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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:
experimental study
Adequacy of study:
key study
Study period:
1983/11/16-1984/02/01
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study performed similarly to the OECD guideline No 476. The purity of the test substance was not indicated. No data on the GLP compliance of the study. Since this study was performed the criteria for the evaluation of the data have changed and using up to date criteria this study is only a borderline or equivocal positive.
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:
study report
Title:
Unnamed
Year:
1986
Report date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
: no detail on the substance
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Chlorine dioxide
EC Number:
233-162-8
EC Name:
Chlorine dioxide
Cas Number:
10049-04-4
Molecular formula:
ClO2
IUPAC Name:
Chlorine dioxide generated from sodium chlorate and hydrogen peroxide in the presence of a strong acid
Details on test material:
- Name of test material (as cited in study report): chlorine Dioxide
- Physical state: Yellow liquid
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: assumed to be stable during the test (sponsor responsibility)
- Storage condition of test material: no data
- Other: received on 1983/11/15, the test item was dissolved in phosphate beffered saline (PBS). TEst chemical are prepared fresh for each mutagenesis experiment. Neutralization with HCl or NaOH is performed if necessary to maintain the desired pH range (between 7.0 and 7.4).

Method

Target gene:
Tymidine Kinase locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Fisher's mouse leukemia medium supplemented with pluronic solutio, L-Glutamine, sodium pyruvate, antibiotics, and horse serum (10% by volume).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes. Cell cultures are exposed to conditions which select against the TK-/- phenotype (exposure to methotrexate) and are then returned to normal growth medium for 3 to 8 days before use.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix of rat liver homogenate and necessary cofactors
Test concentrations with justification for top dose:
1.32, 3.2, 6.73, 11.1, 14.9, 24.3, 36.9 μg/mL without activation.
6.73, 14.9, 18.5, 30.9, 36.9, 48.3, 65.2 μg/mL with metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: phosphate buffered saline (PBS)
- Justification for choice of solvent/vehicle: no data
Controlsopen allclose all
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
3 solvent controls are included in each assay
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Ethylmethane sulfonate (EMS) at 0.25 to 0.4 μL/mL was used as a positive control for non-activation studies.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
3 solvent controls are included in each assay
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
3-methylcholanthrene (MCA) at 2.5 μg/mL was used as a positive control for assays performed with activation.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): no data
- Fixation time (start of exposure up to fixation or harvest of cells): no data

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 1 x 106 cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
- Other:
Evaluation criteria:
- The average absolute cloning efficiency of the negative controls should be between 60 and 130%.
- The normal range of background frequencies for assays performed with different cell stocks is 10x10-6 to 100x10-6.
- The minimum acceptable mutant frequency induced by 0.3 µL/mL EMS is 200x10-6, for 2.5 µg/mL of MCA the minimum is 200x10-6.
- Because of the fact that mutant frequencies increase as a function of lethality, an attempt to obtain treatments in the range of 10 to 20% relative growth must be made for an assay to be considered conclusive.
- An experimental mutant frequency will be considered acceptable for evaluation only if the relative cloning efficiency is 10% or greater and the total number of viable clones exceeds about 30.
The minimum criteria considered necessary to demonstrate mutagenesis for any given treatment will be a mutant frequency that is at least 150% on the concurrent background frequency plus 10x10-6. The background frequency is defined as the average mutant frequency of the solvent negative controls.
Statistics:
No data

Results and discussion

Test resultsopen allclose all
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
The highest concentration assayed, 48.3 μg/mL (31.8% relative growth) induced a mutant frequency of 165.6 × 10-1 and the increase in mutant frequency was accompanied by a significant increase in the mutant colonies.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Lethal at 81.5 μg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
Treatments from 3.2 to 24.3 μg/mL induced significant increases above the minimum criterion and the increases ranged from 1.9-fold to 4.1-fold above the background mutant frequency.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Highly toxic at 81.5 μg/mL.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Chlorine dioxide induced significant increases in the mutant frequency at the TK locus in L5178Y TK +/- mouse lymphoma cells. Under non-activation conditions, dose-dependent increases in the mutant frequency were induced from 3.2 to 24.3 μg/mL. In the presence of S9 metabolic activation system, chlorine dioxide 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 increase in the mutant frequency was induced. Chlorine dioxide is therefore considered active in the mouse lymphoma forward mutation assay in the presence and absence of metabolic activation.

