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Registration Dossier
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EC number: 253-446-5 | CAS number: 37288-54-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
No mutagenic activity of mannanase could be detected in the Ames Assay. Mannanase did not induce chromosomal aberrations in the in vitro mammalian chromosome aberration test performed with human lymphocytes. 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 mannanase.
Link to relevant study records
- 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:
- 19 August 1999 - 13 September 1999
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study was performed in compliance with GLP standards and according to OECD Guidelines No. 471
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- 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 mannanase in amino acid dependent strains of Salmonella typhimurium and Escherichia coli capable of detecting both induced frame-shift (TA1537 and TA98) and base-pair substitution mutations (TA1535, TA100, and WP2uvrA). 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, Escherichia coli WP2uvrA
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix from Aroclor 1254 induced Spraque Dawley rats, obtained from Life Science Denmark Aps, Lot No 5831B, 5832B.
- Test concentrations with justification for top dose:
- Experiment 1: Six concentrations of the test item tested (156, 313, 625, 1250, 2500, 5000 ug dry matter/plate, based on dry matter content of 91 mg/mL), with and without the metabolic activation
Experiment 2: Six concentrations of the test item tested (156, 313, 625, 1250, 2500, 5000 ug dry matter/plate, based on dry matter content of 91 mg/mL), with and without the metabolic activation
Experiment 3 (Treat and Plate): Six concentrations of the test item tested (156, 313, 625, 1250, 2500, 5000 ug dry matter/mL, based on dry matter content of 91 mg/mL), with metabolic activation - Vehicle / solvent:
- Vehicle/solvent used: sterile distilled water
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
- Remarks:
- sterile distilled water
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- N-ethyl-N-nitro-N-nitrosoguanidine
- benzo(a)pyrene
- other: 2-Aminoanthracene
- Remarks:
- Solvent was DMSO, apart from sodium azide where it was water.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: In medium before plating, i.e a liquid culture assay (treat and plate assay – in the 3rd and decisive test).
DURATION
- Exposure duration, pre-incubation: 3 hours (treat & plate)
- Incubation time (selective incubation): 64 hours
DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count
Mannanase was examined for mutagenic activity in four histidine-dependent strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and the tryptophan-dependent strain Escherichia coli WP2uvrA using the direct plate incorporation. In the third experiment the four Salmonella strains were applied in a “Treat and Plate” assay. The study was conducted in the presence and absence of an activating system derived from rat liver (S-9 mix). All tests included solvent (purified water) and positive controls with and without S-9 mix. Bacteria were exposed to 6 doses with 5 mg dry matter per plate as the highest dose level applied and successive 2-fold dilutions hereof.
In the direct “plate incorporation assay”, it was however demonstrated, that mannanase in the presence of the metabolic activation system S-9, significantly supported growth of the histidine requiring S. typhimurium strains and only weakly the tryptophan requiring E. coli strain. Crude enzyme preparations, like the present batch of mannanase, often contain the free amino acid histidine and tryptophan in amounts which exceeds the critical concentration for incorporation in the direct standard assay. Apparently, significant amounts of histidine were released following mixing of mannanase and the S-9 preparation. As a result, the density of the bacterial background lawn became increasingly conspicuous with increasing doses followed by dose related increases in the number of spontaneous revertant colonies in all test series with S. typhimurium strains with metabolic activation.
Histidine in the form of free histidine as well as dipeptides represents the most well documented source of false positives in the Ames test, capable of increasing the number of revertants to at least such an extent that it fulfils the criteria of a mutagenic effect.
Yamasaki and Ames (1977) emphasised the importance of histidine in elevating both background colony counts and lawn densities with TA 98 and TA 100, noting that “this increase in the number of spontaneous revertant can be 3 to 4 fold greater than that observed under standard conditions and one should be attentive to the density of the lawn and be aware of this possible artefact”. These observations are in accordance with the results obtained in this study, when Salmonella strains are applied in the presence of S-9.
It is obvious, that the increases in the number of revertant colonies observed are artificial. Mannanase cannot be tested in Salmonella strains in a standard “plate incorporation assay” with metabolic activation included.
The best approach in testing crude biological samples like enzyme preparations, which contain appreciable amounts of histidine, is to employ a modified pre-incubation procedure of the Ames test - a “treat and plate” assay (Ashby, R. et al., 1987). Test bacteria are pre-incubated with the test substance in liquid culture for 3 hours and subsequently washed extensively before plating on minimal glucose agar plates. The method employed in this study is in accordance with the OECD guideline (1997) and MoL Japan (“Concrete Operation Procedure of Mutagenicity Study Using Bacteria. Ministry of Labour, 1988) concerning the general specifications of the test. But the exposure of test bacteria in liquid culture (“treat and plate”) is not specifically described in any guidelines.
