Oxidase, glucose

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The study used selective agar plates containing fructose instead of glucose.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of Glucose oxidase (EC no. 232-601-0, CAS no. 9001-37-0, EC name: Glucose oxidase, Enzyme Class no. 1.1.3.4)

Molecular formula:

Not applicable, see remarks.

IUPAC Name:

Active enzyme protein of Glucose oxidase (EC no. 232-601-0, CAS no. 9001-37-0, EC name: Glucose oxidase, Enzyme Class no. 1.1.3.4)

Constituent 2

Reference substance name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

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:

liquid

Details on test material:

- Substance type: UVCB
- Name of test material (as cited in study report): Glucose Oxidase
- Lot/batch No.: R-GOx-04004
- Physical state: Clear brown liquid

Method

Target gene:

Operon for synthesis of amino acid histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix prepared from Aroclor®-1254-induced rat livers

Test concentrations with justification for top dose:

50, 160, 500, 1600, and 5000 µg/plate. The highest dose level tested is the maximum required by the OECD Guideline 471 for materials of low toxicity.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: saline (0.9% NaCl)

Controls

Untreated negative controls:

yes

Remarks:

sterile saline solution

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cumene hydroperoxide

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Treat and plate method: Bacterial cultures were centrifuged at 1700 g for 10 minutes, the supernatant was discarded and the bacteria were resuspended in one third of the original volume of fresh broth. Test tubes were prepared containing the test substance solution, vehicle, or positive control solution, S-9 mix or 0.02 M phosphate buffer pH 7.4 and concentrated bacterial suspension. After incubation at 37°C for 30 minutes with gentle shaking, nutrient broth was added to each test tube and the incubation was continued for 3 hours. After incubation, the bacteria were sedimented by centrifugation, the supernatant removed and the bacteria resuspended in buffer. After another centrifugation and removal of supernatant, the bacteria were resuspended in buffer and top agar was added. The contents of each tube were mixed with a vortex mixer and spread on minimal fructose agar plates.
- Cell density at seeding (if applicable): 10E9 bacteria/mL

DURATION
- Preincubation period: 30 minutes at 30°C, nutrient broth added, incubation continued for 3 hours
- Exposure duration: plates were incubated for 72 hours at 37°C

NUMBER OF REPLICATIONS: 2 tests with 3 plates at each test point

Rationale for test conditions:

In preliminary tests with the plate incorporation method, it was observed that the histidine in the test substance was interfering with the test system, preventing adequate assessment of the test substance for mutagenic activity. Therefore, the treat and plate method was carried out to avoid the problem.

Evaluation criteria:

The tests were considered valid if the following criteria were met:
- negative and positive control data were consistent with the historical control data for the 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 for mutagenic activity in the study if all the following were met:
- increases in the number 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
- the increases were statistically significant
- increases were not directly related to increased growth of the non-revertant bacteria

The test substance would be considered to have shown no evidence of mutagenic activity if no dose-related increased in the numbers of revertant colonies which exceeded 1.5 times the negative control value were observed at any test point. The test item would also be considered to have shown no evidence of mutagenic activity if moderately larger increases had been observed that were not reproducible, not statistically significant, or which were sporadic (without a scientifically valid explanation for the dose-response relationship that involved a mutagenic effect of the test substance). Increases between 1.5 and 2-fold greater than the negative control values, which met the other criteria for a positive result, would provide some evidence for a weak effect of the test substance, but would be considered to be clear evidence of mutagenic activity.

Statistics:

The numbers of revertant colonies at each treatment test point in the main tests were compared to the corresponding negative control values using the Analysis of Variance test. When this test showed statistically significant differences in the data, Dunnett's test was used to determine the statistical significance of the increases and decreases in the numbers of revertant colonies for each set of triplicate plates. 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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥500 µg (without S-9); 5000 µg (with S-9)

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥160 µg (without S-9); ≥500 µg (with S-9)

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg (without S-9)

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥1600 µg (without S-9)

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the preliminary test with the treat and plate method, the test substance was toxic to the test bacteria at dose levels of 500, 1600, and 5000 µg/plate without S-9 mix and at 5000 µg/plate with S-9 mix: reduced growth of the background lawn of non-revertant bacteria and reductions in the number of revertant colonies.

