Cobalt chromite green spinel

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics, other

Remarks:

Bioaccessibility

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-04-17 to 2015-12-17

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 Zn concentrations after filtration by ICP-OES and ICP-MS.

Principles of method if other than guideline:

The test was performed on the basis of OECD Series on Testing and Assessment No. 29 as well as according to the bioaccessibility test protocol provided by the monitor. The bioaccessibility protocol has been developed on the basis of relevant published methods ([1], [2], [3], [4] and [5]).

The aim of this test was to assess the dissolution of the pigment IPC-2018-002 (Cobalt chromite green spinel) 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 chromium, cobalt and zinc concentrations after filtration were performed by ICP-OES and ICP-MS. Samples were taken after 2 and 24 hours agitation (100 rpm) at 37 ± 2 °C. The study was performed in triplicate with two additional method blanks per medium.

[1] Hanawa T. 2004. Metal ion release from metal implants. Materials Science and Engineering C 24: 745-752.
[2] Stopford W., Turner J., Cappelini D., Brock T. 2004. Bioaccessibility testing of cobalt compounds. Journal of Environmental Monitoring 5: 675-680.
[3] Midander K., et al. 2007. In vitro studies of copper release from powder particles in synthetic biological media. Environmental Pollution 145: 51-59.
[4] European standard 1998. Test method for release of nickel from products intended to come into direct and prolonged contact with the skin (EN 1811)
[5] ASTM 2003. Standard test method for determining extractability of metals from art materials. ASTM D5517-03

GLP compliance:

yes (incl. 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 and IPC-MS) of dissolved chromium, cobalt and zinc concentrations after filtration
- the study was performed in triplicate

The aim of this test was to assess the dissolution of IPC-2018-002 (Cobalt chromite 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).
- Sodiumhydroxide – pro Analysis quality (Chemsolute, Th. Geyer, Renningen, Germany)

Metal analysis
- Standards: single element standards were used as chromium, cobalt and zinc standards (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. HC388620; Darmstadt, Germany).
- Certified reference materials: quality control standards TM-25.4 (lot no. 0914) and TMDA-53.3 (lot no. 0914) obtained from Environment Canada and a multielement standard (Merck XXI, lot no. HC42984673 and HC55190098, Darmstadt, Germany) were analysed for total dissolved chromium, cobalt and zinc by ICP-OES and ICP-MS along with the samples to determine the accuracy of the applied analytical method. Furthermore the calibration solutions were measured along with the ICP-OES and ICP-MS 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
- Carrier gas flow: 0.75 L/min
- RF power: 1200W
- Wavelengths:
Cr: 205.560 nm, 276.259 nm, 276.653 nm, 283.563 nm, 285.567 nm, 286.257 nm and 357.868 nm
Co: 201.151 nm, 228.615 nm, 230.786 nm, 231.160 nm, 231.497 nm, 235.341 nm, 236.379 nm, 237.863 nm, 238.345 nm, 238.892 nm and 241.765 nm
Zn: 202.548 nm, 206.200 nm and 213.857 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, 2.5 μg/l, 5 μg/L, 7.5 μg/L, 10 μg/L, 25 μg/L, 50 μg/L, 75 μg/L, 100 μg/L, 250 μg/L, 500 μg/L, 750 μg/L and 1000 μg/L.
Correlation coefficients (r): at least 0.999851

Determination of mass balance
To the residual, undissolved test item in the vessels, 80 mL aqua regia (3 : 1 mixture of concentrated hydrochloric and nitric acid) were added to the flasks after the test. The filters used for sampling were extensively rinsed with aqua regia. Solutions were sampled after at least 24h, chromium, cobalt and zinc concentration were measured in at least one mass balance sample (vessel and filter/syringes) for each medium by ICP-OES, and the mass balance was calculated.

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: 0.16 L/min
- RF power: 1500 W
- Isotopes: 52Cr, 54Cr and 103Rh (internal standard)

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

Calibration:blank, 0.1 μg/L, 0.25 μg/l, 0.5 μg/L, 0.75 μg/L, 1 μg/L, 2.5 μg/L, 5.0 μg/L, 7.5 μg/L, 10 μg/L, 12.5 μg/L, 15 μg/L, 17.5 μg/L, 20 μg/L, 22.5 μg/L and 25 μg/L.
Correlation factors (r): at least 0.999671

Details on dosing and sampling:

Loading:
Detailed loadings of the test vessels are given in "Any other information on materials and methods incl. tables".

