Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 213-979-6 | CAS number: 1070-70-8
- 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
Ames: negative (BASF SE, 2006)
HPRT: negative (BASF SE, 2017)
MNT: positive in the absence of metabolic activation only (BASF SE, 2018)
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his and trp gene
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S-9 Mix
- Test concentrations with justification for top dose:
- 22 μg - 5 500 μg/plate (SPT)
11 μg - 2 750 μg/plate (PIT; TA 1535 and TA 100)
2 μg - 550 μg/plate (PIT; TA 1537 and TA 98)
22 μg - 5 500 μg/plate (PIT, E. coli WP2 uvrA) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: [DMSO]
- Justification for choice of solvent/vehicle: Good solubility of the test substance in DMSO - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (2-AA)
- Remarks:
- with S-9 Mix; strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: N-methyl-N'-nitro-N-nitrosoguanine (MNNG)
- Remarks:
- without S-9 Mix; strains TA 1535 and TA 100
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-o-phenylendiamine (NOPD)
- Remarks:
- without S-9 Mix; strains TA 98
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- without S-9 Mix; strains TA 1537
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- without S-9 Mix; E. coli WP2 uvrA
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48-72 hours in the dark
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Evaluation criteria:
- The test chemical is considered positive in this assay if the following criteria are met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. about
doubling of the spontaneous mutation rate in at least one tester strain either without
S-9 mix or after adding a metabolizing system.
A test substance is generally considered nonmutagenic in this test if:
The number of revertants for all tester strains were within the historical negative control
range under all experimental conditions in two experiments carried out independently of
each other. - Statistics:
- not applicable
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- PIT: from about 275 μg/plate onward; SPT: from about 550 μg/plate onward
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- PIT: from about 275 μg/plate onward; SPT: from about 550 μg/plate onward
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed in the standard plate test depending on the strain and test conditions from about 550 μg/plate onward.
In the preincubation assay bacteriotoxicity (reduced his- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed depending on the strain and test conditions from about 275 μg/plate onward. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- 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
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- => no E. coli or TA102 strain tested
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 or phenobarbital-induced S9
- Test concentrations with justification for top dose:
- 30-1000 µg/plate
- Vehicle / solvent:
- Vehicle: DMSO (in 0.1 ml DMSO to top agar)
Justification: Good solubility - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- TA1538, TA98 and TA100; S9 Aroclor-1254-induced
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-Nitro-o-phenylenediamin
- Remarks:
- TA1538 and TA98; without S9
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- TA1537; without S9
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Glycidyl methacrylate
- Remarks:
- TA 1535 and TA 100; without S9
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracen
- Remarks:
- TA 1535 and TA1537; with S9 (phenobarbital-induced)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- TA 1535 and TA100; without S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation);
- Exposure duration: after addition of the test substance in top agar, the plates were incubated for 48-72 hours.
- all test were performed on triplicate plates.
- Compunds were once tested with phenobarbital-induced S9, once with Aroclor-1254-induced S9, and twice without any additional metabolizing system.
- Plated were counted manually
- Controls: Solvent controls, positive controls and sterility controls for S9 mix were run with each experiment. - Evaluation criteria:
- Increase of revertant rate was considered signifcant when their number is more than 2-fold compared to spontaneous revertant rate.
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: toxic in the highest concentration tested
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- July 2016
- Qualifier:
- according to guideline
- Guideline:
- other: EU method B49; No L193: In vitro Mammalian Cell Micronucleus Test
- Version / remarks:
- May 2008
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- human lymphoblastoid cells (TK6)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Cell cycle length, doubling time or proliferation index: 12 -14h
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 containing an L-glutamine source supplemented with 10% FCS, 1% penicillin/streptomycin, 1% amphocericine B
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes - Additional strain / cell type characteristics:
- other: p53 proficient
- Cytokinesis block (if used):
- cytochalasin-B
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbital and β-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- Without S9: 0.16 - 100µg/mL (different concentrations in different experiments)
With S9: 1.56 - 200µg/ml
The concentration was limited by cytotoxicity - Vehicle / solvent:
- Due to the insufficient solubility of the test substance in water, dimethylsulfoxide (DMSO) was used as vehicle, which has been demonstrated to be suitable in the in vitro cytogenetic assay and for which historical control data are available.
