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Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: genome mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Full report; GLP; current guideline method performed on the analogue substance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
1994

Materials and methods

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

Test material

Constituent 1
Reference substance name:
Trisodium bis[3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(3-)
EC Number:
260-906-9
EC Name:
Trisodium bis[3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(3-)
Cas Number:
57693-14-8
Molecular formula:
C40H20CrN6O14S2.3Na
Test material form:
other: solid and liquid preparations

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS- 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

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Vehicle(s)/solvent(s) used: physiol. saline;
Details on exposure:
Dose: preliminary test (toxicity): 100-500 mg/kg bwmain test: 150 mg/kg bwPREPARATION OF DOSING SOLUTIONS: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:
single
Doses / concentrations
Remarks:
Doses / Concentrations:150 mg/kg bwBasis:other: nominal injected
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide; - Justification for choice of positive control(s): routinely used positve control- Route of administration: intraperitoneal- Doses / concentrations: 20 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow (femur)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:The selection of the Telon Echtschwarz LD 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 Telon Echtschwarz LD. 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 Telon Echtschwarz LD was chosen as MTD for this test.TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):DETAILS OF SLIDE PREPARATION: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 smearsThe 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.METHOD OF ANALYSIS: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
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY- 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 hoursRESULTS OF DEFINITIVE STUDY- 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.- Statistical evaluation:

Any other information on results incl. tables

Results of micronucleus test with Telon Echtschwarz LD after acute peritoneal treatment with 150 mg/kg bw

 group

 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 +/-1258  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

Conclusions:
Interpretation of results (migrated information): negativeThe analogue substance was tested in an vivo micronucleus test following OECD 474. Under the experimental conditions the analogue substance did not cause the formation of micronuclei in mice.
Executive summary:

The analogue substance 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.