Mill scale (ferrous metal)

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Reference 2

Reference Type:

publication

Title:

Subchronic inhalation toxicity of iron oxide (magnetite, Fe3O4) in rats: pulmonary toxicity is determined by the particle kinetics typical of poorly soluble particles

Author:

Pauluhn, J.

Year:

2011

Bibliographic source:

J. Appl. Toxicol. 2012; 32:488-504

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

2008-09-30

Principles of method if other than guideline:

The 13-week subchronic inhalation study was conducted also according to OECD guidance 39 (2009).

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Triiron tetraoxide

EC Number:

215-277-5

EC Name:

Triiron tetraoxide

Cas Number:

1317-61-9

Molecular formula:

Fe3O4

IUPAC Name:

Iron oxide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: magnetite, Ferroxide® black 88P
- Average pore diameter of particles: 981 nm
- specific N2-BET surface area: approximately 10 m2/g
- Porosity: 82 %
- Penetrated Hg-pore volume: 1029 mm3/g
- Surface area of particles (based on porosimetry): 11.4 m2/g
- Bulk and apparent density were 0.8 and 4.56 g/cm3, respectively

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Rockwood, Torino, Italia

Test animals

Species:

rat

Strain:

Wistar

Remarks:

HsdCpb:WU

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: 2 months
- Weight at study initiation: approx. 228 g (males); 156 g (females)
- Housing: singly housed in polycarbonate cages, containing low‐dust wood shavings as beddingmaterial.
- Diet (ad libitum): standard fixed‐formula diet (KLIBA 3883 pellets maintenance diet for rats and mice (supplier: PROVIMI KLIBA SA, 4303 Kaiseraugst, Switzerland))
- Water (ad libitum): tap water
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 40 - 60
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks:

conditioned dry air

Mass median aerodynamic diameter (MMAD):

1.3 µm

Geometric standard deviation (GSD):

1.2

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, M A, USA)
- Temperature, humidity, pressure in air chamber: controlled and measured continously
- Air flow rate: monitored and controlled continously by calibrated mass flow meters (Hastings HFCC 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 BERNERTYPE 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:

10 animals/sex/group were exposed for 13 consecutive weeks; 10 animal/sex/group were exposed for 13 - 14 consecutive weeks

Frequency of treatment:

6 hours/, 5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

4.7 mg/m³ air (analytical)

Remarks:

± 0.6; 5 mg/m3 (nominal)

Doses / concentrations 2

Dose / conc.:

16.6 mg/m³ air (analytical)

Remarks:

± 3; 15 mg/m3 (nominal)

Doses / concentrations 3

Dose / conc.:

52.1 mg/m³ air (analytical)

Remarks:

± 6.4; 50 mg/m3 (nominal)

No. of animals per sex per dose:

20 (10 for haematology, clinical pathology, urinalysis, determination of organ weights and histopathology; 10 for analyses in BAL and iron organ burdens)

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 OBSERVATIONS: Yes
- Time schedule: daily; before and after exposure

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly on Mondays and Fridays during the exposure period and once weekly during the post exposure period.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: once per week

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to start and at the end of the exposure period
- Dose groups that were examined: 10 rats/sex/group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the 13‐week study period
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: 10 rats/sex/group
- Parameters examined: erythrocytes (ERY), hematocrit (HCT); hemoglobin (Hb); Hepato Quick (HQUICK); leukocytes (LEUKO); mean corpuscular Hb (MCH); mean corpuscular Hb concentration (MCHC); mean corpuscular volume (MCV); thrombocytes/platelets (THRO).

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the 13‐week study period, uirne was collected over night.
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals: 10 rats/sex/group
- Parameters examined:

NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: at the end of the 13–14 week exposure period
- Dose groups that were examined: control and all dose groups
- Number of animals: 10 rats/group/sex
- Parameters examined: TCC, total cell count in BAL; MCD, mean cellular
diameter; MCV, mean cellular volume; LDH, lactate dehydrogenase; AP, alkaline phosphatase; ACPH, acid phosphatase; PROT, protein; PLIPf, phospholipids in BALF; AM, alveolar macrophages; PMN, polymorphonuclear cells; LYM, lymphocytes; EOS, eosinophils; foamy, foamy; NC, cells not classifiable; NAG, β‐N‐acetyl‐glucosaminidase.

LUNG BURDEN: Yes
- Time schedule for analysis: at the end of the 13 week exposure period
- Dose groups that were examined:
- Number of animals: 10 rats/sex/group
- Parameters examined: Fe3O4 levels in LALN

Sacrifice and pathology:

GROSS PATHOLOGY
clinical pathology was performed at the end of the 13‐week study period using 10 rats per group per sex. 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.

ORGAN WEIGHT:
At the terminal sacrifice, organ weights were determined (adrenal glands, brain, heart, kidneys, liver, lung, lung-associated lymph nodes (LALN), ovaries, spleen, testes, thymus) and organs were persevered for histopathology.

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).

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

Mortality:

no mortality 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:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- 50 mg/m3: significantly increased neutrophil counts in peripheral blood cytodifferentials occurred in all iron oxide male groups and in female rats.

