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Diss Factsheets

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-07-16 to 2012-09-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
2013

Materials and methods

Objective of study:
other: Bioaccessibilty
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The objective of this study was to assess the dissolution of Iron cobalt chromite black spinel (IPC-2013-008) in different artificial physiological media. The test media were selected to simulate relevant human-chemical interactions (as far as practical), i.e. contact of a test substance with skin, a substance entering the human body by inhalation or by ingestion into the gastro-intestinal tract. The dissolved amount of the test item was specified by the mass concentration of the substance in the test media under the applied test conditions. The total dissolved amount was determined by measuring the total concentrations of Co, Cr, Fe, Mn and Ni in solution. The test was performed on the basis of OECD Series on Testing and Assessment No. 29 (2001; ENV/JM/ MONO(2001)9): “Guidance Document on Transformation / Dissolution of Metals and Metal Compounds in Aqueous Media” as well as according to the bioaccessibility test protocol provided by the monitor. The bioaccessibility protocol has been developed on the basis of relevant published methods.

For the experimental setup the test item was weighed into flasks, put to volume with the respective artificial physiological medium (loading of approx. 100 mg/L) and agitated at 100 rpm at 37°C ± 2 °C. Samples were taken after 2h and 24h. The total dissolved chromium, cobalt, iron, manganese and nickel concentrations were determined after filtration (0.2 µm, Supor membrane) by ICP-MS and ICP-OES.

GLP compliance:
yes (incl. QA statement)
Remarks:
2011-02-07

Test material

Constituent 1
Chemical structure
Reference substance name:
Iron cobalt chromite black spinel
EC Number:
269-060-5
EC Name:
Iron cobalt chromite black spinel
Cas Number:
68186-97-0
Molecular formula:
(Co, Fe)(Fe,Cr)2O4
IUPAC Name:
dichromium(3+) diiron(3+) λ²-cobalt(2+) λ²-iron(2+) octaoxidandiide
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Substance name: Iron cobalt chromite black spinel
Appearance: solid, black powder, odourless
Storage: Always keep dry in tightly closed containers in adequately ventilated premises.

Test animals

Species:
other: in vitro (simulated human body fluids)
Details on test animals or test system and environmental conditions:
For the experimental setup the test item was weighed into flasks, put to volume with the respective artificial physiological medium (loading of approx. 100 mg/L) and agitated at 100 rpm at 37°C ± 2 °C. Samples were taken after 2h and 24h. The total dissolved chromium, cobalt, iron, manganese and nickel concentrations were determined after filtration (0.2 µm, Supor membrane) by ICP-MS and ICP-OES.

Administration / exposure

Details on dosing and sampling:
For the experimental setup the test item was weighed into flasks, put to volume with the respective artificial physiological medium (loading of approx. 100 mg/L) and agitated at 100 rpm at 37°C ± 2 °C. Samples were taken after 2h and 24h. The total dissolved chromium, cobalt, iron, manganese and nickel concentrations were determined after filtration (Syringe Filter w / 0.2 µm, polyethersulfon membrane, DIA Nielsen, Dueren, Germany) by ICP-MS and ICP-OES.

Aqueous samples of approx. 20 mL taken for Cr, Co, Fe, Mn and Ni analysis were transferred into disposable scintillation vials (20 mL scintillation tubes, Sarstedt, Nuembrecht, Germany), acidified (target conc. 3% HNO3) and stored at approx. 4°C until analysis.

The maximum storage time until measurement of the samples was less than three months. According to DIN EN ISO 5667-3: 2003 dissolved metals in aqueous samples (waste-, ground- and surface-water) are at least stable for six months. Furthermore the analysed CRMs are stable under these conditions for at least one year from the date of shipment. Acidification is the stabilisation method that is recommended in standard methods for metal analysis (e.g. ISO 11885, DIN). Solutions pHs were measured directly in the test vessel.

