Iron

Endpoint summary

Description of key information

A series of oral acute toxicity studies with iron powders yielded LD50 values that were much higher than the limit below which which a substance needs to be classified for acute oral toxicity.
A series of inhalation and intratracheal instillation studies showed carbonyl iron to be without effects on survival or local sublethal effects. Although the highest air concentration tested (250 mg/m^3) was lower than the upper threshold concentration determining classification, the results indicate that metallic iron needs not to be classified for acute inhalation toxicity.
The lack of systemic bioavailability of iron upon dermal exposure makes testing for acute dermal toxicity redundant and allows for the conclusion that classification for this endpoint is not necessary.  

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: The study does not present sufficient experimental details. Nevertheless, it provides useful information regarding the acute oral toxicity of elemental iron.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was conducted prior to the adoption of OECD and EU guidelines.

GLP compliance:

not specified

Remarks:

The study was performed before the adoption of GLP principles

Test type:

standard acute method

Limit test:

no

Species:

other: 1. white rats and 2. guinea pigs

Strain:

not specified

Sex:

not specified

Route of administration:

oral: gavage

Vehicle:

water

Doses:

no data

No. of animals per sex per dose:

no data

Control animals:

not specified

Statistics:

no data

Preliminary study:

no preliminary study performed

Sex:

not specified

Dose descriptor:

LD50

Effect level:

7.5 other: g/kg bw

Remarks on result:

other: electrolytic iron-white rats

Sex:

not specified

Dose descriptor:

LD50

Effect level:

7.5 other: g/kg bw

Remarks on result:

other: electrolytic iron-guinea pigs

Sex:

not specified

Dose descriptor:

LD50

Effect level:

7.5 other: g/kg bw

Remarks on result:

other: reduced iron-white rats

Sex:

not specified

Dose descriptor:

LD50

Effect level:

8 other: g/kg bw

Remarks on result:

other: reduced iron-guinea pigs

Gross pathology:

yes

Table 1: Acute oral toxicity of the two iron powders for both animal species.

	White rats			Guinea pigs
Substance	LD0(g/kg bw)	LD50(g/kg bw)	LD100(g/kg bw)	LD0(g/kg bw)	LD50(g/kg bw)	LD100(g/kg bw)
Electrolytic iron	5	7.5	10	6	7.5	11
Reduced iron	5	7.5	11	5	8	10

Interpretation of results:

practically nontoxic

Remarks:

Migrated information opinion of the submitter Criteria used for interpretation of results: EU

Conclusions:

The LD50 values for the oral acute toxicity of electrolytic and hydrogen reduced iron for the two animal species were 7.5 and 8 g/kg bw, which is much higher than 2 g/kg bw, which is the highest LD50 that results in classification and labelling. Thus, they were considered practically non-toxic.

Executive summary:

In an acute oral toxicity study, groups of white rats and guinea pigs were given a single oral dose of electrolytic iron powder and hydrogen reduced iron powder in water (50% suspension).The oral LD₅₀value for the electrolytic iron, was determined to be 7.5 g/kg bw for both species tested. For the hydrogen reduced iron it was estimated as 7.5 g/kg bw for white rats and as 8 g/kg bw for guinea pigs. Animals which died were autopsied and no evidence of gross pathology was observed. Both iron powders are practically non toxic when administered via the oral route and need not to be classified for acute oral toxicity.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

7 500 mg/kg bw

Quality of whole database:

The study does not present sufficient experimental details. Nevertheless, it provides useful information regarding the acute oral toxicity of elemental iron.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Before April 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

As most publications, this one does not present all the details that are normally presented in a standard study report. However, a thorough and extensive study is described in the publication that gives valuable information on the acute toxicity upon inhalation of the substance and can thus be used to address the requirement in question. Moreover, the publication is from a well-known peer-reviewed paper and the study was carried out by a laboratory with a good reputation.

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

Rats were exposed nose-only to respirable iron particles (carbonyl iron) for 4 week, 5 days/week and 6 h/day. Endpoints studied were focused at inflammatory reactions in the respiratory tract and clearance.

