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Reference
Endpoint:
basic toxicokinetics in vitro / ex vivo
Remarks:
transformation/dissolution in artificial physiological media
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-08-25 - 2018-01-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Objective of study:
bioaccessibility (or bioavailability)
Qualifier:
equivalent or similar to guideline
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 2 h and 24 h; determination of Cr,Fe, Ti and Zn concentrations after filtration by ICP-OES.
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 that 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 IIPC-2018-010 (Titanium, iron and aluminium pseudobrookite and rutile) 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 titanium, iron and aluminium concentrations after filtration were performed by ICP-OES. 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. QA statement)
Species:
other: in vitro (simulated human body fluids)
Details on test animals or test system 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 ICP-OES) of dissolved aluminium, iron and titanium after filtration
- the study was performed in triplicate

The aim of this test was to assess the dissolution of IPC-2018-010 (Titanium, iron and aluminium pseudobrookite and rutile) 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 24 h.
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:
- Certified reference materials: As quality control standards certified aqueous reference material TM-26.4 (lot no. 1115) and TMDA-52.4 (lot no. 0915) obtained from Environment Canada and a multielement standard (Roth Multielemenstandard, lot no. F38770, Karlsruhe, Germany) were analysed for total dissolved chromium, iron, titanium and zinc by ICP-OES along with the samples to determine the accuracy of the applied analytical method. Furthermore, the calibration solutions were measured along with the ICP-OES measurements as recalibration standards.

Instrumental and analytical set-up for the ICP-OES instrument:
Agilent 720, Agilent Technologies, Waldbronn, Germany
Nebulizer: Sea spray nebulizer from Agilent
Spray chamber: Glass cyclonic spray chamber from Agilent
Plasma stabilization time: at least 30 min before start of the measurements
Plasma gas flow: 15.0 L/min
Additional gas flow: 1.50 L/min
Carrier gas flow: 0.75 L/min
RF power: 1200W
Stabilization time of sample: 15 sec
Repetition time (three internal measurements per sample): 30 sec

Wavelengths: Al: 167.019 nm, 394.401 nm and 396.152 nm
Fe: 238.204 nm, 241.052 nm and 259.837 nm
Ti: 337.280 nm, 368.520 nm and 376.132 nm


The applied LOD/LOQ calculations for the Agilent 720 ICP-OES are (according to DIN 32645):
LOD: 3 x standard deviation of calibration blank/slope of the calibration
LOQ: 3 x LOD

Calibration: blank, 1 µg/L, 2 µg/l, 4 µg/L, 6 µg/L, 8 µg/L, 10 µg/L, 20 µg/L, 40 µg/L, 60 µg/L, 80 µg/L, 100 µg/L, 120 µg/L, 140 µg/L, 150 µg/L, 160 µg/L, 180 µg/L, 200 µg/L, 250 µg/L, 300 µg/L, 350 µg/L, 400 µg/L, 450 µg/L and 500 µg/L, for measurements of test vessel samples and for mass balance sample calibration was extended to 750 µg/L and 1000 µg/L.
Correlation coefficients (r): at least 0.995955
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 100.000 mg/L to 100.178 mg/L in the 15 test vessels.
Type:
other: Bioaccessibility
Results:
Higest dissolution at a loading of 0.1g/L in GST after 24h: Al = 179µg/L, Fe = 215µg/L, Ti =162µg/L
Bioaccessibility (or Bioavailability) testing results:
Concentration of dissolved aluminium in artificial physiological media.
Total Al ± SD in sample vessels with method blank subtraction
GST 2h 87.4 ± 3.41 µg/L
GST 24h 179 ± 8.78 µg/L
GMB 2h 8.67 µg/L
GMB 24h 25.4 µg/L
ALF 2h 52.9 ± 0.41 µg/L
ALF 24h 108 ± 0.70 µg/L
ASW 2h < LOD
ASW 24h 27.5 ± 2.42 µg/L
PBS 2h 2.57 µg/L
PBS 24h 24.2 µg/L

Concentration of dissolved iron in artificial physiological media.
Total Fe ± SD in sample vessels with method blank subtraction
GST 2h 61.2 ± 10.3 µg/L
GST 24h 215 ± 38.2 µg/L
GMB 2h GMB 24h ALF 2h 26.5 ± 0.78 µg/L
ALF 24h 97.7 ± 1.04 µg/L
ASW 2h ASW 24h 11.6 ± 1.35 µg/L
PBS 2h 2.26 ± 1.21 µg/L
PBS 24h 8.03 ± 6.53 µg/L

