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.