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EC number: 304-519-6 | CAS number: 94276-33-2
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
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- Density
- Particle size distribution (Granulometry)
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- Stability: thermal, sunlight, metals
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- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
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- 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
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- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
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- Exposure related observations in humans
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- 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
- Study period:
- 2017-02-13 to 2017-02-24
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997-07-21
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 2008-05-30
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- 1998-08
- Deviations:
- no
- Principles of method if other than guideline:
- Prival Modification
- Short description of test conditions: The Prival preincubation test is a modification of the standard Ames reverse mutation assay (1), in which flavin mononucleotide (FMN), liver S9 mix from uninduced hamsters and a preincubation step are used to facilitate azo reduction and the detection of the resulting mutagenic aromatic amines.
(1) Prival, M.J.; Mitchell, V.D.:
Analysis of a method for testing azo dyes for mutagenicity in Salmonella typhimurium in the presence of flavin monoucleotide and hamster liver S9. Mut. Res., 97, 103 - 116 (1982) - GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Hydrogen [3-[[1-[anilinocarbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzene-1-sulphonato(3-)]hydroxychromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)
- EC Number:
- 304-519-6
- EC Name:
- Hydrogen [3-[[1-[anilinocarbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzene-1-sulphonato(3-)]hydroxychromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)
- Cas Number:
- 94276-33-2
- Molecular formula:
- C16 H12 Cr N4 O9 S . C11 H25 N O . H
- IUPAC Name:
- hydrogen 3-[(2-ethylhexyl)oxy]propan-1-amine 11-methyl-4-nitro-12-(phenylcarbamoyl)-6-sulfonato-8λ³-oxa-10λ³-oxa-1λ⁴,13-diaza-9-chromatricyclo[7.4.0.0²,⁷]trideca-1(13),2(7),3,5,10-pentaene-9,9,9-tris(ylium)-8,12-diid-9-olate
- Test material form:
- solid
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No. of test material: 002-152503
- Expiration date of the batch: 2020-08-06
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. The test substance was dissolved in dimethyl sulfoxide (DMSO). To achieve a clear solution of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and was shaken thoroughly. The further concentrations were diluted from the stock solution according to the planned doses. All test substance formulations were prepared immediately before administration.
OTHER SPECIFICS: solid, orange to brown
Method
- Target gene:
- his/trp
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Remarks:
- uvrA
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/beta-naphthoflavone induced rat liver S9 fraction
- Test concentrations with justification for top dose:
- SPT/Prival: 0, 33, 100, 333, 1000, 2650, and 5300 µg/plate
In agreement with the recommendations of current guidelines 5 mg/plate or 5 µL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 µL/plate might also be tested in repeat experiments for further clarification/substantiation.
In this study, due to the purity of the test substance 5.3 mg/plate was used as top dose in all experiments - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Controlsopen allclose all
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (2-AA)
- Remarks:
- With rat liver S9 mix: 2.5 µg/plate, TA 1535, TA 100, TA 1537, TA 98; 60 µg/plate, Escherichia coli WP2 uvrA With hamster liver S9 mix: 10 µg/plate, TA 1535, TA 100, TA 1537, TA 98, Escherichia coli WP2 uvrA
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- congo red
- Remarks:
- With hamster liver S9 mix: 210 µg/plate, TA98
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
- Remarks:
- Without S9 mix: 5 µg/plate, TA1535, TA 100
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-o-phenylenediamine (NOPD)
- Remarks:
- Without S9 mix: 10 µg/plate, TA 98
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- Without S9 mix: 100 µg/plate, TA 1537
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- Without S9 mix: 5 µg/plate, E. coli WP2 uvrA
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
SPT: in agar (plate incorporation)
Prival: preincubation
DURATION
- Preincubation period: 30 min at 30 °C
- Exposure duration: 48 - 72 h (SPT, Prival) at 37 °C
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
- decrease in the number of revertants (factor < 0.6)
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables. Single values with a factor < 0.6 were not detected as toxicity in low dose groups.
- OTHER: For testing, deep-frozen (-70 °C to -80 °C) bacterial cultures (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) were thawed at room temperature, and 0.1 mL of this bacterial suspension was inoculated in nutrient broth solution (8 g/L Difco nutrient broth + 5 g/L NaCl) and incubated in the shaking water bath at 37 °C for about 12 - 16 hours. The optical density of the fresh bacteria cultures was determined. Fresh cultures of bacteria were grown up to late exponential or early stationary phase of growth (approximately 10E9 cells per mL). These cultures grown overnight were kept in iced water from the beginning of the experiment until the end in order to prevent further growth.
