for Skin Sensitization - ACS Publications - American Chemical Society

Chem. Res. Toxicol. 2010, 23, 1519–1540

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Use of Genotoxicity Information in the Development of Integrated Testing Strategies (ITS) for Skin Sensitization Ovanes Mekenyan,† Grace Patlewicz,*,‡ Gergana Dimitrova,† Chanita Kuseva,† Milen Todorov,† Stoyanka Stoeva,† Stefan Kotov,† and E Maria Donner†,‡ Laboratory of Mathematical Chemistry, “Prof. As. ZlataroV” UniVersity, Bourgas, Bulgaria, and DuPont Haskell Global Centers for Health and EnVironmental Sciences, 1090 Elkton Road, Newark, Delaware 19711 ReceiVed May 11, 2010

Skin sensitization is an end point of concern for various legislation in the EU, including the seventh Amendment to the Cosmetics Directive and Registration Evaluation, Authorisation and Restriction of Chemicals (REACH). Since animal testing is a last resort for REACH or banned (from 2013 onward) for the Cosmetics Directive, the use of intelligent/integrated testing strategies (ITS) as an efficient means of gathering necessary information from alternative sources (e.g., in Vitro, (Q)SARs, etc.) is gaining widespread interest. Previous studies have explored correlations between mutagenicity data and skin sensitization data as a means of exploiting information from surrogate end points. The work here compares the underlying chemical mechanisms for mutagenicity and skin sensitization in an effort to evaluate the role mutagenicity information can play as a predictor of skin sensitization potential. The Tissue Metabolism Simulator (TIMES) hybrid expert system was used to compare chemical mechanisms of both end points since it houses a comprehensive set of established structure-activity relationships for both skin sensitization and mutagenicity. The evaluation demonstrated that there is a great deal of overlap between skin sensitization and mutagenicity structural alerts and their underlying chemical mechanisms. The similarities and differences in chemical mechanisms are discussed in light of available experimental data. A number of new alerts for mutagenicity were also postulated for inclusion into TIMES. The results presented show that mutagenicity information can provide useful insights on skin sensitization potential as part of an ITS and should be considered prior to any in ViVo skin sensitization testing being initiated. Contents 1. Introduction 1.1. Regulatory Landscape and the Use of Alternatives within Intelligent/Integrated Testing Strategies (ITS) 1.2. Skin Sensitization: ITS 1.3. Skin Sensitization: Biological Mechanism 1.4. Skin Sensitization: Chemical Mechanisms 1.5. Mutagenicity As a Surrogate End Point 1.6. Correlation of Mutagenicity and Sensitization Data 2. Materials and Methods 3. Results and Discussion 3.1. Theoretical (Quantum Chemical) Analysis of DNA and Protein Reactivity 3.2. Assumptions Used When Comparing DNA and Protein Binding Alerts 3.3. Mechanistic Comparison between DNA and Protein Binding Alerts 3.3.1. Protein Reactivity of DNA Alerts 3.3.2. DNA Reactivity of Protein Alerts 4. Summary 5. Conclusions

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1. Introduction 1.1. Regulatory Landscape and the Use of Alternatives within Intelligent/Integrated Testing Strategies (ITS). Chemi* To whom correspondence should be addressed. Tel: 1-302-366-5090. Fax: 1-302-451-4531. E-mail: [email protected].

cal regulation and the means by which data are generated and translated into information for the purposes of hazard characterization and risk assessment are undergoing a massive shift. Undoubtedly, the largest impact comes from the move toward nonanimal alternatives. Animal welfare concerns have provided significant impetus to investigate potential alternatives to animal testing which encompass the 3 Rs (refine, reduce, and replace animal testing). Industry has been quick to adopt alternative methods to facilitate cost-effective and efficient screening tools for making decisions on whether or not to advance a new chemical entity/product for further development. In a regulatory context, acceptance of alternative methods by Competent Authorities is slow. The process of adoption of new test methods into OECD guidelines can be a lengthy one; for example, the local lymph node assay (LLNA) required extensive validation and evaluation over a 15 year time period before it officially became adopted as a test guideline within the OECD (1). This apparent barrier is likely to ease as the regulatory landscape evolves. The seventh amendment to the Cosmetics Directive (2) has placed a ban on the in ViVo testing of cosmetics ingredients. The ban for acute testing came into effect in March, 2009, and the ban for repeat dose testing will commence in 2013. The new chemicals legislation within the EU, REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances) specifically calls for the use of nonanimal alternatives (3). Vertebrate testing should only be used as a last resort. REACH advocates the use of intelligent/integrated testing strategies (ITS) as an efficient means of addressing the informa† ‡

“Prof. As. Zlatarov” University. DuPont Haskell Global Centers for Health and Environmental Sciences.

10.1021/tx100161j  2010 American Chemical Society Published on Web 09/16/2010

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tion requirements for a given end point. ITS approaches comprise multiple elements such as optimized in ViVo tests, in Vitro tests, (Q)SARs and chemical categories. The elements themselves are not new, but the integration in a framework where the generated information is nonstandard provides a challenge for interpretation and decision making (4). ITS based on new and emerging technologies are also described in the landmark publication by the National Research Council, Toxicity Testing in the 21st Century (5). Thus, the landscape of gathering information on the hazards of chemical substances is dramatically evolving; opportunities and challenges remain in identifying the critical elements for the evaluation of adverse outcomes. 1.2. Skin Sensitization: ITS. For the purposes of this study, we have chosen to focus on one end point, skin sensitization. This is an end point that will be impacted in the 2013 deadline for in ViVo testing under the seventh amendment, and one where there is a reasonable understanding about the factors involved in skin sensitization induction. Under REACH, skin sensitization is an end point that must be considered for tonnages equal to or greater than 1 ton per annum (3). The information requirements are outlined in Annex VII of the legal text. An ITS framework for skin sensitization is described in the technical guidance in Chapter R7.3 (http:// guidance.echa.europa.eu/docs/guidance_document/information_requirements_en.htm (6)) and elsewhere (7). The first step of the ITS considers what information might already be available from (Q)SAR, chemical category, and/or in Vitro approaches. If the existing information is sufficient for the purposes required, i.e., classification and labeling and risk assessment, then the process stops. If the data requirement is not met, new testing needs to be considered. For REACH, the test of first choice is the LLNA. Surrogate assays are highlighted in the technical guidance as a potential source of information, but little practical guidance is provided on what they entail or how they would be applied. We have considered whether mutagenicity data as surrogate information could be useful in determining sensitization potential. At this stage, it is worthwhile to briefly describe what is understood about the skin sensitization induction process and hence why mutagenicity data is potentially relevant. 1.3. Skin Sensitization: Biological Mechanism. Skin sensitization is a T-cell mediated immunological response specific for the substance comprising two phases, induction and elicitation. During the induction phase, the sensitizing chemical penetrates the stratum corneum to the viable epidermis and binds to skin proteins/peptides to create an immunogenic complex which is then recognized and processed by Langerhans cells (LC) in the epidermis. Upon exposure to the immunogenic complex, the LC begin a maturation process in which they internalize and process the complex to a form that will be recognizable by T-cells. These cells then migrate from the epidermis to the lymph nodes where they present the modified complex to naı¨ve T-cells with receptors that are able to specifically recognize the immunogenic complex and are stimulated to proliferate and circulate throughout the body. Sensitization has now been induced. Upon subsequent exposure to the same sensitizer, protein binding and processing of the immunogenic complex by the LC occurs after which the immunogenic complex is recognized by circulating T-cells triggering a cascade of biochemical and cellular processes which produce the clinical sensitization response, i.e., elicitation (8-12). Thus, a chemical must negotiate a number of steps before sensitization is induced. A sensitizing chemical must penetrate

