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EC number: 305-230-8 | CAS number: 94350-12-6
- 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
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- 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
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- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
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- Endpoint summary
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- 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
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- Additional ecotoxological information
- Toxicological Summary
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- Acute Toxicity
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- Exposure related observations in humans
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- Additional toxicological data
![](https://echa.europa.eu/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/print_toxicological-information.png)
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Based on the three in vitro key studies (Orovecz, Klimisch 1, GLP, 2018, OECD TG 471; Chevallier, Klimisch 1, GLP, 2018, OECD 487 ; Sire, Klimisch 1, GLP, 2018, OECD TG 490), the test substance Saccharomyces Cerevisiae, lysate did not induce mutagenicity and clastogenicity effects on mammalian cells or bacteria strain. Hence, according to CLP criteria, the test substance Saccharomyces Cerevisiae, lysate was not considered as mutagenic substance in vitro.
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 6 October 2017 To 25 April 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- 29 July 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor, batch no. AC17F00560
- Expiration date of the lot/batch: February 2019
- Purity test date: 30 June 2017
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from the light
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: precipitates were in solution at higher than or equal to 125 µg/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Since the stock formulation used in the first experiment was heated at 60°C for 10 minutes whereas heating was not recommended, the corresponding results were not retained for the interpretation and are presented for information purpose only. A second cytogenetic experiment was then undertaken using an unheated formulation.
- Final dilution of a dissolved solid, stock liquid or gel: The test item was suspended in the vehicle at the concentrations of: 10 mg/mL for the preliminary cytotoxicity test, 5 mg/mL for the cytogenetic experiments.
- Final preparation of a solid: not applicable
FORM AS APPLIED IN THE TEST (if different from that of starting material)
In solution with water - Target gene:
- micronuclei
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: ATCC by the intermediate of Biovalley
- Suitability of cells: recommanded by international regulations
- Cell cycle length, doubling time or proliferation index: average cell cycle time : 10-12 hours
- Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: not applicable
- Methods for maintenance in cell culture if applicable: Cell cultures were grown at 37°C in a humidified atmosphere of 5% CO2/95% air in culture medium. The culture medium was RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL),streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL). This medium was supplemented by heat-inactivated horse serum at 10% (v/v).
- Modal number of chromosomes: 40
- Normal (negative control) cell cycle time: 10-12 hours
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640, humidified atmosphere, at 37°C, 5%CO2/95% air
- Properly maintained: not specified
- Periodically checked for Mycoplasma contamination: not specified
- Periodically checked for karyotype stability: not specified
- Periodically 'cleansed' against high spontaneous background: not specified - Additional strain / cell type characteristics:
- other: TK+/-
- Metabolic activation:
- with and without
- Metabolic activation system:
- The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial fraction (S9 fraction) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by intraperitoneal route.
- Test concentrations with justification for top dose:
- Based on available solubility data, the highest achievable dose level to be used in the preliminary cytotoxicity test was 500 µg/mL, dose level limited by the solubility of the test item in the vehicle and by the treatment volume. Thus, using a test item stock formulation at the concentration of 10 mg/mL in the vehicle (i.e. water for injections) and a maximum treatment volume of 5% (v/v) in the culture medium, the dose levels selected for the treatment of the preliminary test were: 1, 10, 50, 100, 250 and 500 µg/mL.
At the highest tested dose level of 500 µg/mL, the pH of the culture medium was approximately 7.4 and the osmolality was 291 mOsm/kg H2O (both as the vehicle control). Therefore, none of the selected dose levels was considered to produce extreme culture conditions and the dose level of 500 µg/mL could be selected as the highest dose level for the main experiment. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used:water
- Justification for choice of solvent/vehicle: According to available solubility data, the vehicle used for the preparation of test item dose formulations and the treatment of vehicle control cultures was water for injections. - Untreated negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: colchicine, without S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 3x10E5 cells/mL
DURATION
- Preincubation period: not applicable
- Exposure duration: 3 hours (with and without S9) and 24 hours (without S9)
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours after treatment or 27 hours after treatment (for 3 hours treatment period)
SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not used
STAIN (for cytogenetic assays): 5% Giemsa
NUMBER OF REPLICATIONS: duplicates were used for each condition
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
After the final cell counting, the cells were washed with culture medium containing 10% inactivated horse serum and 1% pluronic acid. The cells were suspended in 49.5% culture medium containing 10% inactivated horse serum, 50% PBS and 0.5% pluronic acid, before being fixed.
Following the fixation, the cells were kept at 4°C for at least an overnight period.
Depending on the observation at the end of the recovery period (presence or absence of precipitate and/or cytotoxicity), three dose levels of the test item-treated cultures were selected for spreading on slides. Cells were dropped onto clean glass slides. The slides were air-dried before being stained for approximately 15 min in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above.
NUMBER OF CELLS EVALUATED: 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose).
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Analysis was performed under a microscope (1000 x magnification), on the basis of the recommendations of Miller et al. (1995) (e), according to the following criteria:
-micronuclei should be clearly surrounded by a nuclear membrane,
- the micronucleus area should be less than one-third of the area of the main nucleus,
- non-refractility of the micronuclei,
- micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
- micronuclei should be located within the cytoplasma of the cell,
- only mononucleated cells with a number of micronuclei, 5 should be scored to exclude apoptosis and nuclear fragmentation.
Number of cells with micronuclei and number of micronuclei per cell were given separately for each treated and control culture.
DETERMINATION OF CYTOTOXICITY
- Method: Population Doubling
- Any supplementary information relevant to cytotoxicity: For each culture, the Population Doubling (PD) was calculated and used relative to that of the vehicle control. The population doubling is the log of the ratio of the final count at the time of harvesting (N) to the starting count (N0), divided by the log of 2.
PD = [log (N/N0)]/log 2.
Mean PD (%) = (Mean PD treated / Mean PD vehicle control) x 100
The cytotoxicity induced by a treatment was evaluated by the decrease in the PD, when compared to the vehicle control (Mean % PD of the vehicle control set to 100%).
Decrease in PD (%) = 100 - Mean PD as % of control
OTHER EXAMINATIONS:
- Determination of polyploidy: notspecified
- Determination of endoreplication: not specified
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not specified - Evaluation criteria:
- Acceptance criteria
Each main experiment was considered valid if the following criteria were met:
-the mean PD of the vehicle control had to be ≥ 1 (indicating that cells have undergone mitosis),
- the mean frequency of micronucleated cells in the vehicle control should be consistent with (but not necessary within) control historical data of the Laboratory. In any case, this frequency should be ≤ 5‰,
-a statistically significant increase in the frequency of micronucleated cells had to be obtained in the positive controls over the background frequency of the vehicle control cultures. - Statistics:
- For each condition of the cytogenetic experiment, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures.
This comparison was performed using the 2 test, unless treated culture data are lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance. This statistical analysis was performed using a validated Excel sheet.
To assess the dose-response trend, a linear regression was performed between the frequencies of micronucleated cells and the dose levels. This statistical analysis was performed using SAS Enterprise Guide software. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
At the highest tested dose level of 500 µg/mL, the pH of the culture medium was approximately 7.4 and the osmolality was 291 mOsm/kg H2O (both as the vehicle control). Therefore, none of the selected dose levels was considered to produce extreme culture conditions and the dose level of 500 µg/mL could be selected as the highest dose level for the main experiment.
At the end of both 3- and 24-hour treatment periods, a precipitate was observed in the culture medium at dose levels higher than or equal to 100 µg/mL.
No noteworthy cytotoxicity was observed at any dose levels, in any conditions, as shown by the absence of notable decrease in the PD relative to the corresponding vehicle control.
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: treated 83 to 160 x10E4 cells/mL ; control : 67 to 130 x10E4 cells/mL
- Indication whether binucleate or mononucleate where appropriate: not specified
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
See tables in any other information on results incl. tables section
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used:Population Doubling - Conclusions:
- Under the experimental conditions of the study, the test item, Saccharomyces cerevisiae, lysate, did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.
- Executive summary:
The objective of this GLP-compliant study was to evaluate the potential of the test item, Saccharomyces cerevisiae, lysate, to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/- according to OECD TG 487 method.
After a preliminary cytotoxicity test, the potential of the test item Saccharomyces cerevisiae, lysate, suspended in water for injections, to induce an increase in the frequency of micronucleated cells was evaluated in a main cytogenetic experiment, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254 as follows:
-without S9 mix, 3 hours treatment and 24 hours recovery
- without S9 mix, 24 hours treatment, no recovery
-with S9 mix, 3 hours treatment, 24 hours recovery
Each treatment was coupled to an assessment of cytotoxicity at the same dose levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.
After the final cell counting, the cells were washed and fixed. Then, cells from three dose levels of the test item-treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared. For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.
Since the test item was found poorly soluble in the culture medium and non-cytotoxic in the preliminary test, the selection of the highest dose level for the main experiment was based on the presence of precipitate, according to the criteria specified in the international regulations.The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells.
With a treatment volume of 5 % (v/v) in culture medium, the dose levels selected for the treatment were: 7.81, 15.6, 31.3, 62.5, 125 and 250 µg/mL for the 3-hour treatments with and without S9 mix, as well as for the 24-hour treatment without S9 mix. At the end of both 3- and 24-hour treatment periods, a precipitate was observed in the culture medium at dose levels >= 125 µg/mL. No noteworthy cytotoxicity was induced at any dose levels, either following the 3-hour treatments with and without S9 mix or the 24-hour treatment without S9 mix, as shown by the absence of notable decrease in the PD relative to the corresponding vehicle control.
