Disodium 3-(5-chloro-2-hydroxyphenylazo)-4,5-dihydroxynaphthalene-2,7-disulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

26/06/2018 – 03/08/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes

Type of assay:

other: in vitro micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Mordant Blue 013

IUPAC Name:

Mordant Blue 013

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

The human peripheral blood lymphocytes used for testing were obtained from healthy non smoking females (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.

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Concentrations evaluated in analysis of cytotoxicity:
In the 1st experiment (genotoxicity test with and without metabolic activation – 3 hours), the concentrations 125, 250, 500, 1000 and 2000 g/mL have been analysed for cytotoxic effect.
In the 2nd experiment (genotoxicity test with metabolic activation – 3 hours), the concentrations 125, 250, 500, 1000 and 2000 g/mL have been analysed for cytotoxic effect.
In the 3rd experiment (genotoxicity test without metabolic activation – 25.5 hours), the concentrations 125, 250, 500, 1000 and 2000 g/mL have been analysed for cytotoxic effect.

Concentrations evaluated in analysis of genotoxic effect:
In the 1st experiment (genotoxicity test without metabolic activation – 3 hours) the concentrations 2000, 1000 and 500 g/mL have been analysed for genotoxic effect.
In the 2nd experiment (genotoxicity test with metabolic activation – 3 hours), the concentrations 2000, 1000 and 500 g/mL have been analysed for genotoxic effect.
In the 3rd experiment (genotoxicity test without metabolic activation – 25.5 hours), the concentrations 1000, 500 and 250 g/mL have been analysed for genotoxic effect

Vehicle / solvent:

Positive controls:
Colchicine CAS No.: 64-86-8, Sigma-Aldrich Lot. No. SLBX1712 (Exp. 05/2021)
Cyclophosphamide monohydrate: CAS No.: 6055-19-2, Sigma-Aldrich Lot. No. 120M1253V (Exp. 10/2021)


Media:
RPMI 1640 with L-Glutamine (with Sodium Bicarbonate (NaHCO3) and Phenol Red (C19H13NaO5S) as a pH indicator): Sigma Aldrich Lot. No. RNBG4755 (Exp. 12/2018) and Lot. No. RNBG3552 (Exp. 10/2018)
Foetal Bovine Serum: Sigma Aldrich Lot. No. 025M3302 (Exp.2/2020) and Lot. No. BCBT9410 (Exp. 05/2022).
RPMI-M (RPMI 1640 + Foetal Bovine Serum + Penicilin-Streptomycin + PHA-M)
Procedures of media preparation are described in detail in internal SOP M/51.

Other chemicals:
PHA-M (Phytohaemagglutinin M, lectin extracted from red kidney beans): Biological Industries Lot. No. 1707198 (Exp. 2/2020)
Penicilin-Streptomycin: Sigma Aldrich Lot. 037M4878V (Exp. 4/2019)
Cytochalasin B: Sigma Aldrich Lot. No. 037M4083V (Exp. 3/2019)
MgCl2 . 6H2O: Sigma Aldrich Lot. No. SLBP9770V (Exp. 7/2019)
NADP disodium salt: Roth Lot. No. 297260969 (Exp. 1/2021) and Lot. No. 506252868 (Exp. 12/2018)
D-Glucose-6-phosphate disodium salt: Apollo Scientific Lot. No. AS435477 (Exp. 5/2019)
KCl: Lachner Lot. No. 1903280314 (Exp. 3/2019)
Methanol: Lachner Lot. No. PP/2018/00977 (Exp. 7/2020)
Acetic acid: Lachner Lot. No. PP/2017/07314 (Exp. 8/2018) and Lot. No. PP/2018/00116 (Exp. 1/2019)
Giemsa-Romanowski: Penta Lot. No. 1901150118V (Exp. 1/2019)
S9: Lot. No. II (Exp. 28/11/2019)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Colchicine CAS No.: 64-86-8, Sigma-Aldrich Lot. No. SLBX1712 (Exp. 05/2021)

Details on test system and experimental conditions:

Experiments with short exposure
The 1st experiment was performed to assess genotoxicity with time of exposure 3 hours was done.
The final concentrations of 125, 250, 500, 1000 and 2000 µg of test item/mL RPMI M were assessed without and with metabolic activation (S9-mix). Duplicate cultures were used for each concentration and control.
At first, the cytotoxicity of each test concentration and control was evaluated at slide specimens.
The cytotoxicity < 45±5% was observed in every test item concentration in the 1st experiment without metabolic activation. The same result was obtained in 1st experiment with metabolic activation.
At genotoxicity evaluation it was showed:
- In the experiment with metabolic activation the number of cells with micronuclei in negative control was out of range of historical control.
So the experiment with metabolic activation with time of exposure 3 hours was repeated. The cytotoxicity < 45±5% was observed in every test item concentration in the 2nd experiment with metabolic activation.
So that for genotoxicity, the 3 highest concentrations could be evaluated (2000, 1000 and 500 µg/mL) in the 1st experiment without metabolic activation as well as in the 2nd experiment with metabolic activation.
For the 3rd genotoxicity test, the concentration 2000 µg/mL has been chosen as the highest final concentration.


