Amylase, gluco-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No mutagenic activity of glucoamylase could be detected in the Ames Assay. Glucoamylase did not induce chromosomal aberrations in the in vitro mammalian chromosome aberration test performed with human lymphocytes. Glucoamylase did not induce micronuclei in cultured human peripheral blood lymphocytes micronucleus assay. These results are supported by read-across from three in vitro gene mutation studies in L5178Y mouse lymphoma cells performed on three different amylases belonging to the same subclass of glycosidases (IUBMB class 3.2.1.) as glucoamylase.

It can be concluded that glucoamylase does not exhibit any genetic toxicity when tested using in vitro tests.

Link to relevant study records

Reference

Reference 1

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 December 2008 - 30 January 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Principles of method if other than guideline:

Crude enzyme preparations, like the present batch of glucoamylase contain the free amino acid histidine and tryptophan, most often in an amount, which exceeds the critical concentration for incorporation in the direct standard assay.
To overcome this problem, all strains were exposed to glucoamylase in liquid culture (“treat and plate assay”).

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The study describes experiments performed to assess the effect of glucoamylase 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:

bacteria, other: Salmonella typhimurium TA1537, TA98, TA1535, TA100, TA102

Metabolic activation:

with and without

Metabolic activation system:

S9 from Aroclor 1254 induced SPF Wistar rats obtained from Taconic Europe A/S, Ejby, DK-5623 Lille Skensved, Denmark.

Test concentrations with justification for top dose:

50, 160, 500, 1600 and 5000 µg test substance (total protein) per plate, with and without the metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile saline (0.9% NaCl, Fresenius Kabi Norge A/S)
- 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: with S-9 mix: cumene hydroperoxide, sodium azide, 2-nitrofluorene, 9-aminoacridine; without S-9 mix: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium before plating, i.e a liquid culture assay (treat and plate assay).

DURATION
- Exposure duration: 3 hours
- 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 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 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 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

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

Key result

Species / strain:

S. typhimurium TA 1537

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

Key result

Species / strain:

S. typhimurium TA 100

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

Key result

Species / strain:

S. typhimurium TA 98

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

Species / strain:

S. typhimurium TA 102

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

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The number of revertant colonies on the control plates were generally lower than the historical control range for this laboratory, but the data were considered to be adequate to assess the toxicity of the test item. The test item was not toxic to the test bacteria: 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 μg/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. One of the positive control values in TA 102 without S-9 mix in the second test was slightly lower than the historical control range and one plate at this test point had microcolonies. One of the negative control values for TA 1537 without S-9 mix in the second test was higher than the historical control range. These values are considered to be acceptable. The large increases in the number of revertant colonies produced by the positive control treatments demonstrated the sensitivity of the tests and the efficacy of the S-9 mix metabolic activation system.

Conclusions:

Based on the results obtained in this study, it is concluded that the test substance was not mutagenic in the Ames test.

Executive summary:

Glucoamylase was examined for mutagenic activity in the bacterial reverse mutation assay using Salmonella typhimurium strain TA1535, TA100, TA1537, TA98 and TA102.

Crude enzyme preparations, like the present batch contain the free amino acid histidine and tryptophan, which exceeds the critical concentration for incorporation in the direct standard assay. To overcome this problem all strains were exposed to the test substance in liquid culture (“treat and plate assay”).

Bacteria were exposed to 5 doses of the test substance in a phosphate buffered nutrient broth for 3 hours with 5 mg (total protein) per mL as highest concentration. After incubation the test substance was removed by centrifugation prior to plating.

The study was conducted with and without the metabolic activation system S9 - a liver preparation from rats, pre-treated with Aroclor 1254, and the co-factors required for mixed function oxidase activity (S9 mix).

Two identical and independent experiments were conducted.

The treatment of the Salmonella strains with the test substance, in the presence or absence of S9 mix, did not result in any increases in revertant numbers. The test substance was found not mutagenic.

Reference 2

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:

01 December 2008 - 15 March 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate included in the study report.

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

At chromosomal level.

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Highest concentration tested was 5000 µg/ml and 2-fold dilutions herof.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Cell culture medium

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: 3 hours (first experiment); 3 respectively 20 hours (second experiment, with respectively without S9)
- Treatment plus recovery time : 3 + 17 hours, 20 + 0 hours

SPINDLE INHIBITOR (cytogenetic assays): Colchicine

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Duplicate cultures.