Concentration of dissolved titanium in artificial physiological media.
Total Ti ± SD in sample vessels without method blank subtraction
GST 2h 36.2 ± 4.57 µg/L
GST 24h 162 ± 8.75 µg/L
GMB 2h < LOD/LOQ
GMB 24h < LOQ
ALF 2h 17.9 ± 0.66 µg/L
ALF 24h 75.5 ± 1.33 µg/L
ASW 2h 1.44 ± 0.33 µg/L
ASW 24h 20.2 ± 1.13 µg/L
PBS 2h < LOD
PBS 24h 2.62 ± 0.17 µg/L

Method validation summary (ICP-OES)

Limits of detection (LODs), limits of quantification (LOQs) and correlation coefficients (r) are given for each medium and metal measured:

 

ASW medium

date

Chosen wavelength [nm]

LOD
(µg/L)

LOQ
(µg/L)

correlation coefficient

Sum of quality assurance samples

Recovery of quality assurance samples [%]

September 20, 2017; measurement of ASW test samples (2h and 24h) and method blanks

Al: 167.019

Fe: 259.837

Ti: 368.520

Al: 0.356

Fe: 1.13

Ti: 0.068

Al: 1.07

Fe: 3.38

Ti: 0.204

Al: 0.999958

Fe: 0.999979

Ti: 0.999957

Al: 37

Fe: 37

Ti: 37

Al: 95

Fe: 100

Ti: 100

 

PBS medium

date

Chosen wavelength [nm]

LOD
(µg/L)

LOQ
(µg/L)

correlation coefficient

Sum of quality assurance samples

Recovery of quality assurance samples [%]

October 05, 2017; measurement of PBS test samples (2h and 24h) and method blanks

Al: 167.019

Fe: 238.204

Ti: 376.132

Al: 0.252

Fe: 0.350

Ti: 0.201

Al: 0.756

Fe: 1.05

Ti: 0.604

Al: 0.999675

Fe: 0.999932

Ti: 0.999646

Al: 40

Fe: 40

Ti: 40

Al: 100

Fe: 100

Ti: 85

 

GMB medium

date

Chosen wavelength [nm]

LOD
(µg/L)

LOQ
(µg/L)

correlation coefficient

Sum of quality assurance samples

Recovery of quality assurance samples [%]

December 15, 2017; measurement of GMB test samples (2h and 24h) and method blanks

Al: 167.019

Fe: 238.204

Ti: 368.520

Al: 0.237

Fe: 0.432

Ti: 0.139

Al: 0.710

Fe: 1.29

Ti: 0.416

Al: 0.999848

Fe: 0.999860

Ti: 0.999778

Al: 28

Fe: 28

Ti: 28

Al: 100

Fe: 100

Ti: 100

 

GST medium

date

Chosen wavelength [nm]

LOD
(µg/L)

LOQ
(µg/L)

correlation coefficient

Sum of quality assurance samples

Recovery of quality assurance samples [%]

October 11, 2017; measurement of GST test samples (2h and 24h) and method blanks

Al: 396.152

Fe: 238.204

Ti: 376.132

Al: 0.953

Fe: 0.411

Ti: 0.944

Al: 2.86

Fe: 1.23

Ti: 2.83

Al: 0.999938

Fe: 0.999943

Ti: 0.999954

Al: 40

Fe: 40

Ti: 40

Al: 97.5

Fe: 100

Ti: 97.5

 

ALF medium

date

Chosen wavelength [nm]

LOD
(µg/L)

LOQ
(µg/L)

correlation coefficient

Sum of quality assurance samples

Recovery of quality assurance samples [%]

October 11, 2017; measurement of ALF test samples (2h and 24h) and method blanks

Al: 167.019

Fe: 259.837

Ti: 376.132

Al: 0.466

Fe: 1.22

Ti: 0.463

Al: 1.40

Fe: 3.66

Ti: 1.39

Al: 0.999978

Fe: 0.999958

Ti: 0.999953

Al: 40

Fe: 40

Ti: 40

Al: 100

Fe: 85.0

Ti: 100

  

Solution pH values

The target pH in all media before addition of test substance was in the nominal range.

