Chlorine dioxide

Endpoint summary

Description of key information

Repeated dose toxicity (oral): NOAEL = 200 mg/L (11.5 mg/kg bw/d) (K, reliability 2) i.e. the highest dose tested

Repeated dose toxicity (inhalation): LOAEC = 2.8 mg/m³ (1ppm) (WoE, reliability 4)

Repeated dose toxicity (dermal): a repeated dose toxicity study by dermal route does not need to be provided because short term toxicity was already assessed via oral and inhalation route.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions: there is no satellite group; ophtalmological and neurobehavioral examinations were not performed. Details on test material are not available.

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 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

there is no satellite group; ophtalmological and neurobehavioral examinations were not performed

Principles of method if other than guideline:

A subchronic study was performed to assess the effect of chlorine dioxide administered in drinking water to male and female Sprague-Dawley rats for a period of 90 consecutive days.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, Mich.
- Age at study initiation: approximately 70 days old at reception
- Weight at study initiation: males 300-325 g, females: 225-250 g at reception
- Fasting period before study:
- Housing: two per cage by sex in hanging polycarbonate cages containing hardwood chip bedding
- Diet: commercial rodent food ad libitum
- Water: drinking water ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-22 °C
- Humidity: 40-60 %
- Air changes (per hr): no data
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: no data

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

The chlorine dioxide solution was synthesized by purging ClO2 from an acidified sodium chlorite (NaClO2) generator through an absorbent NaClO2 solid column into distilled deionized water.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The chlorine dioxide concentrations were confirmed spectrophotometrically.
The concentration and purity of the chlorine dioxide solutions were determined before the test chemical was given to the animals and at the time of refilling bottles to determine the extent of degradation.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Continuous

Remarks:

Doses / Concentrations:
0, 25, 50, 100 and 200 mg/L
Basis:
nominal in water

Remarks:

Doses / Concentrations:
0; 2.4; 4.6; 8.2; 14.9 mg/kg/day
Basis:
other: actual ingested (females)

Remarks:

Doses / Concentrations:
0; 1.9; 3.6; 6.2 and 11.5 mg/kg bw/d
Basis:
other: actual ingested (males)

No. of animals per sex per dose:

10 rats/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): random

Positive control:

No data

Observations and examinations performed and frequency:

CLINICAL SIGNS AND MORTALITY: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: initially, then weekly throughout the study and at termination

FOOD CONSUMPTION: Yes
- Time schedule: weekly

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: 3 times a week

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: Yes (pentobarbital; 60 mg/kg, intraperitoneally)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 7.5.1/1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 7.5.1/1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 7.5.1/2)
HISTOPATHOLOGY: Yes (see table 7.5.1/2)

Other examinations:

No data

Statistics:

A one factor (dosage) variance (ANOVA) was used to analyze normally distributed measures: body weights, organ weights, organ weight ratios, food and water consumptions, haematology, and clinical chemistry. When a treatment effect was noted (α = 0.05 F test) the difference between control and treatment groups were examined using Tukey’s multiple comparison procedure. A Kruskal-Wallis test was used to determine differences among the groups. Incidence of histopathlogical lesions was analyzed by a Fisher Exact test.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decreased final body weights at 200 mg/L

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

significantly reduced at 200 mg/L

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

dose-related decrease

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no mortalities attributable to 90 days of dosing with any concentration of chlorine dioxide employed.

BODY WEIGHT AND WEIGHT GAIN
Both sexes showed significantly decreased final body weights at 200 mg/L and the mean weight gain was also significantly reduced at 200 mg/L for both sexes.

FOOD CONSUMPTION
Average daily food consumption of the males was significantly reduced at 200 mg/L (26.2 ± 1.0 versus 29.0 ± 1.7 g/day).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
The daily water consumption decreased in both sexes in a dosage related fashion, achieving statistical significance at 25, 50, 100 and 200 mg/L in females and 50, 100 and 200 mg/L in males. Average daily doses calculated from water consumption and body weight data for the 25-, 50-, 100- and 200-mg/L groups were, respectively, 2.4, 4.6, 8.2 and 14.9 mg/kg/d for females, and 1.9, 3.6, 6.2, and 11.5 mg/kg/d for males.

