Mechanistic Applicability Domains for Nonanimal-Based Prediction of

Chemistry-Based Risk Assessment for Skin Sensitization: Quantitative Mechanistic Modeling for the SNAr Domain. D. W. Roberts , A. O. Aptula , and G. Y...
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Chem. Res. Toxicol. 2006, 19, 1097-1105

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Mechanistic Applicability Domains for Nonanimal-Based Prediction of Toxicological End Points: General Principles and Application to Reactive Toxicity Aynur O. Aptula*,† and David W. Roberts‡ SEAC, UnileVer Colworth, Sharnbrook, Bedford, MK44 1LQ, England, and School of Pharmacy and Chemistry, LiVerpool John Moores UniVersity, Byrom Street, LiVerpool, L3 3AF, England ReceiVed May 10, 2006

In light of new legislation (e.g., the REACH program in the European Union), several initiatives have recently emerged to increase acceptance of (quantitative) structure-activity relationships [(Q)SARs] to reduce reliance on animal (in vivo) testing. Among the principles for assessing the validity of (Q)SARs is the need for a defined domain of applicability, i.e., identification of the range of compounds for which the (Q)SAR can confidently be applied for purposes of toxicity prediction. Here, we attempt to develop a “natural” classification into applicability domains based on considering how a compound and the target organism between them “decide” on the nature and extent of the toxic effect. With particular emphasis on reactive toxicity, we present rules, based on organic reaction mechanistic principles, for classifying reactive toxicants into their appropriate mechanistic applicability domains. Introduction Under the European Union (EU) Registration, Evaluation and Authorization of CHemicals (REACH) program, all chemicals produced or imported >1 ton per annum (tpa) in the EU will need to be assessed for human and environmental hazards. If conducted by means of the present data requirements/test strategy, this assessment will use a huge number of test animals and will be neither resource nor time effective. The REACH proposal calls for an increased use of (quantitative) structureactivity relationships [(Q)SARs] and other alternatives for the assessment of, in particular, the lower production volume chemicals, i.e., the categories 1-10 and 10-100 tpa. Although the new and current EU legislation both provide the possibility of using (Q)SARs instead of in vivo testing, there are neither accepted (Q)SARs for human toxicological end points nor detailed guidance on how to develop (Q)SARs for human risk assessment. As a consequence, regulators have scarcely used human toxicological (Q)SARs in decision making. However, as noted, the recently proposed EU REACH program calls for an increased use of (Q)SARs and other nonanimal methods, especially for the assessment of the low production volume chemicals. Therefore, 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 have since been evaluated by OECD [as part of the ad hoc expert group for (Q)SARs]. These are now referred to as the “OECD principles”, which read as follows: “To facilitate the consideration of a (Q)SAR model for regulatory purposes, it should be associated with the following information: 1. a defined endpoint * To whom correspondence should be addressed. Tel: + 44 1234 264823. Fax: + 44 1234 264722. E-mail: [email protected]. † Unilever Colworth. ‡ Liverpool John Moores University.

2. an unambiguous algorithm 3. a defined domain of applicability 4. appropriate measures of goodness-of-fit, robustness and predictivity 5. a mechanistic interpretation, if possible (Q)SARs that fulfill these criteria may, in principle, be applicable within the regulation practice to predict mammalian endpoints.” Further details on these principles and individual case studies are provided by the OECD (1). Principle 3 expresses the need to define an applicability domain for (Q)SARs i.e., the range of compounds for which the (Q)SAR can confidently be applied for purposes of toxicity prediction. Various approaches have been proposed to address the problem of applicability domain definition (2). In the (Q)SAR literature, it is not always apparent whether (or to what extent) the applicability domain concept had been applied. In some cases, the applicability domain concept is implicit in the original publication; for example, the model may have been developed from a training set of closely related (as defined by the authors) compounds. In other cases, the applicability domain may be defined in terms of structural rules and/or a range of parameter values. Here, we attempt to develop a “natural” classification into applicability domains based on considering how a compound and the target organism between them “decide” on the nature and extent of the toxic effect. We present rules, based on organic reaction mechanistic principles, enabling reactive toxicants to be classified into their appropriate mechanistic applicability domains. It is our aim that these rules should facilitate the development of mechanism-based QSARs applicable to broader ranges of compounds than hitherto.

Mechanism and Mode of Action First, we need to define the terms mechanism of action and mode of action. These terms are used widely in the toxicological and (Q)SAR literature (e.g., 3, 4) and not always with the same meanings. Our definitions are as shown and illustrated in Table 1.

10.1021/tx0601004 CCC: $33.50 © 2006 American Chemical Society Published on Web 07/29/2006

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Aptula and Roberts

Table 1. Mechanism and Mode of Action: Definitions examples Chemical mechanism of action Biological mechanism of action Mode of action

definition

skin sensitization

aquatic toxicity

What the toxic chemical and the biological chemicals do together in vivo Events at cellular or subcellular level

Electrophiles react with nucleophilic groups on proteins, e.g., formation of Michael adducts Antigenic modified proteins expressed by Langerhans cells, then T-cell proliferation in lymph nodes Becomes sensitized

Polar narcosis: 2D solvation of chemical in membrane Polar narcosis: reversible (short-term) perturbation of membrane function Hyperactivity, then narcosis

