Genetic toxicity: in vitro.004 to 008

Genetic toxicity: in vitro.004

Administrative data

Study result type:

experimental result

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

Study well conducted that meets basic scientific principles, but study non-GLP and no details on the substance.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2004

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The single cell gel electrophoresis or comet assay was performed on mammalian leukocytes. The comet assay was adapted from Singh et al., 1988. The test allow to detect DNA damage as strand breaks, alkali-labile site and cross-linking, and incomplete excision repair sites in individual cells.

GLP compliance:

no data

Type of genotoxicity:

DNA damage and/or repair

Type of study:

single cell gel/comet assay in mammalian cells for detection of DNA damage

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:

other: leukocytes isolated from whole blood of 3 healthy non-smoking donors

Details on cell lines (if applicable):

- Type and identity of media: RPMI 1640
- Properly maintained: no
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

without

Metabolic activation system:

not applicable

Test concentrations:

0, 0.1, 0.2, 0.5, 1 and 5 ppm

Controls

Negative controls:

no

Solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: 2mM

Results and discussion

Test results

Species / strain / cell line:

other: leukocyte

Metabolic activation:

not applicable

Genotoxicity:

positive

Remarks:

See details in Table 7.6.1/1

Cytotoxicity:

no

Remarks:

See details in Table 7.6.1/1

Vehicle controls valid:

yes

Negative controls valid:

not applicable

Positive controls valid:

yes

Any other information on results incl. tables:

Table 7.6.1/1: Cytotoxic and genotoxic effects in human leukocytes induced by Chlorine Dioxide

Dose (ppm)	Cytotoxicity (cell survival %)	Genotoxicity (median tail length (µm)
0	98 ± 2	7.65 ± 0.32
0.1	97 ± 4	7.82 ± 0.39
0.2	96 ± 2	8.63 ± 0.38*
0.5	94 ± 4	10.21 ± 0.51**
1	95 ± 4	10.57 ± 0.53**
5	91 ± 5	12.45 ± 0.60**

*: P<0.05 (Dunnett’s C multiple comparison analysis)

**: P<0.001 (Dunnett’s C multiple comparison analysis)

The median tail length of the positive control was 53.13 +/- 5.71 µm.

Applicant's summary and conclusion

Conclusions:

No significant cytotoxic effects were observed in the comet assay whereas a significant DNA migration increase was observed from the dose of 0.2 ppm. In conclusion, Chlorine dioxide caused DNA damage in human leukocytes.

Executive summary:

An in vitro DNA damage assay was conducted in using the method of comet assay (also called Single cell gel electrophoresis). Human leukocytes were isolated from whole blood of three non-smoking donors and were exposed to Chlorine Dioxide for 1 hour at 37°C at different doses (0, 0.1, 0.2, 0.5, 1, and 5 ppm). Then the comet assay was performed in order to detect DNA damage after the lysis of the cells and the migration of the damaged DNA. The tail length was measured and used to determine if the test item was genotoxic or not. Ethyl methane sulfonate was used as positive genotoxic control. The results obtained for this positive control were acceptable.

Under the test conditions, chlorine dioxide was not cytotoxic but it was genotoxic as a DNA migration increase was observed from the dose of 0.2 ppm.

However, the COMET assay is not validated according to the EU or OECD guideline, even if the test can measure the DNE strand breaks in any tissue of an animal and may, therefore, also be used in specific site of contact tissues.

Interpretation of results

Interpretation of results:

positive

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment

Genetic toxicity: in vitro.005

Administrative data

Study result type:

experimental result

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

The study is not performed on the substance itself but on reactive product of chlorine dioxide with amino acids or peptides.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1987

Materials and methods

Type of genotoxicity:

gene mutation

Type of study:

bacterial reverse mutation assay (e.g. Ames test)

Results and discussion

Applicant's summary and conclusion

Executive summary:

21 amino acids and 3 peptides were studied for their reactivity with Chlorine dioxide (ClO2). ClO2 reacted only with 6 amino acids in 0.1 mM sodium phosphate buffer, pH 6.0. ClO2 readily reacted with L-glycyl-L-tryptophan and L-tryptophylglycine but not with aspartame. Mutagenicity studies with the Salmonella microsome assay of the reaction mixtures of chlorine dioxide with those 6 reactive amino acids and the 3 peptides indicated that the reaction products of the 3 peptides, hyrdroxyproline, and tyrosine exerted mutagenic activity toward both tester strains of TA98 and TA100 in the presence and absence of rat-liver S9 mix.

