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EC number: 206-735-5 | CAS number: 371-40-4
- 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
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1999-05-05 to 1999-09-06
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 000
- Report date:
- 2000
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted July 21, 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Version / remarks:
- EPA 712-C-98-221, August 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
Test material
- Reference substance name:
- 3,5-difluoroaniline
- EC Number:
- 206-752-8
- EC Name:
- 3,5-difluoroaniline
- Cas Number:
- 372-39-4
- Molecular formula:
- C6H5F2N
- IUPAC Name:
- 3,5-difluoroaniline
- Test material form:
- other: solid
Constituent 1
Method
Species / strain
- Species / strain / cell type:
- lymphocytes: CeII Line L5178Y
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI 1640-HAT and nomral RPMI 1640 medium to reduce the amount of spontaneous mutants
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix - Phenobarbital i.p. and β-Naphthoflavone induced
- Test concentrations with justification for top dose:
- Experiment I (with S9 mix, 4 h): 20.3, 40.6, 81.3, 162.5, 325.0 µg/mL
Experiment I (without S9 mix, 4 h); Experiment II (without S9 mix, 24 h): 40.6, 81.3, 162.5, 325.0, 650.0 µg/mL
Experiment II (with S9 mix, 4 h): 25.0, 50.0, 100.0, 200.0, 400.0 µg/mL - Vehicle / solvent:
- - Solvent: DMSO
- Justification for choice of solvent: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
The final concentration of DMSO in the culture medium did not exceed 0.5 % (v/v).
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- dissolved in DMSO
- Positive control substance:
- 3-methylcholanthrene
- methylmethanesulfonate
- Remarks:
- MMS: 13 µg/mL; 3-MC 3 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: In medium
DURATION
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 72 hours
After the expression period the cultures were seeded into microtiter plates.
Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4x 10^3 cells in selective medium (see below) with TFT (SERVA, D-69042 Heidelberg).
The cloning efficiency (viability) was determined by seeding about 2.5 cells per well into microtiter plates (same medium without TFT). The plates are incubated at 37°C in 4.5 % CO2 / 95.5 % humidified air for 10 - 15 days to determine the plating efficiency and to evaluate mutagenicity. Then the plates were evaluated manually.
Culture medium: RPMI 1640 medium supplemented with 15 % horse serum (HS), 100 U/100 µg/mL Penicilin/ Streptomycin, 220 µg/mL Sodium-Pyruvate, and 1.25 U/mL Amphotericin used as antifungal.
Cloning Medium:
RPMI 1640 (complete culture medium)
Selective Medium:
RPMI 1640 (complete culture medium) by addition of 5 µg/m TFT.
NUMBER OF CELLS EVALUATED: 1 x 10E7
DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth
Test article concentrations between 10.2 and 1300 µg/mL (with and without S9-mix) were used to evaluate toxicity. - Evaluation criteria:
- A test substance is classified as positive if it induces either a reproducible concentration related increase in the mutant frequency or a reproducible positive response for at least one of the test points.
A test substance producing neither a reproducible concentration-related increase in the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
The test substance is classified as mutagenic if it reproducibly induces with at least one of the concentrations a mutation frequency that is two times higher than the mean spontaneous mutation frequency in the experiment.
The test substance is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such evaluation may be considered independently of an enhancement factor for induced mutants.
However, in the evaluation of the test results the historical variability of the mutation rates in negative and solvent controls and the mutation rates of all negative and solvent controls of this study are taken into consideration.
Results of test groups are rejected if either, the relative total growth or relative suspension growth is less than 10 % of the solvent control or the cloning efficiency II after the expression period is less than 20 %. - Statistics:
- No statistical evaluation is required.
Results and discussion
Test results
- Species / strain:
- mouse lymphoma L5178Y cells
- 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
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: No
RANGE-FINDING/SCREENING STUDIES:
After 4 hours of treatment strong toxic effects were observed at 325.0 µg/mL and above in the absence and at 81.3 µg/mL and above in the presence of S9 mix.
COMPARISON WITH HISTORICAL CONTROL DATA:
The highest negative control value (127 colonies) and the highest solvent control value (121 colonies) exceeded the range of our historical control values slightly. This effect was considered as irrelevant since the corresponding solvent or negative control was well within the range of the historical controls.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment I, strong toxic effects occurred at 650 µg/mL in the absence and 81.3 µg/mL and above in both parallel cultures in the presence of metabolic activation (4 h treatment).