What happens to the organism

In deciding the nature and magnitude of the toxic effect, (i) the compound and the target organism together obey the laws of chemistry (including those that we may not know about). (ii) The laws of chemistry determine the chemical mechanism of action. (iii) The chemical mechanism of action determines the biological mechanism of action. (iv) The biological mechanism of action determines the mode of action. The implications are that (i) the mode of action is downstream of the chemicobiological interaction. (ii) To simulate how the compound and the target organism between them “decide” on the nature and extent of the toxic effect, QSARs should be based on chemical mechanisms of action. (iii) The effectiveness of this approach depends critically on how well the chemistry can be characterized, either from structure or from experimental studies. Characterization of the chemistry may involve experiments with nucleophiles to determine the nature of the reactions; measurement of rate constants; use of LFER parameters, e.g., Hammett, Taft σ, σ*, etc., to estimate reactivity; and use of molecular orbital parameters to estimate reactivity. These activities are listed in decreasing order of reliability and of time and cost. The distinction between mode and mechanism is further illustrated by two toxic end points, skin sensitization and acute aquatic toxicity (e.g., to fish). In skin sensitization, electrophiles react with nucleophilic groups on proteins, forming antigenic modified proteins, which are expressed by Langerhans cells, leading to T-cell proliferation in lymph nodes resulting in the development of the sensitized state. Thus, skin sensitization is a single mode of action phenomenon but with several mechanisms of action according to how the electrophiles bind to protein. In contrast, there are several modes of action for aquatic toxicity, e.g., to fish. For example, general narcosis is where the fish become lethargic and then comatose (up to this point reversibly) before death; polar narcosis is where the fish become hyperactive before the onset of narcosis (up to this point reversibly), followed by death; and reactive toxicity is where the fish become hyperactive, followed by death. For the general and polar narcosis modes of action, the mechanism is physical chemistry-based and considered to involve nonspecific binding of the toxicant in membranes, although the exact site of binding has not been determined. For the reactive toxicity mode of action, the mechanism is reaction chemistry-based, involving covalent modification of proteins. Our approach to applicability domain definition is based on the fundamental premise that the physicochemical properties of compounds are causally linked to their toxic properties. In particular, for any given toxic end point, differences in toxicity of different compounds are related only to differences in their physicochemical properties. Here, we apply this approach to electrophilic toxicity. There are various toxic end points dependent on electrophilic reactivity (e.g., skin sensitization, mutagenicity, respiratory allergy, and

the reactive toxicity mode of action in fish and other aquatic organisms). The reaction chemistry principles discussed below are applicable to electrophilic toxicity in general. However, we illustrate the principles by specific reference mainly to skin sensitization but also to aquatic reactive toxicity. Most of the organic chemistry presented here is well-established and can be found in physical organic chemistry textbooks. Although some excellent overviews of electrophilic reactions in the context of aquatic toxicity are available (5-7), the mechanistic chemistry has not previously been put together in a classified way as an overview of electrophilicity from the toxicological perspective. Skin sensitization and aquatic reactive toxicity are very different end points, and the biological mechanisms downstream of the chemical reaction of the toxicant are completely different. However, the protein binding reaction mechanisms are the same, and consequently, dependence on electrophilic reactivity is found for both end points. Mathematical models have been derived for both end points, forming the basis for mechanism-based QSARs (8, 9). A major difference is that for skin sensitization hydrophobicity is also an important parameter [according to the RAI model (8), this can be attributed to the protein binding reaction taking place in a lipid medium] whereas for aquatic reactive toxicity hydrophobicity has little or no influence on toxicity [attributed to the protein binding reaction taking place mainly in an aqueous environment (9)]. However, nonreactive compounds do exhibit aquatic toxicity by general or polar narcosis, for which toxicity is highly correlated with hydrophobicity. Some compounds, although reactive, nevertheless act by a narcosis mechanism: This is because their narcotic toxicity (correlated with hydrophobicity) is greater than their reactive toxicity (correlated with reactivity). There is no equivalent phenomenon in skin sensitization. Examples are primary alkyl halides, which are SN2 electrophiles and act as skin sensitizers (10) but which in aquatic toxicity act as general narcotics (6). Hydrophobicity is the determining parameter for narcotic toxicity but is only weakly, if at all, correlated with reactive aquatic toxicity. Consequently, in an ascending homologous series of reactive compounds, there is a change from reactive to narcotic toxicity on reaching the log P value above which narcotic toxicity is greater than reactive toxicity. The critical log P value varies between different homologous series, being lower for less reactive compounds and higher for more reactive compounds (9). An example is provided by a series of acetylenic alcohols (11) (these can act as reactive toxicants via oxidation to electrophilic acetylenic aldehydes and ketones). At this point, it is appropriate to briefly summarize the nature of skin sensitization and aquatic toxicity data and how these are expressed quantitatively. The mouse local lymph node assay (LLNA) is nowadays the major source of skin sensitization data. In the LLNA, a single dose of the test compound is given, by application of a solution to the skin of the ear. Lymph node

Nonanimal-Based Prediction of Toxicological End Points

uptake of tritiated thymidine, which is an indicator of T-cell proliferation, is measured, and this is effectively the indicator for the sensitization process. The response is recorded as a stimulation index (SI), this being the ratio of tritiated thymidine uptake in treated animals to uptake in control animals. The assay is usually carried out over a range of dosages of test compound, and from dose-response analysis, it is usually possible to derive an EC3 value, this being the dose (expressed as percent concentration by weight) giving SI ) 3. Aquatic toxicity is usually quantified by an EC50 value, this being the concentration of test compound (usually in weight/ volume units for regulatory and risk assessment purposes or in logarithmic molar units for QSAR analysis) that produces the toxic end point (e.g., immobility for test with Daphnia magna, death for fish toxicity tests, etc.) in 50% of the test organisms.

Chem. Res. Toxicol., Vol. 19, No. 8, 2006 1099 Scheme 1. Reaction Mechanistic Applicability Domains

Protein Binding Reaction Mechanisms for Skin Sensitization Skin sensitization is an important end point. It can be a major problem for workers in many industries. It is an effect for which no threshold can yet be established, and it is a lifelong effect. In REACH, the sensitizing potential should therefore be assessed for chemicals below the 10 tpa threshold (Annex V). No in vitro alternative is yet available, nor will it be in near future. According to a European Chemicals Bureau (ECB) assessment of additional testing needs under REACH, the highest number of tests is required for this end point (EC 2003) (12). Skin sensitization to chemicals, in most if not all cases, involves the compound, either as such or after metabolic or abiotic conversion, acting as an electrophile toward nucleophilic groups on skin protein, leading to formation of antigens (13). The biological processes downstream of this reaction will not be discussed here. Important as they clearly are to the induction and elicitation of sensitization, they have no relevance to the question of what makes some compounds strong sensitizers, other compounds weaker sensitizers, and others nonsensitizers. Skin sensitizers fall naturally into several reaction mechanistic domains, the major ones of which are summarized as follows and illustrated in Scheme 1: Michael acceptor, SNAr, SN2, Schiff base, acyl transfer, and nonreactive and nonproreactive. These reaction mechanistic domains, discussed in more detail below, are also the major ones encountered in reactive toxicity to aquatic organisms.

