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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

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

Materials and methods

Objective of study:
bioaccessibility (or bioavailability)
GLP compliance:
yes (incl. QA statement)
Remarks:
2011-02-07

Test material

Constituent 1
Chemical structure
Reference substance name:
Nickel iron chromite black spinel
EC Number:
275-738-1
EC Name:
Nickel iron chromite black spinel
Cas Number:
71631-15-7
Molecular formula:
(Ni,Fe)(Fe,Cr)2O4
IUPAC Name:
Nickel iron chromite black spinel
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Substance name: Nickel iron chromite black spinel
Appearance: solid, black powder, odourless

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, iron, manganese and nickel concentrations of sampled solutions 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, iron, manganese and nickel concentrations of sampled solutions were determined after filtration (0.2 µm, Supor membrane) by ICP-MS and ICP-OES.

Five different artificial physiological media, single loading of test substance of 100 mg/L, measurement of dissolved chromium, iron, manganese and nickel concentrations after 2 and 24 hours agitation (100 rpm) at 37 ± 2°C, two additional method blanks per medium. The study was performed in duplicates.

Two additional control blanks (same procedure) were also prepared. Two replicates and two method blanks per artificial media were tested; solutions were sampled after 2 and 24h, to measure total dissolved chromium, iron, manganese and nickel concentrations (by ICP-OES), temperature and pH.

Solutions were sampled for measuring total dissolved chromium, iron, manganese and nickel by ICP-MS and ICP-OES. All samples were filtered through 0.2 µm filter (Syringe Filter w / 0.2 µm, polyethersulfon membrane, DIA Nielsen, Dueren, Germany) prior to further treatment.

Aqueous samples of approx. 20 mL taken for Cr, 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 fou 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 (see section 15.2). Acidification is the stabilization method that is recommended in standard methods for metal analysis (e.g. ISO 11885, DIN).
Solution pH was measured directly in the test vessel.

The analysis of total dissolved iron in samples and iron and manganese in mass balance samples were measured using an IRIS Intrepid II ICP-OES (Thermo Electron, Dreieich, Germany). Iron was detected at the wavelengths 238.204 nm, 239.526 nm and 259.940 nm. Manganese was detected at the wavelengths 257.610 nm, 260.569 nm and 293.931 nm. Depending on concentrations of the samples, the following solutions were used to calibrate the instrument: blank, 1 µg/L, 5 µg/L, 7,5 µg/L, 10 µg/L, 25 µg/L, 50 µg/L, 100 µg/L, 200 µg/L, 300 µg/L and 400 µg/L. Calibrations were performed before each measurement. The calibration formula was calculated using the linear regression algorithm of the ICP-OES instrument software for both ICP-OES systems. The respective wavelength data with the best correlation for the calibration and the best recoveries for the validation samples (certified reference material and recalibration standards) in the measurements were used for calculating concentrations (i.e. 259.940 nm for iron). Correlation coefficients (r) were at least 0.9999. For each sample, at least three internal measurements were performed and the mean was calculated and printed by the instrument software.

Instrumental and analytical set-up for the ICP-OES instrument:
Thermo IRIS Intrepid II from Thermo Electron Corporation, Germany
Nebulizer: Concentric glass nebulizer, from Thermo
Spray chamber: Glass cyclonic spray chamber, from Thermo
Nebulizer gas flow: 0.68 L/min
Make-up gas flow: 0.5 L/min
RF power: 1150 W

Wavelengths: Fe: 238.204 nm, 239.526 nm and 259.940 nm; Mn: 257.610 nm, 260.569 nm and 293.931 nm
Two measurements were performed for the determination of iron and manganese concentrations in the test item samples, method blanks, mass balance samples and filter samples.

The applied LOD/LOQ calculations for the IRIS Intrepid ICP-OES 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).

