title: Development of a new in vitro skin sensitization assay (Epidermal Sensitization Assay; EpiSensA) using reconstructed human epidermis.
abstract: Recent changes in regulatory requirements and social views on animal testing have accelerated the development of reliable alternative tests for predicting skin sensitizing potential of chemicals. In this study, we aimed to develop a new in vitro skin sensitization assay using reconstructed human epidermis, RhE model, which is expected to have broader applicability domain rather than existing in vitro assays. Microarray analysis revealed that the expression of five genes (ATF3, DNAJB4, GCLM, HSPA6 and HSPH1) related to cellular stress response were significantly up-regulated in RhE model after 6h treatment with representative skin sensitizers, 1-fluoro-2,4-dinitrobenzene and oxazolone, but not a non-sensitizer, benzalkonium chloride. The predictive performance of five genes was examined with eight skin sensitizers (e.g., cinnamic aldehyde), four non-sensitizers (e.g., sodium lauryl sulfate) and four pre-/pro-haptens (e.g., p-phenylenediamine, isoeugenol). When the positive criteria were set to obtain the highest accuracy with the animal testing (LLNA), ATF3, DNAJB4 and GCLM exhibited a high predictive accuracy (100%, 93.8% and 87.5%, respectively). All tested pre-/pro-haptens were correctly predicted by both ATF3 and DNAJB4. These results suggested that the RhE-based assay, termed epidermal sensitization assay (EpiSensA), could be an useful skin sensitization assay with a broad applicability domain including pre-/pro-haptens.
authors.affiliation: Kao Corporation, Safety Science Research Laboratories, 2606 Akabane, Ichikai-Machi, Haga-Gun, Tochigi 321-3497, Japan.
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Saito
authors.forename: Yuko
authors.initials: Y
authors.lastname: Nukada
authors.forename: Osamu
authors.initials: O
authors.lastname: Takenouchi
authors.forename: Masaaki
authors.initials: M
authors.lastname: Miyazawa
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Sakaguchi
authors.forename: Naohiro
authors.initials: N
authors.lastname: Nishiyama
mesh_terms: D000485:Allergens / Q000633:toxicity*; D000826:Animal Testing Alternatives; D001548:Benzalkonium Compounds / Q000633:toxicity; D004139:Dinitrofluorobenzene / Q000633:toxicity; D004817:Epidermis; D020869:Gene Expression Profiling; D006241:Haptens / Q000633:toxicity*; D006801:Humans; D066298:In Vitro Techniques; D020411:Oligonucleotide Array Sequence Analysis; D010081:Oxazolone / Q000633:toxicity; D023422:Skin Irritancy Tests
chemical_list: D000485:Allergens; D001548:Benzalkonium Compounds; D006241:Haptens; D010081:Oxazolone; D004139:Dinitrofluorobenzene
keywords: 1-fluoro-2,4-dinitrobenzene; 2,4-dinitrochlorobenzene; 2-MBT; 2-mercaptobenzothiasole; 4-NBB; 4-nitrobenzyl bromide; ACD; ADH; AOO; ATF3; Alternative method; BKC; CA; CYP; CinA; DAVID; DCs; DNAJB4; DNCB; DNFB; Database for Annotation, Visualization and Integrated Discovery; ECVAM; ER; EpiSensA; Epidermal Sensitization Assay; European Centre for the Validation of Alternative Methods; GCLM; GO; GSH; Gene expression; HSPA6; HSPH1; IEU; IL; KCs; Keratinocytes; LA; LLNA; MDBGN; MTT; Microarray analysis; OXA; PPD; ROS; Reconstructed human epidermis (RhE) model; RhE; SA; SLS; Skin sensitization; TMTD; TNF; acetone: olive oil=4:1; activating transcription factor 3; alcohol dehydrogenase; allergic contact dermatitis; benzalkonium chloride; cinnamic alcohol; cinnamic aldehyde; cytochrome P450; dendritic cells; dnaJ (Hsp40) homolog, subfamily B, member 4; endoplasmic reticulum; gene ontology; glutamate-cysteine ligase, modifier subunit; glutathione; heat shock 105kDa/110kDa protein 1; heat shock 70kDa protein 6; interleukin; isoeugenol; keratinocytes; lactic acid; local lymph node assay; methyldibromo glutaronitrile; methylthiazolydiphenyl-tetrazolium; oxazolone; p-phenylenediamine; reactive oxygen species; reconstructed human epidermis; salicylic acid; sodium lauryl sulfate; tetramethylthiuram disulfide; tumor necrosis factor