Safety evaluation of bacterial melanin as a plant growth stimulant for agricultural and food industry applications

Introduction: Growing global demand for sustainable agriculture requires bio-based inputs with proven safety profiles. Bacterial melanin (BM) from Bacillus thuringiensis enhances plant growth, stress tolerance, and bioinsecticide stability, yet comprehensive toxicological data are lacking. Regulatory acceptance requires evaluation of acute toxicity, genotoxicity, cytotoxicity, and histopathology. Purpose of the Study: This study aimed to establish the acute safety threshold (NOAEL), assess genotoxic potential, evaluate in vitro cytotoxicity, and determine histopathological effects of water-soluble BM under high-dose intramuscular exposure in acute toxicity and genotoxicity rat models. Results: Intramuscular BM administration up to 0.8 g/kg revealed dose-dependent toxicity. At 0.1 g/kg, no mortality, behavioral changes, or weight loss occurred, defining the NOAEL. Doses ≥0.5 g/kg caused neurological signs and mortality. Micronucleus assays at NOAEL showed a non-significant increase in micronucleated polychromatic erythrocytes (BM: 10.0 ± 1.2 vs. control: 1.8 ± 0.5 per 2000 PCEs; p = 0.089) and a slight, non-significant reduction in PCE/NCE ratios (0.72 ± 0.09 vs. 0.85 ± 0.07; p = 0.12). No abnormal erythrocyte morphologies were observed. Histopathology of brain, liver, kidneys, heart, lungs, and injection site muscle showed intact architecture without necrosis, inflammation, or structural damage. In vitro MTT assays using murine fibroblasts (L929 cells) confirmed low cytotoxicity: cell viability remained >90% at ≤0.1 mg/mL and 86.7 ± 3.2% at 0.2 mg/mL after 48 h, with no IC50 reached and no apoptotic or necrotic morphology detected. Novelty of the Study: This is the first comprehensive toxicological evaluation of water-soluble BM from Bacillus thuringiensis intended for agriculture. By establishing NOAEL, conducting OECD-compliant micronucleus assays, and assessing cytotoxicity and histopathology, it addresses critical regulatory data gaps and supports safe use of BM as a biostimulant and bioinsecticide stabilizer. Conclusions: Bacillus thuringiensis-derived melanin exhibits a wide safety margin with no significant acute toxicity, genotoxicity, or histopathological abnormalities at conservative exposure levels. Minimal cytotoxicity supports its biocompatibility, aligning with regulatory guidelines for microbial bio-based products, facilitating its integration in sustainable agriculture. Further subchronic and pharmacokinetic studies are warranted Keywords: Bacterial melanin, toxicological assessment, genotoxicity, phytostimulator, sustainable agriculture, novel toxicological evaluation.