Analysis of mass balance by ICP-OES
Chromium, cobalt, iron, manganese and nickel concentrations of aqueous mass balance samples were measured using an IRIS Intrepid II ICP-OES (Thermo Electron, Dreieich, Germany). Chromium was detected at the wavelengths 205.552 nm, 267.716 nm and 283.563 nm. Cobalt was detected at the wavelengths 228.616 nm, 230.786 nm and 231.160 nm. Iron was detected at the wavelengths 238.204, 239.562 nm and 259.940 nm. Manganese was detected at the wavelengths 257.610 nm, 260.569 nm and 293.931 nm. Nickel was detected at the wavelengths 221.647 nm and 231.604 nm. Depending on concentrations of the mass balance samples the following solutions were used to calibrate the instrument: blank, 1.0 µg/L, 5.0 µg/L, 10 µg/L, 50 µg/L, 100 µg/L, 200 µg/L, 300 µg/L, 400 µg/L and 500 µg/L. Calibrations were performed before the measurement. The calibration formula was calculated using the linear regression algorithm of the ICP-OES instrument software. The respective wavelength data with the best correlation for the calibration in the measurements were used for calculating concentrations (i.e. 283.563 nm for chromium, 259.940 nm for iron and 260.569 nm for manganese). Correlation coefficients (r) were at least 0.9998. For each sample, at least three internal measurements were performed and the mean was calculated and printed by the instrument software.
One measurement was performed for the determination of chromium, cobalt, iron, manganese and nickel concentrations in the mass balance samples.

Analysis of total dissolved Fe concentrations by ICP-OES
The analysis of total dissolved iron in medium samples was performed using an IRIS Intrepid II ICP-OES (Thermo Electron, Dreieich, Germany). Iron detected at the wavelengths 238.204 nm, 239.562 nm and 259.940 nm. The following iron solutions were used to calibrate the instrument: blank, 1.0 µg/L, 2.5 µg/L, 5.0 µg/L, 7.5 µg/L, 10 µg/L, 25 µg/L, 50 µg/L, 75 µg/L and 100 µg/L. Calibration was performed before measurement. The calibration formula was calculated using the linear regression algorithm of the ICP-OES instrument software. The respective wavelength data with the best correlation for the calibration in the measurement was used for calculating concentrations (i.e. 259.940 nm for iron). Correlation coefficient (r) was 0.9995. For each sample, at least three internal measurements were performed and the mean was calculated and printed by the instrument software.
One measurement was performed for the determination of iron concentrations in the media samples.
The applied LOD/LOQ calculations are:

LOD: 3 * method standard deviation from calibration line;
LOQ: 10 * method standard deviation from calibration line.

These data were read directly from the Thermo IRIS Intrepid II ICP-OES instrument output (data calculated by internal algorithms of the instrument software).

July 31, 2012; Measurement of iron in all media samples, blank samples and fortification
LOD(µg/L) LOQ(µg/L) correlation coefficient
3.63 12.1 0.9995

September 19, 2012; Measurement of mass balance all media vessels, syringe and filters
LOD(µg/L) LOQ(µg/L) correlation coefficient
Cr: 6.00 Cr: 20.0 Cr: 0.9998
Fe: 8.48 Fe: 28.3 Fe: 0.9998
Mn: 5.96 Mn: 19.9 Mn: 0.9999

Analysis of total dissolved Cr, Co, Mn and Ni concentrations by ICP-MS
The analysis of total dissolved chromium, cobalt, manganese and nickel in medium samples was performed using an Agilent 7700 ICP-MS with collision cell (Agilent, Waldbronn, Germany). Chromium was detected on the isotopes 52Cr and 54Cr , cobalt on 59Co, manganese on 55Mn and nickel on 60Ni and 61Ni. Depending on the concentration range of the samples, the following standard solutions were used for the calibrations: blank, 0.1 µg/L, 0.25 µg/L, 0.5 µg/L, 0.75 µg/L, 1.0 µg/L, 2.5 µg/L, 5.0 µg/L, 7.5 µg/L, 10.0 µg/L, 25.0 µg/L, 50.0 µg/L, 75.0 µg/L and 100 µg/L. A respective concentration range was selected for calibration to obtain a working range that covers chromium, cobalt, manganese and nickel in samples.
If necessary, samples were diluted to obtain Cr, Co, Mn and Ni concentrations that were in the optimal working range of the method (e.g. 1 mL of media sample diluted with 4 mL of acidified ultrapure water for a dilution factor of 5). The calibration formula was calculated using the linear regression algorithm of the ICP-MS instrument software. The respective isotope and measurement mode (no Gas, Helium or HiHelium mode) for interference free measurement with the best recoveries for certified reference materials were used for calculating concentrations. Correlation factors (r) were at least 0.9977 for measurement series. For each sample, at least three internal measurements were performed and the mean was calculated and printed by the instrument software.