Strong toxic effects were observed in the second experiment at 650 µg/mL without metabolic activation (continuous treatment for 24 h) and at 25 µg/mL and above in both parallel cultures with metabolic activation (4 h treatment). - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Summary of experiment I
|
|
Experiment I, culture I |
|
Experiment I, culture II |
|
|||||
|
Per ml |
mix |
Relative cloning efficiency I |
Relative total growth |
Mutant colonies/ 108 Cells |
Induction factor |
Relative cloning efficiency I |
Relative total growth |
Mutant colonies/ 108 Cells |
Induction factor |
Column |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
Neg. control with medium |
|
- |
100.0 |
100.0 |
29 |
|
100.0 |
100.0 |
43 |
|
Neg. control with DMSO |
|
- |
100.0 |
100.0 |
50 |
1.0 |
100.0 |
100.0 |
70 |
1.0 |
Pos. control with MMS |
13.0 |
- |
53.2 |
82.0 |
124 |
2.5 |
92.8 |
84.9 |
193 |
2.7 |
Test substance |
20.3 |
- |
101.5 |
Culture was not continued |
|
74.5 |
Culture was not continued |
|
||
Test substance |
40.6 |
- |
97.1 |
131.5 |
48 |
1.0 |
84.3 |
127.0 |
55 |
0.8 |
Test substance |
81.3 |
- |
109.5 |
93.7 |
52 |
1.0 |
94.8 |
90.5 |
50 |
0.8 |
Test substance |
162.5 |
- |
133.2 |
88.7 |
46 |
0.9 |
107.9 |
90.3 |
73 |
1.0 |
Test substance |
325.0 |
- |
82.8 |
39.3 |
71 |
1.4 |
103.8 |
53.4 |
56 |
0.8 |
Test substance |
650.0 |
- |
41.4 |
20.0 |
60 |
1.2 |
47.7 |
24.5 |
61 |
0.9 |
|
|
|
|
|
|
|
|
|
|
|
Neg. control with medium |
|
+ |
100.0 |
100.0 |
72 |
|
100.0 |
100.0 |
86 |
|
Neg. control with DMSO |
|
+ |
100.0 |
100.0 |
71 |
1.0 |
100.0 |
100.0 |
47 |
1.0 |
Pos. control with 3-MC |
3.0 |
+ |
96.9 |
68.5 |
221 |
3.1 |
118.7 |
81.2 |
199 |
4.2 |
Test substance |
20.3 |
+ |
61.8 |
43.5 |
63 |
0.9 |
64.9 |
51.3 |
47 |
1.0 |
Test substance |
40.6 |
+ |
49.8 |
44.5 |
91 |
1.3 |
74.1 |
29.0 |
72 |
1.5 |
Test substance |
81.3 |
+ |
32.1 |
22.0 |
110 |
1.6 |
40.0 |
26.2 |
91 |
1.9 |
Test substance |
162.5 |
+ |
34.3 |
14.8 |
132 |
1.9 |
35.6 |
22.7 |
69 |
1.5 |
Test substance |
325.0 |
+ |
16.0 |
13.9 |
93 |
1.3 |
20.3 |
7.5 |
141 |
3.0 |
Test substance |
650.0 |
+ |
1.7 |
Culture was not continued |
|
2.5 |
Culture was not continued |
|
Summary of experiment II
|
|
|
Experiment II, culture I |
|
Experiment II, culture II |
|
||||
|
Per ml |
mix |
Relative cloning efficiency I |
Relative total growth |
Mutant colonies/ 108 Cells |
Induction factor |
Relative cloning efficiency I |
Relative total growth |
Mutant colonies/ 108 Cells |
Induction factor |
Column |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
Neg. control with medium |
|
- |
100.0 |
100.0 |
80 |
|
100.0 |
100.0 |
91 |
|
Neg. control with DMSO |
|
- |
100.0 |
100.0 |
54 |
1.0 |
100.0 |
100.0 |
64 |
1.0 |
Pos. control with MMS |
13.0 |
- |
28.2 |
33.6 |
604 |
11.1 |
47.4 |
36.4 |
690 |
10.8 |
Test substance |
20.3 |
- |
74.2 |
Culture was not continued |
|
133.5 |
Culture was not continued |
|
||
Test substance |
40.6 |
- |
66.1 |
51.0 |
106 |
1.9 |
101.9 |
60.8 |
120 |
1.9 |
Test substance |
81.3 |
- |
60.5 |
69.7 |
69 |
1.3 |
91.3 |
134.1 |
44 |
0.7 |
Test substance |
162.5 |
- |
26.5 |
68.5 |
65 |
1.2 |
83.9 |
88.9 |
65 |
1.0 |
Test substance |
325.0 |
- |
43.1 |
34.1 |
69 |
1.3 |
65.6 |
75.6 |
72 |
1.1 |
Test substance |
650.0 |
- |
13.9 |
14.0 |
133 |
2.4 |
32.2 |
30.8 |
113 |
1.8 |
|
|
|
|
|
|
|
|
|
|
|
Neg. control with medium |
|
+ |
100.0 |
100.0 |
112 |
|
100.0 |
100.0 |
127 |
|
Neg. control with DMSO |
|
+ |
100.0 |
100.0 |
121 |
1.0 |
100.0 |
100.0 |
103 |
1.0 |
Pos. control with 3-MC |
3.0 |
+ |
57.6 |
84.4 |
283 |
2.3 |
86.7 |
77.2 |
280 |
2.7 |
Test substance |
12.5 |
+ |
44.0 |
Culture was not continued |
|
32.5 |
Culture was not continued |
|
||
Test substance |
25.0 |
+ |
27.0 |
35.4 |
117 |
1.0 |
28.8 |
21.4 |
147 |
1.4 |
Test substance |
50.0 |
+ |
28.9 |
18.3 |
187 |
1.6 |
24.6 |
12.3 |
155 |
1.5 |
Test substance |
100.0 |
+ |
9.0 |
8.7 |
330 |
2.7 |
14.0 |
7.3 |
219 |
2.1 |
Test substance |
200.0 |
+ |
12.2 |
5.2 |
344 |
2.9 |
6.4 |
5.2 |
174 |
1.7 |
Test substance |
400.0 |
+ |
1.3 |
0.8 |
438 |
3.6 |
0.9 |
0.3 |
311 |
3.0 |
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative
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.
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