SNAPSHOT

 

Key Event Component

Evidence for Perturbation by Stressor

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Overview for Molecular Initiating Event

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)

Domain of Applicability

Key Event Description

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

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]

References

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.

Key Event Component

Domain of Applicability

Key Event Description

The innate immune system plays a crucial role in the initiation of adaptive immune responses. (Poynter, 2012, Salazar and Ghaemmaghami, 2013) It is a first-line of defense against invading microbial pathogens and is activated via a range of pattern recognition receptors (PRRs) that recognize conserved patterns present on pathogens, that is, the toll-like receptors (TLRs) and the nucleotide binding domain leucine-rich repeat containing receptor (NLR) family. These PRRs can be activated by endogenous danger-associated molecular patterns (DAMPs), released under oxidative stress and cell damage and include components of the extracellular matrix generated after tissue injury, for example, hyaluronic acid fragments, intracellular proteins such as heat shock proteins and nonprotein DAMPs such as uric acid crystals. (Kawai and Akira, 2010, Seong and Matzinger, 2004, Wheeler et al., 2009)

NLR protein-3 (NLRP3) is a PRR that belongs to the NLR family, a group of intracellular receptors activated by mitochondrial oxidative stress, for example, by adenosine triphosphate  and uric acid. (Kawai and Akira, 2009) On activation, TLR and NLRP3 activate innate immunity signaling pathways leading to the release of proinflammatory cytokines and chemokines. In recent years, increasing attention has been paid to the role of the innate immune system in asthma. The sentinel role of the innate immune systems includes the activation of pathways by pathogen-associated molecular patterns and DAMPs. By this, KEs during sensitization such as activation and migration of DCs are set into motion. (Holgate, 2012) Proinflammatory molecules are also known to induce the expression of surface molecules on immune cells such as antigen-presenting cells (APCs), which are greatly involved in the induction of adaptive immune responses. Thus, whether an immune response or tolerance response is induced in APCs depends not only on the presence of antigenic properties of a substance but also on danger signals.

How it is Measured or Detected

There are no predictive markers for cellular danger or proinflammatory responses described for respiratory sensitizers yet. The studies performed up until now did not result in any proteins, genes, or molecular pathways that are consistently regulated by a broad range of respiratory sensitizers or genes; (Remy et al., 2014) however, only a few chemicals have been tested. Cytokine production can be measured by ELISA or Bio-Plex systems either in the supernatants or intracellular matrix. Cell systems that can be used include also complex models such as the 3D epithelial cell models, that is, MucilAir™ and PCLS. (Huang et al., 2013, Lauenstein et al., 2014)

Activation of innate immune response can also be assessed using commercial immunoassays for signal transduction pathways, that is, p38 MAPK, JNK 1/2, and ERK 1/2. Other possible detection methods, focusing on ROS production or the induction of cytoprotective pathways, might be used as well to assess the ability of chemicals to generate endogenous danger signals (DAMPs). For ROS production, commercial assays are available that can be applied. The induction of Nrf2-KEAP1 can be assessed using the Keratinosens® (Natsch et al., 2013, Emter et al., 2010) or LuSens (Ramirez et al., 2014) assays (OECD TG 442D) and by measuring gene expression of Nrf2-dependent genes by quantitative polymerase chain reaction (qPCR), that is, HMOX, (Migdal et al., 2013) although the utility of this pathway for respiratory sensitizers is unclear. The BEAS-2B cell line, coupled with microarray analysis, reveals the PTEN pathway as potentially useful. (Verstraelen et al., 2009) The predictivity of these assays has not been studied with a large number of respiratory sensitizers.

References

EMTER, R., ELLIS, G. & NATSCH, A. 2010. Performance of a novel keratinocyte-based reporter cell line to screen skin sensitizers in vitro. Toxicol Appl Pharmacol, 245, 281-90.

HOLGATE, S. T. 2012. Innate and adaptive immune responses in asthma. Nat Med, 18, 673-83.

