Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

in vitro gene mutation study in bacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
3 (not reliable)
Rationale for reliability incl. deficiencies:
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.
Justification for type of information:
The basis for the read-across concept for this project is the equilibrium between sulfites, hydrogensulfites, and metabisulfites in aqueous solutions depending on pHvalue which is clearly described in published literature and summarised in the following equations:[1],[2]
           SO2+ H2O <->`H2SO3´         H2SO3<->H++ HSO3-<->2H++SO32-    2HSO3-<->H2O +S2O52 -
As the nature of the cation should make no significant difference in this case concerning toxicity and solubility (all compounds are very soluble in water), only the chemical and biological properties of the anion are considered relevant. Based on the described equilibrium correlations, we propose unrestricted read-across between the groups of sulfites, hydrogensulfites and metabisulfites.

Additionally, it is known that sodium dithionite disproportionates in water to form sodium hydrogen sulfite and sodium thiosulfate (equation II) so that this substance can also be added to the read-across concept.[2],[1]It is expected for this case that the substance is not stable enough under physiological conditions to fulfil the requirements of study guidelines and so the products of decomposition have to be considered.
       2 S2O42-+ H2O→2HSO3-+ S2O32 -
Not completely included in this read-across concept is the substance class of thiosulfates. Although thiosulfates may also disproportionate in aqueous solution to form polythionic acids and SO2(HSO3-), the required conditions are somewhat different (more acidic) and are therefore not strictly comparable with physiological conditions, except for the case of oral application where read-across should be considered unrestricted due to the strongly acidic conditions in the stomach:
       HS2O3-+ H2S2O3→HS3O3- + SO2 + H2O
Nevertheless, read-across for all other routes (dermal, inhalation) should also be considered.
The proposed read-across concept only applies to toxicological and ecotoxicological/environmental fate endpoints.
[1]Hollemann Wiberg, Lehrbuch der Anorganischen Chemie, 101.Auflage
[2]Handbook of Chemistry and Physics, Ed. Lide, DR, 88thedition, CRC Press

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 1997-07-21
, please refer to the field "Rationale for reliability incl. deficiencies" above
GLP compliance:
GLP was not compulsory at time of study conduct
Type of assay:
bacterial reverse mutation assay

Test material

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)


Target gene:
not applicable
Species / strain
Species / strain / cell type:
S. typhimurium, 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.
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: Quinacrine or Quinacrine mustard & Dimethylnitrosamine
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.
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.

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.

- Determination of polyploidy:
- Determination of endoreplication:
- Other:

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

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, and TA 1538
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
≥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.

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

Applicant's summary and conclusion

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