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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008
Reference Type:
publication
Title:
No information
Author:
Pauluhn J
Year:
2009
Bibliographic source:
Inhalation Toxicology 21 (S1), 40-54
Reference Type:
publication
Title:
No information
Author:
Ellinger-Ziegelbauer H
Year:
2009
Bibliographic source:
Toxicology 266, 16-29

Materials and methods

Principles of method if other than guideline:
OECD Guideline No. 403 (Acute Inhalation Toxicity)

EU Method B.2 (Acute Toxicity (Inhalation))

EPA OPPTS 870.1300 (Acute inhalation toxicity)


The study was conducted in accordance with the above mentioned test guidelines, as far as relevant for this particular study design. This acute inhalation toxicity study was performed to analyze the pulmonary response to the aerosolized MWCNT (carbon black (Printex 90) and crystalline quartz served as reference groups) in Wistar rats. The exposure was for 6-hours on one single day followed by a postexposure period of maximal 3 months. The results from this study serve the purpose of a dose-range finding study (for a 3 months subchronic inhalation study).
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan-Winkelmann, Borchen, Germany
- Age at study initiation: approx. 2 months
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40 -70
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12


Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
other: conditioned compressed dry air
Details on inhalation exposure:
EXPOSURE CONDITIONS:
- Mode of exposure: Animals were exposed to the aerosolized test substance in Plexiglas exposure tubes applying a directed-flow nose-only exposure principle. Tubes were chosen that accommodated the animals size. These tubes were designed so that the rat's tail remained outside the tube, thus restrained-induced hyperthermia can be avoided.
- Vehicle: The test substance was aerosolized as dust without a carrier or vehicle.

AEROSOL GENERATION AND EXPOSURE TECHNIQUE:

- System of generating particulates:

The test substance was micronized with a Retsch Centrifugal Ball Mill S100. The test substance was characterized before and after micronization. This comparison demonstrated that the test article was not affected by the micronization process to any significant extent. In brief, the reanalysis of the micronized test article showed that the fraction of particulates below 4 µm was increased as called for by the testing requirements. This was achieved without changing the composition and morphology of the agglomerated structures of the actual test substance. A confirmatory analysis was also made from the aerosolized test article (collection of particulates from the inhalation chamber). Again the composition and morphology of the agglomerated structures of the aerosolized test article were essentially identical with the non-dispersed/nonmicronized test substance.

- Description of analytical method used for verification of test substance stability:

Laser Diffraction
Scanning Electron Microscopy (SEM)
Inductive Coupled Plasma - Optical Emission Spectroscopy (ICP-OES)
X-ray Electron Spectroscopy (XPS)
Surface Area Burnauer Emmett-Teller (BET)
Transmission Electron Microscopy (TEM)

- Aerosol generation:

Quartz and Carbon Black: A Rotating Brush Generator (RBG) from Pallas, Germany was used for powder dispersion. The operation principle was as folIows: a compacted mass of powder was loaded into the cylindrical feed stock reservoir (softly compressed using a negligible pressure) and was then brushed-off by a rotating brush feeder. The brush removed a well-defined quantity of powder uniformly across the whole surface area of the compact. This abraded powder was then carried away from the dispersion head by an air stream (approx. 100 kPa dispersion pressure) and then into the inhalation chamber. The Nylon brush rotated at approximately 1200 revolutions/min (maximum range) using a piston driving speed of 10-15 mm/hour. From this device defined amounts of the test article were scraped off and then passed directly into the inner cylinder of the inhalation chamber. Baytubes C150 P: Test atmospheres were generated using a WRIGHT DUST FEE DER system (BGI Inc., Waltham, MA 02154, USA). For dry powder dispersion, conditioned compressed dry air (30 liters/min; generic dispersion pressure: approximately 150 kPa) was used. The principle performance of the WRIGHT DUST FEEDER dust generating system can be described as foliows: the test substance was metered in a reservoir and then was compressed to a pellet using approximately 1 metric ton by a carva laboratory press (F. S. Carver Inc., Wabash, IN 46992, USA). From this pellet defined amounts of test substance were scraped off and entrained into the main air flow. The airbome powder was then conveyed into the inner cylinder
of the inhalation chamber.