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through the stratum corneum and form a stable association with a carrier protein in order to deliver dermal trauma sufficient to induce and upregulate epidermal cytokines. These processes are necessary for the mobilization, migration, and maturation of LC, and for the chemical to be inherently immunogenic in such a way that a T lymphocyte response of sufficient magnitude is stimulated (11). All of these steps cannot be equally important. The step which is dependent on the sensitizing chemical itself, the rate determining step, should provide insight on how to evaluate the skin sensitization potential of a given chemical (13). The relative importance of these different steps has been discussed at some length by Roberts and Aptula (13) and Roberts and Patlewicz (14). The rate determining step is considered to be the covalent binding of a chemical which acts as an electrophile to the skin protein, which acts as a nucleophile, a hypothesis put forward in 1936 by Landsteiner and Jacobs (15). Since the time of their publication, efforts to predict skin sensitizers have largely focused on identifying electrophilic features in chemicals (structural alerts) and relating these back to the skin sensitization potential. 1.4. Skin Sensitization: Chemical Mechanisms. There have been many efforts to develop models that relate the electrophilic features of chemicals to the skin sensitization potential (16). Early efforts relied on the Relative Alkylation Index (RAI) approach developed by Roberts and Williams in the early 1980s (17). In the 1990s and early 2000s, the emphasis was on global statistical models capable of making predictions for sensitization for a wide range of chemicals. These models have been of variable utility and are discussed in references (18, 19). With the regulatory preparations of (Q)SARs under REACH, the shift has switched toward a consideration of mechanisms and trying to encode these into rules. Roberts and Aptula (20) described a set of principles that characterized sensitizers by their reaction mechanisms which have since been implemented into Smiles ARbitrary Target Specification (SMARTS) codes for easy reuse (21). Other initiatives have focused on the development of expert systems. Derek for Windows (DfW) is a well-known knowledge based system that encodes chemistry and toxicity information in the form of toxicophores (22, 23). The hybrid expert system Tissue Metabolism Simulator for Skin Sensitization (TIMESSS) encodes structure toxicity and structure metabolism relationships through a number of transformations simulating skin metabolism and interaction of the generated reactive metabolites with skin proteins. The skin metabolism simulator mimics metabolism using 2D structural information. Metabolic pathways are generated on the basis of a set of 236 hierarchically ordered principal transformations including spontaneous reactions and enzyme catalyzed reactions (phase I and II). The covalent reactions with proteins are described by 47 alerting groups (structural alerts). Some of these alerts are additionally underpinned by mechanistically based 3D-QSARs to refine the predictions. These 3D-QSAR models depend on both the structural alert and factors that influence its reactivity: steric effects, molecular size, shape, solubility, lipophilicity, and electronic properties (24-26). The rules implemented into TIMES-SS have also since been incorporated into the OECD Toolbox (27). 1.5. Mutagenicity As a Surrogate End Point. As discussed above, the rate determining step of skin sensitization induction is thought to be the formation of a covalent bond between the electrophilic chemical and the skin protein nucleophile. Covalent bond formation as a rate determining step is not unique to skin sensitization. Schultz et al. (28) described a conceptual framework for predicting the toxicity of reactive chemicals where

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plausible molecular initiating events were based on covalent reactions with nucleophiles in proteins or DNA and would ultimately lead to a variety of different adverse outcomes such as aquatic fish toxicity, mutagenicity, hepatocyte cytoxicity, or respiratory toxicity. Aptula and Roberts (20) illustrated this concept using aquatic toxicity and sensitization as example end points. Electrophilicity is well-known to be an important factor in driving mutagenicity and carcinogenicity (29). Seminal work by Ashby and Tennant (30-32) described structural alerts for carcinogenicity. They found that the electrophilicity of chemicals correlated very well with mutagenicity in the Ames test. Similar findings have been published by Benigni and co-workers (33). His extensive evaluations on structural alerts developed by Ashby and Tennant (30), Kazius et al. (34), and Woo (35) have been additionally implemented into the software program Toxtree (36). Mekenyan et al. have developed the TIMES system for the prediction of Ames mutagenicity (37) and in Vitro chromosomal aberrations (38). The structure and implementation is similar to that for skin sensitization. Structure toxicity and structure metabolism relationships are encoded through a number of transformations simulating liver metabolism and interaction of the generated reactive metabolites with DNA. 1.6. Correlation of Mutagenicity and Sensitization Data. The concept of relating skin sensitization data to mutagenicity data has been the subject of limited assessment. Ashby et al. (39) explored the mechanistic relationship between mutagenicity, skin sensitization, and skin carcinogenicity. Twenty organic chemicals that were mutagenic in the Ames test, including 7 established skin carcinogens and one welldefined noncarcinogen, were evaluated in the LLNA. Ashby and co-workers focused on the potential utility of skin sensitization data as a screen for skin carcinogenicity. They concluded that the LLNA provided useful information to assist in the early identification of carcinogenicity potential, although some important differences were noted such as penetration, inappropriate skin metabolism, or high water solubility. Their work also cited various other attempts that had been performed earlier on isolated chemical classes such as polyaromatic hydrocarbons and nitrosoureas. Subsequent work by Warbrick et al. (40) acknowledged a correlation between mutagenicity and sensitization, but the use of skin sensitization data as a surrogate for carcinogenicity was not sound in practice. The set of test chemicals under study was very limited, and firm conclusions could not be drawn. Albert (41) performed a comparison between chemicals that had been assayed for carcinogenicity in the National Toxicology Program (NTP, US Department of Health and Human Services) and those that also produced allergic dermatitis (ACD) in humans. The basis of the study was that most contact sensitizers and genotoxic carcinogens were chemically similar in terms of their electrophilic potential and therefore capable of producing adducts on macromolecules including protein and DNA. Of the 209 chemicals with data from bioassays, there were 36 (17%) that were known to be human contact sensitizers; about half of these were positive in tumor bioassays. The addition of a test for contact sensitization to the Ames test as a screening tool was thought to increase the tumorigenic detection efficiency by about 40% because of the nongenotoxic tumorigens. These results are not altogether surprising given the arbitrary selection of the chemicals included in the evaluation. Furthermore, the analysis of data showed that about 80% of Ames positive chemicals were also in Vitro clastogens. This observation confirms the basic principle that was used in modeling chromosomal aberration (CA) potential in TIMES (38), namely, that

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chemicals which directly interacted with DNA should also be inducers of CA since the direct interaction with DNA is one of the mechanisms for eliciting CA (the other main toxicity pathway is initiated by interaction with specific proteins). Another interesting result was that a great proportion of skin sensitizers (86%) were also found to be genotoxic (either Ames or CA positive). Albert and Magee (42) subsequently evaluated 146 chemicals that had NTP data from bioassays for mutagenicity and tumorigenicity, with an analysis of structure-activity relationships for contact sensitization. Using the data from this analysis and from other sources, the proportion of the contact sensitizers that were both mutagenic and tumorigenic was found to range from 20% to 28%. This finding suggested that there may be in the order of 90 genotoxic tumorigens for rodents among the approximately 384 chemicals that have been validated as contact sensitizers for humans. It was also found that only about 30-40% of contact allergens were Ames positive. This is a logical correlation given the CA end point, providing interaction with protein, was not included in the analysis. The analysis of data (not shown in the paper) showed that if Ames was combined with CA, then the correlation between sensitization and genotoxicity would significantly increase. A common feature in these studies is the attempt to correlate carcinogenicity with mutagenicity and/or skin sensitization with the aim of predicting the former. Given the obvious complexity of the carcinogenicity end point, analyzing the relationship between mutagenicity and sensitization alone might have resulted in greater success. Wolfreys and Basketter (43) considered the use of mutagenicity information as part of an ITS approach in the evaluation of skin sensitization. Their evaluation focused on a set of 100 chemicals for which in Vitro mutagenicity and sensitization data were available. They concluded that there was some concordance between the two end points, but neither predicted the other particularly accurately with 32% showing disagreement. No doubt there were other elements missing from their study such as possible detoxification reactions or whether certain chemicals in the set might have been acting by a nongenotoxic mechanism. No detailed evaluation of the chemical mechanisms was performed to rationalize the concordances and disagreements identified. In this work, we have investigated whether mutagenicity information is of practical value in the assessment of skin sensitization potential as part of an ITS approach. Our evaluation has focused on the chemical mechanisms involved in eliciting a response in each of the assays. We have used the structure-activity alerts encoded within TIMES for the mutagenicity (DNA alerts) and sensitization (protein binding alerts) end points to see what commonalities exist in terms of the alerts themselves and their mechanistic rationales. TIMES was selected as a platform since it contains a comprehensive set of alerts for both mutagenicity and sensitization as well as an extensive set of experimental data for both end points. The latter would be used to help validate the analysis performed.

2. Materials and Methods The approach taken for this study can be described in the following 3 steps: (1) theoretical (quantum chemical) analysis of DNA and protein reactivity; (2) evaluating the protein reactivity of DNA alerts and substantiation of the alerts with experimental data; (3) assessing the DNA reactivity of protein binding alerts and substantiation of the alerts with experimental data. Step 1 comprised the identification of a selection of representative amino acids that make up proteins and the nucleobases found in

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the nucleic acids DNA and RNA. The nucleophiles that were used in the theoretical quantum chemical analysis were, namely, guanine, adenine, cytosine, and thymine (to represent nucleobases), and cysteine, lysine, histidine, and tyrosine (to represent amino acids). The two-dimensional (2D) chemical structures for these were imported into OASIS Basic software (44). Structures were then energy minimized, and up to 30 three-dimensional (3D) conformations were generated to cover the conformational space of each chemical (45). The donor superdelocalizabilities, Sµ [(a.u.)2/eV], and charges, Q [a.u.], which quantify soft and hard reactivity, respectively, on the relevant nucleophilic sites were calculated using MOPAC 93 (AM1 Hamiltonian 46, 47) and accounting for molecular flexibility. The strategy for steps 2 and 3 involved comparing the TIMES’s alerts for mutagenicity and skin sensitization and their associated mechanisms. Thus, the DNA alerts referred to here are those alerts extracted from the training set used to develop the TIMES Ames mutagenicity model; similarly, the protein binding alerts are those extracted during the development of the TIMES skin sensitization model. Substantiation of the alerts involved taking the training sets underpinning the TIMES mutagenicity and sensitization models and processing them through their respective models. Substances that fired one of the DNA alerts were then processed through the TIMES sensitization model. Substances which fired one of the sensitization alerts were then processed through the TIMES mutagenicity model. In both cases, the predictions were compared with their experimental data.

