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Genetic toxicity: in vitro

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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 guidelineopen allclose all
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)
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)
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

Constituent 1
Chemical structure
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.
Controlsopen allclose all
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)
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)
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)
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)
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)
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 resultsopen allclose all
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:
not specified
Positive controls validity:
valid
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:
not specified
Positive controls validity:
valid
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:
not specified
Positive controls validity:
valid
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:
not specified
Positive controls validity:
valid
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:
not specified
Positive controls validity:
valid
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:
not specified
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.