Iron manganese trioxide

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics in vitro / ex vivo

Remarks:

Bioaccessibility

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2021 - July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

OECD Series on Testing and Assessment No. 29 (23-Jul-2001): Guidance document on transformation/dissolution of metals and metal compounds in aqueous media

Principles of method if other than guideline:

An internationally agreed guideline does not exist for this test (e.g. OECD). However, similar tests have been conducted with several metal compounds in previous risk assessments (completed under Regulation (EEC) No 793/93) and in recent preparation for REACH regulation (EC) No 1907/2006. The test was conducted on the basis of the guidance for OECD-Series on testing and assessment Number 29 and according to the bioaccessibility test protocol provided by the study monitor. The test media were artificial physiological media: gastric fluid (GST), phosphate-buffered saline (PBS), artificial lysosomal fluid(ALF) and Gamble's solution (GMB).

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Species:

other: in vitro (simulated human body fluids)

Details on test animals or test system and environmental conditions:

Test principle in brief:
- four different artificial physiological media,
- single loading of test substance of 100 mg/L,
- GST and PBS media: samples taken after 2 and 24 hours agitation (100 rpm) at 37 ± 2 °C
- ALF and GMB media: samples taken after 2, 24 and 168 hours agitation (100 rpm) at 37 ± 2 °C
- the study was performed in triplicate
- three vessels with test item and two blanks per medium (measurement in duplicate)
- analysis of two fractions (0.2 µm filtered and 0.2 µm + 3 kDa filtration)

The aim of this test was to assess the dissolution of Iron manganese trioxide (Bayferrox® 645 T) in four artificial physiological media: Phosphate buffered saline (PBS, pH 7.2-7.4), Artificial gastric fluid (GST, 1.5-1.6), Artificial lysosomal fluid (ALF, pH 4.5) and Gamble’s solution (GMB, pH 7.4). The test media were selected to simulate relevant human-chemical interactions (as far as practical), e.g. a substance entering the human body by ingestion into the gastro-intestinal tract (GST).

Duration and frequency of treatment / exposure:

Manganese and Iron concentrations were determined after 2 and 24 hours of incubation in GST, PBS, ALF and GMB. In GMB and ALF media, manganese and iron concentrations were additionally determined after 168 h.

Dose / conc.:

100 other:

Remarks:

mg of test item/L artificial media

Details on study design:

Test setup
Three replicate flasks (500 mL glass flasks) per test medium (PBS, GST, ALF, GMB) were prepared with a loading of 100 mg/L. The test item was weighed into flasks, adjusted to volume with the respective artificial physiological medium and agitated at 100 rpm in the dark at 37 °C ± 1 °C. Furthermore, the test with GMB was performed in a 5% CO2 atmosphere. Samples of GST/ PBS medium were taken after 2 h and 24 h and ALF/GMB samples after 2 h, 24 h and 168 h (7 d). Manganese and iron concentrations were determined after filtration (Syringe Filter w / 0.2 μm, polyethersulfon membrane, DIA Nielsen, Dueren, Germany) and centrifugal filtration (i.e., 0.2 μm filtration and Vivaspin 3kDa centrifugal filtration, Sartorius, Göttingen, Germany) via ICP-OES.

Sample fortification:
In addition, samples of the artificial physiological media were fortified with a known amount of manganese and iron (by standard addition of commercial standards) to determine the standard recovery. For detailed information please refer to "Any other information on materials and methods incl. tables".

Manganese and Iron analysis:
- Standards for metal analysis: Commercially available single element standards were mixed accordingly to prepare a stock solution and subsequent calibration solutions for the ICP-OES measurements. The following single element standards were used for manganese and iron: Merck HC86803426, Merck HC01344369

- Certified reference materials: As quality control standards, certified aqueous reference materials TMDA-52.4 (lot no. 1219) and TMDA-64.3 (lot no. 1219) obtained from Environment Canada and a multi-element standard (Carl Roth ICP standard 100 mg/L, lot no. 723218; Karlsruhe, Germany) were analysed for total dissolved manganese and iron by ICP-OES.

Type:

other: Bioaccessibility GST, 2h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

78.9 µg Fe/L (dissolved)

Type:

other: Bioaccessibility GST, 24h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

735 µg Fe/L (dissolved)

Type:

other: Bioaccessibility PBS, 2h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

< LOD (0.586 µg Fe/L) (dissolved)

Type:

other: Bioaccessibility PBS, 24h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

< LOD (0.586 µg Fe/L) (dissolved)

Type:

other: Bioaccessibility GMB, 2h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

< LOQ (1.38 µg Fe/L) (dissolved)

Type:

other: Bioaccessibility GMB, 24h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

1.20 µg Fe/L (dissolved)

Type:

other: Bioaccessibility GMB, 168h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

0.954 µg Fe/L (dissolved)

Type:

other: Bioaccessibility ALF, 2h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

71.5 µg Fe/L (dissolved)

Type:

other: Bioaccessibility ALF, 24h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

352 µg Fe/L (dissolved)

Type:

other: Bioaccessibility ALF, 168h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

1866 µg Fe/L (dissolved)

