Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 232-615-7 | CAS number: 9001-57-4
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
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
- 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.
- 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. - 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.
- 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.
- 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. - Conclusions:
- Interpretation of results: negative
Referenceopen allclose all
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 |
Table 1: Preliminary cytotoxicity test
Test item concentration (µg drymatter/mL) |
With S-9* |
Without S-9* |
Without S-9** |
|||
Cell count (x106/fl ask) |
% Control |
Cell count (x106/flask) |
% Control |
Cell count (x106/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 (x106/flask) |
% Control |
Cell count (x106/flask) |
% Control |
Cell count (x106/flask) |
% Control |
Cell count (x106/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
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).
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.