StructureâPotency Relationships for Epoxides in Allergic Contact

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Structure-potency relationships for epoxides in allergic contact dermatitis David W. Roberts, Aynur Aptula, and Anne Marie Api Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00241 • Publication Date (Web): 05 Dec 2016 Downloaded from http://pubs.acs.org on December 8, 2016

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Chemical Research in Toxicology

Structure-potency relationships for epoxides in allergic contact dermatitis

David W. Roberts†*, Aynur Aptula‡, Anne Marie Api‡‡

†

School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Liverpool, L3 3AF, United Kingdom

‡

Unilever Safety and Environmental Assurance Centre, Colworth Science Park, Sharnbrook, Bedford, MK44 1LQ, United Kingdom ‡‡

Research Institute for Fragrance Materials, Inc., 50 Tice Boulevard, Woodcliff Lake, NJ 07677, United States

1 ACS Paragon Plus Environment


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Abstract Epoxides are known or proposed to be involved in skin sensitization in various ways. Some are encountered directly, and other have been shown to be formed abiotically and metabolically from various unsaturated chemicals. They can react as SN2 electrophiles.


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To date no quantitative mechanistic models (QMMs) are known for skin sensitization potency of this sub-category of SN2 electrophiles. Here we have considered the reaction mechanistic chemistry of epoxides and combined published experimental kinetic data (rate constants k for reaction with a cysteine-based peptide) together with calculated hydrophobicity data (logP) to derive a QMM correlating potency in the local lymph node assay (LLNA), expressed as EC3, with a relative alkylation index (RAI, calculated as logk + 0.4 logP). The QMM equation, pEC3 = 2.42(±0.26) RAI + 4.0 (±0.25), n = 9, R2 = 0.928, R2(adj) = 0.917, F = 90, s = 0.18, fits the data well, with one positive outlier. The outlier can be rationalised by its exhibiting an alert for oxidation of an amine moiety to give, in this case, the highly reactive glycidaldehyde. The epoxide QMM predicts the potency of a non-epoxide SN2 electrophile (predicted EC3, 0.48%; observed EC3 0.5%), suggesting that it could form the basis for a more general H-polar SN2 QMM that could be a valuable tool in skin sensitization risk assessment for this quite extensive and structurally diverse reaction mechanistic domain.

Introduction Eliminating animal testing is a significant challenge in consumer safety risk assessment. For at least the last 15 years, there has been a great deal of effort to investigate and develop nonanimal approaches to evaluate skin sensitization potential and potency.1-5 It is recognised that the molecular initiating event in skin sensitization is the covalent modification of cutaneous



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protein by reaction with the allergenic chemical or a chemical derived from it by metabolic or abiotic activation.6 Thus, the sensitization process is a function of reaction chemistry. The scope and potential application of chemistry-based approaches in development of non-animal methods for prediction of sensitization potency is discussed in some detail by Roberts et al..7 Qualitative and quantitative understanding of the chemistry underlying the protein modification step is an essential component of approaches based on understanding and modelling the biological mechanism of skin sensitization.8 Elucidation of structure-activity relationships and development of quantitative mechanistic models (QMMs) for already existing skin sensitization data makes an important contribution to such approaches. It is well established that the degree of ability (or lack thereof) of chemicals to act as skin sensitizers is strongly related to their ability to react covalently with skin proteins.9,10 Although the nature and location of the target proteins is not clearly established, reactivity parameters based on simple model nucleophiles have often proved adequate to model structure−potency relationships. A key aspect of chemistry-based modeling of skin sensitization is the application of chemical reaction mechanistic domains,11 based on the concept that different quantitative relationships between skin sensitization potency and chemical properties should apply for each reaction mechanism. The advantage here is that this is the concept that underpins the understanding of the chemical basis of skin sensitization itself12,13 and it has been demonstrated that within reaction mechanistic domains QMMs can be developed. Establishment of QMMs and Structure-Activity Relationship (SAR) principles to model skin sensitization has acquired particular importance in light of the requirement to develop non-animal approaches. Recently QMMs have been applied in the development of rules for predicting high potency chemicals (HPC) to which the Dermal Sensitization Threshold (DST) approach should not be applied.14


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Much of the available data on skin sensitization potency come from animal testing. Although human test data are available for a number of chemicals,15 and are used, as well as for risk assessment purposes, to assess predictive performance of animal tests, routine testing of new chemicals on humans is not an option. The two main test animals are the guinea pig (used in the guinea pig maximization test (GPMT) and the Buehler test) and the mouse (used in the Local Lymph Node Assay (LLNA)).16-20 In the LLNA, skin sensitization hazard is defined as a function of the ability of the test chemical to provoke immune activation (lymphocyte proliferation) in lymph nodes draining the site of topical application.21-23 A substance is classified as a sensitizer if it induces a threefold stimulation index (SI) or greater at one or more test concentrations.24-26 The LLNA is able also to provide a reliable measure of relative skin sensitizing potency. Potency is measured by derivation of an estimated concentration of substance required to induce a threefold SI value (EC3) as compared with concurrent vehicle controls.27 EC3 values are usually considered to be accurate within a factor of ca. 2.25 Human occupational and consumer exposure to epoxides of various sorts can occur from several sources.27 Some epoxides are used in products such as adhesives and epoxy resins, others are implicated as metabolites of naturally occurring fragrance materials.28 They are encountered in the context of several toxicological endpoints, such as aquatic toxicity and mutagenicity, and have been subject to detailed experimental and theoretical studies in these contexts.29-31 Here we consider their sensitization potential and potency from an SAR and QMM perspective. They are known to be, or are proposed to be, involved in skin sensitization in various ways: 1. Several epoxides have been tested as such and are known to be skin sensitizers32,33



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2. Epoxides have been shown to be formed metabolically from various unsaturated chemicals and are invoked as the ultimate sensitizers34 3.