HUANG, S., WISZNIEWSKI, L., CONSTANT, S. & ROGGEN, E. 2013. Potential of in vitro reconstituted 3D human airway epithelia (MucilAir™) to assess respiratory sensitizers. Toxicol In Vitro, 27, 1151-6.

KAWAI, T. & AKIRA, S. 2009. The roles of TLRs, RLRs and NLRs in pathogen recognition. Int Immunol, 21, 317-37.

KAWAI, T. & AKIRA, S. 2010. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol, 11, 373-84.

LAUENSTEIN, L., SWITALLA, S., PRENZLER, F., SEEHASE, S., PFENNIG, O., FÖRSTER, C., FIEGUTH, H., BRAUN, A. & SEWALD, K. 2014. Assessment of immunotoxicity induced by chemicals in human precision-cut lung slices (PCLS). Toxicol In Vitro, 28, 588-99.

MIGDAL, C., BOTTON, J., EL ALI, Z., AZOURY, M. E., GULDEMANN, J., GIMÉNEZ-ARNAU, E., LEPOITTEVIN, J. P., KERDINE-RÖMER, S. & PALLARDY, M. 2013. Reactivity of chemical sensitizers toward amino acids in cellulo plays a role in the activation of the Nrf2-ARE pathway in human monocyte dendritic cells and the THP-1 cell line. Toxicol Sci, 133, 259-74.

NATSCH, A., RYAN, C. A., FOERTSCH, L., EMTER, R., JAWORSKA, J., GERBERICK, F. & KERN, P. 2013. A dataset on 145 chemicals tested in alternative assays for skin sensitization undergoing prevalidation. J Appl Toxicol, 33, 1337-52.

POYNTER, M. E. 2012. Airway epithelial regulation of allergic sensitization in asthma. Pulm Pharmacol Ther, 25, 438-46.

RAMIREZ, T., MEHLING, A., KOLLE, S. N., WRUCK, C. J., TEUBNER, W., ELTZE, T., AUMANN, A., URBISCH, D., VAN RAVENZWAAY, B. & LANDSIEDEL, R. 2014. LuSens: a keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification. Toxicol In Vitro, 28, 1482-97.

REMY, S., VERSTRAELEN, S., VAN DEN HEUVEL, R., NELISSEN, I., LAMBRECHTS, N., HOOYBERGHS, J. & SCHOETERS, G. 2014. Gene expressions changes in bronchial epithelial cells: markers for respiratory sensitizers and exploration of the NRF2 pathway. Toxicol In Vitro, 28, 209-17.

SALAZAR, F. & GHAEMMAGHAMI, A. M. 2013. Allergen recognition by innate immune cells: critical role of dendritic and epithelial cells. Front Immunol, 4, 356.

SEONG, S. Y. & MATZINGER, P. 2004. Hydrophobicity: an ancient damage-associated molecular pattern that initiates innate immune responses. Nat Rev Immunol, 4, 469-78.

VERSTRAELEN, S., BLOEMEN, K., NELISSEN, I., WITTERS, H., SCHOETERS, G. & VAN DEN HEUVEL, R. 2008. Cell types involved in allergic asthma and their use in in vitro models to assess respiratory sensitization. Toxicol In Vitro, 22, 1419-31.

VERSTRAELEN, S., NELISSEN, I., HOOYBERGHS, J., WITTERS, H., SCHOETERS, G., VAN CAUWENBERGE, P. & VAN DEN HEUVEL, R. 2009. Gene profiles of a human alveolar epithelial cell line after in vitro exposure to respiratory (non-)sensitizing chemicals: identification of discriminating genetic markers and pathway analysis. Toxicol Lett, 185, 16-22.

WANNER, R., SONNENBURG, A., QUATCHADZE, M., SCHREINER, M., PEISER, M., ZUBERBIER, T. & STAHLMANN, R. 2010. Classification of sensitizing and irritative potential in a combined in-vitro assay. Toxicol Appl Pharmacol, 245, 211-8.

