4-[[2-[[2,5-dimethoxy-4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1,3-dioxobutyl]amino]-5-methoxy-2-methylbenzenesulphonic acid, sodium salt

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:

October 17th, 1988 to October 19th, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

OECD Guideline For Testing Of Chemicals, 473 "Genetic Toxicology: In vitro Mammalian Cytogenetic Test". Adopted: may 26, 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

4-[[2-[[2,5-dimethoxy-4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1,3-dioxobutyl]amino]-5-methoxy-2-methylbenzenesulphonic acid, sodium salt

EC Number:

303-161-8

EC Name:

4-[[2-[[2,5-dimethoxy-4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1,3-dioxobutyl]amino]-5-methoxy-2-methylbenzenesulphonic acid, sodium salt

Cas Number:

94158-87-9

Molecular formula:

C22H(27-x)N3NaxO14S3 x<=2 C22H27N3O14S3.xNa

IUPAC Name:

4-[[2-[[2,5-dimethoxy-4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1,3-dioxobutyl]amino]-5-methoxy-2-methylbenzenesulphonic acid, sodium salt

Test material form:

solid: particulate/powder

Details on test material:

Reactive Yellow 160

Method

Target gene:

chromosomal aberrations

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the V79 cell line stored in liquid nitrogen in the Laboratory of Genetic Toxicology of Hoechst AG are allowing the repeated use of the same cell culture batch in experiments. Consequently the cellular parameters of the experiments remain similar because of the reproducible characteristics of the cells.
The tawed stock cultures were progagated at 37 °C in 25 cm2 plastic flasks.
Seeding was done with about 1-3 x 105 cells per flask in 5 ml of MEM-medium supplemented with 10% fetal calf serum (FCS). The cells were subcultured twice a week.

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

The concentrations were chosen from the data of the cytotoxicity assay in the preliminary experiment.
For main experiment dose levels of 200, 600, 2000 μg/ml were used, in the absence and in the presence of S9-mix metabolic activation.

In a preliminary experiment Remazol-Brillantgelb 4GL was assayed with respect to its solubility in cell culture medium. The highest concentration at which no visible precipitation was observed, was found to be 2000 ug/ml. The cytotoxicity experiment proved that Remazol-Brillantgelb 4GL was not toxic to the V79 cells in the absence of metabolic activation (S9-mix). Also in the presence of metabolic activation (S9-mix) no indication of toxicity was observed up to the limit of solubility. No cytotoxic effect as reduction of mitotic index was also observed.

Vehicle / solvent:

At the day of the experiment the test substance was dissolved as a solution in aqua bidest at appropriate concentrations.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Two days old exponentially growing stock cultures which were over 50% confluent were trypsinised and a single cell suspension was prepared. The trypsin concentration was 0.5 % in Ca-Mg-free salt solution. 1-2 x 106 cells/flask were seeded into four 80 cm2 plastic flasks containing 15 ml MEM with 10 % FCS (6 h preparation). 4-6 x 105 cells/flask were seeded into four 25 cm2 plastic flasks containing 5 ml MEM with 10% FCS (18 h preparation). 2-4 x 105 cells/flask were seeded into four 25 cm2 plastic flasks containing 5 ml MEM with 10 % FCS (28 h preparation).
After 24 h the medium was replaced with medium containing 5 % FCS and the test substance, both without S9-mix and with 40 ul/ml S9-mix.
After 2 h this medium was replaced with normal medium after rinsing once with physiological saline solution.
Treatment was performed with 3 concentrations of the test substance.
The highest concentration did not reduce the number of scorable metaphases more than 20% of the negative controls. The mitotic index was determined in samples of 1000 cells. The toxicity of the test substance was determined in a preliminary experiment by establishing the concentration-related plating efficiency.
According to these data the concentration range was chosen.
3.5, 15.5 and 25.5 h after the start of the treatment colcemide was added (0.04 μg/ml culture medium) to the cultures. 2.5 h later (6 h, 18 h and 28 h preparation) the cells were trypsinised.
For hypotonic treatment, approximately 5 ml of 0.075 M potassium chloride solution at 37 °C was quickly added and suspended. This suspension was then allowed to incubate for 10 minutes in a water bath at 37 °C. Addition of 1.5 ml fixstive and flow through with air.
After re-centrifuging for five minutes at 1000 rpm, all but one drop of the supernatant was drawn off by pipette. The sediment was carefully covered with a layer composed of 2.5 ml fixative (methanol:glacial acetic acid 3 + 1). After 20 minutes the fixation was removed carefully with a pipette and suspended in 2.5 ml fixative. After another 30 minutes, the mixture was centrifuged, after which the liquid was removed by pipette and fresh fixative added. The tubes were covered and kept for at least 12 hours (overnight) in a refrigerator at 4 °C.
After re-centrifuging for 5 minutes at 1000 rpm, all but one drop of the liquid was removed by pipette and a new suspension formed with a small quantity of freshly prepared fixative. A few drops of this suspension were placed with a pasteur pipette onto clean microscopic slides which had been stored in distilled water at 4 °C, the drops were then briefly passed through a Bunsen flame and air-dried for 24 hours. Staining was performed as follows:
- staining for 10 minutes in 2% orcein solution
- rinsing 3 times in destilled water
- rinsing twice in acetone
- brief rinsing in acetone/xylene
- 2 minutes in acetone/xylene
- 5 minutes in xylene
- 10 minutes in xylene
- embedding in EntellanR or EukittR
2-5 slides were prepared from each flask.
In the same way both negative and positive controls were prepared 16 h after medium change or treatment, respectively.

