Chrome tin orchid cassiterite

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics in vitro / ex vivo

Remarks:

Bioaccessibility

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-04-17 to 2015-10-05

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 and Sn concentrations after filtration by 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-2018-004 (Chrome tin orchid cassiterite) 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 and tin concentrations after filtration were performed by 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)

Remarks:

signed 2016-12-16

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 IPC-MS) of dissolved chromium and tin concentrations after filtration
- the study was performed in triplicate

The aim of this test was to assess the dissolution of IPC-2018-004 (Chrome tin orchid cassiterite) 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, and tin standards to prepare an appropriate stock solution and subsequently calibration solutions for ICP-MS measurements(Merck Certipur Chromiumy ICP standard 1000 mg/L lot no. HC386521; Merck Certipur Tin ICP standard 1000 mg/L lot no. HC375770; Darmstadt, Germany).
- Certified reference materials: quality control standards TM-25.4 (lot no. 0914) obtained from Environment Canada and a multielement standard (Merck XVII, lot no. HC382226 – only tin; Merck XXI, lot no. HC42984673 – only chromium, Darmstadt, Germany) were analyzed for total dissolved chromium and tin by 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-MS measurements as recalibration standards.

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: 0.1 – 0.16 L/min
RF power: 1500 W
Isotopes: 52Cr, 53Cr, 118Sn, 119Sn, 120Sn 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 (for mass balance measurement calibration points were blank, 1 µg/L, 2.5 µg/L, 5.0 µg/L, 7.5 µg/L, 10 µg/L, 25 µg/L, 50 µg/L, 75 µg/L and 100 µg/L). Calibrations were performed before each measurement.
Correlation coefficients (r): at least 0.999912

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, chromium and tin were measured in at least one mass balance sample (vessel and filter/syringes) for each medium by ICP-MS 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 Cr at a loading of 0.1 g/L: ALF (pH 4.5) 2h: 0.097 ± 0.072 μg/L. Dissolution of Sn at a loading of 0.1 g/L: ALF (pH 4.5) 24h: 6.45 ± 0.79 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 and without mean method blank subtraction (mean of 2h and/or 24h)
- GST 2h: 0.023 ± 0.006 μg/L (no subtraction, negative value; method blank: 0.038 ± 0.04 μg/L (two method blanks below LOD, one method blank below LOQ, one method blank above LOQ))
- GST 24h: 0.083 ± 0.005 μg/L (no subtraction, all method blanks below LOD)
- GMB 2h: All samples and method blanks below LOD/LOQ
- GMB 24h: 0.050 ± 0.008 μg/L (no subtraction, all method blanks below LOD/LOQ)
- ALF 2h: 0.097 ± 0.072 μg/L (method blank: 0.178 ± 0.156 µg/L (two method blanks below LOD, one method blank below LOQ, one method blank above LOQ))
- ALF 24h: 0.045 ± 0.099 μg/L (method blank: 0.344 ± 0.216 µg/L (two method blanks below LOQ, two method blank above LOQ))
- ASW 2h: All samples and method blanks below LOD
- ASW 24h: 0.025 ± 0.014 μg/L (no subtraction, all method blanks below LOD)
- PBS 2h: All samples and method blanks below LOD
- PBS 24h: All samples and method blanks below LOD

Concentration of dissolved tin in artificial physiological media
total Sn ± SD in sample vessels (corrected concentrations, corresponding to a loading of exactly 100 mg/L) with and without mean method blank subtraction (mean of 2h and/or 24h)
- GST 2h: 0.081 ± 0.017 μg/L (no subtraction, all method blanks below LOD)
- GST 24h: 0.157 ± 0.006 µg/L (no subtraction, all method blanks below LOD)
- GMB 2h: All samples and method blanks below LOD
- GMB 24h: All samples and method blanks below LOD/LOQ
- ALF 2h: 1.64 ± 0.14 μg/L (no subtraction, all method blanks below LOD)
- ALF 24h: 6.45 ± 0.79 μg/L (no subtraction, all method blanks below LOD)
- ASW 2h: 0.040 ± 0.009 μg/L (method blank: 0.002 ± 0.002 µg/L (one method blanks below LOD, one method blank below LOQ, two method blanks above LOQ))
- ASW 24h: 0.474 ± 0.046 μg/L (method blank: 0.010 ± 0.004 µg/L (all method blanks above LOQ))
- PBS 2h: All samples and method blanks below LOD/LOQ
- PBS 24h: 0.059 ± 0.008 μg/L (no subtraction, all method blanks below LOD)

