Validation of the 3D reconstructed human skin micronucleus (RSMN) assay: an animal-free alternative for following-up positive results from standardin vitrogenotoxicity assays

Abstract

In vitro test batteries have become the standard approach to determine the genotoxic potential of substances of interest across industry sectors. While useful for hazard identification, standard in vitro genotoxicity assays in 2D cell cultures have limited capability to predict in vivo outcomes and may trigger unnecessary follow-up animal studies or the loss of promising substances where animal tests are prohibited or not desired. To address this problem, a team of regulatory, academia and industry scientists was established to develop and validate 3D in vitro human skin-based genotoxicity assays for use in testing substances with primarily topical exposure. Validation of the reconstructed human skin micronucleus (RSMN) assay in MatTek Epi-200™ skin models involved testing 43 coded chemicals selected by independent experts, in four US/European laboratories. The results were analysed by an independent statistician according to predefined criteria. The RSMN assay showed a reproducibly low background micronucleus frequency and exhibited sufficient capacity to metabolise pro-mutagens. The overall RSMN accuracy when compared to in vivo genotoxicity outcomes was 80%, with a sensitivity of 75% and a specificity of 84%, and the between- and within-laboratory reproducibility was 77 and 84%, respectively. A protocol involving a 72-h exposure showed increased sensitivity in detecting true positive chemicals compared to a 48-h exposure. An analysis of a test strategy using the RSMN assay as a follow-up test for substances positive in standard in vitro clastogenicity/aneugenicity assays and a reconstructed skin Comet assay for substances with positive results in standard gene mutation assays results in a sensitivity of 89%. Based on these results, the RSMN assay is considered sufficiently validated to establish it as a ‘tier 2’ assay for dermally exposed compounds and was recently accepted into the OECD’s test guideline development program.

Replacement of animal testing by integrated approaches to testing and assessment (IATA): a call for in vivitrosi

“…An implementation of RhE through new technologies like automation or bioprinting, the improvement of the model through for example the partial or total elimination of animal components from the medium, to allow the application of microfluidic techniques, could bring to a broader application of the RhE model in regulatory pharmacology and toxicology, but also in personalised medicine (Kandarova et al 2021 ). Moreover, the RhE models, found application in other domains, as demonstrated recently for phototoxicity (OECD 2021a ), genotoxicity (Pfuhler et al 2021 ), skin sensitization (McKim et al 2012 ; Saito et al 2013 ; Johansson et al 2019 ) and various pharmacological applications.…”

“…In this respect, the in vitro Comet assay (with or without incorporation of Formamidopyrimidine DNA glycosylase (Fpg)) is widely used to determine DNA effects due to oxidative stress (Kohl 2020 ). It has been validated (though not peer-reviewed by a validation body yet) in vitro using the human lymphoblastoid TK-6 cell line (Muruzabal et al 2021 ) for 3D reconstructed human skin (Pfuhler et al 2021 ). One of the main shortcomings of in vitro genotoxicity assays is that they frequently produce false-positive results, due to the coupling of high sensitivity with a relatively low specificity (Corvi and Madia 2017 ).…”

The BlueScreen HC assay to predict the genotoxic potential of fragrance materials

“…The similarity in histological and physiological parameters of the model with human skin makes the model well suited for genotoxicity testing. This 3D skin model has been successfully validated and was shown to detect the DNA damage of test substances acting via different mechanisms [ 22 , 23 ]. The assay design was partially based on the standards recommended by international expert groups for in vitro and in vivo comet procedures for single-cell preparation and analysis of the nuclei [ 24 , 25 ].…”

Detection and analysis of chemical-induced chromosomal damage for public health: integrating new approach methodologies and non-animal methods

“…In vitro MN Current-TG i) Improvement of in vitro tests to reduce misleading positives 1) Top concentration (Morita et al, 2012(Morita et al, , 2014 2) Cytotoxicity indices (Fowler et al, 2012a) 3) p53 competency (Fowler et al, 2012b) iii) Follow-up evaluation of positive results 1) RS-MN (Pfuhler et al, 2021a(Pfuhler et al, , 2021b 2) Toxicogenomics (Ellinger-Ziegelbauer et al, 2009)…”