The results of the two main tests showed the test substance was toxic to most of the tester strains at the higher does levels, causing reduced growth of background lawn and reductions in the number of revertant colonies. These effects were generally observed over a wider range of dose levels for treatments without S-9 than for treatments with S-9 mix. The amount of toxicity showed some variation between the tester strains.

No biologically significant increases in the number of revertant colonies were observed in any tester strain after treatment with the test substance at any dose level, either in the absence or presence of S-9 mix. A small, but statistically significant increase was observed in strain TA 1535 in the first main test after treatment with the test substance at 5000 µg/plate without S-9 mix. This increase does not meet the criteria for a mutagenic effect because it was too small and it was not reproduced in the second test.

Any other information on results incl. tables

Main Test Strain TA102
Dose	Number of Revertant Colonies / Plate
	Plate 1	Plate 2	Plate 3	Mean	SD	Ratio
Strain TA102, Without S-9 Mix, Main Test 1
Control	436	432	472	446.7	22.0
5000 µg	440T	420T	496T	452.0	39.4	1.01
1600 µg	416T	400T	576T	464.0	97.3	1.04
500 µg	416	368	500	428.0	66.8	0.96
160 µg	424	404	400	409.3	12.9	0.92
50 µg	380	420	324	374.7	48.2	0.84
Cumene hydroperoxide 100 µg	800	1152	1040	997.3	179.8	2.23
Strain TA102, Without S-9 Mix, Main Test 2
Control	648	488	432	522.7	112.1
5000 µg	440T	440T	304T	394.7	78.5	0.76
1600 µg	344T	536T	528T	469.3	108.6	0.90
500 µg	520	472	480	490.7	25.7	0.94
160 µg	352	480	408	413.3	64.2	0.79
50 µg	376	432	448	418.7	37.8	0.80
Cumene hydroperoxide 100 µg	1600	1408	1536	1514.7	97.8	2.90
Strain TA102, With S-9 Mix, Main Test 1
Control	408	189	400	332.3	124.2
5000 µg	352	304	424	360.0	60.4	1.08
1600 µg	476	396	424	432.0	40.6	1.30
500 µg	400	304	576	426.7	137.9	1.28
160 µg	384	432	400	405.3	24.4	1.22
50 µg	432	372	448	417.3	40.1	1.26
2-Aminoanthracene 4 µg	1088	992	358	812.7	396.7	2.45
Strain TA102, With S-9 Mix, Main Test 2
Control	320	360	456	378.7	69.9
5000 µg	544	400	392	445.3	85.5	1.18
1600 µg	376	440	408	408.0	32.0	1.08
500 µg	360	376	448	394.7	46.9	1.04
160 µg	344	440	448	410.7	57.9	1.08
50 µg	384	384	352	373.3	18.5	0.99
2-Aminoanthracene 4 µg	1344	1216	2496	1685.3	705.0	4.45
S.D. = Standard deviation RATIO =Mean number of revertants on treated plates/mean number of revertants on vehicle control plates * = Statistically significant at 5% level ** = Statistically significant at 1% level Otherwise, not statistically significant at 5% level (The positive controls were not included in the statistical analysis) T = Toxicity: reduced growth of background lawn of non-revertant bacteria

 

