Reaction mass of Cobaltate(3-), bis[2-[[[3-[2-[1-[[(2-chlorophenyl)amino]carbonyl]-2-(oxo-κO)propyl]diazenyl-κN1]-4-(hydroxy-κO)phenyl]sulfonyl]amino]benzoato(3-)]-, sodium (1:3) and tetrasodium bis[2-[[[3-[[1-[(2-chloroanilino)carbonyl]-2-oxopropyl]azo]-4-hydroxyphenyl]sulphonyl]amino]benzoato(3-)]cobaltate(4-)

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:

From 29 April, 1993 to 20 July, 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:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

May 26, 1983

Deviations:

yes

Remarks:

(statistical analysis was not performed. However at that time it was not recommended)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

September 19, 1984

Deviations:

yes

Remarks:

(statistical analysis was not performed. However at that time it was not recommended)

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 13, 1987

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Trisodium bis[2-({[3-({1-[(2-chlorophenyl)amino]-3-(hydroxy-kO)-1-oxobut-2-en-2-yl}diazenyl-kN1)-4-(hydroxy-kO)phenyl]sulfonyl}amino)benzoato(3-)]cobaltate(3-) and Trisodium [2-({[3-({1-[(2-chlorophenyl)amino]-3-(hydroxy-kO)-1-oxobut-2-en-2-yl}diazenyl-kN1)-4-(hydroxy-kO)phenyl]sulfonyl}amino)benzoato(3-)][2-({[3-({1-[(2-chlorophenyl)amino]-3-(hydroxy-kO)-1-oxobut-2-en-2-yl}diazenyl-kN2)-4-(hydroxy-kO)phenyl]sulfonyl}amino)benzoato(3-)]cobaltate(3-)

EC Number:

941-792-6

Cas Number:

not given

Molecular formula:

C46H32Cl2CoN8O14S2.3Na

IUPAC Name:

Reaction mass of Trisodium bis[2-({[3-({1-[(2-chlorophenyl)amino]-3-(hydroxy-kO)-1-oxobut-2-en-2-yl}diazenyl-kN1)-4-(hydroxy-kO)phenyl]sulfonyl}amino)benzoato(3-)]cobaltate(3-) and Trisodium [2-({[3-({1-[(2-chlorophenyl)amino]-3-(hydroxy-kO)-1-oxobut-2-en-2-yl}diazenyl-kN1)-4-(hydroxy-kO)phenyl]sulfonyl}amino)benzoato(3-)][2-({[3-({1-[(2-chlorophenyl)amino]-3-(hydroxy-kO)-1-oxobut-2-en-2-yl}diazenyl-kN2)-4-(hydroxy-kO)phenyl]sulfonyl}amino)benzoato(3-)]cobaltate(3-)

Test material form:

solid

Specific details on test material used for the study:

Test material: FAT 92368/A
Batch No.: PLN 13
Purity: 80 %
Expiration date: March 1998
Vehicle: Bidistilled water

Method

Target gene:

Histidine gene

Species / strain

Species / strain / cell type:

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

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 – 5000 µg/plate
Mutagenicity test: 61.7 – 5000 µg/plate

Vehicle / solvent:

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

Controls

Controls 1

Untreated negative controls:

other: (same as solvent control)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

(without metabolic activation for TA 100 and 1535)

Controls 2

Untreated negative controls:

other: (same as solvent control)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

(without metabolic activation for TA 98)

Controls 3

Untreated negative controls:

other: (same as solvent control)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

(without metabolic activation for TA 1537)

Controls 4

Untreated negative controls:

other: same as solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

(Without metabolic activation with strain TA 102)

Controls 5

Untreated negative controls:

other: (same as solvent control)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

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

Controls 6

Untreated negative controls:

other: (same as solvent control)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

(with metabolic activation for TA 1535)

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 L-histidine and 0.5 mM (+)biotin dissolved in water.

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 judgment 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:

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

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 2

Species / strain:

S. typhimurium TA 98

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 3

Species / strain:

S. typhimurium TA 100

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 1537

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 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

positive

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 6

Species / strain:

S. typhimurium TA 102

Metabolic activation:

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

Additional information on results:

Mutagenicity test, original experiment
In the original experiment carried out without metabolic activation, none of the tested concentrations of FAT 92368/A led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. In the experiment with activation performed on strain TA 102 a weak increase in the number of revertant counts was observed at the upper concentrations. No effects were observed with the other strains.

Mutagenicity test, confirmatory experiment
In the confirmatory experiment carried out without metabolic activation, again none of the tested concentrations of FAT 92368/A led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. In the experiment with activation performed on strain TA 102, a marginal increase in the number of revertant counts was observed at a single concentration only. No effects were observed with the other strains. In the mutagenicity tests without and with metabolic activation performed on strains TA 100, TA 102, TA 98 and TA 1537, due to a growth-inhibiting effect of the test material, a reduction in the number of revertant colonies was occasionally observed at the highest concentration.

Remarks on result:

other: all strains/cell types tested were negative

Any other information on results incl. tables

Toxicity test/range finding test:

In the experiment without and with metabolic activation due to an inhibiting effect of the test material on the growth of the bacteria, a slight reduction in the number of revertant colonies was observed at the highest concentration.

Applicant's summary and conclusion

Conclusions:

FAT 92368/A was mutagenic in this reverse mutation assay when tested with S. typhimurium strain TA 102 with metabolic activation.

Executive summary:

An in vitro study was performed to investigate the potential of FAT 92368/A (of ca. 80 % purity) to induce gene mutations according to OECD Guideline 471 and EU Method B.14 in compliance with GLP. The concentration range for the mutagenicity assay was determined in a preliminary test. The substance was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 61.7 to 5000 µg/plate. In order to confirm the results, the experiment was repeated twice. In preliminary toxicity test, In the experiment without and with metabolic activation due to an inhibiting effect of the test material on the growth of the bacteria, a slight reduction in the number of revertant colonies was observed at the highest concentration (5000 µg/plate). In the original as well as confirmatory experiment carried out without metabolic activation, none of the tested concentrations of FAT 92368/A led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. In the experiment with activation performed on strain TA 102 a weak increase in the number of revertant counts was observed at the upper concentrations. No effects were observed with the other strains. In the mutagenicity tests without and with metabolic activation performed on strains TA 100, TA 102, TA 98 and TA 1537, due to a growth-inhibiting effect of the test material, a reduction in the number of revertant colonies was occasionally observed at the highest concentration. Based on the above findings, it can be concluded that FAT 92368/A was mutagenic in this reverse mutation assay when tested with S. typhimurium strain TA 102 with metabolic activation.