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 15 and 50 mg/m3: significantly increased lung and LALN weights were observed in both sexes.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 5, 15 and 50 mg/m3: 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.
- 15 and 50 mg/m3: examinations revealed focal pigmented macrophages in the trachea of many substance-exposed rats. The paracortical area of LALNs appeared stastistically significantly enlarged in all the males and almost all females exposed to the the upper two concentrations.
- 50 mg/m3: 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 and focal inflammatory infiltrates occured in some females. Serius-red staining of the lung resulted in the observation of increased collagenous fibers in all animals of both sexes at the highest concentration.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE FLUID:
- 5, 15 and 50 mg/m3: alveolar macrophages appeared significantly increased already at the dose of 5 mg/m3. A concentration-dependent increase was observed in polymorphonuclear cells (PMNs) at 5 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.
- 15 and 50 mg/m3: A remarkable increase of total cell count (TCC) occured in animals exposed to the upper two concentrations. The LDH measurements revealed signs of cytotoxicity at 15 mg/m3 and above. Beta-N-acetyl-glucosaminidase activity appeared elevated in both sexes; this was the case for acid phosphatase in female rats.
The results of the BAL analysis are summarized in Table 11 (see attachment).

LUNG BURDEN:
- 5, 15 and 50 mg/m3: iron oxide translocation was minimally increased above the background iron levels of this organ at low-dose (approx. 30 times below the LALN iron‐burden observed at mid-dose). However, the respective increase at mid- and high-dose was proportional to the exposure concentration. Lung iron levels were determined but could not be interpreted due to a protocol error (iron analysis in lavaged lungs).

Details on results:

MORTALITY:
- The exposures were tolerated without mortality.

BODY WEIGHT AND WEIGHT CHANGES:
- The exposures were tolerated without concentration‐dependent significant changes in body weights.

FOOD CONSUMPTION:
The exposures were tolerated without concentration‐dependent significant changes in food consumption.

WATER CONSUMPTION:
The exposures were tolerated without concentration‐dependent significant changes water consumption.

OPHTHALMOLOGICAL FINDINGS:
- No abnormality in ophthalmology (performed prior to the start and towards the end of study) was observed.

HAEMATOLOGICAL FINDINGS:
- 5 and 15 mg/m3: PMN counts were still within the range of historical control data.

URINALIS FINDINGS:
- Urinalysis did not demonstrate differences across groups (data not shown).

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS:
- Absolute and relative (vs body weights) organ weights were confounded by the inconsistently higher body weight gains of the male control rats. Toxicologically conclusive changes in the remaining organ weights were not observed when using the organ‐to‐brain weight ratios.

GROSS PATHOLOGICAL FINDINGS:
- General clinical pathology, including hemostasis did not demonstrate differences across groups (data not shown).

HISTOPATHOLOGICAL FINDINGS:
- Adverse extrapulmonary effects of Fe3O4 were not observed at any concentration.
- 5, 15 and 50 mg/m3: diffuse tubular atrophy/degeneration was detected in 1 high-dose 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.
- Testes: (Multi)-focal tubular atrophy and degeneration was seen in all groups including the controls.

Effect levels

Dose descriptor:

NOAEC

Effect level:

4.7 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

immunology

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

LUNG BURDEN - HALF-TIME:

The time- and particle volume-dependence of lung burdens of Fe3O4 in rats exposed for 13 weeks for 6 h/day on 5 day/week in the present study were modeled. The following half-time for each concentrations were calculated: 5 mg/m3: 94 days; 15 mg/m3: 177 days; 50 mg/m3: 392 days. According to the athor, the lung overload treshold was reached after inhalation from a concentration of 15 mg/m3. The exposure concentrations selected for the 13 week study were modeled to attain the overload threshold (NOAEL) at 5 mg/m3 with overload‐dependent pulmonary inflammation at 15 and 50 mg/m3. The overload‐dependent delay in clearance was expected to be in the range of t1/2≈1 year at 50 mg/m3.

 

BRONCHOALVEOLAR LAVAGE FLUID:

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.

Applicant's summary and conclusion

Conclusions:

In the subchronic inhalation toxicity study by Pauluhn (2006a), 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 technique; 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.
In a subchronic inhalation study, 20 male and female Wistar rats were exposed to aerosolized Fe3O4 at concentrations of 4.7, 16.6 and 52.1 mg/m3 (Pauluhn (2006a); Pauluhn (2011)) by the dynamic directed-flow nose-only technique. 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. Clinical signs were recorded daily before and after exposure. Body weights were recorded twice weekly, and food and water consumption were determined once a week. Also, an ophthalmology was performed prior to start and the end of the exposure period. At sacrifice, inflammatory endpoints were determined in bronchoalveolar lavage (BAL). Rats were subjected to gross pathological examination and histopathology (adrenal glands, brain, heart, kidneys, liver, lung, LALNs, ovaries, spleen, testes, thymus).
The exposure was not associated with any specific clinical signs and consistent changes in body weights. Haematology, clinical pathology and urinalysis were unobtrusive.
The NOAEC was 4.7 mg/m3, based on the findings from BAL analysis (increased PMNs, protein levels) 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.

The results of this inhalation RDT study in Wistar rats can generally be regarded as reliable without restrictions, since the study was conducted according to OECD guideline 412 (2008) and under GLP.