Sodium bis[N-(2-chlorophenyl)-2-[[2-hydroxy-5-(N-methylsulphamoyl)phenyl]azo]-3-oxobutyramidato(2-)]cobaltate(1-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Test substance is considered to be non-mutagenic in Salmonella typhimurium reverse mutation assay.

Link to relevant study records

Reference

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:

23 November 1993 to 22 April 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 140200.31
- Expiration date of the lot/batch: October 1998

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Target gene:

None

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

None

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver post mitochondrial supernatant (S9 fraction)

Test concentrations with justification for top dose:

Concentration range in the range finding test: 20.6 to 5000.0 µg/plate
Concentration ranges in the mutagenicity tests:
Original experiment
61.7 to 5000.0 µg/plate
Confirmatory experiment
61.7 to 5000.0 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:Dimethylsulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

TA 100, TA 102, TA 98, TA 1537; with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Bidistilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

TA 1535; wtih metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Bidistilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100, TA 1535; without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Bidistilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

TA 102; without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98; without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537; without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Preliminary range finding test:
A range finding test was carried out with strain TA 100 with and without metabolic activation at six concentrations of the test substance and one negative control according to a Standard Operating Procedure of Genetic Toxicology. The highest concentration applied was 5000 ug/plate. The five lower concentrations decreased by a factor of three. The plates were inverted and incubated for about 48 hours at 37±1.5 °C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration and negative control was used.

Mutagenicity test:
The mutagenicity test was performed with the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535, TA 1537 with and without metabolic activation according to a Standard Operating Procedure of Genetic Toxicology. Each of the five concentrations of the test substance, a negative and a positive control were tested, using three plates per test substance concentration and controls. The highest concentration applied was determined in the preliminary range finding test and the four lower concentrations decreased by a factor of three. The plates were inverted and incubated for about 48 hours at 37± 1.5 °C in darkness. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.

Colony counting and scoring of the plates:
Colonies were counted electronically using an Artek Colony Counter (Fisher Scientific), or manually where minor agar damage or test chemical precipitates might have interfered with automating counting. The results were sent on line to a computer. They were checked on a random basis by the operator. Observations indicating precipitates of the test substance in the top agar or a reduced or absent bacterial background lawn were registered additionally. Means for all mutagenicity assays were calculated and included in the Results section.

Rationale for test conditions:

This test permits the detection of gene mutations induced by the test material or its metabolites in histidine-requiring strains of Salmonella typhimurium.

When the Salmonella strains are exposed to a mutagen, some of the bacteria in the treated population, through chemical interaction with the compound or its metabolites, undergo genetic changes which cause them to revert to a non-histidine-requiring state and thus to grow in the absence of exogenous histidine. Mutagenic effects of the test substance are demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone reverse-mutation to histidine prototrophism. Different tester strains are used because of differing sensitivities to known mutagens.

Evaluation criteria:

The test substance will be considered to be positive in the test system if the following condition is 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 strains tested.
Generally a concentration-related effect should be demonstrable.

Statistics:

A statistical analysis of the test data was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended.

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

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

Species / strain:

S. typhimurium TA 98

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

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

Species / strain:

S. typhimurium TA 102

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

Additional information on results:

None

Remarks on result:

other: all strains/cell types tested

Conclusions:

FAT 20035/C and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium used.

Executive summary:

FAT 20035/C was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium. The following strains of Salmonella typhimurium were used: TA 98, TA 100, TA 102, TA 1535 and TA 1537. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was dissolved in DMSO and tested at five concentrations in the range of 61.7 to 5000.0 µg/plate in the presence and absence of a metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at five concentrations in the range of 61.7 to 5000 µg/plate. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control. In both experiments, performed with and without metabolic activation, none of the tested concentrations of FAT 20035/C led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. Based on the results of these experiments and on standard evaluation criteria, it is concluded that FAT 20035/C and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro:

FAT 20035/C was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium according to OECD 471 guideline. The following strains of Salmonella typhimurium were used: TA 98, TA 100, TA 102, TA 1535 and TA 1537. The compound was dissolved in DMSO and tested at five concentrations in the range of 61.7 to 5000 ug/plate in the presence and absence of a metabolic activation system. In the experimental setup performed with and without metabolic activation, none of the tested concentrations of FAT 20035/C led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control.

Another GLP-compliant study was performed to investigate the potential of FAT 20035/D to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The study was carried out according to OECD guideline 471. The test article was tested at the following concentrations: 33.3; 100; 333.3; 1000; 2500; and 5000 µg/plate. Slight toxic effects, evident as a reduction in the number of revertants, occurred in strains TA 1535 and TA 1537 without metabolic activation (S9 mix) in the first experiment. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No relevant increases in revertant colony numbers of any of the four tester strains were observed following treatment with FAT 20035/D at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, FAT 20035/D is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

Based on the results of these two experiments and on standard evaluation criteria, it is concluded that FAT 20035 and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium used.

Justification for classification or non-classification

Based on the findings of the genetic toxicity studies, the test substance does not considered to be classified as genotoxic according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.