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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:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline conform 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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his
trp
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-mixture
Test concentrations with justification for top dose:
first assay: 3, 10, 33, 100, 333, 1000, 3330 and 5000 pg/plate in the absence and presence of 5% (v/v) S9-mix
second assay: Based on the results of the first mutation assay, TKP 50048 was tested up to concentrations of 100 pg/plate.
Vehicle / solvent:
The test substance was suspended ethanol absolute pro analyse (Labscan Ltd, Dublin, Ireland). The stock solution was treated with ultra-sonication to obtain a homogeneous suspension. Test substance concentrations were prepared directly prior to use.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
sodium azide
Remarks:
TA1535, 1µg/plate

Migrated to IUCLID6: in physiological saline
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
9-aminoacridine
Remarks:
TAl537, 60 µg/plate

Migrated to IUCLID6: in physiological saline
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: daunomycine, in physiological saline
Remarks:
TA98, 4 µg/plate
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
methylmethanesulfonate
Remarks:
TA100, 650 µg/plate

Migrated to IUCLID6: in DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
WP2UvrA, 10 µg/plate

Migrated to IUCLID6: in DMSO
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminoanthracene in DMSO
Remarks:
TAl537, TA1535,TA98 2,5 µg/plate
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminoanthracene in DMSO
Remarks:
TA100 2,5 µg/plate
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminoanthracene in DMSO
Remarks:
WP2UvrA, 5 µg/plate
Details on test system and experimental conditions:
The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments. Top agar in top agar tubes was molten and heated to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (10°cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in ethanol and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml
0.1 M 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 Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.
Evaluation criteria:
The revertant colonies (histidine independent cq. tryptophan independent) 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.
Statistics:
No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) 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.
b) The negative response should be reproducible in at least one independently repeated experiment. A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b) 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
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Precipitate
The test substance precipitated in the top agar at concentrations of 33 pg/plate and upwards. Precipitation of TKP 50048 on the plates was observed at the start of the incubation period at concentrations of 33 pg/plate and upwards. Precipitation on the plates was observed at concentrations of 100 pg/plate and upwards at the end of the incubation period.

Toxicity
To determine the toxicity of TKP 50048, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. The definitions are stated in Appendix 1. No reduction of the bacterial background lawn and no decrease in the number of revertants was observed.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that TKP 50048 is not mutagenic in the
Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation
assay.
Executive summary:

In the first mutation assay, TKP 50048 was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix. TKP 50048 precipitated on the plates at dose levels of 100 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 second mutation assay, TKP 50048 was tested up to concentrations of 100 µg/plate in the absence and presence of S9-mix. TKP 50048 precipitated on the plates 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.

TKP 50048 did not induce a dose-related, two-fold, increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TAl 00) 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 TKP 50048 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.