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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: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
not specified
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 7-benzamido-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate
EC Number:
EC Name:
Disodium 7-benzamido-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate
Cas Number:
Molecular formula:
disodium 7-benzamido-4-hydroxy-3-[(1E)-2-{4-[(1E)-2-(4-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-sulfonate
Test material form:
solid: particulate/powder

Test animals

Details on test animals or test system and environmental conditions:
Source: BRL Tierfarm Fullinsdorf CH-4414 Fullinsdorf/Basel, SwitzerlandNumber of Animals: 84 (42 males/42 females).Initial Age at Start of Acclimatization: minimum 10 weeksAcclimatization: minimiim 5 daysInitial Body Weight at Start of Treatment: approximately 30 gAccording to the suppliers assurance the animals were in healthy condition. The animals were under quarantine in the animal house of teh laboratory for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour.The animals were distributed into the test groups at random and identified by cage number.Housing Cage Type: single; Makrolon Type I, with wire mesh top (EHRET GmbH, D-7830 Eiranendingen)Bedding: granulated soft wood bedding (ALTROMIN, D-4937 Lage/Lippe)Feed: pelleted standard diet (ALTROMIN 1324, D-4937 Lage/Lippe)Water: tap water, ad libitum (Gemeindewerke, D-6101 RoBdorf)Environment! temperature: 21 +. 3°CRelative humidity: 30-70%Artificial light: 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:
oral: drinking water
Deionized water
Details on exposure:
The animals received the tested substance once.
Duration of treatment / exposure:
Approximately 18 hours before treatment with the test article the animals received no food but water ad libitum. At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test article once. Twelve animals, six males and six females, were treated per dose group.Sampling of the bone marrow was done 24, 48 and 72 hours after treatment.
Frequency of treatment:
One time.
Post exposure period:
72 hours
Doses / concentrations
Doses / Concentrations:2500 mg/kg bwBasis:nominal in water
No. of animals per sex per dose:
6 males / 6 females for each group
Control animals:
Positive control(s):
Name: CPA; CyclophosphamideSupplier: SERVA, D-6900 HeidelbergCatalogue no.: 17681Dissolved in: physiological salineDosing: 30 mg/kg b.w.Route and Frequency of Administration: orally, onceVolume Administered: 10 ml/kg b.w.Solution prepared on day of administration.The stability of CPA at room temperature is good. At 20°C only 1 % of CPA is hydrolysed per day in aqueous solution.


Tissues and cell types examined:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow wasflushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 10 minutes and the supernatant was discarded.A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Griinwald (MERCK, D-6100 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg). At least one slide was made from each bone marrow sample.
Evaluation of the slides was performed using NIKON microscopes with lOOx oil immersion objectives. 1000 polychromatic erythrocytes(PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides.Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

Results and discussion

Test results
no effects
Vehicle controls validity:
Negative controls validity:
Positive controls validity:

Applicant's summary and conclusion

Interpretation of results: negative
Executive summary:

The test article was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test article was formulated in deionized water. This vehicle was used as negative control. The volume administered orally was 20 ml/kg b.w.. 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei

analysis. Ten animals (5 male's, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 2500 mg/kg b.w.. In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei

at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with the test article were in the same range as compared to the negative control groups.

30 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronuleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.