In sum, five series of measurements were performed for the determination of total dissolved chromium, cobalt, manganese and nickel concentrations in samples including blanks to determine background levels of elements and fortified samples.
The LOD and LOQ for chromium, cobalt, manganese and nickel were calculated using the internal instrument algorithm. This calculation is according to DIN 32645. For this the standard deviation of calibration blanks is multiplied by 3 and divided by the slope of the calibration line. In Table 9 the LODs and LOQs for chromium, cobalt, manganese and nickel are shown for the different media.

July 23, 2012; Measurement of GST media all sample vessels, blank vessels and fortification
LOD(µg/L) LOQ(µg/L) correlation coefficient
Cr: 0.01 Cr: 0.03 Cr: 0.9999
Co: 0.004 Co: 0.01 Co: 0.9999
Mn: 0.01 Mn: 0.02 Mn: 0.9999
Ni: 0.02 Ni: 0.07 Ni: 0.9999

July 24, 2012; Measurement of GMB media all sample vessels, blank vessels and fortification
LOD(µg/L) LOQ(µg/L) correlation coefficient
Cr: 0.08 Cr: 0.25 Cr: 0.9997
Co: 0.01 Co: 0.02 Co: 0.9998
Mn: 0.02 Mn: 0.06 Mn: 0.9998
Ni: 0.03 Ni: 0.10 Ni: 0.9988

July 25, 2012; Measurement of ALF media all sample vessels, blank vessels and fortification
LOD(µg/L) LOQ(µg/L) correlation coefficient
Cr: 0.06 Cr: 0.17 Cr: 0.9998
Co: 0.02 Co: 0.05 Co: 0.9999
Mn: 0.11 Mn: 0.34 Mn: 0.9999
Ni: 0.06 Ni: 0.18 Ni: 0.9998

July 25, 2012; Measurement of ASW media all sample vessels, blank vessels and fortification
LOD(µg/L) LOQ(µg/L) correlation coefficient
Cr: 0.02 Cr: 0.06 Cr: 0.9997
Co: 0.01 Co: 0.04 Co: 0.9995
Mn: 0.01 Mn: 0.03 Mn: 0.9999
Ni: 0.07 Ni: 0.20 Ni: 0.9993

July 26, 2012; Measurement of PBS media all sample vessels, blank vessels and fortification
LOD(µg/L) LOQ(µg/L) correlation coefficient
Cr: 0.09 Cr: 0.28 Cr: 0.9999
Co: 0.01 Co: 0.04 Co:0.9999
Mn: 0.02 Mn: 0.05 Mn:0.9999
Ni: 0.06 Ni: 0.18 Ni:0.9998

Results and discussion

Any other information on results incl. tables

Solution pH value

After preparation of the artificial physiological media, solution pHs were adjusted to their respective target pH. The target pH in all media before addition of test substance was in the required pH range. In ASW medium, the pH in all vessels (including method blanks) decreased during the time of the test from 6.5 to 6.2 -6.1 (sample vessels) and 6.1- 5.9 (method blank vessels). In GMB medium, the pH in all vessels (including method blanks) increased during the time of the test from 7.4 to 7.7 (sample vessels) and 7.6 -7.7 (method blank vessels). Therefore, an effect of the test substance can be excluded.

pHs of the physiological test media prior to the test

media

target pH

measured pH

temp.
[°C]

ALF

4.5

4.5

30.4

ASW

6.5

6.5

33.2

GMB

7.4

7.4

31.8

GST

1.5 – 1.6

1.5

35.4

PBS

7.2 - 7.4

7.4

35.0

 

Analytical measurements

Chromium, cobalt, iron, manganese and nickel concentrations in method blanks measured by ICP-MS and ICP-OES were below the limit of detection or limit of quantification for mainly all test media. In GST media all method blanks were above the LOD/LOQ nut at least 1.8-fold lower than the concentration in the analysed samples. In ALF media chromium and iron concentration were above the LOD/LOQ but at least 1.4-fold lower than in the analysed samples and for PBS media manganese in the method blanks were above the LOD/LOQ but at least 3.6-fold lower than in the measured samples.

