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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 genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September, 1985 to September 19, 1985
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:
Dilithium 7-acetamido-1-hydroxy-2-(4-((2-sulfonatooxy)ethylsulfonyl)phenylazo)naphthalene-3-sulfonate
EC Number:
EC Name:
Dilithium 7-acetamido-1-hydroxy-2-(4-((2-sulfonatooxy)ethylsulfonyl)phenylazo)naphthalene-3-sulfonate
Cas Number:
Molecular formula:
dilithium 7-acetamido-1-hydroxy-2-(4-((2-sulfonatooxy)ethylsulfonyl)phenylazo)naphthalene-3-sulfonate
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: HOECHST AG, Kastengrund, SPF breeding colony
- Age at study initiation: 8 weeks
- Weight at study initiation: male=32.63 g (27-37 g), female=25.74 g (22-30 g)
- Fasting period before study:
- Housing: in fully air conditioned rooms in Macrolon cages, on softwood granulate in group of 5 animals
- Diet: Altromin 1324, ad libitum
- Water: tap water in plastic bottles, ad libitum
- Acclimation period: at least 5 days

- Temperature (°C): 22±2°C
- Humidity (%): 55±10%
- Lighting time: 12 hours daily

Administration / exposure

Route of administration:
oral: gavage
- Vehicle: water

Details on exposure:

-The test substance dilution were freshly prepared each day. 7500 mg was weighed into a beaker, mixed with deionized water, washed out into a 25 mL flask and topped up to the calibration mark with water, a solution was formed.

- Endoxan stock solution: 25mL distilled water were added to 100mg Endoxan in an injection phial and shaken to form a clear solution. The daily solution for administration were prepared from this stock solution. For this purpose, 2 mL of the 2% stock solution were mixed with 6 mL distilled water.
Duration of treatment / exposure:
- Tratment: single administration
- Duration of exposure: 72 hours
Frequency of treatment:
- once
Post exposure period:
The animals were killed after 24, 48 and 72 hours
Doses / concentrations
Dose / conc.:
3 000 mg/kg bw (total dose)
No. of animals per sex per dose:
5 animals per sex per dose per time point
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: cyclophosphamide
- Route of administration: gavage
- Doses / concentrations: 50 mg/Kg bw.


Tissues and cell types examined:
Details of tissue and slide preparation:
Based on a preliminary study for determinati on of the acute toxicity the dose level for the micronucleus test was selected. Oral application at a dose of 4000 or 5000 mg per kg body weight caused diarrhoe and/or heavy vomiting. The highest dose which could be administered was 3000 mg per kg body weight. This dose level was used for the main study.

The animals were sacrificed according to the test procedure 24, 48 or 72 hours after application by carbon dioxide asphyxiation. For each animal about 3 mL foetal bovine serum was poured into a silicone-coated centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at 1200 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number, air-dried for about 24 hours.

- 5 minutes in methanol
- 3 minutes in May-Grünwalds solution
- 2 minutes in May-Grünwalds solution diluted 1:1 with distilled water
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts distilled water
- rinsing in distilled water
- drying
- providing with Entellan
The number of polychromatic erythrocytes with micronuclei occurring in 1000 counted polychromatic erythrocytes, and the number of normocytes with micronuclei occurring in 1000 counted normocytes, were evaluated statistically; comparison of dose groups with the simultaneous control group was performed according to wilcoxon (paired, one—sided, increase).
The results of the test substance at each killing time and dose were compared with the corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided).

Results and discussion

Test results
Key result
Vehicle controls validity:
Positive controls validity:
Additional information on results:
The incidence of micronucleated polychromatic erythrocytes in each dose group was not significantly different from the data in the negative control groups. The number of erythrocytes with micronuclei did not differ significantly from the values of the simultaneous control animals for each of the three killing times investigated. This is true for the polychromatic erythrocytes as well as for the normocytes. A small increase in the number of polychromatic erythrocytes with micronuclei in female mice 72 hours after dosing was not considered as biologically significant. It was due to an increase of micronuclei in only 1 out of 5 animals and was within the normal range of erythrocytes with micronuclei (mean,value). The ratio of polychromatic erythrocytes to normocytes remained uneffected by the test compound.
Cyclophosphamid induced a marked and statistically significant increase of the number of polychromatic erythrocytes with micronuclei in both males and females. The ratio of polychromatic erythrocytes to normocytes was not shifted (within the normal range).

Any other information on results incl. tables

All animal survived after the application of 0 or 3000 mg per Kg body weight.

Clinical signs of toxicity in test animals: diarrhoe (3/30 red coloured faeces), piloerection, back-arched position, chenge in gait, narrowed eye opening).

Applicant's summary and conclusion

Under the conditions described the application of the test substance did not lead to an elevated occurence of micronuclei in the polychromatic or normochromatic erythrocytes. It is concluded that the test substance is not mutagenic in the micronucleus test.
Executive summary:

The test substance was tested in the micronucleus test. The test compound was administered orally by gavage to male and female mice. The following doses were tested: 0 and 3000 mg per kg bodyweight. The dose level 3000 mg per kg bodyweight was chosen because a preliminary study had shown this dose level to be the maximum tolerated dose.

The animals were treated once with the test substance and sacrificed according to the test procedure 24, 48 or 72 hours after administration of the test compound.

The incidence of micronucleated polychromatic erythrocytes was not significantly effected by the test substance in comparison with the negative control. The number of normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained uneffected by the treatment with the test substance .

The results indicate that the test substance is not mutagenic in the micronucleus test under the conditions of the present study.