To the extent possible under law, AOP-Wiki has waived all copyright and related or neighboring rights to KE:396

Event: 396

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Covalent Binding, Protein

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
Covalent Binding, Protein

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help
Level of Biological Organization
Molecular

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Cell term
eukaryotic cell

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help
Process Object Action
protein binding electrophilic reagant increased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Skin Sensitisation AOP MolecularInitiatingEvent Agnes Aggy (send email) Open for citation & comment TFHA/WNT Endorsed
Covalent binding to proteins leads to Respiratory Sensitisation/Sensitization/Allergy MolecularInitiatingEvent Arthur Author (send email) Under Development: Contributions and Comments Welcome Under Development

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
human Homo sapiens NCBI
guinea pig Cavia porcellus NCBI
mouse Mus musculus NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

The molecular initiating event is covalent binding of electrophilic chemical species with selected nucleophilic molecular sites of action in proteins generating immunogenic neoantigens through a process termed haptenisation[1];[2]. In contrast to receptor-mediated chemical interactions electrophiles are not specific with regard to their molecular target. Moreover, some chemicals are able to react with several different nucleophilic chemical substituents. Therefore, the identification of the specific target protein is not considered to be critical. Moreover, it is recognized that reactivity measured with a particular nucleophilic target or model nucleophile does not necessarily reflect a specific chemical reaction, as many reactions target the same chemical substituent[3]. For toxicological endpoints for which protein binding is important, the biological nucleophile is assumed to be selected amino acids. The exact extent of adduct formation to each amino acid is dependent on the relative hardness / softness of the electrophile and nucleophile[3]. The inability to identify the exact biological nucleophile is deemed less important than information regarding the electrophile. As noted in the hard-soft acid base theory, a soft electrophile will have a relative preference for a soft nucleophile; while a hard electrophile will have a relative preference for a hard nucleophile. As a consequence, for a series of electrophiles assigned to the same mechanistic cluster within a particular domain, the relative rates of reactivity between each electrophile and any nucleophile will remain the same. In other words, while absolute reactivity may vary with protocols, relative reactivity will usually not vary significantly[3]. Binding experiments with small model nucleophiles reveal that, within a particular reaction within a mechanism, the rate of reactivity varies markedly. Moreover, while some compounds appear to bind exclusively with thiol or amine, others bind to a variety of nucleophiles. However, an electrophile is most likely to exhibit a preference for a particular nucleophile. In more complex systems, nucleophilic target preferences may be masked by other factors. It is self-evident that the number of cysteine and lysine residues within a protein will impact target probability. For example, for serum albumin, a major serum protein, 10% of the amino acid residues are lysine but albumin has very few free cysteine residues. Also, it is self-evident that a target site (e.g. cysteine or lysine) which is located on an exposed surface of a protein is more likely to react with an electrophile than one that is located within a grove or fold of a protein. Such steric constraints are imposed by the primary structure (i.e. amino acid sequence) of the peptide or protein, as well as the secondary and tertiary structure of proteins imposed by disulfide bridges, and folding and coiling. Similarly, the microenvironment of the reaction site (e.g. hydrophilic versus hydrophobic) may affect the probability of a particular reaction. Free cysteine residues are more abundant in proteins in the aqueous cytosol than in the non- aqueous biomembranes [4]. An ancillary event in identifying protein-binding is metabolism and/or abiotic transformation (e.g. autoxidation)[5].

How It Is Measured or Detected

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

In silico models, including physiological-based pharmacokinetic models and traditional structure activity ones, as well as in vitro and in vivo experimental approaches exist.

In silico Methods

It is generally recognized that reaction-based methods, as opposed to other means of defining chemical similarity, allow for easier interpretation and provide greater confidence in their use[6]. Chemical reactions related to covalent protein binding have recently been reviewed[7];[8];[9]. Measurements and estimations of reactivity have also recently been reviewed[1];[3]. Computational or in silico techniques to predict chemical reactivity have been developed; they vary in complexity from the relatively simple approach of forming chemical categories from 2D structural alerts (i.e. SARs for qualitative identification of chemical sub-structures with the potential of being reactive), such as used in the Organisation for Economic Co-Operation and Development (OECD)QSAR Toolbox[10] to QSAR models (i.e. quantitative prediction of relative reactivity) as described by Schwöbel et al.[11].

