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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Trisodium 1-amino-4-[[3-[[4-chloro-6-[(sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2,4,6-trimethyl-5-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate
EC Number:
276-481-8
EC Name:
Trisodium 1-amino-4-[[3-[[4-chloro-6-[(sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2,4,6-trimethyl-5-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate
Cas Number:
72214-18-7
Molecular formula:
C32H26ClN7O11S3.3Na
IUPAC Name:
trisodium 1-amino-4-[[3-[[4-chloro-6-[(sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2,4,6-trimethyl-5-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate
Test material form:
not specified
Details on test material:
None
Specific details on test material used for the study:
Test No.: 930844
Test material: Cibacron Blau P-3R roh Lösung für Flüssighandelsform (FAT 41001/F)
Batch No.: Op.Nr.2 2 eingedampft
Purity: about 54 % (active ingredient)
Stability of the test material: see Expiration date
Expiration date: June 1997
Vehicle: Bidistilled water
Study initiation date: September 13, 1993
Experimental start date: September 21, 1993
Experimental termination date: November 15, 1993
Study termination date: December 15, 1993

Method

Target gene:
histidine-requiring strains of Salmonella typhimurium
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
TA 98, TA 102, TA 1535 and TA 1537 - Prof. B. Ames, Berkeley, CA., U.S.A
TA 100 - M. Schüpbach, Hoffmann-La Roche Ltd., Basel, Switzerland.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Range in the cytotoxicity test: 20.58, 61.73, 185.19, 555.56, 1667.67, 5000 µg/plate

Range in the original mutagenicity test: 61.73, 185.19, 555.56, 1667.67, 5000 µg/plate

Range in the confirmatory mutagenicity test: 114.3, 342.93, 1028.78, 3086.33, 9259 µg/plate
Vehicle / solvent:
Bidistilled water
Controlsopen allclose all
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:
without microsomal activation for Salmonella TA 100, TA 1535
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:
without microsomal activation for TA 102
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without microsomal activation; Salmonella TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without microsomal activation; Salmonella TA 1537
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:
with microsomal activation; Salmonella TA 100, 102, 98, 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
bidistilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with microsomal activation; Salmonella TA 1535
Details on test system and experimental conditions:
Preparation of the bacterial cultures:
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.

Preparation of the metabolic activation mixture:
Rat-liver microsomal fraction S9 was prepared in advance from male RAI rats (Tif: RAIf[SPF]), reared at the Animal Farm of CIBA- GEIGY, Sisseln, Switzerland. The animals (150-250 g) were treated with Aroclor 1254 (500 mg/kg, i.p.) 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCl and the 9000x g supernatant (S9) was stored at approximately -80 °C for no longer than one year. The protein contents of the S9 fractions were 30.2 and 28.9 mg/ml.
The activation mixture (Ref. 6) contained:
Rat liver S9 fraction: 100 µl/ml
NADP: 4 µmol/ml
MgCl2: 8 µmol/ml
KCl: 33 µmol/ml
Na-phosphate-buffer, pH 7.4 100 µmol/ml
Glucose-6-phosphate: 5 µmol/ml

Solubilization of the test substance:
The test substance was dissolved in bi-distilled water at the concentrations of 50 mg/ml (toxicity test and original mutagenicity test) and 92.6 mg/ml (confirmatory mutagenicity test). Lower concentrations of the test material were obtained by appropriate dilution of the stock solutions with bi-distilled water. No precipitates or aggregates were noted.

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

Preliminary Toxicity/Ranqe-Finding test:
A toxicity test (check for reduction in the number of revertant colonies) was carried out with strain TA 100 without and with microsomal activation at six concentrations of the test substance and one negative control according to Standard Operating Procedures of Genetic Toxicology. The highest concentration applied was 5000 µg/plate. The five lower concentrations decreased by a factor of 3. 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, as well as each negative control was used.

Mutagenicity test:
The mutagenicity test was performed with strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 without and with microsomal activation according to Standard Operating Procedures 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 as well as each positive and negative control with each tester strain. The highest concentration applied in the first mutagenicity assay was 5000 µg/plate (because of lack of toxicity in the range finding test) and the four lower concentrations were each decreased by a factor of 3. 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.
Evaluation criteria:
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 102, TA 1535 and TA 1537.
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. No appropriate statistical method is available.

Results and discussion

Test results
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Toxicity test/Range finding test:

Normal back-ground growth was observed at all concentrations. The test material exerted no toxic effect on the growth of the bacteria. From the results obtained, the highest concentration suitable for the first mutagenicity test was selected to be 5000 µg/plate without and with activation.

 

Mutagenicity test, original experiment: (concentration range: 61.7 to 5000 µg/plate)

In the experiment performed without and with metabolic activation, treatment with Cibacron Blau P-3R roh Lösung für Flüssighandelsform (FAT 41001/F) did not lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the negative control.

 

Mutagenicity test, confirmatory experiment: (concentration range: 114.3 to 9259 µg/plate)

In the experiment carried out without and with metabolic activation, again none of the tested concentrations of Cibacron Blau P- 3R roh Lösung für Flüssighandelsform (FAT 41001/F) led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. In the mutagenicity tests without and with metabolic activation, normal back-ground growth was observed. The numbers of revertant colonies were not reduced. The test material exerted no toxic effect on the growth of the bacteria. The various mutagens, pro-mutagens, sterility checks, sensitivity and resistance tests, etc., employed to ensure the test system was acceptable, all produced results within our established limits. There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the data.

Applicant's summary and conclusion

Conclusions:
FAT 41001/F and its metabolites did not induce gene mutations in the strains of S. typhimurium used.
Executive summary:

The aim of this test was to evaluate the test compound for mutagenic activity in bacterial test systems (Salmonella typhimurium: TA 98, TA 100, TA 102, TA 1535 and TA 1537) in the absence and presence of a rat liver S9 activity system. This study was conducted in accordance with OECD test guideline 471, EU method B.13/14 and EPA OTS 798.5265 (Bacterial reverse mutation test). The concentration range of FAT 41001/F to be tested in the mutagenicity test was determined in a preliminary toxicity test. Thus, FAT 41001/F was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 61.7 to 5000 µg/plate. An independent repetition of the experiments was performed with the concentrations of 114.3 to 9259 µg/plate. The active ingredient is about 54%. 9259 µg/plate correspond to the concentration of about 5000 µg/plate of pure substance.

Toxicity test/Range finding test

In the experiment without and with metabolic activation no toxic effect of the test material on the growth of the bacteria was observed. Mutagenicity test, original experiment (concentration range: 61.7 to 5000 µg/plate). In the original experiment carried out without and with metabolic activation, none of the tested concentrations of FAT 41001/F led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. Mutagenicity test, confirmatory experiment (concentration range: 114.3 to 9259 µg/plate) In the confirmatory experiment performed without and with metabolic activation, again, none of the tested concentrations of (FAT 41001/F) led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. In the mutagenicity tests without and with metabolic activation, normal background growth was observed. The number of revertant colonies was not reduced. The test substance exerted no toxic effect on the growth of the bacteria. Based on the results of these experiments and on standard evaluation criteria, it is concluded that FAT 41001/F and its metabolites did not induce gene mutations in the strains of S. typhimurium used.