Subacute 28-Day OECD-Guided Oral Toxicity Study of Escherichia coli 5C (LMG S-33222) in Wistar Rats and Immunocompromised Nude Mice

Ensuring the safety of microbial strains intended for probiotic use is essential, particularly for species such as Escherichia coli (E. coli), which include both commensal and pathogenic lineages. E. coli 5C is a recently identified polyketide synthase (pks)-negative strain isolated from healthy infant feces and characterized as free of virulence factors, plasmids, and antimicrobial resistance, suggesting suitability as a probiotic. To confirm its in vivo safety, we evaluated the subacute oral toxicity of E. coli 5C in immunocompetent and immunocompromised rodent models. A GLP-compliant 28-day repeated-dose oral toxicity study was conducted in Wistar rats following OECD Test Guideline 407 (2008 edition), complemented by a parallel non-GLP study in athymic nude mice to assess safety under impaired immune function. Animals received purified water (control) or E. coli 5C at 500, 1000, or 2000 mg/kg/day (~0.5, 1, or 2 × 1011 CFU/kg/day), as low, mid, and high dose, respectively, daily for 28 days. Clinical signs, behaviour, body weight, feed intake, functional observations, haematology, biochemistry, urinalysis, organ weights, gross necropsy, and histopathology were evaluated. Across all treated groups in both species, E. coli 5C produced no mortality, morbidity, or adverse clinical effects. Haematological, biochemical, and behavioural parameters remained comparable to controls, and organ weights, gross pathology, and microscopic examinations revealed no test item-related abnormalities. No systemic infection was observed. No treatment-related adverse effects were observed at any dose level, and the highest tested dose of 2,000 mg/kg/day (2 × 1011 CFU/kg/day) was therefore identified as the No Observed Adverse Effect Level (NOAEL). These findings demonstrate the absence of subacute toxicity of E. coli 5C in both normal and immunodeficient hosts and provide a favourable preclinical safety foundation for its continued development as a candidate probiotic strain.