Fructofuranosidase, β-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Mutagenicity (OECD 471, GLP), Ames: negative with and without metabolic activation

 Read-across from source substances: xylanase (CAS 9025-57-4) and alpha-amylase (CAS 9000-90-2)

Cytogenicity/chromosome aberration in mammalian cells (OECD 473, GLP): negative with and without metablic activation

Read-across from source substances: xylanase (CAS 9025-57-4) and alpha-amylase (CAS 9000-90-2)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Type of assay:

bacterial reverse mutation assay

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

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

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

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

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.

Remarks on result:

other:

Remarks:

Source: CAS 9025-57-4

Conclusions:

The read-across approach is detailed in the analogue justification. The target and source substances are considered unlikely to differ in their in vitro gentic toxicity potential. Based on the results of the available Ames test conducted with the source substance endoxylanase (CAS 9025-57-4), 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) up to 5000 µg/plate with and without metabolic activation. Therefor, the target substance β-fructofuranosidase (CAS 9001-57-4) is not expected to be mutagenic in bacteria.

Reference 2

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

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

Target gene:

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

Species / strain / cell type:

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

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.

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.

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

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

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

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

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.

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

 

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.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Type of assay:

bacterial reverse mutation assay

Key result

Species / strain:

primary culture, other:

Remarks:

human lymphocytes (Experiment 1, 3h exposure)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

blood clotting observed at 4000 and 5000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

primary culture, other:

Remarks:

human lyphocytes (Experiment 2: 3h with metabolic activation and 24 h and 48 h without metabolic activation)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

blood clotting

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH at a concentration of 5000 μg/mlLwas 7.48 (compared to 7.52 in the solvent control).
- Effects of osmolarity: The osmolarity at a concentration of 5000 μg/mL was 273 mOsm/kg respectively (compared to 286 mOsm/kg in the solvent control).
- Water solubility: Yes, test substance is soluble in water
- Precipitation:
Blood clotting was observed in the cultures treated with concentrations of 3330 μg Bacillus subtilis containing alpha-amylase activity/mL and higher, and consequently these concentrations could not be
used for the preparation of slides.

RANGE-FINDING/SCREENING STUDIES:
5000 μg/mL was used as the highest concentration of enzyme preparation of Bacillus subtilis containing alpha-amylase activity.
In the dose range finding test blood cultures were treated with 100, 333, 1000, 3330 and 5000 μg enzyme preparation of Bacillus subtilis containing alpha-amylase activity/mLculture medium with and without S9-mix. For the mitotic index of cultures, Please refer to table 1 in phe '"Any other information on results incl. tables" section. Blood clotting was observed at concentrations of 3330 and 5000 μg/ml. No slides could be prepared from these concentrations

CYTOKINESIS BLOCK: Colchicin was used at 0.5 µg/mL (used during the last 2.5 - 3 h of the culture period)

HISTORICAL CONTROL DATA:
- Positive historical control data:
The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. (Please refer to table 10 and 11 in the '"Any other information on results incl. tables" section).
- Negative (vehicle) historical control data:
Number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range(Please refer to table 10 and 11 in the "Any other information on results incl. tables" section).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used:Mitotic index
- Other observations when applicable: No cytotoxicity observed up to highest concentration of 5000 µg/mL.

OTHER:
During the performance of the test substance used in this experiment was handled as if it had a TOS content of 7.99%. (as initially given by the sponsor) and a correction factor of 12.52 was used in this assay so that all doses mentioned in this report are based on the test substance TOS content. In the course of the development it has been identified that the test substance had a TOS content of 8.91%. Therefore, the actual concentrations were 11.5% higher than stated in the report. The conclusion of the experiments remained unchanged.