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

Genetic toxicity: in vitro.006

Administrative data

Purpose flag:

key study

Study result type:

experimental result

Study period:

1983/11/16-1984/02/01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report Date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to

Guideline:

OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

: no detail on the substance

Principles of method if other than guideline:

Not applicable

GLP compliance:

no data

Type of genotoxicity:

gene mutation

Type of study:

mammalian cell gene mutation 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

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: Yellow liquid
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: assumed to be stable during the test (sponsor responsibility)
- Storage condition of test material: no data
- Other: received on 1983/11/15, the test item was dissolved in phosphate beffered saline (PBS). TEst chemical are prepared fresh for each mutagenesis experiment. Neutralization with HCl or NaOH is performed if necessary to maintain the desired pH range (between 7.0 and 7.4).

Method

Target gene:

Tymidine Kinase locus

Species / strain / cell line

Species / strain / cell line:

mouse lymphoma L5178Y cells

Details on cell lines (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 characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix of rat liver homogenate and necessary cofactors

Test concentrations:

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:

- Vehicle(s)/solvent(s) used: phosphate buffered saline (PBS)
- 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: 4 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): no data
- Fixation time (start of exposure up to fixation or harvest of cells): no data

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

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 1 x 106 cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

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

Evaluation criteria:

- The average absolute cloning efficiency of the negative controls should be between 60 and 130%.
- The normal range of background frequencies for assays performed with different cell stocks is 10x10-6 to 100x10-6. 
- The minimum acceptable mutant frequency induced by 0.3 µL/mL EMS is 200x10-6, for 2.5 µg/mL of MCA the minimum is 200x10-6.
- Because of the fact that mutant frequencies increase as a function of lethality, an attempt to obtain treatments in the range of 10 to 20% relative growth must be made for an assay to be considered conclusive.
- An experimental mutant frequency will be considered acceptable for evaluation only if the relative cloning efficiency is 10% or greater and the total number of viable clones exceeds about 30.
The minimum criteria considered necessary to demonstrate mutagenesis for any given treatment will be a mutant frequency that is at least 150% on the concurrent background frequency plus 10x10-6. The background frequency is defined as the average mutant frequency of the solvent negative controls.

Statistics:

No data

Results and discussion

Test resultsopen allclose all

Test results 1

Test results 2

Additional information on results:

Chlorine dioxide induced significant increases in the mutant frequency at the TK locus in L5178Y TK +/- mouse lymphoma cells. Under non-activation conditions, dose-dependent increases in the mutant frequency were induced from 3.2 to 24.3 μg/mL. In the presence of S9 metabolic activation system, chlorine dioxide was converted to a slightly less toxic and less active form or forms. At 48.3 μg/mL, where the test material was moderately toxic, a 2.7-fold increase in the mutant frequency was induced. Chlorine dioxide is therefore considered active in the mouse lymphoma forward mutation assay in the presence and absence of metabolic activation.

Any other information on results incl. tables:

Table 7.6.1/2:             Table Summarising Mouse Lymphoma Results

Test condition	Daily cell counts		Suspension growth	Total Mutant Colonies	Total Viable Colonies	Cloning Efficiency	Relative Growth	Mutant Frequency (10E-6)
	1	2
Without activation
Solvent Control	9.4	7.37	7.6	51	19	73.0	100.0	23.3
Solvent Control	8.7	7.7	7.4	89	549	83.0	100.0	35.7
Solvent Control	8.3	10.5	9.7	61	204	68.0	100.0	29.9
EMS 0.25 μL/mL	5.9	9.1	6.0	570	121	40.3	39.3	471.1
EMS 0.4 μL/mL	5.2	8.0	4.6	617	96	32.0	24.1	642.7
Test compound			Relative to Control (%)
0.32	6.2	10.9	91.6	56	194	86.6	79.3	28.9
3.2	6.3	6.3	53.8	134	243	108.4	58.3	55.1
6.73	4.9	7.9	52.5	117	194	86.6	45.5	60.3
11.1	5.8	8.3	65.2	181	233	104.0	67.8	77.7
14.9	2.2*	6.6	26.8	174	152	67.8	18.2	114.5
24.3	2.5*	5.0	20.3	180	147	65.6	13.3	122.4
36.9	1.5*	2.7+	Too toxic to clone
With activation
Solvent Control	8.1	8.1	7.3	182	304	101.3	100.0	59.9
Solvent Control	7.3	13.9	11.3	171	264	88.0	100.0	64.8
Solvent Control	10.0	7.8	8.7	162	274	91.3	100.0	59.1
MCA 2.5 μg/mL	9.0	6.0	6.0	506	136	45.3	32.0	372.1
Test compound			Relative to Control (%)
6.73	9.3	7.3	82.9	180	286	102.0	84.6	62.9
14.9	7.8	9.8	93.3	144	224	79.9	74.5	64.3
18.5	9.0	10.1	111.0	122	219	78.1	86.7	55.7
30.9	6.8	7.1	58.9	289	285	101.6	59.8	101.4
36.9	4.6	12.3	69.1	194	237	84.5	58.4	81.9
48.3	4.7	5.7	32.7	452	273	97.3	31.8	165.6
65.2	3.0*	2.0+	Too toxic to clone