HAEMATOLOGY
Haematological analyses indicated the females had decreased Hgb and Hct values at 25 mg/l and the WBC differential showed an increased percentage of lymphocytes (82.5 ± 10 versus 70.3 ± 7.6 %) but a decreased percentage of neutrophils (11.8 ± 8.6 versus 22.9 ± 7.4 %) at 200 mg/l.

CLINICAL CHEMISTRY
Clinical serum chemistry showed several differences in both sexes compared with the controls. Females had significantly decreased Creat levels at 50 and 200 mg/L but exhibited significantly increased PO4 levels after drinking 100 mg/L. In males, Creat was significantly increased at 100 and 200 mg/L and PO4 was increased at 100 mg/L but not at the highest concentration. Decreased levels of AST and LDH at 100 and 200 mg/L and of ALT at 25 and 50 mg/L were also noted in males.

ORGAN WEIGHTS
Mean absolute spleen weights were significantly depressed at all dosages whereas liver weights were depressed in males at 50, 100, 200 mg/L and females at 100 mg/L. At the 200 mg/L concentration, the mean relative kidney weight of females (0.78 ± 0.06 versus 0.67 ± 0.05 g) and the relative brain weight of males (0.43 ± 0.04 versus 0.37 ± 0.04 g) were significantly increased.

GROSS PATHOLOGY AND HISTOPATHOLOGY
Histopathological examination identified the nasal cavity as the target tissue. The histopathological changes seen in both sexes at all chlorine dioxide treatment levels were characterised by various types of inflammation ranging from acute to chronic or active, goblet cell and epithelial cell hyperplasia and squamous metaplasia. Incidences of goblet cell hyperplasia and subacute inflammation of the nasal turbinates in males were significantly increased over controls at all dosage levels. The majority of the changes were present in the most anterior section of the nasal cavity, with the nasal septum the most affected area.

OTHER FINDINGS
The concentration and purity of the chlorine dioxide solutions were determined before the test chemical was given to the animals and at the time of refilling bottles to determine the extent of degradation. The percentage of decomposition for chlorine dioxide during 72 h in the water bottles was 3.6 – 7.3 %.

Dose descriptor:

NOAEL

Effect level:

200 mg/L drinking water

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Male: 11.5 mg/kg bw/d. Female: 14.9 mg/kg bw /d

Critical effects observed:

not specified

Table 7.5.1/3: Selected haematological and clinical chemistry values for rats exposed to chlorine dioxide in drinking water for 90 days

