Non-targeted metabolite profiling reveals substantial equivalence of omega-3 enriched <i>PfFAD3-1</i> transgenic soybeans

The overexpression of the <i>Physaria fendleri</i> ω-3 fatty acid desaturase (<i>PfFAD3-1</i>) in soybean (<i>Glycine max</i> (L.) Merr.) substantially increases seed α-linolenic acid (18:3) content. To evaluate the compositional safety of <i>PfFAD3-1</i> transgenic soybeans, we conducted a three-year, two-location field trial (Jeonju and Gunwi, Republic of Korea) and applied non-targeted metabolite profiling of polar and lipophilic metabolites. Multivariate analyses (PCA, PLS-DA) revealed that environmental factors (site and year) had a strong effect on seed metabolite composition. Volcano plot and variable importance in projection (VIP) analyses indicated that most metabolite differences between <i>PfFAD3-1</i> lines and Kwangan (KA) varied across years and locations, reflecting gene - environment interactions. Importantly, the majority of metabolites in <i>PfFAD3-1</i> lines remained within the natural variation range of non-GM reference cultivars, supporting substantial equivalence. The consistent deviation was an increase in β-amyrin, a triterpenoid precursor of soyasaponins. LC - MS analysis further confirmed line- and environment-dependent increases in specific saponins, particularly soyasaponin I. Given the established safety of triterpenoids in soybean and the biological coherence of fatty acid - triterpenoid pathway cross-talk, these findings suggest that β-amyrin accumulation represents an intended metabolic adjustment rather than an unintended effect. Collectively, our results demonstrate that non-targeted metabolomics complements conventional OECD-recommended compositional analyses, providing a robust framework for the safety assessment of nutritionally enhanced GM soybeans.