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Risk-Based Analysis of Elemental Impurities

Hazard is defined as the potential of a substance to cause damage, while toxicity is the assessment of its ability to poison. Risk is a measure of the probability that harm may result from exposure to a chemical. Thus, if there is no exposure, there is no risk regardless of the magnitude of the hazard. (Duffus, Worth, 2006)

TOXICITY X EXPOSURE = RISK

USP General Chapter <233> describes the test procedures that are allowed for the purpose of compliance with the Elemental Impurities PDE limits in USP General Chapter <232>. These procedures are also expected to become the default procedures for compliance with the Elemental Impurities PDE limits in ICH Q3D. The chapter <233> procedures allow, however, only for total sample digestion such that the entire amount of contained elemental impurities is measured. This can sometimes require the use of Hydrofluoric Acid (HF) in the sample preparation techniques which can create significant analyst safety concerns. In the case of some components and drugs, this approach can significantly overstate the amount of the respective elemental impurities that become available for absorption after ingestion. The analytically measured amounts can indicate a greater level of exposure, and thus risk, than actually exists. Chapter <233> should therefore provide at least the option, and preferably the requirement, for risk-based biorelevant analyses. Chapter <233> should allow determination of physiologically relevant bioaccessible elemental impurity content for pharmaceutical products and components, as opposed to requiring total digestion procedures that report the total content of each element regardless of the physiological fate of the measured elemental concentrations.

BIOACCESSIBILITY→BIOAVAILIBILITY→EXPOSURE

For the purpose of understanding and quantifying the risks associated with oral exposure to elemental impurities in drug products, it is fundamentally important to understand and apply the principles of bioaccessibility and bioavailability.

The bioaccessible amount of the ingested elemental impurity is the fraction that is soluble in digestive juices; this represents the maximum amount of impurity that is available for absorption.

The bioavailable amount of the ingested elemental impurity is the fraction that reaches the central (blood) compartment from the gastrointestinal tract. The bioavailable fraction of an elemental impurity cannot be greater than the bioaccessible fraction and is often less.

Bioaccessibility is one of the principal factors limiting the bioavailable fraction, and is an

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essential consideration for risk assessment purposes. There is, therefore, a clear need for practical analytical methods that measure the bioaccessible fraction of the elemental impurities in drug products and drug components. Consideration solely of the total amount of each elemental impurity can, for some drugs and components, significantly misrepresent the risk posed. – “…if there is no exposure, there is no risk…”.

This distinction between the total and bioaccessible amount of an elemental impurity has not been suitably recognized in the context of risk-based control of elemental impurities in drug products and components. Permissible Daily Exposure (PDE) limits have been proposed based on toxicology reviews but without apparent consideration as to how analyses of drug products and components should be conducted so that the results are relevant to those reviews.

It is interesting to note that although the toxicological reviews that resulted in the oral PDE limits found in both USP General Chapter <232> and the ICH Q3D Step 2b document focused on studies of the risk posed by these elements in bioaccessible and subsequently bioavailable form (mainly based on drinking water standards) the analytical methods proposed in General Chapter <233> have only been designed to allow measurement and use of the total amount of each element in the drug or its components, without regard to exposure.

For substances that are soluble or from which the total elemental impurities are readily extracted by digestive juices, there is no distinction between the total and the bioaccessible content of the elements. However, considerable distinctions can exist for certain naturally-sourced drug components, in particular mineral (e.g., clays and talcs) and mineral-derived products (e.g., titanium dioxide and bismuth compounds). Extraction of trace elements from these materials is generally in proportion to the strength of the extracting acid. For example, in a comparative study of five bioaccessibility methods (Oomen et al. 2002) the authors concluded that pH is probably the one single factor that has the most influence on the final result, with bioaccessibility values inversely proportional to simulated gastric juice pH. Extraction conditions orders of magnitude beyond those experienced physiologically are poorly suited for assessment of risk.

Elemental impurities may be bound within the structure of these components, the structures of residual associated minerals, the complexes adhered to their surfaces, and as cations that balance the negative charge of certain excipients. The amount of each element that becomes solubilized depends on the state in which it exists and its consequent resistance to acid extraction. Particularly aggressive acid digestion is generally required to totally decompose mineral and mineral-derived components so that all elements can be solubilized and measured. This degree of mineral decomposition, as prescribed in chapter <233>, can provide element assays that significantly misrepresent the risk posed to patients by those elements. For risk assessment purposes, acid leach analyses designed to measure the bioaccessible fraction of each element are more useful and relevant than total digestion analyses.

