Mill scale (ferrous metal)

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Remarks:

Bioaccessibility

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-02-26 to 2018-09-26

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:

2018

Materials and methods

Objective of study:

bioaccessibility (or bioavailability)

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

test medium, loading and test duration adopted for toxicokinetics assessment

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)

Remarks:

signed 2016-05-31

Test material

Test material information

Constituent 1

Reference substance name:

Diiron trioxide

EC Number:

215-168-2

EC Name:

Diiron trioxide

Cas Number:

1309-37-1

Molecular formula:

Fe2O3

IUPAC Name:

diiron trioxide

Test material form:

solid: nanoform

Details on test material:

Appearance: red powder, odourless

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
- GMB and ALF media: samples taken after 2, 24 and 168 hours agitation (100 rpm) at 37 ± 2 °C

- two additional method blanks per medium, measurement (ICP-OES) of dissolved Fe concentrations after filtration and centrifugal filtration.
- the study was performed in triplicate

The aim of this test was to assess the dissolution of nano-size diiron trioxide 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) and Gamble’s solution (GMB). 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) or via the respiratory system (ALF).

Administration / exposure

Duration and frequency of treatment / exposure:

Iron concentrations in GST and PBS were determined after 2 and 24 h whereas iron concentrations in GMB and ALF media were assessed after 2, 24 and 168 hours of incubation.

Doses / concentrations

Dose / conc.:

100 other: 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) 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 at 37°C ± 2°C. Two control blank replicates (same procedure) per test medium were also prepared.
Three replicates containing the test item and two method blanks per artificial medium were tested. All solutions were sampled after 2 and 24 h whereas GMB and ALF media were also sampled after 168h to measure total dissolved Fe concentrations (ICP-OES) after 0.2 µm filtration (Syringe Filter w / 0.2 μm, polyethersulfon membrane, DIA Nielsen, Dueren, Germany) and centrifugal filtration (i.e. 0.2 μm filtration and 3kDa centrifugal filtration, Sartorius, Göttingen, Germany). In addition, temperature, pH and observations, including the appearance of the solution (including colour, turbidity and particle film on the surface) were recorded.


Sample fortification:
In addition, samples of the artificial physiological media were fortified with a known amount of 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".

Mass balance:
After the test, aqua regia (3 : 1 mixture of concentrated hydrochloric and nitric acid) was added to the vessels containing the test item to reach a final volume of 500 mL, i.e. 120 mL aqua regia were added to approximately 380 mL GST or PBS medium, 180 mL aqua regia were added to approximately 320 mL ALF or GMB medium. From these solutions, 50 mL were taken after 3 - 14 days of “digestion” for mass balance determination.
The filters (Syringe Filter w / 0.2 μm, polyethersulfon membrane, DIA Nielsen, Dueren, Germany) used for sampling were extensively rinsed with a known volume of aqua regia (ca. 2.5 mL). The added aqua regia was let to drop slowly through the filters and was collected in a clean vial. This procedure was repeated with every syringe and filter used during the study. After collection, the volume was filled up to exactly 10 mL (for the media GST and PBS) and up to 15 mL (for the ALF and GMB media) with aqua regia. Afterwards the concentration of iron in the “filtrated” aqua regia was determined and considered for the determination of the mass balances.

