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    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)