Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-09-23 to 2012-09-21
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
Principles of method if other than guideline:
The objective of this study was to assess the dissolution of silicic acid, zirconium salt, cadmium pigment-encapsulated (IPC-2013-001) 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 Cd, Se and Zr in solution. The test was performed on the basis of OECD Series on Testing and Assessment No. 29 (2001; ENV/JM/ MONO(2001)9): “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.
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 cadmium, selenium and zirconium concentrations were determined after filtration (0.2 µm, Supor membrane) by ICP-MS.
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: Silicic acid, zirconium salt, cadmium pigment-encapsulated (IPC-2013-001)
Appearance: red coloured powder
Storage: Store separate from food and drinks, in closed containers and protected places. Keep containers air-tight.

Test animals

Species:
other: in vitro (simulated human body fluids)
Details on test animals 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 cadmium, selenium and zirconium concentrations were determined after filtration (0.2 µm, Supor membrane) by ICP-MS.

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 cadmium, selenium and zirconium concentrations were determined after filtration (0.2 µm, Supor membrane) by ICP-MS.

Solutions were sampled for measuring total dissolved cadmium, selenium and zirconium by ICP-MS. 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 Cd, Se and Zr 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 three 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. Acidification is the stabilisation method that is recommended in standard methods for metal analysis (e.g. ISO 11885, DIN).
Solutions pHs were measured directly in the test vessel


ICP-OES
Cadmium, selenium and zirconium concentrations of aqueous mass balance and filter/syringe samples were measured using an IRIS Intrepid II ICP-OES (Thermo Electron, Dreieich, Germany). Cadmium was detected at the wavelengths 226.502 nm and 228.802 nm, selenium on the wavelengths 196.090 nm and 203.985 nm and zirconium on the wavelengths 339.198 nm, 343.823 nm and 349.621 nm. Depending on concentrations of the samples, the following cadmium, selenium and zirconium solutions were used to calibrate the instrument: blank, 1.0 µg/L, 5.0 µg/L, 10 µg/L, 50 µg/L, 100 µg/L, 200 µg/L, 300 µg/L, 400 µg/L and 500 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. 226,502 nm for cadmium). 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.

One measurement was performed for the determination of cadmium, selenium and zirconium concentrations in the mass balance and filter/syringe 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).

Date of ICP-OES measurement with corresponding LOD/LOQ and correlation coefficient of external calibration
date LOD(µg/L) LOQ(µg/L) correlation coefficient
September 18, 2012; Cd: 3.30 Cd: 11.0 Cd: 0.9999
Measurement of mass balance all Se: 4.66 Se: 15.5 Se: 0.9999
media vessels, syringe and filters Zr: 3.51 Zr: 11.7 Zr: 0.9999

ICP-MS
The analysis of total dissolved cadmium, selenium and zirconium in medium samples was performed using an Agilent 7700 ICP-MS with collision cell (Agilent, Waldbronn, Germany). Cadmium was detected on the isotopes111Cd and114Cd, selenium on the isotopes78Se and82Se and zirconium on90Zr and91Zr. 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 and 10.0 µg/L. A respective concentration range was selected for calibration to obtain a working range that covers cadmium, selenium and zirconium concentrations in samples.

In sum, six series of measurements were performed for the determination of total dissolved cadmium, selenium and zirconium concentrations in samples including blanks to determine background levels of elements and fortified samples. The LOD and LOQ for cadmium, selenium and 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. InTable9 the LODs and LOQs for cadmium, selenium and zirconium are shown for the different media.

July 30, 2012; Measurement of GST media all sample vessels, blank vessels and fortification
LOD (µg/L) LOQ (µg/L) correlation coefficient
Cd: 0.03 Cd: 0.08 Cd: 0.9999
Se: 0.02 Se: 0.05 Se: 0.9997
Zr: 0.02 Zr: 0.05 Zr: 0.9994

August 02, 2012; Measurement of GMB media all sample vessels, blank vessels and fortification
LOD (µg/L) LOQ (µg/L) correlation coefficient
Cd: 0.02 Cd: 0.05 Cd: 0.9976
Se: 0.06 Se: 0.18 Se: 0.9994
Zr: 0.05 Zr: 0.14 Zr: 0.9989

August 08, 2012; Measurement of PBS media all sample vessels, blank vessels and fortification
LOD (µg/L) LOQ (µg/L) correlation coefficient
Cd: 0.03 Cd: 0.08 Cd: 0.9998
Se: 0.23 Se: 0.68 Se: 0.9996
Zr: 0.15 Zr: 0.44 Zr: 0.9965

August 10, 2012; Measurement of ALF media all sample vessels, blank vessels and fortification
LOD (µg/L) LOQ (µg/L) correlation coefficient
Cd: 0.002 Cd: 0.01 Cd: 0.9981
Se: 0.02 Se: 0.06 Se: 0.9996
Zr: 0.16 Zr: 0.47 Zr: 0.9995

