Rubber closures have been of great concern to regulatory authorities on account of their potential safety risks to patients. The aim of our work is to provide part of data about the compatibility of the injectable powder and its packaging materials for the drug registration. In this report, methodologies were established to study the system of the preparation. Firstly, three major extractables were isolated by semi-preparative HPLC method combined with silica gel-based chromatographic methods. NMR spectra including 1D NMR (1H, 13C, DEPT135) and 2D NMR (COSY, HSQC, HMBC) were introduced to identify the extractables, besides HPLC, GC–MS, ESI–MS/MS and HRMS. The extractables were determined to be N-(2-(2,2,4,4-tetramethylcyclohexyl)allyl) benzo[d]thiazol-2-amine (1), 2,6-Di-tert-butyl-4-methylphenol (2) and sulfur (3) respectively. Then, to address safety concerns, approaches including QSAR analysis, the TTC and comprehensive literature evaluation methods to toxicological safety evaluation of the target compounds were established, where the safety threshold such as TTC and PDE values were developed. Finally, the migration testing of the extractables were performed to assess the leaching behavior of the rubber closures. An optimized analysis method was proposed using SPE and HPLC with an ultraviolet detector, which demonstrated good linearity, acceptable accuracy and precision. The levels of the target compounds in the powder were measured and the calculated worst case exposure of extractable 2 exceeded the TTC limit of 1.5 μg/day, indicating that the products may possess potential health risks to patients. In contrast to previous studies, various NMR techniques, which were rarely applied to identify unknown extractables from rubber closures in the literature, were discussed for the structural elucidation of rubber closures extractables. Among the target compounds, extractable 1 was a new compound, whose isolation and structural elucidation were first reported here.