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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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 Micronucleus Test, Adopted 26^thSeptember, 2014.

The test substance Direct Blue 78 was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The performed test was based on EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria,which is analogous to the OECD Test Guideline No. 471.

The test substance,Direct Blue 78,was assayed for the mutagenicity by the In Vitro Mammalian Cell Gene Mutation Test. The performed test was based on OECD Test Guideline No. 476–In VitroMammalian Cell Gene Mutation Test(2015),which is analogous to the EU methodB.17. V79 hamster fibroblast were used for testing.

These studies are GLP and Klimish score 1.

Link to relevant study records

Reference

Reference 1

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

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

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

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.

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.

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16.02.-24.03.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

A deviation was observed in TA 1535 the second experiment with metabolic activation: average mumber of revertants in positive control was 270 while maximum in historical control range is 264.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

gene for histidine or tryptophan synthesis

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

supernatant of rat liver and a mixture of cofactors.

Test concentrations with justification for top dose:

50, 150, 500, 1500, 5000 μg30, 100, 300, 1000, 3000 μgSelection of doses/toxicity: 2 mL of water for injection was added to 100 mg of the test substance toreach the maximum dose recommended in guidelines - 5000 μg per plate

Vehicle / solvent:

Water for injection, Ardeapharma, Lot. No.: 1501210041, exp. 01/2017- Justification for choice of solvent/vehicle: solubility of the substance

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylenediamine, 2-aminofluorene, 2-aminoanthracene, N-methyl-N´-nitro-N-nitrosoguanidine, 9-aminoacridine hydrochloride monohydrate

Details on test system and experimental conditions:

The bacterial tester strains Salmonella typhimurium TA 1535 (CCM 3814, lot. No. 2101200916917), TA 98 (CCM 3811, lot No. 01022001220053), TA 100 (CCM 3812, lot No. 0102201220054) and TA 1537 (CCM 3815, lot No. 2101200916918) as well as Escherichia coli WP2 uvrA (CCM 4751, lot No. 2104200512732), - were obtained from Czech Collection of Microorganisms (CCM) of Masaryk University, Brno.Strains TA 98 and TA 1537 detect frame shift mutations, strains TA 100 and TA 1535 serve to detectionof base-pair substitution mutations, and strain E.coli WP2 uvrA detects cross-linking mutagensMETHOD OF APPLICATION: in agar (plate incorporation)NUMBER OF REPLICATIONS: two seriesDETERMINATION OF CYTOTOXICITY- Method: relative total growth

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increase rule, which is compatible withthe application of statistical methods (2, 3). After this rule the result is positive, if a reproducible doseresponseeffect occurs and/or a doubling of the ratio Rt/Rc is reached.

Statistics:

For the evaluation of results, the modified two-fold increase rule was used, which is compatible with theapplication of statistical methods:Dunkel V. C.. Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays. inThe Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation. Elsevier North-Holland Biomedical Press. 231 - 417Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests forbacterial mutagenicity. Mutat. Res. 189. 83 - 91

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

THISTORICAL CONTROL DATAEach experiment included corresponding positive (reference mutagens) and negative controls (untreatedcontrol, solvent control). Untreated controls contain no solvent and negative controls contain 0.1 mL ofwater for injection. All the control numbers were compared with historical ranges of mutant frequenciesobtained in our laboratory. The actual numbers were in ranges of the historical numbers.ADDITIONAL INFORMATION ON CYTOTOXICITY:For toxicity experiment, the starting solution (5000 μg/0.1 mL) was diluted to concentration series 10 –5000 μg per plate. The concentration row was tested for toxicityin strain TA 98 without metabolic activation.No toxicity of the test substance or precipitation was observed at evaluation so the dose of 5000 μgper plate was used as maximum for the first mutagenicity experiments as well. The maximum concentration was diluted according to the rules given in guidelines (five different analysable concentrations withapproximately half log ,i.e. approximately #10, intervals between test points). The doses used were 50,150, 500, 1500 and 5000 μg per plateApplicant's summary and co

Conclusions:

Under the above-described experimental design, the test substance, Direct Blue 78, was non mutagenic for all the Salmonella typhimurium as well as Escherichia coli strains with as well as without metabolic activation.

Executive summary:

The test substance Direct Blue 78 was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The performed test was based on EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria,which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimuriumstrains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was diluted in water for injection and assayed in doses of 30-5000mg per plate, which were applied to plates in volume of 0.1 mL.

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

In the arrangement given above, thetest substance, Direct Blue 78, was non-mutagenicfor all the used tester strainswithout as well as with metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17.08.-13.10.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

See Any other...