- Untreated negative controls:
- yes
- Remarks:
- 24h exposure only
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4h (with and without S9), 24h (without S9)
- Expression time (cells in growth medium): 20 or 40h, none in the case of the 24h exposure
- Fixation time (start of exposure up to fixation or harvest of cells): 24 or 44h (with S9 only)
SPINDLE INHIBITOR (cytogenetic assays): cytochalasin-B
STAIN (for cytogenetic assays): DAPI and PI
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION: method according to Fenech: 5x104 cells per slide were centrifuged at 600 rpm for 7 minutes onto labeled slides. At least two slides per replicate were prepared. After drying, the slides were fixed in 90% (v/v) methanol for 10 minutes.
NUMBER OF CELLS EVALUATED: 2000 binucleated cells per test group
CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
- The diameter of the micronucleus is less than 1/3 of the main nucleus.
− The micronucleus and main nucleus retain the same color.
− The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
− Only binucleated cells were scored.
DETERMINATION OF CYTOTOXICITY
- Method: proliferation index: The cytokinesis-block proliferation index (CBPI) is a direct measure of the proliferative activity of the cells and it was determined in at least 1000 cells per culture (at least 2000 cells per test group). This value indicates the average number of cell cycles per cell during the period of exposure to the actin polymerisation inhibitor cytochalasin B. The CBPI is used to calculate the % cytostasis (relative inhibition of cell growth compared to the respective vehicle control group) i.e. a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.
OTHER EXAMINATIONS:
- pH
- Osmolarity
- Precipitation - Evaluation criteria:
- A test substance is considered to be clearly positive if the following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of our laboratory’s historical negative control data
A test substance is considered to be clearly negative if the following criterion is met:
• Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data. - Species / strain:
- human lymphoblastoid cells (TK6)
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- human lymphoblastoid cells (TK6)
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- PH and osmolarity were not influenced.
Precipitation was observed at 100µg/mL -S9 and at 800µg/mL +S9 in the first experiment.
Cytotoxicity
Cell numbers were not reduced to below 50% at any concentration tested. But proliferation was strongly reduced at and above 2.5µg/mL without S9 and at and above 12.5µg/mL with S9. - Remarks on result:
- other: positive results obtained only after 24h of treatment, not after 4h treatment
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- 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:
- July 16
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- May 2008
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Version / remarks:
- August 1998
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell transformation assay
- Target gene:
- HPRT
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Modal number of chromosomes: 20
- Normal (negative control) cell cycle time: 12-16h
MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Ham's F12 supplemented with 10% FCS except during treatment. All media were supplemented with 1% (v/v) penicillin/streptomycin and 1% (v/v) amphotericine B. CO2 concentration was 5%
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbital and β-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- A pretest was performed up to 2200µg/ml (app. 10.7mM). PH was not influenced. Precipitation occured at and above 275µg/ml in the absence at and above 68.75µg/ml in the presence of S9 mix.
After 4 hours of treatment without S9, relative survival of the cells was reduced to below 20% at 8.59µg/ml. In the presence of S9, relative survival was reduced at and above 68.75µg/ml.
Based on these results the following doses were selected for the main study:
-S9, 1st exp. +S9, 1st exp.
0.10 μg/mL 1.56 μg/mL
0.20 μg/mL 3.13 μg/mL
0.39 μg/mL 6.25 μg/mL
0.78 μg/mL 12.50 μg/mL
1.56 μg/mL 25.00 μg/mL
3.13 μg/mL 50.00 μg/mL
6.25 μg/mL 100.00 μg/mL
12.50 μg/mL 200.00 μg/mL
-S9, 2nd exp. +S9, 2nd exp.
0.29 μg/mL 2.34 μg/mL
0.59 μg/mL 4.69 μg/mL
1.17 μg/mL 9.36 μg/mL
2.34 μg/mL 18.75 μg/mL
4.69 μg/mL 37.50 μg/mL
9.36 μg/mL 75.00 μg/mL
18.75 μg/mL 150.00 μg/mL - Vehicle / solvent:
- DMSO
Selected, due to the insolubility of the test substance in aqueous media - Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding: 20x10^6 cells /40mL
DURATION
- Preincubation period: 1 day
- Exposure duration: 4h
- Expression time (cells in growth medium):7-9 days
- Selection time (if incubation with a selection agent): 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 16
SELECTION AGENT (mutation assays): 6-thioguanine
NUMBER OF REPLICATIONS: 2
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- Acceptance criteria
The HPRT assay is considered valid if:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should be within our historical negative control data range (95% control limit).