Therefore, a third experiment with four Salmonella strains and metabolic activation was conducted, applying a “Treat and Plate” assay. Bacteria were exposed to 6 doses of the test substance in a phosphate buffered nutrient broth for 3 hours with 5 mg dry matter per mL as highest dose level followed by successive 2-fold dilutions between doses. After incubation the test substance was removed by centrifugation prior to plating (“treat and plate assay”). The viability of each culture was determined by viable colony count.
References:
Ashby, R. et al. (1987). Tox Letters vol. 36, pp. 23-35.
Yamasaki, E. and Ames, B.N. (1977): Proc. Natl. Acad. Sci. USA 74, pp. 3555-3559. - 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 when 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 is considered to have shown evidence of mutagenic activity if it has induced at least a doubling in the mean number of revertants compared to the appropriate solvent control in one or more of the strains, in the presence or absence of S-9, if this response is dose related and reproducible. - Statistics:
- Statistical evaluation was not judged to provide any added value and was therefore not performed.
- Key result
- Species / strain:
- bacteria, other: Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2uvrA
- 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:
- The test item, Mannanase batch No PPE 6432, was considered to be non-mutagenic under the conditions of this test.
- Conclusions:
- Mannanase is not mutagenic in the Ames assay in both the presence and absence of metabolic activation, when tested under the conditions employed in the study.
- Executive summary:
The objective of this study was to assess the potential of the enzyme mannanase to induce point mutations (frame-shift and base-pair) in four strains of Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2uvrA. The test material was tested both in the presence and absence of a metabolic activation system (Aroclor 1254-induced rat liver; S-9-mix). Two independent tests were performed with all 5 strains in both the presence and absence of S-9-mix using the direct plate incorporation. Triplicate plates were used at each test point. All dose levels were expressed in terms of dry matter.
In the direct “plate incorporation assay”, it was however demonstrated, that mannanase in the presence of the metabolic activation system S-9, significantly supported growth of the histidine requiring S. typhimurium strains and only weakly the tryptophan requiring E. coli strain. Crude enzyme preparations, like the present batch of mannanase, often contain the free amino acid histidine and tryptophan in amounts which exceeds the critical concentration for incorporation in the direct standard assay. Apparently, significant amounts of histidine were released following mixing of mannanase and the S-9 preparation. The first two tests demonstrated that mannanase significantly increased the growth of the histidine requiring Salmonella strains following direct plate incorporation while the part of the study comprising E. coli could be conducted, using the direct plate incorporation assay. As a result, the density of the bacterial background lawn became increasingly conspicuous with increasing doses followed by dose related increases in the number of spontaneous revertant colonies in all test series with S. typhimurium strains with metabolic activation. It was obvious, that these increases in the number of revertant colonies were artificial.
Therefore, in a third experiment the four Salmonella strains were applied in a “Treat and Plate” assay. Bacteria were exposed to 6 doses of the test substance in a phosphate buffered nutrient broth for 3 hours with 5 mg dry matter per mL as highest dose level followed by successive bi-sections between doses. After incubation the test substance was removed by centrifugation prior to plating (“treat and plate assay”). The viability of each culture was determined by viable colony count.
The study was conducted in accordance with OECD Guideline for testing of chemicals, No. 471: Bacterial Reverse Mutation Assay” (July 1997). However, the exposure of test bacteria in liquid culture (“Treat and Plate”), as it was applied in this study, is not specifically described in any guidelines. The study was conducted in compliance with current GLP regulations.
All tests included solvent (purified water) and positive controls with and without S‑9 mix.
No increases in revertant colonies were obtained in any of the test strains in test series without S-9. In the supplementary experiment with metabolic activation, applying four Salmonella strains in a “Treat and Plate” assay with S-9, no increases in the number of revertant colonies were obtained on plates with test substance compared to the solvent control.
It was concluded that the enzyme mannanase did not induce gene mutations in bacteria, in either the absence or presence of S-9, when tested under the conditions employed in the study.
- 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 July 2003 - 11 September 2003
- 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)
- 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:
- Aroclor-incuded S-9 mix
- Test concentrations with justification for top dose:
- Dose selected: 125 - 5000 µg/mL (based on total protein)
- Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile de-ionized water
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Remarks:
- without S-9 mix: Mitomycin C (MMC); with S-9 mix: Cyclophosphamide (CP)
- Details on test system and experimental conditions:
- Exposure duration: 4 or 20 hours at 37+/-1 degrees Celcius.
