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Skin Sensitization: Reaction Mechanistic Applicability Domains for Structure-Activity Relationships Aynur O. Aptula,*,† Grace Patlewicz,‡ and David W. Roberts§ SEAC, Unilever Colworth, Sharnbrook, Bedford, MK44 1LQ, England, European Chemicals Bureau TP582, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Via Fermi, 21020 Ispra (VA), Italy, and School of Pharmacy and Chemistry, Liverpool John Moores University, Byrom Street, Liverpool, L3 3AF, England Received March 16, 2005
The prediction of skin sensitization potential with minimum animal testing is currently of great importance in light of forthcoming legislation. A number of structure-activity relationships for skin sensitization have been published over the years, but their applicability has often been limited to structural classes. The concept of an applicability domain for a quantitative structure-activity relationship [(Q)SAR] is increasingly being viewed as key for the predictive application of (Q)SARs. This is particularly the case for skin sensitization if more widely applicable SARs are to be developed. In this paper, we analyze a recently published chemical data set for skin sensitization, apply reaction mechanistic criteria to domain classification, and evaluate the structure-activity trends observed within each of these mechanistic domains.
Introduction Allergic contact dermatitis (ACD) is an eczematous skin disease affecting a significant minority of the general population worldwide, which can have a serious impact on quality of life (1, 2). At present, the only validated approaches to conclusively identify skin sensitization hazard are in vivo models such as the local lymph node assay (LLNA) (3, 4). There is an urgent need to develop novel approaches or risk assessment strategies to replace animal testing especially in view of legislative initiatives such as REACH (registration, evaluation, authorization of chemicals) as well as the 7th Amendment to the Cosmetics Directive (which poses a ban on animal testing for cosmetic ingredients by 2009, although some tests are exempted until 2013) (5, 6). Quantitative structureactivity relationships [(Q)SARs] are viewed as one of the most cost effective alternatives to estimate toxicity effects of chemicals since they have the potential to save time and money and minimize the use of animal testing. However, although the new and current EU legislation provides the possibility of using (Q)SARs instead of in vivo testing, there are neither accepted (Q)SARs for toxicological end points nor detailed guidance on how to develop (Q)SARs for risk assessment. As a consequence, uptake of (Q)SARs by competent authorities such as regulatory agencies has been very limited. Several initiatives have recently emerged to increase the acceptance of (Q)SARs. The main principles for the validity of (Q)SARs were identified in a workshop organized by CEFIC/ ICCA in Setubal in 2002 and were subsequently evaluated by OECD [as part of the ad hoc expert group for (Q)SARs] (7). These are now referred to as the “OECD principles”, which read as follows: “To facilitate the consideration * To whom correspondence should be addressed. Tel: + 44 1234 264823. Fax: + 44 1234 264722. E-mail:
[email protected]. † Unilever Colworth. ‡ European Commission. § Liverpool John Moores University.
of a (Q)SAR model for regulatory purposes, it should be associated with the following information: -a defined endpoint -an unambiguous algorithm -a defined domain of applicability -appropriate measures of goodness-of-fit, robustness, and predictivity -a mechanistic interpretation, if possible.” (Q)SARs, which fulfill these criteria, may in principle be applicable within the regulation practice to predict mammalian end points. Of these principles, perhaps the one that provokes the most debate is the domain of applicability. The domain of applicability aims to provide some guidance on the scope and boundaries for the use of a given (Q)SAR. Recently, a chemical data set for evaluation of alternative approaches was published (8) listing skin sensitization potential, expressed as LLNA EC3% values, for 41 compounds covering a diverse range of structures. The EC3 is the dose required to give a 3-fold stimulation between treated and control groups in the LLNA. The EC3 is taken as a quantifier of the sensitization potential. These compounds were classified according to chemical class, e.g., ketone, aliphatic aldehyde, halogenated compound, etc., but no clear relationship between structure and sensitization potential was apparent from this classification. The aim of this paper is to apply a chemical mechanism of action approach to reclassify these compounds into appropriate applicability domains.