Some cases of abiotic formation of epoxides have been reported and a role in sensitization for epoxides formed in this way has been suggested28

In this work we first briefly consider the mechanistic principles influencing reactivity of epoxides, then derive a QMM from published reactivity and LLNA data and rationalize, in terms of structure-chemistry principles, patterns of structure-potency for epoxides postulated to be involved in sensitization via abiotic or metabolic activation of non-electrophilic chemicals.

Reaction chemistry of epoxides A skin sensitizing chemical is either directly reactive (electrophilic) with protein nucleophiles or is a pre- or pro-electrophile10,35 requiring activation (usually either metabolic or autoxidative) to convert it to a reactive electrophile. Epoxides are SN2 (in some cases SN1 may be possible) electrophiles. The general SN2 reaction of epoxides is shown in Scheme 1.

Ether oxygen is usually a very poor leaving group, but in the case of epoxides reactivity is enhanced by ring strain. Electronegative groups that can stabilise the developing negative charge on the oxygen atom increase reactivity. Otherwise, the same chemistry principles which apply for SN2 electrophiles in general apply to epoxides: 1. Reaction occurs more readily at primary carbon than at secondary or tertiary. 2. Allylic (and heteroallylic) and benzylic carbon centres are more reactive than saturated. This effect can be very large for SN2 reactions of open chain compounds, but in ring-opening


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reactions, in which in the incoming nucleophile, the carbon reaction centre and the leaving group are unable to be collinear in the transition state, the effect is weaker. 3. Neighbouring group effects can increase reactivity. 4. Point 2 above can outweigh point 1. We will refer to these 4 points as “reactivity SAR principles for SN2”. As regards sensitization potential, on the basis of published work on other SN2 electrophiles with guinea pig data36-38 and to a more limited extent with LLNA data,39 skin sensitization (LLNA and human) potency should be correlated with a combination of reactivity and hydrophobicity parameters.

Quantitative Mechanistic Model for skin sensitization potency of epoxides LLNA and reactivity data have been published for several epoxides, reactivity data having been generated by a modification of the Direct Peptide Reactivity Assay (DPRA) method, using a hexapeptide with a cysteine unit as the major nucleophilic centre.32,33 In some of these cases the reactivity data are in the form of rate constants33 and in others they are presented in the original reference as % reaction (cysteine adduct formation) at determined times.32 For the latter cases, we used the % reaction at 40 min to calculate the rate constant from the equation given by Roberts et al..6 Since the number of data points is quite small, we decided to base the QMM on regression of -log of the molar EC3 (pEC3) against RAI (Relative Alkylation Index), this being a composite parameter defined as logk + 0.4 logP).40 In earlier work the hydrophobicity contribution, relative to reactivity, to QMMs has been found to be well modelled by 0.4 logP,41,42 -0.4 being similar to the coefficient from regression of logP (aqueous methanol/hexane) against logP (octanol/water).38,42



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Table 1 shows the epoxides for which both reactivity and LLNA data are available in the literature. In addition to EC3 and log k values, logP values, calculated by BioByte ClogP v1.6 software (http://www.biobyte.com), are also shown. All of these epoxides are primary, and the reaction centre is not allylic or benzylic in any of them. Consequently, we would expect variations in reactivity to come mostly from variation in the ability of the R group to stabilize the negative charge on the oxygen atom as the ring opens. The only exceptions are the two compounds with R = PhSCH2- and R = PhNHCH2-, for which neighbouring group effects may contribute. From this point, individual epoxides will be referred to as R-epox, e.g. the two compounds above are PhSCH2-epox and PhNHCH2-epox.

At this point we need to discuss the EC3 value of the PhNHCH2-epox derivative. The value given by Niklasson et al.33 is 0.19%, but on examination of the LLNA dose-response data it seems to us that the true EC3 value should be significantly lower. Table 2 shows the dose response data reported by Niklasson et al.33.

There is an irregularity in the dose response below 1%. The SI value at 0.1% was 1.6, and this value together with the SI value of 15.8 at 1% was used to derive the EC3 value of 0.19% by linear interpolation. However, the SI value at the even lower concentration of 0.01% was 2.7. Using the 0.01% SI value of 2.7 together with the SI value of 15.8 at 1%, linear interpolation gives an EC3 value of 0.033%. This value seems to us more realistic. We investigated other ways of deriving an EC3 value from the dose-response data, with the results shown in Table 3.


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From this analysis, we conclude that an EC3 value in the range 0.01 to 0.06 (corresponding to extreme sensitizer classification) is more plausible than the reported value of 0.19% (corresponding to strong sensitizer classification).44 The mean of these EC3 estimates is shown in Table 1 in brackets. It is clear that, although it is not the most reactive of the epoxides listed in Table 1, and several of the other epoxides have larger calculated RAI values, PhNHCH2-epox is substantially more potent as a sensitizer than any of the other epoxides in Table 1 and will be a positive outlier. At this stage therefore, we omit this compound from the exercise to derive a QMM. Later in the paper we consider the mechanistic basis for the high potency of PhNHCH2-epox. We used the first seven epoxides listed in Table 1, for which we had calculated k values from the 40 minute peptide depletion values given in Niklasson et al.32 For these seven epoxides regression analysis gave:

pEC3 = 2.35 (±0.32) RAI + 3.95 (±0.32)

Equation 1

n = 7, R2 = 0.916, R2(adj) = 0.899, F = 55, s = 0.20 We next used equation 1 to predict the EC3 values for the last three epoxides listed in Table 1. For these three epoxides the rate constants reported in Niklasson et al.33 were used to calculate the RAI values. The predicted EC3 values are shown in the right-hand column of Table 1. For the PhSCH2-epox and PhCH2CH2-epox the predicted EC3 values are in good agreement with the experimental values, but PhNHCH2-epox is much more potent than predicted by equation 1 (this applies with the originally reported EC3 value and even more so with the amended EC3 values based on re-analysis of the dose response). We will consider this compound further in the next section. We next repeated the regression analysis, this time with the inclusion of PhSCH2-epox and PhCH2CH2-epox, to give the QMM:



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pEC3 = 2.42 (±0.26) RAI + 4.04 (±0.25)

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Equation 2

n = 9, R2 = 0.928, R2(adj) = 0.917, F = 90, s = 0.18

Since the epoxides are H-polar SN2 electrophiles (H-polar is a term defined by Leo et al.45 to refer to compounds able to hydrogen bond strongly with water molecules) this QMM should predict the potency of other H-polar SN2 electrophiles, based on their log k and logP values. So far we have only found one non-epoxide H-polar SN2 electrophile with a log k value determined for reaction with a peptide similar to the one that was used for the reactivity data shown in Table 1. This is p-nitrobenzyl bromide, with a log k value of 0.059 (after conversion of the second order rate constant, 0.984 [s-1M-1], given by Natsch et al.46 to the pseudo first order rate constant at the concentration (1 mM) and in the units [min-1] used by Niklasson et al.33. Its predicted logP value is 2.67 and its EC3 value is 0.05%.46 In good agreement with this figure, the QMM derived from the epoxides in Table 1 (i.e. Equation 2) predicts an EC3 value of 0.048% for p-nitrobenzyl bromide. If p-nitrobenzyl bromide is included in the regression the QMM equation changes slightly, but not significantly:

pEC3 = 2.41 (±0.16) RAI + 4.03 (±0.15)

Equation 3

n = 10, R2 = 0.965, R2(adj) = 0.960, F = 220, s = 0.17

Figure 1 shows the plot of pEC3 against RAI with p-nitrobenzyl bromide included.

The high potency of PhNHCH2-epox


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Unlike the other epoxides discussed here, PhNHCH2-epox has an additional structural alert for sensitization. This is the “aliphatic amine” alert. In this usage, “aliphatic” applies to any amine in which there is an aliphatic carbon, also bonded to hydrogen, bonded to amino nitrogen. This sub-structure, amino-N-CH, is an alert for in cutaneo oxidation to the aldehyde or ketone O=C. Many aliphatic amines are skin sensitizers, and the rule (implemented in the TIMES-SS software)47 is to assess aliphatic amines based on the reactivity of the corresponding aldehyde or ketone and the hydrophobicity and molecular weight of the parent amine. For example ethylene diamine, NH2CH2CH2NH2, is a sensitizer and its potency is well modelled by considering it as an in cutaneo source of glyoxal, O=CH-CH=O. In the present case, oxidation of the amine would give glycidaldehyde (Figure 2), which is known to be highly reactive.48 We can make a rough estimate of the SN2 reactivity of glycidaldehyde as discussed below.

All of the epoxides in Table 1 have the general structure R-epox. For SN2 reaction at the ring CH2 group, we assume that relative reactivity is determined by the relative ability of R to stabilize the negative charge on the ring oxygen as its bonding to the CH2 group becomes weaker. This can be modelled by Taft σ* values of the R groups. Using the highest and lowest rate constants of the epoxides for which we have σ* values, we estimate the rate constant for the epoxide with R = CHO based on σ* CHO, assuming a linear correlation between logk and σ*: PhOCH2-epox logk = -1.31, σ* PhOCH2 = 0.87 PhCH2CH2-epox logk = -1.82, σ* PhCH2CH2 = -0.06 ∆(σ*) of 0.93 gives ∆(logk) of 0.51 σ* CHO = 2.15, ∆(σ*) (CHO - PhOCH2) = 2.15 - 0.87 = 1.28



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Calculated logk for CHO-epox = logk(PhOCH2-epox) + 1.28*0.51/0.93 = -0.61. Based on the estimated logk value for CHO-epox and the logP value for PhNHCH2-epox, the RAI value is calculated to be -0.09, from which the EC3 value for PhNHCH2-epox, calculated from equation 2, is 0.02%. Bearing in mind the approximate nature of this calculation and the uncertainties in the derivation of the EC3 value from the dose-response, the agreement between the experimental EC3 and the QMM-estimated EC3 is quite close. Figure 1 shows the plot of pEC3 against RAI with PhNHCH2-epox (reacting as glycidaldehyde) included. Kinetic data on further non-epoxide H-polar SN2 electrophiles with existing LLNA data would be useful to confirm Equation 3 as a QMM for H-polar SN2 electrophiles in general. In the meantime, we interpret equation 3 as evidence that epoxides act mainly as H-polar SN2 electrophiles in skin sensitization and that the for this reaction mechanistic domain SAR principles for reactivity, together with those for hydrophobicity, form the basis for reactivity SAR principles for skin sensitization potency. We now apply the reactivity SAR principles to epoxides that have been studied in the context of pre- and pro-hapten mechanisms for naturally occurring chemicals.

