Tetrasodium 4-[[3,5-bis[[4-[(4-nitro-2-sulphonatophenyl)amino]phenyl]azo]-2,4(or 2,6)-dihydroxyphenyl]azo]-5-hydroxynaphthalene-2,7-disulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the evaluation of the available in vitro tests, the substance is considered not mutagen.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

other: experimental study on simillar substance

Adequacy of study:

key study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

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

Target gene:

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.

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 fraction

Test concentrations with justification for top dose:

1 experiment with and without S9: 62.5, 125, 250, 500, 1000 and 2000 µg/ml
2 experiment prolonged exposition without S9: 250, 500, 1000 and 2000 µg/ml
3 experiment prolonged exposition without S9: 62.5 and 125 µg/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: colchicine

Details on test system and experimental conditions:

Media:
RPMI 1640 with L-Glutamine (with Sodium Bicarbonate (NaHCO3) and Phenol Red (C19H13NaO5S) as a pH indicator): Lonza Lot. No. 6MB165 (Exp. 8/2018)
Foetal Bovine Serum: Sigma Aldrich Lot. No. 032M3395 (Exp.3/2017), Lot. No. 124M3337 (Exp. 12/2019)
RPMI-M (RPMI 1640 + Foetal Bovine Serum + Penicilin-Streptomycin + PHA-M)
Procedures of media preparation are described in detail in internal SOP.

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

Species / strain:

lymphocytes: -

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

highest concentration, exposure 23h

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: -

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

lower dose, exposure 23 h

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Three experiment were done.
All of used test concentrations did not show the cytotoxicity higher than 55±5 % in the first experiment with the time of exposure 3 hours.
In the second experiment (time of exposure 23 hours) the two highest concentrations (2000 and 1000 µg/mL) were cytotoxic, so the third experiment (time of exposure 23 hours) with lower concentrations 125 and 62.5 µg/mL had to be done.
In the third experiment both used concentrations did not show the cytotoxicity higher than 55±5 %.
On the basis of these results, the concentration of 2000 g/mL was selected as the highest concentration for the analysis of genotoxic effect (time of exposure 3 hours - see Tables 2 and 3). In the time of exposure 23 hours, the concentration 500 µg/mL was selected as the highest.
The first experiments (time of exposure 3 hours) gave negative results, so the second and the third experiment without metabolic activation had to be done with extended exposure(23 hours) in the presence of cytochalasin B.
The results did not show substantial (biologically significant) increase in the number of binucleated cells with micronuclei.

Remarks on result:

other: exposure time 3h, all doses

Conclusions:

Not genotoxic

Executive summary:

Under the experimental conditions described above, the test substance had no genotoxic effects in the human peripheral blood lymphocytesin experiments both without and with metabolic activation

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

other: experimental study on simillar substance

Adequacy of study:

key study

Study period:

16/05/2018-15/10/2018

Reliability:

2 (reliable with restrictions)

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:

no impact on the outcome of study (see Any other information ...)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

hypoxanthine-guaninephosphoribosyl-transferase (HPRT) gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The lung fibroblasts V79 from male Chinese hamster were used for testing. Frozen permanent cell cultures were obtained from European Collection of Cell Cultures (ECACC). V79 used for experiments: Lot. No.: 15H003. ECACC Certificate of Analysis is a part of archived study documentation. Sponsor declared identity, sterility and absence of Mycoplasma in provided cultures.

- Number of passages if applicable: max. 5

- Methods for maintenance in cell culture if applicable:
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 before cytotoxicity experiments.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
DMEM : FBS : Atb = 500 : 55 : 5.5, prepared in laboratory
Fresh solutions of the test substance were prepared for every experiment

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
As new frozen stock is used for every mutagenicity experiment, no contamination is presumed at the start of experiments. Mycoplasma determination was then performed in samples withdrawn in the end of experiment - usually at withdrawal of mutants - from several plates of random concentration / duplicate in both experiments.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of rat liver homogenate and mixture of cofactors

Test concentrations with justification for top dose:

Based on the cytotoxicity test: 0.25, 0.5, 1.0 and 2.0 mg.mL-1 with/without

Vehicle / solvent:

DMEM, Sigma-Aldrich, RNBG 5578, RNBG 6521, RNBG 7734

Negative solvent / vehicle controls:

yes

Remarks:

9ml of complete medium + 1 ml of DMEM

Positive controls:

yes

Remarks:

9.9 or 9.95 mL of complete medium and 100 or 50 µL of relevant positive control diluted in DMSO

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

- Cell density at seeding (if applicable):
After treatment, approximately 2×10e6 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).