Reagents:
Purified water (resistivity > 18 MΩ·cm, Pure Lab Ultra water purification system from ELGA LabWater, Celle, Germany)
Nitric acid - “Supra” quality (ROTIPURAN® supplied by Roth, Karlsruhe, Germany).
Hydrochloric acid – “Baker-instra-analyzed-plus” quality (J.T. Baker, Griesheim, Germany).
Sodiumhydroxide – pro Analysis quality (Chemsolute, Th. Geyer, Renningen, Germany)
MgCl2 x 6H2O (p.A., Merck, Darmstadt, Germany)
NaCl (p.A., Chemsolute, Renningen, Germany + Merck, Darmstadt, Germany (new GMB medium))
KCl (p.A., Chemsolute, Renningen, Germany + Merck, Darmstadt, Germany (new GMB medium))
Na2HPO4 (p.A. Merck, Darmstadt, Germany)
Na2SO4 (p.A. Merck, Darmstadt, Germany)
CaCl2 x 2H2O (p.A. Merck, Darmstadt, Germany)
NaAcetate (suprapur Merck, Darmstadt, Germany)
NaHCO3 (p.A. Merck, Darmstadt, Germany)
NaOH (p.A., Chemsolute, Renningen, Germany)
Citric acid anhydrous (p.A., Roth, Karlsruhe, Germany)
Glycine (p.A., Merck, Darmstadt, Germany)
Na3Citrate x 2H2O (p.A., Merck, Darmstadt, Germany)
Na2Tartrate x 2H2O (p.A., Merck, Darmstadt, Germany)
NaLactate (98+% Sigma Aldrich, Munich, Germany)
NaPyruvate (p.A., Applichem, Darmstadt, Germany)
KH2PO4 (p.A., Merck, Darmstadt, Germany)
Urea (pure, Applichem, Darmstadt, Germany)
Lactic acid (purum, Fluka, Munich, Germany)
HCl 30% (instra-analyzed, plus J.T. Baker, Griesheim, Germany)


METAL ANALYSIS
- Standards for metal analysis: A commercially available single element standard was used as iron standard (Merck Certipur Iron ICP standard 1000 mg/L lot no. HC68868126; Darmstadt, Germany) to prepare an appropriate stock solution and subsequently calibration solutions for ICP-OES measurements
- Certified reference materials: As quality control standards, certified aqueous reference material TM-DWS.3 (lot no. 0916) and TMDA-70.2 (lot no. 0916 and 0917) obtained from Environment Canada and a multielement standard (Merck Certipur IV ICP standard 1000 mg/L lot no. HC54938555 and HC73962555; Darmstadt, Germany) were analysed for total dissolved iron by ICP-OES.

Instrumental and analytical set-up for the ICP-OES instrument:
Agilent 720, Agilent Technologies, Waldbronn, Germany
Nebulizer: Sea spray nebulizer, from Glass Expansion
Spray chamber: Iso Mist with Twister Helix from Glass Expansion
Plasma stabilization time: at least 30 min before start of the measurements
Plasma gas flow: 15.0 L/min
Additional gas flow: 1.50 L/min
Carrier gas flow: 0.75 L/min
RF power: 1200W
Stabilization time of sample: 15 sec
Repetition time (three internal measurements per sample): 30 sec
Wavelengths: Fe: 238.204 nm, 240.489 nm, 241.052 nm, 258.588 nm and 259.940 nm

- Correlation coefficients (r) for the wavelengths used for evaluation of data were at least >0.999603

The applied LOD/LOQ calculations for the Agilent 720 ICP-OES:
LOD: 3 * standard deviation of calibration blank/slope of the calibration
LOQ: 3 * LOD
The resulting LODs/LOQs are reported in "Any other information on results incl. tables"

Details on dosing and sampling:

Loading:
Detailed loadings of the test vessels are given in "Any other information on materials and methods incl. tables".

Results and discussion

Main ADME results

Main ADME results 1

Type:

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

Results:

16.4 µg Fe/L (dissolved)

Main ADME results 2

Type:

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

Results:

72.1 µg Fe/L (dissolved)

Main ADME results 3

Type:

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

Results:

185 µg Fe/L (dissolved)

Main ADME results 4

Type:

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

Results:

9.27 µg Fe/L (dissolved)

Main ADME results 5

Type:

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

Results:

57.5 µg Fe/L (dissolved)

Main ADME results 6

Type:

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

Results:

0.03 µg Fe/L (dissolved)

Main ADME results 7

Type:

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

Results:

below background

Main ADME results 8

Type:

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

Results:

below background

Main ADME results 9

Type:

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

Results:

< LOD/LOQ (1.54 µg Fe/L)

Main ADME results 10

Type:

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

Results:

< LOD (0.761 µg Fe/L)

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

Iron concentrations in simulated artificial body fluids:

The bioaccessibility of nano-size diiron trioxide 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 iron concentrations were operationally defined as the dissolved Fe fraction after 0.2 µm filtration and centrifugal filtration (~2.1 nm), see Table 3. With a maximum mean released fraction of <0.2% after 168 h, dissolution of Ferroxide 212P E172 was highest in artificial lysosomal fluid (ALF).

In addition, dissolved/dispersed mean iron concentrations (operationally defined as the dissolved Fe fraction after 0.2 µm filtration) are summarized in table 4.

Table 3: Iron concentrations of artificial physiological media (filtrated through a 0.2 µm membrane and centrifugally filtrated) after exposure to diiron trioxide

medium & time	LOD/LOQ of Fe measurement series	Mean Fe±SD of method blanks	Without background correction Mean Fe±SD	With background correction* Mean Fe
GST 2h	LOD:0.677 µg/L LOQ:2.03 µg/L	12.4±1.45 µg/L	21.7±6.40 µg/L	9.27 µg/L
GST 24h(1d)	LOD:0.689 µg/L LOQ:2.07 µg/L	7.77±0.14 µg/L (after outlier exclusion)	65.3±4.46 µg/L	57.5 µg/L
GMB 2h	LOD:0.206 µg/L LOQ:0.619 µg/L	2.49±0.59 µg/L	2.52±1.07 µg/L	0.03 µg/L
GMB 24h (1d)	LOD:0.190 µg/L LOQ:0.570 µg/L	2.33±0.02 µg/L	1.25±0.42 µg/L	Negative value after correction
GMB 7d	LOD:0.242 µg/L LOQ:0.725 µg/L	2.38±1.40 µg/L	1.42±0.54 µg/L	Negative value after correction
ALF 2h	LOD:0.287 µg/L LOQ:0.862 µg/L	10.3±1.01 µg/L	26.7±0.55 µg/L	16.4 µg/L
ALF 24h(1d)	LOD:0.886 µg/L LOQ:2.66 µg/L	5.64±1.83 µg/L	77.7±2.19 µg/L	72.1 µg/L
ALF 7d	LOD:0.434 µg/L LOQ:1.30 µg/L	4.92±0.48 µg/L	190±0.48 µg/L	185 µg/L
PBS 2h	LOD:0.514 µg/L LOQ:1.54 µg/L	All: <LOD(after outlier exclusion)	All: <LOD/LOQ	-
PBS 24h (1d)	LOD:0.761 µg/L LOQ:2.28 µg/L	All: <LOD	All: <LOD	-

*backgroundconcentration=meanofFeconcentrationsmeasuredafter 2h ,24 h or 168h

Table 4: Iron concentrations of artificial physiological media (filtrated through a 0.2 µm membrane) after exposure to diiron trioxide Ferroxide 212P E172