For the three experimental conditions, the dose levels selected for the micronucleus analysis were: 31.3, 62.5 and 125 µg/mL, the latter being the lowest dose level showing precipitate in the culture medium at the end of the treatment periods. Following the 3-hour treatments with and without S9 mix or the 24-hour treatment without S9 mix, neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the analyzed dose levels relative to the corresponding vehicle control.
Under the experimental conditions of the study, the test item, Saccharomyces cerevisiae, lysate, did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 14 August 2017 to 1st February
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AC17F00560
- Expiration date of the lot/batch: February 2019
- Purity test date: 30 June 2017
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25°CC, ≤70 RH%**)
- Solubility and stability of the test substance in the solvent/vehicle: the test item was soluble in distilled water (see attached table "The solubility of the test item in distilled water" in "Any information on material and method including table" section )
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The appropriate vehicle (solvent) and the behaviour of the test item formulations with the solution of top agar and phosphate buffer were examined in a preliminary compatibility test. Distilled water was used as solvent to prepare the stock solution of the test material. Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution (50 mg/mL) using the selected solvent and were used within 4 hours after preparation.
- Final dilution of a dissolved solid, stock liquid or gel: 50, 15.81, 5, 1.581, 0.5, 0.1581, 0.05 mg/mL respectively for 5000, 1581, 500, 158.1, 50, 15.81 and 5 µg/plate
- Final preparation of a solid: not applicable
FORM AS APPLIED IN THE TEST (if different from that of starting material) diluted in distilled water
- Target gene:
- histidine locus
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- other: See table 2 "Genotypes of the strains used for mutagenicity testing" in "additional information on material and methods includ. tables" section
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mitochrondrial section from Wistar rats liver induced with phenobarbital and Beta-Naphthoflavone at 80mg/kg/day by oral gavage during 3 days
- Test concentrations with justification for top dose:
- Based on the results of the preliminary tests, a 50 mg/mL stock solution was prepared in Distilled water. Maximum seven test concentrations were prepared by successive dilutions of the stock solution, to obtain lower doses. The maximum test concentration was 5000 μg test item/plate. Examined concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.
Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500,158.1, 50, 15.81 and 5 μg/plate. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using Distilled water, N,NDimethylformamide (DMF), Acetone and Dimethyl sulfoxide (DMSO). At the 100 mg/mL concentration, suspension with quick sedimentation was observed using DMSO, DMF and Acetone. At this concentration, suspension with slower sedimentation was observed using Distilled water. At the 50 mg/mL concentration, yellow homogeneous suspension with slower sedimentation was observed using Distilled water. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Distilled water
- True negative controls:
- yes
- Remarks:
- Untreated
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- methylmethanesulfonate
- other: 2-Aminoanthracene ; 4-nitro-1,2-phenylenediamine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) (initial); preincubation (confirmatory)
- Cell density at seeding (if applicable): not applicable
DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours
NUMBER OF REPLICATIONS: triplicates
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
NUMBER OF CELLS EVALUATED:
not applicable
DETERMINATION OF CYTOTOXICITY
Cytotoxicity was evaluated in initial mutation test by inhibition of background lawn of auxotrophic cells method. - Evaluation criteria:
- Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains. - Statistics:
- statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not valid
- Positive controls validity:
- not valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
Based on the solubility test, a 50 mg/mL stock solution was prepared in Distilled water. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item, in the absence and presence of metabolic activation.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: inhibition of background lawn of auxotrophic cells: No cytotoxicity was observed. - Conclusions:
- Under the experimental condition of the study, the registered substance saccharomyces cerevisiae, lysate did not induced reverse mutation when incubated with bacteria strain. Hence, the registered substance was considered to be not mutagenic for bacteria strain according to CLP criteria.
- Executive summary:
The purpose of this GLP compliant study was to evaluate the mutagenic potential of the test item by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli WP2 uvrA strain in the presence and absence of activated rat liver S9 fraction (performed according to OECD TG 471 method).
The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/-naphthoflavone- induced rats.
The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).
Based on the results of the Compatibility Test, the test item was dissolved in Distilled water at a concentration of 50 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate, in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50,
15.81 and 5 μg/plate.
Precipitate/slight precipitate was detected on the plates in the Preliminary experiment at Salmonella typhimurium TA98 strain and in the Confirmatory Mutation Test in all examined strains with and without metabolic activation at higher concentrations.
Inhibitory, cytotoxic effect of the test item was not detected in the Initial Mutation Test and Confirmatory Mutation Test.
In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect.
The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.
In conclusion, the test item Saccharomyces cerevisiae, lysate has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 2 November 2017 to 14 December 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- adopted on 29 July 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
supplied by the sponsor, batch no. AC17F00560
- Expiration date of the lot/batch: February 2019
- Purity test date: 30 June 2017
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from humidity (kept in its original packaging)
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: precipitate was observed in the culture medium at dose levels >= 55.6 µg/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was suspended in the vehicle.
- Final dilution of a dissolved solid, stock liquid or gel: -10 mg/mL for the preliminary cytotoxicity test; -2 mg/mL for the first mutagenicity experiment.
- Final preparation of a solid: The stock formulation was sonicated for 10 minutes and then magnetically stirred for at least 15 minutes at room temperature before use. The stock formulations and their dilutions were prepared within one hour before use, and then kept under magnetic stirring, at room temperature, until the end of use.
FORM AS APPLIED IN THE TEST (if different from that of starting material) in solution with water - Target gene:
- Thymidine Kinase Locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: ATCC (USA) through Biovalley (France)
- Suitability of cells: cell line recommended by international regulations for use in the in vitro mammalian cell gene mutation test
- Cell cycle length, doubling time or proliferation index: average cell cycle time : 10-12 hours
- Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: not applicable
- Methods for maintenance in cell culture if applicable: The cells were stored in a cryoprotective medium [10% horse serum and 10% dimethylsulfoxide (DMSO)] at -80°C. 5% CO2 humidified incubator. The cells were maintained in flasks as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 10% v/v, in a 37°C, 5% CO2 humidified incubator.
- Modal number of chromosomes: 40
- Normal (negative control) cell cycle time: average cell cylcle time 10-12 hours
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The cells were maintained in flasks as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 10% v/v, in a 37°C, 5% CO2 humidified incubator.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability:not specified
- Periodically 'cleansed' against high spontaneous background:not specified - Additional strain / cell type characteristics:
- other: TK +/-
- Metabolic activation:
- with and without
- Metabolic activation system:
- The metabolic activation system used in this study, S9 mix, consists of induced enzymatic systems contained in rat liver microsomal fraction (S9 fraction), obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route
- Test concentrations with justification for top dose:
- Preliminary cytotoxicity test : Based on available solubility data, the highest selected dose level was 500 µg/mL since it was expected to produce precipitation in the culture medium. This dose level was obtained using a test item concentration of 10 mg/mL and a treatment volume of 5% (v/v) in the culture medium (i.e. 1000 µL/20 mL culture medium for the 3-hour treatments and 2500 µL/50 mL culture medium for the 24 hour treatment).
The selected dose levels were 2.06, 6.17, 18.5, 55.6, 166.7 and 500 µg/mL.
Main experiments : Since the test item was found non-cytotoxic but poorly soluble in the culture medium, the highest dose level selection for the main experiment was based on the level of precipitate, according to the criteria specified in the international guidelines.
The selected dose levels were 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL for each experiment with or without S9 mix. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: according to available solubility data - Untreated negative controls:
- yes
- Remarks:
- vahicle control : water
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 2x 10E5 cells/mL (for mutagenicity experiment) and were seed in order to average of 1.6 cells per well
DURATION
- Preincubation period: cells were cutured for 2 day before mutagenicity experiment
- Exposure duration: 3 hours and 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days and 3 hours (3 hours exposure period) or 15 days (24 hours exposure period)
SELECTION AGENT (mutation assays): Trifluorothymidine
NUMBER OF REPLICATIONS: duplicates were used per condition
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable
NUMBER OF CELLS EVALUATED: mutant colony were counted
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): not applicable
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: not applicable
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity:
For the evaluation of cytotoxicity, the following parameters were determined: Daily cell growth on Day 1 (or 2)= cell concentration on Day 1 (or 2)/ adjusted concentration on Day 0 (or 1) 2 x 10E5
Cell growth during the 24-hour treatment = post-treatment cell concentration / seeded cell concentration (i.e. = 2 x 10E5 cells/mL)
Suspension growth (SG) for 3-hour treatment = daily growth on Day 1 x daily growth on Day 2
Suspension growth (SG) for 24-hour treatment =cell growth during the 24-hour treatment x daily growth on Day 1 x daily growth on Day 2
RSG (relative SG) = SG treated / SG vehicle control x 100
Adjusted RSG= cell count factor x [(SG treated / SG vehicle control) x 100]
Cell count factor = treated post-treatment cell count/ vehicle control post-treatment cell count
Adjusted relative total growth = Adj. RSG x RCE2 / 100
RCE2 = cloning efficiency treated / cloning efficiency vehicle control x 100
The relative Mutant Frequency (MF) and the induced mutation frequency (IMF) were calculated as follows:
MF = CEmutant x 10E6 / CE2
IMF = MF treated – MF vehicle control
OTHER EXAMINATIONS:
- the clones were counted using a magnifying glass, differentiating small and large colonies:
-size of small colonies: < 25% of the diameter of the well,
-size of large colonies: > 25% of the diameter of the well. - Evaluation criteria:
- IWGT recommendations were followed for the determination of a positive result, which should fulfill the following criteria:
- at least at one dose level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the global evaluation factor (GEF) of 126 x 10-6,
- a dose-response relationship is demonstrated by a statistically significant trend test.
