Iron sinter

Endpoint summary

Description of key information

Acute oral toxicity: LD50 > 5000mg/kg bw

Acute toxicity, inhalation: LC50 > 5 mg/L

Acute toxicity, dermal: There is no information available on acute dermal toxicity but a long history of safe use. In addition, the conduct of acute dermal toxicity testing is not considered to be required since inhalation of the substance is considered the most relevant route of human exposure. Furthermore, physicochemical and toxicological properties of the substance do not suggest a significant rate of absorption through the skin (cf. Annex VIII section 8.5 Column 2 of regulation (EC) 1907/2006)

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

abstract

Qualifier:

no guideline followed

Principles of method if other than guideline:

10 male Wistar rats were dosed with 10000 mg/kg bw of Eisenoxidgelb 920 (FeO(OH) in water by a single oral application by gavage. During post observation time of 14 days, the animals were exanimated for clinical signs and mortality.

GLP compliance:

not specified

Remarks:

Study was conducted prior to implementation of GLP in the EU.

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen
- Age at study initiation: not specified
- Weight at study initiation: 160 - 180 g males
- Housing:5 animals per cage

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

not specified

Doses:

10000 mg/kg bw

No. of animals per sex per dose:

10

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: not specified

Statistics:

No statistical analysis could be performed ( the method used is not intended to allow a calculation of a precise LD50 value).

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

> 10 000 mg/kg bw

Based on:

test mat.

Mortality:

no mortality was observed

Clinical signs:

other: no clinical signs (not specified) were observed

Body weight:

other body weight observations

Remarks:

not specified

Gross pathology:

not specified

Interpretation of results:

GHS criteria not met

Conclusions:

The LD50 cannot be determined, based to the absence of mortality and other effects. Based on the maximal limit dose tested of it could be considered that the LD50 of FeO(OH) is >10000 mg/kg bw/d. According to the Regulation (EC) No 1272/2008 and subsequent adaptions, the substance is not acutely toxic via the oral route.

The study was executed before the establishment of OECD guidelines and the short study report was provided in an abstract like form. Consequently, the study was ranked as “not assignable” (RL4).

Executive summary:

In an acute oral toxicity study, male Wistar rats were given a single oral dose of FeOOH in water, at a dose of  10,000  mg/kg bw (limit test); the treatment was followed by 14 days of observation. No mortality and no signs of toxicity were observed. The study yielded an oral LD50 of > 10000 mg/kg bw in male rats for FeOOH. Based on the results of this study, classification and labelling for acute oral toxicity is not required.

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

abstract

Qualifier:

no guideline followed

Principles of method if other than guideline:

10 male Wistar rats were dosed with 10000 mg/kg bw of Eisenoxidschwarz 318 (Fe3O4) in water by a single oral application by gavage. During post observation time of 14 days, the animals were exanimated for clinical signs and mortality.

GLP compliance:

not specified

Remarks:

Study was conducted prior to implementation of GLP in the EU.

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen
- Age at study initiation: not specified
- Weight at study initiation: 160 - 180 g males
- Housing:5 animals per cage

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

not specified

Doses:

10000 mg/kg bw (based on the active ingredient: Fe3O4)

No. of animals per sex per dose:

10

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: not specified

Statistics:

No statistical analysis could be performed ( the method used is not intended to allow a calculation of a precise LD50 value).

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

> 10 000 mg/kg bw

Based on:

test mat.

Mortality:

No death occured.

Clinical signs:

other: All animals were symptom-free during the 14-day observation at a dose level of 10000 mg/kg bw.

Body weight:

other body weight observations

Remarks:

not specified

Gross pathology:

not specified

Interpretation of results:

GHS criteria not met

Conclusions:

The LD50 cannot be determined, based to the absence of mortality and other effects. Based on the maximal limit dose tested of it could be considered that the LD50 of Fe3O4 is >10000 mg/kg bw/d. According to the Regulation (EC) No 1272/2008 and subsequent adaptions, the substance is not acutely toxic via the oral route.

The study was executed before the establishment of OECD guidelines and the short study report was provided in an abstract like form. Consequently, the study was ranked as “not assignable” (RL4).

Executive summary:

In an acute oral toxicity study, male Wistar rats were given a single oral dose of Fe3O4 in water and 0.2 % Na-Polyacrylate (w/w), 0.1 % Preventol D2 (w/w), at a dose of  10000  mg/kg bw (limit test); the treatment was followed by 14 days of observation. No control animals were used in this study. No mortality was observed. The study yielded an oral LD50 of > 10000 mg/kg bw for male rats for Fe3O4 alone. Based on the results of this study, classification and labelling in terms of acute oral toxicity is not required.

Reference 3

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

abstract

Qualifier:

no guideline followed

Principles of method if other than guideline:

10 male Wistar rats were dosed with 10000 mg/kg bw of Eisenoxidrot (Fe2O3) in water by a single oral application by gavage. During post observation time of 14 days, the animals were exanimated for clinical signs and mortality.

GLP compliance:

not specified

Remarks:

Study was conducted prior to implementation of GLP in the EU.

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen
- Age at study initiation: not specified
- Weight at study initiation: 160 - 180 g males
- Housing:5 animals per cage

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

not specified

Doses:

10000 mg/kg bw

No. of animals per sex per dose:

10

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: not specified

Statistics:

No statistical analysis could be performed ( the method used is not intended to allow a calculation of a precise LD50 value).

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

> 10 000 mg/kg bw

Based on:

test mat.

Mortality:

no mortality was observed

Clinical signs:

other: no clinical signs were observed

Body weight:

other body weight observations

Remarks:

not specified

Gross pathology:

not specified

 

 

Interpretation of results:

GHS criteria not met

Conclusions:

The LD50 cannot be determined, based to the absence of mortality. Based on the maximal limit dose tested of it could be considered that the LD50 of Fe2O3 is >10000 mg/kg bw/d. According to the Regulation (EC) No 1272/2008 and subsequent adaptions, the substance is not acutely toxic via the oral route.

The study was executed before the establishment of OECD guidelines and the short study report was provided in an abstract like form. Consequently, the study was ranked as “not assignable” (RL4).

Executive summary:

In an acute oral toxicity study, male Wistar rats were given a single oral dose of alpha-Fe2O3 in water at a dose of  10000  mg/kg bw (limit test); the treatment was followed by 14 days of observation. No control animals were used in this study. No mortality and clinical abnormalities were observed. The study yielded an oral LD50 of > 10000 mg/kg bw for male rats. Based on the results of this study, classification and labelling for acute oral toxicity is not required.

Reference 4

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Test substance contains 90% iron oxide and 10% other components.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Wistar TNO W74 rats (5 per sex) were dosed with 5000 mg/kg bw of Bayferrox VP AC5100M (Fe3O4) in chremophor / water by a single oral application by gavage. During post observation time of 14 days, the animals were exanimated for clinical signs, mortality and body weight.

GLP compliance:

not specified

Remarks:

Study was conducted prior to implementation of GLP in the EU.

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Remarks:

strain TNO W 74

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen
- Age at study initiation: 9 weeks old male / 14 weeks old female
- Mean Body weight at study initiation: 177 g males / 161 g females
- Housing: Makrolon cage Type III; 5 animals per cage
- Diet (ad libitum): Altromin R1324 (Altromin GmbH, Lage, Germany)
- Water (ad libitum): tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1.5
- Humidity (%): 60 +/- 5
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: water and chremophor

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED:
20 mL/kg bw

Doses:

5000 mg/kg bw

No. of animals per sex per dose:

5 males / 5 females

Control animals:

no

Details on study design:

Duration of observation period following administration: 14 days
- Frequency of observations and weighing: following dosage, the animals were investigated for any clinical signs (not specified) and mortality multiple times (not specified) at the day of dosing and thereafter, at work days: twice and on weekends and holydays once. Body weight was recorded on day 0 (prior treatment) and day 14 (prior to necropsy)
- Necropsy of survivors performed: not specified
- Clinical signs including body weight: yes

Statistics:

No statistical analysis could be performed ( the method used is not intended to allow a calculation of a precise LD50 value).

