Iron hydroxide oxide yellow

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

study was performed with Fe3O4 as a representative for the iron oxide group - see Category approach for Fe2O3, Fe3O4, FeOOH, (Fe,Mn)2O3, (Fe,Mn)3O4, ZnFe2O4

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2004-04-30 to 2004-08-11

Reliability:

other: not rated according Klimisch, selection experiment

Rationale for reliability incl. deficiencies:

other:

Remarks:

This comparative study was conducted according to OECD 412 (1981) and under GLP. However, only male rats were tested without justification, and only one dose was tested which does not allow a dose-response related analysis. A haematological examination and clinical biochemistry in blood were not conducted. Furthermore, the organ weight of the adrenals was missing, and a histopathological examination of adrenals and the heart were not performed. In conclusion, the study is not reliable to draw any hazard conclusions. However, the aim of the study was to provide evidence that different iron oxide substances repesented by two nanoforms of Fe2O3 and FeOOH and one non-nanoform of Fe3O4 have a similar toxicological profile. For this purpose, this well documented study can be used as supporting information.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

1981-05-12

Deviations:

yes

Remarks:

only one dose; only males tested; no haematology and clinical biochemistry; no organ weight of adrenals; no histopathological examination of adrenals and heart.

Principles of method if other than guideline:

Wistar rats (48 male rats per group) were exposed in a subacute 2-week inhalation study to three different aerosolized iron oxide powders: Fe3O4, Fe2O3, FeOOH, abbreviated 'black', 'red', and 'yellow', respectively. Exposure was 6-hours/day on five days/week for two consecutive weeks (days 0-11). The rats were exposed to mean actual concentrations (i.e., breathing zone concentrations) of black, yellow, and red of 185.6, 195.7, and 210.2 mg/m³ air, respectively. As reference dust (positive control for lung damage) served 0012 in a similar concentration (200.4 mg/m³). The mode of exposure was dynamic directed-flow nose-only. Throughout the groups, the aerosol was highly respirable to rats, i.e., the average mass median aerodynamic diameter (MMAD) was in the range of 1.3-1.5 µm, the geometric standard deviation (GSD) ranged from 1.9-2.2. During the course of a 3 months postexposure period subgroups of rats were serially sacrificed on days 15, 29, 58, 107 and examined as follows:
During the study, the body weights were determined twice per week (on Fridays and Mondays) and once per week during the postexposure period. Clinical signs were recorded daily before and after exposure or once per week during the postexposure period. At each sacrifice inflammatory endpoints were determined in bronchoalveolar lavage (BAL), iron was determined in lungs, lung-associated lymph nodes (LALN), spleen, liver, and testes. During each sacrifice, gross pathological examination of rats were made and selected organs were collected for organ weight analysis (lung, LALN, brain, heart, thymus, liver, spleen, kidneys, testes) and histopathology (lung, liver, spleen, kidneys, testes).

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Remarks:

Hsd Cpb:WU (SPF)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Age at study initiation: approx. 2 - 3 months
- Mean body weight at study initiation: 227.61 mg
- Housing: during the study periods the animals were housed singly in conventional Makrolon Type IIIh cages; bedding material: type BK 8/15 low-dust wood granulate, supplier: Ssniff, Soest/Westfalen, Germany.
- Diet (ad libitum): standard fixed-formula diet (KLIBA 3883 = NAFAG 9441 pellets maintenance diet for rats and mice), supplier: PROVIMI KLIBA SA, 4303 Kaiseraugust, Switzerland.
- Water (ad libitum): tap water
- Acclimation period: approx. 1 week

DETAILS OF FOOD AND WATER QUALITY:
Available data provided no evidence of an impact on the study objective. Results of food and water analyses are retained by Bayer HealthCare AG.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 40 - 60 %
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12 (approx. 14 watt/m2 floor area)

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks:

conditioned dry

Mass median aerodynamic diameter (MMAD):

1.31 µm

Geometric standard deviation (GSD):

2.16

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: test atmospheres were generated using a WRIGHT DUST FEEDER system (BGI Inc., Waltham, MA 02154, USA).
- Inhalation chamber: inner diameter: 14 cm; outer diamer: 35 cm (two-chamber system); height 25 cm; internal volume: 3.8 L.
- System of generating particulates/aerosols: test atmospheres were generated using a WRIGHTDUST FEEDER system (BGI Inc., Waltham, MA, USA). For dry powder dispersion, conditioned compressed dry air (30 L/min.; generic dispersion pressure: approx. 200 kPa) was used. Test item was metered in a reservoir and then was compressed to a pellet. From this pellet defined amounts of test item were scraped off and entrained into the main air flow. The airborne powder was then conveyed into the inner cylinder of the inhalation chamber. Then, after humidification, the test atmosphere was forced through openings in the inner concentric cylinder of the chamber, directly towards the rats 'breathing zone (direct-flow). The stability of the test atmosphere was monitored continously using an aerosol real-time device (vide infra).
- Temperature, humidity, pressure in air chamber: controlled and measured continously; 21.7 °C and 21 % rel. humidity.
- Air flow rate: 45 L/min; inlet air flow: 30 L/min.
- Air change rate: 158 air changes per hour (45 L/min. x 60 min./(3 x 3.8 L)), continuous generation of test atmosphere.
- 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 cotton-wool and HEPA filters.

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:

2 consecutive weeks

Frequency of treatment:

6 hr/day, 5 days/week

Dose / conc.:

195.7 mg/m³ air (analytical)

Remarks:

± 19.3 mg/m3

Dose / conc.:

200 mg/m³ air (nominal)

No. of animals per sex per dose:

48

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In the inhalation toxicity study by Warheit et al. (1997), male rats were exposed to TiO2 6 hr/day, 5 days/week, for 4 weeks at actual concentrations of 51.9 and 252 mg/m3 (MMADs: 1.4 - 1.7 μm).* Particle retention half-time was approximately 330 days for 250 mg/m3.
The impact of this TiO2 dust load and similar lung burdens produced a sustained pulmonary inflammatory response measured through a period of 3-6 months postexposure concomitant with increases cell labeling of terminal airway and pulmonary parenchymal cells. The results of this study demonstrate that exposure to high dust concentration of this innocuous particle type 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. With regard to dose-response 51.9 mg/m3 caused minimal toxicity whereas exposure to 252 mg/m3 caused a precipitous increase in the inflammatory endpoints. For this 2-week inhalation study the dosimetrically adjusted concentrations are 104 and 504 mg/m³ air. From the dose-response relationship of the Warheit et al. (1997) study it can be appreciated that a meaningful comparison can only be achieved near the inversion point of the slopes of the respective dose- response curves rather than in the ranges of the amplitudes of maximum responses.* Based on these considerations twice the intermediate concentration of the titanium dioxide study was considered most appropriate.
Therefore, the initial target concentration of this 2-week inhalation study was 250 mg/m³. During the pre-trials and at the commencement of study it was technically difficult to generate this concentration on a day-to-day basis. Therefore, in order to minimize fluctuations in concentrations, all target concentrations were adjusted to approx. 200 mg/m³.
- Post-exposure recovery period in satellite groups: up to 3 months (4, 18, 57 and 96 days)

*Reference:
Warheit, D. B., Hansen, J. F., Yuen, I. S., Kelly, D. P., Snajdr, S. I., and Hartsky, M.A. (1997). Inhalation of High Concentrations of Low Toxicity Dusts in Rats Results in Impaired Pulmonary Clearance Mechanisms and Persistent Inflammation. Toxicol. Appl. Pharmacol. 145, 10-22.

Positive control:

DQ12 (200.4 mg/m3) for lung damage

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: clinical signs were recorded at least daily before and after exposure and once per week during the exposure-free days.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: all animals were measured before exposure, on a twice per week basis on Fridays and Mondays, and after the exposure-free weekends (Mondays).