COMPENDIAL PRECEDENT

Biorelevant analysis has, in fact, established a precedent for the use of dilute acid leach methods for the measurement and control of arsenic and lead in mineral and mineral-derived food and pharmaceutical ingredients, as seen in Table 1.

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Table 1. Current Biorelevant Acid Leach Compendial Methods

As Pb FCC Monographs: Titanium Dioxide Silicon Dioxide Bentonite Talc Kaolin

0.5 N HCl; boil 15 minutes no specification 1:25 HCl (0.5 N); boil 15 minutes 0.5 N HCl; reflux for 30 minutes 0.5 N HCl; reflux for 30 minutes

0.5 N HCl; boil 15 minutes 0.5 N HCl; boil 15 minutes 1:25 HCl (0.5 N); boil 15 minutes 0.5 N HCl; reflux for 30 minutes 0.5 N HCl; reflux for 30 minutes

NF Monographs: Talc Bentonite Purified Bentonite Magnesium Aluminum Silicate Purified Siliceous Earth

no specification 1:25 HCl (0.5 N); boil 15 minutes 1:25 HCl (0.5 N); boil 15 minutes 1:25 HCl (0.5 N); boil 15 minutes 0.5 N HCl; 70oC for 15 minutes

0.5 N HCl; reflux for 30 minutes 1:25 HCl (0.5 N); boil 15 minutes 1:25 HCl (0.5 N); boil 15 minutes 1:25 HCl (0.5 N); boil 15 minutes 0.5 N HCl; 70oC for 15 minutes

USP Monographs: Kaolin Activated Attapulgite Colloidal Activated Attapulgite

no specification 1 N HNO3; boil 30 minutes 1 N HNO3; boil 30 minutes

1 N HNO3; in boiling water 60 min 1 N HNO3; boil 30 minutes 1 N HNO3; boil 30 minutes

Chapter <233> focuses on a state-of-the-art multi-element analytical technique and prescribes total sample digestion for all materials under test. This approach does not allow appropriate application of risk assessment based on the bioaccessible fractions of elemental impurities in materials under test. This paradigm is being set forth despite the uncontested compendial precedents for biorelevant digestions in existing monographs and methods.

NUTRITIONAL ELEMENTS PRECEDENT

While there has been no formal or systematic study of the bioaccessible fraction of the elemental impurities in drugs and drug components, the bioaccessibility of nutritional elements has been studied. The bioaccessible fraction of iron, extracted with simple gastric juice analogues, has been the subject of numerous studies. Similar methods have been generally accepted since at least the late 1960s, as shown below.

• Pepsin in 0.1 N HCl at pH 1.5-2 (Jacobs 1969; Rao and Prabhavathi 1978; Miller et al. 1981)

• Pepsin in 0.1 N HCl at pH 1.5-2, with better correlation to human in vivo results than to rat in vivo results (Schricker et al 1981)

• Pepsin in dilute HCl ranging in pH from1.4-2.8, with comparison to gastric fluid extracted from volunteers (Lock 1980)

• The current common use of pepsin in dilute HCl at pH 2.0 (Etcheverry el al 2012, Patted et al 2013)

ENVIRONMENTAL SCIENCES PRECEDENT

Work on the bioaccessibility of nutrient elements has formed the foundation for the development of methods to measure the bioaccessibility of the environmental elemental impurities that are of particular concern in medical geology and environmental science. Much of this work has been devoted to analysis of the bioaccessible fraction of soils, sediments and even household dust that have been naturally or anthropogenically contaminated with elemental impurities.

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Techniques for biorelevant analysis of soil can be instructive for pharmaceutical analysis because the multicomponent nature of most soils (silica, silicates, clays, organics/humus) often presents analytical challenges similar to those encountered with pharmaceutical products. In the case of contaminated soils, determining the bioaccessible fraction rather than the total content of these elements is fundamental to the risk analysis upon which remediation strategies and costs have been based.