Toxicokinetic parameters:

other: bioaccessibility

Remarks:

Dissolution of Cr at a loading of 0.1 g/L: GST (pH 1.5) 2h: 5.95 ± 0.87 μg/L; dissolution of Co at a loading of 0.1 g/L: ALF (pH 4.5) 24h: 108 ± 1.28 μg/L; dissolution of Zn at a loading of 0.1 g/L: ALF (pH 4.5) 24h: 106 ± 2.61 μg/L.

Bioaccessibility (or Bioavailability) testing results:

Concentration of dissolved chromium in artificial physiological media
Total Cr ± SD in sample vessels (corrected concentrations, corresponding to a loading of exactly 100 mg/L) with mean method blank subtraction (mean of 2h and/or 24h)
- GST 2h: 5.95 ± 0.87 μg/L (method blank: 0.170 ± 0.009 μg/L)
- GST 24h: 6.80 ± 0.07 μg/L (method blank: 0.168 ± 0.019 μg/L)
- GMB 2h: 0.264 ± 0.017 μg/L (no subtraction, all method blanks below LOD)
- GMB 24h: 0.452 ± 0.052 μg/L (no subtraction, all method blanks below LOD)
- ALF 2h: 3.60 ± 0.16 μg/L (method blank: 0.545 ± 0.072 μg/L)
- ALF 24h: 5.39 ± 0.29 μg/L (method blank: 0.466 ± 0.133 μg/L)
- ASW 2h: 0.985 ± 0.16 μg/L (method blank: 0.014 ± 0.014 μg/L (three method blank below LOD, one above LOQ))
- ASW 24h: 3.35 ± 0.06 μg/L (no subtraction, all method blanks below LOD)
- PBS 2h: 0.343 ± 0.138 μg/L (no subtraction, all method blanks below LOD)
- PBS 24h: 2.13 ± 0.16 μg/L (no subtraction, all method blanks below LOD)

Concentration of dissolved cobalt in artificial physiological media
total Co ± SD in sample vessels (corrected concentrations, corresponding to a loading of exactly 100 mg/L) without method blank subtraction (all method blanks below LOD)
- GST 2h: 98.2 ± 0.74 μg/L
- GST 24h: 93.8 ± 0.59 μg/L
- GMB 2h: 17.2 ± 0.49 μg/L
- GMB 24h: 12.8 ± 0.75 μg/L
- ALF 2h: 99.1 ± 2.39 μg/L
- ALF 24h: 108 ± 1.28 μg/L
- ASW 2h: 32.6 ± 8.38 μg/L
- ASW 24h: 89.4 ± 1.03 μg/L
- PBS 2h: 20.8 ± 6.45 μg/L
- PBS 24h: 68.7 ± 1.70 μg/L

Concentration of dissolved zinc in artificial physiological media.
total Zn ± SD in sample vessels (corrected concentrations, corresponding to a loading of exactly 100 mg/L) with mean method blank subtraction (mean of 2h and/or 24h)
- GST 2h: 102 ± 1.65 μg/L (method blank: 4.26 ± 2.95 μg/L)
- GST 24h: 97.0 ± 0.98 μg/L (method blank: 5.47 ± 3.57 μg/L)
- GMB 2h: 2.26 ± 0.32 μg/L (no subtraction, all method blanks below LOD)
- GMB 24h: 0.806 ± 0.850 μg/L (no subtraction, all method blanks below LOD)
- ALF 2h: 102 ± 3.13 μg/L (no subtraction, one method blank below LOD, three method blanks below LOQ)
- ALF 24h: 106 ± 2.61 μg/L (no subtraction, one method blank below LOD, three method blanks below LOQ)
- ASW 2h: 24.3 ± 7.46 μg/L (method blank: 1.47 ± 0.52 μg/L)
- ASW 24h: 87.1 ± 0.87 μg/L (method blank: 2.20 ± 0.58 μg/L)
- PBS 2h: 11.2 ± 4.75 μg/L (no subtraction, all method blanks below LOD)
- PBS 24h: 57.1 ± 2.80 μg/L (no subtraction, all method blanks below LOD)

