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EC number: 253-446-5 | CAS number: 37288-54-3
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- 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
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 999
- Report date:
- 1999
Materials and methods
Test guidelineopen allclose all
- 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
Test material
- Reference substance name:
- Mannanase, endo-1,4-β-
- EC Number:
- 253-446-5
- EC Name:
- Mannanase, endo-1,4-β-
- Cas Number:
- 37288-54-3
- Molecular formula:
- Not available. See Remarks.
- IUPAC Name:
- mannan endo-1,4-beta-mannosidase IUBMB 3.2.1.78
- Reference substance name:
- Protein as a constituent of enzyme deriving from the fermentation or extraction process
- Molecular formula:
- Not available
- IUPAC Name:
- Protein as a constituent of enzyme deriving from the fermentation or extraction process
- Reference substance name:
- Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
- Molecular formula:
- Not available. See remarks.
- IUPAC Name:
- Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
- Reference substance name:
- Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
- Molecular formula:
- Not available. See remarks.
- IUPAC Name:
- Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
- Reference substance name:
- Lipids as a constituent of enzyme deriving from the fermentation or extraction process
- Molecular formula:
- Not available. See remarks.
- IUPAC Name:
- Lipids as a constituent of enzyme deriving from the fermentation or extraction process
- Test material form:
- other: Liquid
- Details on test material:
- Lot/batch No.: PPE 6432
Expiration date of the lot/batch: July 2009
Constituent 1
Constituent 2
Constituent 3
Constituent 4
Constituent 5
Method
- 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
- 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.
Controls
- 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.
Results and discussion
Test results
- 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.
Any other information on results incl. tables
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.
Applicant's summary and conclusion
- 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.
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