Cobalt chromite blue green spinel

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

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:

2012-09-20 to 2013-02-11

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 2h and 24h; determination of Cr, Co and Al concentrations after filtration by ICP-OES and ICP-MS.

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. QA statement)

Remarks:

signed, 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 (by ICP-OES and ICP-MS) of dissolved chromium, aluminium and cobalt 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-2013-011 (Cobalt chromite blue green spinel) 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: single and multielement standards: Merck XXI (lot no. HC075495); Merck VIII (lot no. HC137073); Merck Certipur Cobalt ICP standard 1000 mg/L (lot no. HC242065); Merck Certipur Chromium ICP standard 1000 mg/L (lot. no. HC114885), Merck Certipur Aluminium ICP standard 1000 mg/L (lot no. HC125228, Darmstadt, Germany).
- Certified reference materials: TMDA-70 (lot no. 0310, Environment Canada) and a multielement standard: Merck VIII (lot no. HC137073)

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: Co: 228.616 nm, 230.786 nm and 231.160 nm; Cr: 205.552 nm, 267.716 nm and 284.325 nm
Calibration: blank, 1.0, 2.5, 5.0, 7.5, 10, 25, 50, 75, 100, 200, 300, 400 and 500 µg/L.
Correlation coefficients (r): at least 0.9999
Two measurement series were performed for the determination of cobalt and chromium concentrations in the samples, 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.

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: 52Cr, 53Cr, 54Cr, 59Co, 27Al, 103Rh (internal standard)
Calibrations: blank, 0.1, 0.25, 0.5, 0.75, 1.0, 2.5, 5.0, 7.5, 10.0, 25.0, 50.0, 75.0, 100, 250 and 500 µg/L
Correlation factors (r): at least 0.9989

In sum, five series of measurements were performed for the determination of total dissolved cobalt, chromium and aluminium 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 cobalt, chromium and aluminium 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.

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 100.362 mg/L to 101.934 mg/L in the test vessels.

Type:

other: Bioaccessibility

Results:

Higest dissolution at a loading of 0.1g/L after 24h in ALF: Co: 9.89 ± 0.16 µg/L, Al: 23.5 ± 0.81 µg/L; after 2h in ALF: Cr: 1.11 ± 0.10 µg/L; after 24h in GST: Co: 8.88 ± 0.41 µg/L, Cr: 0.89 ± 0.03 µg/L, Al: 17.5 ± 1.01 µg/L

Method validation summary (ICP-OES)

validation parameter	results	Comment
Selectivity	similar data with two different Co and Cr wavelengths for ICP-OES method	no interferences observed
Linearity	applied calibration functions were linear	correlation coefficient at least 0.9999
Limit of detection	Co: 0.71 -2.58 µg/L Cr: 1.11 – 3.59 µg/L
Limit of quantification	Co: 2.35 – 8.60 µg/L Cr: 8.73 – 12.0 µg/L
Accuracy	mean recovery for CRM TMDA-70: Co: 99.6 ± 2.8 % (n = 9) Cr: 101 ± 2.2 % (n = 9)	high concentration range (285 µg Co/L; 389 µg Cr/L )
Trueness	mean recovery for recalibration standard Merck VIII: Co: 101 ± 1.6 % (n = 5) Cr: 103 ± 1.7 % (n = 5)	low concentration range (10 µg/L)
Trueness	mean recovery for recalibration standard Merck VIII: Co: 95.5 ± 1.5 % (n = 5) Cr: 96.6 ± 1.8 % (n = 5)	mid low concentration range (25 µg/L)
Trueness	mean recovery for recalibration standard Merck VIII: Co: 101 ± 1.2 % (n = 4) Cr: 100 ± 1.2 % (n = 4)	high concentration range (100 µg/L)
Trueness	mean recovery for recalibration standard Merck VIII: Co: 100 ± 0.5 % (n = 4) Cr: 100 ± 0.4 % (n = 4)	high concentration range (250 µg/L)
Reproducibility	mean recovery for CRM TMDA-70: Co: 99.6 ± 2.8 % (n = 9) Cr: 101 ± 2.2 % (n = 9)	high concentration range (285 µg Co/L; 389 µg Cr/L )

Method validation summary (ICP-MS)

validation parameter	results	Comment
Selectivity	Cr: similar on different isotopes and the respective gas modes (NoGas, Helium, HiHelium) Co: similar in Helium and HiHelium mode Al: similar in Helium and HiHelium mode	appropriate Isotope and gas mode were selected for interference free measurements
Linearity	applied calibration functions were linear	correlation coefficients ≥ 0.9989
Limit of detection	Cr: 0.001 – 0.06 µg/L Co: 0.0004 – 0.45 µg/L Al: 0.31 – 3.46 µg/L
Limit of quantification	Cr: 0.003 – 0.08 µg/L Co: 0.001 – 1.38 µg/L Al: 0.92 – 10.4 µg/L
Method blanks	see table below: `Concentration of Cr, Co and Al method blanks in artificial media (ICP-MS measurement series)´	Elevated concentrations of Cr , Co and Al in method blanks origin from the applied chemicals for preparation of different media
Accuracy	mean recovery for CRM TMDA-70: Cr: 103 ± 6.1 % (n = 20) Co: 105 ± 6.4 % (n = 20) Al: 102 ± 14 % (n = 20)	higher concentration range (389 µg Cr/L; 285 µg Co/L, 415 µg Al/L)
Trueness	mean recovery for recalibration standard Merck VIII: Cr: 99.5 ± 6.9 % (n = 20) Co: 98.9 ± 6.2 % (n = 20) Al: 100 ± 14 % (n = 20)	low concentration range (10 µg/L)
Trueness	mean recovery for recalibration standard Merck VIII: Cr: 101 ± 9.4 % (n = 20) Co: 100 ± 4.5 % (n = 20) Al: 101 ± 10 % (n = 20)	mid concentration range (100 µg/L)
Trueness	Fortification of samples: Cr: 89.0– 108 % Ni: 91.2 – 107 % Al: 88.0– 104 %
Reproducibility	mean recovery for CRM TMDA-70: Cr: 103 ± 6.1 % (n = 20) Co: 105 ± 6.4 % (n = 20) Al: 102 ± 14 % (n = 20)	higher concentration range (389 µg Cr/L; 285 µg Co/L, 415 µg Al/L)
Reproducibility	mean recovery for recalibration standard Merck VIII: Cr: 99.5 ± 6.9 % (n = 20) Co: 98.9 ± 6.2 % (n = 20) Al: 100 ± 14 % (n = 20)	low concentration range (10 µg/L)
Reproducibility	mean recovery for recalibration standard Merck VIII: Cr: 101 ± 9.4 % (n = 20) Co: 100 ± 4.5 % (n = 20) Al: 101 ± 10 % (n = 20)	mid concentration range (100 µg/L)