SAR-based interpretation of relative sensitization potency for epoxides derived from autoxidation and/or metabolism of natural products geraniol, geranial and their epoxides Geraniol 1 (Figure 3) is a naturally occurring terpene alcohol which, although not directly reactive, can cause sensitization. Potential activation pathways via abiotic and/or metabolic oxidation have been quite intensively studied.34,28, 49-51 The corresponding aldehyde, geranial


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7 (Figure 3), also naturally occurring, is a direct acting sensitizer as a Michael acceptor, but the evidence does not support oxidation of 1 to 7 as the the main cause of sensitization by 1. In the course of the studies referred to, several epoxides and other derivatives of 1 and 7 have been investigated. These are shown, together with their EC3 values and our mechanistic domain classifications, in Figure 3. The potency trends for the epoxides 2, 3, 6, 8, 9 and 12 (Figure 3) can be explained as follows. In 2, the epoxide carbon atoms are secondary and tertiary so the epoxide has low reactivity (reactivity SAR principle 1). Possibly the weak sensitization results from the weak reactivity of the epoxide, but it is also possible that an impurity (e.g. 8) is responsible. The OH group is not allylic, and therefore an alternative sensitization pathway by oxidation to reactive aldehyde is not available. In 3 the epoxide ring has low reactivity (reactivity SAR principle 1). However, the OH group is allylic so it can be oxidized to 9, which can react as a Michael acceptor, giving rise to the EC3 value of 7% for 3. In 6, the epoxide reactivity is low (reactivity SAR principle 1) and the OH group is not allylic so is not readily oxidized to a reactive aldehyde, so the compound is a non-sensitizer. In 8 the epoxide is heteroallylic, activated by the carbonyl group (C=O), so it is reactive as an SN2 electrophile (reactivity SAR principle 2). In 9, the epoxide ring has low reactivity (reactivity SAR principle 1), but being electronegative it can make the olefinic double bond more reactive as a Michael acceptor. This explains why 9 is more potent than 7. In 12, the epoxide is relatively unreactive (reactivity SAR principle 1), and the observed low skin sensitization potency can be attributed to the C=O group being the reactive site by the Schiff base mechanism.

Cinnamic alcohol, cinnamic aldehyde and their epoxides



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Cinnamic alcohol is a well known contact allergen that is not directly reactive.52,53 Although oxidation to the more potent Michael acceptor allergen cinnamic aldehyde appears an obvious activation mechanism, there is clinical evidence that other mechanisms may be more relevant. The Scheme 2 summarises the findings of Niklasson et al.54 on potential activation pathways. Cinnamic aldehyde can be formed from autoxidation of cinnamyl alcohol and can then be further oxidized to percinnamic acid. Peracids are epoxidising agents and in this case percinnamic acid can react with cinnamyl alcohol or cinnamic aldehyde to give the corresponding epoxides 13 and 14 respectively. Both of these epoxides are benzylic (reactivity SAR principle 2) and in both cases the developing negative charge on the oxygen atom as the ring opens is stabilised – in the case of 14 by the electronegativity of the carbonyl group and in the case of 13 by intramolecular hydrogen bonding (i.e. a neighbouring group effect, reactivity SAR principle 3) as shown in Scheme 3.

Epoxides derived from conjugated dienes Several natural terpenes are known to contain conjugated diene substructures and, although not directly electrophilic, are known to cause sensitization. These have been extensively investigated by the group at Gothenburg University and reactivity SAR principles have been established.34,55 In brief, conjugated dienes in which at least one of the double bonds is cyclic can sensitize via a pro-hapten mechanism involving metabolic activation to allylic epoxides as the ultimate sensitizers. Examples are α-phellandrene, β-phellandrene and α-terpinene. Open chain dienes and cyclic non-conjugated dienes do not give rise to sensitization via metabolism to epoxides. The requirement that the dienes be conjugated may be in part


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because they are more readily oxidized (higher EHOMO value compared to isolated double bonds). The requirement that the conjugated dienes be cyclic may possibly reflect the fact that rotation around the bond connecting the two olefinic units is not possible in a cyclic structure, so that the diene is permanently coplanar with delocalization of the π-electrons. Also, the diene-derived epoxides, being allylic, are more electrophilic than dienes from nonconjugated dienes or mono-olefins, and therefore predicted by equation 2 to be more potent as sensitizers. We note that abietic acid, although usually considered to be a pre-hapten sensitizing via autoxidation to a doubly-allylic hydroperoxide,56 is a cyclic conjugated diene and also meets the criteria for sensitizing as a pro-hapten via metabolic oxidation to an epoxide (Scheme 4).

Conclusions In earlier papers42,57,58 QMMs have been developed for the Schiff base domain,42 the MA domain57 and the SNAr domain.58-60 Another important mechanistic applicability domain is the H-polar SN2 domain. Several sets of closely related chemicals of this type have been successfully modelled for guinea pig data,61 alkane sultones,36 QMMs for other SN2 electrophiles are known both for guinea pig data and to a limited extent for LLNA data.37,38,62,63 We know that for guinea pig, skin sensitization potency of SN2 chemicals depends on a combination of reactivity with hydrophobicity. So far this has not been fully established for LLNA. The alkyl alkanesulphonates studied in Roberts and Basketter63 all had very high logP values (>5) and in this hydrophobicity range the potency was found to depend inversely on hydrophobicity, ascribed to passage through the stratum corneum becoming rate determining. Some evidence for direct dependency on hydrophobicity was obtained from a plot of SI



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values vs RAI for several SN2 electrophiles with logP in the range 5.49 to 8.63, with RAI being based on reactivity (in some cases experimentally measured, in some cases estimated by linear free energy relationship (LFER) methods),39 hydrophobicity and dose. The work of the Karlberg group on, inter alia, epoxides sensitization, has produced valuable qualitative mechanistic and SAR insights, which we have aimed to complement by putting their findings32,33 on a more quantitative basis and thereby increasing their value for quantitative human risk assessment. Specifically, with the publication of the epoxides data32,33 it has now been possible to develop a QMM for the SN2 domain, albeit so far restricted mainly to epoxides. This QMM is based on experimental rate constants as reactivity parameters and on calculated logP values as a hydrophobicity parameter. The observation that the QMM predicts the LLNA potency of an entirely different SN2 electrophile, p-nitrobenzyl bromide, suggests that it could form the basis for a more general H-polar SN2 QMM that could be a valuable tool in skin sensitization risk assessment for this quite extensive and structurally diverse reaction mechanistic domain. Generation of kinetic data on further SN2 electrophiles for which LLNA data are available would be highly desirable for this purpose.