“A test for bioaccessibility of soil contaminants in soil must enable quantification of the dissolution under “realistic worst case conditions”, meaning that the test should simulate the highest bioaccessibility that can be expected without including unrealistic conditions or excessive precaution. To fulfill this, the test must be based upon the properties of the human digestion process, of the contaminants in question and the geochemistry of soils, …”

“If the test shall be used for practical risk assessment, it furthermore needs to fulfill the common, basic requirements for a regulatory method. The method must be:

• simple … • comprehensive … • precise … • reproducible … • interpretable … • consistent … (GrØn and Andersen, 2003)

The human gastrointestinal tract consists of a number of compartments where ingested material undergoes a series of reactions in which fluid composition, pH and reaction time all vary. Table 2 summarizes the order and conditions in each compartment (Oomen et al. 2002). Table 2. Compartments, Residence Time and pH

GI Tract Compartment Residence Time pH

Oral cavity Stomach

Seconds to minutes 8-15 minutes (fasting, half emptying time) 0.5-3 hours (fed, half emptying time)

6.5 1-2 2-5

Small intestine: Duodenum Jejunum Ileum

0.5-0.75 hour 1.5-2 hours 5-7 hours

4-5.5 5.5-7 7-7.5

Colon 15-60 hours 6-7.5

Bioaccessibility test protocols that focus on one (usually lead or arsenic) or more elemental impurities are generally of two types: simulated digestive tract methods and simulated gastric (stomach compartment) methods. The simulated digestive tract methods use extractions that mimic a combination or sequence of the GI tract compartments.

Simulated digestive tract methods and simulated gastric extraction methods are generally run at 37oC, but the extraction time, the pH and the particular composition of the respective simulated digestive juices vary (Wragg, Cave 2003).

Simulated digestive tract methods Physiologically Based Extraction Test (PBET) – This test is a two stage sequential extraction carried out at 37°C. The sample is extracted with pH 1.5 (pH 1.8 and 2.5 have also been used) simulated gastric fluid for one hour followed by four hours in pH 7 simulated intestinal fluid. The gastric fluid is most often a solution of most or all of the following: pepsin, citrate, malate,

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hydrochloric acid, lactic acid, acetic acid and sodium chloride. Simplified gastric fluids, such as USP simulated gastric fluid or SBRC/EPA 9200 gastric fluid (see below) have also been used. The simulated intestinal fluid is most often sodium bicarbonate, bile and pancreatin. This method was developed for Pb and As and has been recommended for Cr and Hg (Shoof 2003). PBET has been used to measure the bioaccessibility of Hg (Koch et al 2013), Pb, As, Cd and Hg (Jayaewardene et al 2010) and Pb and As (Koch et al 2011) in traditional Indian medicines. Table 3 shows an excerpt of the results from the latter study of PBET/ICP-MS gastric bioaccessibility of As and Pb compared to total As and Pb as determined by XRF or boiling aqua regia/ICP-MS. Table 3. As and Pb Bioaccessibility of Selected Traditional Indian Medicines

Traditional Indian Medicine Source

Total, ppm Gastric Bioaccessibility, %

As Pb As Pb Vital Lady minerals (mineral pitch), botanicals 2.1 5.5 52 35 Yog-Raj Guggulu botanicals 31 6 5.1 28 Shilajeet Tablets minerals (mineral pitch) 21 10 19 25

Mahayograj Guggulu bhasmas (calcines) of Sn, Ag, Pb, Fe; HgS; botanicals 89 52,000 45 94

Trifala Guggulu botanicals 27 20.5 5.3 29 Heart Plus botanicals 2.0 7.5 64 72 Mahasudarshan botanicals 140 15 7.1 18 Sugar Fight botanicals 6.4 7.5 29 75