FOOD CONSUMPTION AND COMPOUND INTAKE: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: at the end of the 2-week exposure period and during the course of a 3 months postexposure period subgroups of rats were serially sacrificed on days 15, 29, 58 and 107 and examined.
- Dose groups that were examined: control, positive control and treatment group from the main and recovery studies.
- Number of animals:
- Parameters examined: recovery of lavage fluid, total cell count in BAL, mean cellular diameter, mean cellular volume, lactate dehydrogenase (LDH), alkaline phosphatase, collagen, acid phosphatase, total protein, phospholipids in BALF, β-N-Acetyl-glucosaminidase (β-NAG) in BALF.
LUNG BURDEN: Yes
- Time schedule for analysis: at the end of the 2-week exposure period and during the course of a 3 months postexposure period subgroups of rats were serially sacrificed on days 15, 29, 58, and 107.
- Dose groups that were examined: control group and treatment group from the main and recovery studies.
- Number of animals:
- Parameters examined: iron content in lungs, lung-associated lymph nodes (LALN), spleen, liver, and testes.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All surviving rats were sacrificed at the end of the respective observation period using sodium pentobarbital as anaesthetic and complete exsanguination by severing of the abdominal aorta. All rats, irrespective of the day of death, were given a gross pathological examination. Consideration was given to performing a gross necropsy on animals as indicated by the nature of toxic effects, with particular reference to changes related to the respiratory tract. All gross pathological changes were recorded and evaluated.

ORGAN WEIGHTS:
The following exsanguinated organs were weighed: adrenals, brain, heart, kidneys, liver, lungs, ovaries, spleen, testes and thymus. The organ-to-body relationships are specified in both absolute and relative terms.

HISTOPATHOLOGY: Yes
The following organ tissues were fixed and examined: trachea, lung (left lobe), kidneys, spleen, liver, testes (left), thymus, and all organs of tissues with macroscopic findings. All organs not scheduled for fixation that exhibited gross changes were also fixed if necessary. The lungs were instilled by the intratracheal route with 10 % neutral buffered formalin (20 cm water column) and then postfixed with the other organs in 10 % neutral buffered formalin. All slides were stained with haematoxylin and eosin (H&E) and with Prussian Blue.

Other examinations:

RECTAL TEMPERATURE:
Rectal measurements was made 5 rats/groups on days 0, 4, and 11. shortly after cessation of exposure using a digital thermometer equipped with a rectal probe for rats.

Statistics:

For the statistical evaluation of samples drawn from continuously distributed random variates three types of statistical test were used, the choice of the test being a function of prior knowledge obtained in former studies. The following statistical methods were used: Dunnet test, adjusted Welch test and Kruskal-Wallis test followed by adjusted U test.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

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:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

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):

Iron oxide group:
- Main study: absolute and relative lung weight were statistically significant increased compared to the air control group (p < 0.01).
- Recovery study: relative lung weight was statistically significantly increased in the 1st postexposure period compared to the air control group (p < 0.01). At all time points of postexposure period, the organ weight of lung-associated lymph nodes (LALN) was statistically significant increased compared to air control group (p < 0.01).

Positive control (DQ12):
- absolute and relative organ weights of lung and LALN were statistically significantly increased in the main study and at all time points during postexposure period compared to the control group (p <0.01).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Exposed group: yellow discolouration of lungs and enlarged lung associated lymph nodes (LALN) were observed. Discolouration of the LALN was seen in all rats from this group at the end of exposure and during the whole recovery period (1 - 3 recovery period).
- Positive control (DQ12): grey discolouration of lungs was observed. All lungs from this group during all recovery necropsies were less collapsed upon opening of the thoracic cavity.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Iron oxide group:
- Main study: at the end of the exposure period intraalveolar granular material was seen in all rats. These granules as well as the alveolar macrophages appeared to be yellow.
- Recovery study: at the end of the 13-week recovery period, incidence and intensity of focal inflammatory infiltrates and bronchiolar-alveolar hypercellularity decreased. Focal inflammatory infiltrates and focal septal thickening were detected in almost all animals. At all interim sacrifices iron oxide particles were detected in BALT.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE FLUID
Exposed group:
- At all time points the average recovery of the lavage fluid instilled into the lung was high, i.e., it exceeded 80 % of the instilled volume.

- Main study: statistically significant increases were observed in total cell count in BAL (TCC), mean cellular volume (MCV), lactate dehydrogenase (LDH), total protein (PROT) and β-N-Acetyl-glucosaminidase in BALF NAG compared to the air control group (p < 0.01 and p < 0.05).

- Recovery study: also, TCC were statistically significant increased in all recovery periods compared to air control group (p < 0.01).
The MCV level were also significantly increased in the first and second recovery period compared to air controls. There were statistically significant increases in the levels of LDH and PROT in the second and third recory period, and the mean cellular diameter (MCD) in the third recovery period, compared to air controls (p < 0.01).

- Positive control: DQ12 exposed animals were unequivocally positive at all time points with increasing intensity of changes during the course of the postexposure period. In the main study, statistically significant changes were observed in total cell count in BAL, LDH, AP, ACPH, total protein, and β-NAG compared to the control group (p < 0.01 and p < 0.05). These parameters, as well as the collagen and phospholipids in BALF were significantly increased at all time points during the postexposure period compared to the control group (p < 0.01 and p < 0.05).
For more information, please refer to table 1 in the field "attached background material" field below.

IRON DETERMINATION IN ORGAN TISSUES:
- Exposed group: a remarkable increase of iron was observed in lungs and in LALN with evidence of time related translocation from one compartment to another. The iron content in these organs were statistically significant increased at all time points in the postexposure period compared to the control group (p < 0.01 and p < 0.05).

IRON KINETICS IN LUNG AND LALN:
Assuming a single-compartment 1st order elimination kinetics from the lung, the elimination half-time for the test item was 75 days.

Details on results:

CLINICAL SIGNS:
- air control group: all rats tolerated the exposure without specific signs.
- Iron oxide group: Tachypnea (second exposure day only). As far as clinical signs were observed they were related to incidental and isolated findings without any time-related exacerbation.

MORTALITY:
No mortality occurred.

BODY WEIGHT AND BODY WEIGHT GAIN:
- there was no statistically significant difference in body weights amongst the groups.
- Iron oxide group: statistically significant changes in body weight gain were observed on days 0 – 4 and 63 – 70 in comparison to the air control group (p < 0.01).

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS:
no significant changes were observed in the absolute and relative organ weights of brain, heart, thymus, liver, spleen, kidneys and testes in the main study and at all time points during postexposure period compared to the control group.

HISTOPATHOLOGICAL FINDINGS:
- At all interim sacrifices in the examined extrapulmonary organs (liver, kidneys, and testis) were no evidence of translocated iron particles existed. No iron oxide related findings could be detected in the liver, kidneys and testis at the end of the exposure period and after 2 weeks of recovery.

BALF MEASUREMENTS:
- Lipidperoxidation (TBARS) was measured in BALC and lung tissue and no distinct differences amongst the group was observed. Cytodifferentiation was planned however, omitted due to the extreme dust loading of BALC.

IRON DETERMINATION IN ORGAN TISSUES:
- Iron oxide group: there were no significant changes in the iron content of the liver and testes at all time points in the postexposure period compared to air control group. Splenic iron was examined on days 15 and 29. Due to a lack of any consistent effect and in the light of the spontaneously occurring variability in iron content, iron determinations in spleen were omitted on days 58 and 107.

RECTAL TEMPERATURE:
- Iron oxide group: in comparison to the concurrent air control group, there was no evidence of a conclusive, significant effect on body (rectal) temperature in the iron oxide group, whilst positive control (DQ12) elicited a hypothermic response on days 0 and 4.

