Reaction product of 3,9-dibromobenzanthrone condensed with 2 equivalents of 1-aminoanthraquinone, subsequently further condensed under oxidative conditions

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 20, 1995 to October 24, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

OECD (April 4, 1984)Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Tests.OECD Guideline for Testing of Chemicals 476.

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

EEC Directive 87/302, Annex (November 18, 1987)Part B; Mutagenicity testing and screening for carcinogenicity; In vitro mammalian cell gene mutation test.Official Journal of the European Communities, No L 133, Vol. 31, 61-63, May 30, 1988

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

Version / remarks:

EPA (May 20, 1987)Detection of gene mutations in somatic cells in culture. Environmental Protection Agency Health Effects Testing Guidelines52 FR 19072 (Corn 52 FR 26150, July 13, 1987); 798.5300

Deviations:

not specified

GLP compliance:

yes

Type of assay:

other: forward mutation system in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of 3,9-dibromobenzanthrone condensed with 2 equivalents of 1-aminoanthraquinone, subsequently further condensed under oxidative conditions

EC Number:

944-232-9

Molecular formula:

Not available - UVCB substance

IUPAC Name:

Reaction product of 3,9-dibromobenzanthrone condensed with 2 equivalents of 1-aminoanthraquinone, subsequently further condensed under oxidative conditions

Test material form:

solid

Method

Target gene:

6-TG

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79 Chinese hamster cells were originally derived from embryonic lung tissue. The cells were cultured in Ham's F10 medium supplemented with 10% pre-tested foetal calf serum, 100 U/ml penicillin and 100 ug/ml streptomycin in tissue culture (plastic) flasks. The humidity in the incubator was adjusted to >85% rH, the air was enriched to 5 ± 2.0 Vol% CO2 and the temperature was 37±1°C.Twice per week the growth medium was replaced by fresh one.The laboratory cultures were passaged weekly in low number (about 5e4 cells per 175 cm2) to keep the level of spontaneous mutants low and to prevent the cells of reaching a stationary phase of cell growth. Large stocks of the V79 cell line have been stored in liquid nitrogen allowing the repeated use of the same cell culture batch in experiments. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells. The frozen cell suspension contains 10% dimethylsulfoxide (DMSO). All stock cells were cultured in cleansing medium for three days to purge the cultures of existing hprt" mutants. Cleansing medium was growth medium supplemented with 3 uM aminopterin. The cells have a stable karyotype with a modal chromosome number of 22±1. All stock cells were checked for mycoplasma contamination, using the Hoechst-Dye staining method or the 6-MPDR method, before being frozen. Thawed stock culture cells are kept not longer than for twelve passages (three months) in culture.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction

Test concentrations with justification for top dose:

Cytotoxicity test
Range with metabolic activation: 2.44 to 5000.0 ug/ml
Range without metabolic activation: 2.44 to 5000.0 ug/ml
Mutagenicity test
Original experiment: Range with metabolic activation: 185.19 to 5000.0 ug/ml
Range without metabolic activation: 185.19 to 5000.0 ug/ml
Confirmatory experiment:Range with metabolic activation: 625.0 to 5000.0 ug/ml
Range without metabolic activation: 625.0 to 5000.0 ug/ml
A preliminary range finding test was run assessing cytotoxicity. FAT 45019/E was tested at concentrations up to 5000.0 ug/ml. In the part with metabolic activation no cytotoxic effect could be seen.
Without metabolic activation treatment with FAT 45019/E revealed an acute inhibition of growth of 59.5 and 59.6% at the two highest concentrations. Accordingly, 5000.0 ug/ml with and without metabolic activation was chosen as highest concentration for the first mutagenicity assay.

Vehicle / solvent:

Dimethylsulfoxide (DMSO).FAT 45019/E proved to be insoluble in all common vehicles. Therefore, FAT 45019/E had to be applied as a suspension. DMSO was chosen as vehicle.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

