**Background:** Levamisole (LVM) is an immunomodulatory agent with anti-helminthic properties used in veterinary medicine, but its clinical use in humans was limited due to reports of leukoencephalopathy, neutropenia, agranulocytosis, and skin necrosis. Despite these risks, LVM is still encountered as a cutting agent in cocaine, and its safe dosage remains controversial. This study aimed to evaluate the systemic toxicity and toxicokinetics of LVM in a GLP-compliant setting to determine the no observable adverse effect level (NOAEL) in male beagle dogs.
**Methods:** Twenty male beagle dogs (6–7 months old, 5.5–8.8 kg) were divided into four groups (n=5 each): control (ultrapure water), and LVM at 5, 15, and 30 mg/kg/day via oral gavage for 28 days. Doses were selected based on a preliminary 2-week study where 60 mg/kg caused mortality. Clinical observations, body weight, body temperature, ECG, hematology, serum biochemistry, bone marrow cytology, organ/body weight coefficients, and histopathology (brain H&E staining) were assessed. Toxicokinetic analysis was performed on days 1 and 28 using LC-MS/MS to measure plasma LVM concentrations at 0.5, 1, 2, 4, 8, and 24 hours post-dose.
**Key Results:** All animals survived the 28-day period. In the 30 mg/kg group, transient vomiting, tremor, convulsions, hypoactivity, and gait disorder were observed shortly after gavage. No significant differences in body weight, body temperature, or ECG parameters were noted across groups (p > 0.05). Minor brain hemorrhage was observed in the 5, 15, and 30 mg/kg groups. Hematological examination showed significant decreases in WBC and NEUT counts at 5 mg/kg (WBC: 12.00 ± 2.10 ×10^9/L, p < 0.05; NEUT: 6.55 ± 0.839 ×10^9/L, p < 0.05) and 30 mg/kg (WBC: 10.31 ± 0.99 ×10^9/L, p < 0.01; NEUT: 5.74 ± 0.92 ×10^9/L, p < 0.01) compared to control (WBC: 15.09 ± 2.34; NEUT: 8.36 ± 1.48). MONO (%) increased significantly at 30 mg/kg (9.18 ± 1.97%, p < 0.05 vs control 5.44 ± 1.60%). LYMPH and EO counts decreased significantly only at 30 mg/kg (LYMPH: 3.39 ± 0.92 ×10^9/L, p < 0.05; EO: 0.21 ± 0.11 ×10^9/L, p < 0.05). Serum biochemistry revealed significantly elevated TG at all LVM doses (5 mg/kg: 0.76 ± 0.06 mM, p < 0.01; 15 mg/kg: 0.72 ± 0.11 mM, p < 0.05; 30 mg/kg: 0.79 ± 0.08 mM, p < 0.01 vs control 0.58 ± 0.07 mM) and increased CHOL at 15 mg/kg (6.59 ± 1.24 mM) and 30 mg/kg (8.92 ± 2.54 mM, p < 0.001 vs control 4.06 ± 0.55 mM). Na+ was significantly higher only at 5 mg/kg (141.80 ± 1.08 mM, p < 0.01 vs control 139.62 ± 0.50 mM). Bone marrow cytology showed no significant differences. Organ/body weight coefficients revealed a dose-dependent decrease in thymus weight (15 mg/kg: 0.184 ± 0.065, p < 0.05; 30 mg/kg: 0.155 ± 0.062, p < 0.05 vs control 0.305 ± 0.036) and increased epididymis weight (15 mg/kg: 0.024 ± 0.006, p < 0.05; 30 mg/kg: 0.022 ± 0.007, p < 0.05 vs control 0.010 ± 0.002). Toxicokinetic analysis showed rapid absorption (Tmax 0.5–1.9 h) and complete clearance by 24 h. Cmax and AUC increased proportionally with dose, with no bioaccumulation (Cmax day28/day1: 0.77–1.41; AUC day28/day1: 1.08–1.62).
**Clinical Implications:** The study establishes a NOAEL of 30 mg/kg/day in male beagle dogs, equivalent to 12 times the proposed human clinical dose (150 mg/day for a 60 kg adult). At this dose, LVM did not cause significant systemic toxicity, and the observed hematological and biochemical changes remained within historical normal ranges. The transient neurological signs at the highest dose warrant caution but were reversible. The lack of bioaccumulation supports the safety of repeated dosing. These GLP-compliant data provide a foundation for potential clinical reintroduction of LVM at appropriate doses, though further studies are needed to assess long-term effects and interspecies differences.