3. Results and Discussion 3.1. Theoretical (Quantum Chemical) Analysis of DNA and Protein Reactivity. The nucleophiles representing DNA and protein reactivity are shown in Figures 1a and b, respectively. On inspection, it is evident that there is significant overlap between the nucleophilic sites present in the nucleobases guanine, adenine, thymine, and cytosine (Figure 1a) with those present in the amino acids, lysine, histidine, and tyrosine (Figure 1b). In the majority of these cases, the local superdelocalizabilities and charges are more pronounced on the nucleophilic sites present in the amino acids rather than the nucleobases. The heterocyclic nitrogen present in guanine and adenine possesses some similarity with the nitrogen present in the amino acid histidine. A comparison of the charges and superdelocalizability on this nitrogen reveals stronger values for the charge in the histidine structure (c.f. -0.13 to -0.16 au in histidine, -0.06 au in guanine and -0.11 au in adenine), whereas the superdelocalizabilities are comparable across the structures (∼0.24 (a.u.)2/eV). The amino acid group within lysine bears a striking resemblance to the amino group present within the guanine, adenine, and cytosine structures. The lysine amino group possesses a higher superdelocabilizability (S ) 0.25 to 0.28 (a.u.)2/eV vs 0.24 (a.u.)2/eV) in the nucleobases) and more negative charge (-0.34 to -0.37 au in lysine vs -0.33 au in guanine and -0.36 au in adenine and cytosine). The hydroxyl group within the phenol group in tyrosine reflects the phenol group that exists in the enol forms of guanine and thymine. Tyrosine’s hydroxyl group is associated with a more negative charge (except for 2-OH-thymine having Q ) -0.26 au; Figure 1a), and its superlocalizability is greater (S ) 0.25 (a.u.)2/eV vs 0.24 (a.u.)2/eV). The amino acid cysteine possesses a thiol (-SH) group which is not reflected in the nucleobase structures. The main difference between the above analyzed nucleophilic sites and cysteine thiol group is that the latter has extremely high soft nucleophilicity having superdelocalizability (0.39 to 0.40 (a.u.)2/eV) and very low hard nucleophilicity characterized by a positive charge (0.01 au). The above analysis suggests that nucleophilic sites within nucleic acids of DNA are less reactive

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than the nucleophilic sites that are present in the amino acids found in proteins. Next, a more detailed analysis of reactivity was performed based on a comparison of the spectra of soft and hard reactivity using the computed values of charge and superlocalizability for the nucleophilic functionalities in nucleobases in DNA and amino acid residuals in proteins. The results are illustrated in Figure 2a and b, respectively. As seen, a large overlap between the hard profiles of the nucleophilic sites in DNA and protein (in terms of net atomic charges) is observed (Figure 2a). In contrast, only part of the soft nucleophilicity profile of amino acids (in terms of donor super delocalizabilities) is covered by that of nucleobases; this overlap is observed only in the range of lower soft nucleophilicity (Figure 2b). The main difference in soft nucleophilicity of nucleobases and amino acids is in the extremely high softness of SH-Cys followed by that of the primary NH2-Lys. Hence, the lower reactivity of DNA nucleophilic sites as compared to that of amino acids could be related mainly to their lower soft electrophilicity. This suggests that if substances (possessing DNA alerts) interact with DNA, they could also interact with proteins (i.e., be potential sensitizers); the opposite would not be the case. Exploring the DNA alerts to determine similarity in underlying chemical mechanisms should help inform whether a mutagenic chemical is likely to be a sensitizer. The next step was to compare the DNA and protein binding alerts in terms of their chemical mechanisms to help identify when mutagenicity data would be of most value in the assessment of skin sensitization, i.e., at which point in an ITS. 3.2. Assumptions Used When Comparing DNA and Protein Binding Alerts. The list of alerting groups causing skin sensitization were identified in the parent chemicals only and not after skin metabolism. “Internal” toxicokinetics in bioassays, including bacterial metabolism in Ames and skin metabolism, were accounted for. Some of the alerts causing mutagenicity were only elicited following bacterial metabolism. Similarly, some of the alerts causing mutagenicity could also cause skin sensitization but only after skin metabolism. External toxicokinetic effects, such as exogenous S9 metabolism in mutagenicity, were not accounted for. Analysis with S9 metabolic activation was kept distinct from any analysis without accounting for metabolism. The worst case scenario analysis was taken as the outcome, i.e., if a positive alert with respect to skin sensitization or mutagenicity was observed in at least one case. 3.3. Mechanistic Comparison between DNA and Protein Binding Alerts. 3.3.1. Protein Reactivity of DNA Alerts. There are 19 alerts for DNA binding within TIMES 2.6.2. The alert carbenium and episulfonium ions RX{+} (RX ) C, S) has not been included in the comparative analysis since these ions do not exist in parent molecules, and protein binding alerts are defined as proceeding from the parent chemicals. This alert is included in the TIMES DNA list since it is consistent with the specific metabolic activation simulated by the rat liver S9 metabolism model. The remaining 18 alerts were evaluated with reference to their skin sensitization potential. Fifteen of the alerts could also interact with protein by virtue of the established alerts within TIMES-SS for skin sensitization. Five out of the 15 alerts exhibited the same mechanism of action as hypothesized for skin sensitization, 4 showed partial overlap in mechanism, and 6 had different underlying mechanisms. In addition, two DNA alerts partially overlapped with those alerts present for skin sensitization, and one DNA alert was defined as nonsensitizing. These cases will be discussed in turn and illustrated with examples where feasible.

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Figure 1. Representative nucleophiles that are relevant for DNA (a) and protein binding (b) along with the calculated soft, Sµ [(a.u.)2/eV], and hard, Q [a.u.], nucleophilicity indices (parametric ranges are due to the structural flexibility of the residuals).

3.3.1.1. Alerts Causing DNA Binding and Protein Binding by the Same Mechanism. Table 1 lists the five alerts where binding occurs by the same mechanism. 3.3.1.1.1. Beta-Lactone Group. Lactones are direct-acting alkylating or acylating agents. This alkylating/acylating activity is dependent upon the ring strain in the order 4-membered (βlactone) > 6-membered (δ-lactone) g 5-membered (γ-lactone) . rings with more than 6 atoms. β-Butyrolactone and β-propiolactone are both confirmed experimental rodent carcinogens and Ames mutagens (36). Hemminki described the nucleid acid reactivity of these and other similar compounds (48). Lactones have been studied in the context of skin sensitization and

categorized as acylating agents (20). The reaction scheme is provided as Scheme 1 in the Supporting Information. 3.3.1.1.2. Quinones. Nucleophiles presented by DNA bases can form adducts with the quinone ring by the 1,4-Michaeltype addition reaction (49). For example, the naturally occurring anthraquinone derivative lucidin (1,3-dihydroxy-2-hydroxymethylanthraquinone) has been incubated with DNA in the presence of the S9 mixture forming up to five different DNA adducts. The genotoxic activity of this compound has also been tested in a battery of short-term tests. The compound was found to be mutagenic in five Salmonella typhimurium (S. typhimurium) strains without metabolic activation, but the mutagenicity

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Figure 2. Spectrum of hard (a) and soft (b) nucleophilicity in amino acids and DNA bases.

