2,9-bis(p-methoxybenzyl)anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone

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:

27 Oct 2005 - 13 Apr 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(1997)

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 OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

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:

-

EC Number:

479-300-2

EC Name:

-

Molecular formula:

unspecified

IUPAC Name:

Reaction mass of Bisbenzimidazo[2,1-a:1',2'-b']anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-6,11-dione and Bisbenzimidazo[2,1-a:2',1'-a']anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-10,21-dione

Test material form:

solid: nanoform, no surface treatment

Details on test material:

- State of aggregation: solid, powder
- Particle size distribution (TEM): 30.3 nm (D50)
- Mass median aerodynamic diameter (MMAD): not specified
- Geometric standard deviation (GSD): not specified
- Shape of particles: spherical
- Surface area of particles: 16.8 m²/g
- Crystal structure: crystalline
- Coating: no
- Surface properties: not applicable
- Density: 1515 kg/m³ at 20°C
- Moisture content: refer to IUCLID chapter 1
- Residual solvent: refer to IUCLID chapter 1
- Activation: not applicable
- Stabilisation: not applicable

Specific details on test material used for the study:

- Physical state: Solid, black powder
- Analytical purity: > 99%
- Storage condition of test material: Room temperature

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM medium with glutamine supplemented with − 10% (v/v) fetal calf serum (FCS),
− 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL)
− 1% (v/v) amphotericin B (250 μg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically checked for plating efficiency (= colony forming ability) incl. vital staining: yes

Metabolic activation:

with and without

Metabolic activation system:

The S-9 fraction was prepared according to AMES et al. (Mut. Res. 31, 347-364, 1975) from the livers of at least 5 male Sprague-Dawley rats that had received a single ip injection of 500 mg Aroclor 1254/ kg bw 5 days earlier.

Test concentrations with justification for top dose:

first experiment:
4-hour exposure, 18-hour sampling time, without S-9 mix: 0; 1.56; 3.13; 6.25; 12.5; 25.0; 50.0; 75.0; 100.0 μg/mL
4-hour exposure, 18-hour sampling time, with S-9 mix: 0; 1.56; 3.13; 6.25; 12.5; 25.0; 50.0; 75.0; 100.0 μg/mL

Dose selection was based on the solubility of the test substance, i.e. doses > 12.5 μg/mL both with and without S-9 mix led to strong precipitation which interferes with evaluation of metaphases.

second experiment:
18-hour exposure, 18-hour sampling time, without S-9 mix: 0; 0.78; 1.56; 3.13; 6.25; 12.5; 25.0 μg/mL
18-hour exposure, 28-hour sampling time, without S-9 mix: 0; 3.13; 6.25; 12.5 μg/mL
4-hour exposure, 28-hour sampling time, with S-9 mix: 0; 1.56; 3.13; 6.25; 12.5; 25.0 μg/mL

Again doses ≥ 12.5 μg/mL led to strong test substance precipitation which interferes with the evaluation of metaphases.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was considered the most suitable. Therefore, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the V79 in vitro cytogenetic test and for which historical control data are available.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S-9: 500 µg/mL ethyl methanesulfonate (EMS); With S-9: 500 µg/mL cyclophosphamide (CPP)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium in Quadriperm dishes

DURATION
- Preincubation period: 24 - 30 hrs
- Exposure duration: 4 or 18 hrs

- Expression time (cells in growth medium): 14 hrs or 24 hrs; after continuous treatment, i.e. 18 hours without S-9 mix, cells were treated in culture medium supplemented with 10% FCS and in the case of a sampling time of 28 hrs incubated again for another 10 hours.

- Fixation time (start of exposure up to fixation or harvest of cells): 2 - 3 hours prior to harvesting the cells, 0.2 μg Colcemid/mL culture medium was added to each chamber in order to arrest mitosis in the metaphase. For hypotonic treatment, 5 mL of a 0.4% KCl solution which was at 37°C was added for about 20 min. Subsequently, 5 mL of fixative (methanol : glacial acetic acid/3 : 1) which was added at 4°C and kept for at least 15 min and then replaced. After about another 10 min, the fixative was replaced again and kept for at least 5 min at room temperature for complete fixation.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): solution of Giemsa and Titrisol (15 mL Giemsa, 185 mL Titrisol pH 7.2) for 10 minutes

NUMBER OF REPLICATIONS: 2 chambers of Quadriperm dishes were used per test culture.

