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Source document:
SCCS (2012)

Summary & Details:

Perfume Allergies

3. How can fragrance substance become skin allergens?

    The SCCS opinion states:

    5. Activation of weak or non-sensitising substances into sensitisers - prehaptens and prohaptens

    Fragrance allergens act as haptens, i.e. low molecular weight chemicals that are immunogenic only when attached to a carrier protein. However, not all sensitising fragrance chemicals are directly reactive, but require previous activation. A prehapten is a chemical which by itself is non- or low-sensitising, but that is transformed into a hapten outside the skin by simple chemical transformation (air oxidation, photoactivation) and without the requirement of specific enzymatic systems. A prohapten is a chemical which by itself is non- or low-sensitising but that is transformed into a hapten in the skin (bioactivation) usually via enzyme catalysis. It is not always possible to know whether a particular allergen that is not directly reactive acts as a prehapten or as a prohapten, or both, because air oxidation and bioactivation can often give the same product (geraniol is an example). Some chemicals might act by all three pathways. One example is geranial (an isomer of citral) which is a hapten itself with a moderate sensitisation potency, but can be activated to more potent sensitisers via air oxidation (autoxidation) thus acting as a prehapten and also via bioactivation (metabolic activation) thus acting as a prohapten (118).

    Increased understanding of the importance of activation through interaction with the environment that turns non-sensitising compounds into sensitisers has made it important to distinguish between prehaptens and prohaptens. This distinction facilitates discussions by emphasizing the differences in activation mechanisms between the two types of compounds requiring activation to become haptens. It is important to note that prehapten activation, in contrast to bioactivation, can be prevented to a certain extent by avoidance of air exposure during the handling and storage of the chemicals. This concerns the most prominent haptens formed by autoxidation i.e. the hydroperoxides. In bioactivation, hydroperoxides have not been identified as metabolites, but other allergenic oxidation products (in particular aldehydes and epoxides) have been identified as being formed by both activation routes depending on the structure of the compound. One thoroughly studied example is geraniol which forms the aldehyde geranial, epoxy-geraniol, and also epoxy-geranial via both pathways of activation (autoxidation and metabolic oxidation) (119, 120). When haptens are formed by both pathways, the impact on the sensitisation potency depends on the degree of autoxidation in relation to the amount of metabolic oxidation.

    Human data on established prehaptens are presented in Table 5-1 and Table 5-2. In Table 5-1 the results from patch testing with air exposed samples of the prehaptens are given. Table 5-2 shows the results from testing with the prehaptens themselves without intended air exposure. In addition to the data given in this chapter, animal data (LLNA) on the pure prehaptens or after controlled air exposure are given in Table 8-2. Possible pro- and prehaptens are identified by SAR analyses in chapter 9.

    5.1. Prehaptens

    Autoxidation is a free radical chain reaction in which hydrogen atom abstraction in combination with addition of oxygen forms peroxyl radicals. The reaction shows selectivity for positions where stable radicals can be formed. So far, all fragrance substances that have been investigated with regard to the influence of autoxidation on the allergenic potential, including identification of formed oxidation products, have oxidisable allylic positions that are able to form hydroperoxides and/or hydrogen peroxide as primary oxidation products upon air exposure. Once the hydroperoxides have been formed outside the skin they form specific antigens and act as skin sensitisers (121). Secondary oxidation products such as aldehydes and epoxides can also be allergenic, thus further increasing the sensitisation potency of the autoxidation mixture (122). The process of photoactivation may also play a role, but further research is required to establish whether this activation route is currently underestimated in importance due to insufficient knowledge of the true haptens in this context.

    Most terpenes with oxidisable allylic positions can be expected to autoxidise on air exposure due to their inherent properties. Depending on the stability of the oxidation products that are formed, a difference in the sensitisation potency of the oxidised terpenes can be seen. Oxidation products of commonly used fragrance terpenes (limonene, linalool, geraniol, linalyl acetate) have been identified as potent sensitisers in predictive animal tests (119, 123-128) (see chapter 8). This is also demonstrated for alpha-terpinene (129) and citronellol (AT Karlberg, personal communication 2012). The oxidised fragrance terpenes limonene, linalool and linalyl acetate have been tested in consecutive dermatitis patients and give frequent allergic contact reactions (130-135). Not all oxidised fragrance substances are strong sensitisers, e.g. caryophyllene is readily oxidised but has a low sensitisation potency after autoxidation (136). This is supported by clinical studies showing oxidised caryophyllene to be a less frequent allergen compared to oxidised limonene and oxidised linalool (133). Details are given in Table 5-1 The non-oxidised compounds rarely cause allergic reactions (43-45, 67, 70, 74, 97, 137-139), for details see Table 5-2. As oxidised and non-oxidised fragrance terpenes were not patch tested simultaneously in the same patients, the results are presented in two separate tables (Table 5-1 and Table 5-2).