Concentration of Cr, Co and Al method blanks in artificial media (ICP-MS measurement series)

Artificial media	Element
	Cr	Co	Al
GST	Method blanks <LOD/LOQ; one method blank 24h above LOQ but at least 19 fold lower than the concentrations in samples	<LOD	<LOD/LOQ
GMB	Method blanks above LOQ but at least 2.04 fold lower than the concentrations in samples	Method blanks above LOQ but at least 13 fold lower than the concentrations in samples	Method blanks and samples are in the same concentration range
ALF	Method blanks above LOQ but at least 1.13 lower than the concentrations in samples	Method blanks above LOQ but at least 26 lower than the concentrations in samples	Method blanks above LOQ but at least 1.26 lower than the concentrations in samples
ASW	<LOD	<LOD	<LOD
PBS	<LOD; one method blank 2h above LOQ but at least 1.32 fold lower than concentrations in samples	Two method blanks 2h <LOQ, two method blanks 2h and all method blanks 24 above LOQ but at least 13 fold lower than the concentrations in samples	Method blanks 2h above LOQ but at least 0.19 fold lower than the concentrations in samples; three method blanks 24h <LOQ one above LOQ but at least 3.79 fold lower than concentrations in samples 24h

Concentration of chromium in artificial media, calculated nominal chromium concentration and dissolved amount of chromium:

media and sample	total Cr ± SD in method blanks [µg/L]	total Cr ±SD in sample vessels [µg/L]	Cr ± SD in sample vessels with blank subtraction [µg/L]	calculated nominal Cr concentration in [µg/L]#	dissolved amount Cr in artificial media [%] normalized for measured background in method blank
ALF 2h	7.71 ± 0.17	8.81 ± 0.02	1.11 ± 0.02	30412	0.004 ± <0.001
ALF 24h	7.75 ± 0.20	8.74 ± 0.10	0.99 ± 0.10	30412	0.003 ± <0.001
ASW 2h	<LOD	<LOD	<LOD	30379	-
ASW 24h	<LOD	0.27 ± 0.01	0.27 ± 0.01	30379	0.001 ± <0.001
GMB 2h	0.23	0.47 ± 0.01	0.24 ± 0.01	30309	0.001 ± <0.001
GMB 24h	0.24 ± 0.02	0.51 ± 0.05	0.28 ± 0.05	30309	0.001 ± <0.001
GST 2h	<LOD	0.55 ± 0.03	0.55 ± 0.03	30539	0.002 ± <0.001
GST 24h	0.05	0.94 ± 0.03	0.89 ± 0.03	30539	0.003 ± <0.001
PBS 2h	0.10	0.13 ± <0.01	0.03 ± <0.01	30545	0.0001
PBS 24h	<LOD	0.18 ± 0.03	0.18 ± 0.03	30545	0.001 ± <0.001

# (initial weight (e.g. 50 mg) * 30.04 § (percentage chromium in test item) / 100) * 2 (multiplication to calculate chromium amount in one litre -> 100 mg/L) = nominal chromium concentration in [mg/L] / 1000 = nominal chromium concentration in [µg/L]

§ according to CoA 43.90 % Cr as Cr₂O₃ => 68.42 % chromium in Cr₂O₃ => (43.90 % * 68.42 %) / 100% = 30.04 % Cr in test item

In five different artificial physiological media, between 0.0001 and 0.004 % of chromium was dissolved from the test item IPC-2013-009 cobalt chromite blue green spinel depending on solution parameters and test duration.

Concentration of cobalt in artificial media, calculated nominal cobalt concentration and dissolved amount of cobalt:

media and sample	total Co ± SD in method blanks [µg/L]	total Co ±SD in sample vessels [µg/L]	Co ± SD in sample vessels with blank subtraction [µg/L]	calculated nominal Co concentration in [µg/L]#	dissolved amount Co in artificial media [%] normalized for measured background in method blank
ALF 2h	0.25 ± 0.03	6.71 ± 0.04	6.45 ± 0.04	14973	0.04 ± <0.01
ALF 24h	0.25 ± 0.04	10.1 ± 0.16	9.89 ± 0.16	14973	0.07 ± <0.01
ASW 2h	<LOD	0.62 ± 0.13	0.62 ± 0.13	14957	0.004 ± 0.001
ASW 24h	<LOD	3.51 ± 0.03	3.51 ± 0.03	14957	0.02 ± <0.01
GMB 2h	0.03 ± 0.01	0.43 ± 0.07	0.39 ± 0.07	14923	0.003 ± <0.001
GMB 24h	0.02 ± <0.01	0.40 ± <0.01	0.37 ± <0.01	14923	0.003 ± <0.001
GST 2h	<LOD	5.40 ± 0.29	5.40 ± 0.29	15036	0.04 ± <0.01
GST 24h	<LOD	8.88 ± 0.41	8.88 ± 0.41	15036	0.06 ± <0.01
PBS 2h	0.03	0.37 ± 0.02	0.34 ± 0.02	15038	0.002 ± <0.001
PBS 24h	0.03 ± 0.01	2.15 ± 0.05	2.13 ± 0.05	15038	0.01 ± <0.01

# (initial weight (e.g. 50 mg) * 14.79 § (percentage cobalt in test item) / 100) * 2 (multiplication to calculate cobalt amount in one litre --> 100 mg/L) = nominal cobalt concentration in [mg/L] / 1000 = nominal cobalt concentration in [µg/L]

§ according to CoA 18.80 % Co as CoO => 78.65 % cobalt in CoO => (18.80 % * 78.65 %) / 100 % = 14.79 % Co in test item

In five different artificial physiological media, between 0.002 and 0.07 % of cobalt was dissolved from the test item IPC-2013-009 cobalt chromite blue green spinel depending on solution parameters and test duration.