	Mean ± standard deviation
Parameter	0 mg ClO2/L	25 mg ClO2/L	50 mg ClO2/L	100 mg ClO2/L	200 mg ClO2/L
MALES
RBC x 10^6	8.10 ± 0.39	8.14 ± 0.29	8.06 ± 0.41	7.99 ± 0.33	8.26 ± 0.42
WBC x 10^3	6.55 ± 1.19	6.76 ± 1.26	6.75 ± 1.85	6.56 ± 1.80	6.29 ± 1.87
Hgb-g/dL	14.94 ± 0.79	15.12 ± 0.56	15.06 ± 0.86	15.10 ± 0.74	15.31 ± 0.55
Hct- percent	43.88 ± 2.52	44.36 ± 2.01	44.23 ± 2.71	44.06 ± 2.39	45.25 ± 2.81
MCV-u^3	54.60 ± 1.43	54.80 ± 1.55	55.10 ± 1.91	55.40 ± 1.71	55.30 ± 1.64
Glu-mg/dL	153.50 ± 40.89	141.50 ± 11.04	151.60 ± 31.33	173.10 ± 29.67	176.60 ± 35.33
BUN- mg/dL	19.34 ± 2.22	18.05 ± 2.14	18.94 ± 7.96	23.97 ± 3.69	19.86 ± 1.62
Creat-mg/dL	0.60 ± 0.13	0.59 ± 0.07	0.63 ± 0.07	0.79 ± 0.10*	0.72 ± 0.09*
PO4- mg/dl	7.08 ± 0.93	7.65 ± 1.02	7.72 ± 0.90	8.48 ± 0.63*	8.02 ± 1.21
AST – U/L*	128.30 ± 37.51	92.10 ± 21.90	87.10 ± 25.53	86.00 ± 23.16*	80.90 ± 20.32*
ALT-P-U/L	49.60 ± 13.02	36.80 ± 7.32*	34.00 ± 5.21*	40.40 ± 6.35	39.90 ± 6.52
Chol-mg/dL	61.30 ± 17.03	62.20 ± 12.82	67.50 ± 13.57	57.70 ± 13.06	63.10 ± 14.04
LDH-U/L*	801.4 ± 476.8	53.36 ± 342.9	479.5 ± 161.1	313.1 ± 304.6*	301.6 ± 227.6*
Ca-mg/dL*	10.45 ± 0.70	9.95 ± 0.36	10.15 ± 0.37	10.04 ± 0.36	10.16 ± 0.39
FEMALES
RBC x 10^6	7.48 ± 0.49	6.96 ± 0.25	7.35 ± 0.33	7.39 ± 0.21	7.34 ± 0.63
WBC x 10^3	3.94 ± 1.26	2.96 ± 0.70	3.92 ± 1.21	4.08 ± 1.12	4.97 ± 1.96
Hgb-g/dL	14.83 ± 0.67	13.99 ± 0.45*	14.78 ± 0.41	14.54 ± 0.40	14.27 ± 0.77
Hct-percent	42.50 ± 2.93	39.10 ± 1.67*	41.56 ± 1.98	41.71 ± 1.19	40.89 ± 3.20
MCV-u^3	57.00 ± 1.56	56.50 ± 1.27	56.80 ± 1.48	56.60 ± 1.43	56.10 ± 1.37
Glu-mg/dL	121.10 ± 23.29	124.20 ± 15.43	120.90 ± 16.27	127.10 ± 11.32	144.20 ± 23.47
BUN-mg/dL	19.61 ± 3.44	15.39 ± 2.41	20.32 ± 7.47	17.45 ± 3.00	18.10 ± 2.81
Creat-mg/dL	0.71 ± 0.10	0.63 ± 0.11	0.59 ± 0.03*	0.66 ± 0.11	0.59 ± 0.07*
PO4- mg/dl	3.96 ± 1.20	6.79 ± 0.99	7.17 ± 0.99	7.81 ± 0.68*	6.64 ± 0.93
AST-U/L	86.90 ± 20.78	86.80 ± 20.23	108.40 ± 19.74	73.50 ± 20.49	75.80 ± 14.82
ALT-U/L	40.70 ± 8.37	39.90 ± 7.72	41.60 ± 7.59	35.50 ± 6.42	35.10 ± 8.25
Chol-mg/dL	67.50 ± 14.30	70.20 ± 18.13	83.00 ± 16.95	67.30 ± 12.47	71.40 ± 19.25
LDH-U/L	556.6 ± 431.3	543.7 ± 417.5	629.2 ± 307.7	275.7 ± 220.9	342.4 ± 201.8
Ca-mg/dL	10.88 ± 0.46	10.50 ± 1.45	10.83 ± 0.50	10.58 ± 0.56	10.53 ± 0.68

* significantly different from control group

Conclusions:

The NOAEL for systemic effect is 200 mg/L (11.5 mg/kg bw/day for males and 14.9 mg/kg bw/day for females), i.e. the highest dose tested.

Executive summary:

In a subchronic toxicity study performed similarly to the OECD test guideline No. 408, Chlorine Dioxide (ClO2) was administered in drinking water to male Sprague-Dawley rats (10/sex/group) at dose levels of 0, 25, 50, 100 and 200 mg/L, for a period of 90 consecutive days. A control group received distilled water alone.

Animals were observed daily for clinical signs and mortality, the body weight was determined initially and then weekly, food and water consumption, haematology, clinical chemistry and urinalysis were also examined.

At the end of the experiment, animals were sacrificed and gross autopsy and histopathology were performed, and organ weights were recorded.

 

There were no deaths recorded at any dose level. No treatment-related changes were noted for haematology and biochemistry parameters.

 

Changes in the levels of the enzymes, lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) at 100 and 200 mg/L and AST at 25 and 50 mg/L in male animals were noted. There was a significantly decreased body weight in both sexes at 200 mg/L. A dose-related decrease in liver weight was observed in both sexes and in spleen weights in females only. However all these changes may be explicated by a decrease in the water consumption observed in all the treated groups.

No systemic effect were observed in this study. Therefore the NOAEL is 200 mg/L (14.9 mg/kg bw/day for males and 11.5 mg/kg bw/day for females), i.e. the highest dose tested.

