Chlorine dioxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Table 7.6/1: Summary of in vitro genotoxicity tests

 

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1 Cifone	Mouse lymphoma assay (eq. to OECD 476) K, rel. 2, 1986	Mammalian gene mutation	Mouse lymphoma L5178Y cells	-S9 +S9	1.32 to 36.9 μg/mL without activation and 6.73 to 65.2 μg/mL with metabolic activation	-S9 : mutagenic +S9 : mutagenic
2 Ivett	Chromosomal aberration (eq. to OECD 473) K, rel. 2, 1986	Chromosomal aberration	CHO cells	-S9 +S9	-S9: 2.5 to 30.0 µg/mL +S9: 6.25 to 75.0 µg/mL	-S9 : clastogenic +S9 : clastogenic
3 Bushini	Gene mutation assay in yeast (eq. to OECD 480) S, rel. 2, 2004	Gene mutation	Saccharomyces cerevisiae	-S9 +S9	0, 0.05, 0.1, 0.25, 0.5, 1, 2, 5, and 10 ppm	-S9 : not mutagenic +S9 : not mutagenic
4 Rundell	Cell transformation assay (no guideline followed) S, rel. 2, 1986	in vitro mammalian cell transformation assay	mammalian cell line, other: Clone 1-13 of Balb/c-3T3 mouse cells	-S9	0.5, 1.0, 2.0, 3.0, 5.0, 6.0 μg/mL	inactive in the Balb/3T3

Link to relevant study records

Reference

Reference 1

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.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

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

Target gene:

Tymidine Kinase locus

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

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

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.

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

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).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11-15-1983 to 02-03-1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study performed similarly to OECD guideline No. 473. No detail on the test substance material. No data on the GLP compliance of the study. Cytotoxicity was not determined in the main study and it is possible that dose levels with excessive toxicity were analysed for chromosome aberrations.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

See Remarks on "Rationale for reliability incl. deficiencies" section.

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy's 5a medium supplemented with 10% fetal calf serum, L-Glutamine, and antibiotics
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The in vitro metabolic activation system comprises rat liver enzymes (S9 fraction from rats treated previously with Aroclor) and an energy-producing system necessary for their function (NADP and isocitric acid).

Test concentrations with justification for top dose:

Without metabolic activation: 0, 2.5, 5.0, 10.0 , 15.0 and 30.0 µg/mL.
With metabolic activation: 0, 6.25, 12.50, 25.0 , 50.0 and 75.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: phosphate buffered saline
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

yes

Remarks:

Culture medium

Negative solvent / vehicle controls:

yes

Remarks:

Phosphate buffered saline at the both concentrations 1.41 and 3.5%

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation – mitomycin C (MCC) (dissolved in water) at 80 μg/mL

Untreated negative controls:

yes

Remarks:

Culture medium

Negative solvent / vehicle controls:

yes

Remarks:

Phosphate buffered saline at the both concentrations 1.41 and 3.5%

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation – cyclophosphamide (CP) (dissolved in water) 25 μg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 10 hours (in the absence of metabolic activation), 2 hours (in the presence of metabolic activation)
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 12.5 hours (in the absence of metabolic activation) , 10 hours (in the presence of metabolic activation)

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): methanol:acetic acid, 3:1

NUMBER OF REPLICATIONS: duplicate (2 positive controls, one negative and one solvent control)

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- Method: % of confluence and cell cycle progression analysis

OTHER EXAMINATIONS:
- Determination of polyploidy: no data
- Determination of endoreplication: yes
- Other:

Evaluation criteria:

No data

Statistics:

Statistical analysis employed the Student t-test to compare the aberration frequency in treated cells with pooled results from solvent and negative controls. The difference is considered significant where p<0.05. Using binomial statistics and five comparisons, when 200 cells are read, an increase from background level of 0.01 to 0.05 aberrations per cell in a treated culture is significant at the 1% level with a power of >90%.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Without metabolic activation: at 10 µg/mL. With metabolic activation: at 50 µg/mL

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without metabolic activation: 30 µg/mL. With metabolic activation: 75 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test material chlorine dioxide induced chromosomal aberrations in Chinese Hamster Ovary (CHO) cells cultured with and without an S9 metabolic activation system. The aberration response without metabolic activation was dose responsive and greater in magnitude than with metabolic activation conditions. A higher than expected frequency of complex rearrangements including triradials and quadriradials were observed. Chlorine dioxide is considered positive for chromosomal aberration induction under the conditions of the study.
See details in Tables 7.6.1/2 and 7.6.1/3

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 7.6.1/2: Table for Chromosome Aberration Assay (without activation)
TREATMENT		Cells scored	NUMBER AND TYPE OF ABERRATION															No. of aberrations per cell	% cells with aberrations	% cells with aberrations
			CHROMATID						CHROMOSOME
			TB	TD	F	TR	QR	CR	SB	AF	D	R	MT	PU	E	>	Other
Controls	Negative (medium)	100									2							0.02	2.0	0.0
	Solvent (Phosphate buffered saline, 1.41%)	100																0.00	0.0	0.0
	Positive (mitomycin C, 80 μg/mL)	50	2				1				2							0.10	10.0	0.0
Chlorine Dioxide	2.5 μg/mL                      A	100									1							0.01	1.0	0.0
	B	100					1				1			1				>0.03	3.0	1.0
	5.0 μg/mL                      A	100												1				>0.01	1.0	1.0
	B	100																0.00	0.0	0.0
	10.0 μg/mL                    A	100	3		2	2	2		1	1								0.11	9.0	2.0
	B	100	5			3	4			1							2UC	>0.15	11.0	4.0
	15.0 μg/mL                    A	100	12			6	6	2	3	1	2			1			3ID	0.36	28.0	6.0
	B	100	9			3	4			2	2					1	7ID 2CI	0.39	22.0	4.0
	30.0 μg/mL^a

