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EC number: 403-140-4 | CAS number: 103694-68-4 MAJANTOL; MAJANTOL R
- 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
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- Flash point
- Auto flammability
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- 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
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- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
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- Endpoint summary
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- Environmental data
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- 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
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- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames Test (OECD 471): negative
HPRT Test (OECD 476): negative
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2019-20-18 to 2019-10-30
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted July 21, 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- dated May 30, 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Salmonella typhimurium: histidine (his)
Escherichia coli: tryptophan (trp) - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- Liver homogenate (S9) from Phenobarbital/ß-naphtoflavone pretreated rats (protein concentration: 29.3 mg/mL)
Type and composition of metabolic activation system:
- source of S9 : Phenobarbital/β-naphthoflavone induced rat liver S9 (Lot. No.: 060619K)
- method of preparation of S9 mix: according to Ames et al. (1977); appropriate quantity of S9 supernatant thawed and mixed with S9 cofactor solution (final concentration: approx. 10% (v/v))
- volume of S9 mix in the final culture medium: 500 μL
- quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test. - Test concentrations with justification for top dose:
- The test item was tested at the following concentrations:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II:
Strains TA 1535 and WP2 uvrA: 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate
The remaining strains: 3; 10; 33; 100; 333; 1000; and 2500 μg/plate
The top dose for the main experiments was chosen based on the results of the preliminary study. In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I. Since toxic effects were observed in experiment I seven concentrations were tested in experiment II. Based on toxicity observed in experiment I the maximum concentration of 5000 μg/plate was reduced to 2500 μg/plate in some strains. The concentration range included two logarithmic decades. - Vehicle / solvent:
- - Solvent used: DMSO (purity > 99%)
- Justification for choice of solvent/vehicle: because of its solubility properties and its relative nontoxicity to the bacteria - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-o-phenylene-diamine; 2-aminoanthracene, 2-AA
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2
METHOD OF TREATMENT/ EXPOSURE:
- Pre-experiment for toxicity: in agar (plate incorporation)
- Experiment I: in agar (plate incorporation)
- Experiment II: Preincubation
TREATMENT:
- Preincubation period: 60 minutes
- Exposure duration/duration of treatment: 48 hours
METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Method: Number of revertant colonies - Evaluation criteria:
- A test item was considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control was observed. A dose dependent increase was considered biologically relevant if the threshold is reached or exceeded at more than one concentration. An increase of revertant colonies equal or above the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent controls such an increase was not considered biologically relevant.
- Statistics:
- According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate (experiment I) respectively up to the highest investigated dose (experiment II). No precipitation of the test item was observed in the overlay agar on the incubated agar plates. The plates incubated with the test item showed reduced background growth in experiment I in all strains used with and without S9 mix. In experiment II normal background growth was observed on the incubated agar plates.
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains in the presence and absence of S9 mix.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. - Conclusions:
- The test substance was not mutagenic to Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA in the presence and absence of a metabolizing system.
- Executive summary:
An in vitro reverse mutation assay study (Ames) was conducted under GLP-conditions according to OECD Guideline 471 and EU Method B13/B14 in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. Two separate Experiments were performed, one as a plate incorporation assay (Experiment I) and a second one as a preincubation assay (Experiment II). All tests were conducted in triplicate and concentrations between 3 μg/plate and 5000 μg/plate were tested. Strains were exposed to the test substance with and without metabolic activation with rat liver S9 mix. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Cytotoxicity was observed in all strains in the presence and absence of S9 mix. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Therefore it was concluded that the test substance did not induce gene mutations and it was considered to be non-mutagenic.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1987-03-26
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- yes
- Remarks:
- please refer to principles of method if other than guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1983
- Deviations:
- yes
- Remarks:
- please refer to principles of method if other than guideline
- Principles of method if other than guideline:
- only four strains were tested
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor induced rat liver S9-Mix
- Test concentrations with justification for top dose:
- Experiment I
Concentration range in the main test (with metabolic activation): 0.8; 4; 20; 100; 500 µg/plate
Concentration range in the main test (without metabolic activation): 0.8; 4; 20; 100; 500 µg/plate
Experiment II
Concentration range in the main test (with metabolic activation): 1.6; 8; 40; 200; 1000 µg/plate
Concentration range in the main test (without metabolic activation): 1.6; 8; 40; 200; 1000 µg/plate - Vehicle / solvent:
- Solvent: DMSO
- Untreated negative controls:
- yes
- Remarks:
- sterility control
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- other: 2-aminoanthracene (+S9)
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : two
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added: in agar (plate incorporation)
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Conclusions:
- Based on the results of this bacterial reverse mutation assay according to OECD 471 the test item is not considered to be mutagenic.