Michael Acceptors The basic criteria for a compound to be a Michael acceptor are summarized in Scheme 1. With regard to the activating group X, the effectiveness is related to the ability of X to stabilize a negative charge on the carbon atom to which it is bound. Substituents on the R- and β-carbon atoms can have strong influences on Michael reactivity. Electron-donating substituents such as methyl groups reduce reactivityshence, methacrylates are much weaker sensitizers, if sensitizers at all, than acrylates. This is not a steric effect; it is an electronic effect. Electronattracting substituents increase reactivity. There is a need for comparable reactivity data on Michael acceptors covering a good range of X groups, in order to build good predictive models for reactivity based on parameters such as Hammett/Taft substituent constants or molecular orbital energies. Although so far only for small groups of compounds, some progress has been made in this direction (14-16). R-Methylene-γ,γ-dimethyl-γ-butyrolactone is an interesting and illustrative case. Superficially, it looks similar to a meth-

Scheme 2. Michael Acceptors, r-Methylene-γ,γ-dimethyl-γ-butyrolactone and Methyl Methacrylate

acrylate ester (Scheme 2), but guinea pig data indicate that it is a much stronger sensitizer than the methacrylates (17-19). A more quantitative comparison can be made based on results from the LLNA (20): R-Methylene-γ,γ-dimethyl-γ-butyrolactone (log P ) 1.42) has an EC3 value of 1.8%, whereas ethylene glycol dimethacrylate, which has a similar log P value (1.38), has an EC3 value of 28%. 2-Hydroxypropyl methacrylate, with a slightly lower log P value (1.03), is a nonsensitizer [tested up to 50% in the LLNA (20)]. A key difference is that in a methacrylate the R-substituent is an electron-donating (deactivating) methyl group whereas in the lactone the R-substituent is a β-acyloxyethyl group whose electronic effect is similar to that of hydrogen. Another feature that makes R-methylene-γ-butyrolactones more reactive is the release of bond angle strain1 in the transition state. A large group of phytochemical skin sensitizers, the sesquiterpene lactones, 1 It is appreciated that the concepts of bond angle strain and ring strain are metaphors not to be taken literally. The quantum mechanics interpretation is in terms of effectiveness of orbital overlap between bonded atoms.

1100 Chem. Res. Toxicol., Vol. 19, No. 8, 2006 Scheme 3. Pro-Michael Electrophiles, Activated by Oxidation

Aptula and Roberts Scheme 4. SNAr Reaction Illustrated by 2,4-Dinitrohalobenzenes, Well-Known Sensitizers

as leaving groups. This is because in most cases the ratedetermining step is formation of an intermediate anion (Scheme 4), and the rate is independent of the nature of the leaving group. SNAr electrophiles are reactive toward both hard and soft nucleophiles (23, 26).

SN2 Electrophiles

have an R-methylene-γ-butyrolactone entity as part of a fused ring alicyclic system (21). There is evidence that many proelectrophile sensitizers are activated by conversion to highly reactive Michael acceptors, in particular quinones and quinone methides: Some examples are shown in Scheme 3. Urushiol, the allergenic component of poison ivy and poison oak sap, is a mixture of long chain 3-alkenyl catechols, which are thought to sensitize via oxidation (which may well be abiotic; these compounds are readily oxidized on exposure to air) to Michael electrophilic 3-alkenyl ortho-quinones (21). Similarly, lower molecular weight aromatic compounds with two or more hydroxyl groups ortho or para to each other exhibit reactive toxicity, which can be modeled with electrophilicity parameters calculated for the corresponding quinones or quinone methides, to aquatic organisms (22). A complicating feature with Michael acceptors is that the Michael addition is reversible, and in some cases, the reverse reaction can influence the observed kinetics. Michael acceptors are soft electrophiles. However, this does not mean that they are unreactive toward hard nucleophiles. In fact, in many cases, the strongly basic hard nucleophilic methoxide ion is more reactive than the weakly basic soft nucleophilic thiolate ion. Its basicity advantage outweighs the thiolate’s softness advantage.

SNAr Electrophiles These are usually quite easy to identify from structure (23 and references therein). Two or more activating groups (the Y’s of Scheme 1) in ortho or para positions to the leaving group X are needed. A nitrogen atom in the ring can serve as a Y group. For example, 2,4-dichloropyrimidine is an SNAr skin sensitizer (24, 25). A wider range of substituents can serve as leaving groups X for the SNAr reaction than for the SN2 reaction. For the SNAr reaction, groups such as SO2R (leaves as sulfinate ion), SO3Na (leaves as sulfite ion), and NO2 (leaves as nitrite ion) can act