medium&time	LOD/LOQ of Fe measurement series	Mean Fe±SD of method blanks	Without background correction Mean Fe±SD	With background correction* MeanFe
GST2h	LOD:0.677 µg/L LOQ:2.03 µg/L	All: <LOQ	11.8±0.89 µg/L	No correction
GST24h(1d)	LOD:0.689 µg/L LOQ:2.07 µg/L	2.42±0.91 µg/L (1blank:<LOQ;3blanks:>LOQ)	57.0±0.72 µg/L	54.6 µg/L
GMB 2h	LOD:0.206 µg/L LOQ:0.619 µg/L	0.724±0.023 µg/L (1blank<LOD;3blanks>LOQ)	All: <LOD/LOQ
GMB 24h	LOD:0.190 µg/L LOQ:0.570 µg/L	All: <LOD/LOQ(after outlier exclusion)	All: <LOD (after outlier exclusion)
GMB 168h	LOD:0.242 µg/L LOQ:0.725 µg/L	All: <LOD(after outlier exclusion)	All: <LOD
ALF2h	LOD:0.287 µg/L LOQ:0.862 µg/L	1.96±0.18 µg/L	24.8±0.37 µg/L	22.9 µg/L
ALF24h	LOD:0.886 µg/L LOQ:2.66 µg/L	All: <LOQ(after outlier exclusion)	76.6±0.58 µg/L	No correction
ALF168h	LOD:0.434 µg/L LOQ:1.30 µg/L	2.39±0.54 µg/L	190±1.21 µg/L	188 µg/L
PBS 2h	LOD:0.514 µg/L LOQ:1.54 µg/L	All: <LOD (after outlier exclusion)	All: <LOD/LOW (after outlier exclusion)
PBS 24h	LOD:0.761 µg/L LOQ:2.28 µg/L	All: <LOD	All: <LOD

*background concentration=mean of Fe concentrations measured after 2h ,24 h or 168h

Mass balance

Total mass recoveries were determined by aqua regia digestion for each test item containing vessel at the end of the experiment. Regarding Ferroxide 212P E172, mass recoveries in all media investigated (GST, GMB, ALF, PBS) were > 95.0%.

Solution pH -GST

blank vessels

sample name	target pH	pH prior to the test	pH after 2h	pH after 24 h
GSTblankvessel1	1.5–1.6	1.55	1.57	1.59
GSTblankvessel2	1.5–1.6	1.54	1.58	1.58

Ferroxide212P E172

sample name	target pH	pH prior to the test	pH after 2h		pH after 24h
GSTvessel4	1.5–1.6	1.56		1.62	1.63
GSTvessel5	1.5–1.6	1.55		1.61	1.65
GSTvessel6	1.5–1.6	1.55		1.60	1.64

Solution pH - PBS

blank vessels

samplename	targetpH	pHpriortothetest	pHafter2h	pHafter24h
PBSblankvessel1	7.2–7.4	7.30	7.30	7.33
PBSblankvessel2	7.2–7.4	7.30	7.33	7.33

Ferroxide212P E172

samplename	targetpH	pHpriortothetest	pHafter2h	pHafter24h
PBSvessel4	7.2–7.4	7.31	7.35	7.33
PBSvessel5	7.2–7.4	7.29	7.32	7.33
PBSvessel6	7.2–7.4	7.30	7.32	7.36

Solution pH - GMB

blank vessels

samplename	targetpH	pHpriortothetest	pHafter2h	pHafter24h	pHafter7d
GMBblankvessel1	7.4	7.46	8.10	8.68	9.09
GMBblankvessel2	7.4	7.46	8.14	8.74	9.09

Ferroxide212P E172

samplename	targetpH	pHpriortothetest	pHafter2h	pHafter24h	pHafter7d
GMBvessel4	7.4	7.45	8.19	8.80	9.24
GMBvessel5	7.4	7.46	8.20	8.71	9.24
GMBvessel6	7.4	7.45	8.22	8.86	9.22

Solution pH - ALF

blank vessels

samplename	targetpH	pHpriortothetest	pHafter2h	pHafter24h	pHafter7d
ALFblankvessel1	4.5	4.56	4.57	4.62	4.62
ALFblankvessel2	4.5	4.56	4.59	4.62	4.62

Ferroxide212P E172

samplename	targetpH	pHpriortothetest	pHafter2h	pHafter24h	pHafter7d
ALFvessel4	4.5	4.56	4.59	4.62	4.63
ALFvessel5	4.5	4.55	4.60	4.62	4.62
ALFvessel6	4.5	4.55	4.59	4.62	4.62

Test temperature:

With 37 °C ± 2 °C, the temperature was stable during the test for all solutions

 

Method validation summary (ICP-OES)

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

Limits of detection (LOD) within all measurement series: < 1.24 µg Fe/ L.