- Inhalation chamber: The aluminum inhalation chamber has the following dimensions: inner diameter = 14 cm, outer diameter = 35 cm (two-chamber system), height = 25 cm (internal volume = about 3.8 l).

-Conditioning the compressed air: Compressed air was supplied by Boge compressors and was conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer. Adequate control devices were employed to control supply pressure.

- Inhalation chamber steady-state concentration: The test atmosphere generation conditions provide an adequate number of air exchanges per hour (30 L/min x 60 min/(2 x 3.8 l) = 237, continuous generation of test atmosphere). Under such test conditions steady state is attained within one minute of exposure. At each exposure port a minimal air flow rate of 1.5 l/min was provided. The test atmosphere can by no means be diluted by bias-air-flows.

- Air flows: During the exposure period air flows were monitored continuously and, if necessary, readjusted to the conditions required. Air flows were measured with calibrated flowmeters and/or specialized flow-calaibration devices (DryCal) and were checked for correct performance at regular intervals.

- Treatment of exhaust air: The exhaust air was purified via cotton-wool/HEPA filters. These filters were disposed of by Bayer AG.

ANALYSIS OF THE TEST ATMOSPHERE:
- General remark: A nominal concentration was not calculated since the construction and weight of the dust generator used did not allow for a precise measurement of the powder aerosolized.
- Gravimetric evaluation: The test-substanee concentration was determined by gravimetric analysis (filter: Cellulose-Acetate-Filter, Sartorius, Göttingen, Germany; balance: Mettler AE 100). Chamber samples were taken in the vicinity of the breathing zone. The number of sampIes taken was sufficient to characterize the test atmosphere and was adjusted so as to accommodate the sampling duration and/or the need to confirm specific concentration values. Optimally, samples were collected in hourly intervals. All analytical concentrations reported refer to mg of test substance/m3 air (gravimetrical method).

CHARACTERIZATION OF AERODYNAMIC PARTICLE-SIZE DISTRIBUTATION:
- General remark: The sampIes for the analysis of the particle-size distribution were also taken in the vieinity of the breathing zone.

The particle-size distribution was analyzed using an BERNER-TYPE Aeras low-pressure critical orifice cascade impactor (Hauke, Gmunden, Austria).. The individual impactor stages were covered by aluminum foil and had been subjected to gravimetric analysis. An adhesive stage coating (silicone spray) was used to prevent particle bounce and reentrainmenty. Gravimetric analyses were made using a digital balance.


RESULTS OF PARTICLE-SIZE ANALYSES:
- Particle size distribution
carbon black, 250 mg/m³ exposure group: 69.9 % resp. of particles were < 3 µm.
quartz, 250 mg/m³ exposure group: 66.9 % resp. of particles were < 3 µm.
Baytubes C 150P, 10 and 250 mg/m³ exposure groups: 52.8 and 62.3 % ,resp. of particles were < 3 µm.

- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.):
carbon black, 250 mg/m³ exposure group: MMAD: 1.98 µm; GSD 2.23
quartz, 250 mg/m³ exposure group: MMAD: 2.32 µm; GSD 1.82
Baytubes C 150P, 10 and 250 mg/m³ exposure groups: MMAD: 2.90 and 2.24 µm; GSD: 1.64 and 1.63, resp.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
6 h
Remarks on duration:
followed by a postexposure period of max. 3 months
Concentrations:
MWCNT: 0, 10, 250 mg/m³ (target concentrations)
MWCNT: 11, 241.3 mg/m³ (gravimetrical concentration)
reference substances:
crystalline quartz (= positive control): 0, 250 mg/m³ (target concentration)
crystalline quartz (0 positive control): 2247.5 mg/m³ (gravimetrical concentration)
carbon black (= negative control): 0, 250 mg/m³ (target concentration)
carbon black (= negative control): 228.6 mg/m³ (gravimetrical concentration)
No. of animals per sex per dose:
40
Control animals:
yes
Details on study design:
ADMINISTRATION:
- Type of exposure: dynamic directed-flow nose-only
- Particle size: MMAD 2.2-2.9 µm, GSD approx. 1.8 to 2.6
- Observation period: max, 3 months