Approaches to Chemical Classification There are various ways in which a diverse set of chemicals can be grouped into classes according to some criterion of similarity (9). For example, they can be classified according to similarity in connectivity patterns (this may be useful, for example, where biological activity dependent on noncovalent binding to a specific receptor is being modeled), according to the presence of specific
10.1021/tx050075m CCC: $30.25 © 2005 American Chemical Society Published on Web 08/20/2005
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functional groups and so on. The most appropriate classification depends on the purpose: For example, if the purpose is surfactant formulation, it is appropriate to classify surfactants as anionic, cationic, nonionic, or zwitterionic, possibly with subclassifications within these. As another example, for purposes of compiling a reference table of physical properties, a hierarchical classification based on empirical formulas may be the most appropriate. This raises the question of what is the most appropriate classification approach for skin sensitization. As we will now discuss, enough is known about the factors that influence skin sensitization for a classification based on chemical reaction mechanism to be appropriate.
between the sensitizer remaining at the reaction site for long enough to react and being removed by partition into the polar lymphatic fluid before reacting. Subsequently, the mouse LLNA method was introduced, in which the sensitizer is delivered by topical application to the ear (3, 4). The relevance of penetration and the role of hydrophobicity in determining sensitization potential in the LLNA are quite complex, and we plan to discuss it in more detail in a separate publication. For the present purpose, the following points are worth noting. Some very hydrophilic compounds [e.g., squaric acid, which at physiological pH exists as its dianion, and is a moderate sensitizer in the LLNA (21)] are well able to sensitize, whereas for methyl alkanesulfonates, RSO3Me with R in the range C12-C16, sensitization potential in the LLNA was found to be negatively correlated with log P (20) [although in guinea pig sensitization there is a positive correlation (19)], suggesting that above a certain log P value partitioning of the sensitizer into the lipid bilayers of the stratum corneum starts to restrict the amount of sensitizer reaching the epidermis. The methyl alkane sulfonates have an H-polar (i.e., capable of forming hydrogen bonds) -OSO2- group, which by its interaction with the headgroups in the lipid bilayers may enhance the affinity of the methyl alkane sulfonate for the bilayer. Primary alkyl bromides, which do not have an H-polar group and are more hydrocarbon-like in their solute properties, differ from the methyl alkane sulfonates in showing a positive correlation of LLNA sensitization potential with log P up to the C17 homologue, above which the correlation becomes negative (18). In many cases, where the compounds are less hydrophobic than methyl dodecane sulfonate, there is little evidence for significant correlation of LLNA activity with hydrophobicity. It follows from the above that the key to predicting likely sensitization potential is being able to predict electrophilic reactivity and proelectrophilicity. Electrophilic and proelectrophilic mechanisms of action are wellestablished in several other areas of toxicology (22, 23), and Lipnick (24) has presented a good overview of the underlying reaction chemistry. Qualitatively, recognition of electrophilicity from structure can often be a simple matter for an organic chemist, but in other cases, it may require laboratory investigation to determine whether a compound of an unfamiliar type is electrophilic and how it reacts. However, the number of different electrophilic reaction mechanisms is not large and in our view the most appropriate approach for assessing the applicability domain for skin sensitization (Q)SARs is by chemical mechanism of action.
Physicochemical Basis of Skin Sensitization Research into skin sensitization by low molecular weight chemicals dates back to the pioneering work of Landsteiner and Jacobs in the 1930s (10). They compared the results of guinea pig sensitization on a series of aromatic halides and pseudohalides with the results of experiments in which reactions with aniline (as the model protein) were attempted. This established the connection between the ability of chemicals to react with proteins to form covalently linked conjugates and their sensitization potential (10). In the vast majority of cases, skin sensitization potential is dependent on electrophilic reactivity of the skin sensitizer or a derivative produced (usually by oxidation) in vivo or