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Reference
Endpoint:
basic toxicokinetics, other
Remarks:
Bioaccessibility
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-04-17 to 2015-10-05
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 Cd and Zr concentrations after filtration by ICP-MS.
Principles of method if other than guideline:
The test was performed on the basis of OECD Series on Testing and Assessment No. 29 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 ([1], [2], [3], [4] and [5]).

The aim of this test was to assess the dissolution of the pigment IPC-2018-005 (Zircon, cadmium yellow) in the artificial physiological media GST, GMB, ALF, ASW and PBS. 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 or by inhalation.

Five different artificial physiological media with a single loading of test substance of 100 mg/Lwere used. The measurement of dissolved cadmium and zirconium concentrations after filtration were performed by ICP-MS. Samples were taken after 2 and 24 hours agitation (100 rpm) at 37 ± 2 °C. The study was performed in triplicate with two additional method blanks per medium.

[1] Hanawa T. 2004. Metal ion release from metal implants. Materials Science and Engineering C 24: 745-752.
[2] Stopford W., Turner J., Cappelini D., Brock T. 2004. Bioaccessibility testing of cobalt compounds. Journal of Environmental Monitoring 5: 675-680.
[3] Midander K., et al. 2007. In vitro studies of copper release from powder particles in synthetic biological media. Environmental Pollution 145: 51-59.
[4] European standard 1998. Test method for release of nickel from products intended to come into direct and prolonged contact with the skin (EN 1811)
[5] ASTM 2003. Standard test method for determining extractability of metals from art materials. ASTM D5517-03
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2016-12-16
Species:
other: in vitro (simulated human body fluids)
Details on test animals and environmental conditions:
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 (by IPC-MS) of dissolvedcadmium and zirconium concentrations after filtration
- the study was performed in triplicate

The aim of this test was to assess the dissolution of IPC-2018-005 (zircon, cadmium yellow) 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).
- Sodiumhydroxide – pro Analysis quality (Chemsolute, Th. Geyer, Renningen, Germany)

Metal analysis
- Standards: single element standards were used as chromium, and tin standards to prepare an appropriate stock solution and subsequently calibration solutions for ICP-MS measurements (Merck Certipur cadmium ICP standard 1000 mg/L lot no. HC386977; Merck Certipur Zirconium ICP standard 1000 mg/L lot no. HC388511; Darmstadt, Germany; CPI Multielement standard, lot no. 15C267; CPI, Netherlands).
- Certified reference materials: quality control standards TM-25.4 (lot no. 0914) and TMDA-53.3 (lot no. 0914) obtained from Environment Canada and a multielement standard (Merck XVII, lot no. HC382226; Merck XXI, lot no. HC42984673, Darmstadt, Germany; CPI Multielement standard, lot no. 15C267, CPI, Netherlands) as well as one single element standard for zirconium (CPI Zirconium standard, lot no. 14D050, CPI, Netherlands) were analyzed for total dissolved cadmium and zirconium by ICP-MS along with the samples to determine the accuracy of the applied analytical method. Furthermore the calibration solutions were measured along with the ICP-MS measurements as recalibration standards.

Instrumental and analytical set-up for the ICP-MS instrument:
Agilent 7700ce ICP-MS, Agilent Technologies, Waldbronn Germany
Nebulizer: Conical nebulizer, from Glass Expansion
Spray chamber: Scott Type spray chamber, from Agilent
Carrier gas flow: 0.93 L/min
Dilution Gas flow: 0.1 – 0.16 L/min
RF power: 1500 W
Isotopes: 111Cd, 114Cd, 90Zr, 91Zr, 92Zr and 103Rh (internal standard)

The applied LOD/LOQs were calculated as follows:
LOD: 3 x standard deviation of calibration blank divided by the slope of calibration line;
LOQ: 3 x LOD.
The data for the LODs were read directly from the Agilent 7700 ICP-MS instrument output (data calculated by internal algorithms of the instrument software).