The LOD and LOQ for chromium were 0.01 and 0.03 µg/L for GST, 0.02 and 0.06 µg/L for ASW, 0.08 and 0.25 µg/L for GMB, 0.06 and 0.17 µg/L for ALF and 0.09 and 0.28 µg/L for PBS.

The LOD and LOQ for cobalt were 0.004 and 0.01 µg/L for GST, 0.02 and 0.06 µg/L for ASW, 0.01 and 0.02 µg/L for GMB, 0.02 and 0.05 µg/L for ALF and 0.01 and 0.04 µg/L for PBS.

The LOD and LOQ for iron were 3.63 and 12.1 µg/L for the sample measurement series in all media.

The LOD and LOQ for manganese were 0.01 and 0.02 µg/L for GST, 0.01 and 0.03 µg/L for ASW, 0.02 and 0.06 µg/L for GMB, 0.11 and 0.34 µg/L for ALF and 0.02 and 0.05 µg/L for PBS.

The LOD and LOQ for nickel were 0.02 and 0.07 µg/L for GST, 0.07 and 0.20 µg/L for ASW, 0.03 and 0.10 µg/L for GMB, 0.06 and 0.18 µg/L for ALF and 0.06 and 0.18 µg/L for PBS.

Concentration of chromium in artificial media, calculated nominal chromium concentration in weighted sample mg/L Iron cobalt chromite black spinel and dissolved amount of chromium.

media and sample

total Cr ± SD in method blanks [µg/L]

total Cr ±SD in sample vessels [µg/L]

Cr ± SD in sample vessels with blank subtraction [µg/L]

calculated nominal Cr concentration in [µg/L]#

dissolved amount Cr in artificial media [%] normalizedfor measured background in method blank

ALF 2h

0.21 ± 0.01

2.65 ± 0.08

2.44 ± 0.08

22227

0.01 ± <0.01

ALF 24h

0.23

8.05 ± 0.07

7.82 ± 0.07

22227

0.04 ± <0.01

ASW 2h

0.08

1.51 ± 0.40

1.43 ± 0.40

22281

0.01 ± <0.01

ASW 24h

0.42

2.35 ± 0.20

1.94 ± 0.20

22281

0.01 ± <0.01

GMB 2h

<LOD

0.88 ± 0.24

0.88 ± 0.24

22147

0.004 ± 0.001

GMB 24h

<LOD

1.24 ± 0.30

1.24 ± 0.30

22147

0.01 ± <0.01

GST 2h

0.29 ± 0.06

3.75 ± 0.20

3.46 ± 0.20

22262

0.02 ± <0.01

GST 24h

0.33 ± 0.02

9.39 ± 0.23

9.06 ± 0.23

22262

0.04 ± <0.01

PBS 2h

<LOD

1.29 ± 0.16

1.29 ± 0.16

22196

0.01 ± <0.01

PBS 24h

<LOD

1.93 ± 0.16

1.93 ± 0.16

22196

0.01 ± <0.01

In five different artificial physiological media, between 0.004 and 0.04% of chromium was dissolved from the test item IPC-2013-008 Iron cobalt chromite black spinel depending on solution parameters and test duration.

