Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
15 May to 25 September 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Objective of study:
bioaccessibility
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of the study was to investigate the rate and extent of hydrolysis and condensation reactions that may occur in the stomach of a rat following oral administration of HD4 or HD5. An in vitro test system was established to simulate the conditions of the stomach of a rat using an aqueous phosphate buffer at a pH of 3.14.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
not specified
Radiolabelling:
no

Test animals

Species:
other: in vitro
Details on test animals and environmental conditions:
not applicable

Administration / exposure

Route of administration:
other: in vitro
Vehicle:
other: buffer solution of Sodium Chloride (8.4942 g) and o-Phosphoric acid (1.1724g) were added to one liter of deionized water. This solution was mixed, and then titrated to pH 3.14 using a pH meter and a small amount of sodium hydroxide solution (0.1N).
Duration and frequency of treatment / exposure:
The test materials were allowed to mix for up to 4 hours.
Doses / concentrations
Remarks:
Doses / Concentrations:
0.05g/mL and 0.005g/mL (correlating to assumed doses of 1000 or 100 mg/kg body weight, assuming 250g rat) in phosphate buffer solution.
A stomach volume of 4mL was assumed.
Details on study design:
The reaction vessel with buffer solution was heated to 37 degrees C to mimic the internal body temperature of a potential test animal and stirred with a magnetic stir bar. The test material was then added and allowed to mix for increasing intervals up to 4 hours.
Details on dosing and sampling:
The reaction vessel containing buffer solution was heated using an oil bath to 37 degrees C and was purged with nitrogen prior to test article introduction. A known amount of test material was injected into the gastight apparatus containing pH buffer solution and stirred using a magnetic stir bar.

Measurement of Hydrogen Generation
The water displaced by gas generation was measured using a graduated cylinder and also weighed. As test reaction was done using dilute hydrochloric acid and magnesium turnings.

Sample Extraction for Analysis
After reacting, samples were extracted from the aqueous buffer solution for analysis with chloroform, 1 mL was added to the reaction mixture and the solution was vortex mixed for a minimum of 1 minute. The organic layer was then removed by glass pipette and analyzed by GPC and GC-MS.

GPC Analysis
An Agilent Technologies 1200 Series RID detector coupled to a Hewlett PAckard 1050 Series quaternary pump and autosampler was used for analysis. A solvent blank, original parent material and the solvent extracted test material from the reaction vessel were analysed.

GC-MS Analysis
An HP 6890A GC with an HP 5973N mass selective detector (MSD) was used to verify the major component(s) in each test article and the extract of HD4 4 hour reaction by manual injection.

GC/FID Analysis
A HP 6890 GC with flame ionisation detector (FID) and HP 7683B autosampler was used.
Statistics:
No statistical analysis performed.

Results and discussion

Preliminary studies:
A weighed amount of magnesium turnings was added to the reaction flask and hydrochloric acid (HCl) solution added to produce hydrgen gas. Trial one used 12 M hydrochloric acid and produced an abundance of gas. The water displaced was tested with pH paper and was determined to be acidic. A diluted ~ 3 M HCL solution was used for trials 2 and 3. Displaced water tested during these trials remainined neutral pH. Results demonstrated an error below 10%.
Main ADME results
Type:
other: Hydrolysis of HD4 and HD5 was negligible under the simulated gastric conditions used in this experimental design, they were insoluble in aqueous buffer solution and remained mostly unchanged.

Bioaccessibility

Bioaccessibility testing results:
Tetramethylcyclotetrasiloxane (HD4)
"High Dose" concentration (0.05 g/mL)
No water displacement was observed after 20 minutes. The reaction was continued for 4 hours with no displacement observed. The reaction flask was then removed from heat and extracted with chloroform. A second trial was performed, in which no water displacement was observed after 1 hour. The reaction flask was then removed from heat and extracted with chloroform. The HD4 sample remained immiscible in buffer solution throughout the reaction time.

"Low Dose" concentration (0.005 g/mL)
The test substance concentration was decreased 10-fold for this subsequent experiment. To increase sensitivity the volume of buffer solution was increased to 20mL. A water displacement of 1.2mL was observed after a reaction time of 1 hour. The HD4 sample appeared to be partially undissolved in buffer throughout the reaction time. The reaction flask was removed from heat and extracted with chloroform.

Pentamethylcyclopentasiloxane (HD5)
"High Dose" concentration (0.05 g/mL)
No water displacement was observed after 20 minutes. The reaction was continued for 4 hours with no displacement observed. A second trial was performed in which no water displacement was observed after 1 hour. In both cases the reaction flask was removed from heat and the contents were extracted with chloroform. The HD5 sample was mostly insoluble in buffer solution and manitained as two distinct phases with no rag layer throughout the reaction time.

"Low Dose" concentration (0.005 g/mL)
The test substance concentration was decreased 10-fold. To increase sensitivity the volume of buffer solution was increased to 20mL. A water displacement of 0.48mL was observed after a reaction time of 1 hour. The reaction flask was removed from heat and extracted with chloroform. The HD5 sample appeared to be at least partially insoluble in buffer solution and maintained two distinct phases with no rag layer throughout the reaction time.

GPC Analysis
Tetramethylcyclotetrasiloxane (HD4)
No observable large molecular weight reaction products were detected in eitehr the original HD4 or the reaction samples.

Pentamethylcyclopentasiloxane (HD5)
No observable large molecular weight reaction products were detected in either the original HD5 or the reaction samples.

GC-MS Analysis
The original parent material of HD4 sample consisted of a mixture of HDn cyclics (n = 3, 4, 5, 6 and 7 etc) with the species of interest (HD4) being most abundant. Abundance of cyclic decreased as ring size increased. The extract of the four hour reaction mixture contained a simialr profile. Chloroform and toluene were identified. the chloroform was used as the reaction solvent, and toluene had been previously used to clean the syringe. Cyclics n = 4-9 were identified in the extract as well, with n=4 being most abundant. Certain cyclics, n=3 and n>9 were not identified in the extract. This is possibly due to the variation in concentration between parent material (neat) and extract (~20%). Some peaks were observed at similar retention timeto the larger n-cyclics, but a reliable library match could not be obtained. GC-MS analysis was not completed on HD5 samples as GC-FID results of parent vs extract did not indicate additional components in need of identification.

GC-FID Analysis
GC-FID analysis was focussed on determining if there was a loss of purity, indicating transformation of the main component of the test substance during reaction. Neither HD4 or HD5 demonstrated a discernible change in extractable components after the reaction at either concentration. Parent material was analysued at the same time as the reaction mixture extracts.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: bioaccumulation potential cannot be judged based on study results
In pH and temperature conditions used in the experimental set up in the study to mimic the stomach of a rat during oral dosing, the HD4 and HD5 parent material remained mostly unchanged. Both materials were mostly insoluble in aqueous buffer solution. Rapid and complete hydrolysis of HD4 or HD5 was not observed. No measurable hydrogen release was observed during the time the reaction was allowed to proceed. No major reaction products, either large or small molecule were observed. 99.89% of the original concentration of HD4 (0.05g/ml) remained after four hours. 100.0% of the original concentration of HD5 (0.005g/mL) remained after four hours. 99.54% of the original concentration of HD4 (0.005g/ml) remained after one hour. 99.77% of the original concentration of HD5 (0.005g/mL) remained after one hour. These findings can likely be attributed to the high concentrations of HD4 and HD5 and their limited solubility in aqueous solution.