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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-documented study, carried out according to the guidelines and according to GLP principles.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Qualifier:
equivalent or similar to
Guideline:
other: EC Directive 67/302/EEC
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): iron oxide 'black' or magnetite (ferroxide black 88P; purchased by ROCKWOOD ITALIA)
- Molecular formula (if other than submission substance): Fe3O4
- Physical state: solid; powder
- Analytical purity/impurities:
PURITY DATA
mg/kg Batch Specification
Arsenic (As) <1 Max. 3
Barium (Ba) 6 Max. 50
Cadmium (Cd) <1 Max. 5
Chromium (Cr) 36 Max. 100
Mercury (Hg) <0.1 Max. 1
Nickel (Ni) 42 Max. 50
Lead (Pb) <3 Max. 10
Copper (Cu) 11 Max. 50
Zinc (Zn) 75 Max. 100
Fe content (%) 69.5 Min. 68

- Purity test date: 25/02/2004
- Lot/batch No.: 846
- Storage condition of test material: room temperature
- Other
specific surface area: 10.5 m2/g (BET, DIN 66131, 90% He, 10% N2)
EINECS: 215-277-5
Appearance: powder with characterisitic colour

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: experimental animal breeder Harlan-Winkelmann GmbH, Borchen, Germany
- Age at study initiation: approximately 2 months old
- Weight at study initiation: did not exceed the ±10 % of the mean (data present in Table 3 of attached document below)
- Housing: individually, in conventional Makrolon Type IIIh cages; according to the legal requirements for housing experimental animals (Directive 86/609 EEC)
- Diet: ad libitum
- Water: ad libitum provided in polycarbonate bottles
- Acclimation period: approximately 1.5 weeks before use

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Relative humidity (%): 40-60
- Air changes (per hr): approximately 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12h/12h
- Light density: approximately 14 watt/m2 floor area

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Remarks:
dynamic air-flow
Vehicle:
air
Remarks:
conditioned dry air
Remarks on MMAD:
MMAD / GSD: 1.3 µm/ ~ 2
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The test atmposphere was forced through openings in the inner concentric cylinder of the chamber, towards the rats'breathing zone (direct-flow). The stability of the test atmosphere was monitored continously using an aerosol real-time device (vide infra)
- System of generating particulates/aerosols: WRIGHT DUST FEEDER system (BGI Inc., Waltham, MA, USA)
- Temperature, humidity, pressure in air chamber: controlled and measured continously
- Air flow rate: monitored and controlled continously by calibrated mass flow meters (Hastings HFC-C Mass Flow Controllers, Teledyne Hastings-Raydist, Hampton, VA, USA). TYLAN FC-280 S mass flow controller was used for the analytical sampling.
- Method of particle size determination: samples (from breathing zone) analyzed using a BERNER-TYPE AERAS low pressure critical orifice cascade impactor. A cyclone was used to prevent particles larger than 10 µm to enter in the inhalation chamber.
- Treatment of exhaust air: purification via aerosol and HEPA filters.

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetric analysis
- Samples taken from breathing zone: yes, 3 samples/exposure day/ chamber
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The actual concentrations were determined by gravimetric analysis (filter: Glass-fiber-filter, Sartotius, Gottingen, Germany). Filters were evaluated by gravimetric analysis (balance: Mettler AE 100).
Duration of treatment / exposure:
6h per day; 5 days/week; 10 animals/group/sex were exposed for 13 consecutive weeks; 10 animal/group/ sex were exposed for 14-15 consecutive weeks
Frequency of treatment:
5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (conditioned dry air), 4.7 ±0.6, 16.6 ±3 and 52.1 ±6.4 mg/m3 (target concentrations: 5, 15 and 50 mg/m3)
Basis:
analytical conc.
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: two dose-range finding studies were performed with different Fe oxides (2 weeks) and with Fe3O4 (4 weeks).
** The examined endpoints for the 10 rats/group exposed for 13 weeks were according to the OECD 413, while the remaining 10 rats/group were necropsied 1 -2 weeks later (exposure continued) and were subjected to BAL and analysis of Fe body burden of selected organs.



Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE/CLINICAL OBSERVATIONS: Yes
- Time schedule: daily; before and after exposure, once a day during the exposure-free days. Including changes in the skin and hair-coat, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, sensori- and somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhea, lethargy, somnolence and prostration.

BODY WEIGHT: Yes
- Time schedule for examinations: twice per week

FOOD CONSUMPTION:
- Food consumption for each animal: Yes, determined weekly.

