N,N'-[6,13-diacetamido-2,9-diethoxy-3,10-triphenodioxazinediyl]bis(benzamide)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 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

Test material information

Constituent 1

Reference substance name:

N,N'-[6,13-diacetamido-2,9-diethoxy-3,10-triphenodioxazinediyl]bis(benzamide)

EC Number:

241-734-3

EC Name:

N,N'-[6,13-diacetamido-2,9-diethoxy-3,10-triphenodioxazinediyl]bis(benzamide)

Cas Number:

17741-63-8

Molecular formula:

C40H34N6O8

IUPAC Name:

N,N'-(6,13-diacetamido-2,9-diethoxy[1,4]benzoxazino[2,3-b]phenoxazine-3,10-diyl)dibenzamide

Test material form:

solid: nanoform

Specific details on test material used for the study:

- 98.3%

Method

Species / strain

Species / strain / cell type:

lymphocytes: human lymphocyte cultures prepared from the pooled blood of three male donors in two independent experiments

Details on mammalian cell type (if applicable):

Blood from three healthy, non-smoking male volunteers from a panel of donors was used for each experiment. No donor was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All donors are non-smokers and are not heavy drinkers of alcohol. Donors were not taking any form of medication. HEPES-buffered RPMI medium

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9 mix

Test concentrations with justification for top dose:

Experiment 1:
+ S9 mix: 0, 40, 60, 80 ug/ml
- S9 mix: 0, 80, 100, 150 ug/ml

Experiment 2:
+ S9 mix: 0, 40, 50, 55 ug/ml
- S9 mix: 0, 45, 60, 75, 90 ug/ml

Vehicle / solvent:

DMF

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 S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3h +/- S9 mix experiment I, 20h without S9 mix and 3h with S9 mix experiment II
- Expression time (cells in growth medium): after 3h exposure 17h, after 20h exposure 0h
- Fixation time (start of exposure up to fixation or harvest of cells): after 4h exposure 18-28h, 18h and 28h exposure and fixation direct thereafter

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 independent experiments, samples / cultures in triplicate

NUMBER OF CELLS EVALUATED: 100 well-spread metaphases will be scored per culture

DETERMINATION OF CYTOTOXICITY
- Method: slides will be evaluated for mitotic index and cell numbers, 1000 cells per culture will be scored and values will be expressed as a percentage of the solvent controls

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic events if:
1. A proportion of cells with structural aberrations at one or more concentrations that exceeded the normal range was observed in both replicate cultures
2. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) was observed (p0.05)
3. There was a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
The test article was considered positive in this assay if all of the above criteria were met.

Statistics:

The statistical method used was Fisher's exact test. The proportions of aberrant cells in each replicate were also used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.

Results and discussion

Test results

Species / strain:

lymphocytes: Human Lymphocyte

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

Additional information on results:

Precipitation was observed at 55 and 150 ug/ml

Applicant's summary and conclusion

Conclusions:

It is concluded that the test substance did not induce chromosome aberrations in cultured human peripheral blood lymphocytes following treatment in the absence and presence of rat liver metabolic activation system (S-9). Concentrations were tested up to the limit of solubility within the test system. Therefore, the test substance was considered to be non-clastogenic in this in vitro test system.