Any other information on results incl. tables

Table 7.6.1/2:             Table Summarising Mouse Lymphoma Results

Test condition

Daily

cell

counts

Suspension growth

Total

Mutant

Colonies

Total

Viable

Colonies

Cloning

Efficiency

Relative

Growth

Mutant

Frequency

(10E-6)

1

2

Without activation

Solvent Control

9.4

7.37

7.6

51

19

73.0

100.0

23.3

Solvent Control

8.7

7.7

7.4

89

549

83.0

100.0

35.7

Solvent Control

8.3

10.5

9.7

61

204

68.0

100.0

29.9

EMS 0.25 μL/mL

5.9

9.1

6.0

570

121

40.3

39.3

471.1

EMS 0.4 μL/mL

5.2

8.0

4.6

617

96

32.0

24.1

642.7

Test compound

Relative to Control (%)

0.32

6.2

10.9

91.6

56

194

86.6

79.3

28.9

3.2

6.3

6.3

53.8

134

243

108.4

58.3

55.1

6.73

4.9

7.9

52.5

117

194

86.6

45.5

60.3

11.1

5.8

8.3

65.2

181

233

104.0

67.8

77.7

14.9

2.2*

6.6

26.8

174

152

67.8

18.2

114.5

24.3

2.5*

5.0

20.3

180

147

65.6

13.3

122.4

36.9

1.5*

2.7+

Too toxic to clone

With activation

Solvent Control

8.1

8.1

7.3

182

304

101.3

100.0

59.9

Solvent Control

7.3

13.9

11.3

171

264

88.0

100.0

64.8

Solvent Control

10.0

7.8

8.7

162

274

91.3

100.0

59.1

MCA 2.5 μg/mL

9.0

6.0

6.0

506

136

45.3

32.0

372.1

Test compound

Relative to Control (%)

6.73

9.3

7.3

82.9

180

286

102.0

84.6

62.9

14.9

7.8

9.8

93.3

144

224

79.9

74.5

64.3

18.5

9.0

10.1

111.0

122

219

78.1

86.7

55.7

30.9

6.8

7.1

58.9

289

285

101.6

59.8

101.4

36.9

4.6

12.3

69.1

194

237

84.5

58.4

81.9

48.3

4.7

5.7

32.7

452

273

97.3

31.8

165.6

65.2

3.0*

2.0+

Too toxic to clone

*: not split back

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive With and without metabolic activation

Under the test of conditions, Chlorine dioxide is mutagenic in the mouse lymphoma forward mutation assay in the presence and in the absence of S9 metabolic activation.
Executive summary:

In a in vitro mammalian cell gene mutation assay at the thymidine kinase (TK) locus, performed similarly to the OECD guideline No. 476, cultured mouse lymphoma L5178Y cells were exposed to Chlorine Dioxine, at concentrations of 6.73, 14.9, 18.5, 30.9, 36.9, 48.3, 65.2 µg/mL in the presence of mammalian metabolic activation S9 mix, and at concentrations of 1.32, 3.2, 6.73, 11.1, 14.9, 24.3, 36.9 μg/mL in the absence of mammalian metabolic activation.

3-methylcholanthrene (MCA) at 2.5 μg/mL was used as a positive control for assays performed with S9 metabolic activation. Ethylmethane sulfonate (EMS) at 0.25 to 0.4 μL/mL was used as a positive control for non-activation studies. 3E+06 cells were exposed per dose level and evaluated. The mouse cells were exposed to Chlorine Dioxide for 4 hours and then washed and re-incubated at 37°C for 2 days to allow growth and mutant phenotype expression.

The positive controls induced the appropriate response.

Positive results were recorded for Chlorine Dioxide. The highest concentration assayed, 48.3 μg/mL (31.8% relative growth) induced a mutant frequency of 165.6 × 10-1 and the increase in mutant frequency was accompanied by a significant increase in the mutant colonies. Results for cytotoxicity of Chlorine Dioxide to mouse lymphoma L5178Y cells with mammalian metabolic activation S9 were positive. Chlorine Dioxide was lethal at 81.5 µg/mL. Results for genotoxicity of Chlorine Dioxide to mouse lymphoma L5178Y cells without mammalian metabolic activation were positive. Treatments from 3.2 to 24.3 μg/mL induced significant increases above the minimum criteria and the increases ranged from 1.9-fold to 4.1-fold above the background mutant frequency. Results for cytotoxicity of Chlorine Dioxide to mouse lymphoma L5178Y cells without mammalian metabolic activation were positive. Chlorine Dioxide was highly toxic at 81.5 µg/mL

Under the test conditions, Chlorine dioxide is mutagenic in the mouse lymphoma forward mutation assay in the presence and in the absence of S9 metabolic activation. However, the evaluation criteria used in this study are no longer considered to be appropriate and using upto date evaluation criteria (Global Evaluation Factor, GEF) the data are considered to be only a borderline or equivocal positive.

This study is classified as acceptable and satisfies with the requirements for Test Guideline OECD 476 (In vitro Mammalian Cell Gene Mutation Test).