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EC number: 214-540-1 | CAS number: 1143-72-2
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In vitro gene mutation study in bacteria (similar to OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and E. coli WP2uvrA with and without metabolic activation (RL 1)
In vitro gene mutation study in bacteria (similar to OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and E. coli WP2uvrA with and without metabolic activation (RL 1)
In vitro gene mutation study in bacteria (similar to OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without metabolic activation (RL 1)
In vitro cytogenicity assays in chinese hamster ovary cells (CHO) (similar to OECD 473): positive with and negative without metabolic activation (RL 2)
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:
- 27 Feb - 15 Mar 1985
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted Jul 1997
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon and trp operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- S. typhimurium TA 1538
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
- Test concentrations with justification for top dose:
- Pre-experiment: 4, 20, 100, 500, 2500 and 10000 µg/plate with and without metabolic activation
Main Experiment:
Experiment I:
The pre-experiment served as Experiment I.
Experiment II:
4, 20, 100, 500, 2500 and 5000 µg/plate with and without metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- benzo(a)pyrene
- other: -S9: 2-nitrofluorene (5 µg/plate) for TA1538 and TA98; N-Methyl-N'-nitro-N-nitrosoguanidine (2.5 µg/plate) for WP2uvrA; +S9: 2-aminoanthracene (0.5, 1 or 10 µg/plate) for all strains
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 - 72 h
NUMBER OF REPLICATIONS: 2 and 3 replications each in 2 independent experiments (Experiment 1 and 2), respectively.
DETERMINATION OF CYTOTOXICITY
- Method: A reduced rate of spontaneously occuring colonies and visible thinning of the bacterial lawn were used as indicator for toxicity.
OTHER EXAMINATIONS:
Sterility check: A sterility check was performed with agar plates only containing the test substance with and without S9 mix. - Statistics:
- Mean values were calculated
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1: ≥ 2500 µg/plate without metabolic activation and ≥ 500 µg/plate with metabolic activation; Experiment 2: ≥ 2500 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1 and 2: ≥ 2500 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1 and 2: ≥ 2500 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1 and 2: ≥ 2500 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1 and 2: ≥ 2500 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- 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:
- cytotoxicity
- Remarks:
- Experiment 1 and 2: ≥ 2500 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipiation of the test substance on the plates was observed in the highest concentration of 10000 µg/plate with and without metabolic activation.
RANGE-FINDING/SCREENING STUDIES: The pre-experiment served as Experiment I (all strains were tested in the pre-experiment).
HISTORICAL CONTROL DATA
- Positive historical control data: The historical control data of the positive controls were not provided in the study report, but the control plates with the background control gave the expected number of colonies.
- Negative (solvent/vehicle) historical control data: No historical control data of the vehicle control was provided in the study report, but the control plates with the positive controls gave the expected number of colonies.
OTHER EXAMINATIONS:
Sterility of S-9 Mix and the test compound was indicated by the absence of contamination on the test material and S-9 Mix sterility check plates. - Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 26 Feb - 08 Mar 1985
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted Jul 1997
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon and trp operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- S. typhimurium TA 1538
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
- Test concentrations with justification for top dose:
- Pre-experiment: 4, 20, 100, 500, 2500 and 10000 µg/plate with and without metabolic activation
Main Experiment:
Experiment I:
The pre-experiment served as Experiment I.
Experiment II:
4, 20, 100, 500 and 2500 µg/plate with and without metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- benzo(a)pyrene
- other: -S9: 2-nitrofluorene (5 µg/plate) for TA1538 and TA98; N-Methyl-N'-nitro-N-nitrosoguanidine (2.5 µg/plate) for WP2uvrA; +S9: 2-aminoanthracene (0.5, 1 or 10 µg/plate) for all strains
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 - 72 h
NUMBER OF REPLICATIONS: 2 and 3 replications each in 2 independent experiments (Experiment 1 and 2), respectively.
DETERMINATION OF CYTOTOXICITY
- Method: A reduced rate of spontaneously occuring colonies and visible thinning of the bacterial lawn were used as indicator for toxicity.
OTHER EXAMINATIONS:
Sterility check: A sterility check was performed with agar plates only containing the test substance with and without S9 mix. - Statistics:
- Mean values were calculated.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1 and 2: ≥ 500 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1: ≥ 500 µg/plate with and without metabolic activation; Experiment 2: ≥ 2500 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1 and 2: ≥ 2500 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1: ≥ 100 µg/plate without metabolic activation and ≥ 500 µg/plate with metabolic activation; Experiment 2: ≥ 500 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1: ≥ 500 µg/plate with and without metabolic activation; Experiment 2: at 2500 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- 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:
- cytotoxicity
- Remarks:
- Experiment 1 and 2: ≥ 2500 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance on the plates was observed in concentrations ≥ 2500 µg/plate with and without metabolic activation.
RANGE-FINDING/SCREENING STUDIES: The pre-experiment served as Experiment I (all strains were tested in the pre-experiment).
HISTORICAL CONTROL DATA
- Positive historical control data: The historical control data of the positive controls were not provided in the study report, but the control plates with the background control gave the expected number of colonies.
- Negative (solvent/vehicle) historical control data: No historical control data of the vehicle control was provided in the study report, but the control plates with the positive controls gave the expected number of colonies.
