Acute Toxicity Assessment of Cell-Free Supernatant Derived from Lysinibacillus sp. from Indonesian Peat Soil as a Potential Broad-Spectrum Antibiotic.

&lt;b&gt;Background and Objective:&lt;/b&gt; Antimicrobial resistance (AMR) drives an urgent need for new antibiotics. A &lt;i&gt;Lysinibacillus&lt;/i&gt; sp. isolate from Indonesian peat soil has shown promising broad-spectrum antibacterial activity &lt;i&gt;in vitro&lt;/i&gt;. This study aims to evaluate the acute toxicity profile of its cell-free supernatant (CFS) as an initial &lt;i&gt;in vivo&lt;/i&gt; safety assessment. &lt;b&gt;Materials and Methods:&lt;/b&gt; A single oral dose acute toxicity test was conducted on female mice (n = 3 per group). Treatment groups received CFS at volumes of 0.2 mL, 0.5 mL and 1.0 mL. Control groups received 0.5% Na carboxy methyl cellulose (CMC) and nutrient broth (NB). All animals were observed for 14 days for mortality, behavior and body weight changes, followed by necropsy on day 15. Statistical analysis was conducted using SPSS version 26, applying GLM repeated measures with Shapiro-Wilk normality, Mauchly's sphericity and Bonferroni &lt;i&gt;post-hoc&lt;/i&gt; tests at a significance level of p<0.05, with results expressed as Mean±SD. &lt;b&gt;Results:&lt;/b&gt; There was no mortality or significant behavioral changes. However, macroscopic necropsy revealed dose-dependent toxicity. At a dose of 0.2 mL, heart swelling and tracheal hemorrhage were found. A dose of 0.5 mL caused lung hemorrhage and a pale trachea. At the highest dose of 1.0 mL, all animals showed kidney swelling, lung hemorrhage and pale digestive tract. The Na CMC control group was normal, while the NB control showed mild abnormalities. &lt;b&gt;Conclusion:&lt;/b&gt; The CFS of &lt;i&gt;Lysinibacillus&lt;/i&gt; sp. did not show LD&lt;sub&gt;50&lt;/sub&gt; toxicity or behavioral changes in mice. Some high doses showed organ-specific toxicity.