[No public or meaningful name is available]

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-02-26 to 2013-04-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Objective of study:

bioaccessibility (or bioavailability)

Test guideline

Qualifier:

according 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

Principles of method if other than guideline:

The objective of this study was to assess the dissolution of inorganic glass, solder glass in different artificial physiological media. The test media were selected to simulate relevant human-chemical interactions (as far as practical), i.e. contact of a test substance with skin, a substance entering the human body by inhalation or by ingestion. The dissolved amount of test item was specified by the mass concentration of the substance in the test media under the applied test conditions. The total dissolved amount was determined by measuring the total concentrations of dissolved Pb in solution. The test was performed on the basis of OECD Series on Testing and Assessment No. 29 (2001; ENV/JM/ MONO(2001)9) [1]: “Guidance Document on Transformation / Dissolution of Metals and Metal Compounds in Aqueous Media” as well as according to the bioaccessibility test protocol provided by the monitor (see section A 2). The bioaccessibility protocol has been developed on the basis of relevant published methods ([8], [9], [10], [11] and [12]).

GLP compliance:

yes (incl. QA statement)

Remarks:

signed, 2011-02-07

Test material

Test material information

Constituent 1

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): G017-052 Inorganic glass
- Physical state: colourless, transparent solid, odourless
- Storage condition of test material: Store in dry environment. Avoid high humidity.

Test animals

Species:

other: in vitro (simulated human body fluids)

Details on test animals or test system and environmental conditions:

Five different artificial physiological media, single loading of test substance of 100 mg/L, measurement of dissolved lead concentrations (by ICP-MS and ICP-OES), temperature and pH after filtration, two replicates and two method blanks per artificial media were were sampled after 2 and 24h agitation (100 rpm) at 37 ± 2°C.

Administration / exposure

Details on dosing and sampling:

Preparation, treatment and storage of samples for chemical analysis
Solutions were sampled for measuring total dissolved lead by ICP-MS and ICP-OES. All samples were filtered through 0.2 µm filter (Syringe Filter w / 0.2 µm, polyethersulfon membrane, DIA Nielsen, Dueren, Germany) prior to further treatment.
Aqueous samples of approx. 20 mL taken for Pb analysis were transferred into disposable scintillation vials (20 mL scintillation tubes, Sarstedt, Nuembrecht, Germany), acidified (target conc. 3 % HNO3) and stored at 4 °C until analysis.
The maximum storage time until measurement of the samples was less than two months. According to DIN EN ISO 5667-3: 2003 dissolved metals in aqueous samples (waste-, ground- and surface-water) are at least stable for six months. Furthermore, the analysed CRMs are stable under these conditions for at least one year from the date of shipment. Acidification is the stabilisation method that is recommended in standard methods for metal analysis (e.g. ISO 11885, DIN ).
Solution pH was measured directly in the test vessel.

Analysis of total dissolved lead in test samples (GST, ASW, ALF) and in mass balance samples by ICP-OES
The analysis of total dissolved lead concentrations in test and mass balance samples were measured using an Agilent 720 ICP-OES (Agilent Technologies, Waldbronn, Germany). Lead was detected at the wavelength 167.096 nm, 168.153 nm, 182.143 nm, 217.000 nm, 220.353 nm and 261.417 nm. The following solutions were used to calibrate the instrument: 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, 150 µg/L, 200 µg/L, 250 µg/L, 300 µg/L, 350 µg/L, 500 µg/L, 750 µg/L and 1000 µg/L. Calibrations were performed before each measurement. The calibration formula was calculated using the linear regression algorithm of the ICP-OES instrument. The respective wavelength data with the best recoveries for the validation samples (certified reference material and recalibration / quality control standards) in the measurement series and a correlation coefficient with at least 0.995 were used for calculating concentrations (i.e. 168.153 nm for lead). Correlation coefficients (r) for the wavelengths used for evaluation of data were at least 0.9996. For each sample, at least three internal measurements were performed and the mean was calculated and printed by the instrument software.
Five measurements were performed for the determination of boron concentration in the test item samples, method blanks, mass balance samples and filter samples.

