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EC number: 229-158-0 | CAS number: 6420-33-3
- 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 vivo
Administrative data
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Remarks:
- Comet Assay
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- This in vivo study was performed as part of the OECD 422 study. This has been done as based on the positive outcome of the Ames test, an additional in vivo study is warranted. In order to minimize the number of animals necessary for the assessment and to further investigate the mutagenicity endpoint the assessment was incorporated in the repeated dose study as is also described in the guideline.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
- Principles of method if other than guideline:
- Six males from each dose group will be dosed to Day 45 and six N-Nitroso-N-methylurea dosed controls will kept without dosing during day 0-43 and will be dosed from day 43 to 45. For the males from Groups 1 to 4 of the OECD 422 study as well as all positive control males, samples of the liver (after recording of liver weight), glandular stomach and jejunum were processed to provide single cell suspensions with sufficient numbers of cells for the Comet Assay. The procedure was performed under subdued lighting and the Comet assay tissues/cells were processed as quickly as possible, using ice-cold buffers to maintain the tissues and cell preparations at low temperature.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian comet assay
Test material
- Reference substance name:
- Tetrasodium 3,3'-[carbonylbis[imino(5-methoxy-2-methyl-4,1-phenylene)azo]]bis(naphthalene-1,5-disulphonate)
- EC Number:
- 229-158-0
- EC Name:
- Tetrasodium 3,3'-[carbonylbis[imino(5-methoxy-2-methyl-4,1-phenylene)azo]]bis(naphthalene-1,5-disulphonate)
- Cas Number:
- 6420-33-3
- Molecular formula:
- C37H28N6Na4O15S4
- IUPAC Name:
- tetrasodium 3-({4-[({4-[(4,8-disulfonato-2-naphthyl)diazenyl]-2-methoxy-5-methylphenyl}carbamoyl)amino]-5-methoxy-2-methylphenyl}diazenyl)naphthalene-1,5-disulfonate
- Test material form:
- solid: flakes
- Details on test material:
- dentification: C.I. Direct Yellow 133
Physical state/Appearance: Brown/orange solid flakes
Expiry Date: 01 September 2021
Storage Conditions: Room temperature in the dark
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Strain: Wistar Han™:RccHan™:WIST
- Age at study initiation: 11 weeks
- Weight at study initiation: 274-361 g
- Housing: group housed up to three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK) (except during the mating period of the OECD 422 study)
- Diet: pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) ad libitum
- Water: ad libitum
- Acclimation period: 20 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at lest 15
- Photoperiod (hrs dark / hrs light): 12/12
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- corn oil for treatment groups (4 mL/kg), distilled water for positive control (N-Nitroso-N-methylurea ,10 mL/kg)
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
the test item was prepared at the appropriate concentrations as a suspension in Corn oil. Formulations were made daily from Days 1 to 8 of dosing, and dosed within four hours of being prepared. Following confirmed stability and homogeneity results, formulations were prepared weekly and stored refrigerated in the dark.
N-Nitroso-N-methylurea was dissolved in distilled water at appropriate concentration. A purity adjustment (for 90% purity) was made when preparing dosing formulations. Fresh formulations were prepared each day and dosed within two hours of preparation.
- Duration of treatment / exposure:
- 45 days for treatment groups, 3 days for positive control (undosed during the first 43 days)
- Frequency of treatment:
- daily
Doses / concentrationsopen allclose all
- Dose / conc.:
- 10 mg/kg bw/day (nominal)
- Dose / conc.:
- 30 mg/kg bw/day (nominal)
- Dose / conc.:
- 125 mg/kg bw/day (nominal)
- Remarks:
- initially dosed at 200 mg/kg bw during day 1-22, reduced to 125 mg/kg bw thereafter (due to mortality and sever weight loss)
- Dose / conc.:
- 2.5 mg/kg bw/day (actual dose received)
- Remarks:
- N-Nitroso-N-methylurea (positive control)
- No. of animals per sex per dose:
- 6 males for treatment groups, 5 males for positive controls
- Control animals:
- yes, concurrent vehicle
- yes, historical
- Positive control(s):
- N-Nitroso-N-methylurea
- Justification for choice of positive control(s): according to the guideline and historical control data are available in the laboratory
- Route of administration: gavage
- Doses / concentrations: 2.5 mg/kg bw
Examinations
- Tissues and cell types examined:
- glandular stomach, jejunum and liver
- Details of tissue and slide preparation:
- Liver - A small piece of liver was excised (approximately 1 cm2) and washed in liver buffer, (Hanks balanced salt solution supplemented with EDTA), before being minced and filtered to provide a single cell suspension.