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: equivalent to >4500 Fe3O4 mg/kg bw

Mortality:

No death occured.

Clinical signs:

other: All animals were symptom-free during the 14-day observation at a dose level of 5000 mg/kg bw.

Body weight:

lower than 10% body weight loss

Remarks:

There were no treatment related effects on body weight and body weight gain observed.

Gross pathology:

not specified

Interpretation of results:

GHS criteria not met

Conclusions:

In this acute oral toxicity study the LD50 For Bayferrox V.P. AC5100M was > 5000 mg/kg bw (equivalent to >4500 Fe3O4 mg/kg bw). According to the Regulation (EC) No 1272/2008 and subsequent adaptations, the substance is not acutely toxic via the oral route.

Executive summary:

In an acute oral toxicity study, male and female Wistar rats were given a single oral dose of a mixture containing 90% Fe3O4 in water and chremophor, at a dose of  5000  mg/kg bw (limit test); the treatment was followed by 14 days of observation. No control animals were used in this study. No mortality and clinical abnormalities were observed. The study yielded an oral LD50 of > 4500 mg/kg bw (0.9 Fe3O4*5000) for male and female rats, regarding Fe3O4. Based on the results of this study, GHS criteria are not met for magnetite and classification and labelling in terms of acute oral toxicity is not required.

Reference 5

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1985-11-27 to 1985-12-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Test substance contains 73.2% iron oxide and 26.8% other components.

Justification for type of information:

.

Qualifier:

according to guideline

Guideline:

EU Method B.1 (Acute Toxicity (Oral))

Version / remarks:

cited as Directive 84/449/EEC B.1

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

1981-05-12

GLP compliance:

not specified

Remarks:

not specified in publication

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen
- Age at study initiation: 9 weeks old male / 14 weeks old female
- Mean body weight at study initiation: 178 g males / 174 g females
- Fasting period before study: 16h
- Housing: Makrolon cage Type III; dust free wood granulate; 5 animals per cage
- Diet (ad libitum): Altromin R1324, source Altromin GmbH, Lage, Germany
- Water (ad libitum): tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 50 +/- 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED:
20 mL/kg bw

Doses:

5000 mg/kg bw

No. of animals per sex per dose:

5 males / 5 females

Control animals:

no

Details on study design:

Duration of observation period following administration: 14 days
- Frequency of observations and weighing: following dosage, the animals were investigated for any clinical signs (not specified) and mortality multiple times (not specified) at the day of dosing and thereafter, at work days: twice and on weekends and holydays once. Body weight was recorded on day 0 (prior treatment), day 7 and day 14 (prior to necropsy)
- Necropsy of survivors performed: yes
- Clinical signs including body weight: yes

Statistics:

No statistical analysis could be performed ( the method used is not intended to allow a calculation of a precise LD50 value).

Preliminary study:

not specified

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: equivalent to >3660 Fe2O3 mg/kg bw

Mortality:

No death occured.

Clinical signs:

other: No symptoms of intoxication were noted during the 14-day observation at a dose level of 5000 mg/kg bw.

Body weight:

lower than 10% body weight loss

Remarks:

There were no treatment related effects on body weight and body weight gain observed.

Gross pathology:

There were no macroscopic changes seen at necropsy.

Interpretation of results:

GHS criteria not met

Conclusions:

In this acute oral toxicity study the LD50 For Bayferrox V.P. AC5046 was > 5000 mg/kg bw (equivalent to >3660 Fe2O3 mg/kg bw). According to the Regulation (EC) No 1272/2008 and subsequent adaptations, the substance is not acutely toxic via the oral route.

Executive summary:

In an acute oral toxicity study , male and female Wistar rats were given a single oral dose of a mixture containing 73.2% Fe2O3, 19.5% Al2O3, 3% Mn2O3 in water at a dose of  5000  mg/kg bw (limit test) and observed for14 days. No control animals were used in this study. No mortality, abnormalities or gross pathology signs were observed. The study yielded an oral LD50 of > 5000 mg/kg bw for both male and female rats. Ferric oxide is to be regarded as practically non toxic based on the results of this study, and classification and labelling in terms of acute oral toxicity is not required. Taking into account the presence of the other substances and assuming that these do not diminish the acute toxicity of ferric oxide it may be concluded that the LD50 of ferric oxide is >3660 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

There is no key study available, therefore the acute oral toxicity of the substance was assessed in a weight of evidence approach. One OECD 401 (1981) study with minor restrictions (RL=2) utilizing a test substance containing 73% Fe2O3 was supported by 4 additional studies with insufficient documentation utilizing Fe2O3, FeO(OH), or Fe3O4 up to 10 mg/kg bw. Despite the insufficient documentation, all supportive studies were executed by an established toxicological test centre and therefore can be considered to be relevant. Fe2O3 and Fe3O4 are the main constituents of the substance to be assessed. The overall quality of the database is therefore sufficient.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

2009 - 09 - 08

Deviations:

yes

Remarks:

Bodyweight and individual animal data were not reported.

Principles of method if other than guideline:

640mg/m³ was maximum attainable concentration of Fe3O4NPs.

GLP compliance:

not specified

Remarks:

GLP status not specified in publication

Test type:

traditional method

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Amorphous Materials Inc. (Houston, Texas, USA).
- Purity: 99,5%

INFORMATION ON NANOMATERIALS
- Chemical Composition: Fe3O4
- Particle size distribution: average size 15 - 20 nm (nominal); SEM 48.18 +/- 7.54; DLS 651.2 nm
- Shape of particles: XRD tetrahedral
- Surface area of particles: BET >40 m²/g (nominal)
- Surface properties: zeta potential -15.9

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Rationale for alternative/additional species to rat (if applicable)
- Source: International Institute of Biotechnology and Toxicology (IIBAT)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks
- Weight at study initiation: 185-200g
- Housing: three animals with same sex were housed in stainless steel cages
- Diet (ad libitum): commercial pellet diet (not further specified)
- Water (ad libitum): reverse osmosis water
- Acclimation period: one week period before the initiation of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1°C
- Humidity (%): 54 +/- 4%
- Photoperiod (hrs dark / hrs light): 12 h

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

2.25 µm

Geometric standard deviation (GSD):

2.56

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: inhalation exposure unit (Ch. Technologies,USA). The unit has three major modules: an aerosol generator—Wright Dust Feeder (BGI, Inc.); a rodent head and nose-only inhalation exposure chamber, and aerosol concentration measurement device—gravimetric single filter (BGI, Inc.).
- Method of particle size determination: the average size and crystalline structure of the particles in the dry state was reconfirmed by scanning electron microscopy (SEM; Figure 1(a)) and x-ray diffraction (XRD) analyses, respectively, whereas hydrodynamic particle size and zeta potential measurement in deionized water (wet state) was performed by dynamic light scattering (DLS) using Malvern Zetasizer 3000HS (Malvern, Bangalore, India).
- Temperature, humidity in air chamber: temperature 21 +/- 0.8°C; humidity 52-57%