abiotically. According to the widely accepted biochemical model for skin sensitization, the sensitizer binds covalently as a “hapten” to a skin protein (there is currently some debate as to whether there are specific proteins in specific locations for this role or whether a diverse range of proteins can serve the purpose) leading to it becoming antigenic. The antigenic protein is processed by Langerhans cells, which migrate to a lymph node and express the antigen to T-lymphocytes, as a result of which a population of T-lymphocytes is stimulated to proliferate and circulate, able to recognize the antigen, and on recognition initiate a cascade of biochemical processes leading to the symptoms of ACD (1). For more information on the biological mechanisms affecting skin sensitization, the reader is encouraged to read the excellent review by Pease (11). The first physicochemical mathematical model for skin sensitization, the RAI (relative alkylation index) model (12), was based on the above biochemical model, with the assumption that the reaction between the sensitizer and the protein occurs in a lipid environment such as a cell membrane. On the basis of this model, many QSARs have been demonstrated correlating skin sensitization data with a combination of reactivity {quantified in various ways, e.g., experimental rate constants (13), substituent constants (14, 15), molecular orbital parameters (16), and hydrophobicity [usually modeled by log P, P being the octanol/water partition coefficient (12, 15, 17-20)]}. It is often stated that the role of the hydrophobicity parameter is to model penetration of the skin sensitizer through the stratum corneum to the site of action in the epidermis. However, this was not the basis of the RAI model, which was developed for application to guinea pig sensitization test data for which the sensitizer was delivered directly to the epidermis by epicutaneous injection. The hydrophobicity term was originally incorporated into the RAI model to represent competition
Reaction Mechanism for Skin Sensitization As discussed above, it is well-recognized that skin sensitization to low molecular weight compounds almost invariably involves covalent binding of the sensitizing chemical to protein in skin (12, 25). Usually, this covalent binding involves the sensitizer acting as an electrophile toward nucleophilic groups in proteins. In some cases, the sensitizer is not itself electrophilic but undergoes abiotic or biochemical transformation to an electrophile. Such sensitizers are referred to as proelectrophiles or prohaptens. There are various types of electrophilenucleophile reactions, and in skin sensitization, some of
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Aptula et al.
Table 1. Structural and Reaction Mechanistic Classification of the Gerberick et al. (8) Data Set structural classa aliphatic aldehydes ketones halogenated compounds aromatic esters aliphatic amines phenols aliphatic alcohols miscellaneousb
no. of chemicals 9
reaction mechanistic class
Table 2. Michael Acceptors and Pro-Michael Acceptors no.
no. of chemicals 12
3 4
Michael acceptors and pro-Michael acceptors SNAr electrophiles SN2 electrophiles
2
Schiff base formers
7
1
acylating agents
3
7
no evident electrophilic or pro-electrophilic features
14
1 4
4 11
a
Some compounds are assigned dual class membership by Gerberick et al. (8); for example, methyl salicylate is classified as both “phenol” and “aromatic ester”. In this table, the first class named by these authors is used as the basis for the classification. b Gerberick et al. (8) classify three compounds under “miscellaneous”. Here, we also include compounds classified by them as: coumarins (two compounds), carboxylic acids (two compounds), N ring (three compounds), and aromatic amine (one compound).
the most frequently encountered are Michael type reactions, SN2 reactions, SNAr reactions, acylation reactions, and Schiff base formation. Table 1 summarizes the original (structural) chemical class classification of Gerberick et al. (8) and our reclassification according to reaction mechanism class. Within each structural class, there are both sensitizers and nonsensitizers and no apparent structure-activity pattern. However, the reaction mechanistic classification reveals clear structure-activity trends, which we will discuss class by class.
1 2 3 4 5 6 7 8 9 10 11 12
EC3 (%) pEC3
potency category
3.43b 2.44b 2.43b 3.05 1.96 1.63 1.60 1.41 1.31
extreme strong strong strong moderate moderate moderate moderate moderate
chemical name
CAS
p-benzoquinone 1,4-dihydroquinonea 1,4-phenylene diaminea lauryl gallate isoeugenol cinnamic aldehyde benzylidene acetone R-hexylcinammic aldehyde 1-(p-methoxyphenyl)1-penten-3-one 2-hydroxypropyl methacrylate 6-methyl coumarin 1-chloro-2,4-dinitrobenzenec