 

The main target organ for toxicity was the nasal cavity. The histopathological examination of the nasal epithelium showed increased incidence of goblet cell hyperplasia at 100 and 200 mg/L and at all dose-levels in males. In addition, a sub-acute inflammation of the nasal cavity was significantly increased in males given 25 mg/L and in both sexes at higher dose-levels of chlorine dioxide. These findings were most probably the consequence of chlorine dioxide vapours at the sipper tube. As vapours concentration was not monitored, no LOAEL can be estimated for local effect.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

11.5 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

No data

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study not performed according to a guideline and not GLP. No details concerning material and method and few details on results are available. Animals were only exposed 2 hours for the two upper concentrations tested. Urinalysis, clinical chemistry, ophthalmic and food and water analyses were not performed. Statistical analysis is not reported.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of male and female rats were exposed to chlorine dioxide in air (at 10, 5 or 2.5 ppm) daily for 2 or 4-7 hours per day for 30 days. Clinical signs were monitored daily. Body weights were determined weekly. Blood analyses were performed at the beginning and the end of the tests. All animals were sacrificed at study termination and subjected to histopathological analysis of the lungs and liver.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age: one month old at study initiation
- Weight: 61-64 g (group averages for rats) at study initiation
No other information

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: Not applicable

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: dynamic intoxication chamber in plexiglas
No other information

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During exposure of the animals, samples of the atmosphere were taken to test the concentration of chlorine dioxide present, although the analytical method and results are not stated.

Duration of treatment / exposure:

30 days

Frequency of treatment:

5 and 10 ppm: 2 hours per day, daily. 2.5 ppm: 7 hours per day, daily

Remarks:

Doses / Concentrations:
0; 2.5; 5 and 10 ppm (0; 6.9; 14 and 28 mg/m3)
Basis:
nominal conc.

No. of animals per sex per dose:

10 ppm: 5 rats/sex/group; controls 5 rats/sex/group
5 ppm: 2*5 rats/sex/group; controls 2*5 rats/sex/group
2.5 ppm (30 days): 10 rats/sex/group

Control animals:

yes, sham-exposed

Details on study design:

Post exposure period: 15 days

Positive control:

No

Observations and examinations performed and frequency:

CLINICAL SIGNS AND MORTALITY: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: beginning and end of the study
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all animals
- Parameters examined: red blood cell count, white blood cell count, neutrophil, eosinophil, basophile, lymphocytes, monocytes

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, viscera
HISTOPATHOLOGY: Yes, liver and lungs were examined in all animals
Organs were not weighted.

Other examinations:

Not done

Statistics:

Not done

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
10 ppm: Mucopurulent nasal secretions in exposed animals, irritation of the mucous membrane of the nose. Red eyes especially at the end of exposure.
5 ppm: Slight eye irritation, transitory
2.5 ppm: Normal appearance.
No mortalities at any dose level

BODY WEIGHT AND WEIGHT GAIN (See table 7.5.3/1)
10 ppm: Slight decrease in body weight gain observed.
5 ppm: Normal growth
2.5 ppm: Clear decrease in growth rate.

HAEMATOLOGY (See table 7.5.3/2)
10 ppm: Red blood cell count increased by 40 %. White blood cells count increased by 95 %. No changes in eosinophil, basophile, lymphocyte and monocyte counts.
5 ppm: No change in red blood cell count. White blood cell count increased by 40-50 %. No changes in blood cell differentiation.
2.5 ppm: Blood count remained normal.

GROSS PATHOLOGY and HISTOPATHOLOGY
10 ppm: No macroscopic abnormalities of the internal organs. Some bronchoalveolar lesions: multifocal non-infectuous bronchopneumonia, histologically the affected areas of the lung were mainly peri-vascular and showed alveoli filled with cellular debris from desquamated alveolar epithium.
5 ppm: Histologically the same lesions as at 10 ppm, but less pronounced. Liver not affected
2.5 ppm: Alveolar cavities infiltrated with polynuclear cells and macrophages, vascular congestion. Thickening of Interalveolar septa. Focal haemorrhagic alveolitis. Small epithelial erosions and inflammtory infiltrates in some of the larger bronchi. Recovery from the pulmonary lesions was apparent in rats examined after a 15-day recovery period.