Author Information *Corresponding Author Address: James Parsons Building, Byrom Street, Liverpool, L3 3AF, United Kingdom; Tel: 0151 231 2422; E-mail: [email protected] Funding This work was supported by grant from CEFIC LRI (B14-RIFM: Skin Sensitization – Chemical Applicability Domain of the Local Lymph Node Assay (LLNA) to D.W.R. and A.M.A.).


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Notes The authors declare no competing financial interest.

Abbreviations DPRA, Direct Peptide Reactivity Assay; EC3, an estimated concentration of substance required to induce a three-fold SI value; GPMT, Guinea Pig Maximisation Test; LFER, Linear Free Energy Relationship; LLNA, Local Lymph Node Assay; MA – direct-acting Michael Acceptor; NR – Non-Reactive; pEC3, -log of the molar EC3; Pre-MA, able to be activated by oxidation of the allylic OH group to form a Michael acceptor (α,β-unsaturated aldehyde); QMM, Quantitative Mechanistic Model; RAI, Relative Alkylation Index; SAR, Structure-Activity Relationship; SB, Schiff Base electrophile; SI, Stimulation Index.



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References (1) Jowsey, I. R., Basketter, D. A., Westmoreland, C., and Kimber, I. (2006) A future approach to measuring relative skin sensitizing potency: a proposal. J. Appl. Toxicol. 26, 341–350.

(2) Adler, S., Basketter, D., Creton, S., Pelkonen, O., van Benthem, J., Zuang, V., Andersen, K.E., Angers-Loustau, A., Aptula, A., Bal-Price, A., Benfenati, E., Bernauer, U., Bessems, J., Bois, F.Y., Boobis, A., Brandon, E., Bremer, S., Broschard, T., Casati, S., Coecke, S., Corvi, R., Cronin, M., Daston, G., Dekant, W., Felter, S., Grignard, E., Gundert-Remy, U., Heinonen, T., Kimber, I., Kleinjans, J., Komulainen, H., Kreiling, R., Kreysa, J., Leite, S.B., Loizou, G., Maxwell, G., Mazzatorta, P., Munn, S., Pfuhler, S., Phrakonkham, P., Piersma, A., Poth, A., Prieto, P., Repetto, G., Rogiers, V., Schoeters, G., Schwarz, M., Serafimova, R., Tähti, H., Testai, E., van Delft, J., van Loveren, H., Vinken, M., Worth, A., and Zaldivar, J. M. (2011) Alternative (non-animal) methods for cosmetics testing: current status and future prospects. Arch. Toxicol. 85, 367-485.

(3) Jaworska, J., Dancik, Y., Kern, P., Gerberick, F., and Natsch, A. (2013) Bayesian integrated testing strategy to assess skin sensitization potency: from theory to practice: Integrated testing strategy to assess skin sensitization potency. J. Appl. Toxicol. 33, 13531364.

(4) Teubner, W., Mehling, A., Schuster, P. X., Guth, K., Worth, A., Burton, J., van Ravenzwaay, B., and Landsiedel, R. (2013) Computer models versus reality: How well do in


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silico models currently predict the sensitization potential of a substance. Regul. Toxicol. Pharmacol. 67, 468–485.

(5) Patlewicz, G., Kuseva, C., Kesova, A., Popova, I., Zhechev, T., Pavlov, T., Roberts, D. W., and Mekenyan, O. (2014) Towards AOP application – Implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization. Regul. Toxicol. Pharmacol. 69, 529–545.

(6) Roberts, D.W., Aptula, A.O., Patlewicz, G. and Pease, C. (2008) Chemical reactivity indices and mechanism-based read across for non-animal based assessment of skin sensitization potential, J. Applied Toxicol. 28, 443-454.

(7) Roberts, D.W., Api, A.M., Patlewicz, G., and Schultz, T.W. (2016) Chemical applicability domain of the Local Lymph Node Assay (LLNA) for skin sensitization potency. Part 1. Underlying physical organic chemistry principles and the extent to which they are represented in the LLNA validation dataset. Reg. Toxicol. Pharmacol. 80, 247-254.

(8) Maxwell, G., MacKay, C., Cubberley, R., Davies, M., Gellatly, N., Glavin, S., Gouin, T., Jacquoilleot, S., Moore, C., Pendlington, R., Saib, O., Sheffield, D., Stark, R., and Summerfield, V. (2014) Applying the skin sensitization adverse outcome pathway (AOP) to quantitative risk assessment. Toxicol. in vitro. 28(1), 8-12.



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(9) Roberts, D. W., and Aptula, A. O. (2008) Determinants of skin sensitization potential. J. Appl. Toxicol. 28, 377-387.

(10) Roberts, D. W., and Williams, D. L. (1982) The derivation of quantitative correlations between skin sensitization and physicochemical parameters for alkylating agents and their application to experimental data for sultones. J. Theor. Biol. 99, 807-825.

(11) Aptula, A.O., and Roberts, D.W. (2006) Mechanistic applicability domains for nonanimal-based prediction of toxicological end points: General principles and application to reactive toxicity. Chem. Res. Toxicol. 19(8), 1097-1105.

(12) OECD. 2012. The Adverse Outcome Pathway for skin sensitization initiated by covalent binding

to

proteins.

Part

1.

Series

on

Testing

and

Assessment.

No.

168.

ENV/JM/MONO(2012)10/PART1.

(13) OECD. 2012. The Adverse Outcome Pathway for skin sensitization initiated by covalent binding to proteins. Part 2: Use of the AOP to develop chemical categories and integrated assessment and testing approaches. Series on Testing and Assessment. No. 168. ENV/JM/MONO(2012)/PART2.