Unless an effect is considered as clearly positive, the reproducibility of a positive effect should be confirmed.
Noteworthy increases in the mutation frequency observed only at high levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity at dose levels with Adj. RTG between 10 and 20%, are not considered as positive results.
A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20% if (g):
- there is at least one negative data point between 20 and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20% Adj. RTG,
- there is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also a negative data point between 10 and 1% Adj. RTG. - Statistics:
- To assess the dose-response relationship, a linear regression was performed between dose levels and individual mutation frequencies obtained at dose levels showing a mean Adj. RTG ≥ 10%
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
At the end of the treatment periods (3- or 24-hour treatment), a precipitate was observed in the culture medium at dose levels ≥ 55.6 µg/mL.
No cytotoxicity was induced at any of the tested dose levels, either with or without S9 mix (3- or 24-hour treatment), as shown by the absence of noteworthy decrease in the Adj. RTG.
RESULTS OF THE MAIL TEST:
1) Without S9 mix
- Cytotoxicity: Following either the 3- or 24-hour treatment, no cytotoxicity was observed at any of the tested dose levels, as shown by the absence of any noteworthy decrease in the Adj. RTG.
- Mutagenicity: Following either the 3- or 24-hour treatment, no increase in the mutation frequency exceeding the GEF was observed at any of the tested dose levels relative to the corresponding vehicle control and no dose-response relationship was demonstrated by the linear regression. These results did not meet the criteria for a positive response (Tables 1 and 2 in "Any other information on results incl. Tables" section).
2) With S9 mix
1) Cytotoxicity: No cytotoxicity was observed at any of the tested dose levels, as shown by the absence of any noteworthy decrease in the Adj. RTG
2) Mutagenicity: Relative to the vehicle control, no increase in the mutation frequency exceeding the GEF was observed at any of the tested dose levels and no dose-response relationship was demonstrated by the linear regression. These results did not meet the criteria for a positive response (Table 3 in "Any other information on results incl. Tables" section).
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effect of the pH: 7.4
- Effects of osmolality: No effect on osmolality, Osmolality : 290-299 mOsm/kg H2O
- Evaporation from medium: not applicable
- Water solubility: soluble until 50µg/mL (main terst)
- Precipitation: A precipitate was noted in the culture medium at the end of the treatment periods at dose levels ≥ 50 µg/mL (Main Test).
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: 3 hours treatment without S9 mix MMS : 652-1426 ; mean : 1043±216 ; Lower CL 95% : 935 - Upper CL 95% : 1150
3 hours treatment without S9 mix MMS :401-1013 ; mean : 649±174 ; Lower CL 95% : 563 - Upper CL 95% : 735
CPA with S9 mix 3 hours treatment: 479 - 2269 ; mean : 1113±441 ; Lower CL 95% :938 - Upper CL 95% : 1287
- Negative (solvent/vehicle) historical control data: 3 hours treatment without S9 mix : 71-152 ; mean 109 ±22 ; Lower CL 95% :98 - Upper CL 95% : 120
24 hours treatment without S9 mix : 73 - 143; mean 105 ± 23 ; Lower CL 95% : 94 - Upper CL 95% : 117
3 hours treatment with S9 mix : 59-174 ; mean 110 ±29 ; Lower CL 95% : 99 - Upper CL 95% : 122 - Conclusions:
- Under the experimental conditions of this study, the test item, Saccharomyces cerevisiae, lysate, did notshow any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.
- Executive summary:
The objective of this GLP compliant study was to evaluate the potential of the test item, Saccharomyces cerevisiae, lysate, to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/- mouse lymphoma cells according to OECD TG 490 method.
Material and methods
After a preliminary cytotoxicity test, the test item Saccharomyces cerevisiae, lysate, suspended in water for injections, for was tested in a first mutagenicity experiment, using 3-hour treatments, with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. In a second mutagenicity experiment, the test item was tested at the test site using a 24-hour treatment without metabolic activation system. Cultures of 20 mL at 5 x 105 cells/mL (3-hour treatments) or cultures of 50 mL at 2 x 105 cells/mL (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a 37°C, 5% CO2 humidified incubator.
Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and cloning efficiency following the expression time (CE2). The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.
Results
The cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. For the positive control cultures, the increase in the mutation frequencies met the acceptance criteria. In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%. The study was therefore considered to be valid. Since the test item was found non-cytotoxic but poorly soluble in the culture medium during the preliminary test, the highest dose level selection for the main experiment was based on the level of precipitate, according to the criteria specified in the international guidelines. The selected dose levels were 3.13, 6.25, 12.5, 25, 50 and 100 μg/mL for each experiment with or without S9 mix.
A precipitate was noted in the culture medium at dose levels ≥ 50 μg/mL at the end of the 3-hour treatment period and at dose levels ≥ 6.25 μg/mL at the end of the 24-hour treatment period.
No cytotoxicity was observed at any dose levels in any test conditions, as shown by the absence of any noteworthy decrease in the Adj. RTG.
Following the 3-hour treatments with and without S9 mix or the 24-hour treatment without S9 mix, no increase in the mutation frequency exceeding the GEF was observed at any of the tested dose levels relative to the corresponding vehicle control. Moreover, no dose-response relationship was demonstrated by the linear regression in any test conditions. These results did not meet the criteria for a positive response.
Conclusion
Under the experimental conditions of this study, the test item, Saccharomyces cerevisiae, lysate, did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.
