Disodium 5-[[4-[(2-bromo-1-oxoallyl)amino]-2-sulphonatophenyl]azo]-4-hydroxy-6-(methylamino)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:

January 26, 1993 to February 05, 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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

trisodium 5-({3-[(2-bromoacryloyl)amino]benzoyl}amino)-3-({5-[(2-bromoacryloyl)amino]-2-sulfonatophenyl}diazenyl)-4-hydroxynaphthalene-2,7-disulfonate

EC Number:

941-899-8

Cas Number:

1625603-53-3

Molecular formula:

C29H21Br2N5O13S3.3Na

IUPAC Name:

trisodium 5-({3-[(2-bromoacryloyl)amino]benzoyl}amino)-3-({5-[(2-bromoacryloyl)amino]-2-sulfonatophenyl}diazenyl)-4-hydroxynaphthalene-2,7-disulfonate

Constituent 2

Reference substance name:

Disodium 5-[[4-[(2-bromo-1-oxoallyl)amino]-2-sulphonatophenyl]azo]-4-hydroxy-6-(methylamino)naphthalene-2-sulphonate

EC Number:

274-436-7

EC Name:

Disodium 5-[[4-[(2-bromo-1-oxoallyl)amino]-2-sulphonatophenyl]azo]-4-hydroxy-6-(methylamino)naphthalene-2-sulphonate

Cas Number:

70210-39-8

Molecular formula:

C20H17BrN4O8S2.2Na

IUPAC Name:

disodium 5-({4-[(2-bromoacryloyl)amino]-2-sulfonatophenyl}diazenyl)-4-hydroxy-6-(methylamino)naphthalene-2-sulfonate

Test material form:

other: Solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 287380.26
- Expiration date of the lot/batch: November 1997

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: stable

Method

Target gene:

Preliminary Toxicity/Range-Finding test:
A toxicity test (check for reduction in the number of revertant colonies) was carried out with strain TA 100 without and with microsomal activation at six concentrations of the test substance and one negative control according to Standard Operating Procedures of Genetic Toxicology. The highest concentration applied was 5000 µg/plate. The five lower concentrations decreased by a factor of 3. The plates were inverted and incubated for about 48 hours at 37 ± 1.5 °C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration, as well as each negative control was used.

Mutagenicity test:
The mutagenicity test was performed with strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 without and with microsomal activation according to Standard Operating Procedures of Genetic Toxicology. Each of the five concentrations of the test substance, a negative and a positive control were tested, using three plates per test substance concentration as well as each positive and negative control with each tester strain. The highest concentration applied was 5000 µg/plate (because of lack of toxicity in the range finding test) and the four lower concentrations were each decreased by a factor of 3. The plates were inverted and incubated for about 48 hours at 37 ± 1.5 °C in the dark. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.

Species / strain

Species / strain / cell type:

other: TA 98, TA 100, TA 1535, TA 1537 and TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver microsomal fraction S9

Test concentrations with justification for top dose:

Concentrations for cytotoxicity test:
20.5761 µg/plate
61.7284 µg/plate
185.1852 µg/plate
555.5556 µg/plate
1666.6667 µg/plate
5000.0000 µg/plate

Concentration for mutagenicity test:
61.7284 ug/plate
185.1852 µg/plate
555.5556 µg/plate
1666.6667 µg/plate
5000.0000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100 and TA 1535 without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 and TA 1538 without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA 98, TA 100, TA 1537 and TA 1538 with metabolic activation

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

TA 1535 with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)
Setting up of the test plates
0.1 ml of the overnight cultures were mixed with 2 ml of top agar, either 0.5 ml of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 ml of the activation mixture (experiments with activation) and 0.1 ml of a solution of the test substance, the substance for the positive control or the solvent for the negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20 ml of minimal agar (1.5 % agar supplemented with 2 % salts of the Vogel-Bonner Medium E and 2 % glucose). The top agar was composed of 0.6 % agar and 0.6 % NaCl. It was supplemented with 10 % of 0.5 mM 1-histidine and 0.5 mM (+)biotin dissolved in water.

The mutagenicity test was performed with strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 without and with microsomal activation according to Standard Operating Procedures of Genetic Toxicology. Each of the five concentrations of the test substance, a negative and a positive control were tested, using three plates per test substance concentration as well as each positive and negative control with each tester strain. The highest concentration applied was 5000 /zg/plate (because of lack of toxicity in the range finding test) and the four lower concentrations were each decreased by a factor of 3. The plates were inverted and incubated for about 48 hours at 37 ± 1.5 °C in darkness. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.

Evaluation criteria:

Assay acceptance 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. In either case the final decision is based on the scientific judgement of the Study Director.

Criteria for a positive response:
The test substance is considered to be mutagenic in this test system if the following conditions are met:
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 1535, TA 1537 and TA 1538.
Generally a concentration-related effect should be demonstrable.