^a toxic

Table 7.6.1/3.  Table for Gene Mutation Assay (without activation)
TREATMENT		Cells scored	NUMBER AND TYPE OF ABERRATION															No. of aberrations per cell	% cells with aberrations	% cells with aberrations
			CHROMATID						CHROMOSOME
			TB	TD	F	TR	QR	CR	SB	AF	D	R	MT	PU	E	>	Other
Controls	Negative and solvent	200									2							0.01	1.0	0.0
	Positive (mitomycin C, 80 ng/mL)	50	2				1				2							0.10^**	10.0	0.0
Chlorine Dioxide	2.5 μg/mL	200					1				2			1				>0.02	2.0	0.5
	5.0 μg/mL	200												1				>0.005	0.5	0.5
	10.0 μg/mL	200	8		2	5	6		1	2						2UC		>0.130**	10.0	3.0
	15.0 μg/mL	200	21			9	10	2	3	3	4			1		10ID 2CI		>0.357^**	25.0	5.0
	30.0 μg/mL^a

^a toxic

^** significantly greater than the negative and solvent controls, p0.001

TG: Chromatid Gap

TB: Chromatid Break

TD: Chromatid deletion

TF/F: Chromatif fragment

QR: chromatid interchanges Quadriradial

CR: Chromatid interchanges complex rearrangement

SB: Chromosome Break

AF: Acentric fragment

D: Dicentric chromosome

R: Ring Chromosome

MT: Min Minute Chromosome

PU: Pulverized chromosome

E: Endoreduplication

Conclusions:

Chlorine dioxide is considered to be positive for chromosomal aberration induction, both with and without metabolic activation, under the conditions of the study.

Executive summary:

In an in vitro mammalian chromosome aberration assay performed similarly to the OECD guideline No. 473, Chinese Hamster Ovary (CHO) cells were exposed to Chlorine Dioxide at concentrations of 0, 6.25, 12.50, 25.0 , 50.0 and 75.0 µg/mL with metabolic activation for 2 hours and at concentrations of 0, 2.5, 5.0, 10.0 , 15.0 and 30.0 µg/mL without metabolic activation for 10 hours. The in vitro metabolic activation system comprised rat liver enzymes and an energy-producing system necessary for their function (NADP and isocitric acid).

With metabolic activation, cyclophosphamide (CP) (dissolved in water) 25 μg/mL was used as a positive control whereas without metabolic activation, mitomycin C (MCC) (dissolved in water) at 80 μg/mL was used as a positive control.

Positive controls induced the appropriate response. 

Results for genotoxicity of Chlorine Dioxide to CHO cells were positive without metabolic activation at 10 µg/mL and with metabolic activation at 50 µg/mL. Results for cytotoxicity of Chlorine Dioxide to CHO cells were positive at 30 µg/mL without metabolic activation and at 75 µg/mL with metabolic activation. However, it should be noted that cytotoxicity was not determined in the main study and it is possible that dose levels giving excessive levels of toxicity were analysed for chromosome aberrations.

The test substance Chlorine Dioxide induced chromosomal aberrations in Chinese Hamster Ovary (CHO) cells cultured with and without S9 metabolic activation system. The aberration response without metabolic activation was dose related and greater in magnitude than with metabolic activation conditions. A higher than expected frequency of complex rearrangements including triradials and quadriradials were observed.

Under the test conditions, Chlorine dioxide is positive in the chromosomal aberration assay, both with and without metabolic activation. However, it is not clear that using modern acceptability criteria for appropriate levels of toxicity would provide the same result.

This study is classified as acceptable and satisfies with the requirements for Test Guideline OECD 473 (In vitro Mammalian Chromosome Aberration Test).

Reference 3

Endpoint:

in vitro transformation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 11-15-1983 to 02-14-1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No guideline specified and no detail on the test substance but the study is well conducted and meets basic scientific principles.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline available

Principles of method if other than guideline:

Chlorine dioxide was evaluated for its ability to induce the appearance of transformed foci in cells, recognized by dense, piled-up colonies on a monolayer of normal cells.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

Not applicable

Species / strain / cell type:

mammalian cell line, other: Clone 1-13 of Balb/c-3T3 mouse cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle's minimum essential medium supplemented with heat-inactivated fetal bovine serum, L-Glutamine, penicillin and streptomycin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
- Other: Balb/c-3T3, 1-13 mouse cells multiply in culture until a monolayer is achieved and then cease further division. These cells, when injected into immunosuppressed, syngeneic host animals, did not produce neoplastic tumors. However, cells treated in vitro with chemical carcinogens gave rise to foci of cellular growth super-imposed on the cell monolayer. When these foci were picked from cultures, grown to larger numbers and injected into animals, a malignant tumor was obtained in most cases. Thus, the appearance of piled-up colonies in treated cell cultures at a higher frequency than in control cultures was highly correlated with malignant transformation.

Additional strain / cell type characteristics:

other: a subclone, C-14, selected for its low spontaneous frequency of foci formation

Metabolic activation:

without

Test concentrations with justification for top dose:

0.5, 1.0, 2.0, 3.0, 5.0, 6.0 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: freely soluble in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionized water

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

Migrated to IUCLID6: 2.5 μg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 72 hours
- Expression time (cells in growth medium): 3-5 days for the preliminary assay; 4 weeks for the transformation assay (with refeeding twice a week)
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 4 weeks

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): the assay was terminated by fixing the cell monolayers with methanol and staining with Giemsa.

NUMBER OF REPLICATIONS: 15

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency: a relative survival for each dose was obtained by comparing the number of colonies surviving treatment to the colony counts in negative control dishes.

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
- Other: the stained dishes were examined by eye and by microscope to determine the number of foci of transformed cells.

Evaluation criteria:

At the end for the four-week incubation period, cultures of normal cells yielded a uniformly stained monolayer of round, closely-packed cells. Transformed cells from a dense mass (focus or colony) that stained deeply (usually blue) and were superimposed on the surrounding monolayer of normal cells. The foci were variable in size.
The negative control dishes consisted of a contigous monolayer of cells which may or may not have contained transformed foci. The ngeative control transformation frequency did not exceed an average of about 2-3 foci-dish agter log 10 analysis.
The positive control yielded an average number of foci/dish that was significantly different from the negative control at the 99% confidence level.