Ayu-Hemoridi-Tone botanicals, Na carbonate, Na bicarbonate 7.5 3.9 69 29

GlucoRite™ botanicals 1.8 6.0 86 41 In-Vitro Gastrointestinal Method (IVG) - IVG was developed to estimate the bioaccessibility of arsenic. This test is a two stage anaerobic sequential extraction carried out at 37°C. The sample is extracted with pH 1.8 simulated gastric juice (NaCl, pepsin, HCl) followed by an intestinal phase extraction by adjusting the pH to 5.5 with a saturated sodium bicarbonate solution followed by addition of bile and pancreatin. The IVG gastric juice extraction has been validated for As against in vivo results (Rodriguez et al 1999). A modified IVG method (gastric juice = pepsin/HCl at pH 2) was used to estimate the bioaccessibility of As, Cd, Pb and Hg in two food reference materials (Torres-Escribano et al 2011); excerpted results are shown in Table 4. Table 4. Total vs Leached Elemental Impurities in Two Food Standards ppm, dry weight Seaweed: Fucus sp. (IAEA-140/TM) Lobster hepatopancreas (TORT-2) As Pb Cd Hg As Pb Cd Hg Total 43.3 2.19 0.520 0.036 21.3 0.298 26.6 0.301 Pepsin/HCl, Ph 2 31.0 0.192 0.061 0.001 15.1 0.057 5.18 0.012 Bioaccessibility 72% 9% 12% 3% 71% 19% 21% 4%

DIN 19738 (DIN) - This test is comprised of a two stage extraction: Extraction in pH 2.0 simulated gastric juice for two hours, followed by pH 7.5 simulated intestinal fluid for six hours. The simulated gastric juice is a solution of pepsin, hydrochloric acid, phosphate buffer, sodium chloride, potassium chloride and mucin; whole milk powder is added for “fed state” conditions. Simulated intestinal fluid is a solution of sodium bicarbonate, potassium chloride, calcium chloride, magnesium chloride, trypsin, pancreatin, bile and urea.

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RIVM in-vitro Digestion Model (RIVM) - This test involves a three-stage sequential extraction: five minutes in pH 6.5 simulated saliva, two hours in pH 1.07 simulated gastric fluid and two hours in pH 5.5 simulated intestinal fluid. The compositions of the simulated digestive juices are complex in an effort to closely analogue the biological fluids (Oomen at al 2003a). The RIVM method was originally developed to investigate the bioaccessible fractions of lead and benzo(a)pyrene from ingested soils (Sipps et al 2001; Oomen et al 2003a), but has been subsequently adapted to the investigation of the bioaccessible fractions of lead and target organic chemicals from toy matrices and azo dyes from textiles (Oomen et al 2003b; Oomen et al 2004; Oomen et al 2005; Brandon et al 2006) and cadmium and mycotoxins from food (Versantvoort et al 2004).

Unified Bioaccessibility Method (UBM) – This method, essentially a refinement of RIVM, was developed and validated (Wragg et al 2009, Wragg et al 2011) by the Bioaccessibility Research Group of Europe (BARGE1) after comparing and evaluating various historical models and systems developed to measure bioaccessibility and contaminant exposure. The method involves extraction in complex multicomponent digestive juices: simulated saliva + simulated gastric juice at pH 1.3+0.1 for one hour, plus extraction in simulated gastric juice + simulated intestinal juice (simulated duodenal juice + simulated bile) at pH 6.3 +0.5 for four hours.

Solubility/Bioavailability Research Consortium assay (SBRC) - This method involves two steps. The first step is a stomach phase extraction for one hour at 37°C with a simulated gastric fluid composed of 0.4 M glycine adjusted to pH 1.5 with HCl. This step is followed by intestinal phase extraction for four hours with a simulated intestinal fluid composed of bile and pancreatin at pH 7. Although originally developed and validated for Pb, this method is often used for As and has been recommended for Cd and Ni (Shoof 2003). In a 12-soil comparison study between SBRC, IVG, PBET and DIN, the SBRC simulated gastric fluid extraction provided the best prediction of in vivo relative arsenic bioavailability. (Juhasz et al 2009). This simulated gastric fluid extraction is also referred to as the relative bioaccessibility leaching procedure (RBALP) and has been formalized as an EPA standard operating procedure, EPA 9200.2.

Simulated gastric methods EN 71-3 – The European Standard for Safety of Toys, EN 71-3, measures the bioaccessibility of 17 elements. The method involves milling and extracting toy material in pH 1.5 HCl (5 ml 1 M HCl) at 37°C for 2 hours. The method has been used and comprehensively reviewed (Van Engelen et al 2008) in risk assessments for ingested elements. EN-71 and SBRC gastric fluid extraction studies (see also EPA 9200.2 below) yielded equivalent results for As, Pb, Cd, Cr, Cu, Ni and Zn in four NIST standard reference samples and two dust samples (Dodd et al 2013).

ASTM D5517 – This method was adapted from EN 71-3. The method involves grinding and mixing material with 0.07N HCl, adjusted to pH 1.5, and extracted with one hour of shaking at 37°C followed by one hour of extraction without shaking.