NUMBER OF CELLS EVALUATED: 200 metaphases per dose (100 cells per culture)

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. Evidence of a dose-response relationship would have been considered to support the conclusion.
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:

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.

Key result

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:
Cells treated with the test substance, either in the absence and presence of S-9, had similar numbers of aberrations to those observed in concurrent solvent controls. There were no reproducible increases in aberration frequency that were significantly higher than those observed in the negative controls. The negative controls were within the historical negative control ranges. Normal frequencies of cells with numerical aberrations were seen under all treatment conditions. The positive controls induced satisfactory levels of aberrations.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No significant cytotoxicity was observed.

Conclusions:

It is concluded that the test substance did not cause chromosomal aberrations when tested up to 5000 μg/mL with and without S-9 mix in this in vitro cytogenetic test using cultured human lymphocytes.

Executive summary:

The clastogenic potential of the test substance was evaluated by its effect on chromosomes of human peripheral blood lymphocytes according to OECD guideline 473 (July 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. Two independent tests were performed. 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 20 hours without S-9 mix and three hours with S-9 mix. All cultures were harvested 20 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.

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 both tests were scored for chromosomal aberrations. All the concentrations in this report are expressed in terms of the total protein content of the test item (stated by the Sponsor to be 199.63 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 or statistically significant increases in the frequency of metaphases with chromosomal aberrations were observed in cultures treated with the test item in either test.

The positive control treatments produced statistically significant increases in the frequency of aberrant metaphases, demonstrating the sensitivity of the tests and the efficacy of the S-9 mix.

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 glucoamylase did not cause chromosomal aberrations in this in vitro cytogenetic test using cultured human lymphocytes.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec. 22, 2011 - Jun. 13, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Chromosome defects.

Species / strain / cell type:

lymphocytes: Primary cells from human blood

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver homogenate, S-9 mix.

Test concentrations with justification for top dose:

Highest concentration tested was 5000 ug/mL (weighed out as received) (equivalent to 510 ug enzyme concentrate dry matter/mL) 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.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Vinblastine

Details on test system and experimental conditions:

Glucoamylase was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two male donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254 induced animals. The test article was formulated in water for irrigation (purified water) and the highest concentration tested in the Micronucleus Experiment, 5000 μg/mL (an acceptable maximum concentration for in vitro chromosome aberration studies according to current regulatory guidelines), was determined following a preliminary cytotoxicity Range-Finder Experiment.
Treatments were conducted 48 hours following mitogen stimulation by hytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of glucoamylase on the replication index (RI).

Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate cells (MNBN) in these cultures fell within current historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9. Cells receiving these were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment; all compounds induced statistically significant increases in the proportion of cells with micronuclei.

Evaluation criteria:

Binucleate cells were only included in the analysis if both of the below criteria were met:
1. The cytoplasm remained essentially intact, and
2. The daughter nuclei were of approximately equal size.

A micronucleus was only recorded if it met the following three criteria:
1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth-edged and smaller than approximately one third of the diameter of the main nuclei.

The assay was considered valid if the following four criteria were met:
1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures, particularly where no positive responses were seen.
2. The frequency of MNBN cells in vehicle controls fell within the normal ranges.
3. The positive control chemicals induced statistically significant increases in the proportion of cells with micronuclei. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the current historical vehicle control ranges.
4. A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in negative control cultures at the time of harvest.

The test article was considered to induce clastogenic and/or aneugenic events if all of the following criteria were met:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed.
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed.
3. 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 ≤ 0.05 were accepted as significant. Additionally, the number of micronuclei per binucleate cell were obtained and recorded.

Species / strain:

lymphocytes: from human blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Glucoamylase 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:

Glucoamylase, batch PPY32789 was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two male donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254 induced animals. The test article was formulated in water for irrigation (purified water) and the highest concentration tested in the Micronucleus Experiment, 5000 ug/mL (an acceptable maximum concentration for in vitro chromosome aberration studies according to current regulatory guidelines) (equivalent to 510 ug enzyme concentrate dry matter/mL), was determined following a preliminary cytotoxicity Range-Finder Experiment.