Statistics:

Bailey's modification of Student's t-test.

Species / strain:

mammalian cell line, other: Clone 1-13 of Balb/c-3T3 mouse cells

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Chlorine dioxide did not induce the appearance of a significant number of transformed foci over the concentration range from 6.0 to 1.0 μg/mL. This concentration range corresponded to approximately 0.5% to 103.8% survival in the concomitant cytotoxicity test. Therefore Chlorine dioxide is considered to be inactive in the Balb/3T3 in vitro transformation assay.

Table 7.6.1/1: Table for Raw Data and Data Summary from Concomittant Cytotoxicity Tests

 

Test material Dose (μg/mL)	Flask 1	Flask 2	Flask 3	Average No. of Colonies/Flask	Relative Survival
	Trial 1
6.0	0	0	ND	0	0
5.0	33	45	ND	39.0	38.4
3.0	73	72	ND	72.5	71.4
2.0	77	94	ND	85.5	84.2
1.0	73	83	ND	78.0	76.8
0.5	93	91	ND	92.0	90.6
Conrol	98	105	ND	101.5	100.0
	Trial 2
6.0	0	1	0	0.3	0.5
5.0	33	19	20	24.0	33.8
3.0	54	53	64	57.0	80.2
2.0	65	69	66	66.7	93.9
1.0	82	65	74	73.7	103.8
Conrol	65	74	74	71.0	100.0

ND = Not done

 

Table 7.6.1/2: Statistical Analysis of Transformation Activity – Trial II

	Log10 Analysis* of Foci/Flask
Treatment Condition	Mean	SD	N	SE	T Statistic**	P Values**
Negative Control	0.055	0.119	22	0.025	Control	Control
Positive Control	0.854	0.168	18	0.040	+ 16.996	P0.01
Test Material (mg/mL)
6.0	0.067	0.129	18	0.030	+ 0.0385	0.7 0.8
5.0	0.038	0.103	16	0.026	- 0.474	NS
3.0	0.050	0.115	18	0.027	- 0.121	NS
2.0	0.056	0.145	14	0.039	+ 0.019	0.9 1.0
1.0	0.108	0.253	14	0.068	+ 0.730	0.4 0.5

* Each raw data point was increased by 1.0 and converted to Log10 before statistical analysis was applied

** Bailey, N.J.T. (1959)

Conclusions:

Chlorine dioxide is considered to be inactive in the Balb/3T3 in vitro transformation assay.
(The in vitro cell transformation assay is not specific to genotoxicity. It is an assay developed to be predictive of cell transformation activity which may have both genotoxic and non-genotoxic mechanisms).

Executive summary:

In a mammalian cell transformation assay, Clone 1-13 of Balb/c-3T3 mouse cells cultured in vitro were exposed for 72 hours to Chlorine Dioxide at concentrations of 0.5, 1.0, 2.0, 3.0, 5.0, and 6.0 μg/mL. Then the cells were re-incubed in growth medium for 4 weeks to allow transformation of cells. 3-methylcholanthrene at concentration of 2.5 μg/mL was used as a positive control.

Results for transformation of Chlorine Dioxide to Clone 1-13 of Balb/c-3T3 mouse cells were negative whereas results for cytotoxicity were positive.

Chlorine dioxide did not induce the appearance of a significant number of transformed foci over the concentration range from 6.0 to 1.0 μg/mL. This concentration range corresponded to approximately 0.5% to 103.8% survival in the concomitant cytotoxicity test. Therefore chlorine dioxide is considered to be inactive in the Balb/3T3 in vitro transformation assay.

This study is considered as acceptable.

Reference 4

Endpoint:

genetic toxicity in vitro, other

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed similarly to the OECD guideline No. 480 but no data on the GLP compliance of the experiment and no detail on the test substance.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 480 (Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Type of assay:

other: in vitro gene mutation assay in yeast

Target gene:

Not applicable

Species / strain / cell type:

Saccharomyces cerevisiae

Details on mammalian cell type (if applicable):

- Type and identity of media: medium and agar were from Difco
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

other: D7 strain

Metabolic activation:

with and without

Metabolic activation system:

P450 activation

Test concentrations with justification for top dose:

0, 0.05, 0.1, 0.25, 0.5, 1, 2, 5, and 10 ppm

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

control for non-metabolic activation

Migrated to IUCLID6: 100 mM

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminofuorene 5µg/mL

Remarks:

control for metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethidium bromide 1µg/mL

Remarks:

control for mutation in the mitochondrial DNA

Species / strain:

Saccharomyces cerevisiae

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Cells showed a higher sensitivity to ClO2 toxicity than log cells, with significant effects from 1 ppm. Genotoxic effects were only observed in metabolic activated cells with significance increase of the revertants and number of petite colonies at the highest dose (10 ppm).

 

Table 7.6.1/1 : Cytotoxicity and Genotoxicity of Chlorine Dioxide on Saccharomyces Cerevisiae

 