OTHER EXAMINATIONS:
Sterility of S-9 Mix and the test compound was indicated by the absence of contamination on the test material and S-9 Mix sterility check plates. - Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- Treatment with a mutagen that requires metabolic activation by microsomal enzymes was not used. No strain with an AT base pair at the primary reversion site capable of detection of certain oxidizing mutagens or cross-linking agents was used, e.g. E. coli WP2 or S. typhimurium TA102.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted Jul 1997
- Deviations:
- yes
- Remarks:
- Treatment with a mutagen that requires metabolic activation by microsomal enzymes was not used. No strain with an AT base pair at the primary reversion site was used, e.g. E. coli WP2 or S. typhimurium TA102.
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- other: rfa-deficient, uvrB-deficient and bio-deficient
- Species / strain / cell type:
- S. typhimurium TA 1538
- Additional strain / cell type characteristics:
- other: rfa-deficient, uvrB-deficient and bio-deficient
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
- Test concentrations with justification for top dose:
- Experiment 1 and 2: 10, 50, 100, 500, 1000 and 2500 µg/plate with and without metabolic activation
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- other: -S9 and +S9: 2-aminoanthracene (1 or 2.5 µg/plate) for all strains
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 72 h
NUMBER OF REPLICATIONS: 1 replication each in 2 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: not specified - Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 2500 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 2500 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 2500 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 2500 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 2500 µg/plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- Dec 23, 1983 - Mar, 07 1984
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Principles of method if other than guideline:
- only 100 metaphases per concentration have been evaluated, insufficient substance identity and purity documentation
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Dr. S. Wolff's Laboratory UCSF was the original source. The cells were further cloned at Dr. A. Bloom's Laboratory, Columbia University, New York
- Suitability of cells: This cell line has a high proliferation rate and cloning efficiency. The cells have a stable karyotype and an aberration rate of about 0-5% of the metaphases.
- Methods for maintenance in cell culture if applicable: Large stocks of this clone are stored in liquid nitro gen allowing the repeated use ofthe same cell culture batch in different experiments.
- Modal number of chromosomes: 22 +/- 1
- Normal (negative control) cell cycle time: 12 to 14 hours corresponds to 1.5 normal cell cycle length
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The cells were cultured in supplemented Dulbecco's Minima! Essential Medium (DMEM). The DMEM was supplemented with L-glutamine (4 mM), sodium bicarbonate (0.375 %), antibiotics (neomycine 0.015 %), and 10 % fetal calf serum (FCS). All incubations were performed at +37°C in a 4-5 % carbon dioxide atmosphere (100 % humidity).
- Periodically checked for Mycoplasma contamination: yes
- Other: The cells were cloned to minimize the frequency of mutants, and to stabilize the karyotype. The cells have a spontaneous aberration rate of about 0-5 % aberrant metaphases. - Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver S9 mix induced by Aroclor 1254
- Test concentrations with justification for top dose:
- 100, 150, 200, 250, and 300 µg/mL (+/- S9 mix)
The highest concentration of the test item was chosen based on the toxicity of the test item. - Vehicle / solvent:
- - Vehicle/solvent used: ethanol
- Justification for choice of solvent/vehicle: solubility performance - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
DURATION
- Exposure duration: 16.5 hours (- S9 mix); 2 hours (+ S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 16.5 and 20 hours, respectively
SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Cells were stained in 5 % Giemsa
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The medium is replaced by KCl-solution (0.075 %) for hypotonic treatment. For fixation of cells fixative ( methanol: acetic acid, 3: 1) is added and then renewed 2 times.cells were dropped on slides and air dried.
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 100
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, cell viability
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
The frequnecy determinaton of polyploid cells and endoreduplications (chromosomes with 4, 8, 16 ... chromatids) is based on scoring 100 mitoses per slide, and estimation of the mitotic index on scoring 100 cells per slide.- Evaluation criteria:
- Students T test was applied to compare the aberration frequency in treated cells with pooled results from solvnet and negative controls. The difference was was considered significant wher p>0.05.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolality: not reported
- Precipitation: not reported - Conclusions:
- In conclusion, treatment of CHO cell cultures with the test item, did increase the proportion of cells with aberrant chromosomes.The test item was clastogenic in this in vitro test system.