In Chemico Protocols and Databases

While methionine, histidine, and serine all possess nucleophilic groups that are found in skin proteins, the –SH group of cysteine and the ε-NH2 group of lysine are the most often studied. Soft electrophilic interactions involving the thiol group can be modelled with small molecules. Glutathione (GSH; L-γ-glutamyl-L-cysteinyl-glycine) is the most widely used model nucleophile in soft electrophilic reactivity assays. Typically, chemicals are incubated with GHS and, after a defined reaction time, the concentration of free thiol groups is measured. Such depletion based assays assume adduct formation, which is typically not confirmed. Good relationships between GSH reactivity and toxicity have been demonstrated. Examples of this method can be found in the literature[3];[12];[13];[14]. Recently, OECD adopted the new Test Guideline (TG) No442C: In chemico skin sensitisation – Direct Peptide Reactivity Assay (DPRA). This method quantifies the reactivity of chemicals towards model synthetic peptides containing either lysine or cysteine[15]. The DPRA protocol can be found in the EURL ECVAM Database Service on Alternative Methods to animal experimentation (DB-ALM): Protocol No154 for Direct Peptide Reactivity Assay (DPRA) for skin sensitisation testing[16]. The importance of reaction chemistry for sensitisation indicates that identification of the reaction limited chemical spaces is critical for using the proposed AOP. Systematic databases for reaction-specific chemical spaces are being developed. For example, in chemico databases reporting measurements of reactive potency currently exist for Michael acceptors ([14];[17];[18]). The use of model nucleophiles containing primary amino (–NH2) groups, such as in the amino acids lysine are less well-documented, with the principle of measuring relative reactivity being the same as for thiol[1].

Respiratory Sensitizers

Both respiratory and skin sensitizers are detected by in vitro and in silico methods used to measure electrophilic binding to proteins and peptides. (Basketter et al., 2017) The rate of covalent binding can also be measured. (Natsch and Gfeller, 2008) Dik et al. modified the DPRA protocol to include two peptide depletion measurement time points, and added high-performance liquid chromatography mass spectrometry (MS) analysis of reaction products, which improved predictive capacity. (Dik et al., 2016) Other authors have worked to investigate the binding of diisocyanates in vapor and liquid phases with LC/MS, MS/MS, and ELISA, as well as, Western blot. (Wisnewski et al., 2013a, 2013b, Hettick et al., 2012, Hopkins et al., 2005, Hettick and Siegel, 2011)

Overview table: How it is measured or detected

Method(s) Reference URL Regulatory

Acceptance

Validated Non

Validated

Direct Peptide Reactivity Assay (DPRA) TG 442C [1] X X  
DB-ALM [2]

Domain of Applicability

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

The OECD 2012 document does not indicate in vivo assays that measure covalent protein binding.

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

When a specific MIE can be defined (i.e., the molecular target and nature of interaction is known), in addition to describing the biological state associated with the MIE, how it can be measured, and its taxonomic, life stage, and sex applicability, it is useful to list stressors known to trigger the MIE and provide evidence supporting that initiation. This will often be a list of prototypical compounds demonstrated to interact with the target molecule in the manner detailed in the MIE description to initiate a given pathway (e.g., 2,3,7,8-TCDD as a prototypical AhR agonist; 17α-ethynyl estradiol as a prototypical ER agonist). Depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). Known stressors should be included in the MIE description, but it is not expected to include a comprehensive list. Rather initially, stressors identified will be exemplary and the stressor list will be expanded over time. For more information on MIE, please see pages 32-33 in the User Handbook.

