title: Intra- and inter-laboratory reproducibility and accuracy of the LuSens assay: A reporter gene-cell line to detect keratinocyte activation by skin sensitizers.
abstract: Several non-animal methods are now available to address the key events leading to skin sensitization as defined by the adverse outcome pathway. The KeratinoSens assay addresses the cellular event of keratinocyte activation and is a method accepted under OECD TG 442D. In this study, the results of an inter-laboratory evaluation of the "me-too" LuSens assay, a bioassay that uses a human keratinocyte cell line harboring a reporter gene construct composed of the rat antioxidant response element (ARE) of the NADPH:quinone oxidoreductase 1 gene and the luciferase gene, are described. Earlier in-house validation with 74 substances showed an accuracy of 82% in comparison to human data. When used in a battery of non-animal methods, even higher predictivity is achieved. To meet European validation criteria, a multicenter study was conducted in 5 laboratories. The study was divided into two phases, to assess 1) transferability of the method, and 2) reproducibility and accuracy. Phase I was performed by testing 8 non-coded test substances; the results showed a good transferability to naïve laboratories even without on-site training. Phase II was performed with 20 coded test substances (performance standards recommended by OECD, 2015). In this phase, the intra- and inter-laboratory reproducibility as well as accuracy of the method was evaluated. The data demonstrate a remarkable reproducibility of 100% and an accuracy of over 80% in identifying skin sensitizers, indicating a good concordance with in vivo data. These results demonstrate good transferability, reliability and accuracy of the method thereby achieving the standards necessary for use in a regulatory setting to detect skin sensitizers.
authors.affiliation: BASF SE, Experimental Toxicology and Ecology, Germany.
authors.forename: Tzutzuy
authors.initials: T
authors.lastname: Ramirez
authors.affiliation: BASF SE, Experimental Toxicology and Ecology, Germany.
authors.forename: Nadine
authors.initials: N
authors.lastname: Stein
authors.affiliation: BASF SE, Experimental Toxicology and Ecology, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Aumann
authors.affiliation: DSM Nutritional Products AG, Kaiseraugst, Switzerland.
authors.forename: Tina
authors.initials: T
authors.lastname: Remus
authors.affiliation: Burleson Research Technologies, Morrisville, NC, USA.
authors.forename: Amber
authors.initials: A
authors.lastname: Edwards
authors.affiliation: Institute for In Vitro Sciences, Gaithersburg, MD, USA.
authors.forename: Kimberly G
authors.initials: KG
authors.lastname: Norman
authors.affiliation: The Procter and Gamble Company, Mason, OH, USA.
authors.forename: Cindy
authors.initials: C
authors.lastname: Ryan
authors.affiliation: Institute for In Vitro Sciences, Gaithersburg, MD, USA.
authors.forename: Jackie E
authors.initials: JE
authors.lastname: Bader
authors.affiliation: DSM Nutritional Products AG, Kaiseraugst, Switzerland.
authors.forename: Markus
authors.initials: M
authors.lastname: Fehr
authors.affiliation: Burleson Research Technologies, Morrisville, NC, USA.
authors.forename: Florence
authors.initials: F
authors.lastname: Burleson
authors.affiliation: The Procter and Gamble Company, Mason, OH, USA.
authors.forename: Leslie
authors.initials: L
authors.lastname: Foertsch
authors.affiliation: The Procter and Gamble Company, Mason, OH, USA.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Wang
authors.affiliation: The Procter and Gamble Company, Mason, OH, USA.
authors.forename: Frank
authors.initials: F
authors.lastname: Gerberick
authors.affiliation: DSM Nutritional Products AG, Kaiseraugst, Switzerland.
authors.forename: Paul
authors.initials: P
authors.lastname: Beilstein
authors.affiliation: seh consultancy + services, Paderborn, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Hoffmann
authors.affiliation: BASF Personal Care and Nutrition GmbH, Germany.
authors.forename: Annette
authors.initials: A
authors.lastname: Mehling
authors.affiliation: BASF SE, Experimental Toxicology and Ecology, Germany.
authors.forename: Bennard
authors.initials: B
authors.lastname: van Ravenzwaay
authors.affiliation: BASF SE, Experimental Toxicology and Ecology, Germany. Electronic address: robert.landsiedel@basf.com.
authors.forename: Robert
authors.initials: R
authors.lastname: Landsiedel
mesh_terms: D000485:Allergens / Q000633:toxicity*; D000826:Animal Testing Alternatives; D061745:Antioxidant Response Elements / Q000235:genetics; D001681:Biological Assay; D002460:Cell Line; D003877:Dermatitis, Contact; D017930:Genes, Reporter; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects* / Q000378:metabolism; D007753:Laboratories; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051267:NF-E2-Related Factor 2 / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000485:Allergens; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; D008156:Luciferases