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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 March 2010 - 31 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Principles of method if other than guideline:
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.

GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
The study describes experiments performed to assess the effect of the test material alpha amylase in amino acid dependent strains of Salmonella typhimurium capable of detecting both induced frame-shift (TA1537 and TA98) and base-pair substitution mutations (TA1535, TA100 and TA102). The test system is a reverse mutation of amino acid dependent bacterial strains.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 from Aroclor 1254 induced SPF Wistar rats, manufactured at LAB Research and validated in separate test.
Test concentrations with justification for top dose:
Five dose levels (50, 160, 500, 1600, and 5000 μg/plate) were tested. All dose levels are expressed in terms of the total protein content of the test item supplied, being 46 mg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile saline solution (0.9 % NaCl)
- Justification for choice of solvent/vehicle: The test substance is water-soluble and any human exposure will be in aqueous solutions.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: Cumene hydroperoxide, sodium azide, 2-nitrofluorene, 9-aminoacridine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: All applications were done in medium before plating, i.e. a liquid culture assay (treat and plate assay).

DURATION
- Exposure duration: 3.5 hrs (liquid culture assay)
- Incubation time (selective incubation) : 3 days

DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count

Evaluation criteria:
The numbers of revertant colonies at each treatment test point were compared to the corresponding negative control values for each set of triplicate plates.
The tests were considered to be valid as all the following criteria were met:
- negative and positive control data were consistent with the historical control data for this laboratory
- the positive control data showed marked increases over the concurrent negative control values
- the evaluation of the data was not restricted by loss of plates (e.g. through contamination).
The test item would have been considered to have shown evidence of mutagenic activity in this study if all of the following criteria had been met:
- increases in the numbers of revertant colonies were observed at one or more test points
- the mean number of revertant colonies at the test point showing the largest increase was more than twice the corresponding negative control value
- there was a credible scientific explanation for the observed dose-response relationship that involved a mutagenic effect of the test item
- the increases were reproducible between replicate plates and were observed in both main tests (when treatment conditions were the same)
- the increases were statistically significant
- the increases were not directly related to increased growth of the non-revertant bacteria
Statistics:
The numbers of revertant colonies at each treatment test point in the main tests were compared to the corresponding negative control values using the Analysis of Variance test. When this test showed statistically significant differences in the data, Dunnett’s test was used to determine the statistical significance of increases and decreases in the numbers of revertant colonies for each set of triplicate plates. The statistical analyses were performed with SAS® procedures (version 8.2) described in SAS/STAT® User’s Guide, SAS OnlineDoc®, 1999, SAS Institute Inc., Cary, North Carolina 27513, USA.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The test item was not toxic to the test bacteria, either in the absence or presence of S-9 mix: no marked reductions in the number of revertant colonies or growth of the background lawn of non-revertant bacteria were observed, compared to the negative control plates.
On the basis of these results, 5000 ug test substance/plate was chosen as the highest dose level for the main tests.
COMPARISON WITH HISTORICAL CONTROL DATA: The negative and positive control values were compatible with the historical control values for this laboratory.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
It was concluded, that the results of this Ames test study gave no indication of mutagenic activity of alpha-amylase.
Executive summary:

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.

 

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 March 2010 - 17 October 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate included in the study report.
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
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. All concentrations were expressed in terms of the total protein content of the test item (stated by the Sponsor to be 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 toxicity.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Cell culture medium, RPMI 1640 (Gibco)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: without S-9 mix: Daunomycin; with S-9 mix: cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in suspension

DURATION
- Exposure duration: Cultures, established approx. 48 h before testing, were treated for 3 h with and without S9-mix in the first test. 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.

SPINDLE INHIBITOR (cytogenetic assays): Demecolcine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Duplicate cultures.

NUMBER OF CELLS EVALUATED: at least 1000 cells scored per culture; chromosomal aberrations scored based on 100 metaphases per culture (where ever possible)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes


Evaluation criteria:
It would have been concluded that the test item had shown clastogenic activity in this study if all of the following criteria had been met:
- the increases in the frequency of metaphases with aberrant chromosomes were observed at one or more test concentrations
- the increases were reproducible between replicate cultures and between tests (when treatment conditions were the same)
- the increases were statistically significant
- the increases were not associated with large changes in pH or osmolarity of the treated cultures
The historical negative control data for this laboratory was also considered in the evaluation.
The test item would have been considered to have given a negative response if no reproducible, statistically significant increases were observed.
Results which failed to meet the stated criteria for a negative or positive response would have been considered to be equivocal.
Statistics:
The number of metaphases with aberrant chromosomes at each test concentration was compared to the concurrent negative control value. When appropriate, statistical analysis was performed using Fischer’s Exact Test. The statistical analysis was performed with SAS® procedures (version 8.2) described in SAS/STAT® User’s Guide, SAS OnlineDoc®, 1999, SAS Institute Inc., Cary, North Carolina 27513, USA.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA:
Some of the positive control treatments produced inadequate responses in the first tests and had to be repeated. In the repeated tests, the negative controls were low, the positive control values showed clear increases in levels of aberrations and all the control values were consistent with the historical control values for this laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test item did not cause marked toxicity at any concentration tested in the absence or presence of S-9 mix.
Conclusions:
It is concluded that alpha-amylase did not cause chromosomal aberrations in this in vitro cytogenetic test using cultured human lymphocytes.
Executive summary:

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 toxicity.

