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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 April 1994 to 12 August 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Reaction mass of Disodium [3-{[4-chloro-2-(hydroxy-kO)-5-methoxyphenyl]diazenyl-kN2}-4-(hydroxy-kO)naphthalene-2,7-disulfonato(4-)](hydroxy)chromate(2-) and Trisodium [3-{[4-chloro-2-(hydroxy-kO)-5-methoxyphenyl]diazenyl-kN2}-4-(hydroxy-kO)naphthalene-2,7-disulfonato(4-)](dihydroxy)chromate(3-)
Molecular formula:
C17H10ClCrN2Na2O10S2.C17H11ClCrN2Na3O11S2
IUPAC Name:
Reaction mass of Disodium [3-{[4-chloro-2-(hydroxy-kO)-5-methoxyphenyl]diazenyl-kN2}-4-(hydroxy-kO)naphthalene-2,7-disulfonato(4-)](hydroxy)chromate(2-) and Trisodium [3-{[4-chloro-2-(hydroxy-kO)-5-methoxyphenyl]diazenyl-kN2}-4-(hydroxy-kO)naphthalene-2,7-disulfonato(4-)](dihydroxy)chromate(3-)
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Test material: FAT 20044/B (Neolan Blau 3R roh trocken SFO)
Batch No.: 96
Purity: about 80 %
Stability: July 1999
Appearance: Blue mass
Expiry date: July 1999
Storage: Room temperature

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation system:
With and without metabolic activation
Test concentrations with justification for top dose:
Range finding test: ranging from 20.6 to 5000 µg/plate
From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate with and without metabolic activation.
Main study: 61.7 to 5000 µg/plate
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
with metabolic activation: TA 98, TA100, TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation: TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation: TA100, TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation: TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation: TA 1537
Details on test system and experimental conditions:
The histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 1535, TA 1537) were obtained from Prof. B. Ames, Berkeley, USA. Strain TA 100 was obtained from Dr. M. Schüpbach, Hoffmann-La Roche Limited, Basel, Switzerland.
Evaluation criteria:
Assay acceptance criteria
A test is considered acceptable if the mean colony counts of the negative 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 judgement of the Study Director.

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:
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.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 1535 and TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 100, TA 1535 and TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Four salmonella strains were used

Any other information on results incl. tables

Range finding test


Six concentrations of FAT 20044/B (Neolan Blau 3R roh trocken SFO) ranging from 20.6 to 5000.0 ug/plate were tested with strain Salmonella typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without metabolic activation. Normal background growth was observed. The numbers of revertant colonies were not reduced. From the results obtained the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate with and without metabolic activation.


 


Mutagenicity test, original experiment


In the experiment performed without metabolic activation, treatment of strain TA 98 with FAT 20044/B (Neolan Blau 3R roh trocken SFO) led to a slight increase in the number of histidineprototrophic mutants at the concentration of 5000 µg/plate. No effect was seen in the experiment with activation conducted on this strain and in the experiments with and without activation conducted on strains TA 100, TA 1535 and TA 1537.


 


Mutagenicity test, confirmatory experiment


In the experiment performed without metabolic activation, again, treatment of strain TA 98 with FAT 20044/B (Neolan Blau 3R roh trocken SFO) led to a slight increase in the number of revertant counts at the concentration of 5000.0 µg/plate. No effects occurred in the experiment with activation conducted on this strain and in the experiments with and without activation conducted on strains TA 100, TA 1535 and TA 1537. In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced with increasing concentration. Therefore, the test substance exerted no toxic effect on the growth of the bacteria. There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the test data.


 


SUMMARY OF THE MUTAGENICITY EXPERIMENTS: ORIGINAL EXPERIMENT with metabolic activation


 






























































































































































































Strain



Treatment



Mean Counts



Strain



Treatment



Mean Counts



TA 100



Negative control



121.67



TA 1535



Negative control



11.33



 



61.73 µg/plate



145.00



 



61.73 µg/plate



16.67



 



185.19 µg/plate



143.67



 



185.19 µg/plate



8.33



 



555.56 µg/plate



147.00



 



555.56 µg/plate



12.67



 



1666.67 µg/plate



147.00



 



1666.67 µg/plate



10.67



 



5000.00 µg/plate



158.33



 



5000.00 µg/plate



11.67



 



Positive Control



1893.67



 



Positive Control



358.00



 



 



 



 



 



 



 



 



 



TA 98



Negative control



27.67



 



 



 



 



61.73 µg/plate



33.33



 



 



 



 



185.19 µg/plate



32.0



 



 



 



 



555.56 µg/plate



35.33



 



 



 



 



1666.67 µg/plate



38.67



 



 



 



 



5000.00 µg/plate



39.33



 



 



 



 



Positive Control



1972.33



TA1537



Negative control



7.67



 



 



 



 



61.73 µg/plate



5.33



 



 



 



 



185.19 µg/plate



7.33



 



 



 



 



555.56 µg/plate



9.67



 



 



 



 



1666.67 µg/plate



9.33



 



 



 



 



5000.00 µg/plate



9.33



 



 



 



 



Positive Control



143.33



 



 



 



 


 


SUMMARY OF THE MUTAGENICITY EXPERIMENTS: ORIGINAL EXPERIMENT without metabolic activation


 






























































































































































