Remarks on result:

other: Enzyme preparation of Bacillus subtilis containing alpha-amylase activity is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Conclusions:

The read-across approach is detailed in the analogue justification. The target and source substances are considered unlikely to differ in their in vitro gentic toxicity potential. Based on the results of the available chromosome aberration test conducted with the source alpha-amylase, the substance did not induce chromosome aberrations with and without metabolic activation. Therefore, the target substance β-fructofuranosidase (CAS 9001-57-4) is not expected to induce chromosome aberrations.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

11 -18 February 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted: 21 July 1997

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Adopted: 29 July 2016

Deviations:

yes

Remarks:

instead of 300 only 200 metaphases analysed

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:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1 cell line (Ovary, Chinese hamster, Cricetulus griseus)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, Rockville, Maryland, USA
- Modal number of chromosomes: 20
- Normal (negative control) cell cycle time: 20 h 25 min (about 1.5 cell cycle time)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham's F-12 medium supplemented with 10% fetal bovine serum, sodium bicarbonate, antibiotics and L - glutamine (F-12 FBS 10), 5% CO2, 37 ± 1 °C
- Periodically checked for Mycoplasma contamination: yes

Cytokinesis block (if used):

colchicin 0.2 µg/mL

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Dose-range finding experiment: 75, 150, 300, 600, 1200, 2400 and 5000 µg/mL (3h exposure with and without S-9 mix and 20 h 25 min exposure without S-9 mix)
First experiment: 1250, 2500 and 5000 µg dry matter/mL (3 h exposure with and without S-9 mix (7.5%), tested up to limit concentration)
Second experiment: 1250, 2500 and 5000 µg dry matter/mL (3 h exposure with S-9 mix (10%) and 19 h 35 min without S-9, tested up to limit concentration)

Vehicle / solvent:

- Vehicle used: water (glass distilled)
- Justification for choice of solvent/vehicle:

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Fixation time (start of exposure up to fixation or harvest of cells): 21.05 to 21.30 h

SPINDLE INHIBITOR: colchicin 0.2 µg/mL

STAIN: 5% Giemsa stain in distilled water for 20 min, rinsed with tap water, air dried, immersed in xylene and mounted with DPX

NUMBER OF REPLICATIONS: quintuplicate cultures in 2 independent experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: cell suspension was dropped onto a clean chilled slide, flame dried and dried on a slide warmer maintained at approximately 40°C. The slides were marked (study number, treatment group, activation, trial number and replicate number)

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 200 metaphases

DETERMINATION OF CYTOTOXICITY
- Method: cell count by using a hemocytometer

- OTHER:
- pH was determined at the beginning and end of the treatment period
- Test for protease activity of the test substance on S-9mix

Evaluation criteria:

A positive result is strengthened by the demonstration of a dose-related increase of the effect. This is particularly crucial if only the frequency of gaps is increased. Exchanges are such rare events ( < 1 in 1000 celIs) that they are seldom observed in control samples. Thus, the observation of exchanges in experimental groups, even without a dose-related increase, is a strong indication for a positive response. Biological relevance of the results should be considered first. However, there are certain reservations based on the differences in ranking the types of aberrations. Gaps are ranked lowest and exchange configurations are ranked highest.

Statistics:

Proportions of aberrant metaphases in each sample, both including and excluding gaps as aberrations were analyszed. The pooled data from each test concentration and the positive control data were compared with the water control using one-tailed Fisher exact test. All analysis and comparisons were evaluated at 5% (P < 0.05) level.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

First trial: 3h exposure

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

dose-dependent growth inhibition, but < 50% (For details please refer to Table 2), tested up to limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

Second experiment: 3 h exposure

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

increase of aberrant cells in all test concentrations, aberrations including gaps statistically significant increased at 2500 and 5000 µg/mL (not considered biological relevant)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

dose-dependent growth inhibition, but < 50% (For details please refer to Table 2), tested up to limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

Second expeiment: 24 h exposure

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

increase of aberrant cells at the highest test concentration (not considered biological relevant)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

dose-dependent growth inhibition, but < 50% (For details please refer to Table 2), tested up to limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no (please refer to Table 7 in the 'Any other information on results incl. tables' section)
- Water solubility: yes, test substance is soluble in water
- Precipitation: no precipitates observed

RANGE-FINDING/SCREENING STUDIES: dose-dependent decrease in cell count observed (between 58 and 44% at the hifhest dose tested 5000 µg/mL)(Please refer to Table in the 'Any other information on results invcl. tables' section)