Referenceopen allclose all
Table 1. Second experiment without S9 mix, 3-h treatment + 24-h recovery: cytotoxicity
Treatment |
Cell concentration used for treatment ( x 104cells/mL) |
Culture |
Post-treatment cell count (x 104cells/mL) |
Mean PD |
Mean PD as % of control |
Decrease in (%) |
PD |
|
Vehicle control |
30 |
C1 C2 |
92.0 100.0 |
1.7 |
100 |
|
||
Test item (µg/mL) |
|
|
|
|
|
|
|
|
7.81 |
|
30 |
C1 C2 |
87.0 100.0 |
1.6 |
98 |
2 |
|
15.6 |
|
30 |
C1 C2 |
100.0 102.5 |
1.8 |
105 |
none |
|
31.3 |
|
30 |
C1 C2 |
83.5 105.5 |
1.7 |
99 |
1 |
|
62.5 |
|
30 |
C1 C2 |
100.0 84.0 |
1.6 |
96 |
4 |
|
125 |
P |
30 |
C1 C2 |
113.0 110.0 |
1.9 |
113 |
none |
|
250 |
P |
30 |
C1 C2 |
105.0 102.0 |
1.8 |
106 |
none |
|
Positive controls |
|
|
|
|
|
|
||
MMC ( 1 µg/mL ) |
30 |
C1 C2 |
38.0 38.3 |
0.3 |
21 |
79 |
||
COL ( 0.5 µg/mL) |
30 |
C1 C2 |
34.0 24.2 |
# |
# |
# |
PD: population doubling
Vehicle control: water for injections MMC:MitomycinC
COL:ColchicineC1:Culture1
C2:Culture2
#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment P: precipitate was noted in the culture medium at the end of treatment
Table 2 Second experiment without S9 mix, 3-h treatment + 24-h recovery: cytogenetic results
Treatment |
Mean PD as % of control |
Culture |
Number of cells analysed |
Number of cells with n micronuclei |
Total micronucleatedcells |
Frequency of micronucleatedcells (0/00) |
Ratio treated/control |
||||||||
n = 1 |
n = 2 |
n = 3 |
n = 4 |
n = 5 |
|||||||||||
per culture |
per dose |
||||||||||||||
Vehicle control |
100 |
C1 C2 |
1000 1000 |
1 1 |
0 0 |
0 0 |
0 0 |
0 0 |
1 1 |
2 |
1 |
|
|||
Test item (µg/mL) |
|
|
|
|
|
|
|
|
|
|
|
|
|
||
7.81 |
|
98 |
C1 C2 |
|
|
|
|
|
|
|
|
|
|
||
15.6 |
|
105 |
C1 C2 |
|
|
|
|
|
|
|
|
|
|
||
31.3 |
|
99 |
C1 C2 |
1000 1000 |
3 0 |
0 0 |
0 0 |
0 0 |
0 0 |
3 0 |
3 |
2 |
1.5 |
||
62.5 |
|
96 |
C1 C2 |
1000 1000 |
2 1 |
0 0 |
0 0 |
0 0 |
0 0 |
2 1 |
3 |
2 |
1.5 |
||
125 |
P |
113 |
C1 C2 |
1000 1000 |
3 0 |
0 0 |
0 0 |
0 0 |
0 0 |
3 0 |
3 |
2 |
1.5 |
||
250 |
P |
106 |
C1 C2 |
|
|
|
|
|
|
|
|
|
|
||
Positive controls |
|
|
|
|
|
|
|
|
|
|
|
|
|
||
MMC ( 1 µg/mL ) |
21 |
C1 C2 |
1000 1000 |
52 37 |
13 7 |
5 0 |
0 2 |
0 0 |
70 46 |
116 |
58 |
58.0 |
*** |
||
COL ( 0.5 µg/mL) |
# |
C1 C2 |
1000 1000 |
14 16 |
1 1 |
0 0 |
0 0 |
0 0 |
15 17 |
32 |
16 |
16.0 |
*** |
PD:populationdoubling Statistics:2 x 2 contingencytable:
Vehicle control: waterforinjections ***: p <0.001
MMC:MitomycinCCOL:ColchicineC1:Culture1
C2:Culture2
#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment P: precipitate was noted in the culture medium at the end of treatment
Table 3. Second experiment without S9 mix, 24-h treatment + 0-h recovery: cytotoxicity
Treatment |
Cell concentration used for treatment ( x 104cells/mL) |
Culture |
Post-treatment cell count (x 104cells/mL) |
Mean PD |
Mean PD as % of control |
Decrease in (%) |
PD |
|
Vehicle control |
30 |
C1 C2 |
130.0 113.0 |
2.0 |
100 |
|
||
Test item (µg/mL) |
|
|
|
|
|
|
|
|
7.81 |
|
30 |
C1 C2 |
136.0 119.0 |
2.1 |
103 |
none |
|
15.6 |
|
30 |
C1 C2 |
138.0 128.0 |
2.1 |
106 |
none |
|
31.3 |
|
30 |
C1 C2 |
160.0 134.0 |
2.3 |
114 |
none |
|
62.5 |
|
30 |
C1 C2 |
120.0 135.0 |
2.1 |
103 |
none |
|
125 |
P |
30 |
C1 C2 |
136.0 132.0 |
2.2 |
107 |
none |
|
250 |
P |
30 |
C1 C2 |
133.0 133.0 |
2.1 |
106 |
none |
|
Positive controls |
|
|
|
|
|
|
||
MMC (1 µg/mL ) |
30 |
C1 C2 |
50.7 45.0 |
0.7 |
33 |
67 |
||
COL (0.5 µg/mL) |
30 |
C1 C2 |
28.8 29.5 |
# |
# |
# |
PD: population doubling
Vehicle control: water for injections MMC:MitomycinC
COL:ColchicineC1:Culture1
C2:Culture2
#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment P: precipitate was noted in the culture medium at the end of treatment
Table 4. Second experiment without S9 mix, 24-h treatment + 0-h recovery: cytogenetic results
Treatment |
Mean PD as % of control |
Culture |
Number of cellsanalysed |
Number of cells with n micronuclei |
Total micronucleatedcells |
Frequency of micronucleatedcells (0/00) |
Ratio treated/control |
||||||||
n = 1 |
n = 2 |
n = 3 |
n = 4 |
n = 5 |
|||||||||||
per culture |
per dose |
||||||||||||||
Vehicle control |
100 |
C1 C2 |
1000 1000 |
0 0 |
0 0 |
0 0 |
0 0 |
0 0 |
0 0 |
0 |
0 |
a) |
|||
Test item (µg/mL) |
|
|
|
|
|
|
|
|
|
|
|
|
|
||
7.81 |
|
103 |
C1 C2 |
|
|
|
|
|
|
|
|
|
|
||
15.6 |
|
106 |
C1 C2 |
|
|
|
|
|
|
|
|
|
|
||
31.3 |
|
114 |
C1 C2 |
1000 1000 |
1 1 |
0 0 |
0 0 |
0 0 |
0 0 |
1 1 |
2 |
1 |
2.0 |
||
62.5 |
|
103 |
C1 C2 |
1000 1000 |
2 0 |
0 0 |
0 0 |
0 0 |
1 0 |
3 0 |
3 |
2 |
3.0 |
||
125 |
P |
107 |
C1 C2 |
1000 1000 |
3 1 |
0 0 |
0 0 |
0 0 |
0 0 |
3 1 |
4 |
2 |
4.0 |
||
250 |
P |
106 |
C1 C2 |
|
|
|
|
|
|
|
|
|
|
||
Positive controls |
|
|
|
|
|
|
|
|
|
|
|
|
|
||
MMC (1 µg/mL ) |
33 |
C1 C2 |
1000 1000 |
12 23 |
1 3 |
0 0 |
0 0 |
0 0 |
13 26 |
39 |
20 |
39.0 |
*** |
||
COL (0.5 µg/mL) |
# |
C1 C2 |
1000 1000 |
8 3 |
2 0 |
0 0 |
0 0 |
0 0 |
10 3 |
13 |
7 |
13.0 |
*** |
PD:populationdoubling Statistics:2 x 2 contingencytable:
Vehicle control: waterforinjections ***: p <0.001
MMC:MitomycinCCOL:ColchicineC1:Culture1
C2:Culture2
#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment
a)therawdata obtainedforthevehiclecontrol is 0 but itwaschanged to 1 inorderto allow calculation of ratios P: precipitatewasnoted in the culture medium at the end oftreatment
Table 5. Second experiment with S9 mix, 3-h treatment + 24-h recovery: cytotoxicity
Treatment |
Cell concentration used for treatment ( x 104cells/mL) |
Culture |
Post-treatment cell count (x 104cells/mL) |
Mean PD |
Mean PD as % of control |
Decrease in (%) |
PD |
|
Vehicle control |
30 |
C1 C2 |
116.0 100.0 |
1.8 |
100 |
|
||
Test item (µg/mL) |
|
|
|
|
|
|
|
|
7.81 |
|
30 |
C1 C2 |
110.0 111.0 |
1.9 |
102 |
none |
|
15.6 |
|
30 |
C1 C2 |
123.0 109.0 |
2.0 |
106 |
none |
|
31.3 |
|
30 |
C1 C2 |
129.0 122.0 |
2.1 |
112 |
none |
|
62.5 |
|
30 |
C1 C2 |
120.0 100.0 |
1.9 |
101 |
none |
|
125 |
P |
30 |
C1 C2 |
113.0 111.0 |
1.9 |
103 |
none |
|
250 |
P |
30 |
C1 C2 |
106.0 102.0 |
1.8 |
97 |
3 |
|
Positive controls |
|
|
|
|
|
|
||
CPA (6 µg/mL ) |
30 |
C1 C2 |
69.5 64.0 |
1.2 |
62 |
38 |
PD: population doubling
Vehicle control: water for injections CPA: Cyclophosphamide
C1:Culture1
C2:Culture2