Table 1. Alerts Causing DNA Binding and Protein Binding by the Same Mechanism

was increased after the addition of the rat liver S9 mixture (49-51). 9,10-Anthraquinone, which contains a quinone substructure, is also mutagenic in the Ames test (36). orthoQuinones and para-quinones react with proteins via the 1,4addition of lysine NH2 or cysteine SH groups across the ring system. They often are formed by the oxidation of para- and ortho-dihydroxy aromatics acting as pro-Michael acceptors, for

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example, hydroquinone oxidizes to benzoquinone (52, 53). The reaction scheme is provided as Scheme 2 in the Supporting Information. 3.3.1.1.3. Aliphatic Epoxides, Aziridine, and Epoxyethers. Epoxides are potential alkylating agents. The strained ring system facilitates the opening of the ring to generate a carbonium ion which can alkylate key macromolecules to exert the mutagenic effect. The alkylating activity may be substantially inhibited by ring substitution, particularly by bulky or hydrophilic groups. Typically, epoxides at terminal ends of aliphatic chains are of much greater concern than those embedded inside an aliphatic chain or those embedded in a rigid cycloaliphatic ring (36). All of the simple epoxides react with DNA at the most nucleophilic sites (ring nitrogens) to form 2-hydroxy-2alkyl adducts. These are fairly unstable due to the presence of a charged quaternary nitrogen at the site of alkylation and frequently undergo depurination to remove the charge, thus resulting in the formation of highly mutagenic apurinic sites (54). The same SN2 (bimolecular nucleophilic attack) mechanism occurs in skin sensitizers, i.e., ring-opening to form a reactive cation that can alkylate nucleophilic sites on proteins (55). The reaction scheme is provided as Scheme 3 in the Supporting Information. 3.3.1.1.4. Haloalkanes and Compounds. Aliphatic haloalkanes of the form RCH2X or X-CH2-CH2-X are thought to react by a SN2 pathway. 1,2-Dichloroethane, for example, is carcinogenic in experimental animals, where in ViVo and in Vitro studies in rodents have shown that the primary metabolic pathway for 1,2-dichloroethane probably involves conjugation with glutathione. Acyl halides act as acyl transfer agents, are known to undergo nucleophilic substitution reactions, and readily react with nucleophilic substances such as DNA (54). In the context of skin sensitization, primary alkyl halides have been investigated by Basketter et al. (56) who derived a QSAR model for a series of long chain bromoalkanes. The reaction chemistry categorization of alkyl and acyl halides has been discussed in more detail elsewhere (57, 58). 3.3.1.1.5. Sulfate, Sulfonate, Phosphate, Thiophosphates. Primary alkyl groups bonded to leaving groups such as -OSO2R, -OSO2OR, -OPO(OR)2, or simple groups 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. 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 a part of a ring system (20, 55). The same mechanism is also applicable for mutagenicity. Phosphorylation is another feasible route, i.e., an attack at the P center. Methylparathion, which belongs to the organothiophosphate group of insecticides, exhibits mutagenicity (59). Two chemotoxic mechanisms have been suggested for this: phosphorylation and alkylation. Compared with the carbon atom of the alkyl group, the phosphorus atom is markedly more electron-deficient and susceptible to attack by nucleophiles. 3.3.1.2. Alerts Causing DNA Binding and Protein Binding by Partially the Same Mechanism. These are listed in Table 2. 3.3.1.2.1. r,β-Unsaturated Aldehydes. DNA adducts of crotonaldehyde H3C-CHdCH-C(O)H and other R,β-unsaturated compounds formed by the Michael-type 1,4-addition have been reported by Hecht et al. (60). Eder et al. (61, 62) have studied the mutagenicity of 2-alkylpropenals and DNA adducts of various R,β-unsaturated aldehydes. 2-Hexenal, which is mutagenic and genotoxic, was found to also form cyclic 1,N2-

ReView Table 2. Alerts Causing DNA Binding and Protein Binding by Partially the Same Mechanism

propanodeoxyguanosine adducts (63). The following scheme (Scheme 1) for the formation of the DNA deoxyguanosine adduct has been proposed. This scheme suggests a combination between 1,2-AN and 1,4-AN Michael-type reactions with the formation of cyclic adducts. Benigni et al. (64) have also developed QSAR models for the mutagenicity and carcinogenicity potential of simple and R,β-unsaturated aldehydes. The sensitization potential of unsaturated aldehydes has been extensively studied. Patlewicz et al. (65) categorized R,βunsaturated aldehydes as Michael acceptors. 3.3.1.2.2. Hydrazines. Two mechanisms are proposed for hydrazines: (a) a nucleophilic addition reaction or (b) a free radical mechanism. In the case of reaction (a) direct intercalation of hydrazines with nucleophilic sites (carbonyl groups) of purine and pyrimidine bases by the formation of hydrazones can be expected; for example, phenylhydrazine reacts in this way as described by Gilbert (66) (Scheme 2a). However, the more detailed mechanism of the initial DNA adduct formation for, e.g., hydrazine, appears to be more complicated. It includes an AN-nucleophilic attack on the carbonyl group of the thymine base, resulting in breakage of the C(O)-NH-bond and cyclization to a five-membered heterocycle (66) (Scheme 2b). The proposed radical mechanism (depicted in Scheme 2c) is also implicated in skin sensitization induction (Scheme 2d) (55). 3.3.1.2.3. Nitrogen Mustard Species. The primary mechanism of action involves attack on the nucleophilic sites of macromolecules. These reactions may occur either by an SN1 (substitution nucleophilic unimolecular) mechanism with electrophilic carbocation intermediates or by the SN2 route, involving direct substitution. Most nitrogen and oxygen positions on DNA bases can be alkylated under appropriate conditions, but guanine O6 and N7, cytosine O2, and adenine N1 and N3 are recurrent targets (67). These pathways have been summarized in Scheme 3a and b. For skin sensitization, the SN2 route dominates (68-70). 3.3.1.2.4. Polyaromatic Hydrocarbons. Polycyclic aromatic hydrocarbons (PAHs) are an important class of biologically active compounds. They are mutagenic and carcinogenic, and undergo physical and covalent interactions with DNA. The physical interaction is an intercalation (71-73), and the covalent one is through the generation of electrophilic arene epoxides (74). A critical step in the mechanism is considered to be the epoxide ring-opening to yield a carbenium ion at the benzylic position of the epoxide function. The formation of benzene diol epoxide (or trans-dihydrocatechol epoxide) is crucial in this


respect and is associated with the presence of the so-called bay regions in the PAH’s structure (75). Any compound that contains three or more fused aromatic rings must be considered suspect, including those with nitrogen and even sulfur-containing heterocyclic aromatic fragments. Compounds based on the phenanthrene substructure are often more carcinogenic than those containing the linear anthracene moiety. The fact that quinoline has been reported to be carcinogenic and several derivatives mutagenic suggest that two fused rings are enough to produce an active compound if N-heterocyclic aromatic rings are involved. The skin sensitization behavior of benzo[a]pyrene was discussed by Roberts et al. (57), who classified it as a pro-SN2 electrophile by virtue of forming an electrophilic diol epoxide upon metabolic oxidation. The critical step in the skin sensitization mechanism is also expected to be epoxide ring-opening. 3.3.1.3. DNA Alerts Which Partially Overlap with Alerts Present for Skin Sensitization. These are listed in Table 3. 3.3.1.3.1. Esters/Amides. Activated benzoate esters are categorized as acyl transfer agents. 3-Chloro-phenyl benzoate and 3-methoxy phenyl benzoate are examples and are themselves strong sensitizers (53, 70). Nucleophilic attack occurs at the carbonyl C and is facilitated by the stable leaving group, itself a reasonably acidic conjugate acid. For mutagenicity, direct alkylation can occur for both aliphatic and aromatic esters/ amides (49, 76). 3.3.1.3.2. Ureas. The alerts that are flagged for DNA binding are shown in Figure 3. The proposed mechanism for the mutagenic effect of structure 1 in Figure 3 is illustrated in Scheme 4. The urea is hydrolyzed to the corresponding amine and carboxylate. This amine is often aromatic and can be enzymatically hydroxylated to form a N-hydroxylamine intermediate. This undergoes a phase II conjugation reaction to generate the more reactive N-O sulfate and/or N-O acetyl conjugates. The conjugate acids are themselves excellent leaving groups; they give rise to a highly reactive nitrenium ion. This nitrenium ion is the electrophilic species that is presumed to readily bind covalently to cellular DNA and RNA (49). The pathway is somewhat different in the case of structure 2. Hydrolysis would result in the release of carbon dioxide and the formation of an electrophilic alkyl cation which facilitates direct alkylation. Scheme 5 illustrates this pathway. Structures 1 and 2 shown in Figure 4 are oxophilic SN2 electrophiles. Various pathways exist, which are described in more detail in Roberts et al. (57). Attack may occur on the central carbon atom or the nitroso group. A direct alkylation may also be feasible. The possible reaction pathways are outlined as part of the Supporting Information (Schemes 4a-d). 3.3.1.4. DNA Alerts Which Are Not Active for Skin Sensitization. Only one DNA alert was identified which was not active for skin sensitization. This was an aliphatic nitro group. Aliphatic nitro groups such as 2-nitropropane can form a nitronate which is unstable and goes on to form a nitrite, which can directly attack nucleotide bases in DNA (77). 2-Nitropropane has been found to be mutagenic in several strains of S. typhimurium used in the Ames test, both with and without an exogenous activating system (S9). The following scheme for in Vitro microbial biodegradation and biotransformation of this compound has been proposed (Scheme 6). It is known that this reaction does not occur in skin (78). 3.3.1.5. Alerts Causing DNA Binding and Protein Binding by Different Mechanisms. Six known alerts cause DNA and protein binding by different mechanisms. These are