Statistics:

No appropriate statistical method is available

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

not valid

Additional information on results:

Toxicity test/Range finding test:
In the experiments without and with activation, Lanasol Rot 5B Roh Trocken (FAT 92354/A) led to a slight increase in the number of revertants at the highest concentrations. The test material exerted no toxic effect on the growth of the bacteria.

Any other information on results incl. tables

Toxicity test/range finding test

Six concentrations of FAT 92354/A ranging from 20.6 to 5000 µg/plate were tested with strain S. typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without microsomal activation. Normal background growth was observed. Slightly increased numbers of revertant colonies were observed in the experiments without and with activation at the highest concentration. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate without and with activation.

 

Mutagenicity test, original experiment

In the experiments without microsomal activation performed on strains TA 98, TA 1535, TA 1537 and TA 1538, after treatment with FAT 92354/A no increase in the incidence of histidine-prototrophic mutants was observed in comparison with the negative control. However, treatment of strain TA 100 with the test material lead to a slight increase in the number of revertant colonies at the concentration of 5000 µg/plate. In the experiments with activation performed on strains TA 98, TA 1537 and TA 1538, after treatment with FAT 92354/A no increase in the incidence of histidine prototrophic mutants was observed in comparison with the negative control. Treatment of strains TA 100 and TA 1535 with the test material lead to a slight increase in the number of revertant colonies at the concentration of 5000 µg/plate.

Mutagenicity test, confirmatory experiment

In the experiments without microsomal activation performed on strains TA 1537 and TA 1538, after treatment with FAT 92354/A no increase in the incidence of histidine-prototrophic mutants was observed in comparison with the negative control. However, treatment of strains TA 98 and TA 1535 with the test material lead to a slight increase in the number of revertant colonies at the concentration of 5000 µg/plate. With strain TA 100 this effect occurred at the concentrations of 1666.7 and 5000 µg/plate.

In the experiments with microsomal activation performed on strains TA 98, TA 1535, TA 1537 and TA 1538, after treatment with FAT 92354/A no increase in the incidence of histidine-prototrophic mutants was observed in comparison with the negative control. Treatment of strain TA 100 with the test material lead to a slight increase in the number of revertant colonies at the concentrations of 1250 and 5000 µg/plate.

In the mutagenicity tests, normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced. The test substance exerted no toxic effect on the growth of the bacteria.

The various mutagens, pro-mutagens, sterility checks, sensitivity and resistance tests, etc., employed to ensure the test system was acceptable, all produced results within our established limits.

There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the data.

Applicant's summary and conclusion

Conclusions:

In Ames assay, FAT 92354/A exerted a very weak mutagenic effect.

Executive summary:

The test was carried out to evaluate mutagenic activity of FAT 92354/A in bacterial test systems in the absence and presence of a rat liver S9 activity system. The test was performed following OECD, EEC and EPA guidelines in accordance to GLP principles. This test system permits the detection of gene mutations induced by the test material or its metabolites in histidine-requiring strains of Salmonella typhimurium.

The concentration range of Lanasol Rot 5B Roh Trocken (FAT 92354/A) to be tested in the mutagenicity test was determined in a preliminary toxicity test. Thus, the substance was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 61.7 to 5000 ug/plate. In order to confirm the results, the experiments were repeated in an independent experiment with the same concentrations.

In the toxicity test/range finding test experiments without and with activation Lanasol Rot 5B Roh Trocken (FAT 92354/A) led to a slight increase in the number of revertants at the highest concentrations. The test material exerted no toxic effect on the growth of the bacteria.

Mutagenicity test, original experiment:

In the original experiments without metabolic activation performed on strains TA 98, TA 1535, TA 1537 and TA 1538 none of the tested concentrations of FAT 92354/A led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. However, in the experiment without activation carried out on strain TA 100, treatment with FAT 92354/A led to a slight increase of revertant growth at the highest concentration.

In the original experiments with metabolic activation performed on strains TA 98, TA 1537 and TA 1538 none of the tested concentrations of FAT 92354/A led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. In the experiments with activation on strains TA 100 and TA 1535, treatment with FAT 92354/A led to a slight increase of revertant growth at the highest concentration.

In the confirmatory experiments without metabolic activation performed on strains TA 1537 and TA 1538 none of the tested concentrations of FAT 92354/A led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. While in the experiment without activation carried out on strains TA 98, TA 100 and TA 1535, treatment with FAT 92354/A led to a slight increase of revertant growth at the upper concentrations. In the experiments with activation performed on strains TA 98, TA 1535, TA 1537 and TA 1538 none of the tested concentrations of FAT 92354/A led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. In the experiment with activation on strain TA 100 treatment with FAT 92354/A led to a slight increase of revertant growth at the upper concentrations.

Based on the results of these experiments and on standard evaluation criteria, it is concluded that FAT 92354/A exerted a very weak mutagenic effect in this test system.