Sample	Dose	Without metabolic activation				With metabolic activation
		Cell survival (%)	trp5locus (x10^-8cells)	Ilvllocus (x10^-8cells)	RD (x10^-3cells)	Cell survival (%)	trp5locus (x10^-8cells)	Ilvllocus (x10^-8cells)	RD (x10^-3cells)
ClO2	0 ppm	100	925 ± 155	25 ± 5	4 ± 1	100	1770 ± 170	25 ± 10	3 ± 1
	0.05 ppm	94 ± 6	898 ± 272	33 ± 4	5 ± 1	95 ± 4	1724 ± 84	25 ± 10	4 ± 4
	0.1 ppm	96 ± 5	725 ± 175	37 ± 4	5 ± 1	97 ± 5	1695 ± 95	22 ± 8	4 ± 2
	0.25 ppm	89 ± 5	794 ± 157	37 ± 4	5 ± 1	96 ± 6	1916 ± 76	23 ± 8	3 ± 1
	0.5 ppm	90 ± 4	755 ± 135	35 ± 1	4 ± 2	95 ± 4	1680 ± 420	22 ± 2	2 ± 1
	1 ppm	83 ± 5*	910 ± 150	38 ± 5	5 ± 2	96 ± 4	1540 ± 230	26 ± 10	2 ± 2
	2 ppm	71 ± 4***	913 ± 110	40 ± 5	5 ± 3	97 ± 5	1937 ± 77	32 ± 8	4 ± 3
	5 ppm	54 ± 2***	865 ± 145	40 ± 3	4 ± 1	87 ± 4**	2107 ± 84	34 ± 9	6 ± 3
	10 ppm	10 ± 1***	1100 ± 102	45 ± 5	3 ± 1	61 ± 2***	2675 ± 85***	89 ± 14***	12 ± 4***
EMS	100 mM	94 ± 3	68630 ± 17140	17487 ± 1677	-	-	-	-	-
2AF	5 µg/mL	97 ± 2	930 ± 110	42 ± 7	-	92 ± 7	28590 ± 3230	3129 ± 482	-
EtBr	1 µg/mL	95 ± 6	-	-	134 ± 12	-	-	-	196 ± 17

 

EMS: Ethyl Methanesulfonate

2AF: 2-aminofluorene

EtBr: Ethidium Bromide

* : P<0.05

** : P<0.01

*** : P<0.001

Conclusions:

Under the test conditions, the chlorine dioxide was not genotoxic with or without metabolic activation. The substance was genotoxic at the highest dose with metabolic activation but the substance was also very cytotoxic at this dose level. Therefore, this latter dose in presence of metabolic activation is not relevant and is not considered for the classification of the substance.

Executive summary:

An in vitro mutation assay in yeast was performed similarly to the OECD guideline No. 480. Saccharomyces cerevisiae strain D7 was exposed to chlorine dioxide (0, 0.05, 0.1, 0.25, 0.5, 1, 2, 5 and 10 ppm) at 37 °C for 2 hours with or without metabolic activation. Ethyl methanesulfonate (EMS), 2-aminofluorene (2-AF) and Ethidium Bromide were used as positive control and gave acceptable results.

Several parameters were studied:

-      The reversion of the ilvl-92 locus

-      The mitotic gene conversion at the trp5 locus

-      The mitochondrial mutability

The substance was genotoxic at the highest dose in the presence of metabolic activation but the substance was also very cytotoxic at this dose level

Therefore, this latter dose tested in presence of metabolic activation was not considered as relevant for the classification of the substance.

Under the test conditions, the chlorine dioxide was not mutagenic with or without metabolic activation at the concentrations which were non cytotoxic.

Under the test conditions, the chlorine dioxide is not classified as mutagenic according to the criteria of the Annex VI of the Regulations (CE) N° 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Table 7.6/1: Summary of in vivo genotoxicity tests

 

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1 Moore	Chromosomal aberration (eq. to OECD 478,1984, K, rel. 2)	Germ cell mutations for the mouse dominant lethal assay	Germ cells mouse CD-1	NA	2, 7, 20 mg/kg bw.	Not mutagenic
2 Ivett	Bone marrow chromosome aberration (eq. to OECD 475, 1984, K, rel. 2)	Chromosomal aberration	Bone marrow cells mouse CD-1	NA	1.73, 5.15 and 15.44 mg/kg for the 6 hour groups and 1.72, 5.18 and 16.11 mg/kg for the 24 h and 48 h dose groups.	Not clastogenic
3 Ivett	Sister chromatid exchange assay (eq. to OECD 479, 1984, K, rel. 2)	Sister Chromatid Exchange Assay in Mammalian Cells	Bone marrow cells in ICR male mice	NA	9.04, 21.08, 28.02, 38.55 mg/kg	Negative for inducing sister chromatid exchanges

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 04-30-1984 to 05-14-1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed similarly to OECD guideline with acceptable deviations.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 479

Deviations:

yes

Remarks:

: in vivo study, no detail on the substance

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

sister chromatid exchange assay

Species:

mouse

Strain:

ICR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc. Indianapolis, Indiana, USA.
- Age at study initiation: 9 weeks
- Weight at study initiation: 29.74 g mean weight
- Assigned to test groups randomly: yes, under following basis: according to LBI Standard Operating Procedures (SOP) # 303: "Genetics -- Animal Randomization"
- Fasting period before study: no data
- Housing: up to 5 mice per cage
- Diet (e.g. ad libitum): ad libitum (Purina Laboratory Chow)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
no data

IN-LIFE DATES: no data

Route of administration:

intraperitoneal

Vehicle:

Vehicle: deionised water

Details on exposure:

Total volume applied: 0.15 to 0.41 ml i.p.
Dose applied: 9.04, 21.08, 28.02, 38.55 mg/kg

Approximately 1 h before dosing, all animals received subcutaneous implant of a ~50% paraffin-coated 50 mg BrdU tablet.

Duration of treatment / exposure:

one single acute dose

Frequency of treatment:

Number of applications: 1

Post exposure period:

26 h

Remarks:

Doses / Concentrations:
0.680, 1.586, 2.113, 2.900 mg/mL
Basis:
nominal conc.
in the vehicle

No. of animals per sex per dose:

5/group

Control animals:

yes, concurrent vehicle

Positive control(s):

- Substance used as Positive Control: Cyclophosphamide
- Route of administration: by intraperitoneal injection
- Concentration: 10 mg/kg in 0.9% saline

Tissues and cell types examined:

Tissue: Bone marrow
Number of animals: 5 per group
Number of cells: 25 per animal
Time points: 24 h after treatment
Type of cells bone marrow
Parameters: numbers of sister chromatid exchanges

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:cell cycle kinetics were evaluated from 100 metaphases cells per animal and the appropriate doses and termination times were determined.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION: Slides were stained by a modification of the FPG (Fluorescence plus Giemsa) technique of Perry and Wolff (1974). The slides wre stained in Hoechst 33258.