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 0.999851
Limit of detection	Cr: 0.289 μg/L (only mass balance measurement) Co: 0.209 – 2.21 μg/L Zn: 0.054 – 1.45 μg/L	-
Limit of quantification	Cr: 0.868 μg/L (only mass balance measurement) Co: 0.628 – 6.64 μg/L Zn: 0.163 – 4.35 μg/L	-
Accuracy measurement / Reproducibility test sample and mass balance measurements	Mean recovery for CRM TM- 25.4 (dilution factor 10): Cr: 98.6 ± 3.4 (n = 5; only mass balance) Co: 98.7 ± 6.8 (n = 25) Zn: 105 ± 8.6 (n = 19)	Low concentration range (certified with 24.0 μg Cr/L, diluted 2.40 μg Cr/L; 27.5 μg Co/L, diluted 2.75 μg Co/L; 44.5 μg Zn/L, diluted 4.45 μg Zn/L)
Accuracy measurement / Reproducibility test sample and mass balance measurements	Mean recovery for CRM TMDA- 53.3 (dilution factor 10): Cr: 98.1 ± 2.0 (n = 5; only mass balance) Co: 100 ± 2.4 (n = 29) Zn: 99.7 ± 4.3 (n = 29)	Mid concentration range (certified with 341 μg Cr/L, diluted 34.1 μg Cr/L; 251 μg Co/L, diluted 25.1 μg Co/L; 386 μg Zn/L, diluted 38.6 μg Zn/L)
Trueness test sample measurements	Quality control standard (Merck XXI): Co: 98.0 ± 2.8 (n = 4) Zn: 112 ± 1.5 (n = 4)	Low to mid concentration range (20 μg/L)
Trueness test sample and mass balance measurements	Quality control standard (Merck XXI): Cr: 96.6 ± 1.3 (n = 5; only mass balance) Co: 97.6 ± 2.6 (n = 25) Zn: 99.1 ± 1.6 (n = 25)	Mid to high concentration range (100 μg/L)
Trueness test sample and mass balance measurements	Quality control standard (Merck XXI): Cr: 96.2 ± 1.3 (n = 5; only mass balance) Co: 97.8 ± 3.1 (n = 10) Zn: 99.3 ± 1.3 (n = 10)	High concentration range (500 μg/L)
Trueness test sample measurements	Quality control standard (Merck XXI): Co: 96.6 ± 0.8 (n = 5) Zn: 97.8 ± 0.6 (n = 5)	High concentration range (1000 μg/L)
Trueness test sample and mass balance measurements	Recalibration standard Cr: 96.6 ± 1.2 (n = 5; only mass balance) Co: 97.4 ± 1.6 (n = 20) Zn: 100 ± 4.2 (n = 20)	Mid concentration range (50 μg/L)
Trueness test sample measurements	Recalibration standard Co: 98.5 ± 2.0 (n = 9) Zn: 99.2 ± 3.6 (n = 9)	Mid to high concentration range (100 μg/L)
Trueness test sample and mass balance measurements	Recalibration standard Cr: 97.7 ± 1.0 (n = 5; only mass balance) Co: 99.5 ± 2.4 (n = 15) Zn: 97.4 ± 2.0 (n = 15)	High concentration range (250 μg/L)
Trueness test samples	Fortification of samples: Co: 96.0 - 101% Zn: 99.2 – 115%	-
Trueness mass balance sample measurements	Fortification of samples: Co: 85.5 – 100% Zn: 96.0 – 108%	-

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 0.999671
Limit of detection	Cr: 0.002 – 0.079 μg/L	-
Limit of quantification	Cr: 0.007 – 0.238 μg/L	-
Accuracy measurement / Reproducibility test sample measurements	Mean recovery for CRM TM- 25.4 (dilution factor 10): Cr: 97.1 ± 6.0 (n = 20)	Low concentration range (certified with 24.0 μg Cr/L, diluted 2.40 μg Cr/L)
Accuracy measurement / Reproducibility test sample measurements	Mean recovery for CRM TMDA- 53.3 (dilution factor 20): Cr: 99.2 ± 4.7 (n = 9)	High concentration range (certified with 341 μg Cr/L, diluted 17.1 μg Cr/L)
Accuracy measurement / Reproducibility test sample measurements	Mean recovery for CRM TMDA- 53.3 (dilution factor 50): Cr: 99.2 ± 4.7 (n = 9)	Low to mid concentration range (certified with 341 μg Cr/L, diluted 6.82 μg Cr/L)
Trueness test samples	Quality control standard Merck XXI: Cr: 98.6 ± 5.4 (n = 19)	Low to mid concentration range (5 μg/L)
Trueness test samples	Quality control standard Merck XXI: Cr: 95.3 ± 3.5 (n = 10)	Mid concentration range (10 μg/L)
Trueness test samples	Recalibration standard: Cr: 91.8 ± 11 (n = 10)	Low concentration range (1 μg/L)
Trueness test samples	Recalibration standard: Cr: 97.0 ± 1.3 (n = 10)	Mid concentration range (10 μg/L)
Trueness test samples	Fortification of samples: Cr: 85.1 – 102 %	-