Concentration of aluminium in artificial media, calculated nominal aluminium concentration and dissolved amount of aluminium:

media and sample	total Al ± SD in method blanks [µg/L]	total Al ±SD in sample vessels [µg/L]	Al ± SD in sample vessels with blank subtraction [µg/L]	calculated nominal Al concentration in [µg/L]#	dissolved amount Al in artificial media [%] normalized for measured background in method blank
ALF 2h	20.7 ± 0.80	26.1 ± 0.69	5.43 ± 0.69	9537	0.06 ± 0.01
ALF 24h	6.52 ± 2.19	30.0 ± 0.81	23.5 ± 0.81	9537	0.25 ± 0.01
ASW 2h	<LOD	<LOD	<LOD	9526	-
ASW 24h	<LOD	<LOD	<LOD	9526	-
GMB 2h	30.8 ± 3.40	19.5 ± 1.69	-	9504	-
GMB 24h	18.6 ± 0.02	18.2 ± 1.64	0.76 ± 0.74	9504	0.01
GST 2h	<LOQ	11.0 ± 0.44	11.0 ± 0.44	9576	0.12 ± 0.01
GST 24h	<LOD	17.5 ± 1.01	17.5 ± 1.01	9576	0.18 ± 0.01
PBS 2h	6.75 ± 0.19	1.30	-	9578	-
PBS 24h	1.51	5.71 ± 0.43	4.21 ± 0.43	9578	0.04 ± <0.01

# (initial weight (e.g. 50 mg) * 9.42 § (percentage aluminium in test item) / 100) * 2 (multiplication to calculate aluminium amount in one litre --> 100 mg/L) = nominal aluminium concentration in [mg/L] / 1000 = nominal aluminium concentration in [µg/L]

§ according to CoA 17.80 % Al as Al₂O₃ => 52.93 % aluminium in Al₂O₃ => (17.80 % * 52.93 %) / 100 % = 9.42 % Al in test item

In five different artificial physiological media, between 0.04 and 0.25 % of aluminium was dissolved from the test item IPC-2013-009 cobalt chromite blue green spinel depending on solution parameters and test duration.

Mass balance calculation

Total dissolved chromium and cobalt concentrations in vessels, filters and syringes measured by ICP-OES indicate an incomplete dissolution of the test item in all physiological media after addition of aqua regia to the sample vessels. The results and the recoveries of the nominal amount of the initial weight are compiled only fo cobalt. Chromium concentrations in mass balance samples were always below LOD or LOQ.

Calculation of cobalt mass balance

media	value for dissolved Co after addition ofaqua regia [mg]	nominal concentration [mg] #	recovery [%]
ALF 24h A	0.01	7.47	0.06
ALF 24h B	0.01	7.50	0.07
ASW 24h A	0.02	7.45	0.06
ASW 24h B	0.04	7.50	0.30
GMB 24h A	0.01	7.50	0.06
GMB 24h B	0.02	7.42	0.06
GST 24 A	0.01	7.52	0.08
GST 24h B	0.01	7.51	0.08
PBS 24h A	0.02	7.50	0.07
PBS 24h B	0.01	7.54	0.07

# nominal concentration Co = 18.80 % as CoO in test item = 78.65 % Co in CoO => 7.393 mg Co in 50 mg test item => 7.393 * initial weight / 50 mg

Conclusions:

The bioaccessibility of cobalt chromite blue green spinel 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 Co, Al and Cr concentrations were below 9.89 µg/L, 23.5 µg/L and 1.11 µg/L, respectively, even at the highest loading of 0.1 g/L, referring to a solubility of 0.01 %, 0.024 % and 0.001 %, respectively, the pigment is considered biologically inert.

Executive summary:

The bioaccessibility of Co, Al, and Cr 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 Co, Al, and Cr concentrations were below 9.89 µg/L, 23.5 µg/L and 1.11 µg/L respectively, even at the highest loading of 0.1g/L, referring to a solubility of 0.01 %, 0.024 % and 0.001 %, respectively, the pigment is considered biologically inert.

Reference 2

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:

2016-04-28 to 2016-11-11

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 2h and 24h; determination of Al, Cr, Co and Zn concentrations after filtration by ICP-OES and ICP-MS.

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. QA statement)

Remarks:

signed, 2015-12-16

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 (by ICP-OES and ICP-MS) of dissolved aluminum, chromium, cobalt and zinc concentrations after filtration (0.2 µm, Supor membrane)
- the study was performed in triplicates

The aim of this test was to assess the dissolution of IPC-2013-011 (Cobalt chromite blue green spinel) 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: Single element standards (Merck Certipur Aluminium ICP standard 1000 mg/L lot no. HC388610; Merck Certipur Chromium ICP standard 1000 mg/L lot no. HC386521; Merck Certipur Cobalt ICP standard 1000 mg/L lot no. HC41722713; Merck Certipur Zinc ICP standard 1000 mg/L lot no. HC43663769; Darmstadt, Germany).
- certified reference materials: TM-25.4 (lot no. 0914), TM-26.4 (lot no. 1115), TMDA-52.4 (lot no. 0615) and TMDA-54.5 (lot no. 0815) obtained from Environment Canada and a multielement standard (Merck IV, lot no. HC54938555, Darmstadt, Germany)

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
Carrier gas flow: 0.75 L/min
RF power: 1200W
Wavelengths: Al: 167.019 nm and 396.152 nm / Cr: 267.716 nm, 283.563 nm and 284.325 nm / Co: 231.160 nm, 237.863 nm and 238.892 nm / Zn: 202.548 nm, 206.200 nm and 213.857 nm
Calibration: blank, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 and 100 μg/L.
Correlation coefficients (r): at least 0.996856.
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

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: 0.11 L/min
RF power: 1500 W
Isotopes: 27Al, 52Cr, 53Cr, 59Co, 66Zn, 68Zn and 103Rh (internal standard)
calibrate the instrument: blank, 0.1, 0.25, 0.5, 0.75, 1, 2.5, 5.0, 7.5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μg/L
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).