WHEELER, D. S., CHASE, M. A., SENFT, A. P., POYNTER, S. E., WONG, H. R. & PAGE, K. 2009. Extracellular Hsp72, an endogenous DAMP, is released by virally infected airway epithelial cells and activates neutrophils via Toll-like receptor (TLR)-4. Respir Res, 10, 31.

Key Event Component

Domain of Applicability

Key Event Description

Immature epidermal dendritic cells, known as Langerhans cells, and dermal dendritic cells serve as antigen-presenting cells (^[1];^[2];^[3];^[4]). In this role, they recognize and internalize the hapten-protein complex formed during covalent binding leading to their activation. Subsequently, the dendritic cell loses its ability to seize new hapten-protein complexes and gains the potential to display the allergen-MHC-complex to naive T-cells; this process is often referred to as dendritic cell maturation. Simultaneously, under the influence of fibroblast- blood endothelial- and lymph endothelial chemokines (e.g. CCL19, CCL21) and epidermal cytokines (e.g. interleukin (IL), IL-1 α, IL-1β, IL-18, tumour necrosis factor alpha (TNF-α)) maturing dendritic cells migrate from the epidermis to the dermis of the skin and then to the proximal lymph nodes, where they can present the hapten-protein complex to T-cells via a major histocompatibility complex (MHC) molecule (^[5];^[6]). Dendritic cell activation, upon exposure to hapten-protein complexes also leads to functional changes in the cells. For example, there are changes in chemokine secretion, cytokine secretion and in the expression of chemokine receptors (see^[3]). Additionally, during dendritic cell maturation MHC, co-stimulatory and intercellular adhesion molecules (e.g. CD40, CD86, and DC11 and CD54, respectively) are up-regulated (see^[3];^[4];^[7]). Signal transduction cascades precede changes in expression of surface proteins markers and chemokine or cytokine secretion. In fact, there is evidence that during the response, hapten-protein complexes can react with cell surface proteins and activate mitogen-activated protein kinase signalling pathway. In particular, the biochemical pathway involving extracellulare signal-regulating kinases- the c-jun N-terminal kinases and the p38 kinases have been shown to be activated upon exposure to protein-binding chemicals^[8]. These pathways are of particular importance in keratinocytes and dendritic cell response to protein-hapten complexes. Components of signal transduction pathways are kinases, which phosphorylate and dephosphorylate a variety of substrates in order to elicit a change in the expression or secretion of target molecules. As a result, components of the signal transduction cascade are thought to be biomarkers^[9]. Investigations into the possible role of calcium influx as an early event in dendritic cell activation suggest that calcium influx is a second event following reactive oxygen species induction^[10];^[11].

How it is Measured or Detected

Omic studies

Genomic and proteomic studies also have the potential to reveal biomarkers in dendritic cell-based assays. Custom designed arrays or quantitative polymerase chain reaction (PCR) of selected genes have been used to highlight the reaction of dendritic cells (see^[3]). VITOSENS, an assay that uses human CD34+ progenitor-derived dendritic cells (CD34-DC), is based on the differential expression of the cAMP-responsive element modulator (CREM) and monocyte chemotactic protein-1 receptor (CCR2)^[12]. Genomic signatures have been also developed for the identification of human sensitising chemicals: a biomarker signature, the Genomic Allergen Rapid Detection test (GARD) based on the human myelomonocytic cell line MUTZ-3^[13] and a genomic platform, SENSIS, which consists of measuring the over-expression of 3 sets of genes, that may allow the in vitro assessment of the sensitising potential of a compound^[14].