Treatments were conducted 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of glucoamylase on the replication index (RI). In the Micronucleus Experiment, micronuclei were analysed at three concentrations.

Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate cells (MNBN) in these cultures fell within current historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9. Cells receiving these were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment; all compounds induced statistically significant increases in the proportion of cells with micronuclei.

All acceptance criteria were met and the study was therefore considered as valid.

Pulse 3+21 hour treatment of cells with glucoamylase in the absence and presence of S-9 resulted in frequencies of MNBN cells which were similar to and not significantly (p ≤ 0.05) higher than those observed in concurrent vehicle controls for the majority of concentrations analysed. The single exception to this was for the lowest concentration analysed following treatment in the presence of S-9 (3000 ug/mL) where a small increase was noted (mean MNBN cell frequency = 0.4% compared to 0.15% in the concurrent vehicle control). However, as the MNBN cell frequency of both replicate cultures at this and all other glucoamylase treated cultures fell within historical vehicle control (normal) values, this isolated statistical increase was not considered of biological importance.

Following 24+24 hour –S-9 treatment, a further small but statistically significant increase in MNBN cells was noted at the intermediate concentration analysed (4000 ug/mL). However, there were no instances of MNBN cell values exceeding normal values for any test article treated culture (at this or any other concentration), with higher and lower concentrations analysed showing mean MNBN cell values similar to those observed for the vehicle control. As such, this isolated statistical increase was not considered of biological importance.

It is concluded that glucoamylase did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system (S-9). Concentrations were tested up to 5000 ug/mL

(equivalent to 510 ug enzyme concentrate dry matter/mL), a recommended regulatory maximum concentration for in vitro cytogenetic assays.

Reference 4

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:

Jan. 11, 1990 - Aug. 20, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The results from the present study are used to support the evaluation of the test substance glucoamylase IUB 3.2.1.3 by read-across from the in vitro gene mutation studies in L5178Y mouse lymphoma cells performed on another glucosidase.

See Target Record for futher information on the justification for read-across (Genetic toxicity conclusion read-across ML.00).

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: Two types of Fischer's Medium:
1) FM10 (consisted of 10% horse serum, 100 µg/ml Gentamycin and 2.5 µg/ml Fungizone)
2) FM20 (consisted of 20% horse serum, 100 µg/ml Gentamycin and 2.5 µg/ml Fungizone).
- Periodically checked for Mycoplasma contamination: 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:

Highest concentration tested was 5000 µg/mL (as received, equivalent to 4810 µg enzyme concentrate dry matter/mL) and dilutions hereof.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-1-oxide, benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; growth in suspension; selection phase is performed in microtitre plates

DURATION:
- Exposure duration: 2 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): At the end of the expression time, the culteres were counted and diluted appropriately and placed into microtitre wells. Incubation performed until scorable.

SELECTION AGENT: 6-TG

NUMBER OF REPLICATIONS: Preliminary trial and two independant replicates.

DETERMINATION OF CYTOTOXICITY:
- Method: Cell density by counting viable cells, expressed as relative survival

Evaluation criteria:

A test article was considered to be mutagenic if:
1) The assay was valid, and
2) 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
3) Dose-related increases in mutation could be confirmed by regression analysis in both experiments.

Statistics:

The mutation frequency was expressed as “mutants per 10E6 viable cells”. In order to calculate this, the plating efficiencies of both mutant and viable cells in the same culture were calculated. Confidence limits (95%) were assigned to mutation frequencies 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

Positive controls validity:

valid

Conclusions:

Maltogenic Amylase, PPY 1670, under the conditions of the test, had no mutagenic activity in cultured mouse lymphoma cells when tested to a concentration of 5000 ug/mL (expressed as test material as received, equivalent to 4810 µg enzyme concentrate dry matter/mL) in either the absence or presence of S-9.

Executive summary:

The enzyme IUBMB 3.2.1.133, Maltogenic Amylase, batch PPY 1670, 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 three independent experiments, each conducted in the absence and presence of metabolic activation by Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9 mix).

Following a wide range of treatments, separated by half-log intervals and reaching 5000 µg/ml (equivalent to 4810 µg enzyme concentrate dry matter/mL), cells survived all doses of Maltogenic Amylase giving relative survival values of 109% and 107% at 5000 µg/ml in the absence and in the presence of S-9, respectively. This dose together with the next 3 lower doses, were plated for viability and 6-thioguanine resistance seven days after treatment. In the second and third experiment a narrower dose range was used to maximize the chance of detection 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 95% and 124% survival respectively in experiment 2 and 103% and 96% in experiment 3.

