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Repeated dose toxicity: inhalation

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short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Before April 30, 1996
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
As most publications, this one does not present all the details that are normally presented in a standard study report. However, a thorough and extensive study is described in the publication that gives valuable information on the repeated dose toxicity upon inhalation of the substance and can thus be used to address the requirement in question. Moreover, the publication is from a well-known peer-reviewed paper and the study was carried out by a laboratory with a good reputation. The most serious short coming is the limited number of endpoints investigated. The study was largely restricted to local effects in the respiratory tract, in particular inflammation, cell propliferation and effects on clearance.
Reason / purpose for cross-reference:
reference to same study

Data source

Reference Type:

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
Male rats were exposed by inhalation to carbonyl-iron particles or titanium dioxide particles by nose-only inhalation for 28 days, 6 h/day and 5 days/week.. The effects studied were limited to inflammation and cellular proliferation in the lungs as well as effects on clearance. Due to the limited number of endpoints studied it does not meet the standard guidelines for repeated-dose inhalation testing. Systemic toxicity was not covered at all.
GLP compliance:
not specified
It is not customary to refer to GLP in studies published in peer-reviewed scientific journals.
Limit test:

Test material

Constituent 1
Reference substance name:
carbonyl iron
carbonyl iron
Details on test material:
- Name of test material (as cited in study report): carbonyl iron
- Substance type: monoconstituent
- Physical state: solid particles; 0.2-2 micrometer
- Analytical purity: no data; see "Other"
- Impurities (identity and concentrations): no data; see "Other"
- Composition of test material, percentage of components: no data; see Other"
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Radiochemical purity (if radiolabelling): not labelled
- Specific activity (if radiolabelling): not labelled
- Locations of the label (if radiolabelling): not labelled
- Expiration date of radiochemical substance (if radiolabelling): no labelled
- Stability under test conditions: no data
- Storage condition of test material: no data
- Other: Commercial carbonyl iron was purchased from the GAF Corporation (New York), a well known source of this material. The production of carbonyl iron by reduction of iron pentocarbonyl results in a very high purity. A search on the internet made clear that carbonyl iron of GAF is used as a dietary iron supplement. We further refer to Gordeuk et al., 1987, Am J Clin Nutr 46, 1029-1034. The following is a quote from this publication. "Carbonyl Fe, a pure form of elemental Fe used widely as a food additive (7, 8), has remarkably low toxicity and much larger doses are tolerated when compared with ionized forms of Fe such as FeSO4 (9, 10). Carbonyl does not refer to the composition of the Fe particles but rather to the manufacturing process in which the controlled heating of vaporized Fe pentacarbonyl leads to the deposition of uncharged elemental Fe as microscopic spheres of < 5 /L in diameter (11 ). Although not now approved by the Food and Drug Administration for pharmacologic use, recent studies have demonstrated that carbonyl Fe is effective for the treatment of Fe deficiency anemia (12) and for the replacement of Fe lost in blood donation (13). The low toxicity of carbonyl Fe has permitted a study to test if 10 times the usual daily amount of Fe would be therapeutically effective in a shorter time period." See original publication for references.

Test animals

other: Crl:CDBR
Details on test animals or test system and environmental conditions:
7-8 weeks old; from Charles River Breeding Laboraties, Kingston, NY. No further details provided. The experiment was ended 180 days after the end of exposure.

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Remarks on MMAD:
MMAD / GSD: MMAD was determined during the exposure. The following average values were found: 3.4 µm, 3.2 µm and 2.9 µm for 5, 50 and 250 mg/m^3 (nominal) respectively.
Details on inhalation exposure:
The authors of the publication refer to Warheit et al., 1991 (Toxicology and Applied Toxicology 107: 350-368) for a description of the exposure methods. The section in question in that publication is reproduced below.