Analysis of metaphases
After the slides had been coded (Coding Scheme 1121/88), 100 metaphases per experimental group were examined. The set of chromosomes was examined for completeness and the various chromosomal aberrations were assessed. The chromosomal aberrations were classified. Only metaphases with 22 +/- 1 chromosomes are included in the analysis. The metaphases were examined for the following aberrations: gap (g), break (b), fragment (f), minute (m), deletion (d), exchanges including intrachanges (ex), dicentrics (di), chromosome disintegration (cd) and ring formation (ri). In addition, metaphases with 5 and more aberrations were classified separately as multiple aberrations (ma).
After the metaphases had been evaluated, the code was lifted. The values for the control group were compared with the results from the dose group and the positive control at each preparation time.

Rationale for test conditions:

In accordance with test guidelines.

Evaluation criteria:

The evaluation of the results was performed as follows:
-The test substance is classified as mutagenic if it induces a significantly increased aberration rate as compared with the negative controls with one of the concentrations tested. The significance is obvious either by an enhancement of the rate clearly exceeding the control range or it is proven by adequate biometry (Binomial statistic with Fisher's exact test).
-the test substance is classified as mutagenic if there is a reproducible concentration related increase in the aberration rate.
-the test substance is classified as not mutagenic when it tests negatively both with and without metabolic activation.

Statistics:

Not necessary to perform as all mean chromosome aberration rates after treatment with the test article were in the range of the negative control value.

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:

valid

Positive controls validity:

valid

Additional information on results:

Solubility and toxicity
In a preliminary experiment Remazol-Brillantgelb 4GL was assayed with respect to its solubility in cell culture medium. The highest concentration at which no visible precipitation was observed, was found to be 2000 μg/ml.
The cytotoxicity experiment proved that Remazol-Brillantgelb 4GL was not toxic to the V79 cells in the absence of metabolic activation (S9-mix). Also in the presence of metabolic activation (S9-mix) no indication of toxicity was observed up to the limit of solubility.
No cytotoxic effect as reduction of mitotic index was also observed.
On the basis of these results the preparation of chromosomes was done after 2 h-treatment with 2000 μg/ml at 6, 18 and 28 h, and 18 hours after treatment additionally with 200 and 600 μg/ml, both with and without S9-mix.

Mutagenicity
The test substance Remazol-Brillantgelb 4GL was assessed for its mutagenic potential in vitro in the chromosome-aberration-test with two independent cell cultures without metabolic activation and two independent cell cultures with metabolic activation. No relevant reproducible enhancement of the chromosome aberration rate over the range of the negative control was found with any of the concentrations used, neither with, nor without metabolic activation by S9-mix. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.

Any other information on results incl. tables

Mitotic index

Test group	Dose μg/ml	S9 mix	Fixation interval (h)	Mitotic index per cent*
				Slide		Abs.	Rel.
				1	2
Solvent control Test article	0 2000	- -	6 6	10.9 11.8	11.5 9.9	11.2 10.9	100.0 97.3
Solvent control Test article	0 2000	+ +	6 6	13.3 9.0	10.9 10.9	12.1 10.0	100.0 82.6
Negative control Solvent control Positive control EMS Test article Test article Test article	0 0 2000 200 600 2000	- - - - - -	18 18 18 18 18 18	9.5 13.3 8.6 10.9 10.8 14.1	12.3 12.8 7.8 13.5 8.9 8.6	10.9 13.1 8.2 12.2 9.9 11.4	100.0 100.0 62.6 93.1 75.6 87.0
Negative control Solvent control Positive control CPA Test article Test article Test article	0 0 5 200 600 2000	+ + + + + +	18 18 18 18 18 18	12.0 15.7 13.0 11.5 19.5 11.3	19.5 12.9 11.3 9.5 14.4 10.8	15.8 14.3 12.2 10.5 17.0 11.1	100.0 100.0 85.3 73.4 118.9 77.6
Solvent control Test article	0 2000	- -	28 28	11.4 9.1	8.3 13.3	9.9 11.2	100.0 113.1
Solvent control Test article	0 2000	+ +	28 28	16.6 12.1	11.8 11.3	14.2 11.7	100.0 82.4