106-51-4 123-31-9 106-50-3 1166-52-5 97-54-1 104-55-2 122-57-6 101-86-0 104-27-8
0.01 0.1 0.1 0.3 1.8 3.1 3.7 8.4 9.3
923-26-2
NC
NS
92-48-8 97-00-7
NC 0.04 3.70
NS extreme
a These compounds are considered here as pro-Michael acceptors, but there are other possible reaction pathways for protein binding, including free radical reactions, which cannot be excluded on the basis of the experimental evidence currently available. b pEC3 values calculated as log(MW/EC3) adjusted by subtraction of log 4 to allow for the presence of four equivalent electrophilic reaction sites. c SNAr electrophile. The potency category was determined by the following EC3,% cutoff values: extreme, 5-methyl-2,3-hexanedione > phenylacetaldehyde > cyclamen aldehyde and lilial > hydroxycitronellal. We arrive at this ranking order by application of the following principles. Alkyl chain branching close to the carbonyl group increases electron density at the carbonyl carbon; aromatic groups close to the carbonyl group decrease electron density at the carbonyl carbon; a second carbonyl group decreases electron density at the first and vice versa. It can be seen that the observed EC3 values follow this ranking order closely, with the excep-
Esters of simple aliphatic alcohols are not electrophilic acylating agents, because the corresponding alkoxide ion is too basic to be a good leaving group. Esters of more acidic alcohols such as phenols and carboxylic anhydrides are electrophilic and can acylate nucleophiles (hard nucleophiles more readily than soft nucleophiles) by attack of the nucleophile at the carbonyl group (36). One of the esters in the Gerberick et al. data set (diethylisophthalate) is not activated, and we do not classify it as an acylating agent but assign it into the nonelectrophilic/nonproelectrophilic group. The three acylating agents represented in the Gerberick et al. data set are shown in Table 5, in decreasing order of sensitization potential, oxazolone (extreme sensitizer, EC3 ) 0.003%), 3-propylidenephthalide (moderate sensitizer, EC3 ) 3.7%), and 3,4-dihydrocoumarin (moderate sensitizer, EC3 ) 5.6%). Their chemistries are discussed below. Oxazolone is an azlactone (Scheme 7). Azlactones are used in protein synthesis and react readily with amine nucleophiles by attack at the carbonyl group (37). 3-Propylidenephthalide is a cyclic vinylic ester (Scheme 7), which can react with amine nucleophiles by attack at the carbonyl group. We used ACD-LABS software to calculate pKa for the vinylic/benzylic alcohol corresponding to the leaving group. The calculated pKa is 10.8, similar to that of phenol, and consequently, 3-propylidenephthalide can be considered to be electrophilic. 3,4-Dihydrocoumarin is a phenolic lactone, a cyclic phenol ester that can undergo ring opening by attack of an amine nucleophile at the carbonyl group (Scheme 7). The pKa calculated by ACD-LABS software for the protonated leaving group is 10.2, similar to that of phenol. This reaction is not as easy as for acyclic phenolic esters, since cleavage of the O-CO bond cannot occur
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Chem. Res. Toxicol., Vol. 18, No. 9, 2005 1425
compounds, and we intend to discuss 3-aminophenol and related “meta sensitizers” more fully elsewhere. For dimethylaminopropylamine, we suggest activation by oxidation of the NH2 group followed by hydrolysis to a CHO group (Scheme 8), by analogy to the proposed activation of the sensitizer ethylenediamine by oxidation/ hydrolysis to glyoxal (40).
Scheme 7. Acylating Agents
Conclusions
Table 6. Compounds with No Evident Electrophilic or Proelectrophilic Features no.
chemical name
CAS
1 2 3 4 5 6 7 8 9 10 11 12 13 14
3-dimethylaminopropylamine 3-aminophenol eugenol abietic acid linalool penicillin G chlorobenzene dimethylisophthalate glycerol hexane 1,4-dihydrobenzoic acid 2-propanol lactic acid methyl salicylate
109-55-7 591-27-5 97-53-0 514-10-3 78-70-6 61-33-6 108-90-7 1459-93-4 56-81-5 110-54-3 99-96-7 67-63-0 50-21-5 119-36-8
EC3 potency (%) pEC3 category 2.2 3.2 12.9 14.7 30.4 46.4 NC NC NC NC NC NC NC NC
1.67 1.53 1.10 1.31 0.71 0.86
moderate moderate weak weak weak weak NS NS NS NS NS NS NS NS
Scheme 8. Pro-electrophile Mechanism for 3-Dimethylaminopropylamine
by linear separation of the oxygen and the carbonyl carbon but has to occur by rotation of the other bonds in the ring. This feature, which has been extensively investigated in sultone chemistry (38, 39), leads to a more negative entropy of activation than for analogous acyclic esters and, hence, a lower reaction rate. The corresponding reaction of 6-methyl coumarin is still more difficult, since the rigidity introduced into the ring by the 3,4double bond makes bond rotations more difficult, as referred to in ref 1.