EXAMINATIONS:
- Body weights were recorded immediately prior to exposure on day 0, on postexposure days 1, 3, 7 and  14.

- Clinical observations were made several times on the exposure day and at least once a day up to postexposure day 7, and thereafter weekly.

- Bronchoalveolar lavage (on days 7, 28, and 90 during the postexposure period):
The following prameters were determined in lavage fluid: total cell count, mean cellular diameter, mean cellular volume, lactate dehydrogenase, alkaline phosphatase, collagen, protein, phospholipids, y-glutamyltransferase, ß-n-acetyl-glucosaminidase, and cytodifferentiation of BAL cells.
The following parameters were determined in lavage cells: Rat specific cytokines (interferon-gamma (IFN-y), IL-1a, IL-4, TNFa, granulocyte-macrophage colony-stimulating factor (GM-CSF)) and the chemokine monocyte chemotactic protein-1 (MCP-1).

- Cobalt determinations (restricted to MWCNT exposed rats): right lung lobes, lung-associated Iymph nodes (LALN), liver, kidneys, testes, and brain at the days 7 and 90 serial sacrifices

- Necropsy (during the 3 months postexposure period subgroups of rats (at least 6 per sacrifice) were serially sacrificed on postexposure day 7, 28 and 90): all gross pathological changes were recorded, with particular reference to changes related to the respiratory tract

- Organ weights: lung and lung-associated lymph nodes (LALNs) on postexposure days 7, 28, 90

- Histopathology: lungs (Baytubes: from sacrifices of days 28 and 90; quartz and carbon black: from sacrifices of days 90)

- Gene Expression: lung tissues (MWCNT 10, and 250 mg/m³ and carbon black from sacrifices of days 90)
Statistics:
For the statistical evaluation of sampies drawn from continuously distributed random variates three types of statistical tests are used, the choice of the
test being a function of prior knowledge obtained in former studies. Provided that the variate in question can be considered approximately normally distributed with equal variances across treatments, the Dunnett test is used, if heteroscedasticity appeared to be more likely a p value adjusted Welch test is applied. If the evidence based on experience with historical data indicates that the assumptions for a parametricanalysis of variance cannot be maintained, distribution-free tests in lieu of ANOVA are carried out, i. e. the Kruskal-Wallis test followed by adjusted MWW tests (U tests) where appropriate. Global tests including more than two groups are performed by sex and date, i. e. each sex x date level defines a family of tests in the context of multiple comparison procedures (MILLER RG (1981). Simultaneous statistical inference. 2nd edition, Springer, Berlin, Heidelberg, New York, Tokyo.). Within such a family, the experimentwise
error is controlled. If not otherwise noted, all pair-wise tests are two-sided comparisons.

