Polysodium 4-amino-5-hydroxy-3-[{8-hydroxypolysulfonato-7-[{sulfonato-4-[(sulfonatophenyl)diazenyl]phenyl}diazenyl]-2-naphthyl}diazenyl]-6-[(4-nitro-sulfonatophenyl)diazenyl]naphthalenepolysulfonate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: genome mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 January to 10 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD guidelines in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test animals

Species:

mouse

Strain:

Crj: CD(SD)

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Distilled waterThe test substance was dissolved at 200 mg/mL in distilled water. This solution was considered to be stable from the facts on account of neither change in color, exothermic reaction nor gas generation at room temperature until 4 hours after preparation. Therefore, distilled water was selected as a vehicle.

Duration of treatment / exposure:

Administration was repeated twice with a 24-hour interval by the oral gavage which is generally used for the micronucleus test Administration was carried out at 10 mL/kg based on the body weight at the administration day

Doses / concentrations

Remarks:

Doses / Concentrations:2000, 1000, 500, 250, 125 and 62.5 mg/kg/dayBasis:actual ingested

No. of animals per sex per dose:

Three male and female mice were used.

Control animals:

yes, concurrent vehicle

Positive control(s):

In the positive control group, MMC was intraperitoneally administered at 2 mg/kg/day based on the historical data in the testing facility. Six male mice were used in each control group because it was judged that there was no difference in MTD and toxicity of the test substance between sexes in the preliminary test. Specimens were prepared for all surviving animals at the timing of preparation of specimens and 5 animals of the smaller animal number in each group were used for the evaluation.

Examinations

Tissues and cell types examined:

Animals were observed frequently until 1 hour after each administration. In addition, the animals were observed at 1 day after each administration. In the positive control group, observation of clinical signs was not carried out at the first administration day of the test substance.Body weights were measured before the first and second administrations and at 1 day after the second administration using an electronic balance (Sartorius).Animals were euthanized by a cervical dislocation. The femur was removed and the bone marrow cells were collected with approximately 0.8 mL of a heat-inactived fetal bovine serum into a centrifuge tube, and the tube was centrifuged at 1000 rpm (210xg) for 5 minutes. A small amount of the cell suspension was smeared on a glass slide. The smears were fully air-dried and fixed with methanol. Then, the smears were stained with 3 vo l% Giemsa solution prepared with Sorensen buffer, pH 6.8 and treated with 0.004 w/v% citrate aqueous solution. For all surviving animals at the timing of preparation of specimens, two specimens were prepared per animal.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

At specimen observation, there were 3 doses that PCE/TE of the test substance group was 20% or more compared to that of the negative control group.  Therefore, it was confirmed that the test was conducted appropriately. 

In the test substance group, the MNPCE/PCE at all doses for observation did not show the significant differences compared to the negative control group.  Therefore, it was evaluated that the frequencies of micronucleated polychromatic erythrocytes were not increased by administering the test substance. 

In the PCE/TE, since no statistically significant differences were observed in any doses of the test substance group compared to the negative control group, the exposure of the test substance to bone marrow cells was not proved.  However, the potential to induce the micronuclei was evaluated at the doses up to 2000mg/kg/day described as the maximum dose in the case of no toxicity on the guideline.  Therefore, it was considered that the potential to induce the micronuclei of the test substance could be evaluated adequately in this study.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negativeThe substance did not induce the micronuclei under the conditions of the test

Executive summary:

The test substance was administered to 7 weeks old Crlj:CD1 (ICR) male mice twice at a 24 -hour interval by gavage to investigate the ability of the substance to induce micronuclei according to the OECD 474 test guideline in compliance with GLP. 

The preliminary test was conducted at 62.5 -2000 mg/kg/day of the test substance group in male and female, and it was estimated that each maximum tolerance dose (MTD) determined by death of animals was 2000 mg/kg/day or more in male and female.  Therefore, the highest dose was selected to be 2000 mg/kg/day, and 2 lower doses of 1000 and 500 mg/kg/day were selected based on a geometric progression of 2 for the micronucleus test.  It was found that there was no difference in MTD and toxicity between male and female. Therefore, the only male mice were used for the micronucleus test.  The vehicle, distilled water, was administered at 10mL/kg twice at a 24 -hour interval by gavage as the negative control.   Mitomycin C was administered intraperitoneally at 2 mg/kg/day once as the positive control. 

In the micronucleus test, no animals died at any dose groups until 24 hours after the second administration, therefore, the doses of observation of specimens were selected at setting three doses,  2000,  1000  and  500  mg/kg/day.     The  frequencies  of  the  micronucleated polychromatic erythrocytes to polychromatic erythrocytes (MNPCE/PCE) in bone marrow cells and the ratio of PCE to total erythrocytes (PCE/TE) were examined. 

As a result of observation, the MNPCE/PCE in all doses of the test substance group did not show the significant differences compared to the negative control group.  Therefore, it was evaluated that the frequencies of micronucleated polychromatic erythrocytes were not increased by administering the test substance. 

Consequently, it was concluded that the substance did not induce the micronuclei in mice bone marrow cells under the present test conditions.