The in chemico, in vitro, and in vivo experimental evidence is logical and consistent with the mechanistic plausibility proposed by covalent reactions based on the protein binding theory ([1];[19];[20]). In selected cases, (e.g. 1-chloro-2,4-dinitrobenzene) where the same compound has been examined in a variety of assays (see Annex 1 of[21]), the coherence and consistency of the experimental data is excellent. Alternative mechanism that logically present themselves and the extent to which they may distract from the postulated AOP. It should be noted that alternative mechanisms of action, if supported, require a separate AOP. While covalent reactions with thiol groups and to lesser extent amino groups, are clearly supported by the proposed AOP, reactions targeting other nucleophiles may or may not be supported by the proposed AOP. Limited data on chemical reactivity shows that two competing reactions are possible, the faster reaction dominates. However, this has yet to be proven in vitro or in vivo.

Earlier work on the molecular basis of skin sensitisation was reviewed by Lepoittevin et al. (1998)[22], since then our knowledge of skin sensitisation has continued to expand. Recent reviews (see[3];[9];[20];[22];[23];[24];[25]) repeatedly stress the same key steps leading to sensitisation. These events include hapten formation (i.e., the ability of a chemical to react with skin proteins).

The binding behavior of diisocyanates in particular has been well studied. Wisnewski et al.29,30 demonstrate that hexamethylene diisocyanate (HDI) and 4,4’-diphenylmethane diisocyanate (MDI) react with glutathione (GSH) across an in vitro physiologically relevant vapor/liquid-phase barrier to form conjugates, which may ‘‘shuttle,’’ via a carbamoylating reaction, the chemical to bind with serum albumin. Diisocyanates (MDI) react with GSH across an in vitro physiologically relevant vapor/liquid-phase barrier to form conjugates, which may ‘‘shuttle,’’ via a carbamoylating reaction, the chemical to bind with serum albumin.

In contrast to skin sensitization where cysteine and lysine are both key nucleophiles, experimental work has suggested that some respiratory sensitizers appear to preferentially bind to lysine; (Hettick et al., 2012, Lalko et al., 2012, Holsapple et al., 2006, Hopkins et al., 2005) however, an in chemico analysis of a larger set of respiratory sensitizers indicates lack of a simple division between the reactivity preferences of the two types of sensitizers, showing that certain classes displayed a lysine preference, for example, anhydrides, whereas others, such as diisocyanates, do not. (Dik et al., 2016)

While respiratory sensitizers and skin sensitizers can both bind to cellular and serum proteins in separate cultures, a study comparing the binding profiles of both classes in co-culture systems found that skin sensitizers preferentially bind cellular proteins, while respiratory sensitizers preferentially bind serum proteins. (Hopkins et al., 2005)

