Registration Dossier

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
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
- Principle of test: In vitro investigation of potential hydrolysis and metabolic degradation of pyranyl acetate pure in plasma, simulated liver and skin of rats as well as in simulated gastric
and intestinal fluids

- Parameters analysed / observed:
Enzymatic degradation products of the test substance in different simulated tissues or body fluids of rat.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ZH 146
- Expiration date of the lot/batch: 2015-12

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
Radiolabelling:
no

Test animals

Species:
other: not applicable (in vitro study)
Strain:
other: not applicable (in vitro study)
Details on species / strain selection:
not applicable (in vitro study)

Administration / exposure

Route of administration:
other: not applicable (in vitro study)
Details on exposure:
not applicable (in vitro study)
Details on study design:
The objective of the current study was to investigate potential hydrolysis and metabolic degradation of pyranyl acetate pure in plasma, liver and skin of rats as well as in gastric and intestinal fluids. To determine hydrolysis in either compartment, the test substance was incubated with liver or skin S9 fractions from rats as well as in rat plasma and in gastric and intestinal fluid simulants in duplicates at a target concentration of 250 μM for defined incubation periods at 37°C. Incubation periods were 0.5, 1 and 2 hours for liver S9 fraction and gastric fluid simulant or 2 hours for skin S9 fraction, plasma and intestinal fluid simulant. After incubation, the amounts of remaining substrate were analyzed in the appropriate incubates by GC/MS. Depending on the test system, heat deactivated controls (HDC) and controls, directly stopped after addition of test substance (t=0 controls) served as control samples. A buffer control (BC, test substance in the incubation buffer) was used to calculate consequent recoveries. Defined incubates were additionally analyzed for the potential hydrolysis product pyranol.

Results and discussion

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
For pyranol acetate pure, a series of experiments was conducted to optimize incubations and analyses. Methods and results given within this report concentrated on the final optimized experimental conditions applied for the herein described experiments.
Pyranyl acetate pure was applied to the described experiments in a stock solution of DMSO. Analytical investigations that were also performed within the current study demonstrated the stability of pyranyl acetate pure in this stock solution at least over 24 h.
For experiments in liver and skin S9 fraction and intestinal fluid simulant, BC and t = 0 controls showed generally pyranyl acetate pure with valid recoveries and stabilities. Therefore, it can be concluded that the test substance is stable under the incubation conditions chosen. In HDC, incubated for 2 h at 37 °C, generally lower recoveries were measured. Related to buffer controls, individual values ranged between 46 and 71 %. HDC of liver S9 fraction, analysed for the potential degradation product showed no formation of Pyranol. It may be speculated that the general lower recoveries in HDC than in t=0 controls may be based on potential evaporation of Pyranyl acetate pure in HDC. Taking this hypothesis into account, calculations of metabolic turn over in active incubates were related to HDC (and not to t=0 controls).
 
Liver S9 fraction
In liver S9 fraction of the rat, recoveries of pyranyl acetate pure (t = 0 control versus BC) were between 101 % and 103 %. HDC yielded corresponding values of 65 and 65 %. In active incubates with an incubation period of 2 h, pyranyl acetate pure was metabolized to 79 % (mean value of two incubations). Pyranol could be detected in the active incubations and was attributed to the formed metabolite, since this peak was not present in any of the controls analyzed. In the active incubates 37 up to 49 μmol/L pyranol were measured. These analyzed concentrations of pyranol in active incubates do not correspond stoichiometrically to the amounts of degraded test substance. This demonstrates that beside pyranol further metabolites may occur when pyranyl acetate pure is metabolized in rat liver in vitro systems. Shorter incubation times of 0.5 and 1 h resulted in mean metabolic turn over values of 70 % (0.5 h) and 79 % (1 h), demonstrating that the degradation of pyranyl acetate pure in liver S9 fraction of rats is fast and that about 89 % of the metabolic turn over after an incubation period of 2 h were already reached 0.5 h after start of the incubation.
 
Skin S9 fraction
In skin S9 fraction of rats, recoveries of pyranyl acetate pure (t=0 control versus BC) were between 95 % and 114 %. HDC yielded corresponding values of 53 % and 46 %. Analyzed concentrations of pyranyl acetate pure in active incubates resulted in values that were between the concentrations found in HDC and t = 0 controls. Therewith, the metabolic turnover of pyranyl acetate pure in skin S9 fraction could not be demonstrated and is assessed to be negligible under the test conditions used.
 
