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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
Study period:
1 April 1998 - 8 May 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was performed according to OECD and EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Lot/batch No.: 6090

Method

Target gene:
Histidine gene in Salmonella typhimurium
Tryptophan gene in Escherichia coli
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 rat liver - Aroclor 1254
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 50 ... 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50 ... 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:Dimethyl formamide
- Justification for choice of solvent/vehicle: not indicated
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Dimethyl formamide
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9-mix

Migrated to IUCLID6: 3 µg/plate for TA100; 5µg/plate TA 1535; 2 µg/plate for WP2uvrA
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9-mix

Migrated to IUCLID6: 80 µg/plate for TA 1537
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9-mix

Migrated to IUCLID6: 0.2µg/plate for TA98
Positive controls:
other: non-mutagenic in the absence of metabolic enzymes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9-mix

Migrated to IUCLID6: 5 µg/plate for TA98
Positive controls:
other: non-mutagenic in the absence of metabolic enzymes
Positive control substance:
other: 2-aminoanthracene, 1 µg/plate for TA100; 2 µg/plate for TA 1535 and TA 1537; 10µg/plate for WP2uvrA
Remarks:
with S9-mix
Details on test system and experimental conditions:
Concentration of the test substance resulting in precipitation: 5000 µg/plate
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: 30 min
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 1^9 - 9.9^9 cells/ml

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER:
precipitation of the test substance
Evaluation criteria:
The test substance should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) Significant increase in the revertant count in at least one strain of bacteria. If a greater than twofold increase in revertant count is observed in two experiments then this is taken as evidence of a positive response.
Statistics:
Dunnett's method of linear regression

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
Remarks:
an amber-coloured precipitate was observed at 5000 µg/plate that did not prevent the scoring of revertant colonies
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
an amber-coloured precipitate was observed at 5000 µg/plate that did not prevent the scoring of revertant colonies
Additional information on results:
No toxicity was exhibited to any of the strains of bacteria used. An amber-coloured precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

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

All positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test substance was considered to be non-mutagenic under the conditions of this test.