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EC number: 226-107-4 | CAS number: 5280-80-8
- 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 OECD 471 with four strains; in addition read-across with CAS 5580-57-4, CAS 68516-73-4, CAS 5580-58-5, CAS 79953-85-8 (all negative)
Chromosome aberration: negative; OECD 473
HPRT: negative; OECD 476
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- (mostly due to limited documentation before GLP (only the arithmetic means were shown in tables; no data on test substance purity), only 4 S. typhimurium strains tested; the activation mixture was tested only with strain TA 1535).
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- Batch: EN 44395
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction of liver from rats induced with Aroclor 1254
- Test concentrations with justification for top dose:
- 15, 45, 135, 405 and 1215 µg/0.1 mL
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: see Details on test system and conditions
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
In the experiments in which the substance was metabolically activated, activation mixture was added also. 1 mL activation mixture contains: 0.3 mL S9 fraction and 0.7 mL of a solution of co-factors.
The plates were incubated for about 48 hours at 37 °C in darkness.
NUMBER OF REPLICATIONS: triplicate
Positive controls:
1) for strain TA 98: daunorubicin-HCl (DAUNOBLASTIN), 5 and 10 µg/0.1 mL phosphate buffer;
2) for strain TA 100: 4-nitroquinoline-N-oxide, 0.125 and 0.25 µg/0.1 mL phosphate buffer;
3) for strain TA 1535: N-methyl-N'-nitro-N-nitrosoguanidine, 3 and 5 µg/0.1 mL phosphate buffer;
4) for strain TA 1537: 9(5) aminoacridine hydrochloride monohydrate, 50 and 100 µg/0.1 mL DMSO.
The activation mixture was tested with Strain TA 1535 and cyclophosphamide (ENDOXAN-ASTA), 250 µg/0.1 mL phosphate buffer. - Evaluation criteria:
- A test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration.
- Statistics:
- When the colonies had been counted, the arithmetic mean was calculated.
- Key result
- Species / strain:
- other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- (tested up to precipitating concentrations)
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 45 µg/0.1 mL and above, the substance precipitated in soft agar. - Conclusions:
- negative
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2012-02-07 to 2012-05-25
- 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)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Harlan Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany
- Type of assay:
- mammalian cell gene mutation assay
- Specific details on test material used for the study:
- commercial grade
purity>99% - Target gene:
- HPRT locus
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1 %)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/b-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- Experiment I: 6.6, 13.1, 26.3, 52.5, 105.0, 210.0 µg/mL
Experiment II: 13.1, 26.3, 52.5, 105.0, 210.0 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: the test substance was diluted in medium
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Remarks:
- EMS: without S9 mix; DMBA: with S9 mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
Experiment I: 4 hours without and 4 hours with S9 mix
Experiment II: 24 hours without and 4 hours with S9 mix
The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment as described below.
Three or four days after treatment 1500000 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 300000 - 500000 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days.
SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH
NUMBER OF REPLICATIONS: 2
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration. - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the first experiment precipitation was observed at 105.0 µg/mL and above in the absence and at 26.3 µg/mL and above in the presence of metabolic activation. In the second experiment precipitation was observed at 210.0 µg/mL in the absence and at 52.5 µg/mL and above in the presence of metabolic activation. - Conclusions:
- negative
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted 21st of July, 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- BSL Bioservice Scientific Laboratiories GmbH, Behringstr. 6/8, 82152 Planegg, Germany
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- commercial grade
purity >99% - Target gene:
- not applicable
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Minimum essential medium (MEM) supplemented with 10% fetal bovine serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with penobarbital and b-naphthoflavone
- Test concentrations with justification for top dose:
- Experiment I:
without metabolic activation: 0.975, 7.8, 62.5, 125 and 250 µg/mL
with metabolic activation: 7.8, 250, 500 and 1000 µg/mL
Experiment II:
without metabolic activation: 62.5, 125, 250 and 500 µg/mL
with metabolic activation: 10, 400, 750 and 900 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: none
The test item was prepared in cell culture medium (MEM) followed by ultrasound for 5 – 8 minutes (immediately before treatment). After that the test item was well suspended. The solvent was compatible with the survival of the cells and the S9 activity. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- other: no solvent/vehicle used
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 and 20 hours
- Expression time (cells in growth medium): 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: at least 200
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index in 1000 cells
OTHER EXAMINATIONS:
- Determination of polyploidy: yes - Evaluation criteria:
- There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0% - 4.0% aberrant cells (with and without metabolic activation)).