Dose descriptor:

conc. level: only one concentration tested

Effect level:

195.7 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Focal inflammatory infiltrates, bronchiolo-alveolar hypercellularity

Dose descriptor:

NOAEC

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Critical effects observed:

not specified

The repeated exposure to the aerosolized iron oxid 'yellow' was not associated with any specific

clinical signs, changes in body temperature or body weights. A remarkable increase of iron was

observed in lungs and LALN (lung associated lymph nodes) with time-related translocation from

one compartiment (lung) to another (LALN). There were no toxicologically significant changes in

absolute or relative organ weights except the elevated absolute and relative weights of lungs and

LALNs.

Conclusions:

In this RDT study, aerosolized geothite powder was administered by inhalation at a concentration of 195.7 mg/m3 to male Wistar rats. Exposure was through dynamic directed-flow noseonly for 6-hours/day on 5 days/week for 2 consecutive weeks. During a 3-month postexposure period, subgroups of rats (12 per sacrifice) were serially sacrificed 15, 29, 58, and 107 days and examined. DQ12 was served as reference dust (positive control for lung damage; MMAD (GSD): 2.27 µm (1.84)) in a concentration of 200.4 mg/m3. Body weights and clinical signs were recorded during the study and postexposure period. At each sacrifice inflammatory end points were determined in BAL, rats were examined for gross pathology, histopathology (lung, liver, spleen, kidneys, testes) and organ weights (lung, LALN, brain, heart, thymus, liver, spleen, kidneys, testes), and iron was determined in lungs, LALN, spleen, liver, and testes. aerosol was highly respirable to rats (MMAD: 1.31 μm; GSD: 2.16).
According to the author, histopathological evaluation of rat lungs exposed to three different iron oxides revealed findings consistent with a ‘poorly soluble particle’ effect after the 2- week exposure period, including the 3-month postexposure period. Conclusive evidence of bioavailable iron or iron particles that were translocated to extra- pulmonary organs was not observed. Extrapulmonary effects causally linked to the high-level exposure of iron oxide was not detected at any time point. At the end of the 3-month postexposure period the findings causally linked to the high-level exposure to iron oxides (e.g. focal inflammatory infiltrates, bronchiolo-alveolar hypercellularity) showed a decrease in incidence and/or severity.

This study was conducted as dose range finding study according to OECD 412 (1981) and under GLP.
However, only male rats were tested without justification, and only one dose was tested which does not allow a dose-response related analysis. A haematological examination and clinical biochemistry in blood were not conducted. Furthermore, the organ weight of the adrenals was missing, and a histopathological examination of adrenals and the heart were not performed.

However, the aim of the study was to provide evidence that different iron oxide substances repesented by two nanoforms of Fe2O3 and FeOOH and one non-nanoform of Fe3O4 have a similar toxicological profile. For this purpose, this well documented study can be used as supporting information.

Executive summary:

In a subacute inhalation toxicity study Fe3O4, FeOOH and Fe2O3 particles were administered to Wistar rats (48 male per group) by means of the dynamic directed-flow nose-only technique, with exposure at mean actual concentrations of 185.2, 195.7 and 210.2 mg/m3 air for 6 hours per day, 5 days/week for a total of 2 weeks. As a positive control for lung damage DQ12 was used, administered at 200.4 mg/m3. During the course of a 3-month postexposure period subgroups of rats were sacrified and examined on days 15, 29, 59 and 107. The MMAD of the particles was 1.3 -1.5 µm with a GSD of 1.9 -2.2. The exposure was not associated with any specific clinical signs and consistent changes in body weights. No NOAEC was defined. Iron oxides related findings could not be detected in the extrapulmonary organs (kidneys, testes, liver) both at the end of exposure and after 2 weeks of

recovery. Solubilized Fe was detected within/around the alveolar macrophages, but not in the interstitium and hepatic tissue.

Histopathological evaluations of the lungs demonstrated an effect-pattern consistent with that of poorly soluble particles.

This subacute inhalation toxicity study in the Wistar rats is acceptable and satisfies the guidelines requirement for a subacute inhalation study OECD 412 in rodents.

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

2004-04-30 to 2004-08-11

Reliability:

other: not rated according Klimisch, selection experiment

Rationale for reliability incl. deficiencies:

other:

Remarks:

This comparative study was conducted according to OECD 412 (1981) and under GLP. However, only male rats were tested without justification, and only one dose was tested which does not allow a dose-response related analysis. A haematological examination and clinical biochemistry in blood were not conducted. Furthermore, the organ weight of the adrenals was missing, and a histopathological examination of adrenals and the heart were not performed. In conclusion, the study is not reliable to draw any hazard conclusions. However, the aim of the study was to provide evidence that different iron oxide substances repesented by two nanoforms of Fe2O3 and FeOOH and one non-nanoform of Fe3O4 have a similar toxicological profile. For this purpose, this well documented study can be used as supporting information.

Justification for type of information:

see attachment "Iron oxide category read-across concept-HH " in IUCLID section 13.2.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

1981-05-12

Deviations:

yes

Remarks:

only one dose and only males were tested; no haematology and clinical biochemistry in blood; organ weight of adrenals were missing; histopathology of adrenals and heart were missing.

Principles of method if other than guideline:

Wistar rats (48 male rats per group) were exposed in a subacute 2-week inhalation study to three different aerosolized iron oxide powders: Fe3O4, Fe2O3, FeOOH, abbreviated 'black', 'red', and 'yellow', respectively. Exposure was 6-hours/day on five days/week for two consecutive weeks (days 0-11). The rats were exposed to mean actual concentrations (i.e., breathing zone concentrations) of black, yellow, and red of 185.6, 195.7, and 210.2 mg/m³ air, respectively. As reference dust (positive control for lung damage) served 0012 in a similar concentration (200.4 mg/m³). The mode of exposure was dynamic directed-flow nose-only. Throughout the groups, the aerosol was highly respirable to rats, i.e., the average mass median aerodynamic diameter (MMAD) was in the range of 1.3-1.5 µm, the geometric standard deviation (GSD) ranged from 1.9-2.2. During the course of a 3 months postexposure period subgroups of rats were serially sacrificed on days 15, 29, 58, 107 and examined as follows:
During the study, the body weights were determined twice per week (on Fridays and Mondays) and once per week during the postexposure period. Clinical signs were recorded daily before and after exposure or once per week during the postexposure period. At each sacrifice inflammatory endpoints were determined in bronchoalveolar lavage (BAL), iron was determined in lungs, lung-associated lymph nodes (LALN), spleen, liver, and testes. During each sacrifice, gross pathological examination of rats were made and selected organs were collected for organ weight analysis (lung, LALN, brain, heart, thymus, liver, spleen, kidneys, testes) and histopathology (lung, liver, spleen, kidneys, testes).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Hsd Cpb:WU (SPF)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Age at study initiation: approx. 2 - 3 months
- Mean body weight at study initiation: 227.54 mg
- Housing: during the study periods the animals were housed singly in conventional Makrolon Type IIIh cages; bedding material: type BK 8/15 low-dust wood granulate, supplier: Ssniff, Soest/Westfalen, Germany.
- Diet (ad libitum): standard fixed-formula diet (KLIBA 3883 = NAFAG 9441 pellets maintenance diet for rats and mice), supplier: PROVIMI KLIBA SA, 4303 Kaiseraugust, Switzerland.
- Water (ad libitum): tap water
- Acclimation period: approx. 1 week

DETAILS OF FOOD AND WATER QUALITY:
Available data provided no evidence of an impact on the study objective. Results of food and water analyses are retained by Bayer HealthCare AG.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 40 - 60 %
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12 (approx. 14 watt/m2 floor area)

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks:

conditioned dry air

Mass median aerodynamic diameter (MMAD):

1.43 µm

Geometric standard deviation (GSD):

2.14

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: test atmospheres were generated using a WRIGHT DUST FEEDER system (BGI Inc., Waltham, MA 02154, USA).
- Inhalation chamber: inner diameter: 14 cm; outer diamer: 35 cm (two-chamber system); height 25 cm; internal volume: 3.8 L.
- System of generating particulates/aerosols: test atmospheres were generated using a WRIGHTDUST FEEDER system (BGI Inc., Waltham, MA, USA). For dry powder dispersion, conditioned compressed dry air (30 L/min.; generic dispersion pressure: approx. 200 kPa) was used. Test item was metered in a reservoir and then was compressed to a pellet. From this pellet defined amounts of test item were scraped off and entrained into the main air flow. The airborne powder was then conveyed into the inner cylinder of the inhalation chamber. Then, after humidification, the test atmosphere was forced through openings in the inner concentric cylinder of the chamber, directly towards the rats 'breathing zone (direct-flow). The stability of the test atmosphere was monitored continously using an aerosol real-time device (vide infra).
- Temperature, humidity, pressure in air chamber: controlled and measured continously; 22.7 °C and 14.3 % rel. humidity.
- Air flow rate: 45 L/min; inlet air flow: 30 L/min.
- Air change rate: 158 air changes per hour (45 L/min. x 60 min./(3 x 3.8 L)), continuous generation of test atmosphere.
- 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 cotton-wool and HEPA filters.

