Bisbenzimidazo[2,1-b:1',2'-j]benzo[lmn][3,8]phenanthroline-6,9-dione

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Bisbenzimidazo[2,1-b:2',1'-i]benzo[lmn][3,8]phenanthroline-8,17-dione

EC Number:

224-597-4

EC Name:

Bisbenzimidazo[2,1-b:2',1'-i]benzo[lmn][3,8]phenanthroline-8,17-dione

Cas Number:

4424-06-0

Molecular formula:

C26H12N4O2

IUPAC Name:

bisbenzimidazo[2,1-b:2',1'-i]benzo[lmn][3,8]phenanthroline-8,17-dione

Test material form:

solid: nanoform

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese Hamster (Cricetulus grise us) ovary cell line CHO-Kl, (ATCC CCL-61, Lot 4765275) with a modal chromosome number 20 and a population doubling time of 10 to 14 hours was used.

Metabolic activation:

with and without

Metabolic activation system:

Aroc1or 1254 induced rat liver S9 homogenate

Test concentrations with justification for top dose:

Based on the observations of the preliminary cytotoxicity test, following concentrations of Hostaperm-Orange GR were selected for testing in the initial chromosomal aberration assay.

Experiments No.1 and No.2: 3, 6, and 12 µg/mL

Experiment No.3: 2, 4 , and 8 µg/mL

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 4765275) with a modal chromosome number 20 and a population doubling time of 10 to 14 hours was used.

Batch No. 3 of this CHO-K1 cell line was tested for the absence of mycoplasma contamination at Mycoplasma Laboratory, Statens Serum Institut, Artillerivej 5, Copenhagen S, Denmark and certified free of mycoplasma contamination on 11 August 2014.

Cells were grown in T-75 cm2 flasks at 37 ± 1 °C in a carbon dioxide incubator (5 ± 0.2 % CO2 in air).

Evaluation criteria:

When all the validity criteria are fulfilled:
 
1.        A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test concentrations exhibits a statistically significant increase in aberrant metaphases compared with the concurrent vehicle control
- The increase is dose-dependent when evaluated with an appropriate trend test
- Any of the results are outside the distribution of the historical vehicle control data
 
2.        A test chemical is considered to be clearly negative if, in all experimental conditions examined:
- None of the test concentrations exhibits a statistically significant increase in aberrant metaphases compared with the concurrent vehicle control
- There is no concentration-related increase when evaluated with an appropriate trend test
- All results are inside the distribution of the historical vehicle control data
 
3.        The results are considered equivocal if they do not meet the criteria specified for a positive or negative response. Additional experimental work may be required by the Study Director to clarify such results and the activity, if any, will be documented in a study plan amendment.

Statistics:

Statistical analysis of the experimental data was carried out using validated SYSTAT Statistical package ver.12.0. Data were analyzed for proportions of aberrant metaphases in each sample excluding gaps as aberrations, numerical aberration (Poly) were analyzed. Pooled data from each test concentration and the positive control were compared with the vehicle control using Fisher exact test. All analysis and comparisons were evaluated at 5 % (p < 0.05) level.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

       

  Results

a)  Sterility Check

 

The S-9 homogenate was found to be sterile.

 

b)  Metabolic Activation

 

The S-9 homogenate was characterized for its ability to metabolize the promutagens 2-aminoanthracene and benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 strain and the S-9 homogenate was found to be active.

 

c)  Protein Content

 

The protein content of the S-9 homogenate was 30 mg/mL and was found to be satisfactory.

                      

The test item formeda free flowing suspension in DMSO at 50 mg/mL.

 

1. Solubility Test and Justification for the Selection of Vehicle

DMSO is one of the organic vehicles compatible with this test system. Hence, based on the results of solubility test, DMSO was selected as the vehicle of choice to prepare the stock and dilutions of the test item.

2. Cytotoxicity Test and Justification for the Selection of Test Doses

At the end of 3-hour exposure, Hostaperm-Orange GR did not precipitate in the test medium at any of the tested concentrations.

 

At the end of 3-hour exposure, the pH of the test medium in the presence of metabolic activation ranged from 6.99 to 7.10 with 7.00 in the DMSO control while in the absence of metabolic activation it was between 7.05 and 7.17 with 7.15 in the DMSO control.

 

At the end of 3-hour exposure, osmolality of the test medium at the highest treatment condition (500mg/mL) was 0.412 and 0.386 OSMOL/kg, in the presence and absence of metabolic activation, respectively, whereas the corresponding osmolality in the DMSO control was 0.412 and 0.401 OSMOL/kg, respectively.

 

Hostaperm-Orange GR was toxic to the CHO-K1 cells at and above 20 µg/mL where the cell counts were negligible. At 10 µg/mL, the cell growth inhibition as RICC was 49 and 44% in the presence and absence of metabolic activation with 3-hour exposure and 55% in the absence of metabolic activation with 21-hour exposure.

 

Based on these observations, in the chromosomal aberration assay, a maximum of 12 µg/mL was tested in Experiments 1 and 2, and a maximum of 8 µg/mL was tested in Experiment 3.