In Vitro Assays for Cell Surface Markers, Cytokines, and Chemokines

Alterations in intercellular adhesion molecules, cytokines, and chemokines are part of the immunology response which can serve as biomarkers. Since dendritic cell maturation upon exposure to hapten-protein complexes is accompanied by changes in surface marker expression, these surface markers are perceived as promising candidates as primary biomarkers of dendritic cell activation for the development of cell-based in vitro assays. While a variety of surface markers have been described to be up-regulated upon dendritic cell maturation, a review of the literature reveals that CD86 expression, followed by CD54 and CD40, are the most extensively studied intercellular adhesion and co-stimulator molecules to date. The human Cell Line Activation Test (h-CLAT) reported flow cytometry results for CD86 and CD54 expression in THP-1 cells^[15];^[16]. An OECD Test Guideline for the h-CLAT is currently under review. The h-CLAT protocol can be found in the EURL ECVAM Database Service on Alternative Methods to animal experimentation (DB-ALM): Protocol No158 for human Cell Line Activation Test (h-CLAT)^[17]. Other studies with THP-1 cells include that of An et al. (2009). Another assay, the myeloid U937 skin sensitisation test (U-SENS), is based as well on the measurement of CD86 by flow cytometry^[18];^[19];^[20]). In addition to that, a variety of cytokines have been studied in relationship to skin sensitizers^[4]. IL-8 is a promising chemokine for distinguishing sensitisers from non-sensitisers. Quantification of IL-8 can be performed by Enzyme Linked Immunosorbent Assay, a technique that is far simpler and amenable to high throughput screening than the flow cytometry technique used to measure CD86 expression^[3]. The expression of other cytokines linked to skin sensitisers include IL-1 α, IL-1β, IL-18, and TNF-α form the basis for other dendritic cell assays.

While some respiratory sensitizers have been assessed, it is unclear whether this event is distinct between skin and respiratory sensitizers. (dos Santos et al., 2009) The genomic allergen rapid detection (GARD) test is an MUTZ-3-based assay for assessing chemical sensitizers utilizing genomic biomarker prediction signatures to generate prediction calls of unknown chemicals such as skin sensitizers, respiratory sensitizers, or nonsensitizers, including irritants. (Johannsen et al., 2011) Preliminary data on the performance of the GARD for assessing chemical respiratory sensitizers using transcriptional readouts of a genomic biomarker signature indicated 80% accuracy. (Forreryd, et al., 2015)

There are several in vitro assays available to assess DC maturation; the most advanced is the h-CLAT, which determines changes in CD86 and CD54 levels on THP-1 cell.(Ashikaga, et al., 2006, Sakaguchi, et al., 2006) However, only limited data are available substantiating its performance on chemical respiratory sensitizers. (Basketter, et al. 2017) Several assays similar to the h-CLAT have emerged over time and are currently in the process of being validated (e.g., the MUSST measuring CD86 responses by U937 cells), but again no or minimal information is available to assess assay performance in detecting respiratory sensitizers. The MUTZ-3 cell line is also being investigated for the potential to assess the capacity of a chemical to induce LC migration. The discriminating feature of the assay is that irritant-induced migration is CCL5 dependent, while sensitizer-induced migration is CXCL12 dependent. The readout of the test is the ratio between migration toward CXCL12 or to CCL5. Despite its complexity, the assay seems to be relatively well transferable.(Rees et al., 2011)

 

Overview table: How it is measured or detected

Method(s)	Reference	URL	Regulatory Acceptance	Validated	Non Validated
h-CLAT	draft TG under discussion at OECD	[1]		X
	DB-ALM	[2]
	EURL ECVAM Recommendation	[3]
	Ashiga et al., 2015	[4]
Genomic Allergen Rapid Detection test (GARD)	Johansson et al., 2013	[5]			X
VitroSens	Hooyberghs et al., 2008	[6]			X

