Comprehensive Necropsy and Tissue Evaluation

Complete necropsies, tissue collection, organ weights, and macroscopic tissue evaluation were performed on all animals. Necropsy includes a macroscopic examination of the external surface of the body, the thoracic and abdominal cavities and their contents, and the collection of all major tissues and macroscopic findings (Table S6).
Selected organs from all animals were weighed at the scheduled necropsy (Table S6). Organ-to-body weight and organ-to-brain weight ratios in Study 2 and organ-to-body weight ratios in Study 1 were calculated.
Representative samples of collected tissues were fixed in 10% (Study 2) or 7% (Study 1) neutral buffered formalin except for the eye with optic nerve attached (Davidson’s) and testis and epididymis (modified Davidson’s). All tissues were processed for slide preparation and stained with hematoxylin and eosin.
For the dosing phase, all tissues (excluding the larynx) collected from all dosing phase animals were examined microscopically. In Study 1, all tissues examined at the end of the recovery phase were identical to those evaluated at the end of the dosing phase. In Study 2, microscopic evaluation of recovery phase tissues in all animals was limited to real or anticipated target organs: bone marrow (sternum), joint, liver, draining lymph node, inguinal lymph node, macroscopic findings, skeletal muscle, injection site, and spleen. Microscopic findings were graded on a scale of 1 to 5 as minimal, mild, moderate, marked, or severe; findings not graded were listed as present. The type of infiltrating cells in tissues was based on the morphology of their nucleus, their size, the appearance of cytoplasm, and in the case of granulocytes, how their granules stain.