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes, with the use of an indirect ophthalmoscope (HEINE);
- Time schedule for examinations: prior to the first exposure and towards the end of exposure
- Dose groups that were examined: 10 rats/ dose group and gender

CLINICAL CHEMISTRY/HEMATOLOGY: Both; general clinical chemical tests performed at the end of 13-weeks (during necropsy)
- How many animals: 10 animals/group and gender

URINALYSIS: Yes
- Time schedule for collection of urine: towards the end of the 13th week, collected overnight




Sacrifice and pathology:
GROSS PATHOLOGY
All rats were necropsied (surviving rats were sacrificed) and were given a gross-pathological examination. The general physical condition, body orifices, external and internal organs and tissues were examined. Weight analysis of the following organs was performed: adrenals, brain, heart, kidneys, liver, lungs, lung-associated lymph nodes, ovaries, spleen, testes, thymus.
HISTOPATHOLOGY
Examinations were performed to all the organs mentioned above and to a list of tissues. The histopathological evaluations focused on the entire respiratory tract (nasal passages, trachea, lung, lung-associated lymph nodes); it also included all extrapulmonary organs (OECD Guideline 413).
Other examinations:
At each sacrifice (referring to the remaining 10 rats/gropu/sex that were exposed for 1-2 weeks extra; see details in section 'any other information on materials and methods incl. tables' below):
A.Inflammatory endpoints were determined in bronchoalveolar lavage (BAL); samples of the BAL fluid were collected from 10 rats/group/sex and were analyzed for indicators of inflammatory response, respiratory tract damage and interactions with pulmonary phospholipids (according to Henderson, 1988; Henderson, 1989; Henderson and Belinsky, 1993).
B. Fe content was determined in lungs, lung-associated lymph nodes (LALNs) and ileum (10 rats/group/sex).
Statistics:
Depending on variates: Dunnet test, Adjusted Welch test, Kruskal-Wallis test followed by Adjusted U tests, Analysis of Variance (ANOVAbctic). A detailed description is provided.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No mortality was observed.
Mortality:
mortality observed, treatment-related
Description (incidence):
No mortality was observed.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
Nonetheless, there was no conclusive evidence of test substance- induced changes
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
**The summarized toxicological results after exposure of rats to Fe3O4 for 13 weeks are presented in Table 1 below, in the section "Any other information on results incl. tables".
CLINICAL SIGNS AND MORTALITY
See below, Table 2, section "Any other information on results incl. tables".

BODY WEIGHT AND WEIGHT GAIN
In general, no concentration-dependent significant difference in body weights was found. A distinctly higher body weight gain was observed in the male control rats, which could not be explained by the authors. No significant changes in body weights were observed in female rats. The results are shown in Table 3 and Fig.1, in the attached document below.

FOOD CONSUMPTION
The results are summarized in Table 4 (see attachment below).

WATER CONSUMPTION
The results are shown in Fig. 2 (attachment). No consistent effect of toxicological significance on water consumption was observed for male and female rats.

OPHTHALMOSCOPIC EXAMINATION
No conclusive test substance-induced changes were observed.

HAEMATOLOGY
The summarized results are presented in Table 4 (attachment below). The results show that the major hematological indices were not affected to any toxicologically significant extent in any group. However, an increase in neutrophils occured in males, already from the lowest concentration, that appeared to be concentration-dependent; in females an increase was found only at the highest concentration.

CLINICAL CHEMISTRY
The means of the clinical chemistry parameters are shown in Tables 5, 6 & 7 (attachment below). No significant changes of toxicological relevance were observed in all the parameters measured.

URINALYSIS
No significant changes were observed for both sexes (Table 8, see attachment below).

ORGAN WEIGHTS
The means of organ weights, organ weights vs. body weights and of organ weights vs. brain weights are shown in Tables 9, 10 and 11, respectively (see attachment). As stated in the report on the results obtained with male rats 'the organ weights are somewhat confounded by the unusual increased body weights in the control group. Accordingly, toxicological significance is preferentially defined with regard to concentration-dependence amongst the dust exposure groups. In order to neutralize, body weight-dependent artificial changes in organ weights particular emphasis has been directed toward the analysis of the organ-to-brain weight ratios'. Statistically significant changes were observed in lung and LALN organ/brain weight ratios at the concentration of 15 and 50 mg/m3 in females, while this was the case only for the highest concentration in males. Moreover, there was a decrease in the kidneys/brain weight ratios at the highest two concentrations for female rats.

