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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:
Start: 1999-11-25 End: 1999-12-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(2-{2,4-dihydroxy-5-[2-(4-hydroxyphenyl)propan-2-yl]phenyl}propan-2-yl)phenyl 6-(-lambda5-diazynylidene)-5-oxo-5,6-dihydronaphthalene-1-sulfonate
Molecular formula:
C34H30N2O7S
IUPAC Name:
4-(2-{2,4-dihydroxy-5-[2-(4-hydroxyphenyl)propan-2-yl]phenyl}propan-2-yl)phenyl 6-(-lambda5-diazynylidene)-5-oxo-5,6-dihydronaphthalene-1-sulfonate
Constituent 2
Chemical structure
Reference substance name:
4-[2-(2,4-dihydroxy-5-{2-[4-({[6-(-lambda5-diazynylidene)-5-oxo-5,6-dihydronaphthalen-1-yl]sulfonyl}oxy)phenyl]propan-2-yl}phenyl)propan-2-yl]phenyl 6-(-lambda5-diazynylidene)-5-oxo-5,6-dihydronaphthalene-1-sulfonate
Molecular formula:
C44H34N4O10S2
IUPAC Name:
4-[2-(2,4-dihydroxy-5-{2-[4-({[6-(-lambda5-diazynylidene)-5-oxo-5,6-dihydronaphthalen-1-yl]sulfonyl}oxy)phenyl]propan-2-yl}phenyl)propan-2-yl]phenyl 6-(-lambda5-diazynylidene)-5-oxo-5,6-dihydronaphthalene-1-sulfonate
Test material form:
solid
Details on test material:
Batch PW-9F-29

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
In the dose range finding test, the substance was tested up to concentrations of 5000 µg/plate in the absence and in the rpesence of S9 -mix in the strains TA100 and WP2uvrA. The substance precipitated on the plates at dose levels of 333 µg/plate and upwards. The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed.
Based on the results of the dose range finding study, the substance was tested up to concentrations of 333 µg/plate in the absence and presence of S9-mix.
Vehicle / solvent:
DMSO
Controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
methylmethanesulfonate
other: daunomycine; 2aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
Top agar in top agar tubes was molten and heated to 45°C. The following solutions were successfully added to 3 ml molten top agar : 0.1ml of a fresh bacterial culture (10e9 cells/ml ) of one of the tester strains, 0.1ml of a dilution of the test substance in DMSO and either 0.5ml S9-mix (in case of activation assays) or 0.5 ml 0.1M phosphate buffer (in case of non-activation assays).
The ingredients were mixed on a vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37°C for 48 h. After this period revertant colonies (histidine independent for S. typhimurium bacteria and tryptophan independent for E. coli) were counted.
The revertant colonies were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn, reduction of the number of revertants
Evaluation criteria:
No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if :
1- the total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation
2- the negative response should be reproducible in at least one independently repeated experiment

A test substance is considered positive (mutagenic) in the test if :
1- it induces a number or revertant colonies, dose related, greater than two times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation
However, any mean plate count < 20 is considered to be not significant.
2- the positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
The substance is not mutagenic in this bacterial reverse mutation assay with S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2 uvrA.
Executive summary:

The test substance was tested in the S. typhimurium reverse mutation assay with 4 histidine -requiring strains of S. typhimurium

(TA1535, TA1537, TA98 and TA 100) and in the E. coli reverse mutation assay with a tryptophan-requiring strain of E. coli WP2uvrA in two independent experiments.

In the dose range finding test , the substance was tested up to concentrations of 5000 µg/plate in the absence and in the rpesence of S9 -mix in the strains TA100 and WP2uvrA. The substance precipitated on the plates at dose levels of 333 µg/plate and upwards. The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed.

In the mutation assays, the substance was tested up to concentrations of 333 µg/plate in the presence and in the absence of S9 -mix. The substance precipitated on the plate at this dose level . The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed.

The substance did not induce a dose related , two fold increase in the number of revertant (His+) colonnis in each of the 4 tester strain (TA1535,TA1537, TA100 and TA98) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9 -metabolic activation. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that the substance is not mutagenic in the salmonella typhimurium reverse mutation assay and in the Echerichia coli reverse mutation assay