Repeated dose toxicity: oral.006 to 007

Repeated dose toxicity: oral.006

Administrative data

Study result type:

experimental result

Study period:

no data

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

Hematologic parameters are only evaluated.

Data source

Reference

Reference Type:

publication

Title:

The effects of chlorine dioxide and sodium chlorite on erythrocytes of A/J and C57L/J mice

Author:

Moore, G.S. and Calabrese, E.J.

Year:

1980

Bibliographic source:

Journal of Environmental Pathology and Toxicology, 4(2-3):513-524

Materials and methods

Principles of method if other than guideline:

This study was designated to evaluate the effects of chlorine dioxide on erythrocytes of mouse strains with differential G6PD activity (the A/J strain has 3 times greater activity than the C57L/J strain).

GLP compliance:

no

Remarks:

study conducted before GLP establishment

Test type:

subacute

Limit test:

no

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:

Analytical grade reagents were utilized throughout the generation and determination phases of ClO2 preparation. Water used in the preparation of all reagents and dilutions were processed by a "Continental Water Deionization System", consisting of 2 miwed bed deionizers, one activated carbon unit, a 10 µm prefilter and a 0.3 µM post-filter. Water quality of this system is continuously monitored to insure that electrical resistance never drops below 15 megohms.

Test animals

Species:

mouse

Strain:

other: A/J strain ("normal") and C57L/J stain (low level of G6PD activity)

Sex:

no data

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Jackson Laboratory, Bar Harbor, Maine
- Age at study initiation:
- Weight at study initiation:
- Fasting period before study:
- Housing: individuallu in stainless steel cages equipped with constant feed food hoppers and 50 mL calibrated watering devices.
- Diet: balanced rodent diet (Purina rodent chow) ad libitum
- Water: see bellow
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature: 23.3 +/- 2 °C
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

The watering devices were filled with the appropriate treatment and a reading taken of the volume both before and after the treatment period. The bottles were removed after the end of this period and no other water was provided until the next treatment period.

Analytical verification of doses or concentrations:

no data

Details on analytical verification of doses or concentrations:

Determination of ClO2 levels in solution was made by means of the Palin D.P.D. method (1974). This method utilizes the reagent diethyl-p-phenylene diamine sulphate. Following preparation, the dilutions were tightly sealed and stored under refrigeration. Dilutions were then dispensed and placed on the animal cages at the beginning of the dark cycle.

Duration of treatment / exposure:

30 days

Frequency of treatment:

treatment period of 12 hours, during the dark cycle in order to minimize decomposition of ClO2 by light and to coincide with the nocturnal activity of the mice.

Doses / concentrations

Doses / concentrations:

100 ppm

Basis:

nominal in water

No. of animals per sex per dose:

10 mices/strain/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment: random

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule: no data

BODY WEIGHT: Yes
- Time schedule for examinations:


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of exposure period
- Anaesthetic used for blood collection: No
- Animals fasted: No data
- How many animals: all
- Parameters examined: Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corp. volume (MCV), Mean corp. hemoglobin (MCH), Mean corp. Hb. conc. (MCHC), Reticulocytes. G6PD activity, osmotic fragility, GSH level.


CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

The animals were lightly etherized and then decapited by means of guillotine.

Other examinations:

No other examination

Statistics:

Analysis were performed using the BMDP2V (Larson, 1972) and SPSS ONEWAY (Dixon, 1975) programs.

Results and discussion

Results of examinations

Clinical signs and mortality:

no effects

Body weight and weight gain:

no effects

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

no effects

Ophthalmoscopic examination:

not examined

Haematology:

no effects

Clinical chemistry:

not examined

Urinalysis:

not examined

Neurobehaviour:

not examined

Organ weights:

not examined

Gross pathology:

not examined

Histopathology: non-neoplastic:

not examined

Histopathology: neoplastic:

not examined

Details on results:

HAEMATOLOGY
There were no treatment effects on any hematologic parameter.

Any other information on results incl. tables:

No other information

Applicant's summary and conclusion

Conclusions:

There were no treatment effects on any hematologic parameter in this study.

Executive summary:

In a subacute toxicity study, Chlorine Dioxide (ClO2) was administered in drinking water to A/J strain ("normal") and C57L/J stain (low level of G6PD activity) mice (10/strain/group) at dose levels of 0, and 100 ppm (0 and 0.28 mg/L), for a period of 30 consecutive days. A control group received water alone.

Blood was collected and analyzed at the end of the exposure period. Weight change over 30 days and the average water consumption were also determined.

There were no treatment effects on any hematologic parameter in this study.

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.

Repeated dose toxicity: oral.007

Administrative data

Purpose flag:

supporting study

Study result type:

experimental result

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

Acceptable, well-documented publication which meets basic scientific principles.

Data source

Reference

Reference Type:

publication

Title:

Hematological effects of chlorine dioxide on in vitro exposure in mouse, rat and human blood and on subchronic exposure in mice.

Author:

Ueno H, Sayato Y and Nakamuro K

Year:

2000

Bibliographic source:

Journal of Health Science, 46(2): 110-116

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Subchronic toxicity of ClO2 studied in mouse.

GLP compliance:

no data

Test type:

subchronic

Limit test:

no

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

Details on test material:

Fresh-prepared alklaine 0.5% ClO2 solution containing 0.4% sodium bicarbonate (NaHCO3) as a stabiliser was provided from Sukegawa Chemical Co. Ltd. (Kobe, Japan). The concentration of ClO2 was determined by iodometric method as well as by the DPD method.

Test animals

Species:

mouse

Strain:

other: SPF ddy

Sex:

male

Details on test animals and environmental conditions:

Age: at study initiation: 4 weeks, at time of dosing: 5 weeks
Weight: at study initiation: 10-20 g
Acclimation: Animals were allowed to acclimate to laboratory conditions afor a week prior to initiation of exposures
Drinking water: changed twice weekly
Feed: pelleted basal diet ad libitum

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

Control: no addition of NaHCO3
Exposed: in the presence of stabiliser 1200 mg/L NaHCO3

Analytical verification of doses or concentrations:

no data

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

30, 60 and 90 consecutive days

Frequency of treatment:

Ad libitum

Doses / concentrations

Doses / concentrations:

0, 100, 1000, 1500, 2000 mg/L (corresponding to 0; 5.3; 33.3; 39.7 and 59 mg/kg b.w./day)

Basis:

nominal in water

No. of animals per sex per dose:

10 animals (male) per group

Control animals:

yes, concurrent vehicle

Details on study design:

No data

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

Animals were observed daily for any overt sign of toxicity. 
Body weight and consumption of food and water were measured twice weekly.

Sacrifice and pathology:

Blood was collected from the abdominal aorta of animals under pentobarbital anaesthesia at the termination of the study and used for hematological assays. Organ weight measurements and histopathological examinations were also performed at the termination of the study. Serum clinical chemistry measurements were made for aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), G-6-PD, GSH, albumin, total protein, hemoglobin, serum iron, bilirubin and urea nitrogen (BUN), uric acid, creatine and creatinine.

Other examinations:

No data

Statistics:

Statistical comparisons between control and exposed groups were evaluated by a once-way analysis of variance (ANOVA). Group means were compared with control values using Dunnett's multiple comparison test. The 95% (p<0.05) confidence level was chosen as the criterion of significance.

Results and discussion

Effect levels

Endpoint:

NOAEL

Effect level:

100 mg/L drinking water

Based on:

test mat.

Sex:

male

Basis for effect level / Remarks:

5.3 mg/kg b.w./day

Results of examinations

Clinical signs and mortality:

yes

Body weight and weight gain:

yes

Food consumption and compound intake (if feeding study):

yes

Food efficiency:

no data

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

yes

Ophthalmoscopic examination:

yes

Haematology:

no effects

Clinical chemistry:

no data

Urinalysis:

no data

Neurobehaviour:

yes

Organ weights:

yes

Gross pathology:

no data

Histopathology: non-neoplastic:

no effects

Histopathology: neoplastic:

no data

Details on results:

More than 1000 mg/L ClO2-exposedshowed a significant decrease in rate of mean body weight bain, as well as depressed consumption of food and water. The average daily intake of ClO2 estimated from the water consumption was 0.16, 1.00, 1.19 and 1.77 mg/animal/day for the 100, 1000, 1500 and 2000 mg/L treatment group respectively. 10 out of the 30 mice exposed to 1500 mg/L ClO2 and 26 of the 30 animals exposed to 2000 mg/L ClO2 died before the 60 and 30 day terminal euthanasia, respectively. These mice were characterised by a lack of motile activity, dysbasia and hyposthenia with extermely reduced consumption of food and water prior to death. Although darkish red eyes and cyanosis-like spots in the tail were also observed in the surviving animals, of the 1500 and 2000 mg/L treatment groups, no apparent changes were seen in the 100 and 1000 mg/L groups. Significant increases in absolute weight of organ and organ-to-body weight ratio were observed in spleen for the 1000 mg/L group at 60 d, suggesting hematological effects related to ClO2 exposure . However, histopathological examinations did not reveal exposure-related changes in either liver, kidneys, spleen, lung, heart, pancreas, stomach, duodenum or testes. Haematological examinations resulted in no apparent effects related to ClO2 exposure on AST, ALT, LDH, GSH, albumin, total protein, serum iron, bilirubin, BUN, uric acid, creatine and creatinine. However, a significant increase in methemoglobin was observed in animals exposed to 1000 mg/L ClO2 for 90 days and 15000 mg/L ClO2 for 60 days, whereas a decrease in hemoglobin was shown conversely. Exposure to more than 1000 mg/L ClO2 caused augmentation of G-6-PD activity and a lowering of osmotic fragility in erythrocytes.

Any other information on results incl. tables:

Osmotic fragility is related to the stability of the erythrocyte membrane and this change by subchronic exposure to ClO2 indicates an increased resistance to hemolysis in hypotonic solution. The G-6 -PD supplies NADPH which is used as a cofactor for reduction of oxidised glutathione by GSH reductase. Exogenous GSH and NADPH also stabilise erythrocyte membrane and protect hemoglobin from release to the hypotonic solution.

Applicant's summary and conclusion

Conclusions:

The NOAEL in this study is 5.3 mg/kg bw/d.

Executive summary:

In a subchronic toxicity study, Chlorine Dioxide (ClO2) was administered to male SPF ddy mouse in drinking water at dose levels of 0, 100, 1000, 1500 and 2000 mg/L for 30, 60 and 90 consecutive days. NaHCO3 was used at dose level of 1200mg/L as a stabiliser. A control was performed without NaHCO3.

Animals were observed daily for any overt sign of toxicity. Body weight and consumption of food and water were measured weekly. At the end of the experiment, animals were sacrificed and blood was collected and used for hematological assays. Organ weight measurement and histopathological examinations were also performed at the termination of the study.

The average daily intake of ClO2 estimated from the water consumption was 5.3; 33.3; 39.7 and 59 mg/kg b.w./day for the 100, 1000, 1500 and 2000 mg/L treatment group respectively.

More than 1000 mg/L ClO2-exposed showed a significant decrease in rate of mean body weight gain, as well as depressed consumption of food and water. 10 out of the 30 mice exposed to 1500 mg/L ClO2 and 26 of the 30 animals exposed to 2000 mg/L ClO2 died before the 60 and 30 day terminal euthanasia, respectively.

Significant increases in absolute weight of organ and organ-to-body weight ratio were observed in spleen for the 1000 mg/L group at 60 d, suggesting hematological effects related to ClO2 exposure. However, histopathological examinations did not reveal exposure-related changes in either tissue.

Haematological examinations resulted in no apparent effects related to ClO2 exposure excepted for methemoglobin and hemoglobin: a significant increase in methemoglobin was observed in animals exposed to 1000 mg/L ClO2 for 90 days and 15000 mg/L ClO2 for 60 days, whereas a decrease in hemoglobin was shown conversely.

Based on these observation, the NOAEL is 5.3 mg/kg bw/d.

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.