Calibration: blank, 0.1 µg/L, 0.25 µg/l, 0.5 µg/L, 0.75 µg/L, 1 µg/L, 2.5 µg/L, 5.0 µg/L, 7.5 µg/L, 10 µg/L, 12.5 µg/L, 15 µg/L, 17.5 µg/L, 20 µg/L, 22.5 µg/L and 25 µg/L. For two measurement series only zirconium measurement a special low calibration was performed due to the fact, that the concentration was extremely low: blank, 0.1 µg/L, 0.2 µg/L, 0.3 µg/L, 0.4 µg/L, 0.5 µg/L, 0.6 µg/L, 0.7 µg/L, 0.8 µg/L, 0.9 µg/L, 1 µg/L, 2.5 µg/L, 5 µg/L, 7.5 µg/L and 10 µg/L.
Correlation coefficients (r): at least 0.997539

Determination of mass balance
To the residual, undissolved test item in the vessels, 80 mL aqua regia (3 : 1 mixture of concentrated hydrochloric and nitric acid) were added to the flasks after the test. The filters used for sampling were extensively rinsed with aqua regia. Solutions were sampled after at least 24h, cadmium and zirconium were measured in at least one mass balance sample (vessel and filter/syringes) for each medium by ICP-MS, and the mass balance was calculated.
Details on dosing and sampling:
Loading:
Detailed loadings of the test vessels are given in "Any other information on materials and methods incl. tables".
Toxicokinetic parameters:
other: bioaccessibility
Remarks:
Dissolution of Zr at a loading of 0.1 g/L: ALF (pH 4.5) 24h: 1.44 ± 0.12 μg/L. Dissolution od Cd at a loading of 0.1g/L: ALF (pH 4.5) 24h: 0.459 ± 0.045 μg/L.
Bioaccessibility testing results:
Concentration of dissolved cadmium in artificial physiological media
Total Cd ± SD in sample vessels (corrected concentrations, corresponding to a loading of exactly 100 mg/L) with and without mean method blank subtraction (mean of 2h and/or 24h)
- GST 2h: 0.346 ± 0.029 μg/L (method blank: 0.006 ± 0.007 µg/L (two method blanks below LOD, two method blank above LOQ))
- GST 24h: 0.722 ± 0.050 μg/L (no subtraction, all method blanks below LOD)
- GMB 2h: 0.199 ± 0.038 µg/L (no subtraction, all method blanks below LOD)
- GMB 24h: 0.140 ± 0.020 μg/L (no subtraction, all method blanks below LOD/LOQ)
- ALF 2h: 0.382 ± 0.021 μg/L (method blank: 0.016 ± 0.006 µg/L (two method blanks below LOQ, two method blanks above LOQ))
- ALF 24h: 0.459 ± 0.045 μg/L (method blank: 0.018 ± 0.004 µg/L (one method blanks below LOQ, three method blank sabove LOQ))
- ASW 2h: 0.268 ± 0.038 µg/L (no subtraction, all method blanks below LOD)
- ASW 24h: 0.440 ± 0.023 µg/L (no subtraction, all method blanks below LOD)
- PBS 2h: 0.0.180 ± 0.013 µg/L (no subtraction, all method blanks below LOD)
- PBS 24h: 0.0.180 ± 0.013 µg/L (no subtraction, all method blanks below LOD)

Concentration of dissolved zirconium in artificial physiological media
total Zr ± SD in sample vessels (corrected concentrations, corresponding to a loading of exactly 100 mg/L) with and without mean method blank subtraction (mean of 2h and/or 24h)
- GST 2h: 0.780 ± 0.075 μg/L (method blank: 0.010 ± 0.013 µg/L (two method blanks below LOD, two method blank above LOQ))
- GST 24h: 0.435 ± 0.029 µg/L (no subtraction, all method blanks below LOD)
- GMB 2h: 0.105 ± 0.078µg/L (method blank: 0.169 ± 0.028 µg/L (all method blanks above LOQ))
- GMB 24h: 0.045 ± 0.008 µg/L (method blank: 0.045 ± 0.009 µg/L (all method blanks above LOQ))
- ALF 2h: 0.812 ± 0.073 μg/L (no subtraction, all method blanks below LOD)
- ALF 24h: 1.44 ± 0.12 μg/L (no subtraction, all method blanks below LOD)
- ASW 2h: 0.032 ± 0.032 µg/L (no subtraction, all method blanks below LOD)
- ASW 24h: All samples and method blanks below LOD
- PBS 2h: All samples and method blanks below LOD
- PBS 24h: 0.014 ± 0.012 µg/L (five samples below LOD; no subtraction, all method blanks below LOD)