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Scheme 1. Formation of DNA Deoxyguanosine Adduct from r,β-Unsaturated Aldehydes

Scheme 2. Proposed Reaction Schemes for Hydrazine

summarized in Table 4. The active electrophilic species responsible for mutagenicity is identical in the first four cases. The aromatic nitro group, aromatic nitroso group, aromatic hydroxylamine, and aromatic amine all give rise to the formation of a nitrenium ion. The metabolic steps N-oxidation and nitroreduction to yield N-hydroxyarylamines are crucial for the genotoxic properties of aromatic amines and nitroarenes (79). Nitroaromatic compounds are first converted to their nitroso intermediates which are then reduced to result in the formation of a N-hydroxylamine species, which will undergo a conjugation reaction before generating the electrophilic nitrenium ion. Anilines are oxidized to their hydroxylamine forms first before following the same pathway. The mechanistic path overlaps with that already described for ureas, which is shown in Scheme 4. For skin sensitization, the active species for these four classes of chemicals is the nitroso group. The nitroso group is strongly electron withdrawing and more similar to a weak CdO bond. Additions to the double bond between nitrogen and oxygen consist of two mechanistic steps: a nucleophilic attack on the NdO group followed by a protonation of the anion (80). This pathway is shown in Scheme 5 of the Supporting Information. 3.3.1.5.1. Nitrosoamines. The mechanism is based on metabolic activation, primarily by the cytochrome CYP2E1 enzyme system and is consistent with N-nitrosoamines, containing at least one a-C(sp3)-H bond with respect to the N-nitrosogroup. N-Nitrosodimethylamine, for example, undergoes hydroxylation at the a-C(sp3)-carbon atom (aliphatic C-oxidation), to form an

Scheme 3. Nitrogen Mustard Reactionsa

a (a) SN2 and SN1 reactions at the oxygen nucleophilic site. (b) SN2 and SN1 reaction at the nitrogen nucleophilic site.

Table 3. DNA Alerts Which Partially Overlap with Alerts Present for Skin Sensitization

unstable a-hydroxynitrosoamine. The decomposition products of the latter are formaldehyde and methyl diazohydroxide. The alkyl groups of compounds such as methyl diazohydroxide are good leaving groups and, therefore, are powerful methylating agents that can add a small functional (e.g., CH3- group as methyl cation) at several different sites in DNA (formation of DNA-adducts) (81, 82). The metabolic bioactivation pathway to a highly reactive “ultimate carcinogen” is shown in Scheme 7. For skin sensitization, direct alkylation occurs when the nitrosoamines act as oxophilic SN2 electrophiles. Scheme 4c of the Supporting Information illustrates the pathway (57).

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Chem. Res. Toxicol., Vol. 23, No. 10, 2010 1527 Table 4. Alerts Causing DNA Binding and Protein Binding by Different Mechanisms

Figure 3. Typical DNA urea-like structural alerts.

Scheme 4. Formation of a Nitrenium Species

Scheme 5. Direct Alkylation of N-Alkyl-N-nitrosourea Derivatives

Figure 4. Typical urea structural alerts for skin sensitization.

Scheme 6. Formation of Nitrites for Aliphatic Nitro Compounds

3.3.1.5.2. Azo Groups. The azo linkage is the most labile portion of an azo dye molecule and may easily undergo enzymatic breakdown in mammalian organisms. The majority of azo dyes require metabolic activation for the reduction and cleavage of the azo linkage (azo group reduction) to the component aromatic amines in order to show mutagenicity. For skin sensitization, azo groups are metabolically transformed into nitroso intermediates as discussed already. This theoretical analysis reveals that many of the alerts established for DNA binding derived from the Ames test data have considerable commonality with those derived from skin sensitization data. Analysis of the experimental data identified from the training sets of the TIMES models found that 17 of the 18 DNA alerts were associated with positive skin sensitization data. Table 5 shows the alerts and the statistics of the chemicals in terms of their predicted and experimental outcomes in both the Ames and sensitization tests. Table 5 shows that there is a good agreement between the predicted and experimental results for Ames mutagenicity. For every alert aside from

Scheme 7. Likely Nitrosoamine Metabolic Bioactivation Pathway

that for the hydroxylamine, all of the Ames alerts were substantiated with positive sensitization data. This showed that the majority of Ames positive chemicals that triggered these alerts were also likely to be sensitizers, i.e., practically all of the Ames positive chemicals were also skin sensitizers. The converse analysis was undertaken to consider the protein binding and establish which shared a mechanistic basis for DNA binding. 3.3.2. DNA Reactivity of Protein Alerts. Of the 55 protein binding alerts, 19 could be attributed to DNA binding with the same chemical mechanism, two of which required metabolic activation (S9). Six alerts could be attributed to DNA binding but with a different chemical mechanism, and four of these required metabolic activation (S9). Thirty alerts were identified that could only be attributed to protein binding, but evaluation showed that 8 of these had the potential to be reactive with DNA (though this could not be substantiated with experimental data), and the remaining 22 were thought to be relevant for skin sensitization alone. These will be discussed in turn. In specific cases, evaluation of the protein alerts with respect to their potential relevance for DNA culminated in the creation of new alerts for the TIMES mutagenicity model. These cases are highlighted in the respective discussions below. 3.3.2.1. Protein Alerts That Are Potential DNA Binders with the Same Mechanism. The 19 protein alerts that could be attributed to DNA binding with the same chemical mechanism are listed in Table 6. The first 8 alerts shown in Table 6 (epoxides, alkyl halides, activated halides, quinone, β-lactone, acyl halide, sulfonate/ sulfate, and phosphate/thiophosphates) have been already been discussed in the previous section. 3.3.2.1.1. 1,2-Diketones (New DNA Alert for TIMES). These are established Schiff base formers, and their skin sensitization potential has been discussed by Patlewicz et al. (83). One of the most important chemical properties of the carbonyl group is its tendency to undergo nucleophilic addition reactions. Additions to carbonyl groups generally consist of two mechanistic steps: a nucleophilic attack on the carbonyl group and a protonation of the resulting anion. The nature of the substituents will affect the reactivity of the carbonyl groups

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a

These two chemicals are positive because the DNA alert is part of another protein binding alert (HalCNO2).

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ReView Table 6. List of Protein Binding Alerts with DNA Reactivity and with the Same Active Form


(53, 84). Dorado et al. (85) determined the relative mutagenicity of several R-dicarbonyl compounds in S. typhimurium strain TA100 without or with metabolic activation. The order of mutagenicity of R-diketones was evaluated as follows: 2,3butanedione-diacetyl > 3,4-hexanedione (>camphorquinone). The latter had low or no mutagenic activity. The mutagenic activity of R-diketones was related to the formation of adducts with guanine and/or guanosine bases (85, 86) (Scheme 6 in the Supporting Information shows the proposed pathway). Lee et al. (87) reported that R-dicarbonyl compounds or R-ketoaldehydes were mainly responsible for forming inter- and intramolecular cross-links of proteins by a Schiff base mechanism. 3.3.2.1.2. Simple Aldehydes (New DNA Alert for TIMES). Simple aldehydes are established to undergo Schiff base formation to exert their sensitization effect as described by Roberts et al. (84). Acetaldehyde, for example, reacts with DNA at the guanine fragment (Gua) to give a major Schiff base adduct N(2)-ethylidene-dGua (88, 89). The same or similar mechanism is likely to operate for other low-molecular weight aldehydes such as formaldehyde and 1,3-propanedial (malondialdehyde), which react to form adducts with deoxyguanosine and deoxyadenosine fragments of DNA (see Scheme 7 in the Supporting Information). Due to their reactivity, aldehydes are able to interact with electron-rich biological macromolecules and show general toxicity, mutagenicity, allergenic reactions, and carcinogenicity. The electrophilicity, hydrophobicity, and other parameters exert a certain influence on these toxicity end points (90). 3.3.2.1.3. Peroxides: Organic Hydroperoxides/Diaryl and Dialkyl Peroxides (New DNA Alert for TIMES). Organic hydroperoxides are chemicals that possess a R-OOH structure and are often generated as a product of air oxidation. The -OOH group is not electrophilic and is not protein reactive. It is thought that peroxides undergo one electron reduction in the skin to RO· and OH-. The radicals are formed by the homolytic cleavage of these bonds and are stimulated by heat or light. Diaryl and dialkyl peroxides which contain the very weak O-O bond are also associated with the generation of free radicals. ArO/AlkyO (or other Ar/AlkylO- derived radicals) can act as haptens. Radicals can add to a CdC double bond in a protein residue such as Tyr, Phe, or Trp or abstract a hydrogen from a protonated Lys or His residue (53). Alkoxyl radicals have been detected upon incubation of hydroperoxides and peroxyesters with cytochrome P450 and proposed to be involved in DNA damaging activity. These radicals, similar to hydroxyl ones, are also involved in oxidative stress in ViVo (91). Mutagenicity of various organic peroxy compounds, including tert-butyl hydroperoxide (TBHP), cumene hydroperoxide, 1,2,3,4-tetrahydronaphtyhalene hydroperoxide, etc., has been established (92-94). The following scheme (Scheme 8) for the formation of DNA adducts formed by metabolic activation of organic peroxy compounds can be proposed (91). 3.3.2.1.4. Isocyanates (New DNA Alert for TIMES). Isocyanates are highly reactive with a variety of functional groups found on biological macromolecules. They have a finite lifetime that enables them to react with selected functional groups but do not exist long enough to cause significant, nonspecific modification of the biological macromolecules. Isocyanates are protein modification reagents and are probably also Schiff base sensitizers. The longer aliphatic chain and the phenyl carbon bond to isocyanate groups may lead to a weak skin sensitization effect (58, 95, 96). Methylisocyanate is a direct-acting acylating compound found to react with 2′-deoxyribonucleosides, acylating the exocyclic