Results and discussion

Effect levels
Sex:
male
Dose descriptor:
LC50
Effect level:
> 241 mg/m³ air (analytical)
Exp. duration:
6 h
Remarks on result:
other: Exposure concentration corresponds to the maximum technically attainable concentration of MWCNT
Mortality:
Aerosol (dust) concentrations of 250 mg/m³ (0 target concentration) MWCNT, quartz and carbon blackt were tolerated without mortality (details see table 1).
Clinical signs:
Quartz dust and 250 mg/m³ MWCNT exposed rats displayed irregular and labored breathing patterns, tachypnea. Non-specific signs,
such as piloerection and an ungroomed hair-coat were also observed. Evidence of alopecia existed in some animals. However, this is not considered to be exposure-related.
Body weight:
A significant transient decrease occurred at 250 mg/m³ MWCNT. The exposure to both carbon black and quartz was associated with similar changes in body weights.
Gross pathology:
carbon black: discolorations were detected in lungs and lung-associated Iymph nodes (LALNs)
quartz: LALNs were enlarged, lung were discolorated/slightly collapsed.
MWCNT: At 10 mg/m³ discolorations of the lung (light gray areas) were observed at the day 7 sacrifice, whilst no changes were seen thereafter. Discoloration of the lungs and enlargement and discolorations of LALNs were apparent at all sacrifices at 250 mg/m³; at 10 and 250 mg/m³ the lung weights were significantly increased (day 7 and 90)
Other findings:
- Bronchoalveolar lavage:

Due to the black colour of carbon black and Baytubes phagocytized by cells, the results from cytodifferentiation was predominated by 'non-classifiable'cells (NCs). Nonetheless, it appears to be scientifically justified to assume the most of the NCs are alveolar macrophages, indeed. With regard to the unequivocal classification of neutrophilic granulocytes (PMN) equal concentrations of quartz caused the same influx of PMNs as compared to MWCNT, especially at late time points. At 10 mg/m³ MWCNT elicited still a somewhat higher recruitment of PMNs when compared with the carbon black group exposed at 250 mg/m³. Foamy macrophages, which are indicative of phospholipoproteinosis, occurred in quartz- and MWCNT exposed rats. A time-related exacerbation did not occur in MWCNT-exposed rats. Inflammatory endpoints appeared to be more pronounced in MWCNT exposed rats when compared to the quartz exposure group at a similar concentration. However, while changes increased over time in quartz-exposed rats they decreased in MWCNTexposed rats. Alkaline phosphatase was higher than in carbon black throughout the study, wh ch is suggestive of a sustained increased activity of type II pneumocytes. Pro-inflammatory endpoints suggestive of fibrogenesis and oxidative stress were more elevated in MWCNT-exposed rats when compared to carbon black and quartz.
Nonetheless, the exposure to 10 mg/m³ MWCNT caused more extensive changes in BAL-endpoints as compared to 250 mg/m³ carbon black. Throughout the course of the postexposure period inflammatory endpoints and those suggestive of Iysosomotropic activity were slightly increased at 10 mg/m³ MWCNT and above.

- Cobalt determinations:

A concentration-dependent increase of cobalt occurred in the lung. The determinations in the remaining tissues were unobtrusive. The time-related clearance over a postexposure period of 3 months was 96% and 43% at the intermediate and high exposure level, respectively.

- Histopathology: lungs:

Minimal to slight findings were detected in the lungs of all animals. In controls and 10 mg MWCNT/m³ exposed rats minimally enlarged and/or foamy macrophages were observed. In rats exposed to MWCNT changes were characterized by a more pronounced accumulation of enlarged and/or foamy macrophages at 10 mg/m³and above. At 250 mg/m³, hypercellularity in the bronchiolo-alveolar region with focal septal thickening and focally increased septal collagen occurred. The extent of intraalveolar accumulation of macrophages may be difficult to judge from a quantitative perspective due to prior lung lavage. Therefore, changes in BAL appear to be of higher pathodiagnostic sensitivity than histopathology examinations. Distinct time-dependent differences between recovery days 28 and 90 were not apparent. However, at 250 mg/m³, the grading of some findings (hypercellularity in the bronchiolo-alveolar region, focal septal thickening and focal increased septaI collagen) was minimally increased in some rats after 90 days. After quartz exposure, generally slight inflammatory findings could be detected in the lungs of all animals. These included an accumulation of enlarged and/or foamy macrophages, inflammatory infiltrates, and hypercellularity of the bronchiolo-alveolar region. Furthermore, slight septal thickening occurred in all rats with minimally or slightly increased septal collagen in Sirius-red stained slides. These findings reflect a process of beginning
interstitial fibrosis. As compared to the concurrent air controls, in the carbon black exposed rats macrophages with black cytoplasm (grade 1) were seen and minimal hypercellularity in the bronchiolo-alveolar region. Additionally, black macrophages occurred in the BAL T (bronchial associated Iymphatic tissue).