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results and a statistical evaluation of the results is not regarded as necessary. However, for the interpretation of the data, both biological and thoroughly evaluated statistical significance should be considered together.
A test item is considered to be negative if there is no biologically relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose group. Although most experiments will give clearly positive or negative results, in some cases the data set will preclude making a definitive judgement about the activity of the test substance. - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The test item was suspended in cell culture medium. Consequently, precipitate of the test itern was observed in both experiments (see Tables 1 and 2). Precipitation was assessed after incubation with the test item by unaided eye.
In experiment I without metabolic activation, a biologically relevant decrease of the relative mitotic index (decrease below 70% rel. mitotic index) was noted at 250 µg/mL (42%, Table 1). The cell density was decreased at 125 µg/mL and 250 µg/mL down to 64% and 54%, respectively. With metabolic activation a biologically relevant decrease of the relative mitotic index was noted at 500 µg/mL and higher (54% at 500 µg/mL and 58% at 1000 µg/mL, Table 2). The cell density was decreased at 1000 µg/mL down to 54%.
In experiment II without metabolic activation, a biologically relevant decrease of the relative mitotic index was observed at 500 µg/mL (48%, Table 1). The cell density was not decreased. With metabolic activation, a biologically relevant decrease of the relative mitotic index was noted at 400 µg/mL and higher (49% at 400 µg/mL, 45% at 750 µg/mL and 43% at 900 µg/mL, Table 2). No decrease of the cell density was noted up to the highest concentration evaluated.
Polyploid cells: No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item. - Conclusions:
- negative
Referenceopen allclose all
Standard plate test (15 - 1215 µg/0.1 mL) | ||||||
Strain | Metabolic activation system | mean his+-revertant colonies (negative control) | maximum revertant factor (conc. (µg/0.1 mL)) | dose dependency | Assessment | maximum revertant factor (positive control) |
TA 98 | no | 11 | 1.4 (45) | no | negative | 17.5 / 34.1* (D-HCl) |
yes | 29 | 1.1 (45 / 135) | no | negative | / | |
TA 100 | no | 127 | 1.2 (135) | no | negative | 4.2 / 6.6** (NQNO) |
yes | 140 | 1.1 (405) | no | negative | / | |
TA 1537 | no | 3 | 1.7 (135) | no | negative | 12.8 / 138****(AA) |
yes | 5 | 1.0 (135 / 405) | no | negative | / | |
TA 1535 | no | 5 | 2.2 (1215) | no | negative | 158.8 / 173.8*** (MNNG) |
yes | 6 | 1.8 (15) | no | negative | 37.1 (CPP) | |
* 5 / 10 µg/0.1 mL | ||||||
** 0.125 / 0.25 µg/0.1 mL | ||||||
*** 3 / 5 µg/0.1 mL | ||||||
**** 50 / 100 µg/0.1 mL | ||||||
D-HCl = Daunorubicin | ||||||
NQNO = 4-Nitroquinolin-N-oxide | ||||||
MNNG = N-Methyl-N'-nitro-N-nitroso-guanidine | ||||||
AA = 9 (5) Aminoacridine hydrochloride | ||||||
CPP = Cyclophosphamide |
A slight increase in the number of back-mutant colonies (from 5 to 8) was observed in the experiment without microsomal activation on strain TA 1535. This is attributed to a higher incidence of spontaneously occurring back-mutants.
