Reaction mass of tetrasodium 3-[[4-[[4-[(6-amino-1-hydroxy-3-sulphonato-2-naphthyl)azo]-6-sulphonato-1-naphthyl]azo]-1-naphthyl]azo]naphthalene-1,5-disulphonate and tetrasodium 3-[[4-[[4-[(6-amino-1-hydroxy-3-sulphonato-2-naphthyl)azo]-7-sulphonato-1-naphthyl]azo]-1-naphthyl]azo]naphthalene-1,5-disulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

other: read across from analogue substance

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

performed on analogue substance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

yes

Remarks:

The two different final concentrations (0.007 and 0.008 µg/mL) of colchicine were used in the 3rd experiment. Both concentrations were analysed for cytotoxicity and genotoxicity and the concentration 0.007 µg/mL was used for evaluation.

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:

Sodium 8-phenylamino-5-(4-(3-sulphonatophenylazo)-1-naphthylazo)naphthalenesulphonate

EC Number:

222-111-5

EC Name:

Sodium 8-phenylamino-5-(4-(3-sulphonatophenylazo)-1-naphthylazo)naphthalenesulphonate

Cas Number:

3351-05-1

Molecular formula:

C32H23N5O6S2.2Na

IUPAC Name:

sodium 8-phenylamino-5-(4-(3-sulphonatophenylazo)-1-naphthylazo)naphthalenesulphonate

Test material form:

solid: particulate/powder

Method

Target gene:

Human lymphocites

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

Test concentrations with justification for top dose:

Genotoxicity testing – short exposure:
The 1st experiment to assess genotoxicity with time of exposure 3 hours was done.
The final concentrations 125, 250, 500, 1000 and 2000 µg/mL were used without and with metabolic activation (S9-mix).

Genotoxicity testing – extended exposure:
In the 2nd experiment, the genotoxicity test with time of exposure 23 hours was done. On the basis of results from the 1st experiment as the final concentrationswere used 250, 500, 1000 and 2000 µg/mL.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Remarks:

untreated culture

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Colchicine CAS 64-86-8

Details on test system and experimental conditions:

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 transferred 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 first experiment without and 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 23 hours from the start of exposure).
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.

Test item preparations:
At first, the solubility of the test item was evaluated. The test item was better soluble in DMSO than in medium, so the DMSO was choosed as the solvent for this study.

Test item concentrations:
5 g of the test item was dissolved in 25 mL of DMSO in volumetric flask. The starting stock solution (200 mg/mL) was diluted and concentration line (12.5, 25, 50, 100 mg/mL) was prepared.
Then, 25 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.

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 L of S9 and 18.5 L of cofactors solution (S9-mix = MAS).

Evaluation criteria:

Critetion 1
In the negative control normal cell cycle time or proliferation index should be determined.
Criterion 2
The cytotoxicity of the highest three concentrations selected for genotoxicity evaluation should not be higher than 55±5%. Concurrently, at least 2000 binucleated cells should be scored in each concentration of genotoxicity evaluation.
Criterion 3
Number of micronuclei should be in the limits of historical laboratory data set.
In the case of negative control the upper limit is critical. In the case of positive control the lower limit is critical.
Criterion 4
Negative control value of average number of micronuclei per 1000 binucleated cells will be compared with appropriate positive control value. Concurrently, CBPI of negative control should be in the range 1.3 – 2.2. The result of positive control should be ≥ 2x negative control value.
When the critical limits in criterion 1 will be exceeded and criterion 2 will not be fulfilled the experiment should be repeated. When the critical limits in criterion 1 will be exceeded and criterion 2 will be fulfilled the experiment will not be repeated.

Results and discussion

Test results

Species / strain:

lymphocytes: human

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:

Genotoxicity
No genotoxicity effect was found in any concentration and any treatment (treatment with or without metabolic activation in short and extended time of exposure).
On the basis of the chapter 3.10 – Evaluation and interpretation of results (and OECD TG 487 recommendations), the test item.


Cytotoxicity
The test item was not cytotoxic in the 1st experiment in short exposure in both treatment.
In 2nd experiment the cytotoxicity was found only in test item concentrations 2000 and 1000 µg/mL.
In the 3rd experiment there was no cytotoxicity of the test item:
- did not exhibit a statistically significant increase (two-fold increase rule) compared with concurrently solvent negative control
- did not show evidently the dependence of increasing number of cells with micronuclei on concentration (dose-response relationship)
- did not have any of the results outside the distribution of the historical negative control data

On the basis of informations above, the test item is considered as clearly negative.

Applicant's summary and conclusion

Conclusions:

The analogue substance was tested for in vitro micronucleus folllowing OECD 487. Under the experiemtal conditions the tested substance did not induce chromosome breaks and/or chromosome gain or loss.

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 Vitro MammalianCell MicronucleusTest, Adopted 29^thJuly, 2016. The human peripheral blood lymphocytes from healthy donors were used for testing. The test item was dissolved in DMSO and assayed in concentrations 125 - 2000 mg/mL, which were applied to cultures in volume of 25 mL.

At first, genotoxicity experiment with 3-hour exposure was done to assess the genotoxicity with and without metabolic activation. Because no genotoxicity was observed, the second genotoxicity test was performed to assess the genotoxicityin extended exposure 23 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.