Concentration of manganese in artificial media, calculated nominal manganese concentration in weighted sample mg/L Iron cobalt chromite black spinel and dissolved amount of manganese.

media and sample

total Mn ± SD in method blanks [µg/L]

total Mn ±SD in sample vessels [µg/L]

Mn ± SD in sample vessels with blank subtraction [µg/L]

calculated nominal Mn concentration in [µg/L]#

dissolved amount Mn in artificial media [%] normalizedfor measured background in method blank

ALF 2h

<LOD

6.16 ± 0.10

6.16 ± 0.10

5277

0.12 ± <0.01

ALF 24h

<LOD

17.1 ± 0.22

17.1 ± 0.22

5277

0.32 ± <0.01

ASW 2h

<LOD/LOQ

0.29 ± 0.23

0.29 ± 0.23

5290

0.01 ± <0.01

ASW 24h

<LOD/LOQ

2.40 ± 1.01

2.40 ± 1.01

5290

0.05 ± 0.02

GMB 2h

<LOQ

0.11 ± 0.01

0.11 ± 0.01

5258

0.002 ± <0.001

GMB 24h

<LOD/LOQ

0.31 ± 0.03

0.31 ± 0.03

5258

0.01 ± <0.01

GST 2h

0.18 ± 0.06

6.60 ± 0.15

6.43 ± 0.15

5285

0.12 ± <0.01

GST 24h

0.16 ± 0.07

16.4 ± 0.15

16.2 ± 0.15

5285

0.31 ± <0.01

PBS 2h

<LOQ

0.18 ± 0.03

0.18 ± 0.03

5269

0.003 ± 0.001

PBS 24h

0.05 ± 0.01

0.80 ± 0.12

0.75 ± 0.12

5269

0.01 ± <0.01

In five different artificial physiological media, between 0.002 and 0.32% of manganese was dissolved from the test item IPC-2013-008 Iron cobalt chromite black spinel depending on solution parameters and test duration

Concentration of cobalt in artificial media, calculated nominal cobalt concentration in weighted sample mg/L Iron cobalt chromite black spinel and dissolved amount of cobalt.

media and sample

total Co ± SD in method blanks [µg/L]

total Co ±SD in sample vessels [µg/L]

Co ± SD in sample vessels with blank subtraction [µg/L]

calculated nominal Co concentration in [µg/L]#

dissolved amount Co in artificial media [%] normalizedfor measured background in method blank

ALF 2h

<LOD

5.39 ± 0.09

5.39 ± 0.09

13651

0.04 ± <0.01

ALF 24h

<LOD

13.5 ± 0.11

13.5 ± 0.11

13651

0.10 ± <0.01

ASW 2h

<LOD

0.33 ± 0.31

0.33 ± 0.31

13684

0.002 ± 0.002

ASW 24h

<LOD

2.68 ± 0.92

2.68 ± 0.92

13684

0.02 ± 0.01

GMB 2h

<LOD

0.19 ± 0.01

0.19 ± 0.01

13602

0.001 ± <0.001

GMB 24h

<LOD

0.66 ± 0.05

0.66 ± 0.05

13602

0.01 ± <0.01

GST 2h

2.80 ± 0.11

7.93 ± 0.12

5.13 ± 0.12

13672

0.04 ± <0.01

GST 24h

2.73 ± 0.02

16.4 ± 0.17

13.7 ± 0.17

13672

0.10 ± <0.01

PBS 2h

<LOD

0.25 ± 0.03

0.25 ± 0.03

13632

0.002 ± <0.001

PBS 24h

<LOD/LOQ

1.19 ± 0.17

1.19 ± 0.17

13632

0.01 ± <0.01

In five different artificial physiological media, between 0.001 and 0.10% of cobalt was dissolved from the test item IPC-2013-008 Iron cobalt chromite black spinel depending on solution parameters and test duration

Concentration of nickel in artificial media, calculated nominal nickel concentration in weighted sample mg/L Iron cobalt chromite black spinel and dissolved amount of nickel.

media and sample

total Ni ± SD in method blanks [µg/L]

total Ni ±SD in sample vessels [µg/L]

Ni ± SD in sample vessels with blank subtraction [µg/L]

calculated nominal Ni concentration in [µg/L]#

dissolved amount Ni in artificial media [%] normalizedfor measured background in method blank