Dose descriptor:

LOAEC

Effect level:

6.9 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Pulmonary effects

Dose descriptor:

NOAEC

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Critical effects observed:

not specified

Table 7.5.3/1: Average body weights (males and females)

Body Weights (g)
Dose rate (ppm)	Study initiation	Week 1	Week 2	Week 3	Week 4
0	93	121	149	175	207
5 (A)	86	124	136	174	184
0	79	92	112	121	138
5 (B)	69	82	102	111	125
0	61	86	105	124	149
10	64	80	93	115	131

 

Table 7.5.3/2: Haematology findings

Parameter	2.5ppm		5ppm (A)		5 ppm (B)		10ppm
	Initial	Final	Initial	Final	Initial	Final	Initial	Final
Red blood cell count (10^3/mm^3)	7818	6614	5132	5036	No change		5012	6998
	- 16 %		No change				+ 40 %
White blood cell count	26180	30260	15660	23860	+ 40 %		8990	17580
	+ 16 %		+ 52 %				+ 95 %
Neutrophil			24.6	18.4	25.8	22	18.1	206
			- 25 %		- 15 %		+ 1040 %
Eosinophil			1.2	2.6	1	1	1.8	1.4
			+ 116 %		=		- 23 %
Basophil			0.4	0.2	0	0	0.2	0.4
			- 50 %		=		+ 100 %
Lymphocytes			70.8	76	70.2	73.4	77	74.3
			+ 7.3 %		+ 4.5 %		- 3.5 %
Monocytes			3	2.8	3	3.6	2.9	3.3

Conclusions:

Based on respiratory effects, the LOAEC in this study is 6.9 mg/m3, the lowest concentration tested.

Executive summary:

In subacute repeat-dose study, rats (10/sex/group) were exposed to Chlorine Dioxide (ClO₂) vapors at concentrations of 2.5 ppm 7 h/day or 5 and 10 ppm, 2 h/day, daily, for 30 days (corresponding to nominal concentrations of 6.9; 14 and 28 mg/m³).

Clinical signs were monitored daily, body weights were determined weekly and blood analyses were performed at the beginning and the end of the tests. All surviving animals were sacrificed at study termination and subjected to histopathological analysis of the lungs and liver.

No mortality was observed, at any dose level.

Clinical signs observed were as follow: at 10 ppm dose level, mucopurulent nasal secretion and conjunctival irritation; at 5 ppm dose level, minor eye irritation and at 2.5 ppm dose level, no effects.

A slight and a clear decrease in growth rate were observed at 10 ppm and 2.5 ppm dose levels respectively whereas the animals show normal growth. at 5 ppm dose level.

At 10 ppm, red blood cell count increased by 40 % and white blood cells count increased by 95 %. No changes in eosinophil, basophile, lymphocyte and monocyte counts was noted. At 5 ppm, there was no change in red blood cell count while white blood cell count increased by 40-50 %. No changes in blood cell differentiation was noted. At 2.5 ppm, blood count remained normal.

No macroscopic abnormalities of the internal organs were observed at 10 ppm and 5 ppm dose level. Multifocal non-infectuous broncho pneumonia were observed at 10 ppm dose level and histologically, the affected areas of the lung were mainly peri-vascular and showed alveoli filled with cellular debris from desquamated alveolar epithium. The same histological lesions were obersed at 5 ppm dose level, but less pronounced.

At 2.5 ppm dose level, alveolar cavities were infiltrated with polynuclear cells and macrophages and vascular congestion was observed. Thickening of Interalveolar septa, focal haemorrhagic alveolitis, small epithelial erosions and inflammtory infiltrates in some of the larger bronchi were also observed. Recovery from the pulmonary lesions was apparent in rats examined after a 15-day recovery period.

Based on respiratory effects, the LOAEC in this study is 6.9 mg/m³, the lowest concentration tested.

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

No data

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study not performed according to a guideline and not GLP. No details concerning material and method and few details on results are available. Animals were only exposed 2 hours and 4 hours. Urinalysis, clinical chemistry, ophthalmic and food and water analyses were not performed. Statistical analysis is not reported.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of rabbits were exposed to chlorine dioxide in air (at 5 or 2.5 ppm) daily for 2 or 4 hours per day for 30 or 45 days. Clinical signs were monitored daily. Body weights were determined weekly. Blood analyses were performed at the beginning and the end of the tests. All animals were sacrificed at study termination and subjected to histopathological analysis of the lungs and liver.

GLP compliance:

no

Limit test:

no

Species:

rabbit

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Age: rabbits described as “young animals”
Weight: 679 – 708 g (group averages for rabbits)
No other data

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: No data

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: dynamic intoxication chamber in plexiglas
No other information

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During exposure of the animals, samples of the atmosphere were taken to test the concentration of chlorine dioxide present, although the analytical method and results are not stated.

