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EC number: 232-565-6
CAS number: 9000-90-2
Alpha-amylase was tested in two independent main tests. The ‘treat and
plate’ treatment method was used in each test to avoid the possibility
that bio-available histidine in the test item might compromise the test.
Five dose levels (50, 160, 500, 1600, and 5000 μg/plate) were tested.
Aliquots (200 μl/plate) of formulations of the test item mixed with
sterile saline (0.9 % NaCl) were added to the bacteria. All dose levels
are expressed in terms of the total protein content of the test item
supplied, being 46 mg/ml. Negative control plates were treated with
sterile saline (200 μl/plate). The treatments were performed both with
and without a metabolic activation system (S-9 mix).
The test item was not toxic to the test bacteria at any dose level
tested, either in the absence or presence of S-9 mix: no reductions in
the growth of the background lawn of non-revertant bacteria or the
number of revertant colonies were observed.
No biologically significant increases in the number of revertant
colonies were observed at any dose level of the test item in either main
test. Small, statistically significant increases in the number of
revertant colonies were observed in the first main test in TA 1537
treated at 1600 and 5000 μg/plate in the presence of S-9 mix. These
increases were not considered to be biologically significant and they
did not meet the stated criteria for a mutagenic effect of the test item
because they were too small (just 1.53 and 1.61-fold higher than the
corresponding negative control values, respectively) and they were not
reproduced in the second main test. No other statistically significant
increases in the number of revertant colonies were observed in any test
strain after treatment with the test item at any dose level either with
or without S-9 mix.
The results obtained with the negative and positive controls
demonstrated the sensitivity of the tests and the efficacy of the S-9
mix metabolic activation system.
Based on the results obtained in this study, it is concluded that
alpha-amylase is not mutagenic in the Ames test.
The clastogenic potential of the test substance alpha-amylase was
evaluated by its effect on chromosomes of human peripheral blood
lymphocytes according to OECD guideline 473 (1997).
The test item was tested in human lymphocytes in primary cultures
of whole blood in the absence and presence of S-9 mix. The cultures were
treated with formulations of the test item in cell culture medium.
In the first test, all cultures with and without S-9 mix were
treated for three hours. In the second test, the cultures were treated
for 18 hours without S-9 mix and three hours with S-9 mix. All cultures
were harvested 18 hours (approximately 1.5 normal cell cycles) after the
start of treatment. The final concentration of S-9 homogenate used in
the second test was twice as high as in the first test. Both tests were
repeated because some of the positive control treatments produced
inadequate responses in the original tests and also to investigate two
increases in the frequency of metaphases with aberrant chromosomes.
The test item did not cause marked toxicity at any concentration
tested. Slides from duplicate cultures treated with the test item at
1250, 2500 and 5000 μg/ml with and without S-9 mix in each test were
scored for chromosomal aberrations. The concentrations are expressed in
terms of the total protein content of the test item (46 mg/ml in the
sample supplied). The highest concentration tested (5000 μg/ml) is the
maximum required by the OECD 473 guideline for materials of low
No biologically significant increases in the frequency of
metaphases with chromosomal aberrations were observed in cultures
treated with the test item. Statistically significant increases were
observed at two test points, but they are not considered to be
biologically significant because they were not reproducible between the
replicate cultures at those test points or in the repeat tests.
Two polyploid metaphases were observed in this study, but their
incidence was not dose-related and it is concluded that they were not
caused by the test item. No endoreduplicated metaphases were observed.
It is concluded that alpha-amylase did not cause chromosomal
aberrations in this in vitro cytogenetic test using cultured human
batch PPY2693, was assayed for its ability to induce mutation at the
HGPRT locus (6-thioguanine resistance) in mouse lymphoma cells using a
fluctuation protocol. The study consisted of two independent
experiments, each conducted in the absence and presence of metabolic
activation (S-9 mix).
