Mannanase, endo-1,4-β-

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:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

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

Constituent 2

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

Constituent 3

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

Constituent 4

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

Constituent 5

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

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.