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 lymphocytes.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 June 1989 - 10 October 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HGPRT (6-thioguanine resistance)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Fishers medium (10% horse serum)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
The highest concentration tested was 5000 µg/ml (weighed out as received).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure is in aqueous solutions.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitroquinoline-1-oxide (without S-9) and benzo(a)pyrene (with S-9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium, growth suspension. Selection phase was performed in microtitre plates.

DURATION
- Exposure duration: 2 hours
- Expression time (cells in growth medium): With exception of experiment 1 treatments in the absence of S-9 (where cultures were maintained for eight days) cultures were maintained for 7 days.
- Fixation time (start of exposure up to fixation or harvest of cells): At least 7 days after treatment.

SELECTION AGENT (mutation assays): 6-TG

NUMBER OF REPLICATIONS: duplicate

DETERMINATION OF CYTOTOXICITY
- Method: Cell density by counting viable cells, expressed as percentage relative survival (RS%)
Evaluation criteria:
A test article was considered positive if:
- The assay was valid, and
- Significant induced mutation (i.e. the lower 95 percentile of a treated culture exceeded the upper 95 percentile of a control culture) occurred at consecutive doses in at least one experiment, and
- Dose-related increases in mutation could be confirmed by regression analysis in both experiments.
Statistics:
The mutation frequency was evaluated statistically by using logarithmic transformation of the variances of the number of clones observed on viability and mutation plates as described by E.E. Furth et al., Anal Biochem 110: 1-8, 1981
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Alpha-amylase, batch PPY2693, under the conditions of the test, had no mutagenic activity in cultured mouse lymphoma cells when tested to a concentration of up to 5000 µg/mL in either the absence or presence of S-9.
Executive summary:

Alpha-amylase, 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).

Following a 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 respectively.

Negative (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.

The treatment did not result in any statistically significant increases in mutation frequency, neither in the absence or presence of S-9.

Therefore, 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 independent experiments.

It was concluded that alpha-amylase had no mutagenic activity in this test system.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: other: Chromosome aberration/clastogenicity and aneuploidy
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 25 - Aug. 21, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: Draft OECD guideline 487, adopted 22 July 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: Primary cells from human blood
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Highest concentration tested was 5000 µg/mL (weighed out as received) and dilutions hereof.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Purified water
- Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure will be in aqueous solutions.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: Mitomycin C (MMC) and Vinblastine (VIN) in the absence of rat liver S-9, Cyclophosphamide (CPA) in the presence of S-9.
Details on test system and experimental conditions:
METHOD OF APPLICATION: Whole blood cultures were established in HEPES-buffered RPMI medium containing 20% (v/v) heat inactivated foetal calf serum, 50 μg/mL gentamycin and 2% of the mitogen Phytohaemagglutinin (PHA). These cultures were incubated at approx. 37°C for 48 hours before treatment with test article. 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 by evaluating the effect of the test substance on the replication index. A minimum of 1000 cells per concentration (500 cells from each replicate culture) were scored.

DURATION
- Exposure duration: 3 and 24 hours

NUMBER OF REPLICATIONS: Sets of duplicate cultures were exposed to the test substance.

DETERMINATION OF CYTOTOXICITY
- Method: 5000 μg/mL was determined as max dose following a preliminary cytotoxicity Range-Finder Experiment. Cytotoxicity (%) was expressed as (100 – Relative replication Index (RI)).

Evaluation criteria:
A test article was considered positive if:
- the assay was valid, and
- significant increase in the frequency of MNBN cells at one or more concentrations , and
- the incidence of MNBN cells exceeded the normal range in both replicates, and
- a concentration-related increase in the proportion of MNBN cells was observed.
Statistics:
The proportion of MNBN cells for each treatment condition were compared with the proportion in negative controls by using Fisher's exact test. Probability values of p equal or less than 0.05 were accepted as significant.
Key result
Species / strain:
lymphocytes: from human blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the 24+0 hour treatment without S-9 mix the highest applied concentration was 1000 µg/mL due to cytotoxicity at higher concentrations. No significant cytotoxicity was seen in the 3+21 hour treatment.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH of test material is neutral, 7.4
- Effects of osmolality: no, the test material has the following osmolality in a 30 mg/mL solutionin cell culture medium: 312 mOsm/kg
- Evaporation from medium: no
- Water solubility: yes
- Precipitation: no
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: Preliminary cytotoxicity range-finder performed

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative controls were within the historical negative control ranges. The 3+21 hour treatment of the cells resulted in frequencies of micronucleated binuclear 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) in the absence of S-9 mix and at the intermediate concentration analysed (4000 µg/mL) in the presence of S-9 mix. These increases were considered of no biological importance.



Conclusions:
Alpha-amylase did not show any clastogenic activity, neither in the presence or absence of S-9 mix, when tested in the present in vitro micronucleus assay.
Executive summary:

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 biological importance. 

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 related effect.

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.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

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.

Reference:

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)

Justification for classification or non-classification

Due to the lack of genetic toxicity, alpha-amylase is not classified.