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Acute Toxicity: inhalation

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acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Sufficient information available for assessment; meets basic scientific principles.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference Type:
Pneumotoxicity and pulmonary clearance of different welding fumes after intratracheal instillation in the rat.
Antonini JM, Murthy GGK, Rogers RA, Albert R, Uldrich GD, Brain JD.
Bibliographic source:
Toxicology and applied pharmacology, 140: 188-199.

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
The study shows the effects of different welding fumes, which were instilled intratracheally, on parameters of lung toxicity. Iron(III)oxide (Fe2O3) is used as a negative control as it is considered as a relatively inert dust, and crystalline silica as a positive control. Only one dose was tested.
GLP compliance:
not specified
It is not customary to refer to GLP compliance in publications.
Test type:
other: intratracheal instillation
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
Diiron trioxide
EC Number:
EC Name:
Diiron trioxide
Cas Number:
Molecular formula:
diiron trioxide
Constituent 2
Reference substance name:
iron(III) oxide
iron(III) oxide
Details on test material:
Name of test material (as cited in study report): iron oxide (γ- Fe2O3)
- Molecular formula (if other than submission substance): γ- Fe2O3
- Physical state: solid;particles of about 0.1-0.2 µm in diameter
- Other: magnetic iron particles (γ- Fe2O3) were produced by the combustion of Fe pentacarbonyl (Fe(CO)5) vapors as described by Vlberg and Brain (1979). The Fe(CO)5 is carried as a vapor to 650°C furnace, where air is added to provide oxygen for the formation of γ- Fe2O3. The reducing property of the H2 carrier gas is necessary to prevent oxidation of the Fe before it reaches the furnace, otherwise nonmagnetic hematite will result. The particles produced are crystalline in shape and measure about 0.1-0.2 µm in diameter. The final aerosol is produced from the rapid agglomeration of these individual units immediately after their formation in the furnace. The effluent is diluted with air and cooled to achieve the desired concentration and temperature.

Test animals

other: CD/VAF
Details on test animals or test system and environmental conditions:
- Source: Charles River Laboratories, Wilmington, MA.
- Weight at study initiation: 250-300 g
- Housing: clean air, virus and antigen free room with restricted access
- Diet: ad libitum; conventional laboratory diet
- Water: ad libitum; from the tap

Administration / exposure

Route of administration:
other: intratracheal instillation
Type of inhalation exposure:
other: intratracheal instillation
other: 150 µl of 0.9% sterile saline/100 g bw
Details on inhalation exposure:
- Concentration of test material in vehicle: 6.66 mg/ml

The Fe(III)oxide particle agglomerates had a mean diameter of 0.865 µm ± 0.08
- Rationale for the selection of the starting concentration: only one concentration was used, chosen after a preliminary dose-response study was performed (data not shown).
Analytical verification of test atmosphere concentrations:
non relevant; intratracheal instillations
Remarks on duration:
non relevant; intratracheal instillations
Dose: 10 mg/kg bw particles instilled
No. of animals per sex per dose:
Control animals:
other: Solvent control: saline; negative control: iron(III) oxide (Fe2O3) was used as a negative control because it is considered as a relatively inert dust; positive control: pneumotoxic crystalline silica,
Details on study design:
-The particular dose was selected based on a preliminary dose response study (data not presented in the publication).
-The rats were lightly anesthetized by an intraperitoneal injection (0.6 ml of a 1% solution of sodium methohexital) before the intratracheal instillation. The instillation was performed according to the method of Brain et al. (1976). Each rat was placed on a slanted board and was supported by a rubber band unders its upper incisors. The tongue of the animal was moved aside, and the larynx was transilluminated. Particle suspensions were instilled into the trachea via a No.20 gauge, 4 in. needle.

-Bronchoalveolar lavage (BAL): observations were performed 1, 7, 14 and 35 days post instillation; 4 animals/group at each time point
A. Cellular evaluation: cell numbers were determined with a hemocytometer.
B. Biochemical indicators of pulmonary damage in the cell free supernatant after centrifugation: albumin levels (according to Doumas & Biggs, 1972), β-NAG activity (method of Sellinger et al., 1960) and LDH levels (Perce et al., 1964).
D. Inflammatory cytokines: TNF-a and IL-1β were measured in acellular BALF with the Factor-Test-X TNF-a and the Inter-Test-X IL-1β ELISA kits, respectively.
-Histopathology:on days 14 and 35, additional rats from each group were killed for the histopathological examinations. Microscopic examinations of pulmonary clearance were performed (confocal microscopy).
Use of Statview statistical programme
Analysis of variance (ANOVA) was performed for all parameters.
Fischer's Least Significance Difference post-hoc test was used for analyzing significance between groups (p<0.05).