Conditions of exposure: 37+/-1 degrees Celcius in humidified atmosphere of 5 +/- carbon dioxide in air.
Number of Replications: Duplicate cultures.
Number of Cells Evaluated: 200 metaphases per dose. - Evaluation criteria:
- Toxic effects of treatment are based on mitotic inhibition relative to the solvent-treated control. The following was calculated and examined:
- number and types of aberrations per cell
-percentage of structurally and numerically damaged cells
-frequency of structural aberration per cell in the total population of cells - Statistics:
- Statistical analysis was performed using Fisher's Exact Test. The test was used to compare the percent of aberrant cells of each treatment group with that of the solvent control. When Fisher's exact test is positive at any dose level, the Cochran-Armitage test is used to measure dose-responsiveness.
- 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:
- The percentage of cells with structural or numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control at any dose level (p>0.05, Fisher's exact test). The percentage of structurally damaged cells in the MMC and CP groups were statistically significant (28.0% and 13.5%, respectively).
- Conclusions:
- Mannanase does not induce structural and numerical chromosome aberrations in neither the presence nor the absence of metabolic activation.
- Executive summary:
The objective of this assay was to investigate the potential of mannanase to induce numerical and/or structural changes in the chromosome of mammalian systems (i.e., human peripheral lymphocytes) in both the presence and absence of metabolic activation (Aroclor-induced S-9 mix). This assay was conducted in accordance with OECD guideline No. 473 and complied with all standard GLP.
A preliminary toxicity test was performed to establish the dose range for cytogenetic testing. The maximum dose tested was 5000 µg total protein/mL. Human peripheral blood lymphocytes were treated in the absence and presence of an Aroclor-induced S-9 activation system for 4 hours and continuously for 20 hours in the absence of S-9 activation. Substantial toxicity (defined as at least 50% reduction in mitotic index relative to the solvent control) was observed after administering 5000 µg total protein/ml in the non-activated and S-9 activated 4 hour exposure groups as well as doses greater than or equal to 1500 µg total protein/ml in the non-activated 20-hour exposure group.
In the main study, the dose levels selected for analysis were 625, 1250, and 2500 µg total protein/mL. Duplicate cultures of human peripheral blood lymphocytes were exposed to the dose levels. The cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S-9 activated test system. 20 hours after treatment initiation, the cells were harvested and it was determined that the percentage of cells with clastogenic effects were not significantly increased above that of the solvent control at any dose level.
- 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:
- Due to the similarities between the two enzymes, similar results are expected for mannanase.
- 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 (test substace as is) (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 (test item as is) (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) BS-G-Amylase, 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 (test item as is) (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 (equivalent to 1750 µg enzyme concentrate dry matter/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.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting 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:
- Due to the similarities between the two enzymes, similar results are expected for mannanase.
- 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): 7days
- 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 (test item as is) (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, 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.
Referenceopen allclose all
1. experiment. Without S9 Mix
Test substance concentration |
Number of revertants (number of colonies/plate)
Base-pair substition type Frameshift type |
||||
(ug/plate) |
TA 100 |
TA 1535 |
WP2uvrA |
TA 98 |
TA 1537 |
Solvent control |
167 152 159 (160) 179 143 |
20 17 16 (16) 15 13 |
46 38 47 (44) 53 38 |
28 29 27 (29) 27 35 |
9 11 10 (10) 9 12 |
156 |
172 146 (149) 129 |
15 15 (15) 15 |
50 51 (51) 51 |
35 28 (30) 28 |
12 10 (11) 10 |
313 |
170 160 (160) 150 |
14 15 (15) 17 |
40 51 (44) 42 |
31 36 (35) 38 |
11 12 (12) 13 |
625 |
132 143 (147) 165 |
14 16 (15) 15 |
48 43 (43) 38 |
46 31 (36) 30 |
9 13 (11) 12 |
1250 |
134 181 (159) 163 |
16 11 (13) 11 |
43 49 (48) 51 |
26 38 (30) 27 |
7 9 (10) 13 |
2500 |
175 142 (146) 120 |
12 16 (14) 13 |
51 53 (53) 55 |
46 38 (40) 36 |
8 13 (11) 13 |
5000 |
146 158 (159) 173 |
16 14 (14) 11 |
32 45 (39) 40 |
37 46 (42) 44 |
12 15 (14) 15 |
Positive control not requiring S9-Mix |
Sodium azide |
Sodium azide |
ENNG |
2-Nitro-fluorene |
9-Amino-acridine |
Concentration (ug/mL or plate) |
2 |
0,5 |
2 |
1 |
80 |
Number of colonies/plate |
952 1475 (1392) 1749 |
303 315 (326) 360 |
816 443 (652) 697 |
244 251 (245) 240 |
7923 4971(5541) 3729 |
Notes:
1. ( ) = The average number of colonies.
