Disodium 7-benzamido-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17/06/1994 - 17/08/1994

Reliability:

1 (reliable without restriction)

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:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 7-benzamido-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate

EC Number:

220-028-9

EC Name:

Disodium 7-benzamido-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate

Cas Number:

2610-11-9

Molecular formula:

C29H21N5O8S2.2Na

IUPAC Name:

disodium 7-benzamido-4-hydroxy-3-[(1E)-2-{4-[(1E)-2-(4-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-sulfonate

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rats

Test concentrations with justification for top dose:

First experimental test : 10, 100, 333, 1000, 5000 µg/plateSecond experimental test: 10, 100, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

Distilled water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine (NPD)

Remarks:

Ta 98, TA1537 and TA1538 without S9 fraction

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100 an TA1535 without S9 fraction

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

all strains with S9 fraction

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)PREPARATION OF FRACTION S9: The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats strain WU (SAVO-Ivanovas, med. Versuchstierzuchten Gmbh, D-7964 Kisslegg, FRG; weight approx. 150 - 200 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, D-76275 Ettlingen, F.R.G.) in olive oil 5 days previously.After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate was diluted 1+3 in KCl and centrifuged cold at 9,000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -70° C.Small numbers of the ampoules are kept at -20° C for only several weeks before use. The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-8000 München: Bio-Rad protein assay, Catalogue 500 000 6.The protein concentration in the S9 preparation was 22 mg/ml. PREPARATION OF S9 MIX: Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution in a ratio 3:7. The composition of the co-factor solution was concentrated to yield the following concentrations in the S9 mix:8 mM MgCl233 mM KCl5 mM glucose-6-phosphate5 mM NADPin 100 mM sodium-ortho-phosphate-buffer, pH 7.4.During the experiment the S9 mix was stored in an ice bath.PREPARATION OF PLATES: For each strain and dose level, including the controls three plates were used as a minimum.The following materials were mixed in a test tube and poured onto the selective agar plates:100 ul: Test solution at each dose level, solvent control, 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 After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.DURATION- Expression time (cells in growth medium): 48 to 72 hours

Evaluation criteria:

A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

Statistics:

Kier, L.E., D.J. Brusick, A.E. Auletta, E.S. Von Halle, M.M. Brown, V.F. Simmon, V. Dunkel, J. McCann, K. Mortelmans, M. Privai, T.K. Rao and V. Ray (1986)The Salmonella typhimurium/mammalian microsomal assay A report of the U.S. Environmental Protection Agency Gene-Tox Program Mutation Res. 168, 69-240.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomalactivation. Each concentration, including the controls, was tested in triplicate. In experiment I and II no toxic effects occurred in the test groups with and without metabolic activation in all strains used.The plates incubated with the test article showed normal background growth up to 5000.0 p.g/plate with and without S9 mix in all strains used.Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant niiit±)er exists. The presence of liver microsomal activation did not influence these findings. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Executive summary:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with the tested substance at any concentration level, either in the presence or 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 significance.Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Direct Red 81 is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.