During the study, the pH of GST, ALF and PBS media remained stable in the method blank vessels and the test vessels. 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 7.44 / 7.45 /7.46 to 8.78, 8.84 and 8.96 (test vessels) and 7.46 to 8.79 and 8.84 (method blank 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 5.92 and 5.94 (test vessels) and 6.50 to 5.89 and 5.96 (method blank vessels). Therefore, an effect of the test substance can be excluded.

Temperature control

The test was performed in an incubated laboratory shaker (Shaking incubation cabinet, Minitron, INFORS AG, Bottmingen, Switzerland) at 100 rpm. The temperature was adjusted to 37.5 °C in a thermostatically controlled shaking cabinet to reach a temperature of 37 °C ± 2 °C in the media. The temperature remained stable during the test in all media.

Fortification

Mean recovery of fortified samples: 52.7 - 110 % (Al fortification), 87.7 - 107 % (Fe fortification), and 89.2 – 104% (Ti fortification)

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-2018-010 (Titanium, iron and aluminium pseudobrookite and rutile) 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 aluminium, iron and titanium concentrations after filtration were performed by ICP-OES. The study was performed in triplicate with two additional method blanks per medium.

*Concentration of dissolved aluminium in artificial physiological media.
Total Al ± SD in sample vessels with method blank subtraction
GST 2h 87.4 ± 3.41 µg/L
GST 24h 179 ± 8.78 µg/L
GMB 2h 8.67 µg/L
GMB 24h 25.4 µg/L
ALF 2h 52.9 ± 0.41 µg/L
ALF 24h 108 ± 0.70 µg/L
ASW 2h < LOD
ASW 24h 27.5 ± 2.42 µg/L
PBS 2h 2.57 µg/L
PBS 24h 24.2 µg/L

Concentration of dissolved iron in artificial physiological media.
Total Fe ± SD in sample vessels with method blank subtraction
GST 2h 61.2 ± 10.3 µg/L
GST 24h 215 ± 38.2 µg/L
GMB 2h GMB 24h ALF 2h 26.5 ± 0.78 µg/L
ALF 24h 97.7 ± 1.04 µg/L
ASW 2h ASW 24h 11.6 ± 1.35 µg/L
PBS 2h 2.26 ± 1.21 µg/L
PBS 24h 8.03 ± 6.53 µg/L

Concentration of dissolved titanium in artificial physiological media.
Total Ti ± SD in sample vessels without method blank subtraction
GST 2h 36.2 ± 4.57 µg/L
GST 24h 162 ± 8.75 µg/L
GMB 2h < LOD/LOQ
GMB 24h < LOQ
ALF 2h 17.9 ± 0.66 µg/L
ALF 24h 75.5 ± 1.33 µg/L
ASW 2h 1.44 ± 0.33 µg/L
ASW 24h 20.2 ± 1.13 µg/L
PBS 2h < LOD
PBS 24h 2.62 ± 0.17 µg/L

Description of key information

In conclusion, since the dissolved Al, Fe and Ti concentrations from this pigment under simulated physiological conditions were below 179 µg/L, 215 µg/L and 162 µg/L (GST), respectively even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.6 % 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 Titanium, iron and aluminium pseudobrookite and rutile 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.

 

Solubility of Al from the pigment Titanium, iron and aluminium pseudobrookite and rutile in physiological media was in a range of below LOD and 87.4 µg/L (GST) after 2 hours. After 24 hours a dissolution range from below 24.2 - 179 µg/L (GST) was measured.

Solubility of Fe from the pigment Titanium, iron and aluminium pseudobrookite and rutile in physiological media was in a range of below LOD and 61.2 µg/L (GST) after 2 hours. After 24 hours a dissolution range from below LOD – 215 µg/L (GST) was measured.

Solubility of Ti from the pigment Titanium, iron and aluminium pseudobrookite and rutile in physiological media was in a range of below LOD and 36.2 µg/L (GST) after 2 hours. After 24 hours a dissolution range from below LOD – 162 µg/L (GST) was determined.

In conclusion, since the dissolved Al, Fe and Ti concentrations from this pigment under simulated physiological conditions were below 179 µg/L, 215 µg/L and 162 µg/L (GST), respectively even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.6 % after 24 hours, this pigment may reasonably be considered biologically inert.