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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Trisodium bis[3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(3-)
EC Number:
EC Name:
Trisodium bis[3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(3-)
Cas Number:
Molecular formula:
trisodium bis[3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(3-)
Test material form:
solid: particulate/powder

Test animals

Details on test animals or test system and environmental conditions:
- Source: Harlan Winkelmann Borchen Germany
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 28 to 43 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: males: single; females: groups of - Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 1 week

- Temperature (°C): 22.5 - 23
- Humidity (%): 44 - 52
- Air changes (per hr): ca. 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
Vehicle(s)/solvent(s) used: physiol. saline;
Details on exposure:
preliminary test (toxicity): 100-500 mg/kg bw
main test: 150 mg/kg bw

Telon Echtschwarz LD was dissolved in physiological saline solution, using a magnetic stirrer for 30 minutes, stirred with a magnetic mixer during administration and injected intraperitoneally.

Administration volume was 10 ml/kg bw
Duration of treatment / exposure:
16, 24, or 48 hours
Frequency of treatment:
Doses / concentrations
Dose / conc.:
150 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Positive control(s):
- Substance: cyclophosphamide
- Justification for choice of positive control(s): routinely used positve control
- Route of administration: intraperitoneal
- Doses / concentrations: 20 mg/kg bw


Tissues and cell types examined:
Bone marrow (femur)
Details of tissue and slide preparation:
The selection of the dose was based on a pilot test, in which groups of five animals, including both males and females, were intraperitoneally administered 100 mg/kg, 150 mg/kg, 250 mg/kg and 500 mg/kg test substance. The following symptoms were recorded for up to 48 hours, starting at 100 mg/kg: apathy, black discoloration of hairless parts of skin, staggering gait, sternal recumbency, spasm, difficulty in breathing and eyelids stuck together. In addition, 1 of 5 animals died in the 150 mg/kg group and 5 of 5 animals died in the 250 and 500 mg/kg groups.
Based on these results, 150 mg/kg was chosen as MTD for this test.

At least one intact femur was prepared from each sacrificed animal (not pretreated with a spindle inhibitor). A suitable instrument was used to sever the pelvic bones and lower leg.
The femur was separated from muscular tissue.
The lower-Ieg stump, including the knee and all attached soft parts, was separated in the distal epiphyseal cartilage by a gentle pull at the distal end.
The proximal end of the femur was opened at its extreme end with a suitable instrument, e.g. fine scissors, making visible a small opening in the bone-marrow channel. The femur was then completely immersed in the calf serum and pressed against the wall of the tube, to prevent its slipping off.

A suitable tube was filled with sufficient fetal calf serum. A small amount of serum was drawn from the tube into a suitable syringe with a thin cannula. The cannula was pushed into the open end of the marrow cavity. The femur was then completely immersed in the calf serum and pressed against the wall of the tube, to prevent its slipping off.
The contents were then flushed several times and the bone marrow was passed into the serum as a fine suspension.
Finally, the flushing might be repeated from the other end, after it had been opened.

The tube containing the serum and bone marrow was centrifuged in a suitable centrifuge at approximately 1000 rpm for five minutes.
The supernatant was removed with a suitable pipette (e.g. Pasteur pipette), leaving only a small remainder.
The sediment was mixed to produce a homogeneous suspension.

One drop of the viscou suspension was placed on a well cleaned slide and spread with a suitable object, to allow proper evaluation of the smear.
The labeled slides were dried overnight. If fresh smears needed to be stained, they needed to be dried with heat for a short period.

The staining of smears
The smears were stained automatically with an Ames HemaTek Slide Stainer from the Miles Company. The slides were then "destained" with methanol, rinsed with deionized water and left to dry.

Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. They can be distinguished from artifacts by varying the focus.
Normally, 1000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern.
Evaluation criteria:
A test was considered positive if, at any of the intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control.
A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time.
A test was also considered negative if there was a significant increase in that rate which, according to the laboratory's experience was within the range of negative controls.
In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group. In this case, a second test had to be performed at the most sensitive interval.

An assay was considered acceptable if the figures of negative and positive controls were within the expected range, in accordance with the laboratory's experience and/or the available literature data.

Results and discussion

Test results
Vehicle controls validity:
Positive controls validity:
Additional information on results:
- Dose range: 100 - 500 mg /kg
- Solubility: soluble
- Clinical signs of toxicity in test animals: apathy, black discoloration of hairless parts of skin, staggering gait, sternal recumbency, spasm, difficulty in breathing and eyelids stuck together. In addition, 1 of 5 animals died in the 150 mg/kg group and 5 of 5 animals diedin the 250 and 500 mg/kg groups.
- Harvest times: 48 hours

- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay): see table
- Appropriateness of dose levels and route: intrapreitoneal administration secured target tissue exposure; the dose showed clear signs of systemic toxicity (mortality) in a preliminary test.

Any other information on results incl. tables

Results of micronucleus test with the substance after acute peritoneal treatment with 150 mg/kg bw


 Number of

evaluated PCE

 NCE/1000 PCE

Micronucelated cells

per 1000 NCE

 Micronucleated cells

per 1000 PCE

 negative control   10000

 1056 ± 386

 1,2 ± 1,2  1,3 ± 0,7
 test 16 hours   10000  2370 ± 258  1,2 ± 1,0  1,6 ± 1,6
 test 24 hours   10000  1537 ± 593  1,1 ± 0,8  2,0 ± 1,7
 test 48 hours   10000  1733 ± 1012  0,5 ± 0,7  1,6 ± 0,9
 positive control   10000  674 ± 232  0,4 ± 0,8  14,9 ± 9,8

Applicant's summary and conclusion

The test item did not cause the formation of micronuclei in mice in vivo.
Executive summary:


The test material was examined in mice in vivo for the formation of micronucleated erythrocytes.

Male as well as female mice were treated by intraperitoneal injection with 150 mg/kg bw.

The dose range was 0 (vehicle), 100 to 500 mg/kg bw in a preliminary test and 150 mg/kg bw in the main study. Cyclophosphamide was used as a positive control. Cells were harvested 16, 24 and 40 hours after treatment.


Positive as well as negative controls gave the expected results.

Toxicity was noted at doses of 150 mg/kg bw and above.

No increases of micronucleated cells were observed. The test item is not considered clastogenic in this test.