- Executive summary:
A bacterial reverse mutation assay according to OECD 471 (Ames Test) was conducted with the test item in the four Salmonella Typhimurium strains TA 98, TA 100, TA 1535, TA 1537 with and without metabolic activation. Two independent experiments were conducted as plate incorporation test and each concentration was run in triplicates. The revertant number in the test item treated cultures was not increased when compared to the vehicle control. Thus, under the conditions of this study the test item is considered to not be mutagenic in bacteria.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 17 October 2019 to 07 November 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted July 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- May 30, 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Version / remarks:
- August 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- Japanese Guidelines: "Kanpoan No. 287 - Environment Protection Agency“ “Eisei No. 127 - Ministry of Health & Welfare", "Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry".
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Specific details on test material used for the study:
- - Batch No.: 80100030
- Target gene:
- HPRT locus
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.
- Normal cell cycle time: doubling time 12 - 16 h in stock cultures
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Thawed stock cultures were propagated at 37 °C in 75 cm^2 plastic flasks. About 2-3×10^6 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly.
All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air. - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9 : Phenobarbital/β-naphthoflavone induced rat liver S9
- method of preparation of S9 mix: Phenobarbital/β-naphthoflavone induced rat liver S9 was used as metabolic activation system. An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
The protein concentration of the S9 preparation was 30.7 mg/mL (Lot. No.: 310119) in the pre-experiment and the main experiment.
- concentration or volume of S9 mix and S9 in the final culture medium: 50 µL/mL
- quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test. - Test concentrations with justification for top dose:
- Pre-test: 13.9, 27.9, 55.7, 111.4, 222.9, 445.8, 891.5, 1783.0 µg/mL
Main-test: 12.5; 25.0; 50.0; 100.0; 125.0*, 150.0*, 200.0*, 400.0* μg/mL
* Data not taken into consideration for mutation analysis due to strong cytotoxic effects (rel. adjusted cloning efficiency I below 10%) - Vehicle / solvent:
- - Vehicle used: DMSO; The final concentration of DMSO in the culture medium was 0.5% (v/v).
- Justification for choice of solvent: The solvent was chosen to its solubility properties and as it’s tolerated by the cell cultures. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 5
- Number of independent experiments : 2
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 0.7 to 1.2 x 10^7 cells per flask
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 24 hours
- Exposure duration/duration of treatment: 4 hours
FOR GENE MUTATION:
- Expression time: approx. 7 days
- Selection time: 8-11 days
- Fixation time: The colonies used to determine the cloning efficiency were fixed and stained 6 to 8 days after treatment.
- Selective agent used: 6-thioguanine, 11 μg/mL, 4 hours of exposure
- Number of cells seeded: At least 2.0×10^6 cells per experimental point (concentration series plus controls) were subcultivated in 175 cm^2 flasks containing 30 mL medium .
- Method to enumerate mutated colonies: After 8 – 11 days the colonies were stained with 10% methylene blue in 0.01% KOH solution. Colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival (RS) - Evaluation criteria:
- ACCEPTABILITY OF THE ASSAY
The gene mutation assay is considered acceptable if it meets the following criteria:
a) The mean values of the numbers of mutant colonies per 10^6 cells found in the solvent controls of both parallel cultures remain within the 95% confidence interval of the laboratory historical control data range.
b) Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistical significant increase compared with the concurrent solvent control.
c) Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
d) An adequate number of cells and concentrations (at least four test item concentrations) are analysable even for the cultures treated at concentrations that cause 90% cytotoxicity during treatment.
e) The criteria for the selection of the top concentration are fulfilled.
INTERPRETATION OF RESULTS
A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits). - Statistics:
- A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies (mean values) obtained for the groups treated with the test item was compared to the solvent control groups. A trend was judged as significant whenever the p-value (probability value) was below 0.05. However, both, biological and statistical significance were considered together.
A t-Test was not performed since all mean mutant frequencies were well within the 95% confidence interval of our laboratory’s historical negative control data. - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no relevant shift
- Data on osmolality: no relevant shift
- Precipitation and time of the determination: Phase separation occurred at 445.8 μg/mL and above after 4 hours treatment with and without metabolic activation.
