ALPHA-AMYLASE FROM BACILLUS STEAROTHERMOPHILUS First draft prepared by Dr D.L. Grant, Toxicological Evaluation Division, Health and Welfare Canada 1. EXPLANATION Enzymes used for the hydrolysis of starch, generally called amylases, have a long history of use by the food industry. The amylase catalyzes the hydrolysis of 1,4 alpha-glucosidic linkages in common polysaccharide. Bacterial (Bacillus subtilis) alpha- amylase has been in common use to control the viscosity of chocolate syrup since 1929 and in the brewing industry since 1936. The enzyme preparation derived from these various Bacillus strains is usually added directly to the food to be processed and then removed from the final product by filtration. This alpha-amylase from Bacillus stearothermophilus (ATCC 39,709) has not been previously evaluated by the Joint FAO/WHO Expert Committee on Food Additives. 2. BIOLOGICAL DATA 2.1 Biochemical aspects No information available. 2.2 Toxicological studies 2.2.1 Acute Toxicity studies 2.2.1.1 Rat Groups of 10 male and 10 female rats, (Fischer 344) were dosed by gavage with Bacillus stearothermophilus alpha-amylase as an aqueous suspension at dose levels ranging from 0 to 10 g/kg. There was no mortality and the acute LD50 was determined to be greater than 10 g/kg (Thompson, 1982). 2.2.2 Short term studies 2.2.2.1 Rat Groups of 5 male and 5 female rats (Fischer 344, 28 days of age) were exposed to enzyme levels of 0, 0.84 and 1.68% in the diet for 2 weeks (71.8% protein and alpha-amylase activity of 8660 U/g). All animals were observed at least twice daily and body weights and feed consumption recorded twice weekly. There was no significant differences between treated and control groups in body weight or food consumption, and there was no effect on palatability (Rao, 1981a). 2.2.2.2 Dog Groups of 1 male and 1 female dog (Beagle dogs, 7-8 months of age) were exposed to enzyme levels of 0, 0.84 and 1.68% in the diet for 2 weeks (alpha-amylase 8660 U/g). All animals were observed at least twice daily, feed consumption was recorded daily, and body weights recorded weekly. There was no significant differences between treated and control groups in body weight, a slight reduction in food consumption in the male at 0.84%, and soft stools in all dogs. There was no effect on palatability (Rao, 1981b). Groups of 4 male and 4 female dogs (Beagle dogs, 6-7 months of age) were exposed to enzyme at levels of 0, 0.56 and 1.11% in the diet for 13 weeks (alpha-amylase activity 6540 U/g). All animals were observed at least twice daily, feed consumption was recorded daily, body weights recorded weekly, ophthalmic examinations were preformed prior to dosing and at termination, and haematology, blood chemistry and urinalysis data obtained prior to dosing and monthly thereafter. There were no significant differences between treated and control groups in ophthalmic observations, body weight, food consumption, and haematological parameters. There was a significant reduction in total serum protein concentration in males and females at 2 months in both dose groups, but these values were within normal baseline ranges. There was an increase in urinary protein content in treated females at all times and a slight increase in specific gravity in low dose females at 2 months and in both female dose groups at 3 months. Adrenal weight (both absolute and relative) was reduced at the lower dose level in females compared to controls. There were no treatment-related clinical observations, pathological changes or histopathological observations. The author concluded that the changes observed were not toxicologically significant and determined a NOEL of 1.11% in the diet (277.5 mg/kg b.w./day) (MacWilliams, 1982). 2.2.3 Long-term/carcinogenicity studies No information available. 2.2.4 Reproduction studies 2.2.4.1 Rat Groups of 12 male and 24 female rats (Fischer 344 weanling rats) were exposed to alpha-amylase at levels of 0, 0.56, 1.11% in the diet (alpha-amylase activity 6540 U/g) for 13 weeks and then allowed to mate (one male was placed with two randomly selected females). All animals were observed at least twice daily, feed consumption and body weights were recorded weekly, ophthalmic examinations were performed prior to dosing and at termination, and haematology, blood chemistry and urinalysis data obtained from selected animals prior to dosing and after 6 and 12 weeks of treatment. Pups were culled at random at day 4 to achieve maximum litter size of 10 (5 males and 5 females per litter). Pups were weaned at 28 days of lactation and twenty male and twenty female rats from each group selected at random for continuation for 13 weeks of exposure. Haematology, blood chemistry and urinalysis of the F1 rats were carried out at approximately 45 days post weaning (10/sex/group) and on all rats at termination of treatment. There were no consistent treatment-related effects in the F0 animals in body weight, haematology, blood clinical chemistry, urinalysis, pathology, or histopathology, except for a reduction in food consumption at both treatment levels during weeks 2, 4, 5, 10 and 11. There were no treatment-related reproductive effects and there were no treatment-related effects on the F1 animals for body weight, food consumption, haematology, blood clinical chemistry, urinalysis, pathology or histopathology. The author concluded that a NOEL of 1.11% (approximately 0.98 g/kg b.w./day could be established (MacKenzie, 1982). 2.3 Observations in humans No information available. 3. COMMENTS This alpha-amylase preparation produced no significant toxicological effects in a 13-week feeding study in dogs up to a level of 0.28 g/kg b.w. per day nor in a one-generation (one-litter) rat reproduction study with some of the F1 rats being treated up to a level of 0.98 g/kg b.w. per day for 13 weeks after weaning. 4. EVALUATION The Committee allocated an ADI "not specified" to this enzyme preparation. 5. REFERENCES HAZLETON LABORATORIES AMERICA, INC. Madison, Wisconsin, USA. Submitted to WHO by CPC International, Englewood Cliffs, NJ, USA. MacKENZIE, K.M. (1982). Subchronic oral toxicity study of Bacillus stearothermophilus alpha-amylase in utero exposed F1 rats. Unpublished report No. 81168 from Hazleton Laboratories America, Inc. Madison, Wisconsin, USA. Submitted to WHO by CPC International, Englewood Cliffs, NJ, USA. MacWILLIAMS, P.S. (1982). Ninety-day subchronic oral toxicity study of Bacillus stearothermophilus alpha-amylase in dogs. Unpublished report No. 81170 from Hazleton Laboratories America, Inc. Madison, Wisconsin, USA. Submitted to WHO by CPC International, Englewood Cliffs, NJ, USA. RAO, G.N. (1981a). Fourteen-day palatability study of Bacillus stearothermophilus alpha-amylase in rats. Unpublished report No. 81167 from Hazleton Laboratories America, Inc. Madison, Wisconsin, USA. Submitted to WHO by CPC International, Englewood Cliffs, NJ, USA. RAO, G.N. (1981b). Fourteen-day palatability study of Bacillus stearothermophilus alpha-amylase in dogs. Unpublished report No. 81169 from Hazleton Laboratories America, Inc. Madison, Wisconsin, USA. Submitted to WHO by CPC International, Englewood Cliffs, NJ, USA. THOMPSON, G.W. (1982). Acute oral toxicity study with Bacillus stearothermophilus alpha-amylase in rats. Unpublished report No. 81213 from Hazleton Laboratories America, Inc. Madison, Wisconsin, USA. Submitted to WHO by CPC International, Englewood Cliffs, NJ, USA.
See Also: Toxicological Abbreviations alpha-AMYLASE FROM BACILLUS STEAROTHERMOPHILUS (JECFA Evaluation)