(14) Roberts, D. W., Api, A. M., Safford, R. J., and Lalko, J. F. (2015) Principles for identification of high potency category chemicals for which the dermal sensitization threshold (DST) approach should not be applied. Reg. Toxicol. Pharmacol. 72(3), 683-693.


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(15) Basketter, D.A., Alépée, N., Ashikaga, T., Barroso, J., Gilmour, N., Goebel, C., Hibatallah, J., Hoffmann, S., Kern, P., Martinozzi-Teissier, S., Maxwell, G., Reisinger, K., Sakaguchi, H., Schepky, A., Tailhardat, M., and Templier, M. (2014) Categorization of chemicals according to their relative human skin sensitizing potency. Dermatitis 25(1), 1121.

(16) Buehler, E. V. (1965) Delayed contact hypersensitivity in the Guinea pig. Arch. Dermatol. 91, 171-175.

(17) Magnusson, B., and Kligman, A, M. (1969) The identification of contact allergens by animal assay. The guinea pig maximisation test. J. Invest. Dermatol. 52, 268-276.

(18) Gerberick, G. F., Ryan, C. A., Dearman, R. J., and Kimber, I. (2007) Local lymph node assay (LLNA) for detection of sensitization capacity of chemicals. Methods 41, 54-60.

(19) OECD Test Guideline 406. Skin Sensitization. 1992.

(20) OECD Test Guideline 429. Skin Sensitization: Local Lymph Node Assay. 2002.

(21) Gerberick, G.F., Ryan, C.A., Kimber, I., Dearman R.J., Lea, L.J., and Basketter, D.A. (2000) Local lymph node assay: validation assessment for regulatory purposes. Am. J. Contact Dermat. 11(1), 3-18.



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(22) Basketter, D.A., Gerberick, G.F., Kimber, I., and Loveless, S.E. (1996) The local lymph node assay: a viable alternative to currently accepted skin sensitization tests. Food Chem. Toxicol. 34(10), 985-997.

(23) Basketter, D.A., Evans, P., Fielder, R.J., Gerberick, G.F., Dearman, R.J., and Kimber, I. (2002) Local lymph node assay - validation, conduct and use in practice. Food Chem. Toxicol. 40(5), 3-8.

(24) Kimber, I., Dearman, R.J., Scholes, E.W., and Basketter, D.A. (1994) The local lymph node assay: developments and applications. Toxicology 93(1), 13-31.

(25) Basketter, D.A., Blaikie, L., Dearman, R.J., Kimber, I., Ryan, C.A., Gerberick, G.F., Harvey, P., Evans, P., White, I.R., and Rycroft, R.J. (2000) Use of the local lymph node assay for the estimation of relative contact allergenic potency. Contact Dermatitis 42, 344-348.

(26) Basketter, D.A., Smith, C.K., and Patlewicz, G.Y. (2003) Contact allergy: the local lymph node assay for the prediction of hazard and risk. Clin. Exp. Dermatol. 28(2), 8-21.

(27) Basketter, D.A., Lea, L.J., Cooper, K., Stocks, J., Dickens, A., Pate, I., Dearman, R.J., and Kimber, I. (1999) Threshold for classification as a skin sensitizer in the local lymph node assay: a statistical evaluation. Food Chem. Toxicol. 37(12), 67-74.


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(28) Delaine, T., Ponting, D., Niklasson, I., Emter, R., Hagvall, L., Norrby, PO., Natsch, A., Luthman, K., and Karlberg, AT. (2014) Epoxyalcohols: Bioactivation and conjugation required for skin sensitization. Chem. Res. Toxicol. 27, 1860-1870.

(29) Blaschke, U., Paschke, A., Rensch, I., and Schüürmann, G. (2010) Acute and chronic toxicity toward the bacteria Vibrio fischeri of organic narcotics and epoxides: Structural alerts for epoxide excess toxicity. Chem. Res. Toxicol. 23(12),1936-1946.

(30) Schramm, F., Müller, A., Hammer, H., Paschke, A., and Schüürmann, G. (2011) Epoxide and thiirane toxicity in vitro with the ciliates Tetrahymena pyriformis: Structural alerts indicating excess toxicity. Environ. Sci. Technol. 45(13), 5812-5819.

(31) Thaens, D., Heinzelmann, D., Böhme, A., Paschke, A., and Schüürmann, G. (2012) Chemoassay screening of DNA-reactive mutagenicity with 4-(4-Nitrobenzyl) pyridine Application to epoxides, oxetanes, and sulfur heterocycles. Chem. Res. Toxicol. 25(10), 2092-2102.

(32) Niklasson, I., Broo, K., Jonsson, C., Luthman, K., and Karlberg, AT. (2009) Reduced sensitizing capacity of epoxy resin systems: A structure-activity relationship study. Chem. Res. Toxicol. 22, 1787-1794.



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(33) Niklasson, I., Delaine, T., Luthman, K., and Karlberg, AT. (2011) Impact of a heteroatom in a structure-activity relationship study on analogues of Phenyl Glycidyl Ether (PGE) from epoxy resin systems. Chem. Res. Toxicol. 24, 542-548.

(34) Bergstrӧm, M., Luthman, K., Nilsson, J., and Karlberg, AT. (2006) Conjugated dienes as prohaptens in contact allergy: In vivo and in vitro studies of structure-activity relationships, sensitizing capacity, and metabolic activation. Chem. Res. Toxicol. 19, 760-769.

(35) Aptula, A.O., Roberts, D.W., and Pease, C. (2007) Haptens, prohaptens and prehaptens, or electrophiles and proelectrophiles. Contact Dermatitis 56, 64-65.

(36) Roberts, D. W., and Williams, D. L. (1982) The derivation of quantitative correlations between skin sensitization and physicochemical parameters for alkylating agents and their application to experimental data for sultones. J. Theor. Biol. 99, 807-825.

