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Diss Factsheets

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 22 to May 30, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
July 21, 1997
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
445-280-9
EC Name:
-
Cas Number:
371921-40-3
Molecular formula:
No molecular formula possible, it is a reaction mass.
IUPAC Name:
docosasodium 4-amino-3-[(1E)-2-[4-(ethenesulfonyl)phenyl]diazen-1-yl]-5-hydroxy-6-[(1E)-2-[4-(2-hydroxyethanesulfonyl)-2-sulfonatophenyl]diazen-1-yl]naphthalene-2,7-disulfonate 4-amino-3-[(1E)-2-[4-(ethenesulfonyl)phenyl]diazen-1-yl]-5-hydroxy-6-[(1E)-2-{2-sulfonato-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl]naphthalene-2,7-disulfonate 4-amino-5-hydroxy-6-[(1E)-2-{2-sulfonato-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl]-3-[(1E)-2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl]naphthalene-2,7-disulfonate 4-amino-6-[(1E)-2-[4-(ethenesulfonyl)-2-sulfonatophenyl]diazen-1-yl]-3-[(1E)-2-[4-(ethenesulfonyl)phenyl]diazen-1-yl]-5-hydroxynaphthalene-2,7-disulfonate 4-amino-6-[(1E)-2-[4-(ethenesulfonyl)-2-sulfonatophenyl]diazen-1-yl]-5-hydroxy-3-[(1E)-2-[2-(2-hydroxyethanesulfonyl)phenyl]diazen-1-yl]naphthalene-2,7-disulfonate 4-amino-6-[(1E)-2-[4-(ethenesulfonyl)-2-sulfonatophenyl]diazen-1-yl]-5-hydroxy-3-[(1E)-2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl]naphthalene-2,7-disulfonate

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd. CH4414 Füllinsdorf
- Age at study initiation: males: 5 - 7 weeks; females: 7 - 9 weeks
- Weight at study initiation: males mean value 26.2 g (SD ± 1.6 g); females mean value 24.9 g (SD ± 2.2 g)
- Assigned to test groups randomly: yes
- Fasting period before study: approximately 18 hours before treatment the animals received no food
- Housing: single, in Makrolon Type l, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (ALTROMIN 1 324, D-32791 Lage/Lippe)
- Water (e.g. ad libitum): tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 25 - 75 %
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
deionised water
Details on exposure:
All animals received a single standard volume of 10 ml/kg body weight orally.
Duration of treatment / exposure:
The animals received the test item once
Post exposure period:
Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
48 h preparation interval
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
24 h preparation interval
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
24 h preparation interval
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
24 h preparation interval
No. of animals per sex per dose:
5 per sex per dose
Control animals:
yes, concurrent vehicle
yes, historical
Positive control(s):
40 mg/kg b.w. cyclophosphamide as historical control

Examinations

Tissues and cell types examined:
polychromatic erythrocytes (PCE) from bone marrow
Details of tissue and slide preparation:
The animals were anaesthetized with CO2 and sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390xg) for 10 minutes and-the supernatant was discarded. A small drop of the resuspdended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grunwaid (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.
Evaluation criteria:
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at 2000 mg/kg
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: starvation period, animal strain; vehicle; route, frequency, and volume of administration.
The animals were treated orally with the test item and examined for acute toxic symptoms at intervals of around 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item. 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. Navy MGi 1571 formulated in deionised water. The volume administered was 10 ml/kg b.w. The animals treated with 2000 mg/kg b.w. expressed toxic reactions.

Historical Controls
1999 - 2001
Negative controls:
Percent m icron ucleated polych rom atic eryth rocytes
Range: 0.010 to 0.150
Mean: 0.066* ± 0.032
Positive controls:
Cyclophosphamide; 40 mg/kg b.w. [CPA]
Percent micronucleated polychromatic erythrocytes
Range: 0.910 to 2.975
Mean: 1.644* ± 0.446
*= mean of 95 experiments 0.032

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions reported, the test item did not induce micronuclei
Executive summary:

This study was performed to investigate the potential of Navy MG¡ 1571 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.
The test item was formulated in deionised water, which was also used as vehicle control. The volume administered orally was 10 ml/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.
Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.
To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and total erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.
The following dose levels of the test item were investigated: 
24 h preparation interval: 500, 1000, and 2000 mg/kg b.w
48 h preparation interval: 2000 mg/kg b.w.
The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable.
After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that Navy MGi 1571 did not exert any cytotoxic effects in the bone marrow. However, the urine of the treated animals was blue, indicating the systemic distribution of the test item and thus its bioavailability.
ln comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in tne frequency of the detected micronuclei at any preparation inierval after administration of the test item and with any dose level used.
40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency.
ln conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse
Therefore, Navy MGi 1571 is considered to be non-mutagenic in this micronucleus assay.