Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-01-26 to 2010-03-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed GLP study according to OECD technical guidelines.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
adopted 2009-09-07
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate attached to full study report.
Test type:
standard acute method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Strain Wistar Crl:(WI)WU
- Age at study initiation: 5-6 weeks at delivery
- Weight at study initiation: males 219g-236g, females 162-167g
- Housing: Makrolon, type III cages, two rats of the same sex and dose group per cage; cages and absorbing softwood bedding material were changed twice a week or more often if necessary
- Diet (e.g. ad libitum): Ssniff V1534, Ssniff Spezialdiäten, Soest, Germany, ad libitum
- Water (e.g. ad libitum): Tap water from Hannover city water supplier, ad libitum
- Acclimation period: 2-3 weeks (to be trained to be accustomed to nose-only tubes)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 2°C
- Humidity (%): 55% +/- 15%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
- The rats were placed around the exposure cylinder in tapered acrylic glass tubes with adjustable backstops. The animal's snout protrudes in the anterior end of the tube, which is connected to the exposure cylinder by means of a push fit.
- The aerosol enters the nasal region of the animal through a small tube.
- The exposure cylinder is operated at slightly positive pressure with respect to the surrounding air. This ensures that a continuous air flow is passing through the animal's breathing zone.
- In this system, the aerosol is supplied to each rat individually, and exhaled air is immediately removed. Therefore, oxygen supply is always sufficient and measurement of the oxygen concentration is not necessary.
- The airflow to each rat was approximately 1 l/min which is assumed to be laminar. The total flow rate was approx. 16 l/min; the total volume of the inhalation system including the mixing box ensured that the intended concentration of the test item was reached shortly after start of exposure (50%-value of concentration after approximately 4 min).
- The test item was dispersed using a suitable dispersion system operated with pressurized air.
- A pre-classifier removing bigger particles was used if necessary to adjust the size distribution of the aerosol to an MMAD below or equal to 3 µm. The signal was used to control the feed rate of the dispersion system in order to keep the aerosol concentration in the inhalation unit constant.
- Actual concentrations were measured in the breathing zone of the animals.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
See remarks on results
Duration of exposure:
4 h
Concentrations:
2000mg/m3
No. of animals per sex per dose:
3 males and 3 females, additionally one orienting experiment with one female
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days

DAILY AND WEEKLY OBSERVATIONS
- all animals were observed in their cages daily
- once a week they were carefully examined for abnormalities
- during the exposure and several times afterwards on the the day of exposure the animals were observed in the exposure tubes/cages
- thereafter, the frequency of observation was once a day (or more often if necessary) for the following 14 days
- clinical observation especially comprised: general condition, fur, grooming activity, visible mucous membranes, behavior and locomotor activity, central nervous symptoms, breathing pattern

BODY WEIGHTS
- once a week individual body weights were recorded to the nearest 0.1g for all animals
- in addition the animals were weighed directly prior to exposure

BODY TEMPERATURE BEFORE START AND AFTER END OF EXPOSURE
- was measured rectally to the nearest 0.1°C before and 1hr after the end of exposure

REFLEXES UPON CESSATION OF EXPOSURE
- Grooming activity as detectable by fur condition, mobility, ataxia, visual placing, climbing reflex, pinna reflex, vibrissae reflex, auditory startly, pain sensitivity and seizures were investigated 1, 24 and 48hrs upon cessation of exposure
- all parameters were assessed using a standardised scheme as influenced or not influenced (normal)

GROSS PATHOLOGY/NECROPSY
- all animals were carefully necropsied, comprising examination of the external surface, all orifices and the cranial, thoraic and abdominal cavities and their contents
Statistics:
- differences in rectal temperature and body weight between groups were considered case by case as statistically significant at the level of p<0.05
- t-test was used

Results and discussion

Preliminary study:
In an orienting experiment 1 female rat was exposed nose only for 4 hrs to an aerosol concentration of 2000mg of the test item per m3. Upon cessation of exposure the animals showed slight signs of discomfort (as usually observed after exposure of rats to particle aerosols). However, toxicity effects were not observed at all. Grooming acitivity started directly after the end of exposure. Based on this result, it was decided to start the limit test.
Effect levelsopen allclose all
Sex:
male
Dose descriptor:
LC50
Effect level:
> 2 000 mg/m³ air
Exp. duration:
4 h
Sex:
female
Dose descriptor:
LC50
Effect level:
> 2 000 mg/m³ air
Exp. duration:
4 h
Mortality:
No mortalities.
Clinical signs:
other: - Upon cessation of exposure, none of the rats exposed to the test item showed any signs of toxicity - Only usual signs of discomfort after exposure to particles were observed - Grooming activity started immediately after the end of exposure
Body weight:
See Table 1
- No significant treatment-related effects were observed
Other findings:
BODY TEMPERATURE (See Table 2)
- Body temperature was statistically significant decreased by 2.5°C in the female group, however, not in the male group