(37) Roberts, D.W., and Basketter, D. (1990) A quantitative structure-activity/dose relationship for contact allergenic potential of alkyl group transfer agents. Toxicol. In Vitro, 4(4/5), 686-687.

(38) Roberts, D. W., and Basketter, D. A. (1990) A quantitative structure activity/dose response relationship for contact allergic potential of alkyl group transfer agents. Contact Dermatitis 23, 331-335.


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(39) Roberts, D.W., Aptula, A.O., and Patlewicz, G. (2007) Electrophilic chemistry related to skin sensitization. Reaction mechanistic applicability domain classification for a published data set of 106 chemicals tested in the mouse local lymph node assay. Chem. Res. Toxicol. 20(1), 44-60.

(40) Roberts, D.W., and Williams, D.L. (1982) The derivation of quantitative correlations between skin sensitization and physicochemical parameters for alkylating agents and their application to experimental data for sultones. J. Theor. Biol. 99, 807-825.

(41) Roberts, D.W., Aptula, A.O. and Patlewicz, G. (2006) Mechanistic applicability domains for non-animal based prediction of toxicological endpoints. QSAR analysis of the Schiff Base applicability domain for skin sensitization. Chem. Res. Toxicol. 19(9), 12281233.

(42) Basketter, D. and Roberts, D.W. (1990) Structure/activity relationships in contact allergy. Intern. J. Cosm. Sci. 12, 81-90.

(43) Roberts, D.W. (2015) Estimating skin sensitization potency from a single dose LLNA. Reg. Tox. Pharmacol. 71, 437–443.

(44) Kimber, I., Basketter, D., Butler, M., Gamer, A., Garigue, J.-L., Newsome, C., Steiling, W., and Vohr, H.-W. (2003) Classification of contact allergens according to potency: Proposals. Food Chem. Toxicol. 41,1799-1809.



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(45) Hansch, C., and Leo, A. J. (1979) Substituent Constants for Correlation Analysis in Chemistry and Biology, Wiley and Sons, New York.

(46) Natsch, A., Haupt, T., and Laue, H. (2011) Relating skin sensitizing potency to chemical reactivity: Reactive Michael acceptors inhibit NF-jB signaling and are less sensitizing than SNAr and SN2-reactive chemicals. Chem. Res. Toxicol. 24, 2018-2027.

(47) Patlewicz, G., Dimitrov, S.D., Low, L.K., Kern, P.S., Dimitrova, G.D., Comber, M.I.H., Aptula, A.O., Phillips, R.D., Niemelä, J., Madsen, C., Wedebye, E.B., Roberts, D.W., Bailey, P.T., and Mekenyan, O.G. (2007) TIMES-SS-A promising tool for the assessment of skin sensitization hazard. A characterization with respect to the OECD validation principles for (Q)SARs and an external evaluation for predictivity. Reg. Tox. Pharmacol. 48(2), 225-239.

(48) Williams, P.H., Payne, G.B., Sullivan, W.T., and VanEss, P.R. (1960) Chemistry of glycidaldehyde. J. Am. Chem. Soc. 82, 4883-4888.

(49) Hagvall, L., Bäcktorp, C., Svensson, S., Nyman, G., Bӧrje, A., and Karlberg, AT. (2007) Fragrance compound geraniol forms contact allergens on air exposure. Identification and quantification of oxidation products and effect on skin sensitization. Chem. Res. Toxicol. 20, 807-814.

(50) Hagvall, L., Baron, J. M., Bӧrje, A., Weidolf, L., Merk, H., and Karlberg, AT. (2008) Cytochrome P450-mediated activation of the fragrance compound geraniol forms potent contact allergens. Toxicol. Appl. Pharmacol. 233, 308-313.


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(51) Rudbäck, J., Bergstrӧm, M. A., Bӧrje, A., Nilsson, U., and Karlberg, AT. (2012) αTerpinene, an antioxidant in tea tree oil, autoxidizes rapidly to skin allergens on air exposure. Chem. Res. Toxicol. 25, 713-721.

(52) Cheung, C., Hotchkiss, S. A. M., and Pease, C. K. S. (2003) Cinnamic compound metabolism in human skin and the role metabolism may play in determining relative sensitization potency. J. Dermatol. Sci. 31, 9-19.

(53) Niklasson, I. B., Delaine, T., Islam, M. N., Karlsson, R., Luthman, K., and Karlberg, AT. (2013) Cinnamyl alcohol oxidizes rapidly upon air exposure. Contact Dermatitis 68, 129138.

(54) Niklasson, I. B., Ponting, D. J., Luthman, K., and Karlberg, AT. (2014) Bioactivation of cinnamic alcohol forms several strong skin sensitizers. Chem. Res. Toxicol. 27, 568-575.

(55) Nilsson, A.M., Bergstrӧm, M., Luthman, K., Nilsson, J., and Karlberg, AT. (2005) A conjugated diene identified as a prohapten: Contact allergenic activity and chemical reactivity of proposed epoxide metabolites. Chem. Res. Toxicol. 18, 308-316.

(56) Urbisch, D., Honarvar, N., Kolle, S. N., Mehling, A., Ramirez, T., Teubner, W., and Landsiedel, R. (2016) Peptide reactivity associated with skin sensitization: The QSAR Toolbox and TIMES compared to the DPRA. Toxicol. in Vitro 34, 194-203.



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(57) Roberts, D.W., and Natsch, A. (2009) High throughput kinetic profiling approach for covalent binding to peptides: Application to skin sensitization potency of Michael acceptor electrophiles. Chem. Res. Toxicol. 22, 592-603.

(58) Roberts, D.W., and Aptula, A.O. (2014) Electrophilic reactivity and skin sensitization potency of SNAr electrophiles. Chem. Res. Toxicol. 27, 240-246.