The analysis of total dissolved chromium, manganese and nickel in medium samples was performed using an Agilent 7700 ICP-MS with collision cell (Agilent, Waldbronn, Germany). Chromium was quantified by measuring the isotopes 52Cr and 54Cr, manganese by measuring 55Mn and nickel by measuring 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, manganese and nickel concentrations in samples.
The calibration formula was calculated using the linear regression algorithm of the ICP-MS instrument software. The respective isotope and measurement mode (noGas, Helium or HiHelium mode) for interference free measurement with the best recoveries for certified reference materials were used for calculating the respective test concentrations. Correlation factors (r) were at least 0.9989 in all measurement series. For each sample, at least three internal measurements were performed and the mean was calculated and printed by the instrument software.

Instrumental and analytical set-up for the ICP-MS instrument:
Agilent 7700 ICP-MS, Agilent Technologies, Waldbronn, Germany
Nebulizer: Concentric glass nebulizer, from GlassExpansion
Spray chamber: Scott Type spray chamber, from Agilent
Carrier gas flow: 0.91 L/min
Dilution/Make-up gas flow: 0.13 L/min
RF power: 1500 W
Isotopes: 52Cr, 54Cr, 55Mn, 60Ni, 61N, 103Rh (internal standard)

In sum, five series of measurements were performed for the determination of total dissolved chromium, manganese and nickel concentrations in samples including the test vessels as well as blanks to determine background levels of elements and fortified samples.
The LOD and LOQ for chromium, manganese and nickel were calculated using the internal instrument algorithm.

Results and discussion

Any other information on results incl. tables

Measured pH in method blank and test vessels in ALF medium

sample name

target pH

pH prior to the test

temp. [°C] prior to the test

pH after 2h

temp.[°C]

pH after 24h

temp.[°C]

ALF vessel A

4.5

4.5

32.9

4.5

33.4

4.5

32.7

ALF vessel B

4.5

4.5

32.8

4.5

33.1

4.5

32.7

ALF blank vessel A

4.5

4.5

32.5

4.5

34.8

4.5

34.6

ALF blank vessel B

4.5

4.5

32.7

4.5

34.9

4.5

34.4

 Measured pH in method blank and test vessels in ASW medium

sample name

target pH

pH prior to the test

temp. [°C] prior to the test

pH after 2h

temp.[°C]

pH after 24h

temp.[°C]

ASW vessel A

6.5

6.5

34.9

6.3

34.7

6.0

34.3

ASW vessel B

6.5

6.5

34.7

6.3

34.4

5.9

34.2

ASW blank vessel A

6.5

6.5

35.3

6.3

35.8

6.0

35.7

ASW blank vessel B

6.5

6.5

35.2

6.3

35.9

5.9

35.8

 

Measured pH in method blank and test vessels in GMB medium

sample name

target pH

pH prior to the test

temp. [°C] prior to the test

pH after 2h

temp.[°C]

pH after 24h

temp.[°C]

GMB vessel A

7.4

7.4

33.8

4.5

34.2

7.7

33.6

GMB vessel B

7.4

7.4

33.7

7.5

34.0

7.8

33.5

GMB blank vessel A

7.4

7.4

33.5

7.4

35.3

7.6

35.2

GMB blank vessel B

7.4

7.4

33.4

7.5

35.5

7.7

35.3

 

 Measured pH in method blank and test vessels in GST medium

sample name

target pH

pH prior to the test

temp. [°C] prior to the test

pH after 2h

temp.[°C]

pH after 24h

temp.[°C]

GST vessel A

1.5 – 1.6

1.6

35.5

1.6

34.6

1.6

34.1

GST vessel B

1.5 – 1.6

1.6

35.5

1.6

34.5

1.6

34.1

GST blank vessel A

1.5 – 1.6

1.6

36.0

1.6

36.0

1.6

36.2

GST blank vessel B

1.5 – 1.6

1.6

35.9

1.6

36.2

1.6

36.2

 

 Measured pH in method blank and test vessels inPBS medium

sample name

target pH

pH prior to the test

temp. [°C] prior to the test

pH after 2h

temp.[°C]

pH after 24h

temp.[°C]

