The longevity of pain after surgery is debilitating and limits the recovery of patients. AYX1 is a double-stranded, unprotected, 23 base-pair oligonucleotide designed to reduce acute post-surgical pain and prevent its chronification with a single intrathecal perioperative dose. AYX1 mimics the DNA sequence normally bound by EGR1 on chromosomes, a transcription factor transiently induced in the dorsal root ganglia—spinal cord network following a noxious input. AYX1 binds to EGR1 and prevents it from launching waves of gene regulation that are necessary to maintain pain over time. A formulation suitable for an intrathecal injection of AYX1 was developed, including a specific ratio of AYX1 and calcium so the ionic homeostasis of the cerebrospinal fluid is maintained and no impact on neuromuscular control is produced upon injection. A GLP toxicology study in naïve Sprague Dawley rats was conducted using 3 dose levels up to the maximum feasible dose. Clinical observations, neurobehavioral observations, clinical pathology and histopathology of the nervous system and peripheral tissues were conducted. An additional nonGLP study was conducted in the spared nerve injury model of chronic neuropathic pain in which EGR1 is induced in the dorsal root ganglia and spinal cord. Similar testing was performed, including a modified Irwin test to assess a potential impact of AYX1 on autonomic nervous system responses, locomotion, activity, arousal, sensorimotor, and neuromuscular function. No AYX1-related adverse events were observed in any of the studies and the no-observed-adverse-effect-level was judged to be the maximum feasible dose.