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 100.024 mg/L to 101.014 mg/L in the test vessels.

Type:

other: Bioaccessibility

Results:

Higest dissolution at a loading of 0.1g/L after 24h in ALF: Al: 11.1 ± 0.51 µg/L, Cr: 0.985 ± 0.075 µg/L, Co: 7.60 ± 0.14 µg/L, Zn: 1.59 ± 0.65 µg/L; in GST: Al: 13.1 ± 0.48 µg/L, Cr: 0.697 ± 0.010 µg/L, Co: 8.35 ± 0.29 µg/L, Zn: 4.75 ± 0.23 µg/L

Method validation summary ICP-OES

validation parameter	results	comment
Selectivity	similar data with different wavelengths for ICP-OES method	-
Linearity	applied calibration functions were linear	coefficient at least 0.996856
Limit of detection	Al: 0.099 – 0.387 μg/L Cr: 0.185 μg/L Co: 0.371 – 1.57 μg/L Zn: 0.149 – 0.883 μg/L	-
Limit of quantification	Al: 0.298 – 1.16 μg/L Cr: 0.555 μg/L Co: 1.11 – 4.76 μg/L Zn: 0.448 – 2.65 μg/L	-
Method blanks	Aluminium:  - GST: All samples below LOD - GMB All samples above LOQ: 2h: 5.08 ± 0.06 μg/L; 24h:5.42 ± 1.23 μg/L - PBS: All samples below LOD - ASW All samples below LOD Cobalt: - GST: All samples below LOD Zinc: - GST: All samples above LOQ: 2h: 1.75 ± 0.60 μg/L; 24h: 2.09 ± 0.34 μg/L - GMB: All samples below LOD - PBS: 2h samples below LOD/LOQ; 24h samples one below LOQ, three samples above LOQ: 0.95 ± 0.05 μg/L - ASW: All samples above LOQ: 2h: 1.49 ± 0.15 μg/L; 24h: 2.90 ± 1.05 μg/L
Accuracy measurement / Reproducibility test sample measurements	Mean recovery for CRM TM- 25.4 (dilution factor 10): Al: 104 ± 6.4 (n = 8) Co: 99.9 ± 5.5 (n = 4)	Low concentration range (certified with 30.1 μg Al/L,diluted 3.01 μg Al/L; 27.5 μg Co/L, diluted 2.75 μg Co/L)
Accuracy measurement / Reproducibility test sample measurements	Mean recovery for CRM TMDA- 52.4 (dilution factor 10): Al: 102 ± 2.1 (n = 16) Co: 99.9 ± 1.7 (n = 4) Zn: 101 ± 2.9 (n = 16)	Mid concentration range (certified with 306 μg Al/L, diluted 30.6 μg Al/L; 127 μg Co/L, diluted 12.7 μg Co/L; 306 μg Zn/L, diluted 30.6 μg Zn/L)
Accuracy measurement / Reproducibility test sample measurements	Mean recovery for CRM TMDA- 54.5 (dilution factor 100): Zn: 104 ± 9.1 (n = 16)	Low concentration range (certified with 546 μg Zn/L, diluted 5.46 μg Zn/L)
Accuracy measurement / Reproducibility mass balance sample measurements	Mean recovery for CRM TMDA- 52.4 (dilution factor 10): Al: 104 ± 1.5 (n = 3) Co: 94.7 ± 0.2 (n = 3) Cr: 101 ± 0.6 (n = 3) Zn: 97.2 ± 2.8 (n = 3)	Mid concentration range (certified with 306 μg Al/L, diluted 30.6 μg Al/L; 127 μg Co/L, diluted 12.7 μg Co/L; 158 mg Cr/L, diluted 15.8 μg Cr/L; 306 μg Zn/L, diluted 30.6 μg Zn/L)
Accuracy measurement / Reproducibility mass balance sample measurements	Mean recovery for CRM TMDA- 54.5 (dilution factor 100): Cr: 103 ± 2.8 (n = 3) Zn: 89.4 ± 4.6 (n = 3)	Low concentration range (certified with 546 μg Zn/L, diluted 5.46 μg Zn/L; 438 mg Cr/L, diluted 4.38 μg Cr/L)
Trueness test sample measurements	Quality control standard (Merck IV): Al: 103 ± 2.1 (n = 8) Zn: 100 ± 2.2 (n = 12)	Mid concentration range (20 μg/L)
Trueness test sample measurements	Quality control standard (Merck IV): Al: 103 ± 2.3 (n = 8) Co: 100 ± 2.0 (n = 4) Zn: 95.5 ± 2.3 (n = 4)	Mid concentration range (25 μg/L)
Trueness mass balance sample measurements	Quality control standard (Merck IV): Al: 107 ± 2.9 (n = 4) Cr: 99.4 ± 1.3 (n = 4) Co: 97.1 ± 0.9 (n = 4) Zn: 94.3 ± 1.0 (n = 4)	Mid concentration range (20 μg/L)
Trueness mass balance sample measurements	Quality control standard (Merck IV): Al: 99.2 ± 0.4 (n = 4) Cr: 95.8 ± 0.8 (n = 4) Co: 96.2 ± 0.6 (n = 4) Zn: 97.3 ± 1.6 (n = 4)	High concentration range (75 μg/L)
Trueness test sample measurements	Recalibration standard Al: 108 ± 4.9 (n = 16) Co: 99.1 ± 0.6 (n = 4) Zn: 103 ± 6.1 (n = 16)	Low concentration range (10 μg/L)
Trueness mass balance sample measurements	Recalibration standard Al: 115 ± 3.8 (n = 3) Cr: 100 ± 2.1 (n = 4) Co: 96.0 ± 1.4 (n = 4) Zn: 87.0 ± 3.3 (n = 4)	Low concentration range (10 μg/L)
Trueness mass balance sample measurements	Recalibration standard Al: 102 ± 0.9 (n = 4) Cr: 98.7 ± 1.3 (n = 4) Co: 97.9 ± 1.3 (n = 4) Zn: 96.5 ± 1.3 (n = 4)	High concentration range (50 μg/L)
Trueness test samples	Fortification of samples: Al: 104 – 114 % Co: 101 % Zn: 101 – 115 %	-
Trueness mass balance sample measurements	Fortification of samples: Al: 94.1 – 105 % Cr: 86.1 – 111 % Co: 86.2 – 109 % Zn: 97.5 – 113 %	-

