6,13-dichloro-3,10-bis-{2-[4-fluoro-6-(2-sulfo-phenylamino)-1,3,5-triazin-2-ylamino]-propylamino}-benzo[5,6][1,4]oxazino[2,3-.b.]phenoxazine-4,11-disulphonic acid, lithium, sodium salt.

Genetic toxicity: in vitro

Administrative data

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

6,13-dichloro-3,10-bis-{2-[4-fluoro-6-(2-sulfo-phenylamino)-1,3,5-triazin-2-ylamino]-propylamino}-benzo[5,6][1,4]oxazino[2,3-.b.]phenoxazine-4,11-disulphonic acid, lithium, sodium salt.

EC Number:

418-000-8

EC Name:

6,13-dichloro-3,10-bis-{2-[4-fluoro-6-(2-sulfo-phenylamino)-1,3,5-triazin-2-ylamino]-propylamino}-benzo[5,6][1,4]oxazino[2,3-.b.]phenoxazine-4,11-disulphonic acid, lithium, sodium salt.

Cas Number:

163062-28-0

Molecular formula:

CAS formula: C42 H34 Cl2F2 N14 O14 S4 .xLi. xNa

IUPAC Name:

dilithium(1+) disodium 6,13-dichloro-3,10-bis({[2-({4-fluoro-6-[(2-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)propyl]amino})-5,12-dioxa-7,14-diazapentacene-4,11-disulfonate

Details on test material:

- Name of test material (as cited in study report): FAT 40529/A
- Batch number: P1/95
- Storage: Room temperature

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

In the first assay dose-levels of 5000, 2320, 1080, 500, 232, 108, 50.0 and 23.2 µg/mL were used. At the 20 hour sampling time, in the presence of S9 metabolism, the lower dose of 23.2 µg/mL was not used.
In the second experiment dose-levels of 5000, 2320, 1080, 500, 232 and 108 µg/mL were used. At the 20 hour sampling time in the absence of S9 metabolism, a dose range of 2320, 1080, 500, 232, 108 and 50 µg/mL was selected.

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Assay for chromosomal aberrations: In the presence of S9, the treatment time was 3 hours, and two harvesting times were employed 12 and 20 hours. In the absence of S9 metabolism, the cells were treated for an entire cell cycle, and were harvested after 12 and 20 hours. Two cell cultures were prepared at each test point. Air-dried slides were prepared from each culture and stained with 3% Giemsa. Following treatment, the pH values and osmolality of the treatment media of cultures treated at higher dose-levels were determined.
Assay results: For each experiment, one hundred metaphase spreads were scored for chromosomal aberrations from each culture at the selected test substance treatment-levels.

Evaluation criteria:

In this assay, the test substance is considered to have clastogenic properties if the following criteria are all fulfilled: (i) Statistically significant increases in the incidence of cells bearing aberrations are observed at any dose-level over the concurrent control. (ii) The increases are reproduced in both replicate cultures and must be observed in both experiments.

Statistics:

For the statistical analysis, Fisher's Exact Test is used to compare the number of cells bearing aberrations (assumed to be Poisson distributed) in control and treated cultures. The analysis is performed using sets of data either including or excluding gaps.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS GENOTOXICITY:
In the first main assay, no statistically significant increases in the numbers of cells bearing aberrations, excluding gaps, were observed following treatment with FAT 40529/A at any dose-level selected for scoring either in the absence or presence of S9 metabolism. A statistically significant increase in the number of cells bearing aberrations including gaps, compared with the relevant control value was observed following treatment with FAT 40529/A at the highest dose-level (500 µg/mL) selected for scoring at the 12 hour sampling time in the presence of S9 metabolism. In the second main assay at the 12 hour sampling time, statistically significant increases in the numbers of cells bearing aberrations (both excluding and including gaps), compared with the relevant control values were observed at the highest dose-level (5000 µg/mL) selected for scoring and at the following dose of 2320 µg/mL. For the lowest dose of 1080 µg/mL, a marked increase in the number of cells bearing aberrations excluding gaps, not reaching statistical significance, was also observed. In the second experiment, a single polyploid cell was noted in the 200 cells of the solvent control examined at the 12 hour sampling time in the absence of metabolic activation. A similar incidence was seen in the low dose (108 µg/mL). In the same experiment, polyploid cells were observed at dose levels of 500 and 1080 µg/mL in the presence of metabolic activation with the same level of incidence. No dose-effect relationship was apparent. Where the toxicity of treatment did not inhibit cell proliferation, one hundred metaphase spreads were scored for chromosomal aberrations from each culture of the positive control treatment at the 12 and 20 hours sampling times. When more of 50% of the cells were found to contain aberrations (other than gaps), scoring was terminated at 50 metaphase spreads only. Statistically significant increases in the frequency of aberrant cells compared with the relevant control values, were observed in the cultures treated with the positive control substances, indicating the correct functioning of the assay system. The modal number of chromosomes observed in the cells of the untreated cultures was 21 (approximately 77% of cells).