SN2 electrophiles are also usually easy to identify from structure. Primary alkyl groups bonded to leaving groups such as halide, OSO2R, and OSO2OR are usually easily attacked by nucleophiles in SN2 reactions. The SN2 reaction goes more readily at a methyl group than at a higher alkyl group (27). Benzylic and allylic groups bonded to leaving groups are more susceptible than alkyl groups to SN2 reactions. When the leaving group is bonded to a secondary alkyl group, in most cases, the compound is not reactive enough to sensitize. However, this does not always apply, particularly when the secondary carbon atom is part of a ring system: Alkane γ- and δ-sultones have been shown to be moderate sensitizers in guinea pig tests (8, 28) (Scheme 5). Carboxylate anions are usually not good enough leaving groups for alkyl esters to act as SN2 electrophiles. Thus, methyl acetate and methyl benzoate do not act as SN2 electrophiles and do not sensitize. β-Propiolactone is a rare example of an alkyl ester (in this case alicyclic) that reacts as an SN2 electrophile. Presumably, it is activated by the ring strain that is relieved in the transition state. A halogen substituent β to the leaving group is strongly deactivating, due to adverse nonbonding interaction between the lone pairs of the halogen and the incoming nucleophile. Thus, 1,2-dichloroethane does not react easily with nucleophiles. An alkoxy group in the β-position is similarly deactivating. However, sulfur and nitrogen in the β-position (β-halosulfides and β-haloamines are known as mustards and nitrogen mustards) are activating (27). There is some evidence, although not conclusive, that positive nitrogen groups (e.g., NMe3+) are good enough leaving groups for some quaternary salts to act as sensitizers by the SN2 mechanism. The SN2 reaction can also occur at sulfur, as illustrated by the sensitizers 5-chloro-2-methylisothiazol-3-one and 2-methylisothiazol-3-one, as shown in Scheme 5. These two compounds are the major constituents of the commercial biocide Kathon CG (29, 30). SN2 electrophiles span a wide range of the hard-soft spectrum. Methyl diazonium ion, derived from N-nitroso compounds (more familiar in mutagenicity but also encountered in skin sensitization), is a very hard oxophilic SN2 electrophile. In the carcinogenicity literature, it is often depicted as reacting via an SN1 pathway involving the methyl carbonium ion, but there is no real evidence for CH3+ and its involvement in methylation of biological oxygen nucleophiles by CH3N2+ is chemically very implausible. The diazonium ion CH3N2+ is now widely considered as the alkylating species (31). Toward the other end of the spectrum, the sensitizer p-nitrobenzyl iodide

Nonanimal-Based Prediction of Toxicological End Points Scheme 5. SN2 Electrophiles

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Nature and Location of the Nucleophilic Protein(s) Involved in Skin Sensitization There is controversy but very little firm evidence on this subject. One view is that any protein that the sensitizer encounters in the epidermis or the stratum corneum can serve in the formation of the antigen and that water soluble proteins such as human serum albumin are good models for the proteinbinding reaction. Another view is that the proteins involved are membrane bound and have been “designed” by natural selection to be specially reactive toward electrophiles. Whether the protein-binding reaction takes place in an aqueous environment (e.g., lymphatic fluid) or a more hydrophobic environment (e.g., Langerhans cell membrane) is an important issue in view of the magnitude of medium effects.

Medium Effects

is a soft electrophile. Dialkyl sulfates and alkyl alkanesulfonates are intermediate on the hard-soft spectrum (32).

Schiff Base Formers There is not much to add to what is summarized in Scheme 1 about Schiff base formers. It is not definitively established that sensitization to carbonyl compounds is via Schiff base formation, but the Schiff base model provides the best current rationale for the observed patterns of sensitization/nonsensitization, and mechanism-based QSARs based on this assumption have been developed (16, 33).

Acylating Agents Electrophilic esters, which may be carboxylates R1CO‚OR2 or carbonates R1OCO‚OR2, can be attacked by a nucleophile (e.g., lysine unit of protein) to form a tetrahedral anionic intermediate, R1[O]C(Nu)(O-)OR2, which can lose the R2Oion to give R1[O]CONu (further reactions may then be possible with the carbonate derivatives). Simple alkyl esters, whether carbonates or carboxylates, are not electrophilic because the RO- anion is not easily displaced from the tetrahedral intermediate; the conjugate acid ROH is not sufficiently acidic. Phenols are sufficiently acidic, and numerous cases of skin sensitizing phenolic esters are known (34). Anhydrides RCO‚ O‚COR and acyl halides RCO‚X are particularly reactive acylating electrophiles. Acylating agents are considered to be hard electrophiles.

The rates of many organic reactions, including those between electrophiles and nucleophiles, are significantly influenced by the polarity of the reaction medium. The principle is succinctly expressed in the Hughes-Ingold rule (35), published in 1935 (36): “...an increase in the ion-solvating power of the medium will accelerate the creation and concentration of charges and inhibit their destruction and diffusion.” For example, a reaction of an uncharged SN2 electrophile, such as an alkyl halide, with an uncharged nucelophile, such as an amine, proceeds via a transition state in which a partial positive charge has developed on the nucleophile and a partial negative charge has developed on the leaving group. This transition state is stabilized relative to the reactants more by a polar medium than by a nonpolar medium. If the nucleophile has a negative charge, for example, a thiolate ion, the transition state has part of the negative charge associated with the nucleophile and part with the leaving group, i.e., the charge has become more diffuse than in the reactants. Hence, this transition state is stabilized relative to the reactants more by a nonpolar medium than by a polar solvent. Such effects can be quite large. For example, propane sultone reacts readily and exothermically with tertiary amines RNMe2 (Scheme 5) at room temperature in ethyl acetate but the reaction fails to occur in refluxing toluene (bp 110 °C) (37). It is important to bear medium effects in mind when considering whether reactions of electrophiles in vivo occur in membranes or in aqueous media, whether they react with charged or uncharged nucleophiles, and how to best model their reactivity with in vitro kinetic data. This is made difficult by the absence of clear evidence as to the nature of the medium in which the protein-binding reaction leading eventually to sensitization takes place and the uncertainty as to the nature of the protein nucleophiles involved. Our approach is as follows. Medium Effects. Within a mechanistic applicability domain, a QSAR based on a reactivity parameter (usually in combination with a hydrophobicity parameter) should be applicable, irrespective of the medium in which the reactivity parameter is determined, to compounds, which, with a given nucleophile, undergo similar changes in polarity on going to the transition state. This is based on the premise that, the foregoing condition being met, relative rates in one solvent system can be modeled by relative rates in another solvent system. Where not all compounds undergo similar changes in polarity on going to the transition state, a single QSAR is unlikely to be applicable. Nature of the Nucleophile. Not knowing the nature of the nucleophile involved in cutaneo is not usually a major problem. We arrive at this viewpoint from consideration of the SwainScott relationship (38), according to which the reaction rate k