Table 1: Summary of results
conc. |
P |
S9 mix |
releativ cloning efficiancy I |
relative cell density |
releativ cloning efficiancy II |
mutant colonies/106cells |
induction factor |
releativ cloning efficiancy I |
relative cell density |
releativ cloning efficiancy II |
mutant colonies/106cells |
induction factor |
|
µg/mL |
culture I |
culture II |
|||||||||||
Experiment I / 4 h treatment |
|||||||||||||
solvent control in medium |
- |
100 |
100 |
100 |
12.9 |
1 |
100 |
100 |
100 |
9.7 |
1 |
||
Positive control (EMS) |
150 |
- |
96 |
84.9 |
91.5 |
70.2 |
5.4 |
93.4 |
127.8 |
86.1 |
50.7 |
5.2 |
|
Test item |
6.6 |
- |
96.4 |
culture was not continued# |
98.8 |
culture was not continued# |
|||||||
Test item |
13.1 |
- |
104 |
96.6 |
98.7 |
20.6 |
1.6 |
104.5 |
104.8 |
98.5 |
4.7 |
0.5 |
|
Test item |
26.3 |
- |
95.1 |
94.2 |
90.7 |
10.7 |
0.8 |
99 |
98.1 |
97.2 |
12.1 |
1.2 |
|
Test item |
52.5 |
- |
94.8 |
83.2 |
94.6 |
9.8 |
0.8 |
105.1 |
94.9 |
106.8 |
6.6 |
0.7 |
|
Test item |
105 |
P |
- |
97 |
89.4 |
91 |
10.8 |
0.8 |
96 |
131.6 |
107.3 |
6.4 |
0.7 |
Test item |
210 |
P |
- |
99 |
68.2 |
91 |
21.1 |
1.6 |
98.2 |
126.2 |
101.6 |
29.4 |
3 |
solvent control in medium |
+ |
100 |
100 |
100 |
23.9 |
1 |
100 |
100 |
100 |
9.2 |
1 |
||
Positive control (DMBA) |
1.1 |
+ |
50.6 |
76.3 |
94 |
446 |
18.6 |
58.2 |
107.2 |
86 |
376.5 |
41.1 |
|
Test item |
6.6 |
+ |
97.2 |
96 |
112.7 |
12.6 |
0.5 |
100.6 |
113.8 |
100.8 |
12.7 |
1.4 |
|
Test item |
13.1 |
+ |
94.7 |
95.1 |
122.7 |
17 |
0.7 |
103.1 |
117.2 |
95.4 |
9.5 |
1 |
|
Test item |
26.3 |
P |
+ |
90.1 |
97.2 |
118.1 |
11.7 |
0.5 |
94.3 |
112.1 |
96.3 |
19.6 |
2.1 |
Test item |
52.5 |
P |
+ |
94.8 |
culture was not continued## |
94.9 |
culture was not continued## |
||||||
Test item |
105 |
P |
+ |
93.3 |
87.6 |
110.3 |
14 |
0.6 |
101.6 |
111.5 |
109.2 |
11.8 |
1.3 |
Test item |
210 |
P |
+ |
89.7 |
87.3 |
123.3 |
11 |
0.5 |
101.5 |
115.1 |
92.4 |
10.9 |
1.2 |
Experiment II / 24 h treatment |
|
||||||||||||
solvent control in medium |
- |
100 |
100 |
100 |
6 |
1 |
100 |
100 |
100 |
11 |
1 |
||
Positive control (EMS) |
150 |
- |
95.8 |
95.2 |
92.9 |
405.8 |
67.2 |
78.5 |
81.6 |
63.2 |
524.4 |
47.8 |
|
Test item |
6.6 |
- |
98.4 |
culture was not continued# |
93.3 |
culture was not continued# |
|||||||
Test item |
13.1 |
- |
100.3 |
97.5 |
100.2 |
6.1 |
1 |
98.6 |
93 |
80.2 |
15.4 |
1.4 |
|
Test item |
26.3 |
- |
100 |
99.5 |
103.4 |
15.7 |
2.6 |
95.2 |
73.7 |
78.1 |
12.6 |
1.1 |
|
Test item |
52.5 |
- |
98.7 |
108 |
100.7 |
11.7 |
1.9 |
100 |
99.9 |
84.5 |
7.5 |
0.7 |
|
Test item |
105 |
- |
100.1 |
102 |
98.8 |
9.5 |
1.6 |
98.1 |
81.9 |
72.4 |
18.1 |
1.6 |
|
Test item |
210 |
P |
- |
96.8 |
82.5 |
90 |
29.5 |
4.9 |
97.8 |
76.5 |
57.9 |
14.9 |
1.4 |
Experiment II / 4 h treatment |
|||||||||||||
solvent control in medium |
+ |
100 |
100 |
100 |
8.2 |
1 |
100 |
100 |
100 |
6.1 |
1 |
||
Positive control (DMBA) |
1.1 |
+ |
86.6 |
112.7 |
99.5 |
369.1 |
44.8 |
88.2 |
51 |
63.4 |
495.3 |
81.6 |
|
Test item |
3.3 |
+ |
101.7 |
culture was not continued# |
104.3 |
culture was not continued# |
|||||||
Test item |
6.6 |
+ |
100.3 |
142.2 |
105.1 |
15.1 |
1.8 |
102.4 |
90.9 |
102.3 |
7.9 |
1.3 |
|
Test item |
13.1 |
+ |
100.9 |
204 |
94.7 |
13.1 |
1.6 |
106.2 |
121.7 |
94.2 |
6.5 |
1.1 |
|
Test item |
26.3 |
+ |
102 |
186.1 |
104.1 |
19.5 |
2.4 |
101.6 |
75.6 |
97.1 |
15.3 |
2.5 |
|
Test item |
52.5 |
P |
+ |
100.3 |
195 |
81.2 |
11 |
1.3 |
97.6 |
71.3 |
96.2 |
5.8 |
1 |
Test item |
105 |
P |
+ |
99.9 |
290.9 |
92 |
9.9 |
1.2 |
91.6 |
83.2 |
87.6 |
7.8 |
1.3 |
P = Precipitation
