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Rumen metabolism of Seneciopyrrolizidine alkaloids (PAs) and their N-oxide forms was studied by mass spectrometry in in vitro batch culture incubates and confirmed in in vivo samples. Most N-oxides were found to undergo rapid conversion to their corresponding free bases, followed by biotransformation to metabolites hydrogenated at both the necine base and the necic acid moiety. Therefore, rumen metabolism can be considered a detoxification step, as saturated necine base structures are known as the platyphylline type, which is regarded as less or nontoxic. Individual SenecioPAs, such as jacoline, are metabolized slowly during rumen fermentation. PAs that showed limited biotransformation in the rumen in this study also showed limited transformation and CYP-mediated bioactivation in the liver in other studies. This could not only explain why PAs that are comparatively metabolically stable can pass into milk but also suggest that such PAs might be considered compounds of lesser concern.