2. Abbreviations : ENNG = N-Ethyl-N’-Nitro-Nitrosoguanidine.
1. experiment. With S9 Mix
Test substance concentration |
Number of revertants (number of colonies/plate)
Base-pair substition type Frameshift type |
||||
(ug/mL or plate) |
TA 100 |
TA 1535 |
WP2uvrA |
TA 98 |
TA 1537 |
Solvent control |
125 136 125 (131) 125 146 |
13 7 15 (14) 15 18 |
50 49 50 (50) 53 48 |
48 41 39 (42) 45 37 |
9 7 8 (9) 6 13 |
156 |
162 146 (146) 129 |
18 14 (15) 13 |
61 77 (65) 57 |
56 38 (42) 31 |
9 7 (7) 6 |
313 |
149 168 (159) 159 |
12 17 (16) 18 |
54 61 (57) 57 |
53 41 (46) 45 |
6 13 (9) 8 |
625 |
179 182 (180) 179 |
18 15 (18) 22 |
60 59 (61) 63 |
60 58 (58) 55 |
9 13 (10) 9 |
1250 |
192 194 (192) 189 |
20 16 (19) 21 |
65 70 (63) 53 |
74 66 (73) 78 |
9 4 (6) 6 |
2500 |
198 167 (183) 183 |
21 20 (20) 18 |
60 35 (47) 47 |
66 59 (66) 74 |
10 4 (9) 12 |
5000 |
229 226 (210) 174 |
22 21 (21) 20 |
55 63 (56) 49 |
72 75 (74) 74 |
13 14 (14) 14 |
Positive control requiring S9-Mix |
2-AA |
2-AA |
2-AA |
B(a)P |
B(a)P |
Concentration (ug/mL or plate) |
1 |
2 |
20 |
5 |
5 |
Number of colonies/plate |
259 289 (280) 293 |
96 84 (93) 98 |
788 593 (598) 412 |
130 131 (142) 164 |
40 35 (35) 30 |
Notes:
1. ( ) = The average number of colonies.
2. Abbreviations: B(a)P = Benzo(a)pyrene
3. Abbreviations: 2-AA = 2-Aminoanthracene.
2. experiment. Without S9 Mix
Test substance concentration |
Number of revertants (number of colonies/plate)
Base-pair substition type Frameshift type |
||||
(ug/mL or plate) |
TA 100 |
TA 1535 |
WP2uvrA |
TA 98 |
TA 1537 |
Solvent control |
143 143 123 (139) 163 122 |
15 16 13 (15) 15 14 |
32 21 36 (30) 34 29 |
46 46 36 (47) 56 49 |
5 5 8 (8) 8 12 |
156 |
162 163 (156) 142 |
14 11 (13) 15 |
25 27 (27) 29 |
32 53 (41) 39 |
8 13 (10) 8 |
313 |
162 144 (154) 156 |
14 14 (16) 19 |
27 27 (26) 23 |
48 41 (45) 46 |
10 10 (10) 10 |
625 |
143 163 (152) 150 |
11 15 (12) 11 |
37 25 (28) 22 |
55 57 (52) 44 |
8 10 (8) 7 |
1250 |
168 156 (162) 163 |
12 14 (12) 11 |
32 31 (32) 32 |
55 50 (52) 51 |
11 9 (10) 9 |
2500 |
151 175 (160) 155 |
16 12 (14) 15 |
20 27 (27) 34 |
63 53 (58) 58 |
4 10 (6) 5 |
5000 |
170 170 (170) 169 |
16 11 (14) 16 |
30 19 (24) 23 |
50 51 (50) 50 |
11 7 (9) 10 |
Positive control not requiring S9-Mix |
Sodium azide |
Sodium azide |
ENNG |
2-Nitro-fluorene |
9-Amino-acridine |
Concentration (ug/mL or plate) |
2 |
0,5 |
2 |
1 |
80 |
Number of colonies/plate |
1221 1143 (1229) 1324 |
405 399 (422) 462 |
304 491 (444) 538 |
192 166 (182) 188 |
4728 4092(4301) 4083 |
Notes:
1. ( ) = The average number of colonies.