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 0.998835
Limit of detection	Al: 0.05 – 0.336 μg/L Cr: 0.002 – 0.135 μg/L Co: 0.001 – 0.006 μg/L Zn: 0.474 μg/L	-
Limit of quantification	Al: 0.15 – 1.012 μg/L Cr: 0.006 – 0.404 μg/L Co: 0.003 – 0.019 μg/L Zn: 1.42 μg/L	-
Accuracy measurement / Reproducibility test sample measurements	Mean recovery for CRM TM- 25.4 (dilution factor 2 and 10): Al: 102 ± 7.3 (n = 4) Cr: 90.6 ± 6.2 (n = 12) Co: 99.3 ± 3.1 (n = 12) Zn: 109 ± 1.5 (n = 4)	Low concentration range (certified with 30.1 μg Al/L, diluted 3.01 μg Al/L; 24.0 μg Cr/L, diluted 12.0 and 2.40 μg Cr/L; 27.5 μg Co/L, diluted 13.75 and 2.75 μg Co/L; 44.5 μg Zn/L, diluted 4.45 μg Zn/L)
Accuracy measurement / Reproducibility test sample measurements	Mean recovery for CRM TM- 26.4 (dilution factor 4): Cr: 86.8 ± 4.4 (n = 3) Co: 97.3 ± 2.9 (n = 4)	Low concentration range (certified with 12.4 μg Cr/L, diluted 3.10 μg Cr/L; 8.1 μg Co/L, diluted 2.03 μg Co/L)
Accuracy measurement / Reproducibility test sample measurements	Mean recovery for CRM TMDA- 54.5 (dilution factor 100): Al: 111 (n = 1) Cr: 98.8 ± 0.4 (n = 4)	Low concentration range (certified with 399 μg Al/L, diluted 3.99 μg Al/L; 438 μg Cr/L, diluted 4.38 μg Cr/L)
Accuracy measurement / Reproducibility test sample measurements	Mean recovery for CRM TMDA- 52.4 (dilution factor 10 and 20 only for Al): Al: 103 ± 6.0 (n = 5) Cr: 99.7 ± 3.2 (n = 20) Co: 99.5 ± 3.0 (n = 16) Zn: 101 ± 1.3 (n = 4)	Mid to high concentration range (certified with 306 μg Al/L, diluted 30.6 and 15.3 μg Al/L; 158 μg Cr/L, diluted 15.8 μg Cr/L; 127 μg Co/L, diluted 12.7 μg Co/L; 256 μg Zn/L, diluted 25.6 μg Zn/L)
Trueness test samples	Quality control standard (Merck IV): Al: 99.7 ± 4.3 (n = 5) Cr: 99.2 ± 5.4 (n = 19) Co: 98.4 ± 5.4 (n = 15) Zn: 102 ± 1.8 (n = 4)	High concentration range (25 μg/L)
Trueness test samples	Recalibration standard: Al: 102 ± 4.5 (n = 4) Cr: 99.6 ± 7.0 (n = 20) Co: 98.3 ± 3.2 (n = 16) Zn: 104 ± 1.9 (n = 3)	Mid concentration range (10 μg/L)
Trueness test samples	Fortification of samples: Al: 99.6 – 101 % Cr: 90.0 – 102 % Co: 93.6 – 100 % Zn: 101 – 105 %	-
Method blanks	Chromium: - GST: All samples below LOD - ALF: All samples below LOQ - ASW: All samples below LOD - PBS All samples below LOD - GMB All samples below LOD Cobalt: - ALF: Three 2h samples below LOQ, one sample above LOQ: 0.013 ± 0.003 μg/L; 24h samples all below LOD/LOQ - ASW: All samples below LOD - PBS: All samples above LOQ: 2h: 0.009 ± 0.007 μg/L; 24h: 0.007 ± 0.005 μg/L - GMB: 2h samples all below LOD/LOQ, two samples 24h below LOQ, two samples above LOQ: 0.006 ± 0.002 μg/L Zinc: - ALF: All samples above LOQ: 2h: 3.39 ± 0.32 μg/L; 24h: 4.53 ± 0.79 μg/L

Concentration of dissolved aluminium in artificial physiological media

media and sample	LOD/LOQ of Al measurement series [µg/L]	total Al ± SD in method blanks [µg/L]	Without method blank subtraction		With mean method blank subtraction (mean of 2h and/or 24h)
			total Al ± SD in sample vessels [µg/L]	total Al ± SD in sample vessels [µg/L]*	total Al ± SD in sample vessels [µg/L]	total Al ± SD in sample vessels [µg/L]*
GST 2h	LOD: 0.387 LOQ: 1.16	all method blanks below LOD	10.0 ± 0.28	9.95 ± 0.26	No subtraction	No subtraction
GST 24h	LOD: 0.387 LOQ: 1.16	all method blanks below LOD	13.2 ± 0.51	13.1 ± 0.48	No subtraction	No subtraction
GMB 2h	LOD: 0.193 LOQ: 0.580	5.08 ± 0.06	6.61 ± 1.21	6.61 ± 1.21	2.38 ± 2.44	2.38 ± 2.44
GMB 24h	LOD: 0.193 LOQ: 0.580	5.42 ± 1.23	5.77 ± 0.31	5.76 ± 0.31	0.349 ± 0.309	0.349 ± 0.308
ALF 2h	LOD: 0.336 LOQ: 1.01	all method blanks below LOD/LOQ	8.73 ± 0.35	8.67 ± 0.32	No subtraction	No subtraction
ALF 24h	LOD: 0.336 LOQ: 1.01	0.944 ± 0.316 (two method blanks below LOQ; two above LOQ)	12.1 ± 0.52	12.1 ± 0.51	11.2 ± 0.52	11.1 ± 0.51
ASW 2h	LOD: 0.260 LOQ: 0.779	all method blanks below LOD	All samples below LOD/LOQ	All samples below LOD/LOQ	No subtraction	No subtraction
ASW 24h	LOD: 0.260 LOQ: 0.779	all method blanks below LOD	1.95 ± 0.70	1.94 ± 0.70	No subtraction	No subtraction
PBS 2h	LOD: 0.099 LOQ: 0.298	all method blanks below LOD	all samples below LOD	all samples below LOD	No subtraction	No subtraction
PBS 24h	LOD: 0.099 LOQ: 0.298	all method blanks below LOD	0.346 ± 0.478 (one sample above LOQ; one sample above LOD; four samples below LOD)	0.345 ± 0.476	No subtraction	No subtraction

