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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:
2000
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Reactive Blue 1463

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:
Rat liver S9 mix
Test concentrations with justification for top dose:
- Standard plate test: 0; 36; 180; 900; 4500 and 9000 µg/plate
- Pre-incubation test: 0; 36; 180; 900; 4500 and 9000 µg/plate
Vehicle / solvent:
Water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
other: see Remarks
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min (for preincubation test only)
- Exposure duration: 48-72 h
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):

SPINDLE INHIBITOR (cytogenetic assays):

STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS:

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF CELLS EVALUATED:

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY
- Method:
— decrease in the number of revertants
— clearing or diminution of the background lawn (= reduced his or trp background growth)
— reduction in the titer
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:
Evaluation criteria:
Acceptance criteria
Generally, the experiment is to be considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data.
• The titer of viable bacteria was >10E9/ml.

Evaluation criteria
The test chemical is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e, about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.

A test substance is generally considered nonmutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Results and discussion

Test results
Key result
Species / strain:
other: All tested strains
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

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, the test substance was therefore not mutagenic in the Salmonella typhimurium/ Escherichia coli reverse mutation assay.
Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 and EU Method B13/B14, in compliance with GLP. The tested strains were Salmonella typhimurium TA 1535, TA 100, TA 1537 and TA 98, and Escherichia coli strain WP2 uvrA. A standard plate and a pre-incubation test were run, both in dose range of 36 to 9000 µg test substance / plate, with and without Arochlor-induced rat liver S9 mix. No precipitation of the test substance and no bacterio-toxic effect were found. An increase in the number of his+ or trp+ revertants was not observed in either of the assays, either with or without S9 mix. Under the conditions of the study, the test substance was therefore not mutagenic in the Salmonella typhimurium/ Escherichia coli reverse mutation assay (Engelhardt, 2000).