Calcium 3,5,5-trimethylhexanoate

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics in vitro / ex vivo

Remarks:

Bioelution in simulated gastric medium

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 December 2021 to 12 February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline available

Principles of method if other than guideline:

Bioelution in simulated gastric medium according to Eurometaux (2019): The Standard Operating Procedure for Bioelution Testing of Metals, Inorganic Metal Compounds, and Complex Metal-Containing Materials: Simulated Gastric Fluid (Eurometaux Brussels, November 02, 2019).

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The test item, Calcium 3,5,5-trimethylhexanoate as received, was tested. Therefore, no further preparation of the test item at ECTX was needed.

Vehicle:

other: simulated gastric fluid (0.032 N HCl)

Duration and frequency of treatment / exposure:

1 hour agitation and 1 hour to allow the sample to settle

Positive control reference chemical:

9960 mg/L Calcium standard

Bioaccessibility (or Bioavailability) testing results:

Bioelution refers to the in vitro extraction methods used to measure the degree to which a substance is dissolved in simulated biological fluids. The resulting value is the “bioaccessibility”, and is defined as the “fraction of a substance that is soluble under physiological conditions and therefore potentially available for absorption into systemic circulation”. Simulated biological fluids (e.g. gastric fluid, interstitial fluid, lysosomal fluid,…), represents relevant exposure routes. In this study, the oral route was investigated. Therefore, simulated gastric fluid, as provided in the SOP, was used to measure the release/dissolution of the elements of interest from Calcium 3,5,5-trimethylhexanoate.

The temperature of the sampled test solutions including the Negative and Positive Control vessel at the 2-hours sampling point was between 36.8 °C and 37.0 °C and corresponded to the required test conditions of 37 °C ± 1 °C.

The measured pH of the test medium at the start of the test was 1.50, i.e. within the specifications of pH 1.5 ± 0.1. The pH measured in the additional test item vessel at the beginning of the trial was 1.52 for the 0.2 g/L and 1.56 for the 2 g/L loading, respectively, i.e. within the specifications of pH 1.5 ± 0.1. At the 2 hours endpoint of the test, the pH in the Negative Control vessel was 1.51 and 1.49 in the Positive Control. The pH in the test item vessels was between 1.58 and 1.59 for the vessels at the 0.2 g/L loadings and between 1.65 and 1.67 for the vessels at the 2 g/L loadings

The Negative Control vessel showed no concentrations of calcium above the limit of quantification (i.e. accredited reporting limit) of 0.05 mg/L Ca.

The following observations could be made in the Positive Control item vessel containing a soluble form of calcium:
- The Positive Control item solution remained clear at the tested concentration level. No precipitation could be observed on the filter and in the Erlenmeyer flask at the end of the experiment.
- The recovery of the dissolved calcium concentration in the Positive Control item was 105 % and met the criterion (recovery: 100 % ± 10 %) as indicated in the SOP.

The following observations could be made in the test vessels with a loading of 0.2 g/L Calcium 3,5,5-trimethylhexanoate:
- The test solution was clear at the end of the experiment. No undissolved test item could be observed in the Erlenmeyer flasks at the end of the experiment.
- An average dissolved calcium concentration in solution of 21.7 ± 0.5 mg/L Ca (corresponding to a dissolved calcium concentration per g test item of 108 ± 2 mg/g Ca) was measured after 2 hours of exposure to the simulated gastric fluid (pH 1.5) with a between-vessel variation of 2 % which met the quality criteria at the 2 hours endpoint.
Based on the calcium content of the test item (i.e. 11.78 % Ca) and the average dissolved calcium concentration, a calcium release of 91 % could be calculated at the 2 hours endpoint.

The following observations could be made in the test vessels with a loading of 2 g/L Calcium 3,5,5-trimethylhexanoate:
- The test solution was clear at the end of the experiment. No undissolved test item could be observed in the Erlenmeyer flasks at the end of the experiment.
- An average dissolved calcium concentration in solution of 214 ± 5 mg/L Ca (corresponding to a dissolved calcium concentration per g test item of 107 ± 2 mg/g Ca) was measured after 2 hours of exposure to the simulated gastric fluid (pH 1.5) with a between-vessel variation of 2 % which met the quality criteria at the 2 hours endpoint.
Based on the calcium content of the test item (i.e. 11.78 % Ca) and the average dissolved calcium concentration, a calcium release of 91 % could be calculated at the 2 hours endpoint.

Conclusions:

During this study on Calcium bis(2-ethylhexanoate) at a loading of 0.2 g/L in simulated gastric fluid (pH 1.5), it was shown that for calcium an average dissolved concentration in solution of 21.7 mg/L Ca (CVbetween-vessel = 2 %; N = 3) was found after 2 hours of extraction. This amount corresponds to a dissolved calcium concentration per g test item of 108 mg/g Ca or a calcium release of 91 % of the content.