CYTOKINESIS BLOCK: colchicin was used at 0.2 µg/mL

HISTORICAL CONTROL DATA
- Positive historical control data: Yes (please refer to Table 8 in the 'Any other information on results incl. tables' section)
- Negative (vehicle) historical control data: Yes (please refer to Table 8 in the 'Any other information on results invl. tables' section)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: yes, based on cell counts (please refer to Table 2 in the 'Any other information on results incl. tables' section)

OTHER:
- Protease activity: the test substance at a concentration of 1000 and 10000 µg/mL did not inhibit the activity of S-9 mix
- Statistical significance Experiment 2 (+S-9): statistical increase of aberrant metaphases with gaps at the concentrations of 2500 and 5000 μg dry matter/mLfor experiment 2 (+ S-9) and for the incidence of aberrant metaphases without gaps at the concentration of 5000 μg dry matter/mL for experiment 2 (- S-9). The status of gaps as true chromosome aberrations is a matter of debate and are often ignored. Also in borderline situations, greater significance should be attached to the observation of exchanges in treated cells than to a small numerical increase in breaks. Moreover, these incidences of aberrant metaphases seen lie within the historical control. Taken together, the results of the two trials suggest that the test item does not have the potential to cause chromosome damage either including gaps or excluding gaps and either in the presence or absence of metabolic activation.

Table 1: Preliminary cytotoxicity test 

Test item concentration (µg drymatter/mL)	With S-9*		Without S-9*		Without S-9**
	Cell count (x10^6/fl ask)	% Control	Cell count (x10^6/flask)	% Control	Cell count (x10^6/flask)	% Control
water (0.1 mL)	3.19	100	3.44	100	3.75	100
75	2.57	80. 56	3.20	93.02	3.50	93.33
150	2.54	79.62	3.10	90.12	3.07	81.87
300	2.24	70.22	3.22	93.60	3.25	86.67
600	2.25	70.53	3.30	95.93	3.54	94.40
1200	2.23	69.91	2.99	86.92	3.30	88.00
2400	1.97	61.76	2.97	86.34	2.49	66.40
5000	1.88	58.93	3.05	88.66	2.38	63.47

* 3h treatment

** Prolonged treatment 20 h 25 min

 

Table 2: Cytotoxicity test experiment 1 and 2

Test item concentration (µg dry matter/mL)	Experiment 1		Experiment 2		Experiment 1		Experiment 2
	+ S-9		+ S-9		- S-9		- S 9
	Cell count (x10^6/flask)	% Control	Cell count (x10^6/flask)	% Control	Cell count (x10^6/flask)	% Control	Cell count (x10^6/flask)	% Control
water (0 .3 mL)	6.08	100	6.23	100	5.80	100	4.85	100
1250	5. 80	95.39	5.49	88.12	5.38	92.76	4.45	91.75
2500	5.48	90.13	5.20	83.47	4.83	83.28	4.35	89.69
5000	4.53	74.51	4.83	77.53	4.25	73.28	3.93	81 .03
CPA 55 (+ S-9);  EMS 600 (- S-9)	3.63	59.70	3.90	62.60	3.45	59.48	3.65	75.26

Cyclophosphamide (CPA); Ethylmethanesulfonate (EMS)

 

Table 3: Results experiment 1 (with metabolic activation)

Test Item conecentration (µg dry matter/mL)	No. of MPs scored	No. (%) of metaphases with aberrations							Total No.(%) of MPs with aberrations
		Gaps		Breaks		Exchanges		Ring	With Gaps	Without Gaps
		Cs	Ct	Cs	Ct	Cs	Ct
water (0.3 ml)	200	0 (0)	1 (0.5)	0 (0)	1 (0.5)	0 (0)	0 (0)	0 (0)	2 (1.0)	1 (0.5)
1250	200	0 (0)	2 (1.0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	2 (1.0)	0 (0)
2500	200	0 (0)	0 (0)	0 (0)	1 (0.5)	0 (0)	0 (0)	0 (0)	1 (0.5)	1 (0 5)
5000	200	0 (0)	1 (0.5)	0 (0)	2 (1.0)	0 (0)	0 (0)	0 (0)	3 (1.5)	2 (10)
CPA 55	200	12 (6.0)	77 (38.5)	0 (0)	88 (44.0)	15 (7.5)	93 (46.5)	6 (3.0)	+ 130 (65.0)	+121 (60.5)