*: not split back

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

In a in vitro mammalian cell gene mutation assay at the thymidine kinase (TK) locus, performed similarly to the OECD guideline No. 476, cultured mouse lymphoma L5178Y cells were exposed to Chlorine Dioxine, at concentrations of 6.73, 14.9, 18.5, 30.9, 36.9, 48.3, 65.2µg/mL in the presence of mammalian metabolic activation S9 mix, and at concentrations of1.32, 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).

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 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 informatio

Genetic toxicity: in vitro.007

Administrative data

Data waiving:

study scientifically unjustified

Data source

Materials and methods

Results and discussion

Genetic toxicity: in vitro.008

Administrative data

Study result type:

experimental result

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

Study performed similarly to the OECD guideline No. 471 but there are few informations on the study design and on the test substance.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

: only two strains tested (TA98 and Ta100), no information on the cytotoxicity

Principles of method if other than guideline:

Not applicable

GLP compliance:

no data

Type of genotoxicity:

gene mutation

Type of study:

bacterial reverse mutation assay (e.g. Ames 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

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: 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: The concentrated drinking water samples to be used for toxicological testing were collected and concentrated at a pilot-scale drinking water treatment plant in Jefferson Parish, LA, USA. The influent stream was split into 5 separate process streams. The process streams were untreated or disinfected etiher with ozone, chlorine dioxide (ClO2), monochloramine, or with chlorine. The stream disinfected with ClO2 contained 0.5 mg/L ClO2 residual after the treatment. Then two methods, reverse osmosis and macroreticular resin process were used to collect and concentrate organic compounds from drinking water before toxicological testing.
For the inititation/promotion assay, the study was performed on disinfected water with ClO2 and concentrated via reverse osmosis.

Method

Target gene:

Histidine gene

Species / strain / cell lineopen allclose all

Species / strain / cell line 1

Species / strain / cell line 2

Test concentrations:

0.025, 0.050, 0.100, 0.250, 0.500 and 1.000 mL of the reverse osmosis concentrate

Vehicle:

no data

Details on test system and conditions:

no details

Evaluation criteria:

no data

Statistics:

no data

Results and discussion

Test resultsopen allclose all

Test results 1

Test results 2

Additional information on results:

no additional information

Any other information on results incl. tables:

Table 7.6.1/1:Number of histidine revertants after a chlorine dioxide treatment. Duplicate experiment using 2 plates per dose.

Test compound	Dose level	TA98		TA100
		- S9	+ S9	- S9	+ S9
Negative control	-	24	26	146	135
Sodium azide *	1 µg	-	-	702	-
2-Nitrofluorene *	5 µg	841	-	-	-
2-Aminoanthracene *	1 µg	-	302	-	587
Chlorine dioxide	0.025 mL	26	36	171	150
	0.050 mL	27	29	146	155
	0.100 mL	23	35	159	150
	0.250 mL	25	32	143	140
	0.500 mL	23	28	150	135
	1.000 mL	19	29	130	135

*: used as positive control

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the chlorine dioxide is not mutagenic.

Executive summary:

In an in vitro mutagenic assay, performed similarly to the OECD guideline No. 471, Chlorine dioxide treated water was incubated at different doses (0, 0.025, 0.05, 0.1, 0.250, 0.5 and 1 mL) with two strains of Salmonella typhimurium (TA98 or TA100). The incubation was performed with or without the presence of metabolic activation (S9 mix).

Positive controls were used (sodium azide, 2 -Nitrofluorene, 2 -aminoanthracene) and gave acceptable results in terms of the number of histidine revertants.

Under the test conditions, chlorine dioxide water was not mutagenic.

Interpretation of results

Interpretation of results:

negative

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assig

n without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.