NUMBER OF CELLS EVALUATED: 200 metaphases evaluated

CELL MORPHOLOGY
About 3 hours and 16 - 18 hours after test substance treatment, cultures of all test groups were checked for cell morphology, which is an indication of attachment of the cells to the slides.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
A mitotic index based on 1 000 cells/culture was determined for all evaluated test groups in both experiments.
For the determination of cytotoxicity, additional cell cultures (using 25 cm2 plastic flasks) were treated in the same way as in the main experiment. Growth inhibition was estimated by counting the number of cells in the dose groups in comparison with the concurrent vehicle control at the end of the culture period using a counter.

CELLCYCLE
The cell cycle of the untreated V79 cells lasted for about 13 - 14 hours under the selected culture conditions. Thus, the selected 1st sampling time of 18 hours was within the 1 - 1.5 x the normal cell cycle time, as recommended by the OECD TG 473. The later sampling time of 28 hours was chosen to cover a possible cell cycle delay.

Evaluation criteria:

If there is a clear increase in chromosomally damaged cells, the number of metaphases to be analyzed is reduced from the planned 200 mitoses/test group.
For details concerning evaluation criteria, please refer to "Any other information on materials and methods incl. tables".

Assessment criteria
The test chemical was assessed as “positive” in this assay if the following criteria were met:
• A dose-related and reproducible significant increase in the number of cells with structural / numerical chromosomal aberrations.
• The number of aberrant cells exceeded both the concurrent negative control range and the highest value of the negative historical control range.

A test substance generally was considered as “negative” if the following criteria were met:
• The number of cells with structural / numerical aberrations in the dose groups was not significantly above the concurrent negative control and was within the historical control data.

Statistics:

The proportion of metaphases with aberrations was calculated for each group.
A comparison of each dose group with the vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test were significant, labels (* p < 0.05, ** p < 0.01) are printed in the tables.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At concentrations of 25 µg/mL and above, evaluation was not possible due to strong test substance precipitation which interferes with metaphase evaluation.

Mitotic index:
According to the results of the determination of the mitotic index, no suppression of the mitotic activity was observed under any of the experimental conditions.

Cell counts:
According to the results of the cell count, no growth inhibition was observed under any of the experimental conditions.

Cell morphology:
Cell attachment was not influenced at any dose evaluated for structural chromosomal aberrations.

Any other information on results incl. tables

CHROMOSOME ANALYSIS - 1ST EXPERIMENT

 

- Clastogenic mode of action: After a treatment time of 4 hours no increase in the number of chromosomally damaged cells was observed either without S-9 mix or after the addition of a metabolizing system.

- Aneugenic mode of action: No increase in the number of cells with changes in the number of chromosomes was demonstrated either without S-9 mix or after the addition of a metabolizing system.

 

 

CHROMOSOME ANALYSIS - 2ND EXPERIMENT

 

- Clastogenic mode of action: Both with and without S-9 mix, no increase in the number of structurally damaged metaphases was observed either after a treatment time of 4 hours or after a continuous treatment of 18 hours at both sampling times, i.e. 18 hours and 28 hours.

- Aneugenic mode of action: No increase in the number of cells with changes in the number of chromosomes was demonstrated either without S-9 mix or after the addition of a metabolizing system.

 

SUMMARY TABLE

					Metaphases with Aberrations
					Incl Gaps		Exc. Gaps
Exposure Time	Sampling Time	Dose (µg/ml)	S9-Mix	number of metaphases	n	%	n	%
4 h	18 h	Vehicle	-	200	11	5.5	4	2
4 h	18 h	3.13	-	200	10	5	5	2.5
4 h	18 h	6.25	-	200	7	3.5	2	1
4 h	18 h	12.5	-	200	5	2.5	4	2
4 h	18 h	EMS 500	-	100	19	19	15	15
4 h	18 h	Vehicle	+	200	9	4.5	3	1.5
4 h	18 h	3.13	+	200	12	6	7	3.5
4 h	18 h	6.25	+	200	15	7.5	4	2
4 h	18 h	12.5	+	200	8	4	3	1.5
4 h	18 h	CPP 0.5	+	100	31	31	26	26
18 h	18 h	Vehicle	-	200	6	3	2	1
18 h	18 h	3.13	-	200	13	6.5	7	3.5
18 h	18 h	6.25	-	200	6	3	2	1
18 h	18 h	12.5	-	200	4	2	0	0
18 h	18 h	EMS 500	-	100	19	19	19	19
18 h	28 h	Vehicle	-	200	9	4.5	8	4
18 h	28 h	12.5	-	200	6	3	3	1.5
18 h	28 h	EMS 500	-	100	22	22	19	19
4 h	28 h	Vehicle	+	200	7	3.5	3	1.5
4 h	28 h	3.13	+	200	8	4	4	2
4 h	28 h	6.25	+	200	11	5.5	9	4.5
4 h	28 h	12.5	+	200	7	3.5	4	2
4 h	28 h	CPP 0.5	+	1000	17	17	15	15