Mutation frequencies in negative control cultures fell within normal range, 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.

In the absence of S-9 no significant increases in mutation frequency were obtained following Maltogenic Amylase treatment in experiments 1 and 3. One statistically significant result was observed at the top dose of 5000 µg/ml in experiment 2, but this was not reproducible.

In the presence of S-9 no significant increases in mutation frequency were obtained in experiment 1. In experiments 2 and 3, statistically significant increases in mutation frequency were obtained at intermediate dose levels, but a dose-relationship was not confirmed when analyzed by linear regression analysis. Maltogenic Amylase treatments did not therefore result in reproducible dose-related increases in mutation frequency, which would normally be required to be considered as evidence of mutation induction.

It was concluded that Maltogenic Amylase, under the conditions employed in this study, had no mutagenic activity in this test system.

Reference 5

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:

Oct. 25, 1993 - Sept. 14, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The results from the present study are used to support the evaluation of the test substance glucoamylase IUB 3.2.1.3 by read-across from the in vitro gene mutation studies in L5178Y mouse lymphoma cells performed on another enzyme.

See Target Record for futher information on the justification for read-across (Genetic toxicity conclusion read-across ML.00).

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: Three types of RPMI 1640 Medium was prepared:
1) RPMI A (consisted of 0 % v/v horse serum, 100 µg/ml Gentamycin, 2.5 µg/ml Fungizone and 0.5 µg/ml Pluronic)
2) RPMI 10 (consisted of 10 % v/v horse serum, 100 µg/ml Gentamycin, 2.5 µg/ml Fungizone and 0.5 µg/ml Pluronic)
3) RPMI 30 (consisted of 20 % v/v horse serum, 100 µg/ml Gentamycin and 2.5 µg/ml Fungizone)
- Periodically checked for Mycoplasma contamination: 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:

Highest concentration tested was 5000 µg/mL (as received, equivalent to 1345 µg enzyme concentrate dry matter/mL) and dilutions hereof.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water
- The reference chemical Monopropylene glycol (MPG) was also tested because the test chemical formulation of CTGase contains 24 % MPG.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-1-oxide, benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; growth in suspension; selection phase is performed in microtitre plates

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): At the end of the expression time, the culteres were counted and diluted appropriately and placed into microtitre wells. Incubation performed until scorable

SELECTION AGENT : 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Preliminary trial and two independant replicates.

DETERMINATION OF CYTOTOXICITY
- Method: Cell density by counting viable cells using background illumination, expressed as relative survival

Evaluation criteria:

A test article was considered to be mutagenic if:
1) The assay was valid, and
2) The mutation frequency at 1 or more doses was significantly greater than that of the negative control.
3) There was a significant dose-relationship as indicated by the linear trend analysis
4) The effects described above were reproducible.

Statistics:

The mutation frequency was expressed as “mutants per 10E6 viable cells”. In order to calculate this, the plating efficiencies of both mutant and viable cells in the same culture were calculated.

Statistical significance of mutant frequencies (total wells with clones) was carried out according to the UKEMS guideline (Robison et al. (1990), In Statistical Evaluation of Mutagenicity Test Data, Cambridge University Press, pp. 102-140). Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment dose, and secondly the data were checked for a linear trend in mutant frequency with treatment dose. There tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

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

Positive controls validity:

valid

Additional information on results:

No statistically significant increases in mutant frequency were observed following treatment with MPG at any dose level as well.

Conclusions:

The amylase CGTase, PPA 4357, IUBMB 3.2.1.1, under the conditions of the test, had no mutagenic activity in cultured mouse lymphoma cells when tested to a concentration of 5000 ug/mL (expressed as test material as received, equivalent to 1345 µg enzyme concentrate dry matter/mL) in either the absence or presence of S-9.

Executive summary:

CGTase, PPA 4357 was assayed for its ability to induce mutation at the tk locus in mouse lymphoma cells using a fluctuation protocol. The study consisted of a preliminary experiment and cytotoxicity range-finder experiments followed by 2 independent experiments each conducted in the presence and absence of the S-9 mix. The preliminary experiment established that CGTase did not inactivate the enzymes of S-9 mix and therefore it could be tested as supplied.