Method validation summary ICP-MS

Validation parameter Results Comment
Selectivity similar data with two different gas modes or two different Isotopes and same gas mode -
Linearity applied calibration functions were linear correlation coefficient at least 0.997539
Limit of detection

Zr:0.001 – 0.019 µg/L

Cd: 0.001 – 0.012 µg/L

-
Limit of quantification

Zr: 0.004 – 0.058 µg/L

Cd: 0.003 – 0.036 µg/L

-

Accuracy measurement /

Reproducibility test sample and

mass balance samples measurements

Mean recovery for CRM TM-25.4 (dilution factor 5):

Cd: 102 ± 2.0 % (n = 20)

Low concentration range

(certified with 23.5 µg Cd/L - diluted 4.70 µg Cd/L)

Accuracy measurement /

Reproducibility test sample and

mass balance samples measurements

Mean recovery for CRM TMDA-53.3 (dilution factor 20):

Cd: 100 ± 3.6 % (n = 17)

Low concentration range

(certified with 118 µg Cd/L - diluted 5.90 µg Cd/L)

Accuracy measurement /

Reproducibility test sample measurements

Mean recovery for CRM TMDA-53.3 (dilution factor 10):

Cd: 101 ± 1.7 % (n = 4)

Mid concentration range

(certified with 118 µg Cd/L - diluted 11.8 µg Cd/L)

Trueness test samples

Quality control standard CPI:

Zr: 95.0 ± 3.7 % (n = 7)

Low concentration range (0.25 µg/L)

Trueness test samples

Quality control standard CPI:

Zr: 97.0 ± 1.8 % (n = 3)

Mid concentration range (0.75 µg/L)

Trueness test samples

Quality control standard Merck XVII:

Zr: 105 ± 1.0 % (n = 4)

Mid concentration range (0.75 µg/L)

Trueness test sample and

mass balance samples measurements

Quality control standard Merck XVII:

Zr: 104 ± 7.6 % (n = 12)

High concentration range (20 µg/L)

Trueness test sample and

mass balance samples measurements

Quality control standard Merck XVII:

Cd: 101 ± 3.3 % (n = 19)

High concentration range (20 µg/L)

Trueness test sample

Recalibration standard:

Zr: 98.0 ± 2.1 % (n = 7)

Low concentration range (0.5 µg/L)

Trueness test sample and

mass balance samples measurements

Recalibration standard:

Zr: 99.9 ± 3.6 % (n = 14)

Cd: 98.7 ± 2.7 % (n = 21)

Mid to high (Zr) concentration range (10 µg/L)

Trueness test samples/mass balance samples

Fortification of samples:

Zr: 82.5 (below LOD) – 112 %

Cd: 97.1 – 111 %

Solution pH values

During the study, the pH of GST, ALF and PBS media remained stable in the method blank vessels and the test vessels. A low increase of the pH for GST was measurable but this was measurable for the method blanks, too. Therefore, a possible effect of the test substance can be excluded.

In GMB medium, the pH in all vessels (including method blanks) increased during the time of the test from 7.40 to 8.61 and 8.64 (method blank vessels) and 7.40 to 8.60, 8.66 and 8.68 (test vessels). Therefore, an effect of the test substance can be excluded. In fact, the pH of the GMB media does not seem to be stable under the conditions of the test.

In ASW medium, the pH in all vessels (including method blanks) decreased during the time of the test from 6.50 to 5.98 and 5.99 (method blank vessels) and 6.50 to 5.93 and 5.95 (test vessels). Therefore, an effect of the test substance can be excluded.

Temperature control

The temperature was adjusted to 37.5 in a thermostatically controlled shaking cabinet to reach a temperature of 37 ± 2 °C in the media. The temperature in thermostatically controlled shaking cabinet was confirmed by a certified thermometer and a thermo data logger. In all test vessels, the temperature was constant at 37 ± 2 °C.

Fortification

Selected samples were fortified with a known amount of cadmium and zirconium (by standard addition of commercial standards) to determine the standard recovery. For fortified test samples, recoveries were in the range of 97.1 – 111 % for Cd and 82.5 (below LOD) – 112 % for Zr.