August 14, 2012; Measurement of ASW media all sample vessels, blank vessels and fortification
LOD (µg/L) LOQ (µg/L) correlation coefficient
Cd: 0.02 Cd: 0.06 Cd: 0.9995
Se: 0.05 Se: 0.16 Se: 0.9999

September 05, 2012; Remeasurement of zirconium in ASW media all sample vessels, blank vessels and fortification
LOD (µg/L) LOQ (µg/L) correlation coefficient
Zr: 0.01 Zr: 0.04 Zr: 0.9988

Results and discussion

Any other information on results incl. tables

Solution pH values

After preparation of the artificial physiological media, solution pHs were adjusted to their respective target pH. The target pH in all media before addition of test substance is in the nominal range. During the study, the pH of ALF, GST, GMB and PBS solutions remained stable. The pH of the ASW solutions in all vessels decreased after 24h. The pHs had decreased to 5.8 in all vessels. Therefore, an effect of the test substance can be excluded.

pHs of the physiological test media prior to the test

media

target pH

measured pH

temp. [°C]

ALF

4.5

4.5

30.2

ASW

6.5

6.5

34.0

GMB

7.4

7.4

31.5

GST

1.5 – 1.6

1.5

36.3

PBS

7.2 - 7.4

7.3

35.8

 

Analytical measurements

The results of the ICP-MS measurements of total dissolved Cd, Se and Zr in samples are presented below. Cadmium, selenium and zirconium concentrations in blank samples measured by ICP-MS were below the limit of detection (LOD = 0.002 – 0.03 µg/L for Cd, 0.02 – 0.23 µg/L for Se and 0.01 – 0.16 µg/L for Zr) or limit of quantification (LOQ = 0.01 – 0.08 µg/L for Cd, 0.05 – 0.68 µg/L for Se and 0.04 – 0.47 µg/L for Zr) for mainly all media. In GMB and ALF media the selenium concentrations in blank samples were at least 2.7 fold lower than the concentrations of Se in samples. Furthermore in ASW media the concentrations of Zr in blank vessels are mainly below the LOQ (LOQ = 0.04 µg/L) except for a few blank vessels. The concentrations of these blank vessels are in the range of the Zr concentration in sample vessels. Elevated concentrations of Cd, Se and Zr in method blanks origin from the applied chemicals for preparation of different media.

The LOD and LOQ of dissolved total cadmium (ICP-MS) concentrations were 0.03 and 0.08 µg/L for GST, 0.02 and 0.06 µg/L for ASW, 0.02 and 0.05 µg/L for GMB, 0.002 and 0.01 µg/L for ALF and 0.03 and 0.08 µg/L for PBS. The LOD and LOQ of dissolved total selenium (ICP-MS) concentrations were 0.02 and 0.05 µg/L for GST, 0.05 and 0.16 µg/L for ASW, 0.06 and 0.18 µg/L for GMB, 0.02 and 0.06 µg/L for ALF and 0.23 and 0.68 µg/L for PBS. The LOD and LOQ of dissolved total zirconium (ICP-MS) concentrations were 0.02 and 0.05 µg/L for GST, 0.01 and 0.04 µg/L for ASW, 0.05 and 0.14 µg/L for GMB, 0.16 and 0.47 µg/L for ALF and 0.15 and 0.44 µg/L for PBS.

Concentration of cadmium in artificial media, calculated nominal cadmium concentration in 100 mg/L silicic acid, zirconium salt, cadmium pigment-encapsulated and dissolved amount of cadmium.

media and sample

total Cd ± SD in method blanks [µg/L]

total Cd ±SD in sample vessels [µg/L]

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

calculated nominal Cd concentration in [µg/L]#

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

ALF 2h

<LOD

0.38 ± <0.01

0.38 ± <0.01

4699

0.01 ± <0.01

ALF 24h

<LOD

0.89 ± 0.10

0.89 ± 0.10

4699

0.02 ± <0.01

ASW 2h

<LOD

0.29 ± 0.09

0.29 ± 0.09

4722

0.01 ± <0.01

ASW 24h

<LOD

0.93 ± 0.20

0.93 ± 0.20

4722

0.02 ± <0.01

GMB 2h

<LOD

0.07 ± <0.01

0.07 ± <0.01

4709

0.002 ± <0.001

GMB 24h

<LOD

0.07 ± <0.01

0.07 ± <0.01

4709

0.001 ± <0.001

GST 2h

<LOD

2.58 ± 0.20

2.58 ± 0.20

4733

0.05 ± <0.01

GST 24h

<LOD

6.13 ± 0.42

6.13 ± 0.42

4733

0.13 ± 0.01

PBS 2h

<LOD

0.11 ± 0.02

0.11 ± 0.02

4711

0.002

PBS 24h

<LOD

0.43 ± 0.15

0.43 ± 0.15

4711

0.01 ± <0.01

In five different artificial physiological media, between 0.001 and 0.13% of cadmium was dissolved from the test item IPC-2013-001 Silicic acid, zirconium salt, cadmium pigment-encapsulated depending on solution parameters and test duration.