US Pharmacopoeia Method (USP) – This method is an adaptation of the USP drug dissolution test currently used to determine the bioaccessibility of heavy metals in contaminated soils (Hamel et al. 1998). The methodology uses the USP Simulated Gastric Fluid (NaCl, HCl, pepsin) to extract the elements at 37°C for two hours.

Relative Bioaccessibility Leaching Procedure (RBALP) – This method is essentially the gastric extraction portion of the SBRC method: extraction for one hour at 37°C with a simulated

1 BARGE is a network uniting international institutes and research groups in the study of the oral

bioaccessibility of priority contaminants in soils (As, Pb, Cd) as a tool for risk assessment and policy making (www.bgs.ac.uk/BARGE/).

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gastric fluid composed of 0.4 M glycine adjusted to pH 1.5 with HCl. This method has been validated for Pb (Drexler, Brattin 2007). In an extensive review of in vivo and in vitro methods and evaluations, RBALP was noted as the best bioaccessibility method for childhood exposure to multiple Pb sources (Deshommes et al 2012). The British Geological survey adapted the RBALP as the Simplified Bioaccessibility Extraction Test (SBET).

EPA 9200.2-86 – This method is a validated Standard Operating Procedure for In Vitro Bioaccessibility Assay for Lead in Soil. The extraction takes place at 37°C for one hour using a simulated gastric fluid composed of 0.4 M glycine adjusted to pH 1.5 with HCl. This is the RBALP/SBET/SBRC gastric phase extraction. Although this method best indicates the bioaccessibility of lead, it has been used to estimate the bioaccessibility of other elements, including As, Cd, Cr and Ni (Morman et al 2009). Results for As, Pb, and Cd from this study of uncontaminated soil samples representing a north-south transept across the USA, comparing total (four acid) digestion to simulated gastric fluid extraction, are presented in Figure 1a, 1b and 1c. Note that these uncontaminated soils have element levels comparable to those in mineral and mineral-derived pharmaceutical excipients.

Figure 1a. Total As (mg/kg) and corresponding amount (mg/kg) of As dissolved by the simulated gastric fluid leach (EPA 9200.2-86). Numbers above bars represent percent bioaccessibility.

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Figure 1b. Total Pb (mg/kg) and corresponding amount (mg/kg) of Pb dissolved by the simulated gastric fluid leach (EPA 9200.2-86). Numbers above bars represent percent bioaccessibility.

Figure 1c. Total Cd (mg/kg) and corresponding amount (mg/kg) of Cd dissolved by the simulated gastric fluid leach (EPA 9200.2-86). Numbers above bars represent percent bioaccessibility.

This simulated gastric fluid extraction has also been used to determine the bioaccessibility of Pb in Indian spices, cosmetic powders and religious powders solid in the Boston area (Lin et al 2010).

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“Most previous in vitro test systems have employed a more complex fluid intended to simulate gastric fluid. …. When the bioaccessibility of a series of test substances were compared using 0.4 M glycine buffer (pH 1.5) with and without the inclusion of these enzymes and metabolic acids, no significant difference was observed (p=0.196). This indicates that the simplified buffer employed in the procedure is appropriate, even though it lacks some constituents known to be present in gastric fluid.” (EPA 9200.2-86)

Chemical Extraction Although not a simulated gastric extraction method, a 0.43 M HNO3 leach is commonly used in soil science to assess the geochemical reactivity and oral bioaccessibility of a broad range of elements and elemental impurities (Rodriques et al 2010). Soil studies have reported equivalent results with a 0.43 M HNO3 leach and the SBRC/EPA 9200.2 simulated gastric fluid extraction (Rodrigues et al 2012, Cohelo et al 2012).