Main Test Strain TA100
Dose	Number of Revertant Colonies / Plate
	Plate 1	Plate 2	Plate 3	Mean	SD	Ratio
Strain TA100, Without S-9 Mix, Main Test 1
Control	218	198	272	229.3	38.3
5000 µg	226	184	193	201.0	22.1	0.88
1600 µg	214	201	209	208.0	6.6	0.91
500 µg	173	178	200	183.7	14.4	0.80
160 µg	206	204	212	207.3	4.2	0.90
50 µg	241	218	188	215.7	26.6	0.94
Sodium azide 1 µg	1301	1220	1208	1243.0	50.6	5.42
Strain TA100, Without S-9 Mix, Main Test 2
Control	182	214	200	198.7	16.0
5000 µg	180T	159T	120T	153.0	30.4	0.77
1600 µg	160	154	72	128.7	49.2	0.65
500 µg	137	126	97	120.0	20.7	0.60
160 µg	136	175	131	147.3	24.1	0.74
50 µg	209	188	146	181.0	32.1	0.91
Sodium azide 1 µg	1007	1173	1138	1106.0	87.5	5.57
Strain TA100, With S-9 Mix, Main Test 1
Control	226	238	245	236.3	9.6
5000 µg	198	210	221	209.7	11.5	0.89
1600 µg	221	246	264	243.7	21.6	1.03
500 µg	206	222	246	224.7	20.1	0.95
160 µg	194	199	185	192.7	7.1	0.82
50 µg	233	149	209	197.0	43.3	0.83
2-Aminoanthracene 2 µg	1379	1612	1467	1486.0	117.7	6.29
Strain TA100, With S-9 Mix, Main Test 2
Control	300	238	175	237.7	62.5
5000 µg	184	169	167	173.3	9.3	0.73
1600 µg	179	141	185	168.3	23.9	0.71
500 µg	172	200	168	180.0	17.4	0.76
160 µg	182	172	186	180.0	7.2	0.76
50 µg	217	213	165	198.3	28.9	0.83
2-Aminoanthracene 2 µg	2013	1820	2323	2052.0	253.8	8.63
S.D. = Standard deviation RATIO =Mean number of revertants on treated plates/mean number of revertants on vehicle control plates * = Statistically significant at 5% level ** = Statistically significant at 1% level Otherwise, not statistically significant at 5% level (The positive controls were not included in the statistical analysis) T = Toxicity: reduced growth of background lawn of non-revertant bacteria

 

Main Test Strain TA98
Dose	Number of Revertant Colonies / Plate
	Plate 1	Plate 2	Plate 3	Mean	SD	Ratio
Strain TA98, Without S-9 Mix, Main Test 1
Control	29	28	36	31.0	4.4
5000 µg	12T	23T	24T	19.7*	6.7	0.63
1600 µg	19T	27T	22T	22.7	4.0	0.73
500 µg	31	30	22	27.7	4.9	0.89
160 µg	17	18	15	16.7**	1.5	0.54
50 µg	38	32	31	33.7	3.8	1.09
2-Nitrofluorene 1 µg	121	144	127	130.7	11.9	4.22
Strain TA98, Without S-9 Mix, Main Test 2
Control	31	23	25	26.3	4.2
5000 µg	23T	20T	29T	24.0	4.6	0.91
1600 µg	29T	32T	31T	30.7	1 .5	1.16
500 µg	28T	22T	25T	25.0	3.0	0.95
160 µg	16	16	29	20.3	7.5	0.77
50 µg	25	29	23	25.7	3.1	0.97
2-Nitrofluorene 1 µg	140	104	104	116.0	20.8	4.41
Strain TA98, With S-9 Mix, Main Test 1
Control	40	34	26	33.3	7.0
5000 µg	20T	23T	37T	26.7	9.1	0.80
1600 µg	24	20	24	22.7	2.3	0.68
500 µg	22	27	31	26.7	4.5	0.80
160 µg	27	31	27	28.3	2.3	0.85
50 µg	30	25	36	30.3	5.5	0.91
2-Aminoanthracene 2 µg	600	644	584	609.3	31.1	18.28
Strain TA98, With S-9 Mix, Main Test 2
Control	40	46	37	41.0	4.6
5000 µg	40T	34T	33T	35.7	3.8	0.87
1600 µg	36	36	43	38.3	4.0	0.93
500 µg	33	30	30	31.0	1.7	0.76
160 µg	31	39	26	32.0	6.6	0.78
50 µg	31	34	34	33.0	1.7	0.80
2-Aminoanthracene 2 µg	752	896	704	784.0	99.9	19.12
S.D. = Standard deviation RATIO =Mean number of revertants on treated plates/mean number of revertants on vehicle control plates * = Statistically significant at 5% level ** = Statistically significant at 1% level Otherwise, not statistically significant at 5% level (The positive controls were not included in the statistical analysis) T = Toxicity: reduced growth of background lawn of non-revertant bacteria