GLP compliance:

not specified

Remarks:

It is not customary to provide information on GLP compliance in studies published in regulat scientific journals.

Test type:

other: Actually a repeated-dose study that, due to the high concentrations in the air of the test substance also provides meaningful information about the acute toxicity upon inhalation.

Limit test:

no

Species:

rat

Strain:

other: Charles River Crl:CDBR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, New York
- Age at study initiation: 7-8 weeks
- Weight at study initiation: not data
- Fasting period before study: not data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum):no data
- Acclimation period:no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data


IN-LIFE DATES: From: To: 180 days

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

Animals were placed in cylindrical polycarbonate or stainless steel holders equipped with conical nose pieces. The restrainers were inserted into face plates on the exposure chambers such that the nose of each animal protruded into the chamber. Atmospheres of carbonyl iron were generated with a K-tron bin feeder equipped with twin feed screws. The dust was metered into a polycarbonate transfer tube where high pressures of air swept the test material into the exposure chamber. Chamber concentrations of silica or iron were maintained by controlling the dust-feed rate into the generation apparatus, or by varying the air-flow rate.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

For gravimetric analysis, samples of atmospheric carbonyl iron or silica were taken from the animal breathing zone at approximately 30-min intervals by drawing calibrated volumes of chamber atmosphere through preweighed glass-fiber filters. Filters were w

Duration of exposure:

28 d

Remarks on duration:

4 weeks, 5 days/week, 6 h/day

Concentrations:

Nominal: 0, 5, 50 and 250 mg/m^3; actual: 4.8, 51.8, 243.5 mg/m^3; MMAD (mass median aerodynamic diameter): 3.4, 3.2 and 2.9 micrometer, respectively.

No. of animals per sex per dose:

No data

Control animals:

yes

Details on study design:

The following endpoints were studied in the exposed rats.
- Presence of iron in the lungs;
- Particles in alveolar macrophages obtained by means of bronchoalveolar lavage (BAL);
- Measures for inflammation in BAL;
- Lactate dehydrogenase and protein in BAL;
- Cell proliferation in lungs by means of BrdU labelling;
- Lung clearance;
- Translocation of particles to tracheobronchial lymph nodes;
- Functional responses of alveolar marcophages;
- Lung histopathology.

Statistics:

Two-tailed Student t test

Preliminary study:

No preliminary study performed

Sex:

male

Dose descriptor:

LC50

Effect level:

> 250 mg/m³ air

Exp. duration:

6 h

Remarks on result:

other: 6 h/day, 5 days/week, 4 weeks

Mortality:

Although the presentation of the results does not explicitly states anything about mortality, the exposure for 4 weeks, 5 days/week and 6 h/day to up to 250 mg/m^3 and the extensive description of sublethal effects clearly indicates that no treatment-related mortality occurred. From this study it can safely be concluded that 6 h exposure to 250 mg/m^3 respirable iron powder (carbonyl iron) does not result in mortality.

Clinical signs:

other: No data

Body weight:

No data

Gross pathology:

No data

Other findings:

- Histopathology: The publication presents an extensive and detailed descriptions of the histopathological observations made in the lungs, which was summarized in the abstract as follows (slightly modified). "Free granular pigment (CI = carbonyl iron) was present on the hypertrophic mucosal surfaces of bronchioles and bronchi, and particle-laden macrophages, found individually, were numerous throughout alveoli and within lymphoid tissues immediately after exposure. Aggregates of particle-laden macrophages were present within alveoli and alveolar ducts from 1 week postexposure through the entire 6-month recovery period. Macrophage accumulations increased in size and number from 1 week through 1 month postexposure and then appeared to remain constant through the remaining 5-month postexposure period. Minimal cellular hypertrophy and hyperplasia were evident at alveolar duct bifurcations adjacent to macrophage aggregates, and this effect was most prominent at 3 to 6 months postexposure." These observations were largely restricted to 50 mg/m^3 and 250 mg/m^3.
- Potential target organs: The study was focused entirely on the effects of the exposure on the lungs, in particular effects associated with clearance and inflammation.
- Other observations: What follows is a quote from the abstract of the publication (slightly modified). "Four-week exposures to Ti02 or CI particles at concentrations of 250 mg/m3 resulted in lung burdens of 12 mg titanium and 17 mg iron, respectively, with particle retention half-times ranging from 68 days for 5 mg/m3 Ti02 to approximately 330 days for 250 mg/m3. The impact of this Ti02 dust load and similar lung burdens of CI particles produced a sustained pulmonary inflammatory response measured through a period of 3-6 months postexposure concomitant with increases in BrdU cell labeling of terminal airway and pulmonary parenchymal cells. The impairment of particle clearance mechanisms was accounted for by deficits in in vitro phagocytic and chemotactic potential of alveolar macrophages recovered from the lungs of high-dose, TiO2 or CI-exposed rats." In another part of the publication the observations made are summarized as:
- Pulmonary inflammation;
- Enhanced proliferation of pulmonary cells;
- Impairment of particle clearance mechanisms;
- Deficits in in macrophage function;
- Macrophage aggregation.
No signs of inflammation or affected clearance were found at 5 mg/m^3, while these effects were found at 50 and 250 mg/m^3.

Interpretation of results:

other: Highest concentration tested was 250 mg/m^3; so no final classification possible.

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The study showed that exposure of rats to air concentrations of carbonyl iron up to 250 mg/m^ for 6 h/day, 5 days/week, 4 weeks, does not result in mortality. So an LC50 of > 250 mg/m^3 is established. Fifty and 250 mg/m^3 gave rise to clear effects on particle clearance and resulted in inflammation. Such effects were not found at 5 mg/m^3.

Executive summary:

Although not specifically aimed at establishing the acute inhalation toxicity of iron particles as is done in a standard guideline study, this study shows that up to 250 mg/m^3, respirable iron particles (carbonyl iron) do not cause mortality in males rats when the animals were exposed for 6 h/day, 5 days/week over a period of 4 weeks. So the LC50 lies clearly above the highest air concentration applied of 250 mg/m^3. The other aspects of the study are summarized adequately in the abstract of the original paper, which is, therefore, reproduced below.

This study was carried out to assess the time course of pulmonary clearance impairment and persistence of inflammation following high-dose inhalation exposures to titanium dioxide (Ti0₂) or carbonyl iron (CI) particles. Male rats were exposed to air, Ti0₂ or CI particles 6 hr/day, 5 days/week, for 4 weeks at concentrations of 5, 50, and 250 mg/m³and evaluated at selected intervals through 6 months postexposure. Indices of pulmonary inflammation as well as alveolar macrophage clearance functions (i.e., morphology, in vivo and in vitro phagocytosis, and chemotaxis), cell proliferation, and histopathology endpoints were measured at several post­exposure time periods through 6 months. In addition, amounts of TiO₂ or CI in lungs and tracheobronchial lymph nodes were measured to allow an evaluation of particle clearance and translocation patterns. Four-week exposures to TiO₂ or CI particles at concentrations of 250 mg/m³resulted in lung burdens of 12 mg titanium and 17 mg iron,respectively, with particle retention half-times ranging from 68 days for 5 mg/m³TiO₂ to approximately 330 days for 250 mg/m³. The impact of this TiO₂ dust load and similar lung burdens of CI particles produced a sustained pulmonary inflammatory response measured through a period of 3 -6 months postexposure concomitant with increases in BrdU cell labeling of terminal airway and pulmonary parenchymal cells. The impairment of particle clearance mechanisms was accounted for by deficits in in vitro phagocytic and chemotactic potential of alveolar macrophages recovered from the lungs of high-dose, TiO2or CI-exposed rats. Free granular pigment (TiO₂or CI) was present on the hypertrophic mucosal surfaces of bronchioles and bronchi, and particle­laden macrophages, found individually,were numerous throughout alveoli and within lymphoid tissues immediately after exposure. Aggregates of particle-laden macrophages were present within alveoli and alveolar ducts from 1 week postexposure through the entire 6 -month recovery period. Macrophage accumulations increased in size and number from 1 week through 1 month postexposure and then appeared to remain constant through the remaining 5-month postexposure period. Minimal cellular hypertrophy and hyperplasia were evident at alveolar duct bifurcations adjacent to macrophage aggregates, and this effect was most prominent at 3 to 6 months postexposure. The results of this study clearly demonstrate that exposure to high dust concentrations of two different innocuous particle types 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. In addition,the mass deposition rate determination appears to be a less sensitive indicator of"overload when compared to biomarkers of pulmonary toxicity, such as macrophage function and cellular inflammation and proliferation indices.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating conc.