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help
  1. 1.0 1.1 1.2 1.3 Gerberick F, Aleksic M, Basketter D, Casati S, Karlberg AT, Kern P, Kimber I, Lepoittevin JP, Natsch A, Ovigne JM, Rovida C, Sakaguchi H and Schultz T. 2008. Chemical reactivity measurement and the predictive identification of skin sensitisers. Altern. Lab. Anim. 36: 215-242.
  2. Karlberg AT, Bergström MA, Börje A, Luthman K and Nilsson JL. 2008. Allergic contact dermatitis- formation, structural requirements, and reactivity of skin sensitizers. Chem. Res. Toxicol. 21: 53-69.
  3. 3.0 3.1 3.2 3.3 3.4 3.5 Schwöbel JAH, Koleva YK, Bajot F, Enoch SJ, Hewitt M, Madden JC, Roberts DW, Schultz TW and Cronin MTD. 2011. Measurement and estimation of electrophilic reactivity for predictive toxicology. Chem. Rev. 111: 2562-2596.
  4. Hopkins JE, Naisbitt DJ, Kitteringham NR, Dearman RJ, Kimber I and Park BK. 2005. Selective haptenation of cellular or extracellular proteins by chemical allergens: Association with cytokine polarization. Chem. Res. Toxicol. 18: 375-381.
  5. Lepoittevin JP. 2006. Metabolism versus chemical transformation or pro-versus prehaptens? Contact Dermatitis 54: 73-74.
  6. Freidig AP and Hermens JLM. 2001. Narcosis and chemical reactivity QSARs for acute toxicity. Quant. Struct. Act. Rel. 19: 547-553.
  7. Roberts DW, Aptula AO, Patlewicz G, Pease C. 2008. Chemical reactivity indices and mechanism-based read-across for non-animal based assessment of skin sensitisation potential. J.Appl. Toxicol. 28: 443-454.
  8. Enoch SJ, Ellison CM, Schultz TW, Cronin MTD. 2011. A review of the electrophilic reaction chemistry involved on covalent protein binding relevant to toxicity. Crit. Rev. Toxicol. 41: 783– 802.
  9. 9.0 9.1 OECD 2011. Report of the Expert Consultation on Scientific and Regulatory Evaluation of Organic Chemistry-based Structural Alerts for the Identification of Protein-binding Chemicals. OECD Environment, Health and Safety Publications Series on Testing and Assessment No. 139. ENV/JM/MONO(2011).
  10. Basketter DA, Pease C, Kasting G, Kimber I, Casati S, Cronin MTD, Diembeck W, Gerberick F, Hadgraft J, Hartung J, Marty JP, Nikolaidis E, Patlewicz G, Roberts DW, Roggen E, Rovida C, van de Sandt J. 2007. Skin sensitisation and epidermal disposition: The relevance of epidermal disposition for sensitisation hazard identification and risk assessment. The report of ECVAM workshop 59. Altern. Lab. Anim. 35: 137-154.
  11. Schwöbel J, Wondrousch D, Koleva YK, Madden JC, Cronin MTD, Schüürmann G. 2010. Prediction of Michael type acceptor reactivity toward glutathione. Chem. Res. Toxicol. 23: 1576-1585.
  12. Kato H, Okamoto M, Yamashita K, Nakamura Y, Fukumori Y, Nakai K, Kaneko H. 2003. Peptide-binding assessment using mass spectrometry as a new screening method for skin sensitization. J. Toxicol. Sci. 28: 19-24.
  13. Schultz TW, Yarbrough JW, Woldemeskel M. 2005. Toxicity to Tetrahymena and abiotic thiol reactivity of aromatic isothiocyanates. Cell. Biol. Toxicol. 21: 181-189.
  14. 14.0 14.1 Böhme A, Thaens D, Paschke A, Schüürmann G. 2009. Kinetic glutathione chemoassay to quantify thiol reactivity of organic electrophiles – Application to α, β-unsaturated ketones, acrylates, and propiolates, Chem. Res. Toxicol. 22: 742-750.
  15. OECD. Test No 442C: In chemico skin sensitisation: Direct Peptide Reactivity Assay (DPRA). 2015. OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects, OECD Publishing. Doi 10.1787/9789264229709-en.
  16. EURL ECVAM DB-ALM. Protocol No154: Direct Peptide Reactivity Assay for skin sensitisation testing. Available on: http://ecvam-dbalm.jrc.ec.europa.eu/.
  17. Yarbrough JW and Schultz TW. 2007. Abiotic sulfhydryl reactivity: A predictor of aquatic toxicity for carbonyl-containing α,β-unsaturated compounds. Chem. Res. Toxicol. 20: 558-562.
  18. Roberts DW and Natsch A. 2009. High throughput kinetic profiling approach for covalent binding to peptides: Application to skin sensitisation potency of Michael acceptor electrophiles. Chem. Res. Toxicol. 22: 592-603.
  19. Karlberg AT, Bergström MA, Börje A, Luthman K, Nilsson JL. 2008. Allergic contact dermatitisformation, structural requirements, and reactivity of skin sensitizers. Chem. Res. Toxicol. 21: 53-69.
  20. 20.0 20.1 Adler S, Basketter D, Creton S, Pelkonen O, van Benthem J, Zuang V, Andersen KE, Angers-Loustau A, Aptula A, Bal-Price A, Benfenati E, Bernauer U, Bessems J, Bois FY, Boobis A, Brandon E, Bremer S, Broschard T, Casati S, Coecke S, Corvi R, Cronin M, Daston G, Dekant W, Felter S, Grignard E, Gundert-Remy U, Heinonen T, Kimber I, Kleinjans J, Komulainen H, Kreiling R, Kreysa J, Leite SB, Loizou G, Maxwell G, Mazzatorta P, Munn S, Pfuhler S, Phrakonkham P, Piersma A, Poth A, Prieto P, Repetto G, Rogiers V, Schoeters G, Schwarz M, Serafimova R, Tähti H, Testai E, van Delft J, van Loveren H, Vinken M, Worth A, Zaldivar JM.2011. Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010. Arch Toxicol.85(5):367-485.
  21. OECD. 2012. The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins. Part 1: Scientific Evidence. Series on Testing and Assessment No. 168.
  22. 22.0 22.1 Lepoittevin JP, Basketter DA, Goossens A and Karlberg AT (eds) 1998. Allergic contact dermatitis: the molecular basis. Springer, Berlin.
  23. Vocanson M, Hennino A, Rozieres A, Poyet G, Nicolas JF. 2009. Effector and regulatory mechanisms in allergic contact dermatitis. Allergy 64: 1699-1714.
  24. Aeby P, Ashikaga T, Bessou-Touya S, Schapky A, Geberick F, Kern P, Marrec-Fairley M, Maxwell G, Ovigne JM, Sakaguchi H, Reisinger K, Tailhardat M, Martinozzi-Teisser S and Winkler P. 2010. Identifying and characterizing chemical skin sensitizers without animal testing; Colipa’s research and methods development program. Toxicol. In Vitro 24: 1465-1473.
  25. Basketter DA and Kimber I. 2010. Contact hypersensitivity. In: McQueen, C.A. (ed) Comparative Toxicology Vol. 5, 2nd Ed. Elsevier, Kidlington, UK, pp. 397-411.