Fortification of Cd in test samples

sample

measured concentration

[μg/L]

calculated level after addition

[μg/L]

recovery

[μg/L] / [%]

GST vessel 1 sample a 2h

0.358

0.405

0.409 / 101

GST vessel 1 sample a 24h

0.764

0.843

0.842 / 100

ASW vessel 2 sample a 2h

0.295

0.363

0.363 / 100

ASW vessel 2 sample a 24h

0.454

0.636

0.634 / 99.7

ALF vessel 1 sample a 2h

0.401

0.600

0.599 / 99.7

ALF vessel 1 sample a 24h

0.482

0.655

0.641 / 97.8

PBS vessel 2 sample b 2h

0.194

0.963

0.961 / 99.9

PBS vessel 2 sample b 24h

0.546

2.03

1.97 / 97.1

GMB vessel 1 sample a 2h

0.230

1.74

1.73 / 99.0

GMB vessel 1 sample a 24h

0.146

1.72

1.69 / 98.7

ALF vessel 1 mass balance

3.07

3.71

3.72 / 100

ASW vessel 1 mass balance

2.46

3.30

3.36 / 102

GMB vessel 1 mass balance

5.14

6.76

6.66 / 98.5

GST vessel 1 mass balance

3.33

3.89

4.31 / 111

PBS vessel 1 mass balance

2.99

4.50

4.45 / 99.0

# Solutions with concentrations below the LOD/LOQ were also fortified. However, a recovery of ± 15 % may not be realistic as concentrations in the original (unfortified) sample below the LOD may be lower than the noise ratio and concentrations below the LOQ are between the noise ratio and a real (quantifiable) signal. For solutions with concentrations below the LOD/LOQ, a recovery of ± 25 % - 30 % is more realistic.

Fortification of Zr in test samples

sample

measured concentration

[μg/L]

calculated level after addition

[μg/L]

recovery

[μg/L] / [%]

GST vessel 1 sample a 2h

0.747

0.665

0.594 / 89.3

GST vessel 1 sample a 24h

0.421

0.614

0.535 / 87.2

ASW vessel 2 sample a 2h

0.065

0.210

0.216 / 103

ASW vessel 2 sample a 24h

<LOD#

0.333

0.275 / 82.5

ALF vessel 1 sample a 2h

0.761

0.841

0.864 / 103

ALF vessel 1 sample a 24h

1.39

1.26

1.26 / 99.5

PBS vessel a sample b 2h

<LOD#

0.333

0.285 / 85.5

PBS vessel 1 sample b 24h

<LOD#

0.337

0.330 / 98.0

GMB vessel a sample a 2h

0.343

0.448

0.437 / 97.5

GMB vessel 1 sample a 24h

0.099

0.166

0.166 / 100

ALF vessel 1 mass balance

3.10

3.73

3.94 / 106

ASW vessel 1 mass balance

1.76

2.84

3.08 / 108

GMB vessel 1 mass balance

5.14

6.76

7.05 / 104

GST vessel 1 mass balance

1.88

2.92

3.27 / 112

PBS vessel 1 mass balance

0.270

2.68

2.98 / 111

# Solutions with concentrations below the LOD/LOQ were also fortified. However, a recovery of ± 15% may not be realistic as concentrations in the original (unfortified) sample below the LOD may be lower than the noise ratio and concentrations below the LOQ are between the noise ratio and a real (quantifiable) signal. For solutions with concentrations below the LOD/LOQ, a recovery of ± 25% - 30% is more realistic.

Mass balance calculation

For the mass balance dissolved cadmium and zirconium concentrations in vessels and filters as measured by ICP-MS indicate no complete dissolution of IPC-2018-005 in the physiological media after addition of aqua regia to the sample vessels. Due to the observations during the mass balance test item was still visible which could be a result of insoluble parts of the test item.

Amount of tin in mass balance samples (vessels, filter/syringes and taken samples) for each media for one vessel each

Media

Value for dissolved Cd after addition of aqua regia

In samples

[mg]

Calculated value Cd

[mg]

Nominal amount

[mg] #

Recovery

[%]

Vessels [mg]

Filters/syringes [mg]

GST vessel 1

0.002

0.0000005

0.00005

0.002

1.01

0.169

GMB vessel 3

0.003

0.000001

0.00001

0.003

1.00

0.264

ALF vessel 2

0.002

0.000001

0.00004

0.002

1.01

0.156

ASW vessel 1

0.001

0.000001

0.00003

0.001

1.01

0.124

PBS vessel 3

0.001

0.000002

0.00003

0.002

1.01

0.151

* Measured dissolved Cd includes the amount of cadmium in test solutions after addition of aqua regia, the amount of cadmium rinsed of used filters and syringe and the amount of cadmium removed with samples during the study.