STUDY RESULTS
- Concurrent vehicle negative and positive control data : see tables under section " Any other information on results incl. tables" below
For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : The linear regression analysis showed no significant dose dependent trend of the mutation frequency at any of the experimental groups.
- Statistical analysis; p-value if any : without S9 mix p = 0.294; with S9 mix p= 0.316
HISTORICAL CONTROL DATA
- Positive historical control data:
Positive control EMS (150 and 300 μg/mL, without metabolic activation): Range: 53.9 – 872.3; mean value: 223.3, standard deviation: 101.1; 95% confidence interval: -; number of studies: 199
Positive control DMBA (1.1 and 2.3 μg/mL, with metabolic activation): Range: 55.6 – 739.9; mean value: 188.0, standard deviation: 98.6; 95% confidence interval: -; number of studies: 193
- Solvent control (medium, acetone, water, DMSO, ethanol, THF, EGDE; without metabolic activation): Range: 3.4 – 41.0; mean value: 16.9, standard deviation: 7.0; 95% confidence interval: 2.8 – 30.9; number of studies: 199
- Solvent control (medium, acetone, water, DMSO, ethanol, THF, EGDE; with metabolic activation): Range:2.4 – 40.4; mean value: 16.9, standard deviation: 6.9; 95% confidence interval: 3.1 – 30.7; number of studies: 193 - Conclusions:
- An in vitro gene mutation study (HPRT) was conducted in V79 cells of the Chinese hamster. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.
- Executive summary:
An in vitro gene mutation study (HPRT) according to OECD Guideline 476 was conducted in V79 cells of the Chinese hamster. A pre-experiment was performed with and without metabolic activation for the selection of the test substance concentration. Concentrations between 12.5 and 400.0 μg/mL were tested. For the main experiments a concentration range between 12.5 and 400.0 μg/mL was used. However, a relevant cytotoxic effect indicated by the mean relative adjusted cloning efficiency I below 50% was observed in the presence of metabolic activation at 100.0 μg/mL and above and at 125 μg/mL in the absence of metabolic activation. At the concentration of 125.0 μg/mL the mean relative adjusted cloning efficiency I was far below 10% in the presence and absence of metabolic activation. Consequently, the data at this concentration were not taken into consideration for mutation analysis. The main assay was performed with five replicates for each test concentration and the controls. The cells were exposed to the test item for 4 hours with and without metabolic activation. In the main experiment with and without S9 mix the range of the solvent controls was from 16.1 up to 21.6 mutants per 106 cells and the range of the groups treated with the test item was from 3.8 up to 14.0 mutants per 106 cells. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.
Referenceopen allclose all
Table 1: Summary of Experiment I
Metabolic | Test | Dose Level | Revertant Colony Counts (Mean ± SD) | ||||
TA 1535 | TA 1537 | TA 98 | TA 100 | WP2 uvRA | |||
Without Activation | DMSO |
| 11 ± 2B M | 14 ± 3 | 26 ± 8 | 98 ± 9 | 55 ± 9 |
Untreated |
| 16 ± 3B M | 18 ± 3 | 35 ± 3 | 114 ± 9 | 54 ± 2 | |
Test item | 3 µg | 9 ± 2B M | 14 ± 5 | 35 ± 9 | 97 ± 4 | 51 ± 7 | |