The applied LOD/LOQ calculations for the Agilent 720 ICP-OES are (according to DIN 32645):
LOD: 3 * standard deviation of calibration blank/slope of the calibration
LOQ: 3 * LOD

The resulting LODs/LOQs are reported in the following table:

Date of ICP-OES measurement with corresponding LOD/LOQ and correlation coefficient of external calibration.
Date and type of samples measured LOD (µg/L) LOQ (µg/L) correlation coefficient

January 01, 2013; Mass balance filters (ASW and GMB) 4.02 12.1 0.9996
January 01, 2013; Mass balance filters (PBS, GST, ALF) 5.00 14.7 0.9997
January 08, 2013; Measurement of samples and method
blanks (all media) 5.01 15.0 1.0000
January 09, 2013; Measurement mass balance vessels 2.66 7.99 1.0000
January 10, 2013; Fortification of samples 0.93 2.79 0.9997

Analysis of total dissolved lead in test samples (PBS and GMB) by ICP-MS
The analysis of total dissolved lead in medium samples (PBS and GMB) was performed using an Agilent 7700 ICP-MS with collision cell (Agilent, Waldbronn, Germany). For the determination of lead all three isotope 206Pb, 207Pb and 208Pb were measured. The counts were added automatically by the instrument software and the concentration was given as the lead isotope 208. This procedure is necessary because of the different isotopic ratio of lead in the environment. Depending on the concentration range of the samples, the following standard solutions were used for the calibrations: blank, 0.1 µg/L, 0.25 µg/L, 0.5 µg/L, 0.75 µg/L, 1.0 µg/L, 2.5 µg/L, 5.0 µg/L, 7.5 µg/L, 10.0 µg/L, 25 µg/L, 50 µg/L and 75 µg/L. A respective concentration range was selected for calibration to obtain a working range that covers lead concentrations in samples.

The calibration formula was calculated using the linear regression algorithm of the ICP-MS instrument software. The respective isotope and measurement mode (noGas, Helium mode, HiHelium mode) for interference-free measurement with the best recoveries for certified reference materials were used for calculating the respective test concentrations. Correlation factors (r) were at least 0.9990 for all measurement series. For each sample, at least three internal measurements were performed and the mean was calculated and printed by the instrument software.

In sum, two series of measurements were performed for the determination of total dissolved lead concentrations in samples from test vessels, blanks to determine background levels of elements and fortified samples.
The LOD and LOQ for lead 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.

Date of ICP-MS measurements with corresponding LOD, LOQ and correlation coefficient of external calibration (correlation coefficient shows the linearity of the performed calibration).

date LOD [µg/L] LOQ [µg/L] correlation coefficient
January 17, 2013; Measurement of GMB samples, method blanks and fortification 0.004 0.01 0.9990
January 23, 2013; Measurement of PBS samples, method blanks and fortification 0.01 0.02 0.9999

Results and discussion

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

Under the conditions of this test (flasks with different artificial physiological media; inorganic glass loadings of 50 mg glass per 500 mL (i.e. 100 mg/L), at 37 °C, sampling after 2h and 24h), the measured concentrations of dissolved lead were as follows:
 
Concentration of dissolved lead in artificial physiological media
media and sample Pb ± SD in sample vessels with blank subtraction [µg/L]
ALF 2h 5392 ± 1015
ALF 24h 15085 ± 1751
ASW 2h 187 ± 105
ASW 24h 591 ± 199
GMB 2h 2.27 ± 1.62
GMB 24h 0.65 ± 0.53
GST 2h 34623 ± 1602
GST 24h 85033 ± 871
PBS 2h 0.44 ± 0.29
PBS 24h 1.88 ± 0.67

Any other information on results incl. tables

Solution pH values

After preparation of the artificial physiological media, solution pH was adjusted to their respective target pH. Solution pH data were measured prior to the test, after 2h and 24h in the different media.

The target pH in all media before addition of test substance is in the nominal range. During the study, the pH of ALF, GST and PBS solutions remained stable.

The pH of the ASW media in the sample vessels is decreasing to a pH of 5.7 – 5.8 after 24h. In contrast the pH of ASW media in the method blanks is decreasing during the time of the test from 5.7. Therefore, an effect of the test substance cannot be excluded.