Another chemical extraction study involved preparing and using 3N HCl leach (15 minutes at 70oC) to assess the safety of minerals as food additives (Oshima et al 2007). Eight natural minerals were analyzed for a 28 element suite by total digestion (HF/HNO3) and by the diluted HCl leach. Although this extraction was relatively aggressive in terms of bioaccessibility, the excerpted values in Table 5 demonstrate the significant difference between total and leached values (Oshima et al 2007). Table 5. Total vs Leached Elemental Impurities In Natural Mineral Absorbents

ppm Acid Activated Clay Bentonite Diatomaceous Earth Perlite

Total Leached Total Leached Total Leached Total Leached As 0.9 0.6 1.6 1.2 12.7 2.1 0.4 ND Pb 14.3 8.70 21.6 9.95 8.0 0.03 14.5 0.28 Cd 0.04 0.01 0.10 0.02 0.08 ND 0.07 ND Co 0.6 0.1 1.9 1.0 4.8 ND 0.4 ND Mo 1.0 ND 1.7 1.2 7.7 4.6 1.3 0.1 V 5.2 0.5 9.4 ND 103 20.6 ND ND

RECOMMENDATIONS

With the exception of the pharmaceutical industry, most regulated industries have understood, accepted and documented elemental impurity risk assessments based on biorelevant extraction techniques, rather than total digestion techniques. Even within the pharmaceuticals industry, the use of such extraction techniques for specific control of As and Pb content have been prescribed for many years in certain NF and USP monographs in preference to the non-specific General Chapter <231> Heavy Metals test. There has been no indication that acid leach test methods for As and Pb have been inadequate for the purposes of risk assessment and the protection of public health. Consistent with the position that General Chapter <232> is applicable to drug products only, USP has even stated that acid leach tests for specific elemental impurities will not be removed from excipient and API monographs.

Drug components will be subject, nevertheless, to analysis by General Chapter <233> methods when required by drug product manufacturers who elect to use common concentration limit options or the components summation option to determine or support compliance with chapter <232> and the ICH Q3D standard. Chapter <233>, as currently written, will force drug product manufacturers to use elemental impurities data for some components that can overstate the

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amounts that are actually bioaccessible and misrepresent the risk posed by those components and thus the drug product to patients. Even if they elect to gauge compliance by direct analysis of the formulated drug, drug product manufacturers can likewise measure levels of elemental impurities that are not physiologically relevant either because of the presence of certain components or because of the unforeseen interaction among components.

We recommend, based on toxicological principles and sound risk-assessment practices, the revision of General Chapter <233> such that it prescribes biorelevant extraction techniques rather than total digestion techniques for the purpose of determining compliance with oral PDE limits.

Such techniques can be simply derived from the existing compendial techniques shown in Table 1 (0.5 N HCl or 1 N HNO3 leach), or from an uncomplicated and well researched bioaccessibility technique such as EPA 9200.2-86.

Alternatively, biorelevant extraction techniques such as these should be allowed as an option such that the drug product manufacturer can decide the appropriate technique based on case by case risk assessment considerations and concerns about analyst safety when HF is required for total dissolution of the sample.

REFERENCES

Brandon EF, Oomen AG, Rompelberg CJ, Versantvoort CH, van Engelen JG, Sips AJ (2006) Consumer product in vitro digestion model: Bioaccessibility of contaminants and its application in risk assessment, Regulatory Toxicology and Pharmacology. 2006 Mar;44(2):161-71 Coelho C, Rodrigues S, Cruz N, Carvalho L, Duarte AC (2012) Pereira E, Romkens PF, Can the reactive pool of potentially toxic elements in urban soils give an indication of human exposure?, 9th International Symposium on Environmental Geochemistry Deshommes D, Tardif R, Edwards M, Sauvé S, Prévost M (2012) Experimental determination of the oral bioavailability and bioaccessibility of lead particles, Chemistry Central Journal, 6:138 Dodd M, Rasmussen PE, Chenier M (2013) Comparison of Two In Vitro Extraction Protocols for Assessing Metals’ Bioaccessibility Using Dust and Soil Reference Materials, Human and Ecological Risk Assessment, 19: 1014–1027 Drexler JW, Brattin WJ (2007) An In Vitro Procedure for Estimation of Lead Relative Bioavailability: With Validation, Human and Ecological Risk Assessment: An International Journal Volume 13, Issue 2 Duffus JH, Worth HGJ (2006) Toxicology and the environment: An IUPAC teaching program for chemists, Pure Appl. Chem., Vol. 78, No. 11, pp. 2043–2050 Etcheverry P, Grusak MA, Fleige LE (2012) Application of in vitro bioaccessibility and bioavailability methods for calcium, carotenoids, folate, iron, magnesium, polyphenols, zinc, and vitamins B6, B12, D, and E, Front Physiol., 3: 317 Gron C, Andersen L (2003) Human Bioaccessibility of Heavy Metals and PAH from Soil, Environmental Project No. 840, Technology Programme for Soil and Groundwater Contamination, Danish Environmental Protection Agency Hamel SC, Buckley B, Lioy PJ (1998) Bioaccessibility of metals in soils for different liquid to solid ratios in synthetic gastric fluid. Environmental Science & Technology,32, 358-362