Cyclophosphamide (CPA); Ethylmethanesulfonate (EMS)

Metaphases (MP); Chromosome type (Cs); Chromatid type (Ct)

+ significantly higher than control (P ≤ 0.05)

 *: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

 

Table 4: Results experiment 1 (without metabolic activation)

Test Item cone. (µg dry matter/mL)	No. of MPs scored	No. (%) of metaphases with aberrations							Total No.(%) of MPs with aberrations
		Gaps		Breaks		Exchanges		Ring	With Gaps	Without Gaps
		Cs	Ct	Cs	Ct	Cs	Ct
water (0.3 mL)	200	1 (0 5)	5 (2 5)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0 )	6 (30)	0 (0)
1250	200	1 (05)	3 (1.5)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	4 (2.0)	0 (0)
2500	200	0 (0)	2 (1 0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	2 (1.0)	0 (0)
5000	200	1 (0.5)	9 (4.5)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	10 (5.0)	0 (0)
EMS 600	200	34 (17.0)	83 (41.5)	8 (4.0)	58 (29.0)	16 (8.0)	110 (55.0)	6 (3.0)	+160  (80.0)	+139 (69.5)

Cyclophosphamide (CPA); Ethylmethanesulfonate (EMS)

Metaphases (MP); Chromosome type (Cs); Chromatid type (Ct)

+ significantly higher than control (P ≤ 0.05)

 *: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

 

Table 5: Results experiment 2 (with metabolic activation)

Test Item cone. (µg dry matter/mL)	No. of MPs scored	No. (%) of metaphases with aberrations								Total No.(%) of MPs with aberrations
		Gaps		Breaks		Exchanges		Ring	Endoredu- plication	With Gaps	Without Gaps
		Cs	Ct	Cs	Ct	Cs	Ct
water (0.3 mL)	200	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0 )	0 (0)	0 (0)
1250	200	0 (0)	2 (1.0)	0 (0)	1 (0 5)	0 (0)	0 (0)	0 (0)	0 (0)	3 (1.5)	1 (0.5)
2500	200	0 (0)	3 (1.5)	0 (0)	3 (1.5)	0 (0)	0 (0)	0 (0)	0 (0)	+6 (3.0)	3 (1.5)
5000	200	2 (1.0)	8 (4.0)	0 (0)	3 (1.5)	0 (0)	0 (0)	0 (0)	0 (0)	+12 (6.0)	3 (1.5)
CPA 55	200	18 (9.0)	72 (36.0)	3 (1.5)	60 (30.0)	9 (4.5)	85 (42.5)	2 (1 0)	3 (1.5)	+136 (68.0)	+115 ( 57 5)

Cyclophosphamide (CPA); Ethylmethanesulfonate (EMS)

Metaphases (MP); Chromosome type (Cs); Chromatid type (Ct)

+ significantly higher than control (P ≤ 0.05)

 *: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

 

Table 6: Results experiment 2 (without metabolic activation)

Test Item cone. (µg dry matter/mL)	No. of MPs scored	No. (%) of metaphases with aberrations							Total No.(%) of MPs with aberrations
		Gaps		Breaks		Exchanges		Ring	With Gaps	Without Gaps
		Cs	Ct	Cs	Ct	Cs	Ct
water (0.3 mL)	200	1 (0.5)	4 (2.0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	5 (2.5)	0 (0)
1250	200	0 (0)	3 (1.5)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	3 (1.5)	0 (0)
2500	200	0 (0)	1 (0.5)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (0.5)	0 (0)
5000	200	1 (0.5)	5 (2 . 5)	0 (0)	5 (2.5)	0 (0)	0 (0)	0 (0)	11 (5 5)	+5 (2.5)
EMS 600	200	17 (8.5)	21 (1.5)	0 (0)	41 (20.5)	3 (1.5)	77 (38.5)	1 (0.5)	+ 112 (56.0)	+98 (49.0)