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Table 2. Swain-Scott Parameters for Some Nucleophiles (39) and Electrophiles (38) nucleophile

n

electrophile

sa

aniline n-butylamine HScysteine cysteine ethyl ester serum albumin

4.49 5.13 5.08 5.08 5.24 5.37

ethyl tosylate benzyl chloride methyl bromide

0.66 0.87 1.00

a

Scheme 6. Poison Ivy/r-Methylene-γ-butyrolactone Bifunctional Sensitizer

s values much larger than 1.00 are not usually encountered (38).

depends on the nucleophilicity (n) of the nucleophile (Nu) and on the sensitivity (s) of the electrophile toward Nu.

log k ) ns + log k0

(1)

k0 is the rate constant with a standard nucleophile arbitrarily assigned an n value of zero. Scales of n have been published for water and for methanol as the reference nucleophiles. Suppose the reactivity parameter, expressed as a log k value, is based on a model nucleophile whose nucleophilicity differs from that of the biological nucleophile by ∆n. Suppose the electrophiles being considered have s values covering a range Rs, i.e., Rs is the maximum difference in s between any two of the electrophiles. From the Swain-Scott equation, the relative reactivity toward a given nucleophile, ∆log k, between the two electrophiles at opposite ends of the s range is

∆log k ) nRs + ∆log k0

(2)

If the difference between the n values of the biological nucleophile and the model nucleophile is ∆n, then the difference in ∆log k, i.e., the error resulting from use of the “wrong” nucleophile, is ∆n ‚ Rs. Some n values based on water as the reference nucleophile (39) are listed in Table 2, together with s values for some SN2 electrophiles (38). Thus, for example, the error in log k resulting from using n-butylamine (n ) 5.13) kinetics (measured or calculated) if the in cutaneo nucleophile has n ) 5.37 (as for serum albumin) will be no more than 0.12 if the s values vary over a range of 0.5. Even with aniline as the model nucleophile, the maximum error would only be ca. 0.3 if the s values vary over a range of 0.35.

More Than One Electrophilic Group in the Same Molecule Such situations are manifold and often encountered. No general rules can be given; in some cases, it may be obvious from inspection of the structure which is the most reactive grouping, and in other cases, chemical experiments with model nucleophiles and investigation of the products are the only way to decide. In some cases, both electrophilic centers react sequentially, the second reaction being either intramolecular, with the same nucleophilic center, or intermolecular with a second nucleophile molecule. Some examples are as follows. R,β-Unsaturated aldehydes have both Michael acceptor (the CdC double bond) and Schiff base-forming electrophilic features. Depending on the degree of alkyl substitution on the double bond, either mechanism can predominate. R,β-Unsaturated-γ-sultones have both Michael acceptor and SN2 electrophilic features. With most nucleophiles, the Michael addition occurs first, followed by further reaction involving intramolecular attack at the SN2 center (40). In an interesting study by Benezra et al. in the 1980s (4143), a poison ivy type unit (pro-Michael acceptor) was coupled

to an R-methylene-γ-butyrolactone unit (direct-acting Michael acceptor), and this bifunctional electrophile (Scheme 6) was used in sensitization studies. By analysis of data from crosschallenges with derivatives in which either the poison ivy unit was deactivated (by methylation of the phenolic OH groups) or the R-methylene-γ-butyrolactone unit was deactivated by hydrogenation of the double bond, it was shown that sensitization occurred exclusively via the poison ivy mechanism.

Proelectrophile Mechanisms Some classes of compounds are not directly electrophilic but can readily be converted, either metabolically or abiotically, to electrophiles. For skin sensitization and for aquatic toxicity, the range of such transformations is quite small. Thus, for skin sensitization, transformations have been invoked as follows: (i) quinone or quinoneimine or quinone methide formation from aromatics with ortho- or para-OH and/or NH; (ii) elimination of HX to form Michael acceptors (17, 18, 28); (iii) R-oxidation of aliphatic amines to form aldehydes (Schiff base electrophiles) (21). This is at present a working hypothesis with no firm experimental evidence for or against it; (iv) bay region epoxidation, for PAHs (34); and (v) demethylation of aromatic compounds with an OH group and an OMe group ortho or para to each other (44). This is at present a working hypothesis. Several proelectrophile mechanisms, which are important in carcinogenicity and mutagenicity, are less relevant in skin sensitization. For example, oxidation of aromatic amines and amides and reduction of nitroaromatic compounds are less relevant. Binding to protein via free radical reactions may be the basis of a further mechanistic domain. Some examples of sensitization can be rationalized in terms of radical reactions (21, 45). Further work is needed to determine the applicability of this mechanism to sensitization. There is also the domain of “no reactive or proreactive features”, which is populated by (predicted) nonsensitizers (in other toxicological end points, it is populated by, for example, nonmutagens, general and polar narcotic aquatic toxicants).

Conclusions A major motivation for this paper was to provide a set of guidelines, as summarized in Scheme 1, for applicability domain classification of reactive toxicants. There will inevitably be special cases where compounds do not fit conveniently into one of the domains defined here but are not sufficiently numerous to form a separate mechanistic applicability domain. An example is the skin sensitizer clotrimazole (46), for which an SN1

Nonanimal-Based Prediction of Toxicological End Points

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Figure 1. Nonanimal prediction of skin sensitization potential.