Value:

250 mg/m³ air

Quality of whole database:

As most publications, this one does not present all the details that are normally presented in a standard study report. However, a thorough and extensive study is described in the publication that gives valuable information on the acute toxicity upon inhalation of the substance and can thus be used to address the requirement in question. Moreover, the publication is from a well-known peer-reviewed paper and the study was carried out by a laboratory with a good reputation.

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral exposure

Metallic iron has only been studied for its acute oral toxicity in the form of several iron powders, which are used to treat iron deficiency. Several commercially available iron powders (carbonyl iron, electrolytic iron and hydrogen reduced iron) were tested in different doses. The animals (mostly rats) were exposed by gavage. The LD50 values derived were within the range of 7.5 to 98.6 g/kg bw, which is much higher than 2 g/kg bw, the maximum LD50 that still gives rise to classification and labelling.

It is pointed out here that these iron powders represent a sort of worst case scenario. They can only be used for the treatment of iron deficiency if they are completely or to a large extent converted to iron ions in the acid environment of the stomach. After they have left the stomach, conversion should be large enough to provide for a meaningful dose of absorbable iron ions. This can only be achieved if the particles are small enough, thus making a sufficiently large iron surface available for the chemical reaction in the stomach acid. The tested particles are thus much more reactive than the coarse iron particles that could be produced during various mechanical processes; the iron in the tested particles is much more systemically available. It may, therefore, be assumed, that the larger coarse iron particles cannot be more toxic in terms of an LD50 value than the small particles of carbonyl iron. Therefore, it can be concluded that metallic Fe is practically non-toxic and it does not need to be classified in terms of its acute oral toxicity.

Inhalation

A research group of the DuPont Haskell laboratory published a series of studies in which carbonyl iron was investigated for pulmonary toxicity, in particular effects on particle clearance, macrophage function and inflammation-related endpoints. Biochemical and cytological investigations were carried out with the fluid obtained by means of bronchioalveolar lavage (BAL); macrophages from the BAL were cultured in vitro to investigate chemotaxis, cell proliferation was studied by means labelling with the DNA-base analogue 5-bromodeoxyuridine; the respiratory organs were studied histopathologically. The animals (mostly rats) were either exposed by inhalation or by intratracheal instillation. Both exposure regiments resulted in an iron dose of about 1 mg/animal.

These experiments were not specifically aimed at studying effects on survival. There was no information in any of the publications  on the mortality of the carbonyl-iron exposed groups.  From the description of the study it could be concluded that it indirectly shows that the treatments with carbonyl iron did not result in mortality. Dr. Warheit, one of the principal investigators of the DuPont Haskell group confirmed that this conclusion is correct via a personal communication.  He stated that the treatments with carbonyl iron never resulted in mortality. This implies that the LC50 for inhalation is higher than 250 mg/m^3 (the highest air concentration applied), whereas the LD50 for intratracheal instillation was higher than 5 mg/kg bw. Moreover the treatments had no effects on the endpoints for pulmonary toxicity.

These studies are based on the assumption that there were indeed iron particles that were inhaled or instilled and thus reached the lung. The particle size of carbonyl iron implies a very high surface/mass ratio, which in turn implies a relatively high reactivity. The basic question is whether chemical conversion (oxidation) occurs in the air before inhalation or in the instillation fluid. If so, actual exposure would be to iron oxide. Moreover, carbonyl iron is also available in coated form. Such particles may be protected from chemical reactions and may not represent a good model substance in iron toxicology.

