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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: Modern GLP study conducted in accordance with modern guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

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 (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Solid
- Lot/batch No.: E0011-039E
- Storage condition of test material: Room temperature in the dark

Method

Target gene:
The target genes in the Salmonella strains control the synthesis of histidine.
The target genes in the E. coli strain controls the synthesis of tryptophan.
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 from the livers of male Sprague-Dawley rats treated with phenobarbiton/beta-naphthoflavone
Test concentrations with justification for top dose:
0, 50, 150, 500, 1500 and 5000 micrograms per plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent/vehicle: Based on prior experience with the test material, it was predicted to be soluble in tetrahydrofuran. Preliminary solubility checks confirmed the test material was fully soluble in tetrahydrofuran at 200 mg/ml.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Migrated to IUCLID6: without S9 - TA100, TA1535, WP2uvrA-
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: without S9 - TA1537
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Migrated to IUCLID6: without S9 - TA98
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: with S9 - TA98
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene: with S9 - TA100, TA1535, TA1537, WP2uvrA-
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

EXPOSURE/INCUBATION DURATION: Approximately 48 hours

NUMBER OF REPLICATIONS: Triplicate

Evaluation criteria:
There are several evaluation criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance is the first consideration. Statistical methods are used to aid evaluation, but statistical significance is not the only determining factor for a positive response.
Statistics:
In the absence of any indication of a positive response for the test material, the data were not subjected to statistical analysis.

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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative 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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation: Particulate precipitation was seen at and above 500 micrograms/plate, but this did not prevent scoring of revertant colonies
- Other confounding effects:


RANGE-FINDING/SCREENING STUDIES: The range-finding test was conducted at test material incorporation concentrations of 50, 150, 500, 1500 and 5000 micrograms/plate. The main test was also performed using these concentrations.


COMPARISON WITH HISTORICAL CONTROL DATA: Concurrent negative control data were within the historical control ranges.


ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

No significant increases in the frequency of revertant colonies were recorded for any of the bactrerial strains at any dose level, either with or without metabolic activation.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative
Executive summary:

The test substance was evaluated for mutagenic potential in an Ames test, using the direct plate incorporation method, and conducted in accordance with OECD Test Guideline 471. Salmonella strains TA98, TA100, TA 1535 and TA1537, and E. coli strain WP2uvrA- were used in the test. The test material, in tetrahydrofuran vehicle/solvent was tested at concentrations of 0, 50, 150, 500, 1500 and 5000 micrograms/plate both in the presence and absence of exogenous (S9) metabolic activation. There was no evidence of cytotoxicity. Particulate precipitation of test material was seen at and above 500 micrograms/plate, but this did not prevent scoring of revertant colonies - manual counts were performed at 5000 micrograms/plate due to excessive precipitation. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains and any dose level, either with or without metabolic activation.