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:

2 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

210.2 mg/m³ air (analytical)

Remarks:

± 46.7 mg/m3

Dose / conc.:

200 mg/m³ air (nominal)

No. of animals per sex per dose:

48 males per group: 12 males per group in main study (one dose group, air control and positive control group); 12 males/group/serial sacrifice in recovery study (one dose group, air control and recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In the inhalation toxicity study by Warheit et al. (1997), male rats were exposed to TiO2 6 hr/day, 5 days/week, for 4 weeks at actual concentrations of 51.9 and 252 mg/m3 (MMADs: 1.4 - 1.7 μm).* Particle retention half-time was approximately 330 days for 250 mg/m3. The impact of this TiO2 dust load and similar lung burdens produced a sustained pulmonary inflammatory response measured through a period of 3-6 months postexposure concomitant with increases cell labeling of terminal airway and pulmonary parenchymal cells. The results of this study demonstrate that exposure to high dust concentration of this innocuous particle type 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. With regard to dose-response 51.9 mg/m3 caused minimal toxicity whereas exposure to 252 mg/m3 caused a precipitous increase in the inflammatory endpoints. For this 2-week inhalation study the dosimetrically adjusted concentrations are 104 and 504 mg/m³ air. From the dose-response relationship of the Warheit et al. (1997) study it can be appreciated that a meaningful comparison can only be achieved near the inversion point of the slopes of the respective dose- response curves rather than in the ranges of the amplitudes of maximum responses.* Based on these considerations twice the intermediate concentration of the titanium dioxide study was considered most appropriate. Therefore, the initial target concentration of this 2-week inhalation study was 250 mg/m³. During the pre-trials and at the commencement of study it was technically difficult to generate this concentration on a day-to-day basis. Therefore, in order to minimize fluctuations in concentrations, all target concentrations were adjusted to approx. 200 mg/m³.
- Post-exposure recovery period in satellite groups: up to 3 months (4, 18, 57 and 96 days)

*Reference:
Warheit, D. B., Hansen, J. F., Yuen, I. S., Kelly, D. P., Snajdr, S. I., and Hartsky, M.A. (1997). Inhalation of High Concentrations of Low Toxicity Dusts in Rats Results in Impaired Pulmonary Clearance Mechanisms and Persistent Inflammation. Toxicol. Appl. Pharmacol. 145, 10-22.

Positive control:

DQ12 (Quartz; 200.4 mg/m3) for lung damage

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: clinical signs were recorded at least daily before and after exposure and once per week during the exposure-free days.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: all animals were measured before exposure, on a twice per week basis on Fridays and Mondays, and after the exposure-free weekends (Mondays).

FOOD CONSUMPTION AND COMPOUND INTAKE: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: at the end of the 2-week exposure period and during the course of a 3 months postexposure period subgroups of rats were serially sacrificed on days 15, 29, 58 and 107 and examined.
- Dose groups that were examined: control, positive control and treatment group from the main and recovery studies.
- Number of animals: 6 rats/group/serial sacrifice
- Parameters examined: recovery of lavage fluid, total cell count in BAL, mean cellular diameter (MCD), mean cellular volume (MCV), lactate dehydrogenase (LDH), alkaline phosphatase, collagen, acid phosphatase, total protein, phospholipids in BALF, β-N-Acetyl-glucosaminidase (β-NAG) in BALF.

LUNG BURDEN: Yes
- Time schedule for analysis: at the end of the 2-week exposure period and during the course of a 3 months postexposure period subgroups of rats were serially sacrificed on days 15, 29, 58, and 107.
- Dose groups that were examined: control group and treatment group from the main and recovery studies.
- Number of animals: 6 rats/group/serial sacrifice
- Parameters examined: iron content in lungs, lung-associated lymph nodes (LALN), spleen, liver, and testes.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All surviving rats were sacrificed at the end of the respective observation period using sodium pentobarbital as anaesthetic and complete exsanguination by severing of the abdominal aorta. All rats, irrespective of the day of death, were given a gross pathological examination. Consideration was given to performing a gross necropsy on animals as indicated by the nature of toxic effects, with particular reference to changes related to the respiratory tract. All gross pathological changes were recorded and evaluated.

ORGAN WEIGHTS:
The following exsanguinated organs were weighed: adrenals, brain, heart, kidneys, liver, lungs, ovaries, spleen, testes and thymus. The organ-to-body relationships are specified in both absolute and relative terms.

HISTOPATHOLOGY: Yes
The following organ tissues were fixed and examined: trachea, lung (left lobe), kidneys, spleen, liver, testes (left), thymus, and all organs of tissues with macroscopic findings. All organs not scheduled for fixation that exhibited gross changes were also fixed if necessary. The lungs were instilled by the intratracheal route with 10 % neutral buffered formalin (20 cm water column) and then postfixed with the other organs in 10 % neutral buffered formalin. All slides were stained with haematoxylin and eosin (H&E) and with Prussian Blue.

Other examinations:

RECTAL TEMPERATURE:
Rectal measurements was made 5 rats/groups on days 0, 4, and 11. shortly after cessation of exposure using a digital thermometer equipped with a rectal probe for rats.

Statistics:

For the statistical evaluation of samples drawn from continuously distributed random variates three types of statistical test were used, the choice of the test being a function of prior knowledge obtained in former studies. The following statistical methods were used: Dunnet test, adjusted Welch test and
Kruskal-Wallis test followed by adjusted U test.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

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:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

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):

- Main study: absolute and relative lung weights, and relative lung-associated lymph nodes (LALN) weights were statistically significant increased compared to the air control group (p < 0.01). Relative kidney weights were significantly decreased compared to air controls (p < 0.01).

- Recovery study: relative organ weights of LALN were statistically significantly increased at all time points of the postexposure period compared to air controls (p < 0.01 and p < 0.05).

- Positive control (DQ12): absolute and relative organ weights of lung and LALN were statistically significantly increased in the main study and at all time points during postexposure period compared to the control group (p <0.01).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Exposed group: red discolouration of lungs and enlarged lung associated lymph nodes (LALN) were observed. Discolouration of the LALN was seen in all rats from this group at the end of exposure and during the whole recovery period (1 - 3 recovery period).
- Positive control (DQ12): grey discolouration of lungs was observed. All lungs from this group during all recovery necropsies were less collapsed upon opening of the thoracic cavity.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Exposed group: at the end of the exposure period intraalveolar granular material was seen in all rats exposed to iron-oxide. These granules as well as the alveolar macrophages appeared to be red. At the end of the 13-week recovery period, incidence and intensity of focal inflammatory infiltrates and bronchiolar-alveolar hypercellularity decreased. Focal inflammatory infiltrates and focal septal thickening were detected in almost all animals. At all interim sacrifices iron oxide particles were detected in BALT.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE FLUID
Exposed groups:
- At all time points the average recovery of the lavage fluid instilled into the lung was high, i.e., it exceeded 80 % of the instilled volume.
- Main study: statistically significant increases in were observed in total cell count in BAL (TCC), mean cellular volume (MCV), lactate dehydrogenase (LDH), total protein (PROT) and phospholipids in BALF (PLIPF) compared to the air control group (p < 0.01 and p < 0.05).