The use of the strains mentioned was in accordance with the current scientific recommendations for the conduct of this assay.
The Salmonella strains TA 1535, TA 100, TA 1537 and the Escherichia coli strain were obtained from Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA on 02 Dec 2014. The Salmonella strain TA 98 was obtained from Moltox Molecular Toxicology on 07 Jan 2015. - Evaluation criteria:
- Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- Fresh bacterial culture containing approximately 10E9 cells per mL were used.
Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 2650 μg/plate onward with and without S9 mix.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Toxicity was detected by a decrease in the number of revertants (factor ≤ 0.6) and/or clearing or diminution of the background lawn (= reduced his- or trp- background growth).
- Other observations when applicable: A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions from about 2650 μg/plate onward. In the prival preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions from about 1000 μg/plate onward. - Remarks on result:
- other: SPT, rat S9 mix
Any other information on results incl. tables
Table 1: SPT - without metabolic activation
Strain |
Test group |
Dose (µg/plate) |
Mean revertants per plate |
Standard deviation |
Factor |
TA 1535 |
DMSO Test item
MNNG |
- 33 100 333 1000 2650 5300 5.0 |
12.7 14.0 6.3 12.7 9.3 8.0 4.3 4894.7 |
1.5 1.0 2.3 1.2 1.5 3.0 2.1 91.3 |
- 1.1 0.5 1.0 0.7 P 0.6 B P 0.3 386.4 |
TA 100 |
DMSO Test item
MNNG |
- 33 100 333 1000 2650 5300 5.0 |
105.7 102.3 99.0 88.3 71.3 52.7 9.0 3696.7 |
9.0 9.5 3.5 4.2 8.1 5.7 3.6 115.8 |
- 1.0 0.9 0.8 0.7 P 0.5 P B 0.1 35.0 |
TA 1537 |
DMSO Test item
AAC |
- 33 100 333 1000 2650 5300 100 |
5.7 5.0 5.3 9.3 6.3 7.7 8.0 843.0 |
2.5 1.0 1.2 4.0 0.6 0.6 2.0 197.9 |
- 0.9 0.9 1.6 1.1 P 1.4 P B 1.4 148.8 |
TA 98 |
DMSO Test item
NOPD |
- 33 100 333 1000 2650 5300 10 |
21.3 22.0 24.7 21.3 18.3 21.3 5.3 569.7 |
1.2 1.0 10.6 4.0 1.5 12.3 3.1 22.5 |
- 1.0 1.2 1.0 0.9 P 1.0 P B 0.3 26.7 |
E.coli |
DMSO Test item
4-NQO |
- 33 100 333 1000 2650 5300 5 |
26.7 19.3 21.7 23.7 17.0 18.3 4.3 1851.7 |
7.8 7.8 1.5 5.8 3.6 3.2 3.2 139.5 |
- 0.7 0.8 0.9 0.6 P 0.7 P 0.2 69.4 |
B Reduced background growth
P Precipitation
Table 2: SPT- with metabolic activation
Strain |
Test group |
Dose (µg/plate) |
Mean revertants per plate |
Standard deviation |
Factor |
TA 1535 |
DMSO Test item
2-AA |
- 33 100 333 1000 2650 5300 2.5 |
10.3 10.3 7.7 13.7 14.3 13.3 4.3 243.7 |
1.5 5.5 2.1 3.1 3.2 1.5 3.2 17.8 |
- 1.0 0.7 1.3 1.4 P 1.3 P 0.4 23.6 |
TA 100 |
DMSO Test item
2-AA |
- 33 100 333 1000 2650 5300 2.5 |
93.0 104.3 88.7 83.0 65.7 45.7 17.0 2561.7 |
7.9 2.3 4.0 15.5 6.5 12.1 6.2 208.3 |
- 1.1 1.0 0.9 0.7 P 0.5 P 0.2 27.5 |
TA 1537 |
DMSO Test item
2-AA |
- 33 100 333 1000 2650 5300 2.5 |
