[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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted following the test protocols given in EFSA 'Note for Guidance' 30/7/2008.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Objective of study:

other: hydrolysis

Test guideline

Qualifier:

according to guideline

Guideline:

other: protocols given in EFSA 'Note for Guidance' 30/7/2008

Deviations:

not specified

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Amides, C18, branched and linear

Molecular formula:

Not applicable as UVCB

IUPAC Name:

Amides, C18, branched and linear

Test material form:

not specified

Details on test material:

- Analytical purity: 70% monobranched x-methylheptadecanamide
- Composition of test material, percentage of components: x-methylheptadecanamide, isostearamide

Radiolabelling:

no

Test animals

Species:

other: not applicable

Strain:

other: not applicable

Administration / exposure

Route of administration:

other: not applicable

Vehicle:

other: tetrahydrofuran

Details on exposure:

not applicable

Duration and frequency of treatment / exposure:

4 h (loss of [Tradename])
4 and 24 h (formation of isostearic acid)

Doses / concentrations

Remarks:

Doses / Concentrations:
11.0 - 11.01 mg/L

No. of animals per sex per dose / concentration:

not applicable

Control animals:

other: control blanks were added

Positive control reference chemical:

not applicable

Details on study design:

not applicable

Details on dosing and sampling:

The tests were conducted in triplicate using 160 mL glass vials with PTFE lined septa immersed in a water bath held at 37 °C. Continuous agitation was carried out using submersible magnetic stirrers. It was found that there were interferences in the analysis of isostearic acid originating from the pancreatin. To clean up the pancreatin 100 g was stirred up consecutively with diethyl ether followed by n-pentane (~ 500 mL each) at room temperature and filtered under reduced pressure using a Buchner funnel and glass fibre filters. After rinsing with a further 500 mL of pentane the pancreatin was air dried in a desiccator at ambient temperature under reduced pressure. The simulated intestinal fluid was prepared according to 'Note for Guidance' using 8xUSP pancreatin and the pH adjusted to 7.5. For calibration purposes, denatured intestinal fluid was prepared using the same ingredients but dispersing the pancreatin first in 190 mL of 0.2M sodium hydroxide and storing overnight. The other ingredients were added and the volume made up to 1 L with water. It was found that using the active simulated intestinal fluid for calibration gave a rapid initial loss of [Tradename], therefore the denatured intestinal fluid was used for calibrating the loss of [Tradename].

Analysis of the loss of [Tradename]
- A stock solution of [Tradename] was prepared in propan-2-ol of concentration 11005 mg/L. After pre-warming 100 mL simulant (triplicate) to 37°C, 100 µL of the stock solution was added to each vial giving a starting concentration of 11.0 mg/L. After the 4 h incubation time with stirring, the vials were cooled and 5 g of anhydrous sodium sulphate added. The vials and contents were then extracted with 25 ml of diethyl ether and the top layers removed and centrifuged. 1 mL of the top layer from each vial was then transferred to a fresh vial and the solvent blown down under a stream of nitrogen to dryness. The residue was dissolved in 1 mL cyclohexane and injected for GC/MS analysis. The total area of the main amide peaks [Tradename] was integrated and plotted against the concentration in a range of calibration standards, prepared by diluting 0, 18, 50, 90 and 230 µL of a stock solution of [Tradename] 11005 mg/L to 100 mL aliquots of denatured simulated intestinal fluid and extraction with diethyl ether. These were extracted as described above. The concentration of [Tradename] remaining after the allotted time was interpolated from the graph and the % loss calculated based upon a starting concentration of 11.0 mg/L.

Analysis for the formation of isostearic acid from [Tradename].
- A stock solution of [Tradename] was prepared in propan-2-ol of concentration 11005 mg/L. After pre-warming 100 mL simulant (triplicate) to 37 °C, 100 µL of the stock solution was added to each vial giving a starting concentration of 11.01 mg/L. The solutions were stirred continuously for 4 h. After this time the vials were cooled and the internal marker added (200 µL of a 1000 mg/L solution of heptadecanoic acid, C17 fatty acid). After addition of 5 g of anhydrous sodium sulphate the solutions were extracted with 25 mL of diethyl ether. The top layer was removed, centrifuged and evaporated to dryness under a stream of nitrogen with gentle warming. 1 mL of methanol was added and 100 µL of TMSH (trimethylsulfonium hydroxide). The solutions were further diluted 1:1 with 100 µL of ethyl acetate (to improve the peak shape) and injected for GC/MS. TMSH was used to convert the fatty acid to their methyl esters. The area of the peak for isostearic acid peak was divided by the internal standard peak area and plotted against the concentration in a range of calibration standards (2 sets), prepared by diluting 0, 70(78), 160, 320(330), 640(660), 1000 and 1900 µL of the solution of Isostearic acid in IPA (2532 mg/L) to 100 mL aliquots of blank simulated intestinal fluid. These were extracted as described above. The concentration of isostearic acid formed after 4 h was interpolated from the graph and the % formed calculated based upon a starting concentration of 11.01 mg/L [Tradename]. No correction for molecular weight change in the reaction was made as this is insignificant. Also, the same experiment was carried out to check the % formation of isostearic acid after 24 h incubation time at 37 °C with stirring. To check for stability recovery of isostearic acid, 100 mL triplicate portions of blank simulated intestinal fluid were spiked with 400 µL of 2532 mg/L of isostearic acid and the solutions incubated for 4 h at 37°C, with stirring. After this period of time, the whole amount was extracted with 25 mL diethyl ether and esterified as described above.

Statistics:

Mean values and standard deviations were calculated in % loss of [Tradename] and % formation of isostearic acid.

Results and discussion

Main ADME results

Main ADME results 1

Type:

other: hydrolysis [Tradename]

Results:

79 ± 3% (4 h)

Main ADME results 2

Type:

other: formation (isostearic acid)

Results:

62 ± 7% (4 h)

Main ADME results 3

Type:

other: formation (isostearic acid)

Results:

78 ± 1% (24 h)

Any other information on results incl. tables

Table 1: Loss of [Tradename] (triplicates)

Timepoint	% Loss	% Mean and SD
4 h (1)	76
4 h (2)	78
4 h (3)	82	79 ± 3

SD: Standard deviation

Table 2: Formation of isostearic acid (triplicates)

Timepoint	% Formation	Mean and SD
4 h (1)	68
4 h (2)	65
4 h (3)	55	62 ± 7
24 h (1)	77
24 h (2)	77
24 h (3)	79	78 ± 1

SD: Standard deviation

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
The testing was conducted using simulated intestinal fluid with measurement of the loss of starting substance [Tradename] and measurement of the hydrolysis product isostearic acid. It was decided that the other hydrolysis product (ammonia) would be very difficult to measure at very low levels in this fluid. Difficulties were encountered in measuring isostearic acid in the simulated intestinal fluid owing to the presence of fats/fatty acids in the pancreatin. This was overcome by purifying the pancreatin prior to use by washing with solvent. In calibrating for the loss of [Tradename], a significant initial loss was found on addition of the substance to blank intestinal fluid after mixing well for a few minutes then extraction with solvent. To overcome this, calibration standards were prepared by adding [Tradename] to 100 mL portions of denatured intestinal fluid that was prepared to deactivate the enzymes. Losses of 79% of [Tradename] were found after 4 h incubation with simulated intestinal fluid. The formation of fatty acid was lower at 62% isostearic acid after 4 h and 78% after 24 h.