10 µg | 11 ± 2B M | 11 ± 3 | 33 ± 8 | 114 ± 16 | 54 ± 11 | ||
33 µg | 10 ± 1B M | 14 ± 3 | 30 ± 5 | 99 ± 15 | 58 ± 9 | ||
100 µg | 9 ± 2B M | 15 ± 5 | 29 ± 4 | 95 ± 9 | 48 ± 11 | ||
333 µg | 10 ± 3B M | 11 ± 2 | 26 ± 6 | 100 ± 15 | 50 ± 17 | ||
1000 µg | 10 ± 2B M R | 7 ± 2R | 15 ± 4R | 73 ± 15R | 23 ± 6R | ||
2500 µg | 3 ± 2B M R | 1 ± 1R | 5 ± 2M R | 3 ± 1R | 14 ± 1R M | ||
5000 µg | 3 ± 1B M R | 0 ± 0R | 1 ± 1M R | 1 ± 1R | 11 ± 3R M | ||
NaN3 | 10 µg | 1167 ± 49B M |
|
| 1883 ± 136 |
| |
4-NOPD | 10 µg |
|
| 416 ± 6 |
|
| |
4-NOPD | 50 µg |
|
|
|
|
| |
MMS | 2.0 µL |
|
|
|
| 914 ± 104 | |
With Activation | DMSO |
| 11 ± 2B M | 18 ± 3 | 40 ± 3 | 117 ± 11 | 58 ± 4 |
Untreated |
| 11 ± 2B M | 17 ± 6 | 40 ± 11 | 116 ± 11 | 62 ± 5 | |
Test item | 3 µg | 10 ± 3B M | 17 ± 5 | 41 ± 7 | 115 ± 7 | 60 ± 8 | |
10 µg | 10 ± 2B M | 14 ± 7 | 35 ± 7 | 108 ± 19 | 55 ± 5 | ||
33 µg | 11 ± 3B M | 15 ± 4 | 38 ± 6 | 113 ± 7 | 54 ± 4 | ||
100 µg | 11 ± 2B M | 14 ± 4 | 43 ± 8 | 113 ± 7 | 56 ± 7 | ||
333 µg | 12 ± 3B M | 10 ± 3 | 37 ± 6 | 109 ± 6 | 52 ± 7 | ||
1000 µg | 9 ± 1B M R | 4 ± 1R M | 15 ± 2R M | 50 ± 17R | 23 ± 4R | ||
2500 µg | 1 ± 1B M R | 0 ± 1R M | 2 ± 1M R | 3 ± 1R | 11 ± 3R M | ||
5000 µg | 0 ± 1B M R | 0 ± 0R M | 0 ± 1M R | 0 ± 0R | 5 ± 1R M | ||
2-AA | 2.5 µg | 349 ± 47B M | 434 ± 7 | 3097 ± 218 | 3777 ± 163 |
| |
2-AA | 10 µg |
|
|
|
| 3.22 ± 22 |
Key to Positive Controls
NaN3: sodium azide
2-AA: 2-aminoanthracene
4-NOPD: 4-nitro-o-phenylene-diamine
MMS: methyl methane sulfonate
Key to Plate Postfix Codes
R: Reduced background growth
B: Extensive bacterial growth
M: Manual count
Table 2: Summary of Experiment II
Metabolic | Test | Dose Level | Revertant Colony Counts (Mean ± SD) | ||||
TA 1535 | TA 1537 | TA 98 | TA 100 | WP2 uvRA | |||
Without Activation | DMSO |
| 9 ± 3 | 11 ± 4 | 27 ± 7 | 88 ± 5 | 40 ± 6 |
Untreated |
| 14 ± 5 | 16 ± 4 | 25 ± 10 | 102 ± 4 | 44 ± 3 | |
Test item | 3 µg |
| 15 ± 2 | 25 ± 8 | 90 ± 8 |
| |
10 µg | 11 ± 1 | 17 ± 6 | 28 ± 0 | 89 ± 10 | 41 ± 1 | ||
33 µg | 8 ± 2 | 14 ± 7 | 23 ± 2 | 87 ± 10 | 46 ± 6 | ||
100 µg | 10± 2 | 15 ± 5 | 19 ± 6 | 84 ± 20 | 44 ± 3 | ||
333 µg | 12± 5 | 8 ± 4 | 21 ± 3 | 86 ± 12 | 34 ± 3 | ||
1000 µg | 6 ± 1 | 1 ± 1 | 2 ± 0 | 39 ± 6 | 18 ± 5 | ||
2500 µg | 0 ± 1 | 1 ± 1 | 0 ± 1 | 0 ± 0 | 0 ± 0 | ||
5000 µg | 0 ± 0 |
|
|
| 0 ± 0 | ||
NaN3 | 10 µg | 1106 ± 78 |
|
| 1705 ± 146 |
| |
4-NOPD | 10 µg |
|
| 371 ± 7 |
|
| |
4-NOPD | 50 µg |
| 70 ± 11 |
|
|
| |
MMS | 2.0 µL |
|
|
|
| 820 ± 19 | |
With Activation | DMSO |
| 10 ± 0 | 13 ± 3 | 36 ± 6 | 95 ± 7 | 48 ± 5 |
Untreated |
| 12 ± 4 | 19 ± 3 | 44 ± 3 | 100 ± 6 | 59 ± 6 | |
Test item | 3 µg |
| 14 ± 7 | 33 ± 8 | 84 ± 9 |
| |
10 µg | 14 ± 2 | 14 ± 3 | 36 ± 6 | 87 ± 3 | 54 ± 2 | ||
33 µg | 10 ± 1 | 15 ± 1 | 35 ± 9 | 98 ± 12 | 54 ± 9 | ||
100 µg | 14 ± 3 | 15 ± 3 | 39 ± 6 | 99 ± 3 | 54 ± 8 | ||
333 µg | 13 ± 3 | 17 ± 2 | 28 ± 2 | 68 ± 9 | 49 ± 6 | ||
1000 µg | 9 ± 3 | 1 ± 1 | 1 ± 1 | 1 ± 1 | 31 ± 3 | ||
2500 µg | 0 ± 1 | 0 ± 0 | 0 ± 1 | 0 ± 1 | 2 ± 1 | ||
5000 µg | 0 ± 0 |
|
|
| 0 ± 0 | ||
2-AA | 2.5 µg | 331 ± 23 | 314 ± 11 |
| 3166 ± 109 |
| |
2-AA | 10 µg |
|
|
|
| 306 ± 54 |
Key to Positive Controls
NaN3: sodium azide
2-AA:2-aminoanthracene
4-NOPD:4-nitro-o-phenylene-diamine
MMS: methyl methane sulfonate
Table 1 Experiment I, without S9
Concentration (µg/plate) |