Cyclophosphamide (CPA); Ethylmethanesulfonate (EMS)

Metaphases (MP); Chromosome type (Cs); Chromatid type (Ct)

+ significantly higher than control (P ≤ 0.05)

 *: Metaphase plate with one or more than one aberrations considered as one metaphase plate

with aberrations

 

Table 7 : pH of the medium during precipitation test

Test item concentration (µg dry matter /mL)	pH at the beginning of the treatment period	pH at the end of the treatment period
water (0.1 mL)	7.40	7.35
75	7.36	7.40
150	7.40	7.42
300	7.41	7.44
600	7.43	7.40
1200	7.42	7.39
2400	7.40	7.38
5000	7. 39	7.36

Table 8: Historical control data of solvent and positive control

Parameter	No. of metaphases with aberrations							Total No. of metaphases with aberrations*
	Gaps		Breaks		Exchanges
	Cs	Ct	Cs	Ct	Cs	Ct	Others**	With Gaps	Without Gaps
water (with S-9)
n = 1200
Range	0.1	1-2	0	0-5	0	0	0	1-5	0-5
Mean	0. 17	0.83	0	1.33	0	0	0	2.33	1.33
S.D	0.41	0.75	0	1. 97	0	0	0	1.51	1.97
water (without S-9)
n = 1200
Range	0-1	0-3	0-1	0-3	0	0	0	0-7	0-4
Mean	0.33	1.5	0.33	1.5	0	0	0	3.5	1.83
S.D.	0.52	1.38	0.52	1.38	0	0	0	2.88	1.72
CPA
n = 1524
Range	0-9	9-63	0-12	11-163	0-44	0-2	0-2	17-136	10-114
Mean	4.07	33.7116	3.07	69.93	12.14	0.36	0.36	71.17	56.33
S.D	3.32	.79	3.91	43.11	15.53	0.74	0.74	40 .49	37.1
EMS
n = 1610
Range	0-19	11-74	0.24	6-135	0.71	0-3	0-3	21-128	10-103
Mean	5.21	36.07	4.43	50.0	18 .64	0.5	0.5	71.0	51.58
S.D	6.46	21.96	7.48	36.82	24.1 3	0.85	0.85	41.14	35.22

Cyclophosphamide (CPA); Ethylmethanesulfonate (EMS); Standard deviation (SD)

Metaphases (MP); Chromosome type (Cs); Chromatid type (Ct)

+ significantly higher than control (P ≤ 0.05)

 *: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

** Righ chromosome

Conclusions:

Interpretation of results: negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for read-across:

There are no experimental data available regarding in vitro genotoxicity of β-fructofuranosidase (CAS 9001-57-4). It must be noted however that β-fructofuranosidase is produced by a classical strain of Saccharomyces cerevisiae, which has a long history of safe use in food applications globally. Read-across from two appropriate analogue substances, xylanase (CAS 9025-57-4) and alpha-amylase was conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5 in order to fulfil the standard information requirements defined in Regulation (EC) No 1907/2006, Annex VII and VIII, 8.4.

Common functional groups and structural similarities of the source and target substances are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

 

-Genetic toxicity in bacteria (Ames)

The in-vitro genetic toxicity of the read across substance xylanase (CAS 9025-57-4) was assessed in a bacterial reverse mutation assay (Ames test) according to OECD TG 471 and GLP criteria (Ravi 2001). The mutagenic potential of the test substance was assessed in S. typhimurium tester strains TA 98, 100, 1535, 1537 and E.coli WP2 uvra pKM 101 at concentrations up to 5000 µg/plate in 2 independent plate incorporations experiments with and without metabolic activation.

Based on a dose range finding experiment, the test substance was deemed to be soluble and not cytotoxic. Thus, the maximum recommended concentration was used. Furthermore, the protease activity of the test substance was tested. In this experiment, no inhibiting effect of the test substance on the S-0 mix was observed.

The test item was tested at the concentrations of 50, 158, 500, 1580 and 5000 μg dry matter/plate in the first trial and 100, 266, 707, 1880 and 5000 μg dry matter/plate in the second trial using sterile glass distilled water (GOW) as the solvent.