During this study on Calcium bis(2-ethylhexanoate) at a loading of 2 g/L in simulated gastric fluid (pH 1.5), it was shown that for calcium an average dissolved concentration in solution of 214 mg/L Ca (CVbetween-vessel = 2 %; N = 3) was found after 2 hours of extraction. This amount corresponds to a dissolved calcium concentration per g test item of 107 mg/g Ca or a calcium release of 91 % of the content.

The results are reliable since the test conditions stayed constant during the experiment. The pH in the high loading (2 g/L) test vessels was 1.67 and exceeded the initial range (pH 1.5 ± 0.1) at the end of the experiment. The recovery of Positive Control met the criteria of acceptance.

Executive summary:

Bioelution refers to the in vitro extraction methods used to measure the degree to which a substance (e.g., metal or mineral ion) is dissolved in artificial biological fluids. Simulated biological fluids represent relevant exposure routes. The release/dissolution of calcium ions from Calcium 3,5,5-trimethylhexanoate in the simulated gastric fluid was measured. 

The resulting value is the “bioaccessibility”, and is defined as the “fraction of a substance that is soluble under physiological conditions and therefore potentially available for absorption into systemic circulation”. 

This study was conducted according to The Standard Operating Procedure for Bioelution Testing of Metals, Inorganic Metal Compounds, and Complex Metal-Containing Materials: Simulated Gastric Fluid (Eurometaux, November 2, 2019) in which the gastric medium will be prepared starting from a 0.1 M HCl solution. 

The extent of dissolution of Calcium 3,5,5-trimethylhexanoate in the powder form (as received) was tested in a simulated gastric fluid at 37 °C and pH 1.5 for 2 hours (0.2 g/L and 2 g/L loadings) at an agitation speed of 100 revolutions per minute (rpm) for 1 hour followed by 1 hour of settling (without shaking). The bioelution endpoint is based on the dissolved calcium (Ca) concentrations obtained after 2 hours of extraction. 

The study was performed at ECTX. Analysis of the concentrations of dissolved calcium has been performed at WLN Business B.V. (The Netherlands), an ISO 17025 accredited laboratory, as delegated by ECTX. 

The measured pH of the test medium at the start of the test was 1.50, i.e. within the specifications of pH 1.5 ± 0.1. The pH measured in the additional test item vessel at the beginning of the trial was 1.52 for the 0.2 g/L and 1.56 for the 2 g/L loading, respectively, i.e. within the specifications of pH 1.5 ± 0.1. At the 2 hours endpoint of the test, the pH in the Negative Control vessel was 1.51 and 1.49 in the Positive Control. The pH in the test item vessels was between 1.58 and 1.59 for the vessels at the 0.2 g/L loadings and between 1.65 and 1.67 for the vessels at the 2 g/L loadings. 

The temperature of the sampled test solutions, including the Negative and Positive Control vessel was between 36.8 °C and 37.0 °C and corresponded to the required test conditions of 37 °C ± 1 °C. 

The Negative Control vessel showed no concentrations of calcium above the limit of quantification (i.e. accredited reporting limit) of 0.05 mg/L Ca. 

In the test item vessels with a loading of 0.2 g/L Calcium 3,5,5-trimethylhexanoate, the following average dissolved calcium concentration in solution of: 

• 21.7 ± 0.5 mg/L Ca (CVbetween-vessel = 2 %; N = 3), corresponding to a dissolved calcium concentra-tion per g test item of 108 ± 2 mg/g Ca, was measured after 2 hours of exposure to the simulated gastric fluid (pH 1.5). 

Based on the calcium content (i.e. 11.78 % Ca) in the test item and the average dissolved calcium concentrations in the test solutions, a release of: 

• 91 % of the content could be calculated at the 2 hours endpoint. 

 

In the test item vessels with a loading of 2 g/L Calcium 3,5,5-trimethylhexanoate, the following average dissolved calcium concentration in solution of: 

• 214 ± 5 mg/L Ca (CVbetween-vessel = 2 %; N = 3), corresponding to a dissolved calcium concentration per g test item of 107 ± 2 mg/g Ca, was measured after 2 hours of exposure to the simulated gastric fluid (pH 1.5). 

Based on the calcium content (i.e. 11.78 % Ca) in the test item and the average dissolved calcium concentrations in the test solutions, a release of: 

• 91 % of the content could be calculated at the 2 hours endpoint. 

Description of key information

The dissolution of Calcium from Calcium bis(2-ethylhexanoate)after 2h in simulated gastric acid (pH 1.5) at 0.2 and 2 g/L loading is  both 91%, corresponding to 21.7 and 214 mg Ca/L, respectively.