TEST ATMOSPHERE
- Brief description of analytical method and equipment used: a pilot experiment without rats was preformed continuously generating Fe3O4 NPs for 4 hours and measuring the average actual aerosol concentration with an Mercer–Seven stage cascade impactor (Ch. Technologies, USA).
- Samples taken from breathing zone: yes; samples were assessed periodically for aerosol concentration, aerosol particle size distribution, temperature, relative humidity, and oxygen concentration.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

640 mg/m³ (maximum attainable concentration)

No. of animals per sex per dose:

total 18 male and 18 female animals/dose
6 male and 6 female per time point post exposure

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 24h, 48h and 14 days

- Frequency of observations and weighing: not specified

- Necropsy of survivors performed: yes

- Clinical signs: yes but not specified

- Biochemical analysis: yes, at necropsy
glucose, urea, creatinine, total cholesterol, triglycerides, aspartate transaminase, alanine transaminase, alkaline phosphatase (ALP), total protein (TP), albumin, calcium, and phosphorus

- Haematological analysis: yes, at necropsy
erythrocytes, haemoglobin, haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), MCH concentration (MCHC), platelet count, leukocytes, and leukocyte differential count

- Bronchoalveolar lavage analysis: yes, at necropsy
lactate dehydrogenase (LDH), alkaline phosphatase (ALP), total protein (TP), interleukin-1b (IL-1b), tumor necrosis factor-a (TNF-a), and interleukin-6 (IL-6); cell viability, total and differential cell counts of alveolar macrophages (AMs), neutrophils, and lymphocytes

- Oxidative stress in lungs: yes, at necropsy
activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase activity in the supernatant of lung homegenisates

- Histopathology: : yes, at necropsy
lung along with the lung associated lymph nodes (tracheobronchial and mediastinal lymph nodes), liver, spleen, kidneys, thymus, and brain

Statistics:

Statistical analyses were conducted with NCSS 2007 software. Data were expressed as mean+SD (n = 6).

Student’s t test was carried out to illustrate the significant difference between the treated groups and the respective control groups.

One-way analysis of variance was carried out to compare the differences among the time points within the group.

Spearman rank correlation test was used to analyse correlations of proinflammatory cytokines in BALF and blood.

Key result

Sex:

male/female

Dose descriptor:

LC0

Effect level:

> 640 mg/m³ air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: no mortality was observed, 640 mg/m³ = maximum attainable concentration

Mortality:

no mortality

Clinical signs:

other: no morbidity or clinical signs of toxicity

Body weight:

not specified

Gross pathology:

not specified

Other findings:

Besides the examinations, as described, further investigations were carried out during the study, which do not cover the endpoint. These results were not further discussed.

HISTOPATHOLOGY
male / female:
-Appearance of alveolar macrophages (AMs) overloaded with the phagocytosed discrete single to multiple irregularly gobular intracytoplasmic small agglomerated masses of NPs were observed in the airways and
interstitium early at 24 h of postexposure period along with not phagocytosed free Fe3O4 NPs in alveolar interstium
- NPs emigrated and translocated to the intrapulmonary lymphoid tissues (BALT) and to the extrapulmonary tracheobronchial lymph nodes (lung-associated lymph nodes (LALN))

- Minimal-to moderate, multifocal alveolitis accompanied with perivascular cuffing of mononuclear inflammatory cells were noticed at 48 h and persisted up to 14 days of postexposure period. The lesion was characterized by the presence of both phagocytosed and nonphagocytosed Fe3O4 NPs, moderate alveolar wall hyperplasia with variable interstitial inflammatory reaction and infiltrated masses of mononuclear inflammatory cells in the alveolar lumen

- In the lung multifocal foreign body type low-grade granulomatous inflammation surrounding the agglomerated masses of Fe3O4 NPs were observed in four of six males and five of six females after the 14 days of postexposure, except in the alveolitis.

- No signs of early fibrosis observed in any of the observation period.

- Other investigated organs (liver, spleen, kidneys, thymus, and brain) did not show any Fe3O4 NPs-induced toxic responses or the existence of these NPs.


BIOCHEMICAL ANALYSIS OF BLOOD
male / female:
- no treatment related effects besides different concentration of triglycerides (p<0.5)


HEMATOLOGICAL ANALYSIS OF BLOOD
male / female:
- no treatment related effects


BIOCHEMICAL ASSESSMENTS IN BALF
male / female:
- LDH release was increased significantly throughout the observation period (p < 0.001). LDH activity in BALF of Fe3O4 NPs shows that the animals of either sex showed a time-dependent decreasing manner and found to be around threefold, 2.0-fold, and 1.6-fold increases when compared with control animals at the end of 24 h, 48 h, and 14 days of postexposure periods, respectively.

- TP concentration and ALP activity in BALF reached a maximum at 24 h of postexposure (p < 0.001) and decreased in a time-dependent manner. TP concentration in BALF of Fe3O4 NPs exposed animals of either sex showed around 2.7-fold, 2.0- fold, and 1.4-fold increases when compared with control animals at the end of 24 h, 48 h, and 14 days of


CYTOLOGICAL ASSESSMENTS IN BALF
male / female:
- A significant decrease (p < 0.001) in cell viability appeared in all the three time points with maximum decrease within 24 h of postexposure, where cell viability dropped to about 60% of control animals in either sex. However, a time-dependent recovery was observed with a significant increase (p < 0.001) in cell viability within 48 h of postexposure in males and 14 days of postexposure in female rats.

- BALF cells were significantly increased by about 2.5-fold at 24 h of postexposure period in either sex compared with the respective control animals (p < 0.001). However, these cells showed significant decrease (p < 0.05) in a time-dependent manner, as it reached about 1.3-fold increase at the end of 14 days of postexposure period in either sex.

-Neutrophils showed a time-dependent decrease in BALF upon exposure. The percentage of neutrophils in treated animals showed a maximum of fivefold increase within 24 h of postexposure and decreased in a time-dependent manner, with females showing promising recovery (p < 0.001) than males (p < 0.05) at the end of 14 days of postexposure period when compared with 24 h of postexposure period.

- The percentage of AMs showed a time-dependent recovery in treated animals of either sex.

- The distribution rate of lymphocytes in BALF showed a weak response when compared with neutrophils with a maximum increase at 24 h of postexposure of Fe3O4 NPs

Interpretation of results:

GHS criteria not met

Conclusions:

LC0 (rats; 4 hours) > 640 mg Fe3O4 / m³ air (maximum attainable concentration).

According to information above clinical signs or mortality were not stated in the publication up to the highest attainable concentration of 640 mg/m³ (exposure for 4 hrs) Fe3O4 nano particles the criteria for classification as acutely toxic via inhalation are not met.

Consequently, the substance will not be classified in accordance with EU Regulation 1272/2008, and subsequent adaptations for acute inhalation toxicity.

Executive summary:

In this study, the toxicity responses of rat following a continuous 4 h inhalation exposure of only the head and nose to iron oxide nanoparticles (Fe3O4 NPs, size = 15–20 nm) was investigated. The rats for the investigation were exposed to a concentration of 640mg/m³ (maximum attainable concentration) Fe3O4NPs.

The results showed a significant decrease in the cell viability, with the increase in the levels of lactate dehydrogenase, total protein, and alkaline phosphatase in the BALF. Total leukocyte count and the percentage of neutrophils in BALF increased within 24 h of postexposure. Immediately following acute exposure, rats showed increased inflammation with significantly higher levels of lavage and blood proinflammatory cytokines and were consistent throughout the observation period. Fe3O4 NPs exposure markedly increased malondialdehyde concentration, while intracellular reduced glutathione and antioxidant enzyme activities were significantly decreased in lung tissue within 24-h postexposure period. On histological observation, the lung showed an early activation of pulmonary clearance and a size-dependant biphasic nature of the Fe3O4NPs in causing the structural alteration.Collectively, the data illustrate that Fe3O4NPs inhalation exposure may induce cytotoxicity via oxidative stress and lead to biphasic inflammatory responses inWistar rat.