In the cytotoxicity range-finder experiments 6 doses of CGTase were tested, separated by 2-fold intervals and ranging from 156.25 to 5000 µg/ml (equivalent to 1345 µg enzyme concentrate dry matter/mL). The top dose of CGTase tested yielded 36.1% and 109.6% relative survival in the absence and presence of S-9.

Accordingly, 5 doses of CGTase were chosen for the first experiment, separated by 2-fold intervals and ranging from 312.5 to 5000 µg/ml. Four doses were plated for viability and 5-trifluorothymidine resistance 2 days after treatment. The top dose plated 5000 µg/ml yielded 91.8% and 90.6% relative survival in the absence and presence of S-9, respectively. In the second experiment 5000 µg/ml CGTase was retained as the top dose but the dose range was modified slightly. The top dose tested in this experiment yielded relative survival values of 95.7% in the absence of S-9 and 116.3% in the presence of S-9.

Negative (solvent) and positive control treatments were included in each experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals. Therefore the study was accepted as valid. No statistical significant increases in mutant frequency were observed following treatment with CGTase at any dose level either in absence or presence of S-9 in the two experiments.

It is concluded that, under the conditions employed in this study, that the tested amylase CGTase PPA 4357 is not mutagenic in this test system.

Reference 6

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:

Jun. 14, 1989 - Oct. 10, 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The results from the present study are used to support the evaluation of the test substance glucoamylase IUB 3.2.1.3 by read-across from the in vitro gene mutation studies in L5178Y mouse lymphoma cells performed on another glucosidase.

See Target Record for futher information on the justification for read-across (Genetic toxicity conclusion read-across ML.00).

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: Two types of Fischer's Medium:
1) FM10 (consisted of 10% horse serum, 100 µg/ml Gentamycin and 2.5 µg/ml Fungizone)
2) FM20 (consisted of 20% horse serum, 100 µg/ml Gentamycin and 2.5 µg/ml Fungizone).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Highest concentration tested was 5000 µg/mL (as received) (equivalent to 1750 µg enzyme concentrate dry matter/mL) and dilutions hereof.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled 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: 4-nitroquinoline-1-oxide, benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; growth in suspension; selection phase is performed in microtitre plates.

DURATION
- Exposure duration: 2 hours
- Expression time (cells in growth medium): 7 or 8 days
- Selection time (if incubation with a selection agent): At the end of the expression time, the cultures were counted and diluted appropriately and placed into microtitre wells. Incubation performed until scorable.

SELECTION AGENT: 6-TG

DETERMINATION OF CYTOTOXICITY
- Method: Cell density by counting viable cells, expressed as relative survival.

Evaluation criteria:

A test article was considered to be mutagenic if:
1) The assay was valid, and
2) 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
3) Dose-related increases in mutation could be confirmed by regression analysis in both experiments.

Statistics:

The mutation frequency was expressed as “mutants per 10E6 viable cells”. In order to calculate this, the plating efficiencies of both mutant and viable cells in the same culture were calculated. Confidence limits (95%) were assigned to mutation frequencies 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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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

COMPARISON WITH HISTORICAL CONTROL DATA: Cells treated with the test substance, either in the absence and presence of S-9, had similar mutation frequencies as those observed in concurrent solvent controls. The negative controls were within the historical negative control ranges.

Conclusions:

The test substance, amylase batch PPY2693, under the conditions of the test, had no mutagenic activity in cultured mouse lymphoma cells when tested to a concentration of 5000 ug/mL (provided in test material as received, equivalent to 1750 µg enzyme concentrate dry matter/mL) in either the absence or presence of S-9.

Executive summary:

The amylase (IUBMB 3.2.1.1), batch PPY 2693 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 by Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9 mix).

Following a wide range of treatments, separated by half log intervals and reaching 5000 µg/ml (tested as recived, equivalent to 1750 µg enzyme concentrate dry matter/mL), cultures surviving the top dose of 5000 µg/ml in the absence and in the presence of S-9 showed 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 maximize the chance of detection 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.

