Trisodium 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulphonatonaphthyl]azo]-2,5-dimethylphenyl]azo]benzene-1,4-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:

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:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulphonatonaphthyl]azo]-2,5-dimethylphenyl]azo]benzene-1,4-disulphonate

EC Number:

274-410-5

EC Name:

Trisodium 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulphonatonaphthyl]azo]-2,5-dimethylphenyl]azo]benzene-1,4-disulphonate

Cas Number:

70210-13-8

Molecular formula:

C27H22ClN9O9S3.3Na

IUPAC Name:

trisodium 2-{[4-({4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulfonato-1-naphthyl}diazenyl)-2,5-dimethylphenyl]diazenyl}benzene-1,4-disulfonate

Test material form:

other: solid

Specific details on test material used for the study:

Test material: Cibacron Orange P-4R flüssig (FAT 40075/C)
Batch No.: 289637.26
Purity: 21.4 % (active ingredient)
Expiration date: January 1994

Method

Target gene:

Histidine gene

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 100, TA 102, TA 1535 and TA 1537) were obtained from Prof. B. Ames, Berkeley, CA., U.S.A.
Inoculates from frozen master copies were set up monthly. They were grown in liquid NB-medium overnight and then plated on NB-agar. After incubation, single colonies were taken from the plates, grown overnight in liquid NB-medium and then used for the experiment.
The characteristics of the strains were checked monthly. Histidine-auxotrophy of the strains was demonstrated by the requirement for 1-histidine. The presence of the rfa character was assayed by the sensitivity for crystal-violet. The deletion of the uvrB gene (strains TA 98, TA 100, TA 1535 and TA 1537) was demonstrated by the sensitivity for UV-light. The Salmonella strains containing the R-factor (TA 98 and TA 100) were additionally checked for ampicillin resistance. Strain TA 102 additionally was checked for tetracycline resistance (presence of multicopy plasmid pAQ1). The presence of the uvr+ gene was demonstrated by the resistance of strain TA 102 against UV light. Furthermore, all strains were checked for their characteristic reversion properties with known mutagens (positive controls).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Cytotoxicity test: 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg active ingredient/plate
Mutagenicity tests: 61.7, 185.2, 555.55, 1666.7 and 5000 µg active ingredient/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Bidistilled water
- Justification for choice of solvent/vehicle: Based on solubility

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Bidistilled water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

for TA 100 and TA 1535 without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

bidistilled water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

for TA 102 without metabolic activation

Controls 3

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

for TA 98 without metabolic activation

Controls 4

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

for TA 1537 without metabolic activation

Controls 5

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

for TA 100, TA 102, TA 98 and TA 1537 with metabolic activation

Controls 6

Untreated negative controls:

yes

Remarks:

bidistilled water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: cyclophosphamide H20

Remarks:

for TA 1535 with metabolic activation

Details on test system and experimental conditions:

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.

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 102, TA 1535 and TA 1537 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 in the first and second mutagenicity test 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 h 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.

Statistics:

In deviation to the OECD guideline, a statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended. No appropriate statistical method is available.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535 pSK1002

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

marginal response

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

marginal response

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity test/Range finding test: Six concentrations of the test substance ranging from 20.6 to 5000 µg active ingredient/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 back-ground growth was observed at all concentrations. A slight increase in the number of revertant counts was observed at the upper concentrations, followed by a reduction in the number of revertant colonies at the highest concentration, due to toxicity of the test substance. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg active ingredient/plate without and with activation.

Mutagenicity test, original experiment: In the original experiment carried out without metabolic activation, treatment of strain TA 102 with the test substance led to a slight increase in the number of revertant counts at the concentrations of 185.2 to 1666.7 µg active ingredient/plate. In the experiment with activation, a similar effect occurred on strain TA 100 at concentration of 1666.7 µg active ingredient/plate and on strain TA 102 at concentrations of 185.2 and 1666.7 µg active ingredient/plate. No effects were observed with the other strains.

Mutagenicity test, confirmatory experiment: In the confirmatory experiment carried out without metabolic activation, treatment of strain TA 102 with the test substance led to a slight increase in the number of revertant counts at the concentration of 1666.7 µg active ingredient/plate. In the experiment with activation, a similar effect occurred on strains TA 100 and TA 102 at the same concentration. Again, no effects were observed with the other strains. In the mutagenicity tests normal background growth was observed at all concentrations. In the experiments without and with microsomal activation, due to toxicity of the test substance the numbers of revertant colonies occasionally were slightly reduced with strains TA 100 and TA 102 at the upper concentrations.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

An in vitro study was performed to investigate the potential of the test substance (of ca. 21.4 % purity) to induce gene mutations according to OECD Guideline 471, EU Method B.14 and EPA OTS 798.5265 in compliance with GLP.

Based on the results of a preliminary toxicity/range finding study, the substance was tested for mutagenic effects without and with metabolic activation at 5 doses from 61.7 to 5000 µg active ingredient/plate. An independent repetition of the experiments was performed with the same concentrations. In the preliminary experiment without and with metabolic activation performed on strain TA 100, a slight increase in the number of revertant counts was observed at the upper concentrations, followed by a reduction in the number of revertant colonies at the highest concentration. In the original experiment carried out without metabolic activation, treatment of strain TA 102 with the test substance led to a slight increase in the number of revertant counts at 185.2 to 1666.7 µg active ingredient/plate. In the original experiment with activation, a similar effect occurred on strain TA 100 at 1666.7 µg/plate and on strain TA 102 at 185.2 and 1666.7 µg active ingredient/plate. No effects were observed with the other strains. In the confirmatory experiment carried out without metabolic activation, treatment led to a slight increase in the number of revertant counts at 1666.7 µg active ingredient/plate. In the confirmatory experiment with activation, a similar effect occurred on strains TA 100 and TA 102 at the same concentration. Again, no effects were observed with the other strains. Overall, the test substance exerted only a marginal mutagenic action on strains S. typhimurium TA 100 and TA 102.