Laboratory evaluation of the repellency and oral safety of Azadirachta indica hydromethanol leaf extract against stored-product insects and Amblyomma variegatum

Arthropod pests undermine livestock productivity and food security in tropical regions, while escalating resistance to synthetic pesticides demands safer, plant-derived alternatives. Leaves of Azadirachta indica A. Juss. (Meliaceae) are widely used by smallholder farmers across sub-Saharan Africa for ethnoveterinary ectoparasite management and stored-product pest control, yet leaf-derived preparations remain scientifically under-validated. This study evaluated the repellent efficacy of A. indica hydromethanol leaf extract (AIHMLE) against economically important stored-product insects and a veterinary tick, and assessed its mammalian safety profile. Freshly collected neem leaves were dried, extracted by cold maceration in 70% methanol and characterized via phytochemical screening and GC–MS analysis. Repellent activity was evaluated by area-preference (choice) bioassay against Tribolium castaneum , Alphitobius diaperinus , and Amblyomma variegatum , with DEET as reference. Acute oral toxicity (up to 5000 mg/kg) and 28-day sub-acute toxicity (125–500 mg/kg/day) were assessed in Sprague–Dawley rats per Lorke's method and OECD Guideline 407, with evaluation of clinical, haematological, and serum biochemical parameters. Data were analysed by repeated-measures ANOVA with Greenhouse–Geisser correction and Bonferroni adjustment ( p < 0.05). Phytochemical screening confirmed 11 classes of secondary metabolites, including alkaloids, tannins, saponins, terpenes, and flavonoids. GC–MS profiling of the volatile and semi-volatile fractions revealed a lipoidal profile dominated by fatty acid methyl esters: dodecanoic acid methyl ester (36.26%), 7-hexadecenoic acid methyl ester (19.41%), and hexadecanoic acid methyl ester (14.79%). AIHMLE produced potent, sustained repellency against all three species across 9 h, achieving 100% efficacy at several discrete time points, with mean repellency ranging from 90.0% to 100% across all three species and sustained at or above 90% for the majority of the 9-h observation period. Repellency was comparable to DEET against T. castaneum ( p > 0.05), though this comparison was likely underpowered and significantly superior against A. diaperinus and A. variegatum , exceeding DEET by 10.15% and 20.15%, respectively ( p < 0.05). No mortality or toxicity signs occurred up to 5000 mg/kg (LD₅₀ > 5000 mg/kg), and sub-acute exposure produced no treatment-related alterations in any evaluated parameter. AIHMLE demonstrated potent, sustained repellency against Tribolium castaneum , Alphitobius diaperinus, and Amblyomma variegatum , with efficacy meeting or exceeding DEET at the tested concentration, alongside a favourable oral safety margin. These findings provide rigorous preclinical validation for neem leaf-based pest management and support AIHMLE as a promising botanical repellent for integrated stored-product and ectoparasite control in tropical and subtropical settings.