The Ifakara Ambient Chamber Test (I-ACT) for Evaluation of Indoor Residual Sprays: A Non-Inferiority Test of Sylando® 240SC and SumiShield® 50WG

New insecticides for indoor residual spraying (IRS) are being developed to manage resistance. Chlorfenapyr (Sylando® 240SC), a pro-insecticide, is metabolized by active mosquitoes into the toxic metabolite tralopyril. This mode of action requires adapted “free flying” bioassays (because chlorfenapyr is converted to its toxic metabolite tralopyril when mosquitoes are metabolically active). A miniature-experimental hut (MEH) assay was developed within the Ifakara Ambient Chamber Test (I-ACT) with a rabbit as a host to measure residual efficacy under controlled conditions. Sylando® 240SC was compared with SumiShield® 50WG (clothianidin) for 12-month residual efficacy against malaria and arbovirus vectors. Residual activity was assessed on mud, wood and concrete with two huts per substrate treated with Sylando® 240SC, one with SumiShield® 50WG, and one with untreated control. Five replicates of 20 mosquitoes per strain (malaria vectors: pyrethroid-susceptible Anopheles gambiae and -resistant An. arabiensis and An. funestus; culicines Aedes aegypti and Culex quinquefasciatus) were exposed overnight at one-week post spraying and monthly thereafter. Multivariable mixed-effect logistic regression with binomial errors and log link function assessed non-inferiority with a 7% margin on mosquito mortality as the primary outcome for malaria vectors. Both products induced delayed mortality, with higher effects on malaria vectors than culicines. Across all substrates and malaria species combined over the full 12 months of observation, Sylando® 240SC was non-inferior to SumiShield® 50WG on mortality measured at 72 h (76% vs. 67%, OR = 0.86, 95% CI: 0.77–0.97) and 168 h (89% vs. 82%, OR = 0.74, 95% CI: 0.63–0.87). Sylando® 240SC performed comparably to SumiShield® 50WG, supporting its use as an IRS additional option. The new I-ACT mini-experimental-hut assay provides a practical tool for evaluating pro-insecticides. The importance of free-flight evaluation methods for pro-insecticides is discussed.