 

Main Test Strain TA1537
Dose	Number of Revertant Colonies / Plate
	Plate 1	Plate 2	Plate 3	Mean	SD	Ratio
Strain TA1537, Without S-9 Mix, Main Test 1
Control	15	14	4	11.0	6.1
5000 µg	11T	8T	9T	9.3	1.5	0.85
1600 µg	13T	9T	11T	11.0	2.0	1.00
500 µg	9T	8T	8T	8.3	0.6	0.76
160 µg	11T	8T	10T	9.7	1.5	0.88
50 µg	10	11	9	10.0	1.0	0.91
9-Aminoacridine 80 µg	868	384	728	660.0	249.1	60.00
Strain TA1537, Without S-9 Mix, Main Test 2
Control	17	28	31	25.3	7.4
5000 µg	7T	5T	14T	8.7**	4.7	0.34
1600 µg	9T	10T	9T	9.3**	0.6	0.37
500 µg	6T	18T	10T	11.3*	6.1	0.45
160 µg	11	13	18	14.0	3.6	0.55
50 µg	11	13	20	14.7	4.7	0.58
9-Aminoacridine 80 µg	1115	1196	528	946.3	364.5	37.36
Strain TA1537, With S-9 Mix, Main Test 1
Control	14	8	11	11.0	3.0
5000 µg	13T	14T	9T	12.0	2.6	1.09
1600 µg	9T	8T	5T	7.3	2.1	0.67
500 µg	4T	6T	10T	6.7	3.1	0.61
160 µg	8	11	14	11.0	3.0	1.00
50 µg	10	12	15	12.3	2.5	1.12
2-Aminoanthracene 2 µg	312	304	288	301.3	12.2	27.39
Strain TA1537, With S-9 Mix, Main Test 2
Control	8	19	17	14.7	5.9
5000 µg	8T	10T	13T	10.3	2.5	0.70
1600 µg	8T	13T	8T	9.7	2.9	0.66
500 µg	18	17	11	15.3	3.8	1.05
160 µg	18	23	25	22.0	3.6	1.50
50 µg	10	15	18	14.3	4.0	0.98
2-Aminoanthracene 2 µg	103	93	108	101.3	7.6	6.91
S.D. = Standard deviation RATIO =Mean number of revertants on treated plates/mean number of revertants on vehicle control plates * = Statistically significant at 5% level ** = Statistically significant at 1% level Otherwise, not statistically significant at 5% level (The positive controls were not included in the statistical analysis) T = Toxicity: reduced growth of background lawn of non-revertant bacteria

 