Animals were placed in cylindrical polycarbonate or stainless steel holders equipped with conical nose pieces. The restrainers were inserted into face plates on the exposure chambers such that the nose of each animal protruded into the chamber. Atmospheres of carbonyl iron … were generated with a K-tron bin feeder equipped with twin feed screws. The dust was metered into a polycarbonate transfer tube where high pressures of air swept the test material into the exposure chamber. Chamber concentrations of silica or iron were maintained by controlling the dust-feed rate into the generation apparatus, or by varying the air-flow rate. For gravimetric analysis, samples of atmospheric carbonyl iron … were taken from the animal breathing zone at approximately 30-min intervals by drawing calibrated volumes of chamber atmosphere through preweighed glass-fiber filters. Filters were weighed on a Cahn 26 automatic electrobalance. The atmospheric concentrations of iron … particles were determined from the filter weight differentials before and after sampling. Particle size measurements of airborne particles in the test chamber were determined with a Sierra cascade impactor and reported as the mass median aerodynamic diameter (MMAD) and the percentage of particles with less than a 10 µm aerodynamic diameter.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
See above under "Details on inhalation exposure"
Duration of treatment / exposure:
28 days, 5 days/week, 6 h/day
Frequency of treatment:
one exposure of 6 h per day. Five days with exposure were followed by two day without exposure.
Doses / concentrations
Doses / Concentrations:
0, 4.8±2, 51.8±15 and 243.6±90 mg/m^3
analytical conc.
No. of animals per sex per dose:
It is only stated that "Groups of male ... rats ... were used".
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): no data
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: Groups of rats were investigated for several endpoints 0, 7, 30, 90 and 180 days after termination of exposure.
- Section schedule rationale (if not random): no data

Because the study design differed in various aspects from the standard design of an inhalation study, the submitter reproduced the "Methods" section of the original publication below. The reader is referred to the original publication for the literature references.

"- General experimental design.

Groups of male Crl:CDBR rats (7-8 weeks old, Charles River Breeding Laboratories, Kingston, NY) were used to assess the pulmonary effects of 4-week inhalation exposures to high concentrations of titanium dioxide or carbonyl iron particles. Animals were exposed 6 hr/day, 5 days/week, for 4 weeks at concentrations of 5,50, or 250 mg/m3 Following exposures, the lungs of TiOz- and carbonyl iron¬exposed animals and aged-matched sham controls were subsequently evalu¬ated by bronchoalveolar lavage fluid analysis, BrdU cell labeling, lung clearance analysis, in vitro macrophage function, and histopathology at 0 hr, I week, and 1,3, and 6 months postexposure.

- Inorganic particulates.

Carbonyl iron powder (metallic iron; particle size range 0.2-2.0 µm) was purchased from the GAF Corp. (New York). Pigment-grade titanium dioxide particles (rutile type) were obtained from the DuPont Co. (Wilmington, DE). The mean diameters of individual parti¬cles is 0.25 µm but generally forms 1.0-µm agglomerates. Particles were heated to 200°C for 4 hr to eliminate the possibility of endotoxin contamina¬tion.

- Inhalation exposure and pulmonary lavage.

The methods utilized for aerosol generation of carbonyl iron and titanium dioxide particles have previously been reported (Warheit et al., 1991). Bronchoalveolar lavage procedures and biochemical assays on lavaged fluids were conducted according to methods previously described (Warheit et al., 1991).

- Macrophage cell culture and phagocytosis of iron or latex particles.

Alveolar macrophage cell culture and phagocytosis assay methods have previously been reported (Warheit et al., 1984a,b). For the phagocytic assay for TiOz-exposed macrophages, a suspension of carbonyl iron particles was incubated with normal rat serum for I hr at 40°C and sonicated to reduce aggregations of particles. The iron particles ranged in diameter from 0.4 to 2.0 µm. A final concentration of 1.75 mg/ml (mass per area in the culture dish = 204 µg/cm2) was added to monolayers. For the phagocytic assay for CI-exposed macrophages, a suspension of latex particles was similarly opsonized with serum. The latex particles ranged from 2 to 4 µm in diame¬ter. A similar mass concentration of latex particles (i.e., 1.75 mg/ml) was added to the monolayers.

- Chemotaxis of Ti02- or CI-exposed alveolar macrophages.

Alveolar macrophages were collected from TiOz-, CI-, or sham-exposed rats by lavage as described above. The chemotaxis assay was carried out as described previously using three concentrations (i.e., 1,5, and 10%) of zymosan-activated sera as the chemotactic stimulus (Warheit et al., 1984b, 1992).

- Lung dissection and tissue preparation.

The lungs of rats exposed to Ti02 and carbonyl iron particles for 4 weeks were prepared for light microscopy by airway infusion using methods previously reported (Warheit et al., 1984b, 1991). Analyses of lung and lymph node burdens were conducted by digesting tissue specimens in hydrofluoric acid and analyzing for titanium or iron, using the method of inductively coupled plasma (ICP-AES) spectroscopy.

- Pulmonary cell proliferation studies.