mechanism seems the most likely route to protein binding. However, in our experience, the large majority of known skin sensitizers can be allocated to one of the major mechanistic applicability domains discussed here. Thus, in our analysis of a 41 compound data set (47), there were six compounds that could not be confidently allocated to one of the five major applicability domains by inspection of structure: After checking the literature, the number is reduced to three. In a larger data set of 106 compounds (48), we found only eight compounds that could not be confidently allocated to one of these domains. It is important to point out that being classified in one of the domains of Scheme 1 does not necessarily mean that a compound will be a skin sensitizer. Within a reaction mechanistic applicability domain, skin sensitization potential is a function of reactivity and (usually) hydrophobicity. A compound may be insufficiently reactive or insufficiently hydrophobic to cause sensitization. For example, primary alkyl bromides (SN2 electrophiles) with alkyl chain lengths C11 and above are sensitizers in the murine LLNA but the C4 homologue, which is equally reactive, is a nonsensitizer (10). Much of the debate on alternatives to animal testing has centered on two approaches: in vitro testing and in silico approaches. In vitro test methods involve the use of human or animal primary cells or cell lines in tissue culture. In silico approaches, based on the concept that it is the intrinsic nature of a compound that determines its biological activity (49), encompass a range of computational methods, ranging from transparent mechanism-based (Q)SAR models and expert systems, which use physicochemical properties and structural alerts to predict toxic hazard, to statistical models based on nontransparent structure-derived parameters based on, inter alia, molecular topology and molecular connectivity. Both approaches have their protagonists and their critics. For some end points, good in vitro methods are not available, and there is no guarantee that sufficiently reliable methods will be developed. In silico methods can only be successful insofar as

the parameters on which they are based, whether these are mechanism-based indices, modeling properties such as reactivity and hydrophobicity, or nontransparent structure-derived indices, accurately represent the chemical properties underlying the toxicity. There is a third approach, particularly relevant when the chemicobiological interactions leading to toxicity are wellunderstood mechanistically, which has been largely neglected in the debate over alternatives to animal testing. This is what we will refer to as the in chemico approach, i.e., carrying out laboratory work to determine the relevant chemical properties needed to predict the toxicity. We will discuss it in the context of skin sensitization. Research dating back more than 70 decades has established a very strong mechanistic understanding of skin sensitization. There is a complex network of intercellar interactions downstream of the processes involving the sensitizing compound, and we consider this likely to be a source of problems for development of robust in vitro methods. However, the fundamental chemical basis of skin sensitization is well-understood (despite some gaps, relating to the nature and location of carrier proteins, which have been discussed above): In essence, the ability to sensitize depends on the ability to bind to protein, which can be modeled by reactivity and hydrophobicity parameters. In principle, therefore, nonanimal prediction of sensitization potential could be based on in silico methods using hydrophobicity and reactivity parameters. In silico approaches have had some success, but their limitation is that reactivity cannot always be confidently predictedssometimes not even the nature of the reaction (if any) can be predicted. Organic chemistry and theoretical chemistry are interpretative sciences but still not fully predictive sciences. This is why the in chemico approach is needed. On this basis, we envisage the following approach, represented in Figure 1, for the nonanimal prediction of skin sensitization potential, combining in silico (we include in cerebro

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under this term) and in chemico approaches. When presented with a new compound, (i) the first step is to classify it into its reaction mechanistic domain. This may often be possible by inspection of structure, but inevitably, in some cases, a confident prediction may not be possible. In such situations, experimental work will be needed to determine the reaction chemistry, in particular to determine if the compound is electrophilic or proelectrophilic and the nature of the reactions. An example is the work of Alvarez-Sanchez et al. on 5-chloro-2-methylisothiazol-3-one (MCI) and 2-methylisothiazol-3-one (MI) (29). (ii) Having assigned the compound to its reaction mechanistic applicability domain, the next step is to quantify its reactivity/ hydrophobicity relative to known sensitizers in the same mechanistic applicability domain. These properties may be confidently predictable from structure, using physical organic chemistry approaches such as linear free energy relationships based on substituent constants or on molecular orbital parameters. In other cases, it will be necessary to carry out physical organic chemistry assays, such as determination of reaction kinetics and measurement of partition coefficients. Several methods for experimental electrophilic reactivity parameters suitable for toxicology purposes are available, using model nucleophiles such as n-butylamine (17, 18), glutathione (5053), 4-(4-nitro)benzylpyridine (54), and 2′-deoxyguanosine (50, 51). (iii) Having assigned the compound to its reaction mechanistic applicability domain and quantified its reactivity/hydrophobicity relative to known sensitizers in the same domain, mechanismbased QSAR or mechanistic read-across can be used to predict the sensitization potential. Our vision for skin sensitization prediction is therefore that the animal testing laboratory should be replaced by the physical organic chemistry laboratory. Particularly bearing in mind that many compounds are easily predictable without experimentation, the experimental studies to generate the chemical data required should be no more costly or time-consuming than the animal tests that have hitherto been used. Acknowledgment. This paper originates from discussions at the 1st and 2nd Reactivity Workshops of the International QSAR Foundation, held at the University of Tennessee, Knoxville, May 2005 and April 2006.

References (1) Organisation for Economic Cooperation and Development (OECD) (2004) http://www.oecd.org/document/23/0,2340,en_2649_34365_33957015_1_1_1_1, 00.html. (2) Netzeva, T. I., Worth, A. P., Aldenberg, T., Benigni, R., Cronin, M. T. D., Gramatica, P., Jaworska, J. S., Kahn, S., Klopman, G., Marchant, C. A., Myatt, G., Nikolova-Jeliazkova, N., Patlewicz, G. Y., Perkins, R., Roberts, D. W., Schultz, T. W., and Stanton, D. T. (2005) Current status of methods for defining the applicability domain of (quantitative) structure-activity relationships. The report and recommendations of ECVAM Workshop. ATLA 33, 155-173. (3) Bradbury, S. P., and Lipnick, R. L. (1990) Introduction: Structural properties for determining mechanisms of toxic action. EnViron. Health Perspect. 87, 181-182. (4) Russom, C. L., Bradbury, S. P., Broderius, S. J., Hammermeister, D. E., and Drummond, R. A. (1997) Predicting modes of toxic action from chemical structure: Acute toxicity in the fathead minnow (Pimephales Promelas). EnViron. Toxicol. Chem. 16 (5), 948-967. (5) Hermens, J. L. M. (1990) Electrophiles and acute toxicity to fish. EnViron. Health Perspect. 87, 219-225. (6) Karabunarliev, S., Mekenyan, O. G., Karcher, W., Russom, C. L., and Bradbury, S. P. (1996) Quantum-chemical descriptors for estimating the acute toxicity of electrophiles to the Fathed minnow (Pimephales promelas): An analysis based on molecular mechanisms. Quant. Struct.-Act. Relat. 15, 302-310.

