Cobalt chromite blue green spinel

Basic toxicokinetics

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Objective of study:

bioaccessibility (or bioavailability)

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Cobalt chromite blue green spinel

EC Number:

269-072-0

EC Name:

Cobalt chromite blue green spinel

Cas Number:

68187-11-1

Molecular formula:

Co(Al,Cr)2O4

IUPAC Name:

dialuminium(3+) dichromium(3+) λ²-cobalt(2+) heptaoxidandiide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: Cobalt chromite blue green spinel; Pigment Blue 36 (C.I. 77343)
- Substance type: inorganic pigment
- Physical state: solid, blue green powder, odourless
- Storage condition of test material: at room temperature, kept dry in closed container

Test animals

Species:

other: in vitro (simulated human body fluids)

Administration / exposure

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.

Doses / concentrations

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.

Results and discussion

Main ADME results

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.