ALFERROCK

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Title:

Unnamed

Year:

2021

Materials and methods

Objective of study:

bioaccessibility (or bioavailability)

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

An internationally agreed guideline does not exist yet for this test (e.g. OECD). However, a bioelution test method to assess the relative in vitro bioaccessibility (IVBA) of metals and metalloids in inorganic metal compounds and metal (metalloid)-containing materials was submitted to EURL ECVAM. Further, similar tests have been conducted with several metal compounds incl. steels in previous risk assessments (completed under Regulation (EEC) No 793/93) or in recent preparations for REACH regulation (EC) No 1907/2006.
Therefore, the test was performed on the basis of the guidance for OECD-Series on testing and assessment Number 29 (OECD Guideline 29: 2001; ENV/JM/MONO(2001)9), the bioelution test method reported in the ESAC opinion and bioaccessibility methods reported in published studies.

This report describes the measured bioaccessibility of ALFERROCK in body fluid simulants GST (Artificial gastric fluid), PBS (Phosphate buffered saline), GMB (Gamble´s solution), ALF (Artificial lysosomal fluid) and ASW (Artificial sweat solution) as a surrogate for potential bioavailability of different species contained in ALFERROCK.
The test adopted experimental conditions of the OECD 29 guidance: 24 h agitation at 100 rpm in the dark on an incubated laboratory shaker (Shaking incubation cabinet “Minitron”, INFORS AG, Bottmingen, Switzerland). The temperature in the thermostatically controlled incubation cabinet was adjusted to 37.5°C to obtain a medium temperature of 37 ± 1°C to simulate the human body temperature. Furthermore, the test with GMB was performed in a 5% CO2 atmosphere.
Three replicates and two method blanks per artificial medium were tested. An additional fourth replicate was included for pH measurements during testing. Solutions of three test vessels and two method blanks were sampled after 2 h and 24 h whereas GMB and ALF were also sampled after 168 h. The element concentrations of Al, Ca, Cr, Fe, P, S, Si, Ti, V and Zr were measured after filtration (0.2 µm) and after additional centrifugal filtration (i.e. 0.2 µm and 3 kDa) by ICP-OES. The pH of the three test replicates were measured before test start and at test end. The solution pH of method blanks and the additional fourth replicate was measured at test start, after 2 h, 24 h and 168 h (only ALF and GMB).

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Neutralisation and reduction products of bauxite residue from refinement process

Molecular formula:

The substance is a UVCB for which it is not possible to provide a molecular formula and a molecular weight.

IUPAC Name:

Neutralisation and reduction products of bauxite residue from refinement process

Test material form:

solid

Specific details on test material used for the study:

Lot number: 00-00 05 03
Appearance: reddish brown powder

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- For the test, 1 g of the test item was transferred into 500 mL Erlenmeyer flasks. A volume of 500 mL of test medium was added to the flask to obtain a loading of 2 g/L.

Radiolabelling:

no

Test animals

Species:

other: not applicable

Details on test animals or test system and environmental conditions:

not applicable

Administration / exposure

Route of administration:

application in vitro

Vehicle:

other: artificial body fluids

Details on exposure:

IN VITRO APPLICATION

- Concentration of test material: 2 g/l. 1 g of the test item was transferred into 500 mL Erlenmeyer flasks. A volume of 500 mL of respective test medium was added to the flask to obtain a loading of 2 g/L
- Method of preparation of stock solution(s) of test material and reference chemical:
For the analysis of Si a single element standard was used and diluted with the respective medium to obtain the matrix matched calibration standard solutions.
For the analysis of Al, Ca, Cr, Fe, P, S, Ti, V and Zr the respective single element standards were mixed to prepare a stock solution containing 100 mg/L of Al, Ca, Fe, P, S and Ti and 10 mg/L Cr, V and Zr in 10 % HNO3. This stock solution was further diluted with the respective medium to obtain the matrix matched calibration standard solutions.
For more information on concentrations of standard solutions, please refer to section "Any other information on materials and methods incl. tables".
- Cell culture medium characteristics (temperature, pH): The temperature of the media was hold in the range 37 °C ± 1 °C during the test period. For more information on pH of the test media, please refer to "Any other information on materials and methods, incl. tables".
- Incubation temperature: The temperature in the thermostatically controlled incubation cabinet was adjusted to 37.5°C to obtain a medium temperature of 37 ± 1°C to simulate the human body temperature. Furthermore, the test with GMB was performed in a 5% CO2 atmosphere.
- Number of replicates (if more than one is used per run): Three replicates and two method blanks per artificial medium were tested. An additional fourth replicate was included for pH measurements during testing.
- Number of independent runs: At least three internal measurements for each sample were performed and the mean was calculated and printed by the instrument software.
- Time points: GST, PBS and ASW media were sampled after 2 h and 24 h, whereas GMB and ALF media were sampled additionally after 168 h.