Referenceopen allclose all
Table 1: Summary of Experiment 1
EXPERIMENT 1 | ||||||
S9-Mix | Without | |||||
Test item (µg/plate) | TA100 | TA1535 | TA1537 | TA1538 | TA98 | WP2uvrA |
0 | 144 | 19 | 10 | 12 | 27 | 16 |
4 | 164 | 18 | 8 | 10 | 19 | 27 |
20 | 161 | 14 | 11 | 13 | 24 | 22 |
100 | 175 | 14 | 8 | 13 | 27 | 22 |
500 | 160 | 11 | 7 | 14 | 23 | 17 |
2500 | 9 (ibl) | ibl | nbl | 0 ibl | 0 (ibl) | 4 (ibl) |
10000 | 0 P (nbl) | 0 P (nbl) | 0 P (nbl) | 0 P (nbl) | 0 P (nbl) | 0 P (nbl) |
Positive controls | SA | SA | 9-AA | 2-NF | 2-NF | MNNG |
Concentrations (μg/plate) | 1 | 1 | 50 | 5 | 5 | 2.5 |
Mean No. of colonies/plate | 560 | 384 | 95 | 786 | 450 | 221 |
S9-Mix | With | |||||
Test item (µg/plate) | TA100 | TA1535 | TA1537 | TA1538 | TA98 | WP2uvrA |
0 | 145 | 15 | 12 | 16 | 27 | 25 |
4 | 174 | 13 | 11 | 13 | 31 | 23 |
20 | 185 | 13 | 9 | 18 | 31 | 19 |
100 | 196 | 12 | 15 | 17 | 35 | 25 |
500 | 130 (ibl) | 7 | 11 | 17 | 30 | 19 |
2500 | 23 (ibl) | 2 (ibl) | 0 (ibl) | 0 (ibl) | 1 (ibl) | 4 |
10000 | 0 P (nbl) | 0 P (nbl) | 0 P (ibl) | 0 P (nbl) | 0 P (nbl) | 0 P (nbl) |
Positive control | 2-AA | 2-AA | 2-AA | 2-AA | 2-AA | 2-AA |
Concentrations (μg/plate) | 0.5 | 1 | 1 | 0.5 | 0.5 | 10 |
Mean No. of colonies/plate | 680 | 156 | 109 | 538 | 545 | 272 |
Positive control | B(a)P | B(a)P | B(a)P | B(a)P | B(a)P | B(a)P |
Concentrations (μg/plate) | 10 | 10 | 10 | 10 | 10 | 10 |
Mean No. of colonies/plate | 449 | 29 | 163 | 269 | 704 | 57 |
ibl: incomplete background lawn in all plates
(ibl): incomplete background lawn in individual plates
nbl: no background lawn
(nbl): no background lawn in individual plates
P: precipitation
SA: sodium azide
9-AA: 9 -aminoacridine
2-NF: 2 -nitrofluorene
MNNG: N-methyl-N'-nitro-N-nitrosoguanidine
2-AA: 2 -aminoanthracen
B(a)P: benzo(a)pyrene
Table 2: Summary of Experiment 2
EXPERIMENT 2 | ||||||
S9-Mix | Without | |||||
Test item (µg/plate) | TA100 | TA1535 | TA1537 | TA1538 | TA98 | WP2uvrA |
0 | 134 | 21 | 13 | 16 | 27 | 20 |
4 | 151 | 18 | 14 | 17 | 31 | 23 |
20 | 160 | 22 | 11 | 8 | 26 | 18 |
100 | 199 | 20 | 11 | 14 | 25 | 19 |
500 | 164 | 17 | 15 | 17 | 27 | 20 |
2500 | 0 (nbl) | nbl | 0 (ibl) | 0 (ibl) | 1 (ibl) | 1 (ibl) |
5000 | 0 (nbl) | 0 (nbl) | 0 (nbl) | 0 (nbl) | 0 (nbl) | 0 (nbl) |
Positive controls | SA | SA | 9-AA | 2-NF | 2-NF | MNNG |
Concentrations (μg/plate) | 1 | 1 | 50 | 5 | 5 | 2.5 |
Mean No. of colonies/plate | 601 | 413 | 132 | 192 | 627 | 593 |
S9-Mix | With | |||||
Test item (µg/plate) | TA100 | TA1535 | TA1537 | TA1538 | TA98 | WP2uvrA |
0 | 143 | 17 | 12 | 13 | 27 | 19 |
4 | 157 | 9 | 11 | 17 | 28 | 16 |
20 | 191 | 12 | 15 | 17 | 35 | 21 |
100 | 193 | 11 | 15 | 17 | 34 | 18 |
500 | 131 (ibl) | 11 | 16 | 13 | 36 | 19 |
2500 | 0 (ibl) | 1 (ibl) | 0 (ibl) | 2 (ibl) | 3 (ibl) | 7 (ibl) |
5000 | 0 (nbl) | 0 (nbl) | 0 (nbl) | 0 (nbl) | 0 (nbl) | 0 (nbl) |
Positive control | 2-AA | 2-AA | 2-AA | 2-AA | 2-AA | 2-AA |
Concentrations (μg/plate) | 0.5 | 1 | 1 | 0.5 | 0.5 | 10 |
Mean No. of colonies/plate | 2862 | 246 | 463 | 2520 | 2740 | 324 |
Positive control | B(a)P | B(a)P | B(a)P | B(a)P | B(a)P | B(a)P |
Concentrations (μg/plate) | 10 | 10 | 10 | 10 | 10 | 10 |
Mean No. of colonies/plate | 635 | 30 | 145 | 245 | 734 | 70 |
ibl: incomplete background lawn in all plates
(ibl): incomplete background lawn in individual plates
nbl: no background lawn
(nbl): no background lawn in individual plates
P: precipitation
SA: sodium azide
9-AA: 9 -aminoacridine
2-NF: 2 -nitrofluorene
MNNG: N-methyl-N'-nitro-N-nitrosoguanidine
2-AA: 2 -aminoanthracen
B(a)P: benzo(a)pyrene
Table 1: Summary of Experiment 1
EXPERIMENT 1 | ||||||
S9-Mix | Without | |||||
Test item (µg/plate) | TA100 | TA1535 | TA1537 | TA1538 | TA98 | WP2uvrA |
0 | 166 | 29 | 14 | 13 | 23 | 24 |
4 | 148 | 31 | 16 | 16 | 22 | 31 |
