Iron manganese trioxide

Endpoint summary

Description of key information

A subacute inhalation toxicity study for Fe3O4, Fe2O3 and FeOOH and a subchronic inhalation study for Fe3O4 as representative source substances for the iron oxide category members are available. Additionally, a short-term (5-day) inhalation study with two different grades of nanomaterials was conducted. Rats were exposed to 10 and 30 mg/m³ of smaller nano-sized Fe2O3 and 30 mg/m³ of larger nano-sized Fe2O3.

Sub-chronic repeated dose toxicity studies with Fe3O4, Fe2O3 and FeOOH as representative source substances for the iron oxide category members are available. A NOAEL of greater than 1000 mg/kg bw/day is derived for all three substances, based on a complete absence of adverse effects.

For repeated dose toxicity via the dermal route, no reliable studies are available for the iron oxide category.

Details on the category justification are given in the read-across document attached in IUCLID section 13.2.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: as reported in source record

Justification for type of information:

see attachment "Endpoint-specific read-across justification for the iron oxide category" in IUCLID section 13.2.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: as reported in source record

Justification for type of information:

see attachment "Endpoint-specific read-across justification for the iron oxide category" in IUCLID section 13.2.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Dose descriptor:

NOAEL

Effect level:

4.7 mg/m³ air

Sex:

male/female

Basis for effect level:

other: see "Remarks"

Critical effects observed:

not specified

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

Executive summary:

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Quality of whole database:

GLP guideline study according OECD #413 (Subchronic Inhalation Toxicity: 90-Day)

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: as reported in source record

Justification for type of information:

see attachment "Endpoint-specific read-across justification for the iron oxide category" in IUCLID section 13.2.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Dose descriptor:

NOAEL

Effect level:

4.7 mg/m³ air

Sex:

male/female

Basis for effect level:

other: see "Remarks"

Critical effects observed:

not specified

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

Executive summary:

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

4.7 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP guideline study according OECD #413 (Subchronic Inhalation Toxicity: 90-Day)

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Introductory remark on read-across:

In this dossier, the endpoint repeated dose 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 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, sub-acute or sub-chronic oral or inhalation exposure up to the limit dose of the maximum tolerated concentration of the respective test. Similarly, a lack of systemic effects for the endpoints mutagenicity and reproductive toxicity are anticipated. Further information on the read-across approach is given in the report attached to IUCLID section 13.2.

 

Inhalation:

In a subacute inhalation toxicity study, 48 male rats per group were exposed to three different aerosolized iron oxide powders (Fe2O3, Fe3O4 and FeOOH). Exposure was 6-hours/day on five days/week for two consecutive weeks. The rats were exposed to mean actual concentrations of 185.6 mg/m³ Fe3O4, 210.2 mg/m³ Fe2O3 and 195.7 mg/m³ FeOOH (Pauluhn, 2005). The repeated exposure to the aerosolized iron oxides was not associated with any specific clinical signs, changes in body temperature or body weights. Histopathological evaluation of rat lungs exposed to the different iron oxides revealed findings consistent with a 'poorly soluble particle' effect after the 2-week exposure period, including the 3-month post-exposure period. Conclusive evidence of bioavailable iron or iron particles that were translocated to extrapulmonary organs was not observed (Pauluhn, 2005). This supports the conclusion, that Fe3O4 can serve as a surrogate for FeOOH and Fe2O3.

For Fe3O4 valid subacute and subchronic inhalation studies are available (Pauluhn, 2006a; Pauluhn, 2006b). In the subacute inhalation toxicity study 30 male rats were exposed to 10.1, 19.4, 45.6 and 95.8 mg/m³ Fe3O4 for 6 hours/day, 5 days/week for 4 weeks and serially sacrificed 1, 8, 24 weeks after the 4 weeks exposure period. Clinical signs were recorded daily before and after exposure or once per week during the post-exposure period. At each serial sacrifice, inflammatory endpoints were determined in bronchoalveolar lavage (BAL). Rats were subjected to gross pathological examination and histopathology (nasal passages, trachea, lung, liver, spleen, kidneys, testes and thymus). The repeated 4-week exposure to the aerosolized dry powder was not associated with specific clinical signs or consistent changes in body weights. Changes in organ weights occurred and consisted of increased lung and lung-associated lymph nodes (LALN) weight at 45.6 mg/m³ and above. Histopathological evaluation of rat lungs exposed to Fe3O4 revealed finding consistent with a poorly soluble particle effect. Conclusive evidence of bioavailable iron or iron particles that were translocated to extrapulmonary organs to any appreciable extent was not found. Extrapulmonary effects causally linked to the exposure of Fe3O4 were not found at any exposure concentration and time point (Pauluhn, 2006b). In the subchronic inhalation toxicity study in rats (20 male and 20 female rats per group) the animals were exposed 6 hours/day, 5 days/week for 13 weeks to 4.7, 16.6 and 52.1 mg/m³ Fe3O4. During the study, the body weights were determined twice weekly and clinical signs were recorded daily before and after exposure. At sacrifice, inflammatory endpoints were determined in BAL. Histopathology focused on the entire respiratory tract (nasal passages, trachea, lung, lung associated lymph nodes) and included all extrapulmonary organs as suggested by OECD 413. At sacrifice biological specimens were collected for hematology, clinical pathology and urinalysis (Pauluhn, 2006a). The repeated exposure of rats during a study period of 13 weeks was not associated with any specific clinical signs. Hematology, clinical pathology and urinalysis were unobtrusive. Neither analytical nor toxicological evidence existed that free, biosoluble iron was liberated from the inhaled dust to any appreciable extent. However, the neutrophils in male rats and some biochemical markers were elevated at 4.7 mg/m³ and above (Pauluhn, 2006a). The NOAECs for Fe3O4 are 10.1 mg/m³ for the subacute exposure and 4.7 mg/m³ for subchronic exposure in rats (Pauluhn, 2006a; Pauluhn, 2006b).

