger et al. showed in 2014 that most of the mustard oil samples analysed were well above the European Union limit of 5% erucic acid [114]. Recently, the same group from Germany addresses the intake of erucic acid from different sources including table mustard. According to the authors, teenagers and adults may exceed the tolerable daily intake of 7 mg/kg body weight/day by consuming one serving of mustard (10 g) depending on the contribution of erucic acid to the total fatty acids (>40%) of the seeds and the lipid content of the prepared product (>10%) [117].

It seems obvious that mustard consumption may contribute considerably to the intake of erucic acid. Especially in India and Pakistan, the use of mustard oil as cooking oil is widely distributed and still contain high amount of erucic acid. Several experts from India arguing that the health risks associated with mustard oil, also called the "Olive oil of India" were not observed in the Indian population, probably due to the high alpha-linolenic acid content which might compensate for the erucic acid [140]. Nevertheless, further development of mustard breeds low in erucic acid and a careful choice of the mustard cultivar used in food products will be in favour of consumer protection.

#### *2.4. Allergens*

Mustard seeds have a relatively high protein content, up to 36% depending on the mustard species. The mustard seed storage proteins 2S albumin, referred to as napin, are highly abundant in mustard seeds, and have been identified as major mustard allergens with significantly higher response in allergy tests compared to other storage proteins [141,142].

Allergenic 2S albumins have been characterized from white mustard (*S. alba*) [143,144] and brown mustard (*B. juncea*) [145]. According to the WHO/IUIS Allergen Nomenclature Subcommittee [146] the proteins are termed as Bra j 1 and Sin a 1 which show a close sequence homology [147] which increases the probability that people allergic to one mustard species are also sensitive for the other one [145]. For black mustard (*B. nigra*), one of the progenitor species of *B. juncea*, no information on allergens have been provided by the WHO/IUIS Allergen Nomenclature Subcommittee, however it can be assumed that seeds likewise contain allergenic 2 s albumins [148].

Beside the seed storage protein from the 2S albumin, three more allergens have been identified in *S. alba* including Sin a 2 which belongs to the seed storage 11S globulin and known as cruciferin; Sin a 3 corresponding to a non-specific lipid transfer protein, and Sin a 4, named as profilin (Table 5) [149].


**Table 5.** Mustard allergens.

<sup>a</sup> accessed on 8 June 2021, \* not listed according to the WHO/IUIS Allergen Nomenclature Subcommittee.

Recently, Hocine et al. assessed protein profiles and immunoglobulin E (IgE)-binding patterns of selected mustard varieti es (*S. alba* and *B. juncea*). In addition to proven allergens, the authors identified other new IgE-binding protein bands from *S. alba* and *B. juncea* varieties [150].

At present, no effective preventive treatments exist for mustard allergy. The allergenic proteins are highly resistant to heat treatment. Thermal denaturation for napin and cruciferin occurs upon 80 ◦C depending on the pH-value [151]. Moreover, mustard allergens are poorly digestible. Sin a 1 was shown to be resistant to digestion by trypsin and other proteolytic enzymes [141,152]. Therefore, the allergenic potential cannot be effectively reduced during mustard processing or preparation of table mustard.

Previous studies suggest that allyl-isothiocyanate (AITC), which derives from sinigrin mainly contained in *B. nigra* and *B. juncea* could have the potential to cause allergic contact dermatitis in humans [153,154]. EFSA evaluated the safety of AITC when used as food preservative and concluded that AITC may cause contact hypersensitivity which is an immunologically mediated adverse reaction but mechanistically different from food allergy, although it might be extremely unlikely that AITC acts as a direct food allergen [83]. In the scientific opinion on the evaluation of allergenic foods and food ingredients for labelling purposes, EFSA additionally pointed out that mustard may contain a number of further irritants triggering non-immune mediated reactions mimicking allergic reactions [155].

#### 2.4.1. Clinical Indication

Food allergy can be IgE-mediated or non-IgE-mediated. IgE-mediated reactions have a rapid onset, affecting skin, respiratory and gastrointestinal tract, and in some cases can lead to systemic anaphylaxis, whereas non-IgE-mediated food allergy usually is delayed and affects mainly the skin and the gastrointestinal tract. The allergic symptoms after consuming mustard or mustard-containing products are comparable to other IgE-mediated food allergies. This includes allergic or atopic dermatitis resulting in skin reactions such as rashes or hives, urticaria, swelling of lips and tongue, facial flushing and oedema, chest tightness and respiratory problems, rhinitis, asthma, nausea and vomiting, dizziness and anaphylactic shock. Mustard allergy symptoms were also observed in children under the age of 3 years indicating a primary sensitization to mustard in at least some food allergies [148,156–158].

