Tetrasodium 2-[[4-[[4-[[1-hydroxy-6-(phenylamino)-3-sulphonato-2-naphthyl]azo]-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate

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:

03.05.-01.07.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

yes

Remarks:

See any other information...

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium 2-[[4-[[4-[[1-hydroxy-6-(phenylamino)-3-sulphonato-2-naphthyl]azo]-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate

EC Number:

219-708-8

EC Name:

Tetrasodium 2-[[4-[[4-[[1-hydroxy-6-(phenylamino)-3-sulphonato-2-naphthyl]azo]-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate

Cas Number:

2503-73-3

Molecular formula:

C42H29N7O13S4.4Na

IUPAC Name:

tetrasodium 2-[[4-[[4-[[1-hydroxy-6-(phenylamino)-3-sulphonato-2-naphthyl]azo]-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate

impurity 1

Reference substance name:

Sodium chloride

EC Number:

231-598-3

EC Name:

Sodium chloride

Cas Number:

7647-14-5

Molecular formula:

ClNa

IUPAC Name:

sodium chloride

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Direct Blue 78- Physical state: solid, powder- Analytical purity: 95% (w/w)- Impurities (identity and concentrations): NaCl (CAS: 7647-14-5) 10% (w/w)- Lot/batch No.: 7013/207- Expiration date of the lot/batch: unlisted- Storage condition of test material: The test substance should be stored in dry room in dark in closed container at the room temperature.

Method

Species / strain

Species / strain / cell type:

mammalian cell line, other:

Remarks:

peripheral blood lymphocytes mammalian cell line

Details on mammalian cell type (if applicable):

The human peripheral blood lymphocytes used for testing were obtained from healthy non smokingdonors (up to 35 years of age). Peripheral blood (heparinized) is taken from donors in certified medicallaboratory (MeDiLa) in the morning and as soon as possible transported into the test facility.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of rat liver homogenate and mixture of cofactors

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Colchicine

Details on test system and experimental conditions:

Principle of test is the detection of binucleated cells with micronuclei, which are induced by the test substance in human peripheral blood lymphocytes. Lymphocytes are cultured in growth medium and test substance is added to them. Cell cycle is then stopped by cytochalasin B, cultures are sampled and microscopic preparations are prepared. Preparations are then analysed by microscope. Genotoxicity is indicated by increased incidence of binucleated cells with micronuclei.Experiments with and without metabolic activation with short treatment (3 hours) are done at first. If both experiments with the short treatments are negative or equivocal, subsequently, extended exposure treatment without metabolic activation is performed.

Results and discussion

Test results

Key result

Species / strain:

mammalian cell line, other: peripheral blood lymphocytes

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:

Results overviewResults of experiments are summarized in the tables in the Annexes 1 (cytotoxicity) and 2 (genotoxicity). The tables contain the dose applied per culture in µg/mL (concentrations of test substance were applied to cultures at a volume of 50 µl), amount of S9 per culture in µl, number of mononucleated, binucleated and multinucleated cells, CBPI index and % cytotoxicity, numbers of binucleated cells with micronuclei and average numbers of binucleated cells with micronuclei in 1000 cells, number of micronuclei and average number of micronuclei in 1000 cells, parameter Mt / Mc, i.e. ratio of number of binucleated cells with micronuclei at tested dose (Mt) to number of binucleated cells with micronuclei at negative control (Mc, UTC or S9-mix). Numbers of binucleated cells with micronuclei in negative and positive controls were compared with historical controls in our laboratory. The current ranges are given in Annex 3 (Table 7 and Table 8). Values of negative and positive controls in this study are within the ranges of historical data, so that test system responds adequately and the experiment is acceptable.The cytotoxic effect was characterized as % of cytotoxicity. Results of the cytotoxic effect are given in the Table 1 - 3 (Annex 1). All of test concentrations did not show the cytotoxicity higher than 55±5 % in the time of exposure 3 hours. The second experiment with the prolonged exposition without activation (23 hours) gave in three tested concentrations (2000, 1000 and 500 µg/mL) high cytotoxicity (small and dark cells) therefore microscopic slides could not be analyzed for cytotoxicity and genotoxicity. The concentrations 250 µg/mL was evaluated only for cytotoxicity, but could not be used for genotoxicity evaluation because of poor appearance of microscopic slides (small and dark cells). On the basis of these results, the third experiment without metabolic activation with extended exposure and lower concentrations 125, 62.5 and 31.25 µg/mL had to be done. In the third experiment with the prolonged exposition without activation (23 hours), all of tested concentrations did not show the cytotoxicity higher than 55±5 %. Therefore the concentration of 125 µg/mL was selected as the highest one for the analysis of genotoxic effect.

