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Diss Factsheets

Toxicological information

Genetic toxicity: in vivo

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Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 24, 2002 to August 21, 2002
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
according to guideline
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
according to guideline
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Tetrasodium 4-amino-3,6-bis(2,5-dimethoxy-4-(2-sulfonatooxy- ethylsulfonyl)phenylazo)-5-hydroxy-naphthalene-2,7-disulfonate
Test material form:
solid: particulate/powder
Details on test material:
see below

Test animals

Details on test animals or test system and environmental conditions:
- Source: Harlan Winkelmann GmbH, Gartenstrasse 27, 33178 Borchcn
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: Male animals-mean = 179.7 g (= 100 %) (min = 175 g (-2.6 %), max = 193 g (+7.4 %)); female animals- mean = 140.2 g (=100 %) (min = 135 g (-3.7%), max = 150 g (+7.0%))
- Assigned to test groups randomly: Yes, under following basis: randomization schemes 2002.0403 and 2002.0404
- Housing: Five animals per cage in transparent macrolon cages (type IV) on soft wood granulate in an air conditioned room.
- Diet (e.g. ad libitum): Rat/mice diet ssniff R/M-H (V 1534), ad libitum, ssniff® GmbH, Postbox 2039, 59480 Soest
- Water (e.g. ad libitum): Tap water in plastic bottles, ad libitum
- Acclimation period: 5 d under study conditions
- Animal identification: Fur marking with KMnO4 and cage numbering

- Temperature (°C): 22°C (except short lasting deviations due to disturbances of air condition)
- Humidity (%): 50% (except short lasting deviations due to disturbances of air condition)
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

IN-LIFE DATES: From July 30, 2002 to August 01, 2002

Administration / exposure

Route of administration:
oral: gavage
- Vehicle(s)/solvent(s) used: sesame oil
- Concentration of test material in vehicle: 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SUSPENSION: The test substance was suspended in sesame oil at a appropriate concentration. A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.
Duration of treatment / exposure:
2 d
Frequency of treatment:
twice at an interval of 24 h
Doses / concentrations
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control: Cyclophosphamide
Dissolved in: distilled water
Dose: 40 mg/kg bw
Route and frequency of administration: Oral (gavage), once
Volume Administered: 10 mL/kg bw


Tissues and cell types examined:
- 2,000 polychromatic erythrocytes were counted for each animal.
- The number of cells with micronuclei was recorded, not the number of individual micronuclei.
- The ratio of polychromatic erythrocytes to 200 normochromatic erythrocytes was determined.
- Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on the results of the acute oral toxicity study the dose of 2,000 mg/kg bw was selected as the limit dose, for the main study.

-Extraction of the bone marrow: Animals were killed by carbon dioxide asphyxiation 24 h after dosing. One femora was removed and the bone freed of muscle tissue. The proximal end of the femora was opened, the bone marrow flushed into a centrifuge tube containing about 3 mL of fetal bovine serum and a suspension was prepared. The mixture was then centrifuged for 5 minutes at approximately 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 h.

-Staining procedure: The slides were stained as follows:-
-5 minutes in methanol
-5 minutes in May-Grunwald's solution
-brief rinsing twice in distilled water
-10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
-rinsing in distilled water
-coating with Entellan
Evaluation criteria:
Both biological and statistical significances were considered together for evaluation purposes. A test substance is considered as positive if there is a significant dose- related increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant dose-related increase in the number of micronucleated polychromatic erythrocytes is considered non-clastogenic in this system.
Assuming the study is valid based on a monotone-dose-relationship, one-sided Wilcoxon tests were performed initially comparing control values with those of the highest dose group. A significance level of 5% is adopted for all tests.

Results and discussion

Test results
Key result
no effects
Vehicle controls validity:
Positive controls validity:
Additional information on results:
-All animals survived after treatment. No signs of toxicity were observed.
- All treated animals showed blue-black discolored feces 24 h after the first administration.
-The dissection of the animals revealed an blue-black colored content of the gastro-intestinal tract.

Applicant's summary and conclusion

Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the rat. Therefore, the test substance was not considered to be clastogenic in this micronucleus assay.
Executive summary:

A study was conducted to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat according to OECD Guideline 474, EPA OPPTS 870.5395 and EU Method B.12, in compliance with GLP.

The test substance was suspended in sesame oil and given twice at an interval of 24 h as a oral dose of 2,000 mg/kg bw/d to male and female rats. The dose was selected based on the results of a previous rat acute oral toxicity study. The animals were sacrificed 24 h after the last administration and bone marrow cells were collected for micronuclei analysis. After treatment with the test substance, the number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected and differed less than 20% from the control value.

Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the rat. Therefore, the test substance was not considered to be clastogenic in this micronucleus assay.