References

1. ↑ Ryan CA, Gerberick GF, Gildea LA, Hulette BC, Bettis CJ, Cumberbatch M, Dearman RJ, Kimber I. 2005. Interactions of contact allergens with dendritic cells: opportunities and challenges for the development of novel approaches to hazard assessment. Toxicol. Sci. 88: 4-11.
2. ↑ Ryan CA, Kimber I, Basketter, DA, Pallardy M, Gildea LA, Gerberick GF. 2007. Dendritic cells and skin sensitisation. Biological roles and uses in hazard identification. Toxicol. Appl. Pharmacol. 221: 384-394.
3. ↑ ^{3.0 3.1 3.2 3.3 3.4 3.5} dos Santos GG, Reinders J, Ouwhand K, Rustemeyer T, Scheper RJ, Gibbs S. 2009. Progress on the development of human in vitro dendritic cell based assays for assessment of skin sensitizing potential of compounds. Toxicol. Appl. Pharmacol. 236: 372-382.
4. ↑ ^{4.0 4.1 4.2} Kimber I, Basketter DA, Gerberick GF, Ryan CA, Dearman, R.J. 2011. Chemical allergy: Translating biology into hazard characterization. Toxicol. Sci. 120(S1): S238-S268.
5. ↑ ^{5.0 5.1} Antonopoulos C, Cumberbatch M, Mee JB, Dearman RJ, Wei XQ, Liew FY, Kimber I, Groves RW. 2008. IL-18 is a key proximal mediator of contact hypersensitivity and allergen induced Langerhans cell migration in murine epidermis. J. Leukoc. Biol. 83: 361-367.
6. ↑ Ouwehand K, Santegoets SJAM, Bruynzeel DP, Scheper RJ, de Gruijl TD, Gibbs S. 2008. CXCL12 is essential for migration of activated Langerhans cells for epidermis to dermis. Eur. J. Immunol. 38: 3050-3059.
7. ↑ Vandebriel RJ and van Loveren H. 2010. Non-animal sensitisation testing: State-of-the-art. Crit. Rev. Toxicol. 40: 389-404.
8. ↑ Trompezinski S, Migdal C, Tailhardat M, Le Varlet B, Courtellemont P, Haftek M and Serres M. 2008. Charaterization of early events involved in human dendritic cell maturation induced by sensitizers: cross talk between MAPK signalling pathways. Toxicol. Appl. Pharmacol. 230: 397-406.
9. ↑ Lambrechts N, Vanheel H, Hooyberghs J, De Boever P, Witters H, Van Den Heuval R, Van Tendeloom V, Nelissen I, Schoeters G. 2010. Gene markers in dendritic cells unravel pieces of the skin sensitisation puzzle. Toxicol. Letters 196: 95-103.
10. ↑ Migdal C, Tailhardat M, Courtellemont P, Haftek M, Serres M. 2010. Responsiveness of human monocyte-derived dendritic cells to thimerosal and mercury derivatives. Toxicol. Appl. Pharmacol. 246: 66-73.
11. ↑ 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, 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.
12. ↑ Hooyberghs J, Schoeters E, Lambrechts N, Nelissen I, Witters H, Schoeters G, Van Den Heuvel R. 2008. A cell-based in vitro alternative to identify skin sensitizers by gene expression. Toxicol. Appl. Pharmacol. 231: 103-111.
13. ↑ Borrebaeck CA and Wingren C. 2009. Design of high-density antibody microarrays for disease proteomics: key technological issues. J. Proteomics 72: 928-935.
14. ↑ Groux H and Sabatier JM. 2010. Polypeptides for the in vitro assessment of the sensitising potential of a test compound. International Application Patent No.: PCT/EP2010/055895.
15. ↑ Sakaguchi H, Ashikaga T, Miyazawa M, Kosaka N, Ito Y, Yoneyama K, Sono S, Itagaki H, Toyoda H, Suzuki H. 2009. The relationship between CD86/CD54 expression and THP-1 cell viability in an in vitro skin sensitisation test-human cell line activation test (h-CLAT). Cell Biol. Toxicol. 25: 109-126.
16. ↑ Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa M, Ito Y, Nishiyama N, Itagaki H. 2010. A comparative evaluation of in vitro skin sensitisation tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern. Lab. Anim. 38:275-84.
17. ↑ EURL ECVAM DB-ALM. Protocol No158: Human Cell Line Activation Test (h-CLAT) Available on: http://ecvam-dbalm.jrc.ec.europa.eu/.
18. ↑ Ade N, Martinozzi-Teissier S, Pallaardy M, Rousset F. 2006. Activation of U937 cells by contact sensitizers: CD86 expression is independent of apoptosis. J. Immunotoxicol. 3: 189-197.
19. ↑ Python F, Goebel C, Aeby P. 2007. Assessment of the U937 cell line for detection of contact allergens. Toxicol. Appl. Pharmacol. 220: 113-124.
20. ↑ Ovigne JM, Martinozzi-Teissier S, Verda D, Abdou D, Piroird C, Ade N, Rousset F. 2008. The MUSST for in vitro skin sensitisation prediction: Applicability domains and complementary protocols to adapt to the physico-chemical diversity of chemicals. Toxicology Letters, 180: Supplement 1, 5, S216.