Plasma
In plasma of rats, no buffer control was performed and no recoveries of pyranyl acetate pure were calculated. Analyzed concentrations of pyranyl acetate pure in plasma samples resulted values that were between the concentrations found in HDC and t = 0 controls. Therewith, the metabolic turnover of pyranyl acetate pure in rat plasma could not be demonstrated and is assessed to be negligible under the test conditions used.
 
Intestinal fluid simulant
In intestinal fluid simulant, recoveries of pyranyl acetate pure (t = 0 control versus BC) were between 96 % and 102 %. HDC yielded corresponding values of 63 % and 71 %. The analyzed concentration of pyranyl acetate pure in the active incubate of the first replicate resulted in a value that was between the concentrations found in HDC and t=0 control. The metabolic turn over in replicate 2 was calculated to be 4 %. These results demonstrated, that the metabolism of pyranyl acetate pure in intestinal fluid simulant is negligible under the test conditions used.
 
Gastric juice simulant
In gastric fluid simulant, no buffer controls and no heat deactivated controls were performed. Hence, recoveries cannot be calculated for this test system. However, t = 0 controls yielded analytical concentrations that corresponded to 97 and 95 % of the nominal concentrations of these samples. Analyzed concentrations of pyranyl acetate pure in samples incubated for 0.5, 1 or 2 h, were 9 to 21 % less than in t = 0 controls. Compared to relative amounts of the test substance in HDC of other matrices (HDC are not possible for this matrix) compared to t = 0 controls, it can be assumed that these findings are not due to hydrolytic degradation of pyranyl acetate pure in gastric fluid simulant. This assumption is confirmed by the analysis of pyranol that was not detectable in any of the samples measured. Therewith, these results are assessed to demonstrate that the metabolism of pyranyl acetate pure in gastric fluid simulant is negligible under the test conditions used.

POSITIVE CONTROLS
Benzyl benzoate was used as a positive control in incubates of rat liver S9, plasma and intestinal fluid simulant and fluorescein diacetate was used as positive control in incubates of rat skin S9 fraction. Positive controls were used to demonstrate the validity of the applied in vitro systems and the chosen incubation conditions. Benzyl benzoate, incubated at 250 μM (phosphate buffer, 37 °C for 2 h) was metabolized in rat liver S9 fraction to an extent of 100 % (mean value of two incubations). The metabolic turn over after shorter incubation periods of 0.5 and 1 h ranged from about 99 to 100 % and demonstrates that metabolic degradation of benzyl benzoate was already virtually complete after an incubation period of 0.5 h. Recoveries of t = 0 controls ranged between 105 and 101 % related to the measured buffer controls, HDC yielded values of 49 and 55 % of buffer controls. In rat plasma, benzyl benzoate, incubated at 250 μM (37 °C for 2 h) was metabolized to an extent of about 92 % (mean value of two incubations). Since no buffer control was performed for plasma incubations, no recoveries can be calculated for this in vitro system. However, compared to peak areas of buffer controls of liver S9 fraction and intestinal fluid simulant, peak areas can be assessed to be generally comparable. In intestinal fluid simulant, benzyl benzoate, incubated at 250 μM (37 °C for 2 h) was metabolized to an extent of 70 % (mean value of two incubations). Recoveries of t = 0 controls ranged between about 115 and 110 % related to the measured buffer controls, HDC yielded values of 63 and 47 % of buffer controls. Fluorescein diacetate, incubated at 250 μM (phosphate buffer, 37 °C for 2 h) was metabolized in rat skin S9 fraction to an extent of 86 % (mean value of two incubations). Recoveries of t = 0 controls ranged between 98 and 107 % related to the measured buffer controls, HDC yielded values of 62 and 44 % of buffer controls.
In conclusion, recoveries of positive control incubations of the t = 0 controls are generally between 80 and 120 % and are assessed to be acceptable. The lower recoveries of HDC controls with values below 80 % should be mentioned here. These values are below the internally defined threshold of 80 % for unknown reasons. However, based on received analytical results in active incubates compared to t=0 but also HDC, the stability of appropriate controls and a significant metabolic turn over of positive controls in the test system was demonstrated in principle and therewith the validity of liver and skin S9 fraction, plasma and intestinal fluid simulant including the chosen incubation conditions.

Applicant's summary and conclusion