Preliminary cytotoxicity test
A cytotoxicity test was performed on V79 cells as a preliminary test to determine the highest concentration of the test substance to be applied in the mutagenicity assay. For each concentration and the untreated controls, 2.5e5V79 cells were seeded in 5 ml growth medium into a 25 cm2 tissue culture flask and incubated overnight. The cultures were exposed to the test substance for five hours in the presence and for 21 hours in the absence of a metabolic activation system. In the two parts of the experiment, 12 concentrations of the test substance and two vehicle (DMSO) controls were tested.The highest concentration was determined in a preliminary solubility test. Lower concentrations were prepared by serial dilution by a factor of 0.5. The treatment was terminated by washing the cultures with phosphate buffered saline (PBS). Compound-induced cytotoxicity was estimated by cloning efficiency immediately after treatment. The cultures were counted and diluted so that 100 cells were seeded per 9.6 cm2 in 3 ml of growth medium. After seven to eight days of growth the cultures were fixed and stained with Giemsa and the surviving colonies determined with the aid of an electronic colony counter (Artek Counter®, Fisher Scientific) or by the naked eye. The sensitivity of the colony counter was adjusted to detect clones of about twenty or more cells. The concentration to be selected as the highest for the mutagenicity assay was the one causing about 50-90% reduction of viable cells in comparison with the mean of the two negative controls or corresponds to the substance's solubility limit (precipitates in the culture).
Mutagenicity test
Depending on the toxicity of the test compound 2.5-5.0e6 cells of passage 24 (original experiment) and passage 24 (confirmatory experiment) were plated in 30 ml growth medium into 175 cm2 flasks and incubated overnight. The growth medium was replaced for five hours by 27 ml treatment medium and 3.0 ml S9 activation mixture, or for 21 hours by 30 ml treatment medium alone.In each assay, cultures were treated in duplicate with four test chemical concentrations, a positive and a negative (DMSO) control. In the non-activated part of the experiment, the positive control was the ultimate mutagen Ethylmethansulphonate (EMS) at a concentration of 0.3 ul/ml. In the part with metabolic activation the positive control was the promutagen N-Nitrosodimethylamine (DMN) at a concentration of 1.0 μl/ml.The treatment was terminated by washing the cell layer extensively with PBS. After washing, the cells were suspended by trypsinisation, pelleted, resuspended in fresh growth medium and counted with a haemocytometer or electronic coulter counter (Coulter Counter®, Model ZM), diluted with fresh growth medium and replated into flasks at 2xl06 cells. The cultures were incubated at 37°C for seven to eight days during which the cells could recover and divide to express the mutant phenotype.The cultures were subcultered after the second or third day transferring 2e6 cells to a fresh flask to maintain exponential growth during the expression phase.In parallel cytotoxicity of the compound was estimated from the cloning efficiency immediately after treatment. The counted cell suspension of each concentration level was further diluted so that 100 cells were seeded per 9.6 cm2 in 2.5 ml of growth medium and incubated at 37°C. The number of colonies which developed within seven to eight days in these cultures reflected the viability at the end of the treatment (survival values).At the end of the expression period the cultures were trypsinised, pelleted, resuspended in fresh growth medium and counted with a haemocytometer or electronic coulter counter (Coulter Counter®, Model ZM). The cell suspension of each culture was diluted with fresh growth medium and an aliquot replated into four flasks (75 cm2 growth area) each containing 2e6 cells for the mutant selection. The high-density cultures were subjected to the mutant selection procedure by supplementing the growth medium with 8 μg/ml 6-thioguanine (6-TG). Only cells mutated at the hprt locus could survive the 6-thioguanine treatment. The number of colonies formed in these flasks during the following days reflected the overall number of mutations induced by the treatment with the test substance or the mutagen (positive control). After seven to eight days incubation at 37°C, the cultures were fixed and stained with Giemsa. The mutant clones were counted with the naked eye.
In parallel the viability at the end of the expression period was estimated from the cloning efficiency.The remaining cell suspensions from the various expression cultures were further diluted such that 100 cells were seeded per 9.6 cm2 in 2.5 ml of growth medium and were incubated at 37°C. The number of colonies which developed within these low-density cultures reflected the viability at the end of the expression period (viability values).
Assay acceptance criteria
The results of the experiments should not be influenced by a technical error, contamination or a recognized artifact.From each experiment, at least three concentrations of the test substance, one positive and one solvent control should be evaluated.The mutant frequency of the solvent controls (spontaneous mutant frequency) should not exceed 35e6.
The positive control should fulfil the criteria for a mutagenic substance.The highest concentration of the test substance applied in the mutagenicity test should either reduce the viable cells by about 50-90% or correspond to the test substance's solubility limit (precipitates in the culture). In case of non-toxic freely soluble compounds the highest tested concentration will be 5 mg/ml. In special cases the highest concentration can be determined by the sponsor.

Rationale for test conditions:

The test system allows the detection of base-pair substitutions, frameshift mutations and deletions induced by the test substance or by its metabolites. Mutagenic effects are manifested by the appearance of cells resistant to 6-TG and can be quantified by comparison of the numbers of 6-TG resistant colonies in the treated and control cultures. To ensure that any mutagenic effect of metabolites of the test substance found in mammals is also detected, an experiment is performed, in which the metabolic turnover of the test material is simulated in vitro by the addition of an activation mixture to the cell cultures containing rat-liver post mitochondrial supernatant (S9 fraction) and cofactors.