# cutlture was not continued as a minimum of only four concentrations is required
## culture was not continued to avoid analysis of too many precipitating concentrations
No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration. The mutant frequency remained well within the historical range of solvent controls. An increase of the induction factor reaching or exceeding the threshold of three times the mutation frequency of the corresponding solvent control was observed in the second culture of the first experiment without metabolic activation at 210 µg/mL and in the first culture of the second experiment without metabolic activation at 210 µg/mL. However, the increases were based on a rather low mutation frequency of the corresponding solvent controls of just 9.7 and 6.0 colonies per 106cells, respectively. Furthermore, the effect was not reproduced in the parallel culture under identical experimental conditions. Therefore, the increases of the induction factor were judged as biologically irrelevant fluctuation.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequency. A single significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the second experiment at culture I without metabolic activation. However, the trend was judged as biologically irrelevant as all of the values of the mutation frequency remained within the historical range of solvent controls.
Table 1: Experiment I and II, without metabolic activation
|
Dose Group |
Concentration [µg/mL] |
Relative Mitotic index [%] |
Cell Density [%] |
Mean % Aberrant Cells |
Laboratory Negative Control Range |
Precipitation |
|||||
Incl. Gaps |
Excl. Gaps |
yes |
no |
|||||||||
Experiment I 4h treatment, 20h preparation interval |
C |
0 |
100 |
100 |
1.5 |
1.0 |
0.0% – 4.0% aberrant cells |
|
X |
|||
1 |
0.975 |
94 |
100 |
2.0 |
1.0 |
|
X |
|||||
4 |
7.8 |
81 |
99 |
3.5 |
2.5 |
X |
|
|||||
7 |
62.5 |
76 |
83 |
1.5 |
1.5 |
X |
|
|||||
8 |
125 |
76 |
64 |
0.5 |
0.5 |
X |
|
|||||
9 |
250 |
42 |
54 |
0.5 |
0.5 |
X |
|
|||||
EMS |
900 |
97 |
101 |
10.5 |
8.0 |
|
X |
|||||
|
||||||||||||
Experiment II 20h treatment, 20h preparation interval |
C |
0 |
100 |
100 |
2.5 |
1.0 |
0.0% – 4.0% aberrant cells |
|
X |
|||
6 |
62.5 |
86 |
99 |
2.5 |
0.0 |
|
X |
|||||
7 |
125 |
95 |
100 |
1.0 |
0.5 |
X |
|
|||||
8 |
250 |
74 |
78 |
1.5 |
0.5 |
X |
|
|||||
9 |
500 |
48 |
71 |
1.5 |
0.5 |
X |
|
|||||
EMS |
400 |
79 |
90 |
12.5 |
10.5 |
|
|
|||||
|
C: negative control (culture medium)
EMS: Ethylmethanesulfonate
Table 2: Experiment I and II, with metabolic activation
|
Dose Group |
Concentration [µg/mL] |
Relative Mitotic index [%] |
Cell Density [%] |
Mean % Aberrant Cells |
Laboratory Negative Control Range |
Precipitation |
|||||
Incl. Gaps |
Excl. Gaps |
yes |
no |
|||||||||
Experiment I 4h treatment, 20h preparation interval |
C |
0 |
100 |
100 |
0.5 |
0.0 |
0.0% – 4.0% aberrant cells |
|
X |
|||
4 |
7.8 |
109 |
99 |
2.5 |
0.5 |
|
X |
|||||
9 |
250 |
75 |
80 |
2.0 |
1.5 |
X |
|
|||||
10 |
500 |
54 |
72 |
3.0 |
2.0 |
X |
|
|||||
11 |
1000 |
58 |
54 |
3.0 |
1.0 |
X |
|
|||||
CPA |
0.83 |
70 |
99 |
10.5 |
9.0 |
|
X |
|||||
|
||||||||||||
Experiment II 4h treatment, 20h preparation interval |
C |
0 |
100 |
100 |
4.0 |
2.5 |
0.0% – 4.0% aberrant cells |
|
X |
|||
1 |
10 |
93 |
97 |
3.0 |
2.5 |
|
X |
|||||
5 |
400 |
49 |
83 |
2.5 |
2.0 |
X |
|
|||||
7 |
750 |
45 |
89 |
3.5 |
1.5 |
X |
|
|||||
8 |
900 |
43 |
80 |
1.0 |
0.5 |
X |
|
|||||
CPA |
0.83 |
99 |
100 |
12.0 |
10.5 |
|
X |
|||||
|
C: negative control (culture medium)
CPA: Cyclophosphamide
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Reliable data from several studies on genetic toxicity are summarized in the table below:
| PY 93 | PY 94 | PY 95 | PY 128 | 155 |
5580-57-4 | 5580-58-5 | 5280-80-8 | 79953-85-8 | 68516-73-4 | |
Bacterial mutagenicity | Negative K1 Non Prival | Negative K1 Prival | Negative K2 Non Prival | Negative K1 Non Prival | Negative K2 Non Prival |
Clastogenicity in vitro |
|
|
Non clastogenic K1
|