REFLEXES
- Following treatment, no abnormalities were observed

Any other information on results incl. tables

Summary table of results

Limit test with

Expanded Graphite

Wistar WI (WU) rats

 

Males

Females

Aerosol concentration (mg/m3) - mean (SD)

a aerosol photometer (n=5)

b filter samples (n=5)

 

2020a  (61)

1843b  (145)

MMAD

3.0 µm (n=2)

Number of exposed rats

 3

3

Exposure duration (hrs)

                       4

Number of rats showing affected reflexes 1/24/48 hrs after end of exposure

0 / 0 / 0

0 / 0 / 0

Body temperature decrease measured after end of exposure Δ° C: mean (SD)

-0.2

(0.1)

2.5***

(0.1)

Number of dead ani-mals within 14-day post-exposure period

0

0

Rats showing abnor-malities in gross pa-thology on day 14 of post-exposure period

0

0

significantly different compared to controls * p<0.05 ** p<0.01 *** p<0.001 (Student’s t-test); n = number of measurement

Monitoring and Controlling of the Exposure Atmosphere

Exhaust air flow, temperature and relative humidity were measured continuously and recorded as 10-minute means. Exposure concentrations were measured continuously by an aerosol photometer (by 10-minute intervals). In addition, they were controlled by taking filter samples (5 times per 4-hour inhalation). The MMAD of the aerosol phase was determined twice within the 4-hour exposure period using a cascade impactor (Marple impactor) with subsequent gravimetrical analysis. Measurements during exposure on Jan 27, 2010 resulted in the following data:

Aerosol concentrations:

Aerosol photometer:                           Mean:2.02 x 103+0.06 x 103mg/m3  (n=5)

Filter samples:

·       2021 mg/m3                                                          

·       1827 mg/m3                                                          

·       1592 mg/m3                                                          Mean:1843+145 mg/m3  (n=5)

·       1946 mg/m3                                                          

·       1827 mg/m3

The mean aerosol concentration of 2.02 x 103+0.06 x 103mg/m3, determined by the aerosol photometer, is an optimum for an acute study as guideline OECD 403 concedes a standard deviation of up to 20%. The gravimetrically determined concentrations are within this range as well.

Particle size distributions:

·       MMAD = 2.94 µm ; geometric SD = 2.04

·       MMAD = 3.12 µm ; geometric SD = 1.97                      Mean:3.03 µm (0.09)  (n=2)

Thus, both values are approx. 3 µm; this threshold should not be exceeded in order to ensure a sufficient respirability of the aerosols (OECD guideline 403 recommendations allow the following ranges: MMAD 1-4 µm; geometric SD = 1.5 - 3).

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
From the results observed in this acute 4 -hour nose-only inhalation study the following classification was derived for the test item Expanded Graphite Powder: Unclassified (according to Regulation (EC) No. 1272/2008)
Executive summary:

-        In an acute nose-only inhalation study (limit test) 3 male and 3 female rats were exposed for 4 hours to a target concentration of 2000 mg Expanded Graphite Powder per m3

-        For animal welfare and technical reasons, i.e. to prevent agglomeration of particles and to assure an MMAD in the respirable range from 1 -4μm, an aerosol concentration of 2000 mg test item/ m3 was used in this Limit Test, as recommended by Regulation (EC) No. 1272/2008 and OECD guideline 403.

-        Upon cessation of exposure none of the rats exposed to the test item showed any signs of toxicity. Only usual signs of discomfort after exposure to particles were observed. Grooming activity started immediately after the end of exposure.

-        Body temperature was statiscally significantly decreased by 2.5 °C in the female group, however, not in the male group. Such effect is commonly observed after exposure to large aerosol concentrations indicating a reduction of body functions and reversible.

-        All 6 rats showed a good general health status during the subsequent 14 -day postexposure observation period.

-        All 6 rats did not show any test item-related abnormalities upon necropsy.

-        From the results observed in this acute 4 -hour nose-only inhalation study the following classification was derived for the test item Expanded Graphite Powder: Unclassified (according to Regulation (EC) No. 1272/2008)

-        An LC50 was not determined in this study. An experimentally determined LC50 would be higher than 2000 mg/m3.