(59) Roberts, D. W. (1995) Linear free energy relationships for reactions of electrophilic halo- and pseudohalobenzenes, and their application in prediction of skin sensitization potential for SNAr ectrophiles. Chem. Res. Toxicol. 8, 545-551. (60) Roberts, D., Aptula, A. O., and Patlewicz, G. (2011) Chemistry-based risk assessment for skin sensitization: Quantitative mechanistic modeling for the SNAr domain. Chem. Res. Toxicol. 24, 1003-1011.

(61) Roberts, D. W., Goodwin, B. F. J., Williams, D. L., Jones, K., Johnson, A. W., and Alderson, J. C. E. (1983) Correlations between skin sensitization potential and chemical reactivity for p-nitrobenzyl compounds. Food Chem. Tox. 21(6), 811-813.

(62) Roberts, D. W., and Basketter, D. A. (1997) Further evaluation of the quantitative structure-activity relationship for skin-sensitizing alkyl transfer agents. Contact Dermatitis 37(3), 107-112.


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(63) Roberts, D. W., and Basketter, D. A. (2000) Quantitative structure-activity relationships: Sulfonate esters in the local lymph node assay. Contact Dermatitis 42(3), 154-161.

Table 1. LLNA potency (EC3), reactivity (log k) and hydrophobicity (logP) data for primary epoxides Chemical

EC3calc (Eq. 1)

log k

logP

RAI

EC3, %

PhOCH2-

-1.31

1.62

-0.66

0.46

PhCH2OCH2-

-1.54

1.64

-0.88

2.5

PhOCH2CH2-

-1.62

1.97

-0.83

2.2

cyclohexylOCH2-

-1.62

1.69

-0.94

5.2

PhCH2CH2OCH2-

-1.62

1.75

-0.92

1.5

BuOCH2-

-1.85

1.24

-1.36

28

CH3CH=CHCH2OCH2-

-1.72

0.96

-1.34

14

PhSCH2-*

-1.54

2.12

-0.69

0.58

0.78

PhCH2CH2-*

-1.82

2.19

-0.95

2.3

2.80

R O R=



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

PhNHCH2-*

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0.19 -1.41

1.30

-0.89

2.05

(0.035)**

*rate constants k taken from.33 For the rest, logk is calculated from 40 min peptide depletion values given in.32 Log k based on pseudo first order rate constants in [min-1]. RAI = logk + 0.4 logP. Vehicle used in the LLNA test was Acetone Olive Oil (AOO).32,33 **mean of EC3 values derived from alternative dose-response analyses (see text)

Table 2. Dose response data33 for PhNHCH2-epox Concentration, C (%)

Log C

Stimulation index, SI

0.01

-2

2.7

0.1

-1

1.6

1

0

15.8

10

1

41.8

20

1.3

47.0


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Table 3. Estimation of EC3 for PhNHCH2-epox based on the data in Table 2 Basis of estimate

EC3 (%)

Linear interpolation between (0.01%, 2.7) and (1%, 15.8)

0.033

Logarithmic interpolation between (0.01%, 2.7) and (1%, 15.8)

0.011

Linear regression, all points, of SI vs log C

0.038

Linear regression of probit function* vs log C, all points

0.011

Estimation by rLLNA formula* from SI at: 0.01% 0.012 1% 0.059 10% 0.050 20% 0.064 Mean 0.035 *The probit formula and its use in estimating the EC3 value from the SI at a single dose, are explained in Roberts.43 It is not recommended for use with SI values below 1.8.



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Figure Legends

Figure 1. Graphical respresentation of QMM for skin sensitizing epoxides.

Figure 2. Activation of PhNHCH2-epox to glycidaldehyde.

Figure 3. Geraniol, geranial, and their epoxides investigated in Bergstrӧm et al.34 Pre-MA – able to be activated by oxidation of the allylic OH group to form a Michael acceptor (α,βunsaturated aldehyde); NR – non-reactive; MA – direct-acting Michael acceptor; SB – Schiff base electrophile.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


O OH weak-SN2

OH Pre-MA, 7 1 EC3

2 22%

OH Pre-MA, 10

OH Pre-MA, 9 O

3 57%

4

7%

15% O

OH

OH NR 5

6

NR O

EC3

1.5%

2%

7%

O

MA

MA O

8

O

O 9

7

NS

NS

EC3

O Reactive SN2

O MA

SB

SB

10

12

11 4.5%

60%

Figure 3.


O 57%

O


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme Legends Scheme 1. General reaction of epoxides with a nucleophile

Scheme 2. Abiotic and metabolic transformations of Cinnamic alcohol

Scheme 3. Neighbouring group effect in reactivity of epoxide 13

Scheme 4. Abiotic and metabolic transformations of Abietic acid


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Overall reaction facilitated by: electronegative substituents at position b, stabilising developing negative charge on O-atom absence of substituents at position a, minimizing steric impedance of incoming Nu π-bonded substituents at position a, delocalising transition state electrons neighbouring group effects such as nucleophilic N or S centres at c, H-bond donors at a or c

Scheme 1



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

EC3 0.76%

Cinnamic alcohol Ph

OH

Page 38 of 40

Autoxidation

Ph

O

Metabolism

Autox OOH

Metabolism

Ph O Percinnamic acid

Ph EC3 0.58%

OH O

13

OH

+ Ph O Cinanmic aldehyde EC3 0.76%

Ph

O

Cinnamic acid

Scheme 2.


Ph O O 14 EC3 2.2%

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 3.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Doubly allylic hydroperoxide

Abietic acid

Autoxidation

H HO

Page 40 of 40

HO

H

OOH

H

O

H O

Metabolic epoxidtion

O H HO

Allylic epoxides

H

HO

H

H O

O Scheme 4.


O

StructureâPotency Relationships for Epoxides in Allergic Contact

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