TEST-SPECIFIC CONFOUNDING FACTORS
- The test substance had no obvious effect on the pH or osmolality compared with the control values.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Selection of dose-levels for scoring: For both experiments, the mitotic index (MI) was scored for each of the treatment series. In the first main assay, at the 12 hour sampling time, in the presence of S9 metabolism marked toxicity was observed at 5000 and 2320 µg/mL reducing the mitotic index to 11 and 26 % of the control value respectively. Moderate toxicity was observed at the following dose-levels of 1080 and 500 µg/mL. No remarkable toxicity was observed over the remaining dose-range. In the absence of S9 metabolism, marked toxicity was observed from 5000 µg/mL to 232 µg/mL. No toxicity was observed at the lower three dose-levels. At the 20 hour sampling time, in the presence of S9 metabolism moderate toxicity was observed at the highest doses of 5000 and 2320 µg/mL. Mild toxicity was observed at the following dose of 1080 µg/mL. No toxicity was observed over the remaining dose-range. In the absence of S9 metabolism, severe toxicity was observed at the highest three dose-levels (5000, 2320 and 1080 µg/mL) where few metaphases were recovered (no metaphases at 5000 µg/mL). Moderate toxicity was observed at the following dose of 500 µg/mL, reducing the mitotic index to 34% of the control value. No remarkable toxicity was observed over the remaining dose-range. At the 20 hour sampling time, in the presence of S9 metabolism moderate toxicity was observed at the highest doses of 5000 and 2320 µg/mL. Mild toxicity was observed at the following dose of 1080 µg/mL. No toxicity was observed over the remaining dose-range. In the absence of S9 metabolism, severe toxicity was observed at the highest three dose-levels (5000, 2320 and 1080 µg/mL) where few metaphases were recovered (no metaphases at 5000 µg/mL). Moderate toxicity was observed at the following dose of 500 µg/mL, reducing the mitotic index to 34% of the control value. No remarkable toxicity was observed over the remaining dose-range. In the second main assay, at the 12 hour sampling time in the presence of S9 metabolism, marked toxicity was observed at 5000 µg/ml reducing the mitotic index to 23% of the control value. No toxicity was observed over the remaining dose-range. In the absence of S9 metabolism, marked toxicity was observed at the three highest dose levels (5000, 2320 and 1080 µg/mL). No toxicity was observed over the remaining dose-range. At the 20 hour sampling time in the presence of S9 metabolism, no toxicity was observed at any dose-level. In the absence of S9 metabolism, severe toxicity was observed at the three highest dose-levels (2320, 1080 and 500 µg/mL). No toxicity was observed over the remaining dose-range.
The highest dose-level selected for the scoring of aberrations should be a concentration causing moderate toxicity (ideally the reduction in mitotic index should be approximately 50%) and treatments reducing the mitotic index to below 20% should not be scored. If no toxicity is observed then the highest practicable dose-level should be selected. On the basis of the above results the treatment-levels selected for the scoring of aberrations were as follows: First main assay: 12 hour sampling time: absence of S9 metabolism : 232, 108 and 50 µg/mL and presence of S9 metabolism : 500, 232 and 108 µg/mL, 20 hour sampling time: absence of S9 metabolism : 500, 232 and 108 µg/mL and presence of S9 metabolism: 1080, 500 and 232 µg/mL; Second main assay 12 hour sampling time: absence of S9 metabolism : 500, 232 and 108 µg/mL and presence of S9 metabolism : 5000, 2320 and 1080 µg/mL.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
ambiguous

The test substance did not meet all criteria to be considered positive (eg. reproducibility at the same dose level for both experiments), nevertheless a clear clastogenic effect was observed.

Executive summary:

In a GLP-compliant chromosome aberration test, tested according to OECD guideline 473, Chinese hamster lung fibroblast (V79) were exposed to the test substance with and without metabolic activation.In the first main assay a statistically significant increase in the number of cells bearing aberrations including gaps, compared with the relevant control values, was observed following treatment with the test substance at the highest dose-level (500 µg/mL) selected for scoring at the 12 hour sampling time in the presence of S9 metabolism. In the second main assay at the 12 hour sampling time, statistically significant increases in the number of cells bearing aberrations excluding gaps, compared with the relevant control values were observed at the highest dose-level (5000 µg/mL) selected for scoring and at the following dose of 2320 µg/mL. For the lowest dose of 1080 µg/mL, marked increases in the number of cells bearing aberrations excluding gaps, not reaching statistical significance, were also observed. Although the test substance did not meet all criteria to be considered positive (eg. reproducibility at the same dose level for both experiments), nevertheless a clear clastogenic effect was observed.