- Recovery study: also, TCC were statistically significant increased in all recovery periods compared to air control group. There were statistically significant increases in mean cellular diameter (MCD) and MCV in the first recovery period, and in the levels of LDH and PROT in the second and third recovery period compared to air controls (p < 0.01 and p < 0.05).

- Positive control: DQ12 exposed animals were unequivocally positive at all time points with increasing intensity of changes during the course of the postexposure period. In the main study, statistically significant changes were observed in total cell count
in BAL, LDH, AP, ACPH, total protein, and β-NAG compared to the control group (p < 0.01 and p < 0.05). These parameters, as well as the collagen and phospholipids in BALF were significantly increased at all time points during the postexposure period compared to the control group (p < 0.01 and p < 0.05).
For more information, please refer to table 1 in the field "attached background material" field below.

IRON DETERMINATION IN ORGAN TISSUES:
- Exposed group: a remarkable increase of iron was observed in lungs and in LALN with evidence of time related translocation from one compartment to another. The iron content in these organs were statistically significant increased at all time points in the postexposure period compared to the control group (p < 0.01 and p < 0.05).

IRON KINETICS IN LUNG AND LALN:
Assuming a single-compartment 1st order elimination kinetics from the lung, the elimination half-time for the test item was 87 days.

Details on results:

CLINICAL SIGNS:
air control and exposed group: all rats tolerated the exposure without specific signs. As far as clinical signs were observed in the exposed group they were related to incidental and isolated findings without any time-related exacerbation.

MORTALITY:
No mortality occurred.

BODY WEIGHT AND BODY WEIGHT GAIN:
there was no statistically significant difference in body weights amongst the groups.
Exposed groups: statistically significant changes in body weight gain were observed on days 0 – 4, 21 – 35, 63 - 70 and 77 – 84 compared to the air control group (p < 0.01 and p < 0.05).

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS:
no significant changes were observed in the absolute and relative organ weights of brain, heart, thymus, liver, and spleen in the main study and at all time points during postexposure period compared to the control group. In the 2nd time point of postexposure period, the relative testes weights were statistically different compared to the control group (p < 0.05).

GROSS PATHOLOGICALFINDINGS: ???

HISTOPATHOLOGICAL FINDINGS:
At all interim sacrifices in the examined extrapulmonary organs (liver, kidneys, and testis) were no evidence of translocated iron particles existed. No iron oxide related findings could be detected in the liver, kidneys and testis at the end of the exposure period and after 2 weeks of recovery.

BALF MEASUREMENTS:
Lipidperoxidation (TBARS) was measured in BALC and lung tissue and no distinct differences amongst the group was observed. Cytodifferentiation was planned however, omitted due to the extreme dust loading of BALC.

IRON DETERMINATION IN ORGAN TISSUES:
- Exposed group: during the recovery study (at day 58), statistically significant increase was observed in the testes (p < 0.05) compared to air control group. There were no significant changes in the iron content of the liver at all time points in the postexposure period compared to air control group. Splenic iron was examined on days 15 and 29. Due to a lack of any consistent effect and in the light of the spontaneously occurring variability in iron content, iron determinations in spleen were omitted on days 58 and 107.

RECTAL TEMPERATURE:
Exposed group: in comparison to the concurrent air control group, there was no evidence of a conclusive, significant effect on body (rectal) temperature in the iron oxide group, whilst positive control (DQ12) elicited a hypothermic response on days 0 and 4.

Dose descriptor:

conc. level: only one concentration tested

Effect level:

210.2 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Focal inflammatory infiltrates, bronchiolo-alveolar hypercellularity

Dose descriptor:

NOAEC

Sex:

male

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Critical effects observed:

not specified

In summary, histopathological evaluation of rat lungs exposed to iron oxides revealed findings consistent with a 'poorly suluble particle' effect after the 2 -week exposure period, including the 3 -month postexposure period. Conclusive evidence of bioavailable iron or iron particles that were translocated to extrapulmonary organs was not observed.

Conclusions:

In this RDT study, aerosolized hematite was administered by inhalation at a concentration of 210.2 mg/m3 to male Wistar rats. Exposure was through dynamic directed-flow noseonly for 6-hours/day on 5 days/week for 2 consecutive weeks. During a 3-month postexposure period, subgroups of rats (12 per sacrifice) were serially sacrificed 15, 29, 58, and 107 days and examined. DQ12 was served as reference dust (positive control for lung damage; MMAD (GSD): 2.27 µm (1.84)) in a concentration of 200.4 mg/m3. Body weights and clinical signs were recorded during the study and postexposure period. At each sacrifice inflammatory end points were determined in BAL, rats were examined for gross pathology, histopathology (lung, liver, spleen, kidneys, testes) and organ weights (lung, LALN, brain, heart, thymus, liver, spleen, kidneys, testes), and iron was determined in lungs, LALN, spleen, liver, and testes. aerosol was highly respirable to rats (MMAD: 1.43 μm; GSD: 2.14).
According to the author, histopathological evaluation of rat lungs exposed to three different iron oxides revealed findings consistent with a ‘poorly soluble particle’ effect after the 2-week exposure period, including the 3-month postexposure period. Conclusive evidence of bioavailable iron or iron particles that were translocated to extra-pulmonary organs was not observed. Extrapulmonary effects causally linked to the high-level exposure of iron oxide was not detected at any time point. At the end of the 3-month postexposure period the findings causally linked to the high-level exposure to iron oxides (e.g. focal inflammatory infiltrates, bronchiolo-alveolar hypercellularity) showed a decrease in incidence and/or severity.

This study was conducted as dose range finding study according to OECD 412 (1981) and under GLP.
However, only male rats were tested without justification, and only one dose was tested which does not allow a dose-response related analysis. A haematological examination and clinical biochemistry in blood were not conducted. Furthermore, the organ weight of the adrenals was missing, and a histopathological examination of adrenals and the heart were not performed.

However, the aim of the study was to provide evidence that different iron oxide substances repesented by two nanoforms of Fe2O3 and FeOOH and one non-nanoform of Fe3O4 have a similar toxicological profile. For this purpose, this well documented study can be used as supporting information.

Executive summary:

In a subacute inhalation toxicity study Fe3O4, FeOOH and Fe2O3 particles were administered to Wistar rats (48 male per group) by means of the dynamic directed-flow nose-only technique, with exposure at mean actual concentrations of 185.2, 195.7 and 210.2 mg/m3 air for 6 hours per day, 5 days/week for a total of 2 weeks. As a positive control for lung damage DQ12 was used, administered at 200.4 mg/m3. During the course of a 3-month postexposure period subgroups of rats were sacrified and examined on days 15, 29, 59 and 107. The MMAD of the particles was 1.3 -1.5 µm with a GSD of 1.9 -2.2. The exposure was not associated with any specific clinical signs and consistent changes in body weights. No NOAEC was defined. Iron oxides related findings could not be detected in the extrapulmonary organs (kidneys, testes, liver) both at the end of exposure and after 2 weeks of

recovery. Solubilized Fe was detected within/around the alveolar macrophages, but not in the interstitium and hepatic tissue.

 

Histopathological evaluations of the lungs demonstrated an effect-pattern consistent with that of poorly soluble particles.

This subacute inhalation toxicity study in the Wistar rats is acceptable and satisfies the guidelines requirement for a subacute inhalation study OECD 412 in rodents.