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 0.998812
Limit of detection	Cr: 0.005 – 0.105 µg/L Sn: 0.001 – 0.223 µg/L	-
Limit of quantification	Cr: 0.014 – 0.314 µg/L Sn: 0.002 – 0.669 µg/L	-
Accuracy measurement / Reproducibility test sample and mass balance samples measurements	Mean recovery for CRM TM-25.4 (dilution factor 5): Cr: 94.8 ± 7.4 (n = 32) Sn: 102 ± 4.6 (n = 31)	Low concentration range (certified with 24.0 µg Cr/L - diluted 4.80 µg Cr/L; 23.8 µg Sn/L – diluted 4.76 µg/L)
Trueness test samples	Quality control standard Merck XVII: Sn: 96.6 ± 7.9 (n = 26)	High concentration range (20 µg/L)
Trueness test samples	Quality control standard Merck XXI: Cr: 98.1 ± 5.6 (n = 26)	High concentration range (20 µg/L)
Trueness mass balance samples	Quality control standard Merck XVII: Sn: 95.7 ± 3.8 (n = 5)	High concentration range (50 µg/L)
Trueness mass balance samples	Quality control standard Merck XXI: Cr: 92.1 ± 3.9 (n = 5)	High concentration range (50 µg/L)
Trueness test samples	Recalibration standard: Cr: 97.0 ± 5.8 (n = 27) Sn: 101 ± 7.3 (n = 27)	Low concentration range (5 µg/L)
Trueness mass balance samples	Recalibration standard: Cr: 92.9 ± 3.8 (n = 5) Sn: 96.7 ± 2.5 (n = 5)	Mid concentration range(25 µg/L)
Trueness test samples/ mass balance samples	Fortification of samples: Cr: 75.6 (below LOD) – 107% Sn: 93.2 – 106%	-

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. A low increase of the pH for GST was measurable but this was measurable for the method blanks, too. 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 8.96 and 8.97 (test vessels) and 7.40 to 8.88 and 8.94 (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 5.85 (test vessels) and 6.50 to 5.93 and 5.95 (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.

Fortification

Selected samples were fortified with a known amount of chromium and tin (by standard addition of commercial standards) to determine the standard recovery. For fortified test samples, recoveries were in the range of 75.6 (below LOD) – 107 % for Cr and 93.2 – 106 % for Sn.

Fortification of Cr in test samples

sample	measured concentration [μg/L]	calculated level after addition [μg/L]	recovery [μg/L] / [%]
GST vessel 2 sample b 2h	<LOQ#	0.088	0.088 / 99.6
GST vessel 2 sample b 24h	0.091	0.144	0.143 / 99.6
GMB vessel 1 sample a 24h	0.075	0.383	0.383 / 99.9
ASW vessel 2 sample a 2h	<LOD#	0.834	0.811 / 97.2
ASW vessel 2 sample a 24h	0.067	1.71	1.57 / 92.0
ALF vessel 2 sample a 2h	0.302	1.03	0.921 / 89.0
ALF vessel 2 sample a 24h	0.381	1.92	1.81 / 94.1
PBS vessel 1 sample a 24h	<LOD#	0.667	0.504 / 75.6
PBS vessel 2 sample a 24h	<LOD#	3.33	2.83 / 84.9
Mass balance ALF filter vessel 1 (dilution factor 2)	0.560	25.4	24.5 / 96.7
Mass balance ASW vessel 1 (dilution factor 2)	0.168	33.4	35.9 / 107
Mass balance GMB vessel 1 (dilution factor 2)	0.146	25.1	22.7 / 90.4
Mass balance GST filter vessel 1 (dilution factor 2)	0.342	33.6	31.6 / 94.3

# 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.

Fortification of Sn in test samples

sample	measured concentration [μg/L]	calculated level after addition [μg/L]	recovery [μg/L] / [%]
GST vessel 2 sample b 2h	0.062	0.125	0.122 / 98.1
GST vessel 2 sample b 24h	0.156	0.187	0.185 / 98.8
GMB vessel 1 sample a 24h	<LOD#	1.68	1.58 /94.1
ASW vessel 2 sample a 2h	0.037	0.858	0.857 / 99.8
ASW vessel 2 sample a 24h	0.455	1.97	1.95 / 98.9
ALF vessel 2 sample a 2h	1.47	1.81	1.86 / 102
ALF vessel 2 sample a 24h	5.66	5.44	5.47 / 100
PBS vessel 1 sample a 24h	0.061	0.687	0.728 / 106
PBS vessel 2 sample a 24h	0.061	3.35	3.48 / 104
Mass balance ALF filter vessel 1 (dilution factor 2)	0.095	25.1	24.8 / 98.9
Mass balance ASW vessel 1 (dilution factor 2)	3.92	35.9	36.2 / 101
Mass balance GMB vessel 1 (dilution factor 2)	3.10	27.1	25.2 / 93.2
Mass balance GST filter vessel 1 (dilution factor 2)	0.048	33.4	32.3 / 96.8

