Cobalt zinc silicate blue phenacite

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:

2015-07-07 to 2015-12-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Objective of study:

bioaccessibility (or bioavailability)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Series on Testing and Assessment No. 29 (23-Jul-2001): Guidance document on transformation/dissolution of metals and metal compounds in aqueous media

Deviations:

yes

Remarks:

Bioaccessibility testing: loading of 100 mg/L; five artificial physiological media agitated at 100 rpm, at 37 °C ± 2 °C; sampling after 2 h and 24 h; determination of Co and Zn concentrations after filtration by ICP-OES.

Principles of method if other than guideline:

The test was performed on the basis of OECD Series on Testing and Assessment No. 29 as well as according to the bioaccessibility test protocol 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-2018-006 (cobalt zinc silicate blue phenacite) 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 cobalt and zinc concentrations after filtrationwere performed by ICP-OES. Samples were taken after 2 and 24 hours agitation (100 rpm) at 37 °C ± 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., Odnevall Walinder I., Leygraf C. 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 °C ± 2 °C,
- two method blanks per artificial media were tested; measurement (by ICP-OES) of dissolved cobalt and zinc concentrations after filtration
- the study was performed in triplicate

The aim of this test was to assess the dissolution of IPC-2018-006 (cobalt zinc silicate blue phenacite) 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 = 1.5-1.6), Phosphate buffered saline (PBS, pH = 7.2- 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 artifical 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 to prepare an appropriate stock solution and subsequently calibration solutions for ICP-OES measurements as cobalt and zinc standards (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, Environment Canada) and a multielement standard (Merck XXI, lot no. HC42984673 and HC55190098, Darmstadt, Germany) were analysed for total dissolved cobalt and zinc by ICP-OES along with the samples to determine the accuracy of the applied analytical method. Furthermore the calibration solutions were measured along with the ICP-OES measurements as recalibration standards.

Instrumental and analytical set-up for the ICP-OES instrument:
- Agilent 720, Agilent Technologies, Waldbronn, Germany
- Nebulizer: Sea spray nebulizer from Agilent
- Spray chamber: Glass cyclonic spray chamber from Agilent
- Carrier gas flow: 0.75 L/min
- RF power: 1200W
- Wavelengths:
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 24 h, 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.

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 Co at a loading of 0.1 g/L: ALF 24h: 25097 ± 437 µg/L. Dissolution of Zn at a loading of 0.1 g/L: ALF 24h: 34781 ± 645 µg/L.

Bioaccessibility (or Bioavailability) testing results:

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) with mean method blank subtraction (mean of 2 h and/or 24 h)
- GST 2h: 17389 ± 3552 µg/L (no subtraction, all method blanks below LOD)
- GST 24h: 24288 ± 1267 µg/L (no subtraction, all method blanks below LOD)
- GMB 2h: 29.9 ± 11.5 µg/L (no subtraction, all method blanks below LOD)
- GMB 24h: 34.6 ± 2.23 µg/L (no subtraction, all method blanks below LOD)
- ALF 2h: 11174 ± 7648 µg/L (no subtraction, all method blanks below LOD)
- ALF 24h: 25097 ± 437 µg/L (no subtraction, all method blanks below LOD)
- ASW 2h: 106 ± 42.8 µg/L (no subtraction, all method blanks below LOD)
- ASW 24h: 936 ± 46.8 µg/L (no subtraction, all method blanks below LOD)
- PBS 2h: 55.1 ± 6.78 µg/L (no subtraction, all method blanks below LOD)
- PBS 24h: 181 ± 2.76 µg/L (no subtraction, all method blanks below LOD)