A histopathological evaluation of rat lungs exposed to three different iron oxides revealed findings consistent with a 'poorly soluble particle' effect after the 2- week exposure period, including the 3-month post-exposure period. Conclusive evidence of bioavailable iron or iron particles that were translocated to extrapulmonary organs was not observed. Extrapulmonary effects causally linked to the high-level exposure of iron oxides were not be detected at any time point. At the end of the 3-month post-exposure period the findings causally linked to the high-level exposure to iron oxides (e.g. focal inflammatory infiltrates, bronchioloalveolar hypercellularity) showed a decrease in incidence and/or severity. The comparative assessment of the three different iron oxides revealed the same quality and time course of responses, i.e., marked differences of any toxicological significance between the test specimens were not observed. Nonetheless, there was a consistent trend that the rats exposed to Fe3O4 displayed minimally more pronounced changes when compared to Fe2O3 and FeOOH. This supports the conclusion that from a scientific point of view further testing of Fe2O3 and Fe3O4 does not appear to be justified and that future studies with Fe3O4 can serve as conservative proxy for Fe2O3 and FeOOH (Pauluhn, 2005).

In a short-term (5-day) inhalation study (BASF SE 2015), 8 male Wistar rats (5 animals in the main group and 3 animals in the recovery group) were exposed to 10 and 30 mg/m³ of smaller nano-sized Fe2O3 and 30 mg/m³ of larger nano-sized Fe2O3. Dust Inhalation exposure of rats to 10 and 30 mg/m³ test item 1 and 30 mg/m³ of test item 2 on 5 consecutive days did not cause any adverse effect in the respiratory tract, which was examined by broncho-alveolar lavage and histopathology. There were no changes of hematology and clinical chemistry parameters. A slightly decreased mean body weight and intermittently reduced body weight change were observed during exposure and post-exposure period of the test group with micro-sized Fe2O3. However, the deviations to the control group were not biologically relevant. Thus, under current study conditions, the NOAEC was 30 mg/m³ for both nano-sized samples. No relevant differences between the smaller nano-sized and the larger nano-sized Fe2O3 were observed.

In consideration of the results of the repeated dose inhalation studies with ZnO and the repeated dose inhalation studies in rats and monkeys with Mn3O4 it can be concluded, that the NOEC of Fe2O3, FeOOH, ZnFe2O4, (Fe,Mn)2O3, and (Fe,Mn)3O4 is in the same range as for Fe3O4, both for nano-sized and micro-sized particles (see Category approach justification, IUCLID section 13.2).

 

Oral:

In the repeated dose toxicity study by Hansen (2020, unpublished report), FeOOH (Iron oxide Sicovit® Yellow 10 E172) was administered via diet to groups of 10 male and 10 female Crl:CD (SD) rats at dose levels of 100, 300, and 1000 mg/kg bw/day. The administration occurred daily for a 90-day period. A control group receiving plain diet was run concurrently. Furthermore, recovery groups (n = 10 animals/sex/group; control group and high dose level only; recovery period: 4 weeks) and a satellite group (n = 10 animals/sex/group: treatment period: 91 days) were also run concurrently. During the observation of animals, no test item-related effects were observed in animals for clinical signs, mortality, body weight and weight changes, food consumption, water consumption, ophthalmological findings, haematological findings, clinical chemistry findings, endocrinological findings, urinalysis findings, behaviour (functional findings), organ weights, gross pathology and histopathological findings. Furthermore, iron level analysis of plasma and tissues (spleen and liver) revealed no test item-related influence on iron concentration in plasma and spleen samples. Lastly, visual inspection by microscopy of the complete colon of all submitted rats did not identify aberrant crypt foci (ACF) in any of the submitted tissue samples. Based on the results of this analysis it can be concluded that daily oral administration of the test item to rats up to a daily dose of 1000 mg/kg bw and over a time period of 90 days via diet did not induce ACF in the colon of this species. Based on the findings, the no observed adverse effect level (NOAEL) for general toxicity is greater than 1000 mg/kg bw/day for male and female rats.