DURATION
- Preincubation period: 5 days before treatment
- Exposure duration: 3 h
- Expression time (cells in growth medium): 9 days
- Selection time (if incubation with a selection agent): 6 days
Survival and plating efficiency plates were incubated for at least 6 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).

SELECTION AGENT (mutation assays): 6-thioguanine 98%, CAS No. 154-24-7, Sigma , diluted in 0,5% Na2CO3 (Penta); 5 µg.mL-1 (final concentration) for selection of mutants

STAIN: Methylene blue, CAS No. 122965-43-9, AlfaAesar, 0.1 % solution

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Determination of survival
After treatment period, the cultures were trypsinised and an aliquot (0.3 mL of 10e3/mL cell suspension) was diluted and plated to 6 cm Petri dishes to estimate the viability of the cells.

Subculturing
A sufficient number of live cells (but never less than 2 millions, with respect to cytotoxicity results) were further cultured in 10 cm Petri dishes.
Also on the 3rd, 6th and 8th day the cell populations were sub cultured 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 6 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.

NUMBER OF CELLS EVALUATED: 220000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 300

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Concentratios used in toxicity test were: 2.0, 1.0, 0.5, 0.25, 0.1 and 0.05 mg.mL- 1.
No cytotoxicity was observed.

Evaluation criteria:

Each experiment is separately evaluated 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:

other:

Remarks:

Negative control = Solvent control

Positive controls validity:

valid

Conclusions:

Under the experimental conditions the test item, the tested substance, was non-mutagenic for V79 cells with and without metabolic activation.

Executive summary:

The test substance was assayed for the mutagenicity by the In Vitro Mammalian Cell Gene Mutation Test. The study was performed according to OECD Test Guideline No. 476 – In Vitro Mammalian Cell Gene Mutation Test using the Hprt and Xprt genes (2016), which is analogous to the EU method B.17.

Chinese hamster V79 fibroblasts were used for testing.

The test item is water soluble, so it was dissolved in assay medium (DMEM) and it can be dosed till the highest recommended concentration 2.0 mg per plate.

This concentration was used as maximum for cytotoxicity test without metabolic activation performed in advance. Other concentrations were prepared by dilution and concentrations used were 2.0, 1.0, 0.5, 0.2, 0.1 and 0.01 mg per mL.

No cytotoxicity was observed in the cytotoxicity experiment so concentrations of 0.25, 0.5, 1.0 and 2.0 were used for mutagenicity experiments.

The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors.

Cytotoxicity about 25% was observed in mutagenicity experiments in the highest concentration. No signs of mutagenicity were observed either in experiment with or without metabolic activation.

According to OECD Test Guideline No. 476, UVCB substances should be tested up to 5 mg per mL therefore an extra concentration 5 mg per mL was tested beyond in aditional experiments with and without metabolic activation.

In experiment with metabolic activation the Mt/Msc above two was observed in the second duplicate in concentration of 5.0 (2) mg per mL. No increase was observed in the first duplicate of the same concentration. In experiment without metabolic activation, no increase was observed.

The concurent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations.

Under the experimental conditions indicated above the test item was non-mutagenic for V79 cells with and without metabolic activation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

An Ames test is available on similar substance with positive result.
The OECD 487 (In vitro Mammalian Cell Micronucleus Test) and OECD 476 on the same similar substance do not show any effects.

An in vivo test (OECD 474) is available on another similar substance did not indicate any concern for mutagenicity. Two testing proposal are inserted on similar substances to completeb the mutagenicity assessment.