Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-, disodium

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:

no data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only four strains were tested, no tester strain to detect cross-linking mutagens was included

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

only four strains were tested, no tester strain to detect cross-linking mutagens was included

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-, disodium

EC Number:

274-386-6

EC Name:

Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-, disodium

Cas Number:

70209-87-9

Molecular formula:

C32H21CrN10Na2O11S

IUPAC Name:

Disodium-[2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-chromate(2-)

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): FAT 20'060/E LANACRON ROT S-G ROH TROCKEN
- Physical state: Solid
- Analytical purity: ca. 80 %
- Lot/batch No.: 252
- Expiration date of the lot/batch: July 1999
- Stability under test conditions: Pure- stable until July 1999
- Storage condition of test material: Room temperature

Method

Target gene:

Histidine gene

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The strains are derived from S. typhimurium strain LT2 and due to a mutation in the histidine locus are histidine dependent. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98, TA 100 and TA 102 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. Regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in CCR according to Ames et al. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

33.3, 100, 333.3, 1000, 2500 and 5,000 µg active ingredient/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Aqua bidest
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

(for TA 1535 and TA 100 without metabolic activation)

Controls 2

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

(for TA 1537 and TA 98 without metabolic activation)

Controls 3

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

(for all strains with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test (experiment I) and the pre-incubation test (experiment II). For each strain and dose level, including the controls, a minimum of three plates were used.

Experiment 1: The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 µL: Test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control),
- 500 µL: S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation),
- 100 µL: Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 µL: Overlay agar

Experiment 2: In the pre-incubation assay 100 µL test solution, 500 µL S9 mix /S9 mix substitution buffer and 100 µL bacteria suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 h at 37 °C in the dark.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are corresponding background growth on both negative control and test plates as well as normal range of spontaneous reversion rates. Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time. A test substance is considered as positive if either a dose related or reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced. A test substance producing neither a dose related and reproducible 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 significant response is described as follows:
A test substance is considered mutagenic if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 at least three times higher as compared to the spontaneous reversion rate. Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test substance regardless of whether the highest dose induced the above-described enhancement factors or not.

Statistics:

No appropriate statistical method is available.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

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 98

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

(only in pre-incubation test)

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

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

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 102

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

Toxic effects, evidenced by a reduction in the number of revertants, occurred in strain TA 1535 at 1000, 2500 and 5000 µg/plate with S9 mix in experiment I as well as at 2500 (with S9 mix) and 5000 µg/plate (with and without S9 mix) in experiment II. The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.
Up to the highest dose a significant dose-dependent increase in revertant colony numbers was obtained in tester strain TA 1537 in the presence of metabolic activation in experiment I. In strain TA 98 in experiment I higher values in revertant colony numbers were obtained at 2500 and 5000 µg/plate without S9 mix. Since the obtained effects were less distinct, experiment II was carried out as a pre-incubation assay in order to obtain results from a normally more sensitive assay. In the pre-incubation assay a significant increase in revertant colony numbers was obtained up to 5000 µg/plate with metabolic activation with a higher mutation factor at the highest investigated dose. In tester strain TA 98 a clear dose-dependent increase in revertant colonies was observed up to 5000 µg/plate without S9 mix in experiment II. The results obtained in both independent experiments indicate a mutagenic potential of the test substance in the tester strains TA 1537 and TA 98.

Any other information on results incl. tables

none

Applicant's summary and conclusion

Conclusions:

The test substance is considered to be mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

An in vitro study was performed to investigate the potential of the test substance (at ca. 80 % purity) to induce gene mutations according to OECD Guideline 471 and EU Method B.14 in compliance with GLP with deviation (only four strains were tested). The assay was performed in two independent experiments both with and without liver microsomal activation. Experiment I was performed as a plate incorporation assay and Experiment II as a pre-incubation assay using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. Each concentration, including the controls, was tested in triplicate. The substance was tested up to 5000 µg/plate. Toxic effects, evidenced by a reduction in the number of revertants, occurred in strain TA 1535 at 1000, 2500 and 5000 µg/plate with S9 mix in Experiment I, as well as at 2500 (with S9 mix) and 5000 µg/plate (with and without S9 mix) in Experiment II. The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. A significant dose-dependent increase in revertant colony numbers was observed in strain TA 1537 with S9 mix in Experiments I and II and in strain TA 98 without S9 mix in Experiment II. Based on the findings of the study, it was concluded that the test substance induced gene mutations by frameshifts in the genome of strains TA 1537 and TA 98. Therefore, it is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.