METHOD OF ANALYSIS: the slides were exposed to black-light from a 15 W tube at 60°C for the same time required to detect SCE (15-30 minutes)

OTHER:

Evaluation criteria:

If an increase in SCE is observed, one of the following criteria must normally be met to assess the compound as positive:
- Two-fold increase: approximately a doubling in SCE frequency over the "background" (solvent and negative control) levels, at a minimum of three doses
-Dose response: a positive assessment may be made in the absence of a doubling if there is a statistically significant increase at a minimum of three doses and evidence for a positive dose response

In some cases, statistically significant increases are observed with neither a doubling or a dose response. These results are assessed according to repeatability, the magnitude of the response, and the proportion of the dose levels affected.

Statistics:

The SCE data generated in this study were analyzed by a parametric Analysis of Variance statistical test (Sokal and Rohlf, 1969). Individual animal results will be used as data points in the analysis.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

but the doses tested are high enough (limit of toxicity at 40 mg/kg)

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: the target dose levels of 10, 20, 40 and 60 mg/kg chosen for the preliminary study were based upon toxicity information gathered from a previous study of mouse bone marrow aberrations. 4 groups of 3 male mice per group were dosed with the test compound.
- Solubility: no data
- Clinical signs of toxicity in test animals: one animal was found dead at 40 mg/kg prior to colchicine administration.
- Evidence of cytotoxicity in tissue analyzed: the test compound caused no delay up to and including the target dose level of 40 mg/kg. there was however considerable cell cycle delay at 60 mg/kg.
- Rationale for exposure: based upon the evaluation of cell cycle kinetics data, target doses of 10, 20, 30 and 40 mg/kg were selected for the actual study.
- Harvest times: 24 h after dosing
- High dose with and without activation: no data
- Other:

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): The SCE frequencies were not significantly increased in any of the dose groups relative to the negative controls and were within the range of historical controls. The positive control showed a strongly and significantly increased SCE frequency relative to control (see table 7.6.2/1). Chlorine dioxide did not induce significant increases in SCEs at any of the doses tested.
- Induction of micronuclei (for Micronucleus assay): not applicable
- Ratio of PCE/NCE (for Micronucleus assay): not applicable
- Appropriateness of dose levels and route: no data
- Statistical evaluation:
- other: All animals in the test group exhibited hyperactive behaviour immediately after dosing, but shortly thereafter resumed normal behaviour.

Table 7.6.2/1:Table summarizing SCE frequencies in animals exposedin vivoto chlorine dioxide

 

Treatment	Dose mg/kg	No. of animals	No. of cells scored/animal	Total no. of cells scored	SCE frequency^d x ± S.E.
Deionized water	13.29	4^a	25^b	92	3.89 ± 0.26
Cyclophosphamide	10.00	5	25	125	13.15 ± 0.36*
Chlorine dioxide	9.04	5	25	125	3.50 ± 0.25
Chlorine dioxide	21.08	5	25	125	4.74 ± 1.29
Chlorine dioxide	28.02	4^c	25	100	3.73 ± 0.54
Chlorine dioxide	38.55	5	25	125	5.02 ±0.80

* significantly greater than negative control, p0.01

^aOne animal had cell cycle delay, all cells in M1

^bOne animal only 17 cells were scored

^cone animal died after dosing

^dmean SCE frequency / animal constituted a data point

Conclusions:

Chlorine dioxide is considered negative for inducing sister chromatid exchanges in male mice bone marrow under the test conditions.

Executive summary:

In an in vivo genetic toxicity study, Sister Chromatid Exchange (SCE) frequencies were determined in bone marrow cells in ICR male mice exposed to Chlorine Dioxide administered intraperitoneally as an acute dose, at dose levels of 9.04, 21.08, 28.02, 38.55 mg/kg in deionised water.

Animals were killed at 26 h post implantation, the optimum time for detection of SCE and the cells collected from the bone marrow. M2 cells were scored for the frequency of SCE per cell.

Concurrent vehicle mice were considered as a negative control. Cyclophosphamide was used as a positive control and was administered by injection at 10 mg/kg in 0.9% saline.

Genotoxicity of Chlorine Dioxide was found to be negative to bone marrow cells in ICR male mice and no toxicity effects were found.

Positive controls induced the appropriate response. 

The SCE frequencies were not significantly increased in any of the dose groups relative to the negative controls and were within the range of historical controls. The positive control showed a strongly and significantly increased SCE frequency relative to control. Chlorine dioxide did not induce significant increases in SCEs at any of the doses tested.

Under the test conditions, Chlorine dioxide is considered negative for inducing sister chromatid exchanges in male mice bone marrow.

This study is considered as acceptable as it satisfies with the requirements for Test Guideline OECD 479.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 12-19-1984 to 03-16-1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed similarly to the OECD guideline No. 475. No detail on the substance.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

: no detail on the substance item

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, New York
- Age/weight at study initiation:
Males: 34.19 g; range 35.5 g to 33.0 g, approximately 5 weeks old.
Females: 27.88 g; range 29.9 g to 26.6 g, approximately 5 weeks old.
- Assigned to test groups randomly: yes, under following basis: according to LBI Standard Operating Procedures (SOP).
- Fasting period before study: no data
- Housing: up to 15 mice per cage
- Diet (e.g. ad libitum): ad libitum (Purina Laboratory Chow)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
no data

IN-LIFE DATES: date of birth: 11 November 1983-Dosing date: 17 January 1984 (killed 6, 24 or 48 hrs after dosing)

Route of administration:

intraperitoneal

Vehicle:

Vehicle: Distilled water
Concentration in vehicle:
animals killed 6 hrs after the IP injection: 1.73, 5.15 and 15.44 mg/kg
animals killed 24 or 48 hrs after IP injection: 1.72, 5.18 and 16.11 mg/kg

Details on exposure:

Total volume applied:

Positive control:
males = 0.1 mL
females = 0.08 ml.
Negative control:
males = 0.17 mL
females = 0.14 ml
Test:
males = 0.17 mL
females = 0.14 ml

Duration of treatment / exposure:

No data

Frequency of treatment:

Number of applications: 1

Post exposure period:

6, 24, and 48 h

Remarks:

Doses / Concentrations:
1.73, 5.15 and 15.44 mg/kg for the 6 hour groups and 1.72, 5.18 and 16.11 mg/kg for the 24 h and 48 h dose groups.
Basis:
analytical conc.