P: precipitate was noted in the culture medium at the end of treatment
Table 6. Second experiment with S9 mix, 3-h treatment + 24-h recovery: cytogenetic results
Treatment |
Mean PD as % of control |
Culture |
Number of cells analysed |
Number of cells with n micronuclei |
Totalmicronucleatedcells |
Frequency ofmicronucleatedcells (0/00) |
Ratio treated/control |
||||||||
n = 1 |
n = 2 |
n = 3 |
n = 4 |
n = 5 |
|||||||||||
per culture |
per dose |
||||||||||||||
Vehicle control |
100 |
C1 C2 |
1000 1000 |
1 1 |
0 0 |
0 0 |
0 0 |
0 0 |
1 1 |
2 |
1 |
|
|||
Test item (µg/mL) |
|
|
|
|
|
|
|
|
|
|
|
|
|
||
7.81 |
|
102 |
C1 C2 |
|
|
|
|
|
|
|
|
|
|
||
15.6 |
|
106 |
C1 C2 |
|
|
|
|
|
|
|
|
|
|
||
31.3 |
|
112 |
C1 C2 |
1000 1000 |
2 3 |
0 0 |
0 0 |
0 0 |
0 0 |
2 3 |
5 |
3 |
2.5 |
||
62.5 |
|
101 |
C1 C2 |
1000 1000 |
2 1 |
0 0 |
0 0 |
0 0 |
0 0 |
2 1 |
3 |
2 |
1.5 |
||
125 |
P |
103 |
C1 C2 |
1000 1000 |
2 0 |
0 0 |
0 0 |
0 0 |
0 0 |
2 0 |
2 |
1 |
1.0 |
||
250 |
P |
97 |
C1 C2 |
|
|
|
|
|
|
|
|
|
|
||
Positive controls |
|
|
|
|
|
|
|
|
|
|
|
|
|
||
CPA (6 µg/mL ) |
62 |
C1 C2 |
1000 1000 |
41 33 |
1 2 |
0 0 |
0 0 |
0 0 |
42 35 |
77 |
39 |
38.5 |
*** |
PD:populationdoubling Statistics:2 x 2 contingencytable:
Vehicle control: waterforinjections ***: p <0.001
CPA:CyclophosphamideC1:Culture1
C2:Culture2
P: precipitate was noted in the culture medium at the end of treatment
Table 7: Historical data of experiments without S9
Parameter |
Frequency ofmicronucleatedcells in 1000 cells |
|
|
|||
Treatmentcontrol |
3hourstreatment+ 24hoursrecovery |
24hourstreatment+ 0hoursrecovery |
||||
Control items |
Vehicle(control) |
MMC (1µg/mL) |
COL (0.5 µg/mL) |
Vehicle(control) |
MMC (1µg/mL) |
COL (0.5 µg/mL) |
n |
50 |
50 |
50 |
28 |
28 |
28 |
mean |
1.7 |
133 |
22.9 |
2.1 |
46.4 |
34.5 |
SD |
1 |
67.5 |
13.3 |
1.1 |
19.8 |
17.5 |
LowerCL95% |
1.4 |
113.8 |
19.1 |
1.6 |
38.7 |
27.7 |
UpperCL 95% |
1.9 |
152.2 |
26.6 |
2.5 |
54.1 |
41.2 |
5th percentile |
0.5 |
23 |
7.5 |
0 |
11.5 |
14.5 |
Median |
1.5 |
138.3 |
20 |
2 |
49.5 |
27.0 |
95th percentile |
4 |
238.5 |
54.5 |
3.5 |
72.5 |
65.5 |
Min |
0 |
18 |
5.5 |
0 |
8 |
10.0 |
Max |
4.5 |
306 |
65 |
5 |
97.5 |
75.0 |
Table 8: Historical data of experiments with S9
Parameter |
Frequency of micronucleated cells in 1000 cells |
|||
Treatmentcontrol |
3hourstreatment+ 24hoursrecovery |
|||
Control items |
Vehiclecontrol |
CPA (6µg/mL) |
||
n |
67 |
67 |
||
mean |
1.5 |
101.9 |
||
SD |
0.9 |
49.3 |
||
LowerCL95% |
1.3 |
89.8 |
||
UpperCL 95% |
1.7 |
113.9 |
||
5th percentile |
0.5 |
26 |
||
Median |
1.5 |
105.5 |
||
95th percentile |
3.0 |
184.5 |
||
Min |
0 |
14 |
||
Max |
3.5 |
251 |
Table 3:Summary Table of the Initial Mutation Test
Concentrations (mg/plate) |
Mean values ofrevertants/ Mutation factor (MF) |
Salmonella typhimuriumtester strains |
Escherichia coli |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2uvrA |
|||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||
Untreated control |
Mean |
17.3 |
22.0 |
71.3 |
73.0 |
10.3 |
13.7 |
12.0 |
12.3 |
45.0 |
51.7 |
MF |
1.02 |
0.94 |
0.96 |
1.03 |
0.91 |
1.28 |
0.88 |
1.16 |
1.01 |
0.97 |
|
DMSO control |
Mean |
15.7 |
21.7 |
-- |
76.3 |
-- |
12.7 |
13.0 |
10.0 |
-- |
46.7 |
MF |
0.92 |
0.93 |
-- |
1.08 |
-- |
1.19 |
0.95 |
0.94 |
-- |
0.88 |
|
Distilled water control |
Mean |
-- |
-- |
73.3 |
-- |
11.7 |
-- |
-- |
-- |
47.7 |
-- |
MF |
-- |
-- |
0.99 |
-- |
1.03 |
-- |
-- |
-- |
1.07 |
-- |
|
Distilled water control 100µl |
Mean |
17.0 |
23.3 |
74.0 |
71.0 |
11.3 |
10.7 |
13.7 |
10.7 |
44.7 |
53.3 |
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
|
5000 |
Mean |
13.3 |
22.0 |
62.7 |
78.0 |
12.7 |
11.3 |
13.3 |
11.7 |
64.7 |
69.0 |
MF |
0.78 |
0.94 |
0.85 |
1.10 |
1.12 |
1.06 |
0.98 |
1.09 |
1.45 |
1.29 |
|
1581 |
Mean |
21.0 |
22.7 |
68.3 |
82.3 |
12.3 |
12.0 |
12.0 |
14.0 |
65.3 |
66.3 |
MF |
1.24 |
0.97 |
0.92 |
1.16 |
1.09 |
1.13 |
0.88 |
1.31 |
1.46 |
1.24 |
|
500 |
Mean |
15.3 |
20.3 |
69.0 |
78.7 |
13.0 |
11.7 |
12.0 |
12.0 |
63.0 |
64.7 |
MF |
0.90 |
0.87 |
0.93 |
1.11 |
1.15 |
1.09 |
0.88 |
1.13 |
1.41 |
1.21 |
|
158.1 |
Mean |
16.3 |
22.0 |
73.0 |
80.7 |
14.7 |
11.7 |
10.7 |
10.3 |
64.7 |
66.3 |
MF |
0.96 |
0.94 |
0.99 |
1.14 |
1.29 |
1.09 |
0.78 |
0.97 |
1.45 |
1.24 |
|
50 |
Mean |
18.3 |
21.3 |
79.3 |
84.7 |
14.0 |
9.3 |
11.3 |
15.0 |
58.0 |
67.3 |
MF |
1.08 |
0.91 |
1.07 |
1.19 |
1.24 |
0.88 |
0.83 |
1.41 |
1.30 |
1.26 |
|
15.81 |
Mean |
16.0 |
25.7 |
81.7 |
81.7 |
13.3 |
11.0 |
10.7 |
12.0 |
57.7 |
66.3 |
MF |
0.94 |
1.10 |
1.10 |
1.15 |
1.18 |
1.03 |
0.78 |
1.13 |
1.29 |
1.24 |
|
NPD (4mg) |
Mean |
404.0 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
MF |
25.79 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
2AA (2mg) |
Mean |
-- |
2474.7 |
-- |
2418.7 |
-- |
206.3 |
- |
215.3 |
- |
- |
MF |
-- |
114.22 |
-- |
31.69 |
-- |
16.29 |
- |
21.53 |
- |
- |
|
2AA (50mg) |
Mean |
-- |
|
-- |
-- |
-- |
-- |
-- |
-- |
-- |
257.3 |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
5.51 |
|
SAZ (2mg) |
Mean |
-- |
-- |
1142.0 |
-- |
1026.7 |
-- |
-- |
-- |
-- |
-- |
MF |
-- |
-- |
15.57 |
- |
88.00 |
-- |
-- |
-- |
-- |
-- |
|
9AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
421.3 |
-- |
-- |
-- |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
32.41 |
-- |
-- |
-- |
|
MMS (2mL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
1008.7 |
-- |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
21.16 |
-- |
Table 4:Summary Table of the Confirmatory Mutation Test
Concentrations (mg/plate) |
Mean valuesofrevertants/ Mutation factor(MF) |
Salmonella typhimuriumtester strains |
Escherichia coli |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2uvrA |
|||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||
Untreated control |
Mean |
22.3 |
27.0 |
101.0 |
109.3 |
10.3 |
12.3 |
13.7 |
14.0 |
53.0 |
53.0 |
MF |
1.02 |
1.03 |
1.05 |
1.05 |
0.74 |
0.93 |
0.95 |
1.02 |
0.93 |
1.02 |
|
DMSO control |
Mean |
21.3 |
25.7 |
-- |
99.3 |
-- |
13.7 |
11.3 |
12.3 |
-- |
56.0 |
MF |
0.97 |
0.97 |
-- |
0.96 |
-- |
1.03 |
0.79 |
0.90 |
-- |
1.08 |
|
Distilled water control |
Mean |
-- |
-- |
102.3 |
-- |
13.0 |
-- |
-- |
-- |
53.0 |
-- |
MF |
-- |
-- |
1.06 |
-- |
0.93 |
-- |
-- |
-- |
0.93 |
-- |
|
Distilled water control 100µl |
Mean |
22.0 |
26.3 |
96.3 |
103.7 |
14.0 |
13.3 |
14.3 |
13.7 |