Reference 2

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The study was not aimed at the observation of the endpoints that are typically studied in a standard acute inhalation toxicity study; nevertheless, it provides sufficient evidence on the acute toxicity of the substance as well as the inflammogenic response in the respiratory tract. Some deficiencies were identified, such as: experimental design is insufficiently described, exposure duration was too long, test material was insufficiently characterised (source, purity, impurities) and analytical concentration was missing. Strain and sex of the animals was not specified, number of animals were too low, body weight and body weight gain not reported, gross pathology, clinical sings and mortality was not reported.

Qualifier:

no guideline followed

Principles of method if other than guideline:

5 rats per explorative time point (30 in total) were exposed with very large dust burden of 2100 mg Fe2O3/ m³ air in an inhalation chamber or were instilled intratracheal with 30 mg Fe2O3 in 1 mL water. According to the author 49 control animals were investigated but not further specified if or how they were treated. Pulmonary macrophages counts were made prior to and immediately following the imposition of the particles , as well as 1, 4, 7, 14 and 30 days postexposure. Due to the unphysiological application route of intratracheal instillation, the results of this dosage form are only informative and were not considered in the hazard assessment.

GLP compliance:

not specified

Remarks:

The study was performed before the GLP adoption.

Test type:

other: See above: 'Principles of method if other than guideline'

Limit test:

yes

Species:

rat

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

not specified

Route of administration:

inhalation: dust

Type of inhalation exposure:

whole body

Vehicle:

air

Remark on MMAD/GSD:

not specifed

Details on inhalation exposure:

inhalation chamber

Analytical verification of test atmosphere concentrations:

not specified

Duration of exposure:

12 h

Concentrations:

2100 mg/m³

No. of animals per sex per dose:

5 animals per investigated time point (6) in total 30; control animals: 49

Control animals:

yes

Details on study design:

- Duration of observation period following administration: immediately, as well as 1, 4, 7, 14 and 30 days postexposure. From total 30 animals exposed, 5 were killed at each time point reducing the number of animals observed with advancing observation time points.
- Other examinations performed: pulmonary macrophages counts in bronchial lavage fluid were determined in two animals per group prior exposure and at the observation periods noted above.

Statistics:

paired students t-test

Preliminary study:

not specified

Sex:

not specified

Dose descriptor:

LC0

Effect level:

> 2 100 mg/m³ air (nominal)

Based on:

test mat.

Exp. duration:

12 h

Remarks on result:

other: No direct information on mortality rates was provided in the publication, however, based on the experimental description, the evaluator can indirectly conclude that no mortality was observed 14 days postexposure

Key result

Sex:

not specified

Dose descriptor:

LC0

Effect level:

> 6 300 mg/m³ air (nominal)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: Calculated based on 12 hours exposure period using Haber’s law by toxicologist.

Mortality:

Although the presentation of the results does not explicitly states anything about mortality, the presentation of the results indicates that no treatment-related mortality occurred.

Clinical signs:

other: not specified

Body weight:

not specified

Gross pathology:

not specified

Other findings:

PULMONARY MACROPHAGES COUNTS IN BRONCHIAL LAVAGE FLUID
The findings revealed a wide range in the number and percentages (out of the total cells) of macrophages recovered from both control and experimental animals. The results are depicted in Fig.1, 2, 3 & 4 (see attached background material).

In rats intratracheal instillation or inhalation of Fe2O3 did not result in significant increases in the mean macrophage counts. The results are depicted in Fig.1, 2, 3 & 4 (see attached background material).

As explained by the authors, any interpretation of the significance of the changes in mean macrophage counts must be based on understanding of the factors that may influence the number of macrophages counted: 1.the degree of mobilization, 2.the patency of the air spaces in which the macrophages are situated, 3.the character of the relationship of macrophages to adjoining cells or tissues, 4.the ability of the saline wash fluid to contact the macrophages and 5. the ability of the saline wash fluid to drain from the lung. In view of all uncertainties in the determination of the macrophage counts with this method, interpretations of these results were subject to pitfalls.

 

Although the interpretation of the results regarding the number of macrophages lavaged from the BAL fluid is subject to pitfalls, this study gives important regarding the mortality rates after exposure to Fe2O3.

Interpretation of results:

GHS criteria not met

Conclusions:

No direct information on mortality rates was provided in the publication; however, based on the experimental descriptions, the evaluator can indirectly conclude that no mortality was observed 14 days postexposure.

LC0 (rats; 12 hours) > 2100 mg/m³ air would be equivalent to
LC0 (rats; 4 hours) > 6300 mg/m³ air (extrapolated using Haber's law as detailed in regulation (EU) 1272/2008, Annex I, Table 3.1.1, Note (c) in conjunction with ECHA Guidance on the Application of the CLP Criteria
Version 5.0, July 2017)

According to information above clinical signs or mortality were not stated in the publication up to the highest concentration of 2100 mg/m³ (exposure for 12 hrs), extrapolated to the concentration of 6300 mg/m³ using Haber's law, the criteria for classification as acutely toxic via inhalation are not met.

Consequently, the substance will not be classified in accordance with EU Regulation 1272/2008, and subsequent adaptations for acute inhalation toxicity.

Executive summary:

Dust burdens of Fe2O3 were imposed on rats and hamsters by inhalation and by intratracheal instillation. Pulmonary macrophages counts were made prior to and immediately following the imposition of the particles , as well as 1, 4, 7, 14 and 30 days postexposure. No direct information on mortality rates was provided on the publication; however, the submitter indirectly concludes that no mortality was observed 14 days postexposure. For this reason, this study indicates that the LC50 is > 2100 mg/m3 and > 2800 mg/m3, for rats and hamsters, respectively. After a statistical evaluation of the results, it was concluded that the number of lung macrophages recoverd with the saline wash out method is dependent upon factors that cannot be regulated, nor can their effects be estimated. Hence, these results could not be interpreted.

Reference 3

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The study was not aimed at the observation of the endpoints that are typically studied in a standard acute inhalation toxicity study; nevertheless, it provides sufficient evidence on the acute toxicity of the substance as well as the inflammogenic response in the respiratory tract. Some deficiencies were identified, such as: experimental design is insufficiently described, exposure duration was too long, test material was insufficiently characterised (source, purity, impurities) and analytical concentration was missing. Strain and sex of the animals was not specified, number of animals were too low, body weight and body weight gain not reported, gross pathology, clinical sings and mortality was not reported.

Qualifier:

no guideline followed

Principles of method if other than guideline:

5 hamster per explorative time point (30 in total) were exposed with very large dust burden of 2800 mg Fe2O3/ m³ air in an inhalation chamber or were instilled intratracheal with 15 mg Fe2O3 in 0.5 mL water. According to the author 50 control animals were investigated but not further specified if or how they were treated. Pulmonary macrophages counts were made prior to and immediately following the imposition of the particles , as well as 1, 4, 7, 14 and 30 days postexposure. Due to the unphysiological application route of intratracheal instillation, the results of this dosage form are only informative and were not considered in the hazard assessment.

GLP compliance:

not specified

Remarks:

The study was performed before the GLP adoption.

