Chlorine dioxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

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-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

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

Test material information

Constituent 1

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

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

Controls

Controls 1

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.

Controls 2

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 results

Test results 1

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

Test results 2

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:

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).