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Reference
Endpoint:
basic toxicokinetics in vitro / ex vivo
Remarks:
Bioaccessibility - transformation/dissolution in artificial physiological media
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:
comparable to guideline study
Objective of study:
bioaccessibility
Qualifier:
equivalent or similar to
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
Deviations:
yes
Remarks:
Bioaccessibility testing: loading of 100 mg/L; five artificial physiological media agitated at 100 rpm, at 37 °C ± 2 °C; sampling after 2h and 24h; determination of Zr and Fe concentrations after filtration by ICP-MS and ICP-OES.
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, 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.
GLP compliance:
yes (incl. certificate)
Remarks:
2011-02-07
Species:
other: in vitro (simulated human body fluids)
Details on exposure:
Test principle in brief:
- five different artificial physiological media,
- single loading of test substance of 100 mg/L,
- samples taken after 2 and 24 hours agitation (100 rpm) at 37 ± 2 °C,
- two method blanks per artificial media were tested; measurement (ICP-MS and ICP-OES) of dissolved zirconium and iron concentrations after filtration (0.2 µm, Supor membrane)
- the study was performed in duplicates

The aim of this test was to assess the dissolution of IPC-2013-003 (Zirconium iron pink zircon) in five artificial physiological media: Artificial lysosomal fluid (ALF, pH = 4.5), Artificial sweat solution (ASW, pH = 6.5), Gamble´s solution (GMB, pH = 7.4), Artificial gastric fluid (GST, pH = 1.5), Phosphate buffered saline (PBS, pH = 7.4). The test media were selected to simulate relevant human-chemical interactions (as far as practical), i.e. a substance entering the human body by ingestion into the gastrointestinal tract and by inhalation.
Duration and frequency of treatment / exposure:
Samples were taken after 2 h and 24h.
Dose / conc.:
100 other: mg of the test item /L artificial media
Details on study design:
Reagents
The water (resistivity >18 MΩ·cm.) used for this test was purified with a Pure Lab Ultra water purification system from ELGA LabWater, Celle, Germany.
- Nitric acid - “Supra” quality (ROTIPURAN® supplied by Roth, Karlsruhe, Germany).
- Hydrochloric acid – “instra-analyzed plus” quality (J.T. Baker, Griesheim, Germany).

Metal analysis
- Standards: zirconium and iron standards containing 1000 mg/L Zr and Fe (Fluka TraceCERT, Schnelldorf, Germany lot no. BCBD9418; MerckCertipur Fe lot no. HC093237, Darmstadt, Germany) in 2-3% nitric acid were utilised.
- Reference material: TMDA-70 and TMDWS.2 obtained from Environment Canada (lot no. 0310; lot no. 1010) and a multielement standard (19 elements, CPI, Amsterdam, The Netherlands, lot no.12A089) were analysed for total dissolved iron by ICP-MS and ICP-OES along with the samples to determine the accuracy of the applied analytical method. Further a zirconium single element standard (CPI Zr standard, Amsterdam, The Netherlands, lot no. ZR9301) was analysed for total dissolved zirconium by ICP-MS and ICP-OES along with the samples to determine the accuracy of the applied analytical method. A certified reference material for zirconium was not commercially available.

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
Calibration: 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.
Correlation coefficients (r): at least 0.9991
Four measurement series 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)
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, 100 µg/L, 250 µg/L and 500 µg/L.
Correlation factors (r): at least 0.9977
In sum, three series of measurements were performed for the determination of total dissolved zirconium concentrations in samples from test vessels, blanks to determine background levels of elements and fortified samples.
Details on dosing and sampling:
Loading:
The nominal loading in this test was 100 mg/L. However, due to weighing uncertainties the actual loadings range from 100.336 mg/L to 101.59 mg/L in the test vessels.
Type:
other: Bioaccessibility
Results:
Highest dissolution (loading: 0.1g/L, after 24h) in ALF: Zr: 23.8 ± 4.38 µg/L, Fe: 14.7 ± 1.60 µg/L, in GST: Zr (2h): 67.1 ± 5.32 µg/L, Fe: 25.4 ± 5.02 µg/L

Method validation summary (ICP-OES)

validation parameter

results

Comment

Selectivity

similar data with two different wavelengths for ICP-OES method

no interferences observed

Linearity

applied calibration functions were linear

correlation coefficient at least 0.9991

Limit of detection

Zr: 1.77 – 8.41 µg/L
Fe: 1.78 – 5.22 µg/L

Limit of quantification

Zr: 5.89 – 28.0 µg/L
Fe: 5.95 – 17.4 µg/L

Method blanks

Zr: ALF and GST <LOD/LOQ

Fe: GST, PBS, ASW and GMB <LOD/LOQ; ALF method blanks above LOQ at least 1.2 fold lower than concentration in samples

Elevated concentrations of Fe in method blanks origin from the applied chemicals for preparation of different media

Accuracy

mean recovery for CRM TMDA-70:
Fe: 100 ± 2.5 % (n = 11)

high concentration range (369 µg Fe/L)

Accuracy

mean recovery for CRM TMDWS.2:
Fe: 103 ± 1.3 % (n = 10)

mid concentration range (224 µg V/L)

Trueness iron

mean recovery for recalibration standard:
Fe: 97.0 ± 2.4 % (n = 3)

mid concentration range (250 µg/L)

Trueness iron

mean recovery for recalibration standard:
Fe: 98.4 ± 2.3 % (n = 4)

low concentration range (50 µg/L)

Trueness zirconium

mean recovery for recalibration standard:
Zr: 98.7± 2.2 % (n = 3)

high concentration range (100 µg/L)

Trueness zirconium

mean recovery for recalibration standard:
Zr: 97.2 ± 4.6 % (n = 7)

Mid high concentration range (75 µg/L)

Trueness zirconium

mean recovery for recalibration standard:
Zr: 96.8 ± 2.7 % (n = 4)

Mid low concentration range (10 µg/L)

Trueness zirconium

mean recovery for recalibration standard:
Zr: 94.2 ± 9.8 % (n = 7)

Low concentration range (7.5 µg/L)

Trueness Zr and Fe

Fortification of samples:

Zr: 105 - 110 %

Fe: 89.9 – 98.4 %

Reproducibility Fe

mean recovery for CRM TMDA-70:
Fe: 100 ± 2.5 % (n = 11)

high concentration range (369 µg Fe/L)

Reproducibility Fe

mean recovery for CRM TMDWS.2:
Fe: 103 ± 1.3 % (n = 10)

mid concentration range (224 µg V/L)

Method validation summary (ICP-MS)

validation parameter

results

Comment

Selectivity

Zr: similar on different isotopes and the respective gas modes (NoGas, Helium, HiHelium)

appropriate Isotope and gas mode were selected for interference free measurements

Linearity

applied calibration functions were linear

correlation coefficients 0.9977

Limit of detection

Zr: 0.03 – 0.22 µg/L

Limit of quantification

Zr: 0.08 – 0.67 µg/L

Method blanks

Zr: ASW and PBS <LOD/LOQ;

GMB method blanks 2h <LOD/LOQ, method blanks 24h at least 4.71 fold lower than concentration in samples

Elevated concentrations of Zr in method blanks origin from the applied chemicals for preparation of different media

Trueness

mean recovery for recalibration standard:
Zr: 97.1 ± 12 % (n = 8)

high concentration range (50 µg/L)

Trueness

mean recovery for recalibration standard:
Zr: 101 ± 7.1 % (n = 4)

mid concentration range (10 µg/L)

Trueness

mean recovery for recalibration standard:
Zr: 100 ± 1.4 % (n = 4)

Mid low concentration range (5 µg/L)

Trueness

mean recovery for recalibration standard:
Zr: 110 ± 6.5 % (n = 6)

Low concentration range (1 µg/L)

Trueness Zr

Fortification of samples:

Zr: 101– 107 %

Reproducibility Zr

mean recovery for recalibration standard:
Zr: 97.1 ± 12 % (n = 8)

high concentration range (50 µg/L)

Reproducibility Zr

mean recovery for recalibration standard:
Zr: 101 ± 7.1 % (n = 4)

mid concentration range (10 µg/L)

Reproducibility Zr

mean recovery for recalibration standard:
Zr: 100 ± 1.4 % (n = 4)

Mid low concentration range (5 µg/L)

Reproducibility Zr

mean recovery for recalibration standard:
Zr: 110 ± 6.5 % (n = 6)

Low concentration range (1 µg/L)

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 [%] normalized for 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. 50 mg) * 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 [%] normalized for 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. 50 mg) * 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.

Mass balance calculation

Total dissolved zirconium and iron concentrations in vessels, filters and syringes measured by ICP-OES indicate an incomplete dissolution of Zirconium iron pink zircon in all physiological media after addition of aqua regia to the sample vessels.

Calculation of zirconium mass balance

media

value for dissolved Zr after addition ofaqua regia
[mg]

nominal
concentration
[mg] #

recovery
[%]

ALF 24h A

0.01

19.6

0.05

ALF 24h B

0.01

19.6

0.06

ASW 24h A

0.02

19.4

0.11

ASW 24h B

0.02

19.5

0.10

GMB 24h A

0.02

19.5

0.10

GMB 24h B

0.02

19.5

0.10

GST 24 A

0.03

19.6

0.16

GST 24h B

0.03

19.5

0.15

PBS 24h A

0.005

19.7

0.02

PBS 24h B

0.005

19.5

0.02

# nominal concentration Zr = 52.30 % as ZrO2in test item = 74.03 % Zr in ZrO2 => 37.72 mg Zr in in 50 mg test item =>37.72 * initial weight / 50 mg

Calculation of iron mass balance

media

value for dissolved Fe after addition ofaqua regia
[mg]

nominal
concentration
[mg] #

recovery
[%]

ALF 24h A

0.02

6.26

0.32

ALF 24h B

0.02

6.26

0.30

ASW 24h A

0.01

6.21

0.16

ASW 24h B

0.01

6.22

0.16

GMB 24h A

0.01

6.23

0.19

GMB 24h B

0.01

6.23

0.15

GST 24 A

0.01

6.27

0.19

GST 24h B

0.01

6.24

0.20

PBS 24h A

0.01

6.29

0.21

PBS 24h B

0.02

6.24

0.26

# nominal concentration Fe = 17.70 % as Fe2O3 in test item = 69.94 % Fe in Fe2O3 => 12.38 mg Fe in 50 mg test item =>12.38 * initial weight / 50 mg

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 0.1 g/L, referring to a solubility of 0.067 % and 0.025 %, respectively, 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 0.1 g/L, referring to a solubility of 0.067 % and 0.025 %, the pigment is considered biologically inert.

Description of key information

As dissolved Zr and Fe concentrations were below 67.1 µg/L and 25.4 µg/L, respectively, even at the highest loading of 0.1 g/L, referring to a solubility of 0.067 % and 0.025 %, respectively, the pigment is considered biologically inert.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

The chemical and physiological properties of the pigment zirconium iron pink zircon are characterised by inertness because of the specific synthetic process (calcination at high temperatures, approximately 1000 °C), rendering the substance to be of a unique, stable crystalline structure in which all atoms are tightly bound and not prone to dissolution in environmental and physiological media. This manufacturing process leads to a very low bioaccessibility of the elements contained in the pigment. This 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:

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

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

3.) 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,

4.) 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

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

The dissolution of Zr of the test item is between below LoD (0.22 µg/LL (PBS) and 67.1 µg/L/ 64.2 µg/L (GST) at a loading of 0.1g/L after 2 and 24 hours.

The dissolution of Fe of the test item is in a range of below LoD (1.78 µg/L)/ below LoQ (5.95 µg/L,GMB) and 16.5 µg/L/ 25.4 µg/L (GST) at a loading of 0.1g/L after 2 and 24 hours.

A pHdependent dissolution can be observed.In conclusion, since the dissolved Zr and Fe concentrations were below 65 µg/L even at the highest loading of 0.1g/L, referring to a solubility of 0.065 %, this pigment may reasonably be considered biologically inert.