Duration and frequency of treatment / exposure:

Solutions of test vessels and method blanks were sampled after 2 h and 24 h, whereas GMB and ALF were also sampled after 168 h.

Doses / concentrations

Dose / conc.:

2 other: g/L

Remarks:

1 g of the test item was transferred with 500 mL of test medium was added to a 500 mL flask to obtain a loading of 2 g/L.

No. of animals per sex per dose / concentration:

not applicable

Control animals:

not relevant

Positive control reference chemical:

not applicable

Details on study design:

For the test, 1 g of the test item was transferred into 500 mL Erlenmeyer flasks. A volume of 500 mL of test medium was added to the flask to obtain a loading of 2 g/L. The volume of the media was adjusted as necessary for a precise loading.
Three replicates and two method blanks per artificial medium were tested. An additional fourth replicate was included for pH measurements during testing. The vessels containing the test item were agitated with 100 rpm in the dark on an incubated laboratory shaker (Shaking incubation cabinet “Minitron”, INFORS AG, Bottmingen, Switzerland). Agitation was sufficient to maintain the flow of aqueous medium over the test substance while maintaining the integrity of the surface of the test substance and of any solid reaction product coatings formed during the test. The temperature of the media in the thermostatically controlled incubation cabinet was adjusted to 37.5°C to obtain a medium temperature of 37 ± 1°C to simulate the human body temperature. Furthermore, the test with GMB was performed in a 5% CO2 atmosphere in order to keep the pH stable during the test. Solutions of three test vessels and two method blanks were sampled after 2 h and 24 h whereas GMB and ALF were also sampled after 168 h. The element concentrations were measured after filtration (0.2 µm) and after additional centrifugal filtration (i.e. 0.2 µm and 3 kDa) by ICP-OES. The pH of the three test replicates were measured before test start and at test end. The solution pH of method blanks and the additional fourth replicate was measured at test start, after 2 h, 24 h and 168 h (only ALF and GMB).

Because silicon was analysed in the bioaccessibility tests, the apparatus consisted of open polypropylene flasks in a thermostatically controlled shaking incubator (Minitron-incubation-shaking-device, INFORS AG, Bottmingen, Switzerland) with the possibility for the addition of CO2 to keep the pH stable within the experimental phase. The aluminium foil used standardly was avoided because Al is one of the metals to be analysed. Instead, the exchange with the atmosphere was realized through parafilm with a hole.

Solution pH during the samplings was measured at sampling using a Multi 9430 device equipped with Sentix 940-P electrodes (WTW, Weilheim, Germany) directly in the test vessels. Measurements were taken after readings had stabilized. The pH electrode was calibrated or checked on each sampling day.
Four replicate vessels containing the test material and two control blanks per artificial media were tested. Whereas three replicate vessels containing the test material are used for sampling, one replicate vessel containing the test material and the two control vessels are used for pH measurements at each sampling (blank control vessels were sampled too). However, after the last sampling (24 h for GST, PBS and ASW, 168 h for ALF and GMB), the pH of all test solutions (four replicates plus two control vessels) was measured. A pH meter showing acceptable results within ± 0.01 pH units was used. Before inserting into each vessel, electrode and sensor were rinsed thoroughly with purified water to avoid cross-contamination. Furthermore, a data logger measured the temperature during the test. Solutions were examined for soapy bubbles or floating particles on the surface.

Details on dosing and sampling:

GST, PBS and ASW media were sampled after 2 h and 24 h, whereas GMB and ALF media were sampled additionally after 168 h. Subsequent to the sampling after 24 h, the media GMB and ALF were replenished with new medium in equal volume to that already drawn. Solutions for analytical measurements were sampled with a pipette in duplicate per vessel and sampling point. A total of 4 × 30 mL (ASW medium only 2 × 30 mL sampling because no silicon analysis is necessary) were sampled and filtered through a 0.2 µm polyethersulfone (PES) membrane syringe filter. A subsample of 15 mL of each sample was subsequently filtered through a centrifugal filter (i.e., 3 kDa filter, Vivaspin, Sartorius, Germany). The subsamples of the 0.2 µm filtered and the additionally 3 kDa centrifugally filtered samples were acidified with 69% HNO3 (e.g., 150 µL for 15 mL sample) and stored at approx. 4 °C until analysis.
The two subsamples intended for silicon analysis were alkalized with 10% NaOH (e.g., 150 µL for 15 mL sample) for GST medium and with 50% NaOH (e.g., 150 µL for 15 mL sample) for ALF medium. For PBS medium, neither acidification nor alkalinization was performed. The samples of the test with GMB were acidified with 69% HNO3 (e.g., 150 µL for 15 mL sample). Since it cannot be guaranteed that the dissolved/dispersed Si concentration remains stable in filtrates, the silicon concentration was quantified within 1 day after sampling, if feasible.

