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EC number: 235-476-0 | CAS number: 12239-87-1
- 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
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- Endpoint summary
- Stability
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 001
- Report date:
- 2001
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Test material form:
- solid: particulate/powder
- Remarks:
- An investigation regarding the presence of particles in the nanometer range was not part of the test material characterization at that time.
Constituent 1
- Specific details on test material used for the study:
- - Name of test material (as cited in study report): Heliogen Blau K 6915
- Test substance No.: 00/0503-1
- Analytical purity: >= 98 g / 100 g
- Lot/batch No.: 00-0503-1
- Storage condition of test material: Room temperature
Method
- Target gene:
- Salmonella typhimurium: Determination of the rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+).
Escherichia coli: Determination of the rate of induced back mutations of bacterial mutant from trypthophan auxotrophy (trp-) to tryptophan prototrophy (trp+).
Species / strainopen allclose all
- 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:
- S9 fraction from the liver of male Sprague-Dawley rats (treated i.p. with 500 mg/kg bw Aroclor 1254 5 days before sacrifice), mixed with a series of cofactors (MgCl2, KCl, glucose-6-phosphate, NADP, phosphate buffer pH7.4).
- Test concentrations with justification for top dose:
- 1st experiment:
Standard plate test with and without S9-mix (all strains): 0, 20, 100, 500, 2500 and 5000 µg per plate
2nd experiment:
Preincubation test with and without S9-mix (S. typhimurium T1535 and TA100): 0, 20, 100, 500, 2500 and 5000 µg per plate
3rd experiment:
Preincubation test with and without S9-mix (S. typhimurium TA1535, TA98 and E. coli WP2 uvrA): 0, 20, 100, 500, 2500 and 5000 µg per plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO had been demonstrated to be a suitable vehicle in bacterial reverse mutation tests; historical control data are available for DMSO.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- other: With S-9 mix: 2-aminoanthracene; without S-9 mix: N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
A standard plate test and a preincubation test were conducted, both with and without metabolic activation (S9 mix). Each test was conducted in triplicates.
STANDARD PLATE TEST:
The experimental procedure of the standard plate test (plate incorporation method) is based on the method of Ames et al. (Mut. Res. 31: 347-364, 1975) and Maron and Ames (Mut. Res. 113: 173-215, 1983)
Salmonella typhimurium:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42-45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation).
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
Composition of the minimal glucose agar:
980 mL purified water
20 mL Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.
Escherichia coli:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation).
After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds.
The composition of the minimal agar (SA1 selective agar) is based on the description of Green, M.H.L. and Muriel, W.J. (Mut. Res. 38: 3-32, 1976), with the exception of solution E (tryptophan solution), which has previously been added to the soft agar:
300 mL solution B (agar)
100 mL solution A (saline solution)
8 mL solution C (glucose solution)
10 mL solution D (casein solution)
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.
PREINCUBATION TEST:
The experimental procedure is based on the method described by Yahagi et al. (Mut. Res. 48: 121-130, 1977) and Matsushima et al. (Factors modulating mutagenicity in microbial tests. In: Norpoth, K.H. and R.C. Garner, Short-Term Test Systems for Detecting Carcinogens. Springer Verlag Berlin, Heidelberg, New York, 1980):
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) are incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.
CONTROLS:
Negative control:
Parallel with each experiment with and without S9-mix, negative controls (solvent control, sterility control) were carried out for each tester strain in order to determine the spontaneous mutation rate.
Positive controls:
The following positive control substances were used to check the mutability of the bacteria and the activity of the S9-mix. With S9 mix: 2-aminoanthracene, 2-AA (2.5 µg/plate for all 4 Salmonella strains, 60 µg/plate for E. coli strain); without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine, MNNG (5 µg/plate for TA 1535 and TA100); 4-nitro-o-phenylenediamine, NOPD (10 µg/plate for TA 98), 4-nitroquinoline-1-oxide, 9-Aminoacridine, AAC (100 µg/plate for TA 1537), 4-NQO (5 µg/plate for E. coli WP2 uvrA).
TITER DETERMINATION:
The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.
In the standard plate test, 0.1 mL of the overnight cultures is diluted to 10E-6 in each case. Test tubes containing 2 mL portions of soft agar containing maximal amino acid solution (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 mL vehicle (without and with test substance)
0.1 mL fresh bacterial culture (dilution: 10E-6)
0.5 mL S9 mix
In the preincubation test, 0.1 mL of the overnight cultures is diluted to 10E-6 in each case. 0.1 mL vehicle (with and without test substance), 0.1 mL bacterial suspension and 0.5 mL S9 mix are incubated at 37°C for about 20 minutes using a shaker. Subsequently, 2 mL of soft agar containing maximal amino acid solution for titer determination (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) is added.
After mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted. - Evaluation criteria:
- Assessment 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 S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if the number of revertants for all tester strains are within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.
