Mill scale (ferrous metal)

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The deficits of the study were that details on inhalation exposure missing, the exposure duration was too short, one low concentration only (200 mg/m³) was tested, analytical concentration was missing, the test material was insufficiently characterised (source, purity, impurities), observation periods were too short (<=24h), an uncommon species (rabbit) was used and only lungs were investigated, clinical signs, bodyweight and mortality was missing.

Data source

Reference

Reference Type:

publication

Title:

Lysosomal enzyme activities in pulmonary macrophages from rabbits breathing iron oxide.

Author:

Grant MM, Sorokin SP, and Brain JD.

Year:

1979

Bibliographic source:

American Review of Respiratory disease, 120: 1003-1012

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The aim of the study was to investigate the in vivo pulmonary macrophage response to iron oxide particle (Fe2O3, 200 mg/m³ air) in male New Zealand White rabbits, after 3h of exposure via inhalation. A bronchoalveolar lavage was performed at time points 0, 12, 18 and 24 h after termination of the exposure, for different groups of animals. The lysosomal acid hydrolase activities in the pulmonary macrophages were examined with biochemical and histochemical techniques.

GLP compliance:

not specified

Test type:

other: Inhalation study aimed at the observation of effects on macrophages in the lungs by means of several biochemical and histochemical techniques.

Limit test:

no

Test material

Test material information

Test material form:

solid

Details on test material:

- Name of test material (as cited in study report): iron oxide
- Molecular formula (if other than submission substance): Fe2O3
No further details available in the publication.

Test animals

Species:

rabbit

Strain:

New Zealand White

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Weight at study initiation: 3 to 4 kg
- Diet: ad libitum
- Water: ad libitum

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

head only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 0.17 - <= 0.31 µm

Geometric standard deviation (GSD):

1.61

Details on inhalation exposure:

TEST ATMOSPHERE
- Generation: according to Brain et al. (1974)
- Particle size distribution: determined with an aerosol spectrometer of the Tillery (1974) design

Analytical verification of test atmosphere concentrations:

not specified

Duration of exposure:

3 h

Remarks on duration:

once

Concentrations:

200 mg/m3 (nominal); actual range 186 to 222 mg/m³

No. of animals per sex per dose:

See below 'details on study design'.

Control animals:

yes

Details on study design:

Groups of animals were killed immediately after termination of the exposure (0 h-8 animals) and 12 h (12 animals), 18 h (10 animals), and 24 h (8 animals) thereafter. Pulmonary macrophages were obtained by multiple lung lavages (10 washes of 50 ml) with Dulbecco's Ca+2 and Mg+2 free balanced salt solution warmed at 37°C. Macrophages from 2 animals were pooled for each enzyme analysis. The cell numbers were measured with an American Optical hemocytometer and thereafter, homogenates were prepared and centrifuged at 600 g. The 600 g supernatant was either applied to a sucrose density gradient or centifuged further at 15000g for 15 min to obtain a 15000g supernatant and pellet.

Sucrose density gradient centrifugation: performed in order to examine the density distribution of lysosomes from pulmonary macrophages.
Protein and enzyme analysis: enzymes measured and conditions of the assays are summarized in Table 1 below in section 'any other information on materials and methods incl. tables'.
Histochemical examinations: the cell pellets were used for measurements of acid phosphatase according to Barka and Anderson (1963). Staining with Perl's Prussian was also performed.

After BAL the lungs were dissected and examined for any gross pathologic lesions

Statistics:

not specified

Results and discussion

Preliminary study:

not specified

Effect levels

Dose descriptor:

other: no LC50 established

Remarks on result:

not measured/tested

Remarks:

The study was not aimed at the observation of the endpoints that are typically studied in a standard acute inhalation toxicity study. Mortality rates were not reported and observation periods were too short in the publication and hence, an LC50 value could not be established.

Mortality:

Although the presentation of the results does not explicitly states anything about mortality, the exposure for 3 h up to 200 mg/m³ and the reported results indicates that no treatment-related mortality occurred within the 24 hours post exposure.

Clinical signs:

other: not specified

Body weight:

not specified

Gross pathology:

No gross pathologic lesions in the lung were found in the animals exposed to ferric oxide. However, lesions were observed in three of the control animals, that were for this reason eliminated from the study.

Other findings:

The total cell count increased significantly with time (at 12, 18 and 24 h), after exposure to the aerosol. The protein level in the 600 g- supernatant, did not increase, when expressed per 10^8 cells, whilst the amount of protein recovered after 18 and 24 h after exposure decreased significantly. Macrophages lavaged reacted histochemically for acid-phosphatase and showed increased staining and redestribution of the enzyme within the cytoplasm.

No significant changes on the specific lysosomal enzymic activities, of animals sacrified 0 and 8 h after exposure, were observed in comparison to the controls in the 600g supernatant, except for catepsin D (Table 4, attachment). The activity of β-glycerol-phosphatase and catepsin D did not show any significant changes (expressed per 10^8 cells) in the animals killed at all time points, except for time point 12h, where β-glycerol-phosphatase's activity was decreased. Similarly, catepsin's D activity decreased slightly at 18 h, but not to a statistically significant extent (Table 3, see attachment).

The specific activities of catepsin D and β- glycerol-phosphatase (Fig.1, attachment) remained unchanged or decreased (catepsin D at 18 h), in the 600g supernatant and 15000g pellet fractions of macrophages. On the contrary, in the 15000 g supernatant, the specific activitites were significantly greater than in the controls, suggesting a possible disruption of cells (macrophages).

Any other information on results incl. tables

The study was not aimed at the observation of the endpoints that are typically studied in a standard acute inhalation toxicity study. Mortality rates were not reported in the publication and hence, an LC50 value could not be established.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Although the presentation of the results does not explicitly states anything about mortality, the presentation of the results indicates that no treatment-related mortality occurred. Therefore this study indicates that 3 h exposure to 200 mg/m³ respirable ferric oxide (Fe2O3) particles does not result in mortality within 24 hours post exposure.
Exposure to ferric oxide particles has lead to little effect on the average activities of lysosomal hydrolases in pulmonary macrophages, to an increased lysosomal heterogeneity and redistribution of enzymic activity within the cell cytoplasm.

Executive summary:

Male white New Zeland rabbits were exposed to an iron(III) oxide (Fe2O3) aerosol with an MMAD of 0.17 to 0.31 μm (GSD 1.61) head-only for 3 h, at a concentration of 200 mg/m3. The aim of the study was to investigate the in vivo pulmonary macrophage response after exposure to this aerosol. A bronchoalveolar lavage was performed at time points 0, 12, 18 and 24 h after termination of the exposure, for different groups of animals. The lysosomal acid hydrolase activities in the pulmonary macrophages were examined with biochemical and histochemical techniques. The results suggest that exposure to Fe2O3 may have little effects on the average activities of lysosomal hydrolases of the pulmonary macrophages, although it indeed stimulates their recruitment in the lung, leading to an increase of the total enzyme in the BAL fluid. An increased lysosomal heterogeneity and redistribution of enzymic activity within the cell cytoplasm of the macrophages was observed.