    Oxidised fragrance terpenes with defined content of the major haptens formed after autoxidation have not been commercially available for testing in dermatology clinics. In the published clinical studies testing oxidised fragrence terpenes, the patch test preparations have been obtained specifically for the performed multicentre studies. From 2012, patch test preparations of oxidised limonene and oxidised linalool with defined content of the major allergens in the oxidation mixtures, i.e. the hydroperoxides, are commercially available.

    It should be noted that activation of substances via air oxidation results in various haptens that might be the same or cross-reacting with other haptens (allergens). The main allergens after air oxidation of linalool and linalyl acetate are the hydroperoxides. If linalyl acetate is chemically hydrolysed outside the skin it can thereafter be oxidised to the same haptens as seen for linalool. A corresponding example is citronellol and citronellyl acetate. In clincal studies, concomitant reactions to oxidised linalool and oxidised linalyl acetate have been observed (140, 141). Whether these reactions depend on cross-reactivity or are due to exposure to both fragrance substances cannot be elucidated as both have an allergenic effect themselves.

    Linalool and linalyl acetate are the main components of lavender oil. They autoxidise on air exposure also when present in the essential oil, and form the same oxidation products found in previous studies of the pure synthetic terpenes. Experimental sensitisation studies showed that air exposure of lavender oil increased the sensitisation potency. Patch test results in dermatitis patients showed a connection between positive reactions to oxidised linalool, linalyl acetate and lavender oil (140).

    Air oxidation of prehaptens can be prevented to a certain extent by measures during handling and storage of the ingredients and final products to avoid air exposure, and/or by addition of suitable antioxidants. The autoxidation rate depends not only on the compound itself, but also on its purity. The prevention of autoxidation using antioxidants needs thorough investigation because antioxidants can exert their function by being oxidised instead of the compound that they protect and might thereby be activated to skin sensitising derivatives after oxidation, which is the case for alpha-terpinene from tea tree oil (129). Alpha-Terpinene together with its analogue gamma-terpinene has been suggested as an agent for maintaining the oxidative stability of different matrices, such as food, cosmetics and medicaments (142-144). As antioxidants are now frequently used at elevated concentrations in scented products due to a growing awareness of the problem of autoxidation, there is a risk that sensitisation caused by the antioxidants will rise. One of the most used antioxidants is butylated hydroxytoluene (BHT) which is considered a minimal risk for sensitisation in the concentrations used but nevertheless, with increased concentrations and usage, the risk of sensitisation could increase.

    Due to the complexity of scented products, which are mixtures of many different fragrance substances, there are at present no published data identifying the presence of individual hydroperoxides in cosmetic products containing the above fragrance terpenes. However, clinical studies show a clear connection between contact allergy to oxidised limonene and oxidised linalool, and contact allergy to other markers of fragrance contact allergy (130- 135); see Table 5-3.

    Table 5-1: Contact allergic reactions to the autoxidised fragrance substances limonene, linalool, caryophyllene, myrcene and linalyl acetate in consecutive dermatitis patients. 

    Table 5-2: Contact allergic reactions to limonene, linalool, linalyl acetate and caryophyllene in consecutive dermatitis patient. Please observe that several studies have been performed using the test substances without reporting the autoxidation status but it has been intended to be low. For precise information see the original references. 

    Table 5-3: Concomitant reactions to fragrance markers: Fragrance Mix I and II (FM I, FM II), Myroxylon pereire (MP) and to colophonium (coloph.) in the baseline series in patients with positive or negative patch test reactions to oxidised fragrance substances. 

    5.2. Prohaptens

    Compounds that are bioactivated in the skin and thereby form haptens are referred to as prohaptens. The human skin expresses enzyme systems that are able to metabolise xenobiotics (146), modifying their chemical structure to increase hydrophilicity and allow elimination from the body. Xenobiotic metabolism can be divided into two phases: phase I and phase II. Phase I transformations are known as activation or functionalisation reactions, which normally introduce or unmask hydrophilic functional groups. If the metabolites are sufficiently polar at this point they will be eliminated. However, many phase I products have to undergo subsequent phase II transformations, i.e. conjugation to make them sufficiently water soluble to be eliminated. Although the purpose of xenobiotic metabolism is detoxification, it can also convert relatively harmless compounds into reactive species. Cutaneous enzymes that catalyse phase I transformations include the cytochrome P450 mixed-function oxidase system, alcohol and aldehyde dehydrogenases, monoamine oxidases, flavin-containing monooxygenases and hydrolytic enzymes. Acyltransferases, glutathione S-transferases, UDP-glucuronosyltransferases and sulfotransferases are examples of phase II enzymes that have been shown to be present in human skin (146). These enzymes are known to catalyse both activating and deactivating biotransformations (147), but the influence of the reactions on the allergenic activity of skin sensitisers has not been studied in detail. Skin sensitising prohaptens can be recognised and grouped into chemical classes based on knowledge of xenobiotic bioactivation reactions, clinical observations and/or in vivo and in vitro studies of sensitisation potential and chemical reactivity. Few mechanistic investigations of prohaptens have so far been published. Investigations that are important for the bioactivation of fragrance substances are studies on alkenes, e.g. alpha- terpinene (148-150), the allylic primary alcohols geraniol (120) cinnamyl alcohol (151-155), eugenol and isoeugenol (156).