No. of animals per sex per dose:

3/sex/group

Control animals:

yes, concurrent vehicle

Positive control(s):

Triethylenemelamine (TEM) at 1.5 mg/kg administered as a single intraperitoneal injection.

Tissues and cell types examined:

Tissue: Bone marrow
Number of animals: All animals
Number of cells: 500
Time points: 6, 24, 48 h
Type of cells Not specified
Parameters: Gaps, breaks, fragments, reunion figures
Aberrations included: chromatid gap, chromatid break, chromosome gap, chromosome break, chromatid deletion, fragments, acentric fragments, translocation, triradial, quadriradial, complex rearrangement, pulverised chromosomes, pulverised cells, ring chromosomes, dicentric chromosomes, minute chromosomes, double minute chromosomes, abnormal metacentric chromosomes, polyploidy, aneuploid (both hypo- and hyperploid) and more than 10 aberrations.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Doses were chosen following a range finding study for toxicity. Mortality of animals was measured after intraperitoneal adminsitration of chlorine dioxide and it was decided from the results obtained, to target the top test dose at 20.0 mg/kg and low doses to be one-third and one-tenth of the high dose, 7.0 and 2.0 mg/kg respectively.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION: The centrifugation was repeated and the pellet resuspended in fixative (methanol: acetic acid, 3:1). Cells in fixative were dropped onto glass slides and air-dried. Spreads were stained with 5% Giemsa at pH 6.8. After drying, slides were coverslipped using Depex.

METHOD OF ANALYSIS: Routinely, 50 spreads were read for each animal. The location of cells bearing abberations was identified by the use of coordinates on the mechanical stage. A mitotic index based on at least 500 cells counted was also recorded. It was calculated by scoring the number of cells in mitosis per 500 cells on each slide read.

OTHER:

Evaluation criteria:

A number of general guidelines was established to serve as an aid in determining the meaning of bone marrow chromosomal aberrations.

Statistics:

No data

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: First range finding study: 0.27, 0.96, 3.44, 8.37, 52.49 mg/kg. Second range-finding study: 16.87, 29, and 48.09 mg/kg.
- Solubility: no data
- Clinical signs of toxicity in test animals: in the first range finding study, all animals treated with 52.49 mg/kg died by the third day after dosing. In the second range finding study, all animals treated with 48.09 or 29 mg/kg died by the fifth day after dosing. One animal died at the lowest dose.
- Evidence of cytotoxicity in tissue analyzed: no data
- Rationale for exposure: It was decided considering the results obtained in the both range-finding study, to target the top test dose at 20.0 mg/kg, with the medium and low doses to be one-third and one-tenth of the high dose, 7.0 and 2.0 mg/kg respectively.
- Harvest times: no data
- High dose with and without activation: no data
- Other:

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): no data
- Induction of micronuclei (for Micronucleus assay): not applicable
- Ratio of PCE/NCE (for Micronucleus assay): not applicable
- Appropriateness of dose levels and route: no data
- Statistical evaluation: no data

Immediately after dosing all animals appeared normal. The day after dosing it was noted that the remaining high dose animals (24 and 48 hrs kill groups) appeared to have scruffy fur, however there were no mortalities. Two days after dosing 2 males from the 48 hr high dose group were found dead after administration of cochicine and before harvesting.

 

Table 7.6.2/1: Cytogenetic analysis in bone marrow cells of female mice treated with chlorine dioxide

 

Dose mg/kg	Kill Time (h)	No. of Animals	Total No. of cells	Aberrations Per Cell	Cells with Aberration (No.)	Cells with Aberration (%)	Aberration Frequency	Mitotic Index
1.73	6	5	250	0.000	0	0.0	0.000	4.1
5.15	6	5	250	0.000	0	0.0	0.004	5.9
15.44	6	5	250	0.000	0	0.0	0.004	4.1
1.72	24	5	250	0.000	0	0.0	0.004	7.0
5.18	24	5	250	0.000	0	0.0	0.000	6.5
16.11	24	5	250	0.008	2	0.8	0.008	6.1
1.72	48	5	250	0.008	2	0.8	0.020	4.6
5.18	48	5	250	0.000	0	0.0	0.016	6.2
16.11	48	4	200	0.000	0	0.0	0.010	3.8

 

 

Table 7.6.1/2: Cytogenetic analysis in bone marrow cells of male mice treated with chlorine dioxide

 

Dose mg/kg	Kill Time (h)	No. of Animals	Total No. of cells	Aberrations Per Cell	Cells with Aberration (No.)	Cells with Aberration (%)	Aberration Frequency	Mitotic Index
1.73	6	5	250	0.000	0	0.0	0.004	5.1
5.15	6	5	250	0.000	0	0.0	0.000	4.4
15.44	6	5	250	0.004	1	0.4	0.004	3.1
1.72	24	5	250	0.008	2	0.8	0.008	5.0
5.18	24	5	250	0.008	2	0.8	0.024	6.5
16.11	24	5	250	0.020	4	1.6	0.016	7.6
1.72	48	5	250	0.004	1	0.4	0.000	5.3
5.18	48	5	250	0.000	0	0.0	0.000	5.8
16.11	48	4	150	0.007	1	0.7	0.000	4.5

Conclusions:

Under the test conditions, chlorine dioxide did not induce chromosomal aberrations in the mouse bone marrow cells using the in vivo chromosomal aberration test .