Strain



Treatment



Mean Counts



Strain



Treatment



Mean Counts



TA 100



Negative control



126.33



TA 1535



Negative control



14.00



 



61.73 µg/plate



126.67



 



61.73 µg/plate



8.33



 



185.19 µg/plate



136.67



 



185.19 µg/plate



12.00



 



555.56 µg/plate



121.67



 



555.56 µg/plate



12.00



 



1666.67 µg/plate



142.00



 



1666.67 µg/plate



11.33



 



5000.00 µg/plate



158.67



 



5000.00 µg/plate



10.00



 



Positive Control



1292.67



 



Positive Control



1015.00



 



 



 



 



 



 



 



 



 



TA 98



Negative control



16.00



 



 



 



 



61.73 µg/plate



17.33



 



 



 



 



185.19 µg/plate



16.00



 



 



 



 



555.56 µg/plate



21.33



 



 



 



 



1666.67 µg/plate



29.33



 



 



 



 



5000.00 µg/plate



51.00



 



 



 



 



Positive Control



1750.33



TA1537



Negative control



9.67



 



 



 



 



61.73 µg/plate



5.00



 



 



 



 



185.19 µg/plate



8.00



 



 



 



 



555.56 µg/plate



9.33



 



 



 



 



1666.67 µg/plate



12.00



 



 



 



 



5000.00 µg/plate



11.00



 



 



 



 



Positive Control



2050.33



 



 



 



 


 


SUMMARY OF THE MUTAGENICITY EXPERIMENTS: CONFIRMATORY EXPERIMENT with metabolic activation


 






























































































































































































Strain



Treatment



Mean Counts



Strain



Treatment



Mean Counts



TA 100



Negative control



115.67



TA 1535



Negative control



8.00



 



61.73 µg/plate



121.00



 



61.73 µg/plate



10.33



 



185.19 µg/plate



111.67



 



185.19 µg/plate



10.33



 



555.56 µg/plate



128.33



 



555.56 µg/plate



10.67



 



1666.67 µg/plate



138.67



 



1666.67 µg/plate



10.67



 



5000.00 µg/plate



158.67



 



5000.00 µg/plate



9.00



 



Positive Control



1758.00



 



Positive Control



357.00



 



 



 



 



 



 



 



 



 



TA 98



Negative control



33.00



 



 



 



 



61.73 µg/plate



30.00



 



 



 



 



185.19 µg/plate



39.33



 



 



 



 



555.56 µg/plate



36.33



 



 



 



 



1666.67 µg/plate



34.00



 



 



 



 



5000.00 µg/plate



41.00



 



 



 



 



Positive Control



1743.33



TA1537



Negative control



9.33



 



 



 



 



61.73 µg/plate



5.33



 



 



 



 



185.19 µg/plate



7.00



 



 



 



 



555.56 µg/plate



10.33



 



 



 



 



1666.67 µg/plate



8.67



 



 



 



 



5000.00 µg/plate



16.33



 



 



 



 



Positive Control



176.33



 



 



 



 


 


SUMMARY OF THE MUTAGENICITY EXPERIMENTS: CONFIRMATORY EXPERIMENT without metabolic activation


 






























































































































































































Strain



Treatment



Mean Counts



Strain



Treatment



Mean Counts



TA 100



Negative control



120.33



TA 1535



Negative control



8.67



 



61.73 µg/plate



108.33



 



61.73 µg/plate



11.33



 



185.19 µg/plate



113.00



 



185.19 µg/plate



11.33



 



555.56 µg/plate



124.00



 



555.56 µg/plate



9.33



 



1666.67 µg/plate



139.00



 



1666.67 µg/plate



9.67



 



5000.00 µg/plate



172.00



 



5000.00 µg/plate



11.67



 



Positive Control



1260.33



 



Positive Control



1137.00



 



 



 



 



 



 



 



 



 



TA 98



Negative control



18.33



 



 



 



 



61.73 µg/plate



18.67



 



 



 



 



185.19 µg/plate



15.67



 



 



 



 



555.56 µg/plate



16.00



 



 



 



 



1666.67 µg/plate



24.67



 



 



 



 



5000.00 µg/plate



51.67



 



 



 



 



Positive Control



1792.67



TA1537



Negative control



8.33



 



 



 



 



61.73 µg/plate



9.33



 



 



 



 



185.19 µg/plate



7.00



 



 



 



 



555.56 µg/plate



9.33



 



 



 



 



1666.67 µg/plate



12.67



 



 



 



 



5000.00 µg/plate



13.67



 



 



 



 



Positive Control



2029.67



 



 



 



 


 

Applicant's summary and conclusion

Conclusions:
In the experiments performed without metabolic activation, treatment of strain TA 98 with FAT 20044/B led to a slight increase in the number of back-mutant colonies at the highest concentration only. No effect occurred in the experiments with activation conducted on this strain and on the other strains.
Executive summary:

In a GLP-compliant study, FAT 20044/B, was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium according to OECD guideline 471. The strains of Salmonella typhimurium used were TA 98, TA 100, 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 bi-distilled water and tested at five concentrations in the range of 61.7 to 5000.0 µg/plate. In order to confirm the results, the experiments were repeated with and without metabolic activation in the same concentration range. 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 the experiments performed without metabolic activation, treatment of strain TA 98 with FAT 20044/B led to a slight increase in the number of back-mutant colonies at the highest concentration only. No effect occurred in the experiments with activation conducted on this strain and on the other strains.