Benzenesulfonic acid, 4-(acetylamino)-2-amino-5-[2-[3-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, sodium salt (1:2)

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:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Similar to Guideline Study without GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1982

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Principles of method if other than guideline:

1) B.N. Ames, W.W. Durston, E. Yamasaki and r.D. Lee, Carcinogens are mutagens. A simple test system combining liver homogenate for activation
and bacteria for detection, Proc. Nat. Acad. Sci, USA 70 (1973) 2281 2285
2) B.N. Ames, J. McCann and E. Yamasaki: Methods for detecting carcinogens and mutagens with the Salmonella / mammalian-microsome mutagenicity test, Mutation Res. 31 (1975) 347 - 364.
3) M.H.L. Green, and W.J. Muriel: Mutagen testing using trp+ reversion in Escherichia coli, Mutation Res. 38 (1976) 3 - 32).
4) A. P. Alvares, D. R. Bickers and A. Kappas: Polychlorinated biphenyls: a new type of inducer of cytochrome P 448 in the liver. Proc. Nat. Acad.
Sci USA 70 (1973) 1321 - 1325.
5) J. McCann, N. E. Springarn, J. Kobory and B. N. Ames: Detection of carcinogens as mutagens: bacterial tester strains with R factor plasmids,
Proc. Nat. Acad. Sci. USA, 72 (1975), 979 - 983

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

disodium 2-amino-4-acetamido-5-[(1E)-2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl]benzene-1-sulfonate

EC Number:

940-436-7

Cas Number:

1354632-48-6

Molecular formula:

C16H18N4O10S3.xNa C16H16N4Na2O10S3 as disodium salt

IUPAC Name:

disodium 2-amino-4-acetamido-5-[(1E)-2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl]benzene-1-sulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

see below

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat S9-mix

Test concentrations with justification for top dose:

4 to 5000 µg/plate

Vehicle / solvent:

aqua bidest

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

all strains with S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100, TA1535 w/o S9

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 w/o S9

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98, TA 1538 w/o S9

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Remarks:

WP2uvrA w/o S9

Details on test system and experimental conditions:

Toxicity experiments and dose range finding
Preliminary toxicity tests were performed with all tester strains using a small number of plates to calculate an appropriate dose range. A reduced rate of spontaneously occurring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxicity . Thinning of the bacterial lawn was controlled microscopically. .
In combination with the main experiment, toxicity testing was performed as follows: 0.1 mL of the diffeqsnt dilutions of the test compound were thoroughly mixed with 0.1 mL of 10 dilution of the overnight culture of TA 100 and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control is compared with the number of colonies per plate in the presence of the test compound. Results are given as a ratio of these values. (= surviving fraction).
The test compound was tested at doses of 4 to 10 000 µg/plate and proved to be not toxic to the bacteria. For mutagenicity testing 5000 µg/plate was chosen as the highest dose.

Mutagenicity experiments
Top agar is prepared for the Salmonella strains by mIxmg 100 mL agar (0.6% agar, 0.5%NaCl) with 5 mL of a 1 mM histidine and 5 ml of 1 mM biotin
solution. With E. coli histidine is replaced by tryptophan (5 mL, 0.5 mM). The following ingredients are added (in order) to 2 mL of molten top agar at 45°C:
0.1 mL test compound solution
0.1 mL of an overnight nutrient broth culture of the bacterial tester strain
0.5 mL S-9 Mix (if required) or buffer
After mixing, the liquid is poured into a petridish with minimal agar (1.5% agar, Vogel-Bonner E medium with 2% glucose). After incubation for 48 to
72 hours at 37°C in the dark, colonies (his+ revertants) are counted.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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):
negative

Remazol-Goldgelb RNL is not mutagenic in this bacterial test system either with or without exogenous metabolic activation at the dose levels investigated.

Executive summary:

Remazol-Goldgelb RNL was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA.

The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A dose range of 6 different doses from 4 µg/plate to 5000 µg/plate was used.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similiar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

Toxicity: The test compound proved to be not toxic to the bacteria. 5000 µg/plate was chosen as top dose level for the mutagenicity study.

Mutagenicity: In the absence of the metabolic activation system, the test compound did not show a significant dose dependent influence in the number of revertants in any of the bacterial strains. Also in the presence of metabolic activation system, treatment of the cells with Remazol-Goldgelb RNL did not result in relevant increases in the number of revertant colonies.

Summarizing, it can be stated that Remazol-Goldgelb RNL is not mutagenic in these bacterial test systems neither with nor without exogenous metabolic activation at the dose levels investigated.