Executive summary:

In an in vivo mammalian bone marrow chromosome aberration study, performed similarly to the OECD guideline No. 475, male and female CD-1 mice were exposed to Chlorine Dioxide by intraperitoneal injection at concentrations of 1.73, 5.15 and 15.44 mg/kg for the 6 hour groups and 1.72, 5.18 and 16.11 mg/kg for the 24 h and 48 h groups. Test substance was diluted in distilled water. Animals were sacrificed 6, 24 or 48 hrs after the administration of the substance.

Concurrent vehicle animals were used as negative control and Triethylenemelamine (TEM) at 1.5 mg/kg administered as a single intraperitoneal injection was used as a positive control. The controls gave acceptable results for chromosome aberrations.

500 cells of bone marrow of all animals were examined at 6, 24 and 48 hours for gaps, breaks, fragments and reunion figures.

The results for chlorine dioxide were negative and no toxicity effects were observed.

Under the test conditions, chlorine dioxide did not induce chromosomal aberrations in the mouse bone marrow cells using the in vivo chromosomal aberration test .

This study is classified as acceptable as it satisfies with the requirements for Test Guideline OECD 475 for Mammalian Bone Marrow Chromosome Aberration Test.

Reference 3

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 5-11-1984 to 6-20-1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

Deviations:

yes

Remarks:

: number of males inferior as what is advocated in guideline No.478, females are housed with treated males for a total of 5 days.

Principles of method if other than guideline:

The potential of Chlorine dioxide to produce germ cell mutations in male mice was evaluated for the mouse dominant lethal assay. Chlorine dioxide was given to male mice as an aqueous solution by intraperitoneal injection, at either 2, 7, or 20 mg/kg. Three days after dosing, the males were sequentially mated to two females per week for 4 weeks.

GLP compliance:

not specified

Type of assay:

rodent dominant lethal assay

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, New York, USA.
- Age at study initiation: Young adults, 6 weeks of age when received
- Weight at study initiation: male mice weighed 31.5 g (mean) before the dosing
- Assigned to test groups randomly: yes, under following basis: according to Litton Bionetics, Inc. (LBI) Standard Operating Procedures (SOP) No. 303.
- Fasting period before study: no data
- Housing: up to 15 mice per cage
- Diet (e.g. ad libitum): ad libitum(Purina Certified Laboratory Chow) (unless contraindicated by the particular experimental design).
- Water (e.g. ad libitum): ad libitum (unless contraindicated by the particular experimental design).
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs-12 hrs

IN-LIFE DATES: no data

Route of administration:

intraperitoneal

Vehicle:

Glass-distilled, deionized water

Details on exposure:

Dose applied: 2, 7, 20 mg/kg

Duration of treatment / exposure:

No data

Frequency of treatment:

Number of applications: 1

Post exposure period:

Females were killed 14 days after the midweek of mating.

Remarks:

Doses / Concentrations:
0.290; 1.020 and 2.895 mg/mL
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
2.786; 0.967 and 0.264 mg/mL
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
1.84; 6.76 and 19.46 mg/kg
Basis:
analytical conc.

No. of animals per sex per dose:

12 males /group

Control animals:

yes, concurrent vehicle

Positive control(s):

Triethylenemelamine (TEM) at 0.3 mg/kg as an aqueous solution.

Tissues and cell types examined:

Clinical signs: Premature death at 20 mg/kg

Tissue: Germ cells
Number of animals: 12 or 15 animals per dose level
Number of cells: N/A
Time points: 1, 2, 3 and 4 weeks post dosing
Type of cells male germ cells
Parameters: For each pregnant female, the number of living, dead and total implantations counted and recorded.

Details of tissue and slide preparation:

No data

Evaluation criteria:

No data

Statistics:

No data

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

20 mg/kg was a lethal dose, causing death in 47% of the mice

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Chlorine dioxide at 20 mg/kg was a lethal dose, causing death in 47% of the mice and a significant decrease in male fertility in the surviving mice at mating weeks 1, 2 and 4. It is unknown whether the decrease in male fertility was a direct effect on the male reproductive system or an indirect effect due to systemic toxicity. ClO2 at 2 and 7 mg/kg did not cause any observable signs of toxicity and did not affect male fertility. Compared to the negative control, ClO2 at either 2, 7, or 20 mg/kg did not decrease the number of total implants per female, did not increase the mean number of dead implants per female, did not increase the proportion of females with one or more, or two or more dead implants and did not result in greater mutation index (ratio of dead implants to total implants). Chlorine dioxide was not mutagenic under the conditions tested in the mouse dominant lethal assay.

Table 7.6.2/1: Results of the Mouse dominant lethal assay

 

Week	Parameter	Negative control	ClO2 (2 mg/kg)	ClO2 (7 mg/kg)	ClO2 (20 mg/kg)	Positive control
	Total number of males dosed	12	12	12	15	12
	Number of males dead	0	0	0	7	0
1	Number of mated females	24	24	24	24	24
	Number of pregnant females	21	21	19	3	14
	Mean of implants per female	10.76	10.81	11.47	10.67	7.86*
	Mean of dead implants	0.24	0.81**	0.47	1.33	6.50*
	Proportion of females with 1 or more dead implant^a	5	12	6	3	14
2	Number of mated females	24	24	24	18	24
	Number of pregnant females	22	23	21	6	7
	Mean of implants per female	10.91	12.09	12.29	11.00	3.86*
	Mean of dead implants	0.59	0.61	0.52	0.50	2.86*
	Proportion of females with 1 or more dead implant^a	9	10	8	2	7
3	Number of mated females	24	24	24	18	24
	Number of pregnant females	21	22	23	10	18
	Mean of implants per female	12.52	12.68	13.22	10.80	11.22
	Mean of dead implants	0.62	0.77	0.83	0.80	3.06*
	Proportion of females with 1 or more dead implant^a	9	14	11	6	15
4	Number of mated females	24	24	24	16	24
	Number of pregnant females	22	19	22	9	21
	Mean of implants per female	12.27	12.53	12.73	11.89	11.33
	Mean of dead implants	0.59	1.16	1.23	0.67	0.76
	Proportion of females with 1 or more dead implant^a	10	11	13	5	11

^a: Total number of females with one or more dead implants for all pregnant females in the study group.