BASKETTER, D., POOLE, A. & KIMBER, I. 2017. Behaviour of chemical respiratory allergens in novel predictive methods for skin sensitisation. Regul Toxicol Pharmacol, 86, 101-106.

DIK, S., RORIJE, E., SCHWILLENS, P., VAN LOVEREN, H. & EZENDAM, J. 2016. Can the Direct Peptide Reactivity Assay Be Used for the Identification of Respiratory Sensitization Potential of Chemicals? Toxicol Sci, 153, 361-71.

HETTICK, J. M. & SIEGEL, P. D. 2011. Determination of the toluene diisocyanate binding sites on human serum albumin by tandem mass spectrometry. Anal Biochem, 414, 232-8.

HETTICK, J. M., SIEGEL, P. D., GREEN, B. J., LIU, J. & WISNEWSKI, A. V. 2012. Vapor conjugation of toluene diisocyanate to specific lysines of human albumin. Anal Biochem, 421, 706-11.

HOLSAPPLE, M. P., JONES, D., KAWABATA, T. T., KIMBER, I., SARLO, K., SELGRADE, M. K., SHAH, J. & WOOLHISER, M. R. 2006. Assessing the potential to induce respiratory hypersensitivity. Toxicol Sci, 91, 4-13.

HOPKINS, J. E., NAISBITT, D. J., KITTERINGHAM, N. R., DEARMAN, R. J., KIMBER, I. & PARK, B. K. 2005. Selective haptenation of cellular or extracellular protein by chemical allergens: association with cytokine polarization. Chem Res Toxicol, 18, 375-81.

LALKO, J. F., KIMBER, I., GERBERICK, G. F., FOERTSCH, L. M., API, A. M. & DEARMAN, R. J. 2012. The direct peptide reactivity assay: selectivity of chemical respiratory allergens. Toxicol Sci, 129, 421-31.

NATSCH, A. & GFELLER, H. 2008. LC-MS-based characterization of the peptide reactivity of chemicals to improve the in vitro prediction of the skin sensitization potential. Toxicol Sci, 106, 464-78.

WISNEWSKI, A. V., LIU, J. & REDLICH, C. A. 2013a. Connecting glutathione with immune responses to occupational methylene diphenyl diisocyanate exposure. Chem Biol Interact, 205, 38-45.

WISNEWSKI, A. V., MHIKE, M., HETTICK, J. M., LIU, J. & SIEGEL, P. D. 2013b. Hexamethylene diisocyanate (HDI) vapor reactivity with glutathione and subsequent transfer to human albumin. Toxicol In Vitro, 27, 662-71.