Sodium 1-amino-9,10-dihydro-4-[5-[(2-hydroxyethyl)sulphamoyl]-3,4-xylidino]-9,10-dioxoanthracene-2-sulphonate

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2-​Anthracenesulfonic acid, 1-​amino-​9,​10-​dihydro-​4-​[[3-​[[(2-​hydroxyethyl)​amino]​sulfonyl]​-​4,​5-​dimethylphenyl]​amino]​-​9,​10-​dioxo-​, sodium salt (1:1)

IUPAC Name:

2-​Anthracenesulfonic acid, 1-​amino-​9,​10-​dihydro-​4-​[[3-​[[(2-​hydroxyethyl)​amino]​sulfonyl]​-​4,​5-​dimethylphenyl]​amino]​-​9,​10-​dioxo-​, sodium salt (1:1)

Constituent 2

Reference substance name:

Sodium 1-amino-9,10-dihydro-4-[5-[(2-hydroxyethyl)sulphamoyl]-3,4-xylidino]-9,10-dioxoanthracene-2-sulphonate

EC Number:

247-269-2

EC Name:

Sodium 1-amino-9,10-dihydro-4-[5-[(2-hydroxyethyl)sulphamoyl]-3,4-xylidino]-9,10-dioxoanthracene-2-sulphonate

Cas Number:

25797-81-3

Molecular formula:

C24H23N3O8S2.Na

IUPAC Name:

sodium 1-amino-4-({3-[(2-hydroxyethyl)sulfamoyl]-4,5-dimethylphenyl}amino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Specific details on test material used for the study:

Name: FAT 20033/L
Common Name: TECTILON BLUE 4R CRUDE MOIST (LAB DRIED) Acid Blue 277
Batch No.: AT-PD13-359A1
CAS No.: 25797-81-3
Physical State / Colour: solid / dark blue
Storage Conditions: room temperature, protected from light
Active Components: >95 %
Analysation Date: 07 January 2014
Expiry Date: 07 January 2019
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and ß-naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment:
with and without metabolic activation: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2500 and 5000 µg/mL

Experiment I:
without metabolic activation: 25, 50, and 100 µg/mL
with metabolic activation: 200, 400 and 500 µg/mL

Experiment II:
without metabolic activation: 25, 50, and 100 µg/mL
with metabolic activation: 75, 150 and 350 µg/mL

Vehicle / solvent:

- Vehicle (s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 5 mg/mL. DMSO was used as solvent (1 % DMSO). Different test item stock solutions were prepared and added to the samples. The solvent was compatible with the survival of the cells and the S9 activity.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation (400 and 900 µg/mL)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation (1.11 µg/mL)

Details on test system and experimental conditions:

TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation, experiment II with metabolic activation)
20 hours (Experiment II without metabolic activation)

FIXATION INTERVAL: 20 hours (Experiment I and II with and without metabolic activation)
NUMBER OF REPLICATIONS: 2 independent experiments
NUMBER OF CELLS SEEDED: 1 x 10E4 - 5 x 10E4 cells
NUMBER OF CULTURES: two cultures per concentration
NUMBER OF CELLS SCORED: 200 cells per concentration (100 cells per culture)
DETERMINATION OF CYTOTOXICITY: Mitotic index, cell count

Evaluation criteria:

There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (up to 4 % aberrant cells without and 4.3 % with metabolic activation).

Statistics:

A statistical evaluation was used as an aid for interpretation of the results. Statistical significance at the 5 % level (p <0.05) was evaluated by The Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding negative/solvent control.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

According to the guidelines the highest recommended concentration was 5000 μg/mL. The test item was suspended in an appropriate solvent (DMSO) or in cell culture medium. Precipitation of the test item was noted at concentrations of 2500 μg/mL and higher. The highest dose group evaluated in the pre-experiment was 5000 μg/mL. The relative mitotic index and cell count were used as parameters for toxicity. The concentrations evaluated in the main experiment were chosen on the basis of the results from the pre-experiment. The mitotic index and/or cell count could not be determined at concentrations of 1000 μg/mL and higher due to the high toxicity of the test item.