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 2 h and/or 24 h)
- GST 2h: 24474 ± 4850 µg/L (method blanks: 4.26 ± 2.95 µg/L)
- GST 24h: 33612 ± 2460 µg/L (method blanks: 5.47 ± 3.57 µg/L)
- GMB 2h: 21.7 ± 8.27 µg/L (no subtraction, all method blanks below LOD)
- GMB 24h: All samples below LOD, all method blanks below LOD
- ALF 2h: 17628 ± 10974 µg/L (no subtraction, all method blanks below LOD/LOQ)
- ALF 24h: 34781 ± 645 µg/L (no subtraction, all method blanks below LOD/LOQ)
- ASW 2h: 216 ± 97.0 µg/L (method blanks: 1.47 ± 0.52 µg/L)
- ASW 24h: 1537 ± 76.0 µg/L (method blanks: 2.20 ± 0.58 µg/L)
- PBS 2h: 67.9 ± 8.72 µg/L (no subtraction, all method blanks below LOD)
- PBS 24h: 220 ± 7.87 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	at least 0.999618
Limit of detection	Co: 0.209 – 2.21 µg/L Zn: 0.054 – 1.45 µg/L
Limit of quantification	Co: 0.628 – 6.64 µg/L Zn: 0.163 – 4.35 µg/L
Method blanks	Co: < LOD Zn: - GST 2h: 4.26 ± 2.95 µg/L; 24 h: 5.47 ± 3.57 µg/L - ALF: < LOD/LOQ - GMB and PBS: < LOD - ASW 2h: 1.47 ± 0.52 µg/L; 24 h: 2.20 ± 0.58 µg/L	-
Accuracy measurement / Reproducibility testsample and mass balance measurements	Mean recovery for CRM TM-25.4 (dilution factor 10): Co: 99.0± 6.7 (n = 26) Zn: 105 ± 8.5 (n = 20)	Low concentration range (certified with 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 testsample and mass balance measurements	Mean recovery for CRM TMDA-53.3 (dilution factor 10): Co: 99.9 ± 2.6 (n = 30) Zn: 99.4 ± 4.6 (n = 30)	Low to mid concentration range (certified with 251 µg Co/L, diluted 25.1 µg Co/L; 386 µg Zn/L, diluted 38.6 µg Zn/L)
Truenesstest 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)
Truenesstestsample and mass balance measurements	Quality control standard (Merck XXI): Co: 97.7 ± 2.6 (n = 26) Zn: 99.2 ± 1.7 (n = 26)	Mid to high concentration range (100 µg/L)
Truenesstestsample and mass balance measurements	Quality control standard (Merck XXI): Co: 97.9 ± 3.0 (n = 11) Zn: 99.4 ± 1.3 (n = 11)	High concentration range (500 µg/L)
Truenesstest 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)
Truenesstestsample and mass balance measurements	Recalibration standard Co: 100 (n = 1) Zn: 104 (n = 2)	Low to Mid concentration range (25 µg/L)
Truenesstestsample and mass balance measurements	Recalibration standard Co: 97.5 ± 1.7 (n = 21) Zn: 101 ± 4.2 (n = 21)	Mid concentration range (50 µg/L)
Truenesstest 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)
Truenesstestsample and mass balance measurements	Recalibration standard 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: 98.2 - 102 % Zn: 95.8 – 107 %
Truenessmass balancesample measurements	Fortification of samples: Co: 93.2 – 96.0 % Zn: 99.2 – 101 %

Solution pH values

After preparation of the artificial physiological media, solution pH was adjusted to their respective target pH, which was in the nominal range in all media before addition of test substance.

During the study, the pH of GST (low increase from 1.55 and 1.56 to 1.58 to 1.61), 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 8.73, 8.74 and 8.67 (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 6.36 and 6.37 (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 test was performed in an incubated laboratory shaker (Shaking incubation cabinet, Minitron, INFORS AG, Bottmingen, Switzerland) at 100 rpm. The temperature was adjusted to 37.5 °C in a thermostatically controlled shaking cabinet to reach a temperature of 37 °C ± 2 °C in the media. The temperature remained stable during the test in all media.

Fortification

From selected artificial physiological media, 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 98.2 – 102 % for Co and the recoveries of Zn were in the range of 95.8 – 107 % 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	186	117	118 / 101
GST vessel 1 sample a 24h	244	129	130 / 101
GST vessel 3 sample b 24h (remeasurement series)	261	452	459 / 102
GMB vessel 1 sample a 2h	33.5	45.8	45.0 / 98.4
GMB vessel 2 sample a 24h	33.1	43.2	42.4 / 98.2
ASW vessel 3 sample b 2h (dilution factor 2)	32.1	27.0	26.6 / 98.3
ASW vessel 1 sample a 24h (dilution factor 10)	99.1	39.8	39.5 / 99.1
ALF vessel 1 sample b 2h (dilution factor 100)	192	118	117 / 99.0
ALF vessel 1 sample a 24h (dilution factor 100)	249	130	129 / 99.6
PBS vessel 1 sample a 2h	62.3	84.9	84.6 / 99.6
PBS vessel 1 sample a 24h	188	225	222 / 98.7