• The positive controls both with and without S9 mix should induce a distinct, statistically significant increase in mutant frequencies in the expected range
Assessment criteria
A test substance is considered to be clearly positive if:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
And
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative/vehicle control value and the range of our laboratory’s historical negative control data (95% control limit)
Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
A test substance is considered to be clearly negative if:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit) - Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Cytotoxic effects, as indicated by clearly reduced relative survival of about or below 20% of the respective negative control values were observed in both experiments in the absence and presence of S9 mix at least at the highest applied concentrations. In the absence of S9 mix, there was a strong decrease in the number of colonies at 12.50 μg/mL (RS: 0.0%) in the 1st Experiment and from 9.36 μg/mL (RS: 3.4%) onward in the 2nd Experiment after an exposure period of 4 hours. In addition, in the presence of S9 mix, there was a severe decrease in the number of colonies from 100.00 μg/mL (RS: 1.0%) onward in the 1st Experiment and from 75.00 μg/mL (RS: 1.3%) onward in the 2nd Experiment.
In this study, in the absence of S9 mix, after 4 hours treatment the morphology and attachment of the cells was adversely influenced (grade > 2) from 6.25 μg/mL onward in the 1st Experiment and at 4.69 μg/mL and above in the 2nd Experiment. In addition, in the presence of S9 mix, after 4 hours treatment with 50.00 μg/mL and above in the 1st Experiment and with 75.00 μg/mL and above in the 2nd Experiment, the morphology and attachment of the cells was adversely influenced (grade > 2).
Osmolality and pH values were not influenced by test substance treatment. In this study, in the absence of S9 mix precipitation was not observed up to the highest concentration applied in both experiments. In the presence of S9 mix, test substance precipitation was observed macroscopically in culture medium at the end of treatment at the highest applied concentrations in the 1st and the 2nd Experiment (200.00 or 150.00 μg/mL respectively).
Referenceopen allclose all
The following experiments were invalid due to the indicated reasons. The results from these experiments are not reported, yet will be archived in the raw data of this study:
Experiment I in the presence and absence of S9 mix: Due to strong cytotoxicity in both experimental parts not enough scorable dose groups were provided. Thus, a guideline-conform scoring of three dose groups per experimental part was not given.
Experiment III in the presence of S9 mix: Due to a faulty application of CytB the cultures were abandoned.
Results without S9 mix
Experiment | Exposure / Preparation Interval | Group | Micronucleated cells [%] | Citotoxicity (CBPI) [%] |
2 | 4/24 hrs | neg. control | 1.4 | 0 |
0.63 | 1 | 19.5 | ||
1.25 | 1.1 | 22.9 | ||
2.5 | 1.1 | 47.3 | ||
5 | n.s. | 80.4 | ||
pos. control | 6.1* | 29.6 | ||
3 | 24/24 hrs | neg. control | 1.1 | 0 |
0.63 | 1.2 | 17.2 | ||
1.25 | 1.3 | 19.1 | ||
2.5 | 3.2* | 44.2 | ||
5 | n.s. | n.s. | ||
pos. control | 2.4* | 28.4 | ||
5 | 24/24 hrs | neg. control | 0.6 | 0 |
0.63 | 0.8 | 16.3 | ||
1.25 | 1 | 10.1 | ||
2.5 | 2.6* | 28.9 | ||
3.75 | n.s. | n.s. | ||
pos. control | 2.3* | 15.8 |
* significant differences
n.s.: not scorable due to strong cytotoxicity
Experiments without S9
Experiment | Test Groups | Mutant frequency (per 10^6 cells) | Relative survival | Cloning efficiency2 |
1 | Neg. Control | 1.04 | 100.0 | 100.0 |
0.10 | 2.05 | 124.0 | 101.4 | |
0.20 | 0.70 | 129.0 | 98.6 | |
0.39 | 1.45 | 110.3 | 95.2 | |
0.78 | 4.35 | 124.2 | 95.5 | |
1.56 | 3.17 | 102.3 | 87.2 | |
3.13 | 2.88 | 110.5 | 84.1 | |
6.25 | 2.51 | 62.4 | 68.9 | |
12.50 | n.c. | 0.0 | n.c. | |
Pos. Control | 110.18S | 155.9 | 78.2 | |
2 | Neg. Control | 4.50 | 100.0 | 100.0 |
0.29 | 2.21 | 117.0 | 81.4 | |
0.59 | 3.79 | 117.3 | 95.2 | |
1.17 | 3.69 | 106.1 | 89.5 | |
2.34 | 10.54S | 98.9 | 94.0 | |