Limits of quantification (LOQ) within all measurement series: < 3.73 µg Fe/ L.

Correlation coefficients (r) within all measurement series: >0.999603

 

GST

Mean recovery of fortified samples (n = 20): 98.6 - 104 %

Mean recoveries for certified reference materialTM-DWS.3andTMDA-70.2(concentration range 22.4 – 75.4 µg Fe / L, n = 24): 103 -105 %

Mean recoveries for quality control standard (concentration range 50 -500 µg Fe/ L, n = 24): 98.2 - 105 %

Mean recoveries for internal standard (concentration range 10 -300 µg / L, n = 24): 98.4 - 100 %

 

PBS

Mean recovery of fortified samples (n = 32): 90.5 - 108 %

Mean recoveries for certified reference materialTM-DWS.3andTMDA-70.2(concentration range 22.4 – 75.4 µg Fe / L, n = 20): 98.0-101 %

Mean recoveries for quality control standard (concentration range 5 -50 µg Fe/ L, n = 30): 97.0 – 99.9 %

Mean recoveries for internal standard (concentration range 10 -100 µg / L, n = 30): 97.5 – 99.8 %

 

GMB

Mean recovery of fortified samples (n = 48): 92.2 - 128 %

Mean recoveries for certified reference materialTM-DWS.3andTMDA-70.2(concentration range 22.4 – 75.4 µg Fe / L, n = 59): 95.5-107 %

Mean recoveries for quality control standard (concentration range 5 -50 µg Fe/ L, n = 60): 95.5 – 104 %

Mean recoveries for internal standard (concentration range 10 -100 µg / L, n = 60): 97.0 – 102 %

 

ALF

Mean recovery of fortified samples (n = 24): 85.5 - 106 %

Mean recoveries for certified reference materialTM-DWS.3andTMDA-70.2(concentration range 22.4 – 75.4 µg Fe / L, n = 48): 93.6-103 %

Mean recoveries for quality control standard (concentration range 5 -200 µg Fe/ L, n = 48): 96.1-100 %

Mean recoveries for internal standard (concentration range 10 -100 µg / L, n = 48): 95.0 – 106 %

Method validation – mass balance measurements

Mean recovery of fortified samples (n = 27): 89.4 - 106 %

Mean recoveries for certified reference materialTM-DWS.3andTMDA-70.2(concentration range 22.4 – 75.4 µg Fe / L, n = 30): 100-109 %

Mean recoveries for quality control standard (concentration range 250 -600 µg Fe/ L, n = 30): 100-103 %

Mean recoveries for internal standard (concentration range 100 -400 µg / L, n = 30): 100 – 106 %

Applicant's summary and conclusion

Conclusions:

The bioaccessibility of nano-size diiron trioxide 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. After 2 and 24 h in phosphate buffered saline (PBS, pH 7.2-7.4) solution, dissolved iron concentrations (operationally defined as the dissolved Fe fraction after 0.2 µm filtration and centrifugal filtration (~2.1 nm)) were at least below the LOQ (<1.54 µg/L). With mean concentrations of 0.03 µg Fe/L after 2 h and negative values (after subtraction of background) after 24 and 168 h, dissolved iron concentrations in Gamble´s solution (GMB, pH 7.4) were also very low. In artificial gastric fluid (GST, pH 1.5-1.6), 9.27 µg Fe/L and 57.5 µg Fe/L were detected in the dissolved phase after 2 and 24 h, respectively. Mean iron concentrations were highest in artificial lysosomal fluid (ALF, pH 4.5): 16.4 µg/L, 72.1 µg/L and 185 µg/L of iron were found in the dissolved phase after 2, 24 and 168 h. Therefore, with a maximum mean released fraction of <0.2% after 168 h, the dissolution of diiron trioxide was highest in artificial lysosomal fluid (ALF).