Reaction mass of Sodium 2-(2 or 3-{[(chloroacetyl)amino]methyl}-4-{[4-(cyclohexylamino)-9,10-dioxo-9,10-dihydroanthracen-1-yl]amino}phenoxy)-5-methylbenzenesulfonate and Sodium 2-(3 or 2-{[(chloroacetyl)amino]methyl}-4-{[4-(cyclohexylamino)-9,10-dioxo-9,10-dihydroanthracen-1-yl]amino}phenoxy)-5-methylbenzenesulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A GLP-compliant study was performed to determine the mutagenicity of FAT 20077/C with purity of about65.6%in Salmonella typhimurium according to OECD Guideline 471 (Bacterial Reverse Mutation Assay).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 inDimethylsulfoxide (suspension)and tested at five concentrations in the range of 94.1to 7622.0µg/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 of61.7 to 5000.0µg/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 20077/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 20077/C and its metabolites did not induce any gene mutation in the strains of Salmonella typhimurium.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 25, 1993 to May 20, 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:

Test material: FAT 20077/C (Irganol Brillantblau 7GS roh trocken)
Batch No.: 312901.36
Purity: 65.6 %
Stability: August 1998
Appearance: Dark blue powder
Expiry date: August 1998
Storage: Room temperature

Target gene:

Histidine gene

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 100, TA 102, TA 1535 and TA 1537) were obtained from Prof. B. Ames, Berkeley, CA., U.S.A. Inoculates from frozen master copies were set up monthly. They were grown in liquid NB-medium overnight and then plated on NB-agar. After incubation, single colonies were taken from the plates, grown overnight in liquid NB-medium and then used for the experiment. The characteristics of the strains were checked monthly. Histidine-auxotrophy of the strains was demonstrated by the requirement for 1-histidine. The presence of the rfa character was assayed by the sensitivity for crystal-violet. The deletion of the uvrB gene (strains TA 98, TA 100, TA 1535 and TA 1537) was demonstrated by the sensitivity for UV-light. The Salmonella strains containing the R-factor (TA 98 and TA 100) were additionally checked for ampicillin resistance. Strain TA 102 additionally was checked for tetracycline resistance (presence of multicopy plasmid pAQ1). The presence of the uvr+ gene was demonstrated by the resistance of strain TA 102 against UV light. Furthermore, all strains were checked for their characteristic reversion properties with known mutagens (positive controls).

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:

Cytotoxicity test: 20.6 to 5000 µg active ingredient/plate
Mutagenicity tests: 61.7, 185.2, 555.55, 1666.7 and 5000 µg active ingredient/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (Suspension), bidistilled water
- Justification for choice of solvent/vehicle: Based on solubility

Untreated negative controls:

yes

Remarks:

same as solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cyclophosphamide

mitomycin C

other: 2-Aminoanthracene

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.

Criteria for a positive response
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:

In deviation to the OECD guideline, a statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity test/Range finding test:
Normal back-ground growth was observed at all concentrations. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the first mutagenicity test was selected to be 7622.0 ug/plate with and without metabolic activation. (The purity of the tested batch is 65.6 %. 7622.0 µg/plate correspond to 5000 µg/plate of pure substance). Mutagenicity test, original experiment: Since the purity of the test material is 65.6 %, in this experimental part the concentration range of 94.1 to 7622.0 µg/plate was used. In the experiments performed with and without metabolic activation, treatment of strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 with FAT 20077/C did not lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the negative control. Mutagenicity test, confirmatory experiment: In the experiments performed with and without metabolic activation (concentration range 61.7 to 5000.0 ug/plate) after treatment of strains TA 100, TA 102, TA 1535 and TA 1537 with FAT20077/C no increase in the incidence of either histidine-prototrophic mutants was observed incomparison with the negative control. In the mutagenicity tests normal background growth was observed with all strains at all concentrations. In the confirmatory experiment without activation on strains TA 102 and TA 1537, the numbers of revertant colonies were slightly reduced at the highest concentration. The test substance exerted a weak toxic effect on the growth of these strains.

Conclusions:

FAT 20077/C is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

A GLP compliant study was performed to determine the mutagenicity of FAT 20077/C with purity of about 65.6 % in Salmonella typhimurium according to OECD Guideline 471 (Bacterial Reverse Mutation Assay). 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 Dimethylsulfoxide (Suspension) and tested at five concentrations in the range of 94.1 to 7622 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 20077/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 20077/C and its metabolites did not induce any gene mutation in the strains of Salmonella typhimurium.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

FAT 20077/C is not found to have mutagenic potential in the bacterial reverse mutation assaym, hence it does not warrant classification of mutagenicity according to the criteria of Regulation (EC) No. 1272/2008.