Fructofuranosidase, β-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 - 20 August 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted: 21 July 1997

Deviations:

no

Remarks:

Evaluation criteria different from guideline: positive result 3 concentrations increased number of revertants

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministry of Health Welfare and Sports, Inspectorate for Health Protection, Commodities and Veterinary Public Health, GLP Compliance Monitoring unit, The Netherlands

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Xylanase, endo-1,4-

EC Number:

232-800-2

EC Name:

Xylanase, endo-1,4-

Cas Number:

9025-57-4

Molecular formula:

Not applicable, see remarks.

IUPAC Name:

endo-1,4-beta-xylanase

Method

Target gene:

his operon (for S. typhimurium) and trp operon (for E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers of male Wistar rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Dose-rang finding experiment: 500, 2000, 4000, 8000, 16000 and 32000 μg dry matter/mL (TA 100 strain)
First experiment: 50, 158, 500, 1580 and 5000 μg dry matter/plate (tested up to maximum concentration)
Second experiment: 100, 266, 707, 1880 and 5000 μg dry matter/plate (tested up to maximum concentration, cooncentrations were adapted, since first experiment was negative)

Vehicle / solvent:

- Vehicle used: sterile water
- - Justification for choice of vehicle: The test item is miscible with Glass Distilled Water (GOW) at the tested concentration of 50 mg dry matter/mL.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 4-nitroguinoline-1-oxide; 2-Aminoanthracene (2-AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 67 h at 37.0 - 37.2°C (first experiment) and 36.9 -37.0 °C

NUMBER OF REPLICATIONS: triplicates in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction of bacterial background lawn

Rationale for test conditions:

Based on dose-range finding study and test for effect of protease activity on S-9 mix.
Toxicity and precipitation was tested at the following concentrations: 1000, 2000, 3000, 4000 and 5000 µg dry matter/plate. There was no precipitation, reduction of background lawn as measure of toxicity or an increase in mean number of revertant colonies oberved up to the highest dose tested (5000 µg dry matter/plate) in strain TA 100. Furthermore, the test substance at concentrations of 1000 µg dry matter/mL and 10000 µg dry matter/mL, did not inhibit the activity of S-9 mix.

Evaluation criteria:

For tester strains TA 98, TA 100 and WP2uvrA pKM 101 the test is considered positive when the number of revertants in the treatment groups is twice that of the solvent control and this should be evident at a minimum of three dose levels.
For tester strains TA 1535 and TA 1537 the number of revertants should be thrice that of the solvent control and this should be evident at a minimum of three dose levels.

Statistics:

Means and standard deviations were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: In the dose-range findig test no precipitates or toxicity was observed. Furthermore, the testsfor protease acitivity revealed that the test substance did not inhibit metabolic activation.



In none of the tester strains an oncrease revertant counts were increased 2 or 3 fold

HISTORICAL CONTROL DATA (please refer to Table 5 in the 'Any other information on results incl. tables section')
- Positive historical control data: data were within the range range of historical revertant counts
- Negative (vehicle) historical control data: data were within the range of historical spontaneous revertant counts

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: reduction of background lawn
- In both experiments and for all tester strains a normal background lawn comparable to solvent control plates was observed.

OTHER:
In strain TA 98, trial I (with and without metabolic activation) and trial II with metabolic activation, there was increase in the mean numbers of revertant colonies and in strains TA 100, TA 1535 and WP2 uvrA pKM 101 there was increase, in the mean numbers of revertant colonies in the highest concentration, in both the trials, both in the presence and absence of metabolic activation. But, there was no two fold increase (TA 98, TA 100 and WP2 uvrA pKM 101 ) or three fold increase (TA 1535) when compared to that of solvent control plates.

Any other information on results incl. tables

Table 1: Experiment 1 (without S-9 mix)

Compound	S-9 Mix	concentration	TA 98	TA 100	TA 1535	TA 1537	WP2 uvra pKM 101
Water (0.1 mL)	-	0	20 ± 1	105 ± 2	13 ± 3	10 ± 1	102 ± 5
Test substance	-	50	20 ± 2	104 ± 3	15 ± 2	11 ± 2	99 ± 4
	-	158	22 ± 3	106 ± 1	16 ± 3	8 ± 1	102 ± 5
	-	500	25± 3	107 ± 2	15 ± 1	9 ± 1	99 ± 2
	-	1580	25 ± 2	116 ± 3	17 ± 2	9 ± 1	107 ± 5
	-	5000	26 ± 2	124 ± 3	22 ± 2	11 ± 2	124 ± 3
Positive control	-		197 ± 8 a	506 ± 19 b	130 ± 5 b	102 ± 2 c	618 ± 15 d

Values are averages of three replicates and rounded off to the nearest whole number

a) 2-nitrofluorene: 2 µg/plate; b) sodium azide: 1 µg/plate; c) 9-aminoacridine: 50 µg/plate; d) 4-nitroquinoline-1-oxide: 4 µg/plate

 

Table 2: Experiment 1 (with S-9 mix)

