Genetic toxicity: in vitro.001 to 003

Genetic toxicity: in vitro.001

Administrative data

Purpose flag:

supporting study

Study result type:

experimental result

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2004

Materials and methods

Test guideline

Qualifier:

equivalent or similar to

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

no data

Type of genotoxicity:

gene mutation

Type of study:

other: in vitro gene mutation assay in yeast

Test materials

Identity of test material same as for substance defined in section 1 (if not read-across):

yes

Test material identityopen allclose all

Test material identity 1

Test material identity 2

Test material identity 3

Details on test material:

- Name of test material (as cited in study report): chlorine Dioxide
- Physical state: no data
- 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: source: Caffaro, Brescia, Italy

Method

Target gene:

Not applicable

Species / strain / cell line

Species / strain / cell line:

Saccharomyces cerevisiae

Details on cell lines (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 characteristics:

other: D7 strain

Metabolic activation:

with and without

Metabolic activation system:

P450 activation

Test concentrations:

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

Controlsopen allclose all

Controls 1

Controls 2

Controls 3

Results and discussion

Test results

Species / strain / cell line:

Saccharomyces cerevisiae

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity:

yes

Vehicle controls valid:

yes

Negative controls valid:

not applicable

Positive controls valid:

yes

Any other information on results incl. tables:

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

Applicant's summary and conclusion

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.

Interpretation of results

Interpretation of results:

negative

Remarks:

with and without metabolic activation

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Administrative data

Purpose flag:

key study

Study result type:

experimental result

Study period:

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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.

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 473 (In vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

: no detail on the substance

Principles of method if other than guideline:

Not applicable

GLP compliance:

no data

Type of genotoxicity:

chromosome aberration

Type of study:

in vitro mammalian chromosome aberration test

Test materials

Identity of test material same as for substance defined in section 1 (if not read-across):

yes

Test material identityopen allclose all

Test material identity 1

Identifier:

CAS number

Identity:

10049-04-4

Test material identity 2

Identifier:

EC number

Identity:

233-162-8

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 dissolved in phosphate buffered saline at a final concentration of 2.227 mg/mL.

Method

Target gene:

Not applicable

Species / strain / cell line

Species / strain / cell line:

Chinese hamster Ovary (CHO)

Details on cell lines (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 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:

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:

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

Controlsopen allclose all

Controls 1

Controls 2

Details on test system and 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%.

Results and discussion

Test results

Species / strain / cell line:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Test system:

all strains/cell types tested

Genotoxicity:

positive

Remarks:

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

Cytotoxicity:

yes

Remarks:

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

Vehicle controls valid:

yes

Negative controls valid:

yes

Positive controls valid:

yes

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

Any other information on results incl. tables:

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/mLa

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/mLa

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

Applicant's summary and conclusion

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).

Interpretation of results

Interpretation of results:

positive

Remarks:

with and without metabolic activation

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verifi

Genetic toxicity: in vitro.002

Administrative data

Purpose flag:

supporting study

Study result type:

experimental result

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2004

Materials and methods

Test guideline

Qualifier:

equivalent or similar to

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

no data

Type of genotoxicity:

gene mutation

Type of study:

other: in vitro gene mutation assay in yeast

Test materials

Identity of test material same as for substance defined in section 1 (if not read-across):

yes

Test material identityopen allclose all

Test material identity 1

Test material identity 2

Test material identity 3

Details on test material:

- Name of test material (as cited in study report): chlorine Dioxide
- Physical state: no data
- 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: source: Caffaro, Brescia, Italy

Method

Target gene:

Not applicable

Species / strain / cell line

Species / strain / cell line:

Saccharomyces cerevisiae

Details on cell lines (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 characteristics:

other: D7 strain

Metabolic activation:

with and without

Metabolic activation system:

P450 activation

Test concentrations:

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

Controlsopen allclose all

Controls 1

Controls 2

Controls 3

Results and discussion

Test results

Species / strain / cell line:

Saccharomyces cerevisiae

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity:

yes

Vehicle controls valid:

yes

Negative controls valid:

not applicable

Positive controls valid:

yes

Any other information on results incl. tables:

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

Applicant's summary and conclusion

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.

Interpretation of results

Interpretation of results:

negative

Remarks:

with and without metabolic activation

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Genetic toxicity: in vitro.003

http://www.echa.europa.eu/de/web/guest/registration-dossier/-/registered-dossier/15450/7/7/2

Administrative data

Purpose flag:

supporting study

Study result type:

experimental result

Study period:

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report Date:

1986

Materials and methods

Test guideline

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 genotoxicity:

other:

Type of study:

in vitro mammalian cell transformation assay

Test materials

Identity of test material same as for substance defined in section 1 (if not read-across):

yes

Test material identityopen allclose all

Test material identity 1

Identifier:

CAS number

Identity:

10049-04-4

Test material identity 2

Identifier:

EC number

Identity:

233-162-8

Test material identity 3

Identifier:

IUPAC name

Identity:

Chlorine dioxide

Details on test material:

- Name of test material (as cited in study report): chlorine Dioxide
- Physical state: Clear, pale-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 1983/11/11

Method

Target gene:

Not applicable

Species / strain / cell line

Species / strain / cell line:

other: Clone 1-13 of Balb/c-3T3 mouse cells

Details on cell lines (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 characteristics:

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

Metabolic activation:

without

Test concentrations:

0.5, 1.0, 2.0, 3.0, 5.0, 6.0 μg/mL

Vehicle:

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

Controls

Negative controls:

no

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 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.

Results and discussion

Test results

Species / strain / cell line:

other: Clone 1-13 of Balb/c-3T3 mouse cells

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity:

yes

Vehicle controls valid:

yes

Negative controls valid:

not applicable

Positive controls valid:

yes

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.

Any other information on results incl. tables:

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)

Applicant's summary and conclusion

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.

Interpretation of results

Interpretation of results:

negative