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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:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Significant methodological deficiencies Deviations from guideline: - physical nature and purity of the test substance were missing - only three strains of bacteria were used - TA1538 is not a strain mentioned by the guideline - no historical control data - only one concentration was test in the plate test and two concentrations were tested in the suspension test - only duplicate testing (plate test) - duration of incubation was four days during the plate test.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1975
Report Date:
1975

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 1997-07-21
Deviations:
yes
Remarks:
, please refer to the field "Rationale for reliability incl. deficiencies" above
GLP compliance:
no
Remarks:
GLP was not compulsory at time of study conduct
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): compound FDA 73-43 (sodium sulfite) (Allied Chemicals No. H028)

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
other: TA 1535, TA 1537, and TA 1538
Metabolic activation:
with and without
Metabolic activation system:
tissue homogenates were prepared from liver, lung and testes of mice (ICR random bred; adult males), rat (Sprague-Dawley; adult males), and primate (Macaca mulatta; adult males)
Test concentrations with justification for top dose:
Plate tests: 0.028%
Suspension tests: 2.5% and 5.0%
Vehicle / solvent:
All tests were conducted in an aqueous environment.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation; strain: TA 1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-nitrosofluorene
Remarks:
without metabolic activation; strain: TA 1538
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Quinacrine or Quinacrine mustard
Remarks:
without metabolic activation; strain: TA 1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Dimethylnitrosamine
Remarks:
with metabolic activation; strain: TA 1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Remarks:
with metabolic activation; strains: TA 1537 and TA 1538
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate test & suspension test
1) Plate test
In the non-activation procedure, cells of the bacterial strains were spread over the surface of a minimal plate, and a measured amount of the test chemical was placed in the center of the test plate. In activation tests, the test chemical was added to the cells, and an aliquot of the mixture was spread on the surface of the test plate. The reaction mixture (0.1 mL; tissue homogenate from liver, lung, and testes of mouse, rat and primate) plus tissue extract was then spotted on the surface of the plate. All plates were incubated at 37°C for four days and then scored. Each compound was done in duplicate.

2) Suspension test
Non activation:
Log-phase bacteria of the indicator organisms were grown in complete broth, washed and resuspended in 0.9% saline to densities of 1 X 10^9 cells/mL. This constituted the working stock for tests of a group of test chemicals and their respective controls. Tests were conducted in 30 mL plastic tissue culture flasks. Cells plus appropriate volume(s) of the test chemical were added to the flasks to give a final volume of 2 mL. Solvent replaced the test chemical in the negative controls. Treatment was at 37°C for one hour for bacterial tests. All flasks were shaken during treatment. Following treatment, the flasks were set in ice. Aliquots of cells were removed, diluted in sterile saline (4°C) and plated on the appropriate complete media. Undiluted samples from flasks containing the bacteria were plated on minimal selective medium. Bacterial plates were scored after incubation for 48 hours at 37°C.
Activation:
Bacteria were grown and prepared as described in the non-activation tests except that the cell densities were increased approximately five-fold for working stock suspensions. Measured amounts of the test and control chemicals plus 0.25 ml of the stock cell suspension were added to a 30 mL plastic tissue homogenate (tissue homogenate from liver, lung, and testes of mouse, rat and primate). All flasks (bacteria) were incubated at 37°C with shaking. The treatment times as well as the dilutions, plating procedures and soring of the plates were the same as described for non-activation tests.

Following the specified incubation periods all population plates were scored by an automatic colony counter.

DETERMINATION OF CYTOTOXICITY
The toxicity for all chemicals were determined prior to screening.
Each chemical was tested for survival against strain TA 1537 over a range of doses (test compound: 0.1, 0.5, 1.0, 2.5, and 5.0%) to determine the 50% survival dose. Bacteria were tested in phosphate buffer, pH 7.4, for one hour at 37°C on a shaker. The 50% survival dose was determined from the survival curve and the 1/4 and 1/2 50% doses calculated.
If no toxicity was obtained for a chemical with a given strain, then a maximum dose of 5% (w/v) was used against the strain.

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:
Evaluation criteria:
no data
Statistics:
Frequencies were mechanically calculated and double checked.

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 1537, and TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥0.1% concentration
Vehicle controls validity:
not specified
Untreated negative controls validity:
not examined
Positive controls validity:
not specified
Additional information on results:
Compound FDA 73-43, sodium sulfite, did not exhibit genetic activity.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The following results were obtained when determining the 50% survival level for the test compound:
Control: 100% survival
0.1% concentration: 19% survival
0.5% concentration: 6% survival
1.0% concentration: 0.5% survival
2.5% concentration: 0.8% survival
5.0% concentration: 0% survival
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test substance did not appear to be mutagenic under given test conditions.