Toxicological Safety Evaluation of a Bacillus acidopullulyticus Pullulanase

Abstract

Promozyme®, an amylopectin debranching enzyme produced by Bacillus acidopullulyticus, was studied to evaluate its safety in the food industry. A dietary subchronic toxicity study incorporating fertility and teratogenicity studies was performed in 1-month-old rats at concentrations of 0.5, 1.5 and 5% Promozyme. No adverse effects were seen at the 0.5 and 1.5% dose levels, and at the 5% dose level only minor or equivocal signs of toxicity were recorded. With the exception of a moderate reduction in body weight gain the FIA litters at the 5% dose level, no effects were found in the fertility study, and Promozyme was not teratogenic. In a 13-wk oral toxicity study in dogs, no adverse effects resulted from 0.5 g/kg/d, whereas mild gastrointestinal disturbances were seen clinically at 1.5 and 5.0 g/kg/d. In dogs given 5.0 g/kg/d, terminal investigations showed increased kidney weights and mineralized casts in renal cortical tubules. This was probably due to the high content of ash (phosphorus) in the test material. Lack of mutagenic potential was confirmed in bacterial mutagenic assays with Salmonella typhimurium (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and in an in vivo cytogenetic study in rat bone marrow cells after a single dose and daily dosing for 5 d of up to 8 g/kg/d. In an acute inhalation study with 4 h of exposure of rats, no death occurred at the highest dose level used, i.e., 2 mg/L. The test material was non-irritating to skin and did not produce eye injury in rabbits. A skin sensitization study in guinea pigs revealed no indication that the enzyme is a sensitizer. The pathogenic potential of the enzyme-producing B. acidopullulyticus was investigated by single intraperitoneal and subcutaneous administrations to rats and mice; the microorganism was found to be nonpathogenic (LD50>1010 cells/kg). Tests of culture broths revealed that the microorganism does not produce antibiotics. Results indicated that production and the intended use of Promozyme can be regarded as safe for plant workers and consumers.