Any other information on results incl. tables

 

	Dose Group	Concentration [µg/mL]	Relative Mitotic Index [%]	Relative Cell Count [%]	Mean % Aberrant Cells		Historical Laboratory Negative Control Range	Precipitation^a	Statistical Significance^b
					incl. Gaps	excl. Gaps
Experiment I 4 h treatment, 20 h preparation interval	C	0	111	116	8.0	4.0	0.0 % - 4.0 % aberrant cells	-	-
	S	0	100	100	2.0	0.5		-	-
	3	25	106	80	2.5	1.0		-	-
	4	50	101	74	2.5	1.0		-	-
	5	100	107	54	1.0	0.0		-	-
	EMS	900	94	87	12.5	8.5		-	+
Experiment II 20 h treatment, 20 h preparation interval	C	0	86	96	1.5	0.0	0.0 % - 4.0 % aberrant cells	-	-
	S	0	100	100	2.0	1.5		-	-
	4	25	100	86	5.0	1.5		-	-
	5	50	90	82	4.5	2.0		-	-
	6	100	121	73	2.0	0.0		-	-
	EMS	400	54	72	18.5	16.0		-	+

 

	Dose Group	Concentration [µg/mL]	Relative Mitotic Index  [%]	Relative Cell Count [%]	Mean % Aberrant Cells		Historical Laboratory Negative Control Range	Precipitation^a	Statistical Significance^b
					incl. Gaps	excl. Gaps
Experiment I 4 h treatment, 20 h preparation interval	C	0	91	108	2.5	1.0	0.0 % - 4.3 % aberrant cells	-	-
	S	0	100	100	7.0	3.0		-	-
	3	200	84	102	3.0	1.5		-	-
	5	400	75	68	2.0	1.5		-	-
	6	500	61	38	5.0	2.0		-	-
	CPA	1.1	85	94	10.0	7.0		-	-
Experiment II 4 h treatment, 20 h preparation interval	C	0	100	97	2.5	1.0	0.0 % - 4.3 % aberrant cells	-	-
	S	0	100	100	3.0	2.5		-	-
	1	75	94	81	5.0	2.5		-	-
	2	150	93	78	6.0	3.0		-	-
	4	350	88	46	7.0	3.5		-	-
	CPA	1.11	80	73	12.0	8.5		-	+

Applicant's summary and conclusion

Conclusions:

FAT 20033/L is considered to be non-clastogenic in this chromosome aberration test.

Executive summary:

To investigate the potential of FAT 20033/L to induce structural chromosome aberrations in Chinese hamster V79 cells,an in vitro chromosome aberration assay was carried out according to OECD TG 473. The metaphases were prepared around 20 h after start of treatment with the test item. The treatment interval was 4 h without and with metabolic activation in experiment I. In experiment II, the treatment interval was around 20 h without and 4 h with metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations. DMSO was used as solvent (1 % DMSO). Different test item stock solutions were prepared and added to the samples. The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Experiment I:

without metabolic activation: 25, 50 and 100 µg/mL

with metabolic activation: 200, 400 and 500 µg/mL

 

Experiment II:

without metabolic activation: 25, 50 and 100 µg/mL

with metabolic activation: 75, 150 and 350 µg/mL

 

No precipitation of the test item was observed without and with metabolic activation in all dose groups evaluated in experiment I and II. In experiment I (short-term treatment) without metabolic activation, cytotoxicity of the test item was determined at the highest concentration of 100 µg/mL considering the relative cell count. However, considering the relative mitotic index no cytotoxic effects were observed. With metabolic activation cytotoxic effects of the test item were determined at a concentration of 400 µg/mL and higher considering the relative cell count. However, considering the relative mitotic index cytotoxicity was observed at the highest concentration of 500 µg/mL. In experiment II (long-term treatment) without metabolic activation, cytotoxic effects of the test item were not observed up to a concentration of 100 µg/mL considering the relative mitotic index and cell count. Above this concentration of the test item a very high and steep toxicity was observed considering the relative mitotic index. With metabolic activation, no cytotoxic effects of the test item considering the relative mitotic index were observed up to the highest evaluated concentration. However, considering the relative cell count cytotoxicity was observed at the highest concentration of 350 µg/mL. In both experiments, no biologically relevant increase of the aberration rates was determined after treatment with the test item without and with metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control. In the experiments I and II without and with metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the negative / solvent controls. The distinct increase in the number of structural chromosome aberrations induced by the positive controls (EMS, CPA) clearly demonstrated the sensitivity of the test system. There was no evidence of chromosome aberration induced over background. Based on the above findings, it can be concluded that the test item FAT 20033/L did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, FAT 20033/L  is considered to be non-clastogenic in this chromosome aberration test.