2-Naphthalenesulfonic acid, 4-hydroxy-3-[2-[2-methoxy-5-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-7-[(sulfomethyl)amino]-8-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, sodium salt (1:5)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 07, 2005 to June 09, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ICH Guidelines S2A and S2B

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

464-700-1

EC Name:

-

Cas Number:

607724-42-5

Molecular formula:

Hill formula: C28H24N5Na5O23S7 CAS formula: C28H29N5O23S7.5Na

IUPAC Name:

pentasodium 4-hydroxy-3-(2-{2-methoxy-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)-8-(2-{2-sulfonato-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)-7-[(sulfonatomethyl)amino]naphthalene-2-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

Reactive Red F01-0481

Test animals

Species:

mouse

Strain:

other: CRL: NMRI BR mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Europe) Laboratories, Budapest, HUNGARY
- Age at study initiation: young adult mice
- Weight at study initiation: male: 26.8-32.1 g and female: 22.5-25.8 g
- Assigned to test groups randomly: yes
- Housing: group caging (5 animals/cage), type II polypropylene/polycarbonate cages with laboratory bedding
- Diet: SSNIFF RIM-Z+H diet for rats and mice (ad libitum)
- Water: ad libitum
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3°C
- Humidity: 30-70 %
- Photoperiod: 12 h dark/light

IN-LIFE DATES: From: June 07, 2005 To: June 09, 2005

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Vehicle used for test substance: distilled water
- Concentration of test substance in vehicle: 20% (w/v)
- Batch no.: 9240604
- Date of expiry: June, 2007
- Storage condition: at room temperature

Vehicle of positive control: 0.9% NaCl infusion
- Batch no.: 5671002
- Expiry: October, 2005
- Storage condition: at room temperature

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was dissolved in distilled water for the treatment. The necessary amount of the test substance was weighed into a calibrated volumetric flask and distilled water was added and stirred to obtain a homogenous formulation. It was diluted to the final volume with the distilled water. The solution was prepared fresh each day of dosing and used within 2 h. The test substance was used for treatment at concentration of 20% (w/v). Cyclophosphamide (positive control) was dissolved in 0.9% NaCI infusion for treatment.

Duration of treatment / exposure:

48 h

Frequency of treatment:

twice

Post exposure period:

24 h after the last dosing

Doses / concentrations

Remarks:

Doses / Concentrations:
2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5/sex

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: intraperitoneally
- Doses: 60 mg/kg
- Animals used: 5/sex

Examinations

Tissues and cell types examined:

The slides were examined in a blind manner. Two thousand polychromatic erythrocytes were scored per animal to asses the micronucleatcd cells. The frequency of micronucleated cells were expressed as percent of micronucleated cells based on the first 2000 polychromatic erythrocytes counted in
the optic field. Multiple micronuclei cells were not registered. The proportion of immature among total (irnmature+mature) erythrocytes were
determined for each animal by counting a total of at least 200 immature erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on the results of the acute oral toxicity study the dose of 2,000 mg/kg bw was selected as the limit dose, for the main study.

TREATMENT AND SAMPLING TIMES: The test substance was administered twice at an interval of 24 h orally by gavage to the test animals at a dose 2,000 mg/kg bw. The treatment volume was 0.1 mL/kg bw. The vehicle, distilled water was administered in the same way to the negative control groups. The mice were examined for visible signs of reactions to treatment, immediately after dosing, and at intervals until sacrifice. Sampling was made once at 24 h after the last dosing. Five male and female animals per group were used for sampling.
Cyclophosphamide (positive control) was administered intraperitoneally with a treatment volume: 0.1 mL/kg bw. Sampling was performed 24 h after the beginning of the treatment and five male and female animals were used for sampling.

DETAILS OF SLIDE PREPARATION: Bone marrow was obtained from two exposed femurs of the mice from animals immediately after sacrificing. The bone marrow was flushed with foetal calf serum. The cells were concentrated by a gentle centrifugation, then spread on a standard microscopic slide. Slides were fixed and after that stained with Giemsa. Two slides were prepared from each animal.