20 | 160 | 27 | 12 | 13 | 23 | 24 |
100 | 172 | 24 | 15 | 16 | 26 | 25 |
500 | 71 (nbl) | 17 | 12 | 11 (ibl) | 29 | 28 |
2500 | 0 P (nbl) | P nbl | P nbl | P nbl | 1 (ibl) | 2 (ibl) |
10000 | 0 P (nbl) | 0 P (nbl) | 0 P (nbl) | 0 P (nbl) | 0 P (nbl) | 0 P (nbl) |
Positive controls | SA | SA | 9-AA | 2-NF | 2-NF | MNNG |
Concentrations (μg/plate) | 1 | 1 | 50 | 5 | 5 | 2.5 |
Mean No. of colonies/plate | 469 | 336 | 210 | 2327 | 574 | 329 |
S9-Mix | With | |||||
Test item (µg/plate) | TA100 | TA1535 | TA1537 | TA1538 | TA98 | WP2uvrA |
0 | 166 | 15 | 14 | 16 | 27 | 26 |
4 | 169 | 11 | 14 | 18 | 28 | 26 |
20 | 187 | 10 | 16 | 15 | 30 | 24 |
100 | 182 | 15 | 14 | 27 | 47 | 37 |
500 | 149 | 13 | 10 | 21 | 38 | 32 |
2500 | P nbl | P nbl | 0 P (ibl) | 0 P (ibl) | 3 (ibl) | 1 (ibl) |
10000 | 0 P (nbl) | 0 P (nbl) | 0 P (nbl) | 0 P (nbl) | 0 P (nbl) | 0 P (nbl) |
Positive control | 2-AA | 2-AA | 2-AA | 2-AA | 2-AA | 2-AA |
Concentrations (μg/plate) | 0.5 | 1 | 1 | 0.5 | 0.5 | 10 |
Mean No. of colonies/plate | 658 | 135 | 85 | 535 | 572 | 272 |
Positive control | B(a)P | B(a)P | B(a)P | B(a)P | B(a)P | B(a)P |
Concentrations (μg/plate) | 10 | 10 | 10 | 10 | 10 | 10 |
Mean No. of colonies/plate | 740 | 30 | 143 | 280 | 794 | 60 |
ibl: incomplete background lawn in all plates
(ibl): incomplete background lawn in individual plates
nbl: no background lawn
(nbl): no background lawn in individual plates
SA: sodium azide
9-AA: 9 -aminoacridine
2-NF: 2 -nitrofluorene
MNNG: N-Methyl-N'-nitro-N-nitrosoguanidine
2-AA: 2-Aminoanthracen
B(a)P: Benzo(a)pyrene
Table 2: Summary of Experiment 2
EXPERIMENT 2 | ||||||
S9-Mix | Without | |||||
Test item (µg/plate) | TA100 | TA1535 | TA1537 | TA1538 | TA98 | WP2uvrA |
0 | 148 | 17 | 10 | 14 | 23 | 23 |
4 | 131 | 15 | 10 | 13 | 23 | 21 |
20 | 141 | 17 | 14 | 14 | 24 | 15 |
100 | 140 | 13 | 12 | 11 | 22 | 22 |
500 | 115 | 14 (ibl) | 7 | 9 | 21 | 19 |
2500 | 0 P (nbl) | P nbl | P nbl | 0 (nbl) | P nbl | 1 (ibl) |
Positive controls | SA | SA | 9-AA | 2-NF | 2-NF | MNNG |
Concentrations (μg/plate) | 1 | 1 | 50 | 5 | 5 | 2.5 |
Mean No. of colonies/plate | 505 | 35 | 194 | 935 | 641 | 469 |
S9-Mix | With | |||||
Test item (µg/plate) | TA100 | TA1535 | TA1537 | TA1538 | TA98 | WP2uvrA |
0 | 157 | 14 | 9 | 21 | 34 | 18 |
4 | 155 | 15 | 9 | 19 | 30 | 22 |
20 | 191 | 12 | 10 | 20 | 37 | 16 |
100 | 176 | 13 | 9 | 18 | 39 | 22 |
500 | 129 (ibl) | 12 (ibl) | 10 | 14 | 28 | 18 |
2500 | P nbl | P nbl | 0 P (nbl) | 0 (nbl) | 0 (ibl) | 0 (ibl) |
Positive control | 2-AA | 2-AA | 2-AA | 2-AA | 2-AA | 2-AA |
Concentrations (μg/plate) | 0.5 | 1 | 1 | 0.5 | 0.5 | 10 |
Mean No. of colonies/plate | 748 | 147 | 123 | 498 | 546 | 230 |
Positive control | B(a)P | B(a)P | B(a)P | B(a)P | B(a)P | B(a)P |
Concentrations (μg/plate) | 10 | 10 | 10 | 10 | 10 | 10 |
Mean No. of colonies/plate | 829 | 32 | 163 | 259 | 792 | 57 |
ibl: incomplete background lawn in all plates
(ibl): incomplete background lawn in individual plates
nbl: no background lawn
(nbl): no background lawn in individual plates
SA: sodium azide
9-AA: 9 -aminoacridine
2-NF: 2 -nitrofluorene
MNNG: N-Methyl-N'-nitro-N-nitrosoguanidine
2-AA: 2-Aminoanthracen
B(a)P: Benzo(a)pyrene
Table 1: Revertants per plate after treatment with the test substance (Experiment 1)
EXPERIMENT 1 | |||||
S9-Mix | Without | ||||
Test item (µg/plate) | TA100 | TA1535 | TA1537 | TA1538 | TA98 |
10 | 101 | 24 | 5 | 15 | 25 |
50 | 114 | 10 | 5 | 14 | 28 |
100 | 115 | 9 | 5 | 9 | 27 |
500 | 125 | 18 | 5 | 9 | 40 |
1000 | 110 | 12 | 3 | 5 | 21 |
2500 | T | T | T | T | T |
S9-Mix | With | ||||
Test item (µg/plate) | TA100 | TA1535 | TA1537 | TA1538 | TA98 |
10 | 96 | 12 | 12 | 19 | 20 |
50 | 155 | 8 | 7 | 19 | 43 |
100 | 136 | 8 | 9 | 25 | 42 |
500 | 174 | 4 | 9 | 13 | 41 |
1000 | 129 | 7 | 4 | 8 | 38 |