Glandular Stomach and Jejunum - The stomach was removed and cut longitudinally to allow the stomach contents to be removed. Half the stomach was removed for possible histopathology and the remaining stomach was immersed in stomach buffer (Hanks balanced salt solution supplemented with EDTA and EGTA) and incubated for approximately 15 minutes on ice. The mucosal layer of the stomach was removed by scraping and a single cell suspension obtained by scraping the underlying glandular stomach tissue and suspending it in stomach buffer. The resulting cell suspension
was filtered through gauze prior to use for the comet slides.
A section of jejunum (approximately 2 cm2) was removed, cleaned and then immersed in stomach buffer for approximately 15 minutes on ice. A cell suspension was obtained by scraping the tissue of the jejunum into stomach buffer and filtering it through gauze.
Adequate numbers of slides were pre-coated with 0.5% normal melting point agarose and stored at room temperature. The slides were labelled for animal number, study number and tissue type prior to use for the Comet assay.
Once the cell suspensions were obtained, approximately 30 μL of the cell suspension was added to 270 μL of 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 μL of this agarose/cell suspension mix was placed onto a pre-coated slide. Two gels were placed on
each slide, and 4 gels were prepared for each tissue. The agarose/cell suspension mix was immediately covered with a glass coverslip, transferred to a cold room at approximately 4 °C in the dark for approximately 20 minutes to allow it to solidify.
Once the LMP agarose had set, the coverslips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark overnight. All slides went through the subsequent processing.
After the lysis phase had been completed the slides were removed from the lysing buffer, rinsed with neutralization buffer (0.4M Tris pH 7.5) to remove residual detergents and salts and then placed randomly into an electrophoresis unit. The electrophoresis unit was filled with chilled electrophoresis buffer (pH >13) until the slide surface was just covered. The slides were left for approximately 20 minutes to allow the DNA to unwind, after which electrophoresis proceeded at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for 20 minutes. The buffer in the bath was chilled during the electrophoresis period and the temperature of the electrophoresis buffer was monitored at the start of unwinding, the start of electrophoresis and the end of electrophoresis to ensure the electrophoresis solution was maintained at low temperature (2-10 °C).
At the end of the electrophoresis period the bath was switched off, the slides gently removed and rinsed three times with neutralization buffer for approximately 5 minutes each time. The slides were then carefully drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry, after which they were stored prior to staining and scoring. For each tissue, two processed slide gels were scored blindly. To each dry slide gel, 75 μL of propidium iodide (20 μg/mL) was placed on top of the slide and overlaid with a clean cover slip. After a short period to allow hydration and staining of the DNA, the slide was placed onto the stage of a fluorescence microscope and scored for comets using a CCD camera attached to a PCbased image analysis program (Comet IV version 4.3.1). Two slide gels for each tissue for each animal were scored with a maximum of 100 cells per slide gel giving an accumulative total of 200 cells per tissue per ani. - Evaluation criteria:
- Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if:
a) at least one of the test doses exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is considered to be dose-related
c) the results are substantially outside the distribution of the historical negative control range
The test chemical is when all three conditions are met considered to be able to induce DNA strand breakage in the tissues studied
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no evidene of a dose-related response
c) all results are within historical negative control range
d) direct or indirect evidence to demonstrate exposure of, or toxicity to, the target tissue(s) has been achieved
The test chemical is then considered unable to induce DNA strand breakage in the tissues studied in this test system.