 

In the repeated dose toxicity study by Hansen (2020, unpublished report), Fe₃O₄ (Ferroxide® Black 86) was administered via diet to groups of 10 male and 10 female Crl:CD (SD) rats at dose levels of 100, 300, and 1000 mg/kg bw/day. The administration occurred daily for a 90- day period. A control group without treatment was run concurrently. Recovery groups (n = 10 animals/sex/group; control group and high dose level only; recovery period: 4 weeks) and a satellite group (n = 10 animals/sex/group: treatment period: 91 days) were also run concurrently. During the observation of animals, no test item-related effects were observed in animals for clinical signs, mortality, body weight and weight changes, food consumption, water consumption, ophthalmological findings, haematological findings, clinical chemistry findings, endorcinoloigcal findings, urinalysis findings, behaviour (functional findings), organ weights, gross pathology and histopathological findings. Furthermore, iron level analysis of plasma and tissues (spleen and liver) revealed no test item-related influence on iron concentration in plasma and spleen samples. However, the iron concentrations in liver tissue samples were slightly increased by 35% to 63% for the female animals treated with 100 or 300 mg/kg bw/day of the test item via the diet and for the male and female animals treated with 1000 mg/kg bw/day via the diet for 91 days compared to the control group at the end of the treatment period. All values were statistically significant at p ≤ 0.01 compared to the control group. However, no dose-response relationship was noted. Lastly, visual inspection by microscopy of the complete colon of all submitted rats did not identify aberrant crypt foci (ACF) in any of the submitted tissue samples. Based on the results of this analysis it can be concluded that daily oral administration of the test item to rats up to a daily dose of 1000 mg/kg bw and over a time period of 90 days via diet did not induce ACF in the colon of this species. Based on the lack of any adverse findings, the no observed adverse effect level (NOAEL) for general toxicity is greater than 1000 mg/kg bw/day for male and female rats.

 

A subchronic toxicity study of various orally administered nanoparticles including red iron oxide (Fe₂O₃, 60-118 nm) was performed by Yun et al. (2015) according to the OECD Test Guideline (TG) 408 (OECD, 1998). Sprague-Dawley rats received daily doses of 250, 500 or 1000 mg/kg bw/day for 13 weeks by gavage. Fe₂O₃ nanoparticles had no significant effects on body weight, mean daily food and water consumption, when compared with control groups. There were no treatment-related changes in haematological, serum biochemical parameters or histopathological observations. Some changes in organ weights were observed: decreases in weight of pituitary gland and liver and increases in weight of adrenal gland and testis. According to the authors, ‘these changes were sporadic without dose-dependent trends, indicating that they were not considered toxicologically relevant’. In blood and all tissues tested, including liver, kidney, spleen, lung and brain, the concentration of Fe showed no dose-associated response in comparison to the control groups. Iron concentrations in the urine of Fe2O3 nanoparticle-treated rats showed no significant differences compared to those of control animals. Although not statistically significant, the concentrations of Fe in the faeces of treated animals were found to be higher than those of the control groups. The authors stated that the subchronic oral dosing with Fe2O3 nanoparticles showed no systemic toxicity to rats. The NOAEL was established at 1000 mg/kg bw/day, the highest dose tested in rats receiving Fe2O3 nanoparticles by gavage.

 

 

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

 

In a comparative subacute inhalation toxicity in rats with the iron oxides (FeOOH, Fe2O3 and Fe3O4 also named as  'Black', 'Yellow', 'Red') no significant differences in histopathology and clinical signs were found. Therefore repeated dose studies with longer duration (4 or 13 weeks) were conducted with Fe3O4 as a surrogate for iron manganese oxide - ( see Category approach for Fe2O3, Fe3O4, FeOOH, (Fe,Mn)2O3, (Fe,Mn)3O4, and ZnFe2O4.

 

 

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:

 

In a comparative subacute inhalation toxicity in rats with the iron oxides (FeOOH, Fe2O3 and Fe3O4 also named as  'Black', 'Yellow', 'Red') no significant differences in histopathology and clinical signs were found. Therefore repeated dose studies with longer duration (4 or 13 weeks) were conducted with Fe3O4 as a surrogate for iron manganese oxide - see Category approach for Fe2O3, Fe3O4, FeOOH, (Fe,Mn)2O3, (Fe,Mn)3O4, and ZnFe2O4.

Justification for classification or non-classification

In summary, the members of the iron oxide category are inert particles, therefore no extrapulmonary toxicological effects are expected for repeated oral and dermal toxicity. The NOAECs for Fe3O4 are 10.1 mg/m³ for the subacute exposure and 4.7 mg/m³ for subchronic exposure in rats.

A classification is therefore not justified.