Evaluation criteria:

Assay evaluation criteria
All mutant frequencies are normalized to a virtual cloning efficiency of 100% at the end of the expression period. If the cloning efficiency of the viability cultures is lower than 15%, the corresponding mutant frequency is usually not calculated, owing to the high statistical insignificance of the result. For every concentration a mean mutant factor, which is defined as the ratio of the mean mutant frequencies of the treated cultures with the mean mutant frequencies of the solvent control cultures, will be calculated.
Criteria for a positive response
The test substance will be considered to be mutagenic if:
The assay is valid (see assay acceptance criteria)
The mutant frequency at one or more concentrations is significantly greater than that of the negative control and the number of normalized mutant clones in the treated and untreated cultures differs by more than 20.There is a significant dose-relationship as indicated by the linear trend analysis.The effects described above are reproducible.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Toxicity
In the preliminary toxicity test with and without metabolic activation 12 concentrations of FAT 45019/E were tested. The concentrations selected ranged from 2.44 to 5000.0 μg/ml and separated by 2-fold intervals. In the part with metabolic activation no growth inhibition was seen at any concentration. In the part without metabolic activation FAT 45019/E exerted a growth inhibitory effect of 59.5 and 59.6% at the two highest concentrations.
Accordingly, four concentrations were selected for the original experiment ranging from 185.19 to 5000.0 μg/ml in the presence and absence of metabolic activation.
In the presence and in the absence of metabolic activation no severe toxic effect could be seen at any concentration.
In the confirmatory experiment concentrations ranging from 625.0 to 5000.0 μg/ml were used for both parts. Again, no serious toxicity was seen in the presence and in the absence of metabolic activation at any concentration.
Mutagenicity
The test substance caused the formation of precipitates in the culture medium at all concentrations tested.
In the presence and absence of metabolic activation, no relevant increase in mutant frequency was observed at any concentration level of FAT 45019/E tested in the original or the confirmatory experiment in comparison with the negative control.
The occasional occurrence of statistically significant differences is considered to be purely fortuitous and not related to treatment with the test compound. The respective values were clearly within the historical control range and no concentration dependency could be seen.The positive controls induced a clear increase in mutant frequency.

Any other information on results incl. tables

LEGEND TO TABLES

*	No data
Tx	No data due to high toxicity
nTx	Not toxic
§	One duplicate lost
Ns	Not significant

(All calculations on the following tables were made by a computer using exact values. The calculated value given in tables are rounded to two or three digits).

 

RESULT OF THE CYTOTOXICITY TEST

Experiment with metabolic activation

Treatment	Cell number after treatment (x10E6)	Survival clones after treatment (per well)
Negative control	1.281	84	88	86	82	75	81
Negative control	1.134	76	81	80	84	72	79
FAT 45019/E:
5000.0000 μg/ml	1.715	83	80	75	78	77	83
2500.0000 μg/ml	1.615	67	69	76	70	74	77
1250.0000 μg/ml	1.418	78	74	69	76	68	70
625.0000 μg/ml	1.531	84	71	80	76	72	74
312.0000 μg/ml	1.431	84	81	77	74	90	82
156.2500 μg/ml	1.262	79	79	88	78	81	84
78.1250 μg/ml	1.541	82	77	87	70	74	83
39.0625 μg/ml	1.554	82	77	87	70	74	79
19.5313 μg/ml	1.246	77	74	75	81	80	79
9.7656 μg/ml	1.320	83	88	93	90	93	86
4.8828 μg/ml	1.187	86	90	76	79	85	87
2.4414 μg/ml	1.006	89	84	79	87	90	88
Treatment	Mean of clones	Number of viable cells (x10E6)			Acute cytotoxicity (% of control)
Negative control	82.67	1.06
Negative control	78.67	0.89
FAT 45019/E:
5000.0000 μg/ml	79.33	1.36			nTX
2500.0000 μg/ml	72.17	1.17			nTX
1250.0000 μg/ml	72.50	1.03			nTX
625.0000 μg/ml	76.17	1.17			nTX
312.0000 μg/ml	81.33	1.16			nTX
156.2500 μg/ml	81.50	1.03			nTX
78.1250 μg/ml	80.17	1.24			nTX
39.0625 μg/ml	78.83	1.23			nTX
19.5313 μg/ml	77.67	0.97			0.76
9.7656 μg/ml	88.83	1.17			nTX
4.8828 μg/ml	83.83	1.00			nTX
2.4414 μg/ml	86.17	0.87			11.11