Duration of treatment / exposure:

Duration of treatment: 30 days (5 ppm), 45 days (2.5 ppm)

Frequency of treatment:

5 ppm: 2 hours per day, daily.2.5 ppm: 4 hours per day, daily

Remarks:

Doses / Concentrations:
0; 2.5 and 5 ppm (0; 6.9 and 14 mg/m3)
Basis:
nominal conc.

No. of animals per sex per dose:

5 ppm: 4 rabbits; controls: 5 rabbits
2.5 ppm: 8 rabbits; controls: 7 rabbits

Control animals:

yes, sham-exposed

Details on study design:

Post exposure period: 15 days

Positive control:

No data

Observations and examinations performed and frequency:

CLINICAL SIGNS AND MORTALITY: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: beginning and end of the study
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all animals
- Parameters examined: red blood cell count, white blood cell count, neutrophil, eosinophil, basophile, lymphocytes, monocytes

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, viscera
HISTOPATHOLOGY: Yes, liver and lungs were examined in all animals
Organs were not weighted.

Other examinations:

No data

Statistics:

No data

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
5 ppm: Wet nasal secretions in exposed animals
2.5 ppm: Normal appearance.
No mortalities at any dose level

BODY WEIGHT AND WEIGHT GAIN (See table 7.5.3/1)
5 ppm: Slight decrease in body weight gain observed from the second weeks of treatment.
2.5 ppm: Minor decrease in growth rate was observed during the second week.

HAEMATOLOGY (See table 7.5.3/2)
5 ppm: No change in red blood cell count. White blood cell count increased by 59 %. No changes in blood cell differentiation.
2.5 ppm: Blood count remained normal.

GROSS PATHOLOGY and HISTOPATHOLOGY
5 ppm: No macroscopic abnormalities of the internal organs. Some bronchoalveolar lesions.
2.5 ppm: No macroscopic lesions of internal organs. Lungs showed small foci of haemorrhagic alveolitis with alveolar vascular congestion. Recovery from the pulmonary lesions was apparent in rats examined after a 15-day recovery period.

Dose descriptor:

LOAEC

Effect level:

6.9 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on pulmonary effects

Dose descriptor:

NOAEC

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Critical effects observed:

not specified

Table 7.5.3/1: Average body weights

Body Weights (g)
Dose rate (ppm)	Study initiation	Week 1	Week 2	Week 3	Week 4
0	708	883	1004	1242	1412
5	679	854	1010	1109	1260

 

Table 7.5.3/2: Haematology findings

Parameter	2.5ppm		5ppm
	Initial	Final	Initial	Final
Red blood cell count (10^3/mm^3)	4629	3732	4643	4470
	- 20 %		- 4.1 %
White blood cell count	15400	16250	7800	11433
	+ 5.5 %		+ 59 %
Neutrophil			47.33	47.33
			=
Eosinophil			1.33	1.66
			+ 25 %
Basophil			1.33	1
			- 25 %
Lymphocytes			42.33	46
			+ 8.7 %
Monocytes			7.66	4
			- 48 %

Conclusions:

Based on these observation, the LOAEC in this study is 6.9 mg/m3, the lowest concentration tested.

Executive summary:

In a subacute repeat-dose toxicity study, groups of young rabbits were exposed to Chlorine Dioxide (ClO₂) vapors at concentrations of 2.5 ppm (6.9 mg/m³), 2 h/day, daily, for 45 days, or at dose level of 5 ppm (14 mg/m³), 4 h/day, daily, for 30 days.

Clinical signs were monitored daily. Body weights were determined weekly. Blood analyses were performed at the beginning and the end of the tests. All animals were sacrificed at study termination and subjected to histopathological analysis of the lungs and liver.

No mortalities were observed at any dose level.

At 5 ppm, wet nasal secretions were noted.

A slight decrease in body weight gain was observed from the second weeks of treatment with 5 ppm and minor decrease in growth rate was noted at 2.5 ppm.

At 5 ppm, white blood cell count increased by 59 %. There was no changes in blood cell differentiation and in red blood cell count.At 2.5 ppm, blood count remained normal.

No macroscopic abnormalities of the internal organs. Some bronchoalveolar lesions were observed at 5 ppm. Lungs of 2.5 ppm exposed group showed small foci of haemorrhagic alveolitis with alveolar vascular congestion. Recovery from the pulmonary lesions was apparent in rats examined after a 15-day recovery period.