*: Significantly greater (P<0.01) in comparison to the negative control by Dunett’s T-test

**: Significantly greater (P<0.05) in comparison to the negative control by Dunett’s T-test

Conclusions:

Under the test conditions, the chlorine dioxide was not mutagenic in the mouse dominant lethal assay.

Executive summary:

In an in vivo mouse dominant lethal assay, performed according to the OECD guideline No. 478, male CD-1 mice were exposed to Chlorine Dioxide by a single intraperitoneal injection at doses of 2, 7 and 20 mg/kg bw.

Vehicle was glass-distilled, deionized water. Concurrent vehicle animals were used as a negative control and the positive control was Triethylenemelamine (TEM) at 0.3 mg/kg as an aqueous solution. The controls gaven acceptable results.

Male germ cells were examined at 1, 2, 3 or 4 week post dosing, and for each pregnant female, the number of living, dead and total implantations was counted and recorded.

Genotoxicity results were negative for Chlorine Dioxide to male CD-1 mice whereas toxicity results were positive. Chlorine dioxide at 20 mg/kg was a lethal dose, causing death in 47% of the mice and a significant decrease in male fertility in the surviving mice at mating weeks 1, 2 and 4. It is unknown whether the decrease in male fertility was a direct effect on the male reproductive system or an indirect effect due to systemic toxicity.

ClO2 at 2 and 7 mg/kg did not cause any observable signs of toxicity and did not affect male fertility. Compared to the negative control, ClO2 at either 2, 7, or 20 mg/kg did not decrease the number of total implants per female, did not increase the mean number of dead implants per female, did not increase the proportion of females with one or more, or two or more dead implants and did not result in greater mutation index (ratio of dead implants to total implants).

Under the test conditions, the chlorine dioxide was not mutagenic in the mouse dominant lethal assay.

This study is classified as acceptable as satisfies with the requirements for Test Guideline OECD 478 for Rodent Dominant Lethal Test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

According to the Column 1 of the REACH regulation annex VII, an in vitro gene mutation study in bacteria is required (section 8.4.1).

No reliable bacterial assays for chlorine dioxide were available. However, chlorine dioxide is a powerful oxidising agent and is inherently toxic to bacteria as shown by its use as disinfectant in the drinking water. Considering both the bacteriostatic effects of the chemical and the availability of the whole reliable in vitro and the in vivo tests (see below), it is concluded that the information from a bacterial mutagenic test like the Ames' test is not be required for the assessment of chlorine dioxide genotoxicity. Therefore, an in vitro gene mutation study in bacteria using chlorine dioxide is scientifically unjustified.

 

Regarding the in vitro genotoxicity, two studies were identified as key study:

-      The mouse lymphoma assay (Cifone, 1986) using mice L5178Y cells gave positive results for reverse gene mutations.

-      The chromosome aberration assay (Ivett, 1986) performed on CHO cell lines gave positive results.

 

The conclusion from the results obtained from both of the available in vitro studies was positive. However, considering the strong reactivity of the chlorine dioxide, the positive results observed in vitro were likely due to the in vitro formation of reactive oxygen species which were well known to be genotoxic. In addition, it is also possible that the chromosome aberration test included the evaluation of dose levels that would be considered to be excessively toxic using modern criteria, and the mouse lymphoma assay would be judged as borderline or equivocal positive using modern, globally accepted, evaluation criteria. In this case both results may be considered to be potential false positives caused by excessive toxicity. Therefore, it is not possible to conclude on the genotoxicity potential of chlorine dioxide regarding the in vitro tests only.

 

In vivo, three studies were identified as 'key study'.

-      The sister chromatid exchange (SCE) assay (Ivett, 1984) was performed by the administration of ClO₂ intraperitoneally to male mice. SCE were observed 26 hrs after dosing in the bone marrow tissue of mice. No significant increase in SCE frequency was observed in any of the dose groups relative to the negative controls and the frequencies were within the range of historical controls.

-      The chromosome aberration assay (Ivett, 1984) was performed by the administration of ClO₂ intraperitoneally to mice. Chromosome aberrations were observed 6, 24 and 48 hrs after dosing in the bone marrow tissue of mice. No significant increase in the frequency of chromosomal aberrations was observed.

-      The dominant Rodent lethal assay (Moore, 1984) was performed by the administration of ClO₂ intraperitoneally to mice. Matings were conducted in order to study the consequences of chromosome aberrations in germ cells. Chlorine dioxide was not mutagenic, under the conditions tested, in the mouse dominant lethal assay.

 

The results obtained in vitro and in vivo are different. However according to the ECHA guidance on "information requirements and chemical safety assessment" (R.7a), the results of in vivo tests possess a higher degree of reliability. Indeed, the use of immortalized cell lines such as CHO or L5178Y can give false positive results because of impaired p53 checkpoint and repair pathway processes. In addition, the use of modern evaluation criteria for both of the in vitro assays would likely result in a conclusion of negative, equivocal, or borderline positive. The results obtained in vitro with chlorine dioxide can therefore be considered to be false positive, likely resulting from the reactive oxygen species or from the evaluation of dose levels exhibiting excessive toxicity. The toxicokinetic data on chlorine dioxide indicated that chlorine dioxide and its metabolite, the chlorite, were distributed in the bone marrow. Therefore, the negative results obtained in vivo after an intraperitoneal administration of chlorine dioxide cannot be attributed to a lack in target tissue exposure.

  

In conclusion, chlorine dioxide is not classified as genotoxic considering the overall results from the in vivo studies.

Justification for classification or non-classification

Harmonised classification:

The classification entered in the Annex VI to the CLP Regulation for Chlorine dioxide is not harmonised for the mutagenicity category.

Self-classification:

Based on the above data no additional self-classification is proposed.