No Evident Electrophilic or Proelectrophilic Features This group of chemicals is quite diverse (Table 6), but none has any obvious features corresponding to reactivity. Most of these compounds are either nonsensitizing or only weak sensitizers, in agreement with the view that electrophilic reactivity is required for skin sensitization, but two are classed as moderate. These are 3-aminophenol and 3-dimethylaminopropylamine. Autoxidation and hydrolysis reactions may lead to activation of these
The analysis presented here demonstrates clearly that skin sensitizers are better classified according to chemical reaction mechanisms rather than by similarity of structure. Here, we have shown that the Gerberick et al. (8) data set can be classified in this way into six mechanistic groups (including the nonelectrophilic, nonproelectrophilic group) and that within each mechanistic group there is a clear trend relating electrophilic reactivity to sensitization potential. This reinforces our view that for definition of applicability domains in SARs, domain definition should be mechanistic rather than structural. It may be noted that the concept of intrinsic antigenicity has been ignored in this paper and we have found no necessity to invoke it. This supports an earlier suggestion (15), based on the observation that a structurally diverse set of aldehydes, which all react by the same mechanism but produce diverse determinant structures and would not be expected to show cross-reactivity, fit a simple QSAR, that intrinsic antigenicity may not be an important factor in determining skin sensitization potential.
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unsaturated sultones. In Immunotoxicology (Gibson, G. G., Hubbard, R., and Parke, D. V., Eds.) pp 443-448, Academic Press, London. Roberts, D. W. (1995) Linear free energy relationships for reactions of electrophilic halo- and pseudohalobenzenes, and their application in prediction of skin sensitisation potential for SNAr slectrophiles. Chem. Res. Toxicol. 8, 545-551. Patlewicz, G., Roberts, D. W., and Walker, J. D. (2003) QSARs for the skin sensitization potential of aldehydes and related compounds. QSAR Comb. Sci. 22, 196-203. Mekenyan, O., Roberts, D. W., and Karcher, W. (1997) Molecular orbital parameters as predictors of skin sensitization potential of halo- and pseudohalobenzenes acting as SNAr electrophiles. Chem. Res. Toxicol. 10 (9), 994-1000. Roberts, D. W., York, M., and Basketter, D. A. (1999) Structure activity relationships in the murine local lymph node assay for skin sensitization: R,β-Diketones. Contact Dermatitis 41, 14-17. Basketter, D. A., Roberts, D. W., Cronin, M., and Scholes, E. W. (1992) The value of the local lymph node assay in quantitative structure-activity investigations. Contact Dermatitis 27, 137142. Roberts, D. W., and Basketter, D. A. (1997) Further evaluation of the quantitative structure-activity relationship for skinsensitizing alkyl transfer agents. Contact Dermatitis 37, 107112. Roberts, D. W., and Basketter, D. A. (2000) Quantitative structureactivity relationships: Sulfonate esters in the local lymph node assay. Contact Dermatitis 42, 154-161. Gerberick, G. F., Vassallo, J. D., Bailey, R. E., Chaney, J. G., Morrall, S. W., and Lepoittevin, J.-P. (2004) Development of a peptide reactivity assay for screening contact allergens. Toxicol. Sci. 81 (2), 332-343. Lipnick, R. L., Watson, K. R., and Strausz, A. (1987) A QSAR study of the acute toxicity of some industrial organic chemicals to goldfish. Narcosis, electrophile, and proelectrophile mechanisms. Xenobiotica 17, 1011-1025. Lipnick, R. L., Bentley, J. L., and Bentley, D. L. (1995) QSAR analysis of ocular toxicity. QSAR92: 5th International Workshop on QSAR in Environmental Toxicology and Chemistry, Duluth, MN, July 19-23, 1992. Exploring QSAR (Hansch, C., and Leo, A., Eds.) Chapter 9, pp 349-386, ACS, Washington. Lipnick, R. L. (1991) Outliers: Their origin and use in the classification of molecular mechanisms of toxicity. Sci. Total Environ. 109/110, 131-153. Roberts, D. W., and Lepoittevin, J.-P. (1997) Hapten-protein interactions. In Allergic Contact Dermatitis. The Molecular Basis (Lepoittevin, J.-P., Basketter, D. A., Goosens, A., and Karlberg, A.-T., Eds.) pp 81-111, Springer, Heidelberg. Basketter, D. A., and Goodwin, B. F. J. (1988) Investigation of the prohapten concept. Contact Dermatitis 19, 248-253.
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