 

RESULTS OF THE CYTOTOXICITY TEST

Experiment without metabolic activation

Treatment	Cell number after treatment (x10E6)	Survival clones after treatment (per well)
Negative control	3.075	84	90	78	88	83	85
Negative control	2.450	91	96	84	89	97	95
FAT 45019/E:
5000.0000 μg/ml	2.221	40	43	47	47	41	48
2500.0000 μg/ml	2.096	42	50	48	46	46	49
1250.0000 μg/ml	1.733	89	80	65	68	70	74
625.0000 μg/ml	2.355	67	71	70	67	66	68
312.0000 μg/ml	1.677	74	75	73	69	79	78
156.2500 μg/ml	1.895	74	80	77	81	71	70
78.1250 μg/ml	1.785	73	76	72	70	78	76
39.0625 μg/ml	2.285	85	88	84	84	90	81
19.5313 μg/ml	2.096	97	93	90	93	88	87
9.7656 μg/ml	1.921	86	89	83	88	85	92
4.8828 μg/ml	2.254	90	81	80	69	93	87
2.4414 μg/ml	1.956	99	93	102	96	99	91
Treatment	Mean of clones	Number of viable cells (x10E6)			Acute cytotoxicity (% of control)
Negative control	84.67	2.60
Negative control	92.00	2.25
FAT 45019/E:
5000.0000 μg/ml	44.33	0.98			59.47
2500.0000 μg/ml	46.83	0.98			59.59
1250.0000 μg/ml	74.33	1.29			46.96
625.0000 μg/ml	68.17	1.61			33.90
312.0000 μg/ml	74.67	1.25			48.45
156.2500 μg/ml	75.50	1.42			41.10
78.1250 μg/ml	74.17	1.32			45.49
39.0625 μg/ml	85.33	1.95			19.70
19.5313 μg/ml	91.33	1.91			21.17
9.7656 μg/ml	87.17	1.67			31.05
4.8828 μg/ml	81.67	1.84			24.22
2.4414 μg/ml	96.67	1.89			22.15

 

SUMMARY OF THE MUTAGENICITY EXPERIMENT

Experiment with metabolic activation (Original Experiment)

Treatment	Mean of viability clones per well	Mean of mutants per flask	Normalized mean of mutants per flask
Negative control	77.58	11.75	15.15
Positive control DMN 1 μl/ml	56.83	131.25	230.94
FAT 45019/E:
5000.0000 μl/ml	72.33	14.38	19.87
1666.6667 μl/ml	76.92	14.75	19.18
555.5556 μl/ml	77.33	16.61	21.50
185.1852 μl/ml	74.67	14.25	19.08
Treatment	Mean mutant frequency (x10E-6)	Mean mutant factor	Significance (P)
Negative control	7.57
Positive control DMN 1 μl/ml	115.47	15.25	P<0.001
FAT 45019/E:
5000.0000 μl/ml	9.94	1.31	Ns
1666.6667 μl/ml	9.59	1.27	Ns
555.5556 μl/ml	10.75	1.42	0.02<P<0.05
185.1852 μl/ml	9.54	1.26	Ns
Linear relation : Ns

 

SUMMARY OF THE MUTAGENICITY EXPERIMENT

Experiment without metabolic activation (Original Experiment)

Treatment	Mean of viability clones per well	Mean of mutants per flask	Normalized mean of mutants per flask
Negative control	78.08	7.63	9.77
Positive control EMS 0.3 μl/ml	44.58	1293.50	2901.31
FAT 45019/E:
5000.0000 μl/ml	74.58	10.13	13.58
1666.6667 μl/ml	79.50	8.38	10.53
555.5556 μl/ml	75.33	10.25	13.61
185.1852 μl/ml	75.33	12.50	16.59
Treatment	Mean mutant frequency (x10E-6)	Mean mutant factor	Significance (P)
Negative control	4.88
Positive control Ems 0.3 μl/ml	1450.65	297.11	P<0.001
FAT 45019/E:
5000.0000 μl/ml	6.79	1.39	Ns
1666.6667 μl/ml	5.27	1.08	Ns
555.5556 μl/ml	6.80	1.39	Ns
185.1852 μl/ml	8.30	1.70	Ns
Linear relation : Ns

 

SUMMARY OF THE MUTAGENICITY EXPERIMENT

Experiment with metabolic activation (Confirmatory Experiment)