Pulmonary cell proliferation experiments were conducted according to methods previously described (Warheit et al., 1992). Statistics were carried out using a two-tailed Student t test on a Microsoft Excel software program (p < 0.05)."
Positive control:
No, however, carbonyl iron was compared with another particulate material, viz. titanium dioxide.


Observations and examinations performed and frequency:
See partly under "Details on study design".

- Presence of iron in the lungs;
- Particles in alveolar macrophages obtained by means of bronchoalveolar lavage (BAL);
- Measures for inflammation in BAL;
- Lactate dehydrogenase and protein in BAL;
- Cell proliferation in lungs by means of BrdU labelling;
- Lung clearance;
- Translocation of particles to tracheobronchial lymph nodes;
- Functional responses of alveolar marcophages;
- Lung histopathology.

All examinations were determined immediately after exposure and 7, 30, 90 and 180 days post exposure on groups of rats.
Sacrifice and pathology:
See under "Observations and examinations performed and frequency".
Student t test

Results and discussion

Results of examinations

Clinical signs:
not examined
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See below under "Details on results".
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See below under "Details on results".
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See below under "Details on results".
Details on results:
The exposure to 250 mg/m^3 resulted in a iron lung burden of 2000 µg/g fixed lung tissue or 17 mg/lung. For a detailed presentation of other results, the reader is referred to the attached documents with reproduced figures and tables fron the original publication.

The observations made can be summarized as:

- Pulmonary inflammation;
- Enhanced proliferation of pulmonary cells;
- Impairment of particle clearance mechanisms;
- Deficits in in macrophage function;
- Macrophage aggregation.

No signs of inflammation or affected clearance were found at 5 mg/m^3, while these effects were found at 50 and 250 mg/m^3.

The detailed description in the publications of the histopathology is reproduced in slightly adapted form below, because it allows for a comparison between carbonyl iron with titanium dioxide at equal dose levels.

"Lesions in the respiratory system varied with exposure concentration and duration of postexposure recovery.
The lowest exposure concentration (5 mg/m3) produced only minimal effects. Particle-laden macrophages and a minimal diffuse increase in alveolar macrophages (histiocytosis) were evident at 0 days of recovery. The histiocytosis was no longer evident at 1 week postexposure. However, individual particle-laden macrophages could be found in very low num¬bers within air spaces and lymphoid tissue throughout the entire 6-month postexposure period.
The higher exposure concentrations (50 and 250 mg/m3) produced a wide spectrum of effects within the lung. Free granular pigment of TiO2 and CI were present on the mucosal surfaces of bronchioles and bronchi at 0 days of recovery. Particle-laden macrophages, found individually, were numerous throughout the air spaces at this same time period. Beginning at 1 week postexposure and persisting thereafter, many dense aggregates of particle-laden macrophages were within alveoli and alveolar ducts.
Cellular hypertrophy and hyperplasia were evident at alveolar wall and duct bifurcations that were adjacent to macrophage aggregates. Mucosal hypertrophy and hyperplasia were also observed within the bronchi and bronchioles.
While the type of lesions was similar in animals exposed to 50 and 250 mg/m3, there were significant differences in lesion severity. The number of pigment-laden macrophages found individually and in aggregates was much greater in animals exposed to the highest concentration, and consequently occupied a greater portion of the lung. Also, the severity of cellular hypertrophy and hyperplasia at alveoli and alveolar duct bifurcations was significantly greater in animals exposed to the highest concentration of TiO2 or CI particles. Thus, the cellular reaction and tissue injury was significantly greater in animals exposed to the highest concentration of particles.
The severity and character of the lesions changed with time. Free granular pigment was no longer apparent at 1 week postexposure in any concentration group. Particle-laden macrophages, found individually, decreased in number with time, but were evident in small numbers within the pulmonary air spaces throughout the entire 6-month recovery period. The numbers and size of the dense aggregates of macrophages within alveoli and alveolar ducts, increased during the first month postexposure but did not expand throughout the remaining 5-month postexposure period.
Minimal mucosal hypertrophy and hyperplasia in bronchi and bronchioles were evident at 1 month postexposure in the two highest concen¬tration groups. Focal cellular hypertrophy and hyperplasia were associated with aggregates of pigmented macrophages, and were evident at alveoli and alveolar duct bifurcations for the entire 6-month postexposure period in the two higher concentration groups. Pigmented macrophages could also be observed within pulmonary lymphoid tissue throughout this time period.
Exposure of animals to CI produced essentially the same type of lesions in comparison to TIO2 particles. However, the severity of lesions was less and mucosal hypertrophy and hyperplasia were not detected in bronchi and bronchioles of CI-exposed rats."