Results and discussion

Preliminary study:
non relevant
Effect levels
Dose descriptor:
Effect level:
> 104 mg/m³ air
Based on:
test mat.
Remarks on result:
other: see details for this calculation below
no mortality is mentioned in the publication
Clinical signs:
other: no data
Body weight:
no data
Gross pathology:
no data
Other findings:
see below

Any other information on results incl. tables

Table 2a: Bronchoalveolar Lavage Cell Profiles (total number 10^6).

Time point





1 day

Iron(III) oxide

3.8± 1

6.6± 0.7#

0.3± 0.1#

Solvent control

5.1± 1

0.3± 0.1


Positive control

5.7± 0.5

13.4± 1.8*

0.6± 0.2#

7 days

Iron(III) oxide

5.5± 0.8

1.6± 0.6#

1.3± 0.3#

Solvent control

4.7± 0.6



Positive control

7.2± 0.4

10.3± 0.6*

0.7± 0.1

14 days

Iron(III) oxide

4.1± 0.3

1± 0.6

0.2± 0.1

Solvent control

4.8± 0.2



Positive control

6.8± 1.1

20.4± 1.4*

1.6± 0.2*

35 days

Iron(III) oxide

3.3± 0.5

0.2± 0.1


Solvent control

3.7± 0.2

0.2± 0.1


Positive control

8.7± 0.7*

41.4± 5.2*

2.8± 0.5*

Values are means ± SE, n=4

# significantly greater than saline control group (p<0.05); * significantly greater than all other groups

Results are presented in Table 2, Fig. 1, 2, 3 & 4 (see attachment below).

Iron(III) oxide

- Significant elevations were observed in the number of neutrophils and lymphocytes on days 1 and 7 postinstillation, in comparison to the solvent control.

- Albumine levels appeared increased at the 1 day postinstillation measurement, while at the 14th day, there was no difference anymore in response in comparison to saline control group.

- LDH activity was significantly increased at day 1 postinstillation, whilst it diminished by 7 days and reached the same levels of control group by 14 days.

- None of the two cytokines was detected in the BAL fluid.

- Accumulation of the particles at some areas in the lungs was apparent at both (day 14 and 35) histopathological examinations, usually present inside the macrophages. At 35 day postinstillation lung changes from the Fe oxide were minimal. Macrophages containing oxide particle were observed in the terminal bronchioles and alveolar ducts.

Applicant's summary and conclusion

Interpretation of results:
other: In rats iron(III) oxide (Fe2O3) behaves like an inert, non-toxic dust when instilled intratracheally at 10 mg/kg bw.
In rats iron(III) oxide (Fe2O3) behaves like an inert, non-toxic dust when instilled intratracheally at 10 mg/kg bw. No real evidence of pulmonary injury after exposure to the oxide particles was detected. The observed inflammatory responses were clearly associated with the particle clearance. After 14 days the lungs of the rats appeared fully recovered.
Executive summary:

In an acute inhalation toxicity study groups of young adult male CD/VAF rats were exposed by intratracheal instillation to welding fumes. Iron(III) oxide was chosen as a negative control since it was considered by the investigators as a relatively inert dust and crystalline silica as a positive control. Iron(III) oxide particles were suspended in saline (one dose used, 1 mg suspended in 150 µl of 0.9% sterile saline/100 g bw). Animals then were observed for 14 days. No real evidence of pulmonary injury after exposure to the oxide particles occured. The observed inflammatory responses were clearly associated with particle clearance. Histopathology revealed that by 14 days the lungs of the rats appeared normal. Fe2O3 was indeed found to behave like an inert, non-toxic dust when instilled intratracheally at this dose level. An LC50 value for Fe2O3 was estimated by the conversion of the instilled dose to a concentration in air that was inhaled for 4 h by the rats; this resulted in a value > 104 mg/m3.