Arsenic

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics, other

Remarks:

bioaccessibility / solubility in artificial physiological media

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-11-16 to 2012-02-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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:

In lieu of a harmonised guideline for in-vitro bioaccessibility test, the test was conducted in adaptation of guidance for OECD-Series on testing and assessment Number 29 and according test protocols discussed with the sponsor.

Principles of method if other than guideline:

In lieu of a harmonised guideline for in vitro bioaccessibility test, the test was conducted in adaptation of guidance for OECD-Series on testing and assessment Number 29 and according test protocols discussed with the sponsor. The principle of the test is the introduction of the test material into one or more artificial physiological media at a defined loading, followed by sampling of the solution after certain times, and analysis for dissolved material. The test medium was artificial gastric juice.

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2011-02-07

Test material

Test material information

Constituent 1

Reference substance name:

Arsenic

EC Number:

231-148-6

EC Name:

Arsenic

Cas Number:

7440-38-2

Molecular formula:

As

IUPAC Name:

arsenic

Test material form:

other: solid grains (ca. 1 mm)

Details on test material:

Substance name: Arsenic
Molecular formula: As
Molecular weight: 74.92 g/mol
Appearance: grey metallic grains
Particle size: approx. 1 mm (sieved between two sieves with mesh opening of 0.9 mm and 1.18 mm)
Storage:keep container dry and tightly closed; keep container in a cool, well-ventilated area. Keep away from combustibles and acids. Do not store together with oxidizers
Stability: stable under storage conditions

Test animals

Species:

other: in vitro (simulated human body fluids)

Details on test animals or test system and environmental conditions:

Test principle in brief:
- one artificial physiological medium (GST),
- single loading of test substance of ~100 mg/L,
- the study was performed in triplicate (3 flasks),with 3 additional control blanks,
- two samples were taken after 2 and 24 hours agitation (100 rpm) at 37 ± 2 °C,
- measurement (by ICP-OES) of dissolved arsenic concentrations after filtration. Arsenic (III) and arsenic (V) were separated by HPLC and directly quantified by coupling to ICP-MS


The objective of this study was to assess the dissolution of Arsenic metal (grain size approx. 1 mm) in artificial gastric fluid (GST pH 1.5-1.6). The test medium was selected to simulate a substance entering the human by ingestion into the gastro-intestinal tract.

Administration / exposure

Route of administration:

other: in vitro experiment in artificial gastric juice, simulating oral exposure

Duration and frequency of treatment / exposure:

Single loading of ca. 100 mg/L, sampling and analysis for dissolved metal after 2 and 24 hours.

Details on study design:

PROCEDURE
For the experimental part, three independent flasks were prepared with a loading of 100 mg test item /L GST medium. Three additional control blanks (same procedure, ultrapure water) were also prepared. All flasks (two samples each flask) were tested after 2 and 24 h, to measure total dissolved arsenic concentrations (by ICP-OES), arsenic speciation, temperature and pH. During the study, observations, including the appearance of the solution (color, turbidity, particle film on the surface etc…) were recorded, in addition to measured concentrations, turbidity and pH.

Mass balance calculation:
Total dissolved arsenic concentrations were measured by ICP-OES after the addition of aqua regia to the solution to dissolve the remaining arsenic grain.

Reagents:
- Purified water (resistivity > 18 MΩ·cm, Pure Lab Ultra water purification system from ELGA LabWater, Celle, Germany)
- Nitric acid - “Supra” quality (ROTIPURAN® supplied by Roth, Karlsruhe, Germany).
- Hydrochloric acid – “Baker-instra-analyzed-plus” quality (J.T. Baker, Griesheim, Germany).
- Ammonium hydroxide - p.A. quality (Merck, Darmstadt, Germany)
- Ammoniumdihydrogenphosphate - suprapur (Merck, Darmstadt, Germany)

ARSENIC ANALYSIS
ICP-OES
- Standards (stock solutions and calibration): Arsenic standard containing 1000 mg/L As in nitric acid 2-3 % (lot. No. 09K023, CPI, Amsterdam, The Netherlands)
- Certified aqueous reference material (accuracy and reproducibility of the method): TMDA-70 (Environment Canada, lot no. 0310) and a multielement standard (19 elements, CPI, Amsterdam, The Netherlands, lot no. 009K023)
HPLC-ICP-MS
- Standards (stock solutions, calibration, verification of the method): Arsenic (III) standard (0.05 mol/L, lot no. HC948486, Merck, Darmstadt, Germany) and As (V) standard (1000 mg/L, lot no. HC961324, Merck, Darmstadt, Germany)