Based on these observation, the LOAEC in this study is 6.9 mg/m³, the lowest concentration tested.

Reference 3

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

no data

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study not performed according to a guideline and not GLP. No details concerning material and method and few details on results are available. Animals were only exposed 5 hours/d. No data on actual concentration in air. Urinalysis, clinical chemistry, ophthalmic and food and water analyses were not performed. Statistical analysis is not reported.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Rats were exposed to chlorine dioxide in air (at 1 ppm) for 5 hours per day, 5 days per week for 2 months. Clinical signs were monitored daily. Body weights were determined weekly. Blood analyses were performed at the beginning and the end of the tests. All animals were sacrificed at study termination and subjected to histopathological analysis of the lungs and liver.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

not specified

Details on test animals or test system and environmental conditions:

No data

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

other: air

Remarks on MMAD:

MMAD / GSD: No data

Details on inhalation exposure:

No data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

2 months

Frequency of treatment:

5 hours per day, 5 days per week

Remarks:

Doses / Concentrations:
0 and 1 ppm (0 and 2.8 mg/m3)
Basis:
nominal conc.

No. of animals per sex per dose:

8 rats/dose

Control animals:

yes, sham-exposed

Details on study design:

No data

Positive control:

No data

Observations and examinations performed and frequency:

CLINICAL SIGNS AND MORTALITY: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: beginning and end of the study
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all animals
- Parameters examined: red blood cell count, white blood cell count

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, viscera
HISTOPATHOLOGY: Yes, liver and lungs were examined in all animals
Organs were not weighted.

Other examinations:

No data

Statistics:

No data

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
No effects or mortality observed.

BODY WEIGHT AND WEIGHT GAIN
No effects observed.

HAEMATOLOGY
No effects observed.

GROSS PATHOLOGY and HISTOPATHOLOGY
It was observed on all of the lungs, whatever the duration of the experiment, a congestion of the vessels of all calibres and very small lesions of interstitial oedema afflicting the bronchial mucous membranes and the walls of the bronchi, without perceptibly altering the epithelium. The pulmonary parenchyma on the other hand is absolutely normal. Wilder and orcein stains show that the reticular and elastic structures of the alveoli, the vessels and the bronchi are intact.

Dose descriptor:

LOAEL

Effect level:

2.8 mg/m³ air

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: Based on respiratory effects.

Dose descriptor:

NOAEC

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Critical effects observed:

not specified

No data

Conclusions:

Based on respiratory effects, the LOAEC in this study is 2.8 mg/m3.

Executive summary:

In a subchronic study, Wistar rats were exposed to Chlorine Dioxide by gas inhalation, at dose level of 1ppm (2.8mg/m³), for 5 hours per day, 5 days per week, for 2 months. Control animals were sham-exposed.

Clinical signs were monitored daily. Body weights were determined weekly. Blood analyses were performed at the beginning and the end of the tests. All animals were sacrificed at study termination and subjected to histopathological analysis of the lungs and liver.

No clinical signs due to Chlorine Dioxide or mortalities were observed. No body weight changes or haematologic effects were observed.

It was observed on all of the lungs, whatever the duration of the experiment, a congestion of the vessels of all calibres and very small lesions of interstitial oedema afflicting the bronchial mucous membranes and the walls of the bronchi, without perceptibly altering the epithelium. The pulmonary parenchyma on the other hand is absolutely normal. Wilder and orcein stains show that the reticular and elastic structures of the alveoli, the vessels and the bronchi are intact.

Based on these observation, the LOAEC in this study is 2.8 mg/m³.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

2.8 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral

In a subchronic toxicity study (Daniel et al., 1990) performed similarly to the OECD test guideline No. 408, Chlorine Dioxide (ClO2) was administered in drinking water to rats at dose levels of 0, 25, 50, 100 and 200 mg/L. There were no deaths recorded at any dose level. No treatment-related changes were noted for haematology and biochemistry parameters.

Changes in the levels of the enzymes, lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) at 100 and 200 mg/L and AST at 25 and 50 mg/L in male animals were noted. There was a significant decreased body weight in both sexes at 200 mg/L. A dose-related decrease in liver weight was observed in both sexes and in spleen weights in females only. However, all these changes may be explicated by a decrease in the water consumption observed in all the treated groups.

