Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 1979
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1979
Report Date:
1979

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Target gene:
no specific target gene
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Activation systems - Reaction mixture: TPN (sodium salt); glucose-6-phosphate; sodium phosphate (dibasic); MgCl2; KCl; Homogenate S9 fraction
Test concentrations with justification for top dose:
from 0.5 microg to 1000 microg per plate for S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
strain TA 98 was tested also with a dose of 2000 microg per plate
Controls
Untreated negative controls:
yes
Remarks:
solvent was used as negative control
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
Details on test system and experimental conditions:
Approximately 10^8 cells from an overnight culture of each indicator strain were added to separate test tubes containing 2.0 ml molten agar supplemented with biotin and trace of histidine. For non activation tests, at least 4 dose levels of the test compound were added to the contents of the appropriate tubes and poured over the surface of selective agar plates. In activation tests, at least 4 dose levels of the test chemical were added to the appropriete tubes with cells. Just prior to pouring, an aliquote of reaction mixture (0.5 ml containing the 9,000 x g liver homogenate) was added to each of the activation overlay tubes, which were then mixzed, and the contents poured over the surface of a minimal agar plate and allowed to solidify. The plates were incubated for 48 hours at 37°C and scored for the number of colonies growing on each plate. Positive controls using both directly active positive chemicals and dose that require metabolic activation were run with each assay.
Rationale for test conditions:
Plate test data consist of direct revertant colony counts obtained from a set of selective agar plates seeded with populations of mutant cells suspended in a semisolid overlay. Because the test chemica1 and the cells are incubated in the overlay for 2 days, and a few cell divisions occur during the incubation period, the test is semiquantitative in nature. Although these features of the assay reduce the quantitation of results, they provide certain advantages not contained in a quantitative suspension test:
- The small number of cell divisions permits potential mutagens to act on replicating DNA, which is often more sensitive than nonreplicating DNA.
- The combined incubation of the compound and the cells in the overlay permits constant exposure of the indicator cells for 2 days.
Evaluation criteria:
Because the procedures used to evaluate the mutagenicity of the test chemical are semiquantitative, the criteria used to determine positive effects are inherently subjective and are based primarily on a historical data base. Most data sets are evaluated using the following criteria:
- Strains TA 1535, TA 1537 and TA 1538: if the solvent control value is within the normal range, a chemical that produce a positive dose response over 3 concentrations with the lowest increase equal to twice the solvent control value is considered to be mutagenic.
- Strains TA 98 and TA 100: if the solvent control value is within the normal range, a chemical that produce a positive dose response over 3 concentrations with the highest increase equal to twice the solvent control value for TA 100 and 2-3 times the solvent control value for strain TA 98 is considered to be mutagenic
For these strains, the dose-response increase should start at approximately the solvent control value.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable

Applicant's summary and conclusion

Conclusions:
The results of the tests conduced were all negative for mutagenic activity.
Executive summary:

The test compound was examined for mutagenic activity in a series of in vitro microbial kassay employing Salmonella indicator organism.

The compound was tested directly and in the presence of liver microsomal enzyme prepatarions.

The results of the tests conduced on the compound in the absence and in presence of a metabolic activation system were all negative.