Treatment	Mean of viability clones per well	Mean of mutants per flask	Normalized mean of mutants per flask
Negative control	77.42	3.88	5.01
Positive control DMN 1 μl/ml	57.50	139.75	243.04
FAT 45019/E:
5000.0000 μl/ml	76.67	2.63	3.42
2500.0000 μl/ml	78.50	5.88	7.48
1250.0000 μl/ml	83.50	4.88	5.84
625.0000 μl/ml	79.17	7.13	9.00
Treatment	Mean mutant frequency (x10E-6)	Mean mutant factor	Significance (P)
Negative control	2.50
Positive control DMN 1 μl/ml	121.52	48.56	P<0.001
FAT 45019/E:
5000.0000 μl/ml	1.71	0.68	Ns
2500.0000 μl/ml	3.74	1.50	Ns
1250.0000 μl/ml	2.92	1.17	Ns
625.0000 μl/ml	4.50	1.80	0.01<P<0.02
Linear relation : 0.01<P<0.025

 

SUMMARY OF THE MUTAGENICITY EXPERIMENT

Experiment without metabolic activation (Confirmatory Experiment)

Treatment	Mean of viability clones per well	Mean of mutants per flask	Normalized mean of mutants per flask
Negative control	85.17	2.88	3.38
Positive control EMS 0.3 μl/ml	48.25	1264.75	2621.24
FAT 45019/E:
5000.0000 μl/ml	83.25	4.25	5.11
2500.0000 μl/ml	83.25	3.75	4.50
1250.0000 μl/ml	88.17	5.63	6.38
625.0000 μl/ml	86.67	6.13	7.07
Treatment	Mean mutant frequency (x10E-6)	Mean mutant factor	Significance (P)
Negative control	1.69
Positive control EMS 0.3 μl/ml	1310.62	776.50	P<0.001
FAT 45019/E:
5000.0000 μl/ml	2.55	1.51	Ns
2500.0000 μl/ml	2.25	1.33	Ns
1250.0000 μl/ml	3.19	1.89	Ns
625.0000 μl/ml	3.53	2.09	Ns
Linear relation : Ns

 

Applicant's summary and conclusion

Conclusions:

Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that FAT 45019/E and its metabolites did not show any mutagenic activity in this forward mutation system.

Executive summary:

FAT 45019/E, >90% purity, Ident-Nr. 5546 was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The test substance was suspended in DMSO. The cells were treated in the experiments with metabolic activation for 5 hours and in the experiments without metabolic activation for 21 hours. The results of each experiment were confirmed in a second and independent experiment (confirmatory experiment).

 

This test is in agreement with:

OECD (April 4, 1984); Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Tests. OECD Guideline for Testing of Chemicals 476.

EPA (May 20, 1987); Detection of gene mutations in somatic cells in culture. Environmental Protection Agency Health Effects Testing Guidelines; 52 FR 19072 (Corn 52 FR 26150, July 13, 1987); 798.5300

EEC Directive 87/302, Annex (November 18, 1987); Part B; Mutagenicity testing and screening for carcinogenicity; In vitro mammalian cell gene mutation test. Official Journal of the European Communities, No L 133, Vol. 31, 61-63, May 30, 1988

 

Cytotoxicity test

A preliminary range finding test was run assessing cytotoxicity. FAT 45019/E was tested at concentrations up to 5000.0 μg/ml. In the part with metabolic activation no cytotoxic effect could be seen. Without metabolic activation treatment with FAT 45019/E revealed an acute inhibition of growth of 59.5 and 59.6% at the two highest concentrations. Accordingly, 5000.0 μg/ml with and without metabolic activation was chosen as highest concentration for the first mutagenicity assay.

 

Mutagenicity test with metabolic activation

The original experiment was performed at the following concentrations: 185.19, 555.56, 1666.67 and 5000.0 μg/ml. In the confirmatory experiment the concentrations applied were 625.0, 1250.0, 2500.0 and 5000.0 μg/ml. Both experiments revealed no relevant cytotoxic effects. N-Nitrosodimethylamine (DMN, 1.0 μl/ml) was used as positive control.

In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG).

 

Mutagenicity test without metabolic activation

The original experiment was performed at the following concentrations: 185.19, 555.56, 1666.67 and 5000.0 μg/ml. In the confirmatory experiment the concentrations applied were 625.0, 1250.0, 2500.0 and 5000.0 μg/ml. No severe toxicity was observed in the two experiments. Ethylmethanesulfonate (EMS, 0.3 μl/ml) was used as positive control.

In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-TG.

 

Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that FAT 45019/E and its metabolites did not show any mutagenic activity in this forward mutation system.