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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity potential of BK-659 was assessed with Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2uvrA according to the OECD 471 test guideline compliant with GLP..

The substance did not induce an increase in the number of revertant colonies more than twice in comparison with that of the negative control nor was a dose-related response observed in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation. 

Under the conditions of the test the substance is not mutagenic.

 

The chromosome aberration test was conducted in vitro in CHL lung cells up to and including a maximum dose level of 5000 μg/ml in the presence and absence of rat liver S9 activation according to the OECD 473 test guideline in compliance with GLP.

The frequencies of numerically aberrant cells were below 5% at all observation doses of the test substance in all treatment methods.

The frequencies of cells with structural aberrations were below 5% at all doses observed in the short-term treatments without and with S9 mix.   The frequencies of cells with structural aberrations were over 10% and increased upto 28.5% with a dose-related manner in the 24 hours continuous treatment.   Therefore, structural aberration was judged to be positive.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 January to 10 February 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

1.2, 4.9, 20, 78, 313, 1250 and 5000 µg/plate

Vehicle / solvent:

water.Based on the information from the sponsor that the test substance was soluble at 150000 mg/L in water. Therefore water for injection was used as the solvent for the test substance.

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

benzo(a)pyrene

furylfuramide

other: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine·2HC1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):negativeUnder the conditions of the test the substance is not mutagenic.

Executive summary:

Mutagenicity potential of BK-659 was assessed with Salmonella typhimurium TA100, TA1535, TA98,TA1537and Escherichia coli WP2uvrA according to the OECD 471 test guideline compliant with GLP..

The substance did not induce an increase in the number of revertant colonies more than twice in comparison with that of the negative control nor was a dose-related response observed in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation. 

Under the conditions of the test the substance is not mutagenic.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 August to 31 October 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Short term treatment: 1250, 2500 and 5000 µg/mL24 hours continuous treatment: 313, 1250 and 5000 µg/mL

Vehicle / solvent:

Distilled waterThe test substance was dissolved at 50.0 mg/mL m distilled water. The test substance solution of 50.0 mg/mL prepared with distilled water was considered to be stable from the facts on account of neither change in color, exothermic reaction nor gas generation at room temperature until 2 hours after preparation. Therefore, distilled water was selected as a vehicle.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

At the start and the end of the treatment, the presence or absence of the precipitation of the test substance could not be observed by visual at all doses in all treatment methods because the color of the medium was changed to blue by the test substance.   Therefore, microscope was used for observation, and it was confirmed that there was no precipitation of the test substance at 625µg/mL or below in "-S9 mix" and "+S9 mix", and 313 µg/mL or below in 24 hours treatment method.  The presence or absence of the precipitation of the test substance could not be observed at other doses.   At the end of the culture, the precipitation of the test substance was not observed at all doses in "-S9 mix" and "+S9 mix".

At the start and the end of the treatment, the color change of the medium to red or yellow could not be observed at all doses in all treatment methods because the color of the medium was changed to blue by the test substance.   Therefore, the medium and the test substancesolution were mixed at the same ratio as "-S9 mix" and 24 hours treatment method, and the pH was measured.   The pH was 7.58 at the highest dose, 5000 µg/mL, and it was considered that the pH in the medium was not changed by the test substance.  At the end of the treatment, the color change of the medium was not observed at all doses.

Conclusions:

Interpretation of results (migrated information):ambiguousIt was concluded that BK-659 did not induce numerical aberration but induced structural aberration under the present test conditions.

Executive summary:

The chromosome aberration test was conducted in vitro in CHL lung cells up to and including a maximum dose level of 5000 μg/ml in the presence and absence of rat liver S9 activation according to the OECD 473 test guideline in compliance with GLP.

The frequencies of numerically aberrant cells were below 5% at all observation doses of the test substance in all treatment methods.

The frequencies of cells with structural aberrations were below 5% at all doses observed in the short-term treatments without and with S9 mix.   The frequencies of cells with structural aberrations were over 10% and increased upto 28.5% with a dose-related manner in the 24 hours continuous treatment.   Therefore, structural aberration was judged to be positive.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

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.

Link to relevant study records

Reference

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

Crj: CD(SD)

Sex:

male/female

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

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.

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.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Justification for selection of genetic toxicity endpoint
Data are available from in vitro and in vivo study data conducted to K1 quality.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Mutagenicity potential of BK-659 was assessed with Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2uvrA according to the OECD 471 test guideline compliant with GLP.

The substance did not induce an increase in the number of revertant colonies more than twice in comparison with that ofthenegative control nor was a dose-related response observed in any strains of base-pair substitution type or frame-shift type, with or without metabolicactivation. 

Under the conditions of the test the substance is not mutagenic.

The chromosome aberration test was conducted in vitro in CHL lung cells up to and including a maximum dose level of 5000 μg/ml in the presence and absence of rat liver S9 activation according to the OECD 473 test guideline in compliance with GLP.

The frequencies of numerically aberrant cells were below 5% at all observation doses of the test substance in all treatment methods.

The frequencies of cells with structural aberrations were below 5% at all doses observed in the short-term treatments without and with S9 mix.   The frequencies of cells with structural aberrations were over 10% and increased upto 28.5% with a dose-related manner in the 24 hours continuous treatment.   Therefore, structural aberration was judged to be positive.

The test substance was administered to 7 weeks old Crlj:CD1 (ICR) male mice twice at a24 -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 groupinmale 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.  Itwas 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/kgtwice 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. 

Inthe 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.