The 3D reconstructed skin micronucleus assay: considerations for optimal protocol design

Area of Science:

• In vitro toxicology
• Genotoxicity testing
• Cosmetic safety assessment

Background:

• EU Cosmetics Directive necessitates alternative in vitro assays for risk assessment.
• The reconstructed skin micronucleus (RSMN) assay is a key alternative.
• Previous studies using the RSMN assay with 48-hour treatments yielded mixed results.

Purpose of the Study:

• To evaluate and optimize the RSMN assay at Covance Laboratories.
• To compare the efficacy of 48-hour versus 72-hour treatment protocols.
• To assess the suitability of the 72-hour protocol for detecting various chemical types.

Main Methods:

• Tested nine chemicals using the EpiDerm™ 3D skin model.
• Compared data from standard 48-hour and emerging 72-hour treatment protocols.
• Analyzed micronucleus (MN) induction and Replication Index (RI) data.

Main Results:

• Chemicals requiring metabolic activation (BaP, CPA) were positive only with the 72-hour protocol.
• The 72-hour protocol showed higher Replication Index values, indicating increased cell division.
• A greater magnitude of micronucleus induction was observed with the 72-hour treatment.

Conclusions:

• The 72-hour treatment protocol is more suitable for the RSMN assay.
• This extended protocol effectively detects clastogenic, aneugenic, and metabolically activated chemicals.
• Further optimization, including statistical power analysis, is recommended for the RSMN assay.