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: 189.042 nm, 193.759 nm
Calibration: blank, 10 µg/L, 25 µg/L, 50 µg/L, 100 µg/L, 250 µg/L, 500 µg/L and 1000 µg/L

The separation of As (III) and As (V) in artificial gastric fluid samples was performed using the HPLC system Agilent 1200 coupled to an Agilent ICP-MS 7700 (Agilent Technologies, Waldbronn, Germany). The following analytical and instrumental setup was applied:

HPLC 1200:
column + precolumn: PRP-X100 column (Hamilton, Bonaduz, Switzerland)
flow: 1.5 mL/min
eluent HPLC 1200: 20 mmol/L (NH4)H2PO4, pH = 5.6 (adjusted by NH4OH)
column temp: 22 °C
max. pressure: 100 bar
injection volume: 100 µL

ICP-MS 7500:
power: 1500 W
carrier: 0.91 L/min
dilution gas: 0.15 L/min
nebulizer: concentric
spray chamber: Scott type double
isotopes: 75As, 37Cl (to exclude interferences)
retention As(III): ≈ 2 min
retention As(V): ≈ 8 min

- Calibrations (As(III) and As(V) standards): blank, 0.1 µg/L, 0.5 µg/L, 1 µg/L, 5 µg/L, 10 µg/L, 20 µg/L, 30 µg/L, 40 µg/L and 50 µg/L.

Details on dosing and sampling:

Loading of 100 mg/L: 49.2 mg, 50.4 mg and 49.4 mg were added into 500 mL GST medium.

Sampling:
Solutions were sampled for measuring total dissolved arsenic by ICP-OES. All samples were filtered through 0.2 µm filter prior to further treatment.
Two aqueous subsamples of approx. 20 mL taken for arsenic analysis were transferred into disposable scintillation vials, and stored at approx. 4 °C until analysis. Due to the acid GST medium samples were already acidified.
For speciation analysis of As (III) and As (V) by HPLC-ICP-MS, 20 mL were transferred into disposable scintillation vials, and arsenic species were promptly measured by ICP-MS after HPLC separation.

Results and discussion

Main ADME results

Type:

other: in vitro bioaccessibility: Dissolved fraction (% arsenic dissolved of arsenic added)

Results:

Gastric juice: 0.7 % after 2h; 3.8 % after 24h; effectively all dissolved As present in trivalent form As(III)

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

Final results:
Under the conditions of this test (flasks with artificial gastric fluid; loadings of 100 mg As metal per liter, grain size approx. 1 mm, 37 °C, sampling after 2 h and 24 h), the concentrations of dissolved arsenic, and respective arsenic (III) and arsenic (V) species were as follows:

GST 2 h:
- total As conc. ± SD*: 652 ± 85 µg/L
- As (III) conc. ± SD*: 658 ± 95 µg/L
- As (V) conc. ± SD*: 1 ± 0.1 µg/L
- ∑ As (III) + As (V) ± SD**: 659 ± 95 µg/L

GST 24 h
- total As conc. ± SD*: 3687 ± 873 µg/L
- As (III) conc. ± SD*: 3786 ± 1029 µg/L
- As (V) conc. ± SD*: - ∑ As (III) + As (V) ± SD**: 3786 ± 1029 µg/L
*the mean of vessel A, B and C was calculated
**Original calculations for the data were performed with more digits. Values were rounded

In artificial gastric fluid, approx. 0.7 % of the loaded mass of arsenic metal (grain size approx. 1 mm) were dissolved after 2 h (685 µg/L As (III) and 1 µg/L As (V)).
After 24 h, approx. 3.8 % of the loaded mass of arsenic metal had dissolved. Only the concentration of As (III) was above the limit of quantification in the 24 h samples.
Recovery of total dissolved arsenic when expressed as the sum of measured dissolved tri- and pentavalent arsenic species ranged from 101 - 103 %.