ASHIKAGA T, YOSHIDA Y, HIROTA M, YONEYAMA K, ITAGAKI H, SAKAGUCHI H, MIYAZAWA M, ITO Y, SUZUKI H, TOYODA H. 2006. Development of an in vitro skin sensitization test using human cell lines: the human Cell Line Activation Test (h-CLAT). I. Optimization of the h-CLAT protocol. Toxicol In Vitro. 20(5), 767-73. 

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

DOS SANTOS, G. G., REINDERS, J., OUWEHAND, K., RUSTEMEYER, T., SCHEPER, R. J. & GIBBS, S. 2009. Progress on the development of human in vitro dendritic cell based assays for assessment of the sensitizing potential of a compound. Toxicol Appl Pharmacol, 236, 372-82.

FORRERYD A, JOHANSSON H, ALBREKT AS, BORREBAECK CA, LINDSTEDT M. 2015. Prediction of chemical respiratory sensitizers using GARD, a novel in vitro assay based on a genomic biomarker signature. PLoS One.11;10(3):e0118808.

JOHANSSON H, LINDSTEDT M, ALBREKT AS, BORREBAECK CA. 2011. A genomic biomarker signature can predict skin sensitizers using a cell-based in vitro alternative to animal tests. BMC Genomics. 8;12:399. 

REES B, SPIEKSTRA SW, CARFI M, OUWEHAND K, WILLIAMS CA, CORSINI E, MCLEOD J.D., GIBBS S. 2011. Inter-laboratory study of the in vitro dendritic cell migration assay for identification of contact allergens. Toxicol In Vitro. 25(8), 2124-34.

SAKAGUCHI H, ASHIKAGA T, MIYAZAWA M, YOSHIDA Y, ITO Y, YONEYAMA K, HIROTA M, ITAGAKI H, TOYODA H, SUZUKI H. 2006. Development of an in vitro skin sensitization test using human cell lines; human Cell Line Activation Test (h-CLAT). II. An inter-laboratory study of the h-CLAT. Toxicol In Vitro. 20(5), 774-84. 

Key Event Component

Domain of Applicability

Key Event Description

T-cells are typically affected by protein-hapten complexes presented by dendritic cells on Major Histocompatibility Complex (MHC) molecules. Molecular understanding of this process has improved in recent years (see^[1]). Briefly, MHC molecules are membrane proteins which present the small peptide antigens placed in a “groove” of the MHC molecule during its intracellular synthesis and transport to the cell surface. In the context of the MHC molecular on the cell surface, the small peptide antigen is recognized via the T-cell receptors as self or non-self (e.g. foreign). If this peptide is a foreign peptide, such as part of a protein-hapten complex, the T-cell will be activated to form a memory T-cell, which subsequently proliferates. If reactivated upon presentation by skin dendritic cells, these memory T-cells will induce allergic contact dermatitis^[2].

How it is Measured or Detected

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?

Most protocols recognize the importance of the process of antigen-presentation, so in vitro T-cell-based assays are typically co-cultures of allergen-treated dendritic cells and modified T-lymphocytes with expression of selected biomarkers (e.g. interferon gamma), or T-cell proliferation being the reported outcome. Much of this work has been reviewed by Martin et al^[1]. It should be remembered that lymph node cell proliferation is the basis for the in vivo mouse Local Lymph Node Assay (LLNA). OECD TG 429 is the validated test guideline for the Skin Sensitisation: Local Lymph Node Assay^[3] together with its two non-radioactive modifications (LLNA-DA TG442A^[4] and LLNA-BrdU ELISA TG 442B^[5]).