EMS = ethyl methanesulfonate

CPP = cyclophosphamide

 

Discussion and conclusion:

 

According to the results of the present in vitro cytogenetic study, the test substance did not lead to an increase in the number of structural chromosomal aberrations incl. and excl. gaps either without S-9 mix or after the addition of a metabolizing system in two experiments performed independently of each other selecting different exposure times (4 hours treatment and continuous treatment) and sampling times (18 and 28 hours); the types and frequencies of aberrations were close to the range of that of the concurrent negative control values at both sampling times and in the range of the historical control data.

No increase in the number of cells containing numerical chromosomal aberrations was demonstrated either.

The increase in the frequencies of chromosomal aberrations induced by the positive control agents EMS and CPP clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S-9 mix employed.

 

Thus, under the experimental conditions chosen here, the conclusion is drawn that the test article is neither a clastogenic (chromosome-damaging) nor an aneugenic agent under in vitro conditions using V79 cells.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions chosen here, the conclusion is drawn that the test article is neither a clastogenic (chromosome-damaging) nor an aneugenic agent under in vitro conditions using V79 cells.

Executive summary:

In an GLP and OECD guideline compliant study (No. 473) the test substance was assessed for its potential to induce structural chromosomal aberrations (clastogenic activity) and/or changes in the number of chromosomes (aneugenic activity) in V79 cells in vitro both in the presence and in the absence of a metabolizing system. According to an initial range-finding cytotoxicity test for the determination of the highest experimental doses, the test substance did not exhibit any pronounced toxicity up to the highest possible dose, i.e. 3 000 μg/mL, at which distinct test substance precipitation was observed. However, due to strong test substance precipitation, which interfers with metaphase evaluation, lower doses must be selected and the test groups in bold type were evaluated:

• 1st experiment

4-hour exposure, 18-hour sampling time, without S-9 mix

0; 1.56;3.13; 6.25; 12.5; 25.0; 50.0; 75.0; 100.0 μg/mL

4-hour exposure, 18-hour sampling time, with S-9 mix

0; 1.56;3.13; 6.25; 12.5; 25.0; 50.0; 75.0; 100.0 μg/mL

• 2nd experiment

18-hour exposure, 18-hour sampling time, without S-9 mix

0; 0.78; 1.56;3.13; 6.25; 12.5; 25.0 μg/mL

18-hour exposure, 28-hour sampling time, without S-9 mix

0; 3.13; 6.25;12.5μg/mL

4-hour exposure, 28-hour sampling time, with S-9 mix

0; 1.56;3.13; 6.25; 12.5; 25.0 μg/mL

Doses > 12.5 μg/mL both with and without S-9 mix led to strong precipitation which interferes with evaluation of metaphases. About 2 - 3 hours prior to harvesting the cells, Colcemid was added to arrest cells at a metaphase-like stage of mitosis (c-metaphases). After preparation of the chromosomes and staining with Giemsa, 100 metaphases for each culture in the case of the test substance and vehicle controls, or 50 cells for each culture in the case of the concurrent positive controls, were analyzed for chromosomal aberrations. The negative controls (vehicle controls) gave frequencies of aberrations within the range expected for the V79 cell line. Both of the positive control chemicals, i.e. EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations. On the basis of the results of the present study, the test substance did not cause any increase in the number of structurally aberrant metaphases incl. and excl. gaps at both sampling times either without S-9 mix or after adding a metabolizing system in two experiments performed independently of each other. No increase in the frequency of cells containing numerical aberrations was demonstrated either. Thus, under the experimental conditions of this assay, the test material is considered not to be either a clastogenic or an aneugenic agent under in vitro conditions in V79 cells.