Cross-reference 3

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

2004-04-30 to 2004-08-11

Reliability:

other: not rated according Klimisch, selection experiment

Rationale for reliability incl. deficiencies:

other:

Remarks:

This comparative study was conducted according to OECD 412 (1981) and under GLP. However, only male rats were tested without justification, and only one dose was tested which does not allow a dose-response related analysis. A haematological examination and clinical biochemistry in blood were not conducted. Furthermore, the organ weight of the adrenals was missing, and a histopathological examination of adrenals and the heart were not performed. In conclusion, the study is not reliable to draw any hazard conclusions. However, the aim of the study was to provide evidence that different iron oxide substances repesented by two nanoforms of Fe2O3 and FeOOH and one non-nanoform of Fe3O4 have a similar toxicological profile. For this purpose, this well documented study can be used as supporting information.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

1981-05-12

Deviations:

yes

Remarks:

only one dose and only males were tested; no haematology and clinical biochemistry in blood; organ weight of adrenals were missing; histopathology of adrenals and heart were missing.

Principles of method if other than guideline:

Wistar rats (48 male rats per group) were exposed in a subacute 2-week inhalation study to three different aerosolized iron oxide powders: Fe3O4, Fe2O3, FeOOH, abbreviated 'black', 'red', and 'yellow', respectively. Exposure was 6-hours/day on five days/week for two consecutive weeks (days 0-11). The rats were exposed to mean actual concentrations (i.e., breathing zone concentrations) of black, yellow, and red of 185.6, 195.7, and 210.2 mg/m³ air, respectively. As reference dust (positive control for lung damage) served 0012 in a similar concentration (200.4 mg/m³). The mode of exposure was dynamic directed-flow nose-only. Throughout the groups, the aerosol was highly respirable to rats, i.e., the average mass median aerodynamic diameter (MMAD) was in the range of 1.3-1.5 µm, the geometric standard deviation (GSD) ranged from 1.9-2.2. During the course of a 3 months postexposure period subgroups of rats were serially sacrificed on days 15, 29, 58, 107 and examined as follows:
During the study, the body weights were determined twice per week (on Fridays and Mondays) and once per week during the postexposure period. Clinical signs were recorded daily before and after exposure or once per week during the postexposure period. At each sacrifice inflammatory endpoints were determined in bronchoalveolar lavage (BAL), iron was determined in lungs, lung-associated lymph nodes (LALN), spleen, liver, and testes. During each sacrifice, gross pathological examination of rats were made and selected organs were collected for organ weight analysis (lung, LALN, brain, heart, thymus, liver, spleen, kidneys, testes) and histopathology (lung, liver, spleen, kidneys, testes).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Hsd Cpb:WU (SPF)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Age at study initiation: approx. 2 - 3 months
- Mean body weight at study initiation: 228.84 mg
- Housing: during the study periods the animals were housed singly in conventional Makrolon Type IIIh cages; bedding material: type BK 8/15 low-dust wood granulate, supplier: Ssniff, Soest/Westfalen, Germany.
- Diet (ad libitum): standard fixed-formula diet (KLIBA 3883 = NAFAG 9441 pellets maintenance diet for rats and mice), supplier: PROVIMI KLIBA SA, 4303 Kaiseraugust, Switzerland.
- Water (ad libitum): tap water
- Acclimation period: approx. 1 week

DETAILS OF FOOD AND WATER QUALITY:
Available data provided no evidence of an impact on the study objective. Results of food and water analyses are retained by Bayer HealthCare AG.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 40 - 60 %
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12 (approx. 14 watt/m2 floor area)

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks:

conditioned dry air

Mass median aerodynamic diameter (MMAD):

1.52 µm

Geometric standard deviation (GSD):

1.95

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: test atmospheres were generated using a WRIGHT DUST FEEDER system (BGI Inc., Waltham, MA 02154, USA).
- Inhalation chamber: inner diameter: 14 cm; outer diamer: 35 cm (two-chamber system); height 25 cm; internal volume: 3.8 L.
- System of generating particulates/aerosols: test atmospheres were generated using a WRIGHTDUST FEEDER system (BGI Inc., Waltham, MA, USA). For dry powder dispersion, conditioned compressed dry air (30 L/min.; generic dispersion pressure: approx. 200 kPa) was used. Test item was metered in a reservoir and then was compressed to a pellet. From this pellet defined amounts of test item were scraped off and entrained into the main air flow. The airborne powder was then conveyed into the inner cylinder of the inhalation chamber. Then, after humidification, the test atmosphere was forced through openings in the inner concentric cylinder of the chamber, directly towards the rats 'breathing zone (direct-flow). The stability of the test atmosphere was monitored continously using an aerosol real-time device (vide infra).
- Temperature, humidity, pressure in air chamber: controlled and measured continously; 22.0 °C and 17.3 % rel. humidity.
- Air flow rate: 45 L/min; inlet air flow: 30 L/min.
- Air change rate: 158 air changes per hour (45 L/min. x 60 min./(3 x 3.8 L)), continuous generation of test atmosphere.
- 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 cotton-wool and HEPA filters.

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:

2 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

200 mg/m³ air (nominal)

Dose / conc.:

185.6 mg/m³ air (analytical)

Remarks:

± 31.4 mg/m3

No. of animals per sex per dose:

48 males per group: 12 males per group in main study (one dose group, air control and positive control group); 12 males/group/serial sacrifice in recovery study (one dose group, air control and recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In the inhalation toxicity study by Warheit et al. (1997), male rats were exposed to TiO2 6 hr/day, 5 days/week, for 4 weeks at actual concentrations of 51.9 and 252 mg/m3 (MMADs: 1.4 - 1.7 μm).* Particle retention half-time was approximately 330 days for 250 mg/m3. The impact of this TiO2 dust load and similar lung burdens produced a sustained pulmonary inflammatory response measured through a period of 3-6 months postexposure concomitant with increases cell labeling of terminal airway and pulmonary parenchymal cells. The results of this study demonstrate that exposure to high dust concentration of this innocuous particle type 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. With regard to dose-response 51.9 mg/m3 caused minimal toxicity whereas exposure to 252 mg/m3 caused a precipitous increase in the inflammatory endpoints. For this 2-week inhalation study the dosimetrically adjusted concentrations are 104 and 504 mg/m³ air. From the dose-response relationship of the Warheit et al. (1997) study it can be appreciated that a meaningful comparison can only be achieved near the inversion point of the slopes of the respective dose- response curves rather than in the ranges of the amplitudes of maximum responses.* Based on these considerations twice the intermediate concentration of the titanium dioxide study was considered most appropriate. Therefore, the initial target concentration of this 2-week inhalation study was 250 mg/m³. During the pre-trials and at the commencement of study it was technically difficult to generate this concentration on a day-to-day basis. Therefore, in order to minimize fluctuations in concentrations, all target concentrations were adjusted to approx. 200 mg/m³.
- Post-exposure recovery period in satellite groups: up to 3 months (4, 18, 57 and 96 days)

*Reference:
Warheit, D. B., Hansen, J. F., Yuen, I. S., Kelly, D. P., Snajdr, S. I., and Hartsky, M.A. (1997). Inhalati on of High Concentrations of Low Toxicity Dusts in Rats Results in Impaired Pulmonary Clearance Mechanisms and Persistent Inflammation. Toxicol. Appl. Pharmacol. 145, 10-22.

Positive control:

DQ12 (Quartz; 200.4 mg/m3) for lung damage

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: clinical signs were recorded at least daily before and after exposure and once per week during the exposure-free days.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: all animals were measured before exposure, on a twice per week basis on Fridays and Mondays, and after the exposure-free weekends (Mondays).