Results and discussion
Test results
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- liver degeneration, necrosis and apoptosis, jejunum yellow discolouration; stomach green/yellow coloured contents
- Vehicle controls validity:
- valid
- Remarks:
- within historical control ranges
- Positive controls validity:
- valid
- Remarks:
- within or slightly above historical control values
- Additional information on results:
- At 100 or 200/125 mg/kg bw/day there were small but statistically significant dose related increases (p<0.05 - p<0.01) in the mean and median percentage tail intensities over the vehicle control value for the liver. However, these increases were all within the laboratory historical control range for the vehicle used, and were set against a relatively low vehicle control value and were therefore considered to be due to cytotoxicity rather than DNA strand breakage. This is confirmed by the results of the histopathology on the liver where degeneration, necrosis and apoptosis were observed. The treatment with the test item was therefore considered not to induce any DNA damage in the liver.
Any other information on results incl. tables
Glandular Stomach
Dose level (mg/kg bw) |
Group mean % hedgehogs |
Group mean % tail intensity |
Group mean of median % tail intensity per animal |
Group mean SD % tail intensity |
0 |
4.27 |
2.35 |
0.58 |
4.18 |
30 |
4.74 |
2.08 |
0.49 |
3.66 |
100 |
5.83 |
2.72 |
0.82 |
4.21 |
200/125 |
4.42 |
2.25 |
0.63 |
3.70 |
25 (MNU) |
9.61 |
34.01 |
32.32 |
13.71 |
Jejunum
Dose level (mg/kg bw) |
Group mean % hedgehogs |
Group mean % tail intensity |
Group mean of median % tail intensity per animal |
Group mean SD % tail intensity |
0 |
8.72 |
3.48 |
1.07 |
6.06 |
30 |
11.05 |
4.03 |
1.48 |
6.06 |
100 |
9.25 |
3.63 |
1.18 |
6.01 |
200/125 |
10.74 |
4.00 |
1.32 |
6.79 |
25 (MNU) |
16.59 |
47.53 |
48.22 |
15.44 |
Liver
Dose level (mg/kg bw) |
Group mean % hedgehogs |
Group mean % tail intensity |
Group mean of median % tail intensity per animal |
Group mean SD % tail intensity |
0 |
0 |
0.35 |
0.02 |
0.85 |
30 |
0 |
0.40 |
0.03 |
0.97 |
100 |
0 |
0.52* |
0.03* |
1.45 |
200/125 |
0 |
0.54** |
0.04** |
1.27 |
25 (MNU) |
15.33 |
43.05 |
43.07 |
9.09 |
*P<0.05; ** p<0.01
Historical controls Liver (based on 15-16 experiments)
Dose level (mg/kg bw) |
Group mean % hedgehogs |
Group mean % tail intensity |
Group mean of median % tail intensity per animal |
SD % of tail intensity |
vehicle |
0.00-1.16 |
0.17-0.95 |
0.00-0.08 |
0.55-5.86 |
Positive control |
0.29-10.24 |
13.65-46.47 |
13.49-47.10 |
5.15-13.31 |
Applicant's summary and conclusion
- Conclusions:
- The substance is considered not to induce toxicologically significant increases in the mean or median percentage tail intensity values in the jejunum, glandular stomach and liver and therefore the substance was considered to be non-genotoxic to these tissues.
- Executive summary:
A Comet assay was performed on male rats that were dosed by gavage for 45 days in an OECD 422 study at 0, 30, 100 and 200/125 mg/kg bw. Rats that remained untreated until day 43 and were treated thereafter until day 45 with 2.5 mg/kg bw N-Nitroso-N-methylurea.
The Comet assay assessment revealed there was a small but statistically significant dose related increases in the percentage tail intensity and median percentage tail intensity of the liver but these were all within the background control ranges for the vehicle control. The increases were considered to be due to cytotoxicity rather than DNA strand breakage. This was confirmed by the results of the histopathology on the liver where degeneration, necrosis and apoptosis were observed. Therefore the substance was considered not to induce any DNA damage in the liver. It was concluded that there were no toxicologically significant increases in the mean or median percentage tail intensity values in the jejunum, glandular stomach and liver and therefore the substance was considered to be non-genotoxic to these tissues.
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