wide range of treatments, separated by half-log intervals and reaching
5000 µg/mL, cultures surviving the top dose of 5000 µg/mL in the absence
and in the presence of S-9showed
55% and 53% survival respectively. These, together with the next 3 lower
doses, were plated for viability and 6-thioguanine resistance eight
(treatments in the absence of S-9) or seven (treatments in the presence
of S-9) days after treatment. In the second experiment a narrower dose
range was used to maximise the chance of detecting any dose related
effects. The top dose plated in this experiment was again 5000 µg/mL in
the absence and presence of S-9, which resulted in 50% and 117% survival
(solvent) and positive control treatments were included in each
experiment in the absence and presence of S-9. Mutation frequencies in
negative control cultures fell within normal ranges, and statistically
significant increases in mutation were induced by the positive control
chemicals 4-nitroquinoline-1-oxide (without S-9) and benzo(a)pyrene
(with S-9). Therefore the study was accepted as valid.
did not result in any statistically significant increases in mutation
in the absence or presence of S-9.
when tested to a concentration of 5000 µg/mL in the absence and presence
of S-9, the present alpha-amylase, batch PPY2693 failed to induce
mutation at the HGPRT locus of L5178Y mouse lymphoma cells in two
was concluded that alpha-amylase had no mutagenic activity in this test
The clastogenic and aneugenic activity of alpha-amylase was
investigated in cultured human peripheral blood lymphocytes by effects
on the frequency of micronuclei. Division of the lymphocytes was
stimulated by adding phytohaemagglutinin to the cultures. Sets of
duplicate cultures were exposed to the test substance for 3 hours in the
presence and absence of metabolic activation (S-9 mix) and harvested 24
hours after the beginning of treatment (3+21 hour treatment).
Additionally, a continuous 24-hour treatment without S-9 mix was
included with harvesting at the end of treatment (24+0 hour treatment).
The cultures were treated with cytochalasin-B after removal of the test
substance to block cytokinesis. Three concentrations, covering an
appropriate range of cytotoxicity, were selected for scoring of
micronuclei. A minimum of 1000 cells per concentration were scored.
The proportion of binucleate cells with micronuclei in all
cultures of the vehicle controls (purified water) was within the limits
of the historical ranges. The positive controls induced statistically
significant increases in the proportion of cells with micronuclei,
demonstrating the sensitivity of the test procedure and the metabolic
activity of the S-9 mix employed.
The 3+21 hour treatment of the cells resulted in frequencies of
micronucleated binuclear cells (MNBN cells), which were similar to and
not significantly (p ≤ 0.05) higher than those observed in concurrent
vehicle controls except from an increase at the highest concentration
(5000 µg/mL, test material weighed out as received) in the absence of
S-9 mix and at the intermediate concentration analyzed (4000 µg/mL) in
the presence of S-9 mix. These increases were considered of no
When the cells were treated in the 24+0 hour treatment without S-9
mix, statistically significant elevated frequencies of MNBN cells were
noticed at three of four concentrations analyzed compared to the vehicle
controls. However this was set against a very low concurrent vehicle
control response and with no real evidence of concentration related
response and the values fell within normal historical values. These data
were therefore not considered to represent evidence of a treatment
In conclusion, alpha-amylase
did not induce micronuclei in cultured human peripheral
blood lymphocytes either in the absence or presence of S-9 mix under the
experimental conditions employed for this study.
The genetic toxicity of alpha-amylase has been investigated in the Ames
assay, in the in vitro chromosome aberration assay, in the in
vitro mammalian cell test looking at the induction of micronuclei
and in the mouse lymphoma assay. All tests have been performed according
to OECD and EC guidelines, and in compliance with GLP. No evidence for
genetic toxicity was observed and thus it can be concluded that
alpha-amylase is not mutagenic and does not induce chromosome aberration
in the present test systems.
The safety of the production strain was further fully documented to
belong to a safe strain lineage (Pariza and Johnson, 2001) and the
enzyme test material was well characterized.
Pariza, M. W., and Johnson, E. A. (2001). Evaluating the Safety of
Microbial Enzyme Preparations Used in Food Processing: Update for a New
Century. Regulatory Toxicology and Pharmacology, 33: 173-186.
Short description of key information:
No mutagenic activity of alpha-amylase was observed in the Ames
test, the in vitro micronucleus test, the in vitro human lymphocyte
chromosome aberration test or in the mouse lymphoma assay.
Endpoint Conclusion:No adverse effect observed (negative)
Due to the lack of genetic toxicity, alpha-amylase is not
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