Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
other: Data sharing dispute
Adequacy of study:
key study
Study period:
2010-09-24 to 2010-10-09
Reliability:
1 (reliable without restriction)
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Batch no.: 29004
Appearance: charcoal typic
Purity: C-Fix: 80.5%; dose calculation not adjusted to purity
Expiry: December 2030
Storage condition: At room temperature, moisture protected
Safety precautions: Routine hygienic procedures

The test atmosphere was generated from the micronized powder of test item as supplied by the Sponsor. No vehicle was used.

The original test item was micronized by mill (Fritsch, Laborette, Lot. 09.203.381) to around 150 µm. This particle size was used in the test. The actual particle size distribution in the breathing zone was determined by the parameters of the aerosol generation system employed.

Test animals

Species:
rat
Strain:
other: CRL (WI) BR of Wistar origin Sex
Sex:
male/female
Details on test animals and environmental conditions:
Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90.
Hygienic level: SPF at arrival and kept in good conventional environment during the study.
Age of animals: Young adult rats, 8-12 weeks old
Body weight range at treatment: males: 314-345 g, female: 217-243 g
Number of animals: 5 male and 5 female (nulliparous and non pregnant animals) rats
Number of animals/group: 5 animals/ sex
Acclimatisation time: 15 d
Acclimatisation to the test apparatus: 4 d

Individual identification was performed by numbers on the tail of the animals written with a permanent marker. The numbers were given on the basis of the laboratory master file of the testing laboratory and were remarked as necessary to ensure the correct identification.
The cages were marked by cards holding information about study number, sex of animals, dose group, cage number and individual animal numbers.

All animals were sorted according to body weight by computer and grouped according to weight ranges. There were an equal number of animals from each weight group in each of the experimental groups during the randomisation.


Animal health: Only healthy animals were used for the study. Healthy status was certified by the breeder.
Housing: Group caging (2 or 3 animals, by sex, per cage)
Cage type: Type II polypropylene/polycarbonate.
Bedding: Certified laboratory wood bedding (Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG; D-73494 Rosenberg Holzmühle 1, Germany). The cages and bedding were changed twice a week.
Illumination: Artificial light, from 6 a.m. to 6 p.m.
Temperature: 22±3 °C
Relative humidity: 30-70 %
Ventilation: 8-12 air exchanges/hour by central air-condition system.

Environmental conditions were maintained by an air-condition system. Temperature and relative humidity were verified and recorded daily during the study.

The animals received ssniff® SM R/M-Z+H complete diet (ssniff Spezialdiäten GmbH, D-59494 Soest, Germany) ad libitum and tap water from watering bottles.
The food and water are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The food supplier provided an analytical certificate of the standard diet for the batch used. The drinking water is periodically analysed.

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Anodised aluminium Flow Past Exposure Chamber (CR Equipment SA, Switzerland)
- Exposure chamber volume:
- Method of holding animals in test chamber: The animals were held in polycarbonate restraining tubes and exposed “nose-only” under dynamic air flow conditions. The animals were distributed in two levels of the exposure chamber.
- Source and rate of air: The air was supplied by an oil-free air compressor and was filtered in a two-stage filter set. The temperature of the air was regulated by a heat exchanger. The airflow rate to each animal port was 1.0 L/min.
- System of generating particulates/aerosols:
The test atmosphere was generated by the compressed air dry powder dispersion technique at the pressure of 5 bar. The test item was compacted in the container of the aerosol generator type Wright (TSE Systems GmbH, Bad Homburg, Germany) by the pressure of approximately 1.5 ton/cm². An abrasive disc was used to supply the test item particles into the dispersing tube (diameter: 2.4mm, length: 150mm). The proportion of large particles was decreased by implementing in the system an impactor- and a cyclone-type micronizer/size-selector (TSE Systems GmbH, Bad Homburg, Germany). Constancy of particle size distribution was determined by performing multiple particle size analyses during characterization using a cascade impactor.
- Method of particle size determination: See section "any other information on materials and methods incl. tables".
- Temperature, humidity, pressure in air chamber: Temperature: 20.3–23.3°C, relative humidity: 40.3–62.6%

TEST ATMOSPHERE
- Brief description of analytical method used: See section "Analytical verification of test atmosphere concentrations"

TEST ATMOSPHERE
- Particle size distribution: Inhalable fraction (< 4 µm): 52.3%
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 3.523 µm / 2.46
Analytical verification of test atmosphere concentrations:
yes
Remarks:
The actual concentration of generated atmosphere was measured gravimetrically at regular intervals (about 50-60min) during exposure by pulling a volume of 4L of test atmosphere from the exposure chamber through glass fibre filters Fiberfilm T60A20 (Pall)
Duration of exposure:
4 h
Concentrations:
Mean achieved concentration: 4.968 mg/L
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 d
- Frequency of observations and weighing:
Animals were checked twice daily during the 14-d observation period for morbidity and/or mortality.
All animals were observed for clinical signs at 1st, 2nd and 3rd hours during exposure, as soon as practicable following removal from restraint, 1 h after exposure and subsequently once daily for 14 d.
Individual bodyweights were recorded on the day of exposure prior to exposure (Day 0) and on Days 1, 3, 7 and 14.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,organ weights, histopathology, other:
Statistics:
No statistical evaluation was performed.