*(corrected concentrations, corresponding to a loading of exactly 100 mg/L)

CALCULATION (performed by the monitor)

- calculated nominal concentration of Al: 100 % (test item) / 9.42 % § (Al in test item) * 100 mg/L (initial weight) = 9.42 mg Al/L

- highest dissolved amount in artificial media of Al: 100 % / 9.42 mg/L * 0.0131 mg/L (GST 24h: 13.1 ± 0.48 µg/L) = 0.1391 %

- lowest dissolved amount in artificial media of Al: 100 % / 9.42 mg/L * 0.000345 mg/L (PBS 24h: 0.345 ± 0.476 µg/L) = 0.0037 %

§ according to CoA 17.80 % Al as Al₂O₃=> 52.93 % aluminium in Al₂O₃=> (17.80 % * 52.93 %) / 100 % = 9.42 % Al in test item

In five different artificial physiological media, between 0.0036 and 0.1391 % of aluminium was dissolved from the test item IPC-2013-009 cobalt chromite blue green spinel depending on solution parameters and test duration.

Concentration of dissolved chromium in artificial physiological media (no method blank subtraction – all method blanks below LOD/LOQ)

media and sample	LOD/LOQ of Cr measurement series [µg/L]	total Cr ± SD in method blanks [µg/L]	total Cr ± SD in sample vessels [µg/L] without method blank	total Cr ± SD in sample vessels [µg/L] (corrected concentrations, corresponding to a loading of exactly 100 mg/L)
GST 2h	LOD: 0.003 LOQ: 0.008	all method blanks below LOD	0.450 ± 0.021	0.448 ± 0.021
GST 24h	LOD: 0.003 LOQ: 0.008	all method blanks below LOD	0.701 ± 0.010	0.697 ± 0.010
GMB 2h	LOD: 0.017 LOQ: 0.050	all method blanks below LOD	0.111 ± 0.030	0.125 ± 0.010
GMB 24h	LOD: 0.017 LOQ: 0.050	all method blanks below LOD	0.111 ± 0.030	0.125 ± 0.010
ALF 2h	LOD: 0.135 LOQ: 0.404	all method blanks below LOQ	0.769 ± 0.041	0.763 ± 0.039
ALF 24h	LOD: 0.135 LOQ: 0.404	all method blanks below LOQ	0.991 ± 0.073	0.985 ± 0.075
ASW 2h	LOD: 0.002 LOQ: 0.006	all method blanks below LOD	0.206 ± 0.049	0.205 ± 0.049
ASW 24h	LOD: 0.002 LOQ: 0.006	all method blanks below LOD	0.318 ± 0.027	0.317 ± 0.027
PBS 2h	LOD: 0.025 LOQ: 0.075	all method blanks below LOD	0.145 ± 0.024	0.144 ± 0.025
PBS 24h	LOD: 0.025 LOQ: 0.075	all method blanks below LOD	0.244 ± 0.038	0.243 ± 0.038

CALCULATION (performed by the monitor)

- calculated nominal concentration of Cr: 100 % (test item) / 30.4 % § (Cr in test item) * 100 mg/L (initial weight) = 30.4 mg Cr/L

- highest dissolved amount in artificial media of Cr: 100 % / 30.4 mg/L * 0.000985 mg/L (ALF 24h: 0.985 ± 0.075 µg/L) = 0.0032 %

- lowest dissolved amount in artificial media of Cr: 100 % / 30.4 mg/L * 0.000125 mg/L (GMB 24h: 0.125 ± 0.010 µg/L)= 0.000411 %

§ according to CoA 43.90 % Cr as Cr₂O₃==> 68.42 % chromium in Cr₂O₃==> (43.90 % * 68.42 %) / 100 % = 30.04 % Cr in test item

In five different artificial physiological media, between 0.000411 and 0.0032 % of chromium was dissolved from the test item IPC-2013-009 cobalt chromite blue green spinel depending on solution parameters and test duration.

Concentration of dissolved cobalt in artificial physiological media

media and sample	LOD/LOQ of Co measurement series [µg/L]	total Co ± SD in method blanks [µg/L]	Without method blank subtraction		With mean method blank subtraction (mean of 2h and/or 24h)
			total Co ± SD in sample vessels [µg/L]	total Co ± SD in sample vessels [µg/L]*	total Co ± SD in sample vessels [µg/L]	total Co ± SD in sample vessels [µg/L]*
GST 2h	LOD: 0.371 LOQ: 1.11	all method blanks below LOD	5.89 ± 0.11	5.86 ± 0.12	No subtraction	No subtraction
GST 24h	LOD: 0.371 LOQ: 1.11	all method blanks below LOD	8.39 ± 0.30	8.35 ± 0.29	No subtraction	No subtraction
GMB 2h	LOD: 0.002 LOQ: 0.007	all method blanks below LOD/LOQ	0.357 ± 0.094	0.357 ± 0.094	No subtraction	No subtraction
GMB 24h	LOD: 0.002 LOQ: 0.007	0.006 ± 0.002 (Two method blank above LOQ; two method blanks below LOQ)	0.642 ± 0.035	0.642 ± 0.035	0.636 ± 0.035	0.636 ± 0.035
ALF 2h	LOD: 0.006 LOQ: 0.017	0.013 ± 0.003 (One method blank above LOQ; three method blanks below LOQ)	4.42 ± 0.23	4.39 ± 0.22	4.41 ± 0.23	4.38 ± 0.22
ALF 24h	LOD: 0.006 LOQ: 0.017	all method blanks below LOD/LOQ	7.65 ± 0.16	7.60 ± 0.14	No subtraction	No subtraction
ASW 2h	LOD: 0.006 LOQ: 0.019	all method blanks below LOD	0.641 ± 0.088	0.639 ± 0.088	No subtraction	No subtraction
ASW 24h	LOD: 0.006 LOQ: 0.019	all method blanks below LOD	3.34 ± 0.05	3.32 ± 0.05	No subtraction	No subtraction
PBS 2h	LOD: 0.001 LOQ: 0.003	0.009 ± 0.007 (all method blanks above LOQ)	0.306 ± 0.052	0.304 ± 0.053	0.297 ± 0.052	0.296 ± 0.053
PBS 24h	LOD: 0.001 LOQ: 0.003	0.007 ± 0.005 (all method blanks above LOQ)	1.94 ± 0.07	1.93 ± 0.07	1.93 ± 0.07	1.92 ± 0.07