4.69 | 6.53 | 74.4 | 87.4 | |
9.36 | n.c. | 3.4 | n.c. | |
18.75 | n.c. | 0.0 | n.c. | |
Pos. Control | 109.29S | 94.2 | 80.8 |
Experiments with S9
Experiment | Test Groups | Mutant frequency (per 10^6 cells) | Relative survival | Cloning efficiency2 |
1 | Neg. Control | 1.27 | 100.0 | 100.0 |
1.56 | 1.75 | 122.8 | 108.2 | |
3.13 | 2.90 | 96.7 | 87.3 | |
6.25 | 2.41 | 99.4 | 78.8 | |
12.5 | 2.73 | 99.5 | 81.0 | |
25 | 4.61 | 76.9 | 68.7 | |
50 | 1.67 | 24.6 | 75.6 | |
100 | n.c. | 1.0 | n.c. | |
200 | n.c. | 0.0 | n.c. | |
Pos. Control | 99.02S | 69.3 | 64.9 | |
2 | Neg. Control | 2.92 | 100.0 | 100.0 |
2.34 | 3.51 | 95.7 | 99.7 | |
4.69 | 3.31 | 112.1 | 96.8 | |
9.36 | 2.86 | 137.3 | 91.8 | |
18.75 | 1.67 | 104.7 | 87.5 | |
37.5 | 3.09 | 65.0 | 94.5 | |
75 | n.c. | 1.3 | n.c. | |
150 | n.c. | 0.0 | n.c. | |
Pos. Control | 101.49S | 86.4 | 78.1 |
n.c.: not continued due to strong cytotoxicity
S: significantly different compared to control
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
MNT in vivo: negative (BASF 2018)
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: 5 - 8 weeks
- Assigned to test groups randomly: yes
- Housing: Makrolon cages
- Diet (e.g. ad libitum): ad lib.
- Water (e.g. ad libitum): tap water ad lib.
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Limited solubility of the test substance in water - Duration of treatment / exposure:
- 24 (all treatments), 48 h (control and high dose)
- Frequency of treatment:
- once
- Remarks:
- Females: 1500, 750, 375mg/kg
- Remarks:
- Males: 750, 375, 187.5mg/kg
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide: 20 mg/kg
- Tissues and cell types examined:
- Bone marrow
- Details of tissue and slide preparation:
- DETAILS OF SLIDE PREPARATION
The slides were stained in eosin and methylene blue (modified May-Gruenwald solution or Wrights solution) for about 5 minutes.
After having briefly been rinsed in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
Subsequently, the slides were stained in Giemsa solution (15 ml Giemsa, 185 ml purified water) for about 15 minutes.
After having been rinsed twice in purified water and clarified in xylene, the preparations were mounted in Gorbit-Balsam. - Evaluation criteria:
- Acceptance criteria
The mouse micronucleus test is considered valid if the following criteria are met:
- The quality of the slides must allow the identification and evaluation of a sufficient number of analyzable cells, i.e. >= 2000 PCEs and a clear differentiation between PCEs and NCEs.
- The ratio of PCEs/NCEs in the untreated animals (negative control) has to be within the normal range for the animal strain selected.
- The number of cells containing micronuclei in negative control animals has to be within the range of the historical control data both for PCEs and for NCEs.
- The two positive control substances have to induce a significant increase in the number of PCEs containing small and large micronuclei within the range of the historical control data or above.
Assessment criteria
A finding is considered positive if the following criteria are met:
- Significant and dose-related increase in the number of PCEs containing micronuclei.
- The number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control range.
A test substance is considered negative if the following criteria are met:
- The number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data. - Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- mortality at 1500mg/kg
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Sex:
- female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Since mortality occured in 1500mg/kg males, this dose could not be scored. An additional experiment was performed, adding a new low dose of 187.5mg/kg as well as the 48h evaluation for the new high dose of 750mg/kg for males. Concurrent controls were included in this experiment and valid.
The slight increase observed in the 48h control samples (females) and in the low dose (females) had no relation to dosing and was not confirmed when twice the number of PCEs were recounted.
There was no significant difference in the percentage of PCEs per erythrocytes between groups.
