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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16th November 1983 to 1st February 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
No GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
The assay procedure used is based on reports by Clive and Spector (1975) and Clive, et al. (1979). The cells for the experiment are obtained from logarithmically growing laboratory stock cultures and are seeded into a series of tubes at 3x10E06 cells per tube. The volume during treatment with the test chemical and throughout the expression period is 10 ml per tube. The dosed tubes are placed in a 37±2°C shaker incubator for an exposure period of 4 hours. Afterwards, the cells are washed twice, resuspended in growth medium and returned to the incubator.
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Date Received: November 15, 1983
Physical Description: yellow liquid

Method

Target gene:
Thymidine kinase (TK) locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 activation
Test concentrations with justification for top dose:
without activation: 1.32- 3.2 - 6.73 - 11.1 - 14.9 - 24.3 - 36.9 µg/mLwith metabolic activation: 6.73 - 14.9 - 18.5 - 30.9 - 36.9 - 48.3 - 65.2 µg/mL
Vehicle / solvent:
Water
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
EMS concentration: 0.25 and 0.4 µg/ML, for no activation studies
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
MCA concentration: 2.5 µg/ML, for assays performed with activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: The cells for the experiment are obtained from logarithmically growing laboratory stock cultures and are seeded into a series of tubes at 3x10E06 cells per tube. The dosed tubes are places in a 37 ± 2 ºC shaker incubator for an exposure period of 4 hours. Afterwards, the cells are washed twice, resuspended in growth medium and returned to the incubator.

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days

SELECTION AGENT (mutation assays): selection medium is cloning medium containing 3 μg/ml of TFT.
NUMBER OF REPLICATIONS: Three replicates

DETERMINATION OF CYTOTOXICITY- Method: the cloning efficiency is determined by serially diluting the sample and seeding each of three dishes with approximately 100 cells in cloning medium. All of the dishes are placed in a 37 ± 2 ºC incubator with approximately 5% CO2/humidified air for colony development. After 10 to 14 days in the incubator, the colonies are counted with an electronic colony counter.
Evaluation criteria:
A test material is evaluated as nonmutagenic in a single assay only if the minimum increase in mutant frequency is not observed for: 1) a range of applied concentrations that extends to toxicity causing 10 to 20% relative suspension growth or 2) in the case of relatively nontoxic materials, a range of applied concentrations routinely extending to the maximum of 5 mg/ml (or 5 µg/ml) unlesslimited by solubility.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test material induced significant increases in the mutant frequency at the TK locus in L5178Y TK +/- mouse lymphoma cells. Under nonactivation conditions, dose-dependent increases in the mutant frequency were induced from 3.2 µg/ml to 24.3 µg/ml. In the presence of metabolic activation, the test material was converted to a slightly less toxic and less active form or forms. At 48.3 µg/ml, where the test material was moderately toxic, a 2.7-fold in ease in the mutant frequency was induced.

Applicant's summary and conclusion

Conclusions:
The test substance chlorine dioxide is considered active in the Mouse Lymphoma Forward Mutation Assay in the presence and absence of metabolic activation.
Executive summary:

The aim of the study was to determine the ability of chlorine dioxide to induce forward mutations at the thymidine kinase (TK) locus as assayed by colony growth of L5178Y TK+/- mouse lymphoma cells in the presence of 5-trifluorothymidine (TFT).

The procedure used was based on that reported by Clive and Spector (1975) and Clive et al. (1979).

The test material concentrations were:

-Non activation: 1.32- 3.2 - 6.73 - 11.1 - 14.9 - 24.3 - 36.9 µg/mL

-Activation: 6.73 - 14.9 - 18.5 - 30.9 - 36.9 - 48.3 - 65.2 µg/mL

The test material was considered active in the Mouse Lymphoma Forward Mutation Assay in the presence and absence of metabolic activation.