Tetrasodium 8-[[4-[(4-amino-3-sulphonatophenyl)azo]-6-sulphonatonaphthyl]azo]-5-[[6-(benzoylamino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]naphthalene-2-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There is a reliable in vitro gene mutation study in bacteria available according to Method B.13/14,Mutagenicity: Reverse Mutation Test Using Bacteria,Council Regulation (EC) No.440/2008. Published in O.J. L 142, 2008 with negative result.

In Vitro Mammalian Cell Micronucleus Test was performed according to OECD Test Guideline No. 487 - In Vitro Mammalian Cell Micronucleus Test (Adopted 26th September, 2014). The test substance Direct Blue 85 had genotoxic effects in the human peripheral blood lymphocytes in experiments both without and with metabolic activation. The result of micronucleus test was positive, test substance is then considered able to induce chromosome breaks and/or gain or loss in this test system.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13.01.2017 – 20.02.2017

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)

Version / remarks:

Council Regulation (EC) No.440/2008. Published in O.J. L 142, 2008

Deviations:

no

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 μgSelection of doses/toxicity: The test substance was dissolved in water for injection till the maximum recommended concentration 5000 μg per 0.1 mL. For toxicity experiment the highest concentration was diluted to the other 5 concentrations in 3 digit places interval (10-5000 μg per plate). Although no particles could be observed in higher concentrations due to dark colour of solutions, no particles were observable in top agar and Petri dishes.

Vehicle / solvent:

Water for injection, Ardeapharma, Lot. No.: 1508310536, exp. 08/2017- Justification for choice of solvent/vehicle: solubility of the substance

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

negative controls - 0.1 mL of water for inj.

Positive controls:

yes

Positive control substance:

sodium azide

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

(NPD)

Positive controls:

yes

Positive control substance:

other: 2-aminofluorene

Remarks:

(2-AF)

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

(2-AA)

Positive controls:

yes

Positive control substance:

other: N-methyl-N´-nitro-N-nitrosoguanidine

Remarks:

(MNNG)

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine hydrochloride monohydrate

Remarks:

(9-AAc)

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 detection of base-pair substitution mutations, and strain E.coli WP2uvrA detects cross-linking mutagensMETHOD OF APPLICATION: in medium; 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 with the application of statistical methods (see below). After this rule the result is positive, if a reproducible doseresponse effect 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 the application of statistical methods:Dunkel V. C.. Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays in The 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 for bacterial 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 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:

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:

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:

HISTORICAL CONTROL DATA: Each experiment included corresponding positive (reference mutagens) and negative controls (untreated control, solvent control). Untreated controls contain no solvent and negative controls contain 0.1 mL of water for injection. All the control numbers were compared with historical ranges of mutant frequencies obtained 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.Although no particles could be observed in higher concentrations due to dark colour of solutions, no particles were observable in top agar and Petri dishes. The concentration row was tested for toxicity in strain TA 98 without metabolic activation.No toxicity or precipitation was observed in any dose. The concentration of 5000 µg.0.1 mL-1 was then used as maximum in the first mutagenicity experiments. Further doses were diluted with factor approximately 2-√10.

Conclusions:

Under the above-described experimental design, the test substance, Direct Blue 85, 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 85 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 typhimurium strains 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 at the highest recommended concentration of 5000 μg per plate and tested for toxicity in S. typhimurium strain TA 98. No cytotoxicity or precipitation occurred in any dose.

First mutagenicity experiments were performed with concentration range 50-5000 μg per plate applied to plates in volume of 0.1 mL in experiments without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.

No signs of a positive response were observed in any tester strain, so, to increase the sensitivity of the assay, the second mutagenicity experiments were performed with pre-incubation for 30 minutes at 37±1°C and shaking.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Mean revertant colony counts for the vehicle controls were within the current historical control range for the laboratory

In the arrangement given above, the test substance, Direct Blue 85, was non-mutagenic for all the used tester strains without as well as with metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07.06. – 29.08.2016

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)

Version / remarks:

Adopted 26th September, 2014

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:

lymphocytes: human peripheral blood lymphocytes

Remarks:

primary culture

Details on mammalian cell type (if applicable):

Cells used- Source of cells: human peripheral blood lymphocytes obtained from healthy non smoking female donors (up to 35 years of age).Peripheral blood (heparinized) is taken from donors in certified medical laboratory (MeDiLa) in the morning and transported into the test facility as soon as possible.Media used- Type and identity of media:RPMI 1640 with L-GlutamineFoetal Bovine SerumPHA-M (Phytohaemagglutinin M)RPMI-M (RPMI 1640 + Foetal Bovine Serum + Penicilin-Streptomycin + PHA-M); 5% CO2- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor-supplemented post-mitochondrial fraction (S9)

Test concentrations with justification for top dose:

Selection of concentrations: The test substance was suspended in culture medium. The starting suspension (2000 ug/mL - according OECD TG 487 paragraph 31) was diluted and concentration series (2000, 1000, 500, 250 and 125 ug/mL) was prepared. The concentration series was prepared also according OECD TG 487 (concentration intervals of 2 to 3 fold). At first the cytotoxicity of test substance was determined by measuring of cell proliferation. For the highest concentration of the test substance used for analysis of genotoxic effect the cytotoxicity should not be higher than 55±5 %. Fresh solutions of the test substance were prepared before each experiment. In the highest prepared concentration (2000 ug/mL) the pH was measured. Cytotoxicity: 1st experiment (with/without S9-mix) 125, 250, 500, 1000 and 2000 ug/mL2nd experiment (with/without S9-mix) 125, 250, 500, 1000 and 2000 ug/mL3rd experiment (prolonged exposition without activation with adjusted pH) 250, 500, 1000 and 2000 ug/mLGenotoxicity: 1st experiment (without S9-mix) 125, 250 and 500 ug/mL; (with S9-mix) 125, 250 and 1000 ug/mL2nd experiment (without S9-mix, with adjusted pH) 500, 1000 and 2000 ug/mL; (with S9-mix, with adjusted pH) 250, 500 and 1000 ug/mL3rd experiment (prolonged exposition without activation with adjusted pH) 250, 500 and 1000 ug/mLThe highest concentration 2000 ug/mL in the first and third experiments and 1000 ug/mL in the first experiment without metabolic activation could not be used for genotoxicity evaluation because of high cytotoxicity in this time of exposure.

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: colchicine

Remarks:

colchicine (without metabolic activation), CPA monohydrate (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: suspensions on microscopic slidesDURATION- Exposure duration: 3 h- Expression time (cells in growth medium): 48 h- Fixation time (start of exposure up to fixation or harvest of cells): 23 hSPINDLE INHIBITOR (cytogenetic assays): cytochalasin BSTAIN (for cytogenetic assays): Giemsa Romanowski staining solutionNUMBER OF REPLICATIONS: 2METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cultures were harvested 23 hours after the beginning of treatment (after about 1.5 to 2 cell cycles). Cultures were treated by hypotonic solution (RT, ca 5 min.) and then they were centrifuged (1200 rpm, 10 min.). After removing of hypotonic solution, fixation solution was added to cultures and cultures were centrifuged again (1200 rpm, 10 min.). The addition of fixation solution and centrifugation were repeated three times. Suspensions were then dropped on clear microscopic slides. Preparations were let to dry at laboratory temperature and then slides were stained by Giemsa Romanowski staining solution.NUMBER OF CELLS EVALUATED: 2000 binucleated cells were analysed per each concentration and control divided equally between the duplicates for determination of genotoxicity.CRITERIA FOR MICRONUCLEUS IDENTIFICATION:The genotoxic effect is characterized by numbers of binucleated cells with micronuclei. The results did show substantial (biologically significant) increase in the number of binucleated cells with micronuclei (two-fold increase).The actual numbers of binucleated cells with micronuclei in negative and positive controls were compared with historical controls in testing laboratory. They did not exceed limits of historical controls and the experiment is acceptable. DETERMINATION OF CYTOTOXICITYCBPI index was calculated from ratio of mononucleated, binucleated and multinucleated cell at each culture. The cytotoxic effect was characterized as % of cytotoxicity. At least 1000 cells were scored per each concentration and controls divided equally between the duplicates.

Evaluation criteria:

Genotoxicity: Genotoxic potential is indicated by increasing of number of binucleated cells with micronuclei in comparison to the negative control (two-fold increase rule) and by dependence of number of binucleated cells with micronuclei on dose (dose-response relationship).• at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control (two-fold increase rule)• the dependence of increasing number of cells with micronuclei on concentration (dose-response relationship) is evident• any of the results are outside the distribution of the historical negative control dataCytotoxicity: For the assessment of cell proliferation the CBPI index is calculated using at least 500 cells per culture. If the % cytotoxicity is increased up to more than 50 %, it will refer to cytotoxicity. For the highest concentration of the test substance used for analysis of genotoxic effect the cytotoxicity should not be higher than 55±5 %.

Statistics:

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.

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the experimental conditions described above, the test substance Direct Blue 85 had genotoxic effects in the human peripheral blood lymphocytes in experiments both without and with metabolic activation.

Executive summary:

In Vitro Mammalian Cell Micronucleus Test assayed genotoxicity of the test substance, Direct Blue 85. The test was performed according to OECD Test Guideline No. 487 - In Vitro Mammalian Cell Micronucleus Test, Adopted 26th September, 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 125 - 2000 ug/mL, which were applied to cultures in volume of 50 uL.

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 85, had genotoxic effects in the human peripheral blood lymphocytes in experiments both without and with metabolic activation.

The result of micronucleus test was positive, test substance is then considered able to induce chromosome breaks and/or chromosome gain or loss in this test system.