57.0 |
52.0 |
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
|
5000 |
Mean |
23.0 |
24.7 |
83.3 |
109.3 |
14.3 |
14.0 |
16.0 |
12.7 |
43.3 |
52.7 |
MF |
1.05 |
0.94 |
0.87 |
1.05 |
1.02 |
1.05 |
1.12 |
0.93 |
0.76 |
1.01 |
|
1581 |
Mean |
22.7 |
21.0 |
95.3 |
122.7 |
14.0 |
13.7 |
14.3 |
18.0 |
42.7 |
50.3 |
MF |
1.03 |
0.80 |
0.99 |
1.18 |
1.00 |
1.03 |
1.00 |
1.32 |
0.75 |
0.97 |
|
500 |
Mean |
23.3 |
28.3 |
110.3 |
109.0 |
15.3 |
15.0 |
15.0 |
13.3 |
45.7 |
48.0 |
MF |
1.06 |
1.08 |
1.15 |
1.05 |
1.10 |
1.13 |
1.05 |
0.98 |
0.80 |
0.92 |
|
158.1 |
Mean |
19.0 |
22.7 |
107.7 |
111.7 |
14.7 |
14.3 |
13.0 |
14.3 |
41.7 |
50.7 |
MF |
0.86 |
0.86 |
1.12 |
1.08 |
1.05 |
1.08 |
0.91 |
1.05 |
0.73 |
0.97 |
|
50 |
Mean |
22.3 |
25.0 |
109.3 |
112.0 |
15.3 |
13.3 |
12.7 |
14.3 |
47.0 |
51.0 |
MF |
1.02 |
0.95 |
1.13 |
1.08 |
1.10 |
1.00 |
0.88 |
1.05 |
0.82 |
0.98 |
|
15.81 |
Mean |
23.3 |
26.7 |
110.3 |
110.3 |
9.0 |
13.3 |
14.7 |
14.0 |
43.0 |
46.7 |
MF |
1.06 |
1.01 |
1.15 |
1.06 |
0.64 |
1.00 |
1.02 |
1.02 |
0.75 |
0.90 |
|
5 |
Mean |
17.7 |
27.3 |
102.3 |
116.0 |
15.0 |
14.7 |
13.7 |
17.0 |
44.3 |
47.0 |
MF |
0.80 |
1.04 |
1.06 |
1.12 |
1.07 |
1.10 |
0.95 |
1.24 |
0.78 |
0.90 |
|
NPD (4mg) |
Mean |
394.7 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
MF |
18.50 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
2AA (2mg) |
Mean |
-- |
2402.7 |
-- |
2421.3 |
-- |
209.7 |
-- |
204.0 |
-- |
-- |
MF |
-- |
93.61 |
-- |
24.38 |
-- |
15.34 |
-- |
16.54 |
-- |
-- |
|
2AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
265.0 |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
4.73 |
|
SAZ (2mg) |
Mean |
-- |
-- |
1177.3 |
-- |
1054.7 |
-- |
-- |
-- |
-- |
-- |
MF |
-- |
-- |
11.50 |
-- |
81.13 |
-- |
-- |
-- |
-- |
-- |
|
9AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
425.3 |
-- |
-- |
-- |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
37.53 |
-- |
-- |
-- |
|
MMS (2mL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
1048.0 |
-- |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
19.77 |
-- |
Table 5 Historical Control Data (Period of 2011-2016)
Untreated control data |
||||||||||
|
without metabolic activation (-S9 Mix) |
with metabolic activation (+S9 Mix) |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
22.7 |
103.6 |
11.8 |
7.2 |
33.4 |
29.7 |
111.7 |
11.5 |
8.9 |
39.1 |
St. dev. |
5.8 |
21.4 |
5.1 |
3.3 |
9.7 |
6.8 |
19.6 |
3.9 |
3.8 |
9.9 |
Range |
9-50 |
54-210 |
1-46 |
1-24 |
11-82 |
10-56 |
65-204 |
1-39 |
1-29 |
16-89 |
n |
1371 |
1357 |
1365 |
1371 |
1374 |
1377 |
1365 |
1373 |
1380 |
1371 |
DMSO control data |
||||||||||
|
without metabolic activation (-S9 Mix) |
with metabolic activation (+S9 Mix) |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
21.7 |
98.9 |
12.0 |
7.1 |
32.3 |
28.7 |
109.5 |
11.3 |
8.7 |
38.1 |
St. dev. |
5.7 |
20.7 |
5.0 |
3.3 |
9.6 |
7.0 |
20.7 |
3.8 |
3.7 |
9.7 |
Range |
6-55 |
40-217 |
1-43 |
1-25 |
7-81 |
11-67 |
53-229 |
2-33 |
1-29 |
9-85 |
n |
1482 |
1473 |
1479 |
1485 |
1482 |
1487 |
1476 |
1487 |
1491 |
1482 |
Distilled water control data |
||||||||||
|
without metabolic activation (-S9 Mix) |
with metabolic activation (+S9 Mix) |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
23.5 |
103.2 |
11.9 |
7.8 |
34.5 |
30.8 |
112.2 |
11.3 |
9.3 |
40.3 |
St. dev. |
6.0 |
22.4 |
4.9 |
3.4 |
9.8 |
7.1 |
21.8 |
3.7 |
3.7 |
10.0 |
Range |
11-45 |
45-215 |
2-47 |
2-24 |
12-84 |
10-53 |
64-222 |
3-39 |
1-24 |
13-91 |
n |
267 |
1359 |
1365 |
270 |
1392 |
267 |
1371 |
1380 |
267 |
1383 |
DMF control data |
||||||||||
|
without metabolic activation (-S9 Mix) |
with metabolic activation (+S9 Mix) |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
20.4 |
89.9 |
11.2 |
6.9 |
34.7 |
28.1 |
100.3 |
11.0 |
8.0 |
38.0 |
St. dev. |
5.6 |
17.8 |
4.7 |
3.1 |
12.3 |
7.0 |
19.2 |
3.6 |
3.1 |
10.2 |
Range |
8-38 |
54-152 |
1-34 |
1-19 |
16-99 |
13-49 |
60-156 |
3-21 |
1-23 |
17-76 |
n |
216 |
216 |
216 |
216 |
207 |
216 |
216 |
216 |
213 |
207 |
Acetone control data |
||||||||||
|
without metabolic activation (-S9 Mix) |
with metabolic activation (+S9 Mix) |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
22.6 |
98.1 |
12.1 |
7.4 |
35.0 |
29.1 |
108.1 |
11.1 |
8.6 |
40.5 |
St. dev. |
5.1 |
15.4 |
5.8 |
2.9 |
9.3 |
6.7 |
14.2 |
3.4 |
3.3 |
9.0 |
Range |
11-39 |
62-160 |
4-49 |
1-17 |
17-62 |
15-52 |
66-177 |
4-22 |
1-19 |
17-69 |
n |
278 |
279 |
279 |
279 |
276 |
279 |
279 |
282 |
279 |
279 |
Positive reference control data |
||||||||||
|
without metabolic activation (-S9 Mix) |
with metabolic activation (+S9 Mix) |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
357.2 |
1229.3 |
1169.8 |
454.1 |
1034.3 |
2410.2 |
2429.6 |
235.1 |
221.3 |
257.4 |
St. dev. |
113.8 |
207.5 |
204.2 |
169.7 |
141.7 |
317.7 |
291.6 |
135.9 |
56.2 |
113.4 |
Range |
152-2336 |
536-2120 |
208-2440 |
149-2104 |
488-1708 |
312-4918 |
1192-5240 |
101-2216 |
117-838 |
125-2512 |
n |
1371 |
1359 |
1365 |
1371 |
1377 |
1378 |
1365 |
1377 |
1380 |
1371 |
TA98:SalmonellatyphimuriumTA98, TA100:SalmonellatyphimuriumTA100, TA1535:SalmonellatyphimuriumTA1535, TA1537:
Salmonella typhimuriumTA1537, E. coli:Escherichia coliWP2uvrA;n: number of cases
Table1.Mutagenicity experiment without S9 mix, 3-hour treatment: mutagenicity results
Doses µg/mL |
Adj. RTG % |
CE2 |
Wells* |
MFx10-6 |
Ratio MF |
Mean values of C1and C2 |
|||||||||
|
|
Adj RTG% |
CE2 |
MFx10-6 |
IMF |
||||||||||
0 |
C1 |
100 |
0.64 |
EW |
79 |
78 |
82 |
83 |
138 |
1.0 |
|
|
|
|
|
LC |
9 |
8 |
10 |
7 |
72 |
1.0 |
|
|
|
|
|
||||
SC |
8 |
10 |
5 |
6 |
61 |
1.0 |
|
|
|
|
|
||||
C2 |
100 |
0.76 |
EW |
67 |
72 |
68 |
83 |
185 |
1.0 |
100 |
0.70 |
|
161 |
|
|
LC |
23 |
14 |
18 |
7 |
116 |
1.0 |
|
|
LC |
92 |
|
||||
SC |
7 |
10 |
11 |
6 |
61 |
1.0 |
|
|
SC |
61 |
|
||||
3.13 |
C1 |
127 |
0.68 |
EW |
72 |
76 |
86 |
70 |
173 |
1.3 |
|
|
|
|
|
LC |
14 |
14 |
6 |
12 |
94 |
1.3 |
|
|
|
|
|
||||
SC |
12 |
6 |
4 |
14 |
73 |
1.2 |
|
|
|
|
|
||||
C2 |
125 |
0.85 |
EW |
67 |
71 |
77 |
83 |
149 |
0.8 |
126 |
0.77 |
|
161 |
none |
|
LC |
17 |
17 |
12 |
9 |
91 |
0.8 |
|
|
LC |
92 |
none |
||||
SC |
12 |
8 |
8 |
4 |
51 |
0.8 |
|
|
SC |
62 |
1 |
||||
6.25 |
C1 |
152 |
1.20 |
EW |
66 |
80 |
67 |
75 |
120 |
0.9 |
|
|
|
|
|
LC |
16 |
10 |
20 |
13 |
69 |
1.0 |
|
|
|
|
|
||||
SC |
16 |
7 |
10 |
9 |
