Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-08-14 to 2013-01-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study

Data source

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

Materials and methods

Objective of study:
bioaccessibility
Principles of method if other than guideline:
The objective of this study was to assess the dissolution of zirconium iron pink zircon (IPC-2013-003) 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 Zr and Fe 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. certificate)
Remarks:
2011-02-07

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Substance name: Zirconium iron pink zircon
Appearance: solid, brick red powder, odourless

Test animals

Species:
other: in vitro (simulated human body fluids)
Details on test animals and environmental conditions:
Five different artificial physiological media, single loading of test substance of 100 mg/L, measurement of dissolved zirconium and iron 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.The total dissolved zirconium and iron concentrations in samples were determined after filtration (0.2 µm, Supor membrane) by ICP-MS and ICP-OES.

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 zirconium and iron 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.
The aim of this test was to assess the dissolution of zirconium iron pink zircon (IPC-2013-003) 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 zirconium and iron concentrations in samples were determined after filtration (0.2 µm, Supor membrane) by ICP-MS and ICP-OES.

Aqueous samples of approx. 20 mL taken for Zr and Fe 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 six months. According to DIN EN ISO 5667-3: 2003 [XIII] dissolved metalsin 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 16). 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 zirconium and iron concentrations in medium samples and mass balance samples were measured using an IRIS Intrepid II ICP-OES (Thermo Electron, Dreieich, Germany). Zirconium was detected at the wavelength 339.198 nm and 349.621 nm. Iron was detected at the wavelengths 238.204 nm, 239.562 nm and 259.940 nm. Depending on concentrations of the samples, the following zirconium solutions were used to calibrate the instrument: blank, 2.5 µg/L, 5 µg/L, 7.5 µg/L, 10 µg/L, 25 µg/L, 50 µg/L, 75 µg/L, 100 µg/L, 200 µg/L, 300 µg/L and 400 µg/L. For the calibration, the following iron concentrations were used: blank, 5 µg/L, 7.5 µg/L, 10 µg/L, 25 µg/L, 50 µg/L, 75 µg/L, 100 µg/L, 200 µg/L, 250 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. The respective wavelength data with the best correlation for the calibration in the measurements were used for calculating concentrations (i.e. 259.940 nm for iron). Correlation coefficients (r) were at least 0.9991. For each sample, at least three internal measurements were performed and the mean was calculated and printed by the instrument software.

In sum, three series of measurements were performed for the determination of total dissolved zirconium 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 zirconium 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: Zr: 339.198 nm and 349.621 nm; Fe: 238.204 nm, 239.562 nm and 259.940 nm

Four measurements were performed for the determination of zirconium and iron concentrations in the test item samples, method blanks, 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.
These data were read directly from the Thermo IRIS Intrepid II ICP-OES instrument output (data calculated by internal algorithms of 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: 90Zr, 91Zr, 103Rh (internal standard)

In sum, three series of measurements were performed for the determination of total dissolved zirconium concentrations in samples including the test vessels as well as blanks to determine background levels of elements and fortified samples.

Results and discussion

Any other information on results incl. tables

Concentration of zirconium in artificial media, calculated nominal zirconium concentration in 100 mg/L zirconium iron pink zircon and dissolved amount of zirconium:

media and sample

total Zr ± SD in method blanks [µg/L]

total Zr ±SD in sample vessels [µg/L]

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

calculated nominal Zr concentration in [µg/L]#

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

ALF 2h

<LOQ

15.5 ± 0.33

15.5 ± 0.33

39145

0.04 ± <0.01

ALF 24h

<LOD/LOQ

23.8 ± 4.38

23.8 ± 4.38

39145

0.06 ± 0.01

ASW 2h

<LOD/LOQ

0.71 ± 0.14

0.71 ± 0.14

38886

0.002 ± <0.001

ASW 24h

<LOD

3.02 ± 0.20

3.02 ± 0.20

38886

0.01 ± <0.01

GMB 2h

<LOD/LOQ

0.80 ± 0.21

0.80 ± 0.21

38981

0.002 ± 0.001

GMB 24h

0.17 ± 0.02

0.85 ± 0.10

0.68 ± 0.10

38981

0.002 ± <0.001

GST 2h

<LOQ

67.1 ± 5.32

67.1 ± 5.32

39109

0.17 ± 0.01

GST 24h

<LOD/LOQ

64.2 ± 4.44

64.2 ± 4.44

39109

0.16 ± 0.01

PBS 2h

<LOD

<LOD

-

39171

-

PBS 24h

<LOD

<LOD

-

39171

-

#(initial weight (e.g. 50mg)*38.72§ (percentage zirconium in test item)/100)*2 (multiplication to get zirconium amount in one litre --> 100 mg/L) = nominal zirconium concentration in [mg/L]/1000 = nominal zirconium concentration in [µg/L]

§according to CoA 52.30% Zr as ZrO2==> 74.03% zirconium in ZrO2==>
(52.30% * 74.03%)/100% = 38.72% Zr in test item

In five different artificial physiological media, between 0.002 and 0.17% of zirconium was dissolved from the test item IPC-2013-003 Zirconium iron pink zircon depending on solution parameters and test duration.

Concentration of iron in artificial media, calculated nominal iron concentration in 100 mg/L zirconium iron pink zircon 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

22.9 ± 0.71

28.0 ± 0.14

5.16 ± 0.14

12516

0.04 ± <0.01

ALF 24h

22.4 ± 0.31

37.1 ± 1.60

14.7 ± 1.60

12516

0.12 ± 0.01

ASW 2h

<LOD/LOQ

<LOQ

-

12433

-

ASW 24h

<LOD/LOQ

10.6 ± 0.44

10.6

12433

0.09 ± <0.01

GMB 2h

<LOD

<LOD

-

12463

-

GMB 24h

<LOD

<LOD/LOQ

-

12463

-

GST 2h

<LOD/LOQ

16.5 ± 1.91

16.5 ± 1.91

12504

0.13 ± 0.02

GST 24h

<LOQ

25.4 ± 5.02

25.4 ± 5.02

12504

0.20 ± 0.04

PBS 2h

<LOQ

6.62 ± 0.44

6.62 ± 0.44

12524

0.05 ± <0.01

PBS 24h

<LOQ

21.7 ± 16.3

21.7 ± 16.3

12524

0.17 ± 0.13

#(initial weight (e.g. 50mg)*12.38§ (percentage iron in test item)/100)*2 (multiplication to get iron amount in one litre--> 100 mg/L) = nominal iron concentration in [mg/L]/1000 = nominal iron concentration in [µg/L]

§according to CoA 17.70% Fe as Fe2O3==> 69.94% iron in Fe2O3==> (17.70% * 69.94%)/100% = 12.38% Fe in test item.

In five different artificial physiological media, between 0.04 and 0.20% of iron was dissolved from the test item IPC-2013-003 Zirconium iron pink zircon depending on solution parameters and test duration.

Applicant's summary and conclusion

Conclusions:
The bioaccessibility of zirconium iron pink zircon 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 Zr and Fe concentrations were below 67.1 µg/L and 25.4 µg/L respectively, even at the highest loading of 0.1g/L, referring to a solubility of 0.07 % and 0.03 %, the pigment is considered biologically inert.
Executive summary:

The bioaccessibility of zirconium and iron 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 Zr and Fe concentrations were below 67.1 µg/L and 25.4 µg/L respectively, even at the highest loading of 0.1g/L, referring to a solubility of 0.07 % and 0.03 %, the pigment is considered biologically inert.