Any other information on results incl. tables

Cytotoxic effect

Table No. 1: Evaluation of cytotoxic effect without metabolic activation-3h exposure

- MA I
Culture No.	Treatment/Test substance concentration	Number of MNC	Number of BNC	Number of MTNC	CBPI	Cytotoxicity (%)
1	UTC	756	282	39	1.334	0.0
2	2000 μg/mL	928	182	9	1.179	46.5
3	1000 μg/mL	691	386	26	1.397	-18.8
4	500 μg/mL	811	348	55	1.377	-12.9
5	250 μg/mL	607	422	77	1.521	-55.8
6	125 μg/mL	707	290	56	1.382	-14.2
13	Colchicine	857	176	76	1.296	11.5

 

Table No. 2: Evaluation of cytotoxic effect with metabolic activation (S9-mix) -3h exposure

+MA I
Culture No.	Treatment/Test substance concentration	Number of MNC	Number of BNC	Number of MTNC	CBPI	Cytotoxicity (%)
7	S9-mix	925	298	45	1.306	0.0
8	2000mg/mL + S9-mix	923	204	16	1.206	32.5
9	1000mg/mL + S9-mix	939	183	20	1.195	36.2
10	500mg/mL + S9-mix	828	187	28	1.233	23.9
11	250mg/mL + S9-mix	764	267	69	1.368	-20.3
12	125mg/mL + S9-mix	724	406	67	1.451	-47.4
14	Cyclophosphamide + S9-mix	630	453	35	1.468	-52.9
1	UTC	756	282	39	1.334	-9.2

 

Table No. 3: Evaluation of cytotoxic effect without metabolic activation -23h exposure

- MA III
Culture No.	Treatment/Test substance concentration	Number of MNC	Number of BNC	Number of MTNC	CBPI	Cytotoxicity (%)
15	UTC	528	426	61	1.54	0.0
17	125 μg/mL	642	338	54	1.43	20.1
18	62.5 μg/mL	607	382	50	1.46	14.1
19	31.25 μg/mL	603	497	47	1.52	4.6
16	Colchicine	823	256	67	1.34	37.0

Genotoxic effect

Table No. 4: Evaluation of genotoxicity without metabolic activation-3h exposure

Culture No.	Treatment/Test substance concentration	Number of binucleated cells with MN	Number of MN	Average number of BN cells with MN per 1000 binucleated cells	Average number of MN per 1000 binucleated cells	Mt/Mc
1	UTC	22	24	11	12	1.00
2	2000mg/mL	42	45	21	22.5	1.91
3	1000mg/mL	15	17	7.5	8.5	0.68
4	500mg/mL	35	40	17.5	20	1.59
13	Colchicine	273	323	136.5	161.5	12.41

 

Table No. 5:Evaluation ofgenotoxicitywith metabolic activation (S9-mix) -3h exposure

Culture No.	Treatment/Test substance concentration	Number of binucleated cells with MN	Number of MN	Average number of BN cells with MN per 1000 binucleated cells	Average number of MN per 1000 binucleated cells	Mt/Mc
7	S9-mix	23	24	11.5	12	1.00
8	2000 mg/mL + S9-mix	20	22	10	11	0.87
9	1000mg/mL + S9-mix	20	21	10	10.5	0.87
10	500mg/mL + S9-mix	24	25	12	12.5	1.04
1	UTC	22	24	11	12	0.96
14	Cyclophosphamide + S9-mix	58	64	29	32	2.52

 

Table No. 6: Evaluation of genotoxicity without metabolic activation- 23h exposure

Culture No.	Treatment/Test substance concentration	Number of binucleated cells with MN	Number of MN	Average number of binucleated cells with MN per 1000 binucleated cells	Average number of MN per 1000 binucleated cells	Mt/Mc
15	UTC	17	18	8.5	9	1.00
17	125mg/mL	20	21	10	10.5	1.18
18	62.5mg/mL	22	23	11	11.5	1.29
19	31.25mg/mL	26	29	13	14.5	1.53
16	Colchicine	50	55	25	27.5	2.94

Applicant's summary and conclusion

Conclusions:

Under the experimental design described above, the test substance, Direct Blue 78, had no genotoxic effects in the micronucleus test performed in human peripheral blood lymphocytes in experiments both without and with metabolic activation.The result of micronucleus test was negative, test substance is then considered not able to induce chromosome breaks and/or chromosome gain or loss in this test system.

Executive summary:

In Vitro Mammalian Cell Micronucleus Test assayed genotoxicity of the test substance, Direct Blue 78. The test was performed according to OECD Test Guideline No. 487 -In Vitro Mammalian Cell MicronucleusTest, Adopted 26^thSeptember, 2014.

The human peripheral blood lymphocytes from healthy donors were used for testing. The test substance was suspended in RPMI medium and assayed in five concentrations 31.25-2000  µg/mL, which were applied to cultures in volume of 50 mL.

Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.

Under the experimental design described above, the test substance, Direct Blue 78,had no genotoxic effects in the micronucleus test performed in human peripheral blood lymphocytesin experiments both without and with metabolic activation.

The result of micronucleus test was negative, test substance is then considered not able to induce chromosome breaks and/or chromosome gain or loss in this test system.