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Jacobs A, Greenman DA (1969) Availability of Food Iron, Brit. med. J., 1, 673-676) Jayawardenea I, Saperd R, Lupolia N, Sehgald A, Wright RO, Amarasiriwardena C (2010) Determination of in vitro bioaccessibility of Pb, As, Cd and Hg in selected traditional Indian medicines, J Anal At Spectrom. 25(8): 1275–1282 Juhasz AL, Weber J, Smith E, Naidu R, Rees M, Rofe A, Kuchel T, Sansom L (2009) Assessment of Four Commonly Employed in Vitro Arsenic Bioaccessibility Assays for Predicting in Vivo Relative Arsenic Bioavailability in Contaminated Soils, Environ. Sci. Technol., 43 (24), pp 9487–9494 Koch I, Moriarty M, House K, Sui J, Cullen WR, Saper RB, Reimer KJ (2011) Bioaccessibility of lead and arsenic in traditional Indian medicines, Sci Total Environ. October 1; 409(21): 4545–4552. Koch I, Moriarty M, House K, Sui J, Rutter A, Saper RB, Reimer KJ (2013) Bioaccessibility of mercury in selected Ayurvedic medicines Science of The Total Environment, Volumes 454–455, 1 June, Pages 9–15 Lin CG, Schaider LA, Brabander DJ, Woolf AD (2010) Pediatric Lead Exposure From Imported Indian Spices and Cultural Powders, Pediatrics 125;e828 Lock S, Bender AE (1980) Measurement of chemically-available iron in foods by incubation with human gastric juice in vitro, Br. J. Nutr., 4, 413 Miller DD, Schricker BR, Rasmussen RR, Campen DB (1981) An in-vitro method for estimation of iron availability from meals, American Journal of Clinical Nutrition, 34, 2248–2256 Morman SA, Plumlee GS, Smith DB (2009) Application of in vitro extraction studies to evaluate element bioaccessibility in soils from a transect across the United States and Canada, Applied Geochemistry 24, 1454–1463 Oomen AG, Hack A, Minekus M, Zeijdner E, Cornelis C, Schoeters G, Verstraete W,Van de Wiele T, Wragg J, Rompelberg CJM, Sips A, Van Wijnen JH, (2002) Comparison of five in vitro digestion models to study the bioaccessibility of soil contaminants. Environmental Science & Technology, 36, 3326-3334 Oomen A, Rompelberg CJM, Bruil MA, Dobbe CJG Pereboom DPKH, Sips AJAM (2003a) Development of an In Vitro Digestion Model for Estimating the Bioaccessibility of Soil Contaminants, Arch. Environ. Contam. Toxicol. 44, 281–287 Oomen AG, van Twillert K, Hofhuis MFA, Rompelberg CJM, Versantvoort CHM (2003b) Development and suitability of in vitro digestion models in assessing bioaccessibility of lead from toy matrices, RIVM report 320102001/2003, National Institute for Public Health and the Environment (RIVM), Netherlands Oomen AG, Versantvoort CHM, Duits MR, van de Kamp E, van Twillert K (2004) Application of in vitro digestion models to assess release of lead and phthalate from toy matrices and azo dyes from textile, RIVM report 320102003/2004, National Institute for Public Health and the Environment (RIVM), Netherlands Oomen AG, Rompelberg CJM, Brandon EFA, van de Kamp E, Duits MR, Versantvoort CHM, van Engelen JGM, Sips AJAM (2005) Consumer Product in vitro digestion model: bioaccessibility of contaminants from toys and application in risk assessment, RIVM report 320102004/2005, National Institute for Public Health and the Environment (RIVM), Netherlands Oshima H, Kabashima Y, Ueno E, Ohno T, Oka H, Ito Y, Kakazawa H (2007) Multielement analysis of insoluble minerals as existing food additives by Inductively Coupled Plasma-Mass Spectrometry, Japanese Journal of Food Chemistry, 14(3), 113-120

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