Mutation frequencies in negative control cultures fell within normal range, 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 test substance failed to induce mutation at the HGPRT locus of L5178Y mouse lymphoma cells in two independent experiments when tested to a concentration of 5000 µg/ml in the absence and in the presence of S-9. Hence, it was concluded that this amylase, under the conditions employed in this study, had no mutagenic activity in this test system.

Reference 7

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

other information

Justification for type of information:

According to the ECHA Guidance Chapter R 7a: Endpoint specific guidance (version 2, Nov 2012), the following studies on genetic toxicity are required: In vitro gene mutation study in bacteria and one of the following, in vitro cytogenicity study in mammalian cells or an in vitro micronucleus study. In case these studies are both negative, an in vitro gene mutation study in mammalian cells is requested in addition. The present test substance, glucoamylase, has been investigated in three in vitro test systems, the Ames test, the in vitro chromosome aberration test and in a cultured human peripheral blood lymphocyte micronucleus assay. All tests have been performed according to current OECD guidelines, and in compliance with GLP. No evidence for genetic toxicity was observed. These results are supported by read-across from three in vitro gene mutation studies in L5178Y mouse lymphoma cells performed on three different amylases belonging to the same subclass of glycosidases (IUBMB class 3.2.1.) as glucoamylase. The safety of the production strain is fully documented to belong to a safe strain lineage (Pariza and Johnson, 2001; Enzymes REACH Consortium, 2009) and the enzyme concentrate is well characterized. All enzyme classes are hydrophilic and readily biodegradable and in general, non-protease enzymes exhibit the same toxicological properties and although they are potential respiratory sensitizers, they are considered to be of low toxicity, confirmed by toxicity studies performed and published by the industry (summarized in Basketter et al. 2012a and 2012b). The physico-chemical properties of enzymes including logKow are very similar. They are further proteins built up of amino acids and the type, order and number of the amino acids in the polymer differs between enzymes, determining the 3-dimensional structure, the activity and specificity of the individual enzyme type. Industrial production strains typically have a long history of safe use for many years in the production of technical and also often food grade enzymes. Because all enzymes are built up of the same amino acids the physical and chemical characteristics will be very similar for different enzymes, and hence read-across from other non-proteolytic enzymes (e.g. amylase) should be fully applicable. The overall conclusion is that glucoamylase is not mutagenic and does not induce genotoxicity in the present test systems. References - 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. - Enzymes REACH Consortium: Safety evaluation of technical enzyme products with regards to the REACH legislation. Document from Manufacturers, Importers and/or Only Representatives of one or more enzymes, who are subject to the registration requirements pursuant to REACH, 2009. http://www.enzymes-reach.org/documents.html - D. Basketter; N. Berg; F. Kruszewski; K. Sarlo; B. Concoby. The Toxicology and Immunology of Detergent Enzymes. 2012a. J. Immunotox 9(3): 320-6. - Basketter D., Berg N., Broekhuizen C., Fieldsend M., Kirkwood S., Kluin C., Mathieu S. and Rodriguez C. Enzymes in Cleaning Products: An Overview of Toxicological Properties and Risk Assessment/Management. 2012b. Reg. Toxicol. Pharmacol, 64/1: 117-123

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The genetic toxicity of glucoamylase has been investigated in three test systems, the Ames test, the in vitro chromosome aberration test and in a cultured human peripheral blood lymphocyte micronucleus assay. All tests have been performed according to current OECD guidelines, and in compliance with GLP. No evidence for genetic toxicity was observed. These results are supported by read-across from three in vitro gene mutation studies in mouse lymphoma cells performed on three different amylases belonging to the same subclass as glucoamylase. The safety of the production strain is fully documented to belong to a safe strain lineage (Pariza and Johnson, 2001) and the enzyme concentrate is well characterized.

In conclusion, glucoamylase is not mutagenic and does not induce genotoxicity in any of the presented test systems.

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.

Justification for selection of genetic toxicity endpoint

No genetic toxicity of glucoamylase could be detected in an in vitro gene mutation study in bacteria, in vitro cytogenicity study in mammalian cells or in the in vitro micronucleus test system. The production strains of glucoamylase further meet the criteria for a safe strain production microorganism. The conclusion that glucoamylase is non-genotoxic is supported by the read-across from three in vitro gene mutation studies in mouse lymphoma cells testing other enzymes (alpha-amylases).

Justification for classification or non-classification

Due to the lack of genetic toxicity glucoamylase is not classified.