Human T cell proliferation and DC and T cell cytokine profiles produced in response to chemical respiratory stimuli have been measured in vitro. (Holden et al., 2008, Bernstein et al., 2011)

 

Overview table: How it is measured or detected

Overview

Method(s)	Reference	URL	Regulatory Acceptance	Validated	Non Validated
Local Lymph Node Assay (LLNA)	TG 429	[1]	X	X
	TG 442A LLNA:DA	[2]
	TG 442B LLNA: BrdU-ELISA	[3]

References

1. ↑ ^{1.0 1.1 1.2} Martin SF, Esser PR, Schmucker S, Dietz L, Naisbitt DJ, Park BK, Vocanson M, Nicolas JF, Keller M, Pichler WJ, Peiser M, Luch A, Wanner R, Maggi E, Cavani A, Rustemeyer T, Richter A, Thierse HJ, Sallusto F. 2010. T-cell recognition of chemical, protein allergens and drugs; toward the development of in vitro assays. Cell. Mol. Life Sci. 67: 4171-4184.
2. ↑ Vocanson M, Hennino A, Rozieres A, Poyet G, Nicolas JF. 2009. Effector and regulatory mechanisms in allergic contact dermatitis. Allergy 64: 1699-1714.
3. ↑ OECD 2010. Test No.429: Skin sensitization: Local Lymph Node Assay. OECD Guidelines for the Testing of Chemicals, Section 4: Health effects. OECD Publishing. Doi: 10.1787/9789264071100-en.
4. ↑ OECD 2010. Test No442A: Skin sensitization: Local Lymph Node Assay:DA. OECD Guidelines for the Testing of Chemicals, Section 4: Health effects. OECD Publishing. Doi: 10.1787/9789264090972-en.
5. ↑ OECD 2010. Test No.442B: Skin sensitization: Local Lymph Node Assay: BrdU-ELISA. OECD Guidelines for the Testing of Chemicals, Section 4: Health effects. OECD Publishing. Doi: 10.1787/9789264090996-en.

BERNSTEIN, J. A., GHOSH, D., SUBLETT, W. J., WELLS, H. & LEVIN, L. 2011. Is trimellitic anhydride skin testing a sufficient screening tool for selectively identifying TMA-exposed workers with TMA-specific serum IgE antibodies? J Occup Environ Med, 53, 1122-7.

HOLDEN, N. J., BEDFORD, P. A., MCCARTHY, N. E., MARKS, N. A., IND, P. W., JOWSEY, I. R., BASKETTER, D. A. & KNIGHT, S. C. 2008. Dendritic cells from control but not atopic donors respond to contact and respiratory sensitizer treatment in vitro with differential cytokine production and altered stimulatory capacity. Clin Exp Allergy, 38, 1148-59.

Key Event Component

Domain of Applicability

Key Event Description

The development of an allergic hypersensitivity reaction in the respiratory tract is a two-step process, first requiring induction of the immune response, here as a result of exposure to a low-molecular-weight chemical (Boverhof et al, 2008). Subsequent single or multiple exposures to the same substance result in elicitation of an allergic hypersensitivity reaction, characterized by breathlessness and wheezing, airflow obstruction, bronchoconstriction, and tightness of the chest (Lauenstein et al, 2014). Reactions can be acutely life threatening or lead to chronic occupational asthma (Boverhof et al, 2008).

How it is Measured or Detected

Clinical signs described above can be objectively assessed in humans to confirm diagnosis of respiratory hypersensitivity.

Boverhof et al (2008) reviews various in vivo methods to detect respiratory hypersensitivity.

In rats, respiratory exposure to diisocyanites leads to immediate and delayed airway response (i.e. lung function). Elicitation is confirmed measuring PMN in bronchoalveolar lavage fluid (BAL) one day after inhalation challenge and exhaled NO (Pauluhn 2014).

