Disodium 6-hydroxy-5-[[4-[[4-(phenylamino)-3-sulphonatophenyl]azo]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 (carried out 1994) according to OECD Test Guideline No. 471 - Bacterial Reverse Mutation Assay (1983) 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 Acid Black 26 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 cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18.01. – 25.01.2016

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:

no

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 donors (up to 35 years of age).Peripheral blood (heparinized) is taken from donors in certified medical laboratory (MeDiLa) in the morning and as soon as possible transported into the test facility.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:

Cytotoxicity: 1st experiment ( with/without S9-mix) 312.5; 625; 1250; 2500 and 5000 µg/mLGenotoxicity: 1st experiment ( with/without S9-mix) 1250; 625 and 312.5 µg/mLThe first experiments gave positive results, so the second experiment with extended exposure without metabolic activation had not to be done.The same slides were used for cytotoxicity and genotoxicity evaluation. All concentrations were used for analysis of cytotoxic effect and concentrations 1250, 625 and 312.5 μg/mL were used for analysis of genotoxic effect.Two of test concentrations (5000 and, 2500 μg/mL) did show the cytotoxicity 100%. Test concentrations 1250, 625 and 312.5 μg/mL did not show the cytotoxicity higher than 55±5%. On the basis of this result, the concentration of 1250 μg/mL was selected as the highest concentration to be used for the analysis of genotoxic effect.

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): the first experiments gave positive results, so the second experiment without metabolic activation with extended exposure (23 hours) in the presence of cytochalasin B had not to be done.STAIN (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 at least overnight 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 design described above, the test substance Acid Black 26 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.

Executive summary:

In Vitro Mammalian Cell Micronucleus Test assayed genotoxicity of the test substance, Acid Black 26. 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 concentrations of 312.5 - 5000 µg/mL, which were applied to cultures in volume of 50 µL.

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, Acid Black 26, 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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20.07.1994 – 14.10.1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for Testing of Chemicals, 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

gene for histidine or tryptophan synthesis

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 97

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

supernatant of rat liver and a mixture of cofactors

Test concentrations with justification for top dose:

sample in water 1000 μg/0.1 ml Toxicity test TA 98:doses 10, 100, 500, 1000, 2000 μg /plate (dose was aplicated as 0.2 ml volume)A substance is non-toxic, but from dose 100 μg/plate thesubstance in the background precipitated.Main test TA 98: 500 μg/platedoses tested 10, 100, 250, 500, 1000 μg - 1st and 2nd testThere was 3rd test on TA98:it was necessary to increase the doses to achieve clearer dose-dependent => the same doses as were in toxicity test were aplied i.e. 10, 100, 500, 1000, 2000 μg /plate (dose was aplicated as 0.2 ml volume)

Vehicle / solvent:

sterile distilated water

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;

Details on test system and experimental conditions:

The bacterial tester strains Salmonella typhimurium TA 97, TA 98, TA 100 and TA 1535 were obtained from Czechoslovakia Collection of Microorganisms (CCM) of Masaryk University, Brno.Strains TA 97 and TA 98 detect frame shift mutations, strains TA 100 and TA 1535 serve to detection of base-pair substitution mutations.METHOD OF APPLICATION: in medium; in agar (plate incorporation)NUMBER OF REPLICATIONS: two seriesEach dose was tested on 3 Petri dishesDETERMINATION 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. After this rule the result is positive, if a reproducible dose response 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 97

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

Additional information on results:

HISTORICAL CONTROL DATA: Each experiment included corresponding positive (reference mutagens) and negative controls (solvent control). Negative controls contain 0.2 mL of sterile distiled water.Negative control numbers were compared with historical ranges of spontaneous reversions obtained in our laboratory. Each strain was continuously monitored for the desired genotype (plasmid pKM101, uvrB and rfa mutations)The sample was diluted immediately prior to the experiment.ADDITIONAL INFORMATION ON CYTOTOXICITY:For toxicity experiment, the starting solution (2000 μg/0.2 mL) was diluted to concentration series 10 – 2000 μg per plate. The concentration row was tested for toxicity in strain TA 98 without metabolic activation.No toxicity of the test substance, but weak trend of precipitation from 100 ug dose was observed at evaluation so the dose of 1000 μg per plate was used as maximum for the first mutagenicity experiments. The maximum concentration was diluted according to the rules given in guidelines (five different analysable concentrations with approximately half log ,i.e. approximately #10, intervals between test points). The doses used were 10, 100, 250, 500 and 1000 μg per plate

Conclusions:

Based on the above results the sample Midlonová čerň VL was under given experimental conditions non-mutagenic for strains TA 97, TA 100 and TA 1535. A hint of mutation occurs only in experiments on TA 98 strain without metabolic activation. After the introduction of metabolic activation system did not show any mutagenic effect.

Executive summary:

The test substance Midlonová čerň VL was assayed for the mutagenicity by gene mutation test in the genome of Salmonella typhimurium. The test is based on OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains TA 97, TA 98, TA 100, TA 1535 detecting different types of mutations were used. The test substance was diluted in water and assayed in doses of 50-2000 µg per plate. Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.

Under the above-described experimental design, the test substance, Midlonová čerň VL, was non-mutagenic for all the used bacterial strains TA 97, TA 100, TA 1535 without as well as with metabolic activation. In experiments on strain TA 98 without metabolic activation a weak trend in the number of revertant depending on the dose repeatedly occurred. This effect was not manifested after the introduction of a metabolic activation into system.