ALF 2h

<LOD

1.32 ± 0.02

1.32 ± 0.02

5549

0.02 ± <0.01

ALF 24h

<LOD

4.34 ± 0.03

4.34 ± 0.03

5549

0.08 ± <0.01

ASW 2h

<LOD

<LOD/LOQ

<LOD/LOQ

5563

-

ASW 24h

<LOD

0.73 ± 0.27

0.73 ± 0.27

5563

0.01 ± <0.01

GMB 2h

<LOD

0.12 ± 0.03

0.12 ± 0.03

5529

0.002 ± <0.001

GMB 24h

<LOD

0.29 ± 0.02

0.29 ± 0.02

5529

0.01 ± <0.01

GST 2h

1.41 ± 0.19

2.63 ± 0.04

1.22 ± 0.04

5558

0.02 ± <0.01

GST 24h

1.40 ± 0.20

5.79 ± 0.11

4.39 ± 0.11

5558

0.08 ± <0.01

PBS 2h

<LOD

<LOD

<LOD

5541

-

PBS 24h

<LOD

0.29 ± 0.05

0.29 ± 0.05

5541

0.01 ± <0.01

In five different artificial physiological media, between 0.002% and 0.08% of nickel was dissolved from the test item IPC-2013-008 Iron cobalt chromite black spinel depending on solution parameters and test duration

Concentration of iron in artificial media, calculated nominal iron concentration in weighted sample mg/L Iron cobalt chromite black spinel and dissolved amount of iron.

media and sample

total Fe ± SD in method blanks [µg/L]

total Fe ±SD in sample vessels [µg/L]

Fe ± SD in sample vessels with blank subtraction [µg/L]

calculated nominal Fe concentration in [µg/L]#

dissolved amount Fe in artificial media [%] normalizedfor measured background in method blank

ALF 2h

27.0 ± 0.43

40.5 ± 0.58

13.5 ± 0.58

23992

0.06 ± <0.01

ALF 24h

26.3 ± 0.14

78.4 ± 0.74

52.1 ± 0.74

23992

0.22 ± <0.01

ASW 2h

<LOD

<LOD

<LOD

24051

-

ASW 24h

<LOD

<LOD/LOQ

<LOD/LOQ

24051

-

GMB 2h

<LOD

<LOD

<LOD

23906

-

GMB 24h

<LOD

<LOD

<LOD

23906

-

GST 2h

<LOD/LOQ

22.3 ± 1.10

22.3 ± 1.10

24030

0.09 ± 0.01

GST 24h

<LOQ

64.6 ± 1.92

64.6 ± 1.92

24030

0.27 ± 0.01

PBS 2h

<LOQ

<LOQ

<LOQ

23959

-

PBS 24h

12.3

<LOQ

<LOQ

23959

-

In five different artificial physiological media, between 0.06% and 0.27% of nickel was dissolved from the test item IPC-2013-008 Iron cobalt chromite black spinel depending on solution parameters and test duration

Applicant's summary and conclusion

Conclusions:
The bioaccessibility of iron cobalt chromite black spinel has been investigated experimentally in vitro by simulating dissolution under physiological conditions considered to mimic the most relevant exposure routes (oral, dermal and inhalation). Dissolved Cr, Ni, Co, Mn and Fe concentrations (in GST/ALF) were below 9.06 µg/L, 4.39 µg/L, 13.7 µg/L, 17.1 µg/L and 65 µg/L respectively, even at the highest loading of 0.1g/L, referring to a solubility of 0.009%, 0,005%, 0,014, 0,017 % and 0.07%, the pigment is considered biologically inert.
Executive summary:

The bioaccessibility of iron cobalt chromite black spinel has been investigated experimentally in vitro by simulating dissolution under physiological conditions considered to mimic the most relevant exposure routes (oral, dermal and inhalation), as follows:

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

- phosphate-buffered saline (PBS, pH 7.2), which is a standard physiological solution that mimics the ionic strength of human blood serum,

- artificial sweat (ASW, pH 6.5) which simulates the hypoosmolar fluid, linked to hyponatraemia (loss of Na+ from blood), which is excreted from the body upon sweating,

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

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

Under the conditions of this test (flasks with different artificial physiological media; loadings of 50 mg Iron cobalt chromite black spinel / 500 mL, 37°C, sampling after 2h and 24h), the concentrations of dissolved chromium, cobalt, iron, manganese and nickel were between 0.005% and 0.07%.

Therefore, iron cobalt chromite black spinel may reasonably be considered not bioaccessible.