EVALUATION OF THE RESULTS: The micronucleated polychromatic erythrocytes referring to 2,000 polychromatic erythrocytes and the ratio of polychromatic erythrocytes to normochromatic erythrocytes (PCE/NCE) were listed for each animal. The frequency of micronucleated cells of treated male and female groups were compared with those of the vehicle control groups.

Evaluation criteria:

A positive result was one that gave a significant increase (at p<0.05, p<0.01) of the frequency of micronucleated polychromatic erythrocytes at least in two treated groups in one sex.
This increase would be classified as significant if observed:
- at adjacent dose levels: in the same experiment, dose- dependency,
- at adjacent time points: in the same experiment, same dose level, that is time dependency,
- in two experiments, in the same dose level and time point, that is reproducibility.
Both biological and statistical significance were considered together for evaluation purposes.
The historical range for this laboratory was also considered when evaluating the biological significance of small increases.

Statistics:

Dose dependent increase in the number of micronucleated polychromatic erythrocytes was evaluated by Kruskal-Wallis Non Parametric ANOVA test at 1 and 5% probability levels.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Clinical signs and mortality:

No animals died during the study. Animals did not show any toxicity symptom in the test substance treated group or in the vehicle or positive control groups. Due to the effect of test substance the faeces and tissue of abdominal organs of mice were found to be discoloured with a reddish appearance. The presence of red colour in the tissue after an oral exposure demonstrates systematic exposure to the test substance.

Frequency of micronucleated polychromatic erythrocytes:

The frequency of micronucleated polychromatic erythrocytes for the vehicle control mice was within an acceptable range and compatible with the historical control data. Cyclophosphamide treated mice (60 mg/kg bw) showed large, statistically significant increase in the micronucleated polychromatic erythrocytes number compared to the vehicle control, demonstrating an acceptable sensitivity of the test. The repeated administration of the test substance (twice at an interval of 24 h) at 2,000 mg/kg bw did not induce biologically relevant increase in the frequency of micronucleated polychromatic erythrocytes (MPCEs) in male or female mice at 24 h after the treatment compared to the vehicle control. In the female animals, at 24 h after treatment with 2,000 mg/kg test substance there was small, statistically significant, but biologically not important increase in the number of micronucleated polychromatic erythrocytes (MPCEs). These minor increases were in the range of historical negative control value. No significant difference was found in the ratio of polychromatic and normochromatic erythrocytes after treatment.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
Under the test conditions, the test substance did not induce micronuclei in bone marrow cells of the treated mice. Therefore, the test substance was not considered to be clastogenic in this micronucleus assay.

Executive summary:

A study was conducted to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of mice according to OECD Guideline 474 and ICH Guidelines S2A and S2B, in compliance with GLP.

The substance was dissolved in distilled water and given twice at an interval of 24 h as an oral dose of 2,000 mg/kg bw to male and female rats. The dose was selected based on the results of a preliminary acute oral toxicity study. A vehicle control group treated with distilled water and a positive control group treated with cyclophosphamide (60 mg/kg bw intraperitoneally) were also included. The animals were sacrificed 24 h after the last administration and bone marrow cells were collected for micronuclei analysis. Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) was used to assess the toxicity of the test substance.

The oral administration did not induce any biologically relevant increase in the frequency of micronucleated polychromatic erythrocytes (MPCEs) in male or female mice compared to the vehicle control. No biologically important alteration in the ratio of polychromatic to normochromatic erythrocytes occurred in the treated groups when compared to the vehicle control. Treatment with the positive control, cyclophosphamide, caused significant increase in the number of micronucleated polychromatic erythrocytes (MPCEs), thus validating the test.

Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the treated mice. Therefore, the test substance was not considered to be clastogenic in this micronucleus assay (Beres, 2005).