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EC number: 232-566-1 | CAS number: 9000-91-3
- 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
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- May 1 - Aug. 22, 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 014
- Report date:
- 2014
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- other: Draft OECD guideline 487, adopted 22 July 2010
- Version / remarks:
- 2010
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
Test material
- Reference substance name:
- Active enzyme protein of beta-amylase (EC no.232-566-1, Cas no. 9000-91-3, EC name Beta-amylase, enzyme class 3.2.1.2)
- Molecular formula:
- n.a.
- IUPAC Name:
- Active enzyme protein of beta-amylase (EC no.232-566-1, Cas no. 9000-91-3, EC name Beta-amylase, enzyme class 3.2.1.2)
- Reference substance name:
- Protein as a constituent of enzyme deriving from the fermentation or extraction process
- Molecular formula:
- Not available
- IUPAC Name:
- Protein as a constituent of enzyme deriving from the fermentation or extraction process
- Reference substance name:
- Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
- Molecular formula:
- Not available. See remarks.
- IUPAC Name:
- Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
- Reference substance name:
- Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
- Molecular formula:
- Not available. See remarks.
- IUPAC Name:
- Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
- Reference substance name:
- Lipids as a constituent of enzyme deriving from the fermentation or extraction process
- Molecular formula:
- Not available. See remarks.
- IUPAC Name:
- Lipids as a constituent of enzyme deriving from the fermentation or extraction process
- Test material form:
- liquid
- Details on test material:
- - Lot/batch No.: PPY36295
- Expiration date of the lot/batch: April, 2024
Constituent 1
Constituent 2
Constituent 3
Constituent 4
Constituent 5
Method
Species / strain
- Species / strain / cell type:
- lymphocytes: cultured human peripheral blood lymphocytes
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- The OECD Guideline 487 recommends a maximum test concentration for soluble non-cytotoxic substances of 5000 µg/plate (mL). Thus the test item was tested as 5000 µg total organic solids (TOS)/mL maximum dose, equivalent to 5522 µg enzyme concentrate dry matter/mL, fulfilling the recommendation.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Purified water
- Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure will be in aqueous solutions.
- Solvent for positive control: DMSO
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: Mitomycin C (MMC) and Vinblastine (VIN) in the absence of rat liver S-9, Cyclophosphamide (CPA) in the presence of S-9.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Whole blood cultures were established in HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum, 0.52% penicillin/streptomycin and 2% of the mitogen Phytohaemagglutinin (PHA). These cultures were incubated at approx. 37°C for 48 hours before treatment with test article. Sets of duplicate cultures were exposed to the test substance for 3 hours in the presence and absence of metabolic activation (S-9 mix) and harvested 24 hours after the beginning of treatment (3+21 hour treatment). Additionally, a continuous 24-hour treatment without S-9 mix was included with harvesting 24 hours after the beginning of treatment (24+24 hour treatment). The cultures were treated with cytochalasin-B after removal of the test substance to block cytokinesis. Six concentrations, covering an appropriate range of cytotoxicity, were selected for scoring of micronuclei by evaluating the effect of the test substance on the replication index. A minimum of 1000 cells per concentration (500 cells from each replicate culture) were scored.
DURATION
- Exposure duration: 3 and 24 hours
NUMBER OF REPLICATIONS: Sets of duplicate cultures were exposed to the test substance.
DETERMINATION OF CYTOTOXICITY
- Method: 5522 μg enzyme concentrate dry matter/mL was determined as max dose following a preliminary cytotoxicity Range-Finder Experiment. Cytotoxicity (%) was expressed as (100 – Relative replication Index (RI)). - Evaluation criteria:
- A test article was considered positive if:
- the assay was valid, and
- significant increase in the frequency of MNBN cells at one or more concentrations , and
- the incidence of MNBN cells exceeded the normal range in both replicates, and
- a concentration-related increase in the proportion of MNBN cells was observed.