Concentration of selenium in artificial media, calculated nominal selenium concentration in 100 mg/L silicic acid, zirconium salt, cadmium pigment-encapsulated and dissolved amount of selenium.

media and sample

total Se ± SD in method blanks [µg/L]

total Se ±SD in sample vessels [µg/L]

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

calculated nominal Se concentration in [µg/L]#

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

ALF 2h

0.08 ± 0.01

0.71 ± 0.02

0.64 ± 0.02

1543

0.04 ± <0.01

ALF 24h

0.08 ± 0.01

0.77 ± 0.05

0.69 ± 0.05

1543

0.04 ± <0.01

ASW 2h

<LOD/LOQ

0.59 ± 0.01

0.59 ± 0.01

1550

0.04 ± <0.01

ASW 24h

<LOD/LOQ

0.79 ± 0.1

0.79 ± 0.1

1550

0.05 ± <0.01

GMB 2h

<LOD/LOQ

0.68 ± 0.04

0.68 ± 0.04

1546

0.04 ± <0.01

GMB 24h

0.25

0.90 ± <0.01

0.65 ± <0.01

1546

0.04 ± <0.01

GST 2h

<LOD/LOQ

0.12 ± 0.02

0.12 ± 0.02

1554

0.01 ± <0.01

GST 24h

<LOD/LOQ

0.20 ± 0.02

0.20 ± 0.02

1554

0.01 ± <0.01

PBS 2h

<LOD/LOQ

0.74 ± <0.01

0.74 ± <0.01

1547

0.05 ± <0.01

PBS 24h

<LOD/LOQ

0.82 ± 0.03

0.82 ± 0.03

1547

0.05 ± <0.01

In five different artificial physiological media, between 0.01 and 0.05% of selenium was dissolved from the test item IPC-2013-001 Silicic acid, zirconium salt, cadmium pigment-encapsulated depending on solution parameters and test duration.

Concentration of zirconium in artificial media, calculated nominal zirconium concentration in 100 mg/L silicic acid, zirconium salt, cadmium pigment-encapsulated 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 of Zr in artificial media [%] normalizedfor measured background in method blank

ALF 2h

<LOD

0.52 ± 0.01

0.52 ± 0.01

45913

0.001 ± <0.001

ALF 24h

<LOD

1.25 ± 0.11

1.25 ± 0.11

45913

0.003 ± <0.001

ASW 2h

0.18 ± 0.13

0.07

-

46134

-

ASW 24h

0.47

0.13 ± 0.12

-

46134

-

GMB 2h

<LOD

<LOD

<LOD

46007

-

GMB 24h

<LOD

<LOD

<LOD

46007

-

GST 2h

<LOD

<LOD

<LOD

46244

-

GST 24h

<LOD

<LOD/LOQ

<LOD/LOQ

46244

-

PBS 2h

<LOD

<LOD

<LOD

46033

-

PBS 24h

<LOD

<LOD

<LOD

46033

-

In five different artificial physiological media, between 0.001 and 0.003% of zirconium was dissolved from the test item IPC-2013-001 Silicic acid, zirconium salt, cadmium pigment-encapsulated depending on solution parameters and test duration.

Applicant's summary and conclusion

Conclusions:
The bioaccessibility of Silicic acid, zirconium salt, cadmium pigment-encapsulated has been investigated experimentally in vitro by simulating dissolution under physiological conditions considered to mimic the most relevant exposure routes (oral, dermal and inhalation). Dissolved Cd, Zr and Se concentrations were below 6.13 µg/L, 1.25 µg/L and 0.82 µg/L, respectively even at the highest loading of 0.1g/L, representing a solubility of 0.006 %, 0.001 % and 0.0008 %. Therefore, silicic acid, zirconium salt, cadmium pigment-encapsulated may reasonably be considered not bioaccessible.
Executive summary:

The bioaccessibility of silicic acid, zirconium salt, cadmium pigment-encapsulated 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.

 

Under the conditions of this test (flasks with different artificial physiological media; loadings of 50 mg silicic acid, zirconium salt, cadmium pigment-encapsulated/ 500 mL, 37°C, sampling after 2h and 24h), the concentrations of dissolved cadmium, selenium and zirconium were as follows: In five different artificial physiological media dissolved cadmium concentrations were between 0.001 and 0.13%, dissolved selenium concentrations were between 0.01 and 0.06% and dissolved zirconium concentrations were between 0.001 and 0.003%, based on contained elements.

Therefore, Silicic acid, zirconium salt, cadmium pigment-encapsulated may reasonably be considered not bioaccessible.