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	261	132	131 / 99.1
GST Vessel 1 sample a 24h	338	148	145 / 98.3
GST vessel 3 sample b 24h (remeasurement series)	370	474	478 / 101
GMB Vessel 1 sample a 2h	27.2	40.9	42.9 / 105
GMB vessel 2 sample a 24h	<LOD	29.8 #	29.8 / 100
ASW vessel 3 sample b 2h (dilution factor 2)	68.4	33.7	36.0 / 107
ASW vessel 1 sample a 24h (dilution factor 10)	163	52.6	52.0 / 98.8
ALF vessel 1 sample b 2h (dilution factor 100)	294	139	133 / 95.8
ALF vessel 1 sample a 24h (dilution factor 100)	347	149	146 / 97.8
PBS vessel 1 sample a 2h	76.0	90.4	91.3 / 101
PBS vessel 1 sample a 24h	223	239	239 / 99.8

# 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.

Determination of mass balance

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 93.2 – 96.0 % for Co and 99.2 – 101 % 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 (dilution factor 100)	193	316	302 / 95.8
ASW vessel 1 (dilution factor 100)	227	336	313 / 93.2
GMB vessel 1 (dilution factor 100)	220	332	315 / 94.7
GST vessel 1 (dilution factor 100)	200	320	307 / 96.0
PBS vessel 1 (dilution factor 100)	224	335	316 7 94.4

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 (dilution factor 100)	284	370	370 / 99.9
ASW vessel 1 (dilution factor 100)	325	395	398 / 101
GMB vessel 1 (dilution factor 100)	324	394	395 / 100
GST vessel 1 (dilution factor 100)	273	364	363 / 99.7
PBS vessel 1 (dilution factor 100)	333	400	397 / 99.2

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 cobalt zinc silicate blue phenacite in artificial physiological media with a single loading of 100 mg/L at 37 °C ± 2 °C was determined.
Concentration of dissolved cobalt in artificial physiological media:
- GST 2h: 17389 ± 3552 µg/L
- GST 24h: 24288 ± 1267 µg/L
- GMB 2h: 29.9 ± 11.5 µg/L
- GMB 24h: 34.6 ± 2.23 µg/L
- ALF 2h: 11174 ± 7648 µg/L
- ALF 24h: 25097 ± 437 µg/L
- ASW 2h: 106 ± 42.8 µg/L
- ASW 24h: 936 ± 46.8 µg/L
- PBS 2h: 55.1 ± 6.78 µg/L
- PBS 24h: 181 ± 2.76 µg/L

Concentration of dissolved zinc in artificial physiological media
Total Zn ± SD in sample vessels
- GST 2h: 24474 ± 4850 µg/L
- GST 24h: 33612 ± 2460 µg/L
- GMB 2h: 21.7 ± 8.27 µg/L
- GMB 24h: All samples below LOD
- ALF 2h: 17628 ± 10974 µg/L
- ALF 24h: 34781 ± 645 µg/L
- ASW 2h: 216 ± 97.0 µg/L
- ASW 24h: 1537 ± 76.0 µg/L
- PBS 2h: 67.9 ± 8.72 µg/L
- PBS 24h: 220 ± 7.87 µg/L

Description of key information

In conclusion, the dissolved Co concentrations from this pigment under simulated physiological conditions were below 17.4 mg/L even at the highest loading of 0.1g/L after 2 hours, corresponding to a solubility of less than 18 %. Dissolved Zn concentrations were below 25 mg/L after 2 hours, corresponding to a solubility of less than 25 %.

Therefore, cobalt zinc silicate phenacite blue may reasonably be considered poorly bioaccessible.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

The chemical and physiological properties of pigments in general 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 for this pigment 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.

 

The dissolution of cobalt from the test item Cobalt zinc silicate blue phenacite at a loading of 0.1g/L after 2 hours was between 29.9µg/L (GMB) and 17.39 mg/L (GST) and between 34.6 µg/L (GMB) and 25.1 mg/L (ALF) after 24 hours. Further, the dissolution of zinc from the test item Cobalt zinc silicate blue phenacite was in a range of 21.7 µg/L (GMB) and 24.5 mg/L (GST) at a loading of 0.1g/L after 2 hours and between below LOD (GMB) and 34.8 mg/L (ALF) after 24 hours.

 

In conclusion, the dissolved Co concentrations from this pigment under simulated physiological conditions were below 17.4 mg/L even at the highest loading of 0.1g/L after 2 hours, corresponding to a solubility of less than 18 %. Dissolved Zn concentrations were below 25 mg/L after 2 hours, corresponding to a solubility of less than 25 %.