Reactivation assays

Tobacco accounts for about 90% of lung cancers. Some tobacco carcinogens induce DNA adducts that are repaired by the nucleotide excision repair (NER) pathway (3). The lowest DNA repair capacity (DRC), measured in peripheral blood lymphocytes by the host cell reactivation assay (HCR), was observed in lung cancer patients who were less than 60 years old, female, or lighter smokers, and in those with a family history of cancer (4,5). 

Cross -reactivity Assay validation Single-dose PK (+1 mo) cyno 1 mo (+2 mo) cyno... 

A prevalidation study carried out by Cosmetics Europe on this optimized protocol showed that the SkinEthic HCE method consistently discriminated UN GHS classified from non-classified test substances within and between laboratories [47]. A combination of the two exposure times in a testing strategy that allocates test substances to the short or long exposure time depending on their intrinsic chemical reactivity increased the overall accuracy under GHS. The chemical reactivity, i.e., electrophilic potential to react with cysteine- or lysine-containing peptides, was measured by the direct peptide reactivity assay (DPRA) [48]. Reactive substances (peptide depletion is >5.95 % relative to the control) were allocated to the short exposure time, whereas non-reactive substances (depletion <5.95 %) are allocated to the long exposure time. 

Advances in the understanding of the immunobiology of skin sensitization have led to the establishment of predictive in vivo tests which not only identify sensitizing hazards but also characterize their potency. Recently, appreciation of the underlying biology has also resulted in the development of mechanistically based in vitro alternatives which offer the prospect of the replacement of current in vivo methods. Assays under active validation include the Direct Peptide Reactivity Assay (DPRA), the human Cell Line Activation Test (h-CLAT), and KeratinoSens. None of the methods have a sufficient level of accuracy or freedom from applicability domain limitations to allow them to act as a standalone replacement. Consequently, it will be necessary to consider how to deploy these assays, perhaps in combination and/or in a structured assessment of skin sensitization hazard, to ensure at least the same level of predictive accuracy as the in vivo methods. However, a challenge remains the capacity of these methods to provide potency information on skin-sensitizing chemicals has yet to be assessed. This is an essential requirement for future risk assessment without use of animal models if we are to retain the same level of human health protection that is currently delivered. 

Key words Skin sensitization, Contact allergy, Allergic contact dermatitis, Local lymph node assay, In vitro alternatives, Direct peptide reactivity assay, KeratinoSens, Human cell line activation test... 

Extensive reviews concerning the opportunities for the development of in vitro sensitization methods already exist [41-44], These reviews show that essentially all of the methods address one or other of the key mechanistic steps in the induction of skin sensitization—and these are nicely represented in the OECD adverse outcome pathway description [45], From this large body of work, three methods have emerged whose initial promise has been substantiated by demonstration not only of their predictive merits but also by verification of their robustness in terms of inter-laboratory transferability and within and between laboratory reproducibility [46]. The three methods are the direct peptide reactivity assay (DPRA) [47, 48], KeratinoSens [49, 50], and the human Cell Line Activation Test (h-CLAT) [51-53]. The first of these, the DPRA, addresses the question of chemical reactivity, the second investigates an aspect of keratinocyte activation... 

Gerberick GF, Vassallo JD, Bailey RE, Chaney JG, Morrall SW, Lepoittevin JP (2004) Development of a peptide reactivity assay for screening contact allergens. Toxicol Sci 81 332-343... 

Jeong YH, An S, Shin K, Lee TR (2013) Peptide reactivity assay using spectrophoto-metric method for high-throughput screening of skin sensitization potential of chemical haptens. Toxicol In Vitro 27(1) 264-271... 

Troutman JA, Foertsch LM, Kern PS, Dai HJ, Quijano M, Dobson RL, Lalko JF, Lepoittevin JP, Gerberick GF (2011) The incorporation of lysine into the peroxidase peptide reactivity assay for skin sensitization assessments. Toxicol Sci 122 422-436... 

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