Dr. Warheit was contacted about this. He stated that in his experiments carbonyl iron was stable and that he indeed tested the effects of iron particles. Thus it can be concluded that respirable iron particles were without toxic effects.

If carbonyl iron would be too reactive for testing due to its small particle size, it would be altogether impossible to test iron for toxicity upon exposure via inhalation, because larger particle, which would suffer less from chemical instability would not reach the lower parts of the respiratory tract, i.e., would not be respirable.

Although the highest concentrations tested were lower than the LC50 value that is the upper limit of the range that gives rise to classification, it is not necessary to classify iron for acute inhalation toxicity for the following reasons.

1) The highest concentrations tested did not give rise to any mortality, which means that an LC50 value is much higher;

2) The highest concentrations tested did not give rise to local sublethal effects that may lead to mortality at higher dose levels;

3) The exposures were repeated up to 20 times over a period of 28 days, without mortality, which strengthens the notion that the tested concentration is far below the concentrations that might give rise to mortality;

4) Carbonyl iron can be regarded as a worst-case due to its very small particle size;

5) It may be assumed that metallic iron does not become systemically available, but has to be chemically converted into a soluble form; any systemic toxicity of carbonyl iron after inhalation (or any other non-invasive exposure route) has to be the result of iron ions; it is hard to see how metallic iron can be converted into iron ions under the neutral conditions in the respiratory tract; if anything chemical happens to the particles, it will be oxidation, resulting in the insoluble iron oxides, which are not expected to become systemically available, let alone cause systemic toxicity.

It can thus be concluded that adequate evidence shows that iron needs not to be classified for acute toxicity upon inhalation.

Dermal exposure

No acute dermal toxicity studies with metallic iron are needed owing to the lack of systemic bioavailability of the substance. In its metallic form iron will not pass through the skin. While present on the skin oxidation might occur after some time. Where oxides are formed, these will also not pass through the skin owing to their insolubility. The aquous layer on the skin is not be acidic enough for the formation of iron ions (for this the pH has to be lower than 2) that may pass through the skin. It can thus be concluded that metallic iron need not to be classified for acute dermal toxicity.

Justification for selection of acute toxicity – oral endpoint
Supporting study providing useful information regarding the acute oral toxicity of elemental iron. Full details are provided in the discussion field below.

Justification for selection of acute toxicity – inhalation endpoint
Key study

Justification for classification or non-classification

Oral exposure

LD50 values are much higher than the highest value that still gives rise to classification for acute oral toxicity.

Inhalation

The highest concentrations tested (250 mg/m^3) were lower than the LC50 value that is the upper limit of the range that gives rise to classification. Nevertheless, it is not necessary to classify iron for acute inhalation toxicity for the following reasons.

1) The highest concentrations tested did not give rise to any mortality, which means that an LC50 value is much higher;

2) The highest concentrations tested did not give rise to local sublethal effects that may lead to mortality at higher dose levels;

3) The exposures were repeated up to 20 times over a period of 28 days, without mortality, which strengthens the notion that the tested concentration is far below the concentrations that might give rise to mortality;

4) The carbonyl iron tested can be regarded as a worst-case due to its very small particle size;

5) Metallic iron will not become systemically available, but has to be chemically converted into a soluble form; any systemic toxicity of carbonyl iron after inhalation (or any other non-invasive exposure route) has to be the result of iron ions; it is hard to see how metallic iron can be converted into iron ions under the neutral conditions in the respiratory tract; if anything chemical happens to the particles, it will be oxidation, resulting in the insoluble iron oxides, which are not expected to become systemically available, let alone to cause systemic toxicity.

It can thus be concluded that iron needs not to be classified for acute toxicity upon inhalation.

Dermal exposure

Due to a lack of systemic exposure after dermal exposure of the skin no acute toxicity is to be expected and thus no classification needed.