TA 100 (mean±SD |
TA 1535 (mean±SD |
TA 98 (mean±SD |
TA 1538 (mean±SD |
TA 1537 (mean±SD |
0 |
76± 11 |
11± 2 |
15±3 |
Not tested |
8± 3 |
0.8 |
81± 3 |
13±5 |
20± 9 |
Not tested |
6± 2 |
4.0 |
88± 6 |
11± 5 |
21± 5 |
Not tested |
8± 4 |
20.0 |
88± 12 |
15± 5 |
18± 4 |
Not tested |
5± 3 |
100.0 |
85± 12 |
12± 3 |
19± 8 |
Not tested |
7± 5 |
500.0 |
78± 9 |
11± 2 |
6± 2 |
Not tested |
3±2 |
Na-azide |
739± 58 |
725± 55 |
|
Not tested |
|
2-Nitrofluorene |
|
|
519± 105 |
Not tested |
|
9-Aminoacridine |
|
|
|
Not tested |
554±111 |
Table 2 Experiment I, with S9
Concentration (µg/plate) |
TA 100 (mean +/- SD |
TA 1535 (mean +/- SD |
TA 98 (mean +/- SD |
TA 1538 (mean +/- SD |
TA 1537 (mean +/- SD |
0 |
92± 9 |
25± 7 |
21± 5 |
Not tested |
10± 6 |
0.8 |
97± 6 |
24± 7 |
33± 11 |
Not tested |
7± 2 |
4.0 |
102± 12 |
25± 3 |
27± 5 |
Not tested |
7± 3 |
20.0 |
78± 17 |
24± 2 |
28± 7 |
Not tested |
11± 3 |
100.0 |
97± 7 |
32± 6 |
25± 4 |
Not tested |
7± 2 |
500.0 |
78± 10 |
25± 3 |
27± 4 |
Not tested |
10± 2 |
2-aminoanthracene |
257± 34 |
261± 24 |
121± 10 |
Not tested |
271± 45 |
Table 3 Experiment II, without S9
Concentration (µg/plate) |
TA 100 (mean +/- SD |
TA 1535 (mean +/- SD |
TA 98 (mean +/- SD |
TA 1538 (mean +/- SD |
TA 1537 (mean +/- SD |
0.0 |
83± 3 |
27± 4 |
18± 3 |
Not tested |
7± 2 |
1.60 |
76±11 |
27± 3 |
16± 3 |
Not tested |
8± 3 |
8.0 |
77± 2 |
28± 6 |
10± 4 |
Not tested |
10± 1 |
40.0 |
94± 3 |
36± 2 |
19± 5 |
Not tested |
5± 4 |
200.0 |
85± 10 |
29± 13 |
13± 4 |
Not tested |
6± 2 |
1000.0 |
0± 0 |
0± 0 |
0± 0 |
Not tested |
0± 0 |
Na-azide |
836± 113 |
|
|
Not tested |
|
2-Nitrofluorene |
|
753± 107 |
|
Not tested |
|
9-Aminoacridine |
|
|
599± 81 |
Not tested |
303± 48 |
Table 4 Experiment II, with S9
Concentration (µg/plate) |
TA 100 (mean +/- SD |
TA 1535 (mean +/- SD |
TA 98 (mean +/- SD |
TA 1538 (mean +/- SD |
TA 1537 (mean +/- SD |
0.0 |
85± 5 |
24± 7 |
35± 6 |
Not tested |
10± 2 |
1.60 |
75± 9 |
12± 2 |
30± 11 |
Not tested |
11± 4 |
8.0 |
75± 3 |
14± 4 |
28± 3 |
Not tested |
8± 3 |
40.0 |
80± 2 |
19± 4 |
31± 3 |
Not tested |
10± 3 |
200.0 |
70± 17 |
15±1 |
31± 4 |
Not tested |
13± 6 |
1000.0 |
0± 0 |
0± 0 |
0± 0 |
Not tested |
0± 0 |
2-aminoanthracene |
206± 19 |
195± 8 |
134± 27 |
Not tested |
247± 29 |
Table: Summary of Results
|
|
|
| relative | relative | re. Adjusted | mutant | 95% |
conc. |
| S9 | cloning | cell | cloning | colonies | confidence | |
µg/mL | PS | mix | efficiency I | density | efficiency I | 106 cells | interval | |
|
|
| % | % | % |
|
| |
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
Main Experiment / 4 h treatment | mean values of culture I and II | |||||||
Solvent control with DMSO |
|
| - | 100.0 | 100.0 | 100.0 | 16.0 | 2.8-30.9 |
Positive control (EMS) | 300.0 |
| - | 93.2 | 116.9 | 108.9 | 149.9 | 2.8-30.9 |
Test item | 12.4 |
| - | 94.4 | 86.1 | 80.5 | 8.5 | 2.8-30.9 |
Test item | 25.0 |
| - | 88.4 | 98.7 | 88.0 | 9.6 | 2.8-30.9 |
Test item | 50.0 |
| - | 90.1 | 99.2 | 89.5 | 5.4 | 2.8-30.9 |
Test item | 100.0 |
| - | 73.8 | 97.0 | 71.6 | 9.4 | 2.8-30.9 |
Test item | 125.0 |
| - | 3.1 | 61.4 | 1.9 | 7.1 | 2.8-30.9 |
Test item | 150.0 |
| - | # | 3.7 | culture was not continued # | ||
Test item | 200.0 |
| - | # | 1.8 | culture was not continued # | ||