In both experiments, the test substance did not induce an increase of reversions in any of the tested strains with or without metabolic activation above strain specific thresholds (2 fold increase (TA 98, TA 100 and WP2 uvrA pKM 101 and 3 fold increase (TA 1535) when compared to that of solvent control plates). Cytotoxicity was not observed. The vehicle and positive controls proved the validity of the experiment. Thus 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 pKM101) tested with and without metabolic activation up to 5000 µg/plate.

 

The in-vitro genetic toxicity of the read across substance alpha-amylase was assessed in a bacterial reverse mutation assay (Ames test) according to OECD TG 471 and GLP criteria (Verspeek-Rip, 2013). The mutagenic potential of the test substance was assessed in S. typhimurium tester strains TA 98, 100, 1535, 1537 and E.coli WP2 uvra at concentrations up to 5000 µg/plate in 2 independent plate incorporations experiments with and without metabolic activation. Based on a dose range finding experiment, the test substance did not precipitate and was not cytotoxic and therefore the maximum recommended concentration was used as top concentration. In the dose range finding experiment, the following concentrations were tested in TA 100 and WP2 uvra strains: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate. This test was reported as part of experiment 1. The test item solved in water was tested at the concentrations of 100, 333, 1000, 3330 and 5000 µg/plate (TA 1535, 1537 and TA 98) in the first experiment and 50, 333, 500, 3330 and 5000 µg/plate in the second experiment with (5% S-9 mix experiment 1, 10% S) and without metabolic activation. In both experiments, the test substance did not induce a biological relevant increase of reversions in any of the tested strains with or without metabolic activation. Only in TA 100 strain in the second experiment with metabolic activationm a 2.1-fold increase was observed. Since this increase was not above the historical control data range and just 2-fold and only observed in the second experiment with a low mean solvent control value, it was not considered to be not biologically relevant and Enzyme preparation of Bacillus subtilis containing alpha-amylase activity is considered to be not mutagenic in tester strain TA100. Cytotoxicity was not observed. The vehicle and positive controls proved the validity of the experiment. Thus 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) tested with and without metabolic activation up to 5000 µg/plate.

 

- Cytogenicity/chromosome aberration in mammalian cells:

A GLP conformed chromosome aberration study with the read across substance, xylanase (CAS 9025-57-4), was performed according to OECD guideline 473 (Indrani, 2002). Clastogenicity of the test material, solved in water, was investigated in Chinese hamster ovary cells. Cells were treated with the test material both with and without the addition of cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers of rats treated with Aroclor 1254.

A preliminary test was performed at concentrations of 75, 150, 300, 600, 1200, 2400 and 500 0 µg/mL with and without metabolic activation (3h) and without metabolic activation (20 h 25 min). No precipitates occurred, but dose-dependent cytotoxicity was observed. However, toxicity was < 50% even at the highest dose tested.

In main experiment, 1 and 2 test substance concentrations were 1250, 2500 and 5000 µg/mL. In the main experiment, 1 cell was exposed for a period of 3h with and without metabolic activation (7.5 % S-9). In the main experiment, 2 cells were exposed for 3 h with metabolic activation (10% S-9) and for 19 h 35 min without metabolic activation. Furthermore, the protease activity of the test substance was accessed, but was shown not to inhibit the activity of S-9 mix. Colcemid-solution was added to each culture before the end of the incubation period. The positive controls used were ethylmethansulfonate in the absence and cyclophosphamide in the presence of metabolic activation. Quintuplicate cultures were tested for every concentration of test substance and positive and solvent controls. Cytotoxicity was determined by cell count. From each culture run in parallel 200 metaphases were examined for chromosome aberrations.

In experiment 1, no increase in aberrant cells compared to vehicle control was observed with and without metabolic activation. In experiment 2 with metabolic activation, the incidence of aberrant metaphases both including and excluding gaps were higher than in the respective solvent control at all test concentrations and was statistically significant for that including gaps at the concentrations of 2500 and 5000 μg dry matter/mL.