6.3 8.7 10.0 7.0 10.0 8.0 5.3 228.3 |
0.6 3.2 4.0 2.6 3.6 1.0 2.5 7.0 |
- 1.4 1.6 1.1 1.6 P 1.3 P 0.8 36.1 |
TA 98 |
DMSO Test item
2-AA |
- 33 100 333 1000 2650 5300 2.5 |
19.7 21.7 22.7 30.3 24.7 23.3 6.7 1934.0 |
0.6 1.5 3.8 0.6 8.7 2.9 4.6 359.6 |
- 1.1 1.2 1.5 1.3 P 1.2 P 0.3 98.3 |
E.coli |
DMSO Test item
2-AA |
- 33 100 333 1000 2650 5300 60 |
23.7 29.3 19.3 30.3 22.3 17.0 6.0 96.0 |
2.1 8.6 7.1 2.1 4.9 2.0 1.0 26.3 |
- 1.2 0.8 1.3 0.9 P 0.7 P 0.3 4.1 |
P Precipitation
Table 3: Prival preincubation test - without metabolic activation
Strain |
Test group |
Dose (µg/plate) |
Mean revertants per plate |
Standard deviation |
Factor |
TA 1535 |
DMSO Test item
MNNG |
- 33 100 333 1000 2650 5300 5.0 |
10.0 12.3 9.7 10.7 12.0 9.3 8.3 2495.0 |
1.0 0.6 2.9 5.9 2.6 3.2 3.8 106.5 |
- 1.2 1.0 1.1 1.2 P 0.9 P B 0.8 249.5 |
TA 100 |
DMSO Test item
MNNG |
- 33 100 333 1000 2650 5300 5.0 |
103.3 101.3 111.7 94.3 91.3 62.7 17.7 2183.3 |
1.2 10.8 22.0 3.2 8.6 7.6 7.5 96.4 |
- 1.0 1.1 0.9 0.9 P B 0.6 P B 0.2 21.1 |
TA 1537 |
DMSO Test item
AAC |
- 33 100 333 1000 2650 5300 100 |
5.3 7.3 6.0 5.3 5.0 3.3 0.0 1424.0 |
2.1 2.5 1.0 0.6 1.0 2.3 0.0 726.6 |
- 1.4 1.1 1.0 0.9 P B 0.6 P B 0.0 267.0 |
TA 98 |
DMSO Test item
NOPD |
- 33 100 333 1000 2650 5300 10 |
18.3 17.0 21.3 16.3 6.3 4.0 0.0 588.7 |
5.8 5.6 4.5 3.8 1.5 2.6 0.0 69.1 |
- 0.9 1.2 0.9 B 0.3 P B 0.2 P B 0.0 32.1 |
E.coli |
DMSO Test item
4-NQO |
- 33 100 333 1000 2650 5300 5 |
19.3 18.0 14.3 15.7 10.7 5.7 0.0 376.7 |
4.9 7.0 3.5 0.6 3.2 4.0 0.0 20.0 |
- 0.9 0.7 0.8 0.6 P 0.3 P B 0.0 19.5 |
B Reduced background growth
P Precipitation
Table 4: Prival preincubation test - with metabolic activation
Strain |
Test group |
Dose (µg/plate) |
Mean revertants per plate |
Standard deviation |
Factor |
TA 1535 |
DMSO Test item
2-AA |
- 33 100 333 1000 2650 5300 10 |
10.0 11.7 9.0 9.7 12.0 14.3 7.0 866.7 |
1.0 3.5 1.0 2.1 3.5 1.2 3.5 147.2 |
- 1.2 0.9 1.0 1.2 P 1.4 P 0.7 86.7 |
TA 100 |
DMSO Test item
2-AA |
- 33 100 333 1000 2650 5300 10 |
116.7 113.0 120.0 120.0 87.7 29.3 0.0 2065.3 |
8.1 19.7 12.2 20.8 1.5 10.6 0.0 61.2 |
- 1.0 1.0 1.0 0.8 P B 0.3 P B 0.0 17.7 |
TA 1537 |
DMSO Test item
2-AA |
- 33 100 333 1000 2650 5300 10 |
8.0 7.7 7.7 7.7 6.7 4.0 0.0 100.7 |
2.6 1.2 2.5 2.1 2.3 3.5 0.0 15.3 |
- 1.0 1.0 1.0 0.8 P B 0.5 P B 0.0 12.6 |
TA 98 |
DMSO Test item
2-AA CoR |
- 33 100 333 1000 2650 5300 10 210 |
26.7 21.3 27.7 30.7 34.3 42.3 11.0 625.0 472.7 |
8.7 2.1 0.6 9.1 6.4 5.7 3.0 98.2 61.7 |
- 0.8 1.0 1.2 1.3 P 1.6 P B 0.4 23.4 17.7 |
E.coli |
DMSO Test item
2-AA |
- 33 100 333 1000 2650 5300 10 |
19.3 16.7 14.7 16.0 9.0 5.0 0.0 688.0 |
7.6 7.2 3.5 3.6 3.6 1.7 0.0 76.3 |
- 0.9 0.8 0.8 0.5 P 0.3 P B 0.0 35.6 |
B Reduced background growth
P Precipitation
Applicant's summary and conclusion
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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.