Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-5-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 mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EEC Directive 87/302, L 133, p. 61 - 63

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-5-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:

260-394-7

EC Name:

Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-5-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:

56819-40-0

Molecular formula:

C32H21CrN10O11S.2Na

IUPAC Name:

Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-5-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

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

None

Specific details on test material used for the study:

Name: FAT 20'010/C Lanacron Orange S-2R crude dry
Batch No.: 149
Aggregate State at RT: Solid
Colour: orange
Purity: 78.83 % (active ingredient)
Stability: Pure: stable until December, 1999
Storage: room temperature
Expiration Date: December 1999

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University; D-64293 Darmstadt; F.R.G.) are stored in liquid nitrogen in the cell bank of CCR allowing the repeated use of the same cell culture batch in experimen

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 1, 10, 20, 25, 30, and 40 µg/mL
with S9 mix: 3, 10, 30, 50, 70 and 90 µg/ml

Experiment II:
without S9 mix: 1, 3, 10, 12, 15 and 18 µg/ml
with S9 mix: 3, 10, 30, 50, 70 and 90 µg/ml

Experiment III:
without S9 mix: 12 and 15 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:The solvent was chosen according to its solubility properties and its non-toxicity for the cells.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

nutrient medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With metabolic activation

Evaluation criteria:

A test article is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response for one of the test points. A test article producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system. A significant response is described as follows: The test article is classified as mutagenic if it induces a reproducible mutation frequency that is at least three times higher than the spontaneous mutation frequency in the experiment at one or more of the concentrations.
The test article is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the correspon-ding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0 - 45 mutants per 10E6 cells) a concentration-related increase of the mutations within this range has to be discussed.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Applicant's summary and conclusion

Conclusions:

FAT 20010/C did not induce gene mutations at the HPRT locus in V79 cells.

Executive summary:

A study was performed to investigate the potential of FAT 20010/C to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. This test was performed according to OECD Guideline for Testing of Chemicals, Section 4, No. 476, adopted April 4, 1984, "ln vitro Mammalian Cell Gene Mutation Tests" and EEC Directive 87/302, L 133, p. 61 - 63

The study was performed in two independent main experiments, using identical procedures, both with and without liver microsomal activation.

Experiment I: without S9 mix: 1.0; 10.0; 20.0, 25.0; 30,0 and 40.0 µg/ml

with S9 mix: 3.0; 10.0; 30.0; 50.0; 70.0 and 90.0 µg/ml

Experiment II: without S9 mix: 1.0; 3.0; 10.0; 12,0; 15.0 and 18.0 µg/ml

with S9 mix: 3.0; 10.0; 30.0; 50.0; 70.0 and 90.0 µg/ml

Experiment III: without S9 mix: 12.0 and 15.0 µg/ml

Without metabolic activation strong toxic effects occurred already at concentrations as low as 20 μg/ml. With metabolic activation, the concentration of the test article could be increased up to 90 μg/ml. Although the absolute numbers of colonies in experiment I with metabolic activation indicate no relevant toxicity the colonies per se showed distinct toxic effects at concentrations of 50 μg/ml or above and barely exceeded the limit of 50 cells per colony. In the second experiment these colonies remained almost invisible and clearly did not reach the limit of 50 cells per colony. The cloning efficiency of the cells was reduced below 30 % at the highest concentrations tested. In the first experiment the number of mutant colonies was not substancially increased compared to the frequency of spontaneous mutations at any concentration of the test article. The enhancement factor of three was not reached. There was also no indication of a concentration depend increase of mutant colonies. In the second and third experiments the enhancement factor of three is exceeded in the absence of metabolic activation at almost all concentrations of the test article. This effect however, is due to the rather low values of the solvent controls and does not reflect a true mutagenic effect. The absolute numbers of colonies are well within the historical range of negative controls. In all experiments of this study (with and without S9 mix) the range of the negative controls was from 1.4 up to 16.3 mutants per 10⁶ cells; the range of the groups treated with the test article was from 2.0 up to 26.1 mutants per 10⁶ cells. EMS (0.6 mg/ml) and DMBA (3.85 µg/ml) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion, it can be stated that in this mutagenicity assay and under the experimental conditions reported the test article did not induce gene mutations at the HPRT locus in V79 cells.