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The data matrices obtained from GC-MS were normalized to the total peak area and transferred to the SIMCA-P+ (v13.0, Umetrics, Umea, Sweden) program for multivariate analysis such as principal component analysis (PCA) and partial least squares differentiation analysis (PLS-DA) for the assessment of separability of the groups

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Moreover, emerging evidence indicates that CNMs can be fermented by gut bacteria into bioactive metabolites like butyrate, which then modulate host physiology, such as intestinal stem cell differentiation highlighting not only toxicological but also transformative microbialhost interactions ( in vitro systems such as gut-on-chip platforms are increasingly integrated into tiered risk frameworks to enhance hazard identification without excessive animal use