Benzenesulfonic acid, 2,2'-(1,2-ethenediyl)bis[5-nitro-, reaction products with 4-[(4-aminophenyl)azo]benzenesulfonic acid, reduced, oxidized, potassium sodium salts

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:

1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

1992

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

1987

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzenesulfonic acid, 2,2'-(1,2-ethenediyl)bis[5-nitro-, disodium salt, reaction products with 4-[(4-aminophenyl)azo]benzenesulfonic acid, sodium salts

EC Number:

215-397-8

EC Name:

Benzenesulfonic acid, 2,2'-(1,2-ethenediyl)bis[5-nitro-, disodium salt, reaction products with 4-[(4-aminophenyl)azo]benzenesulfonic acid, sodium salts

Cas Number:

1325-54-8

Molecular formula:

Molecular formula of the main constituents: (1) C38H24N8Na4O12S4 (2) C52H32N10Na6O18S6

IUPAC Name:

Reaction mass of Tetrasodium 5-[[4-[(4-sulfonatophenyl)diazenyl]phenyl]diazenyl]-2-[2-[2-sulfonato-4-[[4-[(4-sulfonatophenyl)diazenyl]phenyl]diazenyl]phenyl]ethenyl]benzenesulfonate and Hexasodium 2-((E)-2-sulfonato-4-((E)-(3-sulfonato-4-((E)-2-sulfonato-4-((E)-(4-((E)-(4-sulfonatophenyl)diazenyl)phenyl)diazenyl)styryl)phenyl)diazenyl)styryl)-5-((E)-(4-((E)-(4- sulfona-tophenyl)diazenyl)phenyl)diazenyl)benzenesulfonate.

Test material form:

solid: particulate/powder

Details on test material:

Direct Orange 39

Method

Target gene:

Histidine-prototrophic

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Test concentrations with justification for top dose:

- Range in the cytotoxicity test*: 20.6-5000 μg /plate
- Range in the orig. mutagenicity test*: 61.7-5000 μg/plate
- Range in the 1st conf. mutagenicity test**: 228.6 - 18519 μg/plate
- Range in the 2nd conf. mutagenicity test**: 228.6 - 18519 μg/plate

* The purity of the tested batch is 27%. Related to 100% purity the highest concentration in these parts of the assay was 1350 μg/plate active ingredient.
** Related to 100% purity the highest concentration in this part of the assay was 5000 μg / plate active ingredient.

Vehicle / solvent:

Bidistilled water (suspension)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cyclophosphamide

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

Colonies were counted electronically with an Artek counter. The results were sent on line to a computer. They were checked on a random basis by the operator. Observations indicating precipitates of the test substance in the top agar or a reduced or absent bacterial background lawn were registered additionally. Means and standard deviations for all mutagenicity assays were calculated by a previously validated computer program and included in the Results section.

Evaluation criteria:

A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and
if the results of the positive controls meet the criteria for a positive response.

Criteria for a positive responses:
At least a reproducible meaningful increase of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains:
S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537.
Generally a concentration-related effect should be demonstrable.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

The highest concentration applied was 5000 ug/plate. The five lower concentrations decreased by a factor of 3

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

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 3

Species / strain:

S. typhimurium TA 1537

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 4

Species / strain:

S. typhimurium TA 98

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 5

Species / strain:

S. typhimurium TA 102

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

Applicant's summary and conclusion

Conclusions:

Based on the results of these experiments and on standard evaluation criteria, it is concluded that the substance exerted a marginal mutagenic action on strain S. typhimurium TA 98. This effect, however, was only observed at the concentration of 18519 µg/plate with a test material of 27% purity. Hence, the test substance was considered not to be mutagenic in the bacteria reverse mutation assay.

Executive summary:

A bacterial reverse mutation test was carried out according to OECD test guideline 471 in Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535, and TA 1537.

The concentration range of Direct Orange 39 (purity 27%) to be tested in the mutagenicity test was determined in a preliminary toxicity test. Thus, Direct Orange 39 was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 61.7 to 5000 µg/plate based on test material. An independent repetition of the experiments was performed with the concentrations of 228.6 to 18519 µg/plate test material (equals to 62 to 5000 µg/plate active ingredient). In-order to confirm the results obtained in the second assay on strain TA 98 without activation and on strain TA 102 with activation, the experiments on these two strains were repeated once more with the concentrations of 228.6 to 18519 µg/plate (62 to 5000 µg/plate active ingredient).

In the experiment without and with metabolic activation no toxic effect of the test material on the growth of the bacteria was observed.

 

Mutagenicitv test, original experiment (61.7 to 5000 µg/plate test mat.)

In the original experiment carried out without and with metabolic activation, none of the tested concentrations of Direct Orange 39 led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control.

 

Mutagenicitv test, first confirmatory experiment (228.6 to 18519 µg/plate test mat.)

In the first confirmatory experiment performed without metabolic activation on strain TA 98, a marginal increase in the number of revertant colonies was observed at the concentration of 18519 µg/plate. No effect was observed with the other strains. In the experiment performed with activation on strain TA 102, a marginal increase (not reaching two-fold) in the number of revertant colonies was observed at the concentration of 685.9 µg/plate only. No effect was seen with the other strains.

 

Mutagenicitv test, second confirmatory experiment (228.6 to 18519 µg/plate test mat.)

In the second confirmatory experiment performed without metabolic activation on strain TA 98, again, a marginal increase in the number of revertant colonies was observed at the concentration of 18519 µg/plate. In the experiment performed with activation on strain TA 102, none of the tested concentrations of Direct Orange 39 led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control.

In the mutagenicity tests without and with metabolic activation, normal background growth was observed. The number of revertant colonies was not reduced. The test substance exerted no toxic effect on the growth of the bacteria.

Based on the results of these experiments and on standard evaluation criteria, it is concluded that Direct Orange 39 exerted a marginal mutagenic action on strain S. typhimurium TA 98. This effect, however, was only observed at the concentration of 18519 µg/plate.

The test was performed under Good Laboratory Practice conditions and was subjected to a periodical quality assurance evaluation.

No circumstances, which may have affected the quality or integrity of the data, have been noted.