GROSS PATHOLOGY/HISTOPATHOLOGY
The summarized data can be found in Tables 9 and 10 (attached document below).
-The respiratory tract findings were correlated to the poor solubility of the substance. Adverse extrapulmonary effects of Fe3O4 were not observed at any concentration.
A. Black discolorations of lungs and lung associated lymph nodes were observed macroscopically in almost all Fe3O4 exposure groups, with increasing grading. Black macrophages were seen in the lungs of all exposed groups and they increased in a dose-dependent manner. In all males and females exposed to the upper two concentrations and in the majority of exposed to the lowest concentration, bronchiolar-alveolar hypercellularity and black macrophages in BAL were detected. Serius-red staining of the lung resulted in the observation of increased collagenous fibers in all animals of both sexes at the highest concentration. The paracortical area of LALNs appeared stastistically significantly enlarged in all the males and almost all females exposed to the the upper two concentrations.
B. The examinations revealed focal pigmented macrophages in the trachea of many substance-exposed rats.
C. In the nasal cavity, eosinophilic epithelial globules occured in both males and females exposed at the highest two concentrations. Epithelial metaplasia was observed in some of the male rats exposed to 50 mg/m3 and focal inflammatory infiltrates occured in some females exposed to the same concentration.
-Testes: (Multi)-focal tubular atrophy and degeneration was seen in all groups including the controls. Diffuse tubular atrophy/degeneration was detected in one high concentration rat and in 3 rats from each of the other concentration groups. As stated by the authors, corresponding to these lesions, spermatic debris occured in the epididymides. These findings are not related to the concentration of the substance and in several animals they occured unilaterally. Therefore, they are not exposure related.

OTHER
A. BRONCHOALVEOLAR LAVAGE
The results of the BAL analysis are summarized in Table 11 (see attachment). Many cells in the BAL fluid could not be clearly differentiated due to extreme loading and were classified as 'non-classifiable cells'. However, most non-classifiable cells were assumed to be alveolar macrophages by the authors of the study report. Alveolar macrophages appeared significantly increased already at the dose of 5 mg/m3. A remarkable increase of total cell count (TCC) occured in animals exposed to the upper two concentrations. A concentration-dependent increase was observed in polymorphonuclear cells (PMNs) at 5 mg/m3 and above. The LDH measurements revealed signs of cytotoxicity at 15 mg/m3 and above. Slight increases of phospholipids in BAL fluid were detected in animals exposed to the highest concentration, while BAL protein levels increased already at 5 mg/m3, albeit slightly. Beta-N-acetyl-glucosaminidase activity appeared elevated at the upper two concentrations in both sexes; this was the case for acid phosphatase in female rats.

B. IRON DISTRIBUTION IN TISSUES
The findings are shown in Table 12 (attachment). A remarkable translocation of Fe was measured from the lungs into the LALNs. Fe was not detected in the ileum.




Effect levels

Dose descriptor:
NOAEC
Effect level:
4.7 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: adverse effects observed in BAL fluid (PMNs, protein levels) and relative lung weights

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Summary of subchronic inhalation toxicity of Fe3O4dry powder.

N Group/sex

Target concentration (mg/m3)

Toxicological result

Onset and duration of signs

Onset and duration of mortality

1/m

0

0/0/20 

-

-

2/m

5

0/0/20

-

-

3/m

15

0/0/20 

-

-

4/m

50

0/0/20 

-

-

1/f

0

 0/0/20

-

-

2/f

5

 0/0/20

-

-

3/f

15

0/0/20 

-

-

4/f

50

0/0/20 

-

-

N= group assignment, m=males, f= females. Values given in the ‘Toxicological Results’ column are: 1st= No of deaths, 2nd= No of animals with signs after cessation of exposure and 3rd= No of animals exposed.

The table below presents the findings from clinical sings and observations. No mortality was found. The clinical results did not occur in any consistent concentration or time-related manner.

Table 2: Signs and observations.

Group 1/m

Palpable mass flank, eschar formation skin

Group 2/m

Nose: red encrustations

Group 3/m

No findings

Group 4/m

Nose: red encrustations

Group 1/f

Nose: red encrustations

Group 2/f

No findings

Group 3/f

No findings

Group 4/f

Nose: red encrustations, discharge from eyes, eyelids reddened, alopecia, eyelids swollen

Applicant's summary and conclusion

Conclusions:
These results were clearly consistent with what would be expected for a poorly soluble particle, indicating their clearance in the lungs, via efficient phagocytosis by the alveolar macrophages and/or movement into the lymphatic system.
Executive summary:

In a subchronic inhalation toxicity study, iron(II,III) oxide (Fe3O4) aerosols were administered to Wistar rats (20 male and 20 female per group) by the dynamic directed-flow nose-only thechnique; actual mean concentrations were 0, 4.7 ±0.6, 16.6 ±3 and 52.1 ±6.4 mg/m3 air; the animals were exposed for 6 h/day, 5 days/week over a period of 13 weeks. Ten rats/group were necropsied 1 -2 weeks later (exposure continued). Particles had a MMAD of 1.3 µm and GSD ~2. The exposure was not associated with any specific clinical signs and consistent changes in body weights. Hematology, clinical pathology and urinanalysis were unobtrusive. The NOAEC was 4.7 mg/m3, based on the findings from BAL analysis and histopathology. Mild and borderline changes were considered to be associated with the exposure to poorly soluble particles rather than specific toxicity of the tested particles. The effects found at higher concentrations appear to be consistent with a particle-overload related inflammatory response.