CARBOHYDRASE (alpha-AMYLASE) FROM BACILLUS LICHENFORMIS EXPLANATION Carbohydrase is an enzyme that catalyzes the hydrolysis of alpha-1,4-glycosidic linkages of starch. The enzyme preparation that is derived from B. lichenformis is added directly to the food to be processed and then it is removed from the final product by filtration. This preparation has not been previously evaluated by the Joint FAO/WHO Expert Committee on Food Additives. BIOLOGICAL DATA Biochemical aspects No information available. Toxicological studies Special studies on genetic toxicity Groups of 20 CD-1 male mice fed diets containing 0, 1.0, 2.0, or 4.0% of the carbohydrase preparation were used in a dominant-lethal study. The animals were fed the test compound for 5 days. These males were then mated 1-to-1 at weekly intervals with 5 different batches of 20 females each. At 14 days, after evidence of copulation, females were sacrificed and the uterine contents examined for implantations, viable embryos, and early and late embryonic deaths. Some weight loss occurred in both the high- and mid-dose males. One total-litter loss occurred in each of the high- and mid-dose groups at the second pairing and in the low-dose group at the third pairing, but the incidence of these losses was too low to be considered treatment- related and there were no other compound-related effects (Palmer & Lowell, 1973a). A dominant-lethal study was carried out using groups of 20 male CD rats given a diet containing 0, 1, 2, or 4% of the carbohydrase preparation for 5 days. The males were then mated on a 1-to-1 basis for 7 days with untreated CD females. A new batch of females were mated with treated males every 7 days for 6 consecutive weeks. Pregnant females were sacrificed on about day 14 of pregnancy and ovaries and uteri were examined for corpora lutea, implantations, viable embryos, and early and late embryonic deaths. During the 5-day treatment period weight loss occurred in the high-dose males, and body-weight gains were reduced at the mid-dose level. There was no significant effect of treatment on mating performance, pregnancy rate, pre- or post-implantation loss, or overall viable litter size. Although 2 females in the high-dose group suffered total-litter losses, the overall incidence did not suggest a treatment-related effect (Palmer & Lowell, 1973b). Special study on teratology Groups of 20 mated female CD rats were fed diets containing 0, 1, 2, or 4% of the carbohydrase preparation from days 6 through 15 of pregnancy. The animals were sacrificed on day 20 of pregnancy and the uterine contents examined for the number of corpora lutea, number of viable foetuses, number of resorption sites, litter weight, and foetal abnormalities. One-third of the foetuses were examined by the Wilson technique for visceral abnormalities and two-thirds were processed for examination of skeletal abnormalities. During the first 4 days of dietary administration of the test compound there was reduced dietary consumption and reduced weight gain at all dose-levels (including weight loss at the high dose). No compound-related effects on foetal or embryological development were reported, although there was a non- significant increase in foetal abnormalities at the high dose (Palmer & Lowell, 1972). Acute toxicity LD50 Species Route (mg/kg b.w.) Reference Mouse oral (gavage) 20,000 Novo, 1973a Rat (male) oral (gavage) 20,500 Novo, 1973b (female) 16,500 Short-term studies Rats Groups of 5 male and 5 female Wistar rats were fed diets containing 0, 0.5, or 2.5% of the carbohydrase preparation in the diet for 4 weeks. Except for a small but statistically-significant increase in absolute and relative kidney weights in the high-dose males, and some fluctuation in weight gain during the initial part of the study, there were no compound-related changes. Gross pathology, clinical chemistry, and feed efficiency were comparable between groups (Novo, 1972). Groups of 15 male and 15 female CFY-strain rats were fed diets containing 0, 1, 2, and 4% of the carbohydrase preparation in the diet for 13 weeks. Thinning of the hair, mainly on the scapular region, was noted in 7 of the high-dose female rats from week 9 onward. There was reduced feed intake in mid-dose females and high-dose males and females, and decreased weight gain in high-dose animals of both sexes. Organ to body-weight ratios for several organs from the high-dose animals differed significantly from control values; however, many of these differences were likely to have arisen because of reduced body- weight gain. When compared to brain weights, only reduced liver weights in high-dose males were found to be significantly differently from controls. Increased adrenal weights in high-dose females were considered to be within the normal range of biological variability. Enlargement of the caecum was noted in mid-dose males and in both sexes at the high dose. No compound-related changes were reported with regard to survival, urinalysis, haematology, clinical chemistry, or microscopic pathology (Rivett et al., 1973a). Dogs Groups of 3 male and 3 female Beagle dogs were given diets containing 0, 1, 2, or 4% carbohydrase preparation for 13 weeks. Reduced mean body-weight gain was observed in high-dose animals of both sexes. Food and water consumption were reduced in high-dose animals of both sexes and in mid-dose females. No compound-related changes were observed with respect to haematology (1 high-dose female had platelet counts greater than the normal range), clinical chemistry, urinalysis, or gross and microscopic pathology. Differences between control and high-dose animals with respect to organ-weight ratios were ascribed to reduced growth of the high-dose animals (Rivett et al., 1973b). Long-term studies No information available. Observations in man No information available. Comments The carbohydrase preparation showed no significant toxicological effects in short-term feeding studies in rats at levels of up to 4% of the diet (40 mg/kg of feed) or in dogs at levels of up to 2% (20 mg/kg of feed). No teratogenic effects were noted in a study in rats. The preparation was also inactive in dominant-lethal tests in rats and mice. EVALUATION Level causing no toxicological effect The no-effect level in a short-term study in dogs was 2% of the diet, equal to 450 mg/kg b.w. Estimate of acceptable daily intake for man ADI "not specified". REFERENCES Novo (1972). Four week oral toxicicity study of Novo alkaline amylase in rats. Unpublished study by Novo Industria A/S. Submitted to WHO by Novo Industria A/S. Novo (1973a). Acute toxicity of Novo amylase to mice. Unpublished study by Novo Industria A/S. Submitted to WHO by Novo Industria A/S. Novo (1973b). Acute toxicity of Novo alkaline amylase to rats. Unpublished study by Novo Industria A/S. Submitted to WHO by Novo Industria A/S. Palmer, A.K. & Lovell, M.R. (1972). Effect of NAA on pregnancy of the rat. Unpublished report of the Huntingdon Research Centre. Submitted to WHO by Novo Industria A/S. Palmer, A.K. & Lovell, M.R. (1973a). Dominant lethal assay of NAA in the male mouse. Unpublished report of the Huntingdon Research Centre. Submitted to WHO by Novo Industria A/S. Palmer, A.K. & Lovell, M.R. (1973b). Dominant lethal assay of NAA in the male rat. Unpublished report of the Huntingdon Research Centre. Submitted to WHO by Novo Industria A/S. Rivett, K.F., Bhutt, A., Street, A.E., Heywood, R., & Newman, A.J. (1973a). Novo alkaline amylase dietary study in rats. Unpublished report of the Huntingdon Research Centre. Submitted to WHO by Novo Industria A/S. Rivett, K.F., Sortwell, R.J., Newman, A.J., & Street, A.E. (1973b). Novo NAA toxicity studies in beagle dogs. Unpublished report of the Huntingdon Research Centre. Submitted to WHO by Novo Industria A/S.
See Also: Toxicological Abbreviations