Type:

other: Bioaccessibility GST, 2h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

361 µg Mn/L (dissolved)

Type:

other: Bioaccessibility GST, 24h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

1388 µg Mn/L (dissolved)

Type:

other: Bioaccessibility PBS, 2h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

32.0 µg Mn/L (dissolved)

Type:

other: Bioaccessibility PBS, 24h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

79.3 µg Mn/L (dissolved)

Type:

other: Bioaccessibility GMB, 2h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

29.7 µg Mn/L (dissolved)

Type:

other: Bioaccessibility GMB, 24h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

33.6 µg Mn/L (dissolved)

Type:

other: Bioaccessibility GMB, 168h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

16.7 µg Mn/L (dissolved)

Type:

other: Bioaccessibility ALF, 2h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

708 µg Mn/L (dissolved)

Type:

other: Bioaccessibility ALF, 24h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

1440 µg Mn/L (dissolved)

Type:

other: Bioaccessibility ALF, 168h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

2908 µg Mn/L (dissolved)

Bioaccessibility (or Bioavailability) testing results:

Please refer to "any other information on results incl. tables" below.

Manganese and iron concentrations in simulated artificial body fluids

All data presented are corrected for background when feasible, i.e. when background manganese or iron concentrations in blank media are > LOQ.

The bioaccessibility of iron manganese trioxide (Bayferrox® 645 T) was determined in vitro by simulating dissolution under physiological conditions considered to mimic artificial body fluids with a loading of 100 mg test item/L. Dissolved manganese and iron concentrations were operationally defined as the dissolved elemental fraction after 0.2 µm filtration and centrifugal filtration (~2.1 nm, see table 2 and 3). In addition, dissolved/dispersed mean iron and manganese concentrations (operationally defined as the dissolved elemental fraction after 0.2 µm filtration) are summarized in table 4 and 5.

 

Tables 2 -5 are provided as an Attachment to this entry.

Solution pH

During the study, the pH of all media incl. blanks remained stable (see Table 6 -9)

Table 6: pH – GST. Target pH = 1.5, n.d. = not determined

Sample name	pH prior to the test	pH after 2 h	pH after 24 h
GST 1	1.501	n.d.	1.5
GST 2	1.507	n.d.	1.517
GST 3	1.501	n.d.	1.517
GST 4	1.506	1.47	1.497
GST blank vessel 1	1.501	1.513	1.521
GST blank vessel 2	1.5	1.504	1.516

Table 7: pH – PBS. Target pH = 7.2 - 7.4, N.d. = not determined

Sample name	pH prior to the test	pH after 2 h	pH after 24 h
PBS 1	7.304	n.d.	7.321
PBS 2	7.308	n.d.	7.327
PBS 3	7.309	n.d.	7.329
PBS 4	7.31	7.293	7.31
PBS blank vessel 1	7.315	7.351	7.36
PBS blank vessel 2	7.314	7.337	7.354

Table 8: pH – GMB. Target pH = 7.4, N.d. = not determined

Sample name	pH prior to the test	pH after 2 h	pH after 24 h	pH after 7 d
GMB 1	7.403	n.d.	n.d.	7.545
GMB 2	7.38	n.d.	n.d.	7.53
GMB 3	7.399	n.d.	n.d.	7.548
GMB 4	7.41	7.486	7.522	7.548
GMB blank vessel 1	7.399	7.486	7.472	7.465
GMB blank vessel 2	7.41	7.497	7.509	7.466

Table 9: pH – ALF. Target pH = 4.5, N.d. = not determined

Sample name	pH prior to the test	pH after 2 h	pH after 24 h	pH after 7 d
ALF 1	4.497	n.d.	n.d.	4.49
ALF 2	4.501	n.d.	n.d.	4.498
ALF 3	4.506	n.d.	n.d.	4.484
ALF 4	4.98	4.452	4.467	4.58
ALF blank vessel 1	4.507	4.529	4.532	4.515
ALF blank vessel 2	4.501	4.508	4.531	4.5

Temperature

The temperature in the thermostatically controlled incubation cabinet was within the limit of 37 °C ± 1 °C to simulate the human body temperature.

Method validation summary (ICP-MS)

Limits of detection (LODs), limits of quantification (LOQs) and correlation coefficients (r)

Manganese:

Limits of detection (LOD) within all measurement series: < 0.281 µg Mn/L.

Limits of quantification (LOQ) within all measurement series: < 0.844 µg Mn/L.

Iron:

Limits of detection (LOD) within all measurement series: < 1.70 µg Fe/L.

Limits of quantification (LOQ) within all measurement series: < 5.10 µg Fe/L.

 

Fe and Mn correlation coefficients (r) within all measurement series: >0.9998

  

Recovery data

Recovery of fortified samples, certified reference materials and quality control standards for both Fe and Mn were in the range of 87.7 – 106 % for all media.

