Trisodium 5-hydroxy-1-(4-sulphophenyl)-4-(4-sulphophenylazo)pyrazole-3-carboxylate

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:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment. GLP compliant.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium 5-hydroxy-1-(4-sulphophenyl)-4-(4-sulphophenylazo)pyrazole-3-carboxylate

EC Number:

217-699-5

EC Name:

Trisodium 5-hydroxy-1-(4-sulphophenyl)-4-(4-sulphophenylazo)pyrazole-3-carboxylate

Cas Number:

1934-21-0

Molecular formula:

C16H12N4O9S2.3Na

IUPAC Name:

trisodium 5-hydroxy-1-(4-sulphophenyl)-4-(4-sulphophenylazo)pyrazole-3-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

No data

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA102

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S-9 mix

Test concentrations with justification for top dose:

312.5; 625; 1250; 2500; and 5000.0 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: water for injections (Batch EVBO3A).- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

1µg/plate: TA1535 and TA100 without S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

50 µg/plate: TA1537 without S9

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

0.5 µg/plate: TA98 without S9

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.5 µg/plate: TA102 without S9

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

2 µg/plate: Escerichia coli WP2 uvra without S9

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: a-anthramine

Remarks:

2 µg/plate: TA1535, TA1537, TA98 and TA100 with S9

Controls 7

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-anthramine

Remarks:

10 µg/plate: TA102 and E.C. WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)NUMBER OF REPLICATIONS: The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.DETERMINATION OF CYTOTOXICITY: Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:- corresponding background growth on both negative control and test plates- normal range of spontaneous reversion rates.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.A test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, 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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

2.3.1 TreatmentThe test item was tested in a preliminary test and two mutagenicity experiments. The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method.The second experiment with S9 mix was performed according to the preincubation method.The direct plate incorporation method was performed as follows: test item solution (0.1 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.The preincubation method ^'^' ^^ was performed as follows: test item solution (0.1 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C under shaking before adding the overlay agar and pouring onto the surface of a minimum agar plate.After 48 to 72 hours of incubation at 37°C, revertants were scored with an automatic counter (Cardinal counter. Perceptive Instruments, Suffolk CB9 7 BN, UK).2.3.2 Preliminary toxicity testTo assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100, TA 102 and WP2 uvrA strains, with and witiiout S9 mix. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.2.3.3 Mutagenicity experimentsIn two independent experiments, using three plates/dose-level, each strain was tested, with and without S9 mix, with:- at least five dose-levels of the test item,- the vehicle control,- the appropriate positive control.The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.

Applicant's summary and conclusion

Conclusions:

Not mutagenic

Executive summary:

Under the experimental conditions, the test item did not show mutagenicity activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia Coli. Acid Yellow 23 could be considered not mutagenic.