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Repeated dose toxicity: oral

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Administrative data

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.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Comparative subchronic toxicity studies of three disinfectants
Author:
Daniel FB, Condie LW, Robinson M, Stober JA, York GR, Olsen RG and Wang SR
Year:
1990
Bibliographic source:
Journal of the American Water Works Association, 82(10): 61-69

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Chlorine dioxide
EC Number:
233-162-8
EC Name:
Chlorine dioxide
Cas Number:
10049-04-4
Molecular formula:
ClO2
IUPAC Name:
Chlorine dioxide generated from sodium chlorate and hydrogen peroxide in the presence of a strong acid
Details on test material:
- Name of test material (as cited in study report): chlorine dioxide

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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 %.

Effect levels

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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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 106

8.10 ± 0.39

8.14 ± 0.29

8.06 ± 0.41

7.99 ± 0.33

8.26 ± 0.42

WBC x 103

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-u3

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 106

7.48 ± 0.49

6.96 ± 0.25

7.35 ± 0.33

7.39 ± 0.21

7.34 ± 0.63

WBC x 103

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-u3

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

Applicant's summary and conclusion

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.