Disodium 3-[[2,4-bis(2-methylphenoxy)phenyl]azo]-4-hydroxy-5-[[(p-tolyl)sulphonyl]amino]naphthalene-2,7-disulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment. GLP compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 3-[[2,4-bis(2-methylphenoxy)phenyl]azo]-4-hydroxy-5-[[(p-tolyl)sulphonyl]amino]naphthalene-2,7-disulphonate

EC Number:

274-402-1

EC Name:

Disodium 3-[[2,4-bis(2-methylphenoxy)phenyl]azo]-4-hydroxy-5-[[(p-tolyl)sulphonyl]amino]naphthalene-2,7-disulphonate

Cas Number:

70210-05-8

Molecular formula:

C37H31N3O11S3.2Na

IUPAC Name:

sodium (E)-3-((2,4-bis(o-tolyloxy)phenyl)diazenyl)-4-hydroxy-5-(4-methylphenylsulfonamido)naphthalene-2,7-disulfonate

Test material form:

other: solid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

experiment 1: 3; 10; 33; 100; 333; 1000; 2500; 5000 um/plateexperiment 2: 33; 100; 333; 1000; 25000; 5000 um/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Remarks:

Concurrent untreated and solvent controls were performed

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent untreated and solvent controls were performed

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene, 4-Nitro-o-phenylene-diamine

Details on test system and experimental conditions:

PreculturesFrom the thawed ampoules of the strains 0.5 ml bacterial suspension were transferred into 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 pl ampicillin (25 pg/ml) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre: 8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt) 5 g NaCI (MERCK, D-64293 Darmstadt) The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C.The overlay agar contains per litre: for Salmonella strains:6.0 g MERCK Agar Agar6.0 g NaCI10.5mg L-Histidine• HCI. H2012.2 mg Biotinfor Escherichia coli: 6.0 g MERCK Agar Agar6.0 g NaCl2.5 mg Tryptophan Sterilisation was performed at 121° C in an autoclaveRat Liver S9 (Preparation by RCC Cytotest Cell Research) Phenobarbital/I3-Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 - 12 weeks old male VVistar Hanlbm rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D-22335 Hamburg) and 13-Naphthoflavone p.o. (Aldrich, 0-89555 Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCI solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at -80° C. Small numbers of the ampoules can be kept at -20°C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo(a)pyrene. The protein content was determined using an analysis kit of Bio-Rad Laboratories, 0-80939 München (Bio-Rad protein assay, Catalogue No. 5000006). The protein concentration in the S9 preparation was 30.6 mg/mL. Hamster Liver S9 (Preparation by RCC Cytotest Cell Research) The S9 liver microsomal fraction was prepared from the liver of 7 - 8 weeks old male Syrian golden hamsters. After decapitation of the anaesthetised animals the livers of the animals was removed, washed in 0.1 M sodium phosphate buffer pH 7.4, 0.25 M sucrose and 1 mM disodium EDTA in deionised water and homogenised. The homogenate, diluted 1+3 in sodium phosphate buffer was centrifuged at 9,000 g for 25 minutes at 4° C. Aliquots of the supernatant were frozen and stored in ampoules at -80° C. Small numbers of the ampoules can be kept at -20°C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as congo red. The protein content was determined using an analysis kit of Bio-Rad Laboratories, 0-80939 München (Bio-Rad protein assay, Catalogue No. 5000006). The protein concentration in the S9 preparation was 42.2 mg/mL.Rat S9 Mix Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 15% v/v. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgC12 33 mM KCI 5 mM glucose-6-phosphate 5 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.(2).Hamster S9 Mix Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 30% v/v. The concentrated cofactor solution yields the following concentrations in the S9 mix: 8.0 mM MgCl2 33.0 mM KCI 20.0 mM Glucose-6-phosphate 2.8 units/ml Glucose-6-phosphate-dehydrogenase 4.0 mM NADP 2.0 mM NADH 2.0 mM FMN in 100 mM Sodium-Ortho-Phosphate-buffer, pH 7.4. During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.(2) and Prival and Mitchell (1).Pre-Experiment for Toxicity To evaluate the toxicity of the test item a pre-experiment was performed with all strains. Eight concentrations were tested for toxicity and mutation induction with 3 plates each. Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. Experimental Performance For each strain and dose level, including the controls three plates were used. The following materials were mixed in a test tube and poured onto the selective agar plates (first experiment): 100 ul Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control), 500 ul S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 ul Bacteria suspension (cf. test system, pre-culture of the strains), 2000 ul Overlay agar According to the pre-incubation method (second experiment) 100 ul test solution, 500 ul S9 mix / S9 mix substitution buffer, and 100 ul bacteria suspension were mixed in a test tube and incubated at 30°C for 30 minutes. After pre-incubation 2.0 ml overlay agar (45°C) was added to each tube. The mixture was poured on selective agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant

Statistics:

No statistical evaluation of the data is required

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxic effects, evident as a reduction in the number of revertants, were observed without metabolic activation in strain TA 1535 at 5000 pg/plate and with metabolic activation in strain TA 1535 at 2500 and 5000 pg/plate, and in strains TA 1537 at 5000 pg/plate and TA 98 at 2500 pg/plate in experiment I.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):negativeThe tested substance is not mutagenic.

Executive summary:

This study was performed to investigate the potential of the substance to induce gene mutations according to the plate incorporation assay with rat liver S9 (experiment l), and the pre-incubation test with hamster liver S9 (experiment II) using the Salmonella typhimuriunn strains TA 1535, TA 1537, TA 98, TA 100 and the Escherichia coli strain VVP2 uvrA.

The assay was performed in two independent experiments with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Experiment I: 3; 10;33; 100; 333; 1000; 2500; and 5000 pg/plate.

Experiment II: 33; 100; 333; 1000; 2500; and 5000 pg/plate

The plates incubated with the test item showed normal background growth up to 5000 ug/plate with and without metabolic activation in both independent experiments.

Toxic effects, evident as a reduction in the number of revertants, were observed without metabolic activation in strain TA 1535 at 5000 ug/plate and with metabolic activation in strain TA 1535 at 2500 and 5000 ug/plate, and in strains TA 1537 at 5000 ug/plate and TA 98 at 2500 ug/plate in experiment I.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Sanolin-Red N-6B at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.