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

See attached document with figures and tables.

Applicant's summary and conclusion

In a subacute inhalation study with carbonyl iron, rats showed a clear inflammatory reaction in the lungs, as well as affected clearance, increased cell proliferation, hypertrophy and hyperplasia at 50 and 250 mg/m^3. The NOAEC was 5 mg/m^3. No systemic endpoints were investigated.
Executive summary:

This executive summary starts with the abstract of the original publication.

This study was carried out to assess the time course of pulmonary clearance impairment and persistence of inflammation following high-dose inhalation exposures to titanium dioxide (Ti02) or carbonyl iron (CI) particles. Male rats were exposed to air, Ti02 or CI particles 6 hr/day, 5 days/week, for 4 weeks at concentrations of 5, 50, and 250 mg/m3 and evaluated at selected intervals through 6 months postexposure.

Indices of pulmonary inflammation as well as alveolar macrophage clearance functions (i.e., morphology, in vivo and in vitro phagocytosis, and chemotaxis), cell proliferation, and histopathology endpoints were measured at several postexposure time periods through 6 months. In addition, amounts of TiO2 or CI in lungs and tracheobronchial lymph nodes were measured to allow an evaluation of particle clearance and translocation patterns.

Four-week exposures to TiO2 or CI particles at concentrations of 250 mg/m3 resulted in lung burdens of 12 mg titanium and 17 mg iron, respectively, with particle retention half-times ranging from 68 days for 5 mg/m3 TiO2 to approximately 330 days for 250 mg/m3.

The impact of this TiO2 dust load and similar lung burdens of CI particles produced a sustained pulmonary inflammatory response measured through a period of 3-6 months postexposure concomitant with increases in BrdU cell labeling of terminal airway and pulmonary parenchymal cells. The impairment of particle clearance mechanisms was accounted for by deficits in in vitro phagocytic and chemotactic potential of alveolar macrophages recovered from the lungs of high-dose, TiO2 or CI-exposed rats. Free granular pigment (TiO2 or CI) was present on the hypertrophic mucosal surfaces of bronchioles and bronchi, and particleladen macrophages, found individually, were numerous throughout alveoli and within lymphoid tissues immediately after exposure.

Aggregates of particle-laden macrophages were present within alveoli and alveolar ducts from 1 week postexposure through the entire 6-month recovery period. Macrophage accumulations increased in size and number from 1 week through 1 month postexposure and then appeared to remain constant through the remaining 5-month postexposure period. Minimal cellular hyper- trophy and hyperplasia were evident at alveolar duct bifurcations adjacent to macrophage aggregates, and this effect was most prominent at 3 to 6 months postexposure.

The results of this study clearly demonstrate that exposure to high dust concentrations of two different innocuous particle types produced sustained pulmonary inflammation, enhanced proliferation of pulmonary cells, impairment of particle clearance, deficits in macrophage function, and the appearance of macrophage aggregates at sites of particle deposition. In addition, the mass deposition rate determination appears to be a less sensitive indicator of "overload" when compared to biomarkers of pulmonary toxicity, such as macrophage function and cellular inflammation and proliferation indices.

Value of the study

The difference between this study and a standard inhalation study is that is is solely focused on the effects the particles have on a number of enpoints in the respiratory tract. Systemic effects are not at all investigated, while the selection of effects studied is influenced by the fact that the authors were interested in dust overload and not so much in chemical factors of toxicity. The omission of systemic effects is not deemed a serious shortcoming in the present context, because is can be assumed that systemic exposure to metallic iron is negligible upon inhalation of particles consisting of this material, while secondary ingestion can only result in a limited exposure to absorbable iron salts in the stomach, which exposure is covered by the oral repeated dose studies summarized in this IUCLID-5 file. The concentration on dust overload is less serious than it seems, because the lungs have meticuously been investigated by means of histopathological techniques.

The value of this study should be judged against the well-known fact that rats are much more sensitive to dust overload than other organisms, including humans (see discussion in the endpoint summary). The study is in particular important in that it indicates whether or not carbonyl iron particles should be regarded as typical poorly soluble particles based on their effects and based on the comparison with another poorly soluble particle, viz. titanium dioxide.


The study yields a NOAEC for carbonyl iron of 5 mg/m^3, based on inflammation, affected clearance, increased cell proliferation and hypertrophy and hyperplasia at 50 mg/m^3 and 250 mg/m^3.

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