2500 | 42 | 7 | 2 | 4 | 9 |
T: Toxic
Table 2: Revertants per plate after treatment with the test substance (Experiment 2)
EXPERIMENT 2 | |||||
S9-Mix | Without | ||||
Test item (µg/plate) | TA100 | TA1535 | TA1537 | TA1538 | TA98 |
10 | 88 | 31 | 16 | 7 | 36 |
50 | 105 | 26 | 17 | 5 | 41 |
100 | 125 | 19 | 15 | 13 | 31 |
500 | 126 | 19 | 13 | 12 | 38 |
1000 | 105 | 19 | 6 | 13 | 20 |
2500 | T | T | T | T | T |
S9-Mix | With | ||||
Test item (µg/plate) | TA100 | TA1535 | TA1537 | TA1538 | TA98 |
10 | 117 | 6 | 30 | 15 | 39 |
50 | 152 | 12 | 25 | 21 | 45 |
100 | 130 | 8 | 24 | 21 | 64 |
500 | 142 | 8 | 19 | 12 | 27 |
1000 | 139 | 8 | 17 | 17 | 24 |
2500 | 56 | 2 | 1 | 11 | 9 |
T: Toxic
Table 3: Revertants per plate (Controls of Experiment 1)
Concentration | TA100 | TA1535 | TA1537 | TA1538 | TA98 | |
without S9-Mix | ||||||
Negative Control | - | 130 | 17 | 6 | 17 | 33 |
- | 100 | 29 | 8 | 18 | 26 | |
Sodium azide | 1 µg/plate | 710 | 390 | - | - | - |
9-Aminoacridine | 50 µg/plate | - | - | 425 | - | - |
2-Nitrofluorene | 5 µg/plate | - | - | - | 686 | 420 |
with S9-Mix | ||||||
Negative Control | - | 90 | 12 | 18 | 21 | 49 |
- | 100 | 14 | 12 | 29 | 32 | |
2-Aminoanthracene | 1 µg/plate | 1275 | - | - | 468 | 1395 |
2.5 µg/plate | - | 590 | 200 | - | - |
Table 4: Revertants per plate (Controls of Experiment 4)
Concentration | TA100 | TA1535 | TA1537 | TA1538 | TA98 | |
without S9-Mix | ||||||
Negative Control | - | 74 | 19 | 24 | 11 | 32 |
- | 81 | 18 | 9 | 10 | 36 | |
Sodium azide | 1 µg/plate | 528 | 414 | - | - | - |
470 | 359 | - | - | - | ||
9-Aminoacridine | 50 µg/plate | - | - | 207 | - | - |
- | - | 147 | - | - | ||
2-Nitrofluorene | 5 µg/plate | - | - | - | 536 | 313 |
- | - | - | 587 | 227 | ||
2-Aminoanthracene | 1 µg/plate | 65 | - | - | 44 | 33 |
88 | - | - | 18 | 49 | ||
2.5 µg/plate | - | 12 | 13 | - | - | |
- | 17 | 26 | - | - | ||
with S9-Mix | ||||||
Negative Control | - | 92 | 4 | 24 | 24 | 55 |
- | 87 | 8 | 32 | 29 | 49 | |
2-Aminoanthracene | 1 µg/plate | 439 | - | - | 246 | 349 |
421 | - | - | 213 | 320 | ||
2.5 µg/plate | - | 171 | 158 | - | - | |
- | 165 | 139 | - | - |
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Description of key information
In vivo mammalian chromosome aberration test (according to OECD 475, GLP): negative in bone marrow cells of Chinese hamster
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- other information
- Study period:
- 09 Jul - 08 Aug 1985
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- significant methodological deficiencies
- Remarks:
- (only one concentration tested, mortality in 1/5 males and clinical signs of systemic toxicity in 2/10 animals; no clear cytotoxicity observed in the bone marrow based on mitotic index; 1000 polychromatic erythrocytes were counted per animal while 4000 is recommended)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- adopted Jul 2016
- Deviations:
- yes
- Remarks:
- (only one concentration tested, mortality in 1/5 males and clinical signs of systemic toxicity in 2/10 animals; no clear cytotoxicity observed in the bone marrow based on MI; 1000 PCEs were counted per animal while 4000 is recommended)
- GLP compliance:
- yes
- Type of assay:
- mammalian erythrocyte micronucleus test
- Species:
- mouse
- Strain:
- NMRI
- Remarks:
- Hoe: NMRKf (SPF71)
- Details on species / strain selection:
- The mouse has been chosen for this study since it provides a convenient in vivo mammalian model, which has been proposed in the literature.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Hoechst AG, Kastengrund, SPF breeding colony
- Age at study initiation: 8 weeks
- Weight at study initiation: Ranges: 28 - 36 g (males); 23 - 29 g (females)
- Housing: Groups of 5 animals were housed in macrolon cages (Type 3) on softwood granulate.