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Scheme 8. Proposed Radical Reaction Mechanism for Peroxides

amino groups and forming methylcarbamoyl DNA adducts (97). Similar observations have been made with 2-naphthylisocyanate (98) and phenylisocyanate (99). The mutagenicity of toluene diisocyanates as important industrial intermediates (100), diphenylmethanediisocyanate (101), and methylisocyanate (102) have also been established and confirmed. In the case of aromatic isocyanates, one mechanism of mutation and increased risk of cancer is the hydrolytic formation of amines which are metabolized to reactive species toward DNA (103). For example, one of the biotransformation products of diphenylmethane diisocyanate is 4,4′-methylenedianiline, formed via hydrolytic cleavage, which is known to be genotoxic. It has been concluded that the positive mutagenicity test results obtained with aromatic isocyanates under certain conditions are due to the formation of aromatic amines, which are further metabolized to Nhydroxylamines and DNA-reactive electrophilic nitrenium ions (101). 3.3.2.1.5. Aromatic Halogenated Compounds (New DNA Alert for TIMES). Halogenated aromatic compounds undergo nucleophilic substitution reactions. Nucleophilic attack occurs at the ring carbon attached to the halide atom and is facilitated by the presence of electron withdrawing groups such as nitro groups in the ortho and para ring positions. Roberts et al. (57) called them SNAr electrophiles, where a classic example is the strong sensitizer, 2,4-dinitrochlorobenzene (DNCB). The mutagenicity of 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4nitrobenzene has been established (104, 105). The 1-halogen substituted 2,4-dinitrobenzenes have also been found to be mutagenic in the S. typhimurium strain TA98 with an activity order of 1-fluoro > 1-chloro > 1-bromo > 1-iodo. These compounds have been suggested to react directly with bacterial DNA through nucleophilic substitution of the halogen atom (106). 3.3.2.1.6. Saturated Sultones (New DNA Alert for TIMES). Ring-opening reactions by an SN2 mechanism has been observed in reactions with model nucleophiles such as butylamine and accounts for their strong sensitization effect (17). 1,3-Propane sultone was found to be mutagenic in S. typhimurium strains TA100 and TA1535 without metabolic activation (107, 108) and clastogenic in Chinese hamster lung (CHL) cells (109, 110). 1,3-Propane sultone is a direct alkylating agent (111), which was shown to react with the guanosine base of DNA (112, 113). 3.3.2.1.7. Conjugated Systems Containing O,S,N-Atoms (New DNA Alert for TIMES). Conjugated systems containing O,S, N-atoms comprise the general case of R,β-unsaturated compounds that are sensitizers by virtue of their ability to behave as Michael acceptors (53, 65). It has been published that acrylonitrile and acrylamide can directly alkylate DNA fragments under physiological pH and temperature conditions,

according to schemes for the formation of DNA adducts given in Solomon and Segal (114). Data for the direct-acting mutagenicity of other R,β-unsaturated functional compounds such as ethylvinyl ketone (115), acrylamides, and vinyl sulfones (116) are also available. For mutagenicity, the Michael addition route is also favored. 3.3.2.1.8. Sulfonyl Azide (New DNA Alert in TIMES). The mechanism of the reaction is the same as the protein acylation by carboxylic acid derivatives (acid halides, acid anhydrides, and esters). Sulfonyl azides may react with protein amines forming the corresponding sulfonic acid amides (117). The mutagenic mechanisms of sulfonyl azides are less clear-cut; the same acylation type route is feasible, but a free radical mechanism is also possible on the basis of experimental data on 4,4′-oxidibenzenesulfonyl azide. 4,4′-Oxidibenzenesulfonyl azide was found to be mutagenic in the Ames test. In cultured mammalian cells and in the in ViVo micronucleus assay, 4,4′oxidibenzenesulfonyl azide showed negative results (118). One possible reason for the suggested mutagenicity in bacterial cells may be assumed by considering the following interactions as evidenced by the chemical modification (functionalization) of poly(propylene) (119), according to the proposed radical mechanism (Scheme 9). 3.3.2.1.9. Conjugated Lactones. For skin sensitization and mutagenicity, the conjugated lactones act by a Michael addition route as already described for R,β-unsaturated compounds. 3.3.2.1.10. Acyl Halide of a Phosphonic Acid (New DNA Alert in TIMES). The acyl halide of a phosphonic acid behaves as an acylating agent as has already been described for acyl halides and phosphates. The skin sensitization effects of such compounds have been discussed by Ashby et al. (120). The mutagenicity behavior in terms of phosphorylation has been outlined in section 3.3.1.1.5 (59). 3.3.3.1. Protein Alerts That Are Potential DNA Binders with a Different Mechanism. Table 7 lists these examples, the first two (nitroso and nitrosoamines) have already been discussed in the previous section. 3.3.3.1.1. N-Trihalomethyl Diacylimides (New DNA Alert in TIMES). The electronegative CHal3 group will stabilize a negative charge on N, making the imide structure more like an anhydride in reactivity; therefore, the reaction will be acylation. Acylation chemistry in the context of skin sensitization is discussed in refs 57 and 58. No direct data about the mutagenicity and clastogenicity of similar compounds have been found. However, the structurally similar compound chloropicrine was found to undergo glutathione bioactivation in the Ames test. It was also evaluated as a strong lacrimator and induced sister chromatid exchanges in cultured human lymphocytes. Chloropicrin became mutagenic on the addition of S9 or glutathione

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Chem. Res. Toxicol., Vol. 23, No. 10, 2010 1531 Scheme 9. Free Radical Mechanism Proposed for Sulphonyl Azides

Table 7. List of Protein Binding Alerts with DNA Reactivity and with a Different Active form

(121). Chloropicrin was also reported to undergo facile metabolic dechlorination on reactions with biological thiols such as glutathione and proteins (122). Chemically or enzymatically generated trichloromethyl radicals are relatively stable due to the delocalization of the single electron with the participation of the adjacent bonds and the other halogens. For example, carbon tetrachloride is metabolized in the cell microsomal membranes to a highly toxic trichloromethyl radical that initiates lipid peroxidation and binds to and destroys cell enzymes (cytochrome P-450), lipids, and proteins in various cell membranes. Its carcinogenicity in animal species has been well documented (123). Many polyhaloalkanes, including CCl4, are procarcinogens that become activated via a sequence of redox reactions, whereby CYP450 acts as the principal catalyst for dehalogenation, according to the following free radical mechanism (124). On the basis of the above information and assuming that the N-CX3 bond in the case of N-trihalomethyl diacetylamines is more likely to be cleaved homolytically, due to the opposite directions of the -I and +Meffects of the three chlorine atoms and the nitrogen one bound to the carbonyl groups, respectively, the following chemical mechanisms for both the protein and DNA reactivity of the structural alert are proposed (Scheme 10). This structural alert is possibly genotoxic, causing both mutagenicity and chromosomal aberration effects. 3.3.3.1.2. Isothiocyanate (New DNA Alert in TIMES). Isothiocyanates are formed by substituting sulfur for oxygen in the isocyanate group. They generally act as electrophiles with the carbon atom between N and S as the electrophilic center and probably are Schiff base sensitizers (58). Electrophilic and radical mechanisms appear to be involved in the mutagenic potential of isothiocyanates. There are conflicting reports,