Statistics:

N/A

Results and discussion

Preliminary studies:

not applicable

Toxicokinetic / pharmacokinetic studies

Details on absorption:

not applicable

Details on distribution in tissues:

not applicable

Metabolite characterisation studies

Metabolites identified:

no

Details on metabolites:

not applicable

Enzymatic activity

Enzymatic activity measured:

N/A

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

The highest concentrations of all elemental species investigated was found for aluminum in artificial gastric fluid and artificial lysosomal fluid, simulating the harsh digestion milieu in the stomach and intracellular conditions in lung cells occurring with phagocytosis, respectively.
In addition, elevated levels of silicon were found after incubation of the test material in these fluids.
Relevant dissolution was also found for Calcium, Iron, Phosphorous, Sulfur, and Titanium.
For more details and quantitative results, please refer to section "Any other information on results incl. tables".

Any other information on results incl. tables

Table 1: Summary of bioaccessibility of various tested compounds

	GST [µg/g Alferrock]		PBS [µg/g Alferrock]		ASW [µg/g Alferrock]		ALF [µg/g Alferrock]			GMB  [µg/g Alferrock]
	2h	24h	2h	24h	2h	24h	2h	24h	168h	2h	24h	168h
Aluminum	17236.5	28094.7	11.6	71.5	33.2	150.3	20118.0	27855.0	24426.0	15.4	334.3	754.5
Calcium	6808.2	10261.7	391.0	872.5	1052.3	3056.7	-	-	-	-	-	-
Chromium	6.8	26.3	0.9	0.7	0.2	0.2	7.5	22.1	47.1	0.3	0.4	0.4
Iron	214.9	1242.5	0.1	0.1	neg value due to higher blank		342.0	1522.0	3744.0	0.3	1.5	0.3
Phosphorous	375.0	215.6	-	-	21.5	20.6	-	-	-	-	-	-
Sulfur	563.7	891.5	49.2	194.0	66.5	117.0	-	-	-	-	-	-
Silicon	12657 .9	11559.7	210.2	2797.6	-	-	18719.0	23735.0	23137.9	102.5	907.8	2487.6
Titanium	85.9	495.3	<LOD	<LOD	0.2	0.1	86.5	566.5	1979.0	0.1	0.1	0.1
Vanadium	10.2	21.9	-	-	0.9	3.7	-	-	-	2.5	12.7	19.6
Zirconium	0.9	2.0	<LOD	<LOD	0.1	0.1	12.2	42.1	110.5	0.2	0.2	0.2

GST: Artificial gastric fluid

GMB: Gamble´s solution

ALF: Artificial lysosomal fluid

ASW: Artificial sweat

PBS: Phosphate buffered saline

Applicant's summary and conclusion

Conclusions:

The objective of this study was to investigate the dissolution rate of the test item "Neutralisation and reduction products of bauxite residue from refinement process" in different artificial physiological media GST, PBS, GMB, ALF and ASW, representing the oral, dermal and inhalation routes of exposure as a measure for bioaccessibility.
The highest concentrations of all elemental species investigated was found for aluminum in GST (artificial gastric fluid) and ALF (artificial lysosomal fluid). In addition, elevated levels of silicon were found after incubation of the test material in these fluids.
Relevant dissolution was also found for Calcium, Iron, Phosphorous, Sulfur, and Titanium.

Executive summary:

In this bioaccessibility study which was performed on the basis of OECD Series on Testing and Assessment No. 29 (2001; ENV/JM/ MONO(2001)9), the test item "Neutralisation and reduction products of bauxite residue from refinement process" was incubated in different artificial physiological media at a concentration of 2 g/l in a total volume of 500 ml in order to determine the dissolution rate of the elemental species aluminium, iron, titanium, calcium, zirconium, sulfur, chromium, phosphorus, vanadium and silicon contained in the material.

 

The media were selected to represent the conditions after exposure via the oral, dermal and inhalation route of exposure (ALF, Artificial lysosomal fluid (pH = 4.5)/ ASW, Artificial sweat solution (pH = 6.5)/ GMB, Gamble´s solution (pH = 7.4)/ GST, Artificial gastric fluid (pH = 1.5) and PBS, Phosphate buffered saline (pH = 7.4)).

For the experimental setup, the test item was incubated in the different artificial physiological media in the dark at 100 rpm and at 37 °C ± 1 °C. Samples of GST, ASW and PBS medium were taken after 2 h and 24 h whereas GMB and ALF samples were taken after 2 h, 24 h and 168 h (7 days). Respective element concentrations of filtered solutions were determined by ICP-OES.

 

The highest concentrations of all elemental species investigated was found for aluminum in artificial gastric fluid and artificial lysosomal fluid, simulating the harsh digestion milieu in the stomach and intracellular conditions in lung cells occurring with phagocytosis, respectively. In addition, elevated levels of silicon were found after incubation of the test material in these fluids. Relevant dissolution was also found for Calcium, Iron, Phosphorous, Sulfur, and Titanium.

For more details and quantitative results, please refer to section "Any other information on results incl. tables".