Test item | 400.0 | PS | - | # | 3.1 | culture was not continued # | ||
Solvent control with DMSO |
|
| + | 100.0 | 100.0 | 100.0 | 12.4 | 3.1-30.7 |
Positive control (DMBA) | 2.3 |
| + | 98.4 | 75.5 | 74.2 | 347.1 | 3.1-30.7 |
Test item | 12.5 |
| + | 101.2 | 101.1 | 102.2 | 16.2 | 3.1-30.7 |
Test item | 25.0 |
| + | 98.5 | 76.0 | 74.9 | 8.4 | 3.1-30.7 |
Test item | 50.0 |
| + | 93.1 | 74.1 | 69.0 | 11.3 | 3.1-30.7 |
Test item | 100.0 |
| + | 75.0 | 54.8 | 40.9 | 10.0 | 3.1-30.7 |
Test item | 125.0 |
| + | 18.3 | 15.4 | 2.8 | 9.9 | 3.1-30.7 |
Test item | 150.0 |
| + | # | 10.3 | culture was not continued # | ||
Test item | 200.0 |
| + | # | 2.2 | culture was not continued # | ||
Test item | 400.0 | PS | + | # | 1.1 | culture was not continued # |
PS = Phase separation at the beginning and at the end of treatment
# culture was not continued due to exceedingly severe cytotoxic effects
* Data printed in bold were not taken into consideration for mutation analysis due to a rel.adjusted CE I below 10%.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
In vivo micronucleus test (OECD 474): negative
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1987-08-26
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- GLP compliance:
- yes
- Type of assay:
- mammalian erythrocyte micronucleus test
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: adult, 3 months old
- Weight at study initiation: males: 28 - 34 g; females: 26 - 32 g
- Assigned to test groups randomly: yes, under following basis: by lot
- Fasting period before study: no
- Housing: collective caging
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11 days
ENVIRONMENTAL CONDITIONS
- Temperature: 20 +/- 2°C
- Humidity: 50 - 80 %
- Air changes: not specified
- Photoperiod: 12 / 12 hrs dark / hrs light - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle/solvent used: isotonic saline
- Justification for choice of solvent/vehicle: recommended by guideline - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test solution was prepared by diluting appropriate amounts with isotonic saline. - Duration of treatment / exposure:
- single intraperitoneal application
- Frequency of treatment:
- 1
- Post exposure period:
- 24, 48, 72 hours, respectively
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- Male: 1000 mg/kg bw; No. of animals: 5; Sacrifice time: 24 hours
Male: 1000 mg/kg bw; No. of animals: 5; Sacrifice time: 48 hours
Male: 1000 mg/kg bw; No. of animals: 5; Sacrifice time: 72 hours
Female: 1000 mg/kg bw; No. of animals: 5; Sacrifice times: 24 hours
Female: 1000 mg/kg bw; No. of animals: 5; Sacrifice times: 48 hours
Female: 1000 mg/kg bw; No. of animals: 5; Sacrifice times: 72 hours - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide
- Justification for choice of positive control: recommended by the guideline
- Route of administration: intraperitoneal
- Doses / concentrations: 40 mg/ kg bw
- Positive control animals were evaluated 24 h after application - Tissues and cell types examined:
- Erythrocytes from femura bone marrow.
- Details of tissue and slide preparation:
- DETAILS OF SLIDE PREPARATION: Preparations were dried, fixed in absolute (99 %) methanol for 5 min. and then allowed to air dry. The slides were stained using a May-Grünwald and Giemsa solution.