- Statistics:
- The proportion of MNBN cells for each treatment condition were compared with the proportion in negative controls by using Fisher's exact test. Probability values of p equal or less than 0.05 were accepted as significant.
Results and discussion
Test results
- Key result
- Species / strain:
- lymphocytes: from human blood
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In the 24+24 hour treatment without S-9 mix the highest applied concentration was 4417 µg enzyme concentrate dry matter/mL due to cytotoxicity at higher concentrations. No significant cytotoxicity was seen in the 3+21 hour treatment.
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH of test material is neutral, 7.4
- Effects of osmolality: no marked changes in osmolality was observed (shifts of greater than 50 mOsm/kg)
- Water solubility: yes
- Precipitation: no
- Other confounding effects: none
RANGE-FINDING/SCREENING STUDIES: Preliminary cytotoxicity range-finder performed
MICRONUCLEUS EXPERIMENT: Treatment of cells with Beta amylase, batch PPY36295 in the absence and presence of S-9 resulted in frequencies of MNBN cells which were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle controls for the majority of all concentrations analysed. Exceptions to this were noted at an intermediate concentration analysed (3313 μg enzyme concentrate dry matter/mL) following 3+21 hour –S-9 treatment and at the highest concentration (4417 μg enzyme concentrate dry matter) following 24+24 hour –S-9 treatment. However, these increases were small such that mean MNBN cell values fell within normal ranges for all concentrations. With the exception of a single replicate culture at 4417 μg enzyme concentrate dry matter/mL (24+24 hour –S-9 treatment), the MNBN cell frequency of all treated cultures (all treatments) fell within the 95th percentile of the current observed historical vehicle control (normal) ranges. The increase above normal observed in replicate ‘B’ at 4417 μg enzyme concentrate dry matter/mL was not observed in either the replicate ‘A’ culture, or, following a second score from a separate prepared slide from the ‘B’ culture (‘B2’). As such, these small statistical increases were not considered of biological importance.
Applicant's summary and conclusion
- Conclusions:
- It was therefore concluded that Beta amylase, batch PPY36295 did not induce biologically relevant increases in micronuclei in cultured human peripheral blood lymphocytes under the experimental conditions employed for this study.
- Executive summary:
Beta amylase, batch PPY36295 was tested in an in vitro micronucleus assay using
duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in water for irrigation (purified water) and the highest concentration tested in the Micronucleus Experiment, 5522 μg enzyme concentrate dry matter/mL (an acceptable maximum concentration for in vitro micronucleus assays according to current regulatory guidelines), was determined following a preliminary cytotoxicity Range-Finder Experiment.Sets of duplicate cultures were exposed to the test substance for 3 hours in the presence and absence of metabolic activation (S-9 mix) and harvested 24 hours after the beginning of treatment (3+21 hour treatment). Additionally, a continuous 24-hour treatment without S-9 mix was included with harvesting 24 hours after the end of treatment (24+24 hour treatment). The cultures were treated with cytochalasin-B after removal of the test substance to block cytokinesis. Appropriatenumber of concentrations, covering an appropriate range of cytotoxicity, were selected for scoring of micronuclei. A minimum of 1000 cells per concentration were scored.
Based on a Range-Finder experiment, three or four concentrations were selected for micronucleus analysis.
Both negative and positive controls were within historical control ranges and the study was accepted as valid.
Treatment of cells with Beta amylase, batch PPY36295 in the absence and presence of S-9 resulted in frequencies of MNBN cells which were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle controls for the majority of all concentrations analysed. A few exceptions to this was noted in the intermediate concentration and with a single replicate culture at 24+24 hours (-S-9) at 4417 μg enzyme concentrate dry matter, but as such, these small statistical increases were not considered of biological importance.
It was therefore concluded that Beta amylase, batch PPY36295 did not induce biologically relevant increases in micronuclei in cultured human peripheral blood lymphocytes under the experimental conditions employed for this study.
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