Test type:

other: See above: 'Principles of method if other than guideline'

Limit test:

yes

Species:

hamster

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

not specified

Route of administration:

inhalation: dust

Type of inhalation exposure:

whole body

Vehicle:

air

Remark on MMAD/GSD:

not specifed

Details on inhalation exposure:

inhalation chamber

Analytical verification of test atmosphere concentrations:

not specified

Duration of exposure:

12 h

Concentrations:

inhalation: 2800 mg/m³
instillation: 15 mg in 0.5 mL water per hamster

No. of animals per sex per dose:

5 animals per investigated time point (6) in total 30; control animals:50

Control animals:

yes

Details on study design:

- Duration of observation period following administration: immediately, as well as 1, 4, 7, 14 and 30 days postexposure. From total 30 animals exposed, 5 were killed at each time point reducing the number of animals observed with advancing observation time points.
- Other examinations performed: pulmonary macrophages counts in bronchial lavage fluid were determined in two animals per group prior exposure and at the observation periods noted above.

Statistics:

paired students t-test with N1 = 2 (exposed) and N2 = 50 (control). Only P > 0.01 was considered as significant

Preliminary study:

not specified

Sex:

not specified

Dose descriptor:

LC0

Effect level:

> 2 800 mg/m³ air

Based on:

test mat.

Exp. duration:

12 h

Remarks on result:

other: No direct information on mortality rates was provided in the publication, however, based on the experimental description, the evaluator can indirectly conclude that no mortality was observed 14 days postexposure

Key result

Sex:

not specified

Dose descriptor:

LC0

Effect level:

> 8 400 mg/m³ air (nominal)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: Calculated based on 12 hours exposure period using Haber’s law by toxicologist.

Mortality:

Although the presentation of the results does not explicitly states anything about mortality, the presentation of the results indicates that no treatment-related mortality occurred.

Clinical signs:

other: not specified

Body weight:

not specified

Gross pathology:

not specified

Other findings:

PULMONARY MACROPHAGES COUNTS IN BRONCHIAL LAVAGE FLUID
The findings revealed a wide range in the number and percentages (out of the total cells) of macrophages recovered from both control and experimental animals. The results are depicted in Fig.1, 2, 3 & 4 (see attached background material).

Intratracheal instillation of Fe2O3 in hamster did result in significant increases in the mean macrophage counts. However, the inhalation of Fe2O3 did not result in any significant increases in mean macrophage counts. The results are depicted in Fig.1, 2, 3 & 4 (see attached background material).

As explained by the authors, any interpretation of the significance of the changes in mean macrophage counts must be based on understanding of the factors that may influence the number of macrophages counted: 1.the degree of mobilization, 2.the patency of the air spaces in which the macrophages are situated, 3.the character of the relationship of macrophages to adjoining cells or tissues, 4.the ability of the saline wash fluid to contact the macrophages and 5. the ability of the saline wash fluid to drain from the lung. In view of all uncertainties in the determination of the macrophage counts with this method, interpretations of these results were subject to pitfalls.

 

Although the interpretation of the results regarding the number of macrophages lavaged from the BAL fluid is subject to pitfalls, this study gives important regarding the mortality rates after exposure to Fe2O3.

Interpretation of results:

GHS criteria not met

Conclusions:

No direct information on mortality rates was provided in the publication; however, based on the experimental descriptions, the evaluator can indirectly conclude that no mortality was observed 14 days postexposure.

LC0 (hamster; 12 hours) > 2800 mg/m³ air would be equivalent to
LC0 (hamster; 4 hours) > approx. 8400 mg/m³ air (extrapolated using Haber's law as detailed in regulation (EU) 1272/2008, Annex I, Table 3.1.1, Note (c) in conjunction with ECHA Guidance on the Application of the CLP Criteria
Version 5.0, July 2017)

According to information above clinical signs or mortality were not stated in the publication up to the highest concentration of 2800 mg/m³ (exposure for 12 hrs), extrapolated to the concentration of 8400 mg/m³ using Haber's law, the criteria for classification as acutely toxic via inhalation are not met.

Consequently, the substance will not be classified in accordance with EU Regulation 1272/2008, and subsequent adaptations for acute inhalation toxicity.

Executive summary:

Dust burdens of Fe2O3 were imposed on rats and hamsters by inhalation and by intratracheal instillation. Pulmonary macrophages counts were made prior to and immediately following the imposition of the particles , as well as 1, 4, 7, 14 and 30 days postexposure. No direct information on mortality rates was provided on the publication; however, the submitter indirectly concludes that no mortality was observed 14 days postexposure. For this reason, this study indicates that the LC50 is > 2100 mg/m3 and > 2800 mg/m3, for rats and hamsters, respectively. After a statistical evaluation of the results, it was concluded that the number of lung macrophages recoverd with the saline wash out method is dependent upon factors that cannot be regulated, nor can their effects be estimated. Hence, these results could not be interpreted.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

There is no key study available, therefore the acute oral toxicity of the substance was assessed in a weight of evidence approach. One study with minor restrictions (RL=2) equvilant to OECD403 utilizing a test substance Fe3O4 (0.64 mg/L maximum attainable concentration) was supported by one additional studies with insufficient documentation utilizing Fe2O3 exposing rats to 2100 mg/m³ and hamster to 2800 mg/m³ for 12 hours. Fe2O3 and Fe3O4 are the main constituents of the substance to be assessed. The overall quality of the database is therefore sufficient.

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Introductory remark on read-across:

In this dossier, the endpoint acute oral toxicity is not addressed by substance-specific information, but instead by a weight of evidence approach based on collected information for all substances of the iron oxide category. The three target substances covered by this read across (Iron sinter; Iron ores, agglomerates; Mill scale) consist primarily of different iron oxides, as described in the technical dossier section 1.2 Composition. Consequently, a read-across from the pure iron oxides to the target substances is justified.

The predominant characteristic of the iron oxide category substances is the inertness being a cause of their chemical stability and very poor reactivity. This is shown by a very low dissolution in water and artificial physiological fluids as well as a very low in vivo bioavailability after oral administration. This very low reactivity, solubility and bioavailability leads to a complete lack of systemic toxicity after acute oral or inhalation exposure up to the limit dose of the maximum tolerated concentration of the respective test. Further information on the read-across approach is given in the report attached to IUCLID section 13.2.

 

Human data

 

Acute oral toxicity

 

One reference describing human data on acute oral toxicity could be identified. The reference does not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The information contained therein were included for information purposes only.

 

Loennemark, M. et al. (1989): based on the fact, that coated iron oxide was used which is outside the scope of this dossier, this reference is considered not relevant and is not used for human health risk assessment but was included for information purposes.

 

 

 

Acute inhalation toxicity

 

One reference describing human data on acute inhalation toxicity could be identified. After a thorough reliability screening, this reference was considered to provide supportive information regarding respiratory irritation.

 

In the study by Lay, J.C. et al. (2001) sixteen healthy (non-smoking) subjects inhaled aerosols of iron oxide particles (MMAD = 1.5 μm) having either high or low water-soluble iron content for 30 min at an average mass concentration of 12.7 mg/m³. The pulmonary clearance (determined with 99mTc-DTPA) was measured and the DLCO and spirometry were performed before and after breathing the iron oxide. No significant difference in DTPA clearance half-times after breathing particles versus particle-free air either at ½ h or 24 h post-inhalation was found for those subjects breathing aerosols with high or low soluble iron content. No significant differences in DLCO between particle exposures and air exposures were found. Minor differences in spirometric measurements were noted but were not statistically significant. The results showed that inhalation of iron oxide particles did not cause an appreciable alteration of alveolar epithelial permeability, lung diffusing capacity, or pulmonary function in healthy subjects under the studied conditions.

Despite the weakness of this study (self-synthesized test material, no information on purity and composition), the reference provides useful information to evaluate respiratory irritation.

