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EC number: 293-693-6 | CAS number: 91081-84-4 By-product, containing primarily carbohydrates, produced by an ethanolic extraction of defatted soybean meal.
- 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 gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 04 October 2017 to 29 October 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
- Version / remarks:
- 24 November 2000
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Soybean meal, protein extn. residue
- EC Number:
- 293-693-6
- EC Name:
- Soybean meal, protein extn. residue
- Cas Number:
- 91081-84-4
- Molecular formula:
- UVCB substance
- IUPAC Name:
- Not determined
- Test material form:
- solid: particulate/powder
- Details on test material:
- - Physical state/Appearance: Beige-yellow/tan powder
- Storage Conditions: Room temperature in the dark
Constituent 1
Method
- Target gene:
- - Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: The bacteria used in the test were obtained from: University of California, Berkeley, on culture discs, on 04 August 1995 and British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.
- Suitability of cells: All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA 98 and TA 100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). The E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).
- In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Top agar was prepared using 0.6 % Bacto agar and 0.5 % sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd.
- Properly maintained: Yes
- All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- EXPERIMENT 1 - Plate Incorporation Method:
- The maximum concentration was 5 000 μg/plate (the maximum recommended dose level).
- Eight concentrations of the test material (1.5, 5, 15, 50, 150, 500, 1 500 and 5 000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
EXPERIMENT 2 - Pre-Incubation Method:
- The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1 500, 5 000 μg/plate.
- Six test material dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test material following the change in test methodology from plate incorporation to pre-incubation. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In solubility checks performed in–house, the test material was noted as insoluble in sterile distilled water, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL but was fully soluble in dimethyl sulphoxide at 50 mg/mL. Dimethyl sulphoxide was, therefore, selected as the vehicle.
- The test material was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 30 minutes at 40 °C. No correction was required for purity allowance. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10^-4 microns. All formulations were used within four hours of preparation and were assumed to be stable for this period.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- benzo(a)pyrene
- other: 4-Nitroquinoline-1-oxide (4NQO): 0.2 μg/plate for TA98 and 2-Aminoanthracene (2AA): 1 μg/plate for TA100, 2 μg/plate for TA1535 and TA1537 and 10 μg/plate for WP2uvrA
- Details on test system and experimental conditions:
- EXPERIMENT 1: PLATE INCORPORATION METHOD
- Eight concentrations of the test material (1.5, 5, 15, 50, 150, 500, 1 500 and 5 000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
- 0.1 mL of the appropriate concentration of test material, solvent vehicle or appropriate positive control was added together with 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer to 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test material, appropriate positive, vehicle and negative controls, and each bacterial strain, were assayed using triplicate plates.
- The procedure with metabolic activation was the same as described previously except that following the addition of the test material formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.
- All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). A single manual count was performed due to colonies spreading, thus distorting the actual plate count.
EXPERIMENT 2: PRE-INCUBATION METHOD
- The dose range used for Experiment 2 was 50, 150, 500, 1 500, 5 000 μg/plate.
- 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test material formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
- The procedure with metabolic activation was the same as described previously except that following the addition of the test material formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.
- All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.
NUMBER OF REPLICATIONS: 3 - Evaluation criteria:
- ACCEPTABILITY CRITERIA
The reverse mutation assay may be considered valid if the following criteria are met:
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.
- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are: TA 1535: 7 to 40, TA 100: 60 to 200, TA 1537: 2 to 30, TA 98: 8 to 60 and WP2uvrA: 10 to 60.
- All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.
- Diagnostic mutagens (positive controls) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
- There should be a minimum of four non-toxic test material dose levels.
- There should be no evidence of excessive contamination.
EVALUATION CRITERIA
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
- A dose-related increase in mutant frequency over the dose range tested.
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS.
- Fold increase greater than two times the concurrent solvent control (three times for TA 1535 and TA 1537) for any tester strain.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test material activity. Results of this type will be reported as equivocal. - Statistics:
- Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium, other: TA 1535, 1537, 98 and 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A fine test material precipitate was observed at 5 000 μg/plate, this observation did not prevent the scoring of revertant colonies.
- Acceptable cells for analysis:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test material formulation was also shown to be sterile.
CONTROL DATA
- Positive control: All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation.
- Negative (solvent/vehicle) control: Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
RESULTS
- The maximum dose level of the test material in the first experiment was selected as the maximum recommended dose level of 5 000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). Consequently, the same maximum dose level was used as the maximum dose in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method).
- There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).
Applicant's summary and conclusion
- Conclusions:
- Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.
- Executive summary:
The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471, EU Method B13/14, EPA OCSPP 870.5100 and the Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries, under GLP conditions using the bacterial reverse mutation assay.
Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2uvrA were treated with the test material using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5 000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5 000 μg/plate. Six test material concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test material following the change in test methodology.
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The maximum dose level of the test material in the first experiment was selected as the maximum recommended dose level of 5 000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). Consequently, the same maximum dose level was used as the maximum dose in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method).
A fine test material precipitate was observed at 5 000 μg/plate, this observation did not prevent the scoring of revertant colonies.
There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).
Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.
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