Solution pH values

During the study, the pH of GST, ALF and PBS media remained stable in the method blank vessels and the test vessels. Therefore, a possible effect of the test substance can be excluded.

In GMB medium, the pH in all vessels (including method blanks) increased during the time of the test from 7.40 to 8.75, 8.76 and 8.80 (test vessels) and 7.40 to 8.79 and 8.81 (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 5.95, 5.96 and 5.97 (test vessels) and 6.50 to 5.96 and 5.99 (method blank vessels). Therefore, an effect of the test substance can be excluded.

Temperature control

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

Analytical measurements

The nominal loading in this test was 100 mg/L. However, due to weighing uncertainties the actual loadings range from 100.342 mg/L to 101.834 mg/L in the 15 test vessels.

Fortification

ICP-OES

For fortified test samples, recoveries were in the range of 96.0 – 101 % for Co and the recoveries of Zn were in the range of 99.2 – 115 % in test samples.

Fortification of test samples with cobalt

sample	measured concentration of Co [μg/L]	calculated level after addition [μg/L]	recovery [μg/L] / [%]
GST vessel 1 sample a 2h	50.0	90.0	88.8 / 98.7
GST vessel 1 sample a 24h	47.7	89.5	90.1/ 101
GMB vessel 1 sample a 2h	16.8	21.7	21.0 / 96.5
GMB vessel 2 sample a 24h	12.1	19.8	19.0 / 96.0
ASW vessel 1 sample a 2h (dilution factor 2)	11.7	23.5	22.7 / 96.4
ASW vessel 1 sample a 24h (dilution factor 2)	45.4	29.1	28.7 / 98.9
ALF vessel 1 sample a 2h	101	40.2	39.9 / 99.2
ALF vessel 1 sample b 24h	109	41.9	41.0 / 98.0
PBS vessel 1 sample a 2h	28.6	26.5	26.3 / 99.3
PBS vessel 1 sample a 24h	71.7	21.7	31.6 / 99.6

Fortification of test samples with zinc

sample	measured concentration of Zn [μg/L]	calculated level after addition [μg/L]	recovery [μg/L] / [%]
GST Vessel 1 sample a 2h	54.9	91.0	90.3 / 99.2
GST Vessel 1 sample a 24h	52.2	90.4	90.7/ 100
GMB Vessel 1 sample a 2h	2.58	16.0	16.1 / 100
GMB vessel 2 sample a 24h	<LOD	14.7#	16.9 / 115
ASW vessel 1 sample a 2h (dilution factor 2)	14.8	23.0	23.2 / 101
ASW vessel 1 sample a 24h (dilution factor 2)	45.8	29.2	29.3 / 100
ALF vessel 1 sample a 2h	105	40.9	40.9 / 100
ALF vessel 1 sample b 24h	109	41.8	42.7 / 102
PBS vessel 1 sample a 2h	16.9	21.8	22.1 / 101
PBS vessel 1 sample a 24h	61.6	27.6	28.7 / 104

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

Furthermore from the mass balance selected samples were fortified with a known amount of cobalt and zinc (by standard addition of commercial standards) to determine the standard recovery. For fortified test samples, recoveries were in the range of 85.5 – 100 % for Co and 96.0 – 108 % for Zn in mass balance samples.

Fortification of mass balance samples with cobalt

sample	measured concentration of Co [μg/L]	calculated level after addition [μg/L]	recovery [μg/L] / [%]
ALF vessel 1	60.8	46.5	46.5 / 100
ASW vessel 1	72.4	83.4	75.6 / 90.6
GMB vessel 1	76.6	146	125 / 85.9
GST vessel 1	82.9	150	141 / 94.1
PBS vessel 1	70.8	142	122 / 85.5

Fortification of mass balance samples with zinc

sample	measured concentration of Zn [μg/L]	calculated level after addition [μg/L]	recovery [μg/L] / [%]
ALF vessel 1	77.7	56.6	61.3 / 108
ASW vessel 1	91.0	94.6	90.8 / 96.0
GMB vessel 1	95.9	158	152 / 96.7
GST vessel 1	98.9	159	165 / 104
PBS vessel 1	91.6	155	154 / 99.6

ICP-MS

Selected samples were fortified with a known amount of chromium (by standard addition of commercial standard) to determine the standard recovery. For fortified test samples, recoveries were in the range of 85.1 – 102 % for Cr.