- Diet: Altromin 1324 (Altromin - GmbH, Lage/Lippe, Germay), ad libitum
- Water: Tap water, ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 10
- Air changes (per hr): fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: polyethylene glycol 400
- Concentration of test material in vehicle: 15% (w/v)
- Amount of vehicle: 10 mL/kg bw - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test substance dilutions were freshly prepared each day. 3750 mg of the test substance was weighed into a beaker. About 15 ml of polyethylenglycol 400 was given into a 25 ml flask. Slowly the substance was given into the flask and solubilized entirely. Then the flask was topped up to the calibration mark with polyethylenglycol 400 and mixed thoroughly until a clear solution was formed. - Duration of treatment / exposure:
- not applicable
- Frequency of treatment:
- single treatment
- Post exposure period:
- 24, 48 or 72 h after last treatment
- Dose / conc.:
- 1 500 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5 animals per sex per dose and per post exposure period
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Positive control: Cyclophosphamide
- Route of administration: Oral
- Doses / concentrations: 50 mg/kg bw in distilled water - Tissues and cell types examined:
- Tissue: bone marrow
Cell type: erythrocytes - Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: Based on a preliminary study (09 – 21 Jul 1985) for determination of the acute toxicity, the dose levels for micronucleus testing were selected. 3 females were dosed at 1500 and 2500 mg/kg bw, respectively, and 3 males were dosed at 2000 mg/kg bw. All females of the highest dose group and one male of the mid-dose group died. Abdominal position, back-arched position, ptosis and gasping were observed in all dose groups. Tachypnoe was noted only in the low-dose group. Hyperreflexia and piloerection was observed in the low- and mid-dose group. Animals of the mid-dose group showed reduced spontaneous activity. Dopiness was seen in the mid- and high-dose group and increased respiratory sounds occurred in the high-dose group. Therefore the highest possible sublethal dose of 1500 mg/kg bw was selected for the main study.
TREATMENT AND SAMPLING TIMES: Sampling was performed 24, 48 and 72 h after administration.
DETAILS OF SLIDE PREPARATION: Slides were fixed with methanol and stained with May-Grünwald for 5 min and with Giemsa-solution for 10 min.
METHOD OF ANALYSIS: 1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition the ratio of polychromatic to normochromatic erythrocytes was determined. - Statistics:
- For evaluation all bone marrow smears are coded to ensure that the group to which they belonged remained unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occuring in 1000 counted polychromatic erythrocytes, and the number of normocytes with micronuclei occuring in 1000 counted normocytes, were evaluated statistically; comparison of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided, increase).
The results of the test substance at each sampling time and dose were compared with the corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided). The statistical evaluations were realized with the "Diamant" computer program Version 2.0, supplied by the Department "Information und Kommunikation" of Hoechst AG. All statistical results are based on the 95 % level of significance. - Key result
- Sex:
- female
- Genotoxicity:
- positive
- Remarks:
- at 1500 mg/kg bw and at all sampling times
- Toxicity:
- yes
- Remarks:
- Irregular breathing, disturbance equilibrium, dopiness, hyperreflexia and backed-arched position
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Sex:
- male
- Genotoxicity:
- positive
- Remarks:
- at 1500 mg/kg bw and at all sampling times
- Toxicity:
- yes
- Remarks:
- Irregular breathing, disturbance equilibrium, dopiness, hyperreflexia and backed-arched position; one male died 5 h after application and was replaced
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 14 Dec 1987 - 06 Jan 1988
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- (2 sampling times instead of the recommended 3; 50 metaphases counted while 200 is recommended; the cell cycle length not given; 500 cells scored for MI while 1000 is recommended; no cytotoxicity observed in bone marrow based on MI)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
- Version / remarks:
- adopted Jul 2016
- Deviations:
- yes
- Remarks:
- (2 sampling times instead of the recommended 3; 50 metaphases counted while 200 is recommended; the cell cycle length not given; 500 cells scored for MI while 1000 is recommended; no cytotoxicity observed in bone marrow based on MI)
- GLP compliance:
- yes
- Type of assay:
- mammalian bone marrow chromosome aberration test
- Species:
- hamster, Chinese
- Strain:
- other: Chinese hamster inbred strain
- Details on species / strain selection:
- The Chinese hamster is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. This species, although little used in other aspects of toxicology, has a karyotype with 22 chromosomes all of which are easily identified. This offers the opportunity to reduce observer errors and the time required for the cytogenetic analysis.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: LMP stock
- Age at study initiation: Minimum 10 weeks
- Weight at study initiation: Approximately 25 g
- Fasting period before study: Yes, animals received no food approximately 18 h beore treatmen with the test substance.
- Housing: The animals were individually housed in Makrolon Type I cages with wire mesh top (EBECO, Castrop-Rauxel, Germany) on granulated soft wood bedding (Altromin, Lage/Lippe, Germany).
- Diet: Pelleted standard diet (Altromin, Lage/Lippe, Germany)
- Water: Tap water, ad libitum
- Acclimation period: Minimum 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: Methocel MC
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative nontoxicity for the animals.
- Amount of vehicle: 20 mL/kg bw - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test substance was suspended in 0.5 % Methocel solution. All animals received a single standard dose volume adjusted to the body weight orally.
- Duration of treatment / exposure:
- not applicable
- Frequency of treatment:
- single treatment
- Post exposure period:
- 24 and 48 h after treatment
- Dose / conc.:
- 4 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 6
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 30 mg/kg bw in physiological saline - Tissues and cell types examined:
- Tissue: bone marrow
Cell type: bone marrow cells - Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: A range finding study was performed to find the maximum tolerated dose of the test substance based on mortality. In the first pre-experiment 2 males and 2 females were treated with 5000 mg/kg bw. In the second pre-experiment 10 males were exposed to 5000 mg/kg bw and in the third pre-experiment 2 males and 2 females received 4000 mg/kg bw.