Experiments with extended exposure
In the 3rd experiment, the testing with time of exposure 25.5 hours was done. On the basis of results from the 1st and 2nd experiments the same concentrations were used (2000, 1000, 500, 250 and 125 µg/mL).
Duplicate cultures (for each concentration and control) were treated with test item without metabolic activation (3rd experiment further in the text).
At first, the cytotoxicity was evaluated at slide specimens. The cytotoxicity ≥ 45±5 % was observed only in the highest concentration.
Therefore for genotoxicity evaluation, the 3 highest concentrations without cytotoxicity ≥ 45±5 % were evaluated (1000, 500 and 250 µg/mL).

Experimental procedures

Test item preparations:
At first, the preliminary preparations of the test item were done. The test item was soluble in RPMI medium. So for the testing the test item was solubled in RPMI.
In the 1st, 2nd and 3rd experiment 300 mg of the test item was dissolved in 3 mL of RPMI medium in volumetric flask. The starting stock solution (100 mg/mL) was diluted and concentration line (100, 50, 25, 12.5 and 6.25 mg/mL) was prepared.
Then, 50 µL of each stock concentration was added to the tube with cultivated blood, so the final concentrations of the test item were 125, 250, 500, 1000, and 2000 µg/mL - according OECD TG 487 paragraph 31. Fresh solutions of the test item were prepared before each experiment.
In the 1st and 2nd experiment the highest prepared concentration (final concentration 2000 g/mL) the pH was measured.

Controls:
Each experiment included corresponding positive controls (known clastogen - cyclophosphamide; and known aneugen – colchicine) and negative controls (untreated culture; in case of experiment with metabolic activation system – untreated culture with S9 mix; solvent control).
The aneugenicity control (colchicine) serves also as the positive control without S9 mix, and the clastogenicity control (cyclophosphamide) is used to test of the adequacy of the metabolic activation system used. The final concentrations of colchicine in cultures were 0.07 µg/mL at 1st and 2nd experiment (3h exposure) and 0.007 g/mL at 3rd experiment (25.5h exposure). The final concentration of cyclophosphamide in cultures is 7.8 µg/mL.

Preparation and using of S9-mix:
The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (mixture of PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg/mL, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames (1983). The liver was removed from each animal and washed in ice cold 0.15 M KCl. The livers washed were mixed with another 0.15 M KCl (3 mL/g wet liver) homogenized in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique at a temperature below –70 C. Fresh solution of the cofactors (1.6 mM MgCl2.6H2O, 0.8 mM NADP and 1 mM glucose-6-phosphate) was prepared before each experiment with metabolic activation. Each culture in experiments with metabolic activation contained 18.5 µL of S9 and 18.5 µL of cofactors solution (S9-mix = MAS).

Cell cycle length determination:
The proliferation of lymphocytes was evaluated by doubling time experiment. Four separate tubes with blood in the medium were prepared in day 2. Every following day (day 3, day 4 and day 5) lymphocytes were isolated and quantified. Then the proliferation curve was constructed and cell cycle length was determined. The lymphocyte`s cell cycle length was 17 hours. This is suitable to experimental design described above according to OECD TG 487 (1).

The cell cycle length determination is done once per 6 months. Last experiment was performed at the date 13.3.-16.3.2018. Results of the experiment are archived by the laboratory.
Treatment and cultivation:
50 L of each stock concentration of test item solution in RPMI medium was added to lymphocyte culture (2.5 mL growth medium RPMI-M + cca 150-210 L human peripheral blood) in the presence and absence of a metabolic activation system (S9-mix = MAS). It was carried out in a laminar box at room temperature. Cultures were treated with the test item 48 hours after mitogenic stimulation.

• Short treatment: After shaking the cultures were cultivated with the test item for 3 hours. After that, they were rinsed up by RPMI-M and then transported to the fresh growth medium (RPMI-M) with 11.25 µL of cytochalasin B (the final concentration in cultures was 4.5 µg/mL). Cultures were then cultivated and sampled after 25.5 hours since the beginning of treatment.

• Extended treatment: After shaking the cultures were treated with test item without metabolic activation for 25.5 hours in the presence of cytochalasin B (7.5 µL; final concentration in cultures was 3 µg/mL). No transport to fresh medium was needed in that experimental design.

Preparation and staining of slides:
Cultures were harvested 25.5 hours after the beginning of treatment (after about 1.5 to 2 cell cycles). Cultures were treated by hypotonic solution (RT, ca 10 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.