Fortification of Cr in test samples

sample	measured concentration [μg/L]	calculated level after addition [μg/L]	recovery [μg/L] / [%]
GST vessel 2 sample a 2h	5.47	3.98	3.97 / 99.8
GST vessel 1 sample a 24h	7.08	4.97	4.70 / 94.5
ASW vessel 1 sample a 2h	1.07	0.88	0.85 / 96.4
ASW vessel 1 sample a 24h	3.43	2.53	2.47 / 97.4
PBS vessel 1 sample b 2h	0.50	1.16	1.03 / 88.8
PBS vessel 2 sample b 24h	2.10	3.07	3.02 / 98.6
GMB vessel 2 sample a 2h	0.23	0.41	0.36 / 88.5
GMB vessel 2 sample a 24h	0.40	0.63	0.54 / 85.1
ALF vessel 1 sample a 2h	4.14	1.71	1.73 / 101
ALF vessel 1 sample a 24h	5.73	2.41	2.47 / 102

Mass balance calculation

The mass balance dissolved chromium, cobalt and zinc concentrations in vessels and filters as measured by ICP-OES indicate no complete dissolution of IPC-2018-002 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 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	0.041	0.00002	0.008	0.049	6.39	0.77
GST vessel 2	0.042	0.00002	0.008	0.049	6.45	0.77
GST vessel 3	0.041	0.00003	0.008	0.049	6.44	0.77
GMB vessel 1	0.038	0.00002	0.001	0.039	6.37	0.62
GMB vessel 2	0.039	0.00002	0.001	0.040	6.42	0.63
GMB vessel 3	0.039	<LOQ	0.001	0.041	6.44	0.63
ALF vessel 1	0.030	0.00002	0.008	0.039	6.43	0.60
ALF vessel 2	0.030	0.00002	0.008	0.038	6.44	0.59
ALF vessel 3	0.030	0.00002	0.009	0.039	6.45	0.60
ASW vessel 1	0.036	0.00008	0.005	0.041	6.40	0.65
ASW vessel 2	0.036	0.00003	0.005	0.042	6.46	0.64
ASW vessel 3	0.037	0.00002	0.005	0.041	6.45	0.64
PBS vessel 1	0.035	0.00002	0.004	0.039	6.45	0.61
PBS vessel 2	0.037	0.00002	0.003	0.040	6.47	0.62
PBS vessel 3	0.037	0.00001	0.003	0.040	6.42	0.62

* Measured dissolved Co includes the amount of cobalt 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: 0.030 mg (amount Co in vessel) + 0.0002 mg (amount Co in syringe/filters) + 0.008 mg (Co in samples) = 0.039 mg

# nominal amount Co = 12.70% in test item (according to CoA) ->6.35 mg Co in 50 mg test item -> nominal amount 6.35 mg * initial weight mg / 50 mg

Example: 6.35 mg Co * 50.626 mg test item / 50 mg = 6.43 mg Co

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

Media	Value for dissolved Zn after addition of aqua regia		In samples [mg]	Calculated value Zn [mg]	Nominal amount [mg] #	Recovery [%]
	Vessels [mg]	Filters/syringes [mg]
GST vessel 1	0.049	0.0001	0.009	0.058	7.50	0.77
GST vessel 2	0.049	0.0002	0.008	0.057	7.57	0.76
GST vessel 3	0.048	0.0001	0.008	0.057	7.55	0.75
GMB vessel 1	0.048	0.0001	0.0001	0.048	7.48	0.64
GMB vessel 2	0.048	0.0002	0.0001	0.048	7.53	0.64
GMB vessel 3	0.048	0.0001	0.0002	0.049	7.55	0.65
ALF vessel 1	0.039	0.0002	0.009	0.048	7.54	0.63
ALF vessel 2	0.037	0.0001	0.008	0.046	7.55	0.61
ALF vessel 3	0.038	0.0004	0.008	0.047	7.57	0.62
ASW vessel 1	0.045	0.0002	0.005	0.050	7.51	0.67
ASW vessel 2	0.043	0.0002	0.005	0.048	7.57	0.64
ASW vessel 3	0.043	0.0001	0.004	0.048	7.57	0.63
PBS vessel 1	0.046	0.0001	0.003	0.049	7.56	0.65
PBS vessel 2	0.046	0.0001	0.003	0.049	7.59	0.65
PBS vessel 3	0.046	0.0001	0.003	0.049	7.54	0.65

* Measured dissolved Zn includes the amount of zinc 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.