In mice, induction of immune response, measured by T-lymphocyte maturation and proliferation in local lymph nodes, can often be detected using a Local Lymph Node Assay protocol (OECD 2010) with subsequent cytokine fingerprinting or IgE testing (Dearman et al 2003; Boverhof et al 2008).

Allergen-specific IgE detection and measurement techniques include skin tests (intradermal and subcutaneous skin prick testing) and blood testing using immune assays such as ELISAs and commercially available tests such as ImmunoCAP™. For example, Bernstein et al. investigated the ability of TMA skin testing to identify sensitized workers and found that skin prick testing was positive in 8 of 11 workers with serum-specific IgE and intradermal testing in a further two. (Bernstein et al., 2011) It is important to note, however, that there are technical challenges associated with detection and measurement of specific IgE and IgG to chemical respiratory allergens, including production of the correct protein conjugate and timing of measurement. (Kimber et al., 2014, Quirce, 2014) Immune assays such as ELISA or ImmunoCAP are also used to investigate allergen-specific antibody isotype profiles. (Movérare et al., 2017) Investigations into direct and indirect class switching involve transcriptomic analyses of IgE heavy chain transcripts and are challenging due to the scarcity of IgE-switched B cells in human blood. (Davies et al., 2013)

Regulatory Significance of the AO

This adverse outcome is of high regulatory interest and relevance, though no test guideline is available. Regulatory agencies and industrial producers are interested in preventing the first step--induction of immune response. Importantly, induction of respiratory sensitisation can be obtained via skin exposure, which is consequential for potential exposure restrictions.

References

Boverhof DR, Billington R, Bhaskar Gollapudi B, Hotchkiss JA, Krieger SM, Poole A, Wiescinski CM, and Woolhiser MR. 2008. Respiratory sensitization and allergy: Current research approaches and needs. Tox Appl Pharm 226:1-13.

Dearman RJ, Betts CJ, Humphreys N, Flanagan BF, Gilmour NJ, Basketter DA, Kimber I. 2003. Chemical allergy: considerations for the practical application of cytokine profiling. Toxicol. Sci. 71, 137–145.

Lauenstein L, Switalla S, Prenzler F, Seehase S, Pfennig O, Förster C, Fieguth H, Braun A and Sewald K. 2014. Assessment of immunotoxicity induced by chemicals in human precision-cut lung slices (PCLS). Tox in Vitro 28:588–599.

OECD (2010) Test No. 429: Skin Sensitisation: Local Lymph Node Assay, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing. doi: 10.1787/9789264071100-en.

Pauluhn J. 2014. Development of a respiratory sensitization/elicitation protocol of toluene diisocyanate (TDI) in Brown Norway rats to derive an elicitation-based occupational exposure level. Toxicology 319: 10–22.

BERNSTEIN, J. A., GHOSH, D., SUBLETT, W. J., WELLS, H. & LEVIN, L. 2011. Is trimellitic anhydride skin testing a sufficient screening tool for selectively identifying TMA-exposed workers with TMA-specific serum IgE antibodies? J Occup Environ Med, 53, 1122-7.

DAVIES, J. M., PLATTS-MILLS, T. A. & AALBERSE, R. C. 2013. The enigma of IgE+ B-cell memory in human subjects. J Allergy Clin Immunol, 131, 972-6.

KIMBER, I., DEARMAN, R. J. & BASKETTER, D. A. 2014. Diisocyanates, occupational asthma and IgE antibody: implications for hazard characterization. J Appl Toxicol, 34, 1073-7.

MOVÉRARE, R., BLUME, K., LIND, P., CREVEL, R., MARKNELL DEWITT, Å. & COCHRANE, S. 2017. Human Allergen-Specific IgG Subclass Antibodies Measured Using ImmunoCAP Technology. Int Arch Allergy Immunol, 172, 1-10.

QUIRCE, S. 2014. IgE antibodies in occupational asthma: are they causative or an associated phenomenon? Curr Opin Allergy Clin Immunol, 14, 100-5.

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List of Adjacent Key Event Relationships