It is suggested that approximately 50% of patients allergic to mustard are also sensitized to mugwort pollen (*Artemisia vulgaris*) and several vegetable foods, mainly from the Rosaceae family, but also from tree nuts, peanuts and legumes [159]. Vereda et al. showed that 21 out of 34 subjects sensitized to mustard had cross-reactivity with fruits from the Rosaceae family (peach, apple, pear, apricot, plum, cherries and strawberries, excluding almond) and 20 suffered from allergy to one or more nuts. Moreover, allergy to mugwort was especially reported [160]. Sin a 1 was the most prevalent allergen and highly correlated with specific IgE levels. Therefore, Sin a 1 is considered the major allergen of white mustard *(S. alba*) and the most suitable marker for a precise diagnostic screening of mustard sensitization.

Figueroa et al. reported that all of the 38 patients with mustard IgE-mediated hypersensitivity showed associated sensitization to other members of Brassicaceae family, and cross-reactivity among them was confirmed. However only 40% of these had symptomatic reactions, mainly to cabbage, cauliflower and broccoli [161].

The reported cross-reactivities with pollens or with members of the Brassicaceae family may influence the positivity of specific IgE and skin-prick tests. This may result in overestimated prevalence of sensitization to mustard, and may possibly influence the occurrence of oral allergy syndrome-like symptoms elicited by mustard. Another factor contributing to a possible overestimation of mustard sensitization and allergy may be the presence of irritants in mustard preparations or mustard containing products. For instance, the neuropeptide-active agent capsaicin may affect the release of substance P causing a non-IgE mediated mast cell degranulation [156,162]. Therefore, irritants such as capsaicin or isothiocyanates may trigger non-immune reactions mimicking allergic reactions which may lead to false positive allergy-like reactions. For example, Morisset et al. have shown that only 23.3% of patients with positive skin prick tests are truly allergic to mustard based on positive oral food challenges [148].

#### 2.4.2. Evaluation of the Allergic Potential

Mustard is one of the priority food allergens declared under several international food allergen labelling regulations, for instance in the European Union or Canada but not in the U.S. [163]. According to Regulation (EU) No 1169/2011 mustard belongs to one of the 14 major allergens that shall be indicated in the list of ingredients. The inclusion of mustard was based on the view that mustard allergy is a serious health problem in certain countries, although limited data are available according to the number of people affected. Mustard allergy is recognized as one of the most frequent spice allergies with a credible cause–effect relationship confirmed by single and double-blind placebo-controlled food challenges (DBPCFC). Several clinical studies and case reports documented severe systemic reactions, including anaphylactic reaction immediately after the ingestion of mustard, even after small quantities [164–166]. Only a small number of DBPCFC or oral food challenges (OFC) studies exist in the literature, which possibly can be justified by technical difficulties of masking the strong taste of mustard limiting the attempts to perform these studies. Previously, it was suggested that mustard allergy accounts for about 1% of food allergies in children and up to 7% of total food allergies based on estimated prevalence in France [152,155,167].

The amount of mustard required to elicit an allergic reaction may be very small. Estimations of the eliciting dose range from a mean cumulative reactive dose of mustard sauce of 891.4 ± 855.2 mg, equivalent to 124.8 ± 119.7 mg of mustard, published by Figueroa et al. to 40 mg of mustard seasoning (roughly equivalent to 0.8 mg of protein) reported by Morisset et al. [148,161]. In 36 children with a positive mustard skin-prick test, the cumulative reactive dose by open challenge or double-blind placebo controlled food challenge (SBPCFC) varied from 1 to 936 mg [156]. Most of the case reports indicate that allergic reactions come from mustard sauce or mustard hidden in other products, such as mayonnaise, dips, ketchup sauces, where mustard is present in small or trace amounts [164].

Using data from clinical studies and mathematical calculations the VITAL Scientific Expert Panel (VSEP) recently published an update on reference doses including mustard allergens. For mustard, 0.05 mg protein has been derived as a reference dose for 'ED01', or 0.4 mg protein for 'ED05' corresponding to a dose where 99% or 95% of people affected by a mustard allergy are protected from developing objectively measurable allergic reactions [168]. This is in line with EFSA's conclusion that 1 mg mustard protein may trigger allergic reactions in patients allergic to mustard [155].

Mustard is widely used in foods due to its sensory attributes, its high protein content and its numerous functional properties. Therefore, foods formulated with mustard are expected to rise in future and the risk of masked allergens in modern food products is increasing. In regions with high consumption, such as France, mustard might be among the most important food allergen for children. Hence, it deserves special attention, particularly as a potent "hidden allergen" provoking unexpected allergic reactions. The seriousness of the allergic reactions argues for informative labelling in countries where mustard is not considered as food allergen, such as the U.S.
