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.