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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:
01-Mar-2006 to 14-Apr-2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

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

Test material

Constituent 1
Reference substance name:
No. 338 Red
IUPAC Name:
No. 338 Red
Details on test material:
- Substance type: Red powder
- Lot/batch No.: 50670



Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain
Species / strain / cell type:
bacteria, other: Salmonella typhimurium : TA1535, TA1537, TA98 et TA100. Escherichia coli : WP2uvrA-
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Concentration range in the range finding test (without and with metabolic activation): 15, 50, 150, 500, 1500 and 5000 µg/plate
Concentration range in the main test (without and with metabolic activation): 15, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9

Migrated to IUCLID6: for WP2uvrA, TA100 and TA1535
Positive control substance:
9-aminoacridine
Remarks:
without S9

Migrated to IUCLID6: for TA1537
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9

Migrated to IUCLID6: for TA98
Positive control substance:
other: 2-aminoanthracene for WP2uvrA, TA100, TA1535 and TA1537
Remarks:
with S9
Positive control substance:
benzo(a)pyrene
Remarks:
with S9

Migrated to IUCLID6: for TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

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

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in the revertant frequency over the dose range tested and/or a reproduceble increase at one or more concentrations in at least one bacterial strain with or without S9.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
( 5000 µg/plate)
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
( 5000 µg/plate)
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
( 5000 µg/plate)
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
( 5000 µg/plate)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at the dose level of 5000 µg/plate.
- A pink colour, becoming darker with increasing concentrations, was noted from 150 µg/plate and above

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at the dose level of 5000 µg/plate in the absence and presence of S9-mix in all tester strains.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

MUTAGENICITY
- In the absence of S9, no increases in the number of revertant colonies were observed

- In the presence of S9 the following dose related increases in the number of revertant colonies were observed
Range finding test
TA1535: 6.6-fold
TA1537: 4.7-fold
TA98: 78-fold
TA100: 9.6-fold
WP2uvrA: 9.5-fold

Main test
TA1535: 4.0-fold
TA1537: 15-fold
TA98: 126-fold
TA100: 11-fold
WP2uvrA: 8.8-fold

Applicant's summary and conclusion

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

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test material was considered to be mutagenic.

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