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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 6, 2007 - January 24, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
484-490-5
EC Name:
-
Molecular formula:
Hill formula:C21 H12 N2 O6 S Sr 5/2(H2O) CAS formula: C21 H14 N2 O6 S . Sr
IUPAC Name:
484-490-5
Test material form:
solid: particulate/powder
Details on test material:
Name: Strontium 3-hydroxy-4-[(l-sulfonato-2-naphthyl)azo]-2-naphthoate 5/2 hydrate
Other name: R507-2
CAS No.: 15792-20-8
LotNo.06-057B
Supplier: DAINIPPON INK AND CHEMICALS, INCORPORATED
Purity: >95.0% (including 8.1% water of crystallization)
Physicochemical properties
Appearance at ordinary temperature:7) Physicochemical properties

Appearance at ordinary temperature: red powder
Molecular weight:553.05
Stability:unknown
Solubility: insoluble
Degree of solubility
Water:<50.0mg/mL
Dimethyl sulfoxide (DMSO):<500mg/mL
Acetone:<500mg/mL

Storage conditions

Stored at room temperature (permissible range: 10-30°C).
It was stored in cabinet No. 1 in the test substance storage room of the experiment building till November 15, 2007, and was stored in cabinet No. 1 in the test substance storage room of the main building after the day.



Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Test concentrations with justification for top dose:
625, 1250, 2500 and 5000 µg/mL in short-term treatments without and with S9 mix.
78.1, 156, 313, 625, 1250, 2500 and 5000 µg/mL in 24 hours continuous treatment.

In the cell growth inhibition test, for the short-term treatments without and with S9 mix, the highest dose of the test substance was selected at 5000 µg/mL and the following 3 doses were set based on a geometric progression of 2, because a cytotoxicity such that the cell growth rate was less than 50% was not obtained. For 24 hours continuous treatment, the highest dose of the test substance was selected at dose of over IC50, because a cytotoxicity that the cell growth rate was less than 50% was obtained. Though IC50 was calculated at 1300 µg/mL, the cell growth rate was 32.2% at 5000 µg/mL. Therefore, the highest dose was selected at 5000 µg/mL and the following 6 doses were set based on a geometric progression of 2.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
- Justification for choice of solvent/vehicle:In the result of solubility test of the test substance, the test substance was not dissolved at 50.0 mg/mL in distilled water and at 500 mg/mL in DMSO or acetone. The results showed that the test substance suspension at 50.0 mg/mL in 0.5 w/v % CMC solution was homogeneous state. This suspension was not indicated any change in color nor exothermic within 2 hours after preparation. Therefore, 0.5 w/v % CMC solution was selected in a solvent.
Controls
Positive controls:
yes
Positive control substance:
other: mitomycin c (MMC), cyclophosphamide monohydrate (CPA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation);
Chromosomal aberration test was carried out using the same procedure as that of the cell growth inhibition test, with the following positive controls. In the preparation of specimens, the cells were completely fixed by 2 to 5 changes with 3 mL of fixative solution. Two specimens per dish (four specimens per dose) were prepared.
In the cell growth inhibition test for 24 hours continuous treatment, it was difficult
to analyze the chromosomes at the doses up 313 µg/mL by precipitation of the test substance, therefore, the medium was exchanged using the same procedure as that of the short-term treatment before the demecolcine solution was added at 2 hours before the end of the culture
In the positive control, after a same volume of distilled water as that of the test substance formulation was added in the treatment medium, each dish was added with 30 µL of a 0.01 mg/mL MMC solution and 18 µL of a 1 mg/mL CPA solution for the short-term treatments without and with S9 mix, respectively, and 25 µL of a 0.01 mg/mL MMC solution for the continuous treatment.
Evaluation criteria:
The findings were judged to be positive when the frequencies of cells with structural aberrations showed 10 % or more with a dose-related increase or the frequencies of cells with structural aberrations showed 5 % or more both in the chromosomal aberration test and the confirmation test. The other cases were judged to be negative. No statistical analyses were used. The frequency of numerical aberration cells was judged according to the same criteria as that of the structural aberration.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Short-term Treatment
1) Without S9 mix
Cell growth rate and IC50
The cell growth rates at 625, 1250, 2500 and 5000 µg/mL of the test substance were 90.1, 91.9, 85.6 and 75.7 %, respectively. The IC50 was calculated more than 5000 µg/mL.
(2) Precipitation of the test substance, color change of medium and corrosion of culture dish
Precipitation of the test substance was observed at all doses at the start and the end
of the treatment and at the end of the culture. The color change of the medium and the corrosion of the culture dish were not observed at all doses.
(3) Frequency of cells with structural aberrations
The frequencies of cells with structural aberrations were 3.0 % in the negative control and 63.0 % in the positive control. The frequencies of cells with structural aberrations at 1250, 2500 and 5000 µg/mL were and 0.5, 0.5 and 0.0 %, respectively, therefore, the results were judged to be negative.
(4) Frequency of numerical aberration cells
The frequencies of numerical aberration cells were Jess than 5 % at all doses including the negative and positive controls, therefore, the results were judged to be negative.