2. Abbreviations : ENNG = N-Ethyl-N’-Nitro-Nitrosoguanidine.
2. experiment. With S9 Mix
Test substance concentration |
Number of revertants (number of colonies/plate)
Base-pair substition type Frameshift type |
||||
(ug/mL or plate) |
TA 100 |
TA 1535 |
WP2uvrA |
TA 98 |
TA 1537 |
Solvent control |
140 162 162 (146) 121 143 |
13 11 18 (13) 11 13 |
42 56 48 (45) 35 46 |
40 39 41 (45) 45 60 |
10 8 12 (11) 12 12 |
156 |
167 163 (164) 161 |
13 14 (14) 14 |
49 51 (52) 56 |
54 63 (59) 60. |
17 11 (13) 10 |
313 |
164 159 (160) 158 |
17 13 (14) 13 |
68 58 (60) 53 |
57 61 (61) 66 |
13 19 (16) 16 |
625 |
194 165 (186) 198 |
14 16 (15) 14 |
49 58 (62) 80 |
66 66 (67) 70 |
16 14 (15) 14 |
1250 |
184 234 (209) 209 |
15 15 (14) 13 |
70 80 (73) 69 |
83 69 (74) 70 |
18 12 (14) 13 |
2500 |
199 222 (213) 217 |
14 14 (14) 15 |
75 60 (73) 85 |
77 88 (83) 84 |
11 18 (16) 20 |
5000 |
192 211 (206) 216 |
18 17 (16) 14 |
67 59 (60) 53 |
94 103 (97) 95 |
17 18 (19) 21 |
Positive control requiring S9-Mix |
2-AA |
2-AA |
2-AA |
B(a)P |
B(a)P |
Concentration (ug/mL or plate) |
1 |
2 |
20 |
5 |
5 |
Number of colonies/plate |
510 615 (582) 621 |
145 119 (134) 137 |
545 362 (412) 330 |
238 155 (193) 187 |
112 120 (132) 165 |
Notes:
1. ( ) = The average number of colonies.
2. Abbreviations: B(a)P = Benzo(a)pyrene
3. Abbreviations: 2-AA = 2-Aminoanthracene.
3. experiment. “Treat and plate” assay.
Results obtained with 4 strains of Salmonella typhimurium exposed to Mannanase (Batch Number: PPE 6432) in the presence of S9.
Test substance concentration |
Number of revertants (number of colonies/plate)
Base-pair substition type Frameshift type |
|||||||||||
(ug/mL) |
TA 100 |
TA 1535 |
TA 98 |
TA 1537 |
||||||||
Solvent control |
94 120 120 111 89 |
(107) |
9 3 10 8 10 |
(8) |
21 28 28 25 31 |
(27) |
8 15 10 9 6 |
(10) |
||||
156 |
129 126 139 |
(131) |
11 9 9 |
(10) |
25 30 28 |
(28) |
10 10 7 |
(9) |
||||
313 |
132 123 124 |
(126) |
9 7 7 |
(8) |
38 26 28 |
(31) |
10 12 11 |
(11) |
||||
625 |
115 130 115 |
(120) |
5 7 11 |
(8) |
19 36 30 |
(28) |
3 7 7 |
(6) |
||||
1250 |
108 111 107 |
(109) |
8 7 8 |
(8) |
38 19 32 |
(30) |
8 11 11 |
(10) |
||||
2500 |
84 115 122 |
(107) |
9 8 14 |
(10) |
27 34 27 |
(29) |
5 14 10 |
(10) |
||||
5000 |
95 101 111 |
(102) |
9 6 13 |
(9) |
25 29 29 |
(28) |
8 11 13 |
(11) |
||||
Positive control requiring S9-Mix |
B(a)P |
2-AA |
2-AA |
2-AA |
||||||||
Concentration (ug/mL or plate) |
10 |
5 |
5 |
5 |
||||||||
Number of colonies/plate |
179 202 208 |
*) (196) |
66 76 84 |
(75) |
1076 1119 1092 |
(1096) |
50 48 56 |
(51) |
||||
Notes:
( ) = The average number of colonies.
Abbreviations: B(a)P = Benzo(a)pyrene; 2-AA = 2-Aminoanthracene.
*) The response of TA100 to 10 ug B(a)P is just below the expected level of at least a doubling of
the number of revertants per plate compared to solvent control.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Additional information from genetic toxicity in vitro:
The genetic toxicity of mannanase has been investigated in two test systems, the Ames test and the in vitro chromosome aberration test. Both 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 group of glycosidases as mannanase. The safety of the production strain is fully documented to belong to a safe strain lineage (Pariza and Johnson, 2001). Because 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) is fully applicable.
In conclusion, mannanase 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 classification or non-classification
Due to the lack of genetic toxicity mannanase should not be classified.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