FOOD CONSUMPTION AND COMPOUND INTAKE: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: at the end of the 2-week exposure period and during the course of a 3 months postexposure period subgroups of rats were serially sacrificed on days 15, 29, 58 and 107 and examined.
- Dose groups that were examined: control, positive control and treatment group from the main and recovery studies.
- Number of animals: 6 rats/group/serial sacrifice
- Parameters examined: recovery of lavage fluid, total cell count in BAL, mean cellular diameter, mean cellular volume, lactate dehydrogenase (LDH), alkaline phosphatase, collagen, acid phosphatase, total protein, phospholipids in BALF, β-N-Acetyl-glucosaminidase (β-NAG) in BALF.

LUNG BURDEN: Yes
- Time schedule for analysis: at the end of the 2-week exposure period and during the course of a 3 months postexposure period subgroups of rats were serially sacrificed on days 15, 29, 58, and 107.
- Dose groups that were examined: control group and treatment group from the main and recovery study.
- Number of animals: 6 rats/group/serial sacrifice
- Parameters examined: iron content in lungs, lung-associated lymph nodes (LALN), spleen, liver, and testes.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All surviving rats were sacrificed at the end of the respective observation period using sodium pentobarbital as anaesthetic and complete exsanguination by severing of the abdominal aorta. All rats, irrespective of the day of death, were given a gross pathological examination. Consideration was given to performing a gross necropsy on animals as indicated by the nature of toxic effects, with particular reference to changes related to the respiratory tract. All gross pathological changes were recorded and evaluated.

ORGAN WEIGHTS:
The following exsanguinated organs were weighed: adrenals, brain, heart, kidneys, liver, lungs, ovaries, spleen, testes and thymus. The organ-to-body relationships are specified in both absolute and relative terms.

HISTOPATHOLOGY: Yes
The following organ tissues were fixed and examined: trachea, lung (left lobe), kidneys, spleen, liver, testes (left), thymus, and all organs of tissues with macroscopic findings. All organs not scheduled for fixation that exhibited gross changes were also fixed if necessary. The lungs were instilled by the intratracheal route with 10 % neutral buffered formalin (20 cm water column) and then postfixed with the other organs in 10 % neutral buffered formalin. All slides were stained with haematoxylin and eosin (H&E) and with Prussian Blue.

Other examinations:

RECTAL TEMPERATURE:
Rectal measurements was made 5 rats/groups on days 0, 4, and 11. shortly after cessation of exposure using a digital thermometer equipped with a rectal probe for rats.

Statistics:

For the statistical evaluation of samples drawn from continuously distributed random variates three types of statistical test were used, the choice of the test being a function of prior knowledge obtained in former studies. The following statistical methods were used: Dunnet test, adjusted Welch test and
Kruskal-Wallis test followed by adjusted U test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- 200 mg/m3: motility reduced, limp (rat no. 138 which succumbed on day 55). This clinical signs were related to incidental and isolated findings without any time-related exacerbation.
- Positive control (DQ12): tachypnea, irregular breathing patterns, labored breathing patterns, hair-coat ungroomed. Signs occurred in a biphasic manner.

Mortality:

mortality observed, treatment-related

Description (incidence):

- 200 mg/m3: 1 single rat (no. 138) succumbed on day 55. Although a clear causal relationship cannot be established.

Body weight and weight changes:

no effects observed

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:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

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):

- 200 mg/m3: absolute organ weights of lung and lung-associated lymph nodes (LALN) were only statistically significantly increased in the main study, and their relative organ weights were increased at all time points during postexposure period compared to the control group (p <0.01 and p < 0.05).
- Positive control (DQ12): absolute and relative organ weights of lung and LALN were statistically significantly increased in the main study and at all time points during postexposure period compared to the control group (p <0.01).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Exposed group: black discolouration of lungs and enlarged lung associated lymph nodes (LALN) were observed. Discolouration of the LALN was seen in all rats from this group at the end of exposure and during the whole recovery period (1 - 3 recovery period).
- Positive control (DQ12): grey discolouration of lungs was observed. All lungs from this group during all recovery necropsies were less collapsed upon opening of the thoracic cavity.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 200 mg/m3: at the end of the exposure period intraalveolar granular material was seen in all rats exposed to iron-oxide. These granules as well as the alveolar macrophages appeared to be black. At the end of the 13-week recovery period, incidence and intensity of focal inflammatory infiltrates and bronchiolar-alveolar hypercellularity decreased. Focal inflammatory infiltrates and focal septal thickening were detected in almost all animals. At all interim sacrifices iron oxide particles were detected in BALT.
Soluble iron staining (Prussian Blue) of the lung (alveolar region) was positive at all time points and this positive staining was evident within/around alveolar macrophages.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE FLUID:
- At all time points the average recovery of the lavage fluid instilled into the lung was high, i.e., it exceeded 80% of the instilled volume.
- 200 mg/m3: in the main study, statistically significant increases were observed in total cell count in BAL, LDH, acid phosphatase, total protein, and β-N-Acetyl-glucosaminidase (β-NAG) compared to the control group (p < 0.01 and p < 0.05). At all time points of the postexposure period, significant increases were observed in total cell count in BAL, mean cellular volume, LDH, alkaline phosphatase (AP), acid phosphatase (ACPH), and total protein compared to controls (p < 0.01 and p < 0.05).
- Positive control: DQ12 exposed animals were unequivocally positive at all time points with increasing intensity of changes during the course of the postexposure period. In the main study, statistically significant changes were observed in total cell count in BAL, LDH, AP, ACPH, total protein, and β-NAG compared to the control group (p < 0.01 and p < 0.05). These parameters, as well as the collagen and phospholipids in BALF were significantly increased at all time points during the postexposure period compared to the control group (p < 0.01 and p < 0.05).
For more information, please refer to table 1 in the field "attached background material" field below.

IRON DETERMINATION IN ORGAN TISSUES:
- 200 mg/m3: a remarkable increase of iron was observed in lungs and in LALN with evidence of time-related translocation from one compartment to another. The iron content in these organs were statistically significantly increased at all time points in the postexposure period compared to the control group (p < 0.01).

Details on results:

CLINICAL SIGNS:
- Control: all rats tolerated the exposure without specific signs.

BODY WEIGHT AND WEIGHT CHANGES:
- there was no statistically significant difference in body weights among the groups.
- 200 mg/m3 and positive control: statistical significances, especially to body weight gais, are considered to be of no toxicological relevance.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS:
- 200 mg/m3: no significant changes were observed in the absolute and relative organ weights of brain, heart, thymus, liver, spleen, and kidneys in the main study and at all time points during postexposure period compared to the control group. The absolute and relative organ weights of testes were not significantly different compared to the controls in the main or postexposure period. In the 1st time point of postexposure period, the relative testes weights were statistically different compared to the control group (p < 0.05).

HISTOPATHOLOGICAL FINDINGS:
No iron oxide related findings could be detected in the liver, kidneys and testis at the end of the exposure period and after 2 weeks of recovery. At all interim sacrifices, in the examined extrapulmonary organs (liver, kidneys, and testis) existed no evidence of translocated iron particles. During the Iron staining with Prussian Blue, the interstitium did not show evidence of solubilized, i.e., potentially bioavailable iron. Prussian Blue staining of hepatic tissue was unobtrusive.

BRONCHOALVEOLAR LAVAGE FLUID:
- Lipidperoxidation measured in BALC and lung tissue showed no distinct differences amongst the groups.
- Cytodifferentiation was planned, however, omitted due to the extreme dust loading of BALC.

RECTAL TEMPERATURE MEASUREMENT:
In comparison to the concurrent air control group, there was no evidence of a conclusive, significant effect on body (rectal) temperature in the iron oxide group, whilst DQ12 (positiv control group) elicited a hyperthermic response on days 0 and 4.

IRON DETERMINATION IN ORGAN TISSUES:
- 200 mg/m3: during the recovery study, statisticially significant increase were observed in the liver tissue at day 29 and in the testes at day 58 (p < 0.05). At the other time points, no significant changes were observed in these two organs. Splenic iron was examined on days 15 and 29. Due to a lack of any consistent effect and in the light of the spontaneously occurring variability in iron content, iron determinations in spleen were omitted on days 58 and 107.