3. Chromosomal Aberration Assay

Experiment 1

 

At the highest concentration tested (12 µg/mL), the reduction in the cell growth was 51 % compared to the vehicle control.

 

The incidence of aberrations in the vehicle control was within the range of the in-house historical control data.

 

The incidence of aberrant metaphases excluding gaps was statistically comparable to the vehicle control value at all the concentrations tested.

 

No incidences of polyploidy, endoreduplication and pulverization observed.

 

The positive control, cyclophosphamide monohydrate caused a statistically significant increase in the aberrant metaphases excluding gaps.

 

 Experiment 2

 

At the highest concentration tested (12 µg/mL), the reduction in the cell growth was 42 % compared to the vehicle control.

 

The incidence of aberrations in the vehicle control was within the range of the in-house historical control data.

 

The incidence of aberrant metaphases excluding gaps was statistically comparable to the vehicle control value at all the concentrations tested.

 

No incidences of polyploidy, endoreduplication and pulverization observed.

 

Experiment 3

 

At the highest concentration tested (8 µg/mL), the reduction in the cell growth was 56 % compared to the respective vehicle control.

 

The incidence of aberrations in the vehicle control was within the range of the in-house historical control data.

 

The incidence of aberrant metaphases excluding gaps was statistically comparable to the vehicle control value at all the three test concentrations. 

 

No incidences of polyploidy, endoreduplication and pulverization observed.

 

Ethyl methanesulphonate caused a statistically significant increase in the aberrant metaphases excluding gaps.

DISCUSSION

The assay conditions fulfilled all the specifications as per the OECD 473 guideline.

 

There was no evidence of an increase in either structural or numerical aberrations in cultures treated with Hostaperm-Orange GR either in the presence or in the absence of metabolic activation.

 

The results of this study suggest that the test item, Hostaperm-Orange GR does not have the potential to cause chromosome damage in the presence or absence of metabolic activation.

 

In each of these experiments, the respective positive controls produced a statistically significant increase in aberrant metaphases, demonstrating that the system was able to detect the effect of known mutagens.

 

Applicant's summary and conclusion

Conclusions:

It was concluded that the test item was not clastogenic in CHO cells at the tested concentrations and under the conditions of testing employed.

Based on these findings the test item has not to be classified as germ cell mutagen according to Regulation (EC) No 1272/2008.

Executive summary:

The clastogenic potential of the test item to induce chromosomal aberrations in mammalian cells was evaluated according to OECD TG 473 using cultured Chinese Hamster Ovary (CHO) cells in the presence and absence of an exogenous metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

 

The study consisted of a preliminary cytotoxicity test and a chromosomal aberration assay. Chromosomal aberration assay consisted of three independent experiments: Experiments 1 and 2 in the presence and absence of metabolic activation system with 3-hour exposure, respectively, and Experiment 3 in the absence of metabolic activation system with 21-hour exposure.

 

The test item formed a free flowing suspension in DMSO at 50 mg/mL.

 

In a preliminary cytotoxicity test for the selection of test concentrations for the chromosomal aberration assay, the test item was toxic to the CHO-K1 cells at and above 20 µg/mL where the cell counts were negligible. At 10 µg/mL, the cell growth inhibition as RICC was 49 and 44% in the presence and absence of metabolic activation with 3-hour exposure and 55% in the absence of metabolic activation with 21-hour exposure. The test item did not precipitate in the test medium and did not cause any appreciable change in the pH and Osmolality of test medium. Based on these observations, in the chromosomal aberration assay, a maximum of 12 µg/mL was tested in Experiments 1 and 2, and a maximum of 8 µg/mL was tested in Experiment 3.

 

In Experiments 1 and 2 of the chromosomal aberration assay, CHO-K1 cells were exposed to the test item in duplicate for 3 hours at concentrations of 3, 6 and 12 µg/mL in the presence and absence of metabolic activation, respectively. In Experiment 3, CHO-K1 cells were exposed to the test item in duplicate for 21 hours at concentrations of 2, 4 and 8 µg/mL in the absence of metabolic activation. Concurrent vehicle (DMSO) and positive controls (cyclophosphamide monohydrate in the presence of metabolic activation and ethyl methanesulfonate in the absence of metabolic activation) were also tested in duplicate. In each case, the cells in C-metaphase were harvested at 21 hours after the start of the treatment and slides were prepared for chromosomal analysis.

 

At the highest concentration tested, the reduction in cell growth as RICC was 51, 42 and 56 % in Experiments 1, 2 and 3, respectively, compared to the vehicle control.

 

A total of 300 metaphases each from the DMSO control, each treatment level and the positive control were evaluated for chromosomal aberrations.

 

There was no evidence of induction of chromosome aberrations, excluding gaps, either in the presence or in the absence of metabolic activation. There was also no incidence of polyploid cells indicating that the test item did not induce numerical chromosomal aberrations. In each of these experiments, under identical conditions, the respective positive control substances produced a large and statistically significant increase in aberrant metaphases.

 

The study indicated that the test item was not clastogenic at the concentrations tested and under the conditions of testing.

Based on these findings the test item has not to be classified as germ cell mutagen according to Regulation (EC) No 1272/2008.