2) With S9 mix
(1) Cell growth rate and IC50
The cell growth rates at 625, 1250, 2500 and 5000 µg/mL of the test substance were 84.0, 83.7, 75.5 and 73.1 %, respectively. The IC50 was calculated more than 5000 µg/mL.
(2) Precipitation of the test substance, color change of medium and corrosion of culture dish
Precipitation of the test substance was observed at all doses at the start and the end of the treatment and at the end of the culture. The color change of the medium and the corrosion of the culture dish were not observed at all doses.
(3) Frequency of cells with structural aberrations
The frequencies of cells with structural aberrations were 2.5 % in the negative control and 33.0 % in the positive control. The frequencies of cells with structural aberrations at 1250, 2500 and 5000 µg/mL were 2.5, 1.5 and 3.0 %, respectively, therefore, the results were judged to be negative.
(4) Frequency of numerical aberration cells
The frequencies of numerical aberration cells were Jess than 5% at all doses including the negative and positive controls, therefore, the results were judged to be negative.
Twenty Four Hours Continuous Treatment
1) Cell growth rate and IC50
The cell growth rates at 78.1, 156, 313, 625, 1250, 2500 and 5000 µg/mL of the test
substance were 104.7, 103.6, 89.9, 77.7, 42.6, 24.9 and 18.0 %, respectively. The IC50 was calculated at 1100 µg/mL.
2) Precipitation of the test substance, color of medium and corrosion of culture dish
Precipitation of the test substance was observed at all doses at the start and the end of the treatment and at the end of the culture. The color change of the medium and the corrosion of the culture dish were not observed at all doses.
3) Frequency of cells with structural aberrations
The frequencies were 1.5 % in the negative control and 62.5 % in the positive control. The frequencies of cells with structural aberrations at 313, 625 and 1250 µg/mL were 1.0, 1.5 and 2.0 %, respectively, therefore, the results were judged to be negative.
4) Frequency of numerical aberration cells
The frequencies of numerical aberration cells were less than 5.0 % at all doses including the negative and positive controls, therefore, the results were judged to be negative.

Applicant's summary and conclusion

Conclusions:
In each treatment method in the chromosomal aberration test, the frequencies of cells with aberrations were less than 5 % in the negative controls, and the frequencies of cells with structural aberrations excluding gaps were more than 20 % in the positive controls, indicating that the present study was appropriately performed.
As a result of chromosomal aberration test, the frequencies of cells with structural aberration and numerical aberration cells showed less than 5 % at all doses of the test substance in the short-term treatments without and with S9 mix and 24 hours continuous treatment, therefore both of structural aberration and numerical aberration were judged to be negative.
Based on the above results, it was considered that R507-2 did not induce the chromosomal aberration under the present test conditions.
Executive summary:

The ability of R507-2 to induce chromosomal aberrations was investigated by using Chinese hamster lung fibroblasts (CHL/IU cells).

Based on the result of cell growth inhibition test, the doses in the chromosomal aberration test were set at 625, 1250, 2500 and 5000 µg/mL in short-term treatments without and with S9 mix and at 78.1, 156, 313, 625, 1250, 2500 and 5000 µg/mL in 24 hours continuous treatment.

In the short-term treatments without and with S9 mix, the observation doses of specimens were selected at 1250, 2500 and 5000 µg/mL as the higher 3 setting doses, because a cytotoxicity such that the cell growth rate was less than 50 % was not obtained. In 24 hours continuous treatment, the observation doses of specimens were selected at 313, 625 and 1250 µg/mL, because cell growth rate of 1250 µg/mL was less than 50 %.Inthe observation, the frequencies of cells with structural aberrations and numerical aberration cells were scored.

As a result of observation of specimens, the frequencies of cells with structural aberration and numerical aberration cells showed less than 5 % at all observation doses of the test substance in all treatment methods, therefore both of structural aberration and numerical aberration were judged to be negative.

On the other hand, the frequencies of cells with structural aberrations or numerical aberration cells in the negative control treated with 0.5 w/v % carboxymethyl cellulose sodium salt solution showed less than 5 %, and the frequencies of cells with structural aberrations in the positive controls treated with mitomycin C or cyclophosphamide monohydrate, showed more than 20 %, indicating the proper performance of the present study.

It was concluded that R507-2 did not induce the chromosomal aberration under the present test conditions.