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-2018-002 (cobalt chromite green spinel) 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 chromium, cobalt and zinc concentrations after filtration were performed by ICP-OES and ICP-MS. The study was performed in triplicate with two additional method blanks per medium.

Concentration of dissolved chromium in artificial physiological media
Total Cr ± SD in sample vessels (corrected concentrations, corresponding to a loading of exactly 100 mg/L) with mean method blank subtraction (mean of 2h and/or 24h)
- GST 2h: 5.95 ± 0.87 μg/L
- GST 24h: 6.80 ± 0.07 μg/L
- GMB 2h: 0.264 ± 0.017 μg/L
- GMB 24h: 0.452 ± 0.052 μg/L
- ALF 2h: 3.60 ± 0.16 μg/L
- ALF 24h: 5.39 ± 0.29 μg/L
- ASW 2h: 0.985 ± 0.16 μg/L
- ASW 24h: 3.35 ± 0.06 μg/L
- PBS 2h: 0.343 ± 0.138 μg/L
- PBS 24h: 2.13 ± 0.16 μg/L

Concentration of dissolved cobalt in artificial physiological media
total Co ± SD in sample vessels (corrected concentrations, corresponding to a loading of exactly 100 mg/L) without method blank subtraction (all method blanks below LOD)
- GST 2h: 98.2 ± 0.74 μg/L
- GST 24h: 93.8 ± 0.59 μg/L
- GMB 2h: 17.2 ± 0.49 μg/L
- GMB 24h: 12.8 ± 0.75 μg/L
- ALF 2h: 99.1 ± 2.39 μg/L
- ALF 24h: 108 ± 1.28 μg/L
- ASW 2h: 32.6 ± 8.38 μg/L
- ASW 24h: 89.4 ± 1.03 μg/L
- PBS 2h: 20.8 ± 6.45 μg/L
- PBS 24h: 68.7 ± 1.70 μg/L

Concentration of dissolved zinc in artificial physiological media.
total Zn ± SD in sample vessels (corrected concentrations, corresponding to a loading of exactly 100 mg/L) with mean method blank subtraction (mean of 2h and/or 24h)
- GST 2h: 102 ± 1.65 μg/L
- GST 24h: 97.0 ± 0.98 μg/L
- GMB 2h: 2.26 ± 0.32 μg/L
- GMB 24h: 0.806 ± 0.850 μg/L
- ALF 2h: 102 ± 3.13 μg/L
- ALF 24h: 106 ± 2.61 μg/L
- ASW 2h: 24.3 ± 7.46 μg/L
- ASW 24h: 87.1 ± 0.87 μg/L
- PBS 2h: 11.2 ± 4.75 μg/L
- PBS 24h: 57.1 ± 2.80 μg/L

Description of key information

In conclusion, since the dissolved Co and Cr concentrations from this pigment under simulated physiological conditions were below 93.8 µg/L and 6.8 µg/L (GST) , respectively even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.106 % 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 Cobalt chromite green spinel 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 Co from the pigment Cobalt chromite green spinel in physiological media was in a range of 17.2µg/L (GMB) and 99.1 µg/L (ALF) after 2 hours. After 24 hours a dissolution range from 12.8 µg/L (GMB) - 108 µg/L (ALF) was measured.

 

Solubility of Cr from the pigment Cobalt chromite green spinel in physiological media was in a range of 0.264µg/L (GMB) and 5.95 µg/L (GST) after 2 hours. After 24 hours a dissolution range from 0.452 µg/L (GMB) - 6.80 µg/L (GST) was measured.

 

In conclusion, since the dissolved Co and Cr concentrations from this pigment under simulated physiological conditions were below 93.8 µg/L and 6.8 µg/L (GST) , respectively even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.106 % after 24 hours, this pigment may reasonably be considered biologically inert.