Comprehensive Genotoxicity and Subchronic Oral Toxicity Evaluation of Kumamolisin from Bacillus sp. MN-32 for Human Use

Abstract

Kumamolisin from Bacillus sp. MN-32 (produced by E. coli B/BL21 background) is an acid-active sedolisin protease developed for food uses; its systemic safety was assessed using GLP (Good-laboratory-practice) in vitro genotoxicity assays and repeated-dose oral studies. A bacterial reverse-mutation assay (OECD TG 471; Salmonella TA98, TA100, TA102, TA1535, TA1537; plate-incorporation and preincubation; {+/-}S9) showed no concentration-related increases in revertants up to 5000 {micro}g total organic solids (TOS)/plate. A mammalian cell micronucleus test (OECD TG 487; human lymphocytes; 4 h {+/-}S9 and 24 h -S9) showed no significant increases in micronucleated binucleated cells at 1250-5000 {micro}g TOS/mL, with cytotoxicity [≤] 21.6% by CBPI (Cytokinesis-Block Proliferation Index). In 28-day and 90-day repeated dose toxicity studies Sprague Dawley rat studies (OECD TG 407/408; n = 10/sex/group) at 0, 500, 1000, and 2000 mg TOS/kg bw/day by gavage (10 mL/kg/day), survival was 100% and in-life observations, ophthalmology, neurobehavior, urinalysis, hematology, coagulation, and clinical chemistry were unremarkable; isolated statistical differences (e.g., male total protein, BUN, glucose, LDL; calcium; female phosphorus) were marginal, within historical ranges, non-monotonic, and lacked histopathology correlates. Organ-weight differences were single-sex and non-dose-dependent. No test-item-related lesions were detected. The NOAEL was 2000 mg TOS/kg bw/day ((highest dose tested) in both the 28-day and 90-day studies, supporting oral safety for food-use exposure.