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    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