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Reference
Endpoint:
basic toxicokinetics in vitro / ex vivo
Remarks:
Bioaccessibility
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-05-31 to 2021-07-20
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 Zinc ferrite Brown Spinel (Bayferrox® 3950) 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:
Zinc and Iron concentrations were determined after 2 and 24 hours of incubation in GST, PBS, ALF and GMB. In GMB and ALF media, zinc 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). Zinc 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 zinc 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".

Zinc 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 zinc and iron: Merck HC86803426, Merck Supelco 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 zinc 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:
54.0 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:
453 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:
< LOQ (0.502 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 (0.167 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:
0.262 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:
0.042 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:
0.100 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:
39.5 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:
223 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:
1965 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:
41.2 µg Zn/L (dissolved)
Type:
other: Bioaccessibility GST, 24h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)
Results:
147 µg Zn/L (dissolved)
Type:
other: Bioaccessibility PBS, 2h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)
Results:
< LOD (0.156 µg Zn/L) (dissolved)
Type:
other: Bioaccessibility PBS, 24h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)
Results:
1.47 µg Zn/L (dissolved)
Type:
other: Bioaccessibility GMB, 2h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)
Results:
< LOD (0.701 µg Zn/L) (dissolved)
Type:
other: Bioaccessibility GMB, 24h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)
Results:
< LOD (0.701 µg Zn/L) (dissolved)
Type:
other: Bioaccessibility GMB, 168h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)
Results:
< LOD (0.701 µg Zn/L) (dissolved)
Type:
other: Bioaccessibility ALF, 2h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)
Results:
33.6 µg Zn/L (dissolved)
Type:
other: Bioaccessibility ALF, 24h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)
Results:
71.2 µg Zn/L (dissolved)
Type:
other: Bioaccessibility ALF, 168h @ 37 °C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)
Results:
887 µg Zn/L (dissolved)
Bioaccessibility (or Bioavailability) testing results:
Please refer to "any other information on results incl. tables" below.

Zinc and iron concentrations in simulated artificial body fluids

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

The bioaccessibility of zinc ferrite brown spinel (Bayferrox® 3950) 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 zinc 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 zinc 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 1.495 n.d. 1.497
GST 2 1.497 n.d. 1.493
GST 3 1.498 n.d. 1.486
GST 4 1.501 1.48 1.482
GST blank vessel 1 1.495 1.514 1.522
GST blank vessel 2 1.491 1.508 1.519

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 7.299 n.d. 7.322
PBS 2 7.301 n.d. 7.323
PBS 3 7.299 n.d. 7.319
PBS 4 7.311 7.313 7.314
PBS blank vessel 1 7.299 7.325 7.332
PBS blank vessel 2 7.326 7.357 7.364

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 7.402 n.d. n.d. 7.524
GMB 2 7.402 n.d. n.d. 7.523
GMB 3 7.403 n.d. n.d. 7.534
GMB 4 7.404 7.487 7.522 7.515
GMB blank vessel 1 7.39 7.497 7.457 7.457
GMB blank vessel 2 7.394 7.48 7.471 7.447

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 4.495 n.d. n.d. 4.502
ALF 2 4.496 n.d. n.d. 4.504
ALF 3 4.496 n.d. n.d. 4.483
ALF 4 4.496 4.479 4.489 4.49
ALF blank vessel 1 4.498 4.519 4.533 4.507
ALF blank vessel 2 4.497 4.514 4.532 4.503

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)

Zinc:

Limits of detection (LOD) within all measurement series: < 0.701 µg Zn/L.

Limits of quantification (LOQ) within all measurement series: < 2.10 µg Zn/L.

Iron:

Limits of detection (LOD) within all measurement series: < 0.652 µg Fe/L.

Limits of quantification (LOQ) within all measurement series: < 1.95 µg Fe/L.

 

Fe and Zn correlation coefficients (r) within all measurement series: >0.9997

  

Recovery data

Recovery of fortified samples, certified reference materials and quality control standards for both Fe and Zn were in the range of 90.8 – 108 % in all media.

Conclusions:
The bioaccessibility of zinc ferrite brown spinel (Bayferrox® 3950) was determined in vitro by examining dissolution in artificial body fluids at 37°C. At a loading of 100 mg test item/L, dissolved zinc concentrations (operationally defined as the dissolved Zn fraction after 0.2 µm filtration and centrifugal filtration (~2.1 nm, < 3 kDa)) were determined with 41.2 and 147 µg Zn/L after 2 and 24 h in artificial gastric fluid (GST, pH = 1.5), respectively. With mean zinc concentrations of 33.6, 71.2 and 887 µg/L, dissolution of zinc ferrite brown 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 54.0 and 453 µg/L after 2 and 24 h in artificial gastric fluid (GST, pH = 1.5), respectively. With mean iron concentrations of 39.5, 223 and 1965 µg/L, the iron release of zinc ferrite brown spinel was maximal in artificial lysosomal fluid (ALF, pH = 4.5) after 2, 24, and 168 hours of incubation, respectively. With Fe and Zn concentrations ranging from < LOD to 1.27 µg/L, very low iron and zinc 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.
In sum, zinc ferrite brown spinel (Bayferrox® 3950) 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 zinc ferrite brown spinel (Bayferrox® 3950) 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 zinc 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 zinc 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 887 µg/L Zn and < 1965 µg/L Fe after 168h. With maximum Zn and Fe concentrations of < 0.701 µg/L Zn (< LOD) and 1.27 µg/L Fe, very low zinc 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 zinc ferrite brown spinel (Bayferrox® 3950) can produce soluble available ionic and other zinc 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.