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Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March-June 2013
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:
2013
Report date:
2013

Materials and methods

Objective of study:
other: hydrolysis and degradation of geranylacetate extra in plasma, liver and gastrointestinal tract
Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
The hydrolysis and degradation of Geranylacetat Extra in plasma, liver and gastrointestinal tract was investigated. To determine hydrolysis in either compartment, the test substance was incubated with plasma and liver-S9 fraction from rat as well as in gastric-juice simulant and intestinal-fluid simulant. After incubation, the proteins were precipitated and the amount of remaining substrate was analysed in the supernatant by GC/FID.
GLP compliance:
yes (incl. QA statement)

Test material

Test material form:
liquid
Details on test material:
CAS 105-87-3
Radiolabelling:
no

Test animals

Species:
other: not applicable; in vitro test

Administration / exposure

Statistics:

For calculation of the hydrolysis (turn over), peak areas of the substrates of active incubation (AI) and heat deactivated control (HDC) were used. Buffer controls were used to calculate the substrate recovery of the HDC and t=0 control.

Calculation of the turn over in plasma, liver- S9 fraction and intestine-fluid simulant:
% turn-over = 100 * (peak area (HDC) – peak area (AI)) / peak area (HDC)

Calculation of the turn-over in gastric-juice simulant:
% turn-over = 100 * (peak area (t=0) – peak area (AI)) / peak area (t=0)

Results and discussion

Any other information on results incl. tables

Geranylacetate Extra hydrolyzed within 0.5h almost completely in rat plasma, liver S9 fraction of rats and intestinal- fluid simulant under the test conditions used. Hydrolysis was demonstrated by the decrease of Geranylacetate Extra as well as by the formation of its hydrolysis product Geraniol Extra. Although the recovery of Geranylacetate Extra was tentatively low in respective controls, the quantitative amount of Geraniol Extra formed was correlated to the nominal substrate concentration of Geranylacetate in incubates and proved a high metabolic turn over. It is assumed that the low recoveries of Geranylacetate in HDC controls are based on potential evaporation of the test substance. In t=0 controls, low recoveries are due to partial hydrolysis, since Geraniol Extra is generally already detectable

in these controls, proving that hydrolysis was faster than the stopping process after addition of the test substance to the system. In gastric fluid simulant, the degradation of Geranylacetate Extra was slower and was calculated to be about 50 % after an incubation period of 2h. The degradation of Geranylacetate Extra yielded maximum degradation rates of ≥211.8 [μmol/L*h], ≥2.7 [μM/g liver equivalent*h], ≥21.3 [μM/g pancreas lipase (15-35 U/mg)*h] and 93.2 [μmol/L*h] for rat plasma, liver S9 fraction of rats, intestinal fluid simulant containing 1 weight% pancreas lipase and gastric fluid simulant, respectively. These maximum degradation rates of Geranylacetate Extra were in the same order of magnitude than the maximum degradation rates determined for the applied positive control Benzyl benzoate and demonstrate a fast hydrolysis of Geranylacetate Extra in gastrointestinal tract, liver and plasma.

Applicant's summary and conclusion

Conclusions:
The current in vitro data demonstrate that Geranylacetate Extra is hydrolyzed within 0.5 h to Geraniol almost completely in rat plasma, liver S9 fraction of rats and intestinal- fluid simulant under the test conditions used. In gastric fluid simulant the degradation of Geranylacetate Extra was slower and was calculated to be about 50 % after an incubation period of 2 h. The degradation of Geranylacetate Extra yielded maximum degradation rates of ≥211.8 [μmol/L*h], ≥2.7 [μM/g liver equivalent*h], ≥21.3 [μM/g pancreas lipase (15-35 U/mg)*h] and 93.2 [μmol/L*h] for rat plasma, liver S9 fraction of rats, intestinal fluid simulant containing 1 weight% pancreas lipase and gastric fluid simulant, respectively.
These maximum degradation rates of Geranylacetate Extra were in the same order of magnitude as the maximum degradation rates determined for the positive control Benzyl benzoate.
Executive summary:

To determine hydrolysis in either compartment, the test substance was incubated in duplicates at a nominal concentration of 250 μM for 0.5, 1 and 2h at 37°C in plasma, liver S9 fraction of rats, gastric-juice simulant and intestinal-fluid simulant including pancreas lipase. After incubation, proteins in incubates were precipitated by the addition of one volume acetone and the amount of remaining

substrate was analysed in the supernatant by GC/FID. Heat deactivated controls (HDC, with the exception of gastric juice simulant) and controls, directly stopped after addition of test substance (t=0 control) served as control samples. A buffer control or medium (BC, test substance in the incubation buffer; MC, test substance in the incubation medium) was used for calculation of recoveries in the HDC and t=0 controls. Additionally recovery of controls was related to the target concentration of the test substance in the in vitro incubate (250 μM).

Positive controls were performed with Benzyl benzoate at a concentration of 250μM. In rat plasma and liver S9 fraction of rats, Benzyl benzoate was hydrolyzed after 0.5 h almost completely, resulting in a metabolic turnover of 97 and 99%, respectively. In the in vitro systems of gastrointestinal tract, hydrolysis was slower, yielding maximum metabolic turn over values of 43 and 51 % after 2h for intestine fluid simulant and gastric fluid simulant, respectively. The maximum degradation rates of Benzyl benzoate were ≥182.3 [μmol/L*h], ≥4.9 [μM/g liver equivalent*h], 27.6 [μM/g pancreas lipase (15-35 U/mg)*h] and 84.6 [μmol/L*h] for rat plasma, liver S9 fraction of rats, intestinal fluid simulant containing 1 weight% pancreas lipase and gastric fluid simulant, respectively. Based on these results, the validity of the applied in vitro systems as well as of the chosen incubation conditions was clearly demonstrated.

The current in vitro data demonstrate that Geranylacetate Extra is hydrolyzed within 0.5 h to Geraniol almost completely in rat plasma, liver S9 fraction of rats and intestinal- fluid simulant under the test conditions used. In gastric fluid simulant the degradation of Geranylacetate Extra was slower and was calculated to be about 50 % after an incubation period of 2 h. The degradation of Geranylacetate Extra yielded maximum degradation rates of ≥211.8 [μmol/L*h], ≥2.7 [μM/g liver equivalent*h], ≥21.3 [μM/g pancreas lipase (15-35 U/mg)*h] and 93.2 [μmol/L*h] for rat plasma, liver S9 fraction of rats, intestinal fluid simulant containing 1 weight% pancreas lipase and gastric fluid simulant, respectively.

These maximum degradation rates of Geranylacetate Extra were in the same order of magnitude as the maximum degradation rates determined for the positive control Benzyl benzoate.

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