In GMB medium, the pH in all vessels (including method blanks) increased during the time of the test from 7.4 to 8.8 (test vessels) and 7.4 to 8.8 – 8.9 (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.

The test was performed in an incubated laboratory shaker (Shaking incubation cabinet, INFORS AG, Bottmingen, Switzerland) at 100 rpm at. The temperature was adjusted to 37 ± 2 °C in a thermostatically controlled room. The temperature in thermostatically controlled room was confirmed by a certified thermometer: 37.0 °C before addition of test item and 30.5 °C at test start; 36.5 °C sampling after 2h; 37.0 °C sampling after 24h.

Mass balance calculation

Total dissolved lead concentrations in vessels, filters and syringes as measured by ICP-OES indicate an incomplete dissolution of inorganic glass in nearly all physiological media after addition of aqua regia to the sample vessels except for GST media.

 

The nominal amount of lead was calculated by:
Added test item (mg) * 72.41[§] (percentage of Pb in test item).

[§] according to the SDS/MSDS 78.0% Pb as lead oxides (considered as PbO) in the raw material for production of glass => 92.8321% lead in PbO => (78.0 % * 92.8321 %) / 100 % = 72.41 % Pb in test item

The results and the recovery of the nominal amount of 50 mg/ 500 mL are compiled following table:

 

Amount of lead in one mass balance sample (vessel and filter/syringes) for each media

media	value for dissolved Pb after addition of aqua regia [mg]	nominal amount [mg]	recovery [%]
ALF vessel B	11.1	36.7	30.3
ASW vessel A	7.77	37.5	20.7
GMB vessel A	5.29	36.9	14.3
GST vessel B	35.8	35.7	100
PBS vessel B	5.45	37.3	14.6

Results

 

Under the conditions of this test (flasks with different artificial physiological media; inorganic glass loadings of 50 mg glass per 500 mL (i.e. 100 mg/L), at 37 °C, sampling after 2h and 24h), the measured concentrations of dissolved lead were as follows:

 

Concentration of dissolved lead in artificial physiological media

media and sample	total Pb ± SD in method blanks [µg/L]	total Pb ±SD in sample vessels [µg/L]	Pb ± SD in sample vessels with blank subtraction [µg/L]
ALF 2h	<LOD	5392 ± 1015	5392 ± 1015
ALF 24h	<LOD	15085 ± 1751	15085 ± 1751
ASW 2h	<LOD/LOQ	187 ± 105	187 ± 105
ASW 24h	<LOD/LOQ	591 ± 199	591 ± 199
GMB 2h	<LOD/LOQ	2.27 ± 1.62	2.27 ± 1.62
GMB 24h	<LOD	0.65 ± 0.53	0.65 ± 0.53
GST 2h	<LOD	34623 ± 1602	34623 ± 1602
GST 24h	<LOD	85033 ± 871	85033 ± 871
PBS 2h	0.08 ± <0.01	0.52 ± 0.29	0.44 ± 0.29
PBS 24h	0.08 ± 0.01	1.96 ± 0.67	1.88 ± 0.67

Applicant's summary and conclusion

Conclusions:

Under the conditions of this test (flasks with different artificial physiological media; inorganic glass loadings of 50 mg glass per 500 mL (i.e. 100 mg/L), at 37 °C, sampling after 2h and 24h), the measured concentrations of dissolved lead were as follows:
 
Concentration of dissolved lead in artificial physiological media
media and sample Pb ± SD in sample vessels with blank subtraction [µg/L]
ALF 2h 5392 ± 1015
ALF 24h 15085 ± 1751
ASW 2h 187 ± 105
ASW 24h 591 ± 199
GMB 2h 2.27 ± 1.62
GMB 24h 0.65 ± 0.53
GST 2h 34623 ± 1602
GST 24h 85033 ± 871
PBS 2h 0.44 ± 0.29
PBS 24h 1.88 ± 0.67

The bioaccessibility of inorganic glass 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 Pb concentrations were between 0.65 µg/L (GMB 24h) and 85033 µg/L (GST 24h)

Executive summary:

The bioaccessibility of lead 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:

- 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.

Based on the high solubility in GST (117.43%) and ALF (20.83%), inorganic glass may reasonably be considered as bioaccessible.