 

Two references were also identified, representing investigations on acute inhalation toxicity in humans. The study designs are not in accordance with accepted guidelines and are therefore of limited relevance for chemical hazard assessment. The references lack significance due to, e.g., missing exposure concentrations or insufficient information about composition of fumes. It is therefore concluded that the references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X).  The references given below were included in the IUCLID for information purposes only:

Ilowite, J.S. et al. (1989): based on the short exposure time (7-8 min), missing exposure concentration and a MMAD of >4 µm of iron oxide and the different breathing pattern of the aerosols, this reference cannot be used for human health risk assessment of iron oxide but was included for information purposes.

Kaye, P.; et al. (2002):  based on the unknown composition and amount of fume exposure, this reference cannot be used for human health risk assessment of iron oxide but was included for information purposes.

 

 

 

 

Animal data

Acute oral toxicity

 

In the study by Ramm, W. et al. (1986) according to OECD guideline 401, groups of five male and five female, albino rats of a Wistar strain were given a single oral dose of 5000 mg/kg bw Bayferrox V.P. AC5046 (nanoform; alpha-Fe2O3 73.2%, Al2O3 19.5%, Mn2O3 3.0%) in water via gavage. The treatment was followed by 14 days of observation. In this acute oral toxicity study the LD50 for Bayferrox V.P. AC5046 was > 5000 mg/kg bw (equivalent to >3660 Fe2O3 mg/kg bw) since no mortality was observed. Furthermore, no clinical signs were observed as well as no changes in body weight, No findings were made during gross pathology.  The study was a guideline study (OECD 401) without detailed documentation on gross pathology.

 

In the study by Loeser, E. et al. (1981), groups of five male and five female, albino rats of a Wistar strain were given a single oral dose of 5000 mg/kg bw Bayferrox VP AC 5100M (nanoform; Fe3O4 90%, Co 2%, ZnO + P2O5 2.5 %) in water and chremophor via gavage. The treatment was followed by 14 days of observation. No control animals were used in this study. No mortality and clinical abnormalities were observed.  The study yielded an oral LD50 > 5000 mg/kg bw for Bayferrox VP AC 5100M, from which can be concluded an oral LD50 of > 4500 mg/kg bw for pure Fe3O4 nanoform.

 

In the studies by Loeser, E. et al. (1977a, b, c), two groups of five male albino rats of a Wistar strain were given a single oral dose of 10000 mg/kg bw Eisenoxidrot 130 (Fe2O3; Loeser, E. et al. 1977a), Eisenoxidschwarz 318 (Fe3O4; Loeser, E. et al. 1977b) and Eisenoxidgelb 920 (nanoform; FeO(OH); Loeser, E. et al. 1977c) in water via gavage. The treatment was followed by 14 days of observation. No control animals were used in this study. No mortality and clinical abnormalities were observed. The studies yielded an oral LD0 > 10,000 mg/kg bw for male rats with Fe2O3, Fe3O4 and FeO(OH). The study was executed before the establishment of OECD guidelines and the short study report was provided in an abstract like form. Consequently, several information was lacking which prevented an evaluation of the reliability of the provided data.

 

Conclusion

The available acute oral toxicity studies equivalent or similar to OECD 401 showed a LD50 > 5000 mg/kg bw in rats after single oral administration of various iron oxides covered by the category approach. It is therefore concluded in a weight of evidence approach, taking into account all above information, that the LD50 for iron oxides covered by the category approach (IUCLID section 13) is > 5,000 mg/kg bw.

 

Acute inhalation toxicity

 

In a study according to OECD TG 403 by Srinivas, A. et al. (2012), the toxicity responses of six male and six female rats per investigation time point following a continuous 4 h inhalation exposure of the head and nose only to iron oxide nanoparticles (Fe3O4 NPs, size = 15–20 nm) was investigated 24 h, 48h and 14 days post exposure. Rats were exposed to a concentration of 640 mg/m³ Fe3O4NPs (maximum attainable concentration; MMAD 2.25 µm). No mortality or clinical signs of toxicity was observed, therefore a LC0 (rats; 4 hours) > 640 mg/m³ air is established. Further, the results showed a significant decrease in the cell viability, with the increase in the levels of lactate dehydrogenase, total protein, and alkaline phosphatase in the BALF. Total leukocyte count and the percentage of neutrophils in BALF increased within 24 h of postexposure. Immediately following acute exposure, rats showed increased inflammation with significantly higher levels of lavage and blood proinflammatory cytokines and were consistent throughout the observation period. Fe3O4 NPs exposure markedly increased malondialdehyde concentration, while intracellular reduced glutathione and antioxidant enzyme activities were significantly decreased in lung tissue within 24-h postexposure period. On histological observation, the lung showed an early activation of pulmonary clearance and a size-dependant biphasic nature of the Fe3O4NPs in causing the structural alteration. Collectively, the data illustrate that Fe3O4NPs inhalation exposure may induce cytotoxicity via oxidative stress and lead to biphasic inflammatory responses in Wistar rat. Some reporting deficiencies of the study were identified, such as: bodyweight and bodyweight development and individual animal data were not reported. The study is considered as reliable with restriction (RL=2) and used in a weight of evidence assessment for acute inhalation toxicity.

 

Gross, P. et al. (1969) treated rats and hamster with Fe2O3 in an inhalation chamber at 2.1 mg/L (rat, MMAD not specified) and 2.8 mg/L (hamster, MMAD not specified) for 12 hours. Each group consisted of 5 animals per investigation time point. One group was examined directly after exposure and other groups were examined 1, 4, 7, 14 and 30 days post exposure. Pulmonary macrophages counts in bronchoalveolar lavage (BAL) fluid were made. No direct information on mortality rates was provided in the publication; however, based on the experimental descriptions, it is concluded that no mortality was observed 14 days postexposure. LC0 (rats; 12 hours) > 2100 mg/m³ air would be equivalent to LC0 (rats; 4 hours) > approx. 6300 mg/m³ air (extrapolated using Haber's law as detailed in regulation (EU) 1272/2008, Annex I, Table 3.1.1, Note (c) in conjunction with ECHA Guidance on the Application of the CLP Criteria Version 5.0, July 2017). The study was not aimed at the observation of the endpoints that are typically studied in a standard acute inhalation toxicity study; nevertheless, it provides sufficient evidence on the acute inhalation toxicity of the substance as well as the inflammogenic response in the respiratory tract. Some deficiencies were identified, such as: experimental design is insufficiently described, test material was insufficiently characterised (source, purity, impurities), strain and sex of the animals was not specified, body weight and body weight gain not reported, gross pathology not reported. The study is considered as not reliable (RL=3) and used in a weight of evidence assessment for acute inhalation toxicity

 

Furthermore, two references on acute inhalation toxicity with iron oxide (Fe2O3) are available. After a thorough reliability screening, these references were considered of limited relevance for hazard assessment purpose and were used as supporting information only.

In the study by Grant, M. M. et al. (1979), male white New Zealand rabbits were exposed to an iron(III) oxide (Fe2O3) aerosol with an MMAD of 0.17 to 0.31 μm (GSD 1.61) head-only at a concentration of 200 mg/m3 for 3 hours. The aim of the study was to investigate the in vivo pulmonary macrophage response after exposure to this aerosol. A bronchoalveolar lavage was performed for different groups of animals at time points 0, 12, 18 and 24 h after termination of the exposure. The lysosomal acid hydrolase activities in the pulmonary macrophages were examined with biochemical and histochemical techniques. The results suggest that exposure to Fe2O3 may have little effects on the average activities of lysosomal hydrolases of the pulmonary macrophages, although it indeed stimulates their recruitment in the lung leading to an increase of the total enzyme in the BAL fluid. An increased lysosomal heterogeneity and redistribution of enzymic activity within the cell cytoplasm of the macrophages was observed. Although the presentation of the results does not explicitly state anything about mortality, the presentation of the results indicates that no treatment-related mortality occurred. Therefore, this study indicates that 3 h exposure to 200 mg/m³ respirable ferric oxide (Fe2O3) particles does not result in mortality within 24 hours post-exposure. Exposure to ferric oxide particles has led to little effect on the average activities of lysosomal hydrolases in pulmonary macrophages, to an increased lysosomal heterogeneity and redistribution of enzymic activity within the cell cytoplasm. The deficits of the study were that the test material was insufficiently characterised (source, purity, impurities), the observation periods were too short (<=24h), an uncommon species (rabbit) was used and only lungs were investigated. The study is considered as not reliable (RL=3) and used as supporting evidence only.