METHOD OF ANALYSIS: From each animal 2 preparations were made. Prior to analysis all the slides were randomized and coded (blind evaluation). The cells were examined at a magnification of 1000 x.
A total of 1000 polychromatic erythrocytes was scored from each slide and the number of micronucleated cells in each sample was recorded. The ratio of polychromatic erythrocytes to normochromatic (mature) erythrocytes was calculated on the base of 1000 cells. - Evaluation criteria:
- Cell counts are based on a total of 1000 cells per anirnal. Due to own laboratory historical data an incidence of (up to) 0.8 %, respectively of micronucleated polychromatic erythrocytes is considered to be within normal limits.
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- clinical signs of toxicity were seen at 1000 mg/kg bw
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- No lethal effects, no cytytoxicity (PCE/NCE).
- Conclusions:
- The intraperitoneal administration of the test item at a dose level of 1000 mg/kg bw to male and female mice did not produce significant increases in the frequency of micronuclei in the polychromatic erythrocyte stem cells. All mean values lay completely within laboratory own normal ranges. Therefore, under the experimental conditions of this study, the test item is considered to be non-mutagenic at any tested time point in this test system.
- Executive summary:
The test item was administered once intraperitoneally at one high dose level of 1000 mg/kg body weight to mice of the NMRI-strain. Samples of the bone marrow were taken after 24 hours, after 48 hours and finally after 72 hours from 5 males and 5 females each. A concurrent control group (C) received the vehicle only and a positive control group (C-pos.) was treated with Cyclophosphamide (EndoxanR) at a dose level of 40 mg/kg body weight. Results from the data analysis show that there was no significant difference in the number of micronucleated polychromatic erythrocytes compared to the control values at any time point. The number of polychromatic and normochromatic erythrocytes and the ratio polychromatic / normochromatic erythrocytes was also not significantly different to the controls in animals treated with the test item at any time point. The positive control group, treated with 40 mg Cyclophosphamide /kg bw revealed a distinct, significant increase in the incidence of micronuclei (62.3‰, p < 0.001). In this group the ratio of polychromatic erythrocytes to normochromatic erythrocytes was significantly decreased in the sum of both sexes in the positive control group, thus indicating signs of toxicity to erythropoiesis. The decrease in the ratio was due to a significant dropping of polychromatic erythrocytes and a significant increase in the number of normochromatic erythrocytes.
The intraperitoneal administration of the test item at a dose level of 1000 mg/kg bw to male and female mice did not produce significant increases in the frequency of micronuclei in the polychromatic erythrocyte stem cells. All mean values lay completely within laboratory own normal ranges. Therefore, under the experimental conditions of this study, the test item is considered to be non-mutagenic at any tested time point in this test system.