    In order to be able to predict the sensitisation potency of prohaptens, steps of bioactivation have to be included in the predictive tests where intrinsic bioactivating systems are lacking. So far, no such predictive non-animal methods have been developed that take account of this.

    When bioactivation occurs, the risk of cross-reactivity also needs to be be considered. Cross-reactivity between certain aldehydes and their corresponding alcohols, e.g. cinnamal - cinnamyl alcohol and geranial - geraniol, due to the metabolic oxidation of the alcohols to the aldehydes in the skin is demonstrated (120, 151-155). When using derivatives of a fragrance substance, it must be taken into account that the derivative could be metabolically transformed in the skin into the parent or cross-reacting compounds. A prominent example of such bioactivation is the hydrolysis of esters by esterases to the corresponding original alcohols. The metabolic product obtained can act as a hapten or a prohapten in exactly the same way as the non-esterified parent compound.

    Isoeugenol and its derivatives are an important example for this mechanism from which general conclusions may be drawn. As only the use of isoeugenol in fragranced products needs to be indicated on the ingredients list, the additional exposure to isoeugenol through its derivatives should also be taken into account. In a study it was shown that several EDP/EDT/aftershave lotions contained high levels of isoeugenyl acetate and isoeugenol methyl ether (Table 5-4) (157). Isoeugenyl acetate will be hydrolysed by esterases in the skin to generate isoeugenol. The situation may be similar for eugenyl acetate and geranyl acetate, which might be used in fragrance formulations instead of eugenol and geraniol, respectively. Moreover, such derivatives will contribute to exceeding any established `acceptable dose/area level’ of the parent compound, i.e., yield unduly high concentrations on the skin.

    Table 5-4: Mean and median content of isoeugenol and its derivatives in the 29 perfume products. 

    5.3. Conclusions

    • Many fragrance substances can act as prehaptens or prohaptens, forming allergens which are more potent than the parent substance by abiotic and/or metabolic activation. Activation can thus increase the risk of sensitisation.
    • Fragrance substances of clinical importance known to be prehaptens and to form sensitising compounds by air oxidation include limonene, linalool, and linalyl acetate.
    • Fragrance substances of clinical importance known to be prohaptens and to form sensitising compounds by metabolic transformation include cinnamyl alcohol, eugenol, isoeugenol and isoeugenol acetate.
    • Fragrance substances of clinical importance with published data known to be both prehaptens and prohaptens and to form sensitising compounds by air oxidation (prehaptens) and by metabolic transformation include geraniol and alpha-terpinene.
    • A fragrance substance that sensitises without activation, but forms more potent sensitising compounds by air oxidation and also by metabolic transformation is, as one example, geranial (one isomer of citral).
    • In the case of prehaptens, it is possible to prevent activation outside the body to a certain extent by different measures, e.g. prevention of air exposure during handling and storage of the ingredients and the final product, and by the addition of suitable antioxidants. When antioxidants are used, care should be taken that they will not be activated themselves and thereby form new sensitisers. It should be noted that the possibility to reduce the sensitisation potency by preventing air oxidation is also important for a direct acting hapten or prohapten, if a further activation by air oxidation to more allergenic compounds has been shown.
    • In the case of prohaptens, the possibility to become activated is inherent to the molecule and activation cannot be avoided by extrinsic measures. Activation processes increase the risk for cross-reactivity between fragrance substances. Cross- reactivity has been shown for certain alcohols and their corresponding aldehydes, i.e. between geraniol and geranial (citral) and between cinnamyl alcohol and cinnamal. Cross-reactivity is also expected between ester derivatives and their parent alcohols, as the esters will be hydrolysed by esterases in the skin. Esters of important contact allergens that can be activated by hydrolysis in the skin are isoeugenol acetate, eugenyl acetate and geranyl acetate all of which are known to be used as fragrance ingredients.
    • Further experimental and clinical research in the area of abiotic and/or metabolic activation of fragrance substances is clearly needed to increase the safety for the consumer. Compounds suspected to act as prehaptens and/or prohaptens should be considered as allergens, unless it could be demonstrated that they do not become activated by one of the described pathways.

    Source & ©: SCCS,   "Opinion on fragrance allergens in cosmetic products",
    26-27 June 2012, 5. Activation of weak or non-sensitising substances into sensitisers - prehaptens and prohaptens. p. 33-39

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