# 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.

Mass balance calculation

For the mass balance dissolved chromium and tin concentrations in vessels and filters as measured by ICP-MS indicate no complete dissolution of IPC-2018-004 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 tin in mass balance samples (vessels, filter/syringes and taken samples) for each media for one vessel each

Media	Value for dissolved Sn after addition of aqua regia		In samples [mg]	Calculated value Sn [mg]	Nominal amount [mg] #	Recovery [%]
	Vessels [mg]	Filters/syringes [mg]
GST vessel 1	0.002	<LOD	0.00001	0.002	39.7	0.004
GMB vessel 3	0.003	<LOD	<LOD	0.003	39.7	0.008
ALF vessel 2	0.005	0.000002	0.0003	0.005	39.2	0.012
ASW vessel 1	0.004	<LOD	0.00002	0.004	39.3	0.010
PBS vessel 3	0.001	<LOD	<LOQ	0.001	39.5	0.003

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

Example ALF: 0.005 mg (amount Sn in vessel) + 0.000002 mg (amount Sn in syringe/filters) + 0.0003 mg (Sn in samples) = 0.005 mg

# nominal amount Sn = 78.3% in test item (according to CoA)  39.15 mg Sn in 50 mg test item  nominal amount 39.15 mg * initial weight mg / 50 mg

Example: 39.15 mg Sn * 50.101 mg test item / 50 mg = 39.2 mg Sn

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-2018-004 (Chrome tin orchid cassiterite) 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 and tin concentrations after filtration were performed by 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 and without mean method blank subtraction (mean of 2h and/or 24h)
- GST 2h: 0.023 ± 0.006 μg/L
- GST 24h: 0.083 ± 0.005 μg/L
- GMB 2h: All samples and method blanks below LOD/LOQ
- GMB 24h: 0.050 ± 0.008 μg/L
- ALF 2h: 0.097 ± 0.072 μg/L
- ALF 24h: 0.045 ± 0.099 μg/L
- ASW 2h: All samples and method blanks below LOD
- ASW 24h: 0.025 ± 0.014 μg/L
- PBS 2h: All samples and method blanks below LOD
- PBS 24h: All samples and method blanks below LOD

Concentration of dissolved tin in artificial physiological media
total Sn ± SD in sample vessels (corrected concentrations, corresponding to a loading of exactly 100 mg/L) with and without mean method blank subtraction (mean of 2h and/or 24h)
- GST 2h: 0.081 ± 0.017 μg/L
- GST 24h: 0.157 ± 0.006 µg/L
- GMB 2h: All samples and method blanks below LOD
- GMB 24h: All samples and method blanks below LOD/LOQ
- ALF 2h: 1.64 ± 0.14 μg/L
- ALF 24h: 6.45 ± 0.79 μg/L
- ASW 2h: 0.040 ± 0.009 μg/L
- ASW 24h: 0.474 ± 0.046 μg/L
- PBS 2h: All samples and method blanks below LOD/LOQ
- PBS 24h: 0.059 ± 0.008 μg/L

Description of key information

In conclusion, since the dissolved Cr and Sn concentrations from this pigment under simulated physiological conditions were below 0.84 µg/L and 6.45 µg/L (GST, ALF), respectively even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.008 % 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 Chrome tin orchid cassiterite 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 Cr from the pigment Chrome tin orchid cassiterite in physiological media was in a range of below LOD (GMB, ASW, PBS) and 0.097 µg/L (ALF) after 2 hours. After 24 hours a dissolution range from below LOD (PBS) - 0.084 µg/L (GST) was determined.

Solubility of Sn from the pigment Chrome tin orchid cassiterite in physiological media was in a range of below LOD (GMB, PBS) and 1.64 µg/L (ALF) after 2 hours. After 24 hours a dissolution range from below LOD (GMB) - 6.45 µg/L (ALF) was measured.

In conclusion, since the dissolved Cr and Sn concentrations from this pigment under simulated physiological conditions were below 0.84 µg/L and 6.45 µg/L (GST, ALF), respectively even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.008% after 24 hours, this pigment may reasonably be considered biologically inert.