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Acute Toxicity: inhalation

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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 December 2011 - 06 February 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study conducted according to OECD Guideline 403 with restrictions: only 3 rats/sex are exposed at the highest concentrations by comparison with the other concentrations (5 rats/sex), 0.6 % aqueous chlorine dioxide (ClO2) solution, inherently stabilized without any explanation about the method used for stabilising the substance. Absence of any reported effects, due to the treatment, except the deaths of animals, according to dose levels. The physical state of the test atmosphere has not been characterized in the study. It is not clear whether or not the test atmosphere consists of gas of chlorine dioxide in liquid droplets. No justification for the selected concentration levels (achievable concentrations or anticipated effects from the acute toxicity by inhalation of gas)
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:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Remarks:
inspected on 25, 26, 27 & 28 November 2008 / signed on 12 November 2009
Test type:
standard acute method
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
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): 0.6 % aqueous chlorine dioxide (ClO2) solution, inherently stabilized
- Physical state: Yellow liquid
- Date of receipt: 25 October 2011
- Storage condition of test material: Stored in a refrigerator

Test animals

Species:
rat
Strain:
other: CD / Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany.
- Age at study initiation: Males: approx. 8 weeks; Females: approx. 9 weeks
- Weight at study initiation: Males: 231-290 g; Females: 218-250 g
- Fasting period before study: Feeding was discontinued approx. 16 h before test item administration
- Housing: During the 14-day observation period the animals were kept by sex in groups of 2-3 animals in MAKROLON cages (type III plus)
- Diet: Commercial diet, ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Drinking water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 55 ± 15 %
- Photoperiod: 12 h dark / 12 h light

Administration / exposure

Route of administration:
inhalation: mist
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Dynamic inhalation chamber (air changes/h (≥ 12 times))
- Exposure chamber volume: 40 L
- Method of holding animals in test chamber: Cylindrical exposure chamber (volume 40 L) which holds a maximum of 20 animals in pyrex tubes at the edge of the chamber in a radial position.
- Source and rate of air: Spray-jet was fed with compressed air (5.0 bar) from a compressor at a flow rate of 900 L/h and with the test item using an infusion pump at flow rates of 12, 20 and 50 mL/h. At the bottom of the exposure chamber, the air was sucked off at a lower rate than created by the spray-jet in order to produce a homogenous distribution and a positive pressure in the exposure chamber (inflow 900 L/h, outflow 800 L/h).
- Air change: 22.5 changes per hour
- Method of conditioning air: A manometer and an air-flow meter were used to control the constant supply of compressed air and the exhaust, respectively. Flow rates were checked hourly and corrected, if necessary.
- The oxygen content in the inhalation chamber was 21 % v/v. It was determined at the beginning and at the end of the exposure with a DRÄGER Oxygen-analysis test set (DRÄGER Tube Oxygen 67 28 081). Carbon dioxide concentration did not exceed 1 %.
- Method of particle size determination: Analysis of the particle size distribution was carried out twice during the exposure period using a cascade impactor according to May, 1975.
- Treatment of exhaust air: Exhaust air was sucked through gas wash-bottles.
- Temperature, humidity, pressure in air chamber: The temperature (20.8-21.5 °C) and humidity (60.4-64.4 %) was checked and noted once every hour during the exposure period of the experiment.

TEST ATMOSPHERE
- Brief description of analytical method used: ClO2-concentrations in the inhalation chamber was determined by iodometric titration
- Aerosol samples were taken once every hour during the 4-hour exposure period. For that purpose, a probe was placed close to the animals’ noses and samples were collected by drawing 7.5 L air at a flow rate of 0.5 L/min for 15 minutes through 2 consecutive gas washing bottles containing approx. 50 mL 0.02 % potassium iodide solution (pH=2.0), each. The contents of both washing bottles were combined in an 1000 mL volumetric flask and filled up to the mark with water. An aliquot of 50 mL was used for the iodometric determination. Each sample was analysed twice.

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: The mist from the exposure chamber was drawn through the cascade impactor for 5 minutes at a constant flow rate of 5 L/min. The slides were removed from the impactor and weighed on an analytical balance (SARTORIUS, type 1601 004, precision 0.1 mg).
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): In the inhalation chamber, close to the animals' noses, the spray-jet generated atmosphere had mass median aerodynamic diameters (MMAD) between 2.388 and 2.467 μm as determined with a cascade impactor. The Geometric Standard Deviations (GSD) of the MMAD were calculated as 2.69, 2.74 and 2.78.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
0.030, 0.047 and 0.074 mg/L air (iodometric determination).


The concentrations are acceptable based on the masse median aerodynamic diameter values of 2.388, 2.429 and 2.467 µm and the corresponding GSD values of 2.78, 2.74 and 2.69. This is inside the required range of 1-4 µm and 1.5-3, respectively as required by the OECD 403 guideline.
However,it is not reported if :
- these concentrations are the technical maximum achievable concentrations knowing that the measured concentrations as pure ClO2 are closed to the nominal concentrations which can be calculated taking into account both the air compressor flow rate (900L/h) and the ClO2 solution infusion pump flow rates (12, 20 et 50 mL/h).
- or if these concentrations are selected based on anticipated effects in rats as showed in the acute toxicity study by inhalation for the gas.
No. of animals per sex per dose:
3 animals/sex/dose at 0.074 mg/L air
5 animals/sex/dose at 0.030 and 0.047 mg/L air
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations: A careful clinical examination was made at least once daily until all symptoms subsided, thereafter each working day. Observations on mortality were made at least once daily to minimize loss of animals to the study, e.g. necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals.
Cageside observations included, but were not limited to: changes in the skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, as well as somatomotor activity and behaviour pattern.
Particular attention was directed to observation of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
- Frequency of weighing: Individual body weights of animals were determined during the acclimatisation period on the day of exposure, prior to exposure and on test days 2, 4, 8 and 15 and at time of death if survival exceeded day 1. Changes in weight were calculated and recorded when survival exceeds one day. At the end of the test, the animals were weighed and sacrificed.
- Necropsy of survivors performed: Yes; Necropsy of all animals was carried out and all gross pathological changes were recorded.
Other examinations performed:
- The weight of the lungs was determined and recorded.
- No microscopic examination was carried out as no pathological findings were noted at necropsy.
Statistics:
No data