48 |
0.8 |
|
|
|
|
|
||||
C2 |
117 |
0.76 |
EW |
69 |
76 |
76 |
72 |
178 |
1.0 |
134 |
0.98 |
|
149 |
none |
|
LC |
15 |
15 |
15 |
17 |
116 |
1.0 |
|
|
LC |
93 |
none |
||||
SC |
12 |
5 |
5 |
7 |
52 |
0.8 |
|
|
SC |
50 |
none |
||||
12.5 |
C1 |
179 |
1.16 |
EW |
74 |
72 |
80 |
72 |
109 |
0.8 |
|
|
|
|
|
LC |
9 |
13 |
8 |
7 |
44 |
0.6 |
|
|
|
|
|
||||
SC |
13 |
11 |
9 |
7 |
47 |
0.8 |
|
|
|
|
|
||||
C2 |
74 |
0.66 |
EW |
73 |
67 |
87 |
84 |
160 |
0.9 |
126 |
0.91 |
|
135 |
none |
|
LC |
16 |
20 |
6 |
7 |
104 |
0.9 |
|
|
LC |
74 |
none |
||||
SC |
8 |
12 |
4 |
5 |
60 |
1.0 |
|
|
SC |
54 |
none |
||||
25 |
C1 |
178 |
1.00 |
EW |
78 |
75 |
69 |
81 |
119 |
0.9 |
|
|
|
|
|
LC |
15 |
14 |
15 |
6 |
70 |
1.0 |
|
|
|
|
|
||||
SC |
4 |
8 |
13 |
9 |
47 |
0.8 |
|
|
|
|
|
||||
C2 |
114 |
0.82 |
EW |
69 |
63 |
70 |
83 |
183 |
1.0 |
146 |
0.91 |
|
151 |
none |
|
LC |
16 |
25 |
16 |
8 |
114 |
1.0 |
|
|
LC |
74 |
none |
||||
SC |
12 |
9 |
10 |
5 |
60 |
1.0 |
|
|
SC |
54 |
none |
||||
50 |
C1 |
161 |
0.91 |
EW |
65 |
80 |
80 |
71 |
144 |
1.0 |
|
|
|
|
|
LC |
17 |
10 |
8 |
13 |
74 |
1.0 |
|
|
|
|
|
||||
SC |
16 |
7 |
8 |
12 |
65 |
1.1 |
|
|
|
|
|
||||
C2 |
94 |
0.71 |
EW |
74 |
13 |
72 |
75 |
351 |
1.9 |
127 |
0.81 |
|
247 |
86 |
|
LC |
12 |
75 |
11 |
13 |
241 |
2.1 |
|
|
LC |
158 |
63 |
||||
SC |
10 |
12 |
14 |
9 |
88 |
1.4 |
|
|
SC |
77 |
16 |
||||
100 |
C1 |
168 |
1.03 |
EW |
73 |
74 |
66 |
77 |
136 |
1.0 |
|
|
|
|
|
LC |
13 |
13 |
17 |
10 |
72 |
1.0 |
|
|
|
|
|
||||
SC |
11 |
10 |
13 |
9 |
58 |
0.9 |
|
|
|
|
|
||||
C2 |
112 |
0.91 |
EW |
56 |
55 |
75 |
82 |
198 |
1.1 |
140 |
0.97 |
|
167 |
6 |
|
LC |
35 |
25 |
13 |
8 |
131 |
1.1 |
|
|
LC |
101 |
7 |
||||
SC |
6 |
16 |
8 |
6 |
54 |
0.9 |
|
|
SC |
56 |
none |
||||
MMS 25 µg/mL |
C1 |
85 |
0.57 |
EW |
40 |
44 |
42 |
39 |
740 |
5.4 |
|
|
|
|
|
LC |
23 |
24 |
24 |
27 |
258 |
3.6 |
|
|
|
|
|
||||
SC |
37 |
34 |
39 |
40 |
434 |
7.1 |
|
|
|
|
|
||||
C2 |
28 |
0.36 |
EW |
44 |
42 |
43 |
48 |
1077 |
5.8 |
56 |
0.47 |
|
908 |
747 |
|
LC |
25 |
22 |
24 |
24 |
395 |
3.4 |
|
|
LC |
327 |
232 |
||||
SC |
31 |
36 |
30 |
24 |
526 |
8.6 |
|
|
SC |
480 |
419 |
Adj. RTG: adjusted relative total growth C1: culture 1
CE2: cloning efficiency C2: culture 2
*: empty wells are counted on a total number of 96wells 0: vehicle control (water for injections)
MF: mutation frequency MMS: methyl methane sulfonate
IMF: Induced Mutation Frequency
P: precipitate observed in the culture medium at the end of treatment
LC: wells with large colonies
SC: wells with small colonies
EW: empty wells
Table2.Mutagenicity experiment without S9 mix, 24-hour treatment: mutagenicity results
Doses µg/mL |
Adj. RTG % |
CE2 |
Wells* |
MFx10-6 |
Ratio MF |
Mean values of C1and C2 |
|||||||||
|
|
Adj RTG% |
CE2 |
MFx10-6 |
IMF |
||||||||||
0 |
C1 |
100 |
0.96 |
EW |
84 |
85 |
85 |
83 |
68 |
1.0 |
|
|
|
|
|
LC |
8 |
7 |
6 |
10 |
44 |
1.0 |
|
|
|
|
|
||||
SC |
4 |
4 |
5 |
3 |
22 |
1.0 |
|
|
|
|
|
||||
C2 |
100 |
1.12 |
EW |
88 |
84 |
85 |
81 |
57 |
1.0 |
100 |
1.04 |
|
62 |
|
|
LC |
4 |
5 |
5 |
12 |
31 |
1.0 |
|
|
LC |
37 |
|
||||
SC |
4 |
7 |
6 |
3 |
24 |
1.0 |
|
|
SC |
23 |
|
||||
3.13 |
C1 |
110 |
1.00 |
EW |
91 |
86 |
89 |
88 |
41 |
0.6 |
|
|
|
|
|
LC |
0 |
6 |
5 |
6 |
23 |
0.5 |
|
|
|
|
|
||||
SC |
5 |
4 |
2 |
2 8 |
17 |
0.8 |
|
|
|
|
|
||||
C2 |
109 |
0.98 |
EW |
86 |
80 |
87 |
91 |
56 |
1.0 |
110 |
0.99 |
|
48 |
none |
|
LC |
4 |
9 |
4 |
4 |
29 |
0.9 |
|
|
LC |
26 |
none |
||||
SC |
6 |
7 |
5 |
1 |
26 |
1.1 |
|
|
SC |
22 |
none |
||||
6.25 |
C1 |
125 |
1.23 |
EW |
81 |
87 |
79 |
82 |
63 |
0.9 |
|
|
|
|
|
LC |
10 |
3 |
11 |
11 |
39 |
0.9 |
|
|
|
|
|
||||
SC |
5 |
6 |
6 |
3 |
22 |
1.0 |
|
|
|
|
|
||||
C2 |
72 |
0.85 |
EW |
87 |
85 |
86 |
87 |
63 |
1.1 |
98 |
1.04 |
|
63 |
1 |
|
LC |
7 |
7 |
4 |
6 |
38 |
1.2 |
|
|
LC |
38 |
1 |
||||
SC |
2 |
4 |
6 |
3 |
23 |
1.0 |
|
|
SC |
23 |
none |
||||
12.5 |
C1 |
123 |
1.23 |
EW |
86 |
88 |
88 |
86 |
40 |
0.6 |
|
|
|
|
|
LC |
3 |
3 |
4 |
6 |
17 |
0.4 |
|
|
|
|
|
||||
SC |
7 |
5 |
4 |
4 |
22 |
1.0 |
|
|
|
|
|
||||
C2 |
114 |
1.01 |
EW |
83 |
91 |
83 |
89 |
51 |
0.9 |
118 |
1.12 |
|
46 |
none |
|
LC |
7 |
2 |
11 |
5 |
33 |
1.1 |
|
|
LC |
25 |
none |
||||
SC |
6 |
3 |
2 |
2 |
17 |
0.7 |
|
|
SC |
19 |
none |
||||
25 |
C1 |
111 |
1.03 |
EW |
88 |
89 |
88 |
82 |
49 |
0.7 |
|
|
|
|
|
LC |
4 |
4 |
5 |
7 |
26 |
0.6 |
|
|
|
|
|
||||
SC |
4 |
3 |
3 |
7 |
22 |
1.0 |
|
|
|
|
|
||||
C2 |
107 |
1.18 |
EW |
85 |
86 |
86 |
82 |
53 |
0.9 |
109 |
1.11 |
|
51 |
none |
|
LC |
9 |
9 |
3 |
10 |
36 |
1.1 |
|
|
LC |
31 |
none |
||||
SC |
2 |
1 |
7 |
4 |
16 |
0.7 |
|
|
SC |
19 |
none |
||||
50 |
C1 |
168 |
1.16 |
EW |
86 |
85 |
89 |
87 |
44 |
0.6 |
|
|
|
|
|
LC |
6 |
9 |
5 |
5 |
29 |
0.7 |
|
|
|
|
|
||||
SC |
4 |
2 |
2 |
4 |
14 |
0.6 |
|
|
|
|
|
||||
C2 |
123 |
1.18 |
EW |
89 |
83 |
85 |
80 |
55 |
1.0 |
145 |
1.17 |
|
49 |
none |
|
LC |
2 |
5 |
9 |
6 |
25 |
0.8 |
|
|
LC |
27 |
none |
||||
SC |
5 |
8 |
2 |
10 |
28 |
1.2 |
|
|
SC |
21 |
none |
||||
100 |
C1 |
131 |
1.33 |
EW |
87 |
83 |
78 |
89 |
49 |
0.7 |
|
|
|
|
|
LC |
5 |
6 |
10 |
3 |
24 |
0.6 |
|
|
|
|
|
||||
SC |
4 |
7 |
8 |
4 |
23 |
1.1 |
|
|
|
|
|
||||
C2 |
95 |
1.05 |
EW |
84 |
85 |
87 |
88 |
53 |
0.9 |
113 |
1.19 |
|
51 |
none |
|
LC |
7 |
5 |
8 |
2 |
28 |
0.9 |
|
|
LC |
26 |
none |
||||
SC |
5 |
6 |
1 |
6 |
23 |
1.0 |
|
|
SC |
23 |
0 |
||||
NQO 0.1 µg /mL |
C1 |
21 |
0.96 |
EW |
24 |
27 |
26 |
26 |
682 |
10.1 |
|
|
|
|
|
LC |
50 |
35 |
40 |
33 |
275 |
6.3 |
|
|
|
|
|
||||
SC |
22 |
34 |
30 |
37 |
200 |
9.1 |
|
|
|
|
|
||||
C2 |
19 |
0.76 |
EW |
19 |
40 |
33 |
32 |
745 |
13.1 |
20 |
0.86 |
|
713 |
651 |
|
LC |
41 |
36 |
40 |
35 |
332 |
10.6 |
|
|
LC |
303 |
266 |
||||
SC |
36 |
20 |
23 |
29 |
218 |
9.1 |
|
|
SC |
209 |
186 |
Adj. RTG: adjusted relative total growth C1: culture 1
CE2: cloning efficiency C2: culture 2
*: empty wells are counted on a total number of 96wells 0: vehicle control (water for injections)
MF: mutation frequency NQO: 4--Nitroquinoline-N-oxide
IMF: Induced Mutation Frequency P: precipitate observed in the culture medium at the end of treatment
SC: wells with small colonies
LC: wells with large colonies
EW: empty wells
Table3.Mutagenicity experiment with S9 mix, 3-hour treatment: mutagenicity results
Doses µg/mL |
Adj. RTG % |
CE2 |
|
Wells* |
MFx10-6 |
Ratio MF |
Mean values of C1and C2 |
||||||||
|
|
Adj RTG% |