No systemic effects were observed in this study. Therefore, the NOAEL is 200 mg/L (11.5 mg/kg bw/day for females and 14.9 mg/kg bw/day for males), i.e. the highest dose tested.

The main target organ for toxicity was the nasal cavity. The histopathological examination of the nasal epithelium showed increased incidence of goblet cell hyperplasia at 100 and 200 mg/L and at all dose-levels in males. In addition, a sub-acute inflammation of the nasal cavity was significantly increased in males given 25 mg/L and in both sexes at higher dose-levels of chlorine dioxide. These findings were most probably the consequence of the inhalation of chlorine dioxide vapours from the sipper tube. As vapours concentration was not monitored, no LOAEL can be estimated for local effect.

 

Repeated dose toxicity: dermal

A repeated dose toxicity study by dermal route does not need to be provided because short term toxicity was already assessed via oral and inhalation route. Moreover, the study is scientifically unjustified considering animal welfare since Chlorine dioxide is classified as corrosive; at non-irritating concentrations (i.e.<3% according to the generic concentration limit), no specific effects are expected.

 

Repeated dose toxicity: inhalation

A weight of evidence approach was used to evaluate the inhalation repeated dose toxicity of Chlorine dioxide.

Paulet and Desbrousses (1970) exposed groups of 5 rats/sex (strain not specified) to chlorine dioxide vapours at concentrations of 0, 2.5, 5 and 10 ppm (0, 6.9, 14 and 28 mg/m3), 7 hours/day (2.5 ppm) or 2 hours/day (5 and 10 ppm) for 30 days. At 10 ppm, the main effect is lung irritation, visible as localised bronchopneumonia. This is the cause of the strong increase (+95 %) in circulating white blood cells. Irritation of the nasal mucosa is responsible for the mucopurulent secretion. The increase (+40 %) in red blood cells is probably due to hypoxia secondary to the lesions in the lung. At 5 ppm, similar, but less pronounced effects to those seen at 10 ppm were noted. At 2.5 ppm, chlorine dioxide-exposed rats exhibited respiratory effects that included lymphocytic infiltration of the alveolar spaces, alveolar vascular congestion, haemorrhagic alveoli, epithelial erosions, and inflammatory infiltrations of the bronchi. The study authors also reported slightly decreased body weight gain, decreased erythrocyte levels, and increased leukocyte levels, relative to controls. Recovery from the pulmonary lesions was apparent in rats examined after a 15-day recovery period.

In a follow-up study designed to examine a lower exposure level (Paulet and Desbrousses, 1971), eight Wistar rats (sex not reported) were exposed to chlorine dioxide vapours at concentrations of 0 and 1 ppm (0 and 2.8 mg/m3), 5 hours/day, 5 days/week for 2 months. The authors stated that weight gain and erythrocyte and leukocyte levels were not affected. Chlorine dioxide-induced respiratory effects included peribronchiolar oedema and vascular congestion in the lungs. No alterations in epithelium or parenchyma were seen.

Collectively, these studies adequately identify a LOAEC of 1 ppm (2.8 mg/m3) based on local adverse effect resulting from the irritant properties of Chlorine dioxide.

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 repeated dose toxicity category.

 

Self classification:

- Repeated oral exposure:

On the basis of the results of the key study, no significant toxic effects were produced at doses up to 200 mg/L (11.5 mg/kg bw/d for males and 14.9 mg/kg bw/day for females). At higher doses, Chlorine dioxide may cause severe toxicological effects and should be evaluated. However, according to the Guidance on the Application of Regulation (EC) No 1272/2008, if the dose is less than half an order of magnitude lower than that mediating the evident corrosivity, then it could not be considered to be a repeated-dose effect distinct from the corrosivity.

Considering the dose level which causes the corrosivity (40 mg/kg bw) and the dose with no systemic effect (11.5 mg/kg bw), ClO2 should not be classified as specific target organ toxicant (repeated exposure). The classification as STOT-RE is warranted by the classification as corrosive.

 

- Repeated dermal exposure:

No self-classification is proposed.

 

- Repeated inhalation exposure:

Local adverse effect resulting from the irritant properties of Chlorine dioxide are observed at concentrations as low as 1 ppm where no systemic effects are noted (Paulet and Debrousses, 1970 & 1971). Therefore, according to the Guidance on the Application of Regulation (EC) No 1272/2008, ClO2 should not be classified as STOT-RE.