Reference
Males | |||||
Interval | Exp. | Micronuclei | Range | PCEs relative to control | |
Vehicle control | 24 | 2 | 1.1 ‰ | 2-6 | 100% |
Positive control | 24 | 2 | 16.9 ‰** | 51-86 | 104% |
187.5mg/kg | 24 | 2 | 1.5 ‰ | 2-10 | 104% |
Vehicle control | 24 | 1 | 1.9 ‰ | 2-10 | 100% |
Positive control | 24 | 1 | 13.5 ‰** | 34-71 | 130% |
375mg/kg | 24 | 1 | 1.7 ‰ | 5-8 | 115% |
750mg/kg | 24 | 1 | 1.9 ‰ | 3-12 | 125% |
Vehicle control | 48 | 2 | 1.1 ‰ | 3-7 | 100% |
750mg/kg | 48 | 2 | 1.3 ‰ | 1-9 | 97% |
Females | |||||
Interval | sample size | Micronuclei | Range | PCEs relative to control | |
Vehicle control | 24 | 4000 | 1.7 ‰ | 3-7 | 100% |
Vehicle control | 24 | 8000 | 1.5 ‰ | 10-14 | 100% |
Positive control | 24 | 4000 | 12.8 ‰** | 28-79 | 94% |
375mg/kg | 24 | 4000 | 2.5 ‰ | 5-16 | 85% |
375mg/kg | 24 | 8000 | 1.7 ‰ | 8-21 | 89% |
750mg/kg | 24 | 4000 | 1.3 ‰ | 3-7 | 97% |
1500mg/kg | 24 | 4000 | 1.5 ‰ | 4-8 | 79% |
Vehicle control | 48 | 4000 | 2.4 ‰ | 6-14 | 100% |
1500mg/kg | 48 | 4000 | 1.8 ‰ | 2-11 | 94% |
** significantly different from control
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
The substance Laromer BDDA was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The test strains are TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. The test concentrations are 22 µg - 5500 µg/plate (SPT), 11 µg - 2750 µg/plate (PIT; TA 1535 and TA 100), 2 µg - 550 µg/plate (PIT; TA 1537 and TA 98) and 22 - 5500 µg/plate (PIT, E.coli WP2 uvrA). No precipitation of the test substance was found. A bacteriotoxic effect was observed depending on the strain and test conditions from about 275 μg/plate onward. An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system. According to the results of the present study, the test substance Laromer BDDA is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.
This result is supported by a publicised plate incorporation Ames Test. Exposure of Salmonelly strains TA 1535, 1537, 100, 98 to up to 1000µg/plate with or without the addition of a metabolizing system led to bacteriotoxicity, but not to an increase in mutant frequencies.
In an HPRT assay in mammalian cells, up to 6.25µg/ml without metabolic activation and up to 50µg/ml with metabolic activation of BDDA were tested. The concentration was limited by the strong cytotoxic effects of the test substance. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations. A single statistically significant mutant frequency observed with the test substance in the 2nd Experiment in the absence of metabolic activation was considered biologically irrelevant. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.
The substance Laromer BDDA was assessed for its potential to induce micronuclei in TK6 cells in vitro (clastogenic or aneugenic activity). Five independent experiments were carried out, with and/or without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). Of these experiment I in the presence and absence of S9 mix was considered invalid due to strong cytotoxicity in both experimental parts, which led to an insufficient number of scorable dose groups. The cultures of experiment III in the presence of S9 mix were abandoned due to a faulty application of CytB. Of the remaining experiments, the highest concentrations that could be scored were 2.5µg/ml in the absence of S9 either for 4 or 24h treatment, and 25µg/mL in the presence of S9. Since no increase in the number of micronuclei was observed after treatment for 4h, an additional experiment was performed with addition of S9 with continous treatment for 24h. A significant increase in the number of micronuclei was observed at 2.5µg/mL in two independent experiments in the presence of strong cytotoxicity. A slightly higher concentration of 3.75µg/mL was not scorable due to excessive cytotoxicity. Consequently, BDDA was judged to be clastogenic in vitro at cytotoxic concentrations in the absence of metabolic activation.
To check the relevance of the results obtained in vitro, an in vivo MNT was subsequently performed.
Five male and female NMRI mice were orally exposed to doses of up to 1500mg/kg of BDDA in corn oil in a study according to OECD guideline 474. The highest dose led to mortality in male mice, so that the maximum evaluable dose in this sex is 750mg/kg. Treatment with BDDA did not lead to an increase in the number of micronuclei, while positive and negative (vehicle) controls gave the expected results. Two marginally (not significantly) increased values, one in the low dose in female animals, which was not confirmed after a higher number of cells were evaluated, and the other in the control group were considered as incidental. Consequently, it can be concluded that BDDA does not possess clastogenic properties.
Justification for classification or non-classification
BDDA did not cause point mutations either in bacteria or mammalian cells. In an MNT in vitro, an increase in the number of micronuclei was observed only without metabolic activation after 24h, and only at cytotoxic concentrations. This result could not be confirmed in an in vivo study. Consequently, there is no need to classify BDDA for mutagenicity according to Regulation (EC) No 1272/2008 (CLP).
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.