Without metabolic activation, there was an increase in the incidence of aberrant metaphases, both including and excluding gaps at 5000 μg dry matter/mL compared to vehicle control. For aberrations excluding gaps, statistical significance was reached.

Since incidences of aberrant metaphases were within historical control data, they were not considered biologically relevant. Positive controls with and without metabolic activation showed a clear and statistically significant increase of metaphases in experiment 1 and 2. Overall, the test substance was evaluated as not clastogenic under the conditions of this test.

 

- Cytogenicity/chromosome aberration in peripheral human lymphocytes:

A GLP conformed chromosome aberration study with the read across substance, alpha-amylase, was performed according to OECD guideline 473 (Verbaan, 2013). Clastogenicity of the test material solved in water was investigated in primary human peripheral lymphocytes. Cells were treated with the test material both with and without the addition of cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers of rats treated with phenobarbital and ß-naphthoflavone induced rat liver S9-mix.

A dose range finding study was performed at concentrations of 100, 333, 1000, 3330 and 5000 µg/mL for 3h (-/+S9) and 24 and 48 h (-S9). Blood clotting was observed at concentrations of 3330 and 5000 μg/ml. Thus, no slides could be prepared from these concentrations. In experiment 1, human lymphocytes were exposed at 100, 1000, 2000, 3000, 4000 and 5000 µg/mL for a period of 3h with and without metabolic activation. Based on blood clotting at 4000 and 5000 µg/mL, the concentrations scored in experiment 1 were 1000, 2000 and 3000 µg/mL. In experiment 2 human lymphocytes were exposed at 100, 333, 1000, 3000, 3500 and 4000 µg/mL for a period of 24 or 48h without metabolic activation and at 100, 1000, 3000, 3500 and 4000 µg/mL for a period of 3h with metabolic activation. During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 μg/ml medium). The positive controls used were mitomycin in the absence and cyclophosphamide in the presence of metabolic activation. Milli-Q water, the vehicle of the test substance served as THE negative control. Duplicate cultures were tested for every concentration of test substance, positive and solvent controls. Cytotoxicity was determined by determination of the mitotic index (100 cells per concentration). From each culture run in parallel 100 metaphases were examined for chromosome aberrations.

In experiment 1 and 2, both in the absence and presence of metabolic activation, alpha-amylase did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. Furthermore, alpha-amylase did not increase the number of polyploid cells and cells with endoreduplicated chromosomes in experiment 1 and 2 in the absence or presence of metabolic activation.

In both main experiments, precipitates did not occur, but dose-dependent cytotoxicity was observed. However, toxicity was < 55% even at the highest dose tested. The number of cells with chromosome aberrations, the number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures were within the laboratory historical control data range. The positive control chemicals both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned adequately.

Thus, it can be concluded that the test was valid and that alpha-amylase is not clastogenic in human lymphocytes under the described experimental conditions.

 

-Genetic toxicity (mutagenicity) in mammalian cells

In general, data on enzymes provides no evidence on the genotoxic or carcinogenic potential of enzymes [1]. (For further details, please refer to the justification for data waiving.

 

Conclusion on genetic toxicity

Overall, the available in vitro studies on genetic toxicity does not indicate that the read-across substances xylanase (CAS 9025-57-4) and alpha-amylase exhibit genotoxic properties. The test substance did not induce gene mutations in bacteria and did not increase the incidence of metaphases with chromosomal aberrations in peripheral human lymphocytes or Chinese hamster ovary cells. Therefore the target substance β-fructofuranosidase (CAS 9001-57-4) is not expected to differ in its genetic toxicity profile. Furthermore, β-fructofuranosidase belongs to a safe strain lineage and in general, enzyme proteins are not regarded as genotoxic substances [1].

References

[1] Reach - Data waiving argumentation for technical enzymes (December 2017, ERC/18/001)

Justification for classification or non-classification

The available genotoxicity data obtained for the read across substances are conclusive but not sufficient for classification according to Regulation (EC) No. 1272/2008 (CLP). Therefore, the target substance is considered not to meet the classification criteria according to Regulation (EC) No. 1272/2008 (CLP).