concerning the possible mutagenicity and carcinogenicity of isothiocyanates. Two isothiocyanates commonly found in the human diet allyl isothiocyanate and phenethyl isocyanate have been tested for genotoxic effects in the Ames test with S. typhimurium strains TA 98 and TA 100. It is suggested that reactive oxygen species might be involved in the genotoxic effects of isothiocyanates. On the other hand, isothiocyanates have been advocated as very promising anticancer agents (125-127). Naturally occurring isothiocyanate compounds are effective chemoprotective agents against chemical carcinogenesis in laboratory animals (128). According to other studies (129), allyl isothiocyanate exhibits carcinogenic potential, and benzyl isothiocyanate and phenethyl isothiocyanate have tumorpromoting activities. Allyl isothiocyanate has caused Cu(II)mediated DNA damage and formation of 8-oxo-7,8-dihydro2′-deoxyguanosine more strongly than benzyl and phenethyl isothiocyanates. H2O2, generating the ultimate reactive oxygen species, is involved in cellular DNA damage and the generation of superoxide and other reactive oxygen species correlated with the yield of -SH groups from isothiocyanates (Scheme 8 in the Supporting Information). The following study proposes an electrophilic pathway. Incubation of 2-naphthylisothiocyanate, microsomes, and NADPH yielded N,N′-dinaphthylurea. This urea derivative was formed by the production of the known genotoxicant, 2-naphthylisocyanate, which reacted with its hydrolysis product, 2-aminonaphthalene, to give the symmetrical urea. The urea formed can then go on to form an electrophilic nitrenium ion as described previously (130). 3.3.3.1.3. Thiol (New DNA Alert in TIMES). Thiols may react with proteins by mechanisms similar to those of protein glutathionylation, where a major path is thiol-disulfide exchange between a free PrSH and GSSG (cysteine). The interchange reactions between protein sulfhydryl groups and the disulfides may be observed when pH is alkaline. Disulfides containing allyl or furfuryl groups were more reactive than saturated disulfides (131-133). A characteristic feature of most thiols is their ability to act as reducing agents and hence accept electrons. In the case of an oxidant-thiol interaction, the oxidant is neutralized to a relatively less toxic byproduct at the expense of the reducing power of thiol, which is oxidized to a disulfide. A thyil radical is produced when a thiol loses the H-atom from the -SH group or loses an electron from sulfur, followed by a proton. Under the conditions of physiological pH, thyil radicals are unstable and may recombine to form the corresponding disulfide. In biological systems, there are specific reductases that recycle disulfides to thiols using cellular reducing equivalents such as NADH, and thus, the power of cellular metabolism is coupled to maintain a favorable redox state of thiols. If imbalance in this process, favoring the generation of prooxidants over antioxidants, occurs for any reason, this results in an oxidative stress and, in some cases, a mutagenic response

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Scheme 10. DNA Mechanism Proposed for N-Trihalomethyl Diacylimides

Scheme 11. Likely Free Radical Route for Dithiocarbamates

(134). The most probable route for the initiation of an oxidative signal is the generation of a superoxide radical anion by oxidoreductase enzymes, which then forms H2O2 through superoxide dismutase-catalyzed disproportionation. Under the same conditions that generate peroxide, cellular thiols can also be oxidized to either a disulfide-S-monooxide or a disulfide-Sdioxide (DSO). Oxidation of thiols either by H2O2 or singlet oxygen has been shown to readily generate mixtures of DSO and other end-products. The one-electron oxidation of GSH to the thyil radicals by oxidoreductase or peroxidase action also opens several free radical pathways by which DSO can be produced. The predominant DSO-producing reaction is assumed to occur by combination of a thyil radical with molecular oxygen to generate thioperoxyl radical, which can combine with a second thyil radical to form DSO. Under such oxidizing conditions, DSO can be formed in significant concentrations. DSOs are considered to be a highly potent and specific thiol oxidizing species with implied cellular targets and redox transformation pathways (135, 136). Moreover, DSOs rapidly oxidize and subsequently inhibit thiol proteins, and enzymes and can be considered as a separate class of oxidative stressors. It has also been stated that oxidative stress arises from the imbalance in the metabolism of redox-active species, and additional reactive sulfur species (RSS) are formed in ViVo under these conditions. RSS are likely to include disulfide S-oxides, sulfenic acids, and thyil radicals which modulate the redox status of biological thiols and disulfides (137, 138). The likely radical mechanisms taking place are illustrated in Scheme 9 of the Supporting Information. 3.3.3.1.4. Dithiocarbamates (New DNA Alert in TIMES). Sulfur is much less electronegative than oxygen; in fact, it has the same electronegativity as that of carbon. It forms reasonably strong bonds to carbon, strong enough for the compounds to be stable but weak enough for selective cleavage. Nucleophilic displacement reactions involving alkyl dithioesters are thought to proceed via the formation of tetrahedral intermediate. The CdS bond is almost nonpolar, but the nucleophilic attack of proteins is realized with ease because of the poor effective interaction between C2p and S3p orbitals that form the π-bond. Dithiocarbamate esters react with protein amino or thiol groups via the addition-elimination process (55). The mutagenicity of dithiocarbamic acid derivatives depends on an indirect effect via oxygen radicals. Dithiocarbamates are capable of exerting

opposing effects on the cellular redox balance by decreasing single-electron radical species (reduction) and causing a twoelectron oxidation of glutathione and protein thiols as a consequence of redox cycling. It has been proposed that thiuram disulfides (oxidized dithiocarbamates) or copper-dithiocarbamate complexes represent the chemical species responsible for the pro-oxidant effects of dithiocarbamates. Oxidation of the dithiocarbamate can be copper-dependent or copper-independent. It is followed by dimerization of dithiocarbamate thiyl radicals generating thiuram disulfide. Dithiocarbamates are also powerful metal chelators, which can additionally induce oxidative damage to biomolecules, particularly as transition metals such as copper, cadmium, and chromium are involved (139). The following scheme of possible bioactivation of dithiocarbamates could be proposed (Scheme 11). 3.3.4.1. Protein Alerts That Are Potential DNA Binders but with No Substantiating Experimental Data. Eight protein alerts were identified as potential DNA binders but lacked concrete experimental data to substantiate the proposed mechanisms. These are listed in Table 8 with their proposed pathways. The remaining 22 alerts that are specific to sensitization alone are provided in Table 9 for completeness, athough they will not be discussed further. Sensitizers that flagged one of the 55 protein binding alerts were evaluated to see whether they were additionally positive in either a chromosomal aberration test or an Ames test. The results are summarized in Table 10. In 24 cases, sensitizers were also positive in both the Ames and CA tests. In 24 further cases, sensitizers were only positive in the CA test. In 6 cases, sensitizers were negative in the other 2 tests, and in one case, a compound was a sensitizer and positive in the Ames test but negative in the CA test. The seven cases are summarized in Table 11. They are evaluated in more detail below to rationalize the absence of mutagenic effects. 3.3.5.1. Alerts That Were Sensitizers but Had Negative Results in Ames and CA. 3.3.5.1.1. Carbodiimides. The mechanism shown in Scheme 12a could explain the observed skin sensitization potential of carbodiimides such as dicyclohexylcarbodiimide and diisopropylcarbodiimide which have been assessed in both the mouse ear swelling test (MEST) and the murine local lymph node assay (LLNA). These chemicals have been identified as both irritants and contact sensitizers (140-143). Carbodiimides are thought

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Table 9. Protein Binding Alerts Evaluated Not to Be DNA Reactive

Table 10. Mutagenic and Chromosomal Aberration Potency of Alerts Causing Skin Sensitization # of alerts/effect

SS

Ames

CA

24 24 6 1

Pos Pos Pos Pos

Pos Neg Neg Pos

Pos Pos Neg Neg

to be nonclastogenic due to the potential of a detoxification hydrolysis reaction occurring as per Scheme 12b (144-148). There were no carbodiimide examples in the TIMES training set, but experimental data found elsewhere supports this detoxification route (149, 150). Carbodiimides might well be Ames mutagenic, though no examples were identified in the training sets to support this assertion. Potential DNA reactivity is thought to occur by way of interaction with the phosphate residue of a nucleoside as shown in the scheme within Table 8. 3.3.5.1.2. Thiosulfonic/Thiosulfonates. The documented protein binding route for these substances are described elsewhere (131-133) (Scheme 10 in the Supporting Information illustrates the pathway). No DNA reactivity is expected under biologically relevant conditions. This is additionally supported by the two negative Ames results identified from the TIMES training set. No examples were identified with CA data, and more work needs to be done to identify additional evidence to support or refute the ability of thiosulfonic acids to be clastogenic (151). 3.3.5.1.3. Sulfonic Acids. The protein binding mechanism for these is described in ref 152 (Scheme 11 illustrates the pathway). No DNA reactivity is expected under biologically relevant conditions, though no Ames negative results were identified from the TIMES training set to support this hypothesis. As far as clastogenic effects are concerned, while no training

Table 11. Sensitization Alerts Not Causing Ames and CA

a

Not causing CA only.

