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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 1,3-dihydroxybenzene, 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid, 4-methoxy-1-aminobenzene, 2-(4-aminoanilino)-5-nitrobenzenesulphonic acid, sodium salt

IUPAC Name:

Reaction products of 1,3-dihydroxybenzene, 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid, 4-methoxy-1-aminobenzene, 2-(4-aminoanilino)-5-nitrobenzenesulphonic acid, sodium salt

Method

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

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

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Applicant's summary and conclusion

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