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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
other: publication
Adequacy of study:
key study
Study period:
1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The publication contains sufficient information for the interpretation of study results.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1988

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
other: mouse lymphoma assay [Clive et al., 1979; Jotz and Mitchell, 1981; Oberly et al., 1984]
Principles of method if other than guideline:
none
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium chloride
EC Number:
231-598-3
EC Name:
Sodium chloride
Cas Number:
7647-14-5
Molecular formula:
ClNa
IUPAC Name:
sodium chloride
Constituent 2
Reference substance name:
231-589-3
IUPAC Name:
231-589-3
Details on test material:
- Name of test material (as cited in study report): Sodium chloride
- Molecular formula (if other than submission substance): NaCI

Method

Target gene:
thymidine kinase (tk) locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells were maintained in culture according to the procedures of Turner et al. [1984].
Cells were maintained in log-phase growth for a 2-day expression period and then cloned with TFT for selection (1 lig/m1) and without TFT for determination of viability in Fischer's medium containing 0.22% Baltimore Biological Laboratory (BBL) agar.
- Properly maintained: no data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
without
Test concentrations with justification for top dose:
Experiment 1
0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 2.5, 3.5, 4.5, 5.5 mg/ml

Experiment 2
4.75, 4.86, 4.97, 5.08, 5.19, 5.30, 5.49, 5.52, 5.63, 5.74

Experiment 3
4.75, 4.86, 4.97, 5.08, 5.19, 5.30, 5.49, 5.52, 5.63, 5.74, 5.85
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
not specified in the report

DURATION

- Exposure duration: 4 hours
- Expression time (cells in growth medium): After 9-11 days of incubation at 37°C, colonies were counted

NUMBER OF REPLICATIONS: not specified

NUMBER OF CELLS EVALUATED: not specified

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
This assay measures forward mutation at the functionally heterozygous thymidine kinase (tk) locus based on resistance to trifluorothymidine (TFT). We have been investigating factors that may impact test design and also influence the interpretation of test results. Factors that effect the magnitude of the quantitated mutant frequency include expression time [Moore and Clive, 1982] and both the growth medium [Moore and Howard, 1982] and the agar used in the cloning medium [Meyer et al., 1986].
Statistics:
no data

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality:
- Evaporation from medium: no data
- Water solubility: yes

RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the first experiment, there was insufficient cytotoxicity to reach a 10-20% survival range, and the mutant frequency (and number of mutants) showed only a very small dose-related increase. It was judged a "no test"—that is, there was insufficient information to make a decision. The second and third* experiments included slightly higher concentrations. Both experiments showed a dose-related increase in both mutant frequency and mutant number.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

In the first experiment, there was insufficient cytotoxicity to reach a 10-20% survival range, andthe mutant frequency (and number of mutants) showed only a very small dose-relatedincrease. It was judged a "no test"—that is, there was insufficient information to make a decision. The second and third experiments included slightly higher concentrations. Both experiments showed a dose-related increase in both mutant frequency and mutant number.

Colony sizing was performed for selected doses.

Colony sizing for weakresponses is some what difficult to interpret. The majority of induced mutant colonieswere small colonies (data not shown), which is consistent with the report by Gallowayet al. [1987] and Ashby and Ishidate [1986] that high-salt concentrations induce chromosome aberrations.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
other: Cells were maintained in culture according to the procedures of Turner et al.[1984]. L5178Y/TICH--3.7.2C cells were treated with sodium chloride for 4 hr inthe absence of exogenous metabolic activation according to procedures described by Turner et al., 1

This experiment with sodium chloride demonstrates the importance of carefully evaluating weak mutagenic responses observed with high concentrations of test compounds. The positive mutagenicity probably is due not to a direct interaction with DNA but to some indirect mechanism resulting from the extremely nonphysiological condition of the test.
Executive summary:

High Concentrations of Sodium Chloride was evaluated for a "Positive" Response at the TK Locus of L5178Y/TK+/- Mouse Lymphoma Cells.

Cells were maintained in culture according to the procedures of Turner et al.[1984]. L5178Y/TICH--3.7.2C cells were treated with sodium chloride for 4 hr inthe absence of exogenous metabolic activation according to procedures described byTurner et al., 1984. Cells were maintained in log-phase growth for a2-day expression period and then cloned with TFT for selection (1 lig/m1) and without TFT for determination of viability in Fischer's medium containing 0.22% BaltimoreBiological Laboratory (BBL)agar. After 9-11 days of incubation at 37°C, colonie swere counted.

Test results generated for the mouse lymphoma assay, the magnitude of the induced mutant frequency is relatively low,and the concentration of test agent required to obtain the response is enormous.

This experiment with sodium chloride demonstrates the importance of carefully evaluating weak mutagenic responses observed with high concentrations of test compounds. The positive mutagenicity probably is due not to a direct interaction with DNA but to some indirect mechanism resulting from the extremely nonphysiological condition of the test.the colony-size distribution determined by an Artek model 880automatic colony counter modified with a 10-turn potentiometer [Moore et al., 1985].

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