Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-08-07 to 2013-03-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study

Data source

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

Materials and methods

Objective of study:
bioaccessibility (or bioavailability)
Principles of method if other than guideline:
The objective of this study was to assess the dissolution of zinc iron chromite brown spinel (IPC-2013-007) 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 Cr, Fe, Ni and Zn in solution. The test was performed on the basis of OECD Series on Testing and Assessment No. 29 (2001; ENV/JM/ MONO(2001)9) [I]: “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
GLP compliance:
yes (incl. QA statement)
Remarks:
2011-02-07

Test material

Constituent 1
Chemical structure
Reference substance name:
Zinc iron chromite brown spinel
EC Number:
269-050-0
EC Name:
Zinc iron chromite brown spinel
Cas Number:
68186-88-9
Molecular formula:
(Zn,Fe)(Fe,Cr)2O4
IUPAC Name:
zinc iron chromite brown spinel
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Substance name: Zinc iron chromite brown spinel
Appearance: solid, brown powder, odourless

Test animals

Species:
other: in vitro (simulated human body fluids)
Details on test animals or test system and environmental conditions:
Five different artificial physiological media, single loading of test substance of 100 mg/L, measurement of dissolved chromium, iron, nickel and zinc concentration after 2 and 24 hours agitation (100 rpm) at 37 ± 2°C, two additional method blanks per medium. The study was performed in duplicates.

Administration / exposure

Details on study design:
An internationally agreed guideline does not exist for this test (e.g. OECD). However, similar tests have been conducted with several metal compounds, including steels, 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 performed 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.

Test conditions: Five different artificial physiological media, single loading of test substance of 100 mg/L, measurement of dissolved chromium, iron, nickel and zinc concentration after 2 and 24 hours agitation (100 rpm) at 37 ± 2°C, two additional method blanks per medium. The study was performed in duplicates.
The aim of this test was to assess the dissolution of zinc iron chromite brown spinel (IPC-2013-007) in the set of artificial physiological media. The test media were selected to simulate relevant human-chemical interactions (as far as practical), i.e. contact of test substance with skin, a substance entering the human body by inhalation or by ingestion into the gastro-intestinal tract.
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, nickel and zinc concentrations of sampled solution were determined after filtration (0.2 µm, Supor membrane) by ICP-MS and ICP-OES.

Aqueous samples of approx. 20 mL taken for Cr, Fe, Ni and Zn 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 four 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). Acidification is the stabilization method that is recommended in standard methods for metal analysis (e.g. DIN EN ISO 11885, DIN).
Solution pH was measured directly in the test vessel.

The analysis of total dissolved chromium 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 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 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 0.9992 in all measurement series. For each sample, at least threeinternal measurements were performed and the mean was calculated and printed by the instrument software.

In sum, six series of measurements were performed for the determination of total dissolved chromium 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 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.

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, 238.204 nm, 239.562 nm and 259.940 nm;
Zn: 202.548 nm, 206.200 nm and 213.856 nm

Four measurements were performed for the determination of iron and zinc concentrations in the samples, mass balance samples and filter samples.
The applied LOD/LOQ calculations are:
LOD: 3 * method standard deviation from calibration line;
LOQ: 10 * method standard deviation from calibration line.

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, 60Ni, 61Ni, 103Rh (internal standard)

In sum, six series of measurements were performed for the determination of total dissolved chromium 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 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.

Results and discussion

Any other information on results incl. tables

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

<LOD

2.11 ± 0.01

2.11 ± 0.01

22193

0.01 ± <0.01

ALF 24h

<LOD

5.81 ± 0.26

5.81 ± 0.26

22193

0.03 ± <0.01

ASW 2h

<LOD

0.48 ± 0.05

0.48 ± 0.05

22088

0.002 ± <0.001

ASW 24h

<LOD

1.04 ± 0.07

1.04 ± 0.07

22088

0.005 ± <0.001

GMB 2h

0.06 ± <0.01

0.57 ± 0.10

0.51 ± 0.10

22064

0.002 ± 0.001

GMB 24h

0.07 ± 0.01

0.73 ± 0.17

0.66 ± 0.17

22064

0.003 ± 0.001

GST 2h

0.10 ± 0.02

1.90 ± 0.09

1.81 ± 0.09

22206

0.01 ± <0.01

GST 24h

0.24 ± 0.02

4.19 ± 0.02

3.95 ± 0.02

22206

0.02 ± <0.01

PBS 2h

<LOD

<LOQ

<LOQ

21962

-

PBS 24h

<LOD

0.31 ± 0.01

0.31 ± 0.01

21962

0.001 ± <0.001

# (initial weight (e.g. 50mg)*21.83§ (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 31.90% Cr as Cr2O3==> 68.42% chromium in Cr2O3==> (31.90% * 68.42%)/100% = 21.83% Cr in test item

In five different artificial physiological media, between 0.001 and 0.03% of chromium was dissolved from the test item I PC-2013-007 zinc iron chromite brown spinel depending 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

24.4 ± 1.61

33.5 ± 0.92

9.07 ± 0.92

23678

0.04 ± <0.01

ALF 24h

20.7 ± 2.26

48.1 ± 8.74

27.4 ± 8.74

23678

0.12 ± 0.04

ASW 2h

<LOD/LOQ

<LOD/LOQ

-

23566

-

ASW 24h

<LOD

<LOQ

-

23566

-

GMB 2h

<LOD/LOQ

<LOD/LOQ

<LOD/LOQ

23540

-

GMB 24h

<LOD/LOQ

11.8 ± 0.30

11.8 ± 0.30

23540

0.05 ± <0.01

GST 2h

<LOD/LOQ

15.8 ± 3.71

15.8 ± 3.71

23691

0.07 ± 0.02

GST 24h

<LOD/LOQ

42.7 ± 2.89

42.7 ± 2.89

23691

0.18 ± 0.01

PBS 2h

9.77

12.5

2.77

23431

0.01

PBS 24h

11.0

11.5 ± 2.80

2.48

23431

0.01

# (initial weight (e.g. 50mg)*23.92§ (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 34.20% Fe as Fe2O3==> 69.94% iron in Fe2O3==> (34.20% * 69.94%)/100% = 23.92% Fe in test item

In five different artificial physiological media, between 0.01 and 0.18% of iron was dissolved from the test item IPC-2013-007 zinc iron chromite brown spinel depending on solution parameters and test duration.

Applicant's summary and conclusion

Conclusions:
The bioaccessibility of zinc iron chromite brown 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 dissolved Cr, Ni, Zn and Fe concentrations (GST, ALF) were below 5.8 µg/L, 6.03 µg/L, 62 µg/L and 43 µg/L respectively, even at the highest loading of 0.1g/L, referring to a solubility of 0.006%, 0,006%, 0,06 and 0,04 %, the pigment is considered biologically inert.
Executive summary:

The bioaccessibility of Cr, Ni, Zn and Fe has been investigated experimentally in vitro by simulating dissolution under physiological conditions considered to mimic the most relevantexposure 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 total dissolved Cr, Ni, Zn and Fe concentrations (GST, ALF) were below 5.8 µg/L, 6.03 µg/L, 62 µg/L and 43 µg/L respectively, even at the highest loading of 0.1g/L, referring to a solubility of 0.006%, 0,006%, 0,06 and 0,04 %, the pigment is considered biologically inert.