- Gene Expression:

The evaluation of the gene expression deregulation revealed, that only one gene, SPP1 (= OPN Osteopontin) was deregulated more than two-fold in at least one sampie. SPP1 appeared robustly increased after inhalation of 10 mg/m³ MWCNT.

Any other information on results incl. tables

Table 1: Acute inhalation toxicity (dust aerosol) of Baytubes C 150P, quartz and carbon black

Group/ Sex Target concentration (mg/m3) Toxicological result  Onset and duration of signs  Onset of mortality
 1/male Control Air 0 / 0 / 40  --- --- 
2/male Quartz 250 0 / 40 / 40 0d - 2d --- 
 3/male carbon black250 0 / 0 / 40 ---  --- 
4/male  Baytubes C 150P10 0 / 0 / 40  --- ---
5/male   Baytubes C150P100 0/ 40 / 40 0d - 2d  ---

Toxicological results:

number of dead animals / number of animals with signs after cessation of exposure / number of animals exposed

Applicant's summary and conclusion

Executive summary:

Rats (40 males/dose group) were exposed to 11 or 241 mg MWCNT/m³ of respirable, solid aerosol for 6-hours on a single day which was followed by a maximum postexposure period of 3 months. Micronization of MWCNT was applied to increase the dustiness of the test material without destroying the assemblage structure of MWCNT. This was verified by characterization the MWCNT before and after micronization. A confimatory analysis was also made from the aerosolized MWCNT which confirmed that the composition and morphology of the aerosolized MWCNT were essentially identical with the non-dispersed/non-micronized MWCNT.

Rats similiary exposed to air, carbon black (229 mg/m³) or crystalline quartz (248 mg/m³) served as control and reference groups. The study was conducted in accordance with OECD TG 403 and expanded to include the determination of inflammatory endpoints in bronchoalveolar lavage, gene expression analysis of eight selected genes with potential role in fibrosis, determination of cobald (lungs, lung-associated lymph nodes, brain, kidneys, testes and liver) and histopathological examination of the lungs.

No specific clinical signs or consistent changes in body weights were observed at 10 mgMWCNT/m³ while the rats exposed to 250 mg MWCNT/m³ revealed transient clinical signs (irregular and labored breathing patterns up to postexposure day 2) and reduced body weights. Mortality did not occur in any group. Therefore the LC50 for MWCNT is described to be > 241 mg/m³, as this is the maximum technically attainable concentration of MWCNT.

Irreversible pulmonary inflammatory response (i.e. influx of polymorphonuclear cells (PMNs), which was more pronounced at 241 mg/m³, was observed at all reading points (7, 28, 90 days after exposure) with peak concentrations shortly after exposure (day 7).

The determination of cobalt as trace impurity of inhaled MWCNT yielded a concentration-dependent increase of cobalt in lungs.

A dose-dependent induction of gene expression 90 days after MWCNT exposure was restricted to the gene encoding SPP1 (= OPN = Osteopontin). During necropsy discolorations of the lungs were detected in rats exposed to 11 mg MWCNT/m³ at the day 7 sacrifice, whilst no changes were seen thereafter. Discolorations of the lungs and enlargement/discolorations of lung-associated lymph nodes (LALNs) were apparent at all sacrifices at 241 mg MWCNT/m³.

Histopathology at recovery days 28 and 90 revealed an accumulation of enlareged and/or foamy marcrophages with dark cytoplasmatic spots in the 11 and 241 mg/m³-groups. Bronchiolo-alveolar hypercellularity, focal septal thickening and focally increased septal collagen were restricted to the 241 mg/m³-dose group. Distinct time-dependent differences between recovery days 28 and 90 were not apparent.