Conclusions:

The bioaccessibility of iron manganese trioxide (Bayferrox® 645 T) was determined in vitro by examining dissolution in artificial body fluids at 37°C. At a loading of 100 mg test item/L, dissolved manganese concentrations (operationally defined as the dissolved Mn fraction after 0.2 µm filtration and centrifugal filtration (~2.1 nm, < 3 kDa)) were determined with 361 and 1388 µg Mn/L after 2 and 24 h in artificial gastric fluid (GST, pH = 1.5), respectively. With mean manganese concentrations of 708, 1440 and 2908 µg/L, dissolution of iron manganese trioxide was maximal in artificial lysosomal fluid (ALF, pH = 4.5) after 2, 24, and 168 hours of incubation, respectively. Accordingly, dissolved iron concentrations (operationally defined as the dissolved Fe fraction after 0.2 µm filtration and centrifugal filtration (~2.1 nm, < 3 kDa)) were determined with 78.9 and 735 µg/L after 2 and 24 h in artificial gastric fluid (GST, pH = 1.5), respectively. With mean iron concentrations of 71.5, 352 and 1866 µg/L, the iron release of iron manganese trioxide was maximal in artificial lysosomal fluid (ALF, pH = 4.5) after 2, 24, and 168 hours of incubation, respectively. With maximum Fe and Mn concentrations of 1.20 µg/L Fe and 79.3 µg/L Mn, iron and manganese release was low in phosphate buffered saline (PBS, pH = 7.2 - 7.4) and Gamble’s solution (GMB, pH 7.4) at a loading of 100 mg/L. In sum, iron manganese trioxide (Bayferrox® 645 T) can be considered poorly soluble in artificial body fluids.

Description of key information

A category approach justification for the iron oxides - Fe2O3, Fe3O4, FeOOH, (Fe,Mn)2O3, (Fe,Mn)3O4, ZnFe2O4 - was developed.

In the updated category approach justification, the category was extended and includes additional information on nano- and/or powder material to justify that the category covers nano- and micro-sized materials.

The members of the category are insoluble, inert particles. Conclusive evidence of bioavailable iron or iron particles that were translocated to extrapulmonary organs after inhalation was not observed in repeated dose toxicity studies (see endpoint summary repeated dose toxicity; Pauluhn, 2005). Due to its structure and physicochemical properties (insoluble in water and organic solvents) absorption and bioaccumulation is negligible if no overload effect of the lung occurs – see Category Approach  Justification - Iron oxides.

Data on the dissolution behaviour of iron oxides in physiological media is available from bioaccessibility testing: The dissolution of iron manganese trioxide (Bayferrox® 645 T) was examined in artificial physiological media selected to simulate relevant human-chemical interactions (as far as practical), i.e. for a substance entering the human body by inhalation or by ingestion. The manganese and iron release was assessed in bioaccessibility tests with a test item loading of 100 mg/L at 37°C after 2 h, 24 h and 168 h (GMB and ALF only) applying the following four media:

- Artificial gastric fluid (GST, pH 1.5) mimics the very harsh digestion milieu of high acidity in the stomach.

- Phosphate-buffered saline (PBS, pH 7.4) is a standard physiological solution that mimics the ionic strength of human blood serum. It is widely used in research and medical health care as reference test solution for comparison with data from simulated physiological conditions.

- Gamble’s solution (GMB, pH 7.4) mimics interstitial fluid within the deep lung under normal health conditions.

- Artificial lysosomal fluid (ALF, pH 4.5)simulates intracellular conditions in lung cells occurring with phagocytosis and represents relatively harsh conditions.

 

In artificial gastric fluid, phosphate buffered saline, Gamble´s solution and artificial lysosomal fluid, maximum dissolved manganese and iron concentrations (operationally defined as the dissolved metal fraction after 0.2 µm filtration and centrifugal filtration (~2.1 nm, < 3 kDa)) at a loading of 100 mg test item/L were determined in ALF media with 2908 µg/L Mn and 1866 µg/L Fe after 168h. With maximum Mn and Fe concentrations of 79.3 µg/L Mn and 1.20 µg/L Fe, very low manganese and iron release was detected in phosphate buffered saline (PBS, pH = 7.2 - 7.4) and Gamble’s solution (GMB, pH 7.4) at a loading of 100 mg/L. Thus, the extent to which iron manganese trioxide (Bayferrox® 645 T) can produce soluble available ionic and other manganese and iron-bearing species in artificial body fluids is limited.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

As demonstrated in the category approach justification, in regard to structure, physicochemical properties, environmental fate characteristics, ecotoxicity and toxicity, the grouping of Fe2O3 (diiron trioxide), Fe3O4 (triiron tetraoxide), FeOOH (iron hydroxide oxide), (Fe,Mn)2O3 (iron manganese trioxide), (Fe,Mn)3O4 (manganese ferrite), and ZnFe2O4 (zinc ferrite) in the "Iron Oxides Category" is justified.

In the updated category approach justification , the category was extended to nano- and/or powder material. Comprehensive and sufficient data are available to conclude that nano- and micro-sized category member behave similar and no further testing is necessary.

Notably, from the results of repeated dose inhalation studies with FeOOH, Fe2O3 or Fe3O4 any size dependent increased translocation of iron outside the lung did not occur. Therefore no systemic toxicity for nano- and powder material of the iron oxides exists.

These repeated dose toxicity studies demonstrated, that the occupational limit value for nano- and powder material is identical.