 

 

In the study of Kavet, R. I. et al. (1979), Syrian golden hamsters were exposed to ferric oxide (Fe2O3) aerosols at a mean concentration of 274±18 (SD) mg/m³ for 3 h. The animals were separated into four groups. Four animals/group of the first two groups received hydrocortisone acetate intramuscularly 18 to 20 h before exposure, in order to inhibit potential recruitment of mononuclear phagocytes into the alveoli. The other four animals not receiving the drug were given injections of saline. Animals were sacrificed at 3 and 24 h. The animals from the last two groups did not receive any injections and they were killed at 3 and 11 d (4 animals/group) postexposure. BAL cells were counted, sized and tested for viability (trypan blue exclusion). The results suggest an inflammatory response (recruitment of polymorphonuclear leukocytes) in the lung after exposure to Fe2O3 that diminishes to control levels after 3 days postexposure. Although the presentation of the results does not explicitly state anything about mortality, the presentation of the results indicates that no treatment-related mortality occurred. Therefore, this study indicates that 3 h exposure to 274 mg/m³ respirable ferric oxide (Fe2O3) particles does not result in mortality within 11 days post exposure. No gross pathological signs were observed in the lung. The results suggest an inflammatory response in the lung after exposure to iron (III) oxide (Fe2O3) that diminishes to normal levels at 3 days postexposure. The study was not aimed at the observation of the endpoints that are typically studied in a standard acute inhalation toxicity study; nevertheless, it provides some information on the toxicity of the substance on the respiratory tract. The experimental design is insufficiently documented, a self-synthesised test material was used, the number of tested animals were too low and the observation period was too short (11 days). The study is considered as not reliable (RL=3) and used as supporting evidence only.

 

Furthermore, nine references were identified, representing mechanistic studies investigating the inflammatory response of iron oxide following single administration via oropharyngeal aspiration, nasal and intratracheal instillation, whole-body or nose-only inhalation. All references do not fulfil the criteria for quality, reliability and adequacy of experimental data for data requirements under REACH and for hazard assessment purposes(ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The non-physiological route of administration via intratracheal instillation is not guideline conform and not suitable to assess acute inhalation toxicity. The references given below were therefore included in the IUCLID for information purposes only.

 

–Inhalation:

Zhou, Y. M. et al. (2003): male rats, of the strain Sprague-Dawley, were treated with self-made nanoparticles of iron oxides (Fe2O3, size 20 – 140 nm). Rats were exposed whole body with 50 and 90 µg/m³ for 6 hours for 3 days. Possible adverse respiratory effects in rats after inhalation of iron(III) oxide (Fe2O3) particles (50 and 90 µg/m3) were examined. Several biochemical indicators of oxidative stress and inflammatory response in the lung were measured. The publication does not fulfil the data requirements (according to OECD 403, Acute inhalation toxicity) for a risk assessment of the test substance: No conclusion can be drawn on the pulmonary toxicity of the substance due to an undefined (self-synthesised) and low concentrated exposure sample. In addition, only male rats were used for the toxicity evaluation. The investigations are restricted to pulmonary effects. Information on number of test animals, food consumption, body weight development is missing.

 

– Instillation:

Antonini, J. M. (1996): after intratracheal installation of 10 mg/kg Fe2O3 following mechanistic parameters in bronchoalveolar lavage (BAL) were investigated, cell numbers, albumin levels, β-NAG activity, LDH levels, TNF-a, IL-1β. In addition, histopathology of the lung was performed. The publication does not fulfil the data requirements (according to OECD 403, Acute inhalation toxicity) for a risk assessment of the test substance: Test substance was administrated by instillation and not as recommended by inhalation. In addition, the test substance was self-made. Only male rats were used for the toxicity evaluation.  The use of only one dose group precludes dose-response relationship evaluations. The investigations are restricted to pulmonary effects. Information on food consumption, body weight development is missing.

 

Gosset, P. et al. (1996): after intratracheal installation of 12 mg/kg Fe2O3 following mechanistic parameters in bronchoalveolar lavage (BAL) were investigated MDA (malondialdehyde) and HHT (hydroxyheptadecatraenoic acid), polymorphonuclear (PMN) elastase determination, cathepsins B and L, immunodetection of a1-antitrypsin (a1AT), inhibitory capacity of a1AT. The publication does not fulfil the data requirements (according to OECD 403, Acute inhalation toxicity) for a risk assessment of the test substance: not sufficient experimental details are presented in the abstract like publication.

 

Garcon, G. et al. (2004): after intratracheal installation of 3 mg/kg Fe2O3 following mechanistic parameters in lung were investigated protein concentration of CYP1A1, catalytic activity of CYP1A1 (EROD), as well as mRNA expressions of CYP1A1, meh (microsomal epoxide hydrolase), GST-ya and GST-toc (glutathione-S-transferases). The publication does not fulfil the data requirements (according to OECD 403, Acute inhalation toxicity) for a risk assessment of the test substance: Test substance was administrated by instillation and not as recommended by inhalation. The purity of the test material was not specified. Only male rats were used. A justification for the concentration tested is not provided. The investigations are restricted to pulmonary effects. Information on food consumption, body weight development is missing.

 

Beck, B. D. (1983): after intratracheal installation of 37.5 mg/kg Fe2O3 following investigated mechanistic parameters were investigated lactate dehydrogenase levels and isoenzyme pattern in in the cell-free supernatant fractions of lung lavage fluid and serum, as well as in lung, liver and heart homogenates. The publication does not fulfil the data requirements (according to OECD 403, Acute inhalation toxicity) for a risk assessment of the test substance: Test substance was administrated by instillation and not as recommended by inhalation. Only male hamsters were used and number of animals per group was too low. A justification for the concentration tested is not provided. The investigations are restricted to lactate dehydrogenase levels and isoenzyme pattern. Information on food consumption, body weight development is missing. The observation period was too short (24 h).

 

Boutin, A.C. (1998): after intratracheal installation of 10 mg/kg Fe2O3 following mechanistic parameters in bronchoalveolar lavage (BAL) were investigated malondialdehyde, alpha-glutathione-S-transferase and Clara cell protein (CC16). The publication does not fulfil the data requirements (according to OECD 403, Acute inhalation toxicity) for a risk assessment of the test substance: Test substance was administrated by instillation and not as recommended by inhalation. Only male animals were used and number of animals per group was too low. A justification for the concentration tested is not provided. Information on food consumption, body weight development is missing. The observation period was only 48h.