Main Test Strain TA1535
Dose	Number of Revertant Colonies / Plate
	Plate 1	Plate 2	Plate 3	Mean	SD	Ratio
Strain TA1535, Without S-9 Mix, Main Test 1
Control	16	23	21	20.0	3.6
5000 µg	36	33	30	33.0**	3.0	1.65
1600 µg	27	26	27	26.7	0.6	1.33
500 µg	29	18	24	23.7	5.5	1.18
160 µg	25	20	25	23.3	2.9	1.17
50 µg	22	15	18	18.3	3.5	0.92
Sodium azide 1 µg	500	352	448	433.3	75.1	21.67
Strain TA1535, Without S-9 Mix, Main Test 2
Control	15	19	25	19.7	5.0
5000 µg	11	14	15	13.3	2.1	0.68
1600 µg	19	25	21	21.7	3.1	1.10
500 µg	13	16	24	17.7	5.7	0.90
160 µg	17	11	13	13.7	3.1	0.69
50 µg	21	20	15	18.7	3.2	0.95
Sodium azide 1 µg	400	408	396	401.3	6.1	20.41
Strain TA1535, With S-9 Mix, Main Test 1
Control	24	34	34	30.7	5.8
5000 µg	31	25	31	29.0	3.5	0.95
1600 µg	31	20	27	26.0	5.6	0.85
500 µg	31	25	31	29.0	3.5	0.95
160 µg	23	29	18	23.3	5.5	0.76
50 µg	26	28	16	23.3	6.4	0.76
2-Aminoanthracene 42 µg	147	176	164	162.3	14.6	5.29
Strain TA1535, With S-9 Mix, Main Test 2
Control	24	22	25	23.7	1 .5
5000 µg	19	18	19	18.7	0.6	0.79
1600 µg	16	22	26	21.3	5.0	0.90
500 µg	20	25	18	21.0	3.6	0.89
160 µg	18	20	20	19.3	1 .2	0.82
50 µg	27	22	20	23.0	3.6	0.97
2-Aminoanthracene 2 µg	184	187	210	193.7	14.2	8.18
S.D. = Standard deviation RATIO =Mean number of revertants on treated plates/mean number of revertants on vehicle control plates * = Statistically significant at 5% level ** = Statistically significant at 1% level Otherwise, not statistically significant at 5% level (The positive controls were not included in the statistical analysis) T = Toxicity: reduced growth of background lawn of non-revertant bacteria

Applicant's summary and conclusion

Conclusions:

The test substance has not shown evidence of mutagenic activity with or without S-9 activation.

Executive summary:

The test substance was tested in the Ames Test using Salmonella typhimurium strains TA 102, TA 100, TA 98, TA 1537, and TA 1535. The test was performed in accordance with OECD Guideline 471. The study was performed using selective agar plates containing fructose instead of glucose (the usual sugar in this type of plate) because glucose is the substrate for the enzymatic activity of this test substance.

A preliminary study was done using the plate incorporation method, but the histidine in the test substance interfered with the test system. A second preliminary study was done with strain TA 98 and the independent two main tests were carried out with the treat and plate method to avoid this problem. The bacteria were treated with solutions of the test substance prepared in sterile saline solution (0.9% NaCl) at five dose levels in the range 50 to 5000 µg/plate. The higest dose is equivalent to 1.87 mg active enzyme protein/ kg bw or 9.71 enzyme concentrate dry matter/kg bw. 

All of the dose levels in this report are expressed in terms of the total protein content of the test substance (stated by the Sponsor to be 36.24 mg/mL in the solution supplied). Negative control plates were treated by the addition of sterile saline solution (200 µL/plate). The treatments were performed both with and without a metabolic activation system (S-9 mix). Triplicate plates were prepared at each test point.

A dose related amount of insoluble material was observed on the plates in some parts of the study. The test substance was toxic to most of the tester strains at the higher dose levels, causing reduced growth of the background lawn and reductions in the number of revertant colonies. These effects were generally observed over a wider range of dose levels for treatments without S-9 mix than for treatments with S-9 mix. The amount of toxicity showed variation between tester strains. No biologically significant increases in the number of revertant colonies were observed in any tester strain after treatment with the test substance at any dose level, either in absence or presence of S-9 mix. Results obtained with the negative and positive controls demonstrated the sensitivity of the tests and efficacy of the S-9 mix metabolic activation system. Based on the results obtained in this study, it is concluded that the test substance has not shown any evidence of mutagenic activity.