DETAILS OF SLIDE PREPARATION: Following treatment and prior to sample collection, animals were injected intraperitoneally with 2 mg/mL Colcemid®, and samples were collected 2.5 hours thereafter. Cells were harvested from the bone marrow, swollen, fixed and stained, and analysed for chromosomal aberrations.
METHOD OF ANALYSIS: At least 50 well spread metaphases were analysed per animal by use of NIKON microscopes with 100x oil immersion objectives and were scored for structural chromosome aberrations (gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations). - Evaluation criteria:
- The test article is classified as mutagenic if it induces a significantly increased aberration rate with at least one of the concentrations tested as compared with the negative control.
This can be confirmed by means of the nonparametric Mann-Whitney test. A test article which produces no significant positive response at any test point is regarded as non-mutagenic in this assay. - Statistics:
- Statistical analysis was not performed due to the obtained results.
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 4000 and 5000 mg/kg bw
- Solubility: Suspension in 0.5% Methocel solution
- Clinical signs of toxicity in test animals: Reduction of spontaneous activity, apathy and abdominal position were observed in both dose groups. 1/2 males of the first pre-experiment and 5/10 males of the second pre-experiment died at 5000 mg/kg bw. None of the exposed animals died at 4000 mg/kg bw.
Referenceopen allclose all
Table 1: Results of the in vivo micronucleus assay in male mice
PCE/NCE | Total Number of PCE with MN of 1000 cells | PCE with MN/1000 cells | |||||||||
Dose group | Number of animals | Dose [mg/kg bw] | 24 h | 48 h | 72 h | 24 h | 48 h | 72 h | 24 h | 48 h | 72 h |
Vehicle control (polyethylenglycol 400 | 5 | - | 1.02 | 0.94 | 0.90 | 1 | 1 | 1 | 0.08 | 0.08 | 0.08 |
Positive control (cyclophosphamide) | 5 | 50 | 0.65 | n.d. | n.d. | 27 | n.d. | n.d. | 2.74 | n.d. | n.d. |
Test substance | 5 | 1500 | 0.85 | 0.83 | 1.00 | 3 | 4 | 5 | 0.34 | 0.38 | 0.52 |
PCE: polychromatic erythrocytes
NCE: normochromatic erythrocytes
MN: micronucleus
n.d.: not determined
Table 2: Results of the in vivo micronucleus assay in female mice
PCE/NCE | Total Number of PCE with MN of 1000 cells | PCE with MN/1000 cells | |||||||||
Dose group | Number of animals | Dose [mg/kg bw] | 24 h | 48 h | 72 h | 24 h | 48 h | 72 h | 24 h | 48 h | 72 h |
Vehicle control (polyethylenglycol 400 | 5 | - | 0.95 | 1.00 | 1.01 | 1 | 1 | 1 | 0.10 | 0.10 | 0.18 |
Positive control (cyclophosphamide) | 5 | 50 | 0.71 | n.d. | n.d. | 24 | n.d. | n.d. | 2.44 | n.d. | n.d. |
Test substance | 5 | 1500 | 0.94 | 1.09 | 1.02 | 2 | 3 | 3 | 0.22 | 0.34 | 0.26 |
PCE: polychromatic erythrocytes
NCE: normochromatic erythrocytes
MN: micronucleus
n.d.: not determined
Table 1: Summary of results
Dose (mg/kg bw) | Postexposure period [h] | Number of cells scored | Aberrant cells incl. gaps (%) | Aberrant cells excl. gaps (%) | Mitotic index (%) |
4000 | 24 | 500 | 0.6 | 0.0 | 5.50 |
4000 | 48 | 500 | 2.2 | 0.4 | 6.51 |
Negative control | 24 | 500 | 2.4 | 1.2 | 5.60 |
Positive control | 24 | 500 | 13.4 | 9.8 | 5.28 |
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Additional information
Genetic toxicity (mutagenicity) in bacteria in vitro
Ames test
A GLP-conform reliable bacterial gene mutation assay (Ames test) was performed with 2,3,4-trihydroxybenzophenone (CAS 1143-72-2) equivalent or similar to OECD 471 (reference 7.6.1-1). The S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and E. coli WP2uvrA were tested conducting the plate incorporation method in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). The experiment was conducted in 2 or 3 replicates each in two independent experiments up to concentrations of 10000 µg/plate in Experiment I and up to concentrations of 5000 µg/plate in Experiment II. Cytotoxicity based on incomplete or no background lawn was observed in all strains from 2500 µg/plate and onwards without metabolic activation. In the presence of S9 mix, cytotoxicity was observed at concentrations ≥ 2500 µg/plate for TA 1535, TA 1537, TA 1538, TA 38 and WP2uvrA, and ≥ 500 µg/plate for TA 100. Precipitation of the test substance on the plates was observed in the highest concentration of 10000 µg/plate with and without metabolic activation. Appropriate positive and solvent controls were included into the test and showed the expected results. No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. Thus, under the conditions of the study, no mutagenic activity was observed in any strain/activation combination in the bacterial Ames-Test for 2,3,4-trihydroxybenzophenone.