Aptula and Roberts (7) Lipnick, R. L. (1991) Outliers: Their origin and use in the classification of molecular mechanisms of toxicity. Sci. Total EnViron. 109/110, 131-153. (8) 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. (9) Roberts, D. W. (1989) Acute lethal toxicity quantitative structureactivity relationships for electrophiles and pro-electrophiles: Mechanistic and toxicokinetic principles. In Aquatic Toxicology and EnVironmental Fate (Suter, G. W., II, and Lewis, M. A., Eds.) Vol. 11, ASTM STP1007, pp 490-506, American Society for Testing and Materials, Philadelphia. (10) Basketter, D., Roberts, D. W., Cronin, M., and Scholes, E. W. (1992) The value of the local lymph node assay in quantitative structureactivity investigations. Contact Dermatitis 27, 137-142. (11) Veith, G. D., Lipnick, R. L., and Russom, C. L. (1989) The toxicity of acetylenic alcohols to the fathead minnow, Pimephales promelas. Narcosis and proelectrophile activation. Xenobiotica 19, 555-565. (12) EC 2003 (2003) 2003/15/EC. Commission Directive of 27 February 2003 amending Council Directive 76/768/EEC on the approximation of laws of the Member States relating to cosmetic products. Off. J. Eur. Union L66, 26-35. (13) Divkovic, M., Pease, C. K., Gerberick, G. F., and Basketter, D. A. (2005) Hapten-protein binding: From theory to practical application in the in vitro prediction of skin sensitization. Contact Dermatitis 53, 189-200. (14) Freidig, A. P., Verhaar, H. J. M., and Hermens, J. L. M. (1999) Comparing the potency of chemicals with multiple modes of action in aquatic toxicology: Acute toxicity due to narcosis versus reactive toxicity of acrylic compounds. EnViron. Sci. Technol. 33, 3038-3043. (15) Freidig, A. P., Verhaar, H. J. M., and Hermens, J. L. M. (1999) Quantitative structure-property relationships for the chemical reactivity of acrylates and methacrylates. EnViron. Toxicol. Chem. 18, 11331139. (16) 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. (17) Franot, C., Roberts, D. W., Smith, R. G., Basketter, D. A., Benezra, C., and Lepoittevin, J.-P. (1994) Structure-activity relationships for contact allergenic potential of γ,γ-dimethyl-γ-butyrolactone derivatives. 1. Synthesis and electrophilic reactivity studies of R-(ωsubstituted-alkyl)-γ,γ-dimethyl-γ-butyrolactones and correlation of skin sensitization potential and cross-sensitization patterns with structure. Chem. Res. Toxicol. 7, 297-306. (18) Franot, C., Roberts, D. W., Basketter, D. A., Benezra, C., and Lepoittevin, J.-P. (1994) Structure-activity relationships for contact allergenic potential of γ,γ-dimethyl-γ-butyrolactone derivatives. 2. Quantititative structure-skin sensitization relationships for R-substitutedR-methyl-γ,γ-dimethyl-γ-butyrolactones. Chem. Res. Toxicol. 7, 307312. (19) Roberts, D. W. (1987) Structure-activity relationships for skin sensitization potential of diacrylates and dimethacrylates. Contact Dermatitis 17, 281-289. (20) Gerberick, G. F., Ryan, C. A., Kern, P. S., Schlatter, H., Dearman, R. J., Kimber, I., Patlewicz, G. Y., and Basketter, D. A. (2005) Compilation of historical local lymph node data for evaluation of skin sensitization alternative methods. Dermatitis 16 (4), 157-202. (21) Roberts, D. W., and Lepoittevin, J.-P. (1998) Hapten-protein interactions. In Allergic Contact Dermatitis. The Molecular Basis. (Lepoittevin, J.-P., Basketter, D. A., Goossens, A., and Karlbert, A.-T., Eds.) pp 81-111, Springer-Verlag, Heidelberg. (22) Aptula, A. O., Roberts, D. W., Cronin, M. T. D., and Schultz, T. W. (2005) Chemistry-toxicity relationships for the effects of di- and trihydroxybenzenes to Tetrahymena pyriformis. Chem. Res. Toxicol. 18 (5), 844-854. (23) 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 electrophiles. Chem. Res. Toxicol. 8, 545-551. (24) Graul, E. H., and Patzschke, K. (1952) Klinischer und experimenteller beitrag zur frage der gewerblichen hautsensibilisierung durch synthesezwishen und nebenproduckte. Arch. Dermatol. Syphilis 193, 582591. (25) Mekenyan, O., Roberts, D. W., and Karcher, W. (1997) Molecular orbital parameters as predictors of skin sensitization potential of haloand pseudohalobenzenes acting as SNAr electrophiles. Chem. Res. Toxicol. 10 (9), 994-1000. (26) Bunnett, J. F. (1958) Mechanism and reactivity in aromatic nucleophilic substitution reactions. Q. ReV. 12, 1-16. (27) Hine, J. (1962) Physical Organic Chemistry, 2nd ed., International Student Edition, p 176, McGraw-Hill, New York and Kogakusha, Tokyo.

