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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:
Nov 1979
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
Refer chapter 13 for the detailed analogue justification.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1979
Report date:
1979

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: AMES, B.N., J. McCANN, and E. YAMASAKI (1975), Methods for Detecting Carcinogens and Mutagens with the Salmonella/ Mammalian-Microsome Mutagenicity Test. Mut. Res. 31, 347-364.
Deviations:
not specified
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Trisodium 5-[[4-chloro-6-(methylphenylamino)-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate
EC Number:
274-417-3
EC Name:
Trisodium 5-[[4-chloro-6-(methylphenylamino)-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate
Cas Number:
70210-20-7
Molecular formula:
C26H20ClN7O10S3.3Na
IUPAC Name:
trisodium 5-({4-chloro-6-[methyl(phenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(2-sulfonatophenyl)diazenyl]naphthalene-2,7-disulfonate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
FAT 40034/B

Method

Target gene:
Histidine auxotrophic strains of Salmonella typhimurium
Species / strain
Species / strain / cell type:
other: Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of liver from rats induced with Arochlor and cofactors
Test concentrations with justification for top dose:
Pre-test: without and with microsomal activation: 15, 45, 135, 405 and 1215 µg/0.1 ml.
Repetition test: without and with microsomal activation: 25, 75, 225, 6 75 and 2025 µg/0.1 ml
Vehicle / solvent:
phosphate buffer
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without metabolic activation: TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunorubicin-HCl
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:
other: N-methyl-N1-nitro-N-nitrosoguanidine
Remarks:
Without metabolic activation: TA 1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 9(5)aminoacridine hydrochloride monohydrate
Remarks:
Without metabolic activation: TA 1537
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 1538
Details on test system and experimental conditions:
The tests were carried out in accordance with the method described by AMES et al. The bacteria on which the tests were performed were the histidineauxotrophic TA 98, TA 100, TA 1535, TA 15 37 and TA 1538, strains of Salmonella typhimurium.
The test was performed with the following concentrations of the trial substance without and with microsomal activation: 15, 45, 135, 405 and 1215 microg/0.1 ml. A repetition of the experiments was performed with the concentrations of 25, 75, 225, 6 75 and 2025 microg/ 0.1 ml. In these experiments tests on Strain TA 15 38 were included. The substance was dissolved in phosphate buffer. Phosphate buffer alone was used for the negative controls. In the experiments in which the substance was metabolically activated, activation mixture was added also. 1 ml activation mixture contains: 0.3 ml S9 fraction of liver from rats induced with Aroclor 1254 and 0.7 ml of a solution of co-factors.

Positive control experiments were carried out simultaneously with the following substances: 1) for Strain TA 98: daunorubicin-HCl (DAUNOBLASTINR), 5 and 10 microg/0.1 ml phosphate buffer; 2) for Strain TA 100: 4-nitroquinoline-N-oxide, 0.125 and 0.25 microg/0.1 ml phosphate buffer; 3) for Strain TA 1535: N-methyl-N1-nitro-N-nitrosoguanidine, 3 and 5 microg/0.1 ml phosphate buffer; 4) for Strain TA 15 37: 9(5) aminoacridine hydrochloride monohydrate, 50 and 100 microg/0.1 ml DMSO; 5) for Strain TA 15 38: 2-nitrofluorene, 5 and 10 microg/0.1 ml DMSO. The activation mixture was tested with Strain TA 15 35 and cyclophosphamide (ENDOXAN-ASTA ), 250 microg/0.1 ml phosphate buffer.
In the experiments with and without the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group).
The plates were incubated for about 48 hours at 37 deg C in darkness. When the colonies had been counted, the arithmetic mean was calculated.
A test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration .

1.AMESf B.N., F.D. LEE, and W.E. DURSTON (1973), An Improved Bacterial Test System for the Detection and Classification of Mutagens and Carcinogens. Proc. Natl. Acad. Sei. USA 70, 782-786.
2.AMES, B.N., W.E. DURSTON, E. YAMASAKI, and F.D. LEE (1973), Carcinogens are Mutagens: A Simple Test System Combining Liver Homogenates for Activation and Bacteria for Detection.
Proc. Natl. Acad. Sei. USA 7_0, 2281-2285.
3.AMES, B.N., J. McCANN, and E. YAMASAKI (1975), Methods for Detecting Carcinogens and Mutagens with the Salmonella/ Mammalian-Microsome Mutagenicity Test. Mut. Res. 31, 347-364.
Rationale for test conditions:
None
Evaluation criteria:
None
Statistics:
None

Results and discussion

Test results
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 1538 and 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
Additional information on results:
In the experiments performed with and without microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment with FAT 40034/B revealed no marked differences.
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:
FAT 40034/B was determined to be non-mutagenic in the bacterial reverse mutation assay.
Executive summary:

FAT 40034/B was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium which included TA 98, TA 100, TA 1535, TA 15 37 and TA 1538. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 15, 45, 135, 405 and 1215 µg/0.1 ml. A repetition of the experiments was performed with the concentrations of 25, 75, 225, 675 and 2025 µg/0.1 ml. In these experiments tests on Strain TA 1538 were included. In the experiments performed with and without microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of FAT 40034/B revealed no marked deviations. Based on the findings of the study, no evidence of the induction of point mutations by FAT 40034/B or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. Hence, FAT 40034/B was determined to be non-mutagenic in the bacterial reverse mutation assay.