Any other information on results incl. tables

Method validation summary (ICP-OES)

validation parameter	results	Comment
selectivity	similar data with two different As wavelengths for ICP-OES method	no interferences due to test media observed
linearity	applied calibration function was linear	correlation coefficient at least0.999937
limit of detection	5.59 - 18.4 µg/L	-
limit of quantification	18.6 - 61.5 µg/L	-
method blanks	< LOD (< 18.4 µg/L)	-
accuracy	mean recovery for CRM TMDA-70: 101 ± 3 % (n = 5)	Lower concentration range (40.7 µg As/L)
trueness	mean recovery for recalibration standard: 98.1 ± 0.7 % (n = 3)	250 µg As/L
trueness	mean recovery for recalibration standard: 98.4± 0.7 % (n = 5)	500 µg As/L
reproducibility	mean recovery for CRM TMDA-70: 101 ± 2.7 % (n = 5)	lower concentration range (40.7 µg As/L)
reproducibility	mean recovery for diluted CPI standard: 100 ± 0.7 % (n = 5)	higher concentration range (250 µg As/L)

Validation of HPLC-ICP-MS:

In addition to the calibration,Recalibration standards of 10 µg/L As(III) and As(V) were analysed in each measurement series

- Recovery rates were 101 ± 4 % for As(III) and 97.3 ± 3.3 % for As(V)

- LOD: 0.120 µg As(III)/L, 0.146 µg As(V)/L

- LOQ: 0.441 µg As(III)/L, 0.534 µg As(V)/L

- correlation coefficient: 0.999842164 As(III), 0.999767349 As(V)

- The As concentrations in method blanks were below the LOD.

To assure reliable HPLC-ICP-MS measurements, a control method standard was included. The control method standard (10 µg/L As (III) and 10 µg/L As (V)) was analysed directly after each calibration, subsequently after three samples and at the end of the measurement series.

- Recovery rates were 97.6-102 % for As (III) and 88.9 -97.8 % for As(V).

Solution pH values

The target pH in GST medium before addition of the test substance is 1.5-1.6. During the study, the pH of all GST solutions increased (samples after 2h: pH 1.59-1.60; after 24h: pH 1.92-1.94 / blank after 2h: pH 1.55 -1.57; after 24h: pH 1.91-1.93)

Mass balance calculation

Total dissolved arsenic concentrations were measured by ICP-OES after the addition of aqua regia to the solution to dissolve the remaining arsenic grain. A complete dissolution of arsenic in vessel 1, 2 and 3 was obtained. The results of the recovery of the nominal loading of 50 mg / 500 mL are compiled in following table:

 

Calculation of mass balance

media	mean value for total As [mg/L]	nominal concentration [mg/L]	recovery [%]
GST Vessel 1	98.5	98.4	100
GST Vessel 2	102	101	101
GST Vessel 2	93.5	98.8	94.7

Applicant's summary and conclusion

Conclusions:

Under the conditions of this test (flasks with artificial gastric fluid; loadings of 100 mg As metal per liter, grain size approx. 1 mm, 37 °C, sampling after 2 h and 24 h), the concentrations of dissolved arsenic, and respective arsenic (III) and arsenic (V) species were as follows:

GST 2 h:
- total As conc. ± SD*: 652 ± 85 µg/L
- As (III) conc. ± SD*: 658 ± 95 µg/L
- As (V) conc. ± SD*: 1 ± 0.1 µg/L
- ∑ As (III) + As (V) ± SD**: 659 ± 95 µg/L

GST 24 h
- total As conc. ± SD*: 3687 ± 873 µg/L
- As (III) conc. ± SD*: 3786 ± 1029 µg/L
- As (V) conc. ± SD*: - ∑ As (III) + As (V) ± SD**: 3786 ± 1029 µg/L
*the mean of vessel A, B and C was calculated
**Original calculations for the data were performed with more digits. Values were rounded

In artificial gastric fluid, approx. 0.7 % of the loaded mass of arsenic metal (grain size approx. 1 mm) were dissolved after 2 h (685 µg/L As (III) and 1 µg/L As (V)).
After 24 h, approx. 3.8 % of the loaded mass of arsenic metal had dissolved. Only the concentration of As (III) was above the limit of quantification in the 24 h samples.
Recovery of total dissolved arsenic when expressed as the sum of measured dissolved tri- and pentavalent arsenic species ranged from 101 - 103 %.