A second GLP-conform Ames test equivalent or similar to OECD 471 was available with 2,3,4-trihydroxybenzophenone (CAS 1143-72-2) (reference 7.6.1-2). The S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and E. coli WP2uvrA were tested conducting the plate incorporation method in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). The experiment was conducted in 2 or 3 replicates each in two independent experiments up to concentrations of 10000 µg/plate in Experiment I and up to concentrations of 2500 µg/plate in Experiment II. Cytotoxicity based on incomplete or no background lawn was observed from 2500 µg/plate and onwards in TA 1535, TA 1537, TA 98 and WP2uvrA and from 500 µg/plate and onwards in TA 100 and TA 1538 without metabolic activation. In the presence of S9 mix, cytotoxicity was observed at concentrations ≥ 2500 µg/plate for TA 1535, TA 1537, TA 1538, TA 38 and WP2uvrA, and ≥ 500 µg/plate for TA 100. Precipitation of the test substance on the plates was observed at concentrations ≥ 2500 µg/plate with and without metabolic activation. Appropriate positive and solvent controls were included into the test and showed the expected results. No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. Thus, under the conditions of the study, no mutagenic activity was observed in any strain/activation combination in the bacterial Ames-Test for 2,3,4-trihydroxybenzophenone.
A third Ames test was conducted with 2,3,4-trihydroxybenzophenone (CAS 1143-72-2) equivalent or similar to OECD 471 (reference 7.6.1-3). The S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were tested by using the plate incorporation method in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). The experiment was conducted in 1 replicate each in two independent experiments up to concentrations of 2500 µg/plate in both experiments. Cytotoxicity was observed in all tester strains from 2500 µg/plate and onwards with and without metabolic activation. Appropriate positive and solvent controls were included into the test and showed the expected results, however treatment with a mutagen that required metabolic activation by microsomal enzymes was not used at the study. No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. Thus, under the conditions of the study, no mutagenic activity was observed in any strain/activation combination in the bacterial Ames-Test for 2,3,4-trihydroxybenzophenone.
Cytogenicity was observed in vitro in CHO cells with metabolic activation. No cytogenicity was evident without metabolic activation. However, the study has significant limitations as relevant information is not provided in the report (e.g. substance purity and identity) or the methods employed do not follow the guideline standards (e.g. number of metaphases analysed, number of replicates for negative controls).
Genetic toxicity (cytogenicity) in vivo mammalian somatic cell study
Micronucleus
Due to methodological deficiencies this study is only regarded as other information with a reliability of 3 (not reliable).
2,3,4-trihydroxybenzophenone (CAS 1143-72-2) was tested in a mammalian erythrocyte micronucleus assay conducted according to OECD guideline 474 and compliant with GLP (reference 7.6.2-1). The maximum tolerated dose (MTD) was determined in a preliminary study, in which 2000 mg/kg bw led to clinical signs and mortality, and 1500 mg/kg bw caused clinical signs. Based on this range finding study, 5 NMRI mice per group were exposed by oral gavage to a dose of 1500 mg/kg bw/day, to the vehicle alone (PEG 400) or to the positive control (50 mg cyclophosphamide per kg bw). The test substance was administered in a single treatment. The frequency of micronucleated polychromatic erythrocytes (% MN PCE) was analysed in the bone marrow, sampled 24, 48 and 72 h after the final administration, respectively. The % MN-PCE values in male and female mice were increased when compared with the values in the vehicle control group at all sampling times. In the main study, clinical signs were observed in all animals at least until 24 h after exposure (males and females), and 1/5 males died (this individual was replaced). The observed clinical signs indicated systemic availability of the test substance. However, no cytotoxicity in the bone marrow was observed based on the mitotic index. Based on the results of the conducted study, 2,3,4-trihydroxybenzophenone (CAS 1143-72-2) induced micronuclei in the polychromatic erythrocytes of the bone marrow of male and female mice treated at 1500 mg/kg bw/day.
Chromosome aberration
An in vivo chromosome aberration test performed in male and female Chinese hamsters with 2,3,4-trihydroxybenzophenone (CAS 1143-72-2) according to OECD TG 475 (dated 1984) and according to GLP was available (reference 7.6.2-2). The use of the hamster (non-standard species) was justified in the study report. A range finding study was performed to find the maximum tolerated dose of the test substance based on mortality. Based on this range finding study, 6 animals per group were exposed by oral gavage to a dose of 4000 mg/kg bw/day, to the vehicle alone (Methocel MC) or to the positive control (30 mg cyclophosphamide per kg bw). In the study, there were 2 sampling times, 24 and 48 h. Furthermore, only 50 metaphases were counted and only 500 cells were scored for determination of the mitotic index. It was unclear if the substance was systemically available and if the bone marrow was exposed, since no cytotoxic effects based on the mitotic index were observed. The included negative and the positive control can be considered as valid. The result of this chromosome aberration study with 2,3,4-trihydroxybenzophenone (CAS 1143-72-2) was negative, since the test substance did not induce the frequency of aberrant cells after both sampling times.
Justification for classification or non-classification
Based on the available information there exist evidence for cytogenicity in vitro. Further results from the in vivo micronucleus test indicate evidence for genotoxicty / clastogenicity in vivo. In addition it should be noted, that the available in vivo data lack the quality standards required for high reliablity, e.g. single dose only and therefore no dose reponse relationsship. As these positive findings are or not supported by data on mutagenicity from in vitro or in vivo assays, the test substance 2,3,4 -trihydroxybenzophenone (CAS 1143 -72 -2), does not meet the classification criteria for Mutagenicity (e.g. Muta 2, H341) according to Regulation (EC) 1272/2008.
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