Results and discussion

Preliminary study:
A sighting exposure was performed with a test item concentration of 5 mg/L on a single male and female animal. Animals were exposed for 4 h. The achieved average concentration of the test atmosphere was 4.7 mg/L, determined by gravimetry and aerosol light scattering photometer. In the breathing zone the MMAD of the dust particles was 3.48 μm. Both animals survived the treatment and the following 72 h.
Mortality:
No animal died during the study.
Clinical signs:
other: All animals were observed for clinical signs at the 1st, 2nd and 3rd hour during exposure, as soon as practicable following removal from restraint, 1 h after exposure and subsequently once daily for 14 d. Individual clinical observations are presented in
Body weight:
Individual bodyweights were recorded on the day of exposure Day 0 (prior to exposure) and Days 1, 3, 7 and 14.

In both genders, body weight loss was observable on the day of inhalation exposure. In both sexes, a compensation of body weight loss was found from the 3rd day of the observation period onwards.
On basis of body weight and body weight gain data, there was no notable test item effect observable in the exposed animals.
Gross pathology:
At the end of the 14-d observation period, the animals were euthanized by exsanguination under anaesthesia and gross necropsies were performed. These included a detailed examination of the abdominal and thoracic cavities with special attention given to the respiratory tract for macroscopic signs of irritancy or local toxicity.

Greyish mottled lungs and grey-coloured peribronchial lymph nodes were observed in the test animals, which may be related to test item but do not have any toxicological relevance.
Other findings:
Deposition of brown-black particles in the lumen of alveoli and in the interstitium were detectable in the investigated lungs, in connection with the macroscopically observable discoloration of the organ.

The localisation of these particles were mainly extracellular but some particles were phagocytized by alveolar macrophages. No particles were visible on the ciliated epithelium of the airways in the bronchial region, trachea or nasal mucosa.

No inflammatory infiltrates, oedema, accumulation of alveolar macrophages or accumulation of phospholipids in alveoli (alveolar proteinosis or lipoproteinosis) was detectable. Hypertrophy or hyperplasia of type II cells in the alveoli was not observed as well.

Deposition of brown-black particles in the sinusoidal lumen of peribronchial lymph nodes was observed, too. No any degenerative, inflammatory or other lesion with toxicological significance in the lymph nodes was detected.

Any other information on results incl. tables

All relevant information on results is provided in the sections above.

Applicant's summary and conclusion

Interpretation of results:
practically nontoxic
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the experimental conditions of this study, no deaths occurred in a group of ten rats exposed to a mean achieved atmosphere of 4.97 mg/L for four hours. The acute inhalation median lethal concentration (4-h LC50) of test item Charcoal (Probe 2: C-Fix=80.5%), in Wistar Crl:(WI) BR rats, was therefore considered to be greater than 4.97 mg/L.
Executive summary:

The acute inhalative toxicity of charcoal (Probe 2: C-Fix=80.5%) was investigated in a study in rats that was performed according to OECD guideline no. 403, EU method B.2 and the US EPA Health Effects Test guideline OPPTS 870.1300, Acute Inhalation Toxicity, as per August 1998.

In this study, a group of 10 Wistar Crl:(WI) BR rats (5 males and 5 females) was exposed to an aerosol atmosphere. The animals were exposed for 4 h using a nose-only exposure system, followed by a 14-d observation period.

The mean achieved concentration of charcoal in the exposure was 4.968 mg/L (standard deviation: 3.624 mg/L; nominal concentration: 18.2 mg/L)

The characteristics of the test atmosphere were as follows: Mean mass median aerodynamic diameter (MMAD) (mm): 3.52 µm; geometric standard seviation: 2.46; inhalable fraction (< 4 µm): 52.3%

No death occurred in the test animals.

In males, test item related clinical signs were found between the third hour of inhalation exposure and first hour of observation period. All animals were symptom –free on first day of observation period.

In females animals, test item related clinical signs were found between the third hour of inhalation exposure and first hour of observation period. All female animals were symptom–free from first day of observation period.

The clinical signs represented the decreased activity and general reaction and dyspnoea.

In both genders, body weight loss was observable on the day of inhalation exposure. In both sexes, a compensation of body weight loss was found from third day of observation period.

On basis of body weight and body weight gain data, there was no notable test item effect observable in the exposed animals.

In conclusion, a single 4-h nose-only exposure to charcoal sample Probe 2 to CRL: (WI) BR rat followed by a 14-day observation period at a dose level 5 mg/L was not associated with mortality or any test item-related toxicological findings on the male and female animals.

Accordingly, the acute inhalation median lethal concentration (4-h LC50) of charcoal rats was therefore considered to be greater than 4.97 mg/L.