Compound	S-9 Mix	concentration	TA 98	TA 100	TA 1535	TA 1537	WP2 uvra pKM 101
Water (0.1 mL)	-	0	21 ± 2	99 ± 3	13 ± 2	9 ± 1	96 ± 3
Test substance	-	50	23 ± 2	102 ± 5	14 ± 1	9 ± 1	100 ± 3
	-	158	24 ± 2	98 ± 2	14 ± 1	10 ± 1	100 ± 6
	-	500	22± 1	93 ± 4	15 ± 2	9 ± 1	97 ± 3
	-	1580	26 ± 1	114 ± 2	18 ± 1	8 ± 1	94 ± 3
	-	5000	28 ± 3	121 ± 2	20 ± 2	9 ± 1	121 ± 2
Positive control	-		745 ± 46 a	919 ± 17 a	140 ± 1 a	94 ± 1 a	525 ± 19 b

Values are averages of three replicates and rounded off to the nearest whole number

a) 2-Aminoanthracene (2-AA): 4 µg/plate; b) 30 µg/plate

 

 

Table 3: Second experiment (without S-9)

Compound	S-9 Mix	concentration	TA 98	TA 100	TA 1535	TA 1537	WP2 uvra pKM 101
Water (0.1 mL)	-	0	17 ± 1	97 ± 3	13 ± 3	11 ± 1	104 ± 4
Test substance	-	100	19 ± 4	96 ± 3	13 ± 2	12 ± 1	105 ± 3
	-	266	16 ± 2	99 ± 4	18 ± 1	12 ± 2	108 ± 5
	-	707	14± 1	115 ± 2	16 ± 1	13 ± 2	114 ± 5
	-	1880	15 ± 2	107 ± 8	19 ± 2	13 ± 2	124 ± 2
	-	5000	18 ± 5	110 ± 5	23 ± 2	13 ± 3	137 ± 2
Positive control	-		171 ± 4 a	469 ± 14 b	126 ± 7 b	115 ± 8 c	626 ± 15 d

Values are averages of three replicates and rounded off to the nearest whole number

a) 2-nitrofluorene: 2 µg/plate; b) sodium azide: 1 µg/plate; c) 9-aminoacridine: 50 µg/plate; d) 4-nitroquinoline-1-oxide: 4 µg/plate

 

Table 4: Second experiment (with S-9)

Compound	S-9 Mix	concentration	TA 98	TA 100	TA 1535	TA 1537	WP2 uvra pKM 101
Water (0.1 mL)	-	0	17 ± 2	108 ± 1	13 ± 2	9 ± 4	117 ± 3
Test substance	-	100	19 ± 4	108 ± 5	13 ± 2	11 ± 2	124 ± 3
	-	266	16 ± 2	116 ± 2	16 ± 1	11 ± 0	129 ± 3
	-	707	14± 1	118 ± 5	17 ± 1	9 ± 3	140 ± 2
	-	1880	15 ± 2	125 ± 5	20 ± 3	9 ± 2	152 ± 4
	-	5000	18 ± 5	145 ± 2	22 ± 1	10 ± 3	166 ± 3
Positive control	-		878 ± 13 a	994 ± 7 a	143 ± 2 a	96 ± 4 a	597 ± 13 b

Values are averages of three replicates and rounded off to the nearest whole number

a) 2-Aminoanthracene (2-AA): 4 µg/plate; b) 30 µg/plate

 

Table 5: Historical control data

Compound	S-9 Mix	substance	TA 98	TA 100	TA 1535	TA 1537	WP2 uvra pKM 101
number of plates	-	vehicle control	309	309	309	309	201
mean	-	vehicle control	15.30097	109.3592	10.35275	7.572816	123.597
standard deviation	-	vehicle control	4. 223854	23.47278	3.294304	3.200858	30.48051
Max	-	vehicle control	30	192	23	16	210
Min	-	vehicle control	4	50	3	2	83
number of plates	+	vehicle control	176	276	276	276	168
mean	+	vehicle control	18.72101	109.6739	10.65942	8.576087	122.4345
standard deviation	+	vehicle control	6.533137	22.67371	3.124091	2.910422	27.47795
Max	+	vehicle control	46	185	19	16	200
Min	+	vehicle control	4	16	2	2	77
number of plates	-	positive control	309	309	309	309	165
mean	-	positive control	190.3135	485.9769	171.6766	91.0297	638.103
standard deviation	-	positive control	106.7323	139.4921	158.5474	36.19149	122.8032
Max	-	positive control	853	1552	2198	229	1080
Min	-	positive control	50	238	18	18	372
number of plates	+	positive control	294	294	294	294	168
mean	+	positive control	592.9626	918.7789	115.9116	36.45918	584.1607
standard deviation	+	positive control	183.8938	216.5313	50.91999	33.08418	138.4984
Max	+	positive control	1108	1433	496	200	1136
Min	+	positive control	161	320	41	20	360

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of the conducted test the substance was not mutagenic in any of the five tester strains (TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA pKM 101) tested with and without metabolic activation up to 5000 µg/plate.