Acceptance criteria:
Generally, the experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls is within the range of the historical negative control data for each tester strain.
- The sterility control reveales no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induce a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- The titer of viable bacteria is >= 1x10exp+8/ml.
Results and discussion
Test resultsopen allclose all
- 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:
- no cytotoxicity
- 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:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TOXICITY:
No bacteriotoxic effect was observed.
SOLUBILITY:
Test substance precipitation was found from about 100 µg/plate onward.
MUTAGENICITY:
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 in any bacterial strain tested.
Any other information on results incl. tables
Table 1: Maximum revertants/plate and corresponding test concentrations in thestandard plate test: |
|||
|
|||
Strain |
Tested compound |
Maximum revertants/plate [corresponding dose unit in µg/plate] |
|
|
|
without S9-mix |
with S9-mix |
S. typhimurium TA1535 |
DMSO |
21 ± 2 |
23 ± 2 |
Test substance |
23 ± 4 [2500] |
25 ± 2 [100] |
|
Positive Control |
639 ± 15 [5; MNNG] |
136 ± 33 [2.5; 2-AA] |
|
S. typhimurium TA100 |
DMSO |
108 ± 6 |
109 ± 3 |
Test substance |
104 ± 4 [20] |
112 ± 5 [500] |
|
Positive Control |
887 ± 73 [5; MNNG] |
809 ± 59 [2.5; 2-AA] |
|
S. typhimurium TA98 |
DMSO |
33 ± 2 |
44 ± 4 |
Test substance |
34 ± 5 [100] |
57 ± 8 [5000] |
|
Positive Control |
712 ± 33 [10; NOPD] |
572 ± 46 [2.5; 2-AA] |
|
S. typhimurium TA1537 |
DMSO |
10 ± 2 |
10 ± 3 |
Test substance |
9 ± 3 [20] |
12 ± 4 [2500] |
|
Positive Control |
609 ± 100 [100; AAC] |
95 ± 3 [2.5; 2-AA] |
|
E. coli WP2 uvrA |
DMSO |
48 ± 7 |
45 ± 3 |
Test substance |
45 ± 13 [20] |
52 ± 11 [500] |
|
Positive Control |
734 ± 46 [5; 4-NQO] |
228 ± 26 [60; 2-AA] |
|
2-AA = 2-aminoanthracene |
|||
MNNG = N-methyl-N'-nitro-N-nitrosoguanidine |
|||
NOPD = 4-nitro-o-phenylenediamine |
|||
AAC = 9-aminoacridine |
|||
4-NQO = 4-nitroquinoline-N-oxide |
|||
|
|||
Table 2: Maximum revertants/plate and corresponding test concentrations in thepreincubation test: |
|||
|
|||
Strain |
Tested compound |
Maximum revertants/plate [corresponding dose unit in µg/plate] |
|
|
|
without S9-mix |
with S9-mix |
S. typhimurium TA1535 |
DMSO |
17 ± 2 |
17 ± 1 |
Test substance |
17 ± 2 [100] |
16 ± 2 [20] |
|
Positive Control |
795 ± 40 [5; MNNG] |
203 ± 6 [2.5; 2-AA] |
|
S. typhimurium TA100 |
DMSO |
106 ± 6 |
109 ± 7 |
Test substance |
108 ± 6 [20] |
107 ± 4 [20] |
|
Positive Control |
1094 ± 105 [5; MNNG] |
889 ± 68 [2.5; 2-AA] |
|
S. typhimurium TA98 |
DMSO |
28 ± 5 |
31 ± 3 |
Test substance |
30 ± 7 [20] |
41 ± 4 [5000] |
|
Positive Control |
877 ± 68 [10; NOPD] |
632 ± 63 [2.5; 2-AA] |
|
S. typhimurium TA1537 |
DMSO |
9 ± 2 |
9 ± 2 |
Test substance |
9 ± 3 [100] |
9 ± 1 [20] |
|
Positive Control |
436 ± 18 [100; AAC] |
94 ± 5 [2.5; 2-AA] |
|
E. coli WP2 uvrA |
DMSO |
38 ± 5 |
29 ± 5 |
Test substance |
39 ± 4 [5000] |
35 ± 6 [2500] |
|
Positive Control |
483 ± 102 [5; 4-NQO] |
223 ± 7 [60; 2-AA] |
|
2-AA = 2-aminoanthracene |
|||
MNNG = N-methyl-N'-nitro-N-nitrosoguanidine |
|||
NOPD = 4-nitro-o-phenylenediamine |
|||
AAC = 9-aminoacridine |
|||
4-NQO = 4-nitroquinoline-N-oxide |
Applicant's summary and conclusion
- Conclusions:
- According to the results of the present study, the test substance did not lead to an increase in the number of his revertant colonies either with or without S9-mix in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.
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