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EC number: 200-806-4 | CAS number: 74-31-7
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
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- Flash point
- Auto flammability
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- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
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- Endpoint summary
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- Environmental data
- 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
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- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
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- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- 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:
- 23 Aug 2016 to 25 Aug 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 21 Jul 1997
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- N,N'-diphenyl-p-phenylenediamine
- EC Number:
- 200-806-4
- EC Name:
- N,N'-diphenyl-p-phenylenediamine
- Cas Number:
- 74-31-7
- Molecular formula:
- C18H16N2
- IUPAC Name:
- N,N'-diphenyl-p-phenylenediamine
- Test material form:
- solid
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: QB-DBF-001
- Date of production/supply: Jul 2016
- Expiration date of the lot/batch: July 2018
- Purity test date: 35%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Physical state, appearance: solid, white to dark grey
- Solubility and stability of the test substance in the vehicle: To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The stability of a comparable batch (20150925) in the vehicle DMSO has been verified analytically.
Method
- Target gene:
- S. typhimurium: his
E. coli: trp
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix from phenobarbital (i.p.) and β-naphthoflavone (oral) induced rats livers
- Test concentrations with justification for top dose:
- JUSTIFICATION
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 14.3 mg/plate was used as top dose.
TEST CONCENTRATIONS
- Without S9 mix: 71.5; 357.5; 715; 3575; 7150; 14300 μg/plate.
- With S9 mix: 71.5; 357.5; 715; 3575; 7150; 14300 μg/plate. - Vehicle / solvent:
- - Vehicle 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.
Controls
- Untreated negative controls:
- yes
- Remarks:
- Sterility control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- other: see section "Any other information on materials and methods incl. tables"
- Details on test system and experimental conditions:
- METHOD OF APPLICATION
In agar (plate incorporation)
DURATION
- Exposure duration: 47 – 72 hours at 37°C in the dark
NUMBER OF REPLICATIONS
3 plates/dose
DETERMINATION OF CYTOTOXICITY
The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.
- decrease in the number of revertants (factor ≤ 0.6)
- clearing or diminution of the background lawn (= reduced his- or trp- background growth) - 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 10^9 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 historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.
Results and discussion
Test resultsopen allclose all
- Species / strain:
- S. typhimurium, other: TA 1535, TA 100, TA 1537, TA 98
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Decreased revertant numbers:TA 1535, TA 1537, and TA 98 3575 – 14300 μg/plate; TA 100 7150 - 14300 μg/plate
- 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:
- Decreased revertant numbers at 3575 – 14300 μg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium, other: TA 1535, TA 1537, TA 98
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- See 'Additional information on results'
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Decreased revertant numbers for TA 1535 at 7150 – 14300 μg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Reduced background growth at 14300 μg/plate
- 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 715 μg/plate onward with and without S9 mix.
ADDITIONAL INFORMATION ON MUTAGENICITY
- TA 1535: Increase in the number of his+ revertants at a concentration of 71.5 μg/plate (factor 3.1).
- TA 1537: Increase in the number of his+ revertants at concentrations of 7150 and 14300 μg/plate (factors 7.3 and 9.8, respectively).
- TA 98: Slight increase in the number of his+ revertants at a concentration of 7150 μg/plate (factor 1.8).
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see Table 1 in section "Any other information on results incl. tables"
- Negative (solvent/vehicle) historical control data: see Table 2 in section "Any other information on results incl. tables"
Any other information on results incl. tables
Table 1. Positive historical control data
Strain |
S9 mix |
Positive control |
Min |
Max |
Mean |
SD |
TA 1535 |
Without |
MNNG |
1174 |
6612 |
4063 |
1308.8 |
|
With |
2-AA |
108 |
367 |
217 |
50.0 |
TA 100 |
Without |
MNNG |
1125 |
5557 |
3331 |
1021.5 |
|
With |
2-AA |
361 |
2819 |
1792 |
448.9 |
TA 1537 |
Without |
AAC |
235 |
1858 |
975 |
339.9 |
|
With |
2-AA |
56 |
241 |
130 |
33.6 |
TA 98 |
Without |
NOPD |
271 |
1119 |
486 |
183.3 |
|
With |
2-AA |
431 |
2427 |
1427 |
361.9 |
E. coli |
Without |
4-NQO |
133 |
1552 |
884 |
442.4 |
|
With |
2-AA |
54 |
169 |
98 |
26.6 |
Abbreviations: MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; 2-AA, 2-aminoanthracene; AAC, 9-aminoacridine; NOPD, 4-nitro-o-phenylenediamine; 4-NQO, 4-nitroquinoline-N-oxide
Table 2. Negative historical control data
Strain |
S9 mix |
Min |
Max |
Mean |
SD |
TA 1535 |
Without |
6 |
17 |
10 |
2.2 |
|
With |
6 |
18 |
10 |
2.1 |
TA 100 |
Without |
69 |
140 |
95 |
9.9 |
|
With |
76 |
150 |
100 |
12.3 |
TA 1537 |
Without |
5 |
12 |
7 |
1.5 |
|
With |
5 |
16 |
8 |
2.2 |
TA 98 |
Without |
12 |
30 |
18 |
3.1 |
|
With |
13 |
38 |
24 |
4.7 |
E. coli |
Without |
12 |
33 |
22 |
4.0 |
|
With |
13 |
35 |
23 |
4.0 |
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