CE2 |
MFx10-6 |
IMF |
||||||||||
0 |
C1 |
100 |
1.01 |
EW |
83 |
83 |
77 |
76 |
92 |
1.0 |
|
|
|
|
|
LC |
9 |
6 |
15 |
13 |
59 |
1.0 |
|
|
|
|
|
||||
SC |
4 |
7 |
5 |
7 |
31 |
1.0 |
|
|
|
|
|
||||
C2 |
100 |
0.69 |
EW |
78 |
80 |
78 |
84 |
133 |
1.0 |
100 |
0.85 |
|
112 |
|
|
LC |
8 |
9 |
6 |
5 |
55 |
1.0 |
|
|
LC |
57 |
|
||||
SC |
10 |
7 |
12 |
7 |
72 |
1.0 |
|
|
SC |
51 |
|
||||
3.13 |
C1 |
82 |
0.96 |
EW |
73 |
84 |
70 |
70 |
133 |
1.5 |
|
|
|
|
|
LC |
11 |
6 |
13 |
11 |
59 |
1.0 |
|
|
|
|
|
||||
SC |
14 |
6 |
13 |
15 |
69 |
2.3 |
|
|
|
|
|
||||
C2 |
97 |
1.05 |
EW |
83 |
73 |
65 |
70 |
133 |
1.0 |
89 |
1.01 |
|
133 |
21 |
|
LC |
8 |
8 |
15 |
15 |
61 |
1.1 |
|
|
LC |
60 |
3 |
||||
SC |
5 |
15 |
16 |
12 |
64 |
0.9 |
|
|
SC |
67 |
15 |
||||
6.25 |
C1 |
108 |
0.79 |
EW |
78 |
80 |
76 |
76 |
135 |
1.5 |
|
|
|
|
|
LC |
13 |
9 |
11 |
15 |
84 |
1.4 |
|
|
|
|
|
||||
SC |
6 |
7 |
9 |
5 |
46 |
1.5 |
|
|
|
|
|
||||
C2 |
106 |
0.95 |
EW |
77 |
74 |
76 |
66 |
142 |
1.1 |
107 |
0.87 |
|
139 |
27 |
|
LC |
13 |
10 |
11 |
17 |
75 |
1.4 |
|
|
LC |
80 |
23 |
||||
SC |
6 |
12 |
10 |
14 |
61 |
0.9 |
|
|
SC |
53 |
2 |
||||
12.5 |
C1 |
117 |
1.14 |
EW |
71 |
80 |
78 |
74 |
104 |
1.1 |
|
|
|
|
|
LC |
12 |
12 |
9 |
10 |
52 |
0.9 |
|
|
|
|
|
||||
SC |
14 |
4 |
10 |
12 |
48 |
1.6 |
|
|
|
|
|
||||
C2 |
110 |
1.08 |
EW |
73 |
75 |
73 |
80 |
113 |
0.8 |
114 |
1.11 |
|
108 |
none |
|
LC |
13 |
11 |
13 |
7 |
56 |
1.0 |
|
|
LC |
54 |
none |
||||
SC |
13 |
10 |
10 |
9 |
54 |
0.7 |
|
|
SC |
51 |
none |
||||
25 |
C1 |
115 |
1.16 |
EW |
70 |
76 |
70 |
78 |
115 |
1.3 |
|
|
|
|
|
LC |
13 |
14 |
18 |
11 |
68 |
1.2 |
|
|
|
|
|
||||
SC |
13 |
8 |
11 |
8 |
47 |
1.6 |
|
|
|
|
|
||||
C2 |
119 |
0.94 |
EW |
78 |
74 |
81 |
78 |
113 |
0.8 |
117 |
1.05 |
|
114 |
2 |
|
LC |
10 |
13 |
13 |
12 |
71 |
1.3 |
|
|
LC |
70 |
13 |
||||
SC |
8 |
10 |
2 |
6 |
37 |
0.5 |
|
|
SC |
42 |
none |
||||
50 |
C1 |
106 |
1.16 |
EW |
74 |
79 |
71 |
82 |
98 |
1.1 |
|
|
|
|
|
LC |
11 |
9 |
15 |
9 |
52 |
0.9 |
|
|
|
|
|
||||
SC |
11 |
8 |
10 |
5 |
40 |
1.3 |
|
|
|
|
|
||||
C2 |
102 |
1.03 |
EW |
74 |
75 |
78 |
68 |
128 |
1.0 |
104 |
1.09 |
|
113 |
1 |
|
LC |
9 |
12 |
10 |
15 |
62 |
1.1 |
|
|
LC |
57 |
0 |
||||
SC |
14 |
9 |
8 |
14 |
61 |
0.8 |
|
|
SC |
50 |
none |
||||
100 |
C1 |
84 |
1.05 |
EW |
74 |
81 |
74 |
68 |
123 |
1.3 |
|
|
|
|
|
LC |
9 |
15 |
14 |
22 |
81 |
1.4 |
|
|
|
|
|
||||
SC |
13 |
10 |
9 |
7 |
51 |
1.7 |
|
|
|
|
|
||||
C2 |
125 |
0.96 |
EW |
74 |
85 |
75 |
74 |
114 |
0.9 |
105 |
1.01 |
|
119 |
6 |
|
LC |
11 |
3 |
12 |
13 |
55 |
1.0 |
|
|
LC |
68 |
11 |
||||
SC |
11 |
8 |
9 |
9 |
52 |
0.7 |
|
|
SC |
52 |
1 |
||||
CPA 3µg/mL |
C1 |
40 |
0.57 |
EW |
30 |
20 |
26 |
17 |
1242 |
13.6 |
|
|
|
|
|
LC |
21 |
31 |
23 |
30 |
280 |
4.8 |
|
|
|
|
|
||||
SC |
55 |
55 |
54 |
65 |
794 |
26.0 |
|
|
|
|
|
||||
C2 |
46 |
0.53 |
EW |
17 |
31 |
24 |
18 |
1364 |
10.3 |
43 |
0.55 |
|
1303 |
1191 |
|
LC |
38 |
29 |
33 |
31 |
392 |
7.1 |
|
|
LC |
336 |
279 |
||||
SC |
56 |
49 |
49 |
54 |
734 |
10.2 |
|
|
SC |
764 |
713 |
Adj. RTG: adjusted relative total growth C1: culture 1
CE2: cloning efficiency C2: culture 2
*: empty wells are counted on a total number of 96wells 0: vehicle control (water for injections)
MF: mutation frequency CPA: Cyclophosphamide
IMF: Induced Mutation Frequency
P: precipitate observed in the culture medium at the end of treatment
SC: wells with small colonies
LC: wells with large colonies
EW: empty wells
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Three in vitro key studies were available for mutagenicity evaluation for the registered substance:
An Ames study was performed (Orovecz, Klimisch 1, 2018, GLP, OECD TG 471). Bacteria strain Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA were treated with test substance in distilled water up to 5000 µg/plate with and without metabolic activation system. Three different test were performed as preliminary cytotoxicity test, initial mutation test (plate incorporation method) and confirmatory test (pre incubation method) and revertant colony number was counted. In these three different experiments, no cytotoxicity or mutagenicity were observed. Hence, the test substance was not considered as mutagenic for bacteria strain under the experimental conditions of the study.
An in vitro micronucleus assay was performed (Chevallier, Klimisch 1, 2018, GLP, OECD TG 487). Mammalian cell line L5178Y TK+/- was exposed to the test substance Saccharomyces Cerevisiae, lysate at concentrations up to 250 µg/mL, with and without metabolic activation system, during 3 or 24 hours period. After the treatment period, 2000 cells per dose were assessed for cytotoxicity (population doubling) and mutagenicity (micronucleus counted per cell). At the end of both 3- and 24-hour treatment periods, a precipitate was observed in the culture medium at dose levels >= 125 µg/mL. No cytotoxicity and no increase or micronucleated cells frequency were observed in the study. Hence, the test item did not induce any chromosome damage, or damage to the cell division apparatus for L5178Y TK +/- cells under the experimental conditions of the study.
An in vitro cytogenicity test was performed (Sire, Klimisch 1, 2018, GLP, OECD TG 480) on mammalian cellsz L5178 TK+/- cells. The cells were incubated with test substance at concentrations up to 1000 µg/mL with and without metabolic activation. Two incubation periods were used, 3- hours and 24- hours. Cytotoxicity was evaluated using Adjusted Relative Total Grown and Cloning Efficiency. The test item was found non-cytotoxic under the test conditions and no mutagenicity was observed under the experimental conditions of the study, after 3 hours of exposure.
Justification for classification or non-classification
Based on the three in vitro key studies (Orovecz, Klimisch 1, GLP, 2018, OECD TG 471; Chevallier, Klimisch 1, GLP, 2018, OECD 487 ; Sire, Klimisch 1, GLP, 2018, OECD TG 490), the test substance Saccharomyces Cerevisiae, lysate did not induce mutagenicity and clastogenicity on mammalian cells or bacteria strain. Hence, according to CLP critera, the test substance Saccharomyces Cerevisiae, lysate was not considered as mutagenic substance in vitro. Further in vivo evaluation was not required according to REACh regulation.
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