 

Wright, J. L. (1988): after intratracheal installation of 50 mg/kg bw iron oxide (obtained by Pfizer, R2999-R) following investigated mechanistic parameters were investigated pulmonary function tests were performed: measurement of pleural pressure, transplumonary pressure (DP 42/32; Validyne Corp., Northridge, CA), FRC, construction of pressure-volume curves. Thereafter, the animals were sacrificed and the lungs were used for morphometric measurements. Light microscopic examinations were performed. The publication does not fulfil the data requirements (according to OECD 403, Acute inhalation toxicity) for a risk assessment of the test substance: Test substance was administrated by instillation and not as recommended by inhalation. Only female animals were used and number of animals per group was too low. A justification for the concentration tested is not provided. The investigations are restricted to pulmonary effects. Information on food consumption, body weight development is missing.

 

Beck, B. D. et al. (1982): after intratracheal installation of 10 mg/kg Fe2O3 following mechanistic parameters in bronchoalveolar lavage (BAL) were investigated beta-N-acetylglucosaminidase (beta-NAG), peroxidase, lactate dehydroganase (LDH) following the oxidation of NADH in the presence of pyruvate, elastase, albumin, macrophage numbers and red blood cells. The publication does not fulfil the data requirements (according to OECD 403, Acute inhalation toxicity) for a risk assessment of the test substance: Test substance was administrated by instillation and not as recommended by inhalation. Only male hamsters were used and the number of animals per group was too low. The investigations are restricted to pulmonary effects. Information on food consumption, body weight development is missing.

 

Garcon, G. et al. (2001): after intratracheal installation of 10 mg/kg Fe2O3 following investigated mechanistic parameters in bronchoalveolar lavage (BAL) were investigated cell viability, number of alveolar macrophages, lipid peroxidation, TNF-a (Tumour necrosis factor-a), IL-1 β (interleukin 1-beta), nitric oxide (NO) and total protein. Total RNA was extracted from lung tissue biopsies and mRNA expressions of TNF-a (TNF-a/ β Actin), IL-l β (IL-l β/ β-Actin) and iNOS (iNOS/ β-Actin) was measured. The publication does not fulfil the data requirements (according to OECD 403, Acute inhalation toxicity) for a risk assessment of the test substance: Test substance was administrated by instillation and not as recommended by inhalation. Only male animals were used and the number of animals per group was too low. A justification for the concentration tested is not provided. Observation period was only 12 hours. The investigations are restricted to pulmonary effects. Information on food consumption, body weight development is missing.

 

Conclusion

One available acute inhalation toxicity study showed no mortality after 4 hours exposure to Fe3O4 nano particles at a maximum attainable concentration of 0.640 mg/L. In an additional publication large number of rats and hamsters were exposed to Fe2O3 dust at a concentration of 2.1 mg/L for 12 hours. No direct information on mortality rates was provided in the publication; however, based on the experimental descriptions, it is concluded that no mortality was observed 14 days postexposure. The concluded LC0 (Fe2O3, rats; 12 hours) > 2100 mg/m³ air is equivalent to LC0 (Fe2O3, rats; 4 hours) > approx. 6300 mg/m³ air (extrapolated using Haber's law as detailed in regulation (EU) 1272/2008, Annex I, Table 3.1.1, Note (c) in conjunction with ECHA Guidance on the Application of the CLP Criteria Version 5.0, July 2017). Based on the lack of clinical signs or mortality, it is therefore concluded that the LC50 (rats, 4h) for iron oxides covered by the category approach (IUCLID section 13) is > 5 mg/L.

 

Acute dermal toxicity

There is no information available on acute dermal toxicity of iron oxide compounds. The conduct of acute dermal toxicity testing is not considered to be required since inhalation of the substance is considered the most relevant route of human exposure. Furthermore, physicochemical and toxicological properties of the substance do not suggest a significant rate of absorption through the skin (cf. Annex VIII section 8.5 Column 2 of regulation (EC) 1907/2006)

Justification for classification or non-classification

Acute oral toxicity

One acute oral toxicity study according to OECD guideline 401 and four additional studies investigating the acute oral toxicity of iron oxide compounds determined in a weight of evidence approach a LD50 > 5,000 mg/kg. The classification criteria according to Regulation (EC) 1272/2008 as acutely toxic are not met, since the ATE is above 2000 mg/kg body-weight, hence no classification and labelling for acute oral toxicity is required.

 

Specific target organ toxicant (STOT) – single exposure: oral

The classification criteria according to Regulation (EC) 1272/2008 as specific target organ toxicant (STOT) – single exposure, oral are not met since no reversible or irreversible adverse health effects were observed immediately or delayed after exposure and no effects were observed at the guidance value, oral for a Category 1 classification of 300 mg/kg bw and at the guidance value, oral for a Category 2 classification of 2000 mg/kg bw. No classification is required.

 

 

Acute inhalation toxicity

One available acute inhalation toxicity study showed no mortality after 4 hours exposure to Fe3O4 nano particles at a maximum attainable concentration of 0.640 mg/L. In an additional publication rats and hamsters were exposed to Fe2O3 dust at a concentration of 2.1 mg/L for 12 hours. No direct information on mortality rates was provided in the publication; however, based on the experimental descriptions, it is concluded that no mortality was observed 14 days post-exposure. The LC0 (Fe2O3, rats; 12 hours) > 2100 mg/m³ air is equivalent to LC0 (Fe2O3, rats; 4 hours) > approx. 6300 mg/m³ air (extrapolated using Haber's law as detailed in regulation (EU) 1272/2008, Annex I, Table 3.1.1, Note (c) in conjunction with ECHA Guidance on the Application of the CLP Criteria Version 5.0, July 2017). Based on the lack of clinical signs or mortality, it is concluded that the LC50 (rats, 4h) for iron oxide category substances is >5 mg/L. The classification criteria according to regulation (EC) 1272/2008 as acutely toxic are not met since the ATE for dusts is above 5.0 mg/L, hence no classification and labelling for acute inhalation toxicity is required.

 

Specific target organ toxicant (STOT) – single exposure: inhalation

 

Animal data show evidence of transient inflammation in the lung after acute inhalation exposure. The observed inflammatory responses in the lung after inhalation can be assigned to particle lung overload, which occurs at exposure concentration above 10 mg/m³ (Pauluhn et al. 2006, 2011, IUCLID section 7.5.2) and was not due to irritating / toxic properties of the test substance.  However, despite the inflammatory response no fibrosis is induced and no marked lung damage was observed in all inhalation studies.

 

The classification criteria acc. to regulation (EC) 1272/2008 as specific target organ toxicant (STOT) – single exposure, inhalation dust/mist/fume are not met since no reversible or irreversible adverse health effects were observed immediately or delayed after exposure and no effects were observed at the guidance value, inhalation dust/mist/fume for a Category 1 classification of 1.0 mg/L/4h and at the guidance value, inhalation dust/mist/fume for a Category 2 classification of 5.0 mg/L/4h. Therefore, no classification and labelling is required.

 

Respiratory irritation (category 3) is primarily to be based on human data. Animal data may be included in the weight of evidence evaluation. The study of Lay J.C. et al. (2001) exposed sixteen healthy human volunteers with iron oxide particles (MMAD = 1.5µm) for 30 min at 12.7 mg/m³ and found no appreciable alteration of alveolar epithelial permeability, lung diffusing capacity, or pulmonary function after 30 minutes or 24 hours post-inhalation. The study shows no pulmonary irritating effects of iron oxides after human inhalation.

Thus, no classification for STOT SE category 3 is required.

 

Acute dermal toxicity & Specific target organ toxicant (STOT) – single exposure: dermal

There is no information available on acute dermal toxicity but a long history of safe use. In addition, the conduct of acute dermal toxicity testing is not considered to be required since inhalation of the substance is considered the most relevant route of human exposure. Furthermore, physicochemical and toxicological properties of the substance do not suggest a significant rate of absorption through the skin (cf. Annex VIII section 8.5 Column 2 of regulation (EC) 1907/2006).