Times schedule of procedures:
Day 1: Blood sampling. Blood was taken and then stored in the fridge until the next day.
Day 2 and 3: Cultivation. The whole human peripheral blood was transfered to the growth medium and mitogenic stimulator (phytohaemagglutinin M) was added. These operations were carried out in a laminar box at room temperature. The cultivation runs without interrupting for 48 hours (37°C ± 1°C; 5% CO2).
Day 4: Exposition. The test item, positive and negative control items were added to the individual cultures (in a laminar box at room temperature). In the 1st experiment without metabolic activation and 2nd with metabolic activation (S9-mix), the cultures were washed (after 3 hours of continuous exposure to the test item in 37°C ± 1°C; 5% CO2) and transferred to fresh culture medium with cytokinesis blocker (cytochalasin B). Washing and transfer were carried out in a laminar box at room temperature. Then the lymphocytes were cultured (37°C ± 1°C; 5% CO2) for remaining period (total 25.5 hours from the start of exposure). In the 3rd experiment without metabolic activation washing of cultures was passed and cultures were cultured (37°C ± 1°C; 5% CO2) 25.5 hours with the test item and cytokinesis blocker (cytochalasin B).
Day 5: Harvesting of cultures. All cultures were processed in a laminar box at room temperature, (with hypotonia, fixation solution). Suspensions were dropped on clear microspcopic slides. The slides were allowed to dry at least overnight.
Day 6: Staining of slides. Slides were stained by Giemsa-Romanowski staining solution.

Evaluation criteria:

Scoring of cells for cytotoxicity and genotoxicity evaluation:
All slides were coded before microscopic analysis.
At least 1000 cells were scored per each concentration and controls to assess cytotoxicity. CBPI index was calculated from ratio of mononucleated, binucleated and multinucleated cell at each concentration. The cytotoxic effect was characterized as % of solvent control (untreated culture or untreated culture with S9 mix respectively).
According to the cytotoxicity of the test item appropriate 3 concentrations were chosen for genotoxicity evaluation according criteria for appropriate concentrations in OECD TG 487. To assess genotoxicity, 2000 binucleated cells were analysed per each concentration and control (divided equally between the duplicates). The genotoxic effect is characterized by numbers of binucleated cells with micronuclei.
At first, the cytotoxicity of test item was determined by measuring of cell proliferation in the preliminary cytotoxicity assay. No precipitatation in any tested concentration was observed, so the highest test concentration was 2 mg/mL.
Cell proliferation is characterized by Cytokinesis-Block Proliferation Index (CBPI).

Then the genotoxicity of the test item was evaluated on the tested concentrations which were choosed on the basis of results from the cytotoxicity evaluation. The genotoxicity was evaluated by scoring of micronuclei in at least 2 000 binucleated cells per concentration and control.
The micronuclei were scored in the binucleate cells only. The micronuclei in the binucleate cells with irregular shapes, in the binucleate cells where the two nuclei differ greatly in size, the binucleate cells without cytoplasm or binucleate cells with poorly spread multi-nucleate cells was not scored.

Statistics:

CBPI = (number of mononuclear cells) + (2. nb. of binucleate c.) + (3. nb. multinuclear c.)
total number of cells

Cytotoxicity (%) = 100 – 100 {(CBPIT – 1) ÷ (CBPIC – 1)}

Where: index T = test item/positive control treated culture
index C = negative control culture

Results and discussion

Test results

Key result

Species / strain:

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:

Cytotoxicity
In the 1st and 2nd experiments in short exposure there was no cytotoxicity of the test item.
In the 3rd experiment in prolonged exposure only the highest concentration 2000 µg/mL was cytotoxic. In concentrations 1000, 500 and 250 µg/mL there was no cytotoxicity.


Genotoxicity
No genotoxicity effect was found in any concentration and any treatment (treatment with or without metabolic activation in short and prolonged time of exposure).

Remarks on result:

other: negative

Applicant's summary and conclusion

Conclusions:

The test item had no genotoxic effects in the human peripheral blood lymphocytes with as well as without metabolic activation.

Executive summary:

In Vitro Mammalian Cell Micronucleus Test assayed genotoxicity of the test item. The test was performed according to OECD Test Guideline No. 487 -In VitroMammalianCell MicronucleusTest, Adopted 29^thJuly, 2016.

The human peripheral blood lymphocytes from healthy donors were used for testing. The test item was dissolved in RPMI medium and assayed in concentrations given further, which were applied to cultures in volume of 50mL.

At first, genotoxicity experiments were done to assess the genotoxicity potential of test item with 3 hours (short) exposure with and without metabolic activation. Because no genotoxicity was observed, the second genotoxicity test was performed to assess the genotoxicity in extended exposure 25.5 hours (without metabolic activation). Under the experimental design described above, the test item, had no genotoxic effects in the human peripheral blood lymphocytesin experiment with metabolic activation as well as without metabolic activation in both times of exposure.

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