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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
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)

Test material

Constituent 1
Chemical structure
Reference substance name:
Manganese ferrite black spinel
EC Number:
269-056-3
EC Name:
Manganese ferrite black spinel
Cas Number:
68186-94-7
Molecular formula:
(Fe,Mn)3O4
IUPAC Name:
diiron(3+) dimanganese(3+) manganese(2+) λ²-iron(2+) octaoxidandiide
Test material form:
solid: particulate/powder
Details on test material:
Trade name: Bayferrox® 303 T
Appearance: black powder
Chemical formula: (Fe,Mn)3O4
EC-No.: 269-056-3
CAS-No.: 68186-94-7
Radiolabelling:
no

Test animals

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 Manganese Ferrite Black Spinel (Bayferrox® 303 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).

Administration / exposure

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.
Doses / concentrations
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.

Results and discussion

Main ADME resultsopen allclose all
Type:
other: Bioaccessibility GST, 2h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)
Results:
104 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:
861 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:
< LOD (0.459 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:
< LOD (0.459 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:
1.18 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:
82.5 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:
486 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:
2375 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:
264 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:
1271 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:
3.86 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:
34.1 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:
1.17 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:
6.00 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:
10.7 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:
354 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:
1258 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:
3938 µg Mn/L (dissolved)

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:
Please refer to "any other information on results incl. tables" below.

Any other information on results incl. tables

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 manganese ferrite black spinel (Bayferrox® 303 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 1.502 n.d. 1.511
GST 2 1.503 n.d. 1.501
GST 3 1.502 n.d. 1.506
GST 4 1.505 1.495 1.475
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 7.31 n.d. 7.336
PBS 2 7.306 n.d. 7.332
PBS 3 7.309 n.d. 7.33
PBS 4 7.307 7.311 7.298
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 7.39 n.d. n.d. 7.541
GMB 2 7.407 n.d. n.d. 7.526
GMB 3 7.409 n.d. n.d. 7.539
GMB 4 7.413 7.498 7.499 7.526
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 4.504 n.d. n.d. 4.493
ALF 2 4.499 n.d. n.d. 4.501
ALF 3 4.496 n.d. n.d. 4.495
ALF 4 4.495 4.477 4.507 4.488
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.

Applicant's summary and conclusion

Conclusions:
The bioaccessibility of manganese ferrite black spinel (Bayferrox® 303 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 264 and 1271 µg Mn/L after 2 and 24 h in artificial gastric fluid (GST, pH = 1.5), respectively. With mean manganese concentrations of 354, 1258 and 3938 µg/L, dissolution of manganese ferrite black spinel 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 104 and 861 µg/L after 2 and 24 h in artificial gastric fluid (GST, pH = 1.5), respectively. With mean iron concentrations of 82.5, 486 and 2375 µg/L, the iron release of manganese ferrite black spinel 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 1.18 µg/L Fe and 34.1 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, manganese ferrite black spinel (Bayferrox® 303 T) can be considered poorly soluble in artificial body fluids.