PBS vessel A

7.2 - 7.4

7.3

35.2

7.3

34.7

7.3

34.3

PBS vessel B

7.2 - 7.4

7.3

35.2

7.3

34.6

7.3

34.1

PBS blank vessel A

7.2 - 7.4

7.3

34.9

7.3

36.0

7.3

36.0

PBS blank vessel B

7.2 - 7.4

7.3

35.3

7.3

36.0

7.3

36.1

Concentration of chromium in artificial media, calculated nominal chromium concentration 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

<LOQ

5.61 ± 1.18

5.61 ± 1.18

18298

0.03 ± 0.01

ALF 24h

<LOQ

8.01 ± 0.24

8.01 ± 0.24

18298

0.04 ± <0.01

ASW 2h

<LOD

3.56 ± 0.58

3.56 ± 0.58

18330

0.02 ± <0.01

ASW 24h

<LOD

5.82 ± 0.10

5.82 ± 0.10

18330

0.03 ± <0.01

GMB 2h

0.05 ± 0.02

1.03 ± 0.07

0.98 ± 0.07

18295

0.01 ± <0.01

GMB 24h

0.06 ± 0.02

2.81 ± 0.10

2.75 ± 0.10

18295

0.02 ± <0.01

GST 2h

<LOD

9.51 ± 0.05

9.51 ± 0.05

18254

0.05 ± <0.01

GST 24h

0.06

18.8 ± 0.89

18.8 ± 0.89

18254

0.10 ± 0.01

PBS 2h

<LOD

0.32 ± 0.03

0.32 ± 0.03

18448

0.002 ± <0.001

PBS 24h

<LOD

1.28 ± 0.42

1.28 ± 0.42

18448

0.01 ± <0.01

# (initial weight (e.g. 50mg)*18.13§ (percentage chromium in test item)/100)*2 (multiplication to calculate chromium amount in one litre --> 100 mg/L) = nominal chromium concentration in [mg/L]/1000 = nominal chromium concentration in [µg/L]

§according to CoA 26.50% Cr as Cr2O3==> 68.42% chromium in Cr2O3==> (26.50% * 68.42%)/100% = 18.13% Cr in test item

In five different artificial physiological media, between 0.002 and 0.10% of chromium was dissolved from the test item IPC-2013-015 nickel iron chromite black spineldepending on solution parameters and test duration.


 

Concentration of iron in artificial media, calculated nominal iron concentration 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

22.2 ± 0.37

27.6 ± 1.11

5.36 ± 1.11

19378

0.03 ± 0.01

ALF 24h

23.3 ± 0.41

37.6 ± 0.24

14.3 ± 0.24

19378

0.07 ± <0.01

ASW 2h

<LOD/LOQ

<LOD

-

19412

-

ASW 24h

<LOD

<LOD/LOQ

-

19412

-

GMB 2h

<LOD

<LOD/LOQ

-

19374

-

GMB 24h

<LOD

<LOD/ Outlier

-

19374

-

GST 2h

<LOD

9.00 ± 0.81

9.00 ± 0.81

19331

0.05 ± <0.01

GST 24h

<LOD/LOQ

30.3 ± 0.50

30.3 ± 0.50

19331

0.16 ± <0.01

PBS 2h

5.85

5.85

-

19536

-

PBS 24h

<LOQ

6.62

6.62

19536

0.03

# (initial weight (e.g. 50mg)*19.02§ (percentage iron in test item)/100)*2 (multiplication to calculate iron amount in one litre --> 100 mg/L) = nominal iron concentration in [mg/L]/1000 = nominal iron concentration in [µg/L]

§according to CoA 27.20% Fe as Fe2O3==> 69.94% iron in Fe2O3==> (27.20% * 69.94%)/100% = 19.02% Fe in test item

In five different artificial physiological media, between 0.03 and 0.16% of iron was dissolved from the test item IPC-2013-015 nickel iron chromite black spineldepending on solution parameters and test duration. 