Reference
Dose-Range-Finding
A preliminary dose-range-finding study was performed applying single doses of 5000, 2500 and 1000 mg/kg body weight, respectively by intraperitoneal injection in 2 mice each. Animals treated at 5000 mg/kg body weight survived, but exhibited signs like marked sedation, ventral position, ataxia, cyanosis and writhing reflex up to six hours p.a. A dose level of 2500 mg/kg body weight produced similar signs less severe and 1000 mg/kg produced only slight, individually barely perceptible reactions. This dose level was therefore considered to be a suitable dosage, representing the area of the MTD (maximally tolerated dosage).
Number of polychromatic erythrocytes with micronuclei
The number of polychromtic erythrocytes with micronuclei was significantly increased 24 h post injection in the positive control animals. This result confirms the sensitivity of the used animal strain. There was no significant difference to control values at any time point in the males of the test compound treated group. All group mean values lay within normal ranges.
Number of polychromatic erythrocvtes
The number of polychromatic erythrocytes was not significantly different in the test item treated animals. It decreased, however, significantly in the positive control group (sum of both sexes).
Number of normochromatic erythrocytes
There was an analogue increase in the number of normochromatic erythrocytes in the positive control group, but none in the test item treated mice.
Ratio polychromatic normochromatic ervthrocytes
Due to the previous results, the ratio dropped significantly only in the positive control group attaining statistical significance in the sum of both sexes.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Ames Test, OECD 471
Key information:
An in vitro reverse mutation assay study (Ames) was conducted under GLP-conditions according to OECD Guideline 471 and EU Method B13/B14 in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia colistrain WP2 uvrA (2019). Two separate Experiments were performed, one as a plate incorporation assay (Experiment I) and a second one as a preincubation assay (Experiment II). All tests were conducted in triplicate and concentrations between 3 μg/plate and 5000 μg/plate were tested. Strains were exposed to the test substance with and without metabolic activation with rat liver S9 mix. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Cytotoxicity was observedin all strains in the presence and absence of S9 mix. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Therefore it was concluded that the test substance did not induce gene mutations and it was considered to be non-mutagenic.
Supporting information:
The result of above mentioned key study is supported by an additional bacterial reverse mutation assay (1987) according to OECD 471 with the test item in the four Salmonella Typhimurium strains TA 98, TA 100, TA 1535, TA 1537 with and without metabolic activation. Two independent experiments were conducted as plate incorporation test and each concentration was run in triplicates. The revertant number in the test item treated cultures was not increased when compared to the vehicle control. Thus, under the conditions of this study the test item is considered to not be mutagenic in bacteria.
HPRT Test, OECD 476
An in vitro gene mutation study (HPRT) according to OECD Guideline 476 was conducted in V79 cells of the Chinese hamster. A pre-experiment was performed with and without metabolic activation for the selection of the test substance concentration. Concentrations between 12.5 and 400.0 μg/mL were tested. For the main experiments a concentration range between 12.5 and 400.0 μg/mL was used. However, a relevant cytotoxic effect indicated by the mean relative adjusted cloning efficiency I below 50% was observed in the presence of metabolic activation at 100.0 μg/mL and above and at 125 μg/mL in the absence of metabolic activation. At the concentration of 125.0 μg/mL the mean relative adjusted cloning efficiency I was far below 10% in the presence and absence of metabolic activation. Consequently, the data at this concentration were not taken into consideration for mutation analysis. The main assay was performed with five replicates for each test concentration and the controls. The cells were exposed to the test item for 4 hours with and without metabolic activation. In the main experiment with and without S9 mix the range of the solvent controls was from 16.1 up to 21.6 mutants per 106 cells and the range of the groups treated with the test item was from 3.8 up to 14.0 mutants per 106 cells. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.
In vivo micronucleus test, OECD 474
The intraperitoneal administration of the test item at a dose level of 1000 mg/kg bw to male and female mice did not produce significant increases in the frequency of micronuclei in the polychromatic erythrocyte stem cells. All mean values lay completely within laboratory own normal ranges. Therefore, under the experimental conditions of this study, the test item is considered to be non-mutagenic at any tested time point in this test system.
Justification for classification or non-classification
Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genotoxicity the test item does not require classification as mutagenic according to Regulation (EC) No 1272/2008 (CLP), as amended for the seventeenth time in Regulation (EU) 2021/849.
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