Dose descriptor:

conc. level: only one concentration tested

Effect level:

185.6 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Focal inflammatory infiltrates, bronchiolo-alveolar hypercellularity

Dose descriptor:

NOAEC

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Critical effects observed:

not specified

Conclusions:

In this RDT study, aerosolized magnetite powder was administered by inhalation at a concentration of 185.6 mg/m3 to male Wistar rats. Exposure was through dynamic directed-flow noseonly for 6-hours/day on 5 days/week for 2 consecutive weeks. During a 3-month postexposure period, subgroups of rats (12 per sacrifice) were serially sacrificed 15, 29, 58, and 107 days and examined. DQ12 was served as reference dust (positive control for lung damage; MMAD (GSD): 2.2.7 µm (1.84)) in a concentration of 200.4 mg/m3. Body weights and clinical signs were recorded during the study and postexposure period. At each sacrifice inflammatory end points were determined in BAL, rats were examined for gross pathology, histopathology (lung, liver, spleen, kidneys, testes) and organ weights (lung, LALN, brain, heart, thymus, liver, spleen, kidneys, testes), and iron was determined in lungs, LALN, spleen, liver, and testes. aerosol was highly respirable to rats (MMAD: 1.52 µm; GSD: 1.95).
According to the author, histopathological evaluation of rat lungs exposed to three different iron oxides revealed findings consistent with a ‘poorly soluble particle’ effect after the 2-week exposure period, including the 3-month postexposure period. Conclusive evidence of bioavailable iron or iron particles that were translocated to extra-pulmonary organs was not observed. Extrapulmonary effects causally linked to the high-level exposure of iron oxide was not detected at any time point. At the end of the 3-month postexposure period the findings causally linked to the high-level exposure to iron oxides (e.g. focal inflammatory infiltrates, bronchiolo-alveolar hypercellularity) showed a decrease in incidence and/or severity.

This study was conducted as a dose range finding study according to OECD 412 (1981) and under GLP. However, only male rats were tested without justification, and only one dose was tested which does not allow a dose-response related analysis. A haematological examination and clinical biochemistry in blood were not conducted. Furthermore, the organ weight of the adrenals was missing, and a histopathological examination of adrenals and the heart were not performed.

However, the aim of the study was to provide evidence that different iron oxide substances repesented by two nanoforms of Fe2O3 and FeOOH and one non-nanoform of Fe3O4 have a similar toxicological profile. For this purpose, this well documented study can be used as supporting information.

Executive summary:

In a subacute inhalation toxicity study Fe3O4, FeOOH and Fe2O3 particles were administered to Wistar rats (48 male per group) by means of the dynamic directed-flow nose-only technique, with exposure at mean actual concentrations of 185.2, 195.7 and 210.2 mg/m3 air for 6 hours per day, 5 days/week for a total of 2 weeks. As a positive control for lung damage DQ12 was used, administered at 200.4 mg/m3. During the course of a 3-month postexposure period subgroups of rats were sacrified and examined on days 15, 29, 59 and 107. The MMAD of the particles was 1.3 -1.5 µm with a GSD of 1.9 -2.2. The exposure was not associated with any specific clinical signs and consistent changes in body weights. No NOAEC was defined. Iron oxides related findings could not be detected in the extrapulmonary organs (kidneys, testes, liver) both at the end of exposure and after 2 weeks of

recovery. Solubilized Fe was detected within/around the alveolar macrophages, but not in the interstitium and hepatic tissue.

 

Histopathological evaluations of the lungs demonstrated an effect-pattern consistent with that of poorly soluble particles.

This subacute inhalation toxicity study in the Wistar rats is acceptable and satisfies the guidelines requirement for a subacute inhalation study OECD 412 in rodents.

Cross-reference 4

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

repeated dose toxicity: inhalation

Remarks:

other: subacute and subchronic

Type of information:

other: Expert opinion_Derivation of OEL based on read-across with Fe3O4 (magnetite)

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Expert opinion

Principles of method if other than guideline:

Repeated dose studies of FeOOH (nano-sized material) and Fe3O4 (micro-sized material) were compared. As a result, a read-across from the data of Fe3O4 to FeOOH is justified.: (Expert opinion by J. Pauluhn)

GLP compliance:

yes

Species:

rat

Route of administration:

inhalation

Analytical verification of doses or concentrations:

yes

Dose descriptor:

other: A read-across fromFe3O4 as a surrogate for other category members is considered to be implicitly conservative and scientifically justified based on the generic, overload dependent effects (inflammogenicity) observed.

Basis for effect level:

other: Expert opinion by J. Pauluhn

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

Critical effects observed:

not specified

PREDICTION OF THE CORNERSTONE OF PULMONARY TOXICITY OF GOETHITE (FeOOH)

The data presented above provide robust evidence that any assessment of the iron-based lung dosimetry and kinetics is subject to errors at cumulative exposure levels resulting in overload-dependent lung inflammation. Therefore, the generic principles established for nano- to micronsized particles is considered to be the method of choice. Suffice it to say, this does not change the overall effect-based conclusion that goethite (FeOOH) is empirically less inflammogenic than magnetite. Therefore, the generic assessment made on magnetite is considered to be implicitly conservative.

Executive summary:

Goethite fulfills the currently applied generic definition of nano-scaled materials with ≤ 0.1 μm in at least one dimension. Despite this characteristic ‘nano-goethite’ was equal-to-less inflammogenic upon repeated inhalation exposure of rats than magnetite under otherwise dosimetrically adjusted similar exposure conditions. The time-course changes of the ‘nano-goethite’ inflammogenicity was typical of nano- /micronsized poorly soluble, low toxicity particles. The apparent faster clearance of ‘nano-goethite’ is considered to be confounded by pulmonary inflammogenicity as endogenous inflammation-related iron’ which cannot be distinguished from particlerelated iron. Therefore, the impact of facilitated dissolution and of goethite relative to magnetite cannot be judged. Translocation of iron is a typical occurrence and sequel of pulmonary inflammation; however any nano-size-dependent increased translocation did not occur. Accordingly, a read-across from magnetite is considered to be implicitly conservative and scientifically justified based on the generic, overload dependent effects observed.

An OEL of 2 mg/m³ (TWA) is considered to be applicable to goethite.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

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

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: MMAD was 1.3 µm, GDS ~2

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day 5 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
4.7, 16.6, 52.1 mg/m³
Basis:
analytical conc.

No. of animals per sex per dose:

20

Control animals:

yes

Results and discussion

Effect levels

Dose descriptor:

NOAEL

Effect level:

4.7 mg/m³ air

Sex:

male/female

Basis for effect level:

other: see "Remarks"

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Rats exposed subchronically to three different concentrations of Fe3O4 revealed findings clearly consistent with and typical for a poorly  soluble particle. Congruent with previous studies addressing the  retention kinetics of inhaled Fe3O4 particles, neither analytical nor  toxicological evidence existed that free, biosoluble iron was liberated  from the inhaled dust to any appreciable extend. Also in this study no  evidence of extrapulmonary toxicity existed. With regard to indices  considered to be adverse, viz. increased counts of cells and especially  PMNs in BAL, elevated LDH as marker of cytotoxicity, and ß-NAG as marker of increased lysosomal activities 4.7 mg/m³ constitute an exposure level  without evidence of adversity. These findings match those observed by  histopathology.

Applicant's summary and conclusion

Executive summary:

Rats exposed subchronically to three different concentrations of Fe3O4 revealed findings clearly consistent with and typical for a poorly soluble particle. The retention kinetics of inhaled Fe3O4 particles revealed neither analytical nor toxicological evidence that free, biosoluble iron was liberated from the inhaled dust to any appreciable extend. Also in this study no evidence of extrapulmonary toxicity existed. The results of this study support the view, that the NOAEL of Fe3O4 is 4.7 mg/m³.