* (corrected concentrations, corresponding to a loading of exactly 100 mg/L)

CALCULATION (performed by the monitor)

- calculated nominal concentration of Co: 100 % (test item) / 14.79 % § (Co in test item) * 100 mg/L (initial weight) = 14.79 mg Co/L

- highest dissolved amount in artificial media of Co: 100 % / 14.79 mg/L * 0.00835 mg/L (GST 24h: 8.35 ± 0.29 µg/L) = 0.0564 %

- lowest dissolved amount in artificial media of Co: 100 % / 14.79 mg/L * 0.000296 mg/L (PBS 2h: 0.296 ± 0.053 µg/L)= 0.0020 %

§ according to CoA 18.80 % Co as CoO ==> 78.65 % cobalt in CoO ==> (18.80 % * 78.65 %) / 100 % = 14.79 % Co in test item

In five different artificial physiological media, between 0.0020 and 0.0564 % of cobalt was dissolved from the test item IPC-2013-009 cobalt chromite blue green spinel depending on solution parameters and test duration.

Concentration of dissolved zinc in artificial physiological media

media and sample	LOD/LOQ of Zn measurement series [µg/L]	total Zn ± SD in method blanks [µg/L]	Without method blank subtraction		With mean method blank subtraction (mean of 2h and/or 24h)
			total Zn ± SD in sample vessels[µg/L]	total Zn ± SD in sample vessels [µg/L]*	total Zn ± SD in sample vessels[µg/L]	total Zn ± SD in sample vessels [µg/L]*
GST 2h	LOD: 0.224 LOQ: 0.672	1.75 ± 0.60	5.26 ± 0.87	5.23 ± 0.87	3.51 ± 0.87	3.49 ± 0.87
GST 24h	LOD: 0.224 LOQ: 0.672	2.09 ± 0.34	6.52 ± 0.24	6.49 ± 0.23	4.77 ± 0.24	4.75 ± 0.23
GMB 2h	LOD: 0.333 LOQ: 0.998	all method blanks below LOD	all samples below LOD	all samples below LOD	No subtraction	No subtraction
GMB 24h	LOD: 0.333 LOQ: 0.998	all method blanks below LOD	all samples below LOD	all samples below LOD	No subtraction	No subtraction
ALF 2h	LOD: 0.474 LOQ: 1.42	3.39 ± 0.32	4.92 ± 0.97	4.88 ± 0.95	1.53 ± 0.97	1.52 ± 0.96
ALF 24h	LOD: 0.474 LOQ: 1.42	4.53 ± 0.79	6.13 ± 0.65	6.09 ± 0.66	1.60 ± 0.65	1.59 ± 0.65
ASW 2h	LOD: 0.162 LOQ: 0.485	1.49 ± 0.15	1.02 ± 0.53 µg/L (four samples above LOQ; one sample below LOQ; one sample below LOD)	1.02 ± 0.53	0.473 ± 0.196 (two sample negative value after subtraction)	0.471 ± 0.195
ASW 24h	LOD: 0.162 LOQ: 0.485	2.90 ± 1.05	0.665 ± 0.267 (four samples above LOQ; two sample below LOQ)	0.662 ± 0.266	0.323 ± <0.001 (four sample negative value after subtraction)	0.322 ± <0.001
PBS 2h	LOD: 0.149 LOQ: 0.448	all method blanks below LOD/LOQ	0.295 ± 0.328 (one sample above LOQ; 5 samples below LOD)	0.293 ± 0.326	No subtraction	No subtraction
PBS 24h	LOD: 0.149 LOQ: 0.448	0.95 ± 0.05 (three method blanks above LOQ; one method blank below LOQ)	all samples below LOD	all samples below LOD	all samples below LOD	all samples below LOD

*(corrected concentrations, corresponding to a loading of exactly 100 mg/L)

CALCULATION (performed by the monitor)

- calculated nominal concentration of Zn: 100 % (test item) / 15.10 % § (Zn in test item) * 100 mg/L (initial weight) = 15.10 mg Zn/L

- highest dissolved amount in artificial media of Zn: 100 % / 15.10 mg/L * 0.0475 mg/L (GST 24h: 4.75 ± 0.23 µg/L) = 0.3146 %

- lowest dissolved amount in artificial media of Zn: 100 % / 15.10 mg/L * 0.000293 mg/L (PBS 2h: 0.293 ± 0.326 µg/L)= 0.00194 %

§ according to CoA 18.80 % Zn as ZnO ==> 80.3411 % zinc in ZnO ==> (18.80 % * 80.3411 %) / 100 % = 15.10 % Zn in test item

In five different artificial physiological media, between 0.00194 and 0.3146 % of zinc was dissolved from the test item IPC-2013-009 cobalt chromite blue green spinel depending on solution parameters and test duration.