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Frozen permanent cell culture was obtained from European Collection of Cell Cultures (ECACC). V79 used for experiments: Lot. No.: 10H016. ECACC

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of rat liver homogenate and mixture of cofactors.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

The lung fibroblasts V79 from male Chinese hamster were used for testing.Frozen permanent cell culture was obtained from European Collection of Cell Cultures (ECACC). V79 used for experiments: Lot. No.: 10H016. ECACC Certificate of Analysis is a part of archived study documentation. The cells were kept at -196 ºC under liquid nitrogen. After activation, cells are grown in DMEM medium with L-glutamine and 10 % FBS in incubator (5 % CO2, 37±1 °C, moistened).Cells underwent maximum 5 passages after thawing the original culture delivered from cell collection before using for test. Cleansing of cultures was performed 5 days before treatment with complete medium supplemented with HAT supplement due to elimination of mutants. Cleansing was not performed in experiment without metabolic activation, because of bad growth of cells in HAT medium.The test substance was then diluted in DMEM. According to the recommendation in the OECD Guideline the maximum concentration used for experiment without metabolic activation was 2 mg·mL-1. The other concentrations were 1.0, 0.5, 0.25 mg·mL-1.At observation after almost 3 hours presence of the undiluted test substance was found in medium, so the maximum concentration was abolished and it was replaced by the dose of 0.125 mg·mL-1. In the test with metabolic activation, the same concentrations (0.125-1.0 mg·mL-1) were used.Concentrations for the experiment without metabolic activation were stated on the base of solubility/Guideline recommendation and were 0.25, 0.5, 1.0 and 2.0 mg.mL-1. Precipitation was observed in the dose of 2.0 mg.mL-1, so the highest dose was omitted and replaced by the dose of 0.125 mg.mL-1. No toxicity or precipitation was observed in the test without metabolic activation, so the same doses were used in experiment with metabolic activation. Every dish with negative control/test substance contained 1.0 mL of application form of the test substance in DMEM so that the final concentrations on dishes were as given above and 9.0 mL of complete medium or activation mixture (S9 mix) - for details see par 3.7.Cells were treated for 3 hours (with as well as without metabolic activation; day 1). After treatment, approximately 2*106 cells were transferred to suitable number of dishes to seed enough cells. At the same time, cells were seeded for detection of number of cells (PE estimation). On the 3rd, 6th and 8th day, approximately 2*106 cells from every culture were transferred and 10th day, extractions of mutants was performed with using selective medium together with PE estimation again (for details see Table 1).Determination of survivalAfter treatment period, the cultures were trypsinised and an aliquot (0.3 mL of 103/mL cell suspension) was diluted and plated to 6 cm Petri dishes to estimate the viability of the cells. A number of cells were then replaced in order to maintain the treated cell populations; the number of cells taken forward was adjusted according to the expected viability of the cultures, to give two millions of viable cells. Cells were grown in 10 cm Petri dishes.SubculturingOn the 3rd, 6th and 8th day the cell populations were subcultured in order to maintain them in exponential growth. The number of cells taken forward was adjusted according to the expected viability, to give two millions viable cells seeded in 10 cm Petri dishes.Incubation, staining and scoring Survival and plating efficiency plates were incubated for at least six days (37±1ºC, 5% CO2, moistened) prior to scoring. Mutant plates were incubated for an appropriate period to ensure adequate colony size (about 10 days). After incubation, the plates were stained with methylene blue and colonies were scored.

Evaluation criteria:

Each experiment is evaluated separately using modified two-fold increase rule according to Claxton L.D. et al, Mutat. Res.,189, 83-91, 1987 (2). The mutagenic potential is indicated by increasing number of mutants in treated groups in comparison to the negative solvent control (modified two-fold increase rule and any of the results outside the distribution of the historical negative control data) and/or by dependence of increasing number of mutants on dose (dose-response relationship).There is no requirement for verification of a clearly positive or negative response.In cases when the response is neither clearly negative nor clearly positive than a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activation conditions i.e. S9 concentration or S9 origin) could be performed.

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

Conclusions:

Under the above-described experimental design. the test substance, Direct Blue 78, was non-mutagenic for V79 cells with as well as without metabolic activation.

Executive summary:

The test substance, Direct Blue 78, was assayed for the mutagenicity by the In Vitro Mammalian Cell Gene Mutation Test. The performed test was based on OECD Test Guideline No. 476–In VitroMammalian Cell Gene Mutation Test (2015), which is analogous to the EU method B.17. V79 hamster fibroblast were used for testing.

The test substance was dissolved in DMEM. No toxicity was supposed, so the maximum recommended concentration (2 mg·mL^-1) was used at first. This concentration produced precipitation in Petri dishes, so concentrations used in the both mutagenicity tests - without as well as with metabolic activation - were1.0, 0.5, 0.25, and 0.125 mg·mL^-1. No toxicity was observed in any dose.

In the arrangement given above, the test substance,Direct Blue 78,wasnon-mutagenicfor V79 cells without as well as with metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, the substance is not classified. All tests are negative.