Nonanimal-Based Prediction of Toxicological End Points (28) Goodwin, B. F. J., and Roberts, D. W. (1986) Structure-activity relationships in allergic contact dermatitis. Food Chem. Toxicol. 24, 795-798. (29) Alvarez-Sa´nchez, R., Basketter, D., Pease, C., and Lepoittevin, J.-P. (2003) Studies of chemical selectivity of hapten, reactivity, and skin sensitization. 3. Synthesis and studies on the reactivity toward model nucleophiles of the 13C-labeled skin sensitizers, 5-chloro-2-methylisothiazol-3-one (MCI) and 2-methylisothiazol-3-one (MI). Chem. Res. Toxicol. 16, 627-636. (30) Aptula, A. O., Roberts, D. W., and Cronin, M. T. D. (2005) From experiment to theory: Molecular orbital parameters to interpret the skin sensitization potential of 5-chloro-2-methylisothiazol-3-one and 2-methylisothiazol-3-one. Chem. Res. Toxicol. 18 (2), 324-329. (31) Lu, X., Heilman, J. M., Blans, P., and Fishbein, J. C. (2005) The structure of DNA dictates purine atom site selectivity in alkylaton by primary diazonium ions. Chem. Res. Toxicol. 18, 1462-1470. (32) Loechler, E. L. (1994) A violation of the Swain-Scott principle, and not SN1 versus SN2 reaction mechanisms, explains why carcinogenic alkylating agents can form different proportions of adducts at oxygen versus nitrogen in DNA. Chem. Res. Toxicol. 7 (3), 277-280. (33) Aptula, A. O., Roberts, D. W., and Patlewicz, G. (2006) Mechanistic applicability domains for nonanimal based toxicological endpoints. QSAR analysis of the Schiff base applicability domain for skin sensitization. Chem. Res. Toxicol. In press. (34) Ashby, J., Basketter, D. A., Paton, D., and Kimber, I. (1995) Structure activity relationships in skin sensitization using the murine local lymph node assay. Toxicology 103, 177-194. (35) Isaacs, N. (1995) Physical Organic Chemistry, 2nd ed., pp 193-214, Longman, Harlow, United Kingdom. (36) Hughes, E. D., and Ingold, C. K. (1935) Mechanism of substitution at a saturated carbon atom. Part IV. A discussion of constitutional and solvent effects on the mechanism, kinetics, velocity, and orientation of substitution. J. Chem. Soc. 244-255. (37) Roberts, D. W. Unpublished. (38) Swain, C. G., and Scott, C. B. (1953) Quantitative correlation of relative rates. Comparison of hydroxide ion with other nucleophilic reagents toward alkyl halides, esters, epoxides and acyl halides. J. Am. Chem. Soc. 75, 141-147. (39) Ogston, A. G., Holiday, E. R., Philpot, J. S., and Stocken, L. A. (1948) The replacement reactions of β,β-dichlorodiethyl sulphide and of some analogues in aqueous solution: The isolation of β-chloro-β′-hydroxy diethyl sulphide. Trans. Faraday Soc. 44, 45-52. (40) Roberts, D. W., Williams, D. L., and Bethell, D. (2006) Electrophilic reactions of skin sensitizing sultones. Chem. Res. Toxicol. Submitted for publication. (41) Marchand, B., and Benezra, C. (1982) Double-head haptens. Synthesis and experimentally induced contact sensitivity to substances containing

Chem. Res. Toxicol., Vol. 19, No. 8, 2006 1105

(42)

(43)

(44) (45) (46) (47) (48)

(49)

(50) (51) (52) (53) (54)

two related haptens, pyrocatechol and R-methylene-γ-butyrolactone. J. Med. Chem. 30, 165-168. Mattes, H., and Benezra, C. (1982) Double-headed haptens with pyrocatechol (poison ivy like) and methylene lactone functional groups: A search for skin tolerance inducers. J. Med. Chem. 25, 650653. Nazih, A., Benezra, C., and Lepoittevin, J.-P. (1993) Bihaptens with 5- and 6-methyl substituted alkylcatechols and methylene lactone functional group: Tools for hapten (allergen or tolerogen)-protein interaction studies. Chem. Res. Toxicol. 6, 215-222. Smith, C. K., and Hotchkiss, S. A. M. (2001) Allergic Contact Dermatitis: Chemical and Metabolic Mechanisms, Taylor & Francis Ltd.,. London, United Kingdom. Gimenez-Arnau, E. Unpublished (presented at ERGECD meeting Germany, 2005). Roberts, D. W., Aptula, A. O., Cronin, M. T. D., Hulzebos, E., and Patlewicz, G. (2006) Global (Q)SARs for skin sensitizations Assessment against OECD principles. SAR QSAR In press. Aptula, A. O., Patlewicz, G. Y., and Roberts, D. W. (2005) Skin sensitization: Reaction mechanistic applicability domains for structureactivity relationships. Chem. Res. Toxicol. 18, 1420-1426. Roberts, D. W., Aptula, A. O., and Patlewicz, G. (2006) Structureactivity relationships in 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. In press. Cronin, M. T. D. (2004) Predicting chemical toxicity and fate in humans and the environmentsAn introduction. In Predicting Chemical Toxicity and Fate (Cronin, M. T. D., and Livingstone, D. J., Eds.) pp 3-13, CRC Press, Boca Raton, FL. Harder, A., Escher, B. I., and Schwarzenbach, R. P. (2003) Applicability and limitation of QSARs for the toxicity of electrophilic chemicals. EnViron. Sci. Technol. 37, 4955-4961. Diss. ETH No. 14966, Angela Harder, http://e-collection.ethbib.ethz.ch/ ecol-pool/diss/fulltext. Schultz, T. W., Yarbrough, J. W., and Johnson, E. L. (2005) Structureactivity relationships for glutathione reactivity of carbonyl-containing compounds. SAR QSAR EnViron. Res. 16, 313-322. Aptula, A. O., Patlewicz, G., Roberts, D. W., and Schultz, T. W. (2006) Non-enzymatic glutathione reactivity and in vitro toxicity: A nonanimal approach to skin sensitisation. Toxicol. in Vitro 20, 239-247. Hermens, J. L. M., Busser, F., Leeuwangh, P., and Musch, A. (1985) Quantiative correlations studies between the acute lethal toxicity of 15 organic halides to the guppy (Poecilla reticulata) and chemical reactivity towards 4-nitrobenzylpyridine. Toxicol. EnViron. Chem. 9, 219-236.

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