 

 

Concentration of manganese in artificial media, calculated nominal manganese concentration 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

<LOQ

20.4 ± 0.75

20.4 ± 0.75

12354

0.16 ± 0.01

ALF 24h

<LOQ

42.0 ± 0.23

42.0 ± 0.23

12354

0.34 ± <0.01

ASW 2h

<LOD

0.96 ± 0.29

0.96 ± 0.29

12375

0.01 ± <0.01

ASW 24h

<LOD

8.49 ± 1.00

8.49 ± 1.00

12375

0.07 ± <0.01

GMB 2h

<LOD

<LOD/LOQ

<LOD/LOQ

12351

-

GMB 24h

<LOD

0.53 ± 0.04

0.53 ± 0.04

12351

0.004 ± <0.001

GST 2h

<LOD

32.6 ± 1.31

32.6 ± 1.31

12324

0.26 ± 0.01

GST 24h

0.04

68.0 ± 0.04

67.9 ± 0.04

12324

0.56 ± <0.01

PBS 2h

<LOD

0.31 ± 0.01

0.31 ± 0.01

12454

0.003 ± <0.001

PBS 24h

<LOD

2.29 ± 0.69

2.29 ± 0.69

12454

0.02 ± 0.01

# (initial weight (e.g. 50mg)*12.24§ (percentage manganese in test item)/100)*2 (multiplication to calculate manganese amount in one litre --> 100 mg/L) = nominal manganese concentration in [mg/L]/1000 = nominal manganese concentration in [µg/L]

§according to CoA 15.80% Mn as MnO ==> 77.45% manganese in MnO ==> (15.80% * 77.45%)/100% = 12.24% Mn in test item

In five different artificial physiological media, between 0.003 and 0.56% of manganese was dissolved from the test item IPC-2013-015 nickel iron chromite black spineldepending on solution parameters and test duration.

 

 

 

Concentration of nickel in artificial media, calculated nominal nickel concentration 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

<LOQ

6.90 ± 0.49

6.90 ± 0.49

21417

0.03 ± <0.01

ALF 24h

<LOD/LOQ

16.7± 0.06

16.7± 0.06

21417

0.08 ± <0.01

ASW 2h

<LOD/LOQ

0.40 ± 0.10

0.40 ± 0.10

21454

0.002 ± 0.001

ASW 24h

<LOD/LOQ

3.70 ± 0.09

3.70 ± 0.09

21454

0.02 ± <0.01

GMB 2h

<LOD

<LOD/LOQ

<LOD/LOQ

21413

-

GMB 24h

<LOD

1.05 ± 0.01

1.05 ± 0.01

21413

0.01 ± <0.01

GST 2h

<LOD

15.2 ± 0.69

15.2 ± 0.69

21365

0.07± <0.01

GST 24h

0.30 ± <0.01

36.1 ± 0.20

35.8 ± 0.20

21365

0.17 ± <0.01

PBS 2h

<LOD

<LOQ

<LOQ

21592

-

PBS 24h

<LOD

1.28 ± 0.27

1.28 ± 0.27

21592

0.01 ± <0.01

# (initial weight (e.g. 50mg)*21.22§ (percentage nickel in test item)/100)*2 (multiplication to calculate nickel amount in one litre --> 100 mg/L) = nominal nickel concentration in [mg/L]/1000 = nominal nickel concentration in [µg/L]

§according to CoA 27.00% Ni as NiO ==> 78.58% nickel in NiO ==> (27.00% * 78.58%)/100% = 21.22% Ni in test item

In five different artificial physiological media, between 0.002 and 0.17% of manganese was dissolved from the test item IPC-2013-015 nickel iron chromite black spineldepending on solution parameters and test duration.

Applicant's summary and conclusion

Conclusions:
As dissolved Cr, Ni, Mn and Fe concentrations were below 18.8 µg/L, 35.8 µg/L, 67.9 µg/L and 30.3 µg/L respectively, even at the highest loading of 0.1g/L, referring to a solubility of 0.019 %, 0.036 %, 0.068 % and 0.03 %, the pigment is considered biologically inert.
Executive summary:

The bioaccessibility of nickel iron 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.

In five different artificial physiological media dissolved chromium concentrations were between 0.01 and 0.10 %, dissolved iron concentrations were between < LoD and 0.16 %, dissolved manganese concentrations were between < LoD and 0.56 %, and dissolved nickel concentrations were between < LoD and 0.17 % based on respective element contained in 100 mg pigment.

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