Validation of the 3D reconstructed human skin Comet assay, an animal-free alternative for following-up positive results from standardin vitrogenotoxicity assays

Abstract

As part of the safety assessment process, all industrial sectors employ genotoxicity test batteries, starting with well-established in vitro assays. However, these batteries have limited predictive capacity for the in vivo situation, which may result in unnecessary follow-up in vivo testing or the loss of promising substances where animal tests are prohibited or not desired. To address this, a project involving regulators, academia and industry was established to develop and validate in vitro human skin-based genotoxicity assays for topically exposed substances, such as cosmetics ingredients. Here, we describe the validation of the 3D reconstructed skin (RS) Comet assay. In this multicenter study, chemicals were applied topically three times to the skin over 48 h. Isolated keratinocytes and fibroblasts were transferred to slides before electrophoresis and the resulting comet formation was recorded as % tail DNA. Before decoding, results of the validation exercise for 32 substances were evaluated by an independent statistician. There was a high predictive capacity of this assay when compared to in vivo outcomes, with a sensitivity of 77 (80)%, a specificity of 88 (97)% and an overall accuracy of 83 (92)%. The numbers reflect the calls of the performing laboratories in the coded phase, whereas those in parenthesis reflect calls according to the agreed evaluation criteria. Intra- and inter-laboratory reproducibility was also very good, with a concordance of 93 and 88%, respectively. These results generated with the Phenion® Full-Thickness skin model demonstrate its suitability for this assay, with reproducibly low background DNA damage and sufficient metabolic capacity to activate pro-mutagens. The validation outcome supports the use of the RS Comet assay to follow up positive results from standard in vitro genotoxicity assays when the expected route of exposure is dermal. Based on the available data, the assay was accepted recently into the OECD test guideline development program.

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

“…In Phase 2c, chemicals were first tested using the 48-h protocol, and any chemical that showed negative or equivocal results were retested using the 72-h protocol. Finally, an additional five coded chemicals were later chosen for testing by the external experts (Phase 2d study) to close a data gap with chemicals that were tested in the RS Comet assay ( 1 , 15 ), bringing the number of chemicals tested in the predictivity Phases 2a–2d of the RSMN assay validation to 40 and increasing the total number of individual coded chemicals tested to 43 overall (for Phases 1 and 2a–2d; see Figure 1 ).…”

“…An experiment with only two valid dose groups could nevertheless be considered valid if: ( 1 ) the first two doses were positive (in terms of genotoxicity) and the thresholds for strong cytotoxicity were not exceeded or ( 2 ) genotoxic effects were absent in all dose groups and cytotoxicity exceeded the thresholds in the third test group only (because the thresholds were set to prevent MP results). Exceptional cases, in which a single tissue may be missing (e.g.…”

“…ICH guidelines for pharmaceuticals) always require testing in animals. As described previously in more detail by Pfuhler et al ( 1 ), for all jurisdictions, positive results from the standard in vitro battery were traditionally investigated further by follow-up testing using animals. However, a paradigm shift away from animal studies is well under way, driven by animal welfare concerns and by questions about human relevance of rodent toxicity data.…”

“…This is because the 3D skin models exhibit xenobiotic metabolism capabilities similar to native human skin, which therefore better reflects metabolism occurring by a dermal route of exposure ( 10–13 ). These efforts resulted in the development of the RS Comet assay ( 1 , 14 , 15 ) and the validation of the reconstructed human skin micronucleus (RSMN) assay ( 7 , 16 , 17 ). The MN endpoint provides a comprehensive basis for investigating chromosome damaging potential in vitro because both aneugens and clastogens can be detected ( 18 ) and is complementary to the Comet assay which can detect primary DNA damage leading to gene mutation ( 17 ).…”

“…Here, we investigated whether the good intra-laboratory reproducibility observed in Phase 0 and 1 with a small number of compounds ( 21 ) could be confirmed with a larger dataset, in addition to further evaluating the predictive capacity of the RSMN. Ultimately, we aimed to establish its usefulness as a ‘tier 2’ assay that can, alongside the RS Comet assay ( 1 ), be used to follow-up on positive results from the standard in vitro test battery.…”