Mass balance calculation

The mass balance dissolved aluminum, chromium, cobalt and zinc concentrations in vessels and filters as measured by ICP-OES indicate no complete dissolution of IPC-2013-009 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 aluminium in mass balance samples (vessels, filter/syringes and taken samples) for each media

Media	Value for dissolved Al after addition of aqua regia		In samples [mg]	Calculated value Al [mg]*	nominal amount [mg] #	recovery [%]
	Vessels [mg]	Filters/syringes [mg]
GST vessel 1	0.009	0.00155	0.0010	0.011	4.746	0.242
GST vessel 2	0.008	0.00083	0.0009	0.010	4.732	0.213
GST vessel 3	0.009	0.00081	0.0009	0.010	4.726	0.219
GMB vessel 1	0.021	0.00097	0.0005	0.023	4.712	0.487
GMB vessel 2	0.032	0.00063	0.0005	0.034	4.717	0.712
GMB vessel 3	0.018	0.00066	0.0004	0.020	4.711	0.414
ALF vessel 1	0.005	0.00079	0.001	0.007	4.758	0.148
ALF vessel 2	0.006	0.00079	0.0009	0.007	4.729	0.156
ALF vessel 3	0.005	0.00062	0.0009	0.007	4.740	0.141
ASW vessel 1	0.009	0.00170	0.00005	0.010	4.728	0.219
ASW vessel 2	0.010	0.00110	0.00009	0.011	4.732	0.226
ASW vessel 3	0.008	0.00068	0.00010	0.009	4.725	0.196
PBS vessel 1	0.013	0.00115	0.00004	0.014	4.729	0.299
PBS vessel 2	0.012	0.00087	0.00000	0.012	4.742	0.263
PBS vessel 3	0.013	0.00061	0.00000	0.013	4.737	0.278

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

Example: 0.009 mg (amount Al in vessels) + 0.00155 mg (amount Al in syringe/filters) + 0.001 mg (Al in samples) = 0.011 mg

# nominal amount Al = 17.80 % in test item (according to CoA) as Al2O3 -> 52.93 % Al in Al2O3 -> 4.71 mg Al in 50 mg test item -> nominal amount 4.71 mg * initial weight mg / 50 mg

Example: 4.71 mg Al * 50.507 mg test item / 50 mg = 4.758 mg Al

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

Media	Value for dissolved Co after addition of aqua regia		In samples [mg]	Calculated value Co [mg]	Nominal amount [mg] #	Recovery [%]
	Vessels [mg]	Filters/syringes [mg]
GST vessel 1	0.005	<LOD	0.0006	0.006	7.449	0.080
GST vessel 2	0.004	<LOD	0.0006	0.005	7.427	0.068
GST vessel 3	0.004	<LOD	0.0006	0.005	7.418	0.064
GMB vessel 1	0.004	<LOD	0.00003	0.004	7.395	0.050
GMB vessel 2	0.003	<LOD	0.00004	0.003	7.404	0.047
GMB vessel 3	0.004	<LOD	0.00004	0.004	7.395	0.048
ALF vessel 1	0.003	<LOD	0.0006	0.004	7.468	0.047
ALF vessel 2	0.003	<LOD	0.0006	0.003	7.422	0.045
ALF vessel 3	0.003	<LOD	0.0006	0.004	7.439	0.050
ASW vessel 1	0.003	<LOD	0.0002	0.004	7.420	0.049
ASW vessel 2	0.004	<LOD	0.0002	0.004	7.428	0.052
ASW vessel 3	0.004	<LOD	0.0002	0.004	7.416	0.051
PBS vessel 1	0.003	<LOD	0.0001	0.004	7.422	0.047
PBS vessel 2	0.003	<LOD	0.0001	0.003	7.442	0.047
PBS vessel 3	0.004	<LOD	0.0001	0.004	7.435	0.048

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

# nominal amount Co = 18.80 % in test item (according to CoA) as CoO -> 78.65 % Co in CoO ->7.39 mg Co in 50 mg test item ->nominal amount 7.39 mg * initial weight mg / 50 mg

Conclusions:

The bioaccessibility of cobalt chromite blue green spinel 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 Al, Cr, Co and Zn concentrations were below 13.1 µg/L, 0.985 µg/L, 8.35 µg/L and 4.75 µg/L, respectively, even at the highest loading of 0.1 g/L, referring to a solubility of 0.013 %, 0.001 %, 0.008 % and 0.005 %, respectively, the pigment is considered biologically inert.

Executive summary:

The bioaccessibility of Al, Cr, Co and Zn 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 Al, Cr, Co and Zn concentrations were below 13.1 µg/L, 0.985 µg/L, 8.35 µg/L and 4.75 µg/L, respectively, even at the highest loading of 0.1 g/L, referring to a solubility of 0.013 %, 0.001 %, 0.008 % and  0.005 %, respectively, the pigment is considered biologically inert.

Description of key information

Dissolved Al, Cr, Co and Zn concentrations were below 13.1 µg/L, 0.985 µg/L, 8.35 µg/L and 4.75 µg/L, respectively, even at the highest loading of 0.1 g/L, referring to a solubility of 0.013 %, 0.001 %, 0.008 % and  0.005 %, respectively, the pigment is considered biologically inert.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Discussion on bioaccumulation potential result:

The chemical and physiological properties of the pigment cobalt chromite blue green spinel are dominated by inertness due to the characteristics of the production process (calcination at high temperatures, approximately 1000 °C), rendering the substance to be of a unique, stable crystalline structure. This fundamental process leads to a very low bioaccessibility for the elements contained in the pigment.

Knopf 2013

The bioaccessibility of Co, Al and Cr 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 Co, Al, and Cr concentrationswere below 9.89 µg/L, 23.5 µg/L and 1.11 µg/L respectively, even at the highest loading of 0.1 g/L, referring to a solubility of 0.01 %, 0.024 % and 0.001 %, the pigment is considered biologically inert.

Knopf 2016

The bioaccessibility of Al, Cr, Co and Zn 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 Al, Cr, Co and Zn concentrationswere below 13.1 µg/L, 0.985 µg/L, 8.35 µg/L and 4.75 µg/L respectively, even at the highest loading of 0.1g/L, referring to a solubility of 0.013 %, 0.001 %, 0.008 % and  0.005 %, the pigment is considered biologically inert.