title: Rapid and simple kinetics screening assay for electrophilic dermal sensitizers using nitrobenzenethiol.
abstract: The need for alternatives to animal-based skin sensitization testing has spurred research on the use of in vitro, in silico, and in chemico methods. Glutathione and other select peptides have been used to determine the reactivity of electrophilic allergens to nucleophiles, but these methods are inadequate to accurately measure rapid kinetics observed with many chemical sensitizers. A kinetic spectrophotometric assay involving the reactivity of electrophilic sensitizers to nitrobenzenethiol was evaluated. Stopped-flow techniques and conventional UV spectrophotometric measurements enabled the determination of reaction rates with half-lives ranging from 0.4 ms (benzoquinone) to 46.2 s (ethyl acrylate). Rate constants were measured for seven extreme, five strong, seven moderate, and four weak/nonsensitizers. Seventeen out of the 23 tested chemicals were pseudo-first order, and three were second order. In three out of the 23 chemicals, deviations from first and second order were apparent where the chemicals exhibited complex kinetics whose rates are mixed order. The reaction rates of the electrophiles correlated positively with their EC3 values within the same mechanistic domain. Nonsensitizers such as benzaldehyde, sodium lauryl sulfate, and benzocaine did not react with nitrobenzenethiol. Cyclic anhydrides, select diones, and aromatic aldehydes proved to be false negatives in this assay. The findings from this simple and rapid absorbance model show that for the same mechanistic domain, skin sensitization is driven mainly by electrophilic reactivity. This simple, rapid, and inexpensive absorbance-based method has great potential for use as a preliminary screening tool for skin allergens.
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505-2888, USA. IChipinda@cdc.gov
authors.forename: Itai
authors.initials: I
authors.lastname: Chipinda
authors.forename: Risikat O
authors.initials: RO
authors.lastname: Ajibola
authors.forename: Moshood K
authors.initials: MK
authors.lastname: Morakinyo
authors.forename: Tinashe B
authors.initials: TB
authors.lastname: Ruwona
authors.forename: Reuben H
authors.initials: RH
authors.lastname: Simoyi
authors.forename: Paul D
authors.initials: PD
authors.lastname: Siegel
mesh_terms: D000485:Allergens / Q000737:chemistry* / Q000633:toxicity; D000826:Animal Testing Alternatives; D000818:Animals; D017449:Dermatitis, Allergic Contact / Q000473:pathology; D006207:Half-Life; D007700:Kinetics; D012882:Skin Tests; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000633:toxicity
chemical_list: D000485:Allergens; D013438:Sulfhydryl Compounds; C014712:4-nitrothiophenolate
references.citation: J Appl Toxicol. 2006 Jul-Aug;26(4):341-50
references.pmid: 16773645
references.citation: Toxicol In Vitro. 2007 Oct;21(7):1220-6
references.pmid: 17513083
references.citation: Chem Res Toxicol. 2006 Mar;19(3):341-50
references.pmid: 16544937
references.citation: Toxicol Sci. 2009 Jan;107(1):78-84
references.pmid: 18936299
references.citation: Toxicol Sci. 2007 Jun;97(2):417-27
references.pmid: 17400584
references.citation: J Invest Dermatol. 1969 Mar;52(3):268-76
references.pmid: 5774356
references.citation: Toxicol Sci. 2005 Nov;88(1):4-11
references.pmid: 16014741
references.citation: Toxicol Sci. 2004 Oct;81(2):332-43
references.pmid: 15254333
references.citation: SAR QSAR Environ Res. 2005 Aug;16(4):313-22
references.pmid: 16234173
references.citation: Chem Res Toxicol. 2009 Mar 16;22(3):592-603
references.pmid: 19206519
references.citation: SAR QSAR Environ Res. 2006 Aug;17(4):413-28
references.pmid: 16920662
references.citation: Contact Dermatitis. 2009 Jan;60(1):21-31
references.pmid: 19125718
references.citation: Toxicol In Vitro. 2009 Apr;23(3):439-46
references.pmid: 19444925
references.citation: Toxicol In Vitro. 2008 Aug;22(5):1169-76
references.pmid: 18440195
references.citation: Contact Dermatitis. 2008 Aug;59(2):79-89
references.pmid: 18759874
references.citation: Contact Dermatitis. 2005 Oct;53(4):189-200
references.pmid: 16191014
references.citation: J Appl Toxicol. 2008 May;28(4):443-54
references.pmid: 17703503
references.citation: Regul Toxicol Pharmacol. 2001 Dec;34(3):258-73
references.pmid: 11754530
references.citation: Toxicol Sci. 2008 Dec;106(2):464-78
references.pmid: 18791182
references.citation: Contact Dermatitis. 2007 Oct;57(4):242-7
references.pmid: 17868217
references.citation: Chem Res Toxicol. 2009 Apr;22(4):742-50
references.pmid: 19317512
references.citation: Arch Dermatol. 1965 Feb;91:171-7
references.pmid: 14237604
references.citation: Toxicology. 2000 Apr 20;146(1):49-59
references.pmid: 10773362
references.citation: SAR QSAR Environ Res. 2007 Jan-Mar;18(1-2):21-9
references.pmid: 17365956
references.citation: Dermatitis. 2005 Dec;16(4):157-202
references.pmid: 16536334