| No indications of clastogenicity seen in the MLA |
Mutagenicity in mammalian cells in vitro |
|
|
Negative K1
|
| Negative K1 |
Micronucleus test in vitro | Negative K1 |
|
| Negative K1 | Negative K1 |
Bacterial gene mutation:
Mutagenicity in bacterial reverse mutation assays (Ames test) have been investigated with all members of 'yellow disazo condensation pigments' (CAS 5580-57-4, 5280-80-8, 68516-73-4, 79953-85-8, and 5580-58-5). Pigment Yellow 94 was tested using the Prival modification for azo substances. The pigments do contain an azo function which is embedded in a larger conjugated system and probably not accessible to enzymes.
Negative results were obtained in all tests with and without metabolic activation. All relevant tester stains were tested and test item concentrations were adequate.
Mammalian gene mutation:
Mutagenicity in mammalian cells has been investigated in two reliable studies according to OECD guideline 476 with CAS 5280-80-8 (Harlan Cytotest Cell Research GmbH, 2012) and with CAS 68516-73-4 (BASF, 2012). Whereas the latter is the smallest molecule of the group, the other is the one containing as building blocks the most critical aromatic amines. The GLP guideline study with CAS 5280-80-8 was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration applied in the pre-experiment (840 µg/mL) was limited by the suspendibility of the test item in aqueous medium. The concentration range of the main experiments was limited by the occurrence of precipitation of the test item. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.
Cytogenicity study in mammalian cells:
Clastogenicity in mammalian cells has been investigated in a reliable study with CAS 5280-80-8 (chromosome aberration test according to OECD 473, BSL, 2012) and indications on the absence of clastogenic properties can also gained from the mouse lymphoma assay with CAS 68516-73-4. As all available information indicates that the pigments are not taken up by cells, having one test was considered sufficient for in-vitro testing of clastogenicity.
CAS 5280-80-8 contains the most problematic amines as building blocks. In the GLP-compliant in-vitro study for clastogenicity, the chromosomes were prepared 20 h after start of treatment with the test item. The treatment interval was 4h with and without metabolic activation in experiment I. In experiment II, the treatment intervalwas 4 h with and 20h without metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations. The following concentrations were evaluated for microscopic analysis:
Experiment I:
without metabolic activation: 0.975, 7.8, 62.5, 125 and 250 µg/mL
with metabolic activation: 7.8, 250, 500 and 1000 µg/mL
Experiment II:
without metabolic activation: 62.5, 125, 250 and 500 µg/mL
with metabolic activation: 10, 400, 750 and 900 µg/mL
The test item was prepared in cell culture medium (MEM). Precipitation of the test item was observed at the end of the treatment by the unaided eye with and without metabolic activation in experiment I and II. Toxic effects of the test item were noted with and without metabolic activation in experiment I and II. In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control. In the experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls. The positive controls induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.
Justification for classification or non-classification
Classification, Labelling, and
Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for
classification purposes under Regulation (EC) No 1272/2008. Based on
available data on genetic toxicity, the test item is not classified
according to Regulation (EC) No 1272/2008 (CLP), as amended for the
tenth time in Regulation (EU) No 2017/776.
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