set data was identified, clastogenicity was thought unlikely due to the instability of these compounds. Arenesulfinic acids can undergo disproportionation in temperatures between 25 and 100 °C, yielding thiosulfonates and arenesulfonic acids (153). Scheme 13 shows the potential mechanism. 3.3.5.1.4. δ-Lactones. The sensitization ability of δ-lactones is described in Payne and Walsh (55) and Gerner et al. (152). Alicyclic, alicylic fused, and aromatic fused ring lactones are

ReView Scheme 12. Reaction Mechanism for Carbodiimides and Their Potential Detoxification Pathway by Hydrolysis

Scheme 13. Proposed Pathway for the Disproportionation of Sulfinic Acids


expected to be negative in chromosomal aberration tests due to a hydrolysis detoxifying mechanism (158). No training set experimental data for mutagenicity and clastogenicity of R,βunsaturated esters was identified. 3.3.5.2. Alerts That Were Sensitizers and Caused Ames but Not CA. There was one sensitization alert, sulfonyl azides, which caused Ames but not CA. This has been discussed in section 3.3.2.1.8. The following justification is proposed for the lack of clastogenicity for sulfonyl azides. The likely mechanism for skin protein reactivity of this alert is possibly conditioned by UV irradiation. Such a irradiation mediated mechanism of interaction with protein is not likely under the test conditions for establishing CA. Overall, of the seven cases, the absence of clastogenicity effects could be rationalized by way of detoxification mechanisms (4 cases): in one case, the alert was found to be relatively unstable in air (sulfonic acids), and in another case, further work is needed (thiosulfonic/thiosulfonates).

4. Summary

used as flavoring agents and are not mutagenic in Vitro in the Ames test or in DNA repair assays. They are rapidly metabolized in ViVo to compounds of lower toxicological potential (154). The predominance of negative results for dihydrocoumarin, for example, in CHO cells in Vitro and in assays in ViVo, suggests a lack of genotoxicity. It is anticipated that dihydrocoumarin could be hydrolyzed to the corresponding o-hydroxyphenylpropionic acid which may either be conjugated with glycine prior to excretion or oxidized and cleaved to yield o-hydroxybenzoic acid (155). Octahydrocoumarine may also be hydrolyzed to a carboxylic acid containing an odd number of carbons in the side chain. Oxidation of this side chain and cleavage yields 2-hydroxycyclohexanecarboxylic acid, which may be subsequently aromatized to a benzoic acid and excreted mainly as hippuric acid (156) (Schemes 12 and 13 in the Supporting Information illustrate these pathways). Negative results in Ames and CA were also identified in the TIMES training sets. 3.3.5.1.5. Activated Esters. The acylation mechanism of these activated esters in terms of their sensitization and mutagenicity potential have already been discussed in section 3.3.1.3.1. For mutagenicity, three Ames negative results were identified and one Ames positive with this alerting group. A CA positive result was also identified. The positive results identified in the training sets were due to benzyl groups which could activate the esters to act as DNA alkylation agents. In the remaining cases, no DNA activity would be expected due to the expected lower nucleophilicity of the primary amino group attached to the purine/pyrimidine bases, compared to the aliphatic primary amino groups in proteins, the lack of alkylating capabilities, and the relatively low electrophilicity of carboxylate ester group, due to conjugation effects. Hydrolysis is catalyzed by classes of enzymes such as carboxyl esterases (see Scheme 14 in the Supporting Information). The final product can be excreted as the corresponding conjugates (156, 157). 3.3.5.1.6. r,β,-Unsaturated Esters. The protein binding effects of these activated esters has already been described above and in Roberts et al. (58). DNA activity is expected. They are

There is an increasing drive to use surrogate data in the ITS evaluation of end points such as skin sensitization particularly as a result of the rapidly changing chemical regulation landscape. Previous efforts in particular by Wolfreys and Basketter (43) have shown some concordance between mutagenicity tests and LLNA sensitization data owing to the same initial molecular initiating events for stable association with a nucleophile. These efforts have prompted our investigation of the underlying assumptions and whether from a chemical mechanism point of view, it is prudent to utilize mutagenicity data as a first screen for sensitization potential. A quantum chemical analysis of typical nucleotides and amino acids showed that there was significant overlap between the nucleophilic sites; moreover, the protein sites were typically more reactive. TIMES is a well established hybrid expert system containing structure-toxicity and structure-metabolism rules for end points including skin sensitization, Ames mutagenicity, and chromosomal aberration. An evaluation of the alerts for DNA binding included in the TIMES knowledge database was conducted to determine the extent to which these overlapped with protein binding alerts. Eighteen alerts were identified of which 15 alerts were shown to overlap with alerts for protein binding. In five instances, the mechanisms were identical, in four cases the mechanism partially overlapped, and in six cases the mechanisms were different. In two cases, the alerts partially overlapped, and in only one case, the alert in DNA did not flag a corresponding alert for protein binding. Since the structure-toxicity rules within TIMES are limited by the underlying training set, the same exercise was conducted on the protein binding alerts, and the potential for mutagenicity to occur was rationalized. Twentytwo alerts could be assigned for protein binding alone. However, 25 alerts were found to be applicable for mutagenicity, of which six required some kind of metabolic activation (S9). The analysis culminated in the development of a number of new alerts (23 in total) for DNA that have now been implemented into TIMES as part of the existing Ames mutagenicity and chromosomal aberration models. On the basis of the alert and training set comparisons, practically all Ames positive chemicals (83-93%) were also found to be skin sensitizers (SS). About 87% of CA active chemicals were skin sensitizers. A positive result in either the Ames test and/or CA test is likely to indicate the potential for a compound to be a sensitizer. Taking into consideration the 7 cases identified where mitigating factors play a role, the use of

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Figure 5. ITS for skin sensitization incorporating mutagenicity (Ames and CA information) (ITS adapted from REACH guidance).

an Ames mutagenicity test and in Vitro chromosomal aberration test as tier 1 and tier 2 screens in the assessment of skin sensitization potential offers significant advantages in terms of cost and animal usage while addressing hazard assessment requirements. This has been summarized in an ITS scheme shown in Figure 5. The first two steps of the ITS remain unchanged from that originally published in the technical guidance (6). Information from all sources (in ViVo, in Vitro, (Q)SAR, etc.) should be gathered, taking into account any potential adaptations to testing as described in column 2 of the legal text in Annex VII. However, before making a weight of evidence assessment, available information from in Vitro mutagenicity assays should be evaluated. This is because mutagenicity information is effectively a useful surrogate for chemical reactivity. On the basis of the analysis performed here, a positive result in either Ames and/or in Vitro CA provides compelling evidence that a substance of interest is a likely sensitizer, and this information could be considered as part of the weight of evidence approach for hazard identification purposes, i.e., classification and labeling. A negative outcome from in Vitro mutagenicity assays should be carefully considered in light of potential mitigating factors. In this study, we identified several cases where mutagenicity was not observed but where a sensitization response was still likely; these cases where known should also be considered in the weight of evidence assessment as they provide useful evidence to support hazard identification. Where no mutagenicity activity is observed and no mitigating alerts are playing a role, then a “no-classification” could be potentially considered. In these cases, the absence of structural alerts together with negative mutagenicity data is an indicator for both a lack of reactivity and sensitization response.

between the DNA and protein alerts taken from the TIMES knowledge database. Given that Ames mutagenicity is mainly conditioned by the direct interactions of chemicals with DNA, this test could be used in a first tier screen for identifying skin sensitizers which in turn are defined by their protein reactivity. Negative results in the Ames test, however, do not necessarily mean that chemicals are not skin sensitizers. In this respect, for their identification one could use another genotoxicity end point, chromosomal aberrations, which is determined by both direct interaction with DNA and/or proteins (histone and topoisomerase). The protein reactivity of chemicals, which is the second reasoning for eliciting chromosomal aberration, could make this genotoxicity end point useful as a second tier for identifying skin sensitization potential. These inferences were largely supported by the experimental data underpinning the TIMES models. In a few cases, there was a lack of correlation between skin sensitization and mutagenicity, but this could be explained through differences in metabolic detoxification in skin versus liver as well as differences in test protocols. Mutagenicity data can play a valuable role in the evaluation of skin sensitization potential as part of an ITS. Careful attention needs to be paid to the chemical mechanisms at play to ensure that results are interpreted in light of the end point of concern. Further work will involve evaluating the mechanisms in light of additional experimental data, in particular the data set from Wolfreys and Basketter (43). The differences in metabolic detoxification will be discussed in more detail in a separate manuscript. Supporting Information Available: Schemes illustrating the reaction pathways. This material is available free of charge via the Internet at http://pubs.acs.org.

5. Conclusions The theoretical and quantum chemical investigations showed a large overlap between the nucleophilic sites in DNA and proteins with predominantly high activity of protein sites. The theoretical commonality was confirmed with significant overlap

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