*The mitotic index was determined in 1000 cells from each of two slides per test group

 

Chromosome aberrations in V79 cells

Preparation: 6 h after administration (100 metaphases were analysed)

Dose μg/ml	CulturewithoutS9 mix	No. of phases with aberrations		No. of aberrations		g	ig	b	ib	f	if	d	id	ma	ex	cd	Others	MI%
		Incl.	Excl.	Incl.	Excl.
		Gaps		Gaps
0 0	L/1 L/2	0 1	0 0	0 1	0 0	1												10.9 11.5
Total		1	0	1	0	1
2000 2000	3/1 3/2	2 2	1 1	2 2	1 1	1 1											1 m 1 di	11.8 9.9
Total		4	2	4	2	2											2

L = solvent control

Dose μg/ml	Culturewith S9 mix	No. of phases with aberrations		No. of aberrations		g	ig	b	ib	f	if	d	id	ma	ex	cd	Others	MI%
		Incl.	Excl.	Incl.	Excl.
		Gaps		Gaps
0 0	L/1 L/2	0 2	0 0	0 2	0 0	2												13.3 10.9
Total		2	0	2	0	2
2000 2000	3/1 3/2	2 2	2 1	2 2	2 1		1								1		1 im 1 m	9.0 10.9
Total		4	3	4	3		1								1		2

L = solvent control

 

Chromosome aberrations in V79 cells

Preparation: 18 h after administration (100 metaphases were analysed)

Dose μg/ml	CulturewithoutS9 mix	No. of phases with aberrations		No. of aberrations		g	ig	b	ib	f	if	d	id	ma	ex	cd	Others	MI%
		Incl.	Excl.	Incl.	Excl.
		Gaps		Gaps
0 0	N/1 N/2	0 0	0 0	0 0	0 0													9.5 12.3
Total		0	0	0	0
0 0	L/1 L/2	0 1	0 0	0 1	0 0	1												13.3 12.8
Total		1	0	1	0	1
2000 2000	P/1 P/2	9 6	5 6	9 6	5 6	1	3					1			3 1		2 di 2 m, 2 di	8.6 7.8
Total		15*	11*	15*	11*	1	3					1			4		6

N = negative control           L = solvent control              P = positive control             *= P <0.05

Dose μg/ml	CulturewithoutS9 mix	No. of phases with aberrations		No. of aberrations		g	ig	b	ib	f	if	d	id	ma	ex	cd	Others	MI%
		Incl.	Excl.	Incl.	Excl.
		Gaps		Gaps
200 200	1/1 1/2	0 0	0 0	0 0	0 0													10.9 13.5
Total		0	0	0	0
600 600	2/1 2/2	0 1	0 0	0 1	0 0	1												10.8 8.9
Total		1	0	1	0	1
2000 2000	3/1 3/2	0 3	0 2	0 3	0 2	1									1		1 di	14.1 8.6
Total		3	2	3	2	1									1		1

 

Dose μg/ml	Culturewith  S9 mix	No. of phases with aberrations		No. of aberrations		g	ig	b	ib	f	if	d	id	ma	ex	cd	Others	MI%
		Incl.	Excl.	Incl.	Excl.
		Gaps		Gaps
0 0	N/1 N/2	0 1	0 0	0 1	0 0	1												12.6 19.5
Total		1	0	1	0	1
0 0	L/1 L/2	1 1	0 1	1 1	0 1	1		1										15.7 12.9
Total		2	1	2	1	1		1
5 5	P/1 P/2	11 15	10 14	12 16	10 15	2 1		1		3 3					5 5		1 di 4 m, 3 di	13.0 11.3
Total		26*	24*	28*	25*	3		1		6					10		8

N = negative control           L = solvent control              P = positive control             *= P <0.05

Dose μg/ml	Culturewith S9 mix	No. of phases with aberrations		No. of aberrations		g	ig	b	ib	f	if	d	id	ma	ex	cd	Others	MI%
		Incl.	Excl.	Incl.	Excl.
		Gaps		Gaps
200 200	1/1 1/2	2 2	0 0	2 2	0 0	1 2	1											11.5 9.5
Total		4	0	4	0	3	1
600 600	2/1 2/2	0 2	0 2	0 2	0 2												1 m, 1 im	19.5 14.4
Total		2	2	2	2												2
2000 2000	3/1 3/2	2 0	0 0	2 0	0 0	2												11.3 10.8
Total		2	0	2	0	2

 

Chromosome aberrations in V79 cells

Preparation: 28 h after administration (100 metaphases were analysed)

Dose μg/ml	Culturewithout S9 mix	No. of phases with aberrations		No. of aberrations		g	ig	b	ib	f	if	d	id	ma	ex	cd	Others	MI%
		Incl.	Excl.	Incl.	Excl.
		Gaps		Gaps
0 0	L/1 L/2	0 0	0 0	0 2	0 0													11.4 8.3
Total		0	0	2	0
2000 2000	3/1 3/2	1 3	1 0	1 3	1 0	2	1								1			9.1 13.3
Total		4	1	4	1	2	1								1

L = solvent control

Dose μg/ml	Culturewith S9 mix	No. of phases with aberrations		No. of aberrations		g	ig	b	ib	f	if	d	id	ma	ex	cd	Others	MI%
		Incl.	Excl.	Incl.	Excl.
		Gaps		Gaps
0 0	L/1 L/2	0 2	0 0	0 2	0 0	2												16.6 11.8
Total		2	0	2	0	2
2000 2000	3/1 3/2	2 2	1 2	2 2	1 2		1		1				1				1 di	12.1 11.3
Total		4	3	4	3		1		1				1				1

L = solvent control

 

Summary of results

Test group	Number of cells analysed	Dose μg/ml	S9-mix	Fixation interval (h)	Per cent aberrant cells
					Incl. gaps	Excl. gaps	Exchanges
Solvent control Test article	200 200	0 2000	- -	6 6	0.5 2.0	0.0 1.0	0.0 0.5
Solvent control Test article	200 200	0 2000	+ +	6 6	1.0 2.0	0.0 1.5	0.0 0.5
Negative control Solvent control Positive control EMS Test article Test article Test article	200 200 200 200 200 200	0 0 2000 200 600 2000	- - - - - -	18 18 18 18 18 18	0.0 0.5 7.5 0.0 0.5 1.5	0.0 0.0 5.5 0.0 0.0 1.0	0.0 0.0 4.0 0.0 0.0 1.0
Negative control Solvent control Positive control CPA Test article Test article Test article	200 200 200 200 200 200	0 0 5 200 600 2000	+ + + + + +	18 18 18 18 18 18	0.5 1.0 13.0 52.0 1.0 1.0	0.0 0.5 12.0 0.0 1.0 0.0	0.0 0.0 7.0 0.0 0.0 0.0
Solvent control Test article	200 200	0 2000	- -	28 28	0.0 2.0	0.0 0.5	0.0 0.5
Solvent control Test article	200 200	0 2000	+ +	28 28	1.0 2.0	0.0 1.5	0.0 0.5

Applicant's summary and conclusion

Conclusions:

The results lead to the conclusion that the test compound is not mutagenic in the chromosome aberration test system in vitro with cells of the V79 Chinese hamster cell line.

Executive summary:

The test substance Remazol-Brillantgelb 4GL was examined for mutagenic activity in V79 Chinese hamster cells. The induction of chromosome aberrations after in vitro treatment was investigated in the presence and absence of a fraction of liver homogenate for metabolic activation (S9-mix).

 

A preliminary cytotoxicity experiment was performed in order to select appropriate dose levels for the mutagenicity study. The test substance produced no significant cytotoxic effect (reduction of plating efficiency) without metabolic activation from 50 μg/ml up to the limit of solubility (2000 μg/ml). No cytotoxic effect was also observed with metabolic activation up to the limit of solubility. For mutagenicity testing two independent cell cultures with and without metabolic activation (S9-mix) up to the limit of solubility (2000 μg/ml) were used.

 

For main experiment dose levels of 200, 600, 2000 μg/ml were used, in the absence and in the presence of S9-mix metabolic activation.

 

The test compound did not induce a significant increase in the number of chromosome aberrations at any preparation time and dose level of the test substance. No relevant cytotoxic effect (reduction of mitotic index) of the compound was observed in the main experiments. Marked increases in the rate of chromosome aberrations were obtained with the positive control substances indicating the sensitivity of the assay.

 

In conclusion Remazol-Brillantgelb 4GL does not induce chromosome mutations (=aberrations) in V79 Chinese hamster cells, neither in the presence nor in the absence of a metabolic activation system, under the experimental conditions described.