Disodium 2-(2,3-dihydro-1,3-dioxosulphonato-1H-inden-2-yl)quinolinesulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

other: Read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From December 11th, 2012 to March 06th, 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

The study is conducted on a read across test material. The complete read across justification is attached in section 13. The reliability of the original study is 1.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

July 2010

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 1

IUPAC Name:

Similar Substance 1

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. J. Thacker, MRC Radiobiology Unit, Harwell, UK
- Suitability of cells:
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable:
- Whether whole blood or separated lymphocytes were used if applicable:
- Number of passages if applicable:
- Methods for maintenance in cell culture if applicable: permanent stocks are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.
- Modal number of chromosomes:
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of culture media:
Minimal medium:
- Eagle's Minimal Essential Medium (10X) 58.7 mL
- L-glutamine (200 mM) 5.9 mL
- Sodium bicarbonate (7.5 %) 15.7 mL
- Non-essential amino acids (100X) 5.9 mL
- Streptomycin sulphate 50.000 UI/mL
- Penicillin G 50,000 IU/mL 1.2 mL
- Sterile distilled water 500.0 mL
Complete medium (10 %):
- Minimal medium 900 mL
- Foetal Calf Serum 100 mL
- Properly maintained: [yes/no]
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: [yes/no)
- Periodically 'cleansed' against high spontaneous background: [yes/no]

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

Cytochalasin-B

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 fraction

Test concentrations with justification for top dose:

- Firts experiment: 5000, 3850, 2960, 2280, 1750, 1350, 1040, 797 and 613 μg/mL both in the absence and presence of S9 metabolism. An additional lower dose level of 471 μg/mL was used in the absence of S9 metabolism.
- Second experiment: 5000, 3850, 2960, 2280, 1750, 1350, 1040, 797 and 613 μg/mL.
- Justification for top dose: determined according to the solubility of test item in the solvent vehicle and culture medium.

Vehicle / solvent:

- Vehicle used: sterile water of injectable grade.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

sterile water of injectable grade

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: colchicine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 500 cells/mL

DURATION
- Preincubation period: 20 hours
- Exposure duration: 3 hours (first experiment), 26 hours (second experiment)
- Treatment time: 3 hours (firts experiment), 26 hours (second experiment)
- Harvest time: 27 hours (first experiment), 26 hours (second experiment)

SELECTION OF DOSE LEVELS FOR SCORING
The highest dose level for genotoxicity assessment is selected as a dose which produces a substantial cytotoxicity compared with the solvent control. Ideally the cytotoxicity should be between 50 % and 60 %. In the absence of cytotoxicity the highest treatment level (5000 µg/mL) is selected as the highest dose level for scoring. Two lower dose levels are also selected for the scoring of micronuclei (3850 and 2960 µg/mL).
For the first experiment, dose levels of 0.300 and 2.50 µg/mL were selected for the scoring of Mitomycin-C and Colchicine, respectively. The dose level of 7.50 µg/mL was selected for the scoring of Cyclophosphamide.
For the second experiment, dose levels of 0.100 and 0.250 µg/mL were selected for the scoring of Mytomycin-C and Colchicine, respectively.

SPINDLE INHIBITOR: Cytochalasin B

STAIN: Acridine Orange

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: slides were prepared using a Cytospin; the cells were fixed in 90 % methanol (-20 °C); the cells were allowed to air dry and were kept at room temperature prior to staining; the slides were stained with a solution of Acridine Orange in PBS (12.5 mg in 100 mL PBS).

NUMBER OF CELLS EVALUATED: 500 cells per culture were analysed to measure the cytotoxic effect; 1000 cells per culture were scored to assess the frequency of micronucleated cells.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): first mitosis, binucleated cells.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
(i) The micronucleus diameter was less than 1/3 of the nucleus diameter;
(ii) The micronucleus diameter was greater than 1/16 of the nucleus diameter;
(iii) No overlapping with the nucleus was observed;
(iv) The aspect was the same as the chromatin.

DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI)
- Any supplementary information relevant to cytotoxicity:
CBPI: [No. mononucleated cells + 2 x No. binucleated cells + 3 x No. multinucleated cells]/total number of cells counted
% Cytotoxicity = 100 – 100 [(CBPIt - 1) / (CBPIc - 1)]
where:
t= test item treated culture
c= untreated/solvent control culture

Evaluation criteria:

The test item is considered as clearly positive if the following criteria are met:
(i) Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
(ii) The proportion of micronucleated cells exceeds the normal range.
(iii) There is a significant dose-relationship.

Statistics:

A modified chi-squared test was used to compare the number of cells bearing micronuclei in control and treated cultures.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No relevant cytotoxicity was observed at any dose level, in the absence or presence of S9 metabolism.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no remarkable variation of pH over the control values was observed.
- Effects of osmolality: no remarkable variation of osmolality values was observed.
- Water solubility: 50 mg/mL.
- Precipitation: no opacity nor precipitation was observed.
- Definition of acceptable cells for analysis: binucleated cells.

SCREENING STUDIES:
- Solubility test: The test item was found to be soluble in sterile water of injectable grade at the concentration of 50 mg/mL.
This concentration was chosen because when this solution was added to culture medium in the ratio 1:10, it gave a final concentration of 5000 μg/mL.
An aliquot of the stock solution at the concentrations of 50 mg/mL added to EMEM complete medium in the ratio 1:10 gave a clear medium without any visible precipitation. On the basis of these results, 5000 µg/mL was selected as the maximum dose level to be used in main experiments.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

Remarks on result:

other: first experiment

Applicant's summary and conclusion

Conclusions:

Based on the results obtained from an in vitro mammalian cell micronucleus test, the test item is not of concern with respect to produce chromosome damage or other effects on cell cycle/cell division to lead to the formation of micronuclei in interphase cells.

Executive summary:

The test item was assayed for the ability to induce micronuclei in Chinese hamster V79 cells, following in vitro treatment in the absence and presence of S9 metabolic activation from rats induced with Phenobarbital-5.6-Benzoflavone.

No statistically significant increase in the incidence of micronucleated cells over the negative control value was observed at any dose level in any treatment series.

Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide, Mitomycin-C and Colchicine indicating the correct functioning of the test system.

On the basis of the results obtained, it is concluded that the test item does not induce micronuclei in Chinese hamster V79 cells after in vitro treatment in the absence or presence of S9 metabolic.