Results and discussion

Preliminary study:
Not applicable
Effect levelsopen allclose all
Sex:
male
Dose descriptor:
LC50
Effect level:
0.048 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Sex:
female
Dose descriptor:
LC50
Effect level:
0.04 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Sex:
male/female
Dose descriptor:
LC50
Effect level:
0.041 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
- Premature death was observed in 3/3 males and 3/3 females at the concentration of 0.074 mg/L air
- Premature death was observed in 2/5 males and 2/5 females at the concentration of 0.047 mg/L air.
- None of 5 male and 1/5 female animals died prematurely at the concentration of 0.030 mg/L air.
Clinical signs:
other:
Body weight:
All surviving animals gained the expected body weight.
Gross pathology:
No pathological findings were noted at necropsy.
Other findings:
None

Any other information on results incl. tables

Table 7.2.2/2: Summary of results

Symptoms/Criteria

Chlorine dioxide

0.074 mg/L air

0.047 mg/L air

0.030 mg/L air

Males (n=3)

Females (n=3)

Males (n=5)

Females (n=5)

Males (n=5)

Females (n=5)

Clinical signs:

Ataxia

+

0’-3 h

(3)

+

0’-3 h

(3)

+

0’-60’

(5)

+

0’-60’

(5)

+

0’-30’

(5)

+

0’-30’

(5)

Tremor

+

0’-60’

(3)

+

0’-60’

(3)

+

0’-60’

(5)

+

0’-60’

(5)

+

0’-30’

(5)

+

0’-30’

(5)

Dyspnoea

+-++

0’-3 h

(3)

+-++

0’-3 h

(3)

+

0’-3 h

(5)

+

0’-3 h

(5)

+

0’-2 d

(5)

+

0’-2 d

(5)

Mortality

Within 3 h

0

0

0

0

0

0

Within 24 h

3

3

2

2

0

1

Within 7 days

3

3

2

2

0

1

Within 14 days

3

3

2

2

0

1

Mean body weight (g)

Start

265.3

246.3

236.2

227.2

278.8

235.6

After 7 days

#

#

299.0

(+26.6)

250.3

(+10.2)

328.0

(+17.6)

249.8

(+6.0)

After 14 days

 

 

335.0

(+43.9)

268.0

(+19.3)

354.2

(+28.6)

259.0

(+11.2)

Inhibition of body weight gain

#

#

None

None

None

None

Necropsy findings

None

None

None

None

None

None

 

+ = slight; ++ = moderate; ' = minutes; h = hours; d = days; 0' = immediately after end of administration; # = all animals died prematurely

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
Chlorine dioxide solutions have to be classified as Category 4 (H332: Harmful if inhaled) for acute inhalation toxicity (ATI4) above a concentration limit of 0.82%.
Executive summary:

In an acute inhalational toxicity study performed in accordance with GLP and OECD Guideline 403, groups of CD / Crl:CD(SD) rats were exposed to 0.6% aqueous chlorine dioxide (ClO2) solution, inherently stabilized *, at actual concentrations of 0.030, 0.047 and 0.074 mg/L air for 4 h by inhalation (mist) using a dynamic nose-only exposure chamber. The exposure concentrations were determined by iodometry.Animals were then observed for mortality, clinical signs and bodyweights for 14 days and necropsy was performed in all animals for macroscopical examination.


 


In the inhalation chamber, close to the animals' noses, the spray-jet generated atmosphere had mass median aerodynamic diameters (MMAD) between 2.388 and 2.467 μm as determined with a cascade impactor. The Geometric Standard Deviations (GSD) of the MMAD were calculated as 2.69, 2.74 and 2.78.


 


At the concentration of 0.074 mg/L air, slight ataxia, slight tremor and slight to moderate dyspnoea and premature death in all 3/3 male and 3/3 female animals. At the concentration of 0.047 mg/L air, slight ataxia, slight tremor and slight dyspnoea in all 5/5 male and 5/5 female animals. Two of 5 male and 2 of 5 female animals died prematurely. At the concentration of 0.030 mg/L air, slight ataxia, slight tremor and slight dyspnoea in all 5/5 male and 5/5 female animals. None of 5 male and 1/5 female animals died prematurely. All surviving animals gained the expected body weight. No pathological findings were noted at necropsy.


 


LC50 for males and females were 0.048 and 0.040 mg/L air/4 h, respectively. LC50 males and females combined (14 days): 0.041 mg/L air/4 h as pure ClO2. Therefore, based on the calculation method of CLP, the LC50value corresponds to 6.83 mg 0.6% ClO2 solution/L air/4h as pure ClO2 which is lower to the classification limit for a mist (5 mg/L) according to the CLP.


 


According to the study results, and applying the classification criteria for acute inhalation toxicity (5 mg/L for mist/aerosol), chlorine dioxide solutions have to be classified as category 4 for acute inhalation toxicity (ATI4) above a concentration limit of 0.82%, based on prorata of concentration according to CLP Regulation (EC) 1272/2008 (6.83 mg/L x 0.6% / 5 mg/L).

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