Example ALF: 0.002 mg (amount Cd in vessel) + 0.000001 mg (amount Cd in syringe/filters) + 0.00004 mg (Cd in samples) = 0.002 mg

# nominal amount Cd = 2.00% in test item (according to CoA)  1.00 mg Cd in 50 mg test item  nominal amount 1.00 mg * initial weight mg / 50 mg

Example: 1.00 mg Cd * 50.524 mg test item / 50 mg = 1.01 mg Cd

Conclusions:
On the basis of OECD Series on Testing and Assessment No. 29 as well as according to a bioaccessibility test protocol, which has been developed on the basis of relevant published methods, the dissolution of the pigment IPC-2018-005 (Zircon, cadmium yellow) in the artificial physiological media (GST, GMB, ALF, ASW and PBS) with a single loading of 100 mg/L, agitation (100 rpm) at 37 °C ± 2 °C and sampling after 2 and 24 h, was determined. The measurement of dissolved cadmium and zirconium concentrations after filtration were performed by ICP-MS. The study was performed in triplicate with two additional method blanks per medium.

Concentration of dissolved cadmium in artificial physiological media
Total Cd ± SD in sample vessels (corrected concentrations, corresponding to a loading of exactly 100 mg/L) with and without mean method blank subtraction (mean of 2h and/or 24h)
- GST 2h: 0.346 ± 0.029 μg/L
- GST 24h: 0.722 ± 0.050 μg/L
- GMB 2h: 0.199 ± 0.038 µg/L
- GMB 24h: 0.140 ± 0.020 μg/L
- ALF 2h: 0.0.382 ± 0.021 μg/L
- ALF 24h: 0.459 ± 0.045 μg/L
- ASW 2h: 0.268 ± 0.038 µg/L
- ASW 24h: 0.440 ± 0.023 µg/L
- PBS 2h: 0.0.180 ± 0.013 µg/L
- PBS 24h: 0.0.180 ± 0.013 µg/L

Concentration of dissolved zirconium in artificial physiological media
total Zr ± SD in sample vessels (corrected concentrations, corresponding to a loading of exactly 100 mg/L) with and without mean method blank subtraction (mean of 2h and/or 24h)
- GST 2h: 0.780 ± 0.075 μg/L
- GST 24h: 0.435 ± 0.029 µg/L
- GMB 2h: 0.105 ± 0.078µg/L
- GMB 24h: 0.045 ± 0.008 µg/L
- ALF 2h: 0.812 ± 0.073 μg/L
- ALF 24h: 1.44 ± 0.12 μg/L
- ASW 2h: 0.032 ± 0.032 µg/L
- ASW 24h: All samples and method blanks below LOD
- PBS 2h: All samples and method blanks below LOD
- PBS 24h: 0.014 ± 0.012 µg/L

Description of key information

In conclusion, since the dissolved Cd and Zr concentrations from this pigment under simulated physiological conditions were below 2.2 µg/L even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.003 % after 24 hours, this pigment may reasonably be considered biologically inert.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

The chemical and physiological properties of the pigment Zircon, cadmium yellow 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 cadmium from the test item Zircon, cadmium yellow at a loading of 0.1g/L after 2 hours was between0.180µg/L (PBS) and 0.398 µg/L (ALF) and between 0.140 µg/L (GMB) - 0.722 µg/L (GST) after 24 hours. Further, the dissolution of zirconium from the test item Zircon, cadmium yellow was in a range ofbelow LODand 0.812 µg/L (ALF) at a loading of 0.1g/L after 2 hours and between below LOD (ASW) - 1.44 µg/L (ALF) after 